Temperature sensitive mutants of matrix metalloproteases and uses thereof

Information

  • Patent Application
  • 20100284995
  • Publication Number
    20100284995
  • Date Filed
    March 05, 2010
    14 years ago
  • Date Published
    November 11, 2010
    14 years ago
Abstract
Provided are modified matrix metalloprotease (MMP) enzymes that exhibit temperature-dependent activity and uses thereof. The MMPs can be used, for example, to treat ECM-mediated diseases or disorders characterized by increased deposition or accumulation of one or more ECM components.
Description

The subject matter of the above-noted related applications is incorporated by reference in its entirety.


Incorporation by Reference of Sequence Listing Provided on Compact Discs

An electronic version on compact disc (CD-R) of the Sequence Listing is filed herewith in duplicate (labeled Copy #1 and Copy #2), the contents of which are incorporated by reference in their entirety. The computer-readable file on each of the aforementioned compact discs, created on Mar. 5, 2010, is identical, 12.8 megabytes in size, and titled 3077SEQ.001.txt.


FIELD OF THE INVENTION

Provided are modified matrix metalloprotease (MMP) enzymes that exhibit temperature-dependent activity and uses thereof. MMPs having a controlled duration of action can be used, for example, to treat ECM-mediated diseases or disorders characterized by increased deposition or accumulation of one or more ECM components.


BACKGROUND

The extracellular matrix (ECM) provides a critical structural support for cells and tissues. Defects or changes in the extracellular matrix as a result of excessive deposition or accumulation of ECM components can lead to ECM-mediated diseases or conditions. Among these are collagen-mediated diseases or conditions characterized by the presence of abundant fibrous septae of collagen. Often the only approved treatment for such diseases or conditions is surgery, which can be highly invasive. Other treatments, such as needle aponeurotomy for the treatment of Dupuytren's syndrome or liposuction for cellulite, also are highly invasive. Bacterial collagenase (also called matrix metalloproteinase-1; MMP-1), an enzyme active at neutral pH that degrades collagen, has been used to treat ECM-mediated conditions such as cellulite (see e.g., published U.S. application serial No. US20070224184); Dupuytren's syndrome (see e.g. U.S. Pat. Nos. RE39941; 5,589,171; 6,086,872); and Peyronie's disease (see e.g., U.S. Pat. No. 6,022,539). Collagenase, however, irreversibly cleaves collagens of type I, II and III. Bacterial collagenase also cleaves type IV collagen, associated with blood vessels, and thus its administration can cause haemorrhage and leaky blood vessels. The prolonged activity of collagenase limits the dosages that can be administered and also risks side effects associated with prolonged activity. Hence, there is a need for alternative treatments of ECM-mediated diseases and conditions. Accordingly, it is among the objects herein to provide alternatives for the treatment of ECM-mediated diseases and conditions.


SUMMARY

Provided are modified matrix metalloprotease (MMP) enzymes and their use, among others, for treating ECM-mediated diseases or conditions. The enzymes include modified MMPs that are modified to exhibit activity at temperatures different from the unmodified enzymes. Hence, provided are temperature-sensitive mutants of MMP. In particular, the mutants are more active at a lower temperature then a higher temperature and typically are substantially inactive at the higher temperature. For example, the mutants are more active at a temperature that is or is about 25° C. then at a higher temperature that is or is about between 34° C. to 37° C. The mutants also retain an activity of the unmodified enzyme at the lower temperature.


Hence, provided herein are modified matrix metalloproteases (MMP). The MMPs contain one or more modification(s) in the sequence of amino acid residues of an MMP polypeptide or modifications in an allelic or species variant of the MMP, or modifications in a mature form thereof, or a catalytically active fragment of the MMP. The modifications, which are in the primary amino acid sequence, include amino acid replacement(s), insertion(s), deletion(s) and combinations thereof. The MMP can include only one modification, only 2, only 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more replacements. The modification be effected on a wildtype MMP, or on an MMP already modified for some other purpose or activity or already mutated. The modification(s) provided herein, confer to the MMP, allelic or species variant thereof or an active fragment thereof, a ratio of enzymatic activity at a permissive temperature compared to at a nonpermissive temperature of at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 20.0, 30, 40, 50, 60, 70, 80, 90, 100 or more. The MMP can include only 1, only 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more replacements to confer a specified ratio of enzymatic activity.


In some embodiments, the modified MMP polypeptide can retain the modified activity of a wildtype MMP at the permissive temperature. For example, it can retain or exhibit at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 140%, 150% or more activity.


The modified MMPs include, but are not limited to, collagenases, gelatinases, stromelysins, matrilysins, metalloelastases, enamelysins and membrane-type MMPs, allelic or species variants thereof and active fragments thereof that include such modification. Exemplary MMPs, include those listed in the Tables herein, such as MMP-1 (collagenase-1), MMP-8 (collagenase-2), MMP-13 (collagenase-3), MMP-18 (collagenase-4), MMP-2 (gelatinase A), MMP-9 (gelatinase B), MMP-3 (stromelysin-1), MMP-10 (stromelysin-2), MMP-11 (atromelysin-3; stromelysin-3), MMP-7 (matrilysin), MMP-26 (matrilysin-2), MMP-12 (metalloelastase), MMP-14 (MT1-MMP), MMP-15 (MT2-MMP), MMP-16 (MT3-MMP), MMP-17 (MT4-MMP), MMP-24 (MT5-MMP), MMP-25 (MT6-MMP), MMP-20 (enamelysin), MMP-19, MMP-21, MMP-23, CA-MMP, MMP-27, CMMP and MMP-28 (epilysin). These include allelic variants and species variants as well as active fragments thereof. The allelic and species variants contain the corresponding modification, which readily can be identified, such as by alignment. The active fragment, includes at least one such modification.


The modified MMPs include those that have lower activity at the nonpermissive temperature than the MMP that does not include the modification at the nonpermissive temperature. The permissive temperature can be lower or higher than the nonpermissive temperature. The modified MMPs can have altered activity compared to the unmodified MMP. The activity can be reduced, such as less than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 1% or less than the activity of the unmodified MMP. The activity also can be increased, such as by the same percentages. Permissive temperatures include, but are not limited to, 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C. or about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C., such as at or about 25° C. Nonpermissive temperatures include, but are not limited to, 34° C., 35° C., 36° C., 37° C., 38° C. or 39° C. or about 34° C., 35° C., 36° C., 37° C., 38° C. or 39° C. For example, in one embodiment, the nonpermissive temperature is or is about 34° C. or 37° C. and the permissive temperature is 25° C. or about 25° C.


In some embodiments, only a catalytically active fragment is provided or used in any of the methods herein. The catalytically active fragment can be linked, such as fusion protein or chemical conjugate to additional amino acids derived from a different protein, or to another moiety, such as a therapeutic agent. When a catalytically active fragment, such as a catalytic domain is provided, it contains at least one of the amino acid replacements that confer the ratio of enzymatic activity.


Provided herein are modified MMP-1 polypeptides. Exemplary modified MMP-1 polypeptides are any provided herein having a sequence of amino acids set forth in any of SEQ ID NOS:3-705, 779-3458, 3507-3536 or allelic or species variants thereof, zymogen forms, mature forms, or catalytically active fragments thereof.


Among the modified MMPs provided herein that contain a modification that confers a ratio as noted above, are those in which the modification is an amino acid replacement(s), and the replacement(s) is at a position corresponding any one or more positions 84, 85, 95, 98, 99, 100, 103, 104, 105, 106, 109, 110, 111, 112, 118, 123, 124, 126, 147, 150, 151, 152, 153, 155, 156, 158, 159, 170, 171, 176, 178, 179, 180, 181, 182, 183, 185, 187, 188, 189, 190, 191, 192, 194, 195, 197, 198, 206, 207, 208, 210, 211, 212, 218, 223, 227, 228, 229, 230, 233, 234, 237, 240, 251, 254, 255, 256, 257 and 258 in an MMP-1 polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:2 or in corresponding residues in an MMP polypeptide. As described herein, corresponding residues can be identified, for example, using standard alignment programs among proteins with substantial homology.


In particular, provided are modified MMP-1 polypeptides, where the unmodified MMP-1 polypeptide contains the sequence of amino acids set forth in SEQ ID NO:2 or is an allelic or species variant thereof or a mature form thereof that contains an amino acid replacement. Such modifications include, but are not limited to, T84F, E85F, L95K, L95I, R98D, 199Q, E100V, E100R, E100S, E100T, E100F, E100I, E100N, T103Y, P104A, P104M, D105A, D105F, D105G, D105I, D105L, D105N, D105R, D105S, D105T, D105W, D105E, L106C, L106S, A109H, D110A, V111R, D112S, A118T, S123V, N124D, T126S, G147P, R150P, R150V, R150D, R150I, R150H, D151G, N152A, N152S, S153T, F155L, F155A, D156H, D156L, D156A, D156W, D156V, D156K, D156T, D156R, D156M, P158T, P158G, P158K, P158N, G159V, G159T, G159M, G159I, G159W, G159L, G159C, P170D, P170A, G171P, G171E, G171D, A176F, A176W, F178T, F178L, D179N, D179V, D179C, E180Y, E180R, E180T, E180F, E180G, E180S, E180N, E180D, E181T, D181L, D181K, D181C, D181G, E182T, E182Q, E182M, E182G, E183G, R183S, T185R, T185Y, T185H, T185G, T185V, T185Q, T185A, T185E, T185D, N187R, N187M, N187W, N187F, N187K, N187I, N187A, N187G, N187C, N187H, F188V, R189N, R189T, R189Q, E190G, E190Y, E190D, Y191V, N192H, N192S, N192D, N192C, H194P, R195C, R195W, R195L, R195G, R195Q, R195A, R195D, R195V, A197V, A197C, A198G, A198L, A198M, G206A, G206S, L207R, L207V, L207I, L207G, S208R, S208L, S210V, S210A, T211L, D212G, D212H, Y218S, F223C, F223E, F223G, F223A, F223S, F223K, F223M, V227C, V227D, V227E, V227L, V227S, V227W, V227G, V227H, V227Q, V227R, Q228P, L229A, L229T, L29I, A230V, D233E, I234A, I234T, I234E, I234Q, I237L, I237W, I237N, I240S, I240A, I240C, I251S, I251W, Q254S, T255H, P256C, K257P, K257T and A258P, such as L95K, D105I, D105N, D105L, D105A, D105G, R150P, D156R, D156H, D156K, D156T, G159V, G159T, D179N, E180T, E180F, E182T, T185Q, N187I, A198L, V227E, I234E and I240S, or L95K, D105N, R150P, D156K, D156T, G159V, D179N, E180T, A198L, V227E, and I240S.


Other modified MMP polypeptides are those where the modification is an amino acid replacement(s) and the replacement(s) is at a position corresponding any one or more of positions 95, 105, 150, 151, 155, 156, 159, 176, 179, 180, 181, 182, 185, 187, 195, 198, 206, 210, 212, 218, 223, 227, 228, 229, 230, 233, 234, and 240 in an MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2 or in corresponding residues in an MMP polypeptide; where the modification(s) confers to the MMP, allelic or species variant thereof or an active fragment thereof, a ratio of enzymatic activity at a permissive temperature compared to at a nonpermissive temperature of at least 1.5. Such modifications, with reference to MMP-1, include, but are not limited to, L95K, D105A, D105F, D105G, D105I, D105L, D105N, D105R, D105S, D105T, D105W, R150P, D151G, F155A, D156K, D156T, D156L, D156A, D156W, D156V, D156H, D156R, G159V, G159T, A176F, D179N, E180Y, E180T, E180F, D181L, D181K, E182T, E182Q, T185R, T185H, T185Q, T185A, T185E, N187R, N187M, N187F, N187K, N187I, R195V, A198L, A198M, G206A, G206S, S210V, Y218S, F223E, V227C, V227E, V227W, Q228P, L229T, L229I, D233E, I234A, I234T, I234E, I240S, and I240C.


Other modified MMP polypeptides are those where the modification is an amino acid replacement(s) and the replacement(s) is at a position corresponding any one or more positions 95, 105, 150, 156, 159, 179, 180, 182, 185, 187, 195, 198, 212, 223, 227, 234, and 240 in an MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2 or in corresponding residues in an MMP polypeptide; and the modified MMP polypeptide retains at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 140%, 150% or more activity at 25° C. compared to wildtype MMP-1 at 25° C. This includes modified MMP polypeptides where a modification is selected from among L95K, D105A, D105G, D105I, D105L, D105N, D105S, D105W, D105T, R150P, D156K, D156T, D156V, D156H, D156R, G159V, G159T, D179N, E180Y, E180T, E180F, E182T, T185H, T185Q, T185E, N187M, N187K, N187I, R195V, A198L, F223E, V227E, I234E and I240S.


Among the modified MMP polypeptides are those in which the activity of the polypeptide is reversible upon exposure to the nonpermissive temperature, such as, for example, where upon exposure to the nonpermissive temperature and return to the permissive temperature the polypeptide exhibits at or about 120%, 125%, 130%, 140%, 150%, 160%, 170%, 180%, 200% or more or the activity compared to at the nonpermissive temperature. These include modified MMP polypeptides where the modification is an amino acid replacement(s) and the replacement(s) is at a position corresponding to any one or more positions D105A, D105F, D105G, D105S, D105T, R150P, G159T, E180Y, E180T, E180F, T185H, T185Q, T185A, T185E, N187R, N187M, N187K, R195V, A198L, A198M, S210V, Y218S, F223E, V227W, L229I and I240C in an MMP polypeptide.


Among the modified MMP polypeptides are those in which the activity of the polypeptide is irreversibly inactive upon exposure to the nonpermissive temperature, such as for example, modified MMP polypeptides, that, upon exposure to the nonpermissive temperature and return to the permissive temperature the polypeptide, exhibit at or about 50%, 60%, 70%, 80%, 90%, 100%, 105%, 110%, 115%, or less than 120% the activity at the non-permissive temperature. These include, but are not limited to, modified MMP polypeptides with a modification in an MMP polypeptide selected from among L95K, D105I, D105L, D105N, D105R, D105W, D151G, F155A, D156K, D156T, D156L, D156A, D156W, D156V, D156H, D156R, G159V, A176F, D179N, D181L, D181K, E182T, E182Q, T185R, N187F, N187I, G206A, G206S, V227C, V227E, Q228E, L229T, D233E, I234A, I234T, I234E and I240S.


Any of the modified MMP-1 polypeptides provided herein above can further include an activity mutation, whereby the mutations confers increased activity compared to the MMP-1 not containing the modification. For example, such a modified MMP-1 polypeptide can include amino acid replacement(s) at a position corresponding to any one or more of positions 81, 84, 85, 86, 87, 89, 104, 105, 106, 107, 108, 109, 124, 131, 133, 134, 135, 143, 146, 147, 150, 152, 153, 154, 157, 158, 160, 161, 164, 166, 167, 180, 183, 189, 190, 207, 208, 211, 213, 214, 216, 218, 220, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 235, 236, 238, 239, 244, 249, 254, 256, 257 and 258 in an MMP-1 polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:2. For example, amino acid replacement can be F81L, F81A, F81G, F81Q, F81R, F81H, T84H, T84L, T84D, T84R, T84G, T84A, E85S, E85V, G86S, N87P, N87R, N87G, N87Q, R89A, R89T, R89G, R89K, P104E, P104D, P104Q, D105V, L106V, P107T, P107S, P107A, R108E, R108A, R108K, R108S, A109S, A109R, A109G, A109M, A109V, N124G, T131D, K132R, V133T, V133L, S134E, S134D, E135M, S143I, R146S, G147R, G147F, R150E, R150G, R150M, T150T, R150A, R150N, R150K, R150L, R150V, R150D, N152G, N152F, N152L, N152I, S153T, S153P, S153F, S153D, S153Y, P154S, P154I, G157F, P158V, P158I, G160Q, N161L, N161R, N161Y, N161E, N161T, N161I, N161V, N161F, N161Q, H164S, F166W, Q167R, Q167A, Q167S, Q167F, Q167P, Q167T, Q167V, Q167M, E180D, R183S, R189N, R189T, R189Q, E190D, L207M, S208K, S208R, S208L, T211N, I213G, G214L, G214E, L216I, Y218W, S220R, S220A, S220Q, S220T, S220G, S220M, S220V, S220N, T222R, T222P, T222S, T222F, T222N, F223Y, F223H, S224Q, S224K, S224D, G225Q, G225E, G225H, D226S, D226E, D226P, D226I, V227T, Q228A, Q228D, Q228E, Q228G, Q228H, Q228K, Q228L, Q228M, Q228N, Q228R, Q228S, Q228T, Q228W, Q228Y, L229Q, L229P, L229V, A230G, A230W, A230D, A230I, A230S, A230C, A230V, A230T, A230M, A230N, A230H, Q231I, Q231A, Q231F, Q231D, Q231G, Q231V, Q231W, Q231S, Q231H, Q231M, D232H, D232G, D232R, D232P, D232Y, D232S, D232F, D232V, D232K, D232W, D232Q, D232E, D232T, D232L, D235G, D235A, D235L, D235E, D235R, D235Q, D235T, D235N, G236M, G236R, G236S, G236T, G236C, G236K, G236E, G236L, G236N, Q238T, A239S, A239V, A239L, A239I, A239G, A239K, A239H, A239R, S244W, S244Q, Q249W, Q254S, P256S, K257E, K257R, or A258P. Exemplary modified MMP-1 polypeptides containing at least one temperature sensitive mutant and at least one activity mutant include those having amino acid replacements S208K/G159V; S208K/D179N; S208K/V227E; G214E/G159V; G214E/D179N; and I213G/D179N.


Also provided herein are modified MMP-1 polypeptides that are activity mutants, whereby the modified MMP-1 polypeptide exhibits increased activity compared to the MMP-1 not containing the modification. Exemplary activity mutants are any having an amino acid replacement in the above paragraph, and further herein in Section D.2.


MMPs that can be modified include, but are not limited to, MMP-1, MMP-8, MMP-13, MMP-18, MMP-2, MMP-9, MMP-3, MMP-10, MMP-7, MMP-6, MMP-12, and allelic or species variants, mature forms, or catalytically active fragments thereof. Exemplary modified MMPs include any in which the unmodified MMP polypeptide has a sequence of amino acids set forth in any of SEQ ID NOS:1, 711, 714, 717, 720, 723, 726, 729, 732, 735, 738, 741, 744, 747, 750, 753, 756, 759, 762, 765, 768, 771, 774 or 777, zymogen forms, allelic or species variants thereof or active fragments thereof. Such modified MMPs can have a modification at a corresponding position in the MMP compared to any of the modifications in MMP-1 provided herein. Exemplary of such corresponding positions are set forth in FIGS. 2 and 3, and exemplary mutations set forth in Section D herein. These include, for example polypeptides containing amino acid replacement(s) at a position corresponding to any two or more positions 95, 105, 151, 156, 159, 176, 179, 180, 181, 182, 185, 195, 198, 206, 210, 212, 218, 223, 228, 229, 233, 234, and 240 in an MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2 or in corresponding residues in an MMP polypeptide.


Provided are modified MMP polypeptides with two or more modifications, where at least one of the modifications confers the ratio, or where two do so, or more do so. The modified MMP polypeptides can contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modifications. Some or all of these can confer or contribute to a desired ratio of activity between the permissive and non-permissive temperature. Exemplary of modified MMP polypeptides are those that contain two or more amino acid replacement(s) and the replacement(s) are at a position corresponding to any two or more of positions 95, 105, 150, 156, 159, 179, 180, 182, 185, 187, 198, 227, 234 and 240 in an MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2 or in corresponding residues in an MMP polypeptide, such as, for example, where the two or more modifications in an MMP polypeptide are selected from among L95K, D105N, R150P, D156K, D156T, G159V, D179N, E180T, A198L, V227E, and I240S, or any where the two or more modifications in an MMP polypeptide are selected from among any set forth in Table 15.


As noted, the modified MMP polypeptide can be a zymogen, an active enzyme, can contain only a catalytically active fragment, such as the catalytic active domain, or can lack all or a portion of a proline rich linker and/or a hemopexin domain.


The modified MMP polypeptides can contain one or more additional modifications in addition to those that confer the activity ratio, such as, but not limited to, modifications that confer increased stability, increased half-life, altered substrate specificity and/or increased resistance to inhibitors. For example, the modified MMP polypeptide can be glycosylated as expressed or can be modified to be glycosylated, or can contain other modifications, such as PEGylation. The modified MMP polypeptide can be a fusion protein with another protein, such as an Fc fusion, or it can be provided as a dimer or a heterodimer or other multimer.


Also provided are nucleic acid molecules and/or vectors that encode any of the modified MMP polypeptides. Vectors include prokaryotic, viral and eukaryotic vectors, including mammalian vector and yeast vectors, such as, for example, adenovirus, an adeno-associated virus, a retrovirus, a herpes virus, a lentivirus, a poxvirus, a cytomegalovirus and Pichia vectors and artificial chromosomes. Cells, including prokaryotic, such as bacterial and algael cells, and eukaryotic, such as mammalian cells, containing the vectors are provided. The cells can express the modified MMP polypeptide, which can be encoded by nucleic acid that directs its secretion or trafficking to other loci in a cell. Methods for producing the MMPs by expressing the encoded MMP in a cell are provided. The MMPs provided herein can be provided in lyophilized or other dried or non-liquid forms.


Also provided are compositions, including pharmaceutical compositions, containing any or mixtures of the modified MMP polypeptides. The pharmaceutical compositions can be formulated for treatment of any disease amenable to treatment by an MMP, and particularly in the methods provided herein, for treatment of disease or conditions of the extracellular matrix (ECM). The compositions can be formulated for single dosage administration and contain multiple dosages or can require dilution or addition of other agents. Amounts per dosage, include for example, 10 μg to 100 mg, 50 μg to 75 mg, 100 μg to 50 mg, 250 μg to 25 mg, 500 μg to 10 mg, 1 mg to 5 mg, or 2 mg to 4 mg per dosage.


Also provided are uses of the modified MMPs for treating a disease or condition of the ECM or formulation of a medicament therefore, and methods for treating a disease or condition of the extracellular matrix (ECM), and processes for treating a disease or condition of the ECM. In practicing the methods, the MMP polypeptide or pharmaceutical compositions containing the MMP polypeptide is administered to the ECM with an activator that when administered or provided to the ECM, provides a temperature activating condition for the enzyme such that the MMP is active. The modified MMP polypeptide is more active at a permissive temperature then at the nonpermissive physiologic temperature, and the activating condition is not present in the ECM prior to administration of the activator.


Also provided herein are methods for treating a disease or condition of the ECM by administering to the ECM a modified MMP-1 polypeptide or composition thereof, or other modified MMP, that exhibits temperature sensitivity, whereby the modified MMP-1 exhibits activity at a permissive temperature that is below the physiologic temperature of the body. In the method, the MMP-1 is administered at or below the permissive temperature. The modified MMP-1 can be mixed with a composition that is at or below the permissive temperature immediately before administration or it can be provided in a composition that is at or below the permissive temperature. In the methods, prior to administration, the ECM can be cooled to below the physiological temperature of the body, for example, by using a cold pack administered at the locus of administration of the MMP. Further, conditional activation of the MMP can be controlled for a predetermined time. For example, the ECM can be maintained at below the physiological temperature of the body for a predetermined time.


Also provided herein are methods similar to above, whereby the modified MMP exhibits is active at a permissive temperature that is above the physiologic temperature of the body. Hence, the MMP, when administered at or above the permissive temperature, can be mixed with a composition that is at or above the permissive temperature immediately before administration or it can be provided in a composition that is at or above the permissive temperature. Conditional activation can be achieved by exposure of the locus of administration by heat to warm the ECM. This can be for a predetermined time.


In the methods, uses and processes herein, the MMP can be a zymogen that is processed, such by a processing agent, before administration. Processing agents include, but are not limited to, plasmin, plasma kallikrein, trypsin-1, trypsin-2, neutrophil elastase, cathepsin G, tryptase, chymase, proteinase-3, proteinase-3, urinary plasminogen activator (uPA), an active MMP, 4-aminophenylmercuric acetate (AMPA), HgCl2, N-ethylmaleimide, sodium dodecyl sulfate (SDS), chaotropic agents, oxidized glutathione, reactive oxygen, Au(I) salts, acidic pH and heat. The modified MMP includes any provided herein, including, but are not limited to, modified MMP-1, MMP-2, MMP-3, MMP-7, MMP-10, MMP-26 and MT1-MMP. The processing agent is purified away from the modified MMP polypeptide before administration as can any non-active cleavage products of the MMP polypeptide. The modified MMP polypeptide is administered in an amount to treat the disease or condition under the activating conditions (i.e., during the period when it is exposed to the permissive temperature). The activator can be administered or provided prior to, simultaneously, subsequently or intermittently from the MMP. Exemplary activator include, a hot pack or a cold pack, a hot or cold liquid, buffer or solution, such as provision of the MMP in chilled buffer, wherein the chilled buffer is the activator. The buffer can be chilled to 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C. or more or about any of these temperatures.


Administration can be effected by any suitable route, including but not limited to, subcutaneous, intramuscular, intralesional, intradermal, topical, transdermal, intravenous, oral and rectal administration, such as for example, sub-epidermal administration, including, subcutaneous administration.


The modified MMP polypeptide can be administered simultaneously, intermittently, sequentially or in the same composition with other active agents, such as a pharmacologic agent, including, for example, a small molecule drug compound (i.e., a compound that is not a macromolecule or biomolecule), dispersing agents, anesthetics and vasoconstrictors and combinations thereof. Exemplary of dispersing agents is a hyaluronan-degrading enzyme, such as, for example, a hyaluronidase. Exemplary of hyaluronidases is PH20, such as a soluble truncated form thereof, including, a hyaluronidase that contains or has a sequence of amino acids set forth in SEQ ID NO:3475, or an allelic or species variant or other variant thereof, including those having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 95% 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino acids set forth in SEQ ID NO:3475, such as 91% or greater sequence identity. The hyaluronidase can be one that is glycosylated. The anesthetics include any suitable anesthetic, such as, for example, lidocaine. The vasoconstrictor can be any suitable vasoconstrictor, such as an alpha adrenergic receptor agonist, such as, for example, levonordefrin, epinephrine or norepinephrine. In the methods, the other agent can be administered prior to administration of the MMP.


The ECM component that is affected by the treatment can include, for example, a collagen, an elastin, a fibronectin or a proteoglycan. The component affected depends upon the MMP selected. Where the ECM component is collagen, the collagen can be selected from among type I, type II, type III or type IV collagen. In any embodiment, the MMP is selected to be one that degrades a particular target, such as selection of a collagenase where the target is collagen. Mixtures of MMP can be used to degrade a plurality of ECM components. Diseases and conditions treated include, collagen-mediated diseases or conditions, such as, but not limited to, cellulite, Dupuytren's disease, Peyronie's disease, Ledderhose fibrosis, stiff joints, existing scars, scleroderma, lymphedema and collagenous colitis, herniated discs, stiff joints, such as a frozen shoulder, scars, such as a scar resulting from among surgical adhesions or keloids, hypertrophic scars and depressed scars.


Also provided are combinations of any modified MMP polypeptide provided herein and an activator thereof. Also provided are kits containing the combinations and one or more of a device for administration and, optionally instructions for administration, and other containers and components, such as reducing agents that increase activity, such as for enzyme with free sulfhydryl groups.





BRIEF DESCRIPTION OF THE FIGURES


FIG. 1: FIG. 1 is an alignment of zymogen MMPs, indicating the propeptide, the catalytic domain, linker region, hemopexin domains 1-4, fibronectin type II repeats, the basic region, the cysteine switch, the calcium (Ca) binding sites I and II, and the zinc binding site. The alignment includes zymogen MMPs, including MMP-1 (SEQ ID NO:2), MMP-8 (amino acids 21-467 of SEQ ID NO:711), MMP-13 (amino acids 20-471 of SEQ ID NO:714), MMP-18 (amino acids 18-467 of SEQ ID NO:717), MMP-2 (amino acids 30-660 of SEQ ID NO:720), MMP-9 (amino acids 20-707 of SEQ ID NO:723), MMP-3 (amino acids 18-477 of SEQ ID NO:726), MMP-10 (amino acids 18-476 of SEQ ID NO:729), MMP-11 (amino acids 32-488 of SEQ ID NO:732), MMP-7 (amino acids 18-267 of SEQ ID NO:735), MMP-26 (amino acids 18-261 of SEQ ID NO:738), MMP-12 (amino acids 17-470 of SEQ ID NO:741), and MMP-19 (amino acids 19-508 of SEQ ID NO:765). A “*” means that the residues or nucleotides in that column are identical in all sequences in the alignment, a “:” means that conserved substitutions have been observed, and a “.” means that semi-conserved substitutions are observed.



FIG. 2: FIG. 2 is an alignment of the catalytic domains of exemplary MMPs, indicating exemplary conserved and conservative amino acid residues. It is understood that other conserved and conservative amino acid residues exist between and among MMPs. Thus, this figure and identification of residues is not intended to limit corresponding residues between and among MMPs. The exemplary MMPs include: MMP-1 (amino acids 81-242 of SEQ ID NO:2), MMP-8 (amino acids 101-242 of SEQ ID NO:711), MMP-13 (amino acids 104-248 of SEQ ID NO:714), MMP-18 (amino acids 100-246 of SEQ ID NO:717), MMP-2 (amino acids 110-417 of SEQ ID NO:720), MMP-9 (amino acids 94-425 of SEQ ID NO:723), MMP-3 (amino acids 100-247 of SEQ ID NO:726), MMP-10 (amino acids 99-246 of SEQ ID NO:729), MMP-11 (amino acids 98-228 of SEQ ID NO:732), MMP-7 (amino acids 95-242 of SEQ ID NO:735), MMP-26 (amino acids 90-236 of SEQ ID NO:738), MMP-12 (amino acids 106-247 of SEQ ID NO:741), and MMP-19 (amino acids 98-239 of SEQ ID NO:765). Exemplary conserved and conservative positions between and among MMPs are highlighted.



FIG. 3: FIG. 3 is an alignment similar to that depicted in FIG. 2. In the alignment, exemplary conserved and conservative positions corresponding to MMP-1 activity mutants are highlighted between and among other MMPs.





DETAILED DESCRIPTION
Outline



  • A. Definitions

  • B. Overview—Temperature Sensitive Matrix Metalloproteases

  • C. Matrix Metalloproteases and the Extracellular Matrix
    • 1. The Extracellular Matrix
      • a. Components of the ECM
        • i. Collagens
        • ii. Elastin
        • iii. Fibronectin
        • iv. Glycosaminoglycans (GAGs)
          • 1) Proteoglycans
          • 2) Hyaluronic Acid
      • b. Histology of the Skin
        • i. The Epidermis
        • ii. The Dermis
        • iii. The Hypodermis
      • c. Diseases of the ECM
    • 2. Matrix Metalloproteases
      • a. Function
      • b. Structure and Activation
    • 3. Matrix Metalloprotease 1 (MMP-1)

  • D. Modified Matrix Metalloprotease-1 Polypeptides
    • 1. Temperature-Sensitive Matrix Metalloprotease-1 (tsMMP-1 Mutants)
    •  Exemplary Temperature Sensitive Modifications
    • 2. Matrix Metalloprotease Activity Mutants
    • 3. Combinations
    • 4. Additional Modifications
    • 5. Other MMPs

  • E. Methods of Producing Nucleic Acids Encoding tsMMPs, and Polypeptides Thereof
    • 1. Vectors and Cells
    • 2. Expression
      • a. Prokaryotic Cells
      • b. Yeast Cells
      • c. Insect Cells
      • d. Mammalian Cells
      • e. Plants
    • 3. Purification Techniques

  • F. Preparation, Formulation and Administration of tsMMPs
    • 1. Injectables, solutions and emulsions
    •  Lyophilized Powders
    • 2. Topical Administration
    • 3. Compositions for other routes of administration
    • 4. Activator
    • 5. Combination Therapies
    •  Hyaluronidases

  • G. Packaging and Articles of Manufacture of tsMMPs
    • 1. Single Chamber Apparatus
    • 2. Dual Chamber Apparatus
    • 3. Kits

  • H. Methods of Assessing Activity of tsMMPs
    • 1. Methods of Assessing Enzymatic Activity
    • 2. Methods of Assessing ECM Degradation
      • a. In vitro assays
      • b. In vivo assays
      • c. Non-human animal models

  • I. Exemplary Methods of Treating Diseases or Defects of ECM
    • 1. Collagen-Mediated Diseases or Conditions
      • a. Cellulite
      • b. Dupuytren's Disease
      • c. Peyronie's Disease
      • d. Ledderhose Fibrosis
      • e. Stiff Joints
      • f. Existing Scars
        • i. Surgical Adhesions
        • ii. Keloids
        • iii. Hypertrophic scars
        • iv. Depressed Scars
      • g. Scleroderma
      • h. Lymphedema
      • i. Collagenous colitis
    • 2. Spinal Pathologies

  • J. Examples



A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, Genbank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the interne can come and go, but equivalent information can be found by searching the interne. Reference thereto evidences the availability and public dissemination of such information.


As used herein, the extracellular matrix (ECM) refers to a complex meshwork structure that surrounds and provides structural support to cells of specialized tissues and organs. The ECM is made up of structural proteins such as collagen and elastin; specialized proteins such as fibronectin; and proteoglycans. The exact biochemical composition varies from tissue to tissue. In the skin, for example, it is the dermal layer that contains the ECM. Reference to the “interstitium” is used interchangeably herein to refer to the ECM.


As used herein, components of the ECM refers to any material produced by cells of connective tissue and secreted into the interstitium. For purposes herein, reference to ECM components refers to proteins and glycoproteins, and not to other cellular components or other components of the ECM. Exemplary ECM components include, but are not limited to, collagen, fibronectin, elastin and proteoglycans.


As used herein, a matrix degrading enzyme refers to any enzyme that degrades one or more components of the ECM. Matrix-degrading enzymes include proteases, which are enzymes that catalyze the hydrolysis of covalent peptide bonds. Matrix-degrading enzyme include any known to one of skill in the art. Exemplary matrix-degrading enzymes include matrix metalloproteases, allelic or species variants or other variants thereof.


As used herein, a matrix metalloprotease (MMP) refers to a type of matrix degrading enzyme that is a zinc-dependent endopeptidase that contain an active site Zn2+ required for activity. MMPs include enzymes that degrade components of the ECM including, but not limited to, collagen, fibronectin, elastin and proteoglycans. MMPs generally contain a propeptide, a catalytic domain, a proline linker and a hemopexin (also called haemopexin-like C-terminal) domain. Some MMPs contain additional domains. Exemplary MMPs are set forth in Table 5. Reference to an MMP includes all forms, for example, the precursor form (containing the signal sequence), the proenzyme form (containing the propeptide), the processed active form, and forms thereof lacking one or more domains. For example, reference to an MMP refers to MMPs containing only the catalytically active domain. Domains of exemplary MMPs are identified in FIG. 1. MMPs also include allelic or species variants or other variants thereof


As used herein, a modified matrix degrading enzyme or a modified MMP (also interchangeably referred to as a variant or mutant) refers to an enzyme that has one or more modifications in primary sequence compared to a wildtype enzyme. The one or more mutations can be one or more amino acids replacements (substitutions), insertions, deletions, and any combination thereof. A modified enzyme includes those with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modified positions. The modifications can provide altered properties of the enzyme. Exemplary of modifications include those described herein that confer temperature-sensitive activity of the enzyme. Other modifications include those that confer altered substrate specificity, stability and/or sensitivity to inhibitors (e.g. TIMPs). A modified enzyme typically has 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding sequence of amino acids of a wildtype enzyme. Typically, a modified enzyme retains an activity or sufficient activity (e.g. degradation of an ECM component) of a wildtype enzyme. It is understood that modifications conferring temperature sensitivity retain an activity or sufficient activity at the requisite temperature compared to a wildtype enzyme at the physiologic temperature.


As used herein, an activity mutant or mutation or variant or modification refers to a modified enzyme, for example a modified matrix metalloprotease such as a modified MMP-1, that exhibits increased enzymatic activity compared to the enzyme that does not contain the particular modification. For example, the enzyme exhibits 1.2-fold to 100-fold or higher increased enzymatic activity, for example, 1.2-fold, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold or more increased enzymatic activity. It is understood that in determining enzymatic activity, the enzymatic activity of the mutant and the unmodified enzyme (e.g. wildtype) is measured under the same assay conditions. Reference to an activity mutant herein is not dependent on temperature. For example, an activity mutant provided herein can exhibit increased activity compared to the enzyme that does not contain the modification at both the permissive and nonpermissive temperature.


As used herein, a temperature sensitive (ts) mutant or mutation or variant or modification conferring temperature sensitivity refers to a polypeptide that is modified to exhibit higher enzymatic activity at some temperatures called permissive temperatures compared to other temperatures called nonpermissive temperatures. Generally, a temperature-sensitive mutant exhibits higher enzymatic activity at lower temperatures then at higher temperatures.


As used herein, permissive temperature is the temperature at which a polypeptide exhibits a higher enzymatic activity then at a second temperature called the nonpermissive temperature. Hence, the modified enzymes provided herein exhibit different activities at different temperatures that is higher at one temperature then at another temperature. The temperature at which it exhibits more activity is the permissive temperature. For example, the permissive temperature is a temperature that is below the physiological temperature of the body, for example, 18° C. to 30° C., and in particular 20° C. to 25° C. Hence, the enzyme exhibits increased activity at a temperature below the physiological temperature of the body then activity at the physiological temperature of the body, such as exists in the interstitium. For example, the permissive temperature is or is about 18° C. 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C.


As used herein, a nonpermissive temperature is the temperature where a polypeptide exhibits lower enzymatic activity then at the permissive temperature and exhibits reduced activity compared to the enzyme that is not modified. Temperature-sensitive mutants provided herein exhibit enzymatic activity at the nonpermissive temperature that is at or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% up to less then 100% the activity at the permissive temperature. The temperature sensitive mutants provided herein also exhibit 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% up to less then 100% of the activity at the nonpermissive temperature compared to the enzyme that is not modified (e.g. wildtype enzyme) at the nonpermissive temperature. For example, the nonpermissive temperature is a temperature that is near to, at or above the physiological temperature of the body, for example, 32° C. to 39° C., for example, 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., or 39° C.


As used herein, the ratio of enzymatic activity at the permissive temperature compared to the nonpermissive temperature refers to the relation of enzymatic activity at the permissive and nonpermissive temperatures. It is expressed by the quotient of the division of the activity at the permissive temperature by the activity at the nonpermissive temperature. It is understood that in determining enzymatic activity and the ratio of enzymatic activity, the enzymatic activity at the permissive and nonpermissive temperatures is measured under the same assay conditions, except for the difference in temperature.


As used herein, physiological temperature refers to temperature conditions maintained in the body, which is approximately 37° C., for example, at or about 34° C., 35° C., 36° C., 37° C., 38° C. or 39° C. It is understood that the normal range of a human body temperature varies depending on factors such as the rate of metabolism, the particular organ and other factors. For purposes herein, physiological temperature is the temperature that exists for a non-fasting, comfortably dressed subject that is indoors in a room that is kept at a normal room temperature (e.g. 22.7 to 24.4° C.).


As used herein, reversible refers to a modified enzyme whose activity at the permissive temperature is capable of being recovered or partially recovered upon exposure to the nonpermissive temperature and reexposure to the permissive temperature. Hence, the activity of a reversible enzyme once it is exposed to the nonpermissive temperature is the same or substantially retained compared to the activity of the enzyme exposed only to the permissive conditions and is greater then the activity of the enzyme exposed only to the nonpermissive temperature. For example, upon return to permissive conditions from nonpermissive conditions, reversible enzymes exhibit at or about 120%, 125%, 130%, 140%, 150%, 160%, 170%, 180%, 200% or more the activity of the enzyme exposed only to the nonpermissive temperatures and retain the activity of the enzyme exposed only to the permissive temperature.


As used herein, irreversible or nonreversible refers to a modified enzyme whose enzymatic activity at the permissive temperature is not recovered upon exposure to the nonpermissive temperature and reexposure to the permissive temperature. Hence, the activity of an irreversible enzyme once it is exposed to the nonpermissive temperature is less then the activity of the enzyme exposed only to the permissive temperature and also is less then or the same or substantially the same as the activity of the enzyme exposed only to the nonpermissive conditions. For example, upon return to permissive conditions, irreversible enzymes exhibit at or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 105%, 110%, 115%, or 120% the activity at nonpermissive temperatures and less then 100% of the activity at the activity of the enzyme exposed only to the permissive temperature.


As used herein, a domain refers to a portion (a sequence of three or more, generally 5 or 7 or more amino acids) of a polypeptide that is a structurally and/or can form an independently folded structure within a protein made up of one or more structural motifs (e.g. combinations of alpha helices and/or beta strands connected by loop regions) and/or that is recognized by virtue of a functional activity, such as kinase activity. A protein can have one, or more than one, distinct domain. For example, a domain can be identified, defined or distinguished by homology of the sequence therein to related family members, such as homology and motifs that define an extracellular domain. In another example, a domain can be distinguished by its function, such as by enzymatic activity, e.g. kinase activity, or an ability to interact with a biomolecule, such as DNA binding, ligand binding, and dimerization. A domain independently can exhibit a function or activity such that the domain independently or fused to another molecule can perform an activity, such as, for example proteolytic activity or ligand binding. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids from the polypeptide. Many polypeptides contain a plurality of domains. For example, the domain structure of MMPs is set forth in FIG. 1. Those of skill in the art are familiar with domains and can identify them by virtue of structural and/or functional homology with other such domains.


As used herein, a catalytic domain refers to any part of a polypeptide that exhibits a catalytic or enzymatic function. Such domains or regions typically interact with a substrate to result in catalyis thereof. For MMPs, the catalytic domain contains a zinc binding motif, which contains the Zn2+ ion bound by three histidine residues and is represented by the conserved sequence HExxHxxGxxH.


As used herein, a proline rich linker (also called the hinge region) refers to a flexible hinge or linker region that has no determinable function. Such a region is typically is found between domains or regions and contributes to the flexibility of a polypeptide.


As used herein, a hemopexin binding domain or haemopexin-like C-terminal domain refers to the C-terminal region of MMP. It is a four bladed β-propeller structure, which is involved in protein-protein interactions. For example, the hemopexin binding domain of MMPs interact with various substrates and also interact with inhibitors, for example, tissue inhibitor of metalloproteases (TIMPs).


As used herein, consisting essentially of or recitation that a polypeptide consists essentially of a particular domain, for example the catalytic domain means that the only MMP portion of the polypeptide is the domain or a catalytically active portion thereof. The polypeptide optionally can include additional non-MMP-derived sequences of amino acids, typically at least 3, 4, 5, 6 or more, such as by insertion into another polypeptide or linkage thereto.


As used herein, a “zymogen” refers to an enzyme that is an inactive precursor of and requires some change, such as chemical modification or proteolysis of the polypeptide, to become active. Some zymogens also require the addition of co-factors such as, but not limited to, pH, ionic strength, metal ions, reducing agents, or temperature for activation. Zymogens include the proenzyme form of enzymes. Hence, zymogens, generally, are inactive and can be converted to a mature polypeptide by chemical modification or catalytic or autocatalytic cleavage of the proregion from the zymogen in the presence or absence of additional cofactors.


As used herein, a prosegment or proregion or propeptide refers to a region or a segment that is cleaved to produce a mature protein. A propeptide is a sequence of amino acids positioned at the amino terminus of a mature polypeptide and can be as little as a few amino acids or can be a multidomain structure. This can include segments that function to suppress the enzymatic activity by masking the catalytic machinery. Propeptides also can act to maintain the stability of an enzyme.


As used herein, a “processing agent” refers to an agent that activates a MMP by facilitating removal of the propeptide or proregion from the zymogen or inactive form of the enzyme. A processing agent includes chemical agents, proteases and other agents such as acidic pH or heat. Exemplary processing agents include, but are not limited to, trypsin, furin, or 4-aminophenylmercuric acetate (AMPA). Other exemplary processing agents are listed in Table 4.


As used herein, a “catalytically active fragment” refers to a polypeptide fragment that contains the catalytically active domain of the enzyme. It is understood that reference to a catalytically active fragment does not necessarily mean that the fragment exhibits activity, but only that is contains the catalytically active domain or portion thereof that is required for activity. Hence, a catalytically active fragment is the portion that, under appropriate conditions (e.g. permissive temperature), can exhibit catalytic activity. For example, a catalytically active fragment of a tsMMP-1 (containing at least one mutation that confers a temperature sensitive phenotype) exhibits activity when it is provided at the requisite permissive temperature (e.g. 18° C. to 25° C.), but exhibits substantially reduced or no activity at the non-permissive temperature (e.g. physiological temperature of the body).


As used herein, an active enzyme refers to an enzyme that exhibits enzymatic activity. For purposes herein, active enzymes are those that cleave any one or more components of the ECM, such as collagen. Active enzymes include those that are processed from the zymogen form into the mature form.


As used herein, reference to the “mature” form or “processed mature” form of an enzyme refers to enzymes that do not include the prosegment or proregion of the enzyme. It can be produced from the zymogen or pro-enzyme by activation cleavage in which a prosegment or proregion of the proenzyme is processed to produce the mature form. Hence, a processed mature enzyme lacks the sequence of amino acids that correspond to the prosegment or proregion. It is understood that reference to a processed mature form of an enzyme includes synthetic sequences, and thus does not necessarily require that the enzyme actually is processed to remove the prosegment or proregion. It is understood that any MMP enzyme that lacks the prosegment or proregion sequence is a mature enzyme. For example, SEQ ID NO:709 is the mature sequence of MMP-1. The processed mature form of an enzyme can exhibit activity, and is thus an active enzyme, under appropriate conditions. For example, under physiological conditions, the mature form of MMP-1 is an active enzyme. In contrast, tsMMP-1 variants provided herein exhibit enzymatic activity at the permissive temperature of 18° C. to 25° C. and substantially reduced or no activity at the physiological temperature of the body.


As used herein, an activating condition refers to any physical condition or combination of conditions that is required for an enzyme's activity. For purposes herein, an activating condition for an activatable matrix-degrading enzyme (AMDE), for example, a matrix metalloprotease (MMP) includes those that are not present at the site of administration, for example, not present in the extracellular matrix, in amounts (i.e. quantity, degree, level or other physical measure) required for activation of the enzyme. Exemplary of activating conditions include temperature. For example, in the case of the interstitium, the physiological temperature is at or about 37° C. An activating condition is a temperature that is not at or about 37° C., but that is cooler or warmer. By virtue of the fact that the activating condition is not present at the site of administration of the enzyme, but must be added exogenously, the activating condition will dissipate over time as the temperature adjusts, such that the activating condition is no longer present to activate the enzyme. Hence, the enzyme will be active for a limited or predetermined time upon administration.


As used herein, an activator refers to any composition or other material or item that provides an activating condition for an activatable matrix-degrading enzyme. For purposes herein, an activator refers to any item that is capable of providing a temperature condition at the permissive temperature of the enzyme. Examples of activators include, but are not limited to hot or cold buffers or hot or cold packs.


As used herein, an “activatable matrix-degrading enzyme (AMDE)” refers to a matrix degrading enzyme that requires an activating condition in order to be active. For purposes herein, for example, an AMDE is substantially inactive in the ECM unless exposed to activators before, with or subsequent to administration of the AMDE, thereby providing an activating condition for the enzyme. Hence, activation of a activatable enzymes is controlled by exogenous conditions so that the period of time at an in vivo locus or site during which the enzyme is active can be predetermined and/or controlled as a result of the dissipation and/or neutralization of the activation condition (i.e. temporally controllable or time-controlled). Thus, by virtue of exposure to an activating condition, the enzymes are active for a limited time and/or to a limited extent in the ECM (i.e. are conditionally active). The extent and time of activation can be controlled by selection of activator or activating conditions, and can be for a predetermined time. For example, temperature sensitive enzyme, such as a tsMMP, is activatable in that it can be activated by exposure to the activating condition of temperature, such as provided by a cold buffer or other liquid solution. Upon administration of the activated enzyme with the activator to the physiologic temperature environment of the ECM, the temperature will adjust to and eventually return to the physiologic temperature in a time period that can be predetermined based upon the initial temperature of the activator, the site of administration, the depth of administration and other factors, such that the enzyme will become inactive or less active.


As used herein, a “therapeutically effective amount” or a “therapeutically effective dose” refers to an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect.


As used herein, an enzyme that is active for a limited time or for limited duration refers to an active enzyme having activity that dissipates and/or is neutralized over time. Thus, by virtue of the absence of an activation condition, the enzyme is rendered inactive.


As used herein, predetermined time means a limited time that is known before and can be controlled. The dissipation and/or neutralization of an activation condition required for an enzyme's activity can be titrated or otherwise empirically determined so that the time required for an active enzyme to become inactive is known. For purposes herein, for example, an enzyme can be active for a predetermined time that is or is about 1 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hour, 3 hour, or 4 hour. The predetermined time can be controlled by the subject or the treating physician, for example, where a cold pack or hot pack is used as the activator. Further, it is understood that reversible enzymes can be re-activated by exposure to permissive conditions, and thereby can be active for an additional predetermined time.


As used herein, sub-epidermal administration refers to any administration that results in delivery of the enzyme under the outer-most layer of the skin. Sub-epidermal administration does not include topical application onto the outer layer of the skin. Examples of sub-epidermal administrations include, but are not limited to, subcutaneous, intramuscular intralesional and intradermal routes of administration.


As used herein, substrate refers to a molecule that is cleaved by an enzyme.


Minimally, a target substrate includes a peptide containing the cleavage sequence recognized by the protease, and therefore can be two, three, four, five, six or more residues in length. A substrate also includes a full-length protein, allelic variant, isoform or any portion thereof that is cleaved by an enzyme. Additionally, a substrate includes a peptide or protein containing an additional moiety that does not affect cleavage of the substrate by the enzyme. For example, a substrate can include a four amino acid peptide, or a full-length protein chemically linked to a fluorogenic moiety.


As used herein, cleavage refers to the breaking of peptide bonds or other bonds by an enzyme that results in one or more degradation products.


As used herein, activity refers to a functional activity or activities of a polypeptide or portion thereof associated with a full-length (complete) protein. Functional activities include, but are not limited to, biological activity, catalytic or enzymatic activity, antigenicity (ability to bind or compete with a polypeptide for binding to an anti-polypeptide antibody), immunogenicity, ability to form multimers, and the ability to specifically bind to a receptor or ligand for the polypeptide.


As used herein, enzymatic activity or catalytic activity or cleavage activity refers to the activity of a protease as assessed in in vitro proteolytic assays that detect proteolysis of a selected substrate.


As used herein, an inactive enzyme refers to an enzyme that exhibits substantially no activity (i.e. catalytic activity or cleavage activity), such as less than 10% of the maximum activity of the enzyme. The enzyme can be inactive by virtue of its conformation, the absence of an activating conditions required for its activity, or the presence of an inhibitor or any other condition or factor or form that renders the enzyme substantially inactive.


As used herein, “retains an activity” refers to the activity exhibited by a modified MMP polypeptide at a particular condition compared to at another condition or to another polypeptide. For example, it is the activity a modified MMP polypeptide exhibits as compared to an unmodified MMP polypeptide of the same form and under the same conditions. It also can be the activity a modified MMP polypeptide exhibits as compared to the modified MMP polypeptide under different conditions, for example, different temperature conditions. Generally, a modified MMP polypeptide that retains an activity exhibits increased or decreased activity compared to an unmodified polypeptide under the same conditions or compared to the unmodified polypeptide under different conditions. For example, the modified MMP polypeptide can retain 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% or more of the enzymatic activity.


As used herein, a human protein is one encoded by a nucleic acid molecule, such as DNA, present in the genome of a human, including all allelic variants and conservative variations thereof. A variant or modification of a protein is a human protein if the modification is based on the wildtype or prominent sequence of a human protein.


As used herein, hyaluronidase refers to an enzyme that degrades hyaluronic acid. Hyaluronidases include bacterial hyaluronidases (EC 4.2.99.1), hyaluronidases from leeches, other parasites, and crustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC 3.2.1.35). Hyaluronidases also include any of non-human origin including, but not limited to, murine, canine, feline, leporine, avian, bovine, ovine, porcine, equine, piscine, ranine, bacterial, and any from leeches, other parasites, and crustaceans. Exemplary non-human hyaluronidases include any set forth in any of SEQ ID NOS: 3482-3505. Exemplary human hyaluronidases include HYAL1 (SEQ ID NO:3469), HYAL2 (SEQ ID NO:3470), HYAL3 (SEQ ID NO:3471), HYAL4 (SEQ ID NO:3472), and PH20 (SEQ ID NO:3473). Also included amongst hyaluronidases are soluble human PH20 and soluble rHuPH20.


Reference to hyaluronidases includes precursor hyaluronidase polypeptides and mature hyaluronidase polypeptides (such as those in which a signal sequence has been removed), truncated forms thereof that have activity, and includes allelic variants and species variants, variants encoded by splice variants, and other variants, including polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the precursor polypeptide set forth any of SEQ ID NO: 3473 or the mature form thereof. Hyaluronidases also include those that contain chemical or posttranslational modifications and those that do not contain chemical or posttranslational modifications. Such modifications include, but are not limited to, PEGylation, albumination, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.


As used herein, soluble human PH20 or sHuPH20 include mature polypeptides lacking all or a portion of the glycosylphospatidylinositol (GPI) attachment site at the C-terminus such that upon expression, the polypeptides are soluble. Exemplary sHuPH20 polypeptides include mature polypeptides having an amino acid sequence set forth in any one of SEQ ID NOS:3476-3481. The precursor polypeptides for such exemplary sHuPH20 polypeptides include an amino acid signal sequence. Exemplary of a precursor is set forth in SEQ ID NO:3473, which contains a 35 amino acid signal sequence at amino acid positions 1-35. Soluble HuPH20 polypeptides can be degraded during or after the production and purification methods described herein.


As used herein, soluble rHuPH20 refers to a soluble form of human PH20 that is recombinantly expressed in Chinese Hamster Ovary (CHO) cells. Soluble rHuPH20 is encoded by nucleic acid that includes the signal sequence and is set forth in SEQ ID NO:3475. Also included are DNA molecules that are allelic variants thereof and other soluble variants. The nucleic acid encoding soluble rHuPH20 is expressed in CHO cells which secrete the mature polypeptide. As produced in the culture medium there is heterogeneity at the C-terminus so that the product includes a mixture of species of SEQ ID NOS:3476-3481. Corresponding allelic variants and other variants also are included. Other variants can have 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NOS:3476-3481 as long they retain a hyaluronidase activity and are soluble.


As used herein, hyaluronidase activity refers to any activity exhibited by a hyaluronidase polypeptide. Such activities can be tested in vitro and/or in vivo and include, but are not limited to, enzymatic activity, such as to effect cleavage of hyaluronic acid, ability to act as a dispersing or spreading agent and antigenicity.


As used herein, the residues of naturally occurring α-amino acids are the residues of those 20 α-amino acids found in nature which are incorporated into protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans.


As used herein, nucleic acids include DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double-stranded. When referring to probes or primers, which are optionally labeled, such as with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that their target is statistically unique or of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids long.


As used herein, a peptide refers to a polypeptide that is from 2 to 40 amino acids in length.


As used herein, the amino acids which occur in the various sequences of amino acids provided herein are identified according to their known, three-letter or one-letter abbreviations (Table 1). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.


As used herein, an “amino acid” is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids include the twenty naturally-occurring amino acids, non-natural amino acids and amino acid analogs (i.e., amino acids wherein the α-carbon has a side chain).


As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are presumed to be in the “L” isomeric form. Residues in the “D” isomeric form, which are so designated, can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243: 3552-3559 (1969), and adopted 37 C.F.R, §§1.821-1.822, abbreviations for amino acid residues are shown in Table 1:









TABLE 1







Table of Correspondence











SYMBOL













1-Letter
3-Letter
AMINO ACID







Y
Tyr
Tyrosine



G
Gly
Glycine



F
Phe
Phenylalanine



M
Met
Methionine



A
Ala
Alanine



S
Ser
Serine



I
Ile
Isoleucine



L
Leu
Leucine



T
Thr
Threonine



V
Val
Valine



P
Pro
proline



K
Lys
Lysine



H
His
Histidine



Q
Gln
Glutamine



E
Glu
glutamic acid



Z
Glx
Glu and/or Gln



W
Trp
Tryptophan



R
Arg
Arginine



D
Asp
aspartic acid



N
Asn
asparagine



B
Asx
Asn and/or Asp



C
Cys
Cysteine



X
Xaa
Unknown or other










It should be noted that all amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase “amino acid residue” is broadly defined to include the amino acids listed in the Table of Correspondence (Table 1) and modified and unusual amino acids, such as those referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, to an amino-terminal group such as NH2 or to a carboxyl-terminal group such as COOH.


As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides.


As used herein, “non-natural amino acid” refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids and include, but are not limited to, the D-isostereomers of amino acids. Exemplary non-natural amino acids are described herein and are known to those of skill in the art.


As used herein, suitable conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p. 224). Such substitutions can be made in accordance with those set forth in TABLE 2 as follows:












TABLE 2







Original residue
Exemplary conservative substitution









Ala (A)
Gly; Ser



Arg (R)
Lys



Asn (N)
Gln; His



Cys (C)
Ser



Gln (Q)
Asn



Glu (E)
Asp



Gly (G)
Ala; Pro



His (H)
Asn; Gln



Ile (I)
Leu; Val



Leu (L)
Ile; Val



Lys (K)
Arg; Gln; Glu



Met (M)
Leu; Tyr; Ile



Phe (F)
Met; Leu; Tyr



Ser (S)
Thr



Thr (T)
Ser



Trp (W)
Tyr



Tyr (Y)
Trp; Phe



Val (V)
Ile; Leu











Other substitutions also are permissible and can be determined empirically or in accord with known conservative substitutions.


As used herein, a DNA construct is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.


As used herein, a DNA segment is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5′ to 3′ direction, encodes the sequence of amino acids of the specified polypeptide.


As used herein, the term polynucleotide means a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated “nt”) or base pairs (abbreviated “bp”). The term nucleotides is used for single- and double-stranded molecules where the context permits. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term base pairs. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide can differ slightly in length and that the ends thereof can be staggered; thus all nucleotides within a double-stranded polynucleotide molecule can not be paired. Such unpaired ends will, in general, not exceed 20 nucleotides in length.


As used herein, “similarity” between two proteins or nucleic acids refers to the relatedness between the sequence of amino acids of the proteins or the nucleotide sequences of the nucleic acids. Similarity can be based on the degree of identity and/or homology of sequences of residues and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those of skill in the art. For example, in one method of assessing sequence similarity, two amino acid or nucleotide sequences are aligned in a manner that yields a maximal level of identity between the sequences. “Identity” refers to the extent to which the amino acid or nucleotide sequences are invariant. Alignment of amino acid sequences, and to some extent nucleotide sequences, also can take into account conservative differences and/or frequent substitutions in amino acids (or nucleotides). Conservative differences are those that preserve the physico-chemical properties of the residues involved. Alignments can be global (alignment of the compared sequences over the entire length of the sequences and including all residues) or local (the alignment of a portion of the sequences that includes only the most similar region or regions).


“Identity” per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exists a number of methods to measure identity between two polynucleotide or polypeptides, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).


As used herein, homologous (with respect to nucleic acid and/or amino acid sequences) means about greater than or equal to 25% sequence homology, typically greater than or equal to 25%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence homology; the precise percentage can be specified if necessary. For purposes herein the terms “homology” and “identity” are often used interchangeably, unless otherwise indicated. In general, for determination of the percentage homology or identity, sequences are aligned so that the highest order match is obtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073). By sequence homology, the number of conserved amino acids is determined by standard alignment algorithms programs, and can be used with default gap penalties established by each supplier. Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.


Whether any two molecules have nucleotide sequences or amino acid sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” or “homologous” can be determined using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48:1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.). Percent homology or identity of proteins and/or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g., Needleman et al. (1970) J. Mol. Biol. 48:443, as revised by Smith and Waterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids), which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.


Therefore, as used herein, the term “identity” or “homology” represents a comparison between a test and a reference polypeptide or polynucleotide. As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference nucleic acid or amino acid sequence of the polypeptide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared, no more than 10% (i.e., 10 out of 100) of the amino acids in the test polypeptide differs from that of the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of a polypeptide or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often by manual alignment without relying on software.


As used herein, an aligned sequence refers to the use of homology (similarity and/or identity) to align corresponding positions in a sequence of nucleotides or amino acids. Typically, two or more sequences that are related by 50% or more identity are aligned. An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.


As used herein, “primer” refers to a nucleic acid molecule that can act as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and a polymerization agent, such as DNA polymerase, RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. It will be appreciated that certain nucleic acid molecules can serve as a “probe” and as a “primer.” A primer, however, has a 3′ hydroxyl group for extension. A primer can be used in a variety of methods, including, for example, polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, RNA PCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′ and 5′ RACE, in situ PCR, ligation-mediated PCR and other amplification protocols.


As used herein, “primer pair” refers to a set of primers that includes a 5′ (upstream) primer that hybridizes with the 5′ end of a sequence to be amplified (e.g. by PCR) and a 3′ (downstream) primer that hybridizes with the complement of the 3′ end of the sequence to be amplified.


As used herein, “specifically hybridizes” refers to annealing, by complementary base-pairing, of a nucleic acid molecule (e.g. an oligonucleotide) to a target nucleic acid molecule. Those of skill in the art are familiar with in vitro and in vivo parameters that affect specific hybridization, such as length and composition of the particular molecule. Parameters particularly relevant to in vitro hybridization further include annealing and washing temperature, buffer composition and salt concentration. Exemplary washing conditions for removing non-specifically bound nucleic acid molecules at high stringency are 0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1% SDS, 50° C. Equivalent stringency conditions are known in the art. The skilled person can readily adjust these parameters to achieve specific hybridization of a nucleic acid molecule to a target nucleic acid molecule appropriate for a particular application. Complementary, when referring to two nucleotide sequences, means that the two sequences of nucleotides are capable of hybridizing, typically with less than 25%, 15% or 5% mismatches between opposed nucleotides. If necessary, the percentage of complementarity will be specified. Typically the two molecules are selected such that they will hybridize under conditions of high stringency.


As used herein, substantially identical to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.


As used herein, it also is understood that the terms “substantially identical” or “similar” varies with the context as understood by those skilled in the relevant art.


As used herein, an allelic variant or allelic variation references any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and can result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides having altered amino acid sequence. The term “allelic variant” also is used herein to denote a protein encoded by an allelic variant of a gene. Typically the reference form of the gene encodes a wildtype form and/or predominant form of a polypeptide from a population or single reference member of a species. Typically, allelic variants, which include variants between and among species typically have at least 80%, 90% or greater amino acid identity with a wildtype and/or predominant form from the same species; the degree of identity depends upon the gene and whether comparison is interspecies or intraspecies. Generally, intraspecies allelic variants have at least about 80%, 85%, 90% or 95% identity or greater with a wildtype and/or predominant form, including 96%, 97%, 98%, 99% or greater identity with a wildtype and/or predominant form of a polypeptide. Reference to an allelic variant herein generally refers to variations n proteins among members of the same species.


As used herein, “allele,” which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide or several nucleotides, and can include substitutions, deletions and insertions of nucleotides. An allele of a gene also can be a form of a gene containing a mutation.


As used herein, species variants refer to variants in polypeptides among different species, including different mammalian species, such as mouse and human.


As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.


As used herein, modification is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively. Methods of Modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.


As used herein, the term promoter means a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5′ non-coding region of genes.


As used herein, isolated or purified polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.


The term substantially free of cellular material includes preparations of proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the term substantially free of cellular material includes preparations of enzyme proteins having less that about 30% (by dry weight) of non-enzyme proteins (also referred to herein as a contaminating protein), generally less than about 20% of non-enzyme proteins or 10% of non-enzyme proteins or less that about 5% of non-enzyme proteins. When the enzyme protein is recombinantly produced, it also is substantially free of culture medium, i.e., culture medium represents less than about or at 20%, 10% or 5% of the volume of the enzyme protein preparation.


As used herein, the term substantially free of chemical precursors or other chemicals includes preparations of enzyme proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. The term includes preparations of enzyme proteins having less than about 30% (by dry weight) 20%, 10%, 5% or less of chemical precursors or non-enzyme chemicals or components.


As used herein, synthetic, with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and/or by chemical synthesis methods.


As used herein, production by recombinant means by using recombinant DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.


As used herein, vector (or plasmid) refers to discrete elements that are used to introduce a heterologous nucleic acid into cells for either expression or replication thereof. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.


As used herein, an expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.


As used herein, vector also includes “virus vectors” or “viral vectors.” Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.


As used herein, operably or operatively linked when referring to DNA segments means that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.


As used herein the term assessing is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of a protease, or a domain thereof, present in the sample, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of the activity. Assessment can be direct or indirect and the chemical species actually detected need not of course be the proteolysis product itself but can for example be a derivative thereof or some further substance. For example, detection of a cleavage product of a substrate, such as by SDS-PAGE and protein staining with Coomasie blue.


As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activities can be observed in in vitro systems designed to test or use such activities. Thus, for purposes herein a biological activity of a protease is its catalytic activity in which a polypeptide is hydrolyzed.


As used herein equivalent, when referring to two sequences of nucleic acids, means that the two sequences in question encode the same sequence of amino acids or equivalent proteins. When equivalent is used in referring to two proteins or peptides, it means that the two proteins or peptides have substantially the same amino acid sequence with only amino acid substitutions that do not substantially alter the activity or function of the protein or peptide. When equivalent refers to a property, the property does not need to be present to the same extent (e.g., two peptides can exhibit different rates of the same type of enzymatic activity), but the activities are usually substantially the same.


As used herein, “modulate” and “modulation” or “alter” refer to a change of an activity of a molecule, such as a protein. Exemplary activities include, but are not limited to, biological activities, such as signal transduction. Modulation can include an increase in the activity (i.e., up-regulation or agonist activity) a decrease in activity (i.e., down-regulation or inhibition) or any other alteration in an activity (such as a change in periodicity, frequency, duration, kinetics or other parameter). Modulation can be context dependent and typically modulation is compared to a designated state, for example, the wildtype protein, the protein in a constitutive state, or the protein as expressed in a designated cell type or condition.


As used herein, a composition refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.


As used herein, a combination refers to any association between or among two or more items. The combination can be two or more separate items, such as two compositions or two collections, can be a mixture thereof, such as a single mixture of the two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related.


As used herein, a kit is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof.


As used herein, “disease or disorder” refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms. Diseases and disorders of interest herein are those involving components of the ECM.


As used herein, an ECM-mediated disease or condition is one where any one or more ECM components is involved in the pathology or etiology. For purposes herein, an ECM-mediated disease or conditions includes those that are caused by an increased deposition or accumulation of one or more ECM component. Such conditions include, but are not limited to, cellulite, Duputyren's syndrome, Peyronie's disease, frozen shoulders, existing scars such as keloids, scleroderma and lymphedema.


As used herein, “treating” a subject with a disease or condition means that the subject's symptoms are partially or totally alleviated, or remain static following treatment. Hence treatment encompasses prophylaxis, therapy and/or cure. Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease. Treatment also encompasses any pharmaceutical use of a modified interferon and compositions provided herein.


As used herein, a pharmaceutically effective agent, includes any therapeutic agent or bioactive agents, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.


As used herein, treatment means any manner in which the symptoms of a condition, disorder or disease or other indication thereof is/are ameliorated or otherwise beneficially altered.


As used herein therapeutic effect means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition. A therapeutically effective amount refers to the amount of a composition, molecule or compound which results in a therapeutic effect following administration to a subject. A therapeutically effective amount effects treatment.


As used herein, the term “subject” refers to an animal, including a mammal, such as a human being.


As used herein, a patient refers to a human subject.


As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic.


As used herein, prevention or prophylaxis refers to methods in which the risk of developing disease or condition is reduced.


As used herein, an effective amount is the quantity of a therapeutic agent necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.


As used herein, unit dose form refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.


As used herein, a single dosage formulation refers to a formulation for direct administration.


As used herein, an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass activatable matrix degrading enzymes contained in articles of packaging.


As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.


As used herein, a “kit” refers to a combination of an activatable matrix-degrading enzyme provided herein and another item for a purpose including, but not limited to, activation, administration, diagnosis, and assessment of a biological activity or property. Kits optionally include instructions for use.


As used herein, a cellular extract or lysate refers to a preparation or fraction which is made from a lysed or disrupted cell.


As used herein, animal includes any animal, such as, but are not limited to primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; ovine, such as pigs and other animals. Non-human animals exclude humans as the contemplated animal. The enzymes provided herein are from any source, animal, plant, prokaryotic and fungal. Most enzymes are of animal origin, including mammalian origin.


As used herein, a control refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be an internal control.


As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a compound, comprising “an extracellular domain” includes compounds with one or a plurality of extracellular domains.


As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 bases” means “about 5 bases” and also “5 bases.”


As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.


As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).


B. OVERVIEW
Temperature Sensitive Matrix Metalloproteases and Other Modified Metalloproteases

Provided herein are modified MMP polypeptides, for example temperature sensitive (ts) mutants of matrix metalloproteases (tsMMPs), that degrade one or more components of the extracellular matrix (ECM). The tsMMPs can degrade one or more components of the ECM in a temperature-dependent manner. In particular, mutants provided herein degrade a collagen. In some examples, the mutants display higher activity at lower temperatures (e.g. 25° C.) then at higher temperatures, for example, physiologic temperatures (e.g. 37° C.). In other examples, the mutants display higher activity at physiologic temperatures then at lower temperatures. Thus, the activation of the tsMMPs, for example upon administration to the body, can be temporally and conditionally controlled by virtue of changes in temperature.


Uncontrolled MMP activity can be highly disruptive to tissue integrity. By virtue of the conditional activation of activatable tsMMPs, temporary activation is achieved, thereby regulating the duration of enzymatic action on extracellular matrix (ECM) components to reduce deleterious side effects associated with unwanted prolonged activation of enzymes. This is an advantage of the present tsMMPs over existing collagenase treatments. Hence, an advantage of such mutants is that their activity can be regulated, thereby permitting the use of tsMMPs to treat diseases and/or conditions of the ECM.


Modified MMP polypeptides provided herein are modified to exhibit temperature sensitivity via increased activity at a permissive temperature compared to a nonpermissive temperature and/or are modified as activity mutants to exhibit increased activity compared to the MMP polypeptide not containing the modification. The modified MMP polypeptides provided herein are modified, for example, by amino acid substitution, insertion or replacement. For example, tsMMPs contain one or more amino acid replacements in their primary sequence rendering the protein more active at permissive temperatures then at non-permissive temperatures. Modified MMP polypeptides provided herein can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid modifications. In particular, modified MMP polypeptides, for example tsMMPs, provided herein contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids modifications.


tsMMPs provided herein are activatable at a permissive temperature, but are less active or inactive at other non-permissive temperatures. The tsMMPs provided herein have a ratio of activity at a permissive temperature compared to a non-permissive temperature that is or is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 30, 40, 50 or more. Thus, the activity of the tsMMPs provided herein at the non-permissive temperature is or is about 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of the activity at a permissive temperature.


For example, MMPs that are normally active at physiological temperature (e.g. 37° C.) are modified and enzymes selected that are active at lower temperatures, i.e. temperatures below the physiological temperature of the body (e.g. less than 37° C.; e.g. at or about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C.), but that are less active or inactive at physiologic temperature. Such modified enzymes can be used as activatable matrix-degrading enzymes (AMDE) where the activation condition is low temperature. The activation of the enzyme is temporally controlled as the in vivo temperature returns to the physiological temperature of 37° C. Thus, for example, tsMMPs provided herein are active at a permissive temperature that is at or about 25° C., but are less active at higher temperatures such as at or about 33° C., 34° C., 35° C., 36° C., 37° C., 38° C. or 39° C. The tsMMPs provided herein have a ratio of activity at the permissive temperature of at or about 25° C. compared to a non-permissive temperature of at or about 34° C. or 37° C., for example, 33° C., 34° C., 35° C., 36° C., 37° C., 38° C. or 39° C., that is or is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 30, 40, 50 or more. Thus, the activity of the tsMMPs provided herein at the non-permissive temperature of at or about 34° C. or 37° C. is or is about 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of the activity at the permissive temperature at or about 25° C.


For example, modified MMPs polypeptides provided herein, in particular modified MMP-1 polypeptides, that exhibit temperature sensitivity are conditionally active and can be used in uses, methods and processes of treating ECM-mediated diseases and disorders. For example, such tsMMP polypeptides are active at a permissive temperature that is below the normal temperature of the ECM. Thus, when administered to the ECM at or below the permissive temperature, the enzymes exhibit activity. In one example, before administration, a tsMMP, for example tsMMP-1, can be reconstituted in a cold buffer and/or can be stored at a cold temperature that that is at or below the permissive temperature. The tsMMP exhibits activity when exposed to the permissive temperature (e.g. 18° C. to 25° C.). As the tsMMP is exposed to a steadily warmer temperature approaching or reaching the nonpermissive temperature, for example upon administration to the body due to the physiologic temperature of the body, the activity of the MMP is reduced. Thus, the tsMMP exhibits conditional activity, conditioned upon maintenance of a permissive temperature. For example, the activity of the ECM can be controlled for a predetermined time by maintaining the ECM below the physiological temperature of the body.


Thus, where the activating condition is temperature, an activator can be provided that exposes the tsMMP to the permissive temperature required for activation. The exposure to the activator can be in vitro or in vivo. The activator can be exposed to the tsMMP prior to, simultaneously, subsequently or intermittently upon in vivo administration. The activator can provide the requisite heat or cold required for activation. For example, where the activating condition is low temperature, the activator can be provided as a cold buffer or as an ice pack to be applied to the site of administration. Where the activating condition is heat, the activator can be provided as a warm buffer or as a heat pack to be applied to the site of administration. The activating condition also can be provided by storage of the tsMMP at the permissive temperature immediately and just prior to use. The duration of exposure to the activator can be continuous, can be for a predetermined time, or can be intermittent (for example, if the tsMMP is reversible). Thus, the time period permitting activation is flexible and can be adapted to the particular enzyme that is used, the disease or condition being treated, the site of administration or other factors. It is within the level of the skilled artisan to determine the duration of exposure to the activator.


In the absence of exposure to the activator providing the activating condition, the tsMMPs present at the non-permissive temperature are inactive or substantially inactive compared to the activity at the permissive temperature. The activating condition of a permissive temperature (e.g. low temperature) not normally present at the site of administration permits the temporal regulation of, and alteration of, the physiological parameters of organs and tissues, such as the interstitium that exhibits a physiologic temperature of approximately 37° C. Under normal physiological conditions, the temperature of the interstitium is approximately 37° C. Thus, for example, tsMMPs active at low temperatures, when present in the interstitium would normally be catalytically inactive because of the physiologic temperature of the interstitium. When the temperature of the interstitium is temporarily rendered cold, for example, by exposure to a cold buffer or to a cold pack administered on the adjacent surface, tsMMPs when administered to the interstitium will become activated. When the temperature increases and returns to physiological levels, then the tsMMPs become inactive or substantially inactive and cease to exert their enzymatic activity. Hence, by taking advantage of the requirement for exogenous activating conditions, tsMMPs are activatable and can be made temporally active for a limited duration during use, such as upon in vivo administration to the body.


The tsMMPs provided herein include those that are irreversibly inactive following exposure to non-permissive temperatures. Such mutants are active when exposed to permissive temperature conditions (e.g. 25° C.), but are less active or inactive when the temperature is altered to a non-permissive temperatures (e.g. 37° C., such as can occur upon in vivo administration to the body and removal of an exogenous activator (e.g. cold pack)). Upon return to permissive conditions, irreversible tsMMP polypeptides provided herein exhibit at or about 50%, 60%, 70%, 80%, 90%, 100%, 105%, 110%, 115%, or 120% the activity at non-permissive temperatures. The activity is not reversible.


Also provided herein are tsMMPs that are reversibly inactive following exposure to a non-permissive temperature. Such mutants are active when exposed to a permissive temperature condition, but are less active or inactive when the temperature is altered to a non-permissive temperatures. Upon renewed exposure to an activating condition providing the permissive temperature (e.g. cold pack), the activity of the tsMMP is restored, thereby rendering the enzyme sufficiently active to degrade one or more components of the ECM. For example, upon return to permissive conditions from nonpermissive conditions, reversible tsMMP polypeptides provided herein exhibit at or about 120%, 125%, 130%, 140%, 150%, 160%, 170%, 180%, 200% or more the activity at non-permissive temperatures.


tsMMPs provided herein retain one or more activities of wildtype MMP, for example, enzymatic activity for cleavage of an ECM component such as collagen. For example, a tsMMP provided herein retains an activity at the permissive temperature that is or is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 140%, 150% or more the activity of wildtype MMP at the permissive temperature. Thus, tsMMPs provided herein include those that are more active than wildtype MMP-1 at the permissive temperature, and also those that are less active than wildtype MMP-1 at the permissive temperature. Generally, tsMMPs provided herein, however, are less active then wildtype MMP-1 at the nonpermissive temperature. For example, tsMMPs provided herein exhibit 95%, 90%, 80%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, generally 40%, 30%, 25%, 20%, 15%, 10%, or 5% residual activity of wildtype MMP-1 at physiologic temperature (e.g. 34 or 37° C.).


Typically, modified MMP polypeptides, for example tsMMPs, provided herein are zymogens (containing a propeptide) or processed enzymes (e.g. mature enzymes, lacking a propeptide), or catalytically active forms thereof. As discussed below, most enzymes, including MMPs, are zymogens and require an initial processing event for activity by removal of a propeptide segment from the N-terminal end of the polypeptide. A processing agent, such as a protease or chemical agent, directly or indirectly initiates one or more cleavage events to generate an active MMP by virtue of removal of the propeptide segment and/or conformational changes that expose the active site of the MMP. Hence, normally, upon processing of an enzyme to a mature form, the enzyme is active. The activity of a processed enzyme is not reversible, thereby leading to uncontrolled degradation of the ECM upon administration of the processed enzyme to the body. It is contemplated herein that modification of the enzyme to additionally confer temperature sensitivity provides a mechanism to conditionally and temporally control activation of the MMP to avoid continued activation of the processed MMP.


Any MMP, whether synthetic or isolated from natural sources, such as those set forth in Table 5 or elsewhere herein, mature forms thereof lacking the propeptide, and catalytically active forms including polypeptides containing only the catalytically active domain or a portion thereof, and allelic or species variants or other variants thereof, or any known to those of skill in the art can be modified as described herein to be temperature sensitive and/or have increased activity and is intended for use in the compositions, combinations, methods and apparatus provided herein. It is understood that any modified enzyme form provided herein exhibits increased activity and/or temperature sensitivity, i.e. the enzyme is activatable due to the requirement of a temperature activating condition. Exemplary MMPs that can be modified, for example to be temperature sensitive, are set forth in Table 1 and include, for example, any of SEQ ID NOS: 1, 711, 714, 717, 720, 723, 726, 729, 732, 735, 738, 741, 744, 747, 750, 753, 756, 759, 762, 765, 768, 771, 774 or 777, zymogen forms or mature forms thereof, catalytically active forms thereof, and allelic or species variants or other variants thereof, so long as the other forms contain the mutation conferring temperature sensitivity and/or increased activity. For example, SEQ ID NO:2 is the zymogen form of SEQ ID NO:1. FIG. 1 exemplifies the zymogen form of other exemplary MMPs. One of skill in the art knows or could identify tsMMPs. For example, one of skill in the art could use routine molecular biology techniques to introduce amino acid mutation(s) herein into an MMP, and test each for enzyme activation under temperature permissive and non-permissive temperatures to assess the requirement of an exogenous activating condition for sustained or reversible activation of any desired enzyme. Exemplary assays for enzyme activation are provided herein and known in the art.


Hence, modified MMP polypeptides, for example tsMMPs, provided herein include zymogen forms (e.g. proenzyme), processed mature forms lacking a propeptide, and polypeptides containing only the catalytically active domains thereof. For example, tsMMPs include zymogen forms (e.g. proenzyme), processed mature forms lacking a propeptide, and polypeptides containing only the catalytically active domains thereof, so long as the tsMMPs exhibits enzymatic activity at the permissive temperature. Exemplary of such a tsMMP is a tsMMP-1. tsMMP-1 provided herein contains one or more amino acid modifications in its primary sequence corresponding to amino acid replacements in a wildtype MMP-1 set forth in SEQ ID NO:2. Exemplary modifications are described elsewhere herein in Section D. The modified MMPs, for example tsMMP-1 mutants or activity mutants, provided herein include those that are zymogens or those that are in a mature form lacking a propeptide. The zymogen or mature polypeptides provided herein include those that are full-length, include all or a portion of the proline rich linker or the hemopexin binding domain, lack all or a portion of the proline rich linker or the hemopexin binding domain, or polypeptides that include only the catalytically active domains thereof (e.g. corresponding to amino acids 81-242 of the sequence of amino acids set forth in SEQ ID NO:1) so long as the tsMMP-1 retains enzymatic activity at the permissive temperature and/or exhibits increased activity.


It is understood that when provided in zymogen form, the modified MMP polypeptides, for example tsMMPs, are inactive and that processing by a processing agent is required for activity. Generally, the processing of the enzyme is effected prior to use, such as prior to administration in vivo. For example, the processing agent can be applied simultaneously, intermittently or subsequently to exposure of the tsMMP to the activating condition (e.g. low temperature) and administration to the body. Generally, the processing agent is chosen that is acceptable for administration to a subject. If desired, the processing agent can be dialyzed or otherwise purified away from the enzyme preparation before administrations. Thus, for zymogen forms of the enzyme, two steps are required for activation: 1) exposure to a processing agent; and 2) exposure to an activating condition. Whether in zymogen or processed form, exposure of the tsMMP to an activator at the permissive temperature temporally controls activity of a tsMMP.


Modified MMP polypeptides, for example tsMMPs, provided herein can be further modified to alter any one or more properties or activities. For example, altered properties or activities include, but are not limited to, modification that render the enzyme more stable, alter the substrate specificity and/or increase resistance to one or more inhibitors. In one example, modified MMP polypeptides, for example tsMMPs, can be modified to alter its substrate specificity. For example, an enzyme can be modified to have increased specificity for a particular substrate. Thus, for example, a modified MMP polypeptide, which exhibits substrate specificity for type I and type IV collagen can be modified so that it has increased substrate specificity for type I collagen, and not type IV collagen, and vice versa. If desired, enzyme stability also can be increased by PEGylation or glycosylation of the enzyme.


Modifications of polypeptides can be achieved by routine molecular biology techniques, and are within the skill of one in the art. For purposes herein, modified MMP polypeptides, for example tsMMPs, retain one or more activities of the wildtype MMP at the permissive temperature. Retained activity can be 40%, 50%, 60%, 70%, 80%, 90%, 95% or more activity of the wildtype MMP at the permissive temperature. Modified enzymes can be tested for their substrate specificity using routine assays for substrate cleavage such as is described herein, or known in the art. For example, substrate cleavage can be assessed on fluorogenic peptides or on purified proteins. Cleavage can be assessed using in vitro or in vivo assays. For example, cleavage can be assessed by incubating the enzyme with the substrate, and then running the mixture on an SDS-PAGE gel. Degradation can be assessed by Western Blot or by using standard protein stains such as Coomasie Blue or Silver Stain reagents.


The modified MMP polypeptides, for example tsMMPs, are provided herein as compositions, combinations and containers. The modified MMPs, for example tsMMP, are provided in a therapeutically effective amount, that when activated, degrade one or more components of the ECM upon administration, such as upon sub-epidermal administration. The resulting modified MMPs, for example tsMMPs, can be used as therapeutics to treat ECM-mediated diseases or conditions. A description of compositions, combinations, containers and methods of using activatable matrix-degrading proteins is provided in related U.S. Provisional Application Nos. 61/068,667 and 61/127,725, U.S. patent application Ser. No. 12/81,063 and International PCT Application No. PCT/US2009/001489, each incorporated by reference in their entirety. Such description of the compositions, combinations, containers and methods can be used for the purpose of preparing and providing compositions, combinations and containers of modified MMPs, for example tsMMPs, and use thereof for treating ECM-mediated diseases and conditions.


For example, the tsMMPs are provided in compositions, combinations and/or containers with an activator that provides the activating condition. In some examples, modified MMPs, for example tsMMPs, also are provided in compositions, combinations and/or containers with a processing agent. The activator and/or processing agent can be in the same composition or in separate compositions and in the same container or separate containers with the tsMMP. In addition, the modified MMPs, for example tsMMP, also can be combined or provided in combination, such as in containers, with other agents such as any one or more of an anesthetic, alpha-adrenergic agent, dispersing agent, or therapeutic agent. The modified MMPs, for example tsMMPs, can be provided in the same or separate composition as other agents and/or can be provided in the same or separate containers.


The modified MMPs, for example tsMMPs, can be provided as a liquid or in lyophilized form at a therapeutically effective concentration. Alternatively, the tsMMPs can be provided as a concentrated liquid, such that addition of a sufficient amount of activator results in a therapeutically effective concentration of enzyme. The enzymes can be provided as a solution or suspension or encapsulated into a suitable delivery vehicle, such as a liposome, glass particle, capillary tube, drug delivery vehicle, gelatin, gel, tablet, capsule, pill, time release coating, as well as transdermal patch preparation and dry powder inhalers or other such vehicle. The activator typically is provided as a liquid solution or suspension for administration into the interstitium either alone or following reconstitution of and/or exposure to the tsMMP. In some examples, the activator is provided exogenously and applied at the site of administration. For example, an activator can be a hot or cold pack that can be applied to the site of administration, e.g. the skin, prior to, simultaneously, subsequently or intermittently following administration of a tsMMP. As described below, kits containing these combinations and also articles of manufacture, such as containers, also are provided.


Thus, when desired, the tsMMP enzyme is subjected to activating conditions in which the enzyme is exposed to an activator to generate an enzyme that is active. Exposure to an activator can be achieved in vitro or in vivo. For example, where an activatable enzyme and activator are separately provided, they can be administered together or separately. Where administered separately, the tsMMP can be administered simultaneously, subsequently or intermittently from the activator. In another example, the tsMMP, in a lyophilized or concentrated liquid form, can be reconstituted with the activator just prior to use. In such an example, the mixture of the tsMMP and activator are administered together. Such methods of activation can be empirically determined by one of skill in the art, and may differ depending on the choice of enzyme and activator, and the method of treatment and treatment regime desired.


The tsMMP, can be provided in an article or manufacture alone or in combination with the activator. For example, if the enzyme is provided in combination with the activator, an article of manufacture can contain an enzyme, either lyophilized or in liquid form, in one compartment, and buffer that is cold or can be rendered cold in an adjacent compartment. The compartments can be separated by a dividing member. Articles of manufacture can additionally contain a processing agent. Such articles of manufacture are described elsewhere herein.


The combinations of also can further contain other agents, discussed in detail below. For example, modified MMP polypeptides, for example tsMMP, are provided in combinations containing one or more of a anesthetic, vasoconstrictor, dispersing agent or other therapeutic agent.


The following sections provide a general overview of the extracellular matrix and diseases thereof, and provide exemplary MMPs for preparation as modified MMPs, for example as temperature-sensitive activatable enzymes; methods of making such modified MMPs; exemplary modified MMPs, for example tsMMPs, that are modified MMP-1 polypeptides; compositions and combinations thereof, and methods of using modified MMP, for example modified MMP-1 polypeptides or compositions to treat ECM-mediated diseases and conditions.


C. MATRIX METALLOPROTEASES AND THE EXTRACELLULAR MATRIX

Provided herein are modified matrix metalloproteases (MMPs). The modified MMPs include those that are activatable by temperature and degrade one or more protein components of the extracellular matrix (ECM) in a temperature controlled manner by virtue of increased activity at a permissive temperature compared to a non-permissive temperature. Hence, the modified MMPs are temperature sensitive. By virtue of such temporal in vivo activation, diseases and/or conditions of the ECM can be treated. In another example, also provided herein are modified MMPs that exhibit increased activity compared to an MMP not containing the modifications. Mutations that confer increased activity can be combined with at least one mutation that confers temperature sensitivity to generate modified MMP polypeptides that have increased activity at the permissive temperature compared to the tsMMP not containing the activity mutation. The modified MMP polypeptides, for example tsMMPs, can degrade any component of the ECM; enzyme selection can depend upon the targeted component and/or the particular disease or condition to be treated.


1. The Extracellular Matrix


The ECM makes up the connective tissue or interstitium that surrounds the spaces outside cells and the vascular and lymphatic system, thereby providing mechanical and structural support with and between different tissues. The complex and dynamic microenvironment of the ECM represents a structural and signaling system within connective tissues, such as the skin. Due to the complex nature of the ECM, it can serve diverse functions such as providing support and anchorage for cells, segregating tissues, regulating intercellular communication, and sequestering cellular growth factors. Defects or changes in the organization, or make-up, of the ECM can contribute to a number of diseases or conditions. For example, changes in the synthesis, degradation and organization of collagen fibers contribute to lipodystrophy (e.g., cellulite) and lymphedema.


The ECM is composed of fibrous structural proteins, such as collagens, polysaccharides, such as proteoglycans and hyaluronic acid, and adhesion proteins that link components of the matrix to each other and to cells. Some connective tissues, such as tendon and cartilage, are principally made up of ECM. The ECM making up the connective tissue of the skin, however, also is distributed with fibroblasts, blood vessels and other components. The ECM also serves as the space where water and its dissolved constituents move from the blood plasma to the lymphatics. The interstitial fluid is nearly isosmotic with the cytoplasm and is bicarbonate buffered providing an extracellular environment that is at neutral pH.


a. Components of the ECM


The ECM (also called the interstitial matrix) is a complex three-dimensional dynamic structure that contains numerous structural macromolecules including fibrous proteins such as collagens, elastin and fibronectin, in which glycosyaminoglycans (GAGs) form a hydrated gel-like substance. The components of the ECM are produced by resident cells, typically fibroblasts or cells of the fibroblast family, and are secreted via exocytosis where they interact with other components of the ECM. It is the variation in the relative amount and the way in which the components organize and form together that give rise to diverse connective tissues such as bone, skin or cornea (Albert et al., “Cell Junctions, Cell Adhesions and the Extracellular Matrix.” Molecular Biology of the Cell. New York: Garland Publishers, 1994. Page 972.)


i. Collagens


Collagen is the major structural constituent of connective tissues, such as the skin, and plays a role in the development and maintenance of tissue architecture, tissue strength and cell-cell interactions. Collagens include a family of structurally-related proteins of the ECM that contain one or more domains having the conformation of a collagen triple helix (Van der Rest et al. (1991) FASEB J., 5:2814-2823). Collagens contain a Gly-X-Y repeating structure, which allows collagen chains to twist into a helical structure. Each collagen molecule contains three chains twisted around each other to form a triple helix, designated α1-α3. The triple helix structure provides a high mechanical strength to a collagen molecule. There are at least 27 different types of collagens, which differ in amino acid sequence and chain composition. For example, depending on the type of collagen, the three chains forming the triple helix can be the same or different. Collagens can be homotrimeric (i.e. all three polypeptide chains of the triple helix are made up of the same collagen) or can be heterotypic (i.e. fibrils made of more than one collagen type). Collagens can be divided into several families depending on the structure they form. These include fibrillar collagens (also called interstitial collagens; e.g., Type I, II, III, V and XI) and non-fibrillar collagens such as facit (e.g., Type IX, XII, XIV), short chain (e.g., Type VIII, X), basement membrane (e.g., Type IV), and other collagens (e.g., Type VI, VII, and XIII). Table 3 below sets forth common collagen types and their representative location (Van der Rest et al. (1991) FASEB J., 5:2814-2823); www.collagenlife.com/page1167323108078.html; www.indstate.edu/thcme/mwking/extracellularmatrix.html).


Among the interstitial collagens, collagen molecules associate to form large fibrils, which have a distinctive banding pattern. The banding pattern results from overlap between adjacent molecules. The strength of collagen fibers is based on a multiplicity of intra- and intermolecular linkages of the collagen fibers that form the dense collagen fiber network of connective tissues. The most common of fibrillar collagens include type I, II and III collagens. Type I collagen is found in most connective tissues such as skin, bone, tendon and cornea, and is a made up of two α1(I) chains and one α2(I) chain ([α1(I)]2 α2(I)). Type II collagen is homotrimeric ([α1(II)]3) and is predominantly found in the cartilage. Type III collagen also is homotrimeric gal ([α1(III)]3) and is predominantly found in the skin and vessels.


Not all collagens form fibril networks. For example, the basement membrane type IV collagen is non-fibrous and has non-helical interruptions in the helix, which acts as a hinge giving the molecule greater flexibility. Thus, type IV collagen forms a sheet made by a meshwork of filaments rather than by linear fibrils.


The most abundant protein of the skin is collagen, which is primarily made up of type I (80-85%) and type III (8-11%) collagen. Type I collagen associates with type III collagen to form the major collagen fibers of the dermis. The tensile strength of skin is due predominantly to these fibrillar collagen molecules, which assemble into microfibrils in a head-to-tail and staggered side-to-side lateral arrangement. Collagen molecules become cross-linked to adjacent collagen molecules, creating additional strength and stability in collagen fibers. For example, type V collagen also associates with type I/III collagen fibers, and regulates the fibril diameter. Other collagen types in the skin include, for example, type IV, type VI, type VII, type XII, type XIV and type XVII.









TABLE 3







Types of Collagens









Type
Molecule Composition
Representative tissue










Fibrillar Collagens









I
[α1(I)]2 [α2(I)]
Skin, bone, tendon, dentin,




ligaments, interstitial tissues


II
[α1(II)]3
Cartilage, vitreous humor


III
[α1(III)]3
Skin, muscle, blood vessels;




frequently associated with type




I


V
[α1(V)][α2(v)][α3(V)]
Similar to Type I, also cell




cultures, fetal tissues;




associates with Type I


XI
[α1(XI)][α2(XI)][α3(XI)]
Cartilage, intervertebral




cartilage and bone enamel







Non-fibrillar collagens









IV
[α1(IV)]2 [α2 (IV)]
Basement membrane


VI
[α1(VI)][α2(VI)][α3(VI)]
Most interstitial tissues;




associates with type I


VII
[α1(VII)]3
epithelia


VIII
[α1(VIII)]3
Unknown, some endothelial




cells


IX
[α1(IX)][α2(IX)][α3(IX)]
Cartilage; associates with Type




II


X
[α1(X)]3
Heterotrophic and mineralizing




cartilage


XII
[α1(XII)]3
Ligaments, tendons and tooth




enamel; interacts with types I




and III









ii. Elastin


A network of elastic fibers in the ECM provides flexibility to tissues that require resilience to recoil after stretching, such as the skin, arteries and lungs. The main component of elastic fibers is the elastin molecule, which creates cross-links to adjacent elastin molecules. These molecules form a core of elastic fibers and are covered by fibrillin, a large glycoprotein that binds to elastin and is important for the integrity of elastic fibers.


iii. Fibronectin


Fibronectin is a glycoprotein that exists as a pair of two large subunits joined by a pair of disulfide bonds near the carboxyl termini. Each subunit contains functionally distinct domains specific for other matrix macromolecules and receptors on the surface of cells. For example, distinct domains on fibronectin bind collagen (separate domains for types I, II and III), heparin, fibrin and cell surface receptors such as integrins. Fibronectin is present in both plasma and tissue. In tissue, fibronectin functions to link together different types of ECM molecules and cells. It also contains an important cell-binding domain made up of the three amino acids, Arg-Gly-Asp (RGD), which is recognized by integrin receptors in the plasma membranes of cells. The binding of fibronectin molecules to integrin receptors on cells leads to the stimulation of signaling pathways that promote cell attachment, migration and differentiation. These characteristics allow fibronectin to play an important role in cell adhesion and to communicate signals between cells and components of the ECM.


iv. Glycosaminoglycans (GAGs)


GAGs are unbranched polysaccharide chains made of repeating disaccharide units that are strongly hydrophilic. GAGs are highly negatively charged and therefore attract osmotically active Na+, causing large amounts of water to be drawn into their structure to keep the ECM hydrated. GAGs, such as dermatan sulfate, typically contain multiple glycosaminoglycan chains of 70-200 sugars long (formed from repeating disaccharide units) that branch from a linear protein core. This results in GAGs occupying a huge volume relative to their mass and forming gels at very low concentrations. The hydrophilic nature of GAGs causes a swelling pressure, or turgor, which allows the ECM to withstand compression forces.


In the ECM, GAGs are attached to ECM proteins to form proteoglycans or, in the case of hyaluronic acid (also called hyaluronan), exist as a non-proteoglycan matrix component. Extracellular proteoglycans are large, highly hydrated molecules that help cushion cells in the ECM. Glycosaminoglycans such as hyaluronan contribute to the “ground substance” by creating a barrier to bulk fluid flow through the interstitial collagenous matrix by way of their viscosity and water of hydration. Proteoglycans and non-proteoglycan GAGs associate to form large polymeric complexes in the ECM. They associate with each other, and also with fibrous proteins such as collagen.


1) Proteoglycans

There are three main types of GAGs that form proteoglycans of the ECM, including dermatan sulfate and chondroitin sulfate, heparin and heparan sulfate, and keratan sulfate. Generally, a proteoglycan is 95% carbohydrate by weight, typically in the form of long unbranched GAG chains. Besides providing hydrated space around cells, proteoglycans also regulate traffic of molecules and cells, bind signaling molecules thereby playing a role in cell activation, and bind other secreted proteins such as proteases and protease inhibitors to regulate the activities of secreted proteins (Albert et al., “Cell Junctions, Cell Adhesions and the Extracellular Matrix” Molecular Biology of the Cell. New York: Garland Publishers, 1994. pp. 972-978). For example, the heparin sulfate chains of proteoglycans bind to several different growth factors, including fibroblast growth factors (FGFs), helping them to bind to their specific cell surface receptors.


Aggrecan is a proteoglycan, which principally contains chondroitin sulfate and heparan sulfate GAGs, and is typically found in cartilage forming large aggregates with hyaluronan to provide mechanical support. Decorin is another exemplary GAG of connective tissues made up primarily of chondroitin sulfate and dermatan sulfate GAGs. It binds to type I collagen fibrils. Perlecan and betaglycan are other exemplary proteoglycans of the ECM. Not all proteoglycans are associated with the ECM: for example, serglycin is associated with secretory vesicles where it helps to package and store secretory molecules, and syndecans are found on the cell surface and act as co-receptors (Albert et al., “Cell Junctions, Cell Adhesions and the Extracellular Matrix” Molecular Biology of the Cell, New York: Garland Publishers, 1994. pp. 972-978).


Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (Wight, T. N., Kinsella, M. G., and Qwarnstromn, E. E. (1992) Curr. Opin. Cell Biol., 4, 793-801; Jackson, R. L., Busch, S. J., and Cardin, A. L. (1991) Physiol. Rev., 71, 481-539; Wight, T. N. (1989) Arteriosclerosis, 9, 1-20; Kjellen, L., and Lindahl, U. (1991) Annu. Rev. Biochem., 60, 443-475; and Ruoslahti, E., and Yamaguchi, Y. (1991) Cell, 64, 867-869). The basic HSPG structure has a protein core to which several linear heparan sulfate chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups. Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPGs in embryonic morphogenesis, angiogenesis, metastasis, neurite outgrowth and tissue repair. The heparan sulfate (HS) chains, which are unique in their ability to bind a multitude of proteins, ensure that a wide variety of effector molecules cling to the cell surface. HSPGs are also prominent components of blood vessels. In large vessels they are concentrated mostly in the intima and inner media, whereas in capillaries they are found mainly in the subendothelial basement membrane where they support proliferating and migrating endothelial cells and stabilize the structure of the capillary wall. The ability of HSPGs to interact with ECM macromolecules such as collagen, laminin and fibronectin, and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion.


2) Hyaluronic Acid

Hyaluronic acid (HA; also called hyaluronan) is a large GAG that attracts water, and when bound to water exists in a viscous, gel-like form. Thus, HA serves as a lubricant, holding together gel-like connective tissues. HA is a polymer of disaccharides (sometimes as many as 25,000 repeats in length) and is composed of repeating units of two modified simple sugars: glucuronic acid and N-acetyl glucosamine. HA is part of the ECM of many connective tissues. HA is found in the greatest amount in the skin with almost 50% of the body's HA found in the skin. The HA provides continuous moisture to the skin by binding up water. Decreased production of HA, such as by age, results in wrinkled and unhealthy skin.


HA, principally through its receptor CD44, also functions to regulate cell behavior during embryonic development and morphogenesis, wound healing, repair and regeneration, inflammation and tumor progression and invasion (Harada et al. (2006) J. Biol. Chem., 8:5597-5607). HA is degraded by hyaluronidases. The degradation products of HA can be found in increased amounts in damaged or growing tissues, and in a variety of inflammatory conditions. HA fragments promote angiogenesis and can stimulate cytokine production by macrophages and dendritic cells in tissue injury and skin transplant.


b. Histology of the Skin


The skin helps to maintain the body's temperature at a physiologic temperature of 37° C. The skin is composed of several distinct layers, principally the epidermis and dermis. The epidermis is a specialized epithelium derived from the ecotoderm, and beneath this is the dermis, which is a derivative of the mesoderm and is a vascular dense connective tissue. These two layers are firmly adherent to one another and form a region which varies in overall thickness form approximately 0.5 to 4 mm in different areas of the body. Beneath the dermis is a layer of loose connective tissue, which varies from areolar to adipose in character. This is referred to as the hypodermis, but is typically considered not to be part of the skin. The dermis is connected to the hypodermis by connect tissue fibers that pass from one layer to the other.


i. The Epidermis


The epidermis is the skin layer directly above the dermis, and is the surface layer of the skin. The principle function of the epidermis is to act as a protective barrier against water loss, chemical injury and invading pathogens. The epidermis is a thin layer of approximately fifteen cell layers that is about 0.1 to 1.5 millimeters thick composed primarily of keratinocytes (Inlander, Skin, New York, N.Y.: People's Medical Society, 1-7 (1998)). The epidermis is itself divided into several layers (e.g., stratum basale, stratum spinosum, stratum granulosum, stratum lucidum, stratum corneum) based on the state of differentiation of the keratinocytes. Keratinocytes originate in the basal layer from keratinocyte stem cells. As the keratinocytes grow and divide, they undergo gradual differentiation eventually reaching the stratum corneum where they form a layer of enucleated, flattened, highly keratinized cells called squamous cells (also called corneocytes). Besides being made up of corneocytes, the stratum corneum also contains sebum. The sebum is secreted by sebaceous glands, which are usually found in hair-covered areas connected to hair follicles. Sebum is a slightly acid layer that helps to hold the corneocytes together and holds moisture in. This acidity is due to the presence of amphoteric amino acids, lactic acid and fatty acids that make up sebum. Thus, the pH of the skin surface is normally between 5 and 6, typically about 5.5. Sebum acts to waterproof hair and skin, and keep them from becoming dry, brittle and cracked, and it also inhibits the growth of microorganisms on skin. The term “acid mantle” refers to the presence of the water-soluble substances on most regions of the skin.


ii. The Dermis


The connective tissue of the skin is called the dermis. The dermis is 1.5 to 4 millimeters thick. In the skin, the dermis contains ECM components; the main protein components are collagen and elastin. The dermis also is home to most of the skin's structures, including sweat and oil glands that secrete substances through openings in the skill called pores, or comedos, hair follicles, nerve endings, and blood and lymph vessels (Inlander, Skin, New York, N.Y.: People's Medical Society, 1-7 (1998)). In addition, the dermis contains blood vessels that play a role in temperature regulation.


iii. The Hypodermis


Below the dermis is the hypodermis, which is a fatty layer and is the deepest layer of the skin. It acts as an insulator for body heat conservation and as a shock absorber for organ protection (Inlander, Skin, New York, N.Y.: People's Medical Society, 1-7 (1998)). In addition, the hypodermis also stores fat for energy reserves.


c. Diseases of the ECM


Certain diseases and conditions result from defects or changes in the architecture of the extracellular matrix due to aberrant expression or production of ECM components. For example, in some inflammatory conditions such as occur upon wound healing, cytokines are secreted, which stimulate fibroblasts to secrete ECM components such as collagen. The ECM components accumulate and become locally deposited, resulting in a wide range of fibrotic conditions. Matrix deposition is a frequent feature in many chronic inflammatory diseases and in other diseases and conditions. Included among these are collagen-mediated disease conditions such as, but not limited to, scars such as keloid and hypertrophic scars, Duputyren's syndrome, Peyronie's disease and lymphedema. Cellulite also is a prominent disease of the ECM that, in addition to increased adipogenicity, is characterized by alterations in the connective tissue matrix resulting in an abnormal fibrous septae network of collagen (Rawlings et al. (2006) Int. J. Cos. Science, 28:175-190).


Diseases and conditions of the ECM that are characterized by aberrant expression or overproduction of matrix components, resulting in their accumulation and unwanted deposition, can be treated by the tsMMPs provided herein. By virtue of the temporal activation of such enzymes upon in vivo administration, the treatment of such diseases and conditions is regulated to limit the enzymatic degradation of the matrix components. For example, by limiting the duration of action of matrix degradation, unwanted side effects associated with uncontrolled protein degradation is minimized.


2. Matrix Metalloproteases


Provided herein are modified MMPs that are temperature sensitive (tsMMPs). The modified MMPs include those that exhibit increased activity at a lower temperature then a higher temperature and also those that exhibit increased activity at a higher temperature then a lower temperature. The tsMMPs are provided as compositions, combinations and containers, and can be used in methods, processes and uses to treat ECM-mediated diseases or conditions. MMPs are matrix-degrading enzymes that degrade protein components of the extracellular matrix (ECM), including, but not limited to, collagen, elastin, fibronectin and proteoglycans. By virtue of their ability to cleave one or more ECM components, activatable tsMMPs provided herein can be used to modify the matrix of tissues, particularly those exhibiting structural defects or changes due to excess of one or more ECM protein or unwanted accumulation of fibrous tissue rich in one or more ECM protein, such as collagen. Thus, such enzymes are useful in treating diseases or conditions in which ECM proteins are involved.


a. Function


Matrix metalloproteinases (MMPs) are a family of zinc-dependent and calcium-dependent endopeptidases. For example, MMPs contain an active site Zn2+ required for activity. Most MMPs are involved in degradation of the extracellular matrix. For example, many of these enzymes can cleave components of the basement membrane and extracellular matrix. They are involved in tissue remodeling, for example, in processes such as wound healing, pregnancy and angiogenesis. In addition, MMPs also can process a number of cell-surface cytokines, receptors and other soluble proteins. The proteolytic activity of MMPs act as an effector mechanism of tissue remodeling in physiologic and pathologic conditions, and as modulator of inflammation. The excess synthesis and production of MMPs leads to accelerated degradation of the ECM which is associated with a variety of diseases and conditions such as, for example, bone homeostasis, arthritis, cancer, multiple sclerosis and rheumatoid arthritis. In the context of neuroinflammatory diseases, MMPs have been implicated in processes such as (a) blood-brain barrier (BBB) and blood-nerve barrier opening, (b) invasion of neural tissue by blood-derived immune cells, (c) shedding of cytokines and cytokine receptors, and (d) direct cellular damage in diseases of the peripheral and central nervous system (Leppert et al. Brain Res. Rev. 36(2-3): 249-57 (2001); Borkakoti et al. Prog. Biophys. Mol. Biol. 70(1): 73-94 (1998)). The enzymes are specifically regulated by endogenous inhibitors called tissue inhibitors of matrix metalloproteases (TIMPs).


b. Structure and Activation


Generally, MMPs contain three common domains: the pro-peptide, the catalytic domain and the hemopexin-like C-terminal domain. MMPs are synthesized as zymogens. Zymogen activation prevents unwanted protein degradation that could occur if proteases were always present in active form. Generally, zymogens contain N-terminal portions (or prosegments or proregions or propeptide) that sterically block the active site of the protease and prevent access of substrates to the active site of the protease. The propeptide also acts to stabilize the polypeptide. The propeptide of zymogen forms of MMPs range in size from about 80-100 residues in length. The propeptide of MMPs contains a cysteine residue generally contained in the conserved sequence PRCxxPD (with the exception of MMP-23, which contains the critical cysteine and different surrounding amino acids). The cysteine residue interacts with the zinc in the active site and prevents binding and cleavage of the substrate, thereby keeping the enzyme in an inactive form. Thus, upon secretion from a preproenzyme form, the proenzyme (containing the propeptide) is inactive. For example, in MMP-1 the propeptide cysteine residue corresponds to amino acid residue 73 in the sequence of amino acids set forth in SEQ ID NO:2.


MMPs require processing for activation. Generally, processing involves removal of the propeptide and/or conformational changes of the enzyme to generate a processed mature form. Processing of the enzyme by removal of the propeptide is required for activity of MMPs. For normal MMPs (e.g. wildtype) that are not conditionally active as provided herein, the processed mature form is an active enzyme. Thus, it is understood that wildtype MMPs in their processed mature form are enzymatically active, and thus for these enzymes this is the active form. tsMMPs provided herein, however, also additionally require the permissive temperature condition to be fully active.


Processing (and thereby activation) can be induced by processing agents such as proteases, including other previously activated MMPs; by chemical activation, such as thiol-modifying agents (4-aminophenylmercuric acetate, HgCl2 and N-ethylmaleimide), oxidized glutathione, SDS, chaotropic agents and reactive oxygens; and by low pH or heat treatment. For example, Table 4 below lists exemplary processing agents (see also Visse et al. (2003) Circ. Res., 92:827-839; Khan et al. (1998) Protein Science, 7:815-836; Okada et al. (1988) Biochem J., 254:731-741; Okada & Nakanashi (1989) FEBS Lett., 249:353-356; Nagase et al. (1990) Biochemistry, 29:5783-5789; Koklitis et al. (1991) Biochem J., 276:217-221; Springman et al. (1990) PNAS, 87:364-8; Murphy et al. (1997) Matrix Biol., 15:511-8).









TABLE 4





Zymogen Activators (i.e. processing agents)







Proteolytic Compounds








Proteases
Plasmin



Plasma kallikrein



Trypsin-1 (Trypsin I)



Trypsin-2 (Trypsin II)



Neutrophil elastase



Cathepsin G



Tryptase



Chymase



Proteinase-3



Furin



uPA



MMPs, including MMP-1, MMP-2,



MMP-3, MMP-7, MMP-10, MMP-26,



and MT1-MMP







Non-Proteolytic Compounds








Thiol-modifying Agents
4-aminophenylmercuric acetate



(AMPA)



HgCl2



N-ethylmaleimide


Conformational Perturbants
Sodium dodecyl sulfate (SDS)



Chaotropic agents


Other Chemical Agents
Oxidized glutathione (GSSG)



Reactive oxygen



Au(I) salts







Other Activating Conditions









Acidic pH



Heat










MMP activation occurs in a stepwise manner. For example, activation by proteases involves a first proteolytic attack of a bait region (corresponding to amino acids 32-38 of proMMP-1 (SEQ ID NO:2)), an exposed loop region found between the first and second helices of the pro-peptide. The sequence of the bait region confers cleavage specificity. Following initial cleavage, the remaining propeptide is destabilized allowing for intermolecular processing by other partially active MMP intermediates or active MMPs. For example, the protease plasmin activates both proMMP-1 and proMMP-3. Once activated, MMP-3 effects the final activation of proMMP-1. Alternatively, activation by chemicals, for example APMA, initially causes the modification of the propeptide cysteine residue, which in turn causes partial activation and intramolecular cleavage of the propeptide. The remaining segment of propeptide is then processed by other proteases or MMPs.


Metalloproteinases contain a Zn2+ ion at the active center of the enzyme required for catalytic activity. Generally, these enzymes have a common zinc binding motif (HExxHxxGxxH) in their active site, and a conserved methionine turn following the active site. The zinc binding motif at the active site of a metalloproteinase includes two histidine residues whose imidazole side-chains are ligands to the Zn2+. During catalysis, the Zn2+ promotes nucleophilic attack on the carbonyl carbon by the oxygen atom of a water molecule at the active site. An active site base (a glutamate residue in carboxypeptidases) facilitates this reaction by extracting a proton from the attacking water molecule. Thus, the glutamate (E) residue activates a zinc-bound H2O molecule, thereby providing the nucleophile that cleaves peptide bonds. Mutation of any one of the histidines ablates catalytic activity. The catalytic domain also contains two calcium binding sites on either side of the zinc binding motif. The Ca2+ binding sites are characterized as being a highly conserved Glu- and Asp-rich region.


Many MMPs also contain a flexible proline-rich hinge region, which is up to about 75 amino acids long, but has no known structure. MMPs also contain a hemopexin-like C-terminal domain that functions in substrate recognition and also interacts with inhibitors, in particular tissue inhibitor of metalloproteinases (TIMPs). MMP-7, MMP-23 and MMP-26 do not contain a hemopexin domain. MMP-2 and MMP-9 also contain an insert in the catalytic domain made up of three tandem repeats of fibronectin type II modules that confer gelatin-binding properties to these enzymes.


There are over 25 MMPs known and they are grouped into different families depending on function, substrate specificity and/or sequence similarity. The families of MMPs include collagenases, gelatinases, stromelysins and matrilysins. Among the various families, some MMPs contain additional domains. For example, membrane-type MMPs contain a transmembrane or a GPI-anchoring domain. Exemplary MMPs are set forth in Table 5. The sequence identifiers (SEQ ID NO) for the nucleotide sequence and encoded amino acid sequence of the precursor polypeptide for each of the exemplary proteases is depicted in the Table. The sequence identifiers (SEQ ID NO) for the amino acid sequence of the preproprotein and the zymogen-activated processed mature form of the protein (lacking the propeptide) also are depicted in the Table. The location of domains also is indicated. Those of skill in the art are familiar with such domains and can identify them by virtue of structural and/or functional homology with other such domains. It is understood that polypeptides and the description of domains thereof are theoretically derived based on homology analysis and alignments with similar polypeptides. Thus, the exact locus can vary, and is not necessarily the same for each polypeptide. Variations of MMPs also exist among allelic and species variants and other variants known in the art, and such variants also are contemplated for modification as activatable tsMMPs as described herein below. The Table also sets forth exemplary ECM target substrates for each enzyme. Reference to such substrates is for reference and exemplification, and are not intended to represent an exhaustive list of all target substrates. One of skill in the art knows or can empirically determine ECM target substrates for a desired enzyme using routine assays, such as any described herein.









TABLE 5







Metalloprotease










Enzyme




databank
SEQ ID NO












access


Mature



code


(processed



(EC)
Genbank
Precursor
form)













Protease
Substrate
www.expasy.ch/sprot/enzyme.html
No.
nt
Aa
aa










Collagenases:













MMP-1
collagen I, II,
3.4.24.7
P03956,
708
 1
709


(collagenase-
III, VII, VIII,

NM_002421

(ss aa 1-19;


1)
X, XI, gelatin,



pp aa 20-99)



proteoglycan,



fibronectin,



glycoprotein


MMP-8
collagen I, II,
3.4.24.34
P22894
710
711
712


(collagenase-
III, aggrecan

NM_002424

(ss aa 1-20;


2)




pp aa 21-100)


MMP-13
collagen I, II,
3.4.24.—
P45452
713
714
715


(collagenase-
III, IV, VI,

NM_002427

(ss aa 1-19;


3)
IX, X, XIV,



pp aa 20-103)



gelatin,



proteoglycan,



fibronectin,



glycoprotein


MMP-18
collagen I
3.4.24.—

Xenopus

716
717
718


(collagenase-



laevis


(ss aa 1-17;


4)


O13065

pp aa 18-99)







Gelatinases:













MMP-2
gelatins,
3.4.24.24
P08253
719
720
721


(gelatinase
collagen I, II,

NM_004530

(ss aa 1-29;


A)
III, IV, V,



pp 30-109)



VII, X, XI,



elastin,



fibronectin,



laminin,



proteoglycan,



glycoprotein


MMP-9
gelatin,
3.4.24.35
P14780
722
723
724


(gelatinase
collagen IV,

NM_004994

(ss aa 1-19;


B)
V, VI, XIV,



pp aa 20-93)



elastin,



laminin,



proteoglycan,



glycoprotein







Stromelysins:













MMP-3
fibronectin,
3.4.24.17
P08254
725
726
727


(stromelysin-
elastin,

NM_002422

(ss aa 1-17;


1)
laminin,



pp aa 18-99)



gelatin,



proteoglycan,



glycoprotein,



collagen III,



IV, V, VII,



IX, X, XI


MMP-10
collagen III,
3.4.24.22
P09238
728
729
730


(stromelysin-
IV, V, elastin,

NM_002425

(ss aa 1-17;


2)
gelatin,



pp aa 18-98)



fibronectin,



aggrecan


MMP-11
Gelatin,
3.4.24.—
P24347
731
732
733


(stromelysin-
fibronectin,

X57766

(ss aa 1-31;


3)
laminin,



pp aa 32-97)



collagen IV







Matrilysins:













MMP-7
fibronectin,
3.4.24.23
P09237
734
735
736


(matrilysin)
laminin,

NM_002423

(ss aa 1-17;



elastin,



pp aa 18-94)



gelatin,



collagen I, IV,



proteoglycan,



glycoprotein


MMP-26
collagen IV,
3.4.24.—
Q9NRE1
737
738
739


(matrilysin-2)
fibronectin,

NM_021801

(ss aa 1-17;



gelatin,



pp aa 18-89)



proteoglycan







Metalloelastase:













MMP-12
elastin,
3.4.24.65
P39900
740
741
742


(metalloelastase)
fibronectin,

NM_002426

(ss aa 1-16;



laminin,



pp aa 17-105)



collagen I, IV,



V, gelatin,



proteoglycan,



glycoprotein







Membrane-type MMPs:













MMP-14
Collagen I, II,
3.4.24.80
P50281
743
744
745


(MT1-MMP)
III, gelatin,

NM_004995

(ss aa 1-20;


Transmembrane
aggrecan,



pp aa 21-111)



fibronectin,



laminin,



proteoglycan,



glycoprotein


MMP-15
aggrecan,
EC
P51511
746
747
748


(MT2-MMP)
fibronectin,
3.4.24.—
NM_002428

(ss aa 1-41;


Transmembrane
laminin,



pp aa 42-131)



glycoprotein


MMP-16
Collagen III,
EC
P51512
749
750
751


(MT3-MMP)
fibronectin,
3.4.24.—
NM_005941

(ss aa 1-31;


Transmembrane
laminin,



pp aa 32-119)



gelatin,



proteoglycan


MMP-17
gelatin
EC
Q9ULZ9
752
753
754


(MT4-MMP)

3.4.24.—
AB021225

(ss aa 1-38;


GPI anchor




pp aa 39-128)


MMP-24
fibronectin,
EC
Q9Y5R2
755
756
757


(MT5-MMP)
gelatin,
3.4.24.—
NM_006690

(ss aa 1-52;


Transmembrane
proteoglycan



pp aa 53-155)


MMP-25
collagen IV,
EC
Q9NPA2
758
759
760


(MT6-MMP)
gelatin,
3.4.24.—
NM_022468

(ss aa 1-21;


GPI anchor
fibronectin,



pp aa 22-107)



proteoglycan







Enamelysin:













MMP-20
aggrecan
EC
O60882
761
762
763


(enamelysin)

3.4.24.—
Y12779

(ss aa 1-22;







pp aa 23-107)







Other:













MMP-19
collagen IV,
EC
Q99542
764
765
766



gelatin,
3.4.24.—
NM_002429

(ss aa 1-18;



laminin,



pp aa 19-97)



aggrecan,



fibronectin,



glycoprotein


MMP-21
gelatin
EC
Q8N119
767
768
769




3.4.24.—
NM_147191

(ss aa 1-24;







pp aa 25-144)


MMP-23
gelatin
EC
O75900
770
771
772


CA-MMP

3.4.24.—
AJ005256


MMP-27
gelatin
EC
Q9H306
773
774
775


CMMP

3.4.24.—
NM_022122

(ss aa 1-17;







pp aa 18-98)


MMP-28

EC
Q9H239
776
777
778


(epilysin)

3.4.24.—
NM_024302

(ss aa 1-22;







pp aa 23-122)









3. Matrix Metalloprotease-1 (MMP-1)


MMP-1 (also called collagenase) is encoded by a nucleic acid molecule set forth in SEQ ID NO:708 resulting in a pre-procollagenase (SEQ ID NO:1), which is co-translationally processed to generate a procollagenase zymogen form (SEQ ID NO:2). Procollagenase contains a propeptide of 80 amino acids (corresponding to amino acid residues 1-80 of the sequence of amino acids set forth in SEQ ID NO:2), a catalytic domain of 162 amino acids (corresponding to amino acid residues 81-242 of the sequence of amino acids set forth in SEQ ID NO:2), a 16-residue linker (corresponding to amino acid residues 243-258 of the sequence of amino acids set forth in SEQ ID NO:2) and a hemopexin (Hpx) domain of 189 amino acid residues (corresponding to amino acid residues 259-450 of the sequence of amino acids set forth in SEQ ID NO:2). Upon processing, the propeptide is removed, resulting in a processed mature form having a sequence of amino acids set forth in SEQ ID NO: 709.


As noted above, MMP-1 cleaves collagen type I and collagen type III, which are the most abundant protein of the skin. These collagen types are associated with many of the conditions of the ECM as described herein in Section I. In contrast, other collagens, for example collagen type IV, is a major component of the basal lamina of blood vessels. Hence, targeting of type IV collagen, for example, can lead to leaky blood vessels, which can be a side effect of treatments that are meant to target the extracellular matrix as described herein. For example, bacterial collagenase, a known treatment for cellulite, can induce haemorrhages (see e.g. Vargaftig et al. (2005) Inflammation Research, 6:627-635). Thus, an advantage of the use of MMP-1, and in particular tsMMP-1 that can be conditionally or temporally controlled, as a therapeutic agent to treat conditions of the ECM is that it does not cleave type IV collagen.


D. MODIFIED MATRIX METALLOPROTEASE-1 POLYPEPTIDES

Provided herein are modified MMP-1 polypeptides. In one example, modified MMP-1 polypeptides provided herein exhibit temperature sensitivity, whereby the modified polypeptide exhibits higher activity at a permissive temperature than a non-permissive temperature. Also provided herein are modified MMP-1 polypeptides that exhibit increased activity compared to the unmodified MMP-1 not containing the modification (e.g. wildtype) at both permissive and non-permissive temperatures. In an additional example, provided herein are modified MMP-1 polypeptides that exhibit modifications that both increase temperature sensitivity and activity.


Modifications provided herein of a starting, unmodified reference polypeptide include amino acid replacements or substitutions, additions or deletions of amino acids, or any combination thereof. For example, modified MMP-1 polypeptides include those with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modified positions. Also provided herein are modified MMP-1 polypeptides with two or more modifications compared to a starting reference MMP-1 polypeptide. Modified MMP-1 polypeptides include those with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modified positions. In some examples, modified MMP-1 polypeptide provided herein contain only a single modification. In other examples, modified MMP-1 polypeptides provided herein contain two, three, four, five or six modifications. In additional examples, any modification(s) provided herein can be combined with any other modification known to one of skill in the art so long as the resulting modified MMP-1 polypeptide retains enzymatic activity when it is in its processed mature form. Where the modified MMP-1 contains a mutation conferring temperature sensitivity, the enzymatic activity of such combination mutant is greater at the permissive temperature compared to the non-permissive temperature. Modified MMP-1 polypeptides provided herein can be assayed for enzymatic activity under various conditions (e.g. permissive and non-permissive temperatures) to identify those that retain enzymatic activity.


Modifications in an MMP-1 polypeptide can be made to any form of an MMP-1 polypeptide, including inactive (e.g. zymogen) or processed mature forms (activated form), allelic and species variants, splice variants, variants known in the art, or hybrid or chimeric MMP-1 polypeptides. For example, modifications provided herein can be made in a precursor MMP-1 polypeptide set forth in SEQ ID NO:1, an inactive pro-enzyme MMP-1 containing the propeptide set forth in SEQ ID NO:2, a mature MMP-1 polypeptide lacking the propeptide set forth in SEQ ID NO:709, or any species, allelic or modified variant and active fragments thereof that has 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the MMP-1 polypeptides set forth in SEQ ID NOS:1, 2 or 709. Modifications also can be in an MMP-1 polypeptide lacking one or more domains, so long as the MMP-1 polypeptide retains enzymatic activity. For example, modifications can be in an MMP-1 polypeptide that includes only the catalytic domain (corresponding to amino acids 81-242) of the proenzyme MMP-1 polypeptide set forth in SEQ ID NO:2). Modifications also can be made in an MMP-1 polypeptide lacking all or a portion of the proline rich linker (corresponding to amino acids 243-258 of the proenzyme MMP-1 polypeptide set forth in SEQ ID NO:2) and/or lacking all or a portion of the hemopexin binding domain (corresponding to amino acids 259-450 of the proenzyme MMP-1 polypeptide set forth in SEQ ID NO:2). Allelic variants and other variants of MMP-1 polypeptides include, but are not limited to, any of MMP-1 polypeptide containing any one or more amino acid variant set forth in SEQ ID NO:3506 and 3549. Exemplary species variants for modification herein include, but are not limited to, pig, rabbit, bovine, horse, rat, and mouse, for example, set forth in any of SEQ ID NOS:3459-3464.


Modifications in an MMP-1 polypeptide provided herein, for example in an MMP-1 containing a modification to confer temperature sensitivity and/or increased activity, can be made to an MMP-1 polypeptide that also contains other modifications, such as those described in the art, including modification of the primary sequence and modifications not in the primary sequence of the polypeptide. It is understood that modifications in an allelic or species variant or other variant include modification in any form thereof such as an active or inactive form, a form including only the catalytic domain, or a form lacking all or a portion of the proline rich linker or the hemopexin binding domain. As discussed herein below, corresponding MMP-1 modifications can be made to similar forms of other MMP polypeptides.


Hence, the resulting modified MMP-1 polypeptides include those that are inactive zymogen proenzymes and those that are processed mature polypeptides. For example, any modified polypeptide provided herein that is a zymogen proenzyme can be activated by a processing agent to generate a processed mature MMP-1 polypeptide. Activity of MMP-1 polypeptides are typically exhibited in its processed mature form following cleavage of the propeptide and/or intermolecular and intramolecular processing of the enzyme to remove the propeptide (see e.g. Visse et al. (2003) Cir. Res., 92:827-839). As noted elsewhere herein, tsMMP's require permissive temperature to be fully active.


The modifications provided herein can be made by standard recombinant DNA techniques such as are routine to one of skill in the art. Any method known in the art to effect mutation of any one or more amino acids in a target protein can be employed. Methods include standard site-directed mutagenesis (using e.g. a kit, such as QuikChange available from Stratagene) of encoding nucleic acid molecules, or by solid phase polypeptide synthesis methods.


Other modifications that are or are not in the primary sequence of the polypeptide also can be included in a modified MMP-1 polypeptide, or conjugate thereof, including, but not limited to, the addition of a carbohydrate moiety, the addition of a polyethylene glycol (PEG) moiety, the addition of an Fc domain, etc. For example, such additional modifications can be made to increase the stability or half-life of the protein.


Exemplary of such modified MMP-1 polypeptides are set forth in any of SEQ ID NOS:3-705, 779-3458 and 3532 and processed mature forms and other forms thereof, and allelic and species variants thereof.


1. Temperature-Sensitive Matrix Metalloprotease-1 (tsMMP-1) Mutants


Provided herein are tsMMP-1 polypeptides that are temperature sensitive by virtue of modifications in the primary sequence of the polypeptide compared to an unmodified MMP-1 polypeptide. The tsMMP-1 polypeptides exhibit increased enzymatic activity at a permissive temperature compared with activity of the tsMMP-1 polypeptide at a non-permissive temperature. For example, tsMMP-1 polypeptides provided herein exhibit increased enzymatic activity at a low temperature that is less then 37° C., for example, that is at or about 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C., in particular at or about 18° C. to 25° C., for example at or about 25° C. compared to a non-permissive high temperature that is at or about 34° C., 35° C., 36° C., 37° C., 38° C. or 39° C., in particular at or about 34° C. or 37° C. Due to the temperature-dependent activity of tsMMP-1 polypeptides, the activity of MMP-1 can be conditionally controlled, thereby temporally regulating activation to prevent prolonged and unwanted degradation of the ECM. In particular, such tsMMP-1 polypeptides can be used in uses, processes or methods to treat diseases or conditions of the ECM, for example, to treat collagen-mediated diseases or conditions such as cellulite.


The tsMMP-1 polypeptides provided herein have a ratio of activity at a permissive temperature compared to a non-permissive temperature that is or is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more. Thus, the activity of tsMMP-1 polypeptides provided herein at the non-permissive temperature is or is about 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of the activity at a permissive temperature. tsMMPs-1 polypeptides provided herein retain one or more activities of wildtype MMP-1 polypeptide at the permissive temperature, for example, enzymatic activity for cleavage of an ECM component such as collagen. Typically, such activity is substantially unchanged (less than 1%, 5%, 10%, 20% or 30% changed) compared to a wildtype or starting protein. In other examples, the activity of a modified MMP-1 polypeptide is increased or is decreased as compared to a wildtype or starting MMP-1 polypeptide. Activity is assessed at the permissive temperature and is compared to the activity of a starting, unmodified MMP-1 polypeptide (i.e. polypeptide not containing the modification) at the permissive temperature or a non-permissive temperature. For example, a tsMMP-1 polypeptide provided herein retains an activity at the permissive temperature that is or is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 140%, 150% or more the activity of wildtype MMP-1 at the permissive temperature or non-permissive temperature. Activity can be assessed in vitro, ex vivo or in vivo and can be compared to that of the unmodified MMP-1 polypeptide, such as for example, an inactive MMP-1 polypeptide set forth in SEQ ID NO:2 activated by a processing agent, or any other MMP-1 polypeptide known to one of skill in the art that is used as the starting material. As discussed elsewhere herein, it is understood that the zymogen inactive form of an MMP-1 or a modified MMP-1 must be processed to a processed mature form required for activity before use or measurement of an activity.


Exemplary Temperature Sensitive Modifications


Provided herein are modified tsMMP-1 polypeptides containing one or more amino acid modifications in a starting, unmodified MMP-1 polypeptide. Typically, the modification is an amino acid replacement. The amino acid replacement or replacements can be at any one or more positions corresponding to any of the following positions: 84, 85, 95, 98, 99, 100, 103, 104, 105, 106, 109, 110, 111, 112, 118, 123, 124, 126, 147, 150, 151, 152, 153, 155, 156, 158, 159, 170, 171, 176, 178, 179, 180, 181, 182, 183, 185, 187, 188, 189, 190, 191, 192, 194, 195, 197, 198, 206, 207, 208, 210, 211, 212, 218, 223, 227, 228, 229, 230, 233, 234, 237, 240, 251, 254, 255, 256, 257, 258, 259 of an unmodified MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2, or at a corresponding position in an allelic or species variant or other variant of an MMP-1 polypeptide that has at least or at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an MMP-1 polypeptide set forth in SEQ ID NO:2. Amino acid replacements include replacement of amino acids to an acidic (D or E); basic (H, K or R); neutral (C, N, Q, T, Y, S, G) or hydrophobic (F, M, W, I V, L A, P) amino acid residue. For example, amino acid replacements at the noted positions include replacement by amino acid residues E, H, R, C, Q, T, S, G, M, W, I, V, L, A, P, N, F, D, Y or K.


Such modified MMP-1 polypeptides include MMP-1 polypeptides that are temperature sensitive by virtue of increased activity at the permissive temperature of 25° C. compared to the non-permissive temperatures of 34° C. or 37° C. For example, modified MMP-1 polypeptides provided herein can include polypeptides having an amino acid modification corresponding to any one or more modifications of T84F (i.e. replacement of T by F at a position corresponding to position 84 of an MMP-1 polypeptide set forth in SEQ ID NO:2), E85F, L95K, L95I, R98D, 199Q, E100V, E100R, E100S, E100T, E100F, E100I, E100N, T103Y, P104A, P104M, D105A, D105F, D105G, D105I, D105L, D105N, D105R, D105S, D105T, D105W, D105E, L106C, L106S, A109H, D110A, V111R, D112S, A118T, S123V, N124D, T126S, G147P, R150P, R150V, R150D, R150I, R150H, D151G, N152A, N152S, S153T, F155L, F155A, D156H, D156L, D156A, D156W, D156V, D156K, D156T, D156R, D156M, P158T, P158G, P158K, P158N, G159V, G159T, G159M, G159I, G159W, G159L, G159C, P170D, P170A, G171P, G171E, G171D, A176F, A176W, F178T, F178L, D179N, D179V, D179C, E180Y, E180R, E180T, E180F, E180G, E180S, E180N, E180D, D181T, D181L, D181K, D181C, D181G, E182T, E182Q, E182M, E182G, R183G, R183S, T185R, T185Y, T185H, T185G, T185V, T185Q, T185A, T185E, T185D, N187R, N187M, N187W, N187F, N187K, N187I, N187A, N187G, N187C, N187H, F188V, R189N, R189T, R189Q, E190G, E190Y, E190D, Y191V, N192H, N192S, N192D, N192C, H194P, R195C, R195W, R195L, R195G, R195Q, R195A, R195D, R195V, A197C, A197V, A198G, A198L, A198M, G206A, G206S, L207R, L207V, L207I, L207G, S208R, S208L, S210V, S210A, T211L, D212G, D212H, Y218S, F223C, F223E, F223G, F223A, F223S, F223K, F223M, V227C, V227D, V227E, V227L, V227S, V227W, V227G, V227H, V227Q, V227R, Q228P, L229A, L229T, L229I, A230V, D233E, I234A, I234T, I234E, I234Q, I237L, I237W, I237N, I240S, I240A, I240C, I251S, I251W, Q254S, T255H, P256C, K257P, K257T, A258P and C259Q. Exemplary modified MMP-1 polypeptides have a sequence of amino acids set forth in any of SEQ ID NOS:6, 18, 22, 25, 27, 29, 31-33, 35-36, 38-39, 41, 43, 55-56, 59, 70, 95-96, 99-101, 105, 110-111, 113-115, 122, 125, 129-133, 148, 150, 159-160, 170, 174, 177, 179, 181-185, 195, 197, 200, 203, 209, 218-219, 222, 224, 231-233, 235, 238-239, 241, 246, 248, 252-255, 260-264, 267, 269, 273, 275, 279, 282, 284-286, 299, 301, 305, 317, 324, 341, 343, 354, 365, 367, 369, 374-376, 381, 383-385, 387-388, 390, 393-394, 397, 399, 420, 429, 436, 438, 440, 460, 466-467, 476, 483, 488, 495, 500, 502, 504, 506, 508, 511-512, 524, 543, 554-555, 572-573, 581, 583, 607, 611, 613, 616, 620, 648, 653, 660, 664-665, 669, 678, 703, 847, 866, 1083, 1109, 1172, 1177, 1183, 1188, 1237, 1271, 1277, 1301, 1414, 1516, 1520, 1567, 1975, 2023, 2031, 2075, 2078, 2080, 2083, 2281, 2299, 2403, 2411, 2423-2424, 2486, 2495-2497, 2552, 2563, 2703, 2715, 2753, 3066, 3074, 3076, 3317, 3321, 3373, 3385, 3407, 3439, 3428, 3458, 3532 and processed mature forms and other forms thereof, and allelic and species variants thereof.


In some examples, such modified MMP-1 polypeptides include polypeptides having an amino acid replacement or replacements at any one or more positions corresponding to any of the following positions: 95, 100, 103, 105, 150, 151, 153, 155, 156, 159, 171, 176, 179, 180, 181, 182, 185, 187, 190, 191, 192, 194, 195, 198, 206, 207, 210, 212, 218, 223, 227, 228, 229, 230, 233, 234, 237, 240 and 259 of an unmodified MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2, or at a corresponding position in an allelic or species variant or other variant of an MMP-1 polypeptide that has at least or at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an MMP-1 polypeptide set forth in SEQ ID NO:2. For example, modified MMP-1 polypeptides provided herein include polypeptides having an amino acid modification corresponding to any one or more modifications of L95K, E100V, T103Y, D105A, D105F, D105G, D105I, D105L, D105N, D105R, D105S, D105T, D105W, R150P, D151G, S153T, F155L, F155A, D156H, D156L, D156A, D156W, D156V, D156K, D156T, D156R, G159V, G159T, G171P, A176F, D179N, E180Y, E180R, E180T, E180F, D181T, D181L, D181K, E182T, E182Q, T185R, T185Y, T185H, T185G, T185V, T185Q, T185A, T185E, N187R, N187M, N187W, N187F, N187K, N187I, N187A, E190G, Y191V, N192H, N192S, N192D, N192C, H194P, R195C, R195W, R195L, R195G, R195Q, R195A, R195D, R195V, A198G, A198L, A198M, G206A, G206S, L207R, L207V, S210V, D212G, Y218S, F223C, F223E, F223G, F223A, F223S, V227C, V227D, V227E, V227L, V227S, V227W, Q228P, L229A, L229T, L229I, A230V, D233E, I234A, I234T, I234E, I234Q, I237L, I240S, I240A, I240C, and C259Q. Such modified MMP-1 polypeptides exhibit at least 1.2 times or more activity at the permissive temperature of 25° C. compared to the non-permissive temperatures of 34° C. or 37° C., for example, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times the activity. Exemplary of such modified MMP-1 polypeptides have a sequence of amino acids set forth in any of SEQ ID NOS:6, 25, 27, 29, 31-33, 35-36, 38-39, 59, 70, 95-96, 99-101, 105, 111, 113-115, 125, 132, 148, 160, 177, 181-182, 185, 195, 200, 209, 218-219, 232-233, 235, 238-239, 241, 246, 248, 253-254, 261-264, 267, 269, 273, 275, 279, 282, 284-286, 299, 301, 305, 317, 324, 341, 354, 365, 369, 374-375, 381, 383-384, 388, 393, 397, 399, 420, 429, 436, 438, 440, 460, 466-467, 476, 483, 488, 495, 512, 524, 543, 572, 583, 607, 611, 613, 616, 620, 648, 653, 665, 678, 703, 3076 and 3532 and processed mature forms and other forms thereof, and allelic and species variants thereof.


In other examples, such modified MMP-1 polypeptides include polypeptides having an amino acid replacement or replacements at any one or more positions corresponding to any of the following positions: 95, 105, 150, 151, 155, 156, 159, 176, 179, 180, 181, 182, 185, 187, 195, 198, 206, 210, 212, 218, 223, 227, 228, 229, 230, 233, 234, 240, 259 of an unmodified MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2, or at a corresponding position in an allelic or species variant or other variant of an MMP-1 polypeptide that has at least or at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an MMP-1 polypeptide set forth in SEQ ID NO:2. For example, modified MMP-1 polypeptides provided herein include polypeptides having an amino acid modification corresponding to any one or more modifications of L95K, D105A, D105F, D105G, D105I, D105L, D105N, D105R, D105S, D105T, D105W, R150P, D151G, F155A, D156K, D156T, D156L, D156A, D156W, D156V, D156H, D156R, G159V, G159T, A176F, D179N, E180Y, E180T, E180F, D181L, D181K, E182T, E182Q, T185R, T185H, T185Q, T185A, T185E, N187R, N187M, N187F, N187K, N187I, R195V, A198L, A198M, G206A, G206S, S210V, Y218S, F223E, V227C, V227E, V227W, Q228P, L229T, L229I, D233E, I234A, I234T, I234E, I240S, I240C and C259Q. Such modified MMP-1 polypeptides exhibit at least 1.5 times or more activity at the permissive temperature of 25° C. compared to the non-permissive temperatures of 34° C. or 37° C., for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times the activity. Exemplary of such modified MMP-1 polypeptides have a sequence of amino acids set forth in any of SEQ ID NOS:6, 25, 27, 29, 31-33, 35-36, 38-39, 59, 70, 96, 99-101, 105, 111, 113-115, 125, 132, 148, 160, 181-182, 185, 195, 209, 218-219, 232-233, 235, 238, 248, 253-254, 261-262, 264, 284, 301, 305, 317, 324, 341, 354, 365, 384, 388, 397, 420, 429, 436, 440, 460, 467, 476, 483, 488, 3532 and processed mature forms and other forms thereof, and allelic and species variants thereof.


In additional examples, modified MMP-1 polypeptides provided herein include modified MMP-1 polypeptides that are temperature sensitive at the permissive temperature of 25° C. and exhibit at least 30%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 140%, 150% or more activity at 25° C. compared to wildtype MMP-1 at 25° C. For example, tsMMP-1 polypeptides that exhibit increased activity compared to wildtype MMP-1 include polypeptides having an amino acid replacement or replacements at any one or more positions corresponding to any of the following positions: 95, 105, 150, 156, 159, 179, 180, 182, 185, 187, 195, 198, 212, 223, 227, 234, and 240 of an unmodified MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2, or at a corresponding position in an allelic or species variant or other variant of an MMP-1 polypeptide that has at least or at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an MMP-1 polypeptide set forth in SEQ ID NO:2. For example, modified tsMMP-1 polypeptides provided herein that have increased activity at the permissive temperature of 25° C. compared to wildtype MMP-1 include polypeptides having an amino acid modification corresponding to any one or more modifications L95K, D105A, D105G, D105I, D105L, D105N, D105S, D105W, D105T, R150P, D156K, D156T, D156V, D156H, D156R, G159V, G159T, D179N, E180Y, E180T, E180F, E182T, T185H, T185Q, T185E, N187M, N187K, N187I, R195V, A198L, F223E, V227E, I234E and I240S. Exemplary of such modified MMP-1 polypeptides have a sequence of amino acids set forth in any of SEQ ID NOS:6, 27, 29, 31-32, 35-36, 38-39, 59, 99-101, 105, 113, 125, 132, 160, 181-182, 185, 219, 232-233, 238, 253, 262, 264, 284, 305, 365, 384, 460, 488 or processed mature forms and other forms thereof, and allelic and species variants thereof.


In particular, modified MMP-1 polypeptides provided herein that are temperature sensitive have an amino acid replacement or replacements at any one or more positions corresponding to any of the following positions: 95, 105, 150, 156, 159, 179, 180, 182, 185, 187, 198, 227, 234 and 240 of an unmodified MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2, or at a corresponding position in an allelic or species variant or other variant of an MMP-1 polypeptide that has at least or at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an MMP-1 polypeptide set forth in SEQ ID NO:2. Such modified MMP-1 polypeptides provided herein include polypeptides having an amino acid modification corresponding to any one or more modifications L95K, D105I, D105N, D105L, D105A, D105G, R150P, D156R, D156H, D156K, D156T, G159V, G159T, D179N, E180T, E180F, E182T, T185Q, N187I, A198L, V227E, I234E and I240S. More particularly, modified MMP-1 polypeptides provided herein include polypeptides having an amino acid modification corresponding to any one or more modifications L95K, D105N, R150P, D156K, D156T, G159V, D179N, E180T, A198L, V227E, and I240S.


Modified MMP-1 polypeptides provided herein include those that exhibit reversible or irreversible (also called non-reversible) temperature-dependent activity. In all cases, modified MMP-1 polypeptides provided herein above exhibit increased activity at a permissive temperature (e.g. 25° C.) compared to a non-permissive temperatures (e.g. 34° C. or 37° C.) For non-reversible polypeptides, exposure to the non-permissive temperature prior to, subsequently or intermittently from exposure to the permissive temperature renders the polypeptide irreversibly inactive. Thus, a modified MMP-1 polypeptide that is returned to temperature permissive conditions, for example 25° C., exhibits the same or similar activity of the MMP-1 polypeptide at non-permissive temperatures, for example, 34° C. or 37° C. For example, upon return to permissive conditions, irreversible modified MMP-1 polypeptides provided herein exhibit at or about 50%, 60%, 70%, 80%, 90%, 100%, 105%, 110%, 115%, or 120% the activity at non-permissive temperatures. Exemplary non-reversible modified MMP-1 polypeptides provided herein include polypeptides having an amino acid modification corresponding to any one or more modifications L95K, D105I, D105L, D105N, D105R, D105W, D151G, F155A, D156K, D156T, D156L, D156A, D156W, D156V, D156H, D156R, G159V, A176F, D179N, D181L, D181K, E182T, E182Q, T185R, N187F, N187I, G206A, G206S, V227C, V227E, Q228E, L229T, D233E, I234A, I234T, I234E, I240S, for example, any set forth in any of SEQ ID NOS:6, 25, 27, 35-36, 38, 70, 96, 99-101, 105, 111, 113-115, 132, 148, 160, 195, 209, 218-219, 235, 261, 264, 317, 324, 384, 388, 403, 429, 440, 460, 467, 476, 488, or processed mature forms and other forms thereof, and allelic and species variants thereof.


For reversible polypeptides, exposure to the non-permissive temperature prior to, subsequently or intermittently from exposure to the permissive temperature renders the polypeptide reversibly active. Thus, a modified MMP-1 polypeptide that is returned to temperature permissive conditions recovers activity, and thereby exhibits increased activity at the permissive temperature compared to the non-permissive temperature. In such examples, the recovered activity can be complete or partial. Thus, a modified MMP-1 polypeptide that is returned to temperature permissive conditions, for example 25° C., exhibits an increased activity compared to activity at non-permissive temperatures, for example, 34° C. or 37° C. For example, upon return to permissive conditions, reversible modified MMP-1 polypeptides provided herein exhibit at or about 120%, 125%, 130%, 140%, 150%, 160%, 170%, 180%, 200% or more of the activity at non-permissive temperatures. Exemplary reversible modified MMP-1 polypeptides provided herein include polypeptides having an amino acid modification corresponding to any one or more modifications D105A, D105F, D105G, D105S, D105T, R150P, G159T, E180Y, E180T, E180F, T185H, T185Q, T185A, T185E, N187R, N187M, N187K, R195V, A198L, A198M, S210V, Y218S, F223E, V227W, L229I and I240C, for example, any set forth in any of SEQ ID NOS: 29, 31-33, 39, 59, 125, 181-182, 185, 232-233, 238, 248, 253-254, 262, 284, 301, 305, 341, 354, 365, 397, 436, 483, or processed mature forms and other forms thereof, and allelic and species variants thereof.


2. Matrix Metalloprotease-1 Activity Mutants


Also provided herein are modified MMP-1 polypeptides that exhibit increased activity compared to wild-type MMP-1 at the permissive and non-permissive temperature. Unlike tsMMP-1 polypeptides provided herein, such activity mutants exhibit increased activity at both the permissive and non-permissive temperature compared to the MMP-1 not containing the modification (e.g. wildtype). For example, modified MMP-1's that are provided herein have increased activity compared to wildtype at a low temperature that is less then 37° C., for example, that is at or about 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C., in particular at or about 18° C. to 25° C., for example at or about 25° C. Modified MMP-1's that are provided herein that have increased activity also exhibit increased activity compared to wild-type at higher temperature that is at or about 34° C., 35° C., 36° C., 37° C., 38° C. or 39° C., in particular at or about 34° C. or 37° C. The modified MMP-1's provided herein exhibit 1.1-fold, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. 3.0. 4.0. 5.0, 6.0, 7.0, 8.0. 9.0, 10.0, 20.0 or more increased activity than an MMP-1 not containing the modification (e.g. wildtype) at the same temperature (permissive or non-permissive). For example, the modified MMP-1's provided herein exhibit 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased activity than an MMP-1 not containing the modification (e.g. wildtype) at the same temperature (permissive or non-permissive).


Typically, the modification is an amino acid replacement. The amino acid replacement or replacements can be at any one or more positions corresponding to any of the following positions: 81, 84, 85, 86, 87, 89, 104, 105, 106, 107, 108, 109, 124, 131, 133, 134, 135, 143, 146, 147, 150, 152, 153, 154, 157, 158, 160, 161, 164, 166, 167, 180, 183, 189, 190, 207, 208, 211, 213, 214, 216, 218, 220, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 235, 236, 238, 239, 244, 249, 254, 256, 257, 258 of an unmodified MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2, or at a corresponding position in an allelic or species variant or other variant of an MMP-1 polypeptide that has at least or at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an MMP-1 polypeptide set forth in SEQ ID NO:2. Amino acid replacements include replacement of amino acids to an acidic (D or E); basic (H, K or R); neutral (C, N, Q, T, Y, S, G) or hydrophobic (F, M, W, I V, L A, P) amino acid residue. For example, amino acid replacements at the noted positions include replacement by amino acid residues E, H, R, C, Q, T, S, G, M, W, I, V, L, A, P, N, F, D, Y or K.


For example, modified MMP-1 polypeptides provided herein can include polypeptides having an amino acid modification corresponding to any one or more modifications of F81L (i.e. replacement of F by L at a position corresponding to position 81 of an MMP-1 polypeptide set forth in SEQ ID NO:2), F81A, F81G, F81Q, F81R, F81H, T84H, T84L, T84D, T84R, T84G, T84A, E85S, E85V, G86S, N87P, N87R, N87G, N87Q, R89A, R89T, R89G, R89K, P104E, P104D, P104Q, D105V, L106V, P107T, P107S, P107A, R108E, R108A, R108K, R108S, A109S, A109R, A109G, A109M, A109V, N124G, T131D, K132R, V133T, V133L, S134E, S134D, E135M, S143I, R146S, G147R, G147F, R150E, R150G, R150M, T150T, R150A, R150N, R150K, R150L, R150V, R150D, N152G, N152F, N152L, N152I, S153T, S153P, S153F, S153D, S153Y, P154S, P154I, G157F, P158V, P158I, G160Q, N161L, N161R, N161Y, N161E, N161T, N161I, N161V, N161F, N161Q, H164S, F166W, Q167R, Q167A, Q167S, Q167F, Q167P, Q167T, Q167V, Q167M, E180D, R183S, R189N, R189T, R189Q, E190D, L207M, S208K, S208R, S208L, T211N, I213G, G214L, G214E, L216I, Y218W, S220R, S220A, S220Q, S220T, S220G, S220M, S220V, S220N, T222R, T222P, T222S, T222F, T222N, F223Y, F223H, S224Q, S224K, S224D, G225Q, G225E, G225H, D226S, D226E, D226P, D226I, V227T, Q228A, Q228D, Q228E, Q228G, Q228H, Q228K, Q228L, Q228M, Q228N, Q228R, Q228S, Q228T, Q228W, Q228Y, L229Q, L229P, L229V, A230G, A230W, A230D, A230I, A230S, A230C, A230V, A230T, A230M, A230N, A230H, Q231I, Q231A, Q231F, Q231D, Q231G, Q231V, Q231W, Q231S, Q231H, Q231M, D232H, D232G, D232R, D232P, D232Y, D232S, D232F, D232V, D232K, D232W, D232Q, D232E, D232T, D232L, D235G, D235A, D235L, D235E, D235R, D235Q, D235T, D235N, G236M, G236R, G236S, G236T, G236C, G236K, G236E, G236L, G236N, Q238T, A239S, A239V, A239L, A2391, A239G, A239K, A239H, A239R, S244W, S244Q, Q249W, Q254S, P256S, K257E, K257R, or A258P.


In particular, modified MMP-1 polypeptides provided herein having increased activity have an amino acid modification corresponding to any one or more modifications of N161I, S208K, I213G, G214E, Q228A, Q228D, Q228E, Q228G, Q228H, Q228K, Q228L, Q228M, Q228N, Q228R, Q228S, Q228W, Q228Y, L229V, A230G, A230D, A230S, A230C, A230T, A230M, A230N, A230H, Q231A, Q231D, Q231G, Q231V, Q231S, D232H, D232G, D232P, D232V, D232K, D232W, D232Q, D232E, or D232T. In one example, activity mutants of MMP-1 provided herein including modified MMP-1 polypeptides having one of more modifications of S208K, I213G, or G214E.


Exemplary modified MMP-1 polypeptides have a sequence of amino acids set forth in any of SEQ ID NOS: 37, 41, 42, 44, 46, 48, 51, 53, 56, 57, 58, 174, 358, 366, 373, 391, 402, 403, 404, 405, 406, 408, 409, 410, 411, 412, 414, 415, 418, 419, 428, 437, 439, 535, 543, 544, 546, 553, 573, 662, 687, 689, 692, 693, 695, 697, 698, 700, 701, 702, 703, 781, 783, 786, 795, 796, 790, 838, 836, 840, 852, 846, 853, 864, 870, 884, 911, 897, 903, 899, 938, 941, 948, 934, 1160, 1159, 1166, 1194, 1205, 1207, 1215, 1217, 1219, 1225, 1233, 1239, 1245, 1246, 1248, 1251, 1530, 1653, 1675, 1699, 1707, 1710, 1711, 1741, 1895, 1947, 1961, 1968, 2024, 2025, 2028, 2030, 2043, 2048, 2087, 2088, 2098, 2111, 2114, 2116, 2117, 2118, 2124, 2125, 2121, 2126, 2176, 2218, 2228, 2241, 2231, 2233, 2235, 2236, 2239, 2242, 2423, 2495, 2496, 2497, 2702, 2703, 2715, 2743 2767, 2776, 2791, 2828, 2874, 2887, 2876, 2877, 2878, 2880, 2882, 2885, 2912, 2914, 2917, 2919, 2926, 2927, 2930, 2934, 2947, 2948, 2953, 2965, 2974, 2979, 2983, 2984, 2986, 2993, 2994, 2995, 2996, 2997, 2998, 2999, 3001, 3003, 3004, 3005, 3006, 3009, 3010, 3011, 3012, 3013, 3014, 3016, 3018, 3019, 3021, 3022, 3025, 3027, 3028, 3029, 3032, 3038, 3039, 3042, 3044, 3046, 3047, 3049, 3051, 3057, 3086, 3100, 3101, 3102, 3108, 3109, 3113, 3114, 3115, 3181, 3187, 3282, 3373, 3412, 3422, 3424, or 3458 and processed mature forms and other forms thereof, and allelic and species variants thereof.


3. Combinations


Provided herein are modified MMP-1 polypeptides that contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modifications compared to a starting or reference MMP-1 polypeptide. Modified MMP-1 polypeptides provided herein can contain any two or more modifications provided above. The two or more modifications can include two or more temperature-sensitive modifications, two or more activity modifications, or at least one temperature sensitive modification and at least one activity modification.


For example, modified MMP-1 polypeptides provided herein contain amino acid replacements at any two or more positions corresponding to any of the following positions: 84, 85, 95, 98, 99, 100, 103, 104, 105, 106, 109, 110, 111, 112, 118, 123, 124, 126, 147, 150, 151, 152, 153, 155, 156, 158, 159, 170, 171, 176, 178, 179, 180, 181, 182, 183, 185, 187, 188, 189, 190, 191, 192, 194, 195, 197, 198, 206, 207, 208, 210, 211, 212, 218, 223, 227, 228, 229, 230, 233, 234, 237, 240, 251, 254, 255, 256, 257, 258 or 259 of an unmodified MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2, or at a corresponding position in an allelic or species variant or other variant of an MMP-1 polypeptide that has at least or at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an MMP-1 polypeptide set forth in SEQ ID NO:2 Generally, such combination mutants are temperature sensitive and exhibit increased enzymatic activity at a permissive temperature compared with activity of the tsMMP-1 polypeptide at a non-permissive temperature. Typically, combination mutants also retain activity at the permissive temperature compared to the single mutant MMP-1 polypeptides alone or compared to an unmodified MMP-1 polypeptide not containing the amino acid, changes (e.g. a wildtype MMP-1 polypeptide set forth in SEQ ID NO:2 or active forms or other forms thereof) at the permissive or non-permissive temperature.


Exemplary MMP-1 combination mutants provided herein contain amino acid replacements at any two or more positions corresponding any of the following positions: 95, 105, 150, 156, 159, 179, 180, 182, 185, 187, 198, 227, 234 and 240 of an unmodified MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2, or at a corresponding position in an allelic or species variant or other variant of an MMP-1 polypeptide that has at least or at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an MMP-1 polypeptide set forth in SEQ ID NO:2. For example, modified MMP-1 polypeptides provided herein include polypeptides having amino acid modification corresponding to any two or more modifications L95K, D105I, D105N, D105L, D105A, D105G, R150P, D156R, D156H, D156K, D156T, G159V, G159T, D179N, E180T, E180F, E182T, T185Q, N187I, A198L, V227E, I234E and I240S. More particularly, modified MMP-1 polypeptides provided herein include polypeptides having amino acid modification corresponding to any two or more modifications L95K, D105N, R150P, D156K, D156T, G159V, D179N, E180T, A198L, V227E, and I240S. It is understood that at least two different positions are modified in the combination mutants provided herein. Exemplary MMP-1 combination mutant polypeptides provided herein are set forth in Table 15 in Example 3. For example, combination mutants provided herein that exhibit temperature sensitivity include D156K/G159V/D179N; R150P/V227E; D156T/V227E; G159V/A198L; D105N/A198L; D179N/V227E; A198L/V227E; E180T/V227E; D179N/A198L; D156K/D179N; D105N/R150P/D156K/G159V/D179N/E180T; D105N/R150P/E180T; G159V/I240S; D156T/D179N/I240S; D156T/G159V; R150P/E180T; D156T/D179N; D179N/I240S; L95K/D156T/D179N; G159V/D179N; L95K/D105N/E180T; R150P/D156T/A198L; L95K/D105N/R150P/D156T/G159V/A198L/V227E/I240S; L95K/R150P; or D105N/E180T. Exemplary modified MMP-1 polypeptides have a sequence of amino acids set forth in any of SEQ ID NOS: 3507-3531 and processed mature forms and other forms thereof, and allelic and species variants thereof.


Combination mutants provided herein also can include amino acid modification C259Q and at least one other modification. The other modification can be another temperature sensitive modification, for example, any of modifications L95K, D105I, D105N, D105L, D105A, D105G, R150P, D156R, D156H, D156K, D156T, G159V, G159T, D179N, E180T, E180F, E182T, T185Q, N187I, A198L, V227E, I234E and I2405. Exemplary of such combination mutants include C259Q/D105N; C259Q/R150P; C259Q/G159V; C259Q/D179N/ or C259Q/E180T, for example, as set forth in SEQ ID NOS: 3533-3537.


Also included among the combination mutants provided herein are MMP-1 polypeptides that contain at least one temperature sensitive modification and at least one activity modification, and retain temperature sensitivity. For example, such combination mutants exhibit increased activity at a permissive temperature compared to a non-permissive temperature as described herein above. Any one or more of the temperature sensitive mutants provided in Section D.1 above can be combined with any one or more of the activity mutants provided in Section D.2 above. For example, a combination mutant provided herein contains at least one modification of L95K, D105I, D105N, D105L, D105A, D105G, R150P, D156R, D156H, D156K, D156T, G159V, G159T, D179N, E180T, E180F, E182T, T185Q, N187I, A198L, V227E, I234E and I240S and at least one modification of N161I, S208K, I213G, G214E, Q228A, Q228D, Q228E, Q228G, Q228H, Q228K, Q228L, Q228M, Q228N, Q228R, Q228S, Q228W, Q228Y, L229V, A230G, A230D, A230S, A230C, A230T, A230M, A230N, A230H, Q231A, Q231D, Q231G, Q231V, Q231S, D232H, D232G, D232P, D232V, D232K, D232W, D232Q, D232E, or D232T. For example, a combination mutant provided herein contains at least one modification of L95K, D105N, R150P, D156K, D156T, G159V, D179N, E180T, A198L, V227E, or I240S and at least one modification of S208K, I213G, or G214E. Exemplary combination mutants provided herein include S208K/G159V; S208K/D179N; S208K/V227E; G214E/G159V; G214E/D179N; or I213G/D179N, for example, as set forth in any of SEQ ID NOS: 3541-3546.


4. Additional Modifications


Any modified MMP-1 polypeptide provided herein also can contain one or more other modifications described in the art. The additional modifications can include, for example, any amino acid substitution, deletion or insertion known in the art. In addition to containing one or more modification(s) described above in Sections D.1 and D.2, any modified MMP-1 polypeptide provided herein can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional modifications. Typically, MMP-1 polypeptides retain enzymatic activity of wildtype MMP-1 at the permissive or non-permissive temperature, or exhibit increased enzymatic activity of wildtype MMP-1. Generally, where at least one modification is a temperature sensitive mutation, the MMP-1 polypeptide also exhibits increased activity at the permissive temperature (e.g. 25° C.) compared to the non-permissive temperature (e.g. 34° C. or 37° C.). The additional modifications can confer additional properties to the enzyme, for example, increased stability, increased half-life and/or increased resistance to inhibitors, for example, TIMP. The additional modifications include modifications to the primary sequence of the polypeptide, as well as other modification such as PEGylation and glycosylation of the polypeptide. Generally, such polypeptides include one or more modifications provided herein and exhibit increased activity at the lower temperature then at the higher temperature. For example, any of the amino acid replacements, including allelic variants and other variants known in the art, as set forth in SEQ ID NO:3506 or 3549, can be including herein. Exemplary modifications that can be included in a polypeptide provided herein include, but are not limited to, modifications T4P, Q10P, R30M, R30S, T96R, A114V, F166C, I172V, D181H, R189T, H199A; E200A, G214E, D232N, D233G, R243S, Q254P, I271A, R272A, T286A, I298T, E314G, F315S, V374M, R386Q, S387T, G391S, and T432A of a polypeptide set forth in SEQ ID NO:2.


5. Other MMPs


Matrix metalloproteases are highly homologous polypeptides and exhibit similar specificities for extracellular matrix components. Exemplary sequences of MMPs are set forth in Table 5, for example, any set forth in SEQ ID NOS:1, 711, 714, 717, 720, 723, 726, 729, 732, 735, 738, 741, 744, 747, 750, 753, 756, 759, 762, 765, 768, 771, 774 or 777 or zymogen forms, processed mature forms or other forms thereof, or allelic or species variants thereof. FIG. 1 provides an alignment of the zymogen form of exemplary MMP polypeptides. Thus, any of the modifications provided herein in an MMP-1 can be made in any other MMP polypeptide. Hence, based on the description herein, any MMP, species, allelic variant or other variant, can be made temporally active (reversible or irreversible) by virtue of activity at a permissive temperature (generally a lower temperature) compared to a nonpermissive temperature (generally a higher temperature). Such tsMMP mutants can be used by one of skill in the art and used in compositions, processes or methods for the treatment of ECM-mediated diseases or conditions.


It is within the level of one of skill in the art to align various MMPs to MMP-1 (for example set forth in SEQ ID NO:2) and identify corresponding residues. Any of the modifications provided herein can be made in any other MMP at the corresponding residue. One of skill in the art can test the activity of the resulting modified polypeptide for enzymatic activity and/or temperature sensitivity at a permissive temperature compared to a non-permissive temperature. In particular, it is understood that conservative amino acid differences at a corresponding position in an MMP are functionally invariant. Thus, where a residue in MMP-1 aligns with a conservative residue thereto in another MMP, it is understood that such a residue is contemplated for modification herein. For example, position 95 in an MMP-1 set forth in SEQ ID NO:2 is a leucine (L). Alignment of SEQ ID NO:2 with other MMPs shows that position 95 in other MMPs is a leucine, isoleucine (I) or valine (V) residue (see FIG. 1). Each of L, I and V are conservative residues.


In particular, provided herein are modified MMP polypeptides that are modified by one or more amino acid replacement to confer temperature sensitivity and/or increased activity by effecting a corresponding MMP-1 modification at a corresponding residue Exemplary modifications provided herein include modification of any MMP, for example, an MMP-8, MMP-13, MMP-18, MMP-2, MMP-9, MMP-3, MMP-10, MMP-11, MMP-7, MMP-26 and MMP-12, at any one or more positions corresponding to any of the following positions: 95, 105, 151, 156, 159, 176, 179, 180, 181, 182, 185, 195, 198, 206, 210, 212, 218, 223, 228, 229, 233, 234, and 240 of an unmodified MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2. In other example, exemplary modifications provided herein include modification of any MMP, for example, an MMP-8, MMP-13, MMP-18, MMP-2, MMP-9, MMP-3, MMP-10, MMP-11, MMP-7, MMP-26 and MMP-12, at any one or more positions corresponding to any of the following positions: 81, 89, 109, 131, 133, 154, 157, 158, 160, 164, 166, 180, 207, 216, 218, 223, 228, 229, 231, 232, 236, 238, 256. The modification includes any one or more of the modifications provided herein in sections D.1 and D.2 at the corresponding position to the recited position in MMP-1. For example, residue 95 in an MMP-1 polypeptide set forth in SEQ ID NO:2 corresponds to residue 113 in an MMP-8 polypeptide set forth in SEQ ID NO:711. Thus, provided herein are modified MMP-8 polypeptides having an amino acid modification L113K of an unmodified MMP-8 polypeptide having a sequence of amino acids set forth in SEQ ID NO:711. Similar modifications are provided herein based on this description.


Any modified MMP polypeptide provided herein also can contain one or more other modifications described in the art. The additional modifications can include, for example, any amino acid substitution, deletion or insertion known in the art. In addition to containing one or modification described above in Sections D.1 and D.2, any modified MMP polypeptide provided herein can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional modifications, so long as the resulting MMP polypeptides exhibits increased activity at the permissive temperature (e.g. 25° C.) compared to the non-permissive temperature (e.g. 34° C. or 37° C.) and retains activity of wildtype MMP at the permissive or non-permissive temperature. The additional modifications can confer additional properties to the enzyme, for example, increased stability, increased half-life and/or increased resistance to inhibitors, for example, TIMP. The additional modifications include modifications to the primary sequence of the polypeptide, as well as other modification such as PEGylation and glycosylation of the polypeptide. Generally, such polypeptides include one or more modifications provided herein and exhibit increased activity at the lower temperature then a higher temperature. Exemplary modifications that can be included in a polypeptide provided herein include, but are not limited to, any modifications set forth in Table 6, below.









TABLE 6







Exemplary modifications in MMPs










SEQ



MMP
ID NO
Amino Acid Modifications





MMP-8
711
S3C; T32I; K87E; E153G; D193V; S229T;




N246Y; L249V; Q251A; Q251D; Q251G;




Q251V; Q251S; D252H; D252G; D252P;




D252V; D252K; D252W; D252Q; D252E;




D252T; K460T


MMP-13
714
H2L; A8V; F75S; D89H; L254V; D257H;




D257G; D257P; D257V; D257K; D257W;




D257Q; D257E; D257T; D390G; I427T


MMP-2
720
A27S; R101H; D210Y; A228T; F239L; E404K;




L433V; Q435A; Q435D; Q435G; Q435V;




Q435S; D436H; D436G; D436P; D436V;




D436K; D436W; D436Q; D436E;




D436T; A447V; T498M; V620I; V621L; S644I


MMP-9
723
A20V; N38S; E82K; N127K; L187F; R239H;




T258I; Q279R; L431V; D434H; D434G; D434P;




D434V; D434K; D434W; D434Q; D434E;




D434T; F571V; P574R; R668Q


MMP-3
726
K45E; H113P; R248W; L251V; Q253A; Q253D;




Q253G; Q253V; Q253S; D254H; D254G;




D254P; D254V; D254K; D254W; D254Q;




D254E; D254T


MMP-10
729
L4V; V8G; R53K; G65R; E142Q; L250V;




Q252A; Q252D; Q252G; Q252V; Q252S;




D253H; D253G; D253P; D253V; D253K;




D253W; D253Q; D253E; D253T; F226L;




G282E; L440F; H475L


MMP-11
732
V38A; E44K; P61L; S86P; D166N; F182S;




L245V; D248H; D248G; D248P; D248V;




D248K; D248W; D248Q; D248E; D248T;




Q323H


MMP-7
735
C7W; R77R; S115T; G137D; P241L; L246V;




Q248A; Q248D; Q248G; Q248V; Q248S;




D249H; D249G; D249P; D249V; D249K;




D249W; D249Q; D249E; D249T


MMP-26
738
K43E; S46L; Q239A; Q239D; Q239E; Q239G;




Q239H; Q239K; Q239L; Q239M; Q239N;




Q239R; Q239S; Q239W; Q239Y; Q239V;




L240V; D243H; D243G; D243P; D243V;




D243K; D243W; D243Q; D243E; D243T;




I260M


MMP-12
741
L250V; D253H; D253G; D253P; D253V;




D253K; D253W; D253Q; D253E; D253T;




N357S; F468L; G469R


MMP-19
765
R103C; L243V; D246H; D246G; D246P;




D246V; D246K; D246W; D246Q; D246E;




D246T; P245S; P488T; T491M









E. METHODS OF PRODUCING NUCLEIC ACIDS ENCODING tsMMPS AND POLYPEPTIDES THEREOF

Modified MMP polypeptides, for example tsMMPs set forth herein, can be obtained by methods well known in the art for protein purification and recombinant protein expression. Any method known to those of skill in the art for identification of nucleic acids that encode desired genes can be used. Any method available in the art can be used to obtain a full length (i.e., encompassing the entire coding region) cDNA or genomic DNA clone encoding a desired MMP, such as from a cell or tissue source. Modified or variant tsMMPs, can be engineered from a wildtype polypeptide, such as by site-directed mutagenesis.


Polypeptides can be cloned or isolated using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include PCR amplification of nucleic acids and screening of libraries, including nucleic acid hybridization screening, antibody-based screening and activity-based screening.


Methods for amplification of nucleic acids can be used to isolate nucleic acid molecules encoding a desired polypeptide, including for example, polymerase chain reaction (PCR) methods. A nucleic acid containing material can be used as a starting material from which a desired polypeptide-encoding nucleic acid molecule can be isolated. For example, DNA and mRNA preparations, cell extracts, tissue extracts, fluid samples (e.g. blood, serum, saliva), samples from healthy and/or diseased subjects can be used in amplification methods. Nucleic acid libraries also can be used as a source of starting material. Primers can be designed to amplify a desired polypeptide. For example, primers can be designed based on expressed sequences from which a desired polypeptide is generated. Primers can be designed based on back-translation of a polypeptide amino acid sequence. Nucleic acid molecules generated by amplification can be sequenced and confirmed to encode a desired polypeptide.


Additional nucleotide sequences can be joined to a polypeptide-encoding nucleic acid molecule, including linker sequences containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, for example, a protein expression vector or a vector designed for the amplification of the core protein coding DNA sequences. Furthermore, additional nucleotide sequences specifying functional DNA elements can be operatively linked to a polypeptide-encoding nucleic acid molecule. Examples of such sequences include, but are not limited to, promoter sequences designed to facilitate intracellular protein expression, and secretion sequences, for example heterologous signal sequences, designed to facilitate protein secretion. Such sequences are known to those of skill in the art. For example, exemplary heterologous signal sequences include, but are not limited to, human kappa IgG heterologous signal sequence set forth in SEQ ID NO:3468. For bacterial expression, and exemplary heterologous signal sequence is the pelB leader sequence, for example, as set forth in SEQ ID NO: 3547. Additional nucleotide residues sequences such as sequences of bases specifying protein binding regions also can be linked to enzyme-encoding nucleic acid molecules. Such regions include, but are not limited to, sequences of residues that facilitate or encode proteins that facilitate uptake of an enzyme into specific target cells, or otherwise alter pharmacokinetics of a product of a synthetic gene. For example, enzymes can be linked to PEG moieties.


In addition, tags or other moieties can be added, for example, to aid in detection or affinity purification of the polypeptide. For example, additional nucleotide residues sequences such as sequences of bases specifying an epitope tag or other detectable marker also can be linked to enzyme-encoding nucleic acid molecules. Exemplary of such sequences include nucleic acid sequences encoding a His tag (e.g., 6×His, HHHHHH; SEQ ID NO:3465) or Flag Tag (DYKDDDDK; SEQ ID NO:3467).


The identified and isolated nucleic acids can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art can be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene, La Jolla, Calif.). Other expression vectors include the pET303CTHis (SEQ ID NO:3466; Invitrogen, CA) or pET-26B vector (SEQ ID NO:3548) expression vector exemplified herein. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. Insertion can be effected using TOPO cloning vectors (INVITROGEN, Carlsbad, Calif.). If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and protein gene can be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of the gene sequence are generated.


In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate the isolated protein gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene can be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.


1. Vectors and cells For recombinant expression of one or more of the desired proteins, such as any described herein, the nucleic acid containing all or a portion of the nucleotide sequence encoding the protein can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence. The necessary transcriptional and translational signals also can be supplied by the native promoter for enzyme genes, and/or their flanking regions.


Also provided are vectors that contain a nucleic acid encoding the enzyme. Cells containing the vectors also are provided. The cells include eukaryotic and prokaryotic cells, and the vectors are any suitable for use therein.


Prokaryotic and eukaryotic cells, including endothelial cells, containing the vectors are provided. Such cells include bacterial cells, yeast cells, fungal cells, Archea, plant cells, insect cells and animal cells. The cells are used to produce a protein thereof by growing the above-described cells under conditions whereby the encoded protein is expressed by the cell, and recovering the expressed protein. For purposes herein, for example, the enzyme can be secreted into the medium.


Provided are vectors that contain a sequence of nucleotides that encodes the proenzyme polypeptide coupled to the native or heterologous signal sequence, as well as multiple copies thereof. The vectors can be selected for expression of the enzyme protein in the cell or such that the enzyme protein is expressed as a secreted protein. The proenzyme (i.e. zymogen) form of the enzyme can be purified for use as an activatable, i.e. conditional active, enzyme herein. Alternatively, upon secretion the prosegment can be cleaved by chemical agents or catalytically or autocatalytically to generate a mature enzyme by the use of a processing agent. This processing step can be performed during the purification step and/or immediately before use of the enzyme. If desired, the processing agent can be dialyzed away or otherwise purified away from the purified protein before use. Alternative or additionally, if necessary, the enzyme can be purified such that the prosegment is removed from the preparation.


A variety of host-vector systems can be used to express the protein coding sequence. These include but are not limited to mammalian cell systems transfected with plasmid DNA or infected with virus (e.g. vaccinia virus, adenovirus and other viruses); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system used, any one of a number of suitable transcription and translation elements can be used.


Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene containing appropriate transcriptional/translational control signals and protein coding sequences. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleic acid sequences encoding protein, or domains, derivatives, fragments or homologs thereof, can be regulated by a second nucleic acid sequence so that the genes or fragments thereof are expressed in a host transformed with the recombinant DNA molecule(s). For example, expression of the proteins can be controlled by any promoter/enhancer known in the art. In a specific embodiment, the promoter is not native to the genes for a desired protein. Promoters which can be used include but are not limited to the SV40 early promoter (Bernoist and Chambon, Nature 290:304-310 (1981)), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the (3-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); see also “Useful Proteins from Recombinant Bacteria”: in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthase promoter (Herrara-Estrella et al., Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res. 9:2871 (1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elements from yeast and other fungi such as the Ga14 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al., Nature 315:115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature 318:533-538 (1985); Alexander et al., Mol. Cell. Biol. 7:1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 45:485-495 (1986)), albumin gene control region which is active in liver (Pinckert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., Genes and Devel. 1:161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al., Nature 315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., Cell 48:703-712 (1987)), myosin light chain-2 gene control region which is active in skeletal muscle (Shani, Nature 314:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 234:1372-1378 (1986)).


In a specific embodiment, a vector is used that contains a promoter operably linked to nucleic acids encoding a desired protein, or a domain, fragment, derivative or homolog, thereof, one or more origins of replication, and optionally, one or more selectable markers (e.g., an antibiotic resistance gene). Exemplary plasmid vectors for transformation of E. coli cells, include, for example, the pQE expression vectors (available from Qiagen, Valencia, Calif.; see also literature published by Qiagen describing the system). pQE vectors have a phage T5 promoter (recognized by E. coli RNA polymerase) and a double lac operator repression module to provide tightly regulated, high-level expression of recombinant proteins in E. coli, a synthetic ribosomal binding site (RBS II) for efficient translation, a 6×His tag coding sequence, t0 and T1 transcriptional terminators, ColE1 origin of replication, and a beta-lactamase gene for conferring ampicillin resistance. The pQE vectors enable placement of a 6×His tag at either the N- or C-terminus of the recombinant protein. Such plasmids include pQE 32, pQE 30, and pQE 31 which provide multiple cloning sites for all three reading frames and provide for the expression of N-terminally 6×His-tagged proteins. Other exemplary plasmid vectors for transformation of E. coli cells, include, for example, the pET expression vectors (see, U.S. Pat. No. 4,952,496; available from NOVAGEN, Madison, Wis.; see, also literature published by Novagen describing the system). Such plasmids include pET 11a, which contains the T71ac promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene; pET 12a-c, which contains the T7 promoter, T7 terminator, and the E. coli ompT secretion signal; and pET 15b and pET19b (NOVAGEN, Madison, Wis.), which contain a His-Tag™ leader sequence for use in purification with a His column and a thrombin cleavage site that permits cleavage following purification over the column, the T7-lac promoter region and the T7 terminator, and pET-26B (SEQ ID NO:3548). An additional pET vector is pET303CTHis (set forth in SEQ ID NO: 3466; Invitrogen, CA), which contains a T71ac promoter, T7 terminator, the inducible E. coli lac operator, a beta-lactamase gene for conferring ampicillin resistance, and also a His-Tag sequence for use in purification.


Exemplary of a vector for mammalian cell expression is the HZ24 expression vector. The HZ24 expression vector was derived from the pCI vector backbone (Promega). It contains DNA encoding the Beta-lactamase resistance gene (AmpR), an F1 origin of replication, a Cytomegalovirus immediate-early enhancer/promoter region (CMV), and an SV40 late polyadenylation signal (SV40). The expression vector also has an internal ribosome entry site (IRES) from the ECMV virus (Clontech) and the mouse dihydrofolate reductase (DHFR) gene.


2. Expression


Modified MMP polypeptides, for example tsMMPs, can be produced by any method known to those of skill in the art including in vivo and in vitro methods. Desired proteins can be expressed in any organism suitable to produce the required amounts and forms of the proteins, such as for example, needed for administration and treatment. Expression hosts include prokaryotic and eukaryotic organisms such as E. coli, yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification.


Many expression vectors are available and known to those of skill in the art and can be used for expression of proteins. The choice of expression vector will be influenced by the choice of host expression system. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector.


Modified MMP polypeptides, for example tsMMPs, also can be utilized or expressed as protein fusions. For example, an enzyme fusion can be generated to add additional functionality to an enzyme. Examples of enzyme fusion proteins include, but are not limited to, fusions of a signal sequence, a tag such as for localization, e.g. a his6 tag or a myc tag, or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and/or membrane association.


Generally, modified MMP polypeptides, for example tsMMPs, are expressed in an inactive zymogen form. Zymogen conversion can be achieved by exposure to chemical agents, to other proteases or to autocatalysis to generate a mature enzyme as described elsewhere herein. Any form of an enzyme is contemplated herein. It is understood that, if provided and expressed in a zymogen form, that it is activated prior to use by a processing agent.


a. Prokaryotic Cells


Prokaryotes, especially E. coli, provide a system for producing large amounts of proteins. Transformation of E. coli is simple and rapid technique well known to those of skill in the art. Expression vectors for E. coli can contain inducible promoters, such promoters are useful for inducing high levels of protein expression and for expressing proteins that exhibit some toxicity to the host cells. Examples of inducible promoters include the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature regulated λPL promoter.


Proteins, such as any provided herein, can be expressed in the cytoplasmic environment of E. coli. The cytoplasm is a reducing environment and for some molecules, this can result in the formation of insoluble inclusion bodies. Reducing agents such as dithiothreotol and β-mercaptoethanol and denaturants, such as guanidine-HCl and urea can be used to resolubilize the proteins. An alternative approach is the expression of proteins in the periplasmic space of bacteria which provides an oxidizing environment and chaperonin-like and disulfide isomerases and can lead to the production of soluble protein. Typically, a leader sequence is fused to the protein to be expressed which directs the protein to the periplasm. The leader is then removed by signal peptidases inside the periplasm. Examples of periplasmic-targeting leader sequences include the pelB leader (SEQ ID NO: 3547) from the pectate lyase gene and the leader derived from the alkaline phosphatase gene. In some cases, periplasmic expression allows leakage of the expressed protein into the culture medium. The secretion of proteins allows quick and simple purification from the culture supernatant. Proteins that are not secreted can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become insoluble and denaturants and reducing agents can be used to facilitate solubilization and refolding. Temperature of induction and growth also can influence expression levels and solubility, typically temperatures between 25° C. and 37° C. are used. Typically, bacteria produce aglycosylated proteins. Thus, if proteins require glycosylation for function, glycosylation can be added in vitro after purification from host cells.


b. Yeast Cells


Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are well known yeast expression hosts that can be used for production of proteins, such as any described herein. Yeast can be transformed with episomal replicating vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters are used to regulate gene expression. Examples of such promoters include GAL1, GAL7 and GAL5 and metallothionein promoters, such as CUP1, AOX1 or other Pichia or other yeast promoter. Expression vectors often include a selectable marker such as LEU2, TRP1, HIS3 and URA3 for selection and maintenance of the transformed DNA. Proteins expressed in yeast are often soluble. Co-expression with chaperonins such as Bip and protein disulfide isomerase can improve expression levels and solubility. Additionally, proteins expressed in yeast can be directed for secretion using secretion signal peptide fusions such as the yeast mating type alpha-factor secretion signal from Saccharomyces cerevisae and fusions with yeast cell surface proteins such as the Aga2p mating adhesion receptor or the Arxula adeninivorans glucoamylase. A protease cleavage site such as for the Kex-2 protease, can be engineered to remove the fused sequences from the expressed polypeptides as they exit the secretion pathway. Yeast also is capable of glycosylation at Asn-X-Ser/Thr motifs.


c. Insect Cells


Insect cells, particularly using baculovirus expression, are useful for expressing polypeptides such as matrix-degrading enzymes. Insect cells express high levels of protein and are capable of most of the post-translational modifications used by higher eukaryotes. Baculovirus have a restrictive host range which improves the safety and reduces regulatory concerns of eukaryotic expression. Typical expression vectors use a promoter for high level expression such as the polyhedrin promoter of baculovirus. Commonly used baculovirus systems include the baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV), and the bombyx mori nuclear polyhedrosis virus (BmNPV) and an insect cell line such as Sf9 derived from Spodoptera frugiperda, Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1). For high-level expression, the nucleotide sequence of the molecule to be expressed is fused immediately downstream of the polyhedrin initiation codon of the virus. Mammalian secretion signals are accurately processed in insect cells and can be used to secrete the expressed protein into the culture medium. In addition, the cell lines Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1) produce proteins with glycosylation patterns similar to mammalian cell systems.


An alternative expression system in insect cells is the use of stably transformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells (Drosophila melanogaster) and C7 cells (Aedes albopictus) can be used for expression. The Drosophila metallothionein promoter can be used to induce high levels of expression in the presence of heavy metal induction with cadmium or copper. Expression vectors are typically maintained by the use of selectable markers such as neomycin and hygromycin.


d. Mammalian Cells


Mammalian expression systems can be used to express proteins including tsMMPs. Expression constructs can be transferred to mammalian cells by viral infection such as adenovirus or by direct DNA transfer such as liposomes, calcium phosphate, DEAE-dextran and by physical means such as electroporation and microinjection. Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translational initiation sequence (Kozak consensus sequence) and polyadenylation elements. IRES elements also can be added to permit bicistronic expression with another gene, such as a selectable marker. Such vectors often include transcriptional promoter-enhancers for high-level expression, for example the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter and the long terminal repeat of Rous sarcoma virus (RSV). These promoter-enhancers are active in many cell types. Tissue and cell-type promoters and enhancer regions also can be used for expression. Exemplary promoter/enhancer regions include, but are not limited to, those from genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin, beta globin, myelin basic protein, myosin light chain 2, and gonadotropic releasing hormone gene control. Selectable markers can be used to select for and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase. For example, expression can be performed in the presence of methotrexate to select for only those cells expressing the DHFR gene. Fusion with cell surface signaling molecules such as TCR-ζ and FcεRI-γ can direct expression of the proteins in an active state on the cell surface.


Many cell lines are available for mammalian expression including mouse, rat human, monkey, chicken and hamster cells. Exemplary cell lines include but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NS0 (nonsecreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are available adapted to serum-free media which facilitates purification of secreted proteins from the cell culture media. Examples include CHO-S cells (Invitrogen, Carlsbad, Calif., cat #11619-012) and the serum free EBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-42.). Cell lines also are available that are adapted to grow in special mediums optimized for maximal expression. For example, DG44 CHO cells are adapted to grow in suspension culture in a chemically defined, animal product-free medium.


e. Plants


Transgenic plant cells and plants can be used to express proteins such as any described herein. Expression constructs are typically transferred to plants using direct DNA transfer such as microprojectile bombardment and PEG-mediated transfer into protoplasts, and with agrobacterium-mediated transformation. Expression vectors can include promoter and enhancer sequences, transcriptional termination elements and translational control elements. Expression vectors and transformation techniques are usually divided between dicot hosts, such as Arabidopsis and tobacco, and monocot hosts, such as corn and rice. Examples of plant promoters used for expression include the cauliflower mosaic virus promoter, the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters.


Selectable markers such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue) or regenerated into whole plants. Transgenic plant cells also can include algae engineered to produce matrix-degrading enzymes. Because plants have different glycosylation patterns than mammalian cells, this can influence the choice of protein produced in these hosts.


3. Purification Techniques


Method for purification of polypeptides, including modified MMP polypeptides such as tsMMPs or other proteins, from host cells will depend on the chosen host cells and expression systems. For secreted molecules, proteins are generally purified from the culture media after removing the cells. For intracellular expression, cells can be lysed and the proteins purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material to make a lysed cell extract. Additionally, transgenic animal production can include the production of polypeptides in milk or eggs, which can be collected, and if necessary, the proteins can be extracted and further purified using standard methods in the art. If there are free cysteines, these can be replaced with other amino acids, such as serine. Replacement of free cysteines can prevent unwanted aggregation.


Generally, modified MMP polypeptides, such as tsMMPs, are expressed and purified to be in an inactive form (zymogen form) for subsequent activation as described in the systems and methods provided herein. Hence, following expression, mature forms can be generated by the use of a processing agent and chemical modification, catalysis and/or autocatalysis to remove the prosegment. Generally, a processing agent is chosen that is acceptable for administration to a subject. If necessary, additional purification steps can be performed to remove the processing agent from the purified preparation. In addition, if necessary, additional purification steps can be performed to remove the prosegment from the purified preparation. Activation can be monitored by SDS-PAGE (e.g., a 3 kilodalton shift) and by enzyme activity (cleavage of a fluorogenic substrate). Where an active enzyme is desired, typically, an enzyme is allowed to achieve >75% activation before purification. Typically, MMPs are rendered active by activation cleavage removing the propeptide or prosegment to generate a mature enzyme from a zymogen form. In some applications under nonpermissive temperatures, however, tsMMPs are inactive in their mature form until exposure to the requisite permissive temperature as described herein. For example, many MMPs provided herein are not active or substantially inactive at the non-permissive temperature.


Proteins, such as modified MMP polypeptides, for example, tsMMPs, can be purified using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation and ionic exchange chromatography, such as anion exchange. Affinity purification techniques also can be utilized to improve the efficiency and purity of the preparations. For example, antibodies, receptors and other molecules that bind MMPs can be used in affinity purification. Expression constructs also can be engineered to add an affinity tag to a protein such as a myc epitope, GST fusion or His6 and affinity purified with myc antibody, glutathione resin and Ni-resin, respectively. Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometric techniques.


F. PREPARATION, FORMULATION AND ADMINISTRATION OF tsMMPS

The pharmaceutical compositions provided herein contain modified MMP polypeptides as described herein, for example tsMMPs and/or activity mutants. The compounds can be formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administrate, as well as transdermal patch preparation and dry powder inhalers. Typically, the compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126). The pharmaceutical compositions are administered prior to, simultaneously, subsequently or intermittently with an activator that provides the requisite temperature for activation.


A selected modified MMP polypeptide, for example tsMMP, is included in an amount sufficient that, when activated to a mature form and, if necessary, exposed to the permissive temperature, exerts a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The composition containing the modified MMP polypeptide, for example tsMMP, can include a pharmaceutically acceptable carrier. Therapeutically effective concentration can be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein. The concentration of a selected modified MMP polypeptide, for example tsMMP, in the composition depends on absorption, inactivation and excretion rates of the complex, the physicochemical characteristics of the complex, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, it is understood that the precise dosage and duration of treatment is a function of the tissue being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the age of the individual treated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope thereof.


The amount of a selected modified MMP polypeptide, for example tsMMP, to be administered for the treatment of a disease or condition, for example an ECM-mediated disease or condition such as cellulite or lymphedema, can be determined by standard clinical techniques. In addition, in vitro assays and animal models can be employed to help identify optimal dosage ranges. The precise dosage, which can be determined empirically, can depend on the particular enzyme, the route of administration, the type of disease to be treated and the seriousness of the disease. Exemplary dosages range from or about 10 μg to 100 mg, particularly 50 μg to 75 mg, 100 μg to 50 mg, 250 μg to 25 mg, 500 μg to 10 mg, 1 mg to 5 mg, or 2 mg to 4 mg. The particular dosage and formulation thereof depends upon the indication and individual. If necessary dosage can be empirically determined. Typically the dosage is administered for indications described herein in a volume of 1-100 ml, particularly, 1-50 ml, 10-50 ml, 10-30 ml, 1-20 ml, or 1-10 ml volumes following reconstitution, such as by addition of an activator (e.g. a cold buffer). Typically, such dosages are from at or about 100 μg to 50 mg, generally 1 mg to 5 mg, in a 10-50 ml final volume.


A modified MMP polypeptide, for example tsMMP, can be administered at once, or can be divided into a number of smaller doses to be administered at intervals of time. Selected modified MMP polypeptides, for example tsMMPs, can be administered in one or more doses over the course of a treatment time for example over several hours, days, weeks, or months. In some cases, continuous administration is useful. It is understood that the precise dosage and course of administration depends on the methods and system of activation contemplated.


Also, it is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values also can vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or use of compositions and combinations containing them. The compositions can be administered hourly, daily, weekly, monthly, yearly or once. Generally, dosage regimens are chosen to limit toxicity. It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney or other tissue dysfunctions. Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects).


Pharmaceutically acceptable compositions are prepared in view of approvals for a regulatory agency or other agency prepared in accordance with generally recognized pharmacopeia for use in animals and in humans. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, and sustained release formulations. A composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and other such agents. The formulation should suit the mode of administration.


Pharmaceutical compositions can include carriers such as a diluent, adjuvant, excipient, or vehicle with which an enzyme is administered. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, generally in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions. Compositions can contain along with an active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polyvinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. A composition, if desired, also can contain minor amounts of wetting or emulsifying agents, or pH buffering agents, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.


Formulations are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof. Pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit dosage forms or multiple dosage forms. Each unit dose contains a predetermined quantity of therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit dose forms can be administered in fractions or multiples thereof. A multiple dose form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dose form. Examples of multiple dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit doses that are not segregated in packaging. Generally, dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier can be prepared.


Compositions can be formulated for administration by any route known to those of skill in the art including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, epidural, nasal, oral, vaginal, rectal, topical, local, otic, inhalational, buccal (e.g., sublingual), and transdermal administration or any route. Administration can be local, topical or systemic depending upon the locus of treatment. Local administration to an area in need of treatment can be achieved by, for example, but not limited to, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant. Compositions also can be administered with other biologically active agents, either sequentially, intermittently or in the same composition. Administration also can include controlled release systems including controlled release formulations and device controlled release, such as by means of a pump.


The most suitable route in any given case depends on a variety of factors, such as the nature of the disease, the progress of the disease, the severity of the disease the particular composition which is used. For purposes herein, it is desired that modified MMP polypeptides, for example tsMMPs, are administered so that they reach the interstitium of skin or tissues. Thus, direct administration under the skin, such as by sub-epidermal administration methods, is contemplated. These include, for example, subcutaneous, intradermal and intramuscular routes of administration. Thus, in one example, local administration can be achieved by injection, such as from a syringe or other article of manufacture containing a injection device such as a needle. Other modes of administration also are contemplated. Pharmaceutical compositions can be formulated in dosage forms appropriate for each route of administration.


In one example, pharmaceutical preparation can be in liquid form, for example, solutions, syrups or suspensions. If provided in liquid form, the pharmaceutical preparation of tsMMP, for example, can be provided as a concentrated preparation to be diluted to a therapeutically effective concentration upon exposure to the permissive temperature, for example, addition of the activator (e.g. a cold buffer). The activator can be added to the preparation prior to administration, or the activator can be added simultaneously, intermittently or sequentially with the tsMMP preparation. Further, if provided in liquid form, the temperature of the preparation can be regulated prior to use in order to achieve a desired temperature for activation. For example, the liquid preparation can be chilled in an ice bucket or in a cold fridge or cold room prior to use and administration. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).


In another example, pharmaceutical preparations can be presented in lyophilized form for reconstitution with water or other suitable vehicle before use. For example, the pharmaceutical preparations of tsMMP can be reconstituted with a solution containing an activator at the requisite temperature, generally a cold buffer or liquid solution or a room temperature buffer or liquid solution. Alternatively, once reconstituted, the preparation can be regulated prior to use in order to achieve a desired temperature for activation. For example, the reconstituted liquid preparation can be stored at temperatures that are below the physiological temperature of the body, e.g. at 4° C. to 25° C.


Typically, modified MMP polypeptides provided herein are prepared in compositions containing requisite metals required for activity. For example, MMPs are Zn-dependent and Ca-dependent polypeptides. It is within the level of one of skill in the art to empirically determine the optimal concentration of zinc and calcium required for activity. Where the modified MMP polypeptide is a tsMMP, the optimal concentration of zinc and calcium is a concentration that maintains the temperature-sensitive phenotype. For example, as described herein (e.g. Examples 13 and 14) the presence of zinc can affect the temperature sensitive phenotype of MMP polypeptides. For example, the optimal concentration of ZnCl2 in MMP compositions provided herein is typically less than 0.01 mM, for example, 0.0005 mM to 0.009 mM, and in particular 0.0005 mM to 0.005 mM, for example 0.001 mM. The optimal concentration of CaCl2 is typically greater than about 1 mM, for example, 2 mM to 50 mM, in particular 5 mM to 20 mM, for example 10 mM to 15 mM, such as 10 mM. Other metals also can be included in the compositions as required for activity.


Administration methods can be employed to decrease the exposure of modified MMP polypeptides to degradative processes, such as proteolytic degradation and immunological intervention via antigenic and immunogenic responses. Examples of such methods include local administration at the site of treatment. PEGylation of therapeutics has been reported to increase resistance to proteolysis, increase plasma half-life, and decrease antigenicity and immunogenicity. Examples of PEGylation methodologies are known in the art (see for example, Lu and Felix, Int. J. Peptide Protein Res., 43: 127-138, 1994; Lu and Felix, Peptide Res., 6-142-6, 1993; Felix et al., Int. J. Peptide Res., 46: 253-64, 1995; Benhar et al., J. Biol. Chem., 269: 13398-404, 1994; Brumeanu et al., J Immunol., 154: 3088-95, 1995; see also, Caliceti et al. (2003) Adv. Drug Deliv. Rev. 55(10):1261-77 and Molineux (2003) Pharmacotherapy 23 (8 Pt 2):3S-8S). PEGylation also can be used in the delivery of nucleic acid molecules in vivo. For example, PEGylation of adenovirus can increase stability and gene transfer (see, e.g., Cheng et al. (2003) Pharm. Res. 20(9): 1444-51).


1. Injectables, Solutions and Emulsions


Parenteral administration, generally characterized by injection, either subcutaneously, intramuscularly or intradermally is contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. The pharmaceutical compositions also may contain other minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins. Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained (see, e.g., U.S. Pat. No. 3,710,795) is also contemplated herein. The percentage of active compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.


Parenteral administration of the compositions generally includes sub-epidermal routes of administration such as intradermal, subcutaneous and intramuscular administrations. If desired, intravenous administration also is contemplated. Injectables are designed for local and systemic administration. For purposes herein, local administration is desired for direct administration to the affected interstitium. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous. If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.


Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multiple-dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEENs 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.


The concentration of the pharmaceutically active compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art. The unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. The volume of liquid solution or reconstituted powder preparation, containing the pharmaceutically active compound, is a function of the disease to be treated and the particular article of manufacture chosen for package. For example, for the treatment of cellulite, it is contemplated that for parenteral injection the injected volume is or is about 10 to 50 milliliters. All preparations for parenteral administration must be sterile, as is known and practiced in the art.


Lyophilized Powders


Of interest herein are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels.


The sterile, lyophilized powder is prepared by dissolving a compound of inactive enzyme in a buffer solution. The buffer solution may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. Briefly, the lyophilized powder is prepared by dissolving an excipient, such as dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent, in a suitable buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art. Then, a selected enzyme is added to the resulting mixture, and stirred until it dissolves. The resulting mixture is sterile filtered or treated to remove particulates and to insure sterility, and apportioned into vials for lyophilization. Each vial will contain a single dosage (1 mg-1 g, generally 1-100 mg, such as 1-5 mg) or multiple dosages of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.


Reconstitution of this lyophilized powder with a buffer solution provides a formulation for use in parenteral administration. The solution chosen for reconstitution can be any buffer. For reconstitution about 1 μg-20 mg, preferably 10 μg-1 mg, more preferably about 100 μg is added per mL of buffer or other suitable carrier. The precise amount depends upon the indication treated and selected compound. Such amount can be empirically determined.


2. Topical Administration


Topical mixtures are prepared as described for the local and systemic administration. The resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.


The compounds or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e. q., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will typically diameters of less than 50 microns, preferably less than 10 microns.


The compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients also can be administered.


Formulations suitable for transdermal administration are provided. They can be provided in any suitable format, such as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches contain the active compound in optionally buffered aqueous solution of, for example, 0.1 to 0.2M concentration with respect to the active compound. Formulations suitable for transdermal administration also can be delivered by iontophoresis (see, e.g., Pharmaceutical Research 3(6), 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.


3. Compositions for Other Routes of Administration


Depending upon the condition treated other routes of administration, such as topical application, transdermal patches, oral and rectal administration are also contemplated herein. For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories include solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used. Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding. The typical weight of a rectal suppository is about 2 to 3 gm. Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.


Formulations suitable for rectal administration can be provided as unit dose suppositories. These can be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.


For oral administration, pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well-known in the art.


Formulations suitable for buccal (sublingual) administration include, for example, lozenges containing the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia.


Pharmaceutical compositions also can be administered by controlled release formulations and/or delivery devices (see, e.g., in U.S. Pat. Nos. 3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566).


Various delivery systems are known and can be used to administer selected tsMMPs, such as but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor mediated endocytosis, and delivery of nucleic acid molecules encoding selected matrix-degrading enzymes such as retrovirus delivery systems.


Hence, in certain embodiments, liposomes and/or nanoparticles also can be employed with administration of matrix-degrading enzymes. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 angstroms containing an aqueous solution in the core.


Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios, the liposomes form. Physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.


Liposomes interact with cells via different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one can operate at the same time. Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use herein, and such particles can be easily made.


4. Activator


Generally, a tsMMP is administered in the presence of an activator that provides the requisite permissive temperature for activation of the enzyme. In other words, tsMMP provided herein are provided for administration at the requisite permissive temperature. Thus, activators provided herein include any that are capable of providing a temperature condition, hot or cold, and that do not exist at the site of administration unless provided exogenously. Thus, tsMMPs can be regulated by controlling the timing and duration of exposure to the temperature condition. An activator is chosen such that it provides a warm or cold temperature depending on the particular enzyme and the permissive temperature requirements provided for activation.


For example where the permissive temperature is 25° C. an activator includes a buffer or other liquid diluent that is at or about 25° C., 24° C., 23° C., 22° C., 21° C., 20° C., 19° C., 18° C., 17° C., 16° C., 15° C., 14° C., 13° C., 12° C., 11° C., 10° C., 9° C., 8° C., 7° C., 6° C., 5° C. or less. In other words, the tsMMP is provided and/or exposed to a buffer or other liquid diluent that is at or about 25° C., 24° C., 23° C., 22° C., 21° C., 20° C., 19° C., 18° C., 17° C., 16° C., 15° C., 14° C., 13° C., 12° C., 11° C., 10° C., 9° C., 8° C., 7° C., 6° C., 5° C. or less. The buffer or liquid can be provided in the same composition as the tsMMP or in a separate composition. When provided separately, it can be administered prior to, simultaneously, subsequently or intermittently from the tsMMP. Upon administration in vivo where the physiologic temperature is at or about 37° C., the temperature of the buffer will warm up to a temperature providing the permissive temperature for activation of the tsMMP (which could occur immediately or almost immediately depending on the temperature of the liquid). Due to the physiologic temperature conditions in vivo, the temperature will warm to non-permissive conditions, thereby resulting in inactivation of the enzyme and temporal control thereof.


In another example, the activator can be a cold pack or a hot pack, depending on the particular enzyme and the permissive temperature provided. Such activators include, but are not limited to ice wraps, gel ice packs, cold therapy, ice packs, cold compress, ice blankets, or other similar items. In other words, the site of locus of administration of the tsMMP can be exposed to the cold pack or hot pack in order to cool or warm the site of administration below or above the physiological temperature of the body, respectively, prior to, concurrently or subsequently with administration of the tsMMP to the same locus. For example, the cold pack can be frozen (e.g. ice pack), or can be a liquid cold pack maintained at a temperature that is 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C. or more. A cold or hot pack can be applied directly to the locus of treatment, and generally is applied locally to the skin at the site of administration of the tsMMP. One of skill in the art can empirically determine the length of time required for application depending of the particular target depth of the tissue that is being treated, the particular enzyme that is being used, and other factors based on known testing protocols or extrapolation from in vivo or in vitro test data. The hot pack or cold pack can be applied prior to, subsequently, simultaneously or intermittently from the tsMMP. For example, if the particular enzyme is reversibly active, the cold pack can be applied intermittently over a course of hours or days. It is understood that it is customary for a subject to feel cold, aching and burning and numbness upon administration of a cold pack, and such symptoms can be monitored by the subject or a treating physician.


In particular embodiments, the tsMMP is exposed to a temperature that is at or below the permissive temperature of the body immediately before administration. For example, the tsMMP is stored at a cold temperature and/or is reconstituted in a cold buffer. In some examples, the locus of administration of the tsMMP also is exposed cold by exposure to a cold pack to cool the site of administration below the physiologic temperature of the body. Upon administration of the tsMMP, the tsMMP is exposed to the permissive temperature, which will steadily warm to the nonpermissive physiologic temperature of the body (e.g. about 37° C.). Where the temperature reaches the nonpermissive temperature, the tsMMP is rendered inactive or substantially inactive. Hence, activation of the tsMMP is conditionally controlled. The duration of time of exposure to a permissive temperature below the physiological temperature of the body can be controlled by continued exposure to a cold pack at the site of administration for a predetermined length of time.


In another embodiment, the tsMMP is exposed to a temperature that is at or above the permissive temperature of the body immediately before administration. For example, the tsMMP is stored at a warm temperature and/or is reconstituted in a warm buffer that is above the physiological temperature of the body. In some examples, the locus of administration of the tsMMP also is warmed by exposure to a hot pack to warm the site of administration above the physiologic temperature of the body. Upon administration of the tsMMP, the tsMMP is exposed to the permissive temperature, which will steadily cool to the nonpermissive physiologic temperature of the body (e.g. about 37° C.). Where the temperature reaches the nonpermissive temperature, the tsMMP is rendered inactive or substantially inactive. Hence, activation of the tsMMP is conditionally controlled. The duration of time of exposure to a permissive temperature above the physiological temperature of the body can be controlled by continued exposure to a hot pack at the site of administration for a predetermined length of time.


5. Combination Therapies


Any of the modified MMP polypeptides, for example tsMMPs, described herein can be further co-formulated or co-administered together with, prior to, intermittently with, or subsequent to, other therapeutic or pharmacologic agents or procedures. Such agents include, but are not limited to, other biologics, small molecule compounds, dispersing agents, anesthetics, vasoconstrictors and surgery, and combinations thereof. For example, for any disease or condition, including all those exemplified above, for which other agents and treatments are available, selected modified MMPs, for example tsMMPs, for such diseases and conditions can be used in combination therewith. In another example, a local anesthetic, for example, lidocaine can be administered to provide pain relief. In some examples, the anesthetic can be provided in combination with a vasoconstrictor to increase the duration of the anesthetic effects. Any of the pharmacological agents provided herein can be combined with a dispersion agent that facilitates access into the tissue of pharmacologic agents, for example, following subcutaneous administration. Such substances are known in the art and include, for example, soluble glycosaminoglycanase enzymes such as members of the hyaluronidase glycoprotein family (US20050260186, US20060104968).


Compositions of modified MMPs, for example tsMMPs, provided herein can be co-formulated or co-administered with a local anesthesia. Anesthesias include short-acting and long-lasting local anesthetic drug formulations. Short-acting local anesthetic drug formulations contain lidocaine or a related local anesthetic drug dissolved in saline or other suitable injection vehicle. Typically, local anesthesia with short-acting local anesthetics last approximately 20-30 minutes. Exemplary anesthetics include, for example, non-inhalation local anesthetics such as ambucaines; amoxecaines; amylocalnes; aptocaines; articaines; benoxinates; benzyl alcohols; benzocaines; betoxycaines; biphenamines; bucricaines; bumecaines; bupivacaines; butacaines; butambens; butanilicaines; carbizocaines; chloroprocaine; clibucaines; clodacaines; cocaines; dexivacaines; diamocaines; dibucaines; dyclonines; elucaines; etidocaines; euprocins; fexicaines; fomocaines; heptacaines; hexylcaines; hydroxyprocaines; hydroxytetracaines; isobutambens; ketocaines; leucinocaines; lidocaines; mepivacaines; meprylcaines; octocaines; orthocaines; oxethacaines; oxybuprocaines; phenacaines; pinolcaines; piperocaines; piridocaines; polidocanols; pramocaines; prilocalnes; procaines; propanocaines; propipocaines; propoxycaines; proxymetacaines; pyrrocaines; quatacaines; quinisocaines; risocaines; rodocaines; ropivacaines; salicyl alcohols; suicaines; tetracaines; trapencaines; and trimecaines; as well as various other non-inhalation anesthetics such as alfaxalones; amolanones; etoxadrols; fentanyls; ketamines; levoxadrols; methiturals; methohexitals; midazolams; minaxolones; propanidids; propoxates; pramoxines; propofols; remifentanyls; sufentanyls; tiletamines; and zolamine. The effective amount in the formulation will vary depending on the particular patient, disease to be treated, route of administration and other considerations. Such dosages can be determined empirically.


Due to the short half-life of local anesthetics, it is often desirable to co-administer or co-formulate such anesthetics with a vasoconstrictor. Examples of vasoconstrictors include alpha adrenergic receptor agonists including catecholamines and catecholamine derivatives. Particular examples include, but are not limited to, levonordefrin, epinephrine and norepinephrine. For example, a local anesthetic formulation, such as lidocaine, can be formulated to contain low concentrations of epinephrine or another adrenergic receptor agonist such as levonordefrin. Combining local anesthetics with adrenergic receptor agonists is common in pharmaceutical preparations (see e.g., U.S. Pat. Nos. 7,261,889 and 5,976,556). The vasoconstrictor is necessary to increase the half-life of anesthetics. The vasoconstrictor, such as epinephrine, stimulates alpha-adrenergic receptors on the blood vessels in the injected tissue. This has the effect of constriction the blood vessels in the tissue. The blood vessel constriction causes the local anesthetic to stay in the tissue much longer, resulting in a large increase in the duration of the anesthetic effect.


Generally, a vasoconstrictor is used herein in combination with an anesthetic. The anesthetic agent and vasoconstrictor can be administered together as part of a single pharmaceutical composition or as part of separate pharmaceutical compositions acting together to prolong the effect of the anesthesia, so long as the vasoconstrictor acts to constrict the blood vessels in the vicinity of the administered anesthetic agent. In one example, the anesthetic agent and vasoconstrictor are administered together in solution. In addition, the anesthetic agent and vasoconstrictor can be formulated together or separate from the activatable matrix-degrading enzyme and activator. Single formulations are preferred. The anesthetic agent and vasoconstrictor can be administered by injection, by infiltration or by topical administration, e.g., as part of a gel or paste. Typically, the anesthetic agent and vasoconstrictor are administered by injection directly into the site to be anesthetized, for example, by subcutaneous administration. The effective amount in the formulation will vary depending on the particular patient, disease to be treated, route of administration and other considerations. Such dosages can be determined empirically. For example, exemplary amounts of lidocaine are or are about 10 mg to 1000 mg, 100 mg to 500 mg, 200 mg to 400 mg, 20 mg to 60 mg, or 30 mg to 50 mg. The dosage of lidocaine administered will vary depending on the individual and the route of administration.


Epinephrine can be administered in amounts such as, for example, 10 μg to 5 mg, 50 μg to 1 mg, 50 μg to 500 μg, 50 μg to 250 μg, 100 mg to 500 μg, 200 μg to 400 μg, 1 mg to 5 mg or 2 mg to 4 mg. Typically, epinephrine can be combined with lidocaine in a 1:100,000 to 1:200,000 dilution, which means that 100 ml of anesthetic contains 0.5 to 1 mg of epinephrine. Volumes administered can be adjusted depending on the disease to be treated and the route of administration. It is contemplated herein that 1-100 ml, 1-50 ml, 10-50 ml, 10-30 ml, 1-20 ml, or 1-10 ml, typically 10-50 ml of an anesthetic/vasoconstrictor formulation can be administered subcutanously for the treatment of an ECM-mediated disease or condition, such as cellulite. The administration can be subsequent, simultaneous or intermittent with administration of an activatable matrix-degrading enzyme and activator.


Compositions of modified MMP polypeptides, for example tsMMPs, provided herein also can be co-formulated or co-administered with a dispersion agent. The dispersion agent also can be co-formulated or co-administered with other pharmacological agents, such as anesthetics, vasoconstrictors, or other biologic agents. Exemplary of dispersion agents are glycosaminoglycanases that open channels in the interstitial space through degradation of glycosaminoglycans. These channels can remain relatively open for a period of 24-48 hours depending on dose and formulation. Such channels can be used to facilitate the diffusion of exogenously added molecules such as fluids, small molecules, proteins (such as matrix degrading enzymes), nucleic acids and gene therapy vectors and other molecules less than about 500 nm in size. In addition, it is thought that the formation of such channels can facilitate bulk fluid flow within an interstitial space, which can in turn promote the dispersion or movement of a solute (such as a detectable molecule or other diagnostic agent, an anesthetic or other tissue-modifying agent, a pharmacologic or pharmaceutically effective agent, or a cosmetic or other esthetic agent) that is effectively carried by the fluid in a process sometimes referred to as “convective transport” or simply convection. Such convective transport can substantially exceed the rate and cumulative effects of molecular diffusion and can thus cause the therapeutic or other administered molecule to more rapidly and effectively perfuse a tissue. Furthermore, when an agent, such as a modified MMP, for example a tsMMP, anesthetic or other agent, is co-formulated or co-administered with a glycosaminoglycanase and both are injected into a relatively confined local site, such as a site of non-intravenous parenteral administration (e.g., intradermal, subcutaneous, intramuscular, or into or around other internal tissues, organs or other relatively confined spaces within the body), then the fluid associated with the administered dose can both provide a local driving force (i.e. hydrostatic pressure) as well as lower impedance to flow (by opening channels within the interstitial matrix), both of which could increase fluid flow, and with it convective transport of the therapeutic agent or other molecule contained within the fluid. As a result, the use of glycosaminoglycanases can have substantial utility for improving the bioavailability as well as manipulating other pharmacokinetic and/or pharmacodynamic characteristics of co-formulated or co-administered agents, such as matrix degrading enzymes.


Hyaluronidases


Exemplary of glycosaminoglycanases are hyaluronidases. Hyaluronidases are a family of enzymes that degrade hyaluronic acid. By catalyzing the hydrolysis of hyaluronic acid, a major constituent of the interstitial barrier, hyaluronidase lowers the viscosity of hyaluronic acid, thereby increasing tissue permeability. There are three general classes of hyaluronidases: Mammalian-type hyaluronidases, (EC 3.2.1.35) which are endo-beta-N-acetylhexosaminidases with tetrasaccharides and hexasaccharides as the major end products. They have both hydrolytic and transglycosidase activities, and can degrade hyaluronan and chondroitin sulfates (CS), generally C4-S and C6-S; Bacterial hyaluronidases (EC 4.2.99.1), which degrade hyaluronan and to various extents, CS and DS. They are endo-beta-N-acetylhexosaminidases that operate by a beta elimination reaction that yields primarily disaccharide end products; and Hyaluronidases (EC 3.2.1.36) from leeches, other parasites, and crustaceans that are endo-beta-glucuronidases that generate tetrasaccharide and hexasaccharide end products through hydrolysis of the beta 1-3 linkage.


There are six hyaluronidase-like genes in the human genome, HYAL1 (SEQ ID NO:3469), HYAL2 (SEQ ID NO: 3470), HYAL3 (SEQ ID NO:3471), HYAL4 (SEQ ID NO:3472), PH20/SPAM1 (SEQ ID NO:3473) and one expressed pseudogene, HYALP1. Among hyaluronidases, PH20 is the prototypical neutral active enzyme, while the others exhibit no catalytic activity towards hyaluronan or any known substrates, or are active only under acidic pH conditions. The hyaluronidase-like enzymes can also be characterized by those which are generally locked to the plasma membrane via a glycosylphosphatidyl inositol anchor such as human HYAL2 and human PH20 (Danilkovitch-Miagkova, et al. (2003) Proc Natl Acad Sci USA. 100(8):4580-5), and those which are generally soluble such as human HYAL1 (Frost et al., (1997) Biochem Biophys Res Commun. 236(1):10-5). N-linked glycosylation of some hyaluronidases can be very important for their catalytic activity and stability. While altering the type of glycan modifying a glycoprotein can have dramatic affects on a protein's antigenicity, structural folding, solubility, and stability, many enzymes are not thought to require glycosylation for optimal enzyme activity. Hyaluronidases are, therefore, unique in this regard, in that removal of N-linked glycosylation can result in near complete inactivation of the hyaluronidase activity. For such hyaluronidases, the presence of N-linked glycans is critical for generating an active enzyme.


Human PH20 (also known as sperm surface protein PH20) is naturally involved in sperm-egg adhesion and aids penetration by sperm of the layer of cumulus cells by digesting hyaluronic acid. The PH20 mRNA transcript (corresponding to nucleotides 1058-2503 of the sequence set forth in SEQ ID NO:3474) is normally translated to generate a 509 amino acid precursor protein containing a 35 amino acid signal sequence at the N-terminus (amino acid residue positions 1-35) and a 19 amino acid GPI anchor at the C-terminus (corresponding to amino acid residues 491-509). The precursor sequence is set forth in SEQ ID NO:3473. An mRNA transcript containing a mutation of C to T at nucleotide position 2188 of the sequence of nucleic acids set forth in SEQ ID NO:3474 also exists and is a silent mutation resulting in the translated product set forth in SEQ ID NO: 3473. The mature PH20 is, therefore, a 474 amino acid polypeptide corresponding to amino acids 36-509 of the sequence of amino acids set forth in SEQ ID NO:3473. There are potential N-linked glycosylation sites required for hyaluronidases activity at N82, N166, N235, N254, N368, N393, N490 of human PH20 exemplified in SEQ ID NO: 3473. Disulfide bonds form between the cysteine residues C60 and C351 and between C224 and C238 (corresponding to amino acids set forth in SEQ ID NO:3473) to form the core hyaluronidase domain. Additional cysteines are required in the carboxy terminus for neutral enzyme catalytic activity such that amino acids 36 to 464 of SEQ ID NO:3473 contain the minimally active human PH20 hyaluronidase domain.


Soluble forms of recombinant human PH20 have been produced and can be used in the methods described herein for co-administration or co-formulation with tsMMPs, activators, anesthetics, vasoconstrictors, other pharmacologic or therapeutic agents, or combinations thereof, to permit the diffusion into tissues. The production of such soluble forms of PH20 is described in related application Ser. Nos. 11/065,716 and 11/238,171. Soluble forms include, but are not limited to, any having C-terminal truncations to generate polypeptides containing amino acid 1 to amino acid 442, 443, 444, 445, 446 and 447 of the sequence of amino acids set forth in SEQ ID NOS:3476-3481. Exemplary of such a polypeptides are those generated from a nucleic acid molecule encoding amino acids 1-482 set forth in SEQ ID NO:3475. Resulting purified rHuPH20 can be heterogenous due to peptidases present in the culture medium upon production and purification. Generally soluble forms of PH20 are produced using protein expression systems that facilitate correct N-glycosylation to ensure the polypeptide retains activity, since glycosylation is important for the catalytic activity and stability of hyaluronidases. Such cells include, for example Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells).


The soluble PH20 can be administered by any suitable route as described elsewhere herein. Typically, administration is by parenteral administration, such as by intradermal, intramuscular, subcutaneous or intravascular administration. The compounds provided herein can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water or other solvents, before use. For example, provided herein are parenteral formulations containing an effective amount of soluble PH20, such as 10 Units to 500,000 Units, 100 Units to 100,000 Units, 500 Units to 50,000 Units, 1000 Units to 10,000 Units, 5000 Units to 7500 Units, 5000 Units to 50,000 Units, or 1,000 Units to 10,000 Units, generally 10,000 to 50,000 Units, in a stabilized solution or suspension or a lyophilized from. The formulations can be provided in unit-dose forms such as, but not limited to, ampoules, syringes and individually packaged tablets or capsules. The dispersing agent can be administered alone, or with other pharmacologically effective agents in a total volume of 1-100 ml, 1-50 ml, 10-50 ml, 10-30 ml, 1-20 ml, or 1-10 ml, typically 10-50 ml.


In one example of a combination therapy, it is contemplated herein that an anesthetic, vasoconstrictor and dispersion agent are co-administered or co-formulated with a tsMMP to be administered subsequently, simultaneously or intermittently therewith. An exemplary formulation is one containing lidocaine, epinephrine and a soluble PH20, for example, a soluble PH20 set forth in SEQ ID NO:3476. Soluble PH20 can be mixed directly with lidocaine (Xylocalne), and optionally with epinephrine. The formulation can be prepared in a unit dosage form, such as in a syringe. For example, the lidocaine/epinephrine/soluble PH20 formulation can be provided in a volume, such as 1-100 ml, 1-50 ml, 10-50 ml, 10-30 ml, 1-20 ml, or 1-10 ml, typically 10-50 ml, prepackaged in a syringe for use.


In the combination therapies, the other pharmacologic agents, such as a lidocaine/epinephrine/soluble PH20 formulation, can be co-administered together with or in close temporal proximity to the administration of an activatable matrix-degrading enzyme (and activator). Typically it is preferred that an anesthetic and/or dispersion agent be administered shortly before (e.g. 5 to 60 minutes before) or, for maximal convenience, together with the pharmacologic agent. As will be appreciated by those of skill in the art, the desired proximity of co-administration depends in significant part on the effective half lives of the agents in the particular tissue setting, and the particular disease being treated, and can be readily optimized by testing the effects of administering the agents at varying times in suitable models, such as in suitable animal models.


G. PACKAGING AND ARTICLES OF MANUFACTURE OF tsMMPS

Pharmaceutical compounds of modified MMPs, for example tsMMPs, or nucleic acids encoding modified MMPs, or a derivative or variant thereof can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for treating the disease or disorder, and a label that indicates that selected modified MMP or nucleic acid molecule is to be used for treating the disease or disorder. Instructions for use can be provided. For example, instructions can be provided that specify that the tsMMP is to be reconstituted with the accompanying liquid buffer or solution, kept cold, immediately before administration. Instructions also can be provided for administration of a cold pack at the site of administration of the tsMMP. Combinations of a modified MMP, for example tsMMP, or derivative or variant thereof and an activator (e.g. cold pack or liquid buffer) also can be packaged in an article of manufacture. In some examples, combinations also can include a processing agent.


The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, for example, U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporated herein in its entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. The articles of manufacture can include a needle or other injection device so as to facilitate administration (e.g. sub-epidermal administration) for local injection purposes. A wide array of formulations of the compounds and compositions provided herein are contemplated as are a variety of treatments for any ECM-mediated disease or disorder.


The choice of package depends on the tsMMP and activator (if included therewith), and whether such compositions will be packaged together or separately. In general, the packaging is non-reactive with the compositions contained therein such that activation of the tsMMP does not occur prior to addition of the activator. In one example, the modified MMP can be packaged in lyophilized form with a buffer or diluent for reconstitution. The buffer or diluent can be stored separately at a temperature providing the activated condition, or can be provided in a form capable of providing the activating condition when desired. For example, instructions can be provided to chill or cool and or warm the buffer or diluent before use. Alternatively, instructions can be provided to activate the enzyme by use of a cold pack or heat pack at the locus of administration, for example, following reconstitution of the enzyme and administration thereof.


Exposure to the activator can occur at any time preceding administration of the tsMMP by exposure of the tsMMP to the requisite permissive temperature. For example, the container can have a single compartment containing the tsMMP and being amenable to addition of the activator (e.g. cold or room temperature liquid buffer or solution) by the user, for example through an opening in the compartment. Any container or other article of manufacture that is amenable to having a defining space for containment of the tsMMP and that is amenable to simple manipulation to permit addition of the final components necessary for activation is contemplated. The activator is added prior to use. Exposure to the activator also can occur following administration to the interstitium. For example, if heat is the activator, a tsMMP can be administered and the local injection site subjected to heat. If colder temperatures are the activator, a tsMMP can be administered and the local injection site subjected to cold, e.g. by a cold pack.


In other examples, the tsMMP is packaged in a container with the activator such that activation of the matrix-degrading enzyme is amenable to activation by the user at will in the container. Generally, examples of such containers include those that have an enclosed, defined space that contains the matrix-degrading enzyme, and a separate enclosed, defined space containing the activator such that the two spaces are separated by a readily removable membrane which, upon removal, permits the components to mix and thereby react, resulting in activation of the protease. The container can be stored under conditions such that the activator is at or near the requisite permissive temperature for activation of the MMP. Alternatively, only the side of the container containing the activator can be cooled or warmed to the desired temperature (e.g. by exposing it to an ice wrap or other temperature condition) just prior to use and reconstitution of the enzyme. Any container or other article of manufacture is contemplated, so long as the tsMMP is separated from the activator. Exposure of the activator to the tsMMP is prior to use. For example, the physical separation means are those that are readily removed by the user, to permit mixing, resulting in activation of the enzyme. For example, an article of manufacture can contain a tsMMP in one compartment and an activator (e.g. cold or room temperature liquid buffer or solution) in an adjacent compartment. The compartments are separated by a dividing member, such as a membrane, that, upon compression of the article or manufacture ruptures permitting separated components to mix. For suitable embodiments see e.g., containers described in U.S. Pat. Nos. 3,539,794 and 5,171,081.


Following are some examples of the packaging requirements of various end uses of activatable matrix-degrading enzymes. These are offered as examples only and in no way are intended as limiting.


1. Single Chamber Apparatus


Among the simplest embodiments herein, are those in which the apparatus contains a single chamber or container and, if needed, ejection means. Single chamber housings or containers include any item in which a tsMMP is included in the container. The tsMMP is housed in the vessel in liquid phase or as a powder or other paste or other convenient composition. The vessel or liquid can be stored at a temperature that is at or below the permissive temperature and/or cooled to at or below the permissive temperature prior to administration. Alternatively, a tsMMP is reconstituted with an appropriate liquid diluent or buffer and the activator is applied locally to the site of administration (e.g. cold pack) or is administered separately at the site of administration. Kits containing the item and the activator also are provided.


2. Dual Chamber Apparatus


An example of an apparatus contemplated for use herein is a dual chamber container. In general, this apparatus has two chambers or compartments thereby maintaining the tsMMP from an activator capable of providing the activating condition until activation is desired. The apparatus can include a mixing chamber to permit mixing of the components prior to dispensing from the apparatus. Alternatively, mixing can occur by ejection of the activator from one chamber into a second chamber containing the tsMMP. For example, the activatable tsMMP can be provided in lyophilized form, and reconstitution can be achieved by ejection of the activator (e.g. e.g. cold or room temperature buffer or liquid solution) from a first chamber into the second chamber containing the lyophilized enzyme. It is understood that the temperature of the entire apparatus can be controlled together and/or the chamber containing the activator can be brought to the desired temperature prior to use and reconstitution of the enzyme.


In one embodiment, a dual chamber apparatus employs a mechanical pump mechanism in its operation. In such an example, the dispensing apparatus maintains the components in separate chambers. A pump mechanism is operated to withdraw the contents from each chamber and into a mixing chamber, or from one chamber into the second chamber. Upon mixing, the mixed composition is activated by reaction of the components in the chambers. The pump mechanism can be manually operated, for example, by a plunger. Exemplary of such dual chamber apparatus include dual chamber syringes (see e.g., U.S. Pat. Nos. 6,972,005, 6,692,468, 5,971,953, 4,529,403, 4,202,314, 421-4584, 4983164, 5788670, 5395326; and Intl. Patent Appl. Nos. WO2007006030, WO2001047584).


Another embodiment of a dual chamber fluid dispensing apparatus contemplated for use herein takes the form of a compressible bottle or tube or other similar device. The device has two compartments within it that keep the components separated. The cap of the device can serve as a mixing chamber, a mixing chamber can be positioned between the two chambers and the cap, or mixing can be achieved within one of the chambers. The components are forced by compression from the separate compartments into the mixing chamber. They are then dispensed from the mixing chamber. For example, the mixed contents can be removed from the device by attaching a plunger/syringe apparatus to the dispensing end and withdrawing the contents therethrough. Such devices are known in the art (see e.g., Intl. Patent Appln. No. WO1994015848).


3. Kits


Selected modified MMP polypeptides, for example tsMMPs, and/or articles of manufacture thereof also can be provided as kits. The kits optionally can include an activator and/or processing agent. Kits can include a pharmaceutical composition described herein and an item for administration provided as an article of manufacture. For example a selected tsMMP can be supplied with a device for administration, such as a syringe, an inhaler, a dosage cup, a dropper, or an applicator. The compositions can be contained in the item for administration or can be provided separately to be added later. Generally, kits contain an item with a tsMMP, and optionally a processing agent and/or an activator capable of providing the activating condition. The kit can, optionally, include instructions for application including dosages, dosing regimens, instructions for using the activator (e.g. warming or cooling the buffer or applying a cold or hot pack), and instructions for modes of administration. Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount or activity of the selected protease in a subject.


H. METHODS OF ASSESSING ACTIVITY OF tsMMPS

1. Methods of Assessing Enzymatic Activity


Modified MMPs, including tsMMPs, can be tested for their enzymatic activity against known substrates. Activity assessment can be performed in the presence or absence of an activator and at varying temperatures. Activity assessments can be performed on conditioned medium or other supernatants or on purified protein.


Enzymatic activity can be assessed by assaying for substrate cleavage using known substrates of the enzyme. The substrates can be in the form of a purified protein or provided as peptide substrates. For example, enzymatic activity of MMP can be assessed by cleavage of collagen. Cleavage of a purified protein by an enzyme can be assessed using any method of protein detection, including, but not limited to, HPLC, SDS-PAGE analysis, ELISA, Western blotting, immunohistochemistry, immunoprecipitation, NH2-terminal sequencing, protein labeling and fluorometric methods. For example, Example 5 describes an assay to assess enzymatic activity for cleavage of a collagen that is FITC-labeled. Fluorescence of the supernatant is an indication of the enzymatic activity of the protein and can be normalized to protein concentration and a standard curve for specific activity assessment.


In addition, enzymatic activity can be assessed on tetrapeptide substrates. The use of fluorogenic groups on the substrates facilitates detection of cleavage. For example, substrates can be provided as fluorogenically tagged tetrapeptides of the peptide substrate, such as an ACC- or 7-amino-4-methyl courmarin (AMC)-tetrapeptide. Other fluorogenic groups are known and can be used and coupled to protein or peptide substrates. These include, for example, 7-amino-4-methyl-2-quinolinone (AMeq), 2-naphthylamine (NHNap) and 7 amino-4-methylcoumarin (NHMec) (Sarath et al. “Protease Assay Methods,” in Proteolytic Enzymes: A


Practical Approach. Ed. Robert J. Beynon and Judith S. Bond. Oxford University Press, 2001. pp. 45-76). Peptide substrates are known to one of skill in the art, as are exemplary fluorogenic peptide substrates. For example, exemplary substrates for MMP include, peptide IX, designated as Mca-K-P-L-G-L-Dpa-A-R-NH2 (SEQ ID NO:707; Mca=(7-Methoxycoumarin-4-yl)acetyl; Dpa=N-3-(2,4,-Dinitrophenyl)-L-2,3-diaminopropionyl; R&D Systems, Minneapolis, Minn., Cat# ES010) and variations thereof such as with different fluorogenic groups. Enzyme assays to measure enzymatic activity by fluorescence intensity are standard and are typically performed as a function of incubation time of the enzyme and substrate (see e.g., Dehrmann et al. (1995) Arch. Biochem. Biophys., 324:93-98; Barrett et al. (1981) Methods Enzymol., 80:536-561). Exemplary assays using fluorescence substrates are described in Example 2 herein.


While detection of fluorogenic compounds can be accomplished using a fluorometer, detection can be accomplished by a variety of other methods well known to those of skill in the art. Thus, for example, when the fluorophores emit in the visible wavelengths, detection can be simply by visual inspection of fluorescence in response to excitation by a light source. Detection also can be by means of an image analysis system utilizing a video camera interfaced to a digitizer or other image acquisition system. Detection also can be by visualization through a filter, as under a fluorescence microscope. The microscope can provide a signal that is simply visualized by the operator. Alternatively, the signal can be recorded on photographic film or using a video analysis system. The signal also can simply be quantified in real time using either an image analysis system or a photometer.


Thus, for example, a basic assay for enzyme activity of a sample involves suspending or dissolving the sample in a buffer (at the pH optima of the particular protease being assayed) adding to the buffer a fluorogenic enzyme peptide indicator, and monitoring the resulting change in fluorescence using a spectrofluorometer as shown in e.g., Harris et al., (1998) J Biol Chem 273:27364. The spectrofluorometer is set to excite the fluorophore at the excitation wavelength of the fluorophore. The fluorogenic enzyme indicator is a substrate sequence of an enzyme (e.g. of a protease) that changes in fluorescence due to a protease cleaving the indicator.


2. Methods of Assessing ECM Degradation


The degradation of extracellular matrix proteins by modified MMPs, for example tsMMPs, including, but not limited to, those described above, such as tsMMP-1, can be assessed in vitro or in vivo. Assays for such assessment are known to those of skill in the art, and can be used to test the activities of a variety of modified MMPs, for example tsMMPs, on a variety of extracellular matrix proteins, including, but not limited to collagen (I, II, III and IV), fibronectin, vitronectin and proteoglycans. Assays can be performed at permissive and non-permissive temperatures. Experiments also can be performed in the presence of an MMP that is not modified to be temperature sensitive. It is understood that assays for enzymatic activity are performed subsequent to activation of the enzyme by a processing agent. As a further control, activity of the zymogen enzyme also can be assessed.


a. In Vitro Assays


Exemplary in vitro assays include assays to assess the degradation products of extracellular matrix proteins following incubation with a modified MMP, for example tsMMP. In some examples, the assays detect a single, specific degradation product. In other examples, the assays detect multiple degradation products, the identity of which may or may not be known. Assessment of degradation products can be performed using methods well known in the art including, but not limited to, HPLC, CE, Mass spectrometry, SDS-PAGE analysis, ELISA, Western blotting, immunohistochemistry, immunoprecipitation, NH2-terminal sequencing, and protein labeling. Extracellular matrix degradation products can be visualized, for example, by SDS-PAGE analysis following incubation with MMPs, such as tsMMPs, for an appropriate amount of time at an appropriate temperature. For example, collagen can be incubated with mature modified MMP, for example tsMMP, and subjected to SDS-PAGE using, for example, a 4-20% Tris/glycine gel to separate the products. Coomassie staining of the gel facilitates visualization of smaller degradation products, or disappearance of collagen bands, compared to intact collagen. Immunoblotting using, for example, a polyclonal Ig specific to the extracellular matrix protein also can be used to visualize the degradation products following separation with SDS-PAGE.


Assays that specifically detect a single product following degradation of an extracellular matrix protein also are known in the art and can be used to assess the ability of a tsMMP to degrade an extracellular matrix protein. For example, the hydroxyproline (HP) assay can be used to measure degradation of collagen. 4-hydroxyproline is a modified imino acid that makes up approximately 12% of the weight of collagen. HP assays measure the amount of solubilized collagen by determining the amount of HP in the supernatant following incubation with a matrix-degrading enzyme (see e.g., Reddy and Enwemeka (1996) Clinical Biochemistry 29:225-229). Measurement of HP can be effected by, for example, colorimetric methods, high performance liquid chromatography, mass spectrometry and enzymatic methods (see e.g., Edwards et al., (1980) Clin. Chim. Acta 104:161-167; Green (1992) Anal. Biochem. 201:265-269; Tredget et al., (1990) Anal. Biochem. 190:259-265; Ito et al., (1985) Anal. Biochem. 151:510-514; Garnero et al. (1998) J. Biol. Chem. 273:32347-32352).


The collagen source used in such in vitro assays can include, but is not limited to, commercially available purified collagen, bone particles, skin, cartilage and rat tail tendon. Collagenolytic activity of a modified MMP, such as tsMMP such as tsMMP-1, can be assessed by incubating the activated enzyme with an insoluble collagen suspension, followed by hydrolysis, such as with HCl. The amount of hydroxyproline derived from the solubilized (degraded) collagen can be determined by spectrophotometric methods, such as measuring the absorbance at 550 nm following incubation with Ehrlich's reagent. In some examples, the collagen source is rat or pig skin explant that is surgically removed from anesthetized animals and then perfused with the tsMMP, for example, tsMMP-1, prior to, subsequently, simultaneously or intermittently with a temperature activator. HP levels in the perfusates can then be assessed. In a modification of this method, the effect on the fibrous septae in the explants can also be assessed. Briefly, following perfusion with the enzyme, the explants are cut into small pieces and embedded in paraffin and analyzed by microscopy following Masson's Trichrome staining for visualization of collagen. The number of collagen fibrous septae can be visualized and compared to tissue that has not been treated with a enzyme.


Assays to detect degradation of specific collagens also are known in the art. Such assays can employ immunological methods to detect a degradation product unique to the specific collagen. For example, the degradation of collagen I by some MMPs releases telopeptides with different epitopes that can be detected using immunoassays. Such assays detect the cross-linked N-telopeptides (NTx) and the cross-linked C-telopeptides (CTx and ICTP), each of which contain unique epitopes. Typically, CTx assays utilize the CrossLaps (Nordic Biosciences) antibodies that recognize the 8 amino acid sequence EKAHD-β-GGR octapeptide, where the aspartic acid is in β-isomerized configuration, in the C-terminal telopeptide region of the al chain (Eastell (2001) Bone Markers: Biochemical and Clinical Perspectives, pg 40). Immunoassays to detect ICTP also are known in the art and can be used to detect degradation of collagen I (U.S. Pat. No. 5,538,853). In other examples, immunoassays, such as, for example, ELISAs, can be used to detect NTx following incubation of collagen type I with proteases such as an MMP (Atley et al., (2000) Bone, 26:241-247). Other antibodies and assays specific for degraded collagens are known in the art and can be used to detect degradation by matrix-degrading enzymes. These include antibodies and assays specific for degraded collagen I (Hartmann et al (1990) Clin. Chem. 36:421-426), collagen II (Hollander et al (1994)J. Clin. Invest. 93:1722-1732), collagen III (U.S. Pat. No. 5,34,2756), and collagen IV (Wilkinson et al (1990) Anal. Biochem. 185:294-6).


b. In Vivo Assays


Assays to detect the in vivo degradation of ECM also are known in the art. Such assays can utilize the methods described above to detect, for example, hydroxyproline and N- and C-telopeptides and degraded collagens or other ECM in biological samples such as urine, blood, serum and tissue. Detection of degraded ECM can be performed following administration to the patient of one or more enzymes. Detection of pyridinoline (PYD) and deoxypyridinoline (DPYD), also can be used to assess degradation of collagen. Also known as hydroxylysylpyridinoline and lysylpyridinoline, respectively, PYD and DPYD are the two nonreducible trivalent cross-links that stabilize type I collagen chains and are released during the degradation of mature collagen fibrils. Pyridinoline is abundant in bone and cartilage, whereas deoxypyridinoline is largely confined to bone. Type III collagen also contains pyridinoline cross-links at the amino terminus. Total PYD and DPYD can be measured, for example, in hydrolyzed urine samples or serum by fluorometric detection after reversed-phase HPLC (Hata et al (1995) Clin. Chimica. Acta. 235:221-227).


c. Non-Human Animal Models


Non-human animal models can be used to assess the activity of matrix-degrading enzymes. For example, non-human animals can be used as models for a disease or condition. Non-human animals can be injected with disease and/or phenotype-inducing substances prior to administration of enzymes. Genetic models also are useful. Animals, such as mice, can be generated which mimic a disease or condition by the overexpression, underexpression or knock-out of one or more genes. For example, animal models are known in the art for conditions including, but not limited to, Peyronie's Disease (Davila et al. (2004) Biol. Reprod., 71:1568-1577), tendinosis (Warden et al., (2006) Br. J. Sports Med. 41:232-240) and scleroderma (Yamamoto (2005) Cur. Rheum. Rev. 1:105-109).


Non-human animals also can be used to test the activity of enzymes in vivo in a non-diseased animal. For example, enzymes can be administered to, non-human animals, such as, a mouse, rat or pig, and the level of ECM degradation can be determined. In some examples, the animals are used to obtain explants for ex vivo assessment of ECM degradation. In other examples, ECM degradation is assessed in vivo. For example, collagen degradation of the skin of anesthetized animals can be assessed. Briefly, an MMP, such as a tsMMP-1, is perfused prior to, simultaneously, subsequently or intermittently with a temperature activator via insertion of a needle into the dermal layer of the skin of the tail. Perfusate fractions are collected from the tail skin and analyzed for collagen degradation by hydroxyproline analysis. Other methods can be used to detect degradation including, but not limited to, any of the assays described above, such as immunoassays to detect specific degradation products.


I. EXEMPLARY METHODS OF TREATING DISEASES OR DEFECTS OF ECM

The modified MMPs, for example tsMMPs, provided herein can be used for treatment of any condition mediated by any one or more ECM components. This section provides exemplary uses of, and administration methods for, modified MMPs, such as tsMMPs. These described therapies are exemplary and do not limit the applications of enzymes. Such methods include, but are not limited to, methods of treatment of any ECM condition or disease that is caused by excess, aberrant or accumulated expression of any one or more ECM component. Exemplary of diseases or conditions to be treated are any mediated by collagen, elastin, fibronectin, or a glycosaminoglycan such as a proteoglycan. For example, exemplary of collagen-mediated diseases or disorders include, but are not limited to, cellulite, Dupuytren's disease (also called Dupuytren's contracture), Peyronie's disease, frozen shoulder, chronic tendinosis or scar tissue of the tendons, localized scleroderma and lymphedema. It is within the skill of a treating physician to identify such diseases or conditions.


The particular disease or condition to be treated dictates the enzyme that is selected. For example, treatment of a collagen-mediated disease or disorder can be effected by administration of a modified MMP, for example tsMMP, that cleaves collagen. For example, a modified MMP-1, for example tsMMP-1, can be selected for cleaving collagen. Such MMPs include modified forms on any MMP listed above in Table 5, and/or known to one of skill in the art. tsMMPs, and systems and methods for activation can be chosen accordingly to treat a particular disease or condition.


Treatment of diseases and conditions with modified MMPs, for example tsMMPs, can be effected by any suitable route of administration using suitable formulations as described herein including, but not limited to, subcutaneous injection, intramuscular, intradermal, oral, and topical and transdermal administration. As described above, a route of administration of modified MMPs, for example tsMMPs, typically is chosen that results in administration under the skin directly to the affected site. Exemplary of such routes of administration include, but are not limited to, subcutaneous, intramuscular, or intradermal.


If necessary, a particular dosage and duration and treatment protocol can be empirically determined or extrapolated. For example, exemplary doses of recombinant and native active MMPs or modified MMPs, for example tsMMPs, can be used as a starting point to determine appropriate dosages. Dosage levels can be determined based on a variety of factors, such as body weight of the individual, general health, age, the activity of the specific compound employed, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, and the patient's disposition to the disease and the judgment of the treating physician. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the particular matrix-degrading enzyme, the host treated, the particular mode of administration, and the activating condition required for activation, and/or the predetermined or length of time in which activation is desired. The pharmaceutical compositions typically should provide a dosage of from about 1 μg/ml to about 20 mg/ml. Generally, dosages are from or about 10 μg/ml to 1 mg/ml, typically about 100 μg/ml, per single dosage administration. It is understood that the amount to administer will be a function of the tsMMP and the activating condition chosen, the indication treated, and possibly side effects that will be tolerated. Dosages can be empirically determined using recognized models for each disorder. Also, as described elsewhere herein, modified MMPs, for example tsMMPs, can be administered in combination with other agents sequentially, simultaneously or intermittently. Exemplary of such agents include, but are not limited to, lidocaine, epinephrine, a dispersing agent such as hyaluronidase and combinations thereof.


Upon improvement of a patient's condition, a maintenance dose of a compound or compositions can be administered, if necessary; and the dosage, the dosage form, or frequency of administration, or a combination thereof can be modified. In some cases, a subject can require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.


Descriptions of the involvement of collagen to collagen-mediated diseases or conditions is provided below as an example of the role of ECM components in diverse disease and conditions. Such descriptions are meant to be exemplary only and are not limited to a particular modified MMP or tsMMP or to a particular ECM-mediated diseases or conditions. One of skill in the art can select a modified MMP, for example, tsMMP and activating condition for activation thereof, to be used in the treatment of any desired ECM-mediated disease, based on the ability of a particular enzyme to cleave or degrade an ECM component involved in the particular disease or condition. For example, as described herein, MMP-1 cleaves type I and type III collagens, such as those abundant in the skin. Hence, a modified MMP-1 can be used for treatments, uses and processes for treating a collagen-mediated disease or condition. The particular treatment and dosage can be determined by one of skill in the art. Considerations in assessing treatment include, for example, the disease to be treated, the ECM component involved in the disease, the severity and course of the disease, whether the modified MMP, for example tsMMP, is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to therapy, and the discretion of the attending physician.


Collagen-Mediated Diseases or Conditions


Collagen is a major structural constituent of mammalian organisms and makes up a large portion of the total protein content of the skin and other parts of the animal body. Numerous diseases and conditions are associated with excess collagen deposition, for example, due to erratic accumulation of fibrous tissue rich in collagen or other causes. Collagen-mediated diseases or conditions (also referred to as fibrotic tissue disorders) are known to one of skill in the art (see e.g., published U.S. Application No. 20070224183; U.S. Pat. Nos. 6,353,028; 6,060,474; 6,566,331; 6,294,350). Excess collagen has been associated with diseases and conditions, such as, but not limited to, fibrotic diseases or conditions resulting in scar formation, cellulite, Dupuytren's syndrome, Peyronie's disease, frozen shoulder, localized scleroderma, lymphedema, Interstitial cystitis (IC), Telangrectase, Barrett's metaplasia, Pneumatosis cytoides intestinalis, collagenous colitis. For example, disfiguring conditions of the skin, such as wrinkling, cellulite formation and neoplastic fibrosis result from excessive collagen deposition, which produces unwanted binding and distortion of normal tissue architecture.


Modified MMP polypeptides, for example tsMMPs described herein, including but not limited to modified MMP-1 and tsMMP-1, can be used to treat collagen-mediated diseases or conditions. Exemplary of tsMMPs for treatment of diseases and conditions described herein is a tsMMP-1 that is more active at a non-permissive temperature that is below the physiological temperature of the body such as at or about 25° C. compared to the nonpermissive physiologic temperature at the site of administration. For example, temporary cooling of the extracellular matrix, such as the skin interstitium, can be achieved by infusing a cold buffered solution or other liquid directly at the affected site and/or applying a cold pack directly to the locus of administration. In one example, a cold buffer can be administered via sub-epidermal administration, i.e. under the skin, such that administration is effected directly at the site where ECM components are present and accumulated. Other methods of activation can be employed, and are known to one of skill in the art in view of the descriptions herein.


a. Cellulite


Modified MMP polypeptides, for example, tsMMPs, such as those described herein, including a modified MMP-1 polypeptide or tsMMP-1, can be used to treat cellulite. In normal adipose tissues, a fine mesh of blood vessels and lymph vessels supplies the tissue with necessary nutrients and oxygen, and takes care of the removal of metabolized products. For example, triglycerides are stored in individual adipocytes that are grouped into capillary rich lobules. Each fat lobule is composed of adipocytes. Vertical strands of collagen fibers named fibrous septae separate the fat lobules and tether the overlying superficial fascia to the underlying muscle.


Cellulite is typically characterized by dermal deterioration due to a breakdown in blood vessel integrity and a loss of capillary networks in the dermal and subdermal levels of the skin. The vascular deterioration tends to decrease the dermal metabolism. This decreased metabolism hinders protein synthesis and repair processes, which results in dermal thinning. The condition is further characterized by fat cells becoming engorged with lipids, swelling and clumping together, as well as excess fluid retention in the dermal and subdermal regions of the skin. The accumulation of fat globules or adipose cells creates a need for a bigger blood supply to provide extra nourishment. To provide the blood to tissues, new capillaries are formed, which release more filtrate resulting in a saturation of tissues with interstitial fluid causing edema in the adipose tissues. Abundant reticular fibers in the interstitial tissues accumulate and thicken around the aggregated adipose cells; they form capsules or septa, which gradually transform into collagen fibers and are felt as nodules. The formation of these septa further occludes fat cells. Collagen fibers are also laid down in the interstitial tissue spaces, rendering the connective tissue sclerotic (hard).


Hence, as the condition further progresses, hard nodules of fat cells and clumps of fats surrounded by septa form in the dermal region. This leads to the surface of the skin displaying considerable heterogeneity and being characterized as having a “cottage cheese” or “orange peel” appearance. The dimpling occurs when the fibrous septae that connect the skin to the dermis and deeper tissue layers tighten and pull in the skin. Thus, the “orange peel” appearance of cellulite is due to the deformation of the fat lobules as a result of outward forces on the adipose tissue. The fat lobules can be large, for example up to 1 cm wide, and easily protrude into the overlying dermis, causing a visible deformation on the surface of the skin. The net result is the undulating appearance of the outer skin as the fat pushes upwards. As the connective septae run in the same direction as these outward forces, they can offer no counter force to keep the adipose from protruding into the dermis.


Cellulite is more prevalent among females than males. The prevalence of cellulite is estimated between 60% and 80% of the female population and its severity tends to worsen with obesity. Recently, a published study showed by in vivo magnetic resonance imaging that women with cellulite have a higher percentage of perpendicular fibrous septae than women without cellulite or men (Querleux et al., (2002) Skin Research and Technology, 8:118-124). Cellulite occurs most often on the hips, thighs and upper arms. For example, premenopausal females tend to accumulate fat subcutaneously, primarily in the gluteal/thigh areas where cellulite is most common. Clinically, cellulite is accompanied by symptoms that include thinning of the epidermis, reduction and breakdown of the microvasculature leading to subdermal accumulations of fluids, and subdermal agglomerations of fatty tissues.


b. Dupuytren's Disease


Modified MMP polypeptides, for example tsMMPs, such as a modified MMP-1 or a tsMMP-1 such as those described herein, can be used to treat Dupuytren's syndrome (also called Dupuytren's contracture). Dupuytren's contracture (also known as Morbus Dupuytren) is a fixed flexion contracture of the hand where the fingers bend towards the palm and cannot be fully extended. A similar lesion sometimes occurs in the foot. The connective tissue within the hand becomes abnormally thick and is accompanied by the presence of nodules containing fibroblasts and collagen, particularly type III collagen. The fibrous cord of collagen is often interspersed with a septa-like arrangement of adipose tissue. These present clinically as mattress-type “lumps” of varying sized and in Dupuytren's disease are termed nodules. This can cause the fingers to curl, and can result in impaired function of the fingers, especially the small and ring fingers. Dupuytren's disease occurs predominantly in men. It is generally found in middle aged and elderly persons, those of Northern European ancestry, and in those with certain chronic illnesses such as diabetes, alcoholism and smoking.


Dupuytren's disease is a slowly progressive disease that occurs over many years causing fixed flexion deformities in the metacarpophalangeal (MP) and proximal interphalangeal (PIP) joints of the fingers. The small and ring fingers are the most often affected. The disease progresses through three stages (Luck et al. (1959) J. Bone Joint Surg., 41A:635-664). The initial proliferative stage is characterized by nodule formation in the palmar fascia in which a cell known as the myofibroblast appears and begins to proliferate. The involutional or mid-disease stage involves myofibroblast proliferation and active type III collagen formation. In the last or residual phase, the nodule disappears leaving acellular tissue and thick bands of collagen. The ratio of type III collagen to type I collagen increases. Treatment of Dupuytren's disease with an activatable-matrix degrading enzyme is typically in the mid-disease and residual disease stages.


c. Peyronie's Disease


Modified MMP-1, for example tsMMPs, such as a modified MMP-1 or a tsMMP-1 such as those described herein, can be used to treat Peyronie's disease. Peyronie's disease is a connective tissue disorder involving the growth of fibrous plaques in the soft tissue of the penis affecting as many as 1-4% of men. Collagen is the major component of the plaque in Peyronie's disease. Specifically, the fibrosing process occurs in the tunica albuginea, a fibrous envelope surrounding the penile corpora cavernosa. The pain and disfigurement associated with Peyronie's disease relate to the physical structure of the penis in which is found two erectile rods, called the corpora cavernosa, a conduit (the urethra) through which urine flows from the bladder, and the tunica which separates the cavernosa from the outer layers of skin of the penis. A person exhibiting Peyronie's disease will have formation(s) of plaque or scar tissue between the tunica and these outer layers of the skin (referred to as “sub-dermal” in this application). The scarring or plaque accumulation of the tunica reduces its elasticity causes such that, in the affected area, it will not stretch to the same degree (if at all) as the surrounding, unaffected tissues. Thus, the erect penis bends in the direction of the scar or plaque accumulation, often with associated pain of some degree. In all but minor manifestations of Peyronie's disease, the patient has some degree of sexual dysfunction. In more severe cases, sexual intercourse is either impossible, or is so painful as to be effectively prohibitive.


Empirical evidence indicates an incidence of Peyronie's disease in approximately one percent of the male population. Although the disease occurs mostly in middle-aged men, younger and older men can acquire it. About 30 percent of men with Peyronie's disease also develop fibrosis (hardened cells) in other elastic tissues of the body, such as on the hand or foot. Common examples of such other conditions include Dupuytren's contracture of the hand and Ledderhose Fibrosis of the foot.


d. Ledderhose Fibrosis


Modified MMP polypeptides, for example tsMMPs, for example, a modified MMP-1 or tsMMP-1 such as those described herein, can be used to treat Ledderhose fibrosis. Ledderhose fibrosis is similar to Dupuytren's disease and Peyronie's disease, except that the fibrosis due to fibroblast proliferation and collagen deposition occurs in the foot. Ledderhose disease is characterized by plantar fibrosis over the medial sole of the foot, and is sometimes referred to as plantar fibrosis.


e. Stiff Joints


Modified MMP polypeptides, for example tsMMPs, such as a modified MMP-1 or a tsMMP-1 such as those described herein, can be used to treat stiff joints, for example, frozen shoulder. Frozen shoulder (adhesive capsulitis) is a chronic fibrozing condition of the capsule of the joint characterized by pain and loss of motion or stiffness in the shoulder. It affects about 2% of the general population. Frozen shoulder results from increased fibroblast matrix synthesis. The synthesis is caused by an excessive inflammatory response resulting in the overproduction of cytokines and growth factors. Fibroblasts and myofibroblasts lay down a dense matrix of collagen in particular, type-I and type-III collagen within the capsule of the shoulder. This results in a scarred contracted shoulder capsule and causes joint stiffness.


Other examples of stiff joints include, but are not limited to, those caused by capsular contractures, adhesive capsulitis and arthrofibrosis, which result from musculoskeletal surgery. Such stiff joints can occur in joints, including, for example, joints of the knees, shoulders, elbows, ankles and hips. Like frozen shoulder, such joint diseases are caused by increased matrix synthesis and scar formation. The stiff joints inevitably can cause abnormally high forces to be transmitted to the articular cartilage of the affected area. Over time, these forces result in the development of degenerative joint disease and arthritis. For example, in arthrofibrosis and capsular contracture, fibroblasts form excessive amounts of matrix in response to local trauma, such as joint dislocation.


f. Existing Scars


Modified MMP-1, for example tsMMPs, such as a modified MMP-1 or tsMMP-1 such as those described herein, can be used to treat existing scars. Collagen is particularly important in the wound healing process and in the process of natural aging, where it is produced by fibroblast cells. In some cases, however, an exaggerated healing response can result in the production of copious amounts of healing tissue (ground substance), also termed scar tissue. For example, various skin traumas such as burns, surgery, infection, wounds and accident are often characterized by the erratic accumulation of fibrous tissue rich in collagen. There also is often an increased proteoglycan content. In addition to the replacement of the normal tissue that has been damaged or destroyed, excessive and disfiguring deposits of new tissue sometimes form during the healing process. The excess collagen deposition has been attributed to a disturbance in the balance between collagen synthesis and collagen degradation. Including among scars are, for example, chronic tendinosis or scar tissue of the tendons, surgical adhesions, keloids, hypertrophic scars, and depressed scars.


i. Surgical Adhesions


Surgical adhesions are attachments of organs or tissues to each other through scar formation, which can cause severe clinical problems. The formation of some scar tissue after surgery or tissue injury is normal. In some cases, however, the scar tissue overgrows the region of injury and creates surgical adhesions, which tend to restrict the normal mobility and function of affected body parts. In particular, fibroblast proliferation and matrix synthesis is increased locally following such soft tissue injury. Adhesions then form when the body attempts to repair tissue by inducing a healing response. For example, this healing process can occur between two or more otherwise healthy separate structures (such as between loops of bowel following abdominal surgery). Alternately, following local trauma to a peripheral nerve, fibrous adhesions can form, resulting in severe pain during normal movement.


ii. Keloids


Keloids are scars of connective tissue containing hyperplastic masses that occur in the dermis and adjacent subcutaneous tissue, most commonly following trauma. Keloids generally are fibrous nodules that can vary in color from pink or red to dark brown. Keloids form in scar tissue as a result of overgrowth of collagen, which participates in wound repair. Keloid lesions are formed when local skin fibroblasts undergo vigorous hyperplasia and proliferation in response to local stimuli. The resulting lesion can result in a lump many times larger than the original scar. In addition to occur as a result of wound or other trauma, keloids also can form from piercing, pimples, a scratch, severe acne, chickenpox scarring, infection at a wound site, repeated trauma to an area, or excessive skin tension during wound closure.


iii. Hypertrophic Scars


Hypertrophic scars are raised scars that form at the site of wounds. They generally do not grow beyond the boundaries of the original wound. Like keloid scars, hypertrophic scars are a result of the body overproducing collagen.


iv. Depressed Scars


Depressed scars generally result from an inflammatory episode and are characterized by contractions of the skin, and leave a cosmetically displeasing and permanent scar. The most common example is scarring that occurs following inflammatory acne. The depression occurs as a normal consequence of wound healing, and the scar tissue causing the depression is predominantly made up of collagen resulting from fibroblast proliferation and metabolism.


g. Scleroderma


Modified MMP polypeptides, for example tsMMPs, for example, a modified MMP-1 or a tsMMP-1 such as those described herein, can be used to treat scleroderma. Scleroderma is characterized by a thickening of the collagen. The more common form of the disease, localized scleroderma, affects only the skin, usually in just a few places, and sometimes the face. It is sometimes referred to as CREST syndrome. Symptoms include hardening of the skin and associated scarring. The skin also appears reddish or scaly, and blood vessels can be more visible. In more serious cases, scleroderma can affect the blood vessels and internal organs. Diffuse scleroderma can be fatal as a result of heart, kidney lung or intestinal damage, due to musculoskeletal, pulmonary, gastrointestinal, renal and other complications.


The condition is characterized by collagen buildup leading to loss of elasticity. The overproduction of collagen has been attributed to autoimmune dysfunction, resulting in accumulation of T cells and production of cytokines and other proteins that stimulate collagen deposition from fibroblasts.


h. Lymphedema


Modified MMP polypeptides, for example tsMMPs, for example, a modified MMP-1 or tsMMP-1 such as those described herein, can be used to treat lymphedema. Lymphedema is an accumulation of lymphatic fluid that causes swelling in the arms and legs. Lymphedema can progress to include skin changes such as, for example, lymphostatic fibrosis, sclerosis and papillomas (benign skin tumors) and swelling. Tissue changes associated with lymphedema include proliferation of connective tissue cells, such as fibroblasts, production of collagen fibers, an increase in fatty deposits and fibrotic changes. These changes occur first at the lower extremities, i.e. the fingers and toes. Lymphedema can be identified based on the degree of enlargement of the extremities. For example, one method to assess lymphedema is based on identification of 2-cm or 3-cm difference between four comparative points of the involved and uninvolved extremities.


i. Collagenous Colitis


Modified MMP polypeptides, for example tsMMPs, such as a modified MMP-1 or a tsMMP-1 such as those described herein, can be used to treat collagenous colitis. Collagenous colitis was first described as chronic watery diarrhea (Lindstrom et al. (1976) Pathol. Eur., 11:87-89). Collagenous colitis is characterized by collagen deposition, likely resulting from an imbalance between collagen production by mucosal fibroblasts and collagen degradation. It results in secretory diarrhea. The incidence of collagenous colitis is similar to primary biliary cirrhosis. The disease has an annual incidence of 1.8 per 100,000 and a prevalence of 15.7 per 100,000, which is similar to primary biliary cirrhosis (12.8 per 100,000) and lower than ulcerative colitis (234 per 100,000), Crohn's disease (146 per 100,000) or celiac disease (5 per 100,000). In patients with chronic diarrhea, about 0.3 to 5% have collagenous colitis. Collagenous colitis is an inflammatory disease resulting in increased production of cytokines and other agents that stimulate the proliferation of fibroblasts, resulting in increased collagen accumulation.


2. Spinal Pathologies


As described herein, the modified MMPs provided herein can be used to treat diseases and conditions of the ECM or involving the ECM. These include spinal pathologies, typically referred to as herniated disc or bulging discs, that can be treated by administering an MMP provided herein and activating as described herein. Herniated discs that can be treated include protruded and extruded discs. A protruded disc is one that is intact but bulging. In an extruded disk, the fibrous wrapper has torn and nucleus pulposus (NP) has oozed out, but is still connected to the disk. While the NP is not the cause of the herniation, the NP contributes to pressure on the nerves causing pain. The NP contains hyaluronic acid, chondrocytes, collagen fibrils, and proteoglycan aggrecans that have hyaluronic long chains which attract water. Attached to each hyaluronic chain are side chains of chondroitin sulfate and keratan sulfate.


Herniated discs have been treated with chemonucleolytic drugs, such as chymopapain and a collagenase, typically by local introduction of the drug into the disc. A chemonucleolytic drug degrades one or more components of the NP, thereby relieving pressure. Chemonucleolysis is effective on protruded and extruded disks. Chemonucleolysis has been used treat lumbar (lower) spine and cervical (upper spine) hernias. Hence, the MMPs provided herein can be used as chemonucleolytic drugs and administered, such as by injection, to the affected disc, under conditions that activate the MMP.


J. EXAMPLES

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.


Example 1
Cloning and Expression of hMMP-1

A. Cloning and High-Throughput Expression of hMMP-1 Library


In this example, a human matrix metalloprotease 1 (hMMP-1) library was created by cloning DNA encoding human MMP-1 into a plasmid followed by transformation and protein expression/isolation. The library was created by introducing mutations in a parent human MMP-1 DNA sequence having the sequence of nucleotides set forth in SEQ ID NO:706, which encodes the inactive zymogen proMMP-1 (set forth in SEQ ID NO:2), to generate single amino acid variants of MMP-1 across the catalytic domain and proline rich linker domain of the polypeptide. The hMMP-1 library was designed to contain at least 15 amino acid variants at each of 178 amino acids positions within the catalytic domain (amino acids 81-242 of SEQ ID NO:2) and the linker region (amino acids 243-258 of SEQ ID NO:2) of human MMP-1 (See Table 7, below).









TABLE 7







hMMP-1 Library









Amino




Acid
Amino Acid Substitutions
SEQ ID NOS





F81
E; H; R; C; Q; T; S; G; M; W; I; V; L;
780-781, 783-784, 786, 787,



A; P
789-797


V82
R; C; N; Q; T; Y; S; G; F; M; W; I; L;
802-816



A; P


L83
D; E; H; R; C; Q; T; Y; S; G; M; W; I;
817-819, 821-822, 824-825,



A; P
826, 827-828, 830-832, 834-835


T84
D; E; H; R; C; Q; Y; S; G; F; I; V; L; A; P
836-838, 840-841, 843-847,




850-854


E85
K; R; C; N; Q; T; Y; S; G; F; M; V; L;
857-867, 870-873



A; P


G86
D; H; K; C; N; T; Y; S; F; M; W; I; V;
874, 876-877, 879-880, 882-890,



L; P
892


N87
E; H; R; C; Q; Y; S; G; F; M; I; V; L;
894-895, 897-899, 901-905,



A; P
907-911


P88
D; E; H; K; R; C; Q; T; Y; G; W; I; V;
912-917, 919-921, 923, 926-930



L; A


R89
E; H; K; N; T; Y; S; G; F; M; W; V; L;
932-934, 936, 938-944, 946-949



A; P


W90
E; H; R; N; Q; T; S; G; F; M; I; V; L; A; P
951-952, 954, 956-958, 960-968


E91
D; H; R; C; N; T; Y; S; G; F; W; I; V;
969-970, 972-974, 976-980,



L; A
982-986


Q92
E; K; R; N; T; Y; S; G; F; W; I; V; L;
989, 991-992, 994-999, 1001-1006



A; P


T93
D; E; K; R; N; S; G; F; M; W; I; V; L;
1007-1008, 1010-1011, 1013,



A; P
1016-1025


H94
D; E; R; N; T; S; G; F; M; W; I; V; L;
1026-1027, 1029, 1031, 1033,



A; P
1035-1044


L95
D; E; H; K; R; C; T; Y; S; G; W; I; V;
3-8, 11-14, 17-21



A; P


T96
E; H; R; C; N; Q; S; G; F; W; I; V; L;
1046-1047, 1049-1052, 1054-1056,



A; P
1058-1063


Y97
D; E; H; K; R; N; Q; T; S; G; W; V; L;
1064-1068, 1070-1074, 1077,



A; P
1079-1082


R98
D; E; H; K; C; Y; S; G; F; M; W; V; L;
1083-1087, 1091-1096, 1098-1101



A; P


I99
E; H; R; C; N; Q; T; Y; S; G; F; W; V;
1103-1104, 1106-1114, 1116-1120



L; A; P


E100
D; H; R; N; T; Y; S; G; F; M; W; I; V;
497-498, 500, 502, 504-513,



L; P
515


N101
D; H; K; R; C; T; Y; S; F; M; W; V; L;
1121, 1123-1126, 1128-1130,



A; P
1132-1134, 1136-1139


Y102
D; E; K; R; C; N; Q; S; G; F; M; V; L;
1140-1141, 1143-1147, 1149-1152,



A; P
1155-1158


T103
D; E; K; R; C; N; Q; Y; S; G; W; V; L;
516-517, 519-526, 529, 531-534



A; P


P104
D; E; H; R; C; Q; T; Y; S; G; F; M; V;
1159-1161, 1163-1164, 1166-1172,



L; A
1175-1177


D105
E; R; C; N; T; S; G; F; M; W; I; V; L;
22, 25-27, 29, 31-40



A; P


L106
D; H; R; C; N; T; Y; S; G; F; M; I; V;
1178, 1180, 1182-1184, 1186-1191,



A; P
1193-1196


P107
D; K; R; C; T; Y; S; G; F; M; W; I; V;
1197, 1200-1202, 1205-1215



L; A


R108
E; K; C; N; T; Y; S; G; F; W; I; V; L;
1217, 1219-1221, 1223-1227,



A; P
1229-1234


A109
D; E; H; R; N; Q; T; Y; S; G; M; W; I;
1235-1237, 1239, 1241-1246,



V; L;
1248-1252


D110
H; R; C; Q; T; Y; S; G; F; M; I; V; L;
1255, 1257-1258, 1260-1266,



A; P
1268-1272


V111
D; E; K; R; C; Q; T; Y; S; G; W; I; L;
1273-1274, 1276-1278, 1280-1284,



A; P
1287-1291


D112
H; K; R; C; Q; T; Y; S; G; F; M; W; I;
1293-1296, 1298-1310



V; L; A; P


H113
D; E; R; N; T; Y; S; G; F; M; W; V; L;
1311-1312, 1314, 1316, 1318-1324,



A; P
1326-1329


A114
E; R; C; N; Q; T; S; G; F; M; W; I; V;
1331, 1334-1338, 1340-1348



L; P


I115
D; E; H; K; R; C; Q; T; S; G; F; W; V;
1349-1354, 1356-1357, 1359-1361,



L; P
1363-1365, 1367


E116
D; H; K; R; C; N; Q; S; G; F; M; I; L;
1368-1374, 1377-1380, 1382,



A; P
1384-1386


K117
D; E; H; R; N; Q; T; Y; S; G; F; W; L;
1387-1390, 1392-1398, 1400,



A; P
1403-1405


A118
D; E; H; K; R; Q; T; S; G; F; W; I; V;
1406-1410, 1413-1414, 1416-1418,



L; P
1420-1424


F119
E; H; K; R; C; N; T; Y; S; G; W; V; L;
1426-1431, 1433-1436, 1438,



A; P
1440-1443


Q120
D; E; H; K; R; C; N; T; Y; G; M; W; V;
1444-1452, 1454, 1456-1457,



A; P
1459, 1461-1462


L121
E; H; K; R; C; N; Q; T; S; G; F; I; V; A; P
1464-1471, 1473-1475, 1478-1481


W122
E; H; K; R; N; Q; T; Y; S; G; F; V; L;
1483-1486, 1488-1494, 1497-1500



A; P


S123
D; H; K; R; C; N; Q; T; Y; G; F; M; W;
1501, 1503-1519



I; V; L; A; P


N124
D; K; R; C; T; S; G; F; M; W; I; V; L;
1520, 1523-1525, 1527, 1529-1538



A; P


V125
D; E; H; R; C; Q; T; Y; S; G; F; M; W;
1539-1541, 1543-1544, 1546-1553,



A; P
1556-1557


T126
E; H; K; R; N; Q; S; G; F; M; W; V; L;
1559-1562, 1564-1565, 1567-1571,



A; P
1573-1576


P127
E; H; K; R; C; Q; T; S; F; M; W; I; V;
1578-1582, 1584-1585, 1587,



L; A
1589-1595


L128
D; K; R; C; Q; T; S; G; F; M; W; I; V;
1596, 1599-1601, 1603-1604,



A; P
1606-1614


T129
E; H; K; R; C; Y; S; G; F; M; I; V; L;
1616-1620, 1623-1627, 1629-1633



A; P


F130
E; H; K; R; C; N; T; Y; S; G; I; V; L; A; P
1635-1640, 1642-1645, 1648-1652


T131
D; E; H; R; C; Q; Y; S; G; F; M; I; L;
1653-1655, 1657-1658, 1660-1665,



A; P
1667, 1669-1671


K132
D; E; H; R; T; Y; S; G; F; M; I; V; L; A; P
1672-1675, 1679-1684, 1686-1690


V133
D; E; H; K; R; C; N; T; S; G; M; W; L;
1691-1697, 1699, 1701-1702,



A; P
1704-1705, 1707-1709


S134
D; E; H; K; R; C; N; Q; T; Y; G; V; L;
1710-1720, 1725-1728



A; P


E135
D; H; R; N; Q; T; S; F; M; W; I; V; L;
1729-1730, 1732, 1734-1736,



A; P
1738, 1740-1747


G136
D; E; H; R; C; N; T; S; M; W; I; V; L;
1748-1750, 1752-1754, 1756,



A; P
1758, 1760-1766


Q137
E; H; K; R; C; N; T; Y; S; G; F; W; L;
1768-1778, 1780, 1783-1785



A; P


A138
D; E; H; R; C; Q; T; S; G; M; W; I; V;
1786-1788, 1790-1791, 1793-1794,



L; P
1796-1797, 1799-1804


D139
E; H; R; C; N; Y; S; G; F; M; W; I; V;
1805-1806, 1808-1810, 1813-1823



L; A; P


I140
D; E; H; K; R; C; T; Y; G; F; M; W; V;
1824-1829, 1832-1833, 1835-1841



L; A


M141
D; E; H; R; C; N; T; Y; S; G; W; I; L;
1843-1845, 1847-1849, 1851-1854,



A; P
1856-1857, 1859-1861


I142
K; R; N; Q; T; Y; S; G; F; M; W; V; L;
1865-1866, 1868-1880



A; P


S143
E; H; R; C; N; Q; T; Y; G; M; W; I; L;
1882-1883, 1885-1891, 1893-1895,



A; P
1897-1899


F144
E; H; K; R; C; N; Q; T; S; G; M; W; V;
1901-1908, 1910-1913, 1915-1916,



L; P
1918


V145
D; E; H; K; R; C; N; Q; T; S; G; W; L;
1919-1927, 1929-1930, 1933,



A; P
1935-1937


R146
D; E; H; K; C; N; Q; T; Y; S; F; V; L;
1938-1947, 1949, 1953-1956



A; P


G147
E; H; R; C; Q; T; S; F; M; W; I; V; L;
1958-1959, 1961-1962, 1964-1965,



A; P
1967-1975


D148
E; K; R; C; N; T; S; G; M; W; I; V; L;
1976, 1978-1981, 1983, 1985-1986,



A; P
1988-1994


H149
E; R; C; N; Q; T; Y; S; G; W; I; V; L;
1996, 1998-2005, 2008-2013



A; P


R150
D; E; H; K; N; T; S; G; M; W; I; V; L;
41-44, 46, 48, 50-51, 53-59



A; P


D151
K; R; N; Q; T; Y; S; G; F; M; W; V; L;
62-63, 65-73, 75-78



A; P


N152
D; H; K; R; C; T; Y; S; G; F; W; I; L;
2014, 2016-2019, 2021-2025,



A; P
2027-2028, 2030-2032


S153
D; H; K; R; C; Q; T; Y; G; F; I; V; L;
535, 537-540, 542-546, 549-553



A; P


P154
H; K; R; C; N; Q; T; Y; S; F; W; I; V;
2035-2043, 2045, 2047-2051



L; A


F155
E; H; R; N; Q; T; Y; S; G; M; W; V; L;
80-81, 83, 85-92, 94-97



A; P


D156
E; H; K; R; C; T; Y; S; G; M; W; V; L;
98-102, 105-108, 110-111,



A; P
113-116


G157
D; H; K; R; N; Q; T; Y; S; F; M; V; L;
2052, 2054-2056, 2059-2065,



A; P
2068-2071


P158
D; K; R; C; N; Q; T; Y; S; G; F; W; I;
2072, 2075-2084, 2086-2090



V; L; A


G159
E; K; R; C; Q; T; Y; S; M; W; I; V; L;
118, 120-122, 124-127, 129-135



A; P


G160
E; H; R; C; N; Q; T; S; M; W; I; V; L;
2092-2093, 2095-2099, 2101,



A; P
2103-2109


N161
E; H; R; C; Q; T; Y; S; G; F; W; I; V; L; P
2111-2112, 2114-2121, 2123-2126,




2128


L162
D; E; R; C; Q; T; Y; S; G; F; M; W; I;
2129-2130, 2133-2134, 2136-2144,



A; P
2146-2147


A163
E; K; R; C; N; Q; T; Y; S; G; F; I; V; L; P
2149, 2151-2160, 2163-2166


H164
E; K; R; C; N; Q; Y; S; G; F; M; V; L;
2168-2173, 2175-2179, 2182-2185



A; P


A165
D; H; K; R; N; Q; T; S; G; F; M; W; V;
2186, 2188-2190, 2192-2194,



L; P
2196-2200, 2202-2204


F166
E; H; K; R; C; N; S; G; M; W; I; V; L;
2206-2211, 2215-2223



A; P


Q167
D; E; K; R; N; T; Y; S; G; F; M; V; L;
2224-2225, 2227-2228, 2230-2236,



A; P
2239-2242


P168
D; H; R; C; N; T; S; G; F; M; W; I; V;
2243, 2245, 2247-2249, 2251,



L; A
2253-2261


G169
D; E; H; R; C; Q; T; S; M; W; I; V; L;
2262-2264, 2266-2267, 2269-2270,



A; P
2272, 2274-2280


P170
D; H; K; R; C; Q; T; S; G; F; M; W; I;
2281, 2283-2286, 2288-2289,



L; A
2291-2296, 2298-2299


G171
D; E; H; K; R; C; N; Q; Y; S; M; W; L;
554-561, 563-564, 566-567,



A; P
570-572


I172
D; E; R; C; N; Q; T; Y; G; M; W; V; L;
2300-2301, 2304-2309, 2311,



A; P
2313-2318


G173
D; K; R; C; N; T; Y; S; F; M; W; V; L;
2319, 2322-2325, 2327-2332,



A; P
2334-2337


G174
D; E; H; R; N; T; Y; S; F; M; W; V; L;
2338-2340, 2342, 2344, 2346-2351,



A; P
2353-2356


D175
E; H; R; C; N; Q; T; Y; S; G; F; I; V; L;
2357-2358, 2360-2368, 2371-2375



A; P


A176
D; E; K; R; C; N; Q; T; S; G; F; W; V;
136-137, 139-144, 146-148,



L; P
150, 152-154


H177
D; R; C; N; Q; T; Y; S; G; W; I; V; L;
2376, 2379-2386, 2389-2394



A; P


F178
E; H; K; R; C; Q; T; Y; S; G; W; I; V;
2396-2400, 2402-2406, 2408-2413



L; A; P


D179
E; K; R; C; N; Q; T; S; G; W; I; V; L;
155, 157-162, 164-165, 168-173



A; P


E180
D; K; R; C; N; Q; T; Y; S; G; F; M; I;
174, 176-186, 188, 191-192



A; P


D181
E; K; R; C; Q; T; Y; S; G; F; M; V; L;
193, 195-197, 199-205, 208-211



A; P


E182
D; R; C; Q; T; Y; S; G; F; M; W; I; L;
212, 215-216, 218-226, 228-230



A; P


R183
E; H; K; C; N; T; S; G; M; W; I; V; L;
2415-2419, 2421, 2423-2424,



A; P
2426-2432


W184
E; H; R; N; Q; T; S; G; F; M; I; V; L; A; P
2434-2435, 2437, 2439-2441,




2443-2451


T185
D; E; H; R; C; N; Q; Y; S; G; W; V; L;
231-233, 235-241, 244, 246-249



A; P


N186
D; E; H; R; C; Q; T; Y; S; G; F; V; L;
2452-2454, 2456-2463, 2467-2470



A; P


N187
D; H; K; R; C; T; S; G; F; M; W; I; L;
250, 252-255, 257, 259-264,



A; P
266-268


F188
D; E; H; K; R; N; Q; S; G; W; I; V; L;
2471-2475, 2477-2478, 2481-2482,



A; P
2484-2489


R189
D; E; H; K; C; N; Q; T; Y; G; W; V; L;
2490-2498, 2500, 2503, 2505-2508



A; P


E190
D; H; K; R; C; T; Y; S; G; M; I; V; L;
573-577, 580-583, 585, 587-591



A; P


Y191
D; E; H; K; R; C; Q; T; S; G; W; V; L;
592-597, 599-602, 605, 607-610



A; P


N192
D; H; K; R; C; Q; T; S; G; M; W; V; L;
611, 613-618, 620-621, 623-624,



A; P
626-629


L193
D; E; K; R; N; Q; T; Y; S; G; F; W; I;
2509-2510, 2512-2513, 2515-2521,



A; P
2523-2524, 2526-2527


H194
E; K; Q; T; Y; S; G; F; M; W; I; V; L;
631-632, 636-648



A; P


R195
D; E; K; C; Q; T; Y; S; G; F; W; V; L;
269-270, 272-273, 275-280,



A; P
282, 284-287


V196
D; E; H; K; R; Q; T; Y; S; G; M; I; L;
2528-2532, 2535-2539, 2541,



A; P
2543-2546


A197
E; H; R; C; N; Q; T; Y; S; G; W; I; V;
2548-2549, 2551-2558, 2561-2565



L; P


A198
D; E; H; K; R; T; Y; S; G; F; M; W; V;
288-292, 296-302, 304-306



L; P


H199
E; K; R; C; N; T; S; G; M; W; I; V; L;
2567-2571, 2573, 2575-2576,



A; P
2578-2584


E200
D; R; C; N; T; Y; S; G; F; M; W; I; V;
2585, 2588-2590, 2592-2600,



A; P
2602-2603


L201
D; E; K; R; N; Q; T; S; G; M; W; I; V;
2604-2605, 2607-2608, 2610-2612,



A; P
2614-2615, 2617-2622


G202
D; E; H; K; R; C; T; Y; S; M; I; V; L;
2623-2628, 2631-2633, 2635,



A; P
2637-2641


H203
D; E; R; C; N; Q; T; Y; S; G; I; V; L; A; P
2642-2643, 2645-2652, 2656-2660


S204
D; H; K; R; N; Q; T; Y; G; W; I; V; L;
2661, 2663-2665, 2667-2671,



A; P
2674-2679


L205
D; E; R; C; N; Q; T; S; G; M; W; I; V;
2680-2681-2684-2688, 2690-2691,



A; P
2693-2698


G206
D; E; H; R; C; Q; T; S; M; W; I; V; L;
307-309, 311-312, 314-315,



A; P
317, 319-325


L207
D; H; K; R; N; Q; Y; S; G; M; W; I; V;
649, 651-653, 655-656, 658-660,



A; P
662-667


S208
D; E; K; R; C; N; Q; T; G; F; W; V; L;
2669-2700, 2702-2707, 2709-2710,



A; P
2712, 2714-2717


H209
D; R; C; N; Q; T; Y; S; G; F; W; V; L;
2718, 2721-2729, 2731, 2733-2736



A; P


S210
H; K; R; C; N; Q; T; G; F; W; I; V; L;
328-334, 336-337, 339-344



A; P


T211
D; H; K; R; N; Q; S; G; F; M; W; V; L;
2737, 2739-2741, 2743-2744,



A; P
2746-2750, 2752-2755


D212
E; H; K; R; N; Q; T; Y; S; G; F; V; L;
668-671, 673-679, 683-686



A; P


I213
D; E; H; K; R; C; N; Q; T; S; G; F; M;
2756-2764, 2766-2769, 2771-2774



V; L; A; P


G214
D; E; R; C; Q; T; Y; S; F; M; I; V; L; A; P
2775-2776, 2779-2780, 2782-2787,




2789-2793


A215
D; H; K; R; C; N; Q; T; S; G; M; W; I;
2794, 2796-2802, 2804-2805,



V; L; P
2807-2812


L216
D; E; K; R; C; Q; T; S; G; M; W; I; V;
2813-2814, 2816-2818, 2820-2821,



A; P
2823-2824, 2826-2831


M217
D; H; K; R; C; N; Q; T; Y; S; G; I; L;
2832, 2834-2843, 2846, 2848-2850



A; P


Y218
D; E; R; C; N; Q; S; G; F; W; I; V; L;
345-346, 349-352, 354-356,



A; P
358-363


P219
D; E; H; K; R; C; Q; T; S; G; F; W; V;
2851-2856, 2858-2859, 2861-2863,



L; A
2865, 2867-2869


S220
E; H; K; R; N; Q; T; G; F; M; I; V; L;
2871-2874, 2876-2878, 2880-2882,



A; P
2884-2888


Y221
E; K; R; C; N; Q; T; S; G; M; W; V; L;
2890, 2892-2899, 2901-2902,



A; P
2904-2907


T222
D; H; R; C; N; Y; S; G; F; M; W; I; V;
2908, 2910, 2912-2914, 2916-2926



L; A; P


F223
E; H; K; R; C; N; Q; T; Y; S; G; M; L;
365-376, 380-382



A; P


S224
D; H; K; R; C; Q; T; G; M; W; I; V; L;
2927, 2929-2932, 2934-2935,



A; P
2937, 2939-2945


G225
D; E; H; K; R; C; N; Q; T; S; M; W; V;
2946-2954, 2956, 2958-2959,



A; P
2961, 2963-2964


D226
E; H; R; C; N; T; S; G; M; W; I; V; L;
2965-2966, 2968-2970, 2972,



A; P
2974-2975, 2977-2983


V227
D; E; H; K; R; C; Q; T; Y; S; G; W; L;
383-388, 390-394, 397, 399-401



A; P


Q228
D; E; H; K; R; N; T; Y; S; G; M; W; L;
402-406, 408-412, 414-415,



A; P
418-420


L229
D; E; H; R; C; Q; T; Y; G; M; W; I; V;
421-423, 425-426, 428-430,



A; P
432, 434-439


A230
D; H; R; C; N; T; Y; S; G; M; W; I; V;
687, 689, 691-693, 695-698,



L; P
700-705


Q231
D; H; R; C; Y; S; G; F; M; W; I; V; L;
2984, 2986, 2988-2989, 2992-3002



A; P


D232
E; H; K; R; N; Q; T; Y; S; G; F; W; V;
3003-3006, 3008-3014, 3016,



L; P
3018-3019, 3021


D233
E; K; R; N; Q; T; S; G; M; W; I; V; L;
440, 442-443, 445-447, 449-450,



A; P
452-458


I234
D; E; H; C; N; Q; T; Y; G; M; W; V; L;
459-461, 464-468, 470, 472-477



A; P


D235
E; H; R; C; N; Q; T; Y; S; G; I; V; L; A; P
3022-3023, 3025-3032, 3036-3040


G236
D; E; K; R; C; N; T; Y; S; F; M; I; V; L; P
3041-3042, 3044-3047, 3049-3053,




3055-3057, 3059


1237
D; E; K; R; C; N; Q; T; Y; S; G; W; L;
3060-3061, 3063-3071, 3074,



A; P
3076-3078


Q238
E; H; K; R; C; N; T; Y; S; G; F; W; I; L; P
3080-3090, 3092-3093, 3095,




3097


A239
D; H; K; R; C; Q; T; Y; S; G; F; W; I;
3099, 3100-3103, 3105-3110,



V; L; P
3112-3116


I240
D; K; R; C; Q; T; Y; S; G; F; M; V; L;
478, 481-483, 485-491, 493-496



A; P


Y241
D; H; R; N; Q; T; S; G; M; W; I; V; L;
3117, 3119, 3121, 3123-3127,



A; P
3129-3135


G242
E; H; K; R; N; T; Y; S; F; W; I; V; L;
3137-3140, 3142, 3144-3147,



A; P
3149-3154


R243
D; H; K; C; N; Q; T; Y; S; G; I; V; L;
3155, 3157-3165, 3169-3173



A; P


S244
D; E; H; R; Q; T; Y; G; F; M; W; V; L;
3174-3176, 3178, 3181-3187,



A; P
3189-3192


Q245
E; H; K; R; C; T; S; G; F; M; W; I; V;
3194-3198, 3200, 3202-3209,



L; P
3211


N246
D; K; R; C; Q; T; Y; S; G; F; W; I; V;
3212, 3215-3223, 3225-3230



L; A; P


P247
D; E; H; K; R; N; Q; T; S; G; F; I; V; L; A
3231-3235, 3237-3239, 3241-3243,




3246-3249


V248
E; H; K; R; C; Q; T; Y; S; G; F; M; W;
3251-3255, 3257-3267



I; L; A


Q249
E; H; K; R; C; N; T; Y; G; W; I; V; L;
3270-3277, 3279, 3282-3287



A; P


P250
D; K; R; N; Q; T; Y; S; G; F; M; W; V;
3288, 3291-3292, 3294-3302,



L; A
3304-3306


I251
D; E; K; R; C; Q; T; Y; S; G; W; V; L;
3307-3308, 3310-3312, 3314-3318,



A; P
3321-3325


G252
D; E; H; K; R; C; T; S; F; M; W; I; V;
3326-3331, 3334, 3336-3344



L; A; P


P253
E; K; R; C; N; Q; T; Y; G; M; W; I; V;
3346, 3348-3354, 3356, 3358-3363



L; A


Q254
D; E; R; C; T; Y; S; G; F; W; I; V; L; A; P
3364-3365, 3368-3369, 3371-3375,




3377-3382


T255
E; H; K; R; C; N; Q; S; G; F; I; V; L; A; P
3384-3390, 3392-3394, 3397-3401


P256
E; K; R; C; N; Q; Y; S; G; F; M; I; V;
3403, 3405-3409, 3411-3415,



L; A
3417-3420


K257
E; R; C; N; T; S; G; F; M; W; I; V; L;
3422, 3424-3426, 3428, 3430-3439



A; P


A258
D; E; R; N; Q; T; Y; G; F; M; W; I; V;
3440-3441, 3444, 3446-3449,



L; P
3451-3458









The cDNA encoding each individual hMMP-1 mutant was generated by changing the wildtype codon, encoding each of the 178 amino acids positions identified in Table 8 below, to a codon encoding the desired amino acid substitution. The wildtype codons are set forth in SEQ ID NO:706. SEQ ID NO:706 also depicts the encoded amino acids. The amino acids substitutions and corresponding mutated codons are listed in Table 8, below.









TABLE 8







Codons encoding each amino acid substitution














Muta-









tion
Codon
Mutation
Codon
Mutation
Codon
Mutation
Codon





F81C
TGT
T84L
TTG
N87S
AGT
W90H
CAT





F81E
GAG
T84D
GAT
N87I
ATT
W90M
ATG





F81I
ATT
T84R
CGG
N87C
TGT
W90R
CGG





F81L
CTG
T84I
ATT
N87A
GCG
W90E
GAG





F81P
CCT
T84S
TCT
N87G
GGT
W90N
AAT





F81S
TCT
T84G
GGT
N87Y
TAT
W90Q
CAG





F81A
GCG
T84Q
CAG
N87E
GAG
E91N
AAT





F81M
ATG
T84P
CCT
N87H
CAT
E91R
CGG





F81G
GGG
T84A
GCG
N87Q
CAG
E91W
TGG





F81T
ACG
T84C
TGT
P88C
TGT
E91G
GGG





F81Q
CAG
T84Y
TAT
P88K
AAG
E91V
GTG





F81R
CGT
T84F
TTT
P88W
TGG
E91Y
TAT





F81W
TGG
E85L
CTG
P88G
GGG
E91C
TGT





F81H
CAT
E85Q
CAG
P88L
CTG
E91H
CAT





F81V
GTG
E85P
CCT
P88Q
CAG
E91T
ACG





V82I
ATT
E85T
ACT
P88A
GCG
E91S
AGT





V82C
TGT
E85K
AAG
P88T
ACG
E91A
GCG





V82A
GCG
E85M
ATG
P88Y
TAT
E91I
ATT





V82P
CCG
E85G
GGT
P88R
CGG
E91D
GAT





V82Y
TAT
E85R
CGT
P88H
CAT
E91F
TTT





V82M
ATG
E85S
TCT
P88I
ATI
E91L
TTG





V82Q
CAG
E85C
TGT
P88V
GTG
Q92V
GTT





V82F
TTT
E85Y
TAT
P88E
GAG
Q92Y
TAT





V82W
TGG
E85A
GCG
P88D
GAT
Q92L
CTG





V82N
AAT
E85N
AAT
R89V
GTG
Q92N
AAT





V82R
CGT
E85V
GTG
R89W
TGG
Q92E
GAG





V82G
GGT
E85F
TTT
R89M
ATG
Q92I
ATT





V82S
TCG
G86L
CTT
R89A
GCG
Q92T
ACT





V82L
TTG
G86P
CCG
R89T
ACG
Q92G
GGT





V82T
ACT
G86I
ATT
R89G
GGG
Q92P
CCG





L83A
GCG
G86T
ACT
R89S
TCT
Q92W
TGG





L83C
TGT
G86H
CAT
R89K
AAG
Q92F
TTT





L83D
GAT
G86D
GAT
R89F
TTT
Q92S
TCG





L83E
GAG
G86N
AAT
R89Y
TAT
Q92R
CGG





L83G
GGT
G86S
AGT
R89N
AAT
Q92K
AAG





L83H
CAT
G86K
AAG
R89H
CAT
Q92A
GCT





L83I
ATT
G86W
TGG
R89L
TTG
T93A
GCG





L83M
ATG
G86Y
TAT
R89E
GAG
T93L
CTT





L83P
CCG
G86V
GTT
R89P
CCT
T93M
ATG





L83Q
CAG
G86C
TGT
W90L
TTG
T93N
AAT





L83R
CGG
G86M
ATG
W90G
GGG
T93V
GTG





L83S
AGT
G86F
TTT
W90P
CCG
T931
ATT





L83T
ACG
N87M
ATG
W90T
ACT
T93D
GAT





L83W
TGG
N87L
CTG
W90S
TCG
T93S
TCG





L83Y
TAT
N87P
CCG
W90V
GTG
T93R
CGG





T84V
GTT
N87V
GTT
W90I
ATT
T93W
TGG





T84E
GAG
N87R
CGT
W90A
GCT
T93F
TTT





T84H
CAT
N87F
TIT
W90F
TTT
T93P
CCT





T93G
GGG
Y97R
CGT
E100L
CTG
T103R
CGG





T93K
AAG
Y97V
GTG
E100H
CAT
T103Y
TAT





T93E
GAG
Y97A
GCT
E100D
GAT
T103N
AAT





H94L
CTG
Y97P
CCT
E100M
ATG
T103C
TGT





H94S
TCG
Y97L
CTT
E100G
GGT
T103Q
CAG





H94M
ATG
Y97T
ACG
E100W
TGG
T103W
TGG





H94R
CGG
Y97K
AAG
E100Y
TAT
T103P
CCG





H94E
GAG
Y97W
TGG
E100R
CGT
T103A
GCG





H94I
ATT
Y97H
CAT
E100S
TCT
T103G
GGG





H94D
GAT
Y97S
TCG
E100T
ACG
T103K
AAG





H94P
CCG
Y97E
GAG
E100F
TTT
P104G
GGG





H94A
GCG
Y97D
GAT
E100I
ATT
P104E
GAG





H94N
AAT
Y97N
AAT
E100N
AAT
P104T
ACT





H94F
TTT
Y97G
GGT
N101M
ATG
P104F
TTT





H94G
GGG
Y97Q
CAG
N101F
TTT
P104R
CGT





H94T
ACT
R98H
CAT
N101L
TTG
P104D
GAT





H94V
GTG
R98K
AAG
N101V
GTG
P104C
TGT





H94W
TGG
R98C
TGT
N101H
CAT
P104Q
CAG





L95E
GAG
R98L
CTG
N101R
CGG
P104V
GTG





L95Y
TAT
R98M
ATG
N101C
TGT
P104Y
TAT





L95R
CGG
R98F
TTT
N101T
ACT
P104H
CAT





L95A
GCT
R98W
TGG
N101P
CCT
P104L
TTG





L95G
GGG
R98Y
TAT
N101W
TGG
P104S
TCG





L95K
AAG
R98P
CCT
N101K
AAG
P104A
GCG





L95S
AGT
R98E
GAG
N101S
TCG
P104M
ATG





L95T
ACG
R98A
GCG
N101D
GAT
D105A
GCT





L95H
CAT
R98G
GGG
N101A
GCG
D105C
TGT





L95W
TGG
R98V
GTT
N101Y
TAT
D105F
TTT





L95V
GTG
R98S
TCG
Y102R
CGT
D105G
GGT





L95C
TGT
R98D
GAT
Y102K
AAG
D105I
ATT





L95P
CCT
I99C
TGT
Y102V
GTG
D105L
CTG





L95D
GAT
I99E
GAG
Y102M
ATG
D105M
ATG





L95I
ATT
I99G
GGG
Y102P
CCG
D105N
AAT





T96E
GAG
I99H
CAT
Y102N
AAT
D105P
CCT





T96R
CGG
I99N
AAT
Y102G
GGG
D105R
CGG





T96P
CCG
I99P
CCT
Y102L
CTG
D105S
TCG





T96S
TCG
I99T
ACG
Y102D
GAT
D105T
ACG





T96A
GCG
I99V
GTT
Y102S
TCG
D105V
GTT





T96L
TTG
I99A
GCG
Y102F
TTT
D105W
TGG





T96W
TGG
I99F
TTT
Y102A
GCT
D105E
GAG





T96N
AAT
I99L
CTG
Y102E
GAG
L106P
CCG





T96G
GGT
199R
CGT
Y102Q
CAG
L106D
GAT





T96F
TTT
I99S
TCG
Y102C
TGT
L106N
AAT





T96Q
CAG
I99Q
CAG
T103E
GAG
L106G
GGT





T96H
CAT
I99W
TGG
T103D
GAT
L106M
ATG





T96V
GTT
I99Y
TAT
T103S
AGT
L106A
GCT





T96I
ATT
E100V
GTT
T103L
CTG
L106R
CGG





T96C
TGT
E100P
CCG
T103V
GTT
L106Y
TAT





L106T
ACG
A109V
GTT
D112I
ATT
E116A
GCG





L106V
GTG
A109E
GAG
D112Y
TAT
E116C
TGT





L106H
CAT
A109L
CTT
D112L
TTG
E116D
GAT





L106F
TIT
A109H
CAT
H113T
ACT
E116F
TTT





L106I
ATT
D110P
CCT
H113L
CTG
E116G
GGT





L106C
TGT
D110F
TTT
H113M
ATG
E116H
CAT





L106S
TCT
D110Q
CAG
H113S
TCG
E116I
ATT





P107L
TTG
D11OR
CGG
H113N
AAT
E116K
AAG





P107W
TGG
D110M
ATG
H113R
AGG
E116L
CTG





P107T
ACT
D110H
CAT
H113A
GCT
E116M
ATG





P107S
TCG
D110I
ATT
H113E
GAG
E116N
AAT





P107R
CGG
D110L
CTT
H113V
GTG
E116P
CCG





P107Y
TAT
D110V
GTG
H113Y
TAT
E116Q
CAG





P107M
ATG
D110T
ACG
H113F
TTT
E116R
AGG





P107V
GTG
Dll0S
TCG
H113D
GAT
E116S
TCT





P107D
GAT
D110Y
TAT
H113W
TGG
K117H
CAT





P107A
GCG
D110G
GGT
H113G
GGG
K117T
ACG





P107C
TGT
D110C
TGT
H113P
CCG
K117Q
CAG





P107K
AAG
D110A
GCG
A114E
GAG
K117E
GAG





P107F
TTT
V111E
GAG
A114S
TCG
K117A
GCG





P107I
ATT
V111A
GCT
A114I
ATT
K117F
TTT





P107G
GGT
V111S
TCT
A114P
CCT
K117D
GAT





R108P
CCT
V111W
TGG
A114N
AAT
K117N
AAT





R108G
GGT
V111G
GGT
A114L
CTT
K117G
GGT





R108T
ACG
V111Y
TAT
A114T
ACT
K117W
TGG





R108E
GAG
V111P
CCG
A114F
TTT
K117Y
TAT





R108A
GCG
V111L
CTG
A114V
GTT
K117L
TTG





R108Y
TAT
V111D
GAT
A114G
GGT
K117S
AGT





R108K
AAG
V111K
AAG
A114C
TGT
K117P
CCG





R108C
TGT
V111T
ACT
A114M
ATG
K117R
AGG





R108S
TCT
V111Q
CAG
A114R
AGG
A118G
GGG





R108F
TTT
V111I
ATT
A114W
TGG
A118R
CGT





R108W
TGG
V111C
TGT
A114Q
CAG
A118W
TGG





R108I
ATT
V111R
CGT
I115F
TTT
A118K
AAG





R108L
CTT
D112A
GCG
I115T
ACT
A118P
CCT





R108N
AAT
D112M
ATG
I115H
CAT
A118V
GTG





R108V
GTT
D112V
AAT
I115G
GGT
A118L
TTG





A109S
TCG
D112R
CGG
I115K
AAG
A118D
GAT





A109R
CGG
D112K
AAG
I115E
GAG
A118S
AGT





A109T
ACG
D112P
CCT
I115S
AGT
A118F
TTT





A109W
TGG
D112Q
CAG
I115P
CCT
A118I
ATT





A1091
ATT
D112F
TTT
I115C
TGT
A118H
CAT





A109Q
CAG
D112G
GGG
I115L
CPT
A118E
GAG





A109N
AAT
D112C
TGT
I115Q
CAG
A118Q
CAG





A109Y
TAT
D112W
TGG
I115R
CGG
A118T
ACT





A109G
GGG
D112T
ACT
I115W
TGG
F119G
GGG





A109M
ATG
D112H
CAT
I115V
GTT
F119T
ACT





A109D
GAT
D112S
TCT
I115D
GAT
F119R
CGG





F119L
TTG
W122G
GGG
V125T
ACG
L128A
GCG





F119N
AAT
W122S
TCG
V125A
GCT
L128D
GAT





F119S
AGT
W122V
OTT
V125C
TGT
L128V
GTG





F119C
TGT
W122H
CAT
V125D
GAT
L128W
TGG





F119P
CCG
W122F
TTT
V125W
TGG
L128C
TGT





F119W
TGG
W122Y
TAT
V125R
CGG
L128K
AAG





F119K
AAG
W122K
AAG
V125E
GAA
T129G
GGT





F119H
CAT
W122Q
CAG
V125F
TTT
T129A
GCT





F119A
GCG
W122E
GAG
V125H
CAT
T129C
TGT





F119V
GTT
S123D
GAT
T126K
AAG
T129K
AAG





F119Y
TAT
S123L
TTG
T126V
GTG
T129F
TTT





F119E
GAG
S123A
GCT
T126G
GGG
T129Y
TAT





Q120K
AAG
S123C
TGT
T126R
CGG
T129S
TCG





Q120N
AAT
S123I
ATT
T126L
TTG
T129R
CGG





Q120A
GCG
S123K
AAG
T126H
CAT
T129V
GTT





Q120V
GTG
S123N
AAT
T126M
ATG
T129L
MT





Q120D
GAT
S123F
TTT
T126P
CCG
T129H
CAT





Q12OR
CGG
S123Y
TAT
T126A
GCG
T129P
CCT





Q120P
CCT
S123M
ATG
T126N
AAT
T129E
GAG





Q120W
TGG
S123H
CAT
T126E
GAG
T129I
ATT





Q120Y
TAT
S123R
CGG
T126F
TTT
T129M
ATG





Q120C
TGT
S123W
TGG
T126W
TGG
F130L
CTG





Q120H
CAT
S123T
ACG
T126Q
CAG
F130P
CCT





Q120T
ACT
S123P
CCT
T126S
AGT
F130C
TGT





Q120M
ATG
S123G
GGG
P127C
TGT
F13OR
CGG





Q120E
GAG
S123Q
CAG
P127F
TTT
F130Y
TAT





Q120G
GGT
S123V
GTT
P127T
ACG
F130H
CAT





L121E
GAG
N124G
GGT
P127E
GAG
F130I
ATT





L121Q
CAG
N124C
TGT
P127W
TGG
F130V
GTT





L121P
CCT
N124V
GTG
P127A
GCT
F130K
AAG





L121R
CGG
N124L
CTT
P127S
AGT
F130T
ACT





L121C
TGT
N124T
ACG
P127H
CAT
F130E
GAG





L121G
GGG
N124R
CGT
P127Q
CAG
F130A
GCG





L121K
AAG
N124M
ATG
P127K
AAG
F130N
AAT





L121F
TTT
N124S
TCG
P127R
CGG
F130G
GGT





L1211
ATT
N124P
CCT
P127I
ATT
F130S
AGT





L121S
TCG
N124A
GCG
P127V
GTG
T131F
TTT





L121V
GTT
N124K
AAG
P127L
CTG
T131P
CCG





L121H
CAT
N124F
AAA
P127M
ATG
T131A
GCG





L121T
ACT
N124W
TGG
L128F
TTT
T131S
TCT





L121A
GCT
N124I
ATT
L128M
ATG
T131G
GGT





L121N
AAT
N124D
GAT
L128T
ACT
T131I
ATT





W122R
CGT
V125G
GGG
L128R
CGT
T131L
CTT





W122A
GCG
V125Q
CAG
L128S
TCG
T131H
CAT





W122N
AAT
V1255
TCG
L128G
GGT
T131Q
CAG





W122P
CCG
V125P
CCG
L128I
ATT
T131D
GAT





W122T
ACG
V125M
ATG
L128Q
CAG
T131E
GAG





W122L
CTT
V125Y
TAT
L128P
CCT
T131C
TGT





T131R
CGT
E135V
GTT
A138C
TGT
M141S
AGT





T131Y
TAT
E135M
ATG
A138T
ACG
M141C
TGT





T131M
ATG
E135S
TCG
A138S
TCT
M141L
CTG





K132G
GGT
E135D
GAT
A138R
CGT
M141A
GCG





K132V
GTG
E135T
ACG
A138G
GGG
M141D
GAT





K132L
TTG
E135L
CTG
A138E
GAG
M141W
TGG





K132A
GCT
E135A
GCG
A138H
CAT
M141G
GGT





K132P
CCG
E135W
TGG
A138M
ATG
M141H
CAT





K132F
TTT
E135F
TTT
A138Q
CAG
M141Y
TAT





K132R
CGG
E135P
CCG
A138I
ATT
M141N
AAT





K132I
ATT
E135R
CGG
A138D
GAT
I142L
CTG





K132H
CAT
E135N
AAT
A138W
TGG
I142M
ATG





K132S
TCT
E135H
CAT
D139R
CGT
I142G
GGT





K132M
ATG
E135Q
CAG
D139V
GTT
I142K
AAG





K132D
GAT
E135I
ATI
D139M
ATG
I142A
GCT





K132T
ACT
G136V
GTG
D139C
TGT
1142N
AAT





K132Y
TAT
G136W
TGG
D139P
CCT
1142W
TGG





K132E
GAG
G136D
GAT
D139S
TCT
I142P
CCG





V133G
GGG
G136M
ATG
D139L
CYT
I142Q
CAG





V133E
GAG
G136N
AAT
D139I
ATT
I142Y
TAT





V133T
ACT
G136A
GCG
D139H
CAT
I142V
GTG





V133N
AAT
G136L
TTG
D139A
GCG
I142T
ACT





V133A
GCG
G136C
TGT
D139G
GGG
I142R
CGG





V133H
CAT
G136P
CCG
D139F
TTT
I142S
AGT





V133P
CCG
G136T
ACG
D139N
AAT
I142F
TTT





V133K
AAG
G136R
CGT
D139W
TGG
S143P
CCG





V133R
CGG
G136S
TCG
D139Y
TAT
S143C
TGT





V133L
CTT
G1361
ATT
D139E
GAG
S143E
GAG





V133W
TGG
G136H
CAT
I140D
GAT
S143G
GGT





V133C
TGT
G136E
GAG
I140K
AAG
S143H
CAT





V133D
GAT
Q137A
GCT
I140A
GCT
S143R
CGT





V133M
ATG
Q137R
CGG
I140G
GGG
S143L
TTG





V133S
AGT
Q137G
GGG
I140C
TGT
S143Q
CAG





S134V
GTT
Q137K
AAG
I140Y
TAT
S143N
AAT





S134H
CAT
Q137H
CAT
I140V
GTT
S143W
TGG





S134P
CCT
Q137P
CCT
I140W
TGG
S143A
GCT





S134G
GGG
Q137S
TCG
I140F
TTT
S143T
ACT





S134N
AAT
Q137L
CTG
I140H
CAT
S143Y
TAT





S134R
CGT
Q137W
TGG
I140L
CTG
S143M
ATG





S134L
CTG
Q137F
TTT
I140R
CGG
S143I
ATT





S134Q
CAG
Q137T
ACG
I140E
GAG
F144K
AAG





5134E
GAG
Q137C
TGT
I140M
ATG
F144M
ATG





S134Y
TAT
Q137Y
TAT
I140T
ACT
F144E
GAG





S134A
GCG
Q137N
AAT
M141E
GAG
F144S
AGT





S134K
AAG
Q137E
GAG
M141I
ATI
F144L
CTG





S134D
GAT
A138V
GTT
M141R
CGG
F144W
TGG





S134T
ACG
A138L
CTT
M141T
ACG
F144P
CCG





S134C
TGT
A138P
CCG
M141P
CCG
F144R
CGG





F144N
AAT
G147V
GTT
R150H
CAT
P154L
C17





F144C
TGT
G147Q
CAG
D151R
CGT
P154C
TGT





F144G
GGT
G147M
ATG
D151F
TTT
P154S
TCT





F144T
ACT
G147P
CCT
D151P
CCG
P154K
AAG





F144Q
CAG
D148R
CGG
D151W
TGG
P154I
ATT





F144H
CAT
D148I
ATT
D151Q
CAG
P154A
GCT





F144V
GTG
D148T
ACG
D151L
CTT
P154T
ACG





V145A
GCG
D148G
GGT
D151S
TCG
P154H
CAT





V145T
ACG
D148L
CTG
D151G
GGT
P154Y
TAT





V145L
CTG
D148V
GTT
D151A
GCT
P154N
AAT





V145P
CCG
D148A
GCG
D151N
AAT
P154F
TTT





V145K
AAG
D148W
TGG
D151K
AAG
P154R
CGT





V145N
AAT
D148P
CCG
D151Y
TAT
P154Q
CAG





V145D
GAT
D148S
TCG
D151V
GTT
F155S
TCT





V145H
CAT
D148K
AAG
D151T
ACT
F155T
ACT





V145R
CGG
D148E
GAG
D151M
ATG
F155G
GGT





V145Q
CAG
D148M
ATG
N152G
GGG
F155N
AAT





V145S
TCT
D148N
AAT
N152C
TGT
F155R
CGG





V145G
GGG
D148C
TGT
N152F
TTT
F155W
TGG





V145W
TGG
H149W
TGG
N152L
TTG
F155L
CTG





V145C
TGT
H149A
GCG
N152P
CCG
F155Q
CAG





V145E
GAG
H149L
TTG
N152R
CGG
F155M
ATG





R146T
ACG
H149C
TGT
N152H
CAT
F155E
GAG





R146L
CTG
H149Q
CAG
N152T
ACG
F155A
GCG





R146N
AAT
H149T
ACT
N152Y
TAT
F155P
CCT





R146H
CAT
H149Y
TAT
N152K
AAG
F155V
GTT





R146Q
CAG
H149P
CCG
N152D
GAT
F155H
CAT





R146K
AAG
H149V
GTT
N152W
TGG
F155Y
TAT





R146C
TGT
H149R
CGG
N1521
ATT
D156H
CAT





R146S
AGT
H149G
GGT
N152A
GCG
D156L
CTT





R146D
GAT
H149E
GAG
N152S
TCT
D156E
GAG





R146A
GCT
H149S
AGT
S153I
ATT
D156A
GCT





R146Y
TAT
H1491
ATI
S153R
CGG
D156W
TGG





R146P
CCT
H149N
AAT
S153K
AAG
D156C
TGT





R146V
GTT
R150S
TCG
S153C
TGT
D156P
CCT





R146E
GAG
R150E
GAG
S153G
GGG
D156V
GTT





R146F
TTT
R150G
GGG
S153H
CAT
D156K
AAG





G147R
CGT
R150M
ATG
S153L
CTT
D156S
TCT





G147F
TTT
R150P
CCG
S153V
GTT
D156G
GGG





G147I
ATT
R150T
ACG
S153T
ACG
D156T
ACT





G147L
CTG
R150W
TGG
S153P
CCT
D156Y
TAT





G147A
GCG
R150A
GCG
S153A
GCG
D156R
CGT





G147E
GAG
R150N
AAT
S153F
TTT
D156M
ATG





G147H
CAT
R150K
AAG
S153D
GAT
G157K
AAG





G147W
TGG
R150L
TTG
S153Q
CAG
G157D
GAT





G147T
ACG
R150V
GTT
S153Y
TAT
G157F
TTT





G147C
TGT
R150D
GAT
P154V
GTT
G157R
CGT





G147S
TCT
R150I
ATT
P154W
TGG
G157H
CAT





G157L
TTG
G160M
ATG
A163E
GAG
F166C
TGT





G157N
AAT
G160C
TGT
A163T
ACG
F166E
GAG





G157Y
TAT
G160Q
CAG
A163Q
CAG
Q167D
GAT





G157S
TCG
G160V
GTT
A163I
ATT
Q167R
CGG





G157T
ACG
G160S
AGT
A163N
AAT
Q167A
GCG





G157A
GCT
G160E
GAG
H164L
CTT
Q167S
AGT





G157Q
CAG
G160L
CTT
H164M
ATG
Q167F
TIT





G157P
CCG
G160T
ACG
H164K
AAG
Q167Y
TAT





G157V
GTG
N161S
AGT
H164P
CCG
Q167P
CCG





G157M
ATG
N161C
TGT
H164C
TGT
Q167T
ACT





P158S
TCT
N161L
TTG
H164R
CGT
Q167V
GTG





P158Y
TAT
N161R
CGT
H164A
GCG
Q167L
CTG





P158R
CGG
N161G
GGT
H164V
GTG
Q167M
ATG





P158L
CTT
N161W
TGG
H164S
TCG
Q167N
AAT





P158V
GTG
N161Y
TAT
H164N
AAT
Q167G
GGG





P158C
TGT
N161E
GAG
H164G
GGG
Q167K
AAG





P158A
GCG
N161P
CCT
H164F
TTT
Q167E
GAG





P158W
TGG
N161T
ACG
H164Y
TAT
P168N
AAT





P158I
ATT
N161H
CAT
H164Q
CAG
P168F
TTT





P158F
TTT
N161I
ATT
H164E
GAG
P168R
CGG





P158Q
CAG
N161V
GTG
A165W
TGG
P168W
TGG





P158T
ACT
N161F
TTT
A165V
GTT
P168A
GCT





P158G
GGT
N161Q
CAG
A165G
GGG
P168T
ACG





P158K
AAG
L162A
GCT
A165K
AAG
P168V
GTT





P158N
AAT
L162G
GGG
A165L
TTG
P168G
GGG





P158D
GAT
L162C
TGT
A165P
CCT
P168C
TGT





G159R
CGG
L162P
CCG
A165Q
CAG
P168M
ATG





G159S
AGT
L162R
CGG
A165D
GAT
P168H
CAT





G159Q
CAG
L162I
ATT
A165H
CAT
P168L
CTT





G159P
CCT
L162S
TCT
A165F
TTT
P168S
AGT





G159V
GTG
L162D
GAT
A165S
AGT
P168I
ATT





G159K
AAG
L162M
ATG
A165T
ACT
P168D
GAT





G159A
GCG
L162E
GAG
A165R
CGG
G169H
CAT





G159Y
TAT
L162T
ACT
A165N
AAT
G169A
GCG





G159E
GAG
L162Y
TAT
A165M
ATG
G169E
GAG





G159T
ACG
L162F
TTT
F166G
GGG
G169C
TGT





G159M
ATG
L162W
TGG
F166S
TCG
G169S
TCG





G159I
ATT
L162Q
CAG
F166L
CTT
G169L
CTG





G159W
TGG
A163R
CGT
F166V
GTG
G169V
GTT





G159L
CTG
A163G
GGG
F166P
CCT
G169T
ACG





G159C
TGT
A163Y
TAT
F166N
AAT
G169R
CGG





G160A
GCG
A163P
CCT
F166R
CGT
G169W
TGG





G160H
CAT
A163S
AGT
F166A
GCG
G169M
ATG





G160N
AAT
A163L
CTT
F166K
AAG
G169I
ATT





G160W
TGG
A163C
TGT
F166H
CAT
G169P
CCG





G16OR
CGG
A163K
AAG
F166W
TGG
G169D
GAT





G160P
CCG
A163V
GTG
F166I
ATT
G169Q
CAG





G160I
ATT
A163F
TTT
F166M
ATG
P170L
CTT





P17OR
CGG
G173S
AGT
A176L
CTG
D179I
ATT





P170I
ATT
G173A
GCG
A176P
CCT
D179R
CGT





P170T
ACG
G173R
AGG
A176N
AAT
D179N
AAT





P17OF
TTT
G173N
AAT
A176G
GGT
D179W
TGG





P170Q
CAG
G173T
ACG
A176S
TCT
D179Q
CAG





P170G
GGG
G173D
GAT
A176R
CGT
D179V
GTG





P170S
TCT
G173V
CTT
A176K
AAG
D179C
TGT





P170H
CAT
G173F
TTT
A176D
GAT
E180M
ATG





P170C
TGT
G173M
ATG
A176W
TGG
E18OP
CCT





P170M
ATG
G173Y
TAT
H177T
ACG
E180K
AAG





P170K
AAG
G173P
CCG
H177P
CCG
E180Y
TAT





P170W
TGG
G174R
CGT
H177Q
CAG
E180Q
CAG





P170D
GAT
G174A
GCG
H177A
GCG
E18OR
CGG





P170A
GCG
G174E
GAG
H177S
TCG
E180A
GCG





G171S
TCT
G174F
TTT
H177G
GGG
E180T
ACT





G171M
ATG
G174H
CAT
H177W
TGG
E180I
ATT





G171N
AAT
G174T
ACT
H177L
CTG
E18OF
TTT





G171P
CCT
G174D
GAT
H177V
GTT
E180C
TGT





G171R
CGG
G174S
AGT
H177I
ATT
E180G
GGG





G171Y
TAT
G174P
CCG
H177R
CGG
E180S
TCG





G171A
GCT
G174W
TGG
H177N
AAT
E18ON
AAT





G171Q
CAG
G174V
CTT
H177Y
TAT
E180D
GAT





G171H
CAT
G174N
AAT
H177C
TGT
D181S
TCG





G171L
CTT
G174Y
TAT
H177D
GAT
D181Q
CAG





G171W
TGG
G174M
ATG
F178G
GGT
D181P
CCT





G171C
TGT
G174L
CTT
F178C
TGT
D181Y
TAT





G171K
AAG
D1751
ATT
F178W
TGG
D181R
CGT





G171E
GAG
D175T
ACG
F178R
CGG
D181V
GTT





G171D
GAT
D175N
AAT
F178K
AAG
D181F
TTT





I172Y
TAT
D175V
CTT
F178S
AGT
D181A
GCT





I172T
ACG
D175S
TCG
F178H
CAT
D181T
ACG





I172P
CCT
D175R
CGG
F178P
CCT
D181L
TTG





I172A
GCG
D175G
GGG
F178V
CTT
D181E
GAG





I172L
CTT
D175A
GCG
F178A
GCT
D181K
AAG





I172Q
CAG
D175F
TTT
F178Q
CAG
D181M
ATG





I172E
GAG
D175C
TGT
F178Y
TAT
D181C
TGT





I172C
TGT
D175Q
CAG
F178I
ATT
D181G
GGT





I172M
ATG
D175Y
TAT
F178T
ACT
E182C
TGT





I172D
GAT
D175L
CTG
F178L
CTG
E182P
CCT





I172V
GTT
D175H
CAT
F178E
GAG
E182S
AGT





I172R
CGT
D175P
CCG
D179P
CCT
E182T
ACG





I172G
GGG
D175E
GAG
D179L
TTG
E182R
CGG





I172W
TGG
A176F
TTT
D179E
GAG
E182D
GAT





I172N
AAT
A176Q
CAG
D179G
GGG
E182A
GCT





G173C
TGT
A176V
GTG
D179S
AGT
E182F
TTT





G173L
CTG
A176E
GAG
D179A
GCT
E182L
CTT





G173K
AAG
A176T
ACT
D179K
AAG
E182I
ATT





G173W
TGG
A176C
TGT
D179T
ACT
E182Y
TAT





E182Q
CAG
T185D
GAT
R189K
AAG
N192S
TCG





E182W
TGG
N186G
GGG
R189P
CCG
N192W
TGG





E182M
ATG
N186A
GCT
R189E
GAG
N192G
GGG





E182G
GGT
N186T
ACT
R189V
GTT
N192D
GAT





R183P
CCT
N186R
CGT
R189D
GAT
N192V
GTG





R183K
AAG
N186L
TAA
R189Y
TAT
N192A
GCT





R183W
TGG
N186P
CCG
R189C
TGT
N192T
ACT





R183E
GAG
N186S
AGT
R189A
GCT
N192K
AAG





R183A
GCT
N186V
GTG
R189H
CAT
N192C
TGT





R183T
ACG
N186Q
CAG
R189W
TGG
N192M
ATG





R183L
CTT
N186H
CAT
R189N
AAT
L193P
CCG





R183N
AAT
N186C
TGT
R189T
ACT
L193G
GGG





R183H
CAT
N186E
GAG
R189Q
CAG
L193F
TTT





R183V
GTG
N186F
TTT
E190A
GCG
L193S
TCG





R183C
TGT
N186Y
TAT
E190H
CAT
L193W
TGG





R183M
ATG
N186D
GAT
E190V
GTG
L193A
GCT





R183I
ATT
N187R
CGG
E190P
CCG
L193R
CGT





R183G
GGT
N187M
ATG
E190C
TGT
L193Q
CAG





R183S
TCT
N187S
TCT
E190G
GGT
L193E
GAG





W184G
GGG
N187T
ACG
E190R
CGG
L193K
AAG





W184H
CAT
N187L
CTG
E190I
ATT
L193N
AAT





W184L
CTG
N187W
TGG
E190S
TCG
L193I
ATT





W184E
GAG
N187F
TTT
E190T
ACT
L193T
ACT





W184P
CCT
N187K
AAG
E190M
ATG
L193D
GAT





W184N
AAT
N1871
ATT
E190L
TTG
L193Y
TAT





W184A
GCG
N187A
GCT
E190K
AAG
H194S
AGT





W184T
ACT
N187P
CCG
E190Y
TAT
H194E
GAG





W184R
CGG
N187D
GAT
E190D
GAT
H194K
AAG





W184Q
CAG
N187G
GGG
Y191T
ACT
H194Q
CAG





W184V
GTG
N187C
TGT
Y191H
CAT
H194V
GTT





W184S
TCT
N187H
CAT
Y191G
GGG
H194T
ACT





W184M
ATG
F188P
CCG
Y191L
TTG
H194L
CTG





W184I
ATT
F188I
ATT
Y191P
CCT
H194Y
TAT





W184F
TTT
F188N
AAT
Y191Q
CAG
H194F
TIT





T185R
CGT
F188S
AGT
Y191K
AAG
H194G
GGT





T185Y
TAT
F188Q
CAG
Y191D
GAT
H194I
ATT





T185W
TGG
F188K
AAG
Y191A
GCG
H194W
TGG





T185H
CAT
F188G
GGG
Y191W
TGG
H194M
ATG





T185G
GGG
F188W
TGG
Y191S
TCT
H194A
GCT





T185P
CCT
F188E
GAG
Y191V
GTT
H194P
CCT





T185S
TCG
F188H
CAT
Y191E
GAG
R195C
TGT





T185V
GTT
F188D
GAT
Y191R
CGT
R195F
TTT





T185Q
CAG
F188A
GCG
Y191C
TGT
R195W
TGG





T185N
AAT
F188L
CTT
N192R
CGG
R195T
ACT





T185C
TGT
F188R
CGT
N192L
CTG
R195L
CTG





T185L
CTT
F188V
GTT
N192Q
CAG
R195G
GGT





T185A
GCG
R189L
TTG
N192P
CCT
R195Q
CAG





T185E
GAG
R189G
GGG
N192H
CAT
R195K
AAG





R195S
TCT
A198F
TTT
L201N
AAT
L205S
TCT





R195A
GCT
A198W
TGG
G202T
ACG
L205G
GGT





R195D
GAT
A198Y
TAT
G202Y
TAT
L205P
CCT





R195P
CCT
A198D
GAT
G202E
GAG
L205E
GAG





R195Y
TAT
H199I
ATT
G202V
GTG
L205V
GTG





R195E
GAG
H199P
CCG
G202S
TCT
L205M
ATG





R195V
GTG
H199G
GGT
G202L
CTG
L205N
AAT





V196T
ACG
H199N
AAT
G202I
ATT
L205C
TGT





V196D
GAT
H199S
TCG
G202M
ATG
L205I
ATT





V196G
GGG
H199L
TTG
G202H
CAT
L205A
GCG





V196E
GAG
H199M
ATG
G202C
TGT
L205R
CGG





V196A
GCG
H199A
GCG
G202R
CGT
L205W
TGG





V196S
AGT
H199C 
TGT
G202P
CCT
L205Q
CAG





V196Q
CAG
H199K
AAG
G202A
GCT
G206I
ATT





V196P
CCG
H199R
CGT
G202K
AAG
G206V
GTG





V196R
CGT
H199V
GTG
G202D
GAT
G206A
GCG





V196H
CAT
H199W
TGG
H203Y
TAT
G206C
TGT





V196Y
TAT
H199T
ACT
H203E
GAG
G206S
TCG





V196I
ATT
H199E
GAG
H203R
CGG
G206P
CCG





V196L
CTG
E200P
CCG
H203Q
CAG
G206L
TTG





V196K
AAG
E200G
GGG
H203P
CCG
G206D
GAT





V196M
ATG
E200A
GCT
H203G
GGG
G206M
ATG





A197G
GGT
E200T
ACG
H203T
ACT
G206R
CGG





A197S
AGT
E200I
ATT
H203D
GAT
G206Q
CAG





A197L
CTT
E200W
TGG
H203L
TTG
G206E
GAG





A197P
CCG
E200R
CGG
H203N
AAT
G206H
CAT





A197V
GTG
E200F
TTT
H203A
GCT
G206T
ACG





A197Y
TAT
E200M
ATG
H203S
TCT
G206W
TGG





A197Q
CAG
E200D
GAT
H203V
GTT
L207S
TCT





A197R
CGG
E200V
GTG
H203I
ATT
L207Y
TAT





A197T
ACT
E200C
TGT
H203C
TGT
L207A
GCG





A197I
ATT
E200S
TCT
S204R
CGG
L207R
CGT





A197H
CAT
E200Y
TAT
S204N
AAT
L207P
CCG





A197E
GAG
E200N
AAT
S204A
GCG
L207Q
CAG





A197W
TGG
L201A
GCG
S204T
ACT
L207N
AAT





A197N
AAT
L201R
CGG
S204Y
TAT
L207K
AAG





A197C
TGT
L201E
GAG
S204V
GTG
L207M
ATG





A198T
ACG
L201P
CCT
S204L
AAT
L207W
TGG





A198K
AAG
L201G
GGT
S204H
CAT
L207H
CAT





A198S
TCG
L201V
GTT
S204D
GAT
L207D
GAT





A198H
CAT
L201T
ACG
S204Q
CAG
L207V
GTT





A198G
GGT
L201I
ATT
S204G
GGG
L207I
ATT





A198E
GAG
L201S
TCT
S204W
TGG
L207G
GGT





A198P
CCG
L201W
TGG
S2041
ATT
S208D
GAT





A198L
TTG
L201Q
CAG
S204K
AAG
S208V
GTT





A198R
CGT
L201D
GAT
S204P
CCT
S208P
CCT





A198V
GTT
L201M
ATG
L205T
ACG
S208G
GGT





A198M
ATG
L201K
AAG
L205D
GAT
S208A
GCG





S208K
AAG
T211Q
CAG
G214A
GCT
M217A
GCG





S208N
AAT
T211S
TCG
G214D
GAT
M217H
CAT





S208F
TTT
T211A
GCG
G214F
TTT
M217I
ATT





S208Q
CAG
T211F
TTT
G214Y
TAT
M217D
GAT





S208W
TGG
T211D
GAT
G214M
ATG
Y218C
TGT





S208T
ACG
T211W
TGG
G214C
TGT
Y218F
TTT





S208E
GAG
T211L
CTG
A215L
CTG
Y218W
TGG





S208C
TGT
D212E
GAG
A215Q
CAG
Y218L
CTG





S208R
CGT
D212A
GCG
A215M
ATG
Y218A
GCG





S208L
CTT
D212K
AAG
A215G
GGT
Y218P
CCG





H209T
ACG
D212R
CGG
A215W
TGG
Y218R
CGG





H209Y
TAT
D212T
ACG
A215S
AGT
Y218N
AAT





H209R
CGG
D212N
AAT
A215T
ACG
Y218V
GTG





H209Q
CAG
D212G
GGG
A215V
GTT
Y218Q
CAG





H209A
GCT
D212S
TCT
A215N
AAT
Y218I
ATT





H209G
GGG
D212P
CCG
A215P
CCG
Y218D
GAT





H209N
AAT
D212Q
CAG
A215H
CAT
Y218S
TCG





H209P
CCT
D212V
GTT
A215K
AAG
Y218G
GGG





H209W
TGG
D212L
TTG
A215I
ATT
Y218E
GAG





H209V
GTT
D212F
TTT
A215R
CGT
P219L
TTG





H209D
GAT
D212H
CAT
A215C
TGT
P219C
TGT





H209S
AGT
D212Y
TAT
A215D
GAT
P219V
GTG





H209F
TTT
I213Q
CAG
L216A
GCT
P219D
GAT





H209L
CTG
I213T
ACT
L216C
TGT
P219F
TTT





H209C
TGT
I213C
TGT
L216D
GAT
P219A
GCG





S210C
TGT
I213P
CCT
L216E
GAG
P219T
ACT





S210G
GGT
I213H
CAT
L216G
GGG
P219E
GAG





S210I
ATT
I213A
GCG
L216I
ATT
P219Q
CAG





S21OR
CGT
I213V
GTT
L216K
AAG
P219R
CGG





S210L
CTG
I213G
GGG
L216M
ATG
P219H
CAT





S210V
GTG
I213N
AAT
L216P
CCT
P219G
GGG





S210H
CAT
I213L
CYT
L216Q
CAG
P219K
AAG





S210N
AAT
I213S
AGT
L216R
CGG
P219S
TCG





S210F
TTT
I213M
ATG
L216S
TCT
P219W
TGG





S210P
CCG
I213R
CGG
L216T
ACT
S220R
CGT





S210W
TGG
I213K
AAG
L216V
GTG
S220A
GCG





S210Q
CAG
I213F
TTT
L216W
TGG
S220Q
CAG





S210T
ACG
I213D
GAT
M217P
CCT
S220T
ACT





S210K
AAG
I213E
GAG
M217Y
TAT
S220L
CTT





S210A
GCG
G214L
TTG
M217T
ACG
S220K
AAG





T211P
CCG
G214Q
CAG
M217C
TGT
S220G
GGG





T211R
CGT
G214S
TCT
M217S
AGT
S220H
CAT





T211K
AAG
G214T
ACT
M217L
CTG
S220E
GAG





T211G
GGG
G214V
GTG
M217N
AAT
S220M
ATG





T211M
ATG
G214I
ATT
M217R
CGG
S220V
GTT





T211N
AAT
G214R
CGT
M217Q
CAG
S220P
CCG





T211V
GTG
G214P
CCG
M217K
AAG
S220I
ATT





T211H
CAT
G214E
GAG
M217G
GGG
S220F
TTT





S220N
AAT
S224T
ACG
V227K
AAG
A230S
TCG





Y221W
TGG
S224Q
CAG
V227L
CTG
A230C
TGT





Y221K
AAG
S224R
CGG
V227P
CCT
A230V
GTT





Y221Q
CAG
S224P
CCG
V227S
TCT
A230T
ACT





Y221C
TGT
S224I
ATT
V227T
ACT
A230Y
TAT





Y221N
AAT
S224V
GTT
V227W
TGG
A230M
ATG





Y221P
CCT
S224L
TTG
V227Y
TAT
A230N
AAT





Y221V
GTT
S224C
TGT
V227G
GGG
A230H
CAT





Y221A
GCG
S224K
AAG
V227H
CAT
Q231I
ATT





Y221G
GGG
S224D
GAT
V227Q
CAG
Q231A
GCT





Y221R
CGG
S224H
CAT
V227R
CGT
Q231F
TTT





Y221S
TCG
S224M
ATG
Q228A
GCT
Q231P
CCT





Y221M
ATG
S224A
GCT
Q228D
GAT
Q231Y
TAT





Y221T
ACG
S224W
TGG
Q228E
GAG
Q231R
CGT





Y221L
CTT
G225D
GAT
Q228G
GGT
Q231L
CTG





Y221E
GAG
G225R
CGT
Q228H
CAT
Q231D
GAT





T222L
TTG
G225Q
CAG
Q228K
AAG
Q231G
GGT





T222Y
TAT
G225M
ATG
Q228L
CTG
Q231V
GTT





T222R
CGT
G225P
CCT
Q228M
ATG
Q231W
TGG





T222V
OTT
G225W
TGG
Q228N
AAT
Q231S
AGT





T222P
CCT
G225S
TCT
Q228P
CCG
Q231H
CAT





T222S
AGT
G225E
GAG
Q228R
CGG
Q231C
TGT





T222A
GCT
G225V
GTT
Q228S
TCT
Q231M
ATG





T222H
CAT
G225T
ACG
Q228T
ACG
D232H
CAT





T222G
GGG
G225K
AAG
Q228W
TGG
D232G
GGG





T222M
ATG
G225N
AAT
Q228Y
TAT
D232R
CGT





T222F
TTT
G225C
TGT
L229R
CGG
D232P
CCT





T222C
TGT
G225H
CAT
L229A
GCG
D232Y
TAT





T222I
ATT
G225A
GCG
L229T
ACG
D232N
AAT





T222N
AAT
D226S
TCT
L229Q
CAG
D232S
TCG





T222W
TGG
D226W
TGG
L229P
CCT
D232F
TTT





T222D
GAT
D226R
CGG
L229E
GAG
D232V
GTG





F223L
TTG
D226A
GCT
L229W
TGG
D232K
AAG





F223T
ACG
D226N
AAT
L229M
ATG
D232W
TGG





F223C
TGT
D226T
ACT
L229I
ATT
D232Q
CAG





F223R
CGT
D226E
GAG
L229G
GGT
D232E
GAG





F223N
AAT
D226L
CTT
L229C
TGT
D232T
ACT





F223P
CCT
D226P
CCT
L229Y
TAT
D232L
CTG





F223E
GAG
D226H
CAT
L229D
GAT
D233Q
CAG





F223G
GGG
D226G
GGT
L229H
CAT
D233P
CCG





F223Q
CAG
D226I
ATT
L229V
GTG
D233S
TCT





F223A
GCG
D226M
ATG
A230L
TTG
D233T
ACG





F223S
TCT
D226V
GTG
A230G
GGT
D233A
GCG





F223Y
TAT
D226C
TGT
A230W
TGG
D233W
TGG





F223H
CAT
V227A
GCT
A230P
CCG
D233G
GGT





F223K
AAG
V227C
TGT
A230D
GAT
D233R
CGT





F223M
ATG
V227D
GAT
A230R
CGT
D233E
GAG





S224G
GGG
V227E
GAG
A230I
ATT
D233N
AAT





D233V
GTG
G236N
AAT
1240G
GGG
R243L
CTT





D233M
ATG
G236F
TTT
1240Q
CAG
R243A
GCG





D233L
CTG
I237S
TCG
1240P
CCG
R243H
CAT





D233K
AAG
I237L
CTG
1240R
CGG
R243Q
CAG





D233I
ATT
I237R
CGT
1240S
TCG
R243S
AGT





I234A
GCT
I237Q
CAG
1240K
AAG
R243I
ATT





I234T
ACG
I237K
AAG
1240V
GTG
R243C
TGT





I234V
GTT
I237D
GAT
1240D
GAT
R243N
AAT





I234W
TGG
I237A
GCG
1240A
GCG
R243Y
TAT





I234E
GAG
I237T
ACG
1240C
TGT
R243G
GGG





I234G
GGT
I237E
GAG
1240L
CTT
R243D
GAT





I234L
CTT
I237C
TGT
1240F
TTT
R243V
GTG





I234H
CAT
I237G
GGG
1240Y
TAT
S244P
CCG





I234M
ATG
1237P
CCT
1240M
ATG
S244L
CTT





I234N
AAT
I237Y
TAT
1240T
ACG
S244W
TGG





I234Y
TAT
I237W
TGG
Y241V
GTT
S244M
ATG





I234P
CCT
I237N
AAT
Y241A
GCT
S244V
GTT





I234D
GAT
Q238G
GGG
Y241G
GGG
S244Q
CAG





I234Q
CAG
Q238H
CAT
Y241H
CAT
S244D
GAT





I234C
TGT
Q238S
TCG
Y241R
CGG
S244E
GAG





D235H
CAT
Q238Y
TAT
Y241P
CCG
S244T
ACG





D235G
GGG
Q238F
TTT
Y241Q
CAG
S244H
CAT





D235H
GCG
Q238E
GAG
Y241L
TTG
S244G
GGT





D235P
CCG
Q238L
TTG
Y241T
ACG
S244A
GCT





D235L
CTT
Q238W
TGG
Y241S
AGT
S244F
TTT





D235V
GTG
Q238P
CCG
Y241W
TGG
S244Y
TAT





D235E
GAG
Q238R
AGG
Y241N
AAT
S244R
CGT





D235R
CGT
Q238C
TGT
Y241M
ATG
Q245P
CCT





D235Q
CAG
Q238N
AAT
Y241I
AAA
Q245I
NTT





D235T
ACG
Q238I
ATT
Y241D
GAT
Q245F
TTT





D235C
TGT
Q238T
ACG
G242A
GCG
Q245V
GTT





D235S
TCG
Q238K
AAG
G242F
TTT
Q245M
ATG





D235N
AAT
A239S
TCT
G242L
AAT
Q245T
ACT





D235Y
TAT
A239Q
CAG
G242N
AAT
Q245E
GAG





D235I
ATT
A239T
ACG
G242P
CCT
Q245S
TCG





G236M
ATG
A239P
CCT
G242W
TGG
Q245R
CGG





G236R
CGG
A239V
GTG
G242T
ACG
Q245G
GGT





G236D
GAT
A239L
CTG
G242R
CGT
Q245H
CAT





G236S
TCT
A239Y
TAT
G242V
AAT
Q245L
CTT





G236T
ACT
A239I
ATT
G242S
TCG
Q245K
AAG





G236C
TGT
A239C
TGT
G242I
ATT
Q245W
TGG





G236K
AAG
A239G
GGG
G242Y
TAT
Q245C
TGT





G236E
GAG
A239W
TGG
G242H
CAT
N246W
TGG





G236P
CCG
A239F
TTT
G242E
GAG
N246R
CGG





G236I
ATT
A239K
AAG
G242K
AAG
N246A
GCG





G236Y
TAT
A239H
CAT
R243P
CCG
N246F
TTT





G236L
CTG
A239R
CGT
R243K
AAG
N246G
GGT





G236V
GTT
A239D
GAT
R243T
ACG
N246P
CCT





N246V
GTT
Q249G
GGT
G252P
CCT
T255L
TTG





N246Q
CAG
Q249N
AAT
G252H
CAT
T255H
CAT





N246P
TAT
Q249K
AAG
G252C
TGT
P256S
AGT





N246C
TGT
Q249I
ATT
G252V
GTT
P256V
GTG





N246I
ATT
Q249Y
TAT
G252I
ATT
P256F
TTT





N246L
TTG
Q249V
GTG
P253C
TGT
P256Y
TAT





N246S
TCT
Q249L
TTG
P253G
GGT
P256I
ATT





N246T
ACT
Q249H
CAT
P253Q
CAG
P256A
GCT





N246K
AAG
P250L
CTG
P253I
ATT
P256L
CTT





N246D
GAT
P250S
TCG
P253L
CTG
P256G
GGT





P247A
GCG
P25OR
CGG
P253R
CGG
P256N
AAT





P247D
GAT
P250Y
TAT
P253A
GCT
P256R
CGG





P247E
GAG
P250M
ATG
P253E
GAG
P256Q
CAG





P247F
TTT
P250F
TTT
P253Y
TAT
P256E
GAG





P247G
GGG
P250A
GCT
P253W
TGG
P256K
AAG





P247H
CAT
P250K
AAG
P253M
ATG
P256M
ATG





P247I
ATT
P250G
GGT
P253V
GTG
P256C
TGT





P247K
AAG
P250N
AAT
P253T
ACT
K257C
TGT





P247L
CTG
P250T
ACT
P253K
AAG
K257M
ATG





P247N
AAT
P250W
TGG
P253N
AAT
K257V
GIT





P247Q
CAG
P250D
GAT
Q254R
CGT
K257A
GCT





P247R
CGT
P250V
GTG
Q254G
GGG
K257E
GAG





P247S
TCG
P250Q
CAG
Q254W
TGG
K257S
TCT





P247T
ACG
I251A
GCG
Q254T
ACT
K257L
CTT





P247V
GTT
I251Q
CAG
Q254A
GCT
K257I
ATT





V248W
TGG
I251G
GGG
Q254F
TTY
K257G
GGG





V248L
CTG
I251L
CTG
Q254D
GAT
I057N
AAT





V248Q
CAG
I251K
AAG
Q254P
CCG
K257F
TTT





V248M
ATG
I251R
CGT
Q254L
CTG
K257W 
TGG





V248Y
TAT
I251E
GAG
Q254C
TGT
K257R
CGG





V248G
GGG
I251D
GAT
Q254Y
TAT
K257P
CCG





V248C
TGT
I251T
ACG
Q254I
ATT
K257T
ACT





V248R
CGG
I251C
TGT
Q254E
GAG
A258Q
CAG





V248A
GCG
1251Y
TAT
Q254V
GTG
A258Y
TAT





V248H
CAT
1251P
CCT
Q254S
TCT
A258W
TGG





V248I
ATT
I251S
TCT
T255I
ATT
A258G
GGG





V248T
ACT
I251W
TGG
T255Q
CAG
A258L
TTG





V248K
AAG
I251V
GTT
T255P
CCG
A258F
TTT





V2485
TCG
G252F
TTT
T255R
CGT
A258M
ATG





V248F
TTT
G252W
TGG
T255C
TGT
A258N
AAT





V248E
GAG
G252A
GCG
T255N
AAT
A258V
GTG





Q249T
ACT
G252R
CGG
T255S
AGT
A258T
ACG





Q249W
TGG
G252L
CTT
T255V
GTG
A258I
ATT





Q249R
CGG
G252E
GAG
T255E
GAG
A258D
GAT





Q249E
GAG
G252D
GAT
T255G
GGG
A258R
CGT





Q249A
GCT
G252K
AAG
T255K
AAG
A258E
GAG





Q249P
CCG
G252S
TCG
T255A
GCT
A258P
CCG





Q249C
TGT
G252T
ACG
T255F
TTT
T255L
TTG









1. Expression


The DNA encoding each individual library member was generated according to standard DNA synthesis protocols and protein was expressed using routine molecular biology techniques. Briefly, the DNA was ligated into vector pET303CTHis (Invitrogen, SEQ ID NO:3466) using routine molecular biology techniques. Plasmid containing one individual hMMP-1 mutant was transformed into BL21 (DE3) E. coli cells (Tigen, Beiging, China) using manufacturers recommendations. The process was repeated for all library members. The transformation culture was used to inoculate 1 mL LB medium containing ampicillin additives. The culture was grown at 37° C. with shaking for 16 hours. Protein expression was induced by the addition of 1 mM isopropyl-β-D-thiogalactoside (IPTG) and the culture was incubated at 25° C. with shaking. After 6 hours, the cells were pelleted by centrifugation at 6,000 g for 10 minutes and the supernatant was removed. The periplasmic protein was enriched by incubating the cells in 50 μl OS buffer (200 mM Tris-HCl, pH 7.5, 20% sucrose, 1 mM EDTA) with 4 μl DNAse (10 μg/ml), 4 μl RNAse (10 μg/ml), and 4 μl lysozyme (10 μg/ml) for 10 minutes at 25° C. 50 μl of water was added to each well followed by centrifugation at 6000 g for 10 minutes to remove cell debris. The supernatant, containing the hMMP-1 protein, was stored at −20° C. Activity of supernatants were screened as described in the following examples. B. Cloning and Expression of Wildtype hMMP-1


In this example, wildtype hMMP-1 was individually expressed in both E. coli and CHO-S cells.


1. Expression in E. coli


Wildtype hMMP-1 (clone BAP00610, having a sequence of nucleotides set forth as nucleotides in SEQ ID NO:706 and containing a pel B signal sequence set forth in SEQ ID NO:3547) was cloned into vector pET303CTHis (Invitrogen, SEQ ID NO:3466) and grown in BL21(DE3) E. coli. The pET303CTHis vector contained a C-terminal His tag (SEQ ID NO:3465). Protein expression was induced upon the addition of 1 mM isopropyl-β-D-thiogalactoside (IPTG) as described above. Following expression, the protein was enriched as described in Example 1A, and subsequently purified using a HiTrap Ni2+ column (GE Healthcare) according to standard molecular biology protocols. Expression and purification were monitored by SDS/PAGE and Western blot analysis.


2. Expression in CHO-S Cells


Wildtype hMMP-1 (clone BAP0062, having a sequence of nucleotides set forth as nucleotides 72-1478 in SEQ ID NO:708 and a sequence encoding a C-terminal His tag) was expressed in CHO-S cells and secreted into the medium. Transfected cells were cultured at 37° C. in CD-CHO serum free media (Invitrogen). The wildtype hMMP-1 protein was purified using a HiTrap Ni2+ column (GE Healthcare) according to standard molecular biology protocols


Example 2
Determination of Enzymatic Activity of the hMMP-1 Mutants Using a Fluorogenic Peptide Substrate

In this example, the hMMP-1 mutant library, generated in Example 1, was screened using a high throughput fluorescence activity assay to identify temperature sensitive hMMP-1 mutants. To screen for temporally sensitive hMMP-1 mutants, the enzymatic activity of each individual mutant was determined at 25° C. and 37° C. and/or 34° C., using a commercially available fluorogenic substrate, peptide IX, designated as Mca-K-P-L-G-L-Dpa-A-R-NH2 (SEQ ID NO:707; Mca=(7-Methoxycoumarin-4-yl)acetyl; Dpa=N-3-(2,4,-Dinitrophenyl)-L-2,3-diaminopropionyl; R&D Systems, Minneapolis, Minn., Cat#ES010). The peptide substrate contains a highly fluorescent 7-methoxycoumarin group that is quenched by resonance energy transfer to the 2,4-dinitrophenyl group. Activated hMMP-1 cleaves the amide bond between glycine and leucine resulting in an increase in released fluorescence. Reactions were initially performed in a 96-well assay and confirmed using a 14 ml tube format.


A. 96-Well Assay

Prior to assessing activity of the supernatants, supernatants were treated with a processing agent to activate the inactive zymogen form into an active enzyme. Briefly, 4 μl of each hMMP-1 mutant supernatant generated in Example 1 was added to 100 μl of TCNB (50 nM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij 35, pH 7.5) with 1 mM of the processing agent p-aminophenylmercuric acetate (APMA) in a 96-well plate. The solution was incubated at the reaction temperature (either 25° C. or 37° C.) for 2 hours. This activation step cleaves the pro-peptide and generates mature hMMP-1.


Following activation, 1.6 μl of TCNB containing 620 μM Mca-K-P-L-G-L-Dpa-A-R-NH2 fluorescent substrate was added to each well to a final concentration of 10 μM, at the indicated reaction temperature (either 25° C. or 37° C.) for 1 hour. Fluorescence was detected by measuring fluorescence in a fluorescent plate reader at 320 nm exitation/405 nm emission. Relative fluorescence units (RFU) were determined. Supernatant from wildtype hMMP-1 and plasmid/vector transformed cells were used as positive and negative controls. Duplicate reactions were performed for each sample, reaction temperature, and positive and negative control.


The results of the initial screen of 2687 hMMP-1 mutants are shown in Table 9. The initial screen resulted in the identification of 199 putative primary hits (see Table 10) with reduced activity at 37° C. as compared to the activity at 25° C.









TABLE 9







Results of Initial Screen for Temperature Sensitive Mutants




















Res.
Res.





Avg.
Avg.

Act.
Act.


Temp.
hMMP-1
SEQ ID
RFU
RFU
Ratio
Mut/wt
Mut/wt


Phenotype
mutation
NO
25° C.
37° C.
25° C./37° C.
25° C.
37° C.

















Down
F81C
784
1740.62
3123.63
0.56
0.35
0.46


Down
F81E
780
871.51
1243.66
0.70
0.18
0.18


Down
F81I
793
4100.22
5376.62
0.76
0.83
0.79


Neutral
F81L
795
8890.68
7913.44
1.12
1.57
1.51


Neutral
F81P
797
1102.23
1043.87
1.06
0.19
0.20


Neutral
F81S
789
2527.30
2312.47
1.09
0.45
0.44


Neutral
F81A
796
8780.53
7784.51
1.13
1.55
1.48


Neutral
F81M
791
2545.25
3095.21
0.82
0.45
0.59


Neutral
F81G
790
8979.05
7773.71
1.16
1.59
1.48


Neutral
F81T
787
1564.49
1373.60
1.14
0.28
0.26


Neutral
F81Q
786
9225.28
7923.69
1.16
1.63
1.51


Neutral
F81R
783
8514.40
7454.74
1.14
1.50
1.42


Neutral
F81W
792
6078.70
5909.04
1.03
1.07
1.12


Neutral
F81H
781
8126.15
7360.21
1.10
1.44
1.40


Neutral
F81V
794
7263.15
6614.17
1.10
1.28
1.26


Neutral
V82I
813
535.78
548.02
0.98
0.06
0.06


Down
V82C
803
4177.57
6476.29
0.65
0.50
0.72


Neutral
V82A
815
9540.61
9240.92
1.03
1.14
1.03


Neutral
V82P
816
599.23
634.69
0.94
0.07
0.07


Down
V82Y
807
3295.59
6173.45
0.53
0.39
0.69


Down
V82M
811
6824.39
8606.64
0.79
0.82
0.96


Neutral
V82Q
805
581.51
652.74
0.89
0.07
0.07


Neutral
V82F
810
7233.54
8739.45
0.83
0.87
0.98


Down
V82W
812
6194.12
8397.19
0.74
0.74
0.94


Neutral
V82N
804
9421.72
8759.51
1.08
1.13
0.98


Down
V82R
802
603.22
781.77
0.77
0.07
0.09


Neutral
V82G
809
8298.42
8911.04
0.93
0.99
0.99


Neutral
V82S
808
8293.03
9022.13
0.92
0.99
1.01


Down
V82L
814
6951.75
8694.05
0.80
0.83
0.97


Neutral
V82T
806
7993.81
8975.05
0.89
0.96
1.00


Neutral
L83A
834
8629.03
9023.51
0.96
1.03
1.01


Neutral
L83C
822
554.26
567.87
0.98
0.07
0.06


Neutral
L83D
817
8705.34
8957.38
0.97
1.04
1.00


Neutral
L83E
818
9212.48
9265.02
0.99
1.10
1.03


Neutral
L83G
828
7713.92
9073.74
0.85
0.92
1.01


Neutral
L83H
819
6449.24
7800.76
0.83
0.77
0.87


Down
L83I
832
4575.76
6963.24
0.66
0.55
0.78


Down
L83M
830
5921.65
8064.61
0.73
0.71
0.90


Neutral
L83P
835
7794.15
8608.36
0.91
0.93
0.96


Neutral
L83Q
824
7291.24
8673.39
0.84
0.87
0.97


Neutral
L83R
821
8509.58
8988.62
0.95
1.02
1.00


Neutral
L83S
827
9261.79
9205.93
1.01
1.11
1.03


Neutral
L83T
825
7549.73
8580.54
0.88
0.90
0.96


Down
L83W
831
4193.18
6044.52
0.69
0.50
0.67


Neutral
L83Y
826
7968.79
9051.39
0.88
0.95
1.01


Down
T84V
851
3169.35
4931.29
0.64
0.64
0.72


Down
T84E
837
498.18
627.84
0.79
0.10
0.09


Neutral
T84H
838
7046.83
6974.20
1.01
1.24
1.33


Neutral
T84L
852
7687.84
6946.59
1.11
1.36
1.32


Neutral
T84D
836
7972.32
7331.43
1.09
1.41
1.39


Neutral
T84R
840
7298.49
6880.17
1.06
1.29
1.31


Neutral
T84I
850
6508.69
5860.75
1.11
1.15
1.11


Neutral
T84S
845
6073.28
5981.85
1.02
1.07
1.14


Neutral
T84G
846
8087.79
7200.99
1.12
1.43
1.37


Neutral
T84Q
843
6275.12
6690.38
0.94
1.11
1.27


Neutral
T84P
854
3528.37
3832.34
0.92
0.62
0.73


Neutral
T84A
853
8718.27
7840.72
1.11
1.54
1.49


Neutral
T84C
841
5177.89
5107.57
1.01
0.91
0.97


Neutral
T84Y
844
4768.51
4818.30
0.99
0.84
0.92


Neutral
T84F
847
6312.72
6453.46
0.98
1.10
1.27


Down
E85L
871
1633.29
2148.43
0.76
0.33
0.31


Down
E85Q
861
2834.50
4068.60
0.70
0.57
0.59


Neutral
E85P
873
2855.52
3389.51
0.84
0.58
0.50


Neutral
E85T
862
401.26
382.58
1.05
0.08
0.06


Down
E85K
857
2293.84
3049.87
0.75
0.46
0.45


Down
E85M
867
2158.30
2821.39
0.76
0.44
0.41


Neutral
E85G
865
1767.69
1734.31
1.02
0.31
0.33


Down
E85R
858
912.46
7286.41
0.13
0.16
1.39


Neutral
E85S
864
7811.54
7488.09
1.04
1.38
1.42


Neutral
E85C
859
6027.10
5938.05
1.01
1.06
1.13


Neutral
E85Y
863
4449.33
3909.71
1.14
0.79
0.74


Neutral
E85A
872
5552.19
5461.08
1.02
0.98
1.04


Down
E85N
860
522.81
7634.45
0.07
0.09
1.45


Neutral
E85V
870
7152.74
7011.60
1.02
1.26
1.33


Neutral
E85F
866
6092.47
6362.37
0.96
1.06
1.26


Down
G86L
890
2452.10
3232.22
0.76
0.50
0.47


Down
G86P
892
2117.46
5219.90
0.41
0.43
0.76


Neutral
G86I
888
1888.26
2293.71
0.82
0.38
0.34


Neutral
G86T
882
363.85
380.61
0.96
0.07
0.06


Neutral
G86H
876
389.15
372.78
1.04
0.08
0.05


Neutral
G86D
874
415.45
406.81
1.02
0.08
0.06


Down
G86N
880
2612.85
3755.02
0.70
0.53
0.55


Neutral
G86S
884
8500.13
7717.19
1.10
1.50
1.47


Neutral
G86K
877
1660.95
2002.39
0.83
0.29
0.38


Neutral
G86W
887
1570.85
1690.05
0.93
0.28
0.32


Neutral
G86Y
883
1829.24
2126.68
0.86
0.32
0.40


Neutral
G86V
889
1830.80
2092.69
0.87
0.32
0.40


Neutral
G86C
879
1784.05
2091.03
0.85
0.32
0.40


Neutral
G86M
886
1687.28
2025.99
0.83
0.30
0.39


Up
G86F
885
1897.87
1483.82
1.28
0.34
0.28


Neutral
N87M
905
418.35
412.23
1.01
0.08
0.06


Down
N87L
909
3385.42
4941.20
0.69
0.69
0.72


Neutral
N87P
911
8762.48
8941.20
0.98
1.55
1.70


Neutral
N87V
908
6199.21
7269.38
0.85
1.09
1.38


Neutral
N87R
897
7761.00
8810.25
0.88
1.37
1.68


Up
N87F
904
6882.19
4428.08
1.55
1.22
0.84


Down
N87S
902
2083.05
3304.46
0.63
0.37
0.63


Neutral
N87I
907
7572.66
8090.13
0.94
1.34
1.54


Neutral
N87C
898
3291.22
3945.40
0.83
0.58
0.75


Down
N87A
910
5482.33
6869.11
0.80
0.97
1.31


Neutral
N87G
903
8060.01
8916.11
0.90
1.42
1.70


Down
N87Y
901
4397.56
5611.87
0.78
0.78
1.07


Up
N87E
894
5876.33
4763.86
1.23
1.04
0.91


Down
N87H
895
5013.05
7306.33
0.69
0.89
1.39


Neutral
N87Q
899
8559.37
9021.72
0.95
1.51
1.72


Down
P88C
917
1255.12
2197.65
0.57
0.15
0.25


Neutral
P88K
915
6857.61
8492.90
0.81
0.82
0.95


Down
P88W
926
664.95
845.70
0.79
0.08
0.09


Down
P88G
923
1694.96
3159.20
0.54
0.20
0.35


Down
P88L
929
2562.59
3576.95
0.72
0.31
0.40


Down
P88Q
919
4499.52
7270.91
0.62
0.54
0.81


Neutral
P88A
930
6549.92
8130.83
0.81
0.78
0.91


Neutral
P88T
920
6576.99
8126.45
0.81
0.79
0.91


Down
P88Y
921
5515.19
7868.29
0.70
0.66
0.88


Down
P88R
916
4209.25
6681.38
0.63
0.50
0.75


Down
P88H
914
2580.97
4465.31
0.58
0.31
0.50


Down
P88I
927
841.81
1249.17
0.67
0.10
0.14


Neutral
P88V
928
1666.69
1915.49
0.87
0.20
0.21


Down
P88E
913
971.61
1460.63
0.67
0.12
0.16


Down
P88D
912
1300.22
1911.83
0.68
0.16
0.21


Down
R89V
946
1163.86
2620.05
0.44
0.24
0.38


Down
R89W
944
1252.89
1744.18
0.72
0.25
0.25


Neutral
R89M
943
402.00
386.98
1.04
0.08
0.06


Neutral
R89A
948
7883.15
8954.83
0.88
1.39
1.70


Neutral
R89T
938
6791.27
6752.46
1.01
1.20
1.28


Neutral
R89G
941
8957.06
8693.72
1.03
1.58
1.65


Up
R89S
940
7342.24
4138.54
1.77
1.30
0.79


Neutral
R89K
934
7679.02
8254.00
0.93
1.36
1.57


Neutral
R89F
942
4764.35
5589.97
0.85
0.84
1.06


Neutral
R89Y
939
5614.23
5949.31
0.94
0.99
1.13


Up
R89N
936
3502.08
1995.86
1.75
0.62
0.38


Neutral
R89H
933
3611.69
4222.47
0.86
0.64
0.80


Neutral
R89L
947
3123.66
3332.30
0.94
0.55
0.63


Neutral
R89E
932
1490.93
1265.89
1.18
0.26
0.24


Down
R89P
949
2659.02
3342.56
0.80
0.47
0.64


Neutral
W90L
966
394.24
411.82
0.96
0.08
0.06


Neutral
W90G
961
448.08
427.28
1.05
0.09
0.06


Neutral
W90P
968
444.72
442.65
1.00
0.09
0.06


Neutral
W90T
958
397.42
365.04
1.09
0.08
0.05


Neutral
W90S
960
443.43
442.72
1.00
0.09
0.06


Neutral
W90V
965
384.57
385.18
1.00
0.08
0.06


Neutral
W90I
964
443.81
432.28
1.03
0.09
0.06


Neutral
W90A
967
497.94
554.07
0.90
0.10
0.08


Neutral
W90F
962
730.98
656.84
1.11
0.15
0.10


Neutral
W90H
952
498.51
493.15
1.01
0.10
0.07


Neutral
W90M
963
512.18
508.03
1.01
0.10
0.07


Neutral
W90R
954
1974.98
1695.11
1.17
0.23
0.19


Up
W90E
951
1537.84
1076.32
1.43
0.18
0.12


Up
W90N
956
1308.11
1001.91
1.31
0.15
0.11


Up
W90Q
957
1392.58
1015.03
1.37
0.16
0.12


Down
E91N
974
4746.43
6166.37
0.77
0.96
0.90


Down
E91R
972
2760.48
3810.12
0.72
0.56
0.56


Down
E91W
982
2595.35
5651.48
0.46
0.53
0.83


Down
E91G
979
4826.02
6684.79
0.72
0.98
0.98


Neutral
E91V
984
454.87
459.17
0.99
0.09
0.07


Neutral
E91Y
977
4885.18
5469.16
0.89
0.99
0.80


Down
E91C
973
3525.68
5567.75
0.63
0.71
0.81


Down
E91H
970
5114.86
6610.88
0.77
1.04
0.97


Neutral
E91T
976
442.21
427.42
1.03
0.09
0.06


Neutral
E91S
978
8147.93
7696.77
1.06
0.94
0.87


Neutral
E91A
986
1140.60
1252.34
0.91
0.13
0.14


Neutral
E91I
983
8414.79
8744.30
0.96
0.97
0.99


Neutral
E91D
969
8482.61
8681.73
0.98
0.98
0.98


Neutral
E91F
980
1159.80
1117.15
1.04
0.13
0.13


Neutral
E91L
985
2012.22
1956.07
1.03
0.23
0.22


Down
Q92V
1003
3748.94
5787.25
0.65
0.76
0.85


Down
Q92Y
996
2141.40
5383.55
0.40
0.43
0.79


Down
Q92L
1004
2422.01
3765.30
0.64
0.49
0.55


Neutral
Q92N
994
8685.91
8183.03
1.06
1.00
0.93


Neutral
Q92E
989
8489.89
8972.33
0.95
0.98
1.02


Neutral
Q92I
1002
7791.35
8518.64
0.91
0.90
0.97


Neutral
Q92T
995
8289.96
8916.74
0.93
0.96
1.01


Neutral
Q92G
998
7218.56
8372.74
0.86
0.83
0.95


Neutral
Q92P
1006
3678.59
4021.57
0.91
0.43
0.46


Neutral
Q92W
1001
7277.76
8042.96
0.90
0.84
0.91


Neutral
Q92F
999
8216.00
8989.59
0.91
0.95
1.02


Neutral
Q92S
997
8760.81
9254.16
0.95
1.01
1.05


Neutral
Q92R
992
8566.65
8894.65
0.96
0.99
1.01


Neutral
Q92K
991
8790.93
9239.36
0.95
1.02
1.05


Neutral
Q92A
1005
8138.84
9037.58
0.90
0.94
1.02


Down
T93A
1024
2321.71
5447.47
0.43
0.47
0.80


Neutral
T93L
1023
541.85
545.89
0.99
0.11
0.08


Down
T93M
1019
5256.54
7088.24
0.74
1.07
1.04


Neutral
T93N
1013
5852.91
7141.52
0.82
1.19
1.04


Neutral
T93V
1022
7976.61
8668.81
0.92
0.92
0.98


Neutral
T93I
1021
9015.76
9426.26
0.96
1.04
1.07


Neutral
T93D
1007
8742.88
9032.61
0.97
1.01
1.02


Neutral
T93S
1016
8832.30
8978.09
0.98
1.02
1.02


Neutral
T93R
1011
8802.98
8782.70
1.00
1.02
1.00


Neutral
T93W
1020
7872.73
8474.20
0.93
0.91
0.96


Down
T93F
1018
4307.35
5656.46
0.76
0.50
0.64


Neutral
T93P
1025
8315.28
8629.67
0.96
0.96
0.98


Down
T93G
1017
4926.38
6453.11
0.76
0.57
0.73


Neutral
T93K
1010
8581.02
8663.14
0.99
0.99
0.98


Neutral
T93E
1008
8081.66
8373.46
0.97
0.93
0.95


Neutral
H94L
1042
509.44
507.83
1.00
0.10
0.07


Down
H94S
1035
3442.98
5184.16
0.66
0.70
0.76


Neutral
H94M
1038
7388.19
8302.74
0.89
0.85
0.94


Neutral
H94R
1029
7237.77
7718.22
0.94
0.84
0.87


Neutral
H94E
1027
8375.45
8466.04
0.99
0.97
0.96


Neutral
H94I
1040
6326.35
7655.54
0.83
0.73
0.87


Neutral
H94D
1026
7358.29
8057.05
0.91
0.85
0.91


Neutral
H94P
1044
2892.06
3183.37
0.91
0.33
0.36


Neutral
H94A
1043
8285.72
8772.41
0.94
0.96
0.99


Neutral
H94N
1031
8497.48
8732.16
0.97
0.98
0.99


Down
H94F
1037
6046.02
7839.76
0.77
0.70
0.89


Neutral
H94G
1036
7671.85
7912.46
0.97
0.89
0.90


Neutral
H94T
1033
7121.14
8100.48
0.88
0.82
0.92


Neutral
H94V
1041
7941.67
8381.81
0.95
0.92
0.95


Neutral
H94W
1039
6520.52
7583.08
0.86
0.75
0.86


Down
L95E
4
4165.44
5381.52
0.77
0.48
0.61


Neutral
L95Y
12
1044.63
1118.92
0.93
0.12
0.13


Neutral
L95R
7
1328.79
1312.13
1.01
0.15
0.15


Neutral
L95A
20
1262.99
1297.06
0.97
0.15
0.15


Neutral
L95G
14
1090.24
1183.93
0.92
0.13
0.13


Up
L95K
6
1333.28
1191.46
1.12
0.15
0.14


Neutral
L95S
13
1077.02
1117.02
0.96
0.12
0.13


Neutral
L95T
11
1407.58
1310.18
1.07
0.16
0.15


Neutral
L95H
5
1270.21
1086.69
1.17
0.15
0.12


Neutral
L95W
17
1133.63
1041.65
1.09
0.13
0.12


Neutral
L95V
19
8390.57
8371.68
1.00
0.97
0.95


Neutral
L95C
8
2189.50
2519.34
0.87
0.25
0.29


Neutral
L95P
21
1084.88
1147.69
0.95
0.13
0.13


Neutral
L95D
3
909.41
933.49
0.97
0.11
0.11


Down
L95I
18
1707.98
2294.02
0.74
0.30
0.45


Neutral
T96E
1046
415.05
397.38
1.04
0.07
0.06


Neutral
T96R
1049
478.81
441.86
1.08
0.08
0.07


Neutral
T96P
1063
589.64
692.90
0.85
0.09
0.11


Down
T96S
1054
3055.53
4011.47
0.76
0.49
0.64


Neutral
T96A
1062
1873.45
2254.28
0.83
0.30
0.36


Down
T96L
1061
2337.67
3156.45
0.74
0.37
0.51


Down
T96W
1058
1194.79
1631.19
0.73
0.19
0.26


Down
T96N
1051
2674.35
3874.07
0.69
0.43
0.62


Neutral
T96G
1055
415.04
387.45
1.07
0.07
0.06


Down
T96F
1056
2640.74
3897.10
0.68
0.42
0.62


Down
T96Q
1052
1865.64
2509.48
0.74
0.30
0.40


Down
T96H
1047
1294.29
1620.22
0.80
0.21
0.26


Down
T96V
1060
1904.14
2730.27
0.70
0.30
0.44


Down
T96I
1059
1814.91
2921.26
0.62
0.29
0.47


Neutral
T96C
1050
701.03
774.48
0.91
0.11
0.12


Neutral
Y97R
1068
447.48
449.81
0.99
0.14
0.09


Neutral
Y97V
1079
637.70
789.90
0.81
0.20
0.16


Neutral
Y97A
1081
507.18
504.63
1.01
0.16
0.10


Neutral
Y97P
1082
488.40
452.67
1.08
0.15
0.09


Neutral
Y97L
1080
510.25
549.53
0.93
0.16
0.11


Neutral
Y97T
1072
538.83
600.97
0.90
0.17
0.12


Up
Y97K
1067
469.55
390.08
1.20
0.15
0.08


Down
Y97W
1077
3115.45
4974.99
0.63
0.98
1.01


Down
Y97H
1066
685.71
879.64
0.78
0.22
0.18


Neutral
Y97S
1073
482.94
471.85
1.02
0.15
0.10


Neutral
Y97E
1065
435.12
432.07
1.01
0.14
0.09


Neutral
Y97D
1064
466.89
455.39
1.03
0.15
0.09


Neutral
Y97N
1070
486.98
490.84
0.99
0.15
0.10


Neutral
Y97G
1074
521.34
516.64
1.01
0.11
0.08


Neutral
Y97Q
1071
567.66
575.73
0.99
0.12
0.08


Down
R98H
1085
1456.51
3257.97
0.45
0.46
0.66


Down
R98K
1086
2994.82
4670.17
0.64
0.95
0.95


Neutral
R98C
1087
761.34
938.43
0.81
0.24
0.19


Down
R98L
1099
3592.72
5087.24
0.71
1.14
1.03


Down
R98M
1095
3551.60
5834.31
0.61
1.12
1.18


Down
R98F
1094
2925.55
4988.31
0.59
0.92
1.01


Down
R98W
1096
833.68
1098.48
0.76
0.26
0.22


Neutral
R98Y
1091
505.91
479.02
1.06
0.16
0.10


Down
R98P
1101
2306.44
3388.72
0.68
0.73
0.69


Down
R98E
1084
1812.72
2769.07
0.65
0.57
0.56


Down
R98A
1100
3006.35
4371.72
0.69
0.95
0.89


Down
R98G
1093
1525.20
2367.66
0.64
0.48
0.48


Down
R98V
1098
1298.78
3330.10
0.39
0.26
0.49


Down
R98S
1092
4646.88
6142.58
0.76
0.94
0.90


Down
R98D
1083
2905.96
3867.31
0.75
0.33
0.47


Neutral
I99C
1107
514.61
514.66
1.00
0.16
0.10


Neutral
I99E
1103
550.80
548.33
1.00
0.17
0.11


Neutral
I99G
1113
588.17
598.60
0.98
0.19
0.12


Neutral
I99H
1104
749.01
834.15
0.90
0.24
0.17


Neutral
I99N
1108
691.55
805.73
0.86
0.22
0.16


Neutral
I99P
1120
567.03
526.02
1.08
0.18
0.11


Down
I99T
1110
1087.94
1583.58
0.69
0.34
0.32


Down
I99V
1117
2373.86
3390.37
0.70
0.75
0.69


Neutral
I99A
1119
654.22
809.57
0.81
0.13
0.12


Down
I99F
1114
2098.09
2958.45
0.71
0.43
0.43


Down
I99L
1118
2592.09
4336.89
0.60
0.53
0.63


Neutral
I99R
1106
561.16
555.21
1.01
0.11
0.08


Neutral
I99S
1112
616.13
673.46
0.91
0.12
0.10


Down
I99Q
1109
3318.21
4623.91
0.72
0.37
0.56


Neutral
I99W
1116
509.03
492.00
1.04
0.06
0.06


Neutral
I99Y
1111
690.55
700.48
0.99
0.08
0.09


Down
E100V
512
3980.72
5009.20
0.79
1.26
1.01


Neutral
E100P
515
727.82
785.14
0.93
0.23
0.16


Down
E100L
513
3370.21
4726.28
0.71
1.06
0.96


Down
E100H
498
1484.00
2354.50
0.63
0.47
0.48


Down
E100D
497
1886.86
3049.67
0.62
0.60
0.62


Down
E100M
509
3046.42
4566.62
0.67
0.96
0.92


Neutral
E100G
507
541.78
567.31
0.95
0.11
0.08


Down
E100W
510
1544.77
3766.06
0.41
0.31
0.55


Down
E100Y
505
2885.60
4167.75
0.69
0.58
0.61


Neutral
E100R
500
7410.11
7964.52
0.93
0.83
0.96


Neutral
E100S
506
3768.09
4664.58
0.81
0.42
0.56


Neutral
E100T
504
6985.28
7478.12
0.93
0.79
0.90


Neutral
E100F
508
6709.27
7436.60
0.90
0.75
0.90


Neutral
E100I
511
8824.19
8458.79
1.04
0.99
1.02


Neutral
E100N
502
8809.68
8215.63
1.07
0.99
0.99


Neutral
N101M
1133
7907.75
7930.91
1.00
0.89
0.96


Neutral
N101F
1132
5045.54
5244.47
0.96
0.57
0.63


Neutral
N101L
1137
6427.09
6656.60
0.97
0.72
0.80


Neutral
N101V
1136
8153.10
7605.57
1.07
0.92
0.92


Neutral
N101H
1123
8863.48
8197.03
1.08
1.00
0.99


Neutral
N101R
1125
8050.92
7576.48
1.06
0.91
0.91


Down
N101C
1126
2651.70
3359.06
0.79
0.30
0.41


Neutral
N101T
1128
9660.15
8437.52
1.14
1.09
1.02


Neutral
N101P
1139
8232.08
7996.53
1.03
0.93
0.96


Neutral
N101W
1134
3302.54
3773.04
0.88
0.37
0.46


Neutral
N101K
1124
7396.60
7283.35
1.02
0.83
0.88


Neutral
N101S
1130
8913.81
8187.44
1.09
1.00
0.99


Neutral
N101D
1121
5424.50
5676.43
0.96
0.61
0.68


Neutral
N101A
1138
6371.04
6423.71
0.99
0.72
0.78


Neutral
N101Y
1129
3878.66
3898.52
0.99
0.44
0.47


Neutral
Y102R
1144
9027.37
8170.55
1.10
1.02
0.99


Neutral
Y102K
1143
5806.60
5157.24
1.13
0.65
0.62


Neutral
Y102V
1155
6412.64
6500.28
0.99
0.72
0.78


Neutral
Y102M
1152
6668.55
6964.91
0.96
0.75
0.84


Neutral
Y102P
1158
4670.45
4431.46
1.05
0.53
0.53


Neutral
Y102N
1146
4618.85
4579.81
1.01
0.52
0.55


Neutral
Y102G
1150
7272.09
6976.34
1.04
0.82
0.84


Neutral
Y102L
1156
3323.14
3802.15
0.87
0.37
0.46


Neutral
Y102D
1140
5174.42
4862.10
1.06
0.58
0.59


Neutral
Y102S
1149
8744.32
8244.11
1.06
0.98
0.99


Neutral
Y102F
1151
7629.25
8362.12
0.91
0.86
1.01


Neutral
Y102A
1157
7177.10
7304.92
0.98
0.81
0.88


Neutral
Y102E
1141
3375.30
3325.21
1.02
0.38
0.40


Neutral
Y102Q
1147
5644.96
5711.44
0.99
0.64
0.69


Neutral
Y102C
1145
1544.43
1842.83
0.84
0.17
0.22


Neutral
T103E
517
619.06
617.97
1.00
0.20
0.13


Neutral
T103D
516
848.44
877.46
0.97
0.27
0.18


Neutral
T103S
525
761.49
855.80
0.89
0.24
0.17


Up
T103L
532
855.65
650.61
1.32
0.27
0.13


Neutral
T103V
531
822.60
1017.88
0.81
0.26
0.21


Neutral
T103R
520
674.37
652.99
1.03
0.21
0.13


Neutral
T103Y
524
1181.09
1423.76
0.83
0.37
0.29


Down
T103N
522
3131.62
4822.91
0.65
0.99
0.98


Neutral
T103C
521
628.62
604.98
1.04
0.20
0.12


Up
T103Q
523
791.61
624.86
1.27
0.25
0.13


Neutral
T103W
529
513.42
548.41
0.94
0.10
0.08


Neutral
T103P
534
513.57
526.91
0.97
0.10
0.08


Neutral
T103A
533
1058.92
950.05
1.11
0.21
0.14


Neutral
T103G
526
749.67
656.69
1.14
0.15
0.10


Neutral
T103K
519
884.09
777.94
1.14
0.18
0.11


Neutral
P104G
1170
602.57
620.78
0.97
0.19
0.13


Down
P104E
1160
4330.78
6029.01
0.72
1.37
1.22


Down
P104T
1167
3213.10
4681.67
0.69
1.02
0.95


Neutral
P104F
1171
2191.45
1923.19
1.14
0.69
0.39


Down
P104R
1163
591.46
5625.37
0.11
0.19
1.14


Down
P104D
1159
4022.87
5896.28
0.68
1.27
1.19


Neutral
P104C
1164
779.25
879.87
0.89
0.25
0.18


Down
P104Q
1166
4140.44
5971.62
0.69
1.31
1.21


Down
P104V
1175
2675.96
4161.77
0.64
0.85
0.84


Down
P104Y
1168
1907.52
2912.52
0.65
0.60
0.59


Down
P104H
1161
3404.74
5009.27
0.68
1.08
1.01


Down
P104L
1176
2981.52
4000.85
0.75
0.60
0.58


Down
P104S
1169
1205.11
2392.05
0.50
0.24
0.35


Neutral
P104A
1177
8861.30
8360.82
1.06
1.00
1.01


Up
P104M
1172
6709.44
7118.65
0.94
0.88
0.75


Up
D105A
39
2674.16
1227.06
2.18
0.65
0.24


Neutral
D105C
26
871.16
737.92
1.18
0.21
0.15


Up
D105F
33
2009.56
1221.58
1.65
0.49
0.24


Up
D105G
32
2407.89
1686.68
1.43
0.58
0.34


Up
D105I
36
1732.38
1105.99
1.57
0.42
0.22


Up
D105L
38
1563.61
859.56
1.82
0.38
0.17


Neutral
D105M
34
2703.51
2920.93
0.93
0.65
0.58


Up
D105N
27
3766.72
1475.08
2.55
0.91
0.29


Up
D105P
40
856.02
604.56
1.42
0.21
0.12


Up
D105R
25
3892.02
2016.90
1.93
0.94
0.40


Up
D105S
31
3646.49
2727.22
1.34
0.88
0.54


Up
D105T
29
2513.64
1729.46
1.45
0.61
0.34


Neutral
D105V
37
5824.43
6784.65
0.86
1.41
1.35


Up
D105W
35
2565.93
1855.05
1.38
0.62
0.37


Neutral
D105E
22
4000.92
3366.64
1.19
0.59
0.45


Up
L106P
1196
793.18
480.45
1.65
0.16
0.10


Neutral
L106D
1178
455.97
436.46
1.04
0.09
0.09


Neutral
L106N
1184
579.84
499.77
1.16
0.12
0.10


Up
L106G
1189
778.24
578.12
1.35
0.16
0.12


Down
L106M
1191
2299.74
3704.96
0.62
0.48
0.74


Down
L106A
1195
3604.47
5633.39
0.64
0.75
1.12


Neutral
L106R
1182
658.60
552.82
1.19
0.14
0.11


Neutral
L106Y
1187
4761.33
5769.09
0.83
0.99
1.15


Neutral
L106T
1186
1604.22
1508.31
1.06
0.33
0.30


Neutral
L106V
1194
8561.50
8230.68
1.04
1.77
1.64


Neutral
L106H
1180
644.13
641.84
1.00
0.13
0.13


Down
L106F
1190
1776.88
2525.65
0.70
0.36
0.37


Down
L106I
1193
2787.16
4408.75
0.63
0.56
0.64


Neutral
L106C
1183
2995.56
3678.33
0.81
0.34
0.44


Neutral
L106S
1188
2730.64
2899.36
0.94
0.31
0.35


Neutral
P107L
1214
3183.54
3874.49
0.82
0.77
0.75


Neutral
P107W
1211
1255.79
1303.70
0.96
0.30
0.25


Neutral
P107T
1205
5673.07
6084.28
0.93
1.37
1.18


Neutral
P107S
1207
5865.31
6191.65
0.95
1.42
1.20


Neutral
P107R
1201
2981.87
3300.34
0.90
0.72
0.64


Neutral
P107Y
1206
2005.11
2383.15
0.84
0.48
0.46


Neutral
P107M
1210
3551.42
4031.55
0.88
0.86
0.78


Neutral
P107V
1213
3499.60
4142.87
0.84
0.85
0.80


Neutral
P107D
1197
3531.02
4095.17
0.86
0.85
0.80


Neutral
P107A
1215
5661.84
6316.88
0.90
1.37
1.23


Neutral
P107C
1202
786.68
776.20
1.01
0.19
0.15


Neutral
P107K
1200
3176.89
3653.27
0.87
0.77
0.71


Neutral
P107F
1209
1603.40
1832.50
0.87
0.39
0.36


Neutral
P107I
1212
2003.91
2369.36
0.85
0.48
0.46


Neutral
P107G
1208
2694.02
3272.63
0.82
0.65
0.64


Up
R108P
1234
4652.14
3388.90
1.37
0.96
0.67


Down
R108G
1226
4168.56
6204.10
0.67
0.86
1.24


Neutral
R108T
1223
1360.40
1652.92
0.82
0.28
0.33


Down
R108E
1217
5311.31
6829.34
0.78
1.10
1.36


Down
R108A
1233
5676.42
7183.19
0.79
1.18
1.43


Down
R108Y
1224
1527.69
2690.78
0.57
0.32
0.54


Down
R108K
1219
7212.78
9049.80
0.80
1.49
1.80


Down
R108C
1220
2092.15
2852.47
0.73
0.43
0.57


Neutral
R108S
1225
8515.31
8202.68
1.04
1.76
1.63


Neutral
R108F
1227
4264.07
5199.96
0.82
0.88
1.04


Down
R108W
1229
1522.39
2152.20
0.71
0.31
0.31


Down
R108I
1230
2968.84
4628.28
0.64
0.60
0.68


Down
R108L
1232
2200.90
3462.10
0.64
0.45
0.51


Down
R108N
1221
2820.25
4415.19
0.64
0.57
0.65


Neutral
R108V
1231
571.77
618.30
0.92
0.12
0.09


Neutral
A109S
1245
6193.70
7627.42
0.81
1.28
1.52


Down
A109R
1239
4933.84
9751.06
0.51
1.02
1.94


Down
A109T
1243
4678.95
6089.37
0.77
0.97
1.21


Down
A109W
1249
5152.58
6447.41
0.80
1.07
1.28


Down
A109I
1250
2587.03
4255.55
0.61
0.54
0.85


Down
A109Q
1242
3475.21
4698.87
0.74
0.72
0.94


Up
A109N
1241
6266.66
4399.73
1.42
1.30
0.88


Up
A109Y
1244
1880.37
1444.85
1.30
0.39
0.29


Down
A109G
1246
5864.62
12111.28
0.48
1.21
2.41


Neutral
A109M
1248
7784.21
8628.31
0.90
1.61
1.72


Down
A109D
1235
4410.30
6431.60
0.69
0.91
1.28


Neutral
A109V
1251
8073.90
8388.34
0.96
1.67
1.67


Down
A109E
1236
2859.74
7453.25
0.38
0.58
1.09


Down
A109L
1252
3649.92
5241.27
0.70
0.74
0.77


Neutral
A109H
1237
7206.01
7536.96
0.96
0.81
0.91


Down
D110P
1272
691.78
937.18
0.74
0.14
0.19


Down
D110F
1265
2469.89
3158.71
0.78
0.51
0.63


Down
D110Q
1260
3028.40
4201.99
0.72
0.63
0.84


Down
D110R
1257
756.25
1109.97
0.68
0.16
0.22


Neutral
D110M
1266
1094.79
917.81
1.19
0.23
0.18


Down
D110H
1255
3327.99
6569.83
0.51
0.69
1.31


Down
D110I
1268
1457.92
2219.69
0.66
0.30
0.44


Down
D110L
1270
1494.01
1991.44
0.75
0.31
0.40


Down
D110V
1269
2494.40
3413.88
0.73
0.52
0.68


Down
D110T
1261
2731.23
4170.98
0.65
0.57
0.83


Down
D110S
1263
1262.77
1714.94
0.74
0.26
0.34


Down
D110Y
1262
2764.78
5378.21
0.51
0.57
1.07


Neutral
D110G
1264
510.14
537.48
0.95
0.10
0.08


Neutral
D110C
1258
827.23
996.83
0.83
0.17
0.15


Neutral
D110A
1271
4179.59
5112.44
0.82
0.47
0.62


Down
V111E
1274
779.81
1134.36
0.69
0.16
0.23


Down
V111A
1290
1964.87
2890.23
0.68
0.41
0.58


Down
V111S
1283
2947.29
4188.33
0.70
0.61
0.83


Neutral
V111W
1287
601.19
580.46
1.04
0.12
0.12


Neutral
V111G
1284
833.15
974.76
0.85
0.17
0.19


Neutral
V111Y
1282
813.12
942.64
0.86
0.17
0.19


Up
V111P
1291
923.36
696.55
1.33
0.19
0.14


Down
V111L
1289
1070.50
1565.39
0.68
0.22
0.31


Neutral
V111D
1273
591.79
576.36
1.03
0.12
0.11


Down
V111K
1276
1017.96
1328.59
0.77
0.21
0.26


Down
V111T
1281
3551.97
4859.95
0.73
0.74
0.97


Down
V111Q
1280
1546.82
2061.98
0.75
0.32
0.41


Down
V111I
1288
4959.51
6699.66
0.74
1.03
1.33


Neutral
V111C
1278
843.17
943.89
0.89
0.17
0.14


Neutral
V111R
1277
2401.69
2925.16
0.82
0.27
0.35


Down
D112A
1309
1419.86
2167.48
0.66
0.29
0.43


Down
D112M
1304
1668.58
2249.91
0.74
0.35
0.45


Down
D112V
1307
2683.45
3699.41
0.73
0.56
0.74


Down
D112R
1295
1072.27
1395.54
0.77
0.22
0.28


Down
D112K
1294
967.53
1261.79
0.77
0.20
0.25


Neutral
D112P
1310
565.23
589.06
0.96
0.12
0.12


Down
D112Q
1298
4681.31
8975.21
0.52
0.97
1.79


Down
D112F
1303
1148.89
1477.74
0.78
0.24
0.29


Down
D112G
1302
1824.01
2601.95
0.70
0.38
0.52


Neutral
D112C
1296
866.83
1034.64
0.84
0.18
0.21


Down
D112W
1305
937.80
1277.50
0.73
0.19
0.25


Neutral
D112T
1299
2538.82
2941.38
0.86
0.53
0.59


Neutral
D112H
1293
480.11
467.40
1.03
0.10
0.07


Neutral
D112S
1301
7203.69
7600.93
0.95
0.81
0.92


Down
D112I
1306
4020.53
5498.90
0.73
0.44
0.72


Down
D112Y
1300
2132.97
2869.86
0.74
0.23
0.38


Down
D112L
1308
2626.71
4159.92
0.63
0.29
0.55


Neutral
H113T
1318
9107.72
8278.01
1.10
1.03
1.00


Neutral
H113L
1327
9479.59
8454.16
1.12
1.07
1.02


Neutral
H113M
1323
9463.40
8759.43
1.08
1.07
1.06


Neutral
H113S
1320
9278.22
9159.47
1.01
1.04
1.11


Neutral
H113N
1316
8609.35
8502.46
1.01
0.97
1.03


Neutral
H113R
1314
7702.30
7852.46
0.98
0.87
0.95


Neutral
H113A
1328
8505.43
8090.18
1.05
0.96
0.98


Neutral
H113E
1312
9118.02
8443.69
1.08
1.03
1.02


Neutral
H113V
1326
9183.53
8450.30
1.09
1.03
1.02


Neutral
H113Y
1319
9688.60
8548.83
1.13
1.09
1.03


Neutral
H113F
1322
9472.51
8729.41
1.09
1.07
1.05


Up
H113D
1311
9304.42
4925.78
1.89
1.05
0.59


Up
H113W
1324
8683.10
5775.24
1.50
0.98
0.70


Neutral
H113G
1321
8953.60
8320.09
1.08
1.01
1.00


Neutral
H113P
1329
2987.12
3102.32
0.96
0.34
0.37


Neutral
A114E
1331
7136.25
7924.97
0.90
0.80
0.96


Neutral
A114S
1340
9211.05
8794.50
1.05
1.04
1.06


Neutral
A114I
1345
7073.18
7475.79
0.95
0.80
0.90


Up
A114P
1348
1691.05
1357.51
1.25
0.19
0.16


Neutral
A114N
1336
9250.51
8746.70
1.06
1.04
1.06


Neutral
A114L
1347
7749.61
8007.88
0.97
0.87
0.97


Neutral
A114T
1338
6242.22
6974.59
0.89
0.70
0.84


Neutral
A114F
1342
605.35
675.10
0.90
0.07
0.08


Neutral
A114V
1346
5527.85
6054.48
0.91
0.62
0.73


Neutral
A114G
1341
7663.26
7892.13
0.97
0.86
0.95


Neutral
A114C
1335
2412.52
3005.83
0.80
0.27
0.36


Neutral
A114M
1343
5287.05
5931.99
0.89
0.60
0.72


Neutral
A114R
1334
4454.65
3915.86
1.14
0.50
0.47


Neutral
A114W
1344
4654.58
5477.95
0.85
0.52
0.66


Neutral
A114Q
1337
8094.57
8337.94
0.97
0.91
1.01


Neutral
I115F
1361
9634.62
9011.34
1.07
1.08
1.09


Neutral
I115T
1357
1935.83
2379.92
0.81
0.22
0.29


Neutral
I115H
1351
805.66
825.35
0.98
0.09
0.10


Neutral
I115G
1360
725.85
626.12
1.16
0.08
0.08


Down
I115K
1352
642.87
920.32
0.70
0.07
0.11


Neutral
I115E
1350
1276.09
1211.16
1.05
0.14
0.15


Neutral
I115S
1359
796.93
780.25
1.02
0.09
0.09


Neutral
I115P
1367
626.77
597.01
1.05
0.07
0.07


Neutral
I115C
1354
1021.21
982.43
1.04
0.11
0.12


Neutral
I115L
1365
8869.57
8467.55
1.05
1.00
1.02


Neutral
I115Q
1356
732.25
652.35
1.12
0.08
0.08


Up
I115R
1353
750.11
575.36
1.30
0.08
0.07


Neutral
I115W
1363
2203.68
2304.27
0.96
0.25
0.28


Neutral
I115V
1364
9365.90
8785.30
1.07
1.05
1.06


Neutral
I115D
1349
694.17
641.97
1.08
0.08
0.08


Neutral
E116A
1385
9273.62
9051.39
1.02
1.04
1.09


Neutral
E116C
1372
5022.73
5732.42
0.88
0.57
0.69


Neutral
E116D
1368
9114.14
8594.45
1.06
1.03
1.04


Neutral
E116F
1379
8569.56
8473.84
1.01
0.96
1.02


Neutral
E116G
1378
8305.07
8358.04
0.99
0.94
1.01


Neutral
E116H
1369
8630.15
8386.63
1.03
0.97
1.01


Neutral
E116I
1382
9386.17
8740.84
1.07
1.06
1.05


Neutral
E116K
1370
9320.21
8760.44
1.06
1.05
1.06


Neutral
E116L
1384
8997.58
8736.18
1.03
1.01
1.05


Neutral
E116M
1380
9046.33
8478.98
1.07
1.02
1.02


Neutral
E116N
1373
8629.15
8503.39
1.01
0.97
1.03


Neutral
E116P
1386
852.91
806.00
1.06
0.10
0.10


Neutral
E116Q
1374
9480.67
8716.72
1.09
1.07
1.05


Neutral
E116R
1371
8871.32
8479.40
1.05
1.00
1.02


Neutral
E116S
1377
9714.89
8843.17
1.10
1.09
1.07


Neutral
K117H
1389
4516.51
4612.42
0.98
0.52
0.55


Neutral
K117T
1394
6149.94
6317.74
0.97
0.71
0.75


Neutral
K117Q
1393
6602.62
6024.00
1.10
0.77
0.72


Neutral
K117E
1388
668.03
667.32
1.00
0.08
0.08


Neutral
K117A
1404
7727.36
7375.30
1.05
0.90
0.88


Neutral
K117F
1398
4020.90
4038.83
1.00
0.47
0.48


Neutral
K117D
1387
5330.37
5924.02
0.90
0.62
0.70


Down
K117N
1392
4666.40
7745.69
0.60
0.54
0.92


Neutral
K117G
1397
7619.16
7218.94
1.06
0.88
0.86


Neutral
K117W
1400
5440.86
4780.56
1.14
0.63
0.57


Neutral
K117Y
1395
5047.23
4760.05
1.06
0.59
0.57


Neutral
K117L
1403
5277.39
5328.70
0.99
0.61
0.63


Neutral
K117S
1396
7278.89
6995.65
1.04
0.85
0.83


Down
K117P
1405
737.96
1153.03
0.64
0.09
0.14


Neutral
K117R
1390
8236.16
7677.40
1.07
0.96
0.91


Down
A118G
1417
2782.31
6427.69
0.43
0.32
0.76


Neutral
A118R
1410
4889.61
5639.79
0.87
0.57
0.67


Up
A118W
1420
652.55
465.07
1.40
0.08
0.06


Neutral
A118K
1409
584.59
543.84
1.07
0.07
0.06


Neutral
A118P
1424
883.04
810.72
1.09
0.10
0.10


Neutral
A118V
1422
869.06
754.10
1.15
0.10
0.09


Neutral
A118L
1423
543.99
523.84
1.04
0.06
0.06


Up
A118D
1406
617.40
468.39
1.32
0.07
0.06


Down
A118S
1416
5502.11
8251.39
0.67
0.64
0.98


Neutral
A118F
1418
7092.17
7315.30
0.97
0.82
0.87


Up
A118I
1421
556.24
456.62
1.22
0.06
0.05


Neutral
A118H
1408
482.33
466.40
1.03
0.06
0.06


Up
A118E
1407
560.55
406.52
1.38
0.07
0.05


Neutral
A118Q
1413
517.05
477.18
1.08
0.06
0.06


Neutral
A118T
1414
745.83
665.63
1.12
0.13
0.13


Down
F119G
1436
2058.01
3284.43
0.63
0.24
0.39


Down
F119T
1433
4492.83
8234.56
0.55
0.52
0.98


Neutral
F119R
1429
648.01
665.21
0.97
0.08
0.08


Neutral
F119L
1441
8529.66
7666.52
1.11
0.99
0.91


Neutral
F119N
1431
1298.98
1614.30
0.80
0.15
0.19


Down
F119S
1435
3021.31
4383.36
0.69
0.35
0.52


Neutral
F119C
1430
2921.13
3375.91
0.87
0.34
0.40


Neutral
F119P
1443
567.10
665.80
0.85
0.07
0.08


Neutral
F119W
1438
4474.41
4610.60
0.97
0.52
0.55


Neutral
F119K
1428
679.32
762.81
0.89
0.08
0.09


Down
F119H
1427
2479.46
3939.67
0.63
0.29
0.47


Neutral
F119A
1442
7345.58
7881.39
0.93
0.85
0.94


Neutral
F119V
1440
7388.01
7712.75
0.96
0.86
0.92


Neutral
F119Y
1434
5832.62
6222.88
0.94
0.68
0.74


Down
F119E
1426
1044.34
1357.80
0.77
0.12
0.16


Neutral
Q120K
1447
8732.08
8385.78
1.04
1.01
1.00


Neutral
Q120N
1450
9186.34
8785.94
1.05
1.07
1.05


Down
Q120A
1461
613.72
979.68
0.63
0.07
0.12


Neutral
Q120V
1459
8711.16
8484.49
1.03
1.01
1.01


Down
Q120D
1444
5912.84
8887.98
0.67
0.69
1.06


Neutral
Q120R
1448
8845.48
8351.59
1.06
1.03
0.99


Neutral
Q120P
1462
1083.78
1186.17
0.91
0.13
0.14


Neutral
Q120W
1457
9339.94
7899.14
1.18
1.08
0.94


Down
Q120Y
1452
4891.24
8236.87
0.59
0.57
0.98


Neutral
Q120C
1449
5241.83
5502.66
0.95
0.61
0.65


Neutral
Q120H
1446
9155.83
8431.63
1.09
1.06
1.00


Neutral
Q120T
1451
9413.75
8645.01
1.09
1.09
1.03


Down
Q120M
1456
5740.33
8861.16
0.65
0.67
1.05


Neutral
Q120E
1445
8896.83
8424.76
1.06
1.03
1.00


Neutral
Q120G
1454
9176.97
8435.97
1.09
1.07
1.00


Up
L121E
1464
3183.74
2155.16
1.48
0.37
0.26


Down
L121Q
1470
2122.18
3128.11
0.68
0.25
0.37


Neutral
L121P
1481
1446.80
1342.92
1.08
0.17
0.16


Up
L121R
1467
1129.37
875.68
1.29
0.13
0.10


Up
L121C
1468
1592.13
1145.83
1.39
0.18
0.14


Down
L121G
1474
2613.21
4720.78
0.55
0.30
0.56


Neutral
L121K
1466
4678.64
4882.50
0.96
0.54
0.58


Up
L121F
1475
1227.52
957.08
1.28
0.14
0.11


Neutral
L121I
1478
7406.57
6937.72
1.07
0.86
0.83


Down
L121S
1473
2463.24
3614.68
0.68
0.29
0.43


Neutral
L121V
1479
7973.31
7244.01
1.10
0.93
0.86


Up
L121H
1465
3156.18
2605.48
1.21
0.37
0.31


Neutral
L121T
1471
7283.06
7372.13
0.99
0.85
0.88


Down
L121A
1480
3311.41
4989.77
0.66
0.38
0.59


Neutral
L121N
1469
6619.84
6504.79
1.02
0.77
0.77


Neutral
W122R
1486
651.20
598.41
1.09
0.08
0.07


Neutral
W122A
1499
699.90
617.84
1.13
0.08
0.07


Neutral
W122N
1488
484.17
598.30
0.81
0.06
0.07


Neutral
W122P
1500
619.39
605.42
1.02
0.07
0.07


Neutral
W122T
1490
621.86
570.65
1.09
0.07
0.07


Neutral
W122L
1498
580.35
563.09
1.03
0.07
0.07


Neutral
W122G
1493
602.75
646.94
0.93
0.07
0.08


Neutral
W122S
1492
602.28
564.94
1.07
0.07
0.07


Neutral
W122V
1497
607.75
532.36
1.14
0.07
0.06


Neutral
W122H
1484
596.81
545.92
1.09
0.07
0.06


Down
W122F
1494
2018.83
3056.56
0.66
0.23
0.36


Neutral
W122Y
1491
667.50
661.98
1.01
0.08
0.08


Neutral
W122K
1485
2724.60
2334.11
1.17
0.32
0.28


Neutral
W122Q
1489
576.75
528.48
1.09
0.07
0.06


Neutral
W122E
1483
564.38
580.16
0.97
0.07
0.07


Neutral
S123D
1501
9453.37
8830.71
1.07
0.92
0.94


Neutral
S123L
1517
9912.51
9431.98
1.05
0.97
1.01


Neutral
S123A
1518
9881.07
9237.14
1.07
0.97
0.99


Neutral
S123C
1506
10654.40
8973.60
1.19
1.04
0.96


Neutral
S123I
1515
9679.91
8521.19
1.14
0.95
0.91


Neutral
S123K
1504
10567.78
9024.26
1.17
1.03
0.96


Neutral
S123N
1507
6481.00
5911.32
1.10
0.63
0.63


Neutral
S123F
1512
7485.79
8458.67
0.88
0.73
0.90


Neutral
S123Y
1510
7667.20
8806.19
0.87
0.75
0.94


Neutral
S123M
1513
9800.43
9159.15
1.07
0.96
0.98


Neutral
S123H
1503
10038.71
9099.05
1.10
0.98
0.97


Down
S123R
1505
5290.53
9248.50
0.57
0.52
0.99


Down
S123W
1514
2039.75
5970.03
0.34
0.20
0.64


Down
S123T
1509
5042.33
9146.80
0.55
0.49
0.98


Neutral
S123P
1519
884.66
799.56
1.11
0.09
0.09


Neutral
S123G
1511
10847.89
9512.32
1.14
1.06
1.02


Neutral
S123Q
1508
10841.56
9551.30
1.14
1.06
1.02


Down
S123V
1516
3220.29
4504.25
0.71
0.41
0.60


Neutral
N124G
1530
5601.70
6396.41
0.88
1.35
1.27


Neutral
N124C
1525
2241.39
2691.13
0.83
0.54
0.53


Neutral
N124V
1535
2966.25
3399.34
0.87
0.72
0.68


Neutral
N124L
1536
2342.72
2849.98
0.82
0.57
0.57


Neutral
N124T
1527
3872.37
4747.39
0.82
0.94
0.94


Neutral
N124R
1524
3795.95
4479.74
0.85
0.92
0.89


Neutral
N124M
1532
2818.81
3511.81
0.80
0.68
0.70


Neutral
N124S
1529
4245.94
5151.63
0.82
1.03
1.02


Down
N124P
1538
3825.40
5084.71
0.75
0.92
1.01


Neutral
N124A
1537
4174.53
4857.21
0.86
1.01
0.96


Neutral
N124K
1523
5006.93
5514.55
0.91
1.21
1.10


Neutral
N124F
1531
3681.53
4406.27
0.84
0.89
0.88


Neutral
N124W
1533
1506.21
1714.90
0.88
0.36
0.34


Neutral
N124I
1534
1663.57
1830.11
0.91
0.40
0.36


Neutral
N124D
1520
6218.73
6620.92
0.94
0.92
0.88


Neutral
V125G
1550
532.18
540.26
0.99
0.09
0.07


Down
V125Q
1546
1480.08
1883.56
0.79
0.26
0.25


Down
V125S
1549
2153.87
2966.73
0.73
0.38
0.39


Down
V125P
1557
1410.46
1873.09
0.75
0.25
0.24


Neutral
V125M
1552
1056.84
1118.42
0.94
0.19
0.15


Down
V125Y
1548
1484.83
2214.89
0.67
0.26
0.29


Down
V125T
1547
1444.16
1850.94
0.78
0.25
0.24


Down
V125A
1556
3246.01
5558.20
0.58
0.57
0.73


Up
V125C
1544
892.38
690.11
1.29
0.16
0.09


Neutral
V125D
1539
727.50
723.91
1.00
0.13
0.09


Neutral
V125W
1553
1537.82
1638.09
0.94
0.27
0.21


Neutral
V125R
1543
1087.69
1057.82
1.03
0.19
0.14


Neutral
V125E
1540
1324.10
1545.82
0.86
0.23
0.20


Down
V125F
1551
1360.07
2068.15
0.66
0.24
0.27


Neutral
V125H
1541
2227.75
2720.50
0.82
0.39
0.36


Neutral
T126K
1561
646.69
546.31
1.18
0.16
0.11


Down
T126V
1573
3034.58
4559.28
0.67
0.73
0.91


Neutral
T126G
1568
970.67
820.16
1.18
0.23
0.16


Neutral
T126R
1562
692.68
612.62
1.13
0.17
0.12


Neutral
T126L
1574
1084.98
970.83
1.12
0.26
0.19


Neutral
T126H
1560
648.90
592.08
1.10
0.16
0.12


Neutral
T126M
1570
1168.66
1078.26
1.08
0.28
0.21


Neutral
T126P
1576
684.23
614.07
1.11
0.17
0.12


Neutral
T126A
1575
2433.37
2923.43
0.83
0.59
0.58


Neutral
T126N
1564
1449.19
1384.47
1.05
0.35
0.28


Up
T126E
1559
697.86
580.78
1.20
0.17
0.12


Neutral
T126F
1569
642.61
550.97
1.17
0.16
0.11


Neutral
T126W
1571
632.89
564.16
1.12
0.15
0.11


Neutral
T126Q
1565
664.00
591.91
1.12
0.16
0.12


Neutral
T126S
1567
7114.42
6856.69
1.04
1.06
0.91


Neutral
P127C
1582
1713.51
1846.56
0.93
0.41
0.37


Neutral
P127F
1589
1444.31
1603.37
0.90
0.35
0.32


Neutral
P127T
1585
2193.26
2519.16
0.87
0.53
0.50


Down
P127E
1578
2480.57
3177.56
0.78
0.60
0.63


Neutral
P127W
1591
1399.71
1476.35
0.95
0.34
0.29


Neutral
P127A
1595
1751.82
1662.47
1.05
0.42
0.33


Neutral
P127S
1587
2842.19
3070.41
0.93
0.69
0.61


Up
P127H
1579
2151.26
1693.77
1.27
0.52
0.34


Neutral
P127Q
1584
1729.40
1882.54
0.92
0.42
0.37


Neutral
P127K
1580
729.23
657.19
1.11
0.18
0.13


Neutral
P127R
1581
1590.44
1491.10
1.07
0.38
0.30


Neutral
P127I
1592
1432.03
1464.78
0.98
0.35
0.29


Neutral
P127V
1593
1214.79
1401.27
0.87
0.29
0.28


Neutral
P127L
1594
1536.18
1604.60
0.96
0.37
0.32


Neutral
P127M
1590
2950.98
3052.79
0.97
0.71
0.61


Neutral
L128F
1608
1165.63
1269.01
0.92
0.28
0.25


Neutral
L128M
1609
1898.38
2135.63
0.89
0.46
0.42


Neutral
L128T
1604
756.63
698.21
1.08
0.18
0.14


Neutral
L128R
1600
919.42
960.28
0.96
0.22
0.19


Neutral
L128S
1606
764.28
672.98
1.14
0.18
0.13


Neutral
L128G
1607
738.26
694.65
1.06
0.18
0.14


Neutral
L128I
1611
1482.67
1715.03
0.86
0.36
0.34


Neutral
L128Q
1603
1042.55
936.43
1.11
0.25
0.19


Neutral
L128P
1614
792.57
760.45
1.04
0.19
0.15


Neutral
L128A
1613
769.15
712.50
1.08
0.19
0.14


Neutral
L128D
1596
682.02
642.58
1.06
0.16
0.13


Down
L128V
1612
1285.36
1696.88
0.76
0.31
0.34


Neutral
L128W
1610
776.89
664.61
1.17
0.19
0.13


Neutral
L128C
1601
856.43
770.10
1.11
0.21
0.15


Neutral
L128K
1599
858.27
846.02
1.01
0.21
0.17


Neutral
T129G
1625
4435.98
5356.41
0.83
1.07
1.06


Down
T129A
1632
2021.18
2774.35
0.73
0.49
0.55


Neutral
T129C
1620
1057.55
1033.96
1.02
0.26
0.21


Neutral
T129K
1618
3686.95
4446.15
0.83
0.89
0.88


Down
T129F
1626
2980.80
3803.12
0.78
0.72
0.76


Neutral
T129Y
1623
2527.88
2885.14
0.88
0.61
0.57


Neutral
T129S
1624
1649.34
1529.13
1.08
0.40
0.30


Neutral
T129R
1619
3334.95
3827.40
0.87
0.81
0.76


Neutral
T129V
1630
4967.86
5698.42
0.87
1.20
1.13


Neutral
T129L
1631
1649.57
1692.51
0.97
0.40
0.34


Down
T129H
1617
3019.81
3803.29
0.79
0.73
0.76


Neutral
T129P
1633
647.52
619.28
1.05
0.16
0.12


Neutral
T129E
1616
3205.16
3919.94
0.82
0.77
0.78


Neutral
T129I
1629
3967.14
4452.39
0.89
0.96
0.88


Down
T129M
1627
4118.98
5214.21
0.79
1.00
1.04


Neutral
F130L
1650
1452.17
1651.58
0.88
0.25
0.23


Neutral
F130P
1652
703.36
797.39
0.88
0.12
0.11


Neutral
F130C
1639
803.88
939.50
0.86
0.14
0.13


Neutral
F130R
1638
613.42
687.58
0.89
0.10
0.09


Down
F130Y
1643
2355.45
3604.50
0.65
0.40
0.49


Down
F130H
1636
1209.72
1960.40
0.62
0.21
0.27


Down
F130I
1648
4480.99
5648.72
0.79
0.76
0.77


Down
F130V
1649
3403.91
4744.33
0.72
0.58
0.65


Neutral
F130K
1637
581.97
670.87
0.87
0.10
0.09


Down
F130T
1642
1529.21
2157.46
0.71
0.26
0.30


Neutral
F130E
1635
571.41
648.15
0.88
0.10
0.09


Down
F130A
1651
1414.89
1990.16
0.71
0.24
0.27


Neutral
F130N
1640
616.91
710.45
0.87
0.10
0.10


Neutral
F130G
1645
1553.35
1726.90
0.90
0.26
0.24


Down
F130S
1644
793.40
1055.93
0.75
0.13
0.14


Down
T131F
1664
2738.64
4500.49
0.61
0.48
0.59


Neutral
T131P
1671
540.49
640.19
0.84
0.10
0.08


Down
T131A
1670
3622.28
6028.39
0.60
0.64
0.79


Down
T131S
1662
3644.14
5779.25
0.63
0.64
0.75


Down
T131G
1663
3345.71
5523.72
0.61
0.59
0.72


Down
1131I
1667
2987.26
4570.78
0.65
0.53
0.60


Down
T131L
1669
3081.92
4518.80
0.68
0.54
0.59


Down
T131H
1655
4201.01
5298.03
0.79
0.74
0.69


Down
T131Q
1660
6169.43
8400.64
0.73
1.08
1.10


Neutral
T131D
1653
8629.30
9616.48
0.90
1.52
1.26


Down
T131E
1654
4396.59
6846.70
0.64
0.77
0.89


Down
T131C
1658
2232.15
3514.32
0.64
0.39
0.46


Down
T131R
1657
4325.73
6209.92
0.70
0.76
0.81


Down
T131Y
1661
2684.82
3916.43
0.69
0.47
0.51


Down
T131M
1665
3101.25
4674.29
0.66
0.55
0.61


Down
K132G
1682
3779.04
5835.32
0.65
0.64
0.80


Down
K132V
1687
3181.94
4834.70
0.66
0.54
0.66


Down
K132L
1688
2407.98
3744.64
0.64
0.41
0.51


Down
K132A
1689
5397.96
7468.39
0.72
0.92
1.02


Down
K132P
1690
4062.71
5742.05
0.71
0.69
0.79


Down
K132F
1683
2012.87
2934.12
0.69
0.34
0.40


Neutral
K132R
1675
7317.48
8467.31
0.86
1.24
1.16


Down
K132I
1686
1811.13
2747.29
0.66
0.31
0.38


Down
K132H
1674
3291.99
4588.07
0.72
0.56
0.63


Neutral
K132S
1681
4947.26
4913.96
1.01
0.84
0.67


Down
K132M
1684
4521.82
6773.06
0.67
0.77
0.93


Down
K132D
1672
2079.75
3166.80
0.66
0.35
0.43


Down
K132T
1679
2515.58
4096.35
0.61
0.43
0.56


Down
K132Y
1680
2363.32
3794.19
0.62
0.40
0.52


Down
K132E
1673
3617.16
5597.32
0.65
0.61
0.77


Down
V133G
1702
3203.88
5198.66
0.62
0.54
0.71


Down
V133E
1692
3621.55
5211.22
0.69
0.62
0.71


Neutral
V133T
1699
7931.49
8920.49
0.89
1.35
1.22


Down
V133N
1697
4321.90
6145.40
0.70
0.73
0.84


Down
V133A
1708
4764.29
6847.44
0.70
0.81
0.94


Down
V133H
1693
3351.37
4739.59
0.71
0.57
0.65


Down
V133P
1709
1405.33
2047.83
0.69
0.24
0.28


Down
V133K
1694
5737.27
7514.38
0.76
0.97
1.03


Down
V133R
1695
5773.82
7252.24
0.80
0.98
0.99


Neutral
V133L
1707
7039.08
8445.43
0.83
1.20
1.16


Down
V133W
1705
2475.35
3564.76
0.69
0.42
0.49


Down
V133C
1696
1863.63
2666.24
0.70
0.32
0.37


Down
V133D
1691
1792.46
2630.40
0.68
0.30
0.36


Down
V133M
1704
4618.45
6302.34
0.73
0.78
0.86


Down
V133S
1701
3077.53
4401.10
0.70
0.52
0.60


Down
S134V
1725
4041.51
5701.67
0.71
0.69
0.78


Neutral
S134H
1712
6079.44
7343.68
0.83
1.03
1.01


Down
S134P
1728
4779.98
6326.78
0.76
0.81
0.87


Down
S134G
1720
5540.33
7442.86
0.74
0.94
1.02


Neutral
S134N
1716
6292.89
7595.95
0.83
1.07
1.04


Down
S134R
1714
5129.73
6824.16
0.75
0.87
0.94


Down
S134L
1726
6015.18
8101.31
0.74
1.02
1.11


Down
S134Q
1717
4325.14
6159.02
0.70
0.73
0.84


Neutral
S134E
1711
7105.19
8577.53
0.83
1.21
1.18


Down
S134Y
1719
5061.86
6645.94
0.76
0.86
0.91


Down
S134A
1727
5179.47
6920.72
0.75
0.88
0.95


Down
S134K
1713
5768.02
7876.85
0.73
0.98
1.08


Neutral
S134D
1710
6958.26
8316.41
0.84
1.18
1.14


Down
S134T
1718
5585.98
7301.80
0.77
0.95
1.00


Down
S134C
1715
1950.58
2625.09
0.74
0.33
0.36


Down
E135V
1744
4036.77
5545.23
0.73
0.69
0.76


Neutral
E135M
1741
8700.42
9297.63
0.94
1.48
1.27


Down
E135S
1738
3895.80
5128.41
0.76
0.66
0.70


Down
E135D
1729
4858.77
6640.34
0.73
0.83
0.91


Down
E135T
1736
4870.41
6518.41
0.75
0.83
0.89


Down
E135L
1745
3276.24
4342.02
0.75
0.56
0.59


Down
E135A
1746
5143.68
7429.20
0.69
0.87
1.02


Down
E135W
1742
3407.93
4761.19
0.72
0.58
0.65


Down
E135F
1740
3206.26
4561.41
0.70
0.54
0.63


Down
E135P
1747
1077.62
1567.43
0.69
0.18
0.21


Neutral
E135R
1732
815.91
921.41
0.89
0.14
0.13


Down
E135N
1734
4626.44
6661.57
0.69
0.79
0.91


Neutral
E135H
1730
6074.22
7339.12
0.83
1.03
1.01


Down
E135Q
1735
5656.70
7144.49
0.79
0.96
0.98


Down
E135I
1743
2140.04
5232.08
0.41
0.36
0.72


Down
G136V
1763
1813.26
2616.78
0.69
0.31
0.36


Down
G136W
1761
993.10
1243.43
0.80
0.17
0.17


Down
G136D
1748
3591.52
5274.69
0.68
0.61
0.72


Down
G136M
1760
3515.40
5367.37
0.65
0.60
0.74


Down
G136N
1754
3503.65
5155.57
0.68
0.60
0.71


Down
G136A
1765
3559.58
5813.34
0.61
0.60
0.80


Down
G136L
1764
2187.68
3866.01
0.57
0.37
0.53


Down
G136C
1753
905.15
1515.22
0.60
0.15
0.21


Down
G136P
1766
3234.60
4934.89
0.66
0.55
0.68


Down
G136T
1756
2555.79
3746.36
0.68
0.43
0.51


Down
G136R
1752
2716.62
4398.06
0.62
0.46
0.60


Down
G136S
1758
3375.11
4670.21
0.72
0.57
0.64


Down
G136I
1762
2006.39
3604.58
0.56
0.34
0.49


Down
G136H
1750
3564.72
4804.54
0.74
0.61
0.66


Down
G136E
1749
5583.49
7289.45
0.77
0.95
1.00


Down
Q137A
1784
3966.25
6312.83
0.63
0.75
0.85


Down
Q137R
1771
3671.71
6256.44
0.59
0.69
0.84


Down
Q137G
1777
4573.46
6739.55
0.68
0.86
0.91


Down
Q137K
1770
6317.49
8133.50
0.78
1.19
1.09


Down
Q137H
1769
5645.75
7063.96
0.80
1.06
0.95


Down
Q137P
1785
5676.99
7744.50
0.73
1.07
1.04


Down
Q137S
1776
5384.46
7395.46
0.73
1.01
1.00


Down
Q137L
1783
5870.03
8003.53
0.73
1.10
1.08


Down
Q137W
1780
3200.77
5519.86
0.58
0.60
0.74


Down
Q137F
1778
3505.51
5883.52
0.60
0.66
0.79


Down
Q137T
1774
6636.95
8394.25
0.79
1.25
1.13


Down
Q137C
1772
1924.03
2898.49
0.66
0.36
0.39


Down
Q137Y
1775
5307.87
7091.79
0.75
1.00
0.95


Down
Q137N
1773
5369.70
7661.25
0.70
1.01
1.03


Down
Q137E
1768
5683.74
7333.42
0.78
1.07
0.99


Down
A138V
1802
1926.65
3043.75
0.63
0.36
0.41


Neutral
A138L
1803
594.07
675.23
0.88
0.11
0.09


Down
A138P
1804
1481.25
2478.85
0.60
0.28
0.33


Down
A138C
1791
1603.91
2981.97
0.54
0.30
0.40


Down
A138T
1794
1740.56
2785.40
0.62
0.33
0.37


Down
A138S
1796
2042.67
2909.34
0.70
0.38
0.39


Down
A138R
1790
759.61
962.30
0.79
0.14
0.13


Down
A138G
1797
3108.95
4692.85
0.66
0.58
0.63


Down
A138E
1787
1450.57
2644.44
0.55
0.27
0.36


Down
A138H
1788
667.58
839.37
0.80
0.13
0.11


Neutral
A138M
1799
626.50
749.79
0.84
0.12
0.10


Down
A138Q
1793
1747.09
2754.75
0.63
0.33
0.37


Down
A138I
1801
1984.57
3124.09
0.64
0.37
0.42


Neutral
A138D
1786
601.71
673.39
0.89
0.11
0.09


Neutral
A138W
1800
595.15
681.04
0.87
0.11
0.09


Neutral
D139R
1808
639.90
725.74
0.88
0.12
0.10


Neutral
D139V
1820
733.94
879.96
0.83
0.14
0.12


Down
D139M
1817
820.44
1093.19
0.75
0.15
0.15


Neutral
D139C
1809
763.17
886.67
0.86
0.14
0.12


Down
D139P
1823
794.89
1048.41
0.76
0.15
0.14


Down
D139S
1814
1060.53
1350.12
0.79
0.20
0.18


Neutral
D139L
1821
802.45
923.25
0.87
0.15
0.12


Neutral
D139I
1819
759.45
884.92
0.86
0.14
0.12


Down
D139H
1806
1442.05
1944.83
0.74
0.27
0.26


Down
D139A
1822
899.50
1179.38
0.76
0.17
0.16


Neutral
D139G
1815
667.47
801.55
0.83
0.13
0.11


Neutral
D139F
1816
670.14
828.73
0.81
0.13
0.11


Down
D139N
1810
1743.46
2795.27
0.62
0.33
0.38


Neutral
D139W
1818
641.04
769.42
0.83
0.12
0.10


Neutral
D139Y
1813
643.83
701.07
0.92
0.12
0.09


Down
D139E
1805
4365.22
7664.89
0.57
0.48
1.01


Neutral
I140D
1824
447.31
470.26
0.95
0.08
0.06


Neutral
I140K
1827
470.80
510.28
0.92
0.08
0.07


Neutral
I140A
1841
521.91
583.80
0.89
0.09
0.08


Neutral
I140G
1835
514.54
519.88
0.99
0.09
0.07


Neutral
I140C
1829
552.10
550.46
1.00
0.10
0.07


Neutral
I140Y
1833
476.67
511.05
0.93
0.08
0.07


Down
I140V
1839
1483.60
2240.10
0.66
0.26
0.29


Neutral
I140W
1838
541.30
540.55
1.00
0.10
0.07


Neutral
I140F
1836
671.22
710.61
0.94
0.12
0.09


Neutral
I140H
1826
568.54
584.67
0.97
0.10
0.08


Down
I140L
1840
4551.18
6862.42
0.66
0.80
0.90


Neutral
I140R
1828
479.35
467.38
1.03
0.08
0.06


Neutral
I140E
1825
480.00
481.92
1.00
0.08
0.06


Down
I140M
1837
1888.65
2695.29
0.70
0.33
0.35


Neutral
I140T
1832
493.59
505.63
0.98
0.09
0.07


Down
M141E
1844
2661.78
3381.08
0.79
0.64
0.66


Neutral
M141I
1857
3206.64
3834.28
0.84
0.77
0.74


Neutral
M141R
1847
3645.85
4050.91
0.90
0.88
0.79


Neutral
M141T
1851
1916.97
2186.65
0.88
0.46
0.42


Neutral
M141P
1861
957.33
1027.91
0.93
0.23
0.20


Neutral
M141S
1853
2578.82
3190.11
0.81
0.62
0.62


Neutral
M141C
1848
1162.41
1355.08
0.86
0.28
0.26


Down
M141L
1859
3257.69
4218.46
0.77
0.79
0.82


Down
M141A
1860
2798.52
3561.95
0.79
0.68
0.69


Down
M141D
1843
1943.58
2586.12
0.75
0.47
0.50


Neutral
M141W
1856
3860.87
4822.22
0.80
0.93
0.94


Neutral
M141G
1854
1252.97
1525.59
0.82
0.30
0.30


Neutral
M141H
1845
2221.73
2624.41
0.85
0.54
0.51


Neutral
M141Y
1852
2117.90
2326.92
0.91
0.51
0.45


Neutral
M141N
1849
3446.02
4175.18
0.83
0.83
0.81


Down
I142L
1878
4079.65
6338.98
0.64
0.72
0.83


Down
I142M
1875
2514.92
3872.66
0.65
0.44
0.51


Neutral
I142G
1873
590.45
573.21
1.03
0.10
0.07


Neutral
I142K
1865
567.58
566.13
1.00
0.10
0.07


Down
I142A
1879
1102.77
1776.73
0.62
0.19
0.23


Neutral
I142N
1868
544.78
582.84
0.93
0.10
0.08


Neutral
I142W
1876
614.88
660.20
0.93
0.11
0.09


Neutral
I142P
1880
517.98
553.16
0.94
0.09
0.07


Neutral
I142Q
1869
561.05
579.03
0.97
0.10
0.08


Neutral
I142Y
1871
535.36
568.63
0.94
0.09
0.07


Down
I142V
1877
2412.99
3835.99
0.63
0.42
0.50


Neutral
I142T
1870
619.92
700.51
0.88
0.11
0.09


Neutral
I142R
1866
592.22
631.35
0.94
0.10
0.08


Neutral
I142S
1872
560.11
608.47
0.92
0.10
0.08


Down
I142F
1874
988.49
1616.93
0.61
0.17
0.21


Neutral
S143P
1899
681.44
714.91
0.95
0.13
0.10


Down
S143C
1886
1242.25
1638.96
0.76
0.23
0.22


Neutral
S143E
1882
679.14
698.14
0.97
0.13
0.09


Down
S143G
1891
2178.31
3221.72
0.68
0.41
0.43


Down
S143H
1883
1946.43
3055.74
0.64
0.37
0.41


Down
S143R
1885
5284.60
7026.38
0.75
0.99
0.95


Down
S143L
1897
1855.11
3143.09
0.59
0.35
0.42


Down
S143Q
1888
4008.07
5922.69
0.68
0.75
0.80


Down
S143N
1887
3447.12
4827.78
0.71
0.65
0.65


Neutral
S143W
1894
1164.49
1414.47
0.82
0.22
0.19


Down
S143A
1898
4862.16
6797.05
0.72
0.91
0.91


Down
S143T
1889
3510.83
4873.23
0.72
0.66
0.66


Down
S143Y
1890
2566.36
3755.42
0.68
0.48
0.51


Down
S143M
1893
3680.60
6112.93
0.60
0.69
0.82


Neutral
S143I
1895
6798.46
8447.06
0.80
1.28
1.14


Neutral
F144K
1903
683.92
723.08
0.95
0.13
0.10


Neutral
F144M
1912
727.58
785.61
0.93
0.14
0.11


Neutral
F144E
1901
684.55
697.38
0.98
0.13
0.09


Neutral
F144S
1910
711.27
779.76
0.91
0.13
0.10


Neutral
F144L
1916
668.14
725.88
0.92
0.13
0.10


Down
F144W
1913
3272.56
4375.61
0.75
0.62
0.59


Neutral
F144P
1918
658.07
729.61
0.90
0.12
0.10


Neutral
F144R
1904
633.36
704.49
0.90
0.12
0.09


Neutral
F144N
1906
648.28
686.56
0.94
0.12
0.09


Neutral
F144C
1905
656.21
698.34
0.94
0.12
0.09


Neutral
F144G
1911
641.29
662.20
0.97
0.12
0.09


Neutral
F144T
1908
704.60
804.24
0.88
0.13
0.11


Neutral
F144Q
1907
679.28
759.34
0.89
0.13
0.10


Neutral
F144H
1902
766.54
861.04
0.89
0.14
0.12


Neutral
F144V
1915
664.73
737.66
0.90
0.12
0.10


Down
V145A
1936
5042.62
7103.17
0.71
0.89
0.93


Down
V145T
1927
3518.22
5408.09
0.65
0.62
0.71


Down
V145L
1935
4048.83
6522.67
0.62
0.71
0.85


Down
V145P
1937
2148.04
3271.70
0.66
0.38
0.43


Down
V145K
1922
4566.52
6542.14
0.70
0.80
0.85


Down
V145N
1925
5756.42
8553.91
0.67
1.01
1.12


Down
V145D
1919
3249.52
5915.18
0.55
0.57
0.77


Down
V145H
1921
3868.79
6370.16
0.61
0.68
0.83


Down
V145R
1923
5093.69
7494.19
0.68
0.90
0.98


Down
V145Q
1926
4550.79
6385.09
0.71
0.80
0.83


Down
V145S
1929
5229.00
7486.54
0.70
0.92
0.98


Down
V145G
1930
2139.70
3072.06
0.70
0.38
0.40


Down
V145W
1933
1735.30
3046.73
0.57
0.31
0.40


Down
V145C
1924
1652.16
3231.89
0.51
0.29
0.42


Down
V145E
1920
4086.60
6893.09
0.59
0.72
0.90


Down
R146T
1945
4145.84
6737.53
0.62
0.78
0.91


Down
R146L
1954
2149.16
3444.38
0.62
0.40
0.46


Down
R146N
1943
4441.83
6346.03
0.70
0.83
0.85


Neutral
R146H
1940
2791.26
3298.03
0.85
0.52
0.44


Down
R146Q
1944
4232.35
6620.08
0.64
0.80
0.89


Down
R146K
1941
5360.91
7104.88
0.75
1.01
0.96


Neutral
R146C
1942
776.54
868.97
0.89
0.15
0.12


Neutral
R146S
1947
8627.00
9288.66
0.93
1.62
1.25


Down
R146D
1938
3803.07
5389.79
0.71
0.71
0.73


Down
R146A
1955
4939.79
6859.12
0.72
0.93
0.92


Down
R146Y
1946
3175.81
5447.89
0.58
0.60
0.73


Neutral
R146P
1956
3019.88
2923.15
1.03
0.57
0.39


Down
R146V
1953
3662.01
6193.78
0.59
0.69
0.83


Down
R146E
1939
2538.71
3832.21
0.66
0.48
0.52


Down
R146F
1949
1272.11
2074.69
0.61
0.24
0.28


Neutral
G147R
1961
7744.82
8412.88
0.92
1.45
1.25


Neutral
G147F
1968
7821.91
7899.45
0.99
1.47
1.18


Neutral
G147I
1971
1451.75
1461.30
0.99
0.27
0.22


Down
G147L
1973
1325.33
1787.45
0.74
0.25
0.27


Neutral
G147A
1974
2288.45
2655.58
0.86
0.43
0.40


Down
G147E
1958
1802.97
2340.36
0.77
0.34
0.35


Neutral
G147H
1959
6234.09
7432.44
0.84
1.17
1.11


Down
G147W
1970
5140.21
6807.71
0.76
0.96
1.01


Down
G147T
1965
5531.13
7240.25
0.76
1.04
1.08


Neutral
G147C
1962
6950.51
7385.23
0.94
1.30
1.10


Down
G147S
1967
3071.28
3887.91
0.79
0.58
0.58


Neutral
G147V
1972
4516.19
5576.27
0.81
0.85
0.83


Neutral
G147Q
1964
6879.67
7686.98
0.89
1.29
1.14


Neutral
G147M
1969
6059.54
7381.69
0.82
1.14
1.10


Up
G147P
1975
494.94
392.93
1.26
0.07
0.05


Neutral
D148R
1979
5874.44
7069.98
0.83
1.10
1.05


Down
D148I
1990
4934.66
6621.95
0.75
0.93
0.99


Neutral
D148T
1983
5534.68
6527.74
0.85
1.04
0.97


Neutral
D148G
1986
5545.91
6487.49
0.85
1.04
0.97


Neutral
D148L
1992
1738.87
2136.70
0.81
0.33
0.32


Neutral
D148V
1991
4521.62
5307.74
0.85
0.85
0.79


Neutral
D148A
1993
7276.18
7955.06
0.91
1.36
1.18


Down
D148W
1989
3622.09
4894.24
0.74
0.68
0.73


Neutral
D148P
1994
7311.56
7404.67
0.99
1.37
1.10


Neutral
D148S
1985
3190.82
3936.89
0.81
0.60
0.59


Down
D148K
1978
2414.59
3115.25
0.78
0.45
0.46


Down
D148E
1976
2457.68
3171.30
0.77
0.46
0.47


Neutral
D148M
1988
928.12
1156.79
0.80
0.17
0.17


Neutral
D148N
1981
5136.96
5810.99
0.88
0.96
0.87


Neutral
D148C
1980
2617.98
3259.70
0.80
0.49
0.49


Neutral
H149W
2008
578.88
610.12
0.95
0.10
0.08


Neutral
H149A
2012
574.51
606.50
0.95
0.10
0.08


Neutral
H149L
2011
562.23
585.57
0.96
0.10
0.08


Neutral
H149C
1999
532.13
536.84
0.99
0.09
0.07


Neutral
H149Q
2001
547.46
565.60
0.97
0.10
0.07


Neutral
H149T
2002
545.99
567.99
0.96
0.10
0.07


Neutral
H149Y
2003
553.52
575.93
0.96
0.10
0.08


Neutral
H149P
2013
502.45
522.17
0.96
0.09
0.07


Neutral
H149V
2010
515.00
521.68
0.99
0.09
0.07


Neutral
H149R
1998
481.87
534.48
0.90
0.08
0.07


Neutral
H149G
2005
492.47
525.75
0.94
0.09
0.07


Neutral
H149E
1996
476.14
472.99
1.01
0.08
0.06


Neutral
H149S
2004
481.76
508.54
0.95
0.08
0.07


Neutral
H149I
2009
510.38
533.47
0.96
0.09
0.07


Neutral
H149N
2000
542.00
555.29
0.98
0.10
0.07


Neutral
R150S
50
4221.17
4687.08
0.90
0.58
0.66


Neutral
R150E
42
9557.47
8282.03
1.15
1.31
1.17


Neutral
R150G
51
10002.15
8470.68
1.18
1.37
1.19


Neutral
R150M
53
8614.46
8306.99
1.04
1.18
1.17


Up
R150P
59
2291.14
828.28
2.77
0.31
0.12


Neutral
R150T
48
9808.17
8294.42
1.18
1.35
1.17


Neutral
R150W
54
8373.53
7574.51
1.11
1.15
1.07


Neutral
R150A
58
10175.13
8554.82
1.19
1.40
1.20


Neutral
R150N
46
10191.05
8571.32
1.19
1.40
1.21


Neutral
R150K
44
9471.29
8346.99
1.13
1.30
1.18


Neutral
R150L
57
9751.98
8444.63
1.15
1.34
1.19


Neutral
R150V
56
6869.28
6604.61
1.04
1.20
1.30


Neutral
R150D
41
7230.41
6033.28
1.20
1.26
1.19


Down
R150I
55
3120.05
4082.34
0.76
0.39
0.55


Neutral
R150H
43
8281.04
8056.17
1.03
1.05
1.08


Neutral
D151R
63
576.24
545.21
1.06
0.11
0.08


Neutral
D151F
71
626.76
601.08
1.04
0.12
0.09


Neutral
D151P
78
670.23
610.90
1.10
0.13
0.09


Neutral
D151W
73
691.38
656.86
1.05
0.13
0.10


Neutral
D151Q
66
634.58
619.91
1.02
0.12
0.09


Neutral
D151L
76
638.24
627.06
1.02
0.12
0.09


Neutral
D151S
69
612.74
579.48
1.06
0.11
0.09


Up
D151G
70
1073.32
733.89
1.46
0.20
0.11


Neutral
D151A
77
635.33
608.12
1.04
0.12
0.09


Neutral
D151N
65
631.72
612.41
1.03
0.12
0.09


Neutral
D151K
62
648.63
635.47
1.02
0.12
0.09


Neutral
D151Y
68
744.90
724.43
1.03
0.14
0.11


Neutral
D151V
75
586.23
585.19
1.00
0.11
0.09


Neutral
D151T
67
589.61
587.04
1.00
0.11
0.09


Up
D151M
72
2945.18
605.81
4.86
0.55
0.09


Neutral
N152G
2024
9852.70
8326.15
1.18
1.35
1.17


Neutral
N152C
2019
6322.44
7849.36
0.81
0.87
1.11


Neutral
N152F
2025
9762.67
8643.34
1.13
1.34
1.22


Neutral
N152L
2030
9176.14
8510.93
1.08
1.26
1.20


Neutral
N152P
2032
7767.98
7907.66
0.98
1.07
1.11


Neutral
N152R
2018
8348.81
7893.25
1.06
1.14
1.11


Down
N152H
2016
3318.01
4257.74
0.78
0.46
0.60


Neutral
N152T
2021
7155.46
7180.94
1.00
0.98
1.01


Neutral
N152Y
2022
8343.23
7992.79
1.04
1.14
1.13


Neutral
N152K
2017
7868.59
7956.82
0.99
1.08
1.12


Neutral
N152D
2014
10221.41
8616.74
1.19
1.40
1.21


Neutral
N152W
2027
5717.25
7086.82
0.81
0.78
1.00


Neutral
N152I
2028
10161.44
8648.89
1.17
1.39
1.22


Neutral
N152A
2031
6669.94
5660.16
1.18
1.17
1.12


Down
N152S
2023
4607.85
8096.31
0.57
0.58
1.08


Neutral
S153I
549
2873.12
2619.74
1.10
0.39
0.37


Neutral
S153R
539
4799.14
4905.35
0.98
0.66
0.69


Neutral
S153K
538
1002.00
1199.78
0.84
0.14
0.17


Down
S153C
540
1934.36
3181.56
0.61
0.27
0.45


Neutral
S153G
545
6175.12
6148.70
1.00
0.85
0.87


Neutral
S153H
537
9759.94
8837.02
1.10
1.34
1.24


Neutral
S153L
551
1285.63
1575.63
0.82
0.18
0.22


Neutral
S153V
550
8993.77
8047.48
1.12
1.23
1.13


Neutral
S153T
543
10530.07
8798.72
1.20
1.44
1.24


Neutral
S153P
553
9442.29
8513.31
1.11
1.29
1.20


Neutral
S153A
552
644.02
569.42
1.13
0.09
0.08


Neutral
S153F
546
10583.60
8979.56
1.18
1.45
1.26


Neutral
S153D
535
8477.40
8662.71
0.98
1.16
1.22


Neutral
S153Q
542
6654.12
7947.98
0.84
0.91
1.12


Neutral
S153Y
544
10164.62
8758.66
1.16
1.39
1.23


Neutral
P154V
2049
1257.75
1273.72
0.99
0.24
0.19


Up
P154W
2047
3838.51
2992.41
1.28
0.72
0.45


Neutral
P154L
2050
5826.55
6782.07
0.86
1.09
1.01


Neutral
P154C
2038
3097.69
3692.51
0.84
0.58
0.55


Neutral
P154S
2043
7417.09
8143.14
0.91
1.39
1.21


Up
P154K
2036
2407.68
1639.28
1.47
0.45
0.24


Neutral
P154I
2048
7298.30
7549.63
0.97
1.37
1.12


Down
P154A
2051
2043.76
2680.62
0.76
0.38
0.40


Neutral
P154T
2041
1763.73
2075.38
0.85
0.33
0.31


Neutral
P154H
2035
1072.51
1021.04
1.05
0.20
0.15


Neutral
P154Y
2042
946.74
834.91
1.13
0.18
0.12


Neutral
P154N
2039
1122.04
1229.36
0.91
0.21
0.18


Neutral
P154F
2045
845.38
757.86
1.12
0.16
0.11


Neutral
P154R
2037
1975.77
1915.36
1.03
0.37
0.29


Neutral
P154Q
2040
2228.56
2374.11
0.94
0.42
0.35


Neutral
F155S
89
894.19
833.57
1.07
0.17
0.12


Neutral
F155T
87
1137.71
1084.01
1.05
0.21
0.16


Neutral
F155G
90
807.68
718.82
1.12
0.15
0.11


Neutral
F155N
85
715.78
688.72
1.04
0.13
0.10


Neutral
F155R
83
702.49
695.27
1.01
0.13
0.10


Neutral
F155W
92
715.40
693.53
1.03
0.13
0.10


Up
F155L
95
1322.13
864.19
1.53
0.25
0.13


Neutral
F155Q
86
731.28
738.56
0.99
0.14
0.11


Neutral
F155M
91
8252.43
8163.55
1.01
1.55
1.22


Neutral
F155E
80
685.90
683.12
1.00
0.13
0.10


Up
F155A
96
1250.93
760.12
1.65
0.23
0.11


Neutral
F155P
97
666.89
658.85
1.01
0.13
0.10


Neutral
F155V
94
681.25
679.13
1.00
0.13
0.10


Neutral
F155H
81
696.34
683.06
1.02
0.13
0.10


Neutral
F155Y
88
676.73
629.34
1.08
0.13
0.09


Up
D156H
99
2722.09
2081.55
1.31
0.51
0.31


Up
D156L
114
2548.30
1597.53
1.60
0.48
0.24


Neutral
D156E
98
6300.50
6871.25
0.92
1.18
1.02


Up
D156A
115
2679.29
1734.45
1.54
0.50
0.26


Up
D156W
111
1575.39
1268.36
1.24
0.30
0.19


Neutral
D156C
102
2842.85
2704.37
1.05
0.53
0.40


Neutral
D156P
116
1002.13
998.80
1.00
0.19
0.15


Up
D156V
113
1400.88
766.80
1.83
0.26
0.11


Up
D156K
100
1292.89
966.62
1.34
0.24
0.14


Neutral
D156S
107
969.70
837.57
1.16
0.18
0.12


Neutral
D156G
108
794.14
709.60
1.12
0.15
0.11


Up
D156T
105
2871.09
1843.03
1.56
0.54
0.27


Neutral
D156Y
106
3406.50
3113.95
1.09
0.64
0.46


Up
D156R
101
2431.23
1545.89
1.57
0.46
0.23


Up
D156M
110
817.96
502.82
1.63
0.12
0.07


Up
G157K
2055
677.09
562.66
1.20
0.09
0.08


Neutral
G157D
2052
603.28
513.64
1.17
0.08
0.07


Neutral
G157F
2064
9535.19
8450.24
1.13
1.31
1.19


Up
G157R
2056
704.56
540.98
1.30
0.10
0.08


Neutral
G157H
2054
608.42
567.38
1.07
0.08
0.08


Up
G157L
2069
582.39
476.09
1.22
0.08
0.07


Up
G157N
2059
721.55
534.46
1.35
0.10
0.08


Up
G157Y
2062
654.13
541.41
1.21
0.09
0.08


Up
G157S
2063
924.62
596.70
1.55
0.13
0.08


Up
G157T
2061
669.55
551.99
1.21
0.09
0.08


Up
G157A
2070
861.29
552.54
1.56
0.12
0.08


Up
G157Q
2060
655.49
522.72
1.25
0.09
0.07


Neutral
G157P
2071
635.63
591.49
1.07
0.09
0.08


Neutral
G157V
2068
654.45
573.19
1.14
0.09
0.08


Neutral
G157M
2065
716.52
615.65
1.16
0.10
0.09


Neutral
P158S
2082
7974.07
7118.62
1.12
1.09
1.00


Neutral
P158Y
2081
7544.63
6885.81
1.10
1.03
0.97


Neutral
P158R
2076
7142.54
6214.55
1.15
0.98
0.88


Up
P158L
2089
9290.77
6775.04
1.37
1.27
0.95


Neutral
P158V
2088
10642.99
8919.30
1.19
1.46
1.26


Up
P158C
2077
6284.97
4792.49
1.31
0.86
0.67


Neutral
P158A
2090
9579.29
8514.00
1.13
1.31
1.20


Up
P158W
2086
5175.38
3078.22
1.68
0.71
0.43


Neutral
P158I
2087
10312.96
8597.26
1.20
1.41
1.21


Up
P158F
2084
6595.54
4090.71
1.61
0.90
0.58


Up
P158Q
2079
10928.51
8709.20
1.25
1.50
1.23


Neutral
P158T
2080
4204.23
3507.76
1.20
0.53
0.47


Neutral
P158G
2083
6277.86
5496.27
1.14
0.79
0.73


Neutral
P158K
2075
6860.82
6680.30
1.03
0.87
0.89


Neutral
P158N
2078
3656.04
3874.48
0.94
0.46
0.52


Up
P158D
2072
8959.02
7355.10
1.22
0.98
0.96


Neutral
G159R
121
6441.49
5914.02
1.09
0.88
0.83


Neutral
G159S
127
6594.46
6573.14
1.00
0.90
0.93


Neutral
G159Q
124
3996.96
4391.10
0.91
0.55
0.62


Neutral
G159P
135
596.30
564.24
1.06
0.08
0.08


Up
G159V
132
2453.98
732.46
3.35
0.34
0.10


Neutral
G159K
120
554.74
515.44
1.08
0.08
0.07


Neutral
G159A
134
5157.14
4685.20
1.10
0.71
0.66


Up
G159Y
126
1029.19
752.76
1.37
0.14
0.11


Neutral
G159E
118
4327.74
4027.23
1.07
0.59
0.57


Up
G159T
125
5059.91
1734.12
2.92
0.69
0.24


Up
G159M
129
5905.06
4874.00
1.21
0.75
0.65


Neutral
G159I
131
5725.99
5357.20
1.07
0.72
0.72


Neutral
G159W
130
6787.40
6287.71
1.08
0.86
0.84


Neutral
G159L
133
8231.62
7638.64
1.08
1.04
1.02


Neutral
G159C
122
2897.77
3053.86
0.95
0.37
0.41


Down
G160A
2108
2080.01
2823.12
0.74
0.60
0.58


Down
G160H
2093
2001.10
3085.22
0.65
0.57
0.63


Down
G160N
2097
3546.69
5339.11
0.66
1.02
1.09


Neutral
G160W
2104
4334.72
3946.12
1.10
1.24
0.80


Down
G160R
2095
2347.11
3791.36
0.62
0.67
0.77


Neutral
G160P
2109
1047.77
929.25
1.13
0.30
0.19


Neutral
G160I
2105
1794.48
1596.11
1.12
0.51
0.33


Down
G160M
2103
2506.95
3576.91
0.70
0.72
0.73


Neutral
G160C
2096
580.68
627.99
0.92
0.17
0.13


Down
G160Q
2098
4740.98
6839.68
0.69
1.36
1.39


Neutral
G160V
2106
3284.36
3030.37
1.08
0.94
0.62


Down
G160S
2101
2991.02
4281.08
0.70
0.86
0.87


Neutral
G160E
2092
3899.28
4071.63
0.96
1.12
0.83


Down
G160L
2107
3396.11
4411.61
0.77
0.97
0.90


Neutral
G160T
2099
3844.32
3943.21
0.97
1.10
0.80


Down
N161S
2119
1251.34
2417.50
0.52
0.36
0.49


Down
N161C
2115
1591.15
2710.58
0.59
0.46
0.55


Down
N161L
2126
4840.46
7275.68
0.67
1.39
1.48


Neutral
N161R
2114
6437.08
7915.53
0.81
1.85
1.61


Down
N161G
2120
3755.93
6728.88
0.56
1.08
1.37


Down
N161W
2123
3135.58
3994.07
0.79
0.90
0.81


Down
N161Y
2118
5507.52
7319.79
0.75
1.58
1.49


Down
N161E
2111
5410.44
8192.46
0.66
1.55
1.67


Down
N161P
2128
1231.02
1593.36
0.77
0.35
0.32


Neutral
N161T
2117
7464.29
9082.12
0.82
2.14
1.85


Down
N161H
2112
2727.71
5034.74
0.54
0.78
1.03


Neutral
N161I
2124
8070.21
9914.56
0.81
2.31
2.02


Down
N161V
2125
5238.40
7429.22
0.71
1.50
1.51


Down
N161F
2121
3890.45
6624.80
0.59
1.12
1.35


Down
N161Q
2116
4690.72
7412.73
0.63
1.35
1.51


Neutral
L162A
2146
584.17
505.94
1.15
0.17
0.10


Neutral
L162G
2140
582.48
602.57
0.97
0.17
0.12


Neutral
L162C
2134
475.91
466.85
1.02
0.14
0.10


Neutral
L162P
2147
514.26
519.71
0.99
0.15
0.11


Neutral
L162R
2133
492.19
498.99
0.99
0.14
0.10


Down
L162I
2144
2948.11
4018.68
0.73
0.85
0.82


Neutral
L162S
2139
473.63
459.28
1.03
0.14
0.09


Neutral
L162D
2129
512.72
487.18
1.05
0.15
0.10


Neutral
L162M
2142
1013.31
1138.86
0.89
0.29
0.23


Neutral
L162E
2130
563.63
631.85
0.89
0.16
0.13


Neutral
L162T
2137
473.46
477.00
0.99
0.14
0.10


Neutral
L162Y
2138
484.26
519.58
0.93
0.14
0.11


Neutral
L162F
2141
484.37
469.30
1.03
0.14
0.10


Neutral
L162W
2143
463.12
457.12
1.01
0.13
0.09


Neutral
L162Q
2136
480.75
481.03
1.00
0.14
0.10


Neutral
A163R
2152
562.68
563.06
1.00
0.16
0.11


Neutral
A163G
2159
819.22
999.88
0.82
0.23
0.20


Neutral
A163Y
2157
562.12
549.69
1.02
0.16
0.11


Neutral
A163P
2166
557.10
559.72
1.00
0.16
0.11


Neutral
A163S
2158
572.70
542.75
1.06
0.16
0.11


Neutral
A163L
2165
532.98
539.88
0.99
0.15
0.11


Neutral
A163C
2153
528.01
546.64
0.97
0.15
0.11


Neutral
A163K
2151
510.99
502.63
1.02
0.15
0.10


Neutral
A163V
2164
567.33
572.66
0.99
0.16
0.12


Down
A163F
2160
931.85
1182.48
0.79
0.27
0.24


Neutral
A163E
2149
560.80
539.21
1.04
0.16
0.11


Neutral
A163T
2156
538.98
537.66
1.00
0.15
0.11


Neutral
A163Q
2155
586.94
586.24
1.00
0.17
0.12


Neutral
A163I
2163
554.29
579.47
0.96
0.16
0.12


Neutral
A163N
2154
575.87
580.49
0.99
0.17
0.12


Neutral
H164L
2183
547.21
565.25
0.97
0.16
0.12


Neutral
H164M
2179
552.91
590.51
0.94
0.16
0.12


Neutral
H164K
2169
575.53
589.33
0.98
0.17
0.12


Neutral
H164P
2185
573.34
570.59
1.00
0.16
0.12


Neutral
H164C
2171
551.45
576.69
0.96
0.16
0.12


Neutral
H164R
2170
558.91
553.87
1.01
0.16
0.11


Neutral
H164A
2184
549.93
598.96
0.92
0.16
0.12


Neutral
H164V
2182
567.08
579.35
0.98
0.16
0.12


Down
H164S
2176
4849.81
6939.34
0.70
1.39
1.41


Down
H164N
2172
437.45
585.42
0.75
0.13
0.12


Neutral
H164G
2177
545.54
547.89
1.00
0.16
0.11


Neutral
H164F
2178
540.67
537.69
1.01
0.16
0.11


Neutral
H164Y
2175
558.66
548.15
1.02
0.16
0.11


Neutral
H164Q
2173
566.62
555.39
1.02
0.16
0.11


Neutral
H164E
2168
569.92
612.16
0.93
0.16
0.12


Neutral
A165W
2200
583.56
591.99
0.99
0.17
0.12


Neutral
A165V
2202
560.38
564.19
0.99
0.16
0.11


Down
A165G
2197
445.09
575.94
0.77
0.13
0.12


Neutral
A165K
2189
537.18
537.57
1.00
0.15
0.11


Neutral
A165L
2203
552.58
553.45
1.00
0.16
0.11


Neutral
A165P
2204
535.50
554.41
0.97
0.15
0.11


Down
A165Q
2193
983.84
1344.06
0.73
0.28
0.27


Neutral
A165D
2186
534.17
577.13
0.93
0.15
0.12


Neutral
A165H
2188
515.90
536.85
0.96
0.15
0.11


Neutral
A165F
2198
493.42
496.39
0.99
0.14
0.10


Down
A165S
2196
390.18
578.00
0.68
0.11
0.12


Neutral
A165T
2194
506.15
502.78
1.01
0.15
0.10


Neutral
A165R
2190
485.08
477.19
1.02
0.14
0.10


Neutral
A165N
2192
509.08
499.01
1.02
0.15
0.10


Neutral
A165M
2199
473.24
523.60
0.90
0.14
0.11


Neutral
F166G
2216
623.89
586.56
1.06
0.11
0.08


Neutral
F166S
2215
724.53
695.67
1.04
0.12
0.10


Neutral
F166L
2221
760.25
829.02
0.92
0.13
0.12


Neutral
F166V
2220
552.68
564.25
0.98
0.09
0.08


Neutral
F166P
2223
530.80
562.94
0.94
0.09
0.08


Neutral
F166N
2211
613.07
589.89
1.04
0.10
0.08


Neutral
F166R
2209
534.62
543.15
0.98
0.09
0.08


Neutral
F166A
2222
638.77
712.81
0.90
0.11
0.10


Neutral
F166K
2208
598.42
615.59
0.97
0.10
0.09


Neutral
F166H
2207
2770.43
2606.89
1.06
0.47
0.37


Neutral
F166W
2218
8234.80
8549.89
0.96
1.40
1.20


Neutral
F166I
2219
617.86
613.36
1.01
0.10
0.09


Neutral
F166M
2217
537.05
571.21
0.94
0.09
0.08


Neutral
F166C
2210
661.10
639.33
1.03
0.11
0.09


Neutral
F166E
2206
616.99
582.48
1.06
0.10
0.08


Neutral
Q167D
2224
4883.56
4579.11
1.07
0.83
0.64


Neutral
Q167R
2228
7660.88
8025.72
0.95
1.30
1.12


Neutral
Q167A
2241
8466.37
8182.70
1.03
1.44
1.15


Neutral
Q167S
2233
7915.08
8512.14
0.93
1.34
1.19


Neutral
Q167F
2235
8209.07
8535.93
0.96
1.39
1.20


Down
Q167Y
2232
5687.17
7642.43
0.74
0.96
1.07


Neutral
Q167P
2242
7513.70
8011.11
0.94
1.27
1.12


Neutral
Q167T
2231
7772.59
8173.09
0.95
1.32
1.15


Neutral
Q167V
2239
7867.97
8191.44
0.96
1.33
1.15


Neutral
Q167L
2240
7174.37
7937.04
0.90
1.22
1.11


Neutral
Q167M
2236
8005.59
8974.74
0.89
1.36
1.26


Down
Q167N
2230
3612.25
5004.93
0.72
0.61
0.70


Neutral
Q167G
2234
6671.99
7782.93
0.86
1.13
1.09


Neutral
Q167K
2227
6453.33
7646.48
0.84
1.09
1.07


Down
Q167E
2225
5009.36
6388.75
0.78
0.85
0.90


Neutral
P168N
2249
638.46
590.71
1.08
0.11
0.08


Neutral
P168F
2255
673.09
638.53
1.05
0.11
0.09


Neutral
P168R
2247
7038.51
7902.50
0.89
1.19
1.11


Neutral
P168W
2257
737.53
666.57
1.11
0.13
0.09


Neutral
P168A
2261
833.88
736.90
1.13
0.14
0.10


Neutral
P168T
2251
646.42
645.28
1.00
0.11
0.09


Neutral
P168V
2259
499.02
557.37
0.90
0.08
0.08


Neutral
P168G
2254
686.51
644.44
1.07
0.12
0.09


Neutral
P168C
2248
568.42
598.90
0.95
0.10
0.08


Neutral
P168M
2256
734.84
652.57
1.13
0.12
0.09


Neutral
P168H
2245
590.54
588.07
1.00
0.10
0.08


Neutral
P168L
2260
715.20
706.06
1.01
0.12
0.10


Neutral
P168S
2253
641.79
605.44
1.06
0.11
0.08


Neutral
P168I
2258
560.15
568.90
0.98
0.09
0.08


Neutral
P168D
2243
530.69
575.63
0.92
0.09
0.08


Neutral
G169H
2264
791.08
828.63
0.95
0.13
0.12


Down
G169A
2279
1556.29
2632.37
0.59
0.26
0.37


Neutral
G169E
2263
789.82
829.24
0.95
0.13
0.12


Neutral
G169C
2267
714.55
744.33
0.96
0.12
0.10


Neutral
G169S
2272
1196.93
1427.18
0.84
0.20
0.20


Neutral
G169L
2278
450.44
534.57
0.84
0.08
0.07


Neutral
G169V
2277
703.56
675.20
1.04
0.12
0.09


Neutral
G169T
2270
676.59
685.16
0.99
0.11
0.10


Neutral
G169R
2266
1119.16
1166.00
0.96
0.19
0.16


Neutral
G169W
2275
802.02
921.44
0.87
0.14
0.13


Neutral
G169M
2274
962.20
1133.87
0.85
0.16
0.16


Neutral
G169I
2276
671.79
677.10
0.99
0.11
0.09


Neutral
G169P
2280
671.60
683.22
0.98
0.11
0.10


Neutral
G169D
2262
714.59
766.96
0.93
0.12
0.11


Neutral
G169Q
2269
977.05
901.01
1.08
0.17
0.13


Down
P170L
2298
5969.84
7995.99
0.75
1.01
1.12


Down
P170R
2285
3566.07
5876.72
0.61
0.60
0.82


Down
P170I
2296
5073.27
7150.78
0.71
0.86
1.00


Neutral
P170T
2289
6734.46
8153.81
0.83
1.14
1.14


Down
P170F
2293
2114.36
3365.04
0.63
0.36
0.47


Down
P170Q
2288
4204.94
6162.63
0.68
0.71
0.86


Down
P170G
2292
5005.05
6924.03
0.72
0.85
0.97


Down
P170S
2291
4526.99
6064.79
0.75
0.77
0.85


Down
P170H
2283
4569.14
6879.10
0.66
0.77
0.96


Down
P170C
2286
931.84
2355.40
0.40
0.16
0.33


Down
P170M
2294
3323.56
6318.16
0.53
0.56
0.89


Down
P170K
2284
4379.75
6206.45
0.71
0.74
0.87


Down
P170W
2295
1794.33
2781.05
0.65
0.30
0.39


Neutral
P170D
2281
1434.38
1462.91
0.98
0.25
0.29


Neutral
P170A
2299
2733.72
2793.24
0.98
0.48
0.55


Down
G171S
564
2129.39
3316.87
0.64
0.36
0.46


Down
G171M
566
2104.33
3308.36
0.64
0.36
0.46


Down
G171N
560
4674.81
6965.17
0.67
0.79
0.98


Up
G171P
572
1570.74
1204.39
1.30
0.27
0.17


Down
G171R
558
1604.06
2486.74
0.65
0.27
0.35


Down
G171Y
563
1519.56
2342.05
0.65
0.26
0.33


Down
G171A
571
3517.89
5269.99
0.67
0.60
0.74


Down
G171Q
561
2361.29
3915.33
0.60
0.40
0.55


Down
G171H
556
1662.65
2616.63
0.64
0.28
0.37


Down
G171L
570
1551.95
2516.17
0.62
0.26
0.35


Down
G171W
567
1068.10
1663.46
0.64
0.18
0.23


Down
G171C
559
1982.45
3409.68
0.58
0.34
0.48


Down
G171K
557
1324.98
1867.15
0.71
0.22
0.26


Neutral
G171E
555
1154.96
1199.65
0.96
0.20
0.24


Neutral
G171D
554
791.81
690.33
1.15
0.14
0.14


Neutral
I172Y
2309
7427.25
7893.50
0.94
0.93
0.95


Neutral
I172T
2308
5861.67
6776.91
0.86
0.73
0.81


Neutral
I172P
2318
6297.28
7073.49
0.89
0.79
0.85


Down
I172A
2317
4666.76
6048.64
0.77
0.58
0.73


Neutral
I172L
2316
9324.48
8876.38
1.05
1.17
1.07


Neutral
I172Q
2307
6906.03
7743.66
0.89
0.87
0.93


Down
I172E
2301
3517.33
4567.20
0.77
0.44
0.55


Down
I172C
2305
1784.43
2422.99
0.74
0.22
0.29


Neutral
I172M
2313
9859.60
9096.03
1.08
1.24
1.09


Neutral
I172D
2300
4276.25
4281.60
1.00
0.54
0.51


Neutral
I172V
2315
9541.02
9174.91
1.04
1.20
1.10


Neutral
I172R
2304
7010.86
7581.48
0.92
0.88
0.91


Neutral
I172G
2311
2169.59
2350.53
0.92
0.27
0.28


Neutral
I172W
2314
5870.25
7244.46
0.81
0.74
0.87


Neutral
I172N
2306
5235.67
6253.19
0.84
0.66
0.75


Down
G173C
2324
816.12
1115.07
0.73
0.10
0.13


Neutral
G173L
2325
454.21
401.73
1.13
0.06
0.05


Neutral
G173K
2322
741.76
685.79
1.08
0.09
0.08


Neutral
G173W
2332
1278.44
1132.78
1.13
0.16
0.14


Neutral
G173S
2329
865.76
793.10
1.09
0.11
0.10


Neutral
G173A
2326
1041.22
1038.66
1.00
0.13
0.12


Neutral
G173R
2323
973.70
877.71
1.11
0.12
0.11


Neutral
G173N
2325
818.38
931.59
0.88
0.10
0.11


Neutral
G173T
2327
481.41
485.39
0.99
0.06
0.06


Neutral
G173D
2319
474.45
424.47
1.12
0.06
0.05


Neutral
G173V
2324
505.39
476.04
1.06
0.06
0.06


Neutral
G173F
2330
670.10
610.91
1.10
0.08
0.07


Neutral
G173M
2331
1085.74
1324.87
0.82
0.14
0.16


Neutral
G173Y
2328
1390.14
1296.93
1.07
0.17
0.16


Neutral
G173P
2327
458.79
435.59
1.05
0.06
0.05


Neutral
G174R
2342
452.76
437.41
1.04
0.06
0.05


Neutral
G174A
2355
491.35
460.84
1.07
0.06
0.06


Neutral
G174E
2339
489.49
459.57
1.07
0.06
0.06


Neutral
G174F
2349
598.90
520.03
1.15
0.08
0.06


Neutral
G174H
2340
577.19
518.90
1.11
0.07
0.06


Neutral
G174T
2346
505.38
483.64
1.04
0.06
0.06


Neutral
G174D
2338
476.79
454.13
1.05
0.06
0.05


Neutral
G174S
2348
641.62
580.36
1.11
0.08
0.07


Neutral
G174P
2356
525.07
505.41
1.04
0.07
0.06


Neutral
G174W
2351
538.38
502.50
1.07
0.07
0.06


Neutral
G174V
2353
504.50
425.17
1.19
0.06
0.05


Neutral
G174N
2344
506.54
460.95
1.10
0.06
0.06


Up
G174Y
2347
722.10
525.97
1.37
0.09
0.06


Neutral
G174M
2350
516.58
464.32
1.11
0.06
0.06


Neutral
G174L
2354
474.83
436.08
1.09
0.06
0.05


Neutral
D175I
2371
697.29
708.65
0.98
0.09
0.09


Neutral
D175T
2364
601.82
560.11
1.07
0.08
0.07


Neutral
D175N
2362
1495.98
1413.58
1.06
0.19
0.17


Neutral
D175V
2372
694.28
652.82
1.06
0.09
0.08


Neutral
D175S
2366
664.04
579.78
1.15
0.08
0.07


Neutral
D175R
2360
593.04
505.98
1.17
0.07
0.06


Down
D175G
2367
3147.62
4207.02
0.75
0.39
0.51


Up
D175A
2374
633.65
519.61
1.22
0.08
0.06


Neutral
D175F
2368
768.41
804.63
0.95
0.10
0.10


Neutral
D175C
2361
535.75
498.44
1.07
0.07
0.06


Neutral
D175Q
2363
702.74
633.64
1.11
0.09
0.08


Neutral
D175Y
2365
574.71
539.79
1.06
0.07
0.06


Neutral
D175L
2373
591.05
549.49
1.08
0.07
0.07


Up
D175H
2358
830.09
564.37
1.47
0.10
0.07


Neutral
D175P
2375
635.08
610.89
1.04
0.08
0.07


Up
D175E
2357
959.14
495.19
1.94
0.10
0.07


Up
A176F
148
10486.82
6516.31
1.61
1.31
0.78


Neutral
A176Q
143
6410.52
6665.27
0.96
0.80
0.80


Neutral
A176V
152
8890.53
8780.42
1.01
1.11
1.05


Neutral
A176E
137
589.82
546.54
1.08
0.07
0.07


Neutral
A176T
144
8471.98
8213.74
1.03
1.06
0.99


Neutral
A176C
141
6777.92
5924.96
1.14
0.85
0.71


Neutral
A176L
153
7190.01
6291.31
1.14
0.90
0.76


Neutral
A176P
154
639.90
596.40
1.07
0.08
0.07


Neutral
A176N
142
1351.92
1250.94
1.08
0.17
0.15


Neutral
A176G
147
2185.25
2395.33
0.91
0.27
0.29


Down
A176S
146
3003.29
3887.12
0.77
0.38
0.47


Neutral
A176R
140
919.15
792.44
1.16
0.12
0.10


Neutral
A176K
139
561.66
522.94
1.07
0.07
0.06


Neutral
A176D
136
863.82
792.98
1.09
0.11
0.10


Neutral
A176W
150
482.38
414.85
1.16
0.06
0.06


Neutral
H177T
2383
600.03
570.23
1.05
0.08
0.07


Neutral
H177P
2394
579.96
544.77
1.06
0.07
0.07


Neutral
H177Q
2382
593.68
549.35
1.08
0.07
0.07


Neutral
H177A
2393
536.01
523.32
1.02
0.07
0.06


Neutral
H177S
2385
561.60
524.64
1.07
0.07
0.06


Neutral
H177G
2386
559.31
519.85
1.08
0.07
0.06


Neutral
H177W
2389
547.21
520.18
1.05
0.07
0.06


Neutral
H177L
2392
486.50
433.60
1.12
0.06
0.05


Neutral
H177V
2391
508.58
447.50
1.14
0.06
0.05


Neutral
H177I
2390
489.45
455.90
1.07
0.06
0.05


Down
H177R
2379
1913.95
2460.77
0.78
0.24
0.30


Neutral
H177N
2381
504.44
478.07
1.06
0.06
0.06


Neutral
H177Y
2384
519.99
467.72
1.11
0.07
0.06


Neutral
H177C
2380
521.67
489.93
1.06
0.07
0.06


Neutral
H177D
2376
534.87
505.07
1.06
0.07
0.06


Neutral
F178G
2406
451.96
391.96
1.15
0.05
0.05


Up
F178C
2400
491.76
403.57
1.22
0.05
0.05


Neutral
F178W
2408
488.21
441.35
1.11
0.05
0.05


Neutral
F178R
2399
492.40
411.21
1.20
0.05
0.05


Neutral
F178K
2398
490.87
494.09
0.99
0.05
0.06


Neutral
F178S
2405
489.84
507.26
0.97
0.05
0.06


Neutral
F178H
2397
525.63
500.02
1.05
0.06
0.06


Neutral
F178P
2413
441.78
397.05
1.11
0.05
0.05


Neutral
F178V
2410
742.61
814.06
0.91
0.08
0.10


Neutral
F178A
2412
421.25
367.26
1.15
0.05
0.04


Neutral
F178Q
2402
409.62
360.29
1.14
0.04
0.04


Neutral
F178Y
2404
861.20
830.80
1.04
0.09
0.10


Neutral
F178I
2409
1118.23
1329.96
0.84
0.12
0.16


Neutral
F178T
2403
560.54
487.01
1.15
0.10
0.10


Up
F178L
2411
1788.95
1314.38
1.36
0.31
0.26


Neutral
F178E
2396
524.72
515.62
1.02
0.06
0.07


Neutral
D179P
173
526.54
527.98
1.00
0.06
0.06


Neutral
D179L
171
444.31
410.22
1.08
0.05
0.05


Neutral
D179E
155
520.82
438.27
1.19
0.06
0.05


Neutral
D179G
165
470.21
426.64
1.10
0.05
0.05


Neutral
D179S
164
461.51
421.09
1.10
0.05
0.05


Neutral
D179A
172
464.49
431.24
1.08
0.05
0.05


Neutral
D179K
157
483.67
456.75
1.06
0.05
0.05


Neutral
D179T
162
451.18
419.76
1.07
0.05
0.05


Neutral
D179I
169
425.91
372.56
1.14
0.05
0.04


Neutral
D179R
158
473.21
450.10
1.05
0.05
0.05


Up
D179N
160
2433.73
812.01
3.00
0.26
0.10


Neutral
D179W
168
465.31
423.56
1.10
0.05
0.05


Neutral
D179Q
161
446.51
414.79
1.08
0.05
0.05


Up
D179V
170
604.63
490.35
1.23
0.11
0.10


Up
D179C
159
613.81
503.76
1.22
0.11
0.10


Neutral
E180M
186
9630.23
8513.41
1.13
1.04
1.00


Neutral
E180P
192
523.92
492.75
1.06
0.06
0.06


Neutral
E180K
176
4017.43
3889.45
1.03
0.43
0.46


Up
E180Y
182
6655.19
5379.42
1.24
0.72
0.63


Neutral
E180Q
180
5146.93
4568.90
1.13
0.56
0.54


Neutral
E180R
177
6932.51
6309.81
1.10
0.75
0.74


Neutral
E180A
191
9562.37
8450.18
1.13
1.04
1.00


Up
E180T
181
3718.16
2425.13
1.53
0.40
0.29


Neutral
E180I
188
9126.95
7770.14
1.17
0.99
0.92


Up
E180F
185
7014.78
5382.78
1.30
0.76
0.63


Neutral
E180C
178
2926.15
2569.75
1.14
0.32
0.30


Up
E180G
184
5952.65
4547.28
1.31
1.04
0.90


Up
E180S
183
5217.80
3977.60
1.31
0.91
0.78


Up
E180N
179
6534.65
4843.84
1.35
1.14
0.96


Up
E180D
174
7738.70
6277.22
1.23
1.35
1.24


Neutral
D181S
202
9064.64
8368.97
1.08
0.98
0.99


Neutral
D181Q
199
7875.57
7127.19
1.11
0.85
0.84


Neutral
D181P
211
753.20
639.76
1.18
0.08
0.08


Up
D181Y
201
1137.94
716.86
1.59
0.12
0.08


Up
D181R
196
997.11
712.77
1.40
0.11
0.08


Up
D181V
208
945.65
721.77
1.31
0.10
0.09


Up
D181F
204
933.48
670.40
1.39
0.10
0.08


Neutral
D181A
210
7936.89
7854.96
1.01
0.86
0.93


Neutral
D181T
200
6867.00
6057.09
1.13
0.74
0.71


Up
D181L
209
1727.20
1274.09
1.36
0.19
0.15


Neutral
D181E
193
8647.28
8246.36
1.05
0.94
0.97


Up
D181K
195
1087.36
696.83
1.56
0.12
0.08


Up
D181M
205
3805.65
2986.75
1.27
0.41
0.35


Up
D181C
197
549.29
447.40
1.23
0.10
0.09


Up
D181G
203
2764.20
2056.56
1.34
0.48
0.41


Neutral
E182C
216
601.45
561.21
1.07
0.07
0.07


Neutral
E182P
230
606.01
574.24
1.06
0.07
0.07


Up
E182S
221
967.49
642.27
1.51
0.10
0.08


Up
E182T
219
2995.97
1779.42
1.68
0.32
0.21


Neutral
E182R
215
661.10
622.75
1.06
0.07
0.07


Neutral
E182D
212
2078.47
2140.28
0.97
0.23
0.25


Neutral
E182A
229
619.23
531.55
1.16
0.07
0.06


Neutral
E182F
223
1484.85
1677.82
0.88
0.16
0.20


Neutral
E182L
228
569.35
524.25
1.09
0.06
0.06


Neutral
E182I
226
606.88
519.75
1.17
0.07
0.06


Neutral
E182Y
220
593.61
561.88
1.06
0.06
0.07


Up
E182Q
218
1393.28
804.84
1.73
0.15
0.09


Neutral
E182W
225
556.78
536.32
1.04
0.06
0.06


Up
E182M
224
649.73
524.43
1.24
0.11
0.10


Neutral
E182G
222
604.92
543.78
1.11
0.11
0.11


Neutral
R183P
2432
9143.00
8148.29
1.12
0.99
0.96


Neutral
R183K
2417
9843.98
8685.25
1.13
1.07
1.02


Neutral
R183W
2427
8144.07
7669.02
1.06
0.88
0.90


Neutral
R183E
2415
9873.25
8403.44
1.17
1.07
0.99


Neutral
R183A
2431
9386.14
8368.29
1.12
1.02
0.99


Down
R183T
2421
4841.94
8385.09
0.58
0.52
0.99


Neutral
R183L
2430
517.07
532.72
0.97
0.06
0.06


Neutral
R183N
2419
10062.02
8456.13
1.19
1.09
1.00


Neutral
R183H
2416
9434.01
8295.55
1.14
1.02
0.98


Neutral
R183V
2429
9252.08
7954.42
1.16
1.00
0.94


Neutral
R183C
2418
6603.93
6597.30
1.00
0.72
0.78


Neutral
R183M
2426
9679.52
8250.27
1.17
1.05
0.97


Down
R183I
2428
495.34
8009.63
0.06
0.05
0.94


Up
R183G
2424
7326.36
6021.39
1.22
1.28
1.19


Up
R183S
2423
7896.17
6240.74
1.27
1.38
1.23


Neutral
W184G
2444
430.62
391.79
1.10
0.05
0.05


Neutral
W184H
2435
440.24
428.35
1.03
0.05
0.05


Neutral
W184L
2449
476.77
428.90
1.11
0.06
0.05


Neutral
W184E
2434
463.88
438.97
1.06
0.05
0.05


Neutral
W184P
2451
437.57
387.71
1.13
0.05
0.05


Neutral
W184N
2439
467.91
468.52
1.00
0.06
0.06


Neutral
W184A
2450
452.63
451.66
1.00
0.05
0.06


Neutral
W184T
2441
421.08
419.51
1.00
0.05
0.05


Neutral
W184R
2437
457.42
390.02
1.17
0.05
0.05


Neutral
W184Q
2440
450.92
448.33
1.01
0.05
0.05


Neutral
W184V
2448
454.60
407.30
1.12
0.05
0.05


Neutral
W184S
2443
486.70
485.16
1.00
0.06
0.06


Neutral
W184M
2446
447.30
395.61
1.13
0.05
0.05


Neutral
W184I
2447
478.17
503.24
0.95
0.06
0.06


Neutral
W184F
2445
455.86
427.51
1.07
0.05
0.05


Up
T185R
235
1728.04
851.07
2.03
0.20
0.10


Up
T185Y
239
937.75
540.66
1.73
0.11
0.07


Neutral
T185W
244
577.54
501.10
1.15
0.07
0.06


Up
T185H
233
1448.04
783.89
1.85
0.17
0.10


Up
T185G
241
3922.30
1990.15
1.97
0.46
0.24


Neutral
T185P
249
1773.27
1542.44
1.15
0.21
0.19


Neutral
T185S
240
9554.77
8267.62
1.16
1.12
1.01


Up
T185V
246
1648.14
897.66
1.84
0.19
0.11


Up
T185Q
238
1594.81
583.93
2.73
0.19
0.07


Up
T185N
237
790.61
546.44
1.45
0.09
0.07


Up
T185C
236
1554.42
1248.58
1.24
0.18
0.15


Neutral
T185L
247
483.25
463.52
1.04
0.06
0.06


Up
T185A
248
1599.64
711.08
2.25
0.19
0.09


Up
T185E
232
1324.02
703.76
1.88
0.16
0.09


Neutral
T185D
231
485.86
418.67
1.16
0.06
0.06


Neutral
N186G
2462
7592.31
6944.43
1.09
0.89
0.85


Neutral
N186A
2469
7466.07
7519.13
0.99
0.88
0.92


Neutral
N186T
2459
8897.05
8063.02
1.10
1.05
0.98


Neutral
N186R
2456
3212.69
3085.21
1.04
0.38
0.38


Neutral
N186L
2468
8097.42
7286.32
1.11
0.95
0.89


Neutral
N186P
2470
2173.37
1948.86
1.12
0.26
0.24


Neutral
N186S
2461
6854.56
6735.79
1.02
0.81
0.82


Neutral
N186V
2467
6303.91
6575.96
0.96
0.74
0.80


Neutral
N186Q
2458
4834.56
4621.18
1.05
0.57
0.56


Neutral
N186H
2454
3390.53
3309.97
1.02
0.40
0.40


Neutral
N186C
2457
3139.47
3113.35
1.01
0.37
0.38


Neutral
N186E
2453
3801.36
3332.52
1.14
0.45
0.41


Neutral
N186F
2463
3794.65
3316.48
1.14
0.45
0.40


Neutral
N186Y
2460
6301.09
7570.59
0.83
0.74
0.92


Neutral
N186D
2452
6853.09
6333.37
1.08
0.81
0.77


Up
N187R
254
1042.36
709.74
1.47
0.12
0.09


Up
N187M
262
1731.67
995.07
1.74
0.20
0.12


Neutral
N187S
259
9538.59
8971.12
1.06
1.12
1.10


Neutral
N187T
257
9856.38
8855.58
1.11
1.16
1.08


Neutral
N187L
266
505.93
464.62
1.09
0.06
0.06


Neutral
N187W
263
1694.86
1425.68
1.19
0.20
0.17


Up
N187F
261
1240.41
731.98
1.69
0.15
0.09


Up
N187K
253
2331.93
1140.19
2.05
0.27
0.14


Up
N187I
264
1444.98
683.03
2.12
0.17
0.08


Up
N187A
267
4379.80
2616.49
1.67
0.52
0.32


Neutral
N187P
268
644.27
572.98
1.12
0.08
0.07


Neutral
N187D
250
9843.65
8801.57
1.12
1.16
1.07


Neutral
N187G
260
535.06
514.10
1.04
0.07
0.07


Neutral
N187C
255
1804.28
1860.67
0.97
0.23
0.25


Neutral
N187H
252
1143.07
1071.67
1.07
0.14
0.14


Neutral
F188P
2489
10012.21
8943.91
1.12
1.18
1.09


Neutral
F188I
2485
7342.21
6782.40
1.08
0.86
0.83


Neutral
F188N
2477
10024.22
8961.63
1.12
1.18
1.09


Neutral
F188S
2481
9564.51
8841.98
1.08
1.13
1.08


Neutral
F188Q
2478
9591.39
8664.63
1.11
1.13
1.06


Neutral
F188K
2474
8347.12
7497.38
1.11
0.98
0.92


Neutral
F188G
2482
9891.61
9065.43
1.09
1.16
1.11


Neutral
F188W
2484
9389.97
8774.36
1.07
1.10
1.07


Neutral
F188E
2472
10235.38
8984.46
1.14
1.20
1.10


Neutral
F188H
2473
2065.12
1901.41
1.09
0.24
0.23


Neutral
F188D
2471
10087.61
8889.75
1.13
1.19
1.09


Up
F188A
2488
1502.70
1231.99
1.22
0.18
0.15


Neutral
F188L
2487
8309.64
7501.30
1.11
0.98
0.92


Neutral
F188R
2475
8182.64
7750.05
1.06
0.96
0.95


Up
F188V
2486
7116.29
5860.00
1.21
1.24
1.16


Neutral
R189L
2506
9236.08
8947.54
1.03
1.09
1.09


Neutral
R189G
2500
10307.88
9096.35
1.13
1.21
1.11


Neutral
R189K
2493
9365.15
9033.15
1.04
1.10
1.10


Neutral
R189P
2508
3200.68
3533.96
0.91
0.38
0.43


Neutral
R189E
2491
9552.57
8789.28
1.09
1.12
1.07


Neutral
R189V
2505
9150.17
8088.39
1.13
1.08
0.99


Neutral
R189D
2490
9506.16
8933.41
1.06
1.12
1.09


Neutral
R189Y
2498
9893.14
8946.52
1.11
1.16
1.09


Neutral
R189C
2494
5318.06
5457.78
0.97
0.63
0.67


Neutral
R189A
2507
9718.09
8798.13
1.10
1.14
1.07


Neutral
R189H
2492
1360.90
1350.33
1.01
0.16
0.16


Neutral
R189W
2503
7657.12
7070.92
1.08
0.90
0.86


Neutral
R189N
2495
7842.39
6675.36
1.17
1.37
1.32


Neutral
R189T
2497
7610.10
6459.94
1.18
1.33
1.27


Neutral
R189Q
2496
7465.37
6396.79
1.17
1.30
1.26


Down
E190A
590
1510.06
2116.94
0.71
0.21
0.23


Neutral
E190H
574
6276.13
7564.04
0.83
0.88
0.83


Down
E190V
588
643.37
1658.11
0.39
0.09
0.18


Up
E190P
591
2420.68
1767.43
1.37
0.34
0.19


Neutral
E190C
577
1827.25
2083.88
0.88
0.26
0.23


Up
E190G
583
5313.99
4365.93
1.22
0.75
0.48


Down
E190R
576
1185.26
1810.53
0.65
0.17
0.20


Down
E190I
587
1880.80
2886.28
0.65
0.27
0.32


Down
E190S
582
4542.61
5987.33
0.76
0.64
0.66


Down
E190T
580
2293.47
4444.68
0.52
0.32
0.49


Up
E190M
585
2557.21
1317.73
1.94
0.36
0.15


Neutral
E190L
589
2542.38
2986.91
0.85
0.36
0.33


Down
E190K
575
2960.37
4343.12
0.68
0.42
0.48


Up
E190Y
581
7243.54
5742.33
1.26
1.27
1.13


Up
E190D
573
7910.21
6468.78
1.22
1.38
1.28


Neutral
Y191T
600
611.75
535.95
1.14
0.07
0.06


Neutral
Y191H
594
2333.85
2191.64
1.06
0.28
0.24


Neutral
Y191G
602
428.17
432.65
0.99
0.05
0.05


Neutral
Y191L
608
379.02
357.82
1.06
0.05
0.04


Up
Y191P
610
1359.30
1046.33
1.30
0.16
0.12


Neutral
Y191Q
599
451.92
403.46
1.12
0.05
0.05


Neutral
Y191K
595
464.62
406.52
1.14
0.06
0.05


Neutral
Y191D
592
392.24
370.67
1.06
0.05
0.04


Neutral
Y191A
609
452.13
418.53
1.08
0.05
0.05


Neutral
Y191W
605
395.63
411.91
0.96
0.05
0.05


Neutral
Y191S
601
530.80
447.13
1.19
0.06
0.05


Up
Y191V
607
1553.58
1254.11
1.24
0.19
0.14


Neutral
Y191E
593
395.04
407.49
0.97
0.05
0.05


Neutral
Y191R
596
652.95
725.68
0.90
0.08
0.08


Neutral
Y191C
597
530.42
463.90
1.14
0.06
0.05


Up
N192R
615
640.72
482.61
1.33
0.09
0.05


Neutral
N192L
627
591.92
571.56
1.04
0.08
0.06


Neutral
N192Q
617
1089.41
1020.23
1.07
0.15
0.11


Neutral
N192P
629
685.62
856.11
0.80
0.10
0.09


Up
N192H
613
2274.24
1058.80
2.15
0.32
0.12


Up
N192S
620
2043.65
1630.74
1.25
0.29
0.18


Neutral
N192W
624
548.30
538.86
1.02
0.08
0.06


Up
N192G
621
899.47
659.29
1.36
0.13
0.07


Up
N192D
611
4213.33
2216.40
1.90
0.59
0.24


Neutral
N192V
626
588.02
537.64
1.09
0.08
0.06


Neutral
N192A
628
574.26
543.66
1.06
0.08
0.06


Neutral
N192T
618
536.50
576.21
0.93
0.08
0.06


Neutral
N192K
614
685.26
633.89
1.08
0.10
0.07


Up
N192C
616
1310.46
987.31
1.33
0.18
0.11


Neutral
N192M
623
547.98
537.29
1.02
0.08
0.06


Neutral
L193P
2527
388.57
381.15
1.02
0.05
0.04


Neutral
L193G
2520
437.84
478.44
0.92
0.05
0.05


Neutral
L193F
2521
481.49
491.33
0.98
0.06
0.05


Neutral
L193S
2519
448.35
449.03
1.00
0.05
0.05


Neutral
L193W
2523
481.79
460.74
1.05
0.06
0.05


Neutral
L193A
2526
510.96
468.83
1.09
0.06
0.05


Neutral
L193R
2513
481.08
477.55
1.01
0.06
0.05


Neutral
L193Q
2516
417.53
412.01
1.01
0.05
0.05


Neutral
L193E
2510
401.70
409.23
0.98
0.05
0.05


Neutral
L193K
2512
417.39
426.26
0.98
0.05
0.05


Neutral
L193N
2515
432.35
434.95
0.99
0.05
0.05


Down
L193I
2524
2767.03
3467.03
0.80
0.33
0.39


Neutral
L193T
2517
679.48
638.88
1.06
0.08
0.07


Neutral
L193D
2509
419.37
424.41
0.99
0.05
0.05


Neutral
L193Y
2518
3022.00
2706.28
1.12
0.36
0.30


Neutral
H194S
639
5518.27
6112.38
0.90
0.78
0.67


Neutral
H194E
631
7667.53
8295.22
0.92
1.08
0.91


Neutral
H194K
632
5130.62
6124.27
0.84
0.72
0.67


Neutral
H194Q
636
6399.62
7113.56
0.90
0.90
0.78


Down
H194V
645
1611.06
5696.43
0.28
0.23
0.63


Up
H194T
637
3884.64
2598.12
1.50
0.55
0.29


Neutral
H194L
646
5710.11
6872.56
0.83
0.80
0.76


Neutral
H194Y
638
4922.31
5688.29
0.87
0.69
0.63


Down
H194F
641
3787.65
5388.18
0.70
0.53
0.59


Neutral
H194G
640
4636.22
5437.23
0.85
0.65
0.60


Down
H194I
644
2901.13
3777.68
0.77
0.41
0.42


Down
H194W
643
5434.60
7448.23
0.73
0.77
0.82


Down
H194M
642
2941.85
9057.43
0.32
0.41
1.00


Up
H194A
647
4681.45
2746.90
1.70
0.66
0.30


Neutral
H194P
648
5264.79
5058.19
1.04
0.74
0.56


Up
R195C
273
4231.32
1853.20
2.28
0.60
0.20


Neutral
R195F
280
687.70
720.42
0.95
0.10
0.08


Neutral
R195W
282
5099.23
4524.84
1.13
0.72
0.50


Neutral
R195T
276
1101.98
1175.85
0.94
0.16
0.13


Neutral
R195L
285
5073.57
4520.73
1.12
0.72
0.50


Up
R195G
279
5269.21
3025.93
1.74
0.74
0.33


Up
R195Q
275
1958.69
1361.83
1.44
0.28
0.15


Down
R195K
272
3839.86
7080.78
0.54
0.54
0.78


Neutral
R195S
278
642.14
649.21
0.99
0.09
0.07


Up
R195A
286
5605.90
3852.81
1.46
0.79
0.42


Up
R195D
269
2724.53
1907.81
1.43
0.38
0.21


Neutral
R195P
287
571.50
615.21
0.93
0.08
0.07


Neutral
R195Y
277
763.31
794.42
0.96
0.11
0.09


Neutral
R195E
270
7597.55
8468.35
0.90
1.07
0.93


Up
R195V
284
1711.48
1037.62
1.65
0.24
0.11


Neutral
V196T
2536
1040.90
1268.04
0.82
0.12
0.14


Neutral
V196D
2528
443.04
446.39
0.99
0.05
0.05


Neutral
V196G
2539
490.83
494.67
0.99
0.06
0.06


Neutral
V196E
2529
488.55
489.74
1.00
0.06
0.05


Neutral
V196A
2545
452.36
452.12
1.00
0.05
0.05


Up
V196S
2538
1186.52
949.52
1.25
0.14
0.11


Neutral
V196Q
2535
412.17
430.91
0.96
0.05
0.05


Neutral
V196P
2546
576.83
620.88
0.93
0.07
0.07


Neutral
V196R
2532
493.29
474.38
1.04
0.06
0.05


Neutral
V196H
2530
465.64
479.66
0.97
0.06
0.05


Neutral
V196Y
2537
462.28
474.94
0.97
0.06
0.05


Neutral
V196I
2543
1125.67
1229.87
0.92
0.13
0.14


Neutral
V196L
2544
464.80
491.01
0.95
0.06
0.05


Neutral
V196K
2531
455.84
482.44
0.94
0.05
0.05


Neutral
V196M
2541
479.36
518.00
0.93
0.06
0.06


Down
A197G
2558
1238.39
2552.91
0.49
0.17
0.28


Up
A197S
2557
3959.39
2633.91
1.50
0.56
0.29


Up
A197L
2564
1013.13
809.32
1.25
0.14
0.09


Neutral
A197P
2565
857.06
933.29
0.92
0.12
0.10


Down
A197V
2563
2549.12
4355.70
0.59
0.36
0.48


Neutral
A197Y
2556
650.21
722.49
0.90
0.09
0.08


Neutral
A197Q
2554
658.64
652.52
1.01
0.09
0.07


Neutral
A197R
2551
635.08
640.91
0.99
0.09
0.07


Down
A197T
2555
1933.94
4482.59
0.43
0.27
0.49


Up
A197I
2562
1440.69
1060.51
1.36
0.20
0.12


Neutral
A197H
2549
604.11
638.63
0.95
0.09
0.07


Neutral
A197E
2548
686.96
624.22
1.10
0.10
0.07


Down
A197W
2561
1448.83
2588.64
0.56
0.20
0.29


Down
A197N
2553
623.17
840.56
0.74
0.09
0.09


Up
A197C
2552
4012.80
3140.52
1.28
0.70
0.62


Neutral
A198T
296
761.19
700.22
1.09
0.11
0.08


Down
A198K
291
490.20
1179.92
0.42
0.07
0.13


Up
A198S
298
4061.28
3136.65
1.29
0.57
0.35


Neutral
A198H
290
581.41
575.73
1.01
0.08
0.06


Neutral
A198G
299
2610.82
2368.26
1.10
0.37
0.26


Down
A198E
289
485.45
662.62
0.73
0.07
0.07


Neutral
A198P
306
656.48
580.71
1.13
0.09
0.06


Up
A198L
305
1339.94
726.74
1.84
0.19
0.08


Neutral
A198R
292
570.33
565.56
1.01
0.08
0.06


Down
A198V
304
3026.36
7305.85
0.41
0.43
0.80


Up
A198M
301
1384.46
999.55
1.39
0.20
0.11


Neutral
A198F
300
572.48
559.57
1.02
0.08
0.06


Neutral
A198W
302
560.48
547.72
1.02
0.08
0.06


Down
A198Y
297
486.57
612.28
0.79
0.07
0.07


Up
A198D
288
633.49
474.50
1.34
0.09
0.05


Neutral
H199I
2580
520.35
496.48
1.05
0.08
0.07


Neutral
H199P
2584
437.57
404.21
1.08
0.07
0.05


Neutral
H199G
2576
436.53
392.94
1.11
0.07
0.05


Neutral
H199N
2571
420.26
375.18
1.12
0.07
0.05


Neutral
H199S
2575
411.09
377.36
1.09
0.06
0.05


Neutral
H199L
2582
531.61
530.53
1.00
0.08
0.07


Neutral
H199M
2578
413.37
384.23
1.08
0.07
0.05


Neutral
H199A
2583
391.56
381.36
1.03
0.06
0.05


Neutral
H199C
2570
404.49
366.35
1.10
0.06
0.05


Neutral
H199K
2568
402.34
383.95
1.05
0.06
0.05


Neutral
H199R
2569
422.19
387.94
1.09
0.07
0.05


Neutral
H199V
2581
421.16
378.71
1.11
0.07
0.05


Neutral
H199W
2579
377.01
345.02
1.09
0.06
0.05


Neutral
H199T
2573
399.21
382.65
1.04
0.06
0.05


Neutral
H199E
2567
399.49
385.83
1.04
0.06
0.05


Neutral
E200P
2603
414.11
409.55
1.01
0.07
0.06


Neutral
E200G
2595
440.94
402.85
1.09
0.07
0.05


Neutral
E200A
2602
448.41
413.61
1.08
0.07
0.06


Neutral
E200T
2592
461.19
418.51
1.10
0.07
0.06


Neutral
E200I
2599
457.88
419.19
1.09
0.07
0.06


Neutral
E200W
2598
418.40
403.05
1.04
0.07
0.05


Neutral
E200R
2588
449.83
425.86
1.06
0.07
0.06


Neutral
E200F
2596
446.49
417.58
1.07
0.07
0.06


Neutral
E200M
2597
448.32
428.16
1.05
0.07
0.06


Neutral
E200D
2585
428.91
401.64
1.07
0.07
0.05


Neutral
E200V
2600
426.45
407.13
1.05
0.07
0.06


Neutral
E200C
2589
413.11
384.79
1.07
0.07
0.05


Neutral
E200S
2594
422.57
391.02
1.08
0.07
0.05


Neutral
E200Y
2593
412.07
393.97
1.05
0.07
0.05


Neutral
E200N
2590
430.94
412.07
1.05
0.07
0.06


Down
L201A
2621
754.66
957.77
0.79
0.12
0.13


Neutral
L201R
2608
442.35
442.57
1.00
0.07
0.06


Neutral
L201E
2605
464.22
443.89
1.05
0.07
0.06


Neutral
L201P
2622
494.97
471.92
1.05
0.08
0.06


Neutral
L201G
2615
574.82
590.26
0.97
0.09
0.08


Down
L201V
2620
3359.21
4623.67
0.73
0.53
0.62


Down
L201T
2612
1509.22
2175.97
0.69
0.24
0.29


Up
L201I
2619
2861.66
2231.87
1.28
0.45
0.30


Neutral
L201S
2614
859.79
964.65
0.89
0.14
0.13


Neutral
L201W
2618
1258.36
1335.56
0.94
0.20
0.18


Neutral
L201Q
2611
657.51
749.98
0.88
0.10
0.10


Neutral
L201D
2604
486.09
471.81
1.03
0.08
0.06


Down
L201M
2617
5637.84
7147.36
0.79
0.89
0.97


Neutral
L201K
2607
484.89
467.23
1.04
0.08
0.06


Neutral
L201N
2610
440.03
432.42
1.02
0.07
0.06


Neutral
G202T
2631
556.53
546.05
1.02
0.09
0.07


Neutral
G202Y
2632
533.64
530.73
1.01
0.08
0.07


Neutral
G202E
2624
558.69
543.68
1.03
0.09
0.07


Neutral
G202V
2638
569.22
572.58
0.99
0.09
0.08


Neutral
G202S
2633
512.82
503.35
1.02
0.08
0.07


Neutral
G202L
2639
513.71
508.34
1.01
0.08
0.07


Neutral
G202I
2637
535.96
516.37
1.04
0.08
0.07


Neutral
G202M
2635
507.94
500.04
1.02
0.08
0.07


Neutral
G202H
2625
567.88
547.25
1.04
0.09
0.07


Neutral
G202C
2628
508.19
499.05
1.02
0.08
0.07


Neutral
G202R
2627
537.10
511.28
1.05
0.08
0.07


Neutral
G202P
2641
544.39
535.24
1.02
0.09
0.07


Neutral
G202A
2640
580.75
571.95
1.02
0.09
0.08


Neutral
G202K
2626
531.07
520.45
1.02
0.08
0.07


Neutral
G202D
2623
559.64
544.50
1.03
0.09
0.07


Down
H203Y
2650
910.26
1218.02
0.75
0.14
0.16


Neutral
H203E
2643
7284.23
7937.91
0.92
1.15
1.07


Neutral
H203R
2645
545.70
545.36
1.00
0.09
0.07


Neutral
H203Q
2648
570.55
541.42
1.05
0.09
0.07


Neutral
H203P
2660
547.00
527.67
1.04
0.09
0.07


Neutral
H203G
2652
558.88
576.91
0.97
0.09
0.08


Neutral
H203T
2649
534.16
535.33
1.00
0.08
0.07


Neutral
H203D
2642
542.85
530.31
1.02
0.09
0.07


Down
H203L
2658
1224.67
1746.14
0.70
0.19
0.24


Neutral
H203N
2647
547.92
532.11
1.03
0.09
0.07


Neutral
H203A
2659
513.18
515.49
1.00
0.08
0.07


Neutral
H203S
2651
534.50
507.56
1.05
0.08
0.07


Neutral
H203V
2657
565.64
554.43
1.02
0.09
0.07


Neutral
H203I
2656
568.56
613.73
0.93
0.09
0.08


Neutral
H203C
2646
504.41
522.69
0.97
0.08
0.07


Neutral
S204R
2665
557.42
544.69
1.02
0.09
0.07


Neutral
S204N
2667
733.30
754.34
0.97
0.12
0.10


Neutral
S204A
2678
3654.83
3972.28
0.92
0.58
0.54


Down
S204T
2669
1697.49
3586.11
0.47
0.27
0.48


Neutral
S204Y
2670
550.01
538.07
1.02
0.09
0.07


Up
S204V
2676
3063.02
1827.71
1.68
0.48
0.25


Neutral
S204L
2677
501.10
594.44
0.84
0.08
0.08


Neutral
S204H
2663
486.78
508.13
0.96
0.08
0.07


Neutral
S204D
2661
507.05
489.81
1.04
0.08
0.07


Neutral
S204Q
2668
530.67
472.92
1.12
0.08
0.06


Neutral
S204G
2671
1483.41
1333.79
1.11
0.23
0.18


Neutral
S204W
2674
487.01
504.11
0.97
0.08
0.07


Up
S204I
2675
634.82
516.30
1.23
0.10
0.07


Neutral
S204K
2664
484.92
471.83
1.03
0.08
0.06


Neutral
S204P
2679
483.87
506.90
0.95
0.08
0.07


Neutral
L205T
2688
1304.89
1099.25
1.19
0.13
0.12


Neutral
L205D
2680
774.29
830.37
0.93
0.08
0.09


Neutral
L205S
2690
686.11
601.35
1.14
0.07
0.07


Neutral
L205G
2691
792.45
790.93
1.00
0.08
0.09


Neutral
L205P
2698
592.15
673.32
0.88
0.06
0.07


Neutral
L205E
2681
473.89
446.64
1.06
0.05
0.05


Down
L205V
2696
5589.64
7308.12
0.76
0.57
0.80


Neutral
L205M
2693
8334.85
8229.20
1.01
0.85
0.90


Neutral
L205N
2686
1426.11
1322.80
1.08
0.15
0.15


Down
L205C
2685
1903.14
2394.15
0.79
0.20
0.26


Down
L205I
2695
5644.28
7817.06
0.72
0.58
0.86


Neutral
L205A
2697
1796.22
1704.85
1.05
0.18
0.19


Neutral
L205R
2684
508.62
575.22
0.88
0.05
0.06


Neutral
L205W
2694
497.92
427.60
1.16
0.05
0.05


Neutral
L205Q
2687
2191.83
2399.54
0.91
0.22
0.26


Neutral
G206I
321
467.21
460.72
1.01
0.05
0.05


Neutral
G206V
322
619.10
682.58
0.91
0.06
0.07


Up
G206A
324
4554.61
2702.11
1.69
0.47
0.30


Neutral
G206C
312
491.44
469.90
1.05
0.05
0.05


Up
G206S
317
1226.37
919.66
1.33
0.13
0.10


Neutral
G206P
325
503.21
497.87
1.01
0.05
0.05


Neutral
G206L
323
499.74
469.53
1.06
0.05
0.05


Neutral
G206D
307
490.08
451.61
1.09
0.05
0.05


Neutral
G206M
319
478.55
451.47
1.06
0.05
0.05


Neutral
G206R
311
677.07
831.95
0.81
0.07
0.09


Neutral
G206Q
314
805.32
851.38
0.95
0.08
0.09


Neutral
G206E
308
469.86
447.60
1.05
0.05
0.05


Neutral
G206H
309
463.25
437.73
1.06
0.05
0.05


Neutral
G206T
315
475.20
491.10
0.97
0.05
0.05


Neutral
G206W
320
472.91
437.66
1.08
0.05
0.05


Up
L207S
659
657.07
501.03
1.31
0.07
0.05


Neutral
L207Y
658
1032.96
1142.52
0.90
0.11
0.13


Neutral
L207A
666
6302.90
5614.64
1.12
0.65
0.62


Up
L207R
653
3476.88
1332.44
2.61
0.36
0.15


Neutral
L207P
667
528.87
508.95
1.04
0.05
0.06


Up
L207Q
656
671.72
518.36
1.30
0.07
0.06


Neutral
L207N
655
551.03
476.05
1.16
0.06
0.05


Up
L207K
652
860.90
594.92
1.45
0.09
0.07


Neutral
L207M
662
11903.05
12984.69
0.92
1.22
1.42


Neutral
L207W
663
509.40
470.26
1.08
0.05
0.05


Neutral
L207H
651
620.20
595.55
1.04
0.06
0.07


Neutral
L207D
649
523.82
473.80
1.11
0.05
0.05


Neutral
L207V
665
656.95
550.54
1.19
0.08
0.07


Neutral
L207I
664
645.37
550.32
1.17
0.08
0.07


Up
L207G
660
610.01
484.35
1.26
0.08
0.06


Neutral
S208D
2699
10064.82
9325.26
1.08
1.03
1.02


Neutral
S208V
2714
10469.49
9334.16
1.12
1.07
1.02


Neutral
S208P
2717
9922.26
9236.91
1.07
1.02
1.01


Neutral
S208G
2709
10452.64
9295.93
1.12
1.07
1.02


Neutral
S208A
2716
10553.22
9517.15
1.11
1.08
1.04


Neutral
S208K
2702
22659.58
19984.18
1.13
2.32
2.19


Neutral
S208N
2705
9993.85
9327.07
1.07
1.02
1.02


Neutral
S208F
2710
8826.28
9040.21
0.98
0.91
0.99


Neutral
S208Q
2706
10196.89
9183.58
1.11
1.05
1.01


Neutral
S208W
2712
9229.04
9226.75
1.00
0.95
1.01


Neutral
S208T
2707
9241.73
8912.77
1.04
0.95
0.98


Neutral
S208E
2700
10198.81
9401.75
1.08
1.05
1.03


Down
S208C
2704
10497.72
16287.64
0.64
1.08
1.79


Neutral
S208R
2703
7639.06
6465.10
1.18
1.34
1.28


Up
S208L
2715
7811.78
6354.14
1.23
1.37
1.25


Neutral
H209T
2725
466.30
415.72
1.12
0.11
0.08


Neutral
H209Y
2726
471.70
455.15
1.04
0.11
0.09


Neutral
H209R
2721
489.49
463.09
1.06
0.12
0.09


Neutral
H209Q
2724
513.42
476.96
1.08
0.12
0.09


Neutral
H209A
2735
511.91
469.64
1.09
0.12
0.09


Neutral
H209G
2728
495.58
466.25
1.06
0.12
0.09


Neutral
H209N
2723
455.09
424.90
1.07
0.11
0.08


Neutral
H209P
2736
526.85
480.73
1.10
0.13
0.09


Neutral
H209W
2731
516.05
484.16
1.07
0.12
0.09


Neutral
H209V
2733
499.35
465.99
1.07
0.12
0.09


Neutral
H209D
2718
479.48
442.06
1.08
0.12
0.09


Neutral
H209S
2727
490.77
438.98
1.12
0.12
0.09


Neutral
H209F
2729
490.42
437.68
1.12
0.12
0.09


Neutral
H209L
2734
491.46
441.89
1.11
0.12
0.09


Neutral
H209C
2722
471.56
420.60
1.12
0.11
0.08


Neutral
S210C
331
634.06
565.38
1.12
0.15
0.11


Neutral
S210G
336
643.08
581.11
1.11
0.16
0.11


Up
S210I
340
778.38
625.00
1.25
0.19
0.12


Neutral
S210R
330
644.74
565.67
1.14
0.16
0.11


Neutral
S210L
342
737.60
623.25
1.18
0.18
0.12


Up
S210V
341
1190.35
856.63
1.39
0.29
0.17


Neutral
S210H
328
605.43
521.90
1.16
0.15
0.10


Neutral
S210N
332
615.29
556.38
1.11
0.15
0.11


Neutral
S210F
337
529.93
487.42
1.09
0.13
0.09


Neutral
S210P
344
544.94
513.59
1.06
0.13
0.10


Neutral
S210W
339
527.32
486.97
1.08
0.13
0.09


Neutral
S210Q
333
593.74
548.93
1.08
0.14
0.11


Neutral
S210T
334
2977.61
3427.71
0.87
0.72
0.67


Neutral
S210K
329
625.14
573.41
1.09
0.15
0.11


Neutral
S210A
343
1682.05
1546.97
1.09
0.25
0.21


Neutral
T211P
2755
3493.13
3774.82
0.93
0.84
0.73


Neutral
T211R
2741
4636.24
5429.67
0.85
1.12
1.05


Neutral
T211K
2740
4457.25
5411.31
0.82
1.08
1.05


Neutral
T211G
2747
3443.93
3543.72
0.97
0.83
0.69


Down
T211M
2749
3806.80
4871.37
0.78
0.92
0.95


Neutral
T211N
2743
5924.95
6170.25
0.96
1.43
1.20


Neutral
T211V
2752
5095.76
5335.63
0.96
1.23
1.04


Neutral
T211H
2739
1885.69
1829.82
1.03
0.46
0.36


Neutral
T211Q
2744
4868.86
5772.70
0.84
1.18
1.12


Neutral
T211S
2746
4641.02
4565.80
1.02
1.12
0.89


Neutral
T211A
2754
2696.88
2830.43
0.95
0.65
0.55


Neutral
T211F
2748
1412.47
1277.53
1.11
0.34
0.25


Neutral
T211D
2737
2442.24
2154.48
1.13
0.59
0.42


Neutral
T211W
2750
1362.99
1207.40
1.13
0.33
0.23


Neutral
T211L
2753
2376.23
2102.07
1.13
0.35
0.28


Neutral
D212E
668
4877.68
4473.75
1.09
1.18
0.87


Neutral
D212A
685
2710.82
2417.65
1.12
0.66
0.47


Neutral
D212K
670
2296.16
2049.97
1.12
0.55
0.40


Neutral
D212R
671
2273.87
2004.06
1.13
0.55
0.39


Neutral
D212T
675
2923.39
2699.75
1.08
0.71
0.52


Neutral
D212N
673
4575.59
5229.75
0.87
1.11
1.02


Up
D212G
678
1011.62
657.28
1.54
0.24
0.13


Neutral
D212S
677
5035.28
4894.92
1.03
1.22
0.95


Neutral
D212P
686
3270.81
2918.36
1.12
0.79
0.57


Neutral
D212Q
674
2823.54
2576.63
1.10
0.68
0.50


Neutral
D212V
683
2000.60
1876.86
1.07
0.48
0.36


Neutral
D212L
684
517.72
497.60
1.04
0.13
0.10


Neutral
D212F
679
2378.07
2185.27
1.09
0.57
0.42


Neutral
D212H
669
4696.49
4001.41
1.17
0.70
0.53


Neutral
D212Y
676
5489.99
5319.27
1.03
0.49
0.55


Neutral
I213Q
2763
9326.77
8702.80
1.07
0.96
0.95


Neutral
I213T
2764
9396.39
8742.82
1.07
0.96
0.96


Neutral
I213C
2761
9396.24
8859.11
1.06
0.96
0.97


Neutral
I213P
2774
10248.90
9319.88
1.10
1.05
1.02


Neutral
I213H
2758
9826.58
9076.23
1.08
1.01
1.00


Neutral
I213A
2773
10044.30
9249.07
1.09
1.03
1.01


Neutral
I213V
2771
10260.18
9459.80
1.08
1.05
1.04


Neutral
I213G
2767
21327.14
19706.88
1.08
2.19
2.16


Neutral
I213N
2762
8790.33
7995.03
1.10
0.90
0.88


Neutral
I213L
2772
9974.73
9208.92
1.08
1.02
1.01


Neutral
I213S
2766
9599.72
9004.65
1.07
0.98
0.99


Neutral
I213M
2769
9987.31
9083.77
1.10
1.02
1.00


Neutral
I213R
2760
9253.06
8997.34
1.03
0.95
0.99


Neutral
I213K
2759
9682.80
9286.32
1.04
0.99
1.02


Neutral
I213F
2768
9368.13
8940.38
1.05
0.96
0.98


Neutral
I213D
2756
7017.06
7368.43
0.95
0.77
0.97


Neutral
I213E
2757
8169.74
7234.77
1.13
0.90
0.95


Neutral
G214L
2791
13500.23
13135.46
1.03
1.38
1.44


Neutral
G214Q
2782
9914.48
9182.63
1.08
1.02
1.01


Neutral
G214S
2785
9503.68
9036.70
1.05
0.97
0.99


Neutral
G214T
2783
9940.86
9214.25
1.08
1.02
1.01


Neutral
G214V
2790
8185.36
7785.72
1.05
0.84
0.85


Neutral
G214I
2789
6068.79
5773.01
1.05
0.62
0.63


Neutral
G214R
2779
9720.43
9083.04
1.07
1.00
1.00


Neutral
G214P
2793
8763.31
8875.24
0.99
0.90
0.97


Neutral
G214E
2776
21602.30
19851.77
1.09
2.22
2.18


Neutral
G214A
2792
10063.30
9154.93
1.10
1.03
1.00


Neutral
G214D
2775
9967.49
9121.71
1.09
1.02
1.00


Neutral
G214F
2786
9750.30
9157.75
1.06
1.00
1.00


Neutral
G214Y
2784
9886.62
9025.45
1.10
1.01
0.99


Neutral
G214M
2787
9472.77
8919.05
1.06
0.97
0.98


Neutral
G214C
2780
6716.52
7097.60
0.95
0.69
0.78


Neutral
A215L
2811
454.59
428.41
1.06
0.07
0.06


Neutral
A215Q
2801
765.06
739.09
1.04
0.12
0.10


Neutral
A215M
2807
672.22
624.41
1.08
0.10
0.09


Down
A215G
2805
4240.44
6854.29
0.62
0.66
0.96


Neutral
A215W
2808
377.79
348.04
1.09
0.06
0.05


Neutral
A215S
2804
559.99
538.20
1.04
0.09
0.08


Neutral
A215T
2802
664.02
711.35
0.93
0.10
0.10


Neutral
A215V
2810
473.67
492.63
0.96
0.07
0.07


Neutral
A215N
2800
4328.77
4488.89
0.96
0.67
0.63


Neutral
A215P
2812
638.50
596.48
1.07
0.10
0.08


Neutral
A215H
2796
3954.04
4447.65
0.89
0.61
0.62


Neutral
A215K
2797
420.46
402.71
1.04
0.07
0.06


Neutral
A215I
2809
413.93
386.28
1.07
0.06
0.05


Neutral
A215R
2798
421.35
389.00
1.08
0.07
0.05


Neutral
A215C
2799
437.44
425.03
1.03
0.07
0.06


Neutral
A215D
2794
1031.48
913.25
1.13
0.11
0.12


Neutral
L216A
2830
808.93
759.54
1.07
0.13
0.12


Neutral
L216C
2818
473.05
462.23
1.02
0.08
0.07


Neutral
L216D
2813
497.61
457.15
1.09
0.08
0.07


Neutral
L216E
2814
480.72
458.21
1.05
0.08
0.07


Neutral
L216G
2824
473.61
452.00
1.05
0.08
0.07


Neutral
L216I
2828
7525.06
8586.88
0.88
1.20
1.37


Neutral
L216K
2816
478.52
460.66
1.04
0.08
0.07


Neutral
L216M
2826
4641.29
5160.67
0.90
0.74
0.83


Neutral
L216P
2831
466.46
475.96
0.98
0.07
0.08


Neutral
L216Q
2820
693.10
638.62
1.09
0.11
0.10


Neutral
L216R
2817
458.77
437.34
1.05
0.07
0.07


Neutral
L216S
2823
454.85
441.50
1.03
0.07
0.07


Neutral
L216T
2821
1484.74
1392.55
1.07
0.24
0.22


Down
L216V
2829
5022.20
6281.17
0.80
0.80
1.01


Neutral
L216W
2827
479.55
454.07
1.06
0.08
0.07


Neutral
M217P
2850
458.79
440.59
1.04
0.07
0.06


Neutral
M217Y
2841
459.96
427.87
1.07
0.07
0.06


Neutral
M217T
2840
699.27
663.16
1.05
0.11
0.09


Down
M217C
2837
5441.67
7486.86
0.73
0.85
1.05


Neutral
M217S
2842
470.92
424.03
1.11
0.07
0.06


Neutral
M217L
2848
443.33
403.49
1.10
0.07
0.06


Neutral
M217N
2838
462.12
424.21
1.09
0.07
0.06


Neutral
M217R
2836
454.58
442.20
1.03
0.07
0.06


Neutral
M217Q
2839
449.94
427.47
1.05
0.07
0.06


Neutral
M217K
2835
506.96
458.74
1.11
0.08
0.06


Neutral
M217G
2843
746.17
728.78
1.02
0.12
0.10


Neutral
M217A
2849
437.36
410.18
1.07
0.07
0.06


Neutral
M217H
2834
442.18
398.29
1.11
0.07
0.06


Neutral
M217I
2846
483.00
449.94
1.07
0.08
0.06


Neutral
M217D
2832
503.49
491.20
1.03
0.04
0.05


Neutral
Y218C
350
511.72
486.65
1.05
0.08
0.07


Down
Y218F
356
4555.92
6084.93
0.75
0.71
0.85


Neutral
Y218W
358
8521.86
9311.36
0.92
1.32
1.30


Neutral
Y218L
361
834.41
743.23
1.12
0.13
0.10


Neutral
Y218A
362
1935.94
1652.76
1.17
0.30
0.23


Neutral
Y218P
363
503.58
469.06
1.07
0.08
0.07


Neutral
Y218R
349
508.52
465.38
1.09
0.08
0.07


Neutral
Y218N
351
704.77
640.35
1.10
0.11
0.09


Neutral
Y218V
360
527.03
480.30
1.10
0.08
0.07


Neutral
Y218Q
352
513.25
468.66
1.10
0.08
0.07


Up
Y218I
359
698.50
542.67
1.29
0.11
0.08


Neutral
Y218D
345
835.29
885.84
0.94
0.13
0.12


Up
Y218S
354
3702.49
3099.73
1.19
0.58
0.43


Neutral
Y218G
355
504.65
460.32
1.10
0.08
0.06


Neutral
Y218E
346
511.24
471.64
1.08
0.08
0.07


Neutral
P219L
2868
578.24
550.18
1.05
0.09
0.08


Neutral
P219C
2856
622.59
613.51
1.01
0.10
0.09


Neutral
P219V
2867
586.82
583.21
1.01
0.09
0.08


Neutral
P219D
2851
819.59
881.94
0.93
0.13
0.12


Neutral
P219F
2863
571.45
542.25
1.05
0.09
0.08


Neutral
P219A
2869
1749.52
1799.14
0.97
0.27
0.25


Neutral
P219T
2859
870.52
853.07
1.02
0.14
0.12


Neutral
P219E
2852
895.73
858.50
1.04
0.14
0.12


Neutral
P219Q
2858
601.64
557.23
1.08
0.09
0.08


Neutral
P219R
2855
580.05
533.83
1.09
0.09
0.07


Neutral
P219H
2853
595.81
592.49
1.01
0.09
0.08


Neutral
P219G
2862
625.62
619.20
1.01
0.10
0.09


Neutral
P219K
2854
647.47
633.20
1.02
0.10
0.09


Neutral
P219S
2861
1549.48
1669.93
0.93
0.24
0.23


Neutral
P219W
2865
929.41
912.72
1.02
0.14
0.13


Down
S220R
2874
7949.20
10460.71
0.76
1.23
1.46


Neutral
S220A
2887
9804.98
9347.41
1.05
1.52
1.31


Neutral
S220Q
2878
9804.83
9328.79
1.05
1.52
1.30


Neutral
S220T
2877
9371.43
9378.23
1.00
1.46
1.31


Down
S220L
2886
1688.62
2607.71
0.65
0.26
0.36


Down
S220K
2873
2607.58
3704.87
0.70
0.40
0.52


Neutral
S220G
2880
9916.14
9356.60
1.06
1.54
1.31


Down
S220H
2872
1496.17
1874.12
0.80
0.23
0.26


Neutral
S220E
2871
3553.14
3992.18
0.89
0.55
0.56


Neutral
S220M
2882
7913.94
8545.54
0.93
1.23
1.20


Neutral
S220V
2885
10179.81
9414.03
1.08
1.58
1.32


Neutral
S220P
2888
592.32
587.51
1.01
0.09
0.08


Down
S220I
2884
6596.23
8678.69
0.76
1.02
1.21


Down
S220F
2881
2458.37
3612.28
0.68
0.38
0.51


Neutral
S220N
2876
10548.94
9399.76
1.12
1.64
1.31


Up
Y221W
2902
1201.23
891.46
1.35
0.19
0.12


Neutral
Y221K
2892
595.72
575.31
1.04
0.09
0.08


Neutral
Y221Q
2896
592.45
568.96
1.04
0.09
0.08


Neutral
Y221C
2894
583.88
558.96
1.04
0.09
0.08


Neutral
Y221N
2895
607.96
599.09
1.01
0.09
0.08


Neutral
Y221P
2907
575.23
546.02
1.05
0.09
0.08


Neutral
Y221V
2904
600.84
608.45
0.99
0.09
0.09


Neutral
Y221A
2906
613.20
571.57
1.07
0.10
0.08


Neutral
Y221G
2899
558.30
544.78
1.02
0.09
0.08


Neutral
Y221R
2893
508.18
483.59
1.05
0.08
0.07


Neutral
Y221S
2898
551.66
511.82
1.08
0.09
0.07


Up
Y221M
2901
733.99
576.28
1.27
0.11
0.08


Neutral
Y221T
2897
552.92
554.37
1.00
0.09
0.08


Neutral
Y221L
2905
600.40
544.47
1.10
0.09
0.08


Neutral
Y221E
2890
585.19
609.28
0.96
0.09
0.09


Down
T222L
2924
1251.44
1749.83
0.72
0.21
0.25


Down
T222Y
2916
3088.86
4344.09
0.71
0.52
0.61


Neutral
T222R
2912
7857.83
8130.34
0.97
1.33
1.14


Neutral
T222V
2923
6050.08
7520.37
0.80
1.03
1.06


Neutral
T222P
2926
9566.57
8477.71
1.13
1.62
1.19


Neutral
T222S
2917
8669.64
8464.76
1.02
1.47
1.19


Neutral
T222A
2925
5927.34
6623.26
0.89
1.00
0.93


Down
T222H
2910
4207.02
5413.49
0.78
0.71
0.76


Neutral
T222G
2918
7265.81
7630.73
0.95
1.23
1.07


Neutral
T222M
2920
4765.98
5354.04
0.89
0.81
0.75


Neutral
T222F
2919
8084.32
8023.96
1.01
1.37
1.13


Neutral
T222C
2913
1134.10
1047.66
1.08
0.19
0.15


Neutral
T222I
2922
489.93
514.09
0.95
0.08
0.07


Neutral
T222N
2914
8082.92
8215.81
0.98
1.37
1.15


Down
T222W
2921
4390.84
5903.99
0.74
0.74
0.83


Neutral
T222D
2908
4859.28
5584.93
0.87
0.53
0.73


Neutral
F223L
380
2776.43
3305.29
0.84
0.47
0.46


Neutral
F223T
372
7801.97
7792.62
1.00
1.32
1.09


Up
F223C
369
3115.11
2488.91
1.25
0.53
0.35


Neutral
F223R
368
5508.50
5094.99
1.08
0.93
0.71


Neutral
F223N
370
7434.46
6650.94
1.12
1.26
0.93


Neutral
F223P
382
8466.83
7678.71
1.10
1.43
1.08


Up
F223E
365
7194.34
5884.03
1.22
1.22
0.83


Up
F223G
375
3236.56
2599.04
1.25
0.55
0.36


Neutral
F223Q
371
8100.68
7468.16
1.08
1.37
1.05


Up
F223A
381
5226.86
3982.92
1.31
0.89
0.56


Up
F223S
374
6006.80
4916.07
1.22
1.02
0.69


Neutral
F223Y
373
9072.25
8479.33
1.07
1.54
1.19


Neutral
F223H
366
8573.59
8056.97
1.06
1.45
1.13


Neutral
F223K
367
4021.97
3712.91
1.08
0.60
0.49


Neutral
F223M
376
525.66
441.29
1.19
0.08
0.06


Neutral
S224G
2937
5580.59
6030.81
0.93
0.89
0.97


Neutral
S224T
2935
6189.79
7398.93
0.84
0.99
1.18


Neutral
S224Q
2934
7258.89
8221.79
0.88
1.16
1.32


Neutral
S224R
2931
4718.67
4984.94
0.95
0.76
0.80


Neutral
S224P
2945
475.19
459.57
1.03
0.08
0.07


Neutral
S224I
2941
5653.45
6319.33
0.89
0.90
1.01


Neutral
S224V
2942
4074.45
5042.87
0.81
0.65
0.81


Down
S224L
2943
4272.54
5590.35
0.76
0.68
0.89


Neutral
S224C
2932
4057.16
4912.59
0.83
0.65
0.79


Neutral
S224K
2930
7286.24
8122.32
0.90
1.17
1.30


Neutral
S224D
2927
7201.97
8490.41
0.85
1.15
1.36


Neutral
S224H
2929
5928.85
6787.33
0.87
0.95
1.09


Neutral
S224M
2939
5967.51
6770.07
0.88
0.95
1.08


Neutral
S224A
2944
469.39
427.21
1.10
0.08
0.07


Down
S224W
2940
4323.69
5971.55
0.72
0.69
0.96


Neutral
G225D
2946
4925.13
4615.89
1.07
0.83
0.65


Neutral
G225R
2950
6317.32
6775.84
0.93
1.07
0.95


Neutral
G225Q
2953
8693.50
8267.06
1.05
1.47
1.16


Neutral
G225M
2958
3626.70
3585.88
1.01
0.61
0.50


Neutral
G225P
2964
4775.00
4558.87
1.05
0.81
0.64


Neutral
G225W
2959
6452.91
7515.31
0.86
1.09
1.05


Neutral
G225S
2956
4811.54
4789.30
1.00
0.82
0.67


Neutral
G225E
2947
9174.21
8356.85
1.10
1.55
1.17


Neutral
G225V
2961
3525.03
3330.02
1.06
0.60
0.47


Neutral
G225T
2954
7463.15
7841.71
0.95
1.26
1.10


Neutral
G225K
2949
7135.01
7721.15
0.92
1.21
1.08


Neutral
G225N
2952
5858.96
5807.35
1.01
0.99
0.81


Neutral
G225C
2951
1631.86
1835.77
0.89
0.28
0.26


Neutral
G225H
2948
8719.92
8448.61
1.03
1.48
1.19


Neutral
G225A
2963
6048.29
5768.91
1.05
1.03
0.81


Neutral
D226S
2974
8608.26
8605.77
1.00
1.46
1.21


Neutral
D226W
2978
1817.34
2172.37
0.84
0.31
0.30


Down
D226R
2968
5584.63
7070.57
0.79
0.95
0.99


Neutral
D226A
2982
6987.67
7786.46
0.90
1.18
1.09


Neutral
D226N
2970
6464.01
7314.30
0.88
1.10
1.03


Neutral
D226T
2972
3450.45
4219.45
0.82
0.58
0.59


Neutral
D226E
2965
9308.62
8744.05
1.06
1.58
1.23


Neutral
D226L
2981
3411.80
4254.22
0.80
0.58
0.60


Neutral
D226P
2983
8574.77
8325.60
1.03
1.45
1.17


Neutral
D226H
2966
4217.71
4180.90
1.01
0.71
0.59


Neutral
D226G
2975
6320.31
7359.33
0.86
1.07
1.03


Neutral
D226I
2979
7753.45
8016.92
0.97
1.31
1.12


Neutral
D226M
2977
6501.53
7210.62
0.90
1.10
1.01


Neutral
D226V
2980
3680.55
4504.86
0.82
0.62
0.63


Neutral
D226C
2969
7227.15
7735.09
0.93
1.22
1.09


Neutral
V227A
400
5109.18
5056.19
1.01
0.87
0.71


Up
V227C
388
4040.96
3278.65
1.23
0.68
0.46


Up
V227D
383
1190.09
731.34
1.63
0.20
0.10


Up
V227E
384
5381.63
2605.20
2.07
0.91
0.37


Neutral
V227K
386
580.24
550.24
1.05
0.10
0.08


Up
V227L
399
4883.98
4000.68
1.22
0.83
0.56


Neutral
V227P
401
3682.22
3644.94
1.01
0.62
0.51


Up
V227S
393
3863.33
3131.47
1.23
0.65
0.44


Neutral
V227T
391
9817.63
8523.33
1.15
1.66
1.20


Up
V227W
397
1845.46
1374.06
1.34
0.31
0.19


Up
V227Y
392
657.68
542.07
1.21
0.11
0.08


Neutral
V227G
394
1040.74
883.01
1.18
0.15
0.12


Up
V227H
385
689.20
504.65
1.37
0.10
0.07


Up
V227Q
390
696.97
506.11
1.38
0.10
0.07


Neutral
V227R
387
664.31
561.06
1.18
0.10
0.07


Neutral
Q228A
419
9710.68
9175.62
1.06
4.50
3.13


Neutral
Q228D
402
10931.89
9274.00
1.18
5.06
3.16


Neutral
Q228E
403
9825.63
9396.31
1.05
4.55
3.20


Neutral
Q228G
412
9400.23
9058.34
1.04
4.35
3.09


Neutral
Q228H
404
9748.08
9288.74
1.05
4.51
3.17


Neutral
Q228K
405
9999.23
9262.11
1.08
4.63
3.16


Neutral
Q228L
418
9199.01
8900.02
1.03
4.26
3.03


Neutral
Q228M
414
9510.07
8915.79
1.07
4.40
3.04


Neutral
Q228N
408
8774.26
8679.09
1.01
4.06
2.96


Up
Q228P
420
2862.74
1291.55
2.22
1.33
0.44


Neutral
Q228R
406
7443.02
8091.71
0.92
3.45
2.76


Neutral
Q228S
411
8188.30
8162.27
1.00
3.79
2.78


Neutral
Q228T
409
4335.26
5179.47
0.84
2.01
1.77


Neutral
Q228W
415
6169.39
6508.89
0.95
2.86
2.22


Neutral
Q228Y
410
7426.87
7840.08
0.95
3.44
2.67


Neutral
L229R
425
485.95
489.74
0.99
0.22
0.17


Up
L229A
438
2627.78
2118.07
1.24
1.22
0.72


Up
L229T
429
3780.54
1464.25
2.58
1.75
0.50


Neutral
L229Q
428
5328.09
5303.89
1.00
2.47
1.81


Neutral
L229P
439
4795.14
5009.73
0.96
2.22
1.71


Neutral
L229E
422
737.30
657.34
1.12
0.34
0.22


Neutral
L229W
435
577.28
520.84
1.11
0.27
0.18


Neutral
L229M
434
3207.73
2829.20
1.13
1.49
0.96


Up
L229I
436
1158.56
828.94
1.40
0.54
0.28


Neutral
L229G
432
552.22
520.30
1.06
0.26
0.18


Up
L229C
426
633.99
516.61
1.23
0.29
0.18


Neutral
L229Y
430
549.90
504.60
1.09
0.25
0.17


Neutral
L229D
421
498.06
485.50
1.03
0.23
0.17


Neutral
L229H
423
551.54
501.77
1.10
0.26
0.17


Neutral
L229V
437
6249.58
6487.57
0.96
2.89
2.21


Up
A230L
704
3437.91
2154.62
1.60
1.59
0.73


Neutral
A230G
698
6804.87
8304.63
0.82
3.15
2.83


Neutral
A230W
701
4773.24
5118.69
0.93
2.21
1.74


Up
A230P
705
699.78
568.62
1.23
0.32
0.19


Neutral
A230D
687
7281.83
8033.59
0.91
3.37
2.74


Up
A230R
691
2986.52
2304.10
1.30
1.38
0.79


Up
A230I
702
4609.64
3490.44
1.32
2.13
1.19


Neutral
A230S
697
9181.71
8982.15
1.02
4.25
3.06


Neutral
A230C
692
5061.18
5781.86
0.88
2.34
1.97


Up
A230V
703
5030.94
3433.18
1.47
2.33
1.17


Neutral
A230T
695
8822.74
9169.52
0.96
4.08
3.13


Neutral
A230Y
696
3327.47
2858.53
1.16
1.54
0.97


Neutral
A230M
700
9543.01
9520.33
1.00
4.42
3.25


Neutral
A230N
693
9217.40
9384.02
0.98
4.27
3.20


Up
A230H
689
8514.47
6763.46
1.26
3.94
2.31


Neutral
Q231I
2998
4161.47
3993.24
1.04
1.93
1.36


Neutral
Q231A
3001
6899.50
6650.48
1.04
3.19
2.27


Neutral
Q231F
2995
4049.29
4120.56
0.98
1.87
1.40


Neutral
Q231P
3002
613.73
591.34
1.04
0.28
0.20


Neutral
Q231Y
2992
2460.04
2960.32
0.83
1.14
1.01


Down
Q231R
2988
366.16
1013.08
0.36
0.17
0.35


Up
Q231L
3000
3744.09
2834.26
1.32
1.73
0.97


Neutral
Q231D
2984
7507.92
7957.26
0.94
3.48
2.71


Neutral
Q231G
2994
5743.70
6012.88
0.96
2.66
2.05


Neutral
Q231V
2999
6114.28
6172.88
0.99
2.83
2.10


Neutral
Q231W
2997
4910.80
4767.26
1.03
2.27
1.62


Neutral
Q231S
2993
6593.10
7180.32
0.92
3.05
2.45


Neutral
Q231H
2986
4961.12
5622.05
0.88
2.30
1.92


Up
Q231C
2989
970.74
697.37
1.39
0.45
0.24


Down
Q231M
2996
3314.86
4166.20
0.80
1.53
1.42


Neutral
D232H
3004
6046.51
7174.55
0.84
2.80
2.45


Down
D232G
3013
5492.29
7079.04
0.78
2.54
2.41


Neutral
D232R
3006
5077.01
5692.86
0.89
2.35
1.94


Neutral
D232P
3021
7665.95
8291.54
0.92
3.55
2.83


Neutral
D232Y
3011
3001.62
3628.15
0.83
1.39
1.24


Neutral
D232N
3008
825.42
739.97
1.12
0.38
0.25


Up
D232S
3012
14389.74
5104.26
2.82
6.66
1.74


Neutral
D232F
3014
3599.26
3719.64
0.97
1.67
1.27


Neutral
D232V
3018
7938.31
9176.75
0.87
3.68
3.13


Neutral
D232K
3005
4844.31
5829.58
0.83
2.24
1.99


Neutral
D232W
3016
8404.13
9037.60
0.93
3.89
3.08


Neutral
D232Q
3009
7550.58
8008.46
0.94
3.50
2.73


Neutral
D232E
3003
9294.39
9251.91
1.00
4.30
3.15


Neutral
D232T
3010
9434.20
9583.53
0.98
4.37
3.27


Up
D232L
3019
7603.68
4213.70
1.80
3.52
1.44


Neutral
D233Q
446
653.34
640.95
1.02
0.30
0.22


Neutral
D233P
458
629.51
626.42
1.00
0.29
0.21


Neutral
D233S
449
637.89
623.07
1.02
0.30
0.21


Neutral
D233T
447
621.24
615.06
1.01
0.29
0.21


Neutral
D233A
457
650.58
634.46
1.03
0.30
0.22


Neutral
D233W
453
644.19
649.94
0.99
0.30
0.22


Neutral
D233G
450
657.96
666.27
0.99
0.30
0.23


Up
D233R
443
715.14
467.03
1.53
0.33
0.16


Up
D233E
440
2881.17
1918.57
1.50
1.33
0.65


Neutral
D233N
445
580.50
572.32
1.01
0.27
0.20


Neutral
D233V
455
609.36
603.42
1.01
0.28
0.21


Neutral
D233M
452
581.45
593.79
0.98
0.27
0.20


Neutral
D233L
456
584.42
597.47
0.98
0.27
0.20


Neutral
D233K
442
608.53
615.71
0.99
0.28
0.21


Neutral
D233I
454
682.66
661.78
1.03
0.32
0.23


Up
I234A
476
1458.10
1018.50
1.43
0.31
0.18


Up
I234T
467
1451.51
1188.67
1.22
0.31
0.21


Down
I234V
474
3474.82
5245.94
0.66
0.73
0.91


Up
I234W
473
743.35
570.02
1.30
0.16
0.10


Up
I234E
460
1301.06
840.09
1.55
0.27
0.15


Neutral
I234G
470
498.38
467.78
1.07
0.10
0.08


Down
I234L
475
2584.47
3312.61
0.78
0.54
0.57


Up
I234H
461
684.95
503.63
1.36
0.14
0.09


Down
I234M
472
3478.87
4732.62
0.74
0.73
0.82


Up
I234N
465
633.30
513.69
1.23
0.13
0.09


Neutral
I234Y
468
749.28
930.94
0.80
0.16
0.16


Neutral
I234P
477
470.41
431.33
1.09
0.10
0.07


Neutral
I234D
459
428.28
397.06
1.08
0.09
0.07


Up
I234Q
466
1095.18
837.53
1.31
0.23
0.15


Up
I234C
464
702.09
483.22
1.45
0.15
0.08


Neutral
D235H
3023
5217.44
6443.75
0.81
1.10
1.12


Neutral
D235G
3032
5966.03
6875.44
0.87
1.26
1.19


Down
D235A
3039
5874.20
9191.04
0.64
1.24
1.59


Neutral
D235P
3040
488.90
464.91
1.05
0.10
0.08


Neutral
D235L
3038
6353.97
6868.10
0.93
1.34
1.19


Down
D235V
3037
4167.59
6418.00
0.65
0.88
1.11


Neutral
D235E
3022
8377.14
8154.10
1.03
1.76
1.41


Neutral
D235R
3025
7249.34
7013.16
1.03
1.53
1.22


Neutral
D235Q
3028
6969.55
7752.26
0.90
1.47
1.34


Neutral
D235T
3029
6608.45
7282.18
0.91
1.39
1.26


Down
D235C
3026
3805.25
5237.38
0.73
0.80
0.91


Down
D235S
3031
3798.36
6310.00
0.60
0.80
1.09


Neutral
D235N
3027
6427.98
6780.43
0.95
1.35
1.18


Neutral
D235Y
3030
3539.06
3728.70
0.95
0.75
0.65


Neutral
D235I
3036
5390.78
5499.20
0.98
1.14
0.95


Neutral
G236M
3053
8157.95
7452.06
1.09
1.72
1.29


Neutral
G236R
3045
8890.85
8115.23
1.10
1.87
1.41


Neutral
G236D
3041
3820.99
4576.94
0.83
0.80
0.79


Neutral
G236S
3051
9887.69
8558.48
1.16
2.08
1.48


Down
G236T
3049
6244.13
7949.24
0.79
1.32
1.38


Neutral
G236C
3046
8441.80
7993.25
1.06
1.78
1.39


Neutral
G236K
3044
9473.18
8370.29
1.13
2.00
1.45


Neutral
G236E
3042
7240.97
7573.43
0.96
1.53
1.31


Up
G236P
3059
969.12
668.23
1.45
0.20
0.12


Neutral
G236I
3055
5356.96
5189.19
1.03
1.13
0.90


Down
G236Y
3050
4511.53
5725.61
0.79
0.95
0.99


Neutral
G236L
3057
8099.87
7699.72
1.05
1.71
1.34


Down
G236V
3056
4448.72
6422.58
0.69
0.94
1.11


Neutral
G236N
3047
8477.40
8088.17
1.05
1.79
1.40


Neutral
G236F
3052
5761.86
5560.83
1.04
1.21
0.96


Neutral
I237S
3070
587.31
536.19
1.10
0.12
0.09


Up
I237L
3076
2880.14
2240.61
1.29
0.61
0.39


Neutral
I237R
3064
572.70
548.66
1.04
0.12
0.10


Neutral
I237Q
3067
552.21
543.69
1.02
0.12
0.09


Neutral
I237K
3063
571.56
531.05
1.08
0.12
0.09


Neutral
I237D
3060
567.39
512.64
1.11
0.12
0.09


Down
I237A
3077
1512.93
2138.58
0.71
0.32
0.37


Neutral
I237T
3068
572.40
524.74
1.09
0.12
0.09


Neutral
I237E
3061
555.97
535.94
1.04
0.12
0.09


Neutral
I237C
3065
565.11
532.36
1.06
0.12
0.09


Neutral
I237G
3071
620.23
586.38
1.06
0.13
0.10


Neutral
I237P
3078
554.04
497.24
1.11
0.12
0.09


Neutral
I237Y
3069
688.92
602.65
1.14
0.15
0.10


Down
I237W
3074
4188.38
5663.94
0.74
0.62
0.75


Neutral
I237N
3066
5368.49
6271.59
0.86
0.80
0.83


Down
Q238G
3089
1382.45
2524.64
0.55
0.29
0.44


Down
Q238H
3081
3150.20
5045.01
0.62
0.66
0.88


Down
Q238S
3088
3298.60
4524.89
0.73
0.69
0.78


Down
Q238Y
3087
2078.90
2953.44
0.70
0.44
0.51


Down
Q238F
3090
1342.33
1916.87
0.70
0.28
0.33


Down
Q238E
3080
4075.95
5719.51
0.71
0.86
0.99


Down
Q238L
3095
3030.44
4771.52
0.64
0.64
0.83


Neutral
Q238W
3092
3649.81
4317.00
0.85
0.77
0.75


Neutral
Q238P
3097
568.68
548.50
1.04
0.12
0.10


Down
Q238R
3083
4199.78
5952.76
0.71
0.88
1.03


Down
Q238C
3084
3179.60
4072.46
0.78
0.67
0.71


Neutral
Q238N
3085
3119.13
3894.89
0.80
0.66
0.68


Down
Q238I
3093
3863.21
5191.15
0.74
0.81
0.90


Neutral
Q238T
3086
7425.48
7998.67
0.93
1.56
1.39


Down
Q238K
3082
4717.87
5952.01
0.79
0.99
1.03


Neutral
A239S
3108
7235.02
7630.43
0.95
1.52
1.32


Down
A239Q
3105
4817.25
8226.13
0.59
1.01
1.43


Down
A239T
3106
1232.13
2351.52
0.52
0.26
0.41


Neutral
A239P
3116
606.39
564.14
1.07
0.13
0.10


Neutral
A239V
3114
6075.94
6919.38
0.88
1.28
1.20


Neutral
A239L
3115
7174.28
7878.58
0.91
1.51
1.37


Neutral
A239Y
3107
5570.87
6668.58
0.84
1.17
1.16


Neutral
A239I
3113
6821.36
7628.60
0.89
1.44
1.32


Neutral
A239C
3103
4986.74
5916.10
0.84
1.05
1.03


Neutral
A239G
3109
6430.98
7617.20
0.84
1.35
1.32


Down
A239W
3112
2215.28
4554.04
0.49
0.47
0.79


Neutral
A239F
3110
719.92
750.33
0.96
0.15
0.13


Neutral
A239K
3101
8365.39
8161.53
1.02
1.76
1.42


Neutral
A239H
3100
6013.93
6892.53
0.87
1.27
1.20


Neutral
A239R
3102
8860.20
8322.26
1.06
1.87
1.44


Neutral
A239D
3098
9256.74
8197.40
1.13
1.02
1.08


Up
I240G
489
550.59
455.20
1.21
0.09
0.06


Neutral
I240Q
485
1050.40
921.68
1.14
0.16
0.12


Down
I240P
496
2259.38
3251.71
0.69
0.35
0.42


Down
I240R
482
2771.00
3465.26
0.80
0.43
0.44


Up
I240S
488
2033.91
1204.66
1.69
0.32
0.15


Neutral
I240K
481
5557.21
6183.54
0.90
0.87
0.79


Down
I240V
493
4682.76
6307.59
0.74
0.73
0.81


Neutral
I240D
478
480.83
456.39
1.05
0.08
0.06


Neutral
I240A
495
2099.13
1776.41
1.18
0.33
0.23


Up
I240C
483
970.78
650.04
1.49
0.15
0.08


Neutral
I240L
494
8303.04
8506.66
0.98
1.30
1.09


Down
I240F
490
1345.29
2090.14
0.64
0.21
0.27


Up
I240Y
487
1910.61
1482.53
1.29
0.30
0.19


Neutral
I240M
491
8056.10
7463.56
1.08
1.26
0.95


Neutral
I240T
486
2147.14
1862.29
1.15
0.34
0.24


Neutral
Y241V
3132
568.18
567.15
1.00
0.09
0.07


Neutral
Y241A
3134
514.00
498.25
1.03
0.08
0.06


Neutral
Y241G
3127
484.83
493.78
0.98
0.08
0.06


Neutral
Y241H
3119
555.41
547.86
1.01
0.09
0.07


Neutral
Y241R
3121
479.35
491.61
0.98
0.08
0.06


Neutral
Y241P
3135
542.62
468.74
1.16
0.09
0.06


Neutral
Y241Q
3124
494.42
468.65
1.05
0.08
0.06


Neutral
Y241L
3133
486.82
484.95
1.00
0.08
0.06


Neutral
Y241T
3125
574.08
548.16
1.05
0.09
0.07


Neutral
Y241S
3126
512.62
498.70
1.03
0.08
0.06


Neutral
Y241W
3130
592.83
556.32
1.07
0.09
0.07


Neutral
Y241N
3123
438.44
443.51
0.99
0.07
0.06


Neutral
Y241M
3129
488.24
448.13
1.09
0.08
0.06


Neutral
Y241I
3131
469.78
446.31
1.05
0.07
0.06


Neutral
Y241D
3117
454.99
443.57
1.03
0.07
0.06


Neutral
G242A
3153
1948.43
1965.86
0.99
0.31
0.25


Neutral
G242F
3147
2367.13
2861.31
0.83
0.37
0.37


Down
G242L
3152
4261.36
6043.02
0.71
0.67
0.77


Neutral
G242N
3142
1712.51
1893.88
0.90
0.27
0.24


Neutral
G242P
3154
2683.19
3311.87
0.81
0.42
0.42


Down
G242W
3149
1200.76
1522.34
0.79
0.19
0.19


Neutral
G242T
3144
1845.15
1926.23
0.96
0.29
0.25


Neutral
G242R
3140
1425.79
1462.84
0.97
0.22
0.19


Neutral
G242V
3151
1864.42
2075.51
0.90
0.29
0.27


Down
G242S
3146
3463.26
4491.54
0.77
0.54
0.57


Down
G242I
3150
881.04
2441.87
0.36
0.14
0.31


Neutral
G242Y
3145
895.61
928.34
0.96
0.14
0.12


Neutral
G242H
3138
1038.60
1063.68
0.98
0.16
0.14


Neutral
G242E
3137
1039.40
1200.19
0.87
0.16
0.15


Neutral
G242K
3139
1259.85
1404.80
0.90
0.20
0.18


Down
R243P
3173
3936.04
7438.61
0.53
0.62
0.95


Neutral
R243K
3158
8397.44
8514.77
0.99
1.32
1.09


Neutral
R243T
3162
7451.28
7306.32
1.02
1.17
0.93


Neutral
R243L
3171
6953.44
7458.28
0.93
1.09
0.95


Neutral
R243A
3172
8253.02
8378.15
0.99
1.29
1.07


Neutral
R243H
3157
6757.06
7710.25
0.88
1.06
0.99


Neutral
R243Q
3161
7563.55
8367.33
0.90
1.19
1.07


Neutral
R243S
3164
7872.26
8367.98
0.94
1.23
1.07


Down
R243I
3169
4421.12
8337.68
0.53
0.69
1.07


Neutral
R243C
3159
6128.24
6907.63
0.89
0.96
0.88


Neutral
R243N
3160
7064.71
7808.36
0.90
1.11
1.00


Neutral
R243Y
3163
6415.10
7427.20
0.86
1.01
0.95


Neutral
R243G
3165
9279.36
8697.39
1.07
1.46
1.11


Neutral
R243D
3155
5769.78
6318.28
0.91
0.90
0.81


Neutral
R243V
3170
6349.88
7531.41
0.84
1.00
0.96


Neutral
S244P
3192
7394.47
7844.16
0.94
1.16
1.00


Down
S244L
3190
3480.19
7154.31
0.49
0.55
0.91


Neutral
S244W
3187
10346.96
9035.98
1.15
1.62
1.15


Neutral
S244M
3186
728.14
748.02
0.97
0.11
0.10


Neutral
S244V
3189
6842.04
7456.02
0.92
1.07
0.95


Neutral
S244Q
3181
9318.66
8746.49
1.07
1.46
1.12


Neutral
S244D
3174
6915.01
7609.44
0.91
1.08
0.97


Neutral
S244E
3175
8814.39
8156.16
1.08
1.38
1.04


Neutral
S244T
3182
7442.34
8205.48
0.91
1.17
1.05


Down
S244H
3176
5019.42
8528.78
0.59
0.79
1.09


Neutral
S244G
3184
888.96
801.21
1.11
0.14
0.10


Neutral
S244A
3191
9174.68
8474.43
1.08
1.44
1.08


Neutral
S244F
3185
962.91
1017.49
0.95
0.15
0.13


Neutral
S244Y
3183
6333.20
6595.86
0.96
0.99
0.84


Neutral
S244R
3178
10483.23
9488.64
1.10
0.93
0.99


Neutral
Q245P
3211
8046.62
8690.91
0.93
1.26
1.11


Neutral
Q245I
3207
7611.43
8270.47
0.92
1.19
1.06


Down
Q245F
3204
3940.03
8048.83
0.49
0.62
1.03


Neutral
Q245V
3208
7785.27
8186.90
0.95
1.22
1.05


Up
Q245M
3205
494.18
323.62
1.53
0.08
0.04


Neutral
Q245T
3200
8684.29
8676.53
1.00
1.36
1.11


Neutral
Q245E
3194
10044.47
8646.78
1.16
1.58
1.10


Neutral
Q245S
3202
8700.39
8695.56
1.00
1.36
1.11


Neutral
Q245R
3197
8323.06
8629.37
0.96
1.31
1.10


Neutral
Q245G
3203
8495.47
8561.87
0.99
1.33
1.09


Neutral
Q245H
3195
8236.63
8640.32
0.95
1.29
1.10


Neutral
Q245L
3209
6762.99
6774.37
1.00
1.06
0.87


Neutral
Q245K
3196
347.86
290.28
1.20
0.05
0.04


Neutral
Q245W
3206
7517.93
8157.63
0.92
1.18
1.04


Neutral
Q245C
3198
7377.19
7707.00
0.96
1.16
0.98


Down
N246W
3225
3998.57
5256.41
0.76
0.55
0.68


Neutral
N246R
3216
6324.43
7263.78
0.87
0.87
0.93


Neutral
N246A
3229
7162.60
7821.64
0.92
0.98
1.00


Neutral
N246F
3223
5961.78
6704.16
0.89
0.82
0.86


Neutral
N246G
3222
7132.52
7954.86
0.90
0.98
1.02


Neutral
N246P
3230
5753.82
6382.30
0.90
0.79
0.82


Neutral
N246V
327
7113.82
7563.12
0.94
0.98
0.97


Neutral
N246Q
3218
8249.22
7962.93
1.04
1.13
1.02


Neutral
N246Y
3220
6460.04
7091.47
0.91
0.89
0.91


Neutral
N246C
3217
3668.07
4131.97
0.89
0.50
0.53


Neutral
N246I
3226
6761.84
6684.68
1.01
0.93
0.86


Neutral
N246L
3228
7470.87
7558.25
0.99
1.03
0.97


Neutral
N246S
3221
7681.59
7872.41
0.98
1.05
1.01


Neutral
N246T
3219
7476.51
7504.31
1.00
1.03
0.96


Neutral
N246K
3215
8008.77
7820.31
1.02
1.10
1.00


Neutral
N246D
3212
7062.38
7827.99
0.90
0.78
1.03


Neutral
P247A
3249
8242.00
7947.96
1.04
1.13
1.02


Neutral
P247D
3231
6640.01
7179.97
0.92
0.91
0.92


Neutral
P247E
3232
8181.45
7231.43
1.13
1.12
0.93


Neutral
P247F
3243
8964.42
7929.76
1.13
1.23
1.02


Neutral
P247G
3242
7256.65
7455.20
0.97
1.00
0.96


Neutral
P247H
3233
8093.84
7667.72
1.06
1.11
0.99


Neutral
P247I
3246
7375.24
7729.05
0.95
1.01
0.99


Neutral
P247K
3234
8454.74
7912.16
1.07
1.16
1.02


Neutral
P247L
3248
8316.29
8009.70
1.04
1.14
1.03


Neutral
P247N
3237
8142.72
8006.73
1.02
1.12
1.03


Neutral
P247Q
3238
8231.43
7739.72
1.06
1.13
0.99


Neutral
P247R
3235
7029.19
7443.10
0.94
0.96
0.96


Neutral
P247S
3241
8040.91
7895.89
1.02
1.10
1.01


Neutral
P247T
3239
7243.03
7527.94
0.96
0.99
0.97


Neutral
P247V
3247
7907.19
7717.20
1.02
1.08
0.99


Neutral
V248W
3264
6631.29
6916.87
0.96
0.91
0.89


Neutral
V248L
3266
8767.88
8252.54
1.06
1.20
1.06


Neutral
V248Q
3257
6709.44
6735.33
1.00
0.92
0.87


Neutral
V248M
3263
7437.73
7338.43
1.01
1.02
0.94


Neutral
V248Y
3259
6509.63
6927.69
0.94
0.89
0.89


Neutral
V248G
3261
6438.97
6744.48
0.95
0.88
0.87


Neutral
V248C
3255
3692.16
3816.99
0.97
0.51
0.49


Neutral
V248R
3254
7253.81
7153.37
1.01
1.00
0.92


Neutral
V248A
3267
8420.73
7739.03
1.09
1.16
0.99


Neutral
V248H
3252
8578.28
7867.88
1.09
1.18
1.01


Neutral
V248I
3265
8473.10
8209.35
1.03
1.16
1.05


Neutral
V248T
3258
8100.54
7751.03
1.05
1.11
1.00


Neutral
V248K
3253
7147.53
7653.87
0.93
0.98
0.98


Neutral
V248S
3260
6899.65
6729.67
1.03
0.95
0.86


Neutral
V248F
3262
6736.68
6651.01
1.01
0.92
0.85


Neutral
V248E
3251
9210.34
8235.44
1.12
1.01
1.08


Neutral
Q249T
3276
6909.47
7370.27
0.94
0.95
0.95


Neutral
Q249W
3282
10145.55
8691.89
1.17
1.39
1.12


Neutral
Q249R
373
7102.73
7103.74
1.00
0.97
0.91


Down
Q249E
3270
3987.22
7583.88
0.53
0.55
0.97


Neutral
Q249A
3286
8992.77
8414.65
1.07
1.23
1.08


Neutral
Q249P
3287
8376.56
8108.11
1.03
1.15
1.04


Neutral
Q249C
3274
5978.93
5496.03
1.09
0.82
0.71


Neutral
Q249G
3279
7612.71
7662.25
0.99
1.04
0.98


Neutral
Q249N
3275
7180.54
7257.66
0.99
0.99
0.93


Neutral
Q249K
3272
7772.72
7296.54
1.07
1.07
0.94


Neutral
Q249I
3283
7262.56
7159.06
1.01
1.00
0.92


Neutral
Q249Y
3277
6047.16
6053.08
1.00
0.83
0.78


Neutral
Q249V
3284
8717.93
8059.04
1.08
1.20
1.04


Neutral
Q249L
3285
6532.65
6824.78
0.96
0.90
0.88


Neutral
Q249H
3271
8441.70
7557.69
1.12
1.16
0.97


Neutral
P250L
3305
9455.47
8580.12
1.10
1.30
1.10


Neutral
P250S
3298
7684.90
7513.77
1.02
1.05
0.97


Neutral
P250R
3292
7701.81
7566.23
1.02
1.06
0.97


Neutral
P250Y
3297
7886.68
7534.22
1.05
1.08
0.97


Neutral
P250M
3301
8416.52
8221.73
1.02
1.15
1.06


Neutral
P250F
3300
8150.35
7703.24
1.06
1.12
0.99


Neutral
P250A
3306
8963.20
8460.94
1.06
1.23
1.09


Neutral
P250K
3291
7830.18
7732.04
1.01
1.07
0.99


Neutral
P250G
3299
7623.88
7834.34
0.97
1.05
1.01


Neutral
P250N
3294
7600.44
7961.88
0.95
1.04
1.02


Down
P250T
3296
1147.37
1489.99
0.77
0.16
0.19


Neutral
P250W
3302
7431.76
7755.84
0.96
1.02
1.00


Neutral
P250D
3288
7767.77
7525.09
1.03
1.07
0.97


Neutral
P250V
3304
7355.32
7719.82
0.95
1.01
0.99


Neutral
P250Q
3295
7797.52
8203.80
0.95
1.07
1.05


Down
I251A
3324
4953.41
8984.79
0.55
0.48
0.96


Neutral
I251Q
3314
10910.92
9221.40
1.18
1.07
0.98


Neutral
I251G
3318
11041.83
9640.57
1.15
1.08
1.03


Neutral
I251L
3323
11028.53
9408.72
1.17
1.08
1.00


Neutral
I251K
3310
11050.61
9421.73
1.17
1.08
1.01


Neutral
I251R
3311
10950.25
9220.62
1.19
1.07
0.98


Neutral
I251E
3308
10262.05
9115.62
1.13
1.00
0.97


Neutral
I251D
3307
10582.82
9557.71
1.11
1.04
1.02


Neutral
I251T
3315
10884.22
9485.20
1.15
1.06
1.01


Neutral
I251C
3312
10348.04
9428.04
1.10
1.01
1.01


Neutral
I251Y
3316
10319.00
9450.22
1.09
1.01
1.01


Neutral
I251P
3325
10762.38
9410.57
1.14
1.05
1.00


Neutral
I251S
3317
8445.88
7160.96
1.18
1.07
0.96


Neutral
I251W
3321
7305.95
6974.26
1.05
0.92
0.93


Neutral
I251V
3322
8343.83
7350.61
1.14
0.91
0.98


Neutral
G252F
3337
7921.80
7529.24
1.05
1.09
0.97


Neutral
G252W
3339
6989.36
7313.18
0.96
0.96
0.94


Neutral
G252A
3343
8567.46
8300.90
1.03
1.18
1.07


Neutral
G252R
3330
7756.55
7447.08
1.04
1.06
0.96


Neutral
G252L
3342
8684.63
8094.21
1.07
1.19
1.04


Neutral
G252E
3327
7651.86
7211.52
1.06
1.05
0.93


Neutral
G252D
3326
7977.50
7049.47
1.13
1.09
0.91


Neutral
G252K
3329
9685.27
8502.04
1.14
1.33
1.09


Neutral
G252S
3336
7596.71
6986.94
1.09
1.04
0.90


Neutral
G252T
3334
7242.98
7147.95
1.01
0.99
0.92


Neutral
G252P
3344
8175.79
8226.12
0.99
1.12
1.06


Neutral
G252H
3328
8030.53
7802.24
1.03
1.10
1.00


Neutral
G252C
3331
5540.29
5421.44
1.02
0.76
0.70


Neutral
G252V
3341
7910.50
7997.71
0.99
1.09
1.03


Neutral
G252I
3340
7702.75
7964.05
0.97
1.06
1.02


Neutral
P253C
3350
7906.13
8213.73
0.96
0.99
1.04


Neutral
P253G
3356
9640.00
8446.66
1.14
1.20
1.07


Neutral
P253Q
3352
9482.36
8631.24
1.10
1.18
1.09


Neutral
P253I
3360
6906.18
7721.21
0.89
0.86
0.97


Neutral
P253L
3362
8851.11
8489.29
1.04
1.10
1.07


Neutral
P253R
3349
9020.78
8580.86
1.05
1.12
1.08


Neutral
P253A
3363
8697.23
8410.29
1.03
1.08
1.06


Neutral
P253E
3346
9074.45
8476.99
1.07
1.13
1.07


Neutral
P253Y
3354
7935.28
8171.53
0.97
0.99
1.03


Neutral
P253W
3359
6635.85
7293.26
0.91
0.83
0.92


Neutral
P253M
3358
6895.66
7648.23
0.90
0.86
0.96


Neutral
P253V
3361
7058.87
7756.04
0.91
0.88
0.98


Neutral
P253T
3353
6728.25
7541.00
0.89
0.84
0.95


Neutral
P253K
3348
6929.49
7400.65
0.94
0.86
0.93


Neutral
P253N
3351
7354.73
7533.05
0.98
0.92
0.95


Neutral
Q254R
3368
9454.92
8474.29
1.12
1.18
1.07


Neutral
Q254G
3374
3549.45
3806.63
0.93
0.44
0.48


Neutral
Q254W
3377
3389.45
3326.38
1.02
0.42
0.42


Neutral
Q254T
3371
7491.28
7853.86
0.95
0.93
0.99


Neutral
Q254A
3381
7226.25
7451.70
0.97
0.90
0.94


Neutral
Q254F
3375
6263.95
6007.53
1.04
0.78
0.76


Neutral
Q254D
3364
9098.08
8154.92
1.12
1.13
1.03


Neutral
Q254P
3382
6827.99
7340.40
0.93
0.85
0.93


Neutral
Q254L
3380
7602.15
7940.64
0.96
0.95
1.00


Neutral
Q254C
3369
9284.18
8479.77
1.09
1.16
1.07


Neutral
Q254Y
3372
8847.02
7831.28
1.13
1.10
0.99


Neutral
Q254I
3378
9340.36
8662.75
1.08
1.16
1.09


Neutral
Q254E
3365
9466.76
8516.08
1.11
1.18
1.07


Neutral
Q254V
3379
9803.92
8575.31
1.14
1.22
1.08


Neutral
Q254S
3373
7768.13
8801.19
0.88
1.15
1.17


Neutral
T255I
3397
9880.58
8415.65
1.17
1.23
1.06


Neutral
T255Q
3390
9537.20
8410.86
1.13
1.19
1.06


Neutral
T255P
3401
7468.08
7296.37
1.02
0.93
0.92


Neutral
T255R
3387
5740.42
4974.50
1.15
0.72
0.63


Neutral
T255C
3388
2626.79
2503.21
1.05
0.33
0.32


Neutral
T255N
3389
5128.08
4479.75
1.14
0.64
0.57


Neutral
T255S
3392
7334.60
6905.71
1.06
0.91
0.87


Neutral
T255V
3398
5463.42
5187.78
1.05
0.68
0.65


Neutral
T255E
3384
7691.31
7194.23
1.07
0.96
0.91


Neutral
T255G
3393
8166.77
7682.14
1.06
1.02
0.97


Neutral
T255K
3386
6636.15
5647.18
1.18
0.83
0.71


Neutral
T255A
3400
4436.98
4322.98
1.03
0.55
0.55


Neutral
T255F
3394
3562.89
3107.64
1.15
0.44
0.39


Neutral
T255L
3399
4904.06
4266.71
1.15
0.61
0.54


Neutral
T255H
3385
8243.01
7352.60
1.12
1.22
0.98


Up
P256S
3412
10876.81
9018.60
1.21
1.36
1.14


Up
P256V
3418
10408.68
8594.11
1.21
1.30
1.08


Neutral
P256F
3414
6020.49
5181.94
1.16
0.75
0.65


Neutral
P256Y
3411
10270.90
8699.77
1.18
1.28
1.10


Neutral
P256I
3417
9089.54
7980.23
1.14
1.13
1.01


Neutral
P256A
3420
9426.67
8868.67
1.06
1.18
1.12


Neutral
P256L
3419
8342.08
7217.69
1.16
1.04
0.91


Neutral
P256G
3413
4631.84
4679.24
0.99
0.58
0.59


Neutral
P256N
3408
4406.75
3946.90
1.12
0.55
0.50


Neutral
P256R
3406
4975.17
4155.27
1.20
0.62
0.52


Neutral
P256Q
3409
6177.77
5546.92
1.11
0.77
0.70


Neutral
P256E
3403
9266.75
8366.07
1.11
1.16
1.06


Neutral
P256K
3405
5919.72
5928.31
1.00
0.74
0.75


Neutral
P256M
3415
8787.02
8554.52
1.03
1.10
1.08


Neutral
P256C
3407
4674.45
4633.67
1.01
0.69
0.62


Neutral
K257C
3425
4327.83
4267.45
1.01
0.54
0.54


Neutral
K257M
3433
5985.85
5236.20
1.14
0.75
0.66


Neutral
K257V
3436
7316.42
7115.65
1.03
0.91
0.90


Neutral
K257A
3438
9355.23
8528.52
1.10
1.17
1.08


Neutral
K257E
3422
10237.19
9141.73
1.12
1.28
1.15


Neutral
K257S
3430
9952.97
8464.79
1.18
1.24
1.07


Neutral
K257L
3437
10053.73
8711.61
1.15
1.25
1.10


Neutral
K257I
3435
8609.80
7806.76
1.10
1.07
0.98


Neutral
K257G
3431
8280.79
7718.36
1.07
1.03
0.97


Neutral
K257N
3426
8528.22
7707.49
1.11
1.06
0.97


Neutral
K257F
3432
7720.51
6633.90
1.16
0.96
0.84


Neutral
K257W
3434
7039.69
7120.56
0.99
0.88
0.90


Neutral
K257R
3424
9688.77
9114.18
1.06
1.21
1.15


Neutral
K257P
3439
8039.60
7464.88
1.08
1.19
0.99


Neutral
K257T
3428
9346.88
8849.42
1.06
1.39
1.18


Neutral
A258Q
3447
7000.31
6977.33
1.00
0.87
0.88


Neutral
A258Y
3449
6636.02
5998.83
1.11
0.83
0.76


Neutral
A258W
3454
9438.05
8527.86
1.11
1.18
1.08


Neutral
A258G
3451
7204.05
7778.97
0.93
0.90
0.98


Neutral
A258L
3457
1222.62
1226.40
1.00
0.15
0.15


Neutral
A258F
3452
9548.91
8531.04
1.12
1.19
1.08


Neutral
A258M
3453
8161.79
8061.20
1.01
1.02
1.02


Neutral
A258N
3446
7808.83
6968.56
1.12
0.97
0.88


Neutral
A258V
3456
8395.80
8391.43
1.00
1.05
1.06


Neutral
A258T
3448
8674.71
7958.00
1.09
1.08
1.00


Neutral
A258I
3455
8452.43
7509.34
1.13
1.05
0.95


Up
A258D
3440
7741.51
6346.88
1.22
0.97
0.80


Neutral
A258R
3444
9008.56
7908.51
1.14
1.12
1.00


Neutral
A258E
3441
10198.40
8709.16
1.17
1.27
1.10


Neutral
A258P
3458
10414.06
9178.82
1.13
1.55
1.22









The 199 hMMP-1 putative hit mutants were rescreened, using the same assay, and 104 primary hits were confirmed (see Table 11, below). hMMP-1 mutants that were active at 25° C. and had at least a 16% decrease in activity at 37° C. (e.g., the ratio of the activities at 25° C. or 37° C. (25° C./37° C.) is greater than or equal to 1.2) were deemed to be confirmed primary temperature sensitive hits.


Table 10, below, lists the hMMP-1 mutation, the average RFU at 25° C. and 37° C., and the ratio of the activities (25° C./37° C.). The Table also lists the temperature phenotype:DOWN, indicates the ratio (25° C./37° C.) of the activity of the mutant is decreased compared to the ratio (25° C./37° C.) of the activity of the wildtype, i.e. decreased greater than 16% the activity of the wildtype; NEUTRAL, indicates the ratio (25° C./37° C.) of the activity of the mutant is similar to the ratio (25° C./37° C.) of the activity of wildtype, i.e. within 16% of the activity of the wildtype; and UP, indicates the ratio (25° C./37° C.) of the activity of the mutant is increased compared to the ratio (25° C./37° C.) of the activity of the wildtype, i.e. increased more than 16% the activity of the wildtype.


Table 10, below, also lists the residual activities at 25° C. and 37° C., as compared to wild type hMMP-1. The residual activity is the ratio of the hMMP-1 mutant activity versus the wildtype hMMP-1 activity at the indicated temperature, either 25° C. or 37° C. A ratio of less than one indicates that a given mutant has less activity than the wildtype at the indicated temperature and a ratio of greater than one indicates that the mutant has more activity than the wildtype. Several of the hMMP-1 primary hit mutants exhibited activities that were comparable to or greater than wildtype hMMP-1 at 25° C. All of the hMMP-1 confirmed as primary hits exhibited decreated activities at 37° C., thereby reconfirming their decreased activity at elevated temperatures.









TABLE 10







Results of Initial Screen for Temperature Sensitive hMMP-1 mutants




















Res.






Avg.
Avg.

Act.
Res. Act.


Temp.
hMMP-1
SEQ
RFU
RFU
Ratio
Mut/wt
Mut/wt


Phenotype
mutation
ID NO
25° C.
37° C.
25° C./37° C.
25° C.
37° C.

















Neutral
T84F
847
6312.72
6453.46
0.98
1.10
1.27


Neutral
E85F
866
6092.47
6362.37
0.96
1.06
1.26


Up
L95K
6
1333.28
1191.46
1.12
0.15
0.14


Down
L95I
18
1707.98
2294.02
0.74
0.30
0.45


Down
R98D
1083
2905.96
3867.31
0.75
0.33
0.47


Down
I99Q
1109
3318.21
4623.91
0.72
0.37
0.56


Down
E100V
512
3980.72
5009.20
0.79
1.26
1.01


Neutral
E100R
500
7410.11
7964.52
0.93
0.83
0.96


Neutral
E100S
506
3768.09
4664.58
0.81
0.42
0.56


Neutral
E100T
504
6985.28
7478.12
0.93
0.79
0.90


Neutral
E100F
508
6709.27
7436.60
0.90
0.75
0.90


Neutral
E100I
511
8824.19
8458.79
1.04
0.99
1.02


Neutral
E100N
502
8809.68
8215.63
1.07
0.99
0.99


Neutral
T103Y
524
1181.09
1423.76
0.83
0.37
0.29


Neutral
P104A
1177
8861.30
8360.82
1.06
1.00
1.01


Up
P104M
1172
6709.44
7118.65
0.94
0.88
0.75


Up
D105A
39
2674.16
1227.06
2.18
0.65
0.24


Up
D105F
33
2009.56
1221.58
1.65
0.49
0.24


Up
D105G
32
2407.89
1686.68
1.43
0.58
0.34


Up
D105I
36
1732.38
1105.99
1.57
0.42
0.22


Up
D105L
38
1563.61
859.56
1.82
0.38
0.17


Up
D105N
27
3766.72
1475.08
2.55
0.91
0.29


Up
D105R
25
3892.02
2016.90
1.93
0.94
0.40


Up
D105S
31
3646.49
2727.22
1.34
0.88
0.54


Up
D105T
29
2513.64
1729.46
1.45
0.61
0.34


Up
D105W
35
2565.93
1855.05
1.38
0.62
0.37


Neutral
D105E
22
4000.92
3366.64
1.19
0.59
0.45


Neutral
L106C
1183
2995.56
3678.33
0.81
0.34
0.44


Neutral
L106S
1188
2730.64
2899.36
0.94
0.31
0.35


Neutral
A109H
1237
7206.01
7536.96
0.96
0.81
0.91


Neutral
D110A
1271
4179.59
5112.44
0.82
0.47
0.62


Neutral
V111R
1277
2401.69
2925.16
0.82
0.27
0.35


Neutral
D112S
1301
7203.69
7600.93
0.95
0.81
0.92


Neutral
A118T
1414
745.83
665.63
1.12
0.13
0.13


Down
S123V
1516
3220.29
4504.25
0.71
0.41
0.60


Neutral
N124D
1520
6218.73
6620.92
0.94
0.92
0.88


Neutral
T126S
1567
7114.42
6856.69
1.04
1.06
0.91


Up
G147P
1975
494.94
392.93
1.26
0.07
0.05


Up
R150P
59
2291.14
828.28
2.77
0.31
0.12


Neutral
R150V
56
6869.28
6604.61
1.04
1.20
1.30


Neutral
R150D
41
7230.41
6033.28
1.20
1.26
1.19


Down
R150I
55
3120.05
4082.34
0.76
0.39
0.55


Neutral
R150H
43
8281.04
8056.17
1.03
1.05
1.08


Up
D151G
70
1073.32
733.89
1.46
0.20
0.11


Neutral
N152A
2031
6669.94
5660.16
1.18
1.17
1.12


Down
N152S
2023
4607.85
8096.31
0.57
0.58
1.08


Neutral
S153T
543
10530.07
8798.72
1.20
1.44
1.24


Up
F155L
95
1322.13
864.19
1.53
0.25
0.13


Up
F155A
96
1250.93
760.12
1.65
0.23
0.11


Up
D156H
99
2722.09
2081.55
1.31
0.51
0.31


Up
D156L
114
2548.30
1597.53
1.60
0.48
0.24


Up
D156A
115
2679.29
1734.45
1.54
0.50
0.26


Up
D156W
111
1575.39
1268.36
1.24
0.30
0.19


Up
D156V
113
1400.88
766.80
1.83
0.26
0.11


Up
D156K
100
1292.89
966.62
1.34
0.24
0.14


Up
D156T
105
2871.09
1843.03
1.56
0.54
0.27


Up
D156R
101
2431.23
1545.89
1.57
0.46
0.23


Up
D156M
110
817.96
502.82
1.63
0.12
0.07


Neutral
P158T
2080
4204.23
3507.76
1.20
0.53
0.47


Neutral
P158G
2083
6277.86
5496.27
1.14
0.79
0.73


Neutral
P158K
2075
6860.82
6680.30
1.03
0.87
0.89


Neutral
P158N
2078
3656.04
3874.48
0.94
0.46
0.52


Up
G159V
132
2453.98
732.46
3.35
0.34
0.10


Up
G159T
125
5059.91
1734.12
2.92
0.69
0.24


Up
G159M
129
5905.06
4874.00
1.21
0.75
0.65


Neutral
G159I
131
5725.99
5357.20
1.07
0.72
0.72


Neutral
G159W
130
6787.40
6287.71
1.08
0.86
0.84


Neutral
G159L
133
8231.62
7638.64
1.08
1.04
1.02


Neutral
G159C
122
2897.77
3053.86
0.95
0.37
0.41


Neutral
P170D
2281
1434.38
1462.91
0.98
0.25
0.29


Neutral
P170A
2299
2733.72
2793.24
0.98
0.48
0.55


Up
G171P
572
1570.74
1204.39
1.30
0.27
0.17


Neutral
G171E
555
1154.96
1199.65
0.96
0.20
0.24


Neutral
G171D
554
791.81
690.33
1.15
0.14
0.14


Up
A176F
148
10486.82
6516.31
1.61
1.31
0.78


Neutral
A176W
150
482.38
414.85
1.16
0.06
0.06


Neutral
F178T
2403
560.54
487.01
1.15
0.10
0.10


Up
F178L
2411
1788.95
1314.38
1.36
0.31
0.26


Up
D179N
160
2433.73
812.01
3.00
0.26
0.10


Up
D179V
170
604.63
490.35
1.23
0.11
0.10


Up
D179C
159
613.81
503.76
1.22
0.11
0.10


Up
E180Y
182
6655.19
5379.42
1.24
0.72
0.63


Neutral
E180R
177
6932.51
6309.81
1.10
0.75
0.74


Up
E180T
181
3718.16
2425.13
1.53
0.40
0.29


Up
E180F
185
7014.78
5382.78
1.30
0.76
0.63


Up
E180G
184
5952.65
4547.28
1.31
1.04
0.90


Up
E180S
183
5217.80
3977.60
1.31
0.91
0.78


Up
E180N
179
6534.65
4843.84
1.35
1.14
0.96


Up
E180D
174
7738.70
6277.22
1.23
1.35
1.24


Neutral
D181T
200
6867.00
6057.09
1.13
0.74
0.71


Up
D181L
209
1727.20
1274.09
1.36
0.19
0.15


Up
D181K
195
1087.36
696.83
1.56
0.12
0.08


Up
D181C
197
549.29
447.40
1.23
0.10
0.09


Up
D181G
203
2764.20
2056.56
1.34
0.48
0.41


Up
E182T
219
2995.97
1779.42
1.68
0.32
0.21


Up
E182Q
218
1393.28
804.84
1.73
0.15
0.09


Up
E182M
224
649.73
524.43
1.24
0.11
0.10


Neutral
E182G
222
604.92
543.78
1.11
0.11
0.11


Up
R183G
2424
7326.36
6021.39
1.22
1.28
1.19


Up
R183S
2423
7896.17
6240.74
1.27
1.38
1.23


Up
T185R
235
1728.04
851.07
2.03
0.20
0.10


Up
T185Y
239
937.75
540.66
1.73
0.11
0.07


Up
T185H
233
1448.04
783.89
1.85
0.17
0.10


Up
T185G
241
3922.30
1990.15
1.97
0.46
0.24


Up
T185V
246
1648.14
897.66
1.84
0.19
0.11


Up
T185Q
238
1594.81
583.93
2.73
0.19
0.07


Up
T185A
248
1599.64
711.08
2.25
0.19
0.09


Up
T185E
232
1324.02
703.76
1.88
0.16
0.09


Neutral
T185D
231
485.86
418.67
1.16
0.06
0.06


Up
N187R
254
1042.36
709.74
1.47
0.12
0.09


Up
N187M
262
1731.67
995.07
1.74
0.20
0.12


Neutral
N187W
263
1694.86
1425.68
1.19
0.20
0.17


Up
N187F
261
1240.41
731.98
1.69
0.15
0.09


Up
N187K
253
2331.93
1140.19
2.05
0.27
0.14


Up
N187I
264
1444.98
683.03
2.12
0.17
0.08


Up
N187A
267
4379.80
2616.49
1.67
0.52
0.32


Neutral
N187G
260
535.06
514.10
1.04
0.07
0.07


Neutral
N187C
255
1804.28
1860.67
0.97
0.23
0.25


Neutral
N187H
252
1143.07
1071.67
1.07
0.14
0.14


Up
F188V
2486
7116.29
5860.00
1.21
1.24
1.16


Neutral
R189N
2495
7842.39
6675.36
1.17
1.37
1.32


Neutral
R189T
2497
7610.10
6459.94
1.18
1.33
1.27


Neutral
R189Q
2496
7465.37
6396.79
1.17
1.30
1.26


Up
E190G
583
5313.99
4365.93
1.22
0.75
0.48


Up
E190Y
581
7243.54
5742.33
1.26
1.27
1.13


Up
E190D
573
7910.21
6468.78
1.22
1.38
1.28


Up
Y191V
607
1553.58
1254.11
1.24
0.19
0.14


Up
N192H
613
2274.24
1058.80
2.15
0.32
0.12


Up
N192S
620
2043.65
1630.74
1.25
0.29
0.18


Up
N192D
611
4213.33
2216.40
1.90
0.59
0.24


Up
N192C
616
1310.46
987.31
1.33
0.18
0.11


Neutral
H194P
648
5264.79
5058.19
1.04
0.74
0.56


Up
R195C
273
4231.32
1853.20
2.28
0.60
0.20


Neutral
R195W
282
5099.23
4524.84
1.13
0.72
0.50


Neutral
R195L
285
5073.57
4520.73
1.12
0.72
0.50


Up
R195G
279
5269.21
3025.93
1.74
0.74
0.33


Up
R195Q
275
1958.69
1361.83
1.44
0.28
0.15


Up
R195A
286
5605.90
3852.81
1.46
0.79
0.42


Up
R195D
269
2724.53
1907.81
1.43
0.38
0.21


Up
R195V
284
1711.48
1037.62
1.65
0.24
0.11


Up
A197C
2552
4012.80
3140.52
1.28
0.70
0.62


Neutral
A198G
299
2610.82
2368.26
1.10
0.37
0.26


Up
A198L
305
1339.94
726.74
1.84
0.19
0.08


Up
A198M
301
1384.46
999.55
1.39
0.20
0.11


Up
G206A
324
4554.61
2702.11
1.69
0.47
0.30


Up
G206S
317
1226.37
919.66
1.33
0.13
0.10


Up
L207R
653
3476.88
1332.44
2.61
0.36
0.15


Neutral
L207V
665
656.95
550.54
1.19
0.08
0.07


Neutral
L207I
664
645.37
550.32
1.17
0.08
0.07


Up
L207G
660
610.01
484.35
1.26
0.08
0.06


Neutral
S208R
2703
7639.06
6465.10
1.18
1.34
1.28


Up
S208L
2715
7811.78
6354.14
1.23
1.37
1.25


Up
S210V
341
1190.35
856.63
1.39
0.29
0.17


Neutral
S210A
343
1682.05
1546.97
1.09
0.25
0.21


Neutral
T211L
2753
2376.23
2102.07
1.13
0.35
0.28


Up
D212G
678
1011.62
657.28
1.54
0.24
0.13


Neutral
D212H
669
4696.49
4001.41
1.17
0.70
0.53


Up
Y218S
354
3702.49
3099.73
1.19
0.58
0.43


Up
F223C
369
3115.11
2488.91
1.25
0.53
0.35


Up
F223E
365
7194.34
5884.03
1.22
1.22
0.83


Up
F223G
375
3236.56
2599.04
1.25
0.55
0.36


Up
F223A
381
5226.86
3982.92
1.31
0.89
0.56


Up
F223S
374
6006.80
4916.07
1.22
1.02
0.69


Neutral
F223K
367
4021.97
3712.91
1.08
0.60
0.49


Neutral
F223M
376
525.66
441.29
1.19
0.08
0.06


Up
V227C
388
4040.96
3278.65
1.23
0.68
0.46


Up
V227D
383
1190.09
731.34
1.63
0.20
0.10


Up
V227E
384
5381.63
2605.20
2.07
0.91
0.37


Up
V227L
399
4883.98
4000.68
1.22
0.83
0.56


Up
V227S
393
3863.33
3131.47
1.23
0.65
0.44


Up
V227W
397
1845.46
1374.06
1.34
0.31
0.19


Neutral
V227G
394
1040.74
883.01
1.18
0.15
0.12


Up
V227H
385
689.20
504.65
1.37
0.10
0.07


Up
V227Q
390
696.97
506.11
1.38
0.10
0.07


Neutral
V227R
387
664.31
561.06
1.18
0.10
0.07


Up
Q228P
420
2862.74
1291.55
2.22
1.33
0.44


Up
L229A
438
2627.78
2118.07
1.24
1.22
0.72


Up
L229T
429
3780.54
1464.25
2.58
1.75
0.50


Up
L229I
436
1158.56
828.94
1.40
0.54
0.28


Up
A230V
703
5030.94
3433.18
1.47
2.33
1.17


Up
D233E
440
2881.17
1918.57
1.50
1.33
0.65


Up
I234A
476
1458.10
1018.50
1.43
0.31
0.18


Up
I234T
467
1451.51
1188.67
1.22
0.31
0.21


Up
I234E
460
1301.06
840.09
1.55
0.27
0.15


Up
I234Q
466
1095.18
837.53
1.31
0.23
0.15


Up
I237L
475
2880.14
2240.61
1.29
0.61
0.39


Down
I237W
3074
4188.38
5663.94
0.74
0.62
0.75


Neutral
I237N
3066
5368.49
6271.59
0.86
0.80
0.83


Up
I240S
488
2033.91
1204.66
1.69
0.32
0.15


Neutral
I240A
495
2099.13
1776.41
1.18
0.33
0.23


Up
I240C
483
970.78
650.04
1.49
0.15
0.08


Neutral
I251S
3317
8445.88
7160.96
1.18
1.07
0.96


Neutral
I251W
3321
7305.95
6974.26
1.05
0.92
0.93


Neutral
Q254S
3373
7768.13
8801.19
0.88
1.15
1.17


Neutral
T255H
3385
8243.01
7352.60
1.12
1.22
0.98


Neutral
P256C
3407
4674.45
4633.67
1.01
0.69
0.62


Neutral
K257P
3439
8039.60
7464.88
1.08
1.19
0.99


Neutral
K257T
3428
9346.88
8849.42
1.06
1.39
1.18


Neutral
A258P
3458
10414.06
9178.82
1.13
1.55
1.22
















TABLE 11







Reconfirmed HITs











Temperature
hMMP-1
SEQ ID



Phenotype
mutation
NO















Up
L95K
6



Down
E100V
512



Neutral
T103Y
524



Up
D105A
39



Up
D105F
33



Up
D105G
32



Up
D105I
36



Up
D105L
38



Up
D105N
27



Up
D105R
25



Up
D105S
31



Up
D105T
29



Up
D105W
35



Up
R150P
59



Up
D151G
70



Neutral
S153T
543



Up
F155L
95



Up
F155A
96



Up
D156H
99



Up
D156L
114



Up
D156A
115



Up
D156W
111



Up
D156V
113



Up
D156K
100



Up
D156T
105



Up
D156R
101



Up
G159V
132



Up
G159T
125



Up
G171P
572



Up
A176F
148



Up
D179N
160



Up
E180Y
182



Neutral
E180R
177



Up
E180T
181



Up
E180F
185



Neutral
D181T
200



Up
D181L
209



Up
D181K
195



Up
E182T
219



Up
E182Q
218



Up
T185R
235



Up
T185Y
239



Up
T185H
233



Up
T185G
241



Up
T185V
246



Up
T185Q
238



Up
T185A
248



Up
T185E
232



Up
N187R
254



Up
N187M
262



Neutral
N187W
263



Up
N187F
261



Up
N187K
253



Up
N187I
264



Up
N187A
267



Up
E190G
583



Up
Y191V
607



Up
N192H
613



Up
N192S
620



Up
N192D
611



Up
N192C
616



Neutral
H194P
648



Up
R195C
273



Neutral
R195W
282



Neutral
R195L
285



Up
R195G
279



Up
R195Q
275



Up
R195A
286



Up
R195D
269



Up
R195V
284



Neutral
A198G
299



Up
A198L
305



Up
A198M
301



Up
G206A
324



Up
G206S
317



Up
L207R
653



Up
S210V
341



Up
D212G
678



Up
Y218S
354



Up
F223C
369



Up
F223E
365



Up
F223G
375



Up
F223A
381



Up
F223S
374



Up
V227C
388



Up
V227D
383



Up
V227E
384



Up
V227L
399



Up
V227S
393



Up
V227W
397



Up
Q228P
420



Up
L229A
438



Up
L229T
429



Up
L229I
436



Up
A230V
703



Up
D233E
440



Up
I234A
476



Up
I234T
467



Up
I234E
460



Up
I234Q
466



Up
I237L
475



Up
I240S
488



Neutral
I240A
495



Up
I240C
483










B. 14-mL Protein Expression

In this example, the hMMP-1 mutants that were identified as temperature sensitive primary hits in Example 2 were expressed in 14 ml culture tubes and their enzymatic activity was measured at 25° C., 34° C. and 37° C. for 1 hour, 2 hours or overnight in order to verify the desired phenotype of decreased activity at elevated temperatures. Protein was expressed and purified as in Example 1 with the exception that the expression was performed in 14 ml tubes rather than a 96-well plate.


Four (4) μl of each hMMP-1 mutant supernatant was transferred to a 96-well microplate. Supernatants were activated with APMA as described in Example 2A above, except that the solution was incubated at the reaction temperature of 25° C., 34° C., or 37° C. for 2 hours. As above, following activation, 100 μl of TCNB containing 10 μM Mca-K-P-L-G-L-Dpa-A-R-NH2 fluorescent substrate was added to each tube at the indicated reaction temperature (25° C., 34° C. or 37° C.) for one hour. Wildtype hMMP-1 was used as a positive control and supernatant from cells transformed with the vector was used as a negative control. Fluorescence was detected by measuring fluorescence in a fluorescent plate reader at 320 nm exitation/405 nm emission. Relative fluorescence units (RFU) were determined. Duplicate reactions were performed for each sample, reaction temperature, and positive and negative control.


The data is shown in Table 12A (1 hour incubation); Table 12B (2 hour incubations) and Table 12C (overnight incubation), below. Mutants that were active at 25° C. but demonstrated at least a 33% decreased activity at 34° C. or 37° C. (i.e. had a ratio of activity at 25° C. and 34° C. or a ratio of activity of 25° C. and 37° C. equal to or greater than 1.5 under any of the time point conditions tested were identified as temperature sensitive hits. Tables 12A-12C, below, list the hMMP-1 mutation, the RFU at 25° C., 34° C. and 37° C., and the ratio of the activities (at both 25° C./34° C. and 25° C./37° C.) of 64 hMMP-1 mutants whose decreased enzymatic activity at elevated temperatures were confirmed. Some of the hMMP-1 mutants, were noticeably more active at 25° C. than at an elevated temperature. For example, hMMP-1 mutant D179N (SEQ ID NO:160) was 87.5% more active at 25° C. than 37° C. after an overnight incubation (see e.g. Table 12C). Additionally, although expression levels, and therefore overall RFU values, varied in different experiments, the ratios of the activities remained the same. For example, mutant D156T was tested twice (see Table 12C below) and although each test gave different data RFU values the ratio of the values were similar and consistently within the 1.5 ratio parameter.









TABLE 12A







Temperature Sensitive hMMP-1 Mutants, 1 hour incubation













hMMP-1
SEQ ID
RFU
RFU
RFU
Ratio
Ratio


mutation
NO
25° C.
34° C.
37° C.
25° C./34° C.
25° C./37° C.
















L95K
6
2677.64
553.00
572.70
4.84
4.68


D105A
39
3496.48
697.79
1119.92
5.01
3.12


D105F
33
1749.85
554.69
685.49
3.15
2.55


D105G
32
7450.35
2196.32
3514.50
3.39
2.12


D105I
36
4720.96
638.42
943.44
7.39
5.00


D105L
38
2636.80
490.04
552.90
5.38
4.77


D105N
27
7487.95
776.33
1513.73
9.65
4.95


D105R
25
1732.70
641.23
736.92
2.70
2.35


D105S
31
8637.40
3782.36
6510.05
2.28
1.33


D105W
35
4263.51
1321.69
2422.77
3.23
1.76


D105T
29
2666.45
770.72
1685.33
3.46
1.58


R150P
59
7568.19
1678.59
2010.33
4.51
3.76


D151G
70
973.47
517.98
595.63
1.88
1.63


F155A
96
1800.92
592.07
596.31
3.04
3.02


D156K
100
8718.91
1733.90
1839.60
5.03
4.74


D156T
105
8034.06
2216.02
2255.25
3.63
3.56


D156L
114
1825.01
528.43
619.10
3.45
2.95


D156A
115
1495.21
450.17
496.04
3.32
3.01


D156W
111
1006.97
463.48
493.84
2.17
2.04


D156V
113
1140.60
484.30
504.38
2.36
2.26


D156T
105
2796.00
581.90
743.53
4.80
3.76


D156H
99
3489.60
578.59
711.59
6.03
4.90


D156R
101
4983.67
678.23
734.95
7.35
6.78


G159V
132
3416.77
705.80
739.87
4.84
4.62


G159T
125
4081.99
1732.63
1865.15
2.36
2.19


A176F
148
967.31
539.31
517.16
1.79
1.87


D179N
160
4105.85
492.00
513.37
8.35
8.00


E180Y
182
8803.90
3904.31
5268.18
2.25
1.67


E180T
181
5957.38
1155.89
1430.72
5.15
4.16


E180F
185
7484.41
2677.89
3141.69
2.79
2.38


D181L
209
1629.22
559.04
549.09
2.91
2.97


D181K
195
844.40
570.98
569.44
1.48
1.48


E182T
219
2244.96
653.93
668.01
3.43
3.36


E182Q
218
1066.68
583.87
582.84
1.83
1.83


T185R
235
1599.19
867.00
872.66
1.84
1.83


T185H
233
3616.30
1601.20
1842.01
2.26
1.96


T185Q
238
4365.21
1512.02
1899.46
2.89
2.30


T185A
248
1374.00
567.04
608.05
2.42
2.26


T185E
232
2145.28
1263.20
1399.76
1.70
1.53


N187R
254
1659.90
955.75
1054.91
1.74
1.57


N187M
262
2842.50
1343.95
1464.36
2.12
1.94


N187F
261
1846.10
716.62
786.07
2.58
2.35


N187K
253
2428.31
1703.73
1914.84
1.43
1.27


N187I
264
2455.44
717.51
773.59
3.42
3.17


R195V
284
3121.02
1947.80
2132.94
1.60
1.46


A198L
305
4547.61
1570.19
2061.87
2.90
2.21


A198M
301
1948.92
1101.86
1535.22
1.77
1.27


G206A
324
667.50
543.90
540.79
1.23
1.23


G206S
317
608.46
427.44
412.07
1.42
1.48


S210V
341
1952.12
961.54
1791.55
2.03
1.09


Y218S
354
1674.47
1531.03
1573.00
1.09
1.06


F223E
365
5837.16
2747.99
4955.08
2.12
1.18


V227C
388
1138.96
684.05
722.68
1.67
1.58


V227E
384
5892.76
653.81
803.12
9.01
7.34


V227W
397
716.50
607.92
646.75
1.18
1.11


Q228P
420
676.11
488.99
495.88
1.38
1.36


L229T
429
768.59
492.66
491.49
1.56
1.56


L229I
436
1470.04
753.87
1231.17
1.95
1.19


D233E
440
1195.07
959.25
1056.45
1.25
1.13


I234A
476
1402.15
1014.61
1127.63
1.38
1.24


I234T
467
857.79
644.52
712.49
1.33
1.20


I234E
460
2281.82
591.10
762.52
3.86
2.99


I240S
488
2678.36
776.88
1314.40
3.45
2.04


I240C
483
1540.91
474.82
666.63
3.25
2.31
















TABLE 12B







Temperature Sensitive hMMP-1 Mutants, 2 hours incubation













hMMP-1
SEQ ID
RFU
RFU
RFU
Ratio
Ratio


mutation
NO
25° C.
34° C.
37° C.
25° C./34° C.
25° C./37° C.
















L95K
6
4650.42
748.29
746.89
6.21
6.23


D105A
39
5669.31
824.07
1336.14
6.88
4.24


D105F
33
2980.00
623.89
818.63
4.78
3.64


D105G
32
8821.81
2759.24
4313.40
3.20
2.05


D105I
36
6832.34
780.32
1110.07
8.76
6.15


D105L
38
4206.38
534.24
607.46
7.87
6.92


D105N
27
8920.05
918.13
1727.44
9.72
5.16


D105R
25
2821.20
722.46
813.68
3.90
3.47


D105S
31
9355.63
4607.18
7274.97
2.03
1.29


D105W
35
6663.80
1690.93
3081.59
3.94
2.16


D105T
29
4457.16
974.63
2220.03
4.57
2.01


R150P
59
8750.30
2315.11
2497.86
3.78
3.50


D151G
70
1264.62
589.27
616.51
2.15
2.05


F155A
96
2824.01
779.72
746.59
3.62
3.78


D156K
100
8576.47
2210.63
2310.30
3.88
3.71


D156T
105
8727.27
2679.17
2752.35
3.26
3.17


D156L
114
2916.24
576.84
688.08
5.06
4.24


D156A
115
2299.63
533.68
554.21
4.31
4.15


D156W
111
1502.86
539.74
575.12
2.78
2.61


D156V
113
1593.06
534.71
542.36
2.98
2.94


D156T
105
4469.68
690.87
848.14
6.47
5.27


D156H
99
5387.79
698.77
819.82
7.71
6.57


D156R
101
7020.81
793.83
872.40
8.84
8.05


G159V
132
4673.44
856.78
838.46
5.45
5.57


G159T
125
6704.95
2294.40
2347.74
2.92
2.86


A176F
148
1609.85
654.43
618.72
2.46
2.60


D179N
160
5660.69
644.51
656.31
8.78
8.63


E180Y
182
8557.09
4979.24
6079.36
1.72
1.41


E180T
181
7870.99
1532.35
1794.15
5.14
4.39


E180F
185
8508.13
3597.75
3975.22
2.36
2.14


D181L
209
2710.97
619.39
611.92
4.38
4.43


D181K
195
1130.63
625.01
608.68
1.81
1.86


E182T
219
3702.08
791.23
826.28
4.68
4.48


E182Q
218
1331.50
639.84
623.11
2.08
2.14


T185R
235
2637.31
1187.63
1183.37
2.22
2.23


T185H
233
5593.77
2278.26
2534.15
2.46
2.21


T185Q
238
7006.87
2250.58
2642.74
3.11
2.65


T185A
248
2474.96
663.82
707.09
3.73
3.50


T185E
232
3948.43
2088.15
2091.32
1.89
1.89


N187R
254
3006.08
1352.97
1421.87
2.22
2.11


N187M
262
4934.44
1811.35
1893.07
2.72
2.61


N187F
261
3227.96
877.21
931.04
3.68
3.47


N187K
253
4182.49
2425.34
2652.79
1.72
1.58


N187I
264
4218.55
849.11
887.80
4.97
4.75


R195V
284
4847.81
2724.92
2984.10
1.78
1.62


A198L
305
6756.76
2056.50
2642.76
3.29
2.56


A198M
301
3777.50
1708.61
2155.58
2.21
1.75


G206A
324
872.27
603.01
586.57
1.45
1.49


G206S
317
932.69
492.65
463.60
1.89
2.01


S210V
341
3349.95
1249.47
2314.86
2.68
1.45


Y218S
354
2878.50
2373.98
2350.27
1.21
1.22


F223E
365
8318.70
3685.68
6209.93
2.26
1.34


V227C
388
1998.67
950.01
992.19
2.10
2.01


V227E
384
7904.54
839.00
1015.12
9.42
7.79


V227W
397
996.55
729.20
787.87
1.37
1.26


Q228P
420
1082.56
607.78
586.63
1.78
1.85


L229T
429
1221.05
580.15
564.49
2.10
2.16


L229I
436
2790.27
1050.86
1803.44
2.66
1.55


D233E
440
2195.02
1393.95
1454.71
1.57
1.51


I234A
476
2375.42
1473.70
1594.08
1.61
1.49


I234T
467
1199.18
713.83
796.81
1.68
1.50


I234E
460
3920.02
705.86
923.57
5.55
4.24


I240S
488
3867.71
973.97
1575.05
3.97
2.46


I240C
483
2688.75
561.91
853.66
4.78
3.15
















TABLE 12C







Temperature Sensitive hMMP-1 Mutants, Overnight


incubation













hMMP-1
SEQ ID
RFU
RFU
RFU
Ratio
Ratio


mutation
NO
25° C.
34° C.
37° C.
25° C./34° C.
25° C./37° C.
















L95K
6
7744.34
1803.12
1677.96
4.29
4.62


D105A
39
8466.62
1302.84
1931.17
6.50
4.38


D105F
33
6725.59
938.60
1173.23
7.17
5.73


D105G
32
8940.06
3560.75
5390.32
2.51
1.66


D105I
36
8394.32
1614.57
1958.96
5.20
4.29


D105L
38
6546.78
957.95
1070.51
6.83
6.12


D105N
27
9119.04
1459.16
2347.74
6.25
3.88


D105R
25
5775.25
1407.06
1499.57
4.10
3.85


D105S
31
9300.85
5584.70
8234.95
1.67
1.13


D105W
35
8617.36
2851.22
4593.06
3.02
1.88


D105T
29
7910.47
1899.25
3292.01
4.17
2.40


R150P
59
9011.11
3533.16
3559.66
2.55
2.53


D151G
70
1956.65
959.80
1097.68
2.04
1.78


F155A
96
4891.89
2016.76
1843.31
2.43
2.65


D156K
100
8696.27
3968.92
3858.90
2.19
2.25


D156T
105
8972.20
3971.43
3854.84
2.26
2.33


D156L
114
5254.55
972.64
1232.94
5.40
4.26


D156A
115
3585.37
1098.25
1110.73
3.26
3.23


D156W
111
2570.24
1091.27
1206.22
2.36
2.13


D156V
113
2208.99
954.21
997.64
2.31
2.21


D156T
105
7229.28
1256.02
1540.11
5.76
4.69


D156H
99
7587.19
1451.49
1763.27
5.23
4.30


D156R
101
8622.23
1735.02
1846.71
4.97
4.67


G159V
132
6555.27
1821.53
1683.20
3.60
3.89


G159T
125
9105.95
3210.57
3160.07
2.84
2.88


A176F
148
4191.69
1414.21
1336.32
2.96
3.14


D179N
160
7317.57
1504.84
1485.28
4.86
4.93


E180Y
182
9281.77
6080.89
6894.61
1.53
1.35


E180T
181
8475.04
2585.89
2809.15
3.28
3.02


E180F
185
9360.74
5183.25
5335.15
1.81
1.75


D181L
209
4534.34
1078.98
1000.80
4.20
4.53


D181K
195
1869.47
946.27
928.55
1.98
2.01


E182T
219
6752.25
1483.52
1496.55
4.55
4.51


E182Q
218
2212.75
1065.07
1035.24
2.08
2.14


T185R
235
6281.97
2425.71
2300.61
2.59
2.73


T185H
233
8531.85
3164.69
3515.59
2.70
2.43


T185Q
238
9044.23
3639.00
4012.93
2.49
2.25


T185A
248
6156.97
1110.68
1059.61
5.54
5.81


T185E
232
8479.18
3868.06
3892.33
2.19
2.18


N187R
254
7593.11
2415.63
2370.01
3.14
3.20


N187M
262
8605.76
2769.52
2720.28
3.11
3.16


N187F
261
7352.85
1612.23
1704.23
4.56
4.31


N187K
253
8667.36
3458.94
3709.62
2.51
2.34


N187I
264
8306.40
1459.25
1465.77
5.69
5.67


R195V
284
8634.05
4648.03
4960.91
1.86
1.74


A198L
305
8795.36
3469.36
4181.78
2.54
2.10


A198M
301
8352.73
3215.69
3637.79
2.60
2.30


G206A
324
2492.53
1038.14
974.96
2.40
2.56


G206S
317
2845.84
908.82
808.42
3.13
3.52


S210V
341
7104.17
2441.96
3939.90
2.91
1.80


Y218S
354
7740.61
4057.37
4093.29
1.91
1.89


F223E
365
9650.44
4849.58
7645.34
1.99
1.26


V227C
388
5833.84
2207.20
2432.82
2.64
2.40


V227E
384
8630.90
2283.07
2152.81
3.78
4.01


V227W
397
3070.92
1370.13
1456.45
2.24
2.11


Q228P
420
3673.33
1162.95
1081.32
3.16
3.40


L229T
429
3543.75
1103.34
1030.05
3.21
3.44


L229I
436
7333.92
1832.18
3268.93
4.00
2.24


D233E
440
6694.93
2570.71
2661.43
2.60
2.52


I234A
476
6250.56
3890.90
4043.80
1.61
1.55


I234T
467
3507.08
1099.58
1228.23
3.19
2.86


I234E
460
7541.73
1365.08
1901.96
5.52
3.97


I240S
488
4376.99
2108.15
2592.19
2.08
1.69


I240C
483
6170.51
1174.96
2223.23
5.25
2.78









Table 13 below depicts the residual activity (the ratio of hMMP-1 mutant RFU/wt hMMP-1 RFU) of the hMMP-1 mutants following overnight incubation with the fluorescent peptide. The activity of mutants at 25° C., 34° C., or 37° C. were compared to the activity of wildtype hMMP-1 at the respective temperatures. At 25° C., five hMMP-1 mutants (E180F, E180Y, D156T, D156K, R150P) were more active than wildtype hMMP-1 as indicated by a residual activity >1. At elevated temperatures, all of the hMMP-1 mutants exhibited an overall decrease in activity when compared to wildtype hMMP-1 at the same temperature, thus confirming the phenotype of the hMMP-1 mutants as temperature sensitive mutants.









TABLE 13







Residual Activity of hMMP-1 Temperature Sensitive Mutants,


Overnight Incubation















Residual
Residual
Residual



hMMP-1
SEQ ID
Activity
Activity
Activity



mutation
NO
25° C.
34° C.
37° C.

















L95K
6
0.80
0.20
0.20



D105A
39
0.93
0.15
0.22



D105F
33
0.74
0.11
0.13



D105G
32
0.99
0.42
0.60



D105I
36
0.93
0.19
0.22



D105L
38
0.72
0.11
0.12



D105N
27
1.01
0.17
0.26



D105R
25
0.64
0.16
0.17



D105S
31
1.03
0.65
0.92



D105W
35
0.95
0.33
0.51



D105T
29
0.87
0.22
0.37



R150P
59
0.99
0.41
0.44



D151G
70
0.22
0.11
0.12



F155A
96
0.51
0.22
0.22



D156K
100
0.97
0.46
0.46



D156T
105
1.00
0.46
0.46



D156L
114
0.58
0.11
0.14



D156A
115
0.40
0.13
0.12



D156W
111
0.28
0.13
0.14



D156V
113
0.24
0.11
0.11



D156T
105
0.80
0.15
0.17



D156H
99
0.84
0.17
0.20



D156R
101
0.95
0.20
0.21



G159V
132
0.73
0.21
0.20



G159T
125
1.00
0.37
0.39



A176F
148
0.43
0.16
0.16



D179N
160
0.81
0.17
0.18



E180Y
182
1.02
0.70
0.85



E180T
181
0.93
0.30
0.35



E180F
185
1.03
0.60
0.66



D181L
209
0.50
0.12
0.12



D181K
195
0.21
0.11
0.11



E182T
219
0.74
0.17
0.18



E182Q
218
0.24
0.12
0.13



T185R
235
0.69
0.28
0.28



T185H
233
0.94
0.37
0.43



T185Q
238
1.00
0.42
0.49



T185A
248
0.68
0.13
0.13



T185E
232
0.93
0.45
0.48



N187R
254
0.84
0.28
0.29



N187M
262
0.95
0.32
0.33



N187F
261
0.81
0.19
0.21



N187K
253
0.95
0.40
0.46



N187I
264
0.92
0.17
0.18



R195V
284
0.96
0.54
0.59



A198L
305
0.98
0.40
0.49



A198M
301
0.87
0.36
0.42



G206A
324
0.27
0.12
0.12



G206S
317
0.31
0.10
0.10



S210V
341
0.78
0.29
0.44



Y218S
354
0.85
0.47
0.50



F223E
365
1.07
0.57
0.86



V227C
388
0.64
0.26
0.27



V227E
384
0.95
0.27
0.24



V227W
397
0.34
0.16
0.16



Q228P
420
0.38
0.13
0.13



L229T
429
0.37
0.12
0.12



L229I
436
0.76
0.20
0.38



D233E
440
0.69
0.28
0.31



I234A
476
0.69
0.45
0.45



I234T
467
0.39
0.13
0.14



I234E
460
0.83
0.16
0.21



I240S
488
0.48
0.25
0.29



I240C
483
0.68
0.14
0.25











C. hMMP-1 Top Mutant Hits


Fourteen (14) positions were identified as top hit positions: 95, 105, 150, 156, 159, 179, 180, 182, 185, 187, 198, 227, 234 and 240. Twenty three (23) hMMP-1 mutants at 14 positions were selected as top hits based on two criteria, including:1) the ratio of the activities (25° C. to 37° C. and 25° C. to 34° C.); and 2) the activity (in RFUs). All of the mutants listed in Table 14 below had an activity greater than 2000 and a ratio of 25° C. to 37° C. greater than 2. The eleven hits identified with a ** are the hits that ranked high for both the ratio or activities and the activity level, and were used to develop a combinatorial library as described in Example 3.









TABLE 14





Top Hits




















L95K**
D105I
D105N**
D105L



D105A
D105G
R150P**
D156R



D156H
D156K**
D156T**
G159V**



G159T
D179N**
E180T**
E180F



E182T
T185Q
N187I
A198L**



V227E**
I234E
I240S**










Example 3
Combinatorial hMMP-1 Variant Libraryanapec

1. Generation


In this example, a combinatorial hMMP-1 variant library was generated from the mutants selected in Example 2C and shown in Table 14 with a double asterix (**). Mutants at positions 182, 185 and 187 were excluded in the generation of the combinatorial library because of the importance of these positions for hMMP-1 catalytic activity. The library was generated to contain every possible combination of amino acid variants for each of the selected mutants. Table 15 depicts all mutant combinations theoretically contained in the library. The theoretical diversity of the library is 1536 mutants, which includes wild type, the 11 single mutants and all possible combinations of the mutants. The positions indicated are with respect to positions corresponding to amino acid residues of hMMP-1 set forth in SEQ ID NO:2. Each row and column indicates one polypeptide containing the noted mutations. For example, 156K 179N 227E, refers to a polypeptide containing three amino acid replacements at positions corresponding to positions set forth in SEQ ID NO:2: D by K at position 156, D by N at position 179 and V by E at position 227. The library was generated and expressed as described in Example 1.


The constructed library (designated CPS library) contained a total of 1238 mutants, including the wildtype and 9 individual hits. The distribution of the number of mutations in the library was determined. The constructed and screened library contained 81% of the maximal diversity.









TABLE 15





Combinatorial Library Mutants


















95K
150P 156T
156K 179N 227E
150P 156T 240S


105N
105N 240S
156K 179N 198L
150P 156T 227E


150P
105N 227E
156K 179N 180T
150P 156T 198L


156K
105N 198L
156K 159V 240S
150P 156T 180T


156T
105N 180T
156K 159V 227E
150P 156T 179N


159V
105N 179N
156K 159V 198L
150P 156T 159V


179N
105N 159V
156K 159V 180T
105N 227E 240S


180T
105N 156K
156K 159V 179N
105N 198L 240S


198L
105N 156T
156T 227E 240S
105N 198L 227E


227E
105N 150P
156T 198L 240S
105N 180T 240S


240S
95K 240S
156T 198L 227E
105N 180T 227E


227E 240S
95K 227E
156T 180T 240S
105N 180T 198L


198L 240S
95K 198L
156T 180T 227E
105N 179N 240S


198L 227E
95K 180T
156T 180T 198L
105N 179N 227E


180T 240S
95K 179N
156T 179N 240S
105N 179N 198L


180T 227E
95K 159V
156T 179N 227E
105N 179N 180T


180T 198L
95K 156T
156T 179N 198L
105N 159V 240S


179N 240S
95K 150P
156T 179N 180T
105N 159V 227E


179N 227E
95K 105N
156T 159V 240S
105N 159V 198L


179N 198L
95K 156K
156T 159V 227E
105N 159V 180T


179N 180T
180T 227E 240S
156T 159V 198L
105N 159V 179N


159V 240S
180T 198L 240S
156T 159V 180T
105N 156K 240S


159V 227E
180T 198L 227E
156T 159V 179N
105N 156K 227E


159V 198L
179N 227E 240S
150P 227E 240S
105N 156K 198L


159V 180T
179N 198L 240S
198L 227E 240S
105N 156K 180T


159V 179N
179N 198L 227E
150P 198L 240S
105N 156K 179N


156K 240S
179N 180T 240S
150P 198L 227E
105N 156K 159V


156K 227E
179N 180T 227E
150P 180T 240S
105N 156T 240S


156K 198L
179N 180T 198L
150P 180T 227E
105N 156T 227E


156K 180T
159V 227E 240S
150P 180T 198L
105N 156T 198L


156K 179N
159V 198L 240S
150P 179N 240S
105N 156T 180T


156K 159V
159V 198L 227E
150P 179N 227E
105N 156T 179N


156T 240S
159V 180T 240S
150P 179N 198L
105N 156T 159V


156T 227E
159V 180T 227E
150P 179N 180T
105N 150P 240S


156T 198L
159V 180T 198L
150P 159V 240S
105N 150P 227E


156T 180T
159V 179N 240S
150P 159V 227E
105N 150P 198L


156T 179N
159V 179N 227E
150P 159V 198L
105N 150P 180T


156T 159V
159V 179N 198L
150P 159V 180T
105N 150P 179N


150P 240S
159V 179N 180T
150P 159V 179N
105N 150P 159V


150P 227E
156K 227E 240S
150P 156K 240S
105N 150P 156K


150P 198L
156K 198L 240S
150P 156K 227E
105N 150P 156T


150P 180T
156K 198L 227E
150P 156K 198L
95K 227E 240S


150P 179N
156K 180T 240S
150P 156K 180T
95K 198L 240S


150P 156K
156K 180T 227E
156K 180T 198L
150P 156K 179N


150P 159V
156K 179N 240S
150P 156K 159V
95K 180T 240S









95K 180T 227E
179N 180T 198L 240S
156T 159V 180T 198L


95K 180T 198L
179N 180T 198L 227E
156T 159V 179N 240S


95K 179N 240S
159V 198L 227E 240S
156T 159V 179N 227E


95K 179N 227E
159V 180T 227E 240S
156T 159V 179N 198L


95K 179N 198L
159V 180T 198L 240S
156T 159V 179N 180T


95K 179N 180T
159V 180T 198L 227E
150P 198L 227E 240S


95K 159V 240S
159V 179N 227E 240S
150P 180T 227E 240S


95K 159V 227E
159V 179N 198L 240S
150P 180T 198L 240S


95K 159V 198L
159V 179N 198L 227E
150P 180T 198L 227E


95K 159V 180T
159V 179N 180T 240S
150P 179N 227E 240S


95K 159V 179N
159V 179N 180T 227E
150P 179N 198L 240S


95K 156K 240S
159V 179N 180T 198L
150P 179N 198L 227E


95K 156K 227E
156K 198L 227E 240S
150P 179N 180T 240S


95K 156K 198L
156K 180T 227E 240S
150P 179N 180T 227E


95K 156K 180T
156K 180T 198L 240S
150P 179N 180T 198L


95K 156K 179N
156K 180T 198L 227E
150P 159V 227E 240S


95K 156K 159V
156K 179N 227E 240S
150P 159V 198L 240S


95K 198L 227E
156K 179N 198L 240S
150P 159V 198L 227E


95K 156T 240S
156K 179N 198L 227E
150P 159V 180T 240S


95K 156T 227E
156K 179N 180T 240S
150P 159V 180T 227E


95K 156T 198L
156K 179N 180T 227E
150P 159V 180T 198L


95K 156T 180T
156K 179N 180T 198L
150P 159V 179N 240S


95K 156T 179N
156K 159V 227E 240S
150P 159V 179N 227E


95K 156T 159V
156K 159V 198L 240S
150P 159V 179N 198L


95K 150P 240S
156K 159V 198L 227E
150P 159V 179N 180T


95K 150P 227E
156K 159V 180T 240S
150P 156K 227E 240S


95K 150P 198L
156K 159V 180T 227E
150P 156K 198L 240S


95K 150P 180T
156K 159V 180T 198L
150P 156K 198L 227E


95K 150P 179N
156K 159V 179N 240S
150P 156K 180T 240S


95K 150P 159V
156K 159V 179N 227E
150P 156K 180T 227E


95K 150P 156K
156K 159V 179N 198L
150P 156K 180T 198L


95K 150P 156T
156K 159V 179N 180T
150P 156K 179N 240S


95K 105N 240S
156T 198L 227E 240S
150P 156K 179N 227E


95K 105N 227E
156T 180T 227E 240S
150P 156K 179N 198L


95K 105N 198L
156T 180T 198L 240S
150P 156K 179N 180T


95K 105N 180T
156T 180T 198L 227E
150P 156K 159V 240S


95K 105N 179N
156T 179N 227E 240S
150P 156K 159V 227E


95K 105N 159V
156T 179N 198L 240S
156T 179N 180T 240S


95K 105N 156K
156T 179N 198L 227E
156T 179N 180T 227E


95K 105N 156T
156T 159V 227E 240S
156T 179N 180T 198L


95K 105N 150P
156T 159V 198L 240S
150P 156T 227E 240S


180T 198L 227E 240S
156T 159V 198L 227E
150P 156T 198L 240S


179N 198L 227E 240S
156T 159V 180T 240S
150P 156T 198L 227E


179N 180T 227E 240S
156T 159V 180T 227E
150P 156T 180T 240S


150P 156T 180T 227E
105N 156T 198L 240S
95K 180T 198L 227E


150P 156T 180T 198L
105N 156T 180T 227E
95K 179N 180T 227E


150P 156T 179N 240S
105N 156T 180T 198L
95K 179N 180T 198L


150P 156T 179N 227E
105N 156T 179N 240S
95K 159V 227E 240S


150P 156T 179N 198L
105N 156T 179N 227E
95K 159V 198L 240S


150P 156T 179N 180T
105N 156T 179N 198L
95K 159V 198L 227E


150P 156T 159V 240S
105N 156T 179N 180T
95K 159V 180T 240S


150P 156T 159V 227E
105N 156T 159V 240S
95K 159V 180T 227E


150P 156T 159V 198L
105N 156T 159V 227E
95K 159V 180T 198L


150P 156T 159V 180T
105N 156T 159V 198L
95K 159V 179N 240S


150P 156T 159V 179N
105N 156T 159V 180T
95K 159V 179N 227E


105N 198L 227E 240S
105N 156T 159V 179N
95K 159V 179N 198L


105N 180T 227E 240S
105N 150P 227E 240S
95K 159V 179N 180T


105N 180T 198L 240S
105N 150P 198L 240S
95K 156K 227E 240S


105N 180T 198L 227E
105N 150P 198L 227E
95K 156K 198L 240S


105N 179N 227E 240S
105N 150P 180T 240S
95K 156K 198L 227E


105N 179N 198L 240S
105N 150P 180T 227E
95K 156K 180T 240S


105N 179N 198L 227E
105N 150P 180T 198L
95K 156K 180T 227E


105N 179N 180T 240S
105N 150P 179N 240S
95K 156K 180T 198L


105N 179N 180T 227E
105N 150P 179N 227E
95K 156K 179N 240S


105N 179N 180T 198L
105N 150P 179N 198L
95K 156K 179N 227E


105N 159V 227E 240S
105N 150P 179N 180T
95K 156K 179N 198L


105N 159V 198L 240S
105N 150P 159V 240S
95K 156K 179N 180T


105N 159V 198L 227E
105N 150P 159V 227E
95K 156K 159V 240S


105N 159V 180T 240S
105N 150P 159V 198L
95K 156K 159V 227E


105N 159V 180T 227E
105N 150P 159V 180T
95K 156K 159V 198L


105N 159V 180T 198L
105N 150P 159V 179N
95K 156K 159V 180T


105N 159V 179N 240S
105N 150P 156K 240S
95K 156K 159V 179N


105N 159V 179N 227E
105N 150P 156K 227E
95K 156T 227E 240S


105N 159V 179N 198L
105N 150P 156K 198L
95K 156T 198L 240S


105N 159V 179N 180T
105N 150P 156K 180T
95K 156T 198L 227E


105N 156K 227E 240S
105N 156K 180T 227E
95K 156T 180T 240S


105N 156K 198L 240S
105N 156K 180T 198L
105N 150P 156K 179N


105N 156K 198L 227E
105N 156K 179N 240S
105N 150P 156K 159V


105N 156K 180T 240S
105N 156K 179N 227E
105N 150P 156T 240S


150P 156K 159V 198L
105N 156K 179N 198L
105N 150P 156T 227E


150P 156K 159V 180T
105N 156K 179N 180T
105N 150P 156T 198L


150P 156K 159V 179N
105N 150P 156T 179N
105N 150P 156T 180T


105N 156K 159V 240S
105N 150P 156T 159V
95K 156T 179N 198L


105N 156K 159V 227E
95K 198L 227E 240S
95K 156T 179N 180T


105N 156K 159V 198L
95K 180T 227E 240S
95K 156T 159V 240S


105N 156K 159V 180T
95K 180T 198L 240S
95K 156T 159V 227E


105N 156K 159V 179N
95K 179N 227E 240S
95K 156T 159V 198L


105N 156T 227E 240S
95K 179N 198L 240S
95K 156T 159V 180T


105N 156T 198L 227E
95K 179N 198L 227E
95K 156T 159V 179N


105N 156T 180T 240S
95K 179N 180T 240S
95K 150P 227E 240S








95K 150P 198L 240S
95K 105N 156K 179N


95K 150P 198L 227E
95K 105N 156K 159V


95K 150P 180T 240S
95K 105N 156T 240S


95K 150P 180T 227E
95K 105N 156T 227E


95K 150P 180T 198L
95K 105N 156T 198L


95K 150P 179N 240S
95K 105N 156T 180T


95K 150P 179N 227E
95K 105N 156T 179N


95K 150P 179N 198L
95K 105N 156T 159V


95K 150P 179N 180T
95K 105N 150P 240S


95K 150P 159V 240S
95K 105N 150P 227E


95K 150P 159V 227E
95K 105N 150P 198L


95K 150P 159V 198L
95K 105N 150P 180T


95K 150P 159V 180T
95K 105N 150P 179N


95K 150P 159V 179N
95K 105N 150P 159V


95K 150P 156K 240S
95K 105N 150P 156K


95K 150P 156K 227E
95K 105N 150P 156T


95K 150P 156K 198L
95K 105N 179N 227E


95K 150P 156K 180T
95K 105N 179N 198L


95K 150P 156K 179N
95K 105N 179N 180T


95K 150P 156K 159V
95K 105N 159V 240S


95K 150P 156T 240S
156K 159V 179N 198L 227E


95K 150P 156T 227E
179N 180T 198L 227E 240S


95K 150P 156T 198L
159V 180T 198L 227E 240S


95K 150P 156T 180T
159V 179N 198L 227E 240S


95K 150P 156T 179N
159V 179N 180T 227E 240S


95K 150P 156T 159V
159V 179N 180T 198L 240S


95K 105N 227E 240S
159V 179N 180T 198L 227E


95K 105N 198L 240S
156K 180T 198L 227E 240S


95K 105N 198L 227E
156K 179N 198L 227E 240S


95K 105N 180T 240S
156K 179N 180T 227E 240S


95K 105N 180T 227E
156K 179N 180T 198L 240S


95K 105N 180T 198L
156K 179N 180T 198L 227E


95K 105N 179N 240S
156K 159V 198L 227E 240S


95K 156T 180T 227E
156K 159V 180T 227E 240S


95K 156T 180T 198L
156K 159V 180T 198L 240S


95K 156T 179N 240S
156K 159V 180T 198L 227E


95K 156T 179N 227E
156K 159V 179N 227E 240S


95K 105N 159V 227E
156K 159V 179N 198L 240S


95K 105N 159V 198L
150P 156K 159V 198L 227E


95K 105N 159V 180T
156K 159V 179N 180T 240S


95K 105N 159V 179N
156K 159V 179N 180T 227E


95K 105N 156K 240S
156K 159V 179N 180T 198L


95K 105N 156K 227E
156T 180T 198L 227E 240S


95K 105N 156K 198L
156T 179N 198L 227E 240S


95K 105N 156K 180T
156T 179N 180T 227E 240S


156T 179N 180T 198L 240S
150P 156K 159V 179N 180T


156T 179N 180T 198L 227E
150P 156T 198L 227E 240S


156T 159V 198L 227E 240S
150P 156T 180T 227E 240S


156T 159V 180T 227E 240S
150P 156T 180T 198L 240S


156T 159V 180T 198L 240S
150P 156T 180T 198L 227E


156T 159V 180T 198L 227E
150P 156T 179N 227E 240S


156T 159V 179N 227E 240S
150P 156T 179N 198L 240S


156T 159V 179N 198L 240S
150P 156T 179N 198L 227E


156T 159V 179N 198L 227E
150P 156T 179N 180T 240S


156T 159V 179N 180T 240S
150P 156T 179N 180T 227E


156T 159V 179N 180T 227E
150P 156T 179N 180T 198L


156T 159V 179N 180T 198L
150P 156T 159V 227E 240S


150P 180T 198L 227E 240S
150P 156T 159V 198L 240S


150P 179N 198L 227E 240S
150P 156T 159V 198L 227E


150P 179N 180T 227E 240S
150P 156T 159V 180T 240S


150P 179N 180T 198L 240S
150P 156T 159V 180T 227E


150P 179N 180T 198L 227E
150P 156T 159V 180T 198L


150P 159V 198L 227E 240S
150P 156T 159V 179N 240S


150P 159V 180T 227E 240S
150P 156T 159V 179N 227E


150P 159V 180T 198L 240S
150P 159V 180T 198L 227E


150P 159V 179N 227E 240S
150P 156T 159V 179N 180T


150P 159V 179N 198L 240S
105N 180T 198L 227E 240S


150P 159V 179N 198L 227E
105N 179N 198L 227E 240S


150P 159V 179N 180T 240S
105N 179N 180T 227E 240S


150P 159V 179N 180T 227E
105N 179N 180T 198L 240S


150P 159V 179N 180T 198L
105N 179N 180T 198L 227E


150P 156K 198L 227E 240S
105N 159V 198L 227E 240S


150P 156K 180T 227E 240S
105N 159V 180T 227E 240S


150P 156K 180T 198L 240S
105N 159V 180T 198L 240S


150P 156K 180T 198L 227E
105N 159V 180T 198L 227E


150P 156K 179N 227E 240S
105N 159V 179N 227E 240S


150P 156K 179N 198L 240S
105N 159V 179N 198L 240S


150P 156K 179N 198L 227E
105N 159V 179N 198L 227E


150P 156K 179N 180T 240S
105N 159V 179N 180T 240S


150P 156K 179N 180T 227E
105N 159V 179N 180T 227E


150P 156K 179N 180T 198L
105N 159V 179N 180T 198L


150P 156K 159V 227E 240S
105N 156K 198L 227E 240S


150P 156K 159V 198L 240S
105N 156K 180T 227E 240S


105N 156K 180T 198L 240S
105N 150P 179N 180T 240S


150P 156K 159V 180T 240S
105N 156K 180T 198L 227E


150P 156K 159V 180T 227E
105N 156K 179N 227E 240S


150P 156K 159V 180T 198L
105N 156K 179N 198L 240S


150P 156K 159V 179N 240S
105N 156K 179N 198L 227E


150P 156K 159V 179N 227E
105N 156K 179N 180T 240S


150P 156K 159V 179N 198L
105N 156K 179N 180T 227E


105N 156K 179N 180T 198L
105N 150P 159V 180T 227E


105N 156K 159V 227E 240S
105N 150P 159V 180T 198L


105N 156K 159V 198L 240S
105N 150P 159V 179N 240S


105N 156K 159V 198L 227E
105N 150P 159V 179N 227E


105N 156K 159V 180T 240S
105N 150P 159V 179N 198L


105N 156K 159V 180T 227E
105N 150P 159V 179N 180T


105N 156K 159V 180T 198L
105N 150P 156K 227E 240S


105N 156K 159V 179N 240S
105N 150P 156K 198L 240S


105N 156K 159V 179N 227E
105N 150P 156K 198L 227E


105N 156K 159V 179N 198L
105N 150P 156K 180T 240S


105N 156K 159V 179N 180T
105N 150P 156K 180T 227E


105N 156T 198L 227E 240S
105N 150P 156K 180T 198L


105N 156T 180T 227E 240S
105N 150P 156K 179N 240S


105N 156T 180T 198L 240S
105N 150P 156K 179N 227E


105N 156T 180T 198L 227E
105N 150P 156K 179N 198L


105N 156T 179N 227E 240S
105N 150P 156K 179N 180T


105N 156T 179N 198L 240S
105N 150P 156K 159V 240S


105N 156T 179N 198L 227E
105N 150P 156K 159V 227E


105N 156T 179N 180T 240S
105N 150P 156K 159V 198L


150P 156T 159V 179N 198L
105N 156T 179N 180T 227E


105N 156T 179N 180T 198L
105N 150P 156K 159V 179N


105N 156T 159V 227E 240S
105N 150P 156T 227E 240S


105N 156T 159V 198L 240S
105N 150P 156T 198L 240S


105N 156T 159V 198L 227E
105N 150P 156T 198L 227E


105N 156T 159V 180T 240S
105N 150P 156T 180T 240S


105N 156T 159V 180T 227E
105N 150P 156T 180T 227E


105N 156T 159V 180T 198L
105N 150P 156T 180T 198L


105N 156T 159V 179N 240S
105N 150P 156T 179N 240S


105N 156T 159V 179N 227E
105N 150P 156T 179N 227E


105N 156T 159V 179N 198L
105N 150P 156T 179N 198L


105N 156T 159V 179N 180T
105N 150P 156T 179N 180T


105N 150P 198L 227E 240S
105N 150P 156T 159V 240S


105N 150P 180T 227E 240S
105N 150P 156T 159V 227E


105N 150P 180T 198L 240S
105N 150P 156T 159V 198L


105N 150P 180T 198L 227E
105N 150P 156T 159V 180T


105N 150P 179N 227E 240S
105N 150P 156T 159V 179N


105N 150P 179N 198L 240S
95K 180T 198L 227E 240S


105N 150P 179N 198L 227E
95K 179N 198L 227E 240S


95K 179N 180T 227E 240S
95K 156T 159V 198L 227E


105N 150P 179N 180T 227E
95K 179N 180T 198L 240S


105N 150P 179N 180T 198L
95K 179N 180T 198L 227E


105N 150P 159V 227E 240S
95K 159V 198L 227E 240S


105N 150P 159V 198L 240S
95K 159V 180T 227E 240S


105N 150P 159V 198L 227E
95K 159V 180T 198L 240S


105N 150P 159V 180T 240S
95K 159V 180T 198L 227E


95K 159V 179N 227E 240S
95K 156T 159V 179N 180T


95K 159V 179N 198L 240S
95K 150P 198L 227E 240S


95K 159V 179N 198L 227E
95K 150P 180T 227E 240S


95K 159V 179N 180T 240S
95K 150P 180T 198L 240S


95K 159V 179N 180T 227E
95K 150P 180T 198L 227E


95K 159V 179N 180T 198L
95K 150P 179N 227E 240S


95K 156K 198L 227E 240S
95K 150P 179N 198L 240S


95K 156K 180T 227E 240S
95K 150P 179N 198L 227E


95K 156K 180T 198L 240S
95K 150P 179N 180T 240S


95K 156K 180T 198L 227E
95K 150P 179N 180T 227E


95K 156K 179N 227E 240S
95K 150P 179N 180T 198L


95K 156K 179N 198L 240S
95K 150P 159V 227E 240S


95K 156K 179N 198L 227E
95K 150P 159V 198L 240S


95K 156K 179N 180T 240S
95K 150P 159V 198L 227E


95K 156K 179N 180T 227E
95K 150P 159V 180T 240S


95K 156K 179N 180T 198L
95K 150P 159V 180T 227E


95K 156K 159V 227E 240S
95K 150P 159V 180T 198L


95K 156K 159V 198L 240S
95K 150P 159V 179N 240S


95K 156K 159V 198L 227E
95K 150P 159V 179N 227E


105N 150P 156K 159V 180T
95K 156K 159V 180T 240S


95K 156K 159V 180T 227E
95K 150P 159V 179N 180T


95K 156K 159V 180T 198L
95K 150P 156K 227E 240S


95K 156K 159V 179N 240S
95K 150P 156K 198L 240S


95K 156K 159V 179N 227E
95K 150P 156K 198L 227E


95K 156K 159V 179N 198L
95K 150P 156K 180T 240S


95K 156K 159V 179N 180T
95K 150P 156K 180T 227E


95K 156T 198L 227E 240S
95K 150P 156K 180T 198L


95K 156T 180T 227E 240S
95K 150P 156K 179N 240S


95K 156T 180T 198L 240S
95K 150P 156K 179N 227E


95K 156T 180T 198L 227E
95K 150P 156K 179N 198L


95K 156T 179N 227E 240S
95K 150P 156K 179N 180T


95K 156T 179N 198L 240S
95K 150P 156K 159V 240S


95K 156T 179N 198L 227E
95K 150P 156K 159V 227E


95K 156T 179N 180T 240S
95K 150P 156K 159V 198L


95K 156T 179N 180T 227E
95K 150P 156K 159V 180T


95K 156T 179N 180T 198L
95K 150P 156K 159V 179N


95K 156T 159V 227E 240S
95K 150P 156T 227E 240S


95K 156T 159V 198L 240S
95K 150P 156T 198L 240S


95K 150P 156T 198L 227E
95K 105N 156K 159V 198L


95K 156T 159V 180T 240S
95K 150P 156T 180T 240S


95K 156T 159V 180T 227E
95K 150P 156T 180T 227E


95K 156T 159V 180T 198L
95K 150P 156T 180T 198L


95K 156T 159V 179N 240S
95K 150P 156T 179N 240S


95K 156T 159V 179N 227E
95K 150P 156T 179N 227E


95K 156T 159V 179N 198L
95K 150P 156T 179N 198L


95K 150P 156T 179N 180T
95K 105N 156T 180T 227E


95K 150P 156T 159V 240S
95K 105N 156T 180T 198L


95K 150P 156T 159V 227E
95K 105N 156T 179N 240S


95K 150P 156T 159V 198L
95K 105N 156T 179N 227E


95K 150P 156T 159V 180T
95K 105N 156T 179N 198L


95K 150P 156T 159V 179N
95K 105N 156T 179N 180T


95K 105N 198L 227E 240S
95K 105N 156T 159V 240S


95K 105N 180T 227E 240S
95K 105N 156T 159V 227E


95K 105N 180T 198L 240S
95K 105N 156T 159V 198L


95K 105N 180T 198L 227E
95K 105N 156T 159V 180T


95K 105N 179N 227E 240S
95K 105N 156T 159V 179N


95K 105N 179N 198L 240S
95K 105N 150P 227E 240S


95K 105N 179N 198L 227E
95K 105N 150P 198L 240S


95K 105N 179N 180T 240S
95K 105N 150P 198L 227E


95K 105N 179N 180T 227E
95K 105N 150P 180T 240S


95K 105N 179N 180T 198L
95K 105N 150P 180T 227E


95K 105N 159V 227E 240S
95K 105N 150P 180T 198L


95K 105N 159V 198L 240S
95K 105N 150P 179N 240S


95K 105N 159V 198L 227E
95K 105N 150P 179N 227E


95K 150P 159V 179N 198L
95K 105N 159V 180T 240S


95K 105N 159V 180T 227E
95K 105N 150P 179N 180T


95K 105N 159V 180T 198L
95K 105N 150P 159V 240S


95K 105N 159V 179N 240S
95K 105N 150P 159V 227E


95K 105N 159V 179N 227E
95K 105N 150P 159V 198L


95K 105N 159V 179N 198L
95K 105N 150P 159V 180T


95K 105N 159V 179N 180T
95K 105N 150P 159V 179N


95K 105N 156K 227E 240S
95K 105N 150P 156K 240S


95K 105N 156K 198L 240S
95K 105N 150P 156K 227E


95K 105N 156K 198L 227E
95K 105N 150P 156K 198L


95K 105N 156K 180T 240S
95K 105N 150P 156K 180T


95K 105N 156K 180T 227E
95K 105N 150P 156K 179N


95K 105N 156K 180T 198L
95K 105N 150P 156K 159V


95K 105N 156K 179N 240S
95K 105N 150P 156T 240S


95K 105N 156K 179N 227E
95K 105N 150P 156T 227E


95K 105N 156K 179N 198L
95K 105N 150P 156T 198L


95K 105N 156K 179N 180T
95K 105N 150P 156T 180T


95K 105N 156K 159V 240S
95K 105N 150P 156T 179N


95K 105N 156K 159V 227E
95K 105N 150P 156T 159V


95K 105N 150P 179N 198L
150P 156T 159V 179N 198L 240S


95K 105N 156K 159V 180T
159V 179N 180T 198L 227E 240S


95K 105N 156K 159V 179N
156K 179N 180T 198L 227E 240S


95K 105N 156T 227E 240S
156K 159V 180T 198L 227E 240S


95K 105N 156T 198L 240S
156K 159V 179N 198L 227E 240S


95K 105N 156T 198L 227E
156K 159V 179N 180T 227E 240S


95K 105N 156T 180T 240S
156K 159V 179N 180T 198L 240S


150P 156T 159V 179N 198L 227E
105N 159V 179N 198L 227E 240S


150P 156T 159V 179N 180T 240S
105N 159V 179N 180T 227E 240S


150P 156T 159V 179N 180T 227E
105N 159V 179N 180T 198L 240S


150P 156T 159V 179N 180T 198L
105N 159V 179N 180T 198L 227E


105N 179N 180T 198L 227E 240S
105N 156K 180T 198L 227E 240S


105N 159V 180T 198L 227E 240S
105N 156K 179N 198L 227E 240S


156K 159V 179N 180T 198L 227E
105N 156K 179N 180T 227E 240S


156T 179N 180T 198L 227E 240S
105N 156K 179N 180T 198L 240S


156T 159V 180T 198L 227E 240S
105N 156K 179N 180T 198L 227E


156T 159V 179N 198L 227E 240S
105N 156K 159V 198L 227E 240S


156T 159V 179N 180T 227E 240S
105N 156K 159V 180T 227E 240S


156T 159V 179N 180T 198L 240S
105N 156K 159V 180T 198L 240S


156T 159V 179N 180T 198L 227E
105N 156K 159V 180T 198L 227E


150P 179N 180T 198L 227E 240S
105N 156K 159V 179N 227E 240S


150P 159V 180T 198L 227E 240S
105N 156K 159V 179N 198L 240S


150P 159V 179N 198L 227E 240S
105N 156K 159V 179N 198L 227E


150P 159V 179N 180T 227E 240S
105N 156K 159V 179N 180T 240S


150P 159V 179N 180T 198L 240S
105N 156K 159V 179N 180T 227E


150P 159V 179N 180T 198L 227E
105N 156K 159V 179N 180T 198L


150P 156K 180T 198L 227E 240S
105N 156T 180T 198L 227E 240S


150P 156K 179N 198L 227E 240S
105N 156T 179N 198L 227E 240S


150P 156K 179N 180T 227E 240S
105N 156T 179N 180T 227E 240S


150P 156K 179N 180T 198L 240S
105N 156T 179N 180T 198L 240S


150P 156K 179N 180T 198L 227E
105N 156T 179N 180T 198L 227E


150P 156K 159V 198L 227E 240S
105N 156T 159V 198L 227E 240S


150P 156K 159V 180T 227E 240S
105N 156T 159V 180T 227E 240S


150P 156K 159V 180T 198L 240S
105N 156T 159V 180T 198L 240S


150P 156K 159V 180T 198L 227E
105N 156T 159V 180T 198L 227E


150P 156K 159V 179N 227E 240S
105N 156T 159V 179N 227E 240S


150P 156K 159V 179N 198L 240S
105N 156T 159V 179N 198L 240S


150P 156K 159V 179N 198L 227E
105N 156T 159V 179N 198L 227E


150P 156K 159V 179N 180T 240S
105N 156T 159V 179N 180T 240S


150P 156K 159V 179N 180T 227E
105N 156T 159V 179N 180T 227E


150P 156K 159V 179N 180T 198L
105N 156T 159V 179N 180T 198L


150P 156T 180T 198L 227E 240S
105N 150P 180T 198L 227E 240S


150P 156T 179N 198L 227E 240S
105N 150P 179N 198L 227E 240S


150P 156T 179N 180T 227E 240S
105N 150P 179N 180T 227E 240S


150P 156T 179N 180T 198L 240S
105N 150P 179N 180T 198L 240S


150P 156T 179N 180T 198L 227E
105N 150P 156T 159V 180T 227E


150P 156T 159V 198L 227E 240S
105N 150P 159V 179N 227E 240S


150P 156T 159V 180T 227E 240S
105N 150P 159V 179N 198L 240S


150P 156T 159V 180T 198L 240S
105N 150P 159V 179N 198L 227E


150P 156T 159V 180T 198L 227E
105N 150P 159V 179N 180T 240S


150P 156T 159V 179N 227E 240S
105N 150P 159V 179N 180T 227E


105N 150P 179N 180T 198L 227E
105N 150P 159V 179N 180T 198L


105N 150P 156K 198L 227E 240S
95K 159V 180T 198L 227E 240S


105N 150P 156K 180T 227E 240S
95K 159V 179N 198L 227E 240S


105N 150P 156K 180T 198L 240S
95K 159V 179N 180T 227E 240S


105N 150P 156K 180T 198L 227E
95K 159V 179N 180T 198L 240S


105N 150P 156K 179N 227E 240S
95K 159V 179N 180T 198L 227E


105N 150P 156K 179N 198L 240S
95K 156K 180T 198L 227E 240S


105N 150P 156K 179N 198L 227E
95K 156K 179N 198L 227E 240S


105N 150P 156K 179N 180T 240S
95K 156K 179N 180T 227E 240S


105N 150P 156K 179N 180T 227E
95K 156K 179N 180T 198L 240S


105N 150P 156K 179N 180T 198L
95K 156K 179N 180T 198L 227E


105N 150P 156K 159V 227E 240S
95K 156K 159V 198L 227E 240S


105N 150P 156K 159V 198L 240S
95K 156K 159V 180T 227E 240S


105N 150P 156K 159V 198L 227E
95K 156K 159V 180T 198L 240S


105N 150P 156K 159V 180T 240S
95K 156K 159V 180T 198L 227E


105N 150P 156K 159V 180T 227E
95K 156K 159V 179N 227E 240S


105N 150P 156K 159V 180T 198L
95K 156K 159V 179N 198L 240S


105N 150P 156K 159V 179N 240S
95K 156K 159V 179N 198L 227E


105N 150P 156K 159V 179N 227E
95K 156K 159V 179N 180T 240S


105N 150P 156K 159V 179N 198L
95K 156K 159V 179N 180T 227E


105N 150P 156K 159V 179N 180T
95K 156K 159V 179N 180T 198L


105N 150P 159V 198L 227E 240S
95K 156T 180T 198L 227E 240S


105N 150P 159V 180T 227E 240S
95K 156T 179N 198L 227E 240S


105N 150P 159V 180T 198L 240S
95K 156T 179N 180T 227E 240S


105N 150P 159V 180T 198L 227E
95K 156T 179N 180T 198L 240S


105N 150P 156T 198L 227E 240S
95K 156T 179N 180T 198L 227E


105N 150P 156T 180T 227E 240S
95K 156T 159V 198L 227E 240S


105N 150P 156T 180T 198L 240S
95K 156T 159V 180T 227E 240S


105N 150P 156T 180T 198L 227E
95K 156T 159V 180T 198L 240S


105N 150P 156T 179N 227E 240S
95K 156T 159V 180T 198L 227E


105N 150P 156T 179N 198L 240S
95K 156T 159V 179N 227E 240S


105N 150P 156T 179N 198L 227E
95K 156T 159V 179N 198L 240S


105N 150P 156T 179N 180T 240S
95K 156T 159V 179N 198L 227E


105N 150P 156T 179N 180T 227E
95K 156T 159V 179N 180T 240S


105N 150P 156T 179N 180T 198L
95K 156T 159V 179N 180T 227E


105N 150P 156T 159V 227E 240S
95K 156T 159V 179N 180T 198L


105N 150P 156T 159V 198L 240S
95K 150P 180T 198L 227E 240S


105N 150P 156T 159V 198L 227E
95K 150P 179N 198L 227E 240S


105N 150P 156T 159V 180T 240S
95K 150P 179N 180T 227E 240S


95K 150P 179N 180T 198L 240S
95K 150P 156T 159V 180T 240S


105N 150P 156T 159V 180T 198L
95K 150P 179N 180T 198L 227E


105N 150P 156T 159V 179N 240S
95K 150P 159V 198L 227E 240S


105N 150P 156T 159V 179N 227E
95K 150P 159V 180T 227E 240S


105N 150P 156T 159V 179N 198L
95K 150P 159V 180T 198L 240S


105N 150P 156T 159V 179N 180T
95K 150P 159V 180T 198L 227E


95K 179N 180T 198L 227E 240S
95K 150P 159V 179N 227E 240S


95K 150P 159V 179N 198L 240S
95K 105N 179N 198L 227E 240S


95K 150P 159V 179N 198L 227E
95K 105N 179N 180T 227E 240S


95K 150P 159V 179N 180T 240S
95K 105N 179N 180T 198L 240S


95K 150P 159V 179N 180T 227E
95K 105N 179N 180T 198L 227E


95K 150P 159V 179N 180T 198L
95K 105N 159V 198L 227E 240S


95K 150P 156K 198L 227E 240S
95K 105N 159V 180T 227E 240S


95K 150P 156K 180T 227E 240S
95K 105N 159V 180T 198L 240S


95K 150P 156K 180T 198L 240S
95K 105N 159V 180T 198L 227E


95K 150P 156K 180T 198L 227E
95K 105N 159V 179N 227E 240S


95K 150P 156K 179N 227E 240S
95K 105N 159V 179N 198L 240S


95K 150P 156K 179N 198L 240S
95K 105N 159V 179N 198L 227E


95K 150P 156K 179N 198L 227E
95K 105N 159V 179N 180T 240S


95K 150P 156K 179N 180T 240S
95K 105N 159V 179N 180T 227E


95K 150P 156K 179N 180T 227E
95K 105N 159V 179N 180T 198L


95K 150P 156K 179N 180T 198L
95K 105N 156K 198L 227E 240S


95K 150P 156K 159V 227E 240S
95K 105N 156K 180T 227E 240S


95K 150P 156K 159V 198L 240S
95K 105N 156K 180T 198L 240S


95K 150P 156K 159V 198L 227E
95K 105N 156K 180T 198L 227E


95K 150P 156K 159V 180T 240S
95K 105N 156K 179N 227E 240S


95K 150P 156K 159V 180T 227E
95K 105N 156K 179N 198L 240S


95K 150P 156K 159V 180T 198L
95K 105N 156K 179N 198L 227E


95K 150P 156K 159V 179N 240S
95K 105N 156K 179N 180T 240S


95K 150P 156K 159V 179N 227E
95K 105N 156K 179N 180T 227E


95K 150P 156K 159V 179N 198L
95K 105N 156K 179N 180T 198L


95K 150P 156K 159V 179N 180T
95K 105N 156K 159V 227E 240S


95K 150P 156T 198L 227E 240S
95K 105N 156K 159V 198L 240S


95K 150P 156T 180T 227E 240S
95K 105N 156K 159V 198L 227E


95K 150P 156T 180T 198L 240S
95K 105N 156K 159V 180T 240S


95K 150P 156T 180T 198L 227E
95K 105N 156K 159V 180T 227E


95K 150P 156T 179N 227E 240S
95K 105N 156K 159V 180T 198L


95K 150P 156T 179N 198L 240S
95K 105N 156K 159V 179N 240S


95K 150P 156T 179N 198L 227E
95K 105N 156K 159V 179N 227E


95K 150P 156T 179N 180T 240S
95K 105N 156K 159V 179N 198L


95K 150P 156T 179N 180T 227E
95K 105N 156K 159V 179N 180T


95K 150P 156T 179N 180T 198L
95K 105N 156T 198L 227E 240S


95K 150P 156T 159V 227E 240S
95K 105N 156T 180T 227E 240S


95K 150P 156T 159V 198L 240S
95K 105N 156T 180T 198L 240S


95K 150P 156T 159V 198L 227E
95K 105N 156T 179N 227E 240S


95K 150P 156T 159V 180T 227E
95K 105N 156T 179N 198L 240S


95K 150P 156T 159V 180T 198L
95K 105N 156T 179N 198L 227E


95K 150P 156T 159V 179N 240S
95K 105N 156T 179N 180T 240S


95K 150P 156T 159V 179N 227E
95K 105N 156T 179N 180T 227E


95K 150P 156T 159V 179N 198L
95K 105N 156T 179N 180T 198L


95K 150P 156T 159V 179N 180T
95K 105N 156T 159V 227E 240S


95K 105N 180T 198L 227E 240S
95K 105N 156T 159V 198L 240S







95K 105N 156T 159V 198L 227E


95K 105N 156T 159V 180T 240S


95K 105N 156T 159V 180T 227E


95K 105N 156T 159V 180T 198L


95K 105N 156T 159V 179N 240S


95K 105N 156T 159V 179N 227E


95K 105N 156T 159V 179N 198L


95K 105N 156T 159V 179N 180T


95K 105N 150P 198L 227E 240S


95K 105N 150P 180T 227E 240S


95K 105N 150P 180T 198L 240S


95K 105N 150P 180T 198L 227E


95K 105N 150P 179N 227E 240S


95K 105N 150P 179N 198L 240S


95K 105N 150P 179N 198L 227E


95K 105N 150P 179N 180T 240S


95K 105N 150P 179N 180T 227E


95K 105N 150P 179N 180T 198L


95K 105N 150P 159V 227E 240S


95K 105N 150P 159V 198L 240S


95K 105N 150P 159V 198L 227E


95K 105N 150P 159V 180T 240S


95K 105N 150P 159V 180T 227E


95K 105N 150P 159V 180T 198L


95K 105N 150P 159V 179N 240S


95K 105N 150P 159V 179N 227E


95K 105N 150P 156K 179N 198L


95K 105N 150P 156K 179N 180T


95K 105N 150P 156K 159V 240S


95K 105N 150P 156K 159V 227E


95K 105N 150P 156K 159V 198L


95K 105N 150P 156K 159V 180T


95K 105N 150P 156K 159V 179N


95K 105N 150P 159V 179N 198L


95K 105N 150P 159V 179N 180T


95K 105N 150P 156K 227E 240S


95K 105N 150P 156K 198L 240S


95K 105N 150P 156K 198L 227E


95K 105N 150P 156K 180T 240S


95K 105N 150P 156K 180T 227E


95K 105N 150P 156K 180T 198L


95K 105N 150P 156T 227E 240S


95K 105N 150P 156T 198L 240S


95K 105N 150P 156T 198L 227E


95K 105N 150P 156T 180T 240S


95K 105N 150P 156T 180T 227E


95K 105N 150P 156T 180T 198L


95K 105N 150P 156T 179N 240S


95K 105N 150P 156T 179N 227E


95K 105N 150P 156T 179N 198L


95K 105N 150P 156T 179N 180T


95K 105N 150P 156T 159V 240S


95K 105N 150P 156T 159V 227E


95K 105N 150P 156T 159V 198L


95K 105N 150P 156T 159V 180T


95K 105N 150P 156T 159V 179N


95K 105N 150P 156K 179N 240S


95K 105N 150P 156K 179N 227E


95K 105N 156T 180T 198L 227E


156K 159V 179N 180T 198L 227E 240S


156T 159V 179N 180T 198L 227E 240S


150P 159V 179N 180T 198L 227E 240S


150P 156K 179N 180T 198L 227E 240S


150P 156K 159V 180T 198L 227E 240S


150P 156K 159V 179N 198L 227E 240S


150P 156K 159V 179N 180T 227E 240S


150P 156K 159V 179N 180T 198L 240S


150P 156K 159V 179N 180T 198L 227E


150P 156T 179N 180T 198L 227E 240S


150P 156T 159V 180T 198L 227E 240S


150P 156T 159V 179N 198L 227E 240S


150P 156T 159V 179N 180T 227E 240S


150P 156T 159V 179N 180T 198L 240S


150P 156T 159V 179N 180T 198L 227E


105N 156K 179N 180T 198L 227E 240S


105N 156K 159V 180T 198L 227E 240S


105N 156K 159V 179N 198L 227E 240S


105N 156K 159V 179N 180T 198L 240S


105N 156K 159V 179N 180T 198L 227E


105N 159V 179N 180T 198L 227E 240S


105N 156K 159V 179N 180T 227E 240S


105N 156T 159V 179N 180T 198L 227E


105N 156T 179N 180T 198L 227E 240S


105N 150P 179N 180T 198L 227E 240S


105N 150P 159V 180T 198L 227E 240S


105N 150P 159V 179N 198L 227E 240S


105N 150P 159V 179N 180T 227E 240S


105N 150P 159V 179N 180T 198L 240S


105N 150P 159V 179N 180T 198L 227E


105N 150P 156K 180T 198L 227E 240S


105N 150P 156K 179N 198L 227E 240S


105N 150P 156K 179N 180T 227E 240S


105N 150P 156K 179N 180T 198L 240S


105N 150P 156K 179N 180T 198L 227E


105N 150P 156K 159V 198L 227E 240S


105N 150P 156K 159V 180T 227E 240S


105N 150P 156K 159V 180T 198L 240S


105N 150P 156K 159V 180T 198L 227E


105N 150P 156K 159V 179N 227E 240S


105N 150P 156K 159V 179N 198L 240S


105N 150P 156K 159V 179N 198L 227E


105N 150P 156K 159V 179N 180T 240S


105N 150P 156K 159V 179N 180T 227E


105N 150P 156K 159V 179N 180T 198L


105N 150P 156T 179N 198L 227E 240S


105N 150P 156T 179N 180T 227E 240S


105N 150P 156T 179N 180T 198L 240S


105N 150P 156T 179N 180T 198L 227E


105N 150P 156T 159V 198L 227E 240S


105N 150P 156T 159V 180T 227E 240S


105N 150P 156T 159V 180T 198L 240S


105N 150P 156T 159V 180T 198L 227E


105N 150P 156T 159V 179N 198L 240S


105N 150P 156T 159V 179N 198L 227E


105N 150P 156T 180T 198L 227E 240S


105N 156T 159V 179N 198L 227E 240S


105N 156T 159V 179N 180T 198L 240S


105N 156T 159V 179N 180T 227E 240S


105N 156T 159V 180T 198L 227E 240S


95K 156T 159V 179N 198L 227E 240S


95K 156T 159V 179N 180T 227E 240S


95K 156T 159V 179N 180T 198L 240S


95K 156T 159V 179N 180T 198L 227E


95K 150P 179N 180T 198L 227E 240S


95K 150P 159V 180T 198L 227E 240S


95K 150P 159V 179N 198L 227E 240S


95K 150P 159V 179N 180T 227E 240S


95K 150P 159V 179N 180T 198L 240S


95K 150P 159V 179N 180T 198L 227E


105N 150P 156T 159V 179N 227E 240S


105N 150P 156T 159V 179N 180T 240S


105N 150P 156T 159V 179N 180T 227E


105N 150P 156T 159V 179N 180T 198L


95K 159V 179N 180T 198L 227E 240S


95K 156K 179N 180T 198L 227E 240S


95K 156K 159V 180T 198L 227E 240S


95K 156K 159V 179N 198L 227E 240S


95K 156K 159V 179N 180T 227E 240S


95K 156K 159V 179N 180T 198L 240S


95K 156K 159V 179N 180T 198L 227E


95K 156T 179N 180T 198L 227E 240S


95K 156T 159V 180T 198L 227E 240S


95K 150P 156K 159V 179N 180T 227E


95K 150P 156K 159V 179N 180T 198L


95K 150P 156T 180T 198L 227E 240S


95K 150P 156T 179N 198L 227E 240S


95K 150P 156T 179N 180T 227E 240S


95K 150P 156T 179N 180T 198L 240S


95K 150P 156T 179N 180T 198L 227E


95K 150P 156T 159V 198L 227E 240S


95K 150P 156T 159V 180T 227E 240S


95K 150P 156T 159V 180T 198L 240S


95K 150P 156T 159V 180T 198L 227E


95K 150P 156T 159V 179N 227E 240S


95K 150P 156T 159V 179N 198L 240S


95K 150P 156T 159V 179N 198L 227E


95K 150P 156T 159V 179N 180T 240S


95K 150P 156T 159V 179N 180T 227E


95K 150P 156K 180T 198L 227E 240S


95K 150P 156K 179N 198L 227E 240S


95K 150P 156K 179N 180T 227E 240S


95K 150P 156K 179N 180T 198L 240S


95K 150P 156K 179N 180T 198L 227E


95K 150P 156K 159V 198L 227E 240S


95K 150P 156K 159V 180T 227E 240S


95K 150P 156K 159V 180T 198L 240S


95K 150P 156K 159V 180T 198L 227E


95K 150P 156K 159V 179N 227E 240S


95K 150P 156K 159V 179N 198L 240S


95K 150P 156K 159V 179N 198L 227E


95K 150P 156K 159V 179N 180T 240S


95K 105N 156K 180T 198L 227E 240S


95K 105N 156K 179N 198L 227E 240S


95K 105N 156K 179N 180T 227E 240S


95K 105N 156K 179N 180T 198L 240S


95K 105N 156K 179N 180T 198L 227E


95K 105N 156K 159V 198L 227E 240S


95K 105N 156K 159V 180T 227E 240S


95K 105N 156K 159V 180T 198L 240S


95K 105N 156K 159V 180T 198L 227E


95K 105N 156K 159V 179N 227E 240S


95K 105N 156K 159V 179N 198L 240S


95K 105N 156K 159V 179N 198L 227E


95K 105N 156K 159V 179N 180T 240S


95K 105N 156K 159V 179N 180T 227E


95K 105N 156K 159V 179N 180T 198L


95K 105N 156T 180T 198L 227E 240S


95K 150P 156T 159V 179N 180T 198L


95K 105N 179N 180T 198L 227E 240S


95K 105N 159V 180T 198L 227E 240S


95K 105N 159V 179N 198L 227E 240S


95K 105N 159V 179N 180T 227E 240S


95K 105N 159V 179N 180T 198L 240S


95K 105N 159V 179N 180T 198L 227E


95K 105N 156T 159V 180T 198L 227E


95K 105N 156T 159V 179N 227E 240S


95K 105N 156T 159V 179N 198L 240S


95K 105N 156T 159V 179N 198L 227E


95K 105N 156T 159V 179N 180T 240S


95K 105N 156T 159V 179N 180T 227E


95K 105N 156T 159V 179N 180T 198L


95K 105N 150P 180T 198L 227E 240S


95K 105N 150P 179N 198L 227E 240S


95K 105N 150P 179N 180T 227E 240S


95K 105N 150P 179N 180T 198L 240S


95K 105N 150P 179N 180T 198L 227E


95K 105N 150P 159V 198L 227E 240S


95K 105N 150P 159V 180T 227E 240S


95K 105N 150P 159V 180T 198L 240S


95K 105N 150P 159V 180T 198L 227E


95K 105N 150P 159V 179N 227E 240S


95K 105N 150P 159V 179N 198L 240S


95K 105N 150P 159V 179N 198L 227E


95K 105N 150P 159V 179N 180T 240S


95K 105N 150P 159V 179N 180T 227E


95K 105N 150P 159V 179N 180T 198L


95K 105N 150P 156K 198L 227E 240S


95K 105N 150P 156T 179N 227E 240S


95K 105N 150P 156T 179N 198L 240S


95K 105N 150P 156T 179N 198L 227E


95K 105N 150P 156T 179N 180T 240S


95K 105N 150P 156T 179N 180T 227E


95K 105N 150P 156T 179N 180T 198L


95K 105N 150P 156T 159V 227E 240S


95K 105N 150P 156T 159V 198L 240S


95K 105N 150P 156T 159V 198L 227E


95K 105N 150P 156T 159V 180T 240S


95K 105N 150P 156T 159V 180T 227E


95K 105N 150P 156T 159V 180T 198L


95K 105N 150P 156T 159V 179N 240S


95K 105N 150P 156T 159V 179N 227E


95K 105N 150P 156T 159V 179N 198L


95K 105N 150P 156T 159V 179N 180T


95K 105N 156T 179N 198L 227E 240S


95K 105N 156T 179N 180T 227E 240S


95K 105N 156T 179N 180T 198L 240S


95K 105N 156T 179N 180T 198L 227E


95K 105N 156T 159V 198L 227E 240S


95K 105N 156T 159V 180T 227E 240S


95K 105N 156T 159V 180T 198L 240S


95K 105N 150P 156K 180T 227E 240S


95K 105N 150P 156K 180T 198L 240S


95K 105N 150P 156K 180T 198L 227E


95K 105N 150P 156K 179N 227E 240S


95K 105N 150P 156K 179N 198L 240S


95K 105N 150P 156K 179N 198L 227E


95K 105N 150P 156K 179N 180T 240S


95K 105N 150P 156K 179N 180T 227E


95K 105N 150P 156K 179N 180T 198L


95K 105N 150P 156K 159V 227E 240S


95K 105N 150P 156K 159V 198L 240S


95K 105N 150P 156K 159V 198L 227E


95K 105N 150P 156K 159V 180T 240S


95K 105N 150P 156K 159V 180T 227E


95K 105N 150P 156K 159V 180T 198L


95K 105N 150P 156K 159V 179N 240S


95K 105N 150P 156K 159V 179N 227E


95K 105N 150P 156K 159V 179N 198L


95K 105N 150P 156K 159V 179N 180T


95K 105N 150P 156T 198L 227E 240S


95K 105N 150P 156T 180T 227E 240S


95K 105N 150P 156T 180T 198L 240S


95K 105N 150P 156T 180T 198L 227E


105N 150P 156K 159V 179N 198L 227E 240S


105N 150P 156K 159V 179N 180T 227E 240S


105N 150P 156K 159V 179N 180T 198L 240S


105N 150P 156K 159V 179N 180T 198L 227E


105N 150P 156T 179N 180T 198L 227E 240S


105N 150P 156T 159V 180T 198L 227E 240S


105N 150P 156T 159V 179N 198L 227E 240S


105N 150P 156T 159V 179N 180T 227E 240S


105N 150P 156T 159V 179N 180T 198L 240S


105N 150P 156T 159V 179N 180T 198L 227E


95K 150P 156T 159V 179N 180T 198L 240S


95K 150P 156T 159V 179N 180T 198L 227E


95K 105N 159V 179N 180T 198L 227E 240S


95K 105N 156K 179N 180T 198L 227E 240S


95K 105N 156K 159V 180T 198L 227E 240S


95K 105N 156K 159V 179N 198L 227E 240S


95K 105N 156K 159V 179N 180T 227E 240S


95K 105N 156K 159V 179N 180T 198L 240S


95K 105N 156K 159V 179N 180T 198L 227E


95K 105N 156T 179N 180T 198L 227E 240S


95K 156K 159V 179N 180T 198L 227E 240S


95K 156T 159V 179N 180T 198L 227E 240S


95K 150P 159V 179N 180T 198L 227E 240S


95K 150P 156K 179N 180T 198L 227E 240S


95K 150P 156K 159V 180T 198L 227E 240S


95K 150P 156K 159V 179N 198L 227E 240S


95K 105N 150P 159V 180T 198L 227E 240S


95K 105N 150P 156T 179N 180T 198L 227E


95K 105N 150P 156T 159V 198L 227E 240S


95K 105N 150P 156T 159V 180T 227E 240S


95K 105N 150P 156T 159V 180T 198L 240S


95K 105N 150P 156T 159V 180T 198L 227E


95K 105N 150P 156T 159V 179N 180T 227E


105N 150P 156K 159V 180T 198L 227E 240S


105N 150P 156K 179N 180T 198L 227E 240S


105N 150P 159V 179N 180T 198L 227E 240S


105N 156K 159V 179N 180T 198L 227E 240S


105N 156T 159V 179N 180T 198L 227E 240S


150P 156K 159V 179N 180T 198L 227E 240S


150P 156T 159V 179N 180T 198L 227E 240S


95K 105N 150P 156T 179N 180T 198L 240S


95K 105N 150P 179N 180T 198L 227E 240S


95K 105N 156T 159V 179N 180T 198L 240S


95K 105N 156T 159V 179N 180T 198L 227E


95K 105N 156T 159V 179N 180T 227E 240S


95K 105N 156T 159V 179N 198L 227E 240S


95K 105N 156T 159V 180T 198L 227E 240S


95K 150P 156K 159V 179N 180T 198L 240S


95K 150P 156K 159V 179N 180T 198L 227E


95K 150P 156K 159V 179N 180T 227E 240S


95K 150P 156T 159V 179N 180T 227E 240S


95K 150P 156T 159V 179N 198L 227E 240S


95K 150P 156T 159V 180T 198L 227E 240S


95K 150P 156T 179N 180T 198L 227E 240S


95K 105N 150P 156T 159V 179N 180T 198L


95K 105N 150P 159V 179N 198L 227E 240S


95K 105N 150P 159V 179N 180T 227E 240S


95K 105N 150P 159V 179N 180T 198L 240S


95K 105N 150P 159V 179N 180T 198L 227E


95K 105N 150P 156K 180T 198L 227E 240S


95K 105N 150P 156K 179N 198L 227E 240S


95K 105N 150P 156K 179N 180T 227E 240S


95K 105N 150P 156K 179N 180T 198L 240S


95K 105N 150P 156K 179N 180T 198L 227E


95K 105N 150P 156K 159V 198L 227E 240S


95K 105N 150P 156K 159V 180T 227E 240S


95K 105N 150P 156K 159V 180T 198L 240S


95K 105N 150P 156K 159V 180T 198L 227E


95K 105N 150P 156K 159V 179N 227E 240S


95K 105N 150P 156K 159V 179N 198L 240S


95K 105N 150P 156K 159V 179N 198L 227E


95K 105N 150P 156K 159V 179N 180T 240S


95K 105N 150P 156K 159V 179N 180T 227E


95K 105N 150P 156K 159V 179N 180T 198L


95K 105N 150P 156T 180T 198L 227E 240S


95K 105N 150P 156T 179N 198L 227E 240S


95K 105N 150P 156T 179N 180T 227E 240S


95K 105N 150P 156T 159V 179N 227E 240S


95K 105N 150P 156T 159V 179N 198L 240S


95K 105N 150P 156T 159V 179N 198L 227E


95K 105N 150P 156T 159V 179N 180T 240S


105N 150P 156K 159V 179N 180T 198L 227E 240S


105N 150P 156T 159V 179N 180T 198L 227E 240S


95K 150P 156K 159V 179N 180T 198L 227E 240S


95K 150P 156T 159V 179N 180T 198L 227E 240S


95K 105N 156K 159V 179N 180T 198L 227E 240S


95K 105N 156T 159V 179N 180T 198L 227E 240S


95K 105N 150P 159V 179N 180T 198L 227E 240S


95K 105N 150P 156K 179N 180T 198L 227E 240S


95K 105N 150P 156K 159V 180T 198L 227E 240S


95K 105N 150P 156K 159V 179N 198L 227E 240S


95K 105N 150P 156K 159V 179N 180T 227E 240S


95K 105N 150P 156K 159V 179N 180T 198L 240S


95K 105N 150P 156K 159V 179N 180T 198L 227E


95K 105N 150P 156T 179N 180T 198L 227E 240S


95K 105N 150P 156T 159V 180T 198L 227E 240S


95K 105N 150P 156T 159V 179N 198L 227E 240S


95K 105N 150P 156T 159V 179N 180T 227E 240S


95K 105N 150P 156T 159V 179N 180T 198L 240S


95K 105N 150P 156T 159V 179N 180T 198L 227E


95K 105N 150P 156K 159V 179N 180T 198L 227E 240S


95K 105N 150P 156T 159V 179N 180T 198L 227E 240S









2. Screening



E. coli BL21 cells were transformed with the 1238 hMMP-1 CPS mutants in 96-well format and induced with IPTG at 25° C. as described in Example 1. The proteolytic activity of the hMMP-1 mutants were measured at 25° C. and 37° C. using fluorogenic peptide IX (R&D Systems, Minneapolis, Minn., Cat#ES010) as described in Example 2. Mutants that were active at 25° C. but showed minimal activity at 37° C. were identified as putative hits. The identified hits are set forth in Table 15B. The hits are sorted from the mutant with the lowest activity at 37° C. to the mutant with the highest activity at 37° C., i.e. wildtype. The results showed that many of the mutants, including combination mutants, exhibited 10% or less the activity of wildtype hMMP-1 at 25° C. The low activity was not due to problems with expression, since many of the mutants with no or low activities at 25° C. were expressed well. Several mutants (single and combination) exhibited substantial activity at 25° C. and also showed the best temperature profile (25° C./37° C.).









TABLE 15B







Temperature Profiles of Combination Mutants


















25° C.:
37° C.:






Ratio
% Act.
% Act.



SEQ ID
Avg. RFU
Avg. RFU
(25° C./
of wt
of wt


Variant
NO
25° C.
37° C.
37° C.)
25° C.
25° C.
















D156K/G159V/D179N
3507
1261.31
786.28
1.60
4.73
2.95


R150P/V227E
3508
1801.03
859.01
2.10
6.44
3.07


D156T/V227E
3509
2021.29
864.71
2.34
7.22
3.09


G159V/A198L
3510
1684.53
863.78
1.95
6.06
3.11


D105N/A198L
3511
1422.45
919.80
1.55
5.34
3.45


L95K
6
1389.81
969.67
1.43
5.00
3.49


D179N/V227E
3512
1446.86
948.41
1.53
5.43
3.56


A198L/V227E
3513
2740.04
1036.69
2.64
9.79
3.70


E180T/V227E
3514
2549.76
1038.44
2.46
9.11
3.71


D179N/A198L
3515
1411.89
968.14
1.46
5.45
3.74


D156K/D179N
3516
1227.63
973.51
1.26
4.74
3.76


D105N/R150P/D156K/
3517
1668.82
1002.65
1.66
6.26
3.76


G159V/D179N/E180T


D105N/R150P/E180T
3518
1846.75
1003.36
1.84
6.93
3.76


G159V/I240S
3519
2565.48
1031.27
2.49
9.45
3.80


D156T/D179N/I240S
3520
1326.33
774.68
1.71
6.53
3.81


D156T/G159V
3521
1521.88
1048.30
1.45
5.71
3.93


R150P/E180T
3522
1636.14
1112.37
1.47
5.85
3.98


D156T/D179N
3523
3855.30
1049.65
3.67
14.72
4.01


D179N/I240S
3524
1890.16
826.28
2.29
9.30
4.07


L95K/D156T/D179N
3525
2075.52
1194.20
1.74
7.79
4.48


D156T
125
5564.55
1304.31
4.27
26.15
6.13


G159V
132
6330.31
1716.35
3.49
24.17
6.94


G159V/D179N
3526
4741.70
1896.45
2.50
17.79
7.12


A198L
305
4888.05
1555.23
3.14
22.97
7.31


L95K/D105N/E180T
3527
3640.58
2177.79
1.67
13.66
8.17


R150P/D156T/A198L
3528
2554.33
1770.29
1.44
12.00
8.32


V227E
384
21170.85
2439.36
9.01
76.14
8.45


I240S
488
5525.59
1486.79
3.72
33.21
8.94


L95K/D105N/R150P/
3529
2930.99
2217.79
1.32
14.58
11.03


D156T/G159V/A198L/


V227E/I240S


L95K/R150P
3530
6360.67
3108.26
2.05
30.68
14.99


D105N/E180T
3531
13018.08
4994.85
2.61
46.52
17.85


R150P
59
11979.01
4261.20
2.81
56.29
20.02


D105N
27
12356.79
4628.13
2.67
58.06
21.75


E180T
181
26456.92
11205.01
2.36
94.55
40.04


Wildtype
2
26316.84
22348.45
1.18
94.64
80.37









The results showing low activity at 25° C. for many of the combination mutants suggested that the combination mutants were altering the protein, such that their optimal temperature for activity was shifted below 25° C. To test this, the proteolytic activity of some of the combination mutants against the fluorogenic peptide IX was tested at 20° C., 25° C. and 37° C. Included among the combination mutants that were tested were: G159V/A198L; D156T/D179N; G159V/D179N; D179N/V227E; A198L/V227E; D156K/D179N; 179/240; and D156T/D179N/I240S. The results showed that several of the combination mutants had slightly higher activity at 20° C. than at 25° C., and little activity at 37° C. All of the mutants tested exhibited less activity (only about 33% of the activity or less) than wildtype MMP-1 at the corresponding temperature. One of the mutants, D156T/D179N, was tested and exhibited higher activity at 18° C. than wildtype.


Example 4
Reversibility of Enzymatic Activity Following Decrease in Temperature

In this example, the temperature sensitive hMMP-1 mutants that were confirmed in Example 2B were further assayed to determine whether enzymatic activity at 25° C. was reversible or irreversible following subsequent exposure to elevated temperatures followed by a return to 25° C. The hMMP-1 mutants were expressed in 14 ml culture tubes, as described in Example 2B. The putative hits were tested for their activities under five conditions: at 25° C., 34° C. or 37° C., and at 34° C. or 37° C. and subsequent re-exposure to the requisite temperature of 25° C. (see Table 16 for reaction conditions). Mutants that were active at 25° C., showed decreased activity when raised to 34° C. or 37° C. (i.e. the ratio of the activities at 25° C./34° C. or 25° C./37° C. is equal to or greater than 1.5), and exhibited a baseline activity when lowered again to 25° C. were scored as “Reversible Hits.” Mutants that were active at 25° C., showed decreased activity when raised to 34° C. or 37° C. (i.e. the ratio of the activities at 25° C./34° C. or 25° C./37° C. is equal to or greater than 1.5), and exhibited the same amount of decreased activity when lowered again to 25° C. were scored as “Irreversible Hits.”


A. Reaction Conditions

The reversibility of enzymatic activity of each hMMP-1 mutant was determined using the previously described fluorescence assay as modified below. In short, the 4 μl of the supernatant of each hMMP-1 mutant was diluted in TCNB with 1 mM APMA and transferred to a 96-well plate. Five different wells were prepared for each hMMP-1 mutant as set forth in Table 16. The solution was incubated at the initial reaction temperature (25° C., 34° C., or 37° C.) for 2 hours. This activation step cleaves the pro-peptide and generates mature hMMP-1.


Following activation, 100 μl of TCNB with 10 μM Mca-K-P-L-G-L-Dpa-A-R-NH2 fluorescent substrate was added to each well and reaction conditions were as summarized in Table 16, below. Briefly, each hMMP-1 mutant was exposed to each of the five reaction conditions by incubation of the hMMP-1 mutant in the presence of the fluorogenic substrate for an hour at the initial temperature. For each mutant, baseline activity at 25° C., 34° C., or 37° C. was assessed by incubation with the substrate for an additional 1 hour (2 hour condition) or overnight (overnight condition), followed by fluorescence measurement. To assess the reversibility/irreversibility of activity, samples incubated for an initial 1 hour at 34° C., or 37° C. were lowered to 25° C. and allowed to incubate for either an hour (2 hour condition) or 16 hours (overnight condition), followed by fluorescence measurement. Wildtype hMMP-1 was used as a positive control and supernatant from cells transformed with only vector was used as a negative control. Fluorescence was detected by measuring fluorescence in a fluorescent plate reader at 320 nm exitation/405 nm emission. Relative fluorescence units (RFU) were determined. Duplicate reactions were performed for each sample, reaction temperature, and positive and negative control.









TABLE 16







Reaction Conditions












Initial
Incubation




Condition
Temperature
at 25° C.
2 Hours
Overnight





25° C.
25° C.

2 hours
overnight


34° C.
34° C.

2 hours
overnight


34° C. to
34° C.
25° C.
a) 34° C. for 1
a) 34° C. for 1


25° C.


hour
hour





b) 25° C. for 1
b) 25° C. for 16





hour
hours


37° C.
37° C.

2 hours
overnight


37° C. to
37° C.
25° C.
a) 37° C. for 1
a) 37° C. for 1


25° C.


hour
hour





b) 25° C. for 1
b) 25° C. for 16





hour
hours










B. Results: Partially Reversible hMMP-1 Mutants


Twenty six hMMP-1 mutants were determined to be partially reversible. Although the activity (in RFU) did not return to baseline activity observed at 25° C., an overall increase in activity was observed when the temperature was returned to 25° C. compared to activity at 34° C. or 37° C. The results are shown in Tables 17-20 below, which list the activities (in RFUs) and the ratios of the activities. Tables 17 and 18 summarize the results of reversibility at 34° C. or 37° C., respectively, of the hMMP-1 partially reversible mutants under the 2 hour condition. Tables 19 and 20 summarize the results of reversibility at 34° C. or 37° C., respectively, of the partially reversible hMMP-1 mutants under the overnight condition. The results are similar under all reaction conditions, temperature and time. The activity at 34° C. or 37° C. overnight is lower than the activity when incubated at 34° C. or 37° C. for one hour then 25° C. overnight. For example, the activity of E180Y at 34° C. is 6080 RFU but its activity at 34° C. then overnight at 25° C. increased to 8570 RFU (see Table 19, below).









TABLE 17







Partially Reversible hMMP-1 mutants (2 Hours, 34° C.)



















Ratio






RFU
Ratio
25° C./


hMMP-1
SEQ ID
RFU
RFU
34 to
25° C./
34 to


mutation
NO
25° C.
34° C.
25° C.
34° C.
25° C.
















D105A
39
5669.31
824.07
922.97
6.88
6.14


D105F
33
2980.00
623.89
725.03
4.78
4.11


D105G
32
8821.81
2759.24
2966.37
3.20
2.97


D105S
31
9355.63
4607.18
6681.63
2.03
1.40


D105T
29
4457.16
974.63
1534.71
4.57
2.90


R150P
59
8750.30
2315.11
2506.15
3.78
3.49


G159T
125
6704.95
2294.40
2344.57
2.92
2.86


E180Y
182
8557.09
4979.24
6224.87
1.72
1.37


E180T
181
7870.99
1532.35
1852.46
5.14
4.25


E180F
185
8508.13
3597.75
3915.71
2.36
2.17


T185H
233
5593.77
2278.26
2429.05
2.46
2.30


T185Q
238
7006.87
2250.58
2397.60
3.11
2.92


T185A
248
2474.96
663.82
822.83
3.73
3.01


T185E
232
3948.43
2088.15
1862.83
1.89
2.12


N187R
254
3006.08
1352.97
1343.94
2.22
2.24


N187M
262
4934.44
1811.35
1793.14
2.72
2.75


N187K
253
4182.49
2425.34
2415.57
1.72
1.73


R195V
284
4847.81
2724.92
2517.49
1.78
1.93


A198L
305
6756.76
2056.50
2046.15
3.29
3.30


A198M
301
3777.50
1708.61
1725.14
2.21
2.19


S210V
341
3349.95
1249.47
1622.57
2.68
2.06


Y218S
354
2878.50
2373.98
2187.48
1.21
1.32


F223E
365
8318.70
3685.68
5283.08
2.26
1.57


V227W
397
996.55
729.20
834.38
1.37
1.19


L229I
436
2790.27
1050.86
1738.46
2.66
1.61


I240C
483
2688.75
561.91
884.15
4.78
3.04
















TABLE 18







Partially Reversible hMMP-1 mutants (2 Hours, 37° C.)



















Ratio






RFU
Ratio
25° C./


hMMP-1
SEQ ID
RFU
RFU
37 to
25° C./
37 to


mutation
NO
25° C.
37° C.
25° C.
37° C.
25° C.
















D105A
39
5669.31
1336.14
1509.52
4.24
3.76


D105F
33
2980.00
818.63
1004.23
3.64
2.97


D105G
32
8821.81
4313.40
4643.53
2.05
1.90


D105S
31
9355.63
7274.97
7453.42
1.29
1.26


D105T
29
4457.16
2220.03
2177.84
2.01
2.05


R150P
59
8750.30
2497.86
3115.73
3.50
2.81


G159T
125
6704.95
2347.74
2530.78
2.86
2.65


E180Y
182
8557.09
6079.36
6421.56
1.41
1.33


E180T
181
7870.99
1794.15
1824.99
4.39
4.31


E180F
185
8508.13
3975.22
3981.79
2.14
2.14


T185H
233
5593.77
2534.15
2693.25
2.21
2.08


T185Q
238
7006.87
2642.74
2589.77
2.65
2.71


T185A
248
2474.96
707.09
730.58
3.50
3.39


T185E
232
3948.43
2091.32
2106.55
1.89
1.87


N187R
254
3006.08
1421.87
1476.42
2.11
2.04


N187M
262
4934.44
1893.07
1998.97
2.61
2.47


N187K
253
4182.49
2652.79
2902.79
1.58
1.44


R195V
284
4847.81
2984.10
3555.03
1.62
1.36


A198L
305
6756.76
2642.76
2540.07
2.56
2.66


A198M
301
3777.50
2155.58
2802.78
1.75
1.35


S210V
341
3349.95
2314.86
2277.32
1.45
1.47


Y218S
354
2878.50
2350.27
2383.67
1.22
1.21


F223E
365
8318.70
6209.93
7415.02
1.34
1.12


V227W
397
996.55
787.87
850.67
1.26
1.17


L229I
436
2790.27
1803.44
2453.07
1.55
1.14


I240C
483
2688.75
853.66
872.62
3.15
3.08
















TABLE 19







Partially Reversible hMMP-1 mutants (Overnight, 34° C.)



















Ratio






RFU
Ratio
25° C./


hMMP-1
SEQ ID
RFU
RFU
34 to
25° C./
34 to


mutation
NO
25° C.
34° C.
25° C.
34° C.
25° C.
















D105A
39
8466.62
1302.84
1532.38
6.50
5.53


D105F
33
6725.59
938.60
1172.86
7.17
5.73


D105G
32
8940.06
3560.75
5314.44
2.51
1.68


D105S
31
9300.85
5584.70
9413.56
1.67
0.99


D105T
29
7910.47
1899.25
3254.16
4.17
2.43


R150P
59
9011.11
3533.16
4443.96
2.55
2.03


G159T
125
9105.95
3210.57
4179.05
2.84
2.18


E180Y
182
9281.77
6080.89
8570.48
1.53
1.08


E180T
181
8475.04
2585.89
3901.87
3.28
2.17


E180F
185
9360.74
5183.25
7022.64
1.81
1.33


T185H
233
8531.85
3164.69
5520.76
2.70
1.55


T185Q
238
9044.23
3639.00
5467.27
2.49
1.65


T185A
248
6156.97
1110.68
1585.53
5.54
3.88


T185E
232
8479.18
3868.06
4836.97
2.19
1.75


N187R
254
7593.11
2415.63
3156.74
3.14
2.41


N187M
262
8605.76
2769.52
4008.68
3.11
2.15


N187K
253
8667.36
3458.94
5465.35
2.51
1.59


R195V
284
8634.05
4648.03
5966.81
1.86
1.45


A198L
305
8795.36
3469.36
5027.30
2.54
1.75


A198M
301
8352.73
3215.69
4220.51
2.60
1.98


S210V
341
7104.17
2441.96
3664.23
2.91
1.94


Y218S
354
7740.61
4057.37
5769.79
1.91
1.34


F223E
365
9650.44
4849.58
9311.40
1.99
1.04


V227W
397
3070.92
1370.13
1632.51
2.24
1.88


L229I
436
7333.92
1832.18
4427.24
4.00
1.66


I240C
483
6170.51
1174.96
2389.06
5.25
2.58
















TABLE 20







Partially Reversible hMMP-1 mutants (Overnight, 37° C.)



















Ratio






RFU
Ratio
25° C./


hMMP-1
SEQ ID
RFU
RFU
37 to
25° C./
37 to


mutation
NO
25° C.
37° C.
25° C.
37° C.
25° C.
















D105A
39
8466.62
1931.17
2589.08
4.38
3.27


D105F
33
6725.59
1173.23
1759.31
5.73
3.82


D105G
32
8940.06
5390.32
7139.57
1.66
1.25


D105S
31
9300.85
8234.95
8615.33
1.13
1.08


D105T
29
7910.47
3292.01
4482.74
2.40
1.76


R150P
59
9011.11
3559.66
5181.30
2.53
1.74


G159T
125
9105.95
3160.07
4338.35
2.88
2.10


E180Y
182
9281.77
6894.61
8986.47
1.35
1.03


E180T
181
8475.04
2809.15
3649.72
3.02
2.32


E180F
185
9360.74
5335.15
7183.36
1.75
1.30


T185H
233
8531.85
3515.59
6101.91
2.43
1.40


T185Q
238
9044.23
4012.93
5623.60
2.25
1.61


T185A
248
6156.97
1059.61
1315.46
5.81
4.68


T185E
232
8479.18
3892.33
5330.81
2.18
1.59


N187R
254
7593.11
2370.01
3425.18
3.20
2.22


N187M
262
8605.76
2720.28
4400.27
3.16
1.96


N187K
253
8667.36
3709.62
6374.32
2.34
1.36


R195V
284
8634.05
4960.91
7212.05
1.74
1.20


A198L
305
8795.36
4181.78
5395.22
2.10
1.63


A198M
301
8352.73
3637.79
5914.49
2.30
1.41


S210V
341
7104.17
3939.90
4626.58
1.80
1.54


Y218S
354
7740.61
4093.29
6181.92
1.89
1.25


F223E
365
9650.44
7645.34
9149.09
1.26
1.05


V227W
397
3070.92
1456.45
1695.81
2.11
1.81


L229I
436
7333.92
3268.93
5729.00
2.24
1.28


I240C
483
6170.51
2223.23
2050.31
2.78
3.01










C. Results: Non Reversible hMMP-1 Mutants


Thirty eight hMMP-1 mutants were determined to be non reversible. The activity of these mutants at 34° C. or 37° C., which is decreased compared to the activity at 25° C., remained decreased when lowered to 25° C. The results are shown in Tables 21-24 below, which list the activities (in RFUs) and the ratios of the activities. Tables 21 and 22 summarize the results at 34° C. or 37° C., respectively, of the hMMP-1 irreversible mutants under the two hour condition. Tables 23 and 24 summarize the results of reversibility at 34° C. or 37° C., respectively, of the irreversible hMMP-1 mutants under the overnight condition. The results are similar under all reaction conditions, temperature and time. The activity at 34° C. or 37° C. overnight is the same or similar to the activity when incubated at 34° C. or 37° C. for one hour then 25° C. overnight. For example, the activity of D105R at 34° C. is 1407 RFU and its activity at 34° C. then overnight at 25° C. is 1424 RFU (see Table 23, below).









TABLE 21







Non Reversible hMMP-1 mutants (2 Hours, 34° C.)



















Ratio






RFU
Ratio
25° C./


hMMP-1
SEQ ID
RFU
RFU
34 to
25° C./
34 to


mutation
NO
25° C.
34° C.
25° C.
34° C.
25° C.
















L95K
6
4650.42
748.29
833.29
6.21
5.58


D105I
36
6832.34
780.32
908.39
8.76
7.52


D105L
38
4206.38
534.24
630.66
7.87
6.67


D105N
27
8920.05
918.13
1128.03
9.72
7.91


D105R
25
2821.20
722.46
843.19
3.90
3.35


D105W
35
6663.80
1690.93
2266.26
3.94
2.94


D151G
70
1264.62
589.27
664.86
2.15
1.90


F155A
96
2824.01
779.72
735.02
3.62
3.84


D156K
100
8576.47
2210.63
2318.28
3.88
3.70


D156T
105
8727.27
2679.17
2770.95
3.26
3.15


D156L
114
2916.24
576.84
655.46
5.06
4.45


D156A
115
2299.63
533.68
635.67
4.31
3.62


D156W
111
1502.86
539.74
637.12
2.78
2.36


D156V
113
1593.06
534.71
634.83
2.98
2.51


D156H
99
5387.79
698.77
784.55
7.71
6.87


D156R
101
7020.81
793.83
881.39
8.84
7.97


G159V
132
4673.44
856.78
789.92
5.45
5.92


A176F
148
1609.85
654.43
633.13
2.46
2.54


D179N
160
5660.69
644.51
644.98
8.78
8.78


D181L
209
2710.97
619.39
645.65
4.38
4.20


D181K
195
1130.63
625.01
609.58
1.81
1.85


E182T
219
3702.08
791.23
805.48
4.68
4.60


E182Q
218
1331.50
639.84
623.88
2.08
2.13


T185R
235
2637.31
1187.63
1158.47
2.22
2.28


N187F
261
3227.96
877.21
823.16
3.68
3.92


N187I
264
4218.55
849.11
869.19
4.97
4.85


G206A
324
872.27
603.01
592.13
1.45
1.47


G206S
317
932.69
492.65
507.75
1.89
1.84


V227C
388
1998.67
950.01
1115.17
2.10
1.79


V227E
384
7904.54
839.00
906.06
9.42
8.72


Q228P
420
1082.56
607.78
617.33
1.78
1.75


L229T
429
1221.05
580.15
605.83
2.10
2.02


D233E
440
2195.02
1393.95
1332.07
1.57
1.65


I234A
476
2375.42
1473.70
1456.58
1.61
1.63


I234T
467
1199.18
713.83
775.40
1.68
1.55


I234E
460
3920.02
705.86
829.15
5.55
4.73


I240S
488
3867.71
973.97
1027.84
3.97
3.76
















TABLE 22







Non Reversible hMMP-1 mutants (2 Hours, 37° C.)



















Ratio






RFU
Ratio
25° C./


hMMP-1
SEQ ID
RFU
RFU
37 to
25° C./
37 to


mutation
NO
25° C.
37° C.
25° C.
37° C.
25° C.
















L95K
6
4650.42
746.89
1092.61
6.23
4.26


D105I
36
6832.34
1110.07
1104.96
6.15
6.18


D105L
38
4206.38
607.46
624.88
6.92
6.73


D105N
27
8920.05
1727.44
1820.97
5.16
4.90


D105R
25
2821.20
813.68
846.09
3.47
3.33


D105W
35
6663.80
3081.59
3123.49
2.16
2.13


D151G
70
1264.62
616.51
628.65
2.05
2.01


F155A
96
2824.01
746.59
867.76
3.78
3.25


D156K
100
8576.47
2310.30
2080.22
3.71
4.12


D156T
105
8727.27
2752.35
2251.21
3.17
3.88


D156L
114
2916.24
688.08
652.06
4.24
4.47


D156A
115
2299.63
554.21
606.45
4.15
3.79


D156W
111
1502.86
575.12
582.43
2.61
2.58


D156V
113
1593.06
542.36
544.49
2.94
2.93


D156H
99
5387.79
819.82
881.23
6.57
6.11


D156R
101
7020.81
872.40
944.17
8.05
7.44


G159V
132
4673.44
838.46
932.14
5.57
5.01


A176F
148
1609.85
618.72
741.21
2.60
2.17


D179N
160
5660.69
656.31
636.18
8.63
8.90


D181L
209
2710.97
611.92
668.31
4.43
4.06


D181K
195
1130.63
608.68
646.77
1.86
1.75


E182T
219
3702.08
826.28
746.25
4.48
4.96


E182Q
218
1331.50
623.11
629.01
2.14
2.12


T185R
235
2637.31
1183.37
1158.87
2.23
2.28


N187F
261
3227.96
931.04
856.03
3.47
3.77


N187I
264
4218.55
887.80
879.78
4.75
4.80


G206A
324
872.27
586.57
654.37
1.49
1.33


G206S
317
932.69
463.60
552.97
2.01
1.69


V227C
388
1998.67
992.19
1130.51
2.01
1.77


V227E
384
7904.54
1015.12
1127.74
7.79
7.01


Q228P
420
1082.56
586.63
777.28
1.85
1.39


L229T
429
1221.05
564.49
747.87
2.16
1.63


D233E
440
2195.02
1454.71
1976.42
1.51
1.11


I234A
476
2375.42
1594.08
1460.23
1.49
1.63


I234T
467
1199.18
796.81
833.55
1.50
1.44


I234E
460
3920.02
923.57
867.78
4.24
4.52


I240S
488
3867.71
1575.05
1594.10
2.46
2.43
















TABLE 23







Non Reversible hMMP-1 mutants (Overnight, 34° C.)



















Ratio






RFU
Ratio
25° C./


hMMP-1
SEQ ID
RFU
RFU
34 to
25° C./
34 to


mutation
NO
25° C.
34° C.
25° C.
34° C.
25° C.
















L95K
6
7744.34
1803.12
1892.59
4.29
4.09


D105I
36
8394.32
1614.57
1736.52
5.20
4.83


D105L
38
6546.78
957.95
988.23
6.83
6.62


D105N
27
9119.04
1459.16
1822.40
6.25
5.00


D105R
25
5775.25
1407.06
1424.59
4.10
4.05


D105W
35
8617.36
2851.22
4709.94
3.02
1.83


D151G
70
1956.65
959.80
1013.03
2.04
1.93


F155A
96
4891.89
2016.76
1493.70
2.43
3.28


D156K
100
8696.27
3968.92
4371.25
2.19
1.99


D156T
105
8972.20
3971.43
4480.62
2.26
2.00


D156L
114
5254.55
972.64
1011.27
5.40
5.20


D156A
115
3585.37
1098.25
1057.84
3.26
3.39


D156W
111
2570.24
1091.27
1126.01
2.36
2.28


D156V
113
2208.99
954.21
954.54
2.31
2.31


D156H
99
7587.19
1451.49
1440.25
5.23
5.27


D156R
101
8622.23
1735.02
1760.60
4.97
4.90


G159V
132
6555.27
1821.53
1524.05
3.60
4.30


A176F
148
4191.69
1414.21
1181.99
2.96
3.55


D179N
160
7317.57
1504.84
1458.70
4.86
5.02


D181L
209
4534.34
1078.98
984.43
4.20
4.61


D181K
195
1869.47
946.27
841.77
1.98
2.22


E182T
219
6752.25
1483.52
1570.77
4.55
4.30


E182Q
218
2212.75
1065.07
929.49
2.08
2.38


T185R
235
6281.97
2425.71
2808.30
2.59
2.24


N187F
261
7352.85
1612.23
1533.32
4.56
4.80


N187I
264
8306.40
1459.25
1598.90
5.69
5.20


G206A
324
2492.53
1038.14
906.63
2.40
2.75


G206S
317
2845.84
908.82
816.00
3.13
3.49


V227C
388
5833.84
2207.20
2739.65
2.64
2.13


V227E
384
8630.90
2283.07
2096.30
3.78
4.12


Q228P
420
3673.33
1162.95
1213.48
3.16
3.03


L229T
429
3543.75
1103.34
1105.90
3.21
3.20


D233E
440
6694.93
2570.71
3171.20
2.60
2.11


I234A
476
6250.56
3890.90
3608.10
1.61
1.73


I234T
467
3507.08
1099.58
1194.99
3.19
2.93


I234E
460
7541.73
1365.08
1817.16
5.52
4.15


I240S
488
4376.99
2108.15
2290.56
2.08
1.91
















TABLE 24







Non Reversible hMMP-1 mutants (Overnight, 37° C.)



















Ratio






RFU
Ratio
25° C./


hMMP-1
SEQ ID
RFU
RFU
37 to
25° C./
37 to


mutation
NO
25° C.
37° C.
25° C.
37° C.
25° C.
















L95K
6
7744.34
1677.96
2463.18
4.62
3.14


D105I
36
8394.32
1958.96
1925.73
4.29
4.36


D105L
38
6546.78
1070.51
939.53
6.12
6.97


D105N
27
9119.04
2347.74
2813.87
3.88
3.24


D105R
25
5775.25
1499.57
1312.01
3.85
4.40


D105W
35
8617.36
4593.06
5698.08
1.88
1.51


D151G
70
1956.65
1097.68
900.59
1.78
2.17


F155A
96
4891.89
1843.31
1882.95
2.65
2.60


D156K
100
8696.27
3858.90
4126.13
2.25
2.11


D156T
105
8972.20
3854.84
3990.29
2.33
2.25


D156L
114
5254.55
1232.94
1008.08
4.26
5.21


D156A
115
3585.37
1110.73
940.62
3.23
3.81


D156W
111
2570.24
1206.22
997.15
2.13
2.58


D156V
113
2208.99
997.64
777.35
2.21
2.84


D156H
99
7587.19
1763.27
1536.01
4.30
4.94


D156R
101
8622.23
1846.71
1764.13
4.67
4.89


G159V
132
6555.27
1683.20
1842.91
3.89
3.56


A176F
148
4191.69
1336.32
1553.01
3.14
2.70


D179N
160
7317.57
1485.28
1378.59
4.93
5.31


D181L
209
4534.34
1000.80
1020.08
4.53
4.45


D181K
195
1869.47
928.55
895.45
2.01
2.09


E182T
219
6752.25
1496.55
1319.53
4.51
5.12


E182Q
218
2212.75
1035.24
916.32
2.14
2.41


T185R
235
6281.97
2300.61
2829.34
2.73
2.22


N187F
261
7352.85
1704.23
1533.08
4.31
4.80


N187I
264
8306.40
1465.77
1560.83
5.67
5.32


G206A
324
2492.53
974.96
1057.32
2.56
2.36


G206S
317
2845.84
808.42
908.44
3.52
3.13


V227C
388
5833.84
2432.82
2707.71
2.40
2.15


V227E
384
8630.90
2152.81
2615.26
4.01
3.30


Q228P
420
3673.33
1081.32
1681.57
3.40
2.18


L229T
429
3543.75
1030.05
1488.58
3.44
2.38


D233E
440
6694.93
2661.43
4531.45
2.52
1.48


I234A
476
6250.56
4043.80
3433.03
1.55
1.82


I234T
467
3507.08
1228.23
1397.18
2.86
2.51


I234E
460
7541.73
1901.96
1783.16
3.97
4.23


I240S
488
4376.99
2592.19
3417.53
1.69
1.28









Example 5
Proteolytic Activity of hMMP-1 on Insoluble Collagen

In this example, the collagenase activity of hMMP-1 was assessed for the protein substrate collagen using SDS-PAGE analysis. Wildtype hMMP-1 cleaves insoluble collagen (α1(I) and α2(I) chains) into three-quarter and one-quarter length digestion products. In this assay, a fluorescein isothiocyanate (FITC)-conjugated collagen was used as the substrate and the reaction was monitored by SDS-PAGE of the reaction products. Cleavage of α1(I) and α2(I) collagen chains results in ¾ and ¼ length digestion products which are distinguishable from full length collagen by separation on SDS polyacrylamide gels. Alternatively, cleavage was assessed by fluorometric analysis. A similar assay can be used to assess the activity of mutant hMMPs for cleavage activity at 25° C. versus 34° C. or 37° C.


A. SDS-PAGE Analysis


In this Example, wild-type MMP-1 was tested for cleavage of insoluble collagen and assessed by SDS-PAGE. In short, 2 μg of hMMP-1 (purchased from R&D Systems, #901-MP; or BAP0062 and BAP00610 purified as described in Example 1.B) was diluted in TCNB containing 1 mM AMPA and incubated at the reaction temperature (25° C. or 37° C.) for 2 hours. This activation step cleaves the pro-peptide and generates mature hMMP-1. Subsequently, 6 μg of insoluble collagen conjugated to fluorescein isothiocyanate (FITC) (Anaspec #85111 or Sigma Collagen #C4361) in 20 μl TCNB was added to each activated hMMP-1 aliquot and the mixture was incubated at 25° C. or 37° C. for 24 hours or 6 days.


Cleavage of the insoluble collagen was observed by SDS/PAGE. The reaction mixture was separated on a 7.5 SDS polyacrylamide gel and visualized by staining with Coomassie Blue dye. SDS/PAGE results show that after 24 hours incubation at 25° C. or 37° C., hMMP-1 partially cleaved the α1(I) and α2(I) collagen chains into ¾ and ¼ length digestion products for all hMMP-1 proteins tested. After 6 days at 25° C., complete cleavage into ¾ and ¼ length digestion products was observed. After 6 days at 37° C., the collagen was digested completely. The ¾ and ¼ length collagen digestion products are thermally unstable at body temperature.


B. Fluorometric Analysis


Alternatively, collagenase activity was measured using a fluorescence assay. 5 μg hMMP-1 (purchased from R&D Systems, #901-MP; or BAP0062 and BAP00610 purified as described in Example 1.B) was diluted in TCNB containing 1 mM AMPA to a final concentration and incubated at 37° C. for 2 hours. The activity of hMMP-1 for FITC-labeled collagen (Sigma #C4361 or Elastin #CF308) was assessed using a protocol adapted from Baici A et al. (1980) Anal. Biochem., 108: 230-232). Briefly, hMMP-1 was incubated with the substrate for 144 hours at 37° C. As a negative control, the substrate was incubated with buffer only. Following incubation, the reaction mixture was first centrifuged to remove insoluble particles. Fluorescence of the supernatant was detected by measuring fluorescence in a fluorescent plate reader at 495 nm excitation/520 nm emission. Relative fluorescence units (RFU) were determined. Duplicate reactions were performed for each sample.


The results (see Tables 25 and 26 below) show that incubation of insoluble collagen with wildtype hMMP-1 at 37° C. for 144 hours resulted in cleavage of collagen as indicated by high RFU values compared to buffer only control. For example, for cleavage of collagen from Sigma, all hMMPs tested had an RFU between about 1000.00-1200.00 compared to buffer only with an RFU value of about 400.00. The activity of purified collagens from CHO-S (BAP0062) and BL21 cells (BAP00610) for cleavage of Sigma insoluble collagen was comparable to hMMP-1 purchased from R&D systems. For cleavage of Elastin collagen, the activity of recombinant hMMP-1 purchased from R&D and BAP00610 were about 3000.00 RFU, while the activity of BAP0062 was about 2000.00 RFU. Buffer only exhibited a background fluorescence for cleavage of Elastin collagen of about 1500.00 RFU.









TABLE 25







Cleavage of Collagen (Sigma Insoluble Substrate)











hMMP-1
37° C.
37° C.
Avg 37° C.
St Dev














R&D systems
1163.17
1137.81
1150.49
17.93


Buffer only
481.49
490.57
486.03
6.42


BAP006_2 (CHO)
1265.61
1275.17
1270.39
6.76


BAP006_10 (BL21)
1292.36
1335.14
1313.75
30.25
















TABLE 26







Cleavage of Collagen (Elastin Insoluble Substrate)











hMMP-1
37° C.
37° C.
Avg 37° C.
St Dev





R&D systems
3488.224
2981.417
3235.32
357.66


Buffer only
1312.511
1807.479
1560.00
350.00


BAP006_2 (CHO)
1729.757
2297.573
2013.67
401.51


BAP006_10 (BL21)
2669.758
3056.381
2863.07
273.38









Example 6
Identification of Temperature Sensitive Mutants in the Hemopexin Binding Domain

A hMMP-1 mutant library was generated similar to Example 1 by introducing mutations in the parent human MMP-1 DNA to generate single amino acid variants of MMP- in the hemopexin domain at amino acid positions 259, 260, 261, 262, 263, 264, 301, 302, 303, 304, 305, 306, 441, 442, 443, 444, 445 and 446. The mutants were expressed as described in Example 1 and tested for enzymatic activity against a fluorogenic peptide substrate as described in Example 2. One mutant, C259Q (set forth in SEQ ID NO:3532), was identified as a hit with increased activity at 25° C. compared to 37° C.


Next, 11 double mutants were generated containing C259Q and one of L95K; D105N; R150P; D156K; D156T; G159V; D179N; E180T; A198L; V227E or I240S. These double mutants were expressed as described in Example 1 and tested for enzymatic activity against a fluorogenic peptide substrate as described in Example 2. Five (5) double mutants were identified that are active at 25° C. but show decreased activity at 37° C. The identified double mutants were C259Q/D105N (SEQ ID NO:3533); C259Q/R150P (SEQ ID NO:3534); C259Q/G159V (SEQ ID NO:3535); C259Q/D179N (SEQ ID NO:3536); and C259Q/E180T (SEQ ID NO:3537). The mutants exhibited a ratio of activity (25° C./37° C.) of 10-fold to almost 25-fold, with the C259Q/D179N exhibiting the greatest ratio of activity at almost 25-fold.


Example 7
Generation of Activity & Temperature-Sensitive Combination Mutants

Three (3) hMMP-1 variant activity mutants (S208K set forth in SEQ ID NO:3538; I213G set forth in SEQ ID NO:3539; and G214E set forth in SEQ ID NO:3540), identified in Table 9 as having higher activity at 37° C. and 25° C., were used to generate double mutants with the temperature-sensitive hits set forth in Table 14. Each activity mutant was combined with each of the 11 temperature-sensitive hits set forth in Table 14 (**) to generate double mutants. Wildtype hMMP-1 and 31 double mutants were transformed into E. coli BL21 (DE3) competent cells in 14 mL tubes as described in Example 1. Protein was expressed as described in Example 1 upon the addition of 1 mM IPTG at 25° C. Cells were collected 6 hours post-induction. Periplasmic proteins were prepared by incubating the cells in OS buffer (200 mM Tris-HCl, pH 7.5, 20% sucrose, 1 mM EDTA) with DNAse, RNAse and lysozyme. After addition of H2O to the cells in OS buffer, the cells were centrifuged. The supernatants which contain the periplasmic fractions were transferred to another tube. Supernatants were used to measure the proteolytic activity of hMMP-1 produced by BL21 cells transformed with the wildtype and the double mutants using the assay described in Example 2. The supernatants were incubated with APMA at 37° C. and 25° C. to activate the enzymes. Fluorogenic peptide IX was used as the substrate to determine the activity of hMMP-1. Fluorescence was measured using wavelengths of 320 nm (excitation) and 405 nm (emission) with a microtiter plate fluorescence reader. Duplicate reactions were done for each sample. The ratios were determined by dividing the activities at 25° C. to the activities at 37° C. The value of background activities were substrated from the activities of the wildtype and double mutants. The results showed that incorporation of the activity mutation did not increase the activity of the temperature-sensitive mutants at 25° C. Six (6) double mutants, however, were identified as exhibit activity at 25° C., but show decreased activity at 37° C. These double mutants include: S208K/G159V (SEQ ID NO:3541); S208K/D179N (SEQ ID NO:3542); S208K/V227E (SEQ ID NO:3543); G214E/G159V (SEQ ID NO:3544); G214E/D179N (SEQ ID NO:3545); and I213G/D179N (SEQ ID NO:3546). The ratio of activity (25° C./37° C.) of the mutants were as follows: almost 14-fold for the S208K/G159V mutant; about 14-fold for the S208K/D179N mutant; about 13-fold for the S208K/C227E mutant; about 8-fold for the G214E/G159V mutant; almost 14-fold for the G214E/D179N mutant; and about 14-fold for the I213G/D179N mutant. As expected, wild-type hMMP-1 exhibited a ratio of activity of about 1-fold.


Example 8
Proteolytic Activity of hMMP-1 Variants on Collagens

Cleavage activity of wild-type and various mutant hMMP-1's for Collagen Type I and Type IV at 25° C. or 37° C. was tested by separation on SDS polyacrylamide gels and analysis of digestion products. Wild-type hMMP-1 used in these experiments included mammalian expressed purchased from R&D systems (R&D Systems, Catalog #901-MP; NSO cells) or E. coli expressed (BL21 cells) as described in Example 1B. hMMP-1 variants were expressed in E. coli BL21 cells as described in Example 1A, and E. coli supernatant lysates were further purified using Q-Fast Flow Resin (GE Healthcare) to remove some contaminating proteins as described in Example 11. Briefly, 0.025 mL of wildtype hMMP-1 or hMMP-1 TS variant E. coli lysates were diluted into 0.175 mL TCNB buffer containing 1 mM APMA. The preparations were incubated for 2 hours at 25° C. to activate the MMP. Activation was confirmed by Western Blot, by a downward shift in MMP-1 molecular weight. The activated preparation was divided into 0.1 mL aliquots, then pre-incubated for another 2 hours at either 25° C. or 37° C. prior to addition to purified soluble or insoluble collagens. Then, 20 μg soluble Human Collagen Type I (BD Biosciences), 10 μg soluble Human Collagen Type IV (Millipore) after lyophilization to remove acetic acid, or 30 μg pH neutralized Gelled-Insoluble Rat Collagen Type I (BD Biosciences) were incubated in the presence of the activated and preincubated wildtype or variant hMMP-1's for 24 hours at 25° C. Digestion products were analyzed by SDS-PAGE. The results are depicted in Table 27. A (+) indicates that digestion products were present, while a (−) indicates that no digestion product of the collagen was observed. The results show that, as expected, each of the wildtype hMMP-1 tested digested Collagen I (both soluble and insoluble) whether preincubated at 25° C. or 37° C. In contrast, for the hMMP-1 variants, digestion products of collagen I were observed from both gelled and lyophilized collagen I, only when the variants were preincubated at 25° C. prior to exposure to collagen I. No collagen I digestion was observed, after 37C pre-incubation of the hMMP-1 variants. No Collagen IV digestion products were detected, confirming that, like wildtype hMMP-1, the variant hMMP-1's do not cleave collagen IV.









TABLE 27







MMP-1 Digestion of Purified Collagens















R&D

E. coli-








Pre-
Systems
expressed


incubation
WT MMP-1
WT MMP-1
D179N
G159V
S208K/G159V
D156R/D179N
V227E










Digestion of Collagen Type I Lyophilized














25° C.
+
+
+
+
+
+
+


37° C.
+
+












Digestion of Collagen Type IV Lyophilized














25° C.









37° C.














Digestion of Collagen Type I Gels














25° C.
+
+
+
+
+
+
+


37° C.
+
+














Example 9
Kinetic Assay of hMMP-Variant Enzymatic Activity

Activity of wildtype or variant hMMP-1's expressed from E. coli lysates (Example 1) or enriched by Q-Fast Flow Resin (GE Healthcare) to remove some contaminating proteins (Example 11) was measured in a kinetic assay for cleavage of its substrate from the linear portion of the kinetic curve. Wildtype MMP-1 purchased from AnaSpec also was tested (catalog No. 72004).


Briefly, 0.01 mL of wildtype or variant hMMP-1's were diluted into 0.19 mL TCNB buffer containing 1 mM APMA. The preparations were incubated for 2 hours at 25° C. to activate the MMP. The preparations were then split (into two 100 μl aliquots) and pre-incubated at either 25° C. or 37° C. for 2 hours. Then, activated and pre-incubated hMMP-1 samples were added to a 96-well microplate to which Mca-K-P-L-G-L-Dpa-A-R-NH2 fluorescent substrate was added to wells of the microplate.


Kinetic analysis of enzymatic activity was performed in a SpectraMax® florescent microplate reader at 25° C. Readings were taken once every ˜23 seconds from 0 to 3600 seconds (1 hour), and analyzed using Softmax® Pro Software (Molecular Devices). Based on the extended substrate digestion times monitored for the amount of substrate added to wells, the maximal processable substrate observed to be released is about 17000 RFU. The half maximal substrate processed (about 8500 RFU), by the fastest enzyme, released the 8500 RFU after 500 seconds into the reaction; therefore, the timepoint of 500 seconds was used as endpoint to determine Vmax, just before half substrate was used. The maximum slope of the kinetic display of relative fluorescence units released versus time was calculated with SOFTmax PRO software and is reported as V. units per second. Vmax units per second values at the 500 sec time point were used as end points for sample comparisons, which, as described above, is the timepoint where less than 50% of the substrate was utilized in the assay by all samples tested. Thus, the substrate has not become limiting in any well assayed. Higher Vmax values correspond to an increased presence of the processed substrate.


Table 28 sets forth the results of the analysis for hMMP-1 and variants produced in E. coli lysates or Q-Ft Enriched E. coli lysates. The kinetic results confirm the temperature-sensitivity of the variants at 25° C. as measured by end-point methods for screening.









TABLE 28







Kinetic Assay











E. coli Lysates

Q-FT Enriched












Vmax per second
Ratio
Vmax per second
Ratio














25° C.
37° C.
(25/37)
25° C.
37° C.
(25/37)

















Ananspec
7.208
8.879
0.8
8.042
9.177
0.9


wildtype
13.000
10.621
1.2
12.304
10.145
1.2


D179N
3.319
0.262
12.7
1.598
0.087
18.4


G159V
0.611
0.026
23.5
5.629
0.468
12.0


S208K/G159V
0.392
0.011
35.6
4.729
0.187
25.3


D156T/D179N
0.662
0.116
5.7
1.439
0.039
36.9


V227E
0.846
0.087
9.7
1.309
0.595
2.2









Example 10
Comparison of Expression and Specific Activity of hMMP-1 Variants with or without His Tag

hMMP-1 mutants were expressed in E. coli without a His tag using the pET base vector described in Example 1A.1. The proteins were expressed in E. coli BL21 cells as described in Example 1A.1. Expression of each mutant was assessed from Western blot analysis of periplasmic extracts of BL21 cells transformed with the constructs using a primary goat anti-hMMP1 antibody (R&D System) followed by detection with a secondary HRP-anti-goat IgG antibody (CalBioChem). The expression levels of each mutant with or without a His tag was normalized by dividing the value of their expression level by the value of the expression level of the wildtype hMMP-1 without a His tag. The normalized expression level of wild type hMMP-1 without a His tag is 1. The normalized expression level of the other tested proteins is set forth in Table 29.









TABLE 29







Normalized Expression Level












Without a His tag

With a His tag














Normalized to

Normalized



Variant
wildtype
Variant
to wildtype
















Wildtype
1.00
Wildtype-his
0.51



D105N
0.57
D105N-his
0.77



R150P
0.39
R150P-his
0.46



D156K
0.68
D156K-his
2.52



D156T
0.66
D156T-his
0.75



G159V
0.30
G159V-his
0.11



D179N
1.21
D179N-his
1.22



E180T
0.98
E180T-his
1.05



A198L
0.07
A198L-his
n/a



V227E
0.20
V227E-his
0.04



I240S
−0.01
I240S-his
0.03










The normalized expression levels were used to determine the specific activity of the mutants. Activity was assessed similar to Example 2 using a fluorogenic substrate. Each mutant was activated at the indicated temperature (25° C. or 37° C.) with APMA for 2 hours. Following activation, fluorogenic substrate peptide IX was added at 25° C. and incubated at the indicated temperature (25° C. or 37° C.) for four hours. Fluorescence was detected by measuring fluorescence in a fluorescent plate reader at 320 nm exitation/405 nm emission. Relative fluorescence units (RFU) were determined. Specific activities at 25° C. and 37° C. was determined by dividing the activities at 25° C. or 37° C. to the normalized expression level. Data was normalized to vector only and background RFU was subtracted. The therapeutic index (TI; ratio of normalized activity at 25° C./37° C.) was determined. The TI of wildtype with or without a His tag was about 1-fold.


The results show that the mutants without a His tag exhibited a TI ranging from almost 5-fold to about 30-fold. For example, the TI of variant D105N was about 5-fold; R150P was almost 5-fold; D156K was about 11-fold; D156K was about 10-fold; G159V was about 16-fold; D179N was about 30-fold; E180T was about 5-fold; A198L was about 10-fold; and V227E was almost 25-fold. The results show that the presence of the His tag had a decreasing effect on some of the mutants activity. For example, the results show that the mutants with a His tag exhibited a TI ranging from just greater than wild-type to about 10-fold. Most mutants with a His tag exhibited a TI that was less than 5-fold. The highest TI observed for the mutants containing a his tag was for D179N-his exhibiting a TI of about 10-fold compared to a TI of D179N without a His tag of about 30-fold.


The percentage of normalized activity of the variant MMPs without a his tag at the indicated temperature (25° C. or 37° C.) was compared to the activity of wildtype hMMP-1 without a his tag. For percentage of activity at 25° C., normalized activities of mutants activated and incubated with substrate at 25° C. were divided by the normalized activity of wildtype MMP-1 activated and incubated with substrate at 25° C. The results show that the mutants D105N, D156T, and E180T exhibited about 120% of the activity of wildtype; mutants G159V, S208K/G159V, V227E exhibited similar activity as wildtype, i.e. about 100% of the activity of wildtype; mutants D156T/D179N, R150P and D156K exhibited about 80% of the activity of wildtype; D179N exhibited about 50% of the activity of wildtype; and mutant D179N/I240S exhibited about 35% of the activity of wildtype.


For the percentage of activity at 37° C., normalized activities of mutants activated at 25° C., preincubated at 37° C. for 2 hours and incubated at 37° C. with substrate were divided by the normalized activity of wildtype MMP-1 activated at 25° C. and incubated with substrate at 25° C. The results show that mutants D179N, S208K/G159V, D156T/D179N, and D179N/I240S exhibited less than 5% of the activity of wildtype; mutant G159V exhibited just over 5% of the activity of wildtype; mutants V227E, D105N, D156K and D156T exhibited about 10% to about 12% the activity of wildtype; mutants R150P exhibited about 20% the activity of wildtype; and mutant E180T exhibited almost 30% the activity of wildtype.


Example 11
100 mL Scale Expression and Purification of hMMP-1 Mutants with Q-Sepharose Fast Flow Resin

hMMP-1 and variants were purified and enriched from periplasmic preparation using a Q-Sepharose Fast Flow (FF) Resin (GE Healthcare). Briefly, wildtype hMMP-1 s and mutants were cloned into pET303CHis to either be expressed with or without a His tag using routine molecular biology techniques. The tested wildtype hMMP-1 included clone BAP006-09 (without a His tag; having a sequence of nucleotides set forth as nucleotides in SEQ ID NO:706 and containing a pel B signal sequence encoding amino acids set forth in SEQ ID NO:3547) and clone BAP006-10 (having a sequence of nucleotides set forth as nucleotides in SEQ ID NO:706 and containing a pel B signal sequence encoding amino acids set forth in SEQ ID NO:3547 and sequence encoding a C-terminal His tag as described in Example 1. B). Plasmids were transformed into BL21 (DE3) E. coli cells and the transformation culture was used to inoculate 15 mL LB medium containing ampicillin additives (in a 50 mL conical) and grown overnight at 37° C. LB without antibiotics was pre-warmed to 37° C. by incubating 100 mL LB medium (in a 500 mL or 100 mL Erlenmeyer flask) overnight. The OD600 of the inoculated culture was measured the next morning until the OD600 was 0.05-0.1. Ampicillin antibiotics were added to the pre-warmed LB. The 100 mL pre-warmed LB culture with antibiotics was inoculated with the 15 mL overnight culture. The OD600 was measured after 60 and 120 minutes, and then every 30 minutes until the OD600 reached about 0.6. At OD600˜0.6, 1 mL was removed and spun down and periplasmic proteins were prepared as described below for use in analysis. The remaining culture was placed in a 25° C. incubator for 30 minutes (20° C. for combination mutants). The cultures were induced with IPTG at a final concentration of 1 mM and the culture was incubated at 25° C. (or 20° C.) with shaking for 6 hours. After 6 hours, the OD600 was measured.


To prepare periplasmic proteins from the 100 mL culture, the induced culture was transferred to 250 mL conicals and the cells were spun down at 1500 g for 10 minutes at room temperature. An enzyme mix was prepared containing 10 mg DNAase and 10 mg lyzozyme dissolved in 1 mL RNAase (10 mg/mL). The mix was filter sterilized and stored at 4° C. Immediately before use, 50 μl of the enzyme mix was added to Buffer I (200 mM Tris/HcL pH7.5, 20% sucrose, 1 mM EDTA). From the cell culture, supernatant was removed and the pellet was carefully resuspended in 2.5 mL Buffer I/enzyme mixture per tube. The mixture was incubated at room temperature for 5 minutes. 2.5 mL of ice cold water tube was added, mixed by inversion, incubated on ice for 10 minutes, and centrifuged at 5000 g for 15 minutes at room temperature to spin down cell debris. Supernatant, containing the hMMP-1 proteins, was combined in a fresh tube as periplasmic proteins and stored in 500 μl aliquots at −20° C. or was purified further using Q Sepharose FF as described below.


Prior to further purifying the protein with Q Sepharose FF, the Q Sepharose FF material was prepared and equilibrated from the original stock by resuspending the contents of the entire bottle and then transferring 10 mL into a 50 mL conical and centrifuging at 4000 g for 3 minutes. The supernatant was discarded and the pellet was resuspended in 20 mL buffer Q-bind (100 mM Tris/HCl, pH 7.5, 10% sucrose, 10 mM CaCl2, 0.5 mM EDTA). The mixture was centrifuged at 4000 g for 3 minutes and supernatant was removed. This was repeated two times, and after the final spin the pellet was resuspended in 10 mL buffer Q-bind.


To purify the periplasmic prep with Q-Sepharose, 1 mL of the equilibrated Q-Sepharose was centrifuged in a 1.5 mL Eppendorf tube in a microcentrifuge for 2 minutes at full speed. The supernatant was carefully removed. 25 μl 2 M NaCl and 10 μl 1 M CaCl2 was added to 1 mL periplasmic prep. The 1 mL of perimplasmic prep was used to resuspend the Q-Sepharose, and the mixture was incubated on ice for 10 minutes with occasional mixing. The mixture was centrifuged in a microcentrifuge for 3 minutes at full speed, and the supernatant was transferred to a new tube and saved as “Q-FT 1.” The pellet was resuspended in 1 mL buffer Q bind, and the mixture was centrifuged in a microcentrifuge for 3 minutes at full speed. The supernatant was transferred to a new tube and saved as “Q-FT2.” The pellet was resuspended in 1 mL buffer Q bind, and the mixture was centrifuged in a microcentrifuge for 3 minutes at full speed. The supernatant was transferred to a new tube and saved as “Q-FT3.” The pellet was resuspended in 1 mL buffer Q Elute (100 mM Tris/HCl pH 7.5, 10% sucrose, 10 mM CaCl2, 1 M NaCl, 0.5 mM EDTA) and the mixture was centrifuged in a microcentrifuge for 3 minutes at full speed. The supernatant was transferred to a new tube and saved as “Q-ET.”


The eluted supernatant was concentrated using an Amicon 30K spin filter (Millipore). The Amicon 30K filter was rinsed with 1 mL Q-bind buffer, and centrifuged at 3000 g in an SW rotor for 5 minutes at room temperature. The buffer was removed from both compartments. 800 μl of the Q-FT1 was added to the filter, and the filter was centrifuged at 3000 g in an SW rotor for 5 minutes at room temperature. The retentate (about 250 μl) was collected.


The various preparations and fractions were analyzed on SDS-PAGE for purity. The activities also were tested following activation by adding 4 μl of lysate, purified Q-FT1, or purified and concentrated Q-FT1 to 96 μl APMA in TCNB. The reaction mixture was incubated for 2 hours at 37° C. or 25° C., followed by the addition of 10 μM fluorescent peptide substrate and incubation for 4 hours at 37° C. or 25° C. In one experiment for the tested proteins (wildtype, D179N, and D156T/D179N), the results show that each of the proteins exhibited activity whether the lysate, purified protein or purified concentrated protein was tested. For wildtype and D179N, the activity of each was substantially the same whether the lysate, purified protein, or purified concentrated protein was tested. For the D156T/D179N double mutant, the activity of the lysate preparation was about half of the activity exhibited by the purified and concentrated preparation. For the Q-Sepharose purified and concentrated preparations, the activity of the D179N mutant and D156T/D179N double mutant at 25° C. was similar to wildtype with an RFU value of about 10,000.00 observed for each condition. The activity of the wildtype was similar at 37° C. or 25° C. In contrast, under all purification conditions tested, the D179N and D156T/D179N mutants exhibited greater activity at 25° C. (about 10,000.00 RFU) than at 37° C. (about 1000 RFU or less), thereby exhibiting greater than 40-fold activity at 25° C. compared to 37° C. Similar results were obtained for other tested mutants (D179N/I240S, G159V, S208K/G159V, V227E, D105N, R150P, D156K, D156T, E180T), with greater activity observed when the tested protein was Q-Sepharose purified compared to when the tested protein was a lysate preparation for many of the proteins tested. Thus, the results show that purification with Q-sepharose in the presence of 10 mM CaCl2 retain the activities and temperature sensitive phenotype of the mutants.


Purification with Q-Sepharose FF was in the presence of 10 mM CaCl2. There is no addition of ZnCl2 in the purification process. If the purification is performed in the absence of 10 mM CaCl2, the activity of the mutants was reduced.


Example 12
Bacterial Expression and Ni-NTA Purification

DNA encoding wildtype hMMP-1 or variants as described in Example 1 were cloned into vector pET-26b containing a C-terminal 6×-His tag (Catalog No. 69862-3, Novagen; SEQ ID NO:3548) at restriction sites NdeI and XhoI. The respective pET26b-hMMP1 vector was transformed into competent BL21(DE3) cells using standard molecular biology techniques and transformants were plated on Kan-LB-agar plates. Two colonies were picked and grown overnight in 50 mL LB media with Kanamycin (50 μg/mL, final concentration) at 37° C. overnight with shaking (200 rpm). For each overnight culture, 20-22 mL of culture was used to inoculate 800 mL of LB media in a 2 L flask (2 baffeled flasks per colony) containing 0.1% glucose, 0.0005% antifoam and 50 μg/mL Kanamycin. The culture was grown at 37° C. with shaking (200 rpm), and the OD600 measured. When the OD600 reached 0.8-1.3 (about 4.5 hours), the temperature was reduced to 25° C. and IPTG was added to a final concentration of 0.4 mM. Growth was continued overnight (about 12-15 hours) at 25° C. with shaking. Cells were harvested by centrifugation at 4000 g using a JA-5.3 rotor, at 4° C. for 20 minutes for generation of the periplasmic fraction as described below. To confirm protein induction, 1 mL of the culture was centrifuged and resuspended in 200 μl PBS and sonicated to lyse the bacteria. 6×SDS sample buffer containing β-mercaptoethanol (BME) was added to the lysed bacteria, boiled for 10 minutes, and 20 μl was loaded onto a 4-20% TG PAGE Gel. The gel was stained with Simply Blue (Invitrogen) to visualize protein and to determine the degree of protein induction.


For generation of the periplasmic fraction, the harvested cell pellet was re-suspended in 5 mL/gram of lysis buffer (0.5M NaCl, 50 mM Tris-HCL pH 7.9, 10 mM Imidazole, 10% glycerol). To every 40 mL of cell suspension, 1 mM EDTA, 0.5 mg/mL lysozyme, and 50 μl DNase I from a 1 mg/mL stock was added and the suspension was shaken at room temperature for one hour to lyse the bacteria. The cell debris was pelleted by centrifugation at 6000×g at 4° C. for 30 minutes. The supernatant was collected and transferred to new tubes for purification. The pellet was frozen at −80° C. for extraction/solubilization and re-folding of insoluble protein, if desired.


To purify the protein from the supernatant, 5 mL Ni-NTA SuperFlow resin (Qiagen, Cat. No. 30430; 60% slurry) was added to the clarified periplasmic fraction and stirred for 1 hour at 4° C. The mixture was passed through an Econo-column (Biorad) to retain beads and the flow through (FT) and 3 mL bed volume of Ni-NTA resin was collected. The Ni-NTA resin was washed in the column with 3×50 mL of 0.5 M NaCl, 20 mM Tris-HCl pH 7.9, and 10 mM Imidazole. The washes were saved and collected for SDS-PAGE analysis. The MMP-1 was eluted with sequential steps of elution from the column by washing with 6×3 ml of 0.3 M imidazole, 0.5 M NaCl, 20 mM Tris-HCL at pH7.9, and then 4×3 mL of 1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl at pH 7.9. The resin was incubated for 5 minutes with each elution step before spinning down the resin. The supernatants after each wash were collected and saved. About 32 μl of each supernatant fraction was run on 4-20% TG PAGE gel as described above to analyze purification efficiency and yield. Also, the proteins were transferred to PVDF membrane using iBlot® (Invitrogen), and Western Blot was performed using goat anti-hMMP1 antibody (R & D Systems, 0.5 μg/mL) and HRP-anti-goat 1 gG μg/mL).


Based on the overall yield and purity determined by SDS-PAGE analysis, 300 mM imidazole eluents were combined into two pools (typically #2 and 3 and #4-6). Each sample was dialyzed using a 30-KDa Molecular weight cut-off (MWCO) slide-a-lyzer cassette against TCNB buffer in 2 L with one change (2×2 L) at 4° C. overnight. The collected and dialyzed material was stored at 4° C. or −80° C. for longer term storage. The protein concentration was determined by Bradford. For wildtype hMMP-1, typically, about 9-10 mg of protein was purified at about 80% purity obtained from 3.2 L culture.


Example 13
Effect of Zinc
Biochemical and Activity Analysis of Enriched MMP-1 Variants

Periplasmic preparations of wildtype hMMP-1 and variants were generated by hypotonic lysis as described in Example 1 with the addition of 3 freeze/thaw/probe sonication steps prior to bacterial debris removal by centrifugation. The clarified bacterial lysate produced with the additional free/thaw sonication steps was further purified using Q-Sepharose Fast Flow Resin as described in Example 11. The resulting proteins were further enriched using Mimetic Green 1 ligand affinity purification bead columns (ProMetic™ Biosciences; Cat. No. A6XL). MMP-1 proteins were eluted from the green mimetic affinity resin, in the presence or absence of 1 mM Zinc, by increasing the NaCl concentration in step elution buffers. Protein was resolved on a 4-20% TG PAGE Gel, and visualized using Simply Blue and by Western Blot.


Activity of the variant MMP-1's purified in the presence of 1 mM Zinc was assessed using a kinetic assay as described in Example 9. Vmax units per second values at the 500 sec time point were used as end points for sample comparisons. The results are set forth in Table 30. The results show that there is no temperature sensitivity displayed by the variants when purified in the presence of zinc.












TABLE 30









Vmax per second











25° C.
37° C.















Ananspec
4.450
5.387



wildtype
1.744
1.989



D179N
3.688
3.723



D156T/D179N
3.996
3.972



V227E
2.332
2.216










To restore activity, zinc was removed from variants purified in the presence of 1 mM Zinc by chelation with EDTA. P-30 gel filtration spin columns (exclusion 40,000 molecular weight; BioRad) were equilibrated by 4 washes with 0.5 mL 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2 and 0.05% Brij35. 0.1 mL of each enriched MMP-1 (purified in the presence of Zn) was mixed with 0.002 mL 500 mM EDTA, pH 8.0 and then loaded onto a buffer-equilibrated spin column. The column was centrifuged for 10-15 seconds at 2000×g and the flow-thru was assayed for MMP-1 activity. By the chelation, the zinc was removed and the NaCl was lowered from 1 M to 150 mM.


To assess activity, 0.01 mL of the flow-thru was added to 0.19 mL TNBC in the presence or absence of 1 mM APMA to activate the protein, and incubated at room temperature (20° C. to 25° C.) for 2 hours. The mixture was split into two with 0.1 mL of the sample removed to a new tube and incubated at 37° C. for 2 hours, with the remaining mixture remaining at room temperature. Then, 0.045 mL TNCB in the presence or absence of 1 mM APMA was added to wells of a 96-well black plate. 0.05 mL of activated/pre-incubated MMP-1 samples at the respective temperature was added to corresponding wells of the assay plate. 0.005 mL of fluorogenic peptide 1× substrate was added to each well to initiate the assay. Vmax units per second values at the 30 minute time point (1200 sec) were used as end points for sample comparisons. The results are set forth in Table 31. The results show that after EDTA and spin column treatment to chelate zinc, the temperature-sensitivity phenotype was restored.












TABLE 31









Vmax per second
Ratio:











25° C.
37° C.
25° C./37° C.
















wildtype
5.401
5.115
1.05



G159V
1.309
−0.003
1.3



R150P
1.514
0.171
8.8



V227E
3.268
0.773
4.2



D179N
5.569
0.143
38.9



D156T/D179N
1.706
0.011
155.4










Example 14
Effects of Metals on the Activity of Mutants

In this example, the activity of wild-type MMP-1 and various mutants was tested in the presence of varying concentrations of ZnCl2, CaCl2, MgCl2, and NaCl2, and the optimal concentration of each for activity determined.


The effect of ZnCl2 and CaCl2 was assessed by testing the activity of wild-type MMP-1 and various mutants (D179N, G159V, D156T/D179N) after activating the enzyme by incubation with APMA for 2 hours at 20° C., 25° C. or 37° C. Specifically, 4 μl of periplasmic extract as described in Example 1 was added to 96 μl of APMA in the following solutions: 1) TCNB (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij 3 at pH7.5); 2) TCNB with 1 mM ZnCl2; 3) TNB (50 mM Tris, 150 mM NaCl, 0.05% Brij 35 at pH 7.5); or TNB with 1 mM ZnCl2. After 2 hours, 10 μM fluorogenic peptide IX substrate was added to the reaction mixture and incubated for 4 hours at 20° C., 25° C. or 37° C. Fluorescence was detected by measuring fluorescence in a fluorescent plate reader at 320 nm exitation/405 nm emission. Relative fluorescence units (RFU) were determined. The results show that calcium was required for activity of all of the enzymes, with little to no activity observed under conditions where activation and substrate reaction occurred in TNB buffer. For wild-type MMP-1, the presence of ZnCl2 slightly reduced activity, suggesting that there was residual zinc present in the periplasmic extracts and/or reaction buffer. For the temperature-sensitive mutants, the presence of 1 mM ZnCl2 affected the temperature-sensitive phenotypes of the mutants. In the presence of 1 mM ZnCl2, the ratio of activity at 20° C./37° C. or 25° C./37° C. was dramatically reduced, approaching wild-type levels of about 1.0.


To assess the optimal concentration of ZnCl2 necessary to retain a temperature-sensitive phenotype, a titration experiment was performed for wild-type MMP-1 and mutant D179N in the presence of 0.001 mM, 0.01 mM, 0.1 mM or 1 mM ZnCl2 Activity was assessed by adding 4 μl of periplasmic extract to 96 μl APMA in TCNB in the presence or absence of the indicated concentrations of ZnCl2. The reaction mixture was incubated at 25° C. or 37° C. for 2 hours. After 2 hours, 10 μM fluorogenic peptide IX substrate was added to the reaction mixture and incubated for 4 hours at 25° C. or. Fluorescence was detected by measuring fluorescence in a fluorescent plate reader at 320 nm exitation/405 nm emission, and RFU determined. For wild-type, the results show that activity was substantially the same under all of the tested conditions, with slightly less activity observed at 37° C. than 25° C. Also, at 37° C., activity was slightly lower at 0.1 or 1 mM ZnCl2 compared to lower concentrations. For the mutant D179N, the activity detected at 25° C. was greatest in the presence of zinc than if zinc was absent (about 9000 RFU in the presence of zinc, compared to about 4000 RFU in the absence of zinc). This activity of the mutant D179N at 25° C. was comparable to wild-type at 25° C., and also was the same in the presence of 0.001 mM, 0.01 mM, or 0.1 mM zinc. The activity of mutant D179N was reduced to about 6000 RFU in the presence of 1 mM zinc. The greatest temperature-sensitive phenotype was observed at 0.001 mM ZnCl2 (about 13-fold 25° C./37° C. ratio of activity), with decreasing temperature sensitivity detected with increasing concentrations of zinc. In the presence of 0.1 mM and 1 mM ZnCl2, the D179N exhibited no temperature sensitive phenotype (ratio 25° C./37° C. of about 1.0). Thus, the optimal ZnCl2 concentration was observed to be at or about 0.001 mM.


A similar experiment was performed to determine the optimal concentration of CaCl2 necessary to retain activity and a temperature-sensitive phenotype. Activity was assessed by adding 4 μl of periplasmic extract to 96 μl APMA in TCNB in the presence or absence of the indicated concentrations of CaCl2. The reaction mixture was incubated at 25° C. or 37° C. for 2 hours. After 2 hours, 10 μM fluorogenic peptide IX substrate was added to the reaction mixture and incubated for 4 hours at 25° C. or. Fluorescence was detected by measuring fluorescence in a fluorescent plate reader at 320 nm exitation/405 nm emission, and RFU determined. For wild-type MMP-1. little activity was observed at calcium levels less than 1 mM. Activity was observed at 1 mM CaCl2, but the activity was greatest at 10 mM CaCl2 (9000-10,000 RFU). For the D179N MMP-1 variant, activity was only observed in the presence of 10 mM CaCl2 The activity observed was less than for wild-type, although the sample that was tested was subjected to repeated freezing/thawing, which might affect the activity of the mutant lysate. Thus, the optimal CaCl2 concentration was observed to be at or about 10 mM or greater than 10 mM.


Similar experiments as above also were performed in the presence or absence of MgCl2 (0, 0.01 mM, 0.2 mM, 0.2 mM, 1 mM and 10 mM) or NaCl (0, 0.0625 M, 0.125 M, 0.25 M and 0.5M). The results showed that the tested concentrations had no effect on the activities or temperature sensitive phenotypes of the mutants.


Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

Claims
  • 1. A modified matrix metalloprotease-1 (MMP-1), comprising one or more modification(s) in the sequence of amino acid residues of an MMP-1 polypeptide or a catalytically active fragment thereof, wherein: the modification(s) confer to the MMP-1 or the catalytically active fragment thereof, a ratio of enzymatic activity at a permissive temperature compared to at a nonpermissive temperature of at least 1.2; andif the modified MMP-1 is a catalytically active fragment thereof, the active fragment exhibits the ratio of enzymatic activity.
  • 2. The modified MMP-1 of claim 1, wherein the modification is selected from among an amino acid replacement(s), insertion, deletion and combinations thereof.
  • 3. The modified MMP-1 of claim 1, wherein the unmodified polypeptide comprises the sequence of amino acids set forth in SEQ ID NO:1, or is an allelic or species variant thereof, a zymogen, a mature form, or a catalytically active fragment that contains the modification.
  • 4. The modified MMP-1 polypeptide of claim 3, wherein the unmodified MMP-1 polypeptide comprises the sequence of amino acids set forth in SEQ ID NO:2, or is an allelic and species variant thereof, a mature form, or a catalytically fragment thereof that contains the modification.
  • 5. The modified MMP-1 polypeptide of claim 3, wherein the catalytically active fragment comprises the catalytic domain or a catalytically active portion of the catalytic domain.
  • 6. A modified MMP-1 of claim 1 that has lower activity at the nonpermissive temperature than the MMP-1 that does not include the modification has at the nonpermissive temperature.
  • 7. A modified MMP-1 of claim 1, wherein the permissive temperature is lower than the nonpermissive temperature.
  • 8. The modified MMP-1 polypeptide of claim 1, wherein the permissive temperature is between 18° C., 19° C. or 20° C. and 30° C.
  • 9. The modified MMP-1 polypeptide of claim 8, wherein the permissive temperature is or is about 25° C.
  • 10. The modified MMP-1 polypeptide of claim 1, wherein the non-permissive temperature is between 34° C. and 39° C.
  • 11. The modified MMP-1 polypeptide of claim 10, wherein the nonpermissive temperature is or is about 34° C. or 37° C.
  • 12. The modified MMP-1 polypeptide of claim 1, wherein: the modification is an amino acid replacement(s) and the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 replacements.
  • 13. The modified MMP-1 polypeptide of claim 1 that contains only one amino acid replacement to confer the ratio of enzymatic activity.
  • 14. The modified MMP-1 polypeptide of claim 1 that contains only two amino acid replacements to confer the ratio of enzymatic activity.
  • 15. The modified MMP-1 polypeptide of claim 1 that contains only the catalytic domain of an MMP-1 or a catalytically active portion thereof, wherein the catalytic domain contains at least one of the amino acid replacements that confers the ratio of enzymatic activity.
  • 16. A fusion protein, comprising the modified MMP-1 polypeptide of claim 15 with a second but different polypeptide that is not an MMP-1.
  • 17. The modified MMP-1 polypeptide of claim 1 wherein: a modification is an amino acid replacement(s) and the replacement(s) is at a position corresponding to any one or more of positions 84, 85, 95, 98, 99, 100, 103, 104, 105, 106, 109, 110, 111, 112, 118, 123, 124, 126, 147, 150, 151, 152, 153, 155, 156, 158, 159, 170, 171, 176, 178, 179, 180, 181, 182, 183, 185, 187, 188, 189, 190, 191, 192, 194, 195, 197, 198, 206, 207, 208, 210, 211, 212, 218, 223, 227, 228, 229, 230, 233, 234, 237, 240, 251, 254, 255, 256, 257 and 258 in an MMP-1 polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:2.
  • 18. The modified MMP-1 polypeptide of claim 17, wherein the modification is selected from among T84F, E85F, L95K, L95I, R98D, I99Q, E100V, E100R, E100S, E100T, E100F, E100I, E100N, T103Y, P104A, P104M, D105A, D105F, D105G, D105I, D105L, D105N, D105R, D105S, D105T, D105W, D105E, L106C, L106S, A109H, D110A, V111R, D112S, A118T, S123V, N124D, T126S, G147P, R150P, R150V, R150D, R150I, R150H, D151G, N152A, N152S, S153T, F155L, F155A, D156H, D156L, D156A, D156W, D156V, D156K, D156T, D156R, D156M, P158T, P158G, P158K, P158N, G159V, G159T, G159M, G159I, G159W, G159L, G159C, P170D, P170A, G171P, G171E, G171D, A176F, A176W, F178T, F178L, D179N, D179V, D179C, E180Y, E180R, E180T, E180F, E180G, E180S, E180N, E180D, D181T, D181L, D181K, D181C, D181G, E182T, E182Q, E182M, E182G, E183G, R183S, T185R, T185Y, T185H, T185G, T185V, T185Q, T185A, T185E, T185D, N187R, N187M, N187W, N187F, N187K, N187I, N187A, N187G, N187C, N187H, F188V, R189N, R189T, R189Q, E190G, E190Y, E190D, Y191V, N192H, N192S, N192D, N192C, H194P, R195C, R195W, R195L, R195G, R195Q, R195A, R195D, R195V, A197V, A197C, A198G, A198L, A198M, G206A, G206S, L207R, L207V, L207I, L207G, S208R, S208L, S210V, S210A, T211L, D212G, D212H, Y218S, F223C, F223E, F223G, F223A, F223S, F223K, F223M, V227C, V227D, V227E, V227L, V227S, V227W, V227G, V227H, V227Q, V227R, Q228P, L229A, L229T, L229I, A230V, D233E, I234A, I234T, I234E, I234Q, I237L, I237W, I237N, I240S, I240A, I240C, I251S, I251W, Q254S, T255H, P256C, K257P, K257T and A258P.
  • 19. The modified MMP-1 polypeptide of claim 1 wherein: the modification is an amino acid replacement(s) and the replacement(s) is at a position corresponding to any one or more of positions 95, 105, 150, 151, 155, 156, 159, 176, 179, 180, 181, 182, 185, 187, 195, 198, 206, 210, 212, 218, 223, 227, 228, 229, 230, 233, 234, and 240 in an MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2; andthe modification(s) confer to the MMP-1, allelic or species variant thereof or an active fragment thereof, a ratio of enzymatic activity at a permissive temperature compared to at a nonpermissive temperature of at least 1.5.
  • 20. The modified MMP-1 polypeptide of claim 19, wherein the modification is selected from among L95K, D105A, D105F, D105G, D105I, D105L, D105N, D105R, D105S, D105T, D105W, R150P, D151G, F155A, D156K, D156T, D156L, D156A, D156W, D156V, D156H, D156R, G159V, G159T, A176F, D179N, E180Y, E180T, E180F, D181L, D181K, E182T, E182Q, T185R, T185H, T185Q, T185A, T185E, N187R, N187M, N187F, N187K, N187I, R195V, A198L, A198M, G206A, G206S, S210V, Y218S, F223E, V227C, V227E, V227W, Q228P, L229T, L229I, D233E, I234A, I234T, I234E, I240S, and I240C.
  • 21. The modified MMP-1 polypeptide of claim 1, wherein the polypeptide retains the activity of the unmodified MMP-1 at the permissive temperature.
  • 22. The modified MMP-1 polypeptide of claim 1, wherein the activity of the polypeptide, following exposure to the nonpermissive temperature, is reversible upon exposure to the permissive temperature.
  • 23. The modified MMP-1 polypeptide of claim 22, wherein the modification is selected from among D105A, D105F, D105G, D105S, D105T, R150P, G159T, E180Y, E180T, E180F, T185H, T185Q, T185A, T185E, N187R, N187M, N187K, R195V, A198L, A198M, S210V, Y218S, F223E, V227W, L229I and I240C.
  • 24. The modified MMP-1 polypeptide of claim 1, wherein the activity of the polypeptide is irreversibly inactive upon exposure to the nonpermissive temperature.
  • 25. The modified MMP-1 polypeptide of claim 24, wherein the modification is selected from among L95K, D105I, D105L, D105N, D105R, D105W, D151G, F155A, D156K, D156T, D156L, D156A, D156W, D156V, D156H, D156R, G159V, A176F, D179N, D181L, D181K, E182T, E182Q, T185R, N187F, N187I, G206A, G206S, V227C, V227E, Q228E, L229T, D233E, I234A, I234T, I234E and I240S.
  • 26. The modified MMP-1 polypeptide of claim 1 that has a sequence of amino acids set forth in any of SEQ ID NOS: 3-705, 779-3458 3507-3531 and 3541-3546.
  • 27. The modified MMP-1 polypeptide of claim 1, wherein the polypeptide comprises two or more amino acid replacement(s) and the replacement(s) is at a position corresponding to any two or more of positions 95, 105, 150, 156, 159, 179, 180, 182, 185, 187, 198, 227, 234 and 240 in an MMP-1 polypeptide having a sequence of amino acids set forth in SEQ ID NO:2.
  • 28. The modified MMP-1 polypeptide of claim 27, wherein the two or more modifications are selected from among L95K, D105N, R150P, D156K, D156T, G159V, D179N, E180T, A198L, V227E, and I240S.
  • 29. The modified MMP-1 polypeptide of claim 28, wherein the modified MMP-1 polypeptide is selected from among a polypeptide having amino acid replacements D156K/G159V/D179N; R150P/V227E; D156T/V227E; G159V/A198L; D105N/A198L; D179N/V227E; A198L/V227E; E180T/V227E; D179N/A198L; D156K/D179N; D105N/R150P/D156K/G159V/D179N/E180T; D105N/R150P/E180T; G159V/I240S; D156T/D179N/I240S; D156T/G159V; R150P/E180T; D156T/D179N; D179N/I240S; L95K/D156T/D179N; G159V/D179N; L95K/D105N/E180T; R150P/D156T/A198L; L95K/D105N/R150P/D156T/G159V/A198L/V227E/I240S; L95K/R150P; and D105N/E180T.
  • 30. The modified MMP-1 polypeptide of claim 1, further comprising at least one amino acid replacement(s) that confers increased activity compared to the MMP-1 polypeptide not containing the amino acid replacement(s).
  • 31. The modified MMP-1 polypeptide of claim 30, wherein the amino acid replacement(s) is at a position corresponding to any one or more of positions 81, 84, 85, 86, 87, 89, 104, 105, 106, 107, 108, 109, 124, 131, 133, 134, 135, 143, 146, 147, 150, 152, 153, 154, 157, 158, 160, 161, 164, 166, 167, 180, 183, 189, 190, 207, 208, 211, 213, 214, 216, 218, 220, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 235, 236, 238, 239, 244, 249, 254, 256, 257 and 258 in an MMP-1 polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:2.
  • 32. The modified MMP-1 polypeptide of claim 31, wherein the amino acid replacement is selected from among F81L, F81A, F81G, F81Q, F81R, F81H, T84H, T84L, T84D, T84R, T84G, T84A, E85S, E85V, G86S, N87P, N87R, N87G, N87Q, R89A, R89T, R89G, R89K, P104E, P104D, P104Q, D105V, L106V, P107T, P107S, P107A, R108E, R108A, R108K, R108S, A109S, A109R, A109G, A109M, A109V, N124G, T131D, K132R, V133T, V133L, S134E, S134D, E135M, S143I, R146S, G147R, G147F, R150E, R150G, R150M, T150T, R150A, R150N, R150K, R150L, R150V, R150D, N152G, N152F, N152L, N152I, S153T, S153P, S153F, S153D, S153Y, P154S, P154I, G157F, P158V, P158I, G160Q, N161L, N161R, N161Y, N161E, N161T, N161I, N161V, N161F, N161Q, H164S, F166W, Q167R, Q167A, Q167S, Q167F, Q167P, Q167T, Q167V, Q167M, E180D, R183S, R189N, R189T, R189Q, E190D, L207M, S208K, S208R, S208L, T211N, I213G, G214L, G214E, L216I, Y218W, S220R, S220A, S220Q, S220T, S220G, S220M, S220V, S220N, T222R, T222P, T222S, T222F, T222N, F223Y, F223H, 2224Q, S224K, S224D, G225Q, G225E, G225H, D226S, D226E, D226P, D226I, V227T, Q228A, Q228D, Q228E, Q228G, Q228H, Q228K, Q228L, Q228M, Q228N, Q228R, Q228S, Q228T, Q228W, Q228Y, L229Q, L229P, L229V, A230G, A230W, A230D, A230I, A230S, A230C, A230V, A230T, A230M, A230N, A230H, Q231I, Q231A, Q231F, Q231D, Q231G, Q231V, Q231W, Q231S, Q231H, Q231M, D232H, D232G, D232R, D232P, D232Y, D232S, D232F, D232V, D232K, D232W, D232Q, D232E, D232T, D232L, D235G, D235A, D235L, D235E, D235R, D235Q, D235T, D235N, G236M, G236R, G236S, G236T, G236C, G236K, G236E, G236L, G236N, Q238T, A239S, A239V, A239L, A239I, A239G, A239K, A239H, A239R, S244W, S244Q, Q249W, Q254S, P256S, K257E, K257R, and A258P.
  • 33. The modified MMP-1 polypeptide of claim 32, wherein the modified MMP-1 polypeptide is selected from a polypeptide having amino acid replacements S208K/G159V; S208K/D179N; S208K/V227E; G214E/G159V; G214E/D179N; and I213G/D179N.
  • 34. A modified MMP-1 polypeptide, comprising one or more amino acid replacement(s) in the sequence of amino acid residues of an MMP-1 polypeptide or a catalytically active fragment thereof, wherein the replacement(s) confer to the MMP-1 or the catalytically active fragment thereof increased activity compared to the MMP-1 polypeptide not containing the amino acid replacement(s).
  • 35. A modified MMP-1 polypeptide, comprising at least two amino acid replacement(s) in the sequence of amino acid residues of an MMP-1 polypeptide or a catalytically active fragment thereof, wherein: at least one amino acid replacement confers to the MMP-1 or the catalytically active fragment thereof, a ratio of enzymatic activity at a permissive temperature compared to at a nonpermissive temperature of at least 1.2; andat least one amino acid replacement confers to the MMP-1 or the catalytically active fragment thereof increased activity compared to the MMP-1 polypeptide not containing the amino acid replacement.
  • 36. The modified MMP-1 polypeptide of claim 1 that is a zymogen.
  • 37. The modified MMP-1 polypeptide of claim 1 that is a mature enzyme.
  • 38. The modified MMP-1 polypeptide of claim 1 that contains only the catalytically active domain or a catalytically active portion of the catalytic domain.
  • 39. The modified MMP-1 polypeptide of claim 1 that lacks all or a portion of a proline rich linker and/or a hemopexin domain.
  • 40. The modified MMP-1 polypeptide of claim 1 that comprises one or more additional modifications, wherein the one or more additional modifications confer increased stability, increased half-life, altered substrate specificity and/or increased resistance to inhibitors.
  • 41. The modified MMP-1 polypeptide of claim 1 that is glycosylated or PEGylated.
  • 42. The modified MMP-1 polypeptide of claim 1 that is a fusion protein.
  • 43. The modified MMP-1 polypeptide of claim 42 that is fused to an Fc domain or other multimerization domain.
  • 44. A nucleic acid molecule, comprising a sequence of nucleotides encoding a modified MMP polypeptide of claim 1.
  • 45. A vector, comprising the nucleic acid molecule of claim 44.
  • 46. The vector of claim 45, wherein the vector is a prokaryotic vector, viral vector or a eukaryotic vector.
  • 47. The vector of claim 46, wherein the vector is a mammalian vector or a yeast vector.
  • 48. A cell, comprising the vector of claim 45.
  • 49. The cell of claim 48 that is a prokaryotic cell or a mammalian cell.
  • 50. A method of producing a modified MMP-1 polypeptide, comprising: culturing a cell of claim 48 under conditions whereby the cell expresses the modified MMP-1 polypeptide; and purifying the MMP-1 polypeptide.
  • 51. A pharmaceutical composition, comprising a modified MMP-polypeptide of claim 1.
  • 52. A method of treating a disease or condition of the extracellular matrix (ECM), comprising administering to the ECM a pharmaceutical composition of claim 51, wherein: the permissive temperature is below the normal temperature of the ECM; andthe MMP-1 is administered at or below the permissive temperature.
  • 53. The method of claim 52, wherein the MMP-1 is provided in a composition that is at or below the permissive temperature.
  • 54. The method of claim 52, wherein the MMP-1 is mixed with a composition that is at or below the permissive temperature immediately before administration.
  • 55. The method of claim 52, wherein, prior to administration, the ECM is cooled to below the physiological temperature of the body.
  • 56. The method of claim 52, wherein following administration, the ECM is maintained below the physiological temperature of the body for a predetermined time.
  • 57. A method of treating a disease or condition of the extracellular matrix (ECM), comprising administering to the ECM a pharmaceutical composition of claim 51, wherein: the permissive temperature is above the normal temperature of the ECM; andthe MMP-1 is administered at or above the permissive temperature.
  • 58. The method of claim 57, wherein the MMP-1 is provided in a composition that is at or above the permissive temperature.
  • 59. The method of claim 57, wherein the MMP-1 is mixed with a composition that is at or above the permissive temperature immediately before administration.
  • 60. The method of claim 57, wherein, prior to administration, the ECM is heated to above the physiological temperature of the body.
  • 61. The method of claim 57, wherein following administration, the ECM is maintained at above the physiological temperature of the body for a predetermined time.
  • 62. The method of claim 52, wherein the MMP-1 is a zymogen and is processed before administration.
  • 63. The method of claim 62, wherein the MMP-1 is processed by a processing agent.
  • 64. The method of claim 63, wherein the processing agent is selected from among plasmin, plasma kallikrein, trypsin-1, trypsin-2, neutrophil elastase, cathepsin G, tryptase, chymase, proteinase-3, proteinase-3, furin, urinary plasminogen activator (uPA), an active MMP, 4-aminophenylmercuric acetate (AMPA), HgCl2, N-ethylmaleimide, sodium dodecyl sulfate (SDS), chaotropic agents, oxidized glutathione, reactive oxygen, Au(I) salts, acidic pH and heat.
  • 65. The method of claim 64, wherein the active MMP is selected from among an MMP-1, MMP-2, MMP-3, MMP-7, MMP-10, MMP-26 and MT1-MMP.
  • 66. The method of claim 64, wherein the processing agent is AMPA.
  • 67. The method of claim 63, wherein the processing agent is purified away from the modified MMP-1 polypeptide before administration.
  • 68. The method of claim 52, wherein the modified MMP-1 polypeptide is administered at a therapeutically effective amount to treat the disease or condition.
  • 69. The method of claim 52, wherein administration is selected from among subcutaneous, intramuscular, intralesional, intradermal, topical, transdermal, intravenous, oral and rectal.
  • 70. The method of claim 52, wherein administration is sub-epidermal administration.
  • 71. The method of claim 52, wherein administration is subcutaneous administration.
  • 72. The method of claim 52, further comprising administering a pharmacologic agent selected from among other biologics, small molecule compounds, dispersing agents, anesthetics and vasoconstrictors or combinations thereof.
  • 73. The method of claim 72, wherein the dispersing agent is a hyaluronan-degrading enzyme.
  • 74. The method of claim 73, wherein the hyaluronan degrading enzyme is a hyaluronidase.
  • 75. The method of claim 72, wherein the other pharmacologic agent(s) is administered simultaneously, sequentially or intermittently from the MMP-1.
  • 76. The method of claim 72, wherein the other agent(s) is administered prior to administration of the MMP-1.
  • 77. The method of claim 52, wherein the disease or condition of the ECM is a collagen-mediated disease or condition.
  • 78. The method of claim 77, wherein the collagen-mediated disease or condition is selected from among cellulite, Dupuytren's disease, Peyronie's disease, Ledderhose fibrosis, stiff joints, existing scars, scleroderma, lymphedema and collagenous colitis.
  • 79. The method of claim 78, wherein the collagen-mediated disease or condition is stiff joints that is frozen shoulder.
  • 80. The method of claim 78, wherein the collagen-mediated disease or condition is existing scars that is selected from among surgical adhesions, keloids, hypertrophic scars and depressed scars.
  • 81. The method of claim 52, wherein the ECM-mediated disease or condition is herniated protruding discs.
RELATED APPLICATIONS

Benefit of priority is claimed to U.S. Provisional Application Ser. No. 61/209,366, to Louis Bookbinder, Gregory I. Frost, Gilbert Keller, Gerhard Johann Frey, Hwai Wen Chang and Jay Milton Short, entitled “Temperature Sensitive Mutants of Matrix Metalloproteases and Uses Thereof,” filed Mar. 6, 2009. The subject matter of the above-noted application is incorporated by reference in its entirety. This application is related to International PCT Application Serial No. (Attorney Dkt. No. 3800020.00252/3077PC), entitled “Temperature Sensitive Mutants of Matrix Metalloproteases and Uses Thereof,” which claims priority to U.S. Provisional Application Ser. No. 61/209,366. This application is related to International PCT Application Serial No. PCT/US2009/001486 to Gilbert Keller and Gregory Frost, and to U.S. application Ser. No. 12/381,063 to Gilbert Keller and Gregory Frost, each entitled “In Vivo Temporal Control of Activatable Matrix-Degrading Enzymes,” and each which claim priority to U.S. Provisional Application Ser. No. 61/068,667 and to U.S. Provisional Application Ser. No. 61/127,725.

Provisional Applications (1)
Number Date Country
61209366 Mar 2009 US