Patent Application
20080124362
1. Field of the Invention
The present invention in the fields of biochemistry, genetics and medicine is directed to mutants of anthrax Lethal Factor (LF) in domain II of the molecule that lack toxicity and are therefore useful in screening methods and as an immunogenic compositions against anthrax.
2. Description of the Background Art
Anthrax toxin is derived from an exotoxin produced by the gram-positive bacterium Bacillus anthracis. The toxin is composed of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). PA, by itself, is not toxic but rather plays the role of translocating EF and LF to a target cell's cytosol (Klimpel, K R et al., (1992) Proc Natl Acad Sci USA 89:10277-81; Molloy, S S et al. (1992) J Biol Chem 267:16396-402; Singh, Y et al., (1989) J. Biol. Chem. 264:11099-11102; Petosa, C et al., (1997) Nature 385:833-838). Two cell surface receptors for PA (anthrax toxin receptor or ANTXR) have recently been identified (Bradley, K A et al., (2001) Nature 414:225-9; Scobie, H M et al., (2003) Proc Natl Acad Sci USA 100:5170-4). Following binding to ANTXR, PA is cleaved by cell surface-associated furin, that removes a 20 kDa fragment and leaving a 63 kDa fragment PA63) bound to ANTXR. This step is necessary to expose a binding site for EF or LF (Mogridge, J et al. (2002) Biochemistry 41:1079-82) as well as to remove steric hindrances to PA's subsequent oligomerization into a heptamer (Petosa et al., supra; Leppla, S H, In: Sourcebook of Bacterial Protein Toxins, Freer, A, ed., Academic Press, 1991, pp. 277-302; Milne, J C et al. (1994) J. Biol. Chem. 269:20607-12). After EF or LF binds to heptameric PA63, the toxin complex is internalized via the endosomal pathway (Friedlander, A M (1986) J. Biol. Chem. 261:7123-26; Gordon, V M et al. (1988) Infec Immun 56:1066-9; Leppla, S H (1982) Proc Natl Acad Sci USA 79:3162-6). The acidic environment of the endosome induces a conformational change in the PA structure, causing it to form a pore through which EF or LF apparently transits into the cytosol.
EF is an adenylate cyclase (Leppla, supra). EF+PA (=edema toxin or EdTx) is not lethal but causes edema when injected subcutaneously (s.c.) (Beall, F A et al. (1962) J. Bacteriol. 83:1274-80; Stanley, J L et al. (1961) J. Gen. Microbiol. 26:49-66).
LF is a Zn2+-metalloprotease which specifically cleaves the NH2-termini of several mitogen-activated protein kinase kinases (MAPKK=MEK, including MEKs 1, 2, 3, 4, 6 and 7) (Duesbery, N S et al. (1998) Science 280:734-7; Vitale, G et al. (1998) Biochem Biophys Res Commun 248:706-11; Pellizzari, R et al. (1999) FEBS Lett 462:199-204; Vitale, G et al. (2000) Biochem J 352 Pt 3:739-45). resulting in their inactivation (Duesbery et al., supra; Chopra, A P et al. (2003) J. Biol Chem 278:9402-6; Bardwell, A J et al. (2004) Biochem J 378:569-77). LF does not cleave MEK-5 (Vitale et al., supra). Although the combination of PA+LF (=lethal toxin or “LeTx”) does not cause edema, when injected intravenously (i.v.), it rapidlys induce hypotensive shock leading to death.
Mature LF is a large 776-amino-acid (90.2 kDa) protein (Bragg, T S et al. (1989) Gene 81:45-54). The full length protein shown below with the leader sequence present has 809 amino acids (SEQ ID NO:2). The crystal structure of LF has been solved to a resolution of 2.2 Å ((Pannifer, A D et al. (2001) Nature 414:229-33) and is depicted in
The present inventor and his colleagues demonstrated the existence of an LF-interacting region (LFIR) located in C-terminal region of MEK1, adjacent to a proline-rich region where other regulatory molecules, including B-Raf, interact with MEK (Chopra, A P et al. (2003) J Biol Chem 278:9402-6). Mutation of conserved residues within this region prevented LF proteolysis of MEKs without altering MEK's kinase activity. The precise function of the LFIR is not certain, though it was hypothesized that it is required for MEK association with LF.
Again, MEKs are upstream activators of members of the MAPK family. These members comprise extracellular-signal-regulated kinases (ERKs) also known as mitogen-activated protein kinases (MAPKs), for example, ERK 1 or ERK 2 which are the same as MAPK 1 or MAPK 2). Seven different MEK enzymes have been described. MEKs 1 and 2 phosphorylate and activate ERK 1 and 2 (=MAPK 1 and 2) in response to activation by the ras pathway. MEKs 1 and 2 are stimulated by mitogens or growth factors. Mitogen-induced entry of cells into S-phase of the cell cycle is blocked by antisense ERK mRNA (Pages G et al., Proc Natl Acad Sci USA, 1993, 90:8319-23) dominant negative ERK mutants (Troppmair J et al., J Biol Chem, 1994, 269:7030-5; Frost J A et al., Proc Natl Acad Sci USA, 1994, 91:3844-8), and small molecule inhibitors of MEK1/2 such as PD98059 (Dudley D T et al., Proc Natl Acad Sci USA, 1995, 92:7686-7689) or PD184352 (Sebolt-Leopold J S et al., Nat Med, 1999, 5:810-6). MEKs also play a role in programmed cell death (see WO 02/076496, by the present inventor and colleagues, and references cited therein).
MEKs regulate cellular responses to mitogens as well as environmental stress. Inappropriate activation of these kinases contributes to tumorigenesis. Activated MAPK or elevated MAPK expression has been detected in a variety of human tumors including breast carcinoma and glioblastoma, as well as primary tumor cells derived from kidney, colon, and lung tissues (see, for example). MEK-ERK signalling has also been shown to play a critical role in tumor metastasis and in tumor angiogenesis (WO 02/076496, supra).
Abbreviations used; ATP, adenosine triphosphate; ANTXR, anthrax toxin receptor; COOH, carboxy; CHO, Chinese hamster ovary; df, degrees of freedom; EC50, 50% effective concentration; EF, edema factor; ERK, extracellular regulated kinase; FPLC, fast pressure liquid chromatography; kDa, kilodalton; LeTx, lethal toxin; LF, lethal factor; LFIR, LF-interacting region; MEK, mitogen activated protein kinase kinase (MAPKK); NH2, amino; p, probability; PA, protective antigen; SDS, sodium lauryl sulfate.
The present inventor has discovered that a number of specific mutants of LF lose the ability or have reduced ability to bind to and interact productively with MEK-1 or MEK-2, the substrate of LF action. It is through proteolysis of MEK that the LF exerts its toxic effects.
Thus, the present invention is directed to a mutant or variant anthrax lethal factor (LF) polypeptide in which between one and five amino acid residues in domain II that is important for interaction with the LF substrates MEK-1 or MEK-2 (as well as MEKs-3, 4, 6 and 7), are either substituted, deleted, or chemically derivatized such that the polypeptide is inhibited compared to normal LF in binding to and interacting with said MEK, the residues selected from the group consisting of L293, K294, R491, L514 and N516. These position correspond to residues L326, K327, R524, L547 and N549 of SEQ ID NO:2.
In one embodiment of the mutant or variant LF, at least two amino acid residues in domain II is substituted or mutated, which two residues are selected from the group consisting of L514/L293, L514/K294 and L514/R491.
Preferably, in the above mutants or variants, one or more amino acid residues is substituted with Ala or Gly, most preferably with Ala.
A preferred group of mutants is L293A, K294A, R491A, L514A, and N516A, and double mutants L514A/L293A, L514A/K294A and L514A/R491A.
Also provided is a fragment of the above mutant or variant corresponding to domain IIa or domain IIb of LF, or a mixture of such fragments. Preferably, the sequence of the fragments is SEQ ID NO:4 or SEQ ID NO:6.
The present invention is directed to an isolated nucleic acid molecule that encodes the above mutant or variant LF polypeptide, or fragment. These nucleic acids may be used to produce the LF polypeptide or as immunogenic DNA vaccines by administration to a subject using methods and routes well-known in the art.
As a result of the recognition by the inventor that particular conformational epitopes of domain II of LF are rendered incapable or less capable of binding MEK as a result of the mutations in the above positions, it has become possible to design screening assays that examine the effect of a potential inhibitor of LF-MEK binding based solely on inhibition of binding (vs. inhibition of proteolysis which was the only disclosed basis for testing inhibitors prior to this invention. Any type of binding assay known in the art, using labeled or unlabeled components (LF, MEK) may be used. Other assays that are well-known for this purpose are those that do not require labels, such as with the BiaCore technology, or isothermal calorimetric assays, or an assay based on labeled reactants, the “AlphaScreen™ assay. Exemplified herein are assays that examine competition of B-Raf binding or in vitro MEK proteolysis (the latter of which, alone does not distinguish the binding phase from the proteolysis phase and would identify inhibitors of either or both phases).
The invention provides method for screening a test sample comprising an agent or compound being tested for its ability to inhibit the binding interaction of LF and MEK independent of any effect on LF-mediated proteolysis of MEK, comprising
(a) contacting a test sample with LF and a MEK protein; and
(b) assaying for the binding of LF to MEK;
(c) comparing the binding to the binding of LF in the absence of the test sample,
wherein, if the binding measured in (a) is lower than the binding measure in (b), the agent or compound is an inhibitor of LF-MEK binding. This method may further comprise the step of comparing the binding in step (b) with the binding to MEK of an LF mutant, variant or fragment as described herein.
The above method of claim may also comprise testing the ability of the sample to inhibit MEK proteolysis, wherein if the compound is positive in inhibiting the binding and negative in inhibiting the proteolysis, it is a pure binding inhibitor
Also provides is a method for screening a sample or multiplicity of samples comprising an agent or compound (or agents/compounds) being tested for (i) the ability to inhibit the binding interaction of LF and MEK and (ii) the ability to inhibit LF-mediated proteolysis of MEK and, comprising
The present invention includes an immunogenic or vaccine composition comprising: (a) the mutant or variant LF as above, and (b) an immunologically acceptable carrier or excipient. Also included are DNA vaccines, well-known in the art, that comprise (a) the nucleic acid molecule as above encoding the mutant of variant LF, and (b) an immunologically acceptable carrier or excipient.
The invention is further directed to a method of inducing LF-specific immunity in a subject comprising administering to the subject an immunogenically effective amount of the above polypeptide or nucleic acid immunogenic composition. The method can be used to generate LF-specific antibodies which may be stored, isolated, etc., and used in passive immunization.
a-4d: Toxicity and proteolytic activity of purified LF and LF double mutants.
b shows results where His6-tagged wild-type MEK1 (0.2 μg) was incubated with wild-type LF or LF mutants (0.2 μg) at 30° C.C for 1 or 5 min., proteins were separated by SDS-PAGE and immunoblotted with an antibody raised against residues 216-233 of human MEK1. MEK1 not reacted with LF (control) or reacted with inactive LF (E687C) are included as negative controls. MEK1 cleavage is indicated by increased electrophoretic mobility following proteolytic removal of the His6-tag as well as the NH2-terminus of MEK1.
Based on the conclusion that LF and MEK interact outside the active site complex (Chopra et al., supra), the present inventor conceived that LF must have a corresponding region in which the introduction of mutations at key residues should disrupt toxicity. The present invention identifies a cluster of residues in domain II of LF that play a key role in LF-mediated toxicity. Site directed mutagenesis was the preferred approach to achieve such identification. Once the existence of such a site or sites was known, and the existence of separate binding of LF to MEK, it opened the way to development of new screening methods that focus on inhibitors of this interactions, as distinct from the proteolysis function. This is different from the interactions of most proteases with their targets, where the binding and recognition functions all occur via the enzyme's catalytic/active site.
Ala substitution of the residues in this cluster substantially reduced LF toxicity and blocked proteolysis of MEK in cells. It is noteworthy that these residues are not contiguous in the primary sequence, but rather are a “sensitive” positions in the tertiary structure of the LF protein. In other words, the important regions of the molecule behave like “conformational” epitopes vs. linear epitopes.
Functional tests of these mutations indicated that loss of toxicity was not caused by interference with the ability of LF to bind PA, translocate across the membrane, or to cleave MEK in vitro. Rather, the loss of toxicity was related to a reduction in the ability of LF to interact with MEK.
The region containing this cluster of residues could is a useful therapeutic target for discovery and development of small molecule inhibitors that disrupt LF-MEK association and thereby block LF-mediated proteolysis of MEK, which would result in the inhibition of LF toxicity to cells. A drug discovered in this manner could be used to treat infections with natural or weaponized B. anthracis bacterial, or the impact of contact with isolated anthrax lethal toxin molecules.
In another embodiment, mutant LF molecules as described herein are useful as vaccine immunogens, because their administration to a subject to induce immunity to various protective epitopes of the molecule would not be accompanied by toxic effects of the LF.
The complete nucleotide and amino acid sequence of LF are shown below. The nucleotide sequence is SEQ ID NO:1 and the amino acid sequence is SEQ ID NO:2. This sequence is annotated by underscoring to show the nt and aa sequences corresponding to the two segment of domain II (which is made up of two regions, domains IIa and IIb). (The sequence between IIa and IIb is referred to as domain III, and, at the protein level, comprises a series of imperfect repeats of a motif found in domain II that together are considered to form a distinct region). Thus domain II comprises:
(a) the nt (SEQ ID NO:3 and aa (SEQ ID NO:4) sequence of domain IIa
(b) the nt (SEQ ID NO:5) and aa acid (SEQ ID NO:6) of domain IIb.
Domain IIa coding sequence (SEQ ID NO:3) is from nt 885-1125 of the LF DNA (SEQ ID NO:1).
Domain IIb coding sequence (SEQ ID NO:5) is from nt 1157-1749 of the LF DNA (SEQ ID NO:1).
Domain IIa aa sequence (SEQ ID NO:4) is from aa 296-375 of the LF protein (SEQ ID NO:2).
Domain IIb aa sequence (SEQ ID NO:6) is from aa 419-583 of the LF protein (SEQ ID NO:2).
The codons and amino acid residues that are bolded and italicized in domain II (
atg aat ata aaa aaa gaa ttt ata aaa gta att
M N I K K E F I K V I
agt atg tca tgt tta gta aca gca att
S M S C L V T A I
act ttg agt ggt ccc gtc ttt atc ccc ctt gta
T L S G P V F I P L V
cag ggg gcg ggc ggt cat ggt gat gta
Q G A G G H G D V
tat gaa aaa tgg gaa aag ata aaa cag cac tat
Y E K W E K I K Q H Y
caa cac tgg agc gat tct tta tct gaa
Q H W S D S L S E
gaa gga aga gga ctt aag ctg cag att
E G R G L L Q I
cct att gag cca aag aaa gat gac ata
P I E P K K D D I
att cat tct tta tct caa gaa gaa aaa gag ctt
I H S L S Q E E K E L
cta aaa aga ata caa att gat agt agt
L K R I Q I D S S
gat ttt tta tct act gag gaa aaa gag ttt tta
D F L S T E E K E F L
aaa aag cta caa att gat att cgt gat
K K L Q I D I R D
att aat caa agg ttg caa gat aca gga ggg tta
I N Q R L Q D T G G L
att gat agt ccg tca att aat ctt gat
I D S P S I N L D
gta aga aag cag tat aaa agg gat att caa aat
V R K Q Y K R D I Q N
att gat gct tta tta cat caa tcc att
I D A L L H Q S I
gga agt acc ttg tac aat aaa att tat ttg tat
G S T L Y N K I Y L Y
gaa aat atg aat atc aat aac ctt aca
E N M N I N N L T
gca acc cta ggt gcg gat tta gtt gat tcc act
A T L G A D L V D S T
gat aat act aaa att aat aga ggt att
D N T K I N R G I
ttc aat gaa ttc aaa aaa aat ttc aaa tat agt
F N E F K K N F K Y S
att tct agt aac tat atg att gtt gat
I S S N Y M I V D
ata aat gaa cct gca tta gat aat gag cgt
P A L D N E R
ttg aaa tgg aga atc caa tta tca cca
L K W R I Q L S P
gat act cga gca gga tat gaa gga aag
D T R A G Y E G K
ctt ata tta caa aga aac atc ggt ctg
L I L Q R N I G L
gaa ata aag gat gta caa ata att aag caa tcc
E I K D V Q I I K Q S
gaa aaa gaa tat ata agg att gat gcg
E K E Y I R I D A
aaa gta gtg cca aag agt aaa ata gat aca aaa
K V V P K S K I D T K
The separate amino acid sequence of LF (SEQ ID NO:2) is shown separately below and is annotated as follows:
MNIKKEFIKV ISMSCLVTAI TLSGPVFIPL VQGAGGHGDV
YEKWEKIKQH YQHWSDSLSE EGRGL KLQ IPIEPKKDDI
IHSLSQEEKE
LLKRIQIDSS DFLSTEEKEF LKKLQIDIRD SLSEEEKELL
VRKQYKRDIQ
NIDALLHQSI GSTLYNKIYL YENMNINNLT ATLGADLVDS
TDNTKINRGI
FNEFKKNFKY STSSNYMIVD INE PALDNE RLKWRIQLSP
DTRAGY E G
KLILQRNIGL EIKDVQIIKQ SEKEYIRIDA KVVPKSKIDT
Below, the nucleotide and amino acid sequences of domain IIa and IIb are shown “removed” from the full length LF sequences above (with the annotations of codons/amino acids of particular importance to this invention still annotated as bold and italic.
KLQIPIEP
PALDN ERLKWRIQLS
GKLILQRNIG LEIKDVQIIK QSEKEYIRID AKVV
aag ctg cag att
cct gca tta
gaa
gga aag
In the following sections, the positions to be mutated were described using a somewhat modified numbering system than above, in which the first 33 residues of SEQ ID NO:2 were omitted from numbering as they represent the leader sequence of the polypeptide (shown as double underscored above. Thus, the following list shows the equivalent (identical) amino acid residues
Thus, all references to L293, K294, R491, L514 or N516 and mutations at those sites in this application, particularly in the Examples, refer to positions L326, K327, R524, L547 and N549, respectively, of SEQ ID NO:2. Of course, and their coding sequences of SEQ ID NO:1)
Preferred mutants are amino acid substitution variants at L293, K 294, R491, N516; any combination thereof is also intended, with the combinations of L514/L293, L514/K294 and L514/491 preferred. Preferred substitution is with Ala or Gly. Other substitutions can be made in accordance with this invention. Similarly, deletion of any one, two, three, four or all five of these residues are also included in the scope of this invention.
It would be a matter of routine testing to make such substitutions or deletions and test them using the methods described herein, to determine which results in a polypeptide having the desired property, namely an LF molecule with a reduced or no ability (within the limits of the testing systems) to interact productively with MEK, such that these LF mutants have reduced or no toxicity.
The polypeptides of the present invention including not only full length LF molecules that comprise the domain II mutations described herein, but also shorter molecules, such as domain II peptides themselves that include one or more of the mutations described herein. Preferred examples are mutated forms of SEQ ID NO:4 and SEQ ID NO:6 which can be use in the screening assays (of inhibition of binding) described below, alone or in combination, in place of the full length LF molecules.
In addition the mutants and variants described herein, the present invention includes LF polypeptides in which have been chemically modified or derivatized
Covalent modifications of the LF polypeptides may be introduced by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. The preferred derivatives are those that mimic the mutations by inhibiting the ability of the LF chemical derivative to bind to and productively interact with MEK leading to MEK proteolysis.
For examples lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents reverses the charge of the lysinyl residues. Other suitable reagents for derivatizing α-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Such derivatization requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine ε-amino group.
Another type of chemical derivative is one in which a mutant LF of the present invention is further derivatized in order to improve its immunogenicity when used as a vaccine composition. Such derivatization are used to cross-link the polypeptide to itself (to make conjugates with improved immunogenic properties as is known in the art) or to various water-insoluble support matrices or other macromolecular carriers. Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R′—N—C—N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Commonly used cross-linking agents include 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane.
Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
These clustered residues define a surface epitope of LF in domain II which is required for LF toxicity. Small molecules which occlude this site can thus serve as LF inhibitors and be used as drugs to treat the effects of B. anthracis infection or other effects of Anthrax lethal toxin in a subject. Thus, one embodiment of the present invention is a method to identify such inhibitors of LF-MEK binding/interaction. This method involves incubating a test or candidate molecule or agent with LF and MEK and measuring the ability of the candidate molecule/agent to prevent binding of MEK—using any binding assay. Alternatively, it is possible to assay for the inhibition of the cleavage of MEK and independently assaying for inhibition of LF-mediated proteolysis, such that inhibitors can be found that inhibit binding only, proteolysis only, or both, depending how the screening assays are combined.
Methods described in the Examples below and in the references cited herein provide certain appropriate techniques to use for such measurements. These techniques are considered conventional and routine in the art and need not be detailed here any further. Others are well-known in the art. Duesbery et al., U.S. Pat. No. 6,485,925 describes a method for evaluating agents for their ability to inhibit LF proteolysis of MEK. However, prior to the making of the present invention, there was no basis for evaluating a compounds ability to inhibit LF/MEK interactions at a stage prior to the proteolysis step.
The present invention thus includes a “pharmaceutical” or “immunogenic” composition comprising a domain II mutant of LF as described, or a chemical derivative, analogue, or mimetic thereof, along with a pharmaceutically or immunologically acceptable excipient. Thus, the term “therapeutic composition” includes immunogenic or vaccine compositions and any other pharmaceutical comprising the LF mutant polypeptide, derivative, analogue, or mimetic (or nucleic acid if a DNA vaccine composition is to be used) and a therapeutically acceptable carrier or excipient. General methods to prepare immunogenic or vaccine compositions are described in Remington's Pharmaceutical Science; Mack Publishing Company Easton, Pa. (latest edition).
The invention provides a method of treating a subject, preferably a human, by immunizing or vaccinating the subject to induce an antibody response and any other accompanying protective form of immune reactivity against anthrax LF or lethal toxin.
The immunogenic material may be adsorbed to or conjugated to beads such as latex or gold beads, ISCOMs, and the like. Immunogenic compositions may comprise adjuvants, which are substance that can be added to an immunogen or to a vaccine formulation to enhance the immune-stimulating properties of the immunogenic moiety. Liposomes are also considered to be adjuvants (Gregoriades, G. et al., Immunological Adjuvants and Vaccines, Plenum Press, New York, 1989) Examples of adjuvants or agents that may add to the effectiveness of proteineaceous immunogens include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, and oil-in-water emulsions. A preferred type of adjuvant is muramyl dipeptide (MDP) and various MDP derivatives and formulations, e.g., N-acetyl-D-glucosaminyl-(β1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) (Hornung, R L et al. Ther Immunol 1995 2:7-14) or ISAF-l (5% squalene, 2.5% pluronic L121, 0.2% Tween 80 in phosphate-buffered solution with 0.4 mg of threonyl-muramyl dipeptide; see Kwak, L W et al. (1992) N. Engl. J. Med., 327: 1209-38). Other useful adjuvants are, or are based on, bacterial endotoxin, lipid X, whole organisms or subcellular fractions of the bacteria Propionobacterium acnes or Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin and saponin derivatives such as QS21 (White, A. C. et al. (1991) Adv. Exp. Med. Biol., 303:207-210) which is now in use in the clinic (Helling, F et al. (1995) Cancer Res., 55:2783-88; Davis, T A et al. (1997) Blood, 90:509). QS21 is a triterpene glycoside from the South American tree Quillaja saponaria (Soltysik S et al., 1993, Ann N Y Acad Sci 690:392-5). Other adjuvants include levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. A number of other adjuvants are available commercially from various sources, for example
(c) Alhydrogel®-aluminum hydroxide gel, one of the oldest adjuvants known and approved for humans
(d) Amphigen (which may not be a registered trademark) and is an oil in water preparation defined in more detail in, for example, US Pat Publication 20050058667A1 (Mar. 17, 2005) which further cites U.S. Pat. No. 5,084,269 which states that “AMPHIGEN™ consists of de-oiled lecithin dissolved in an oil, usually light liquid paraffin.”. Veterinary Practice (at the world-wide web URL of .“vpmag.co.uk/news/article.php?article=1483020736.html”) refers to it as consisting of oil micelles coated with lecithin; or
(e) a mixture of Amphigen and Alhydrogel®.
A preferred effective dose for treating a subject in need of the present treatment, preferably a human, is an amount of up to about 100 milligrams of active compound per kilogram of body weight. A typical single dosage of the peptide, chimeric protein or peptidomimetic is between about 1 ng and about 100 mg/kg body weight, and preferably from about 10 μg to about 50 mg/kg body weight. A total daily dosage in the range of about 0.1 milligrams to about 7 grams is preferred for intravenous administration. A useful dose of an antibody for passive immunization is between 10-100 mg/kg. These dosages can be determined empirically in conjunction with the present disclosure and state-of-the-art.
The foregoing ranges are, however, suggestive, as the number of variables in an individual treatment regime is large, and considerable excursions from these preferred values are expected. As is evident to those skilled in the art, the dosage of an immunogenic composition may be higher than the dosage of the compound used to treat. Not only the effective dose but also the effective frequency of administration is determined by the intended use, and can be established by those of skill without undue experimentation. The total dose required for each treatment may be administered by multiple doses or in a single dose.
Pharmaceutically acceptable acid addition salts of certain compounds of the invention containing a basic group are formed where appropriate with strong or moderately strong, non-toxic, organic or inorganic acids by methods known to the art. Exemplary of the acid addition salts that are included in this invention are maleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide, sulfate, phosphate and nitrate salts. Pharmaceutically acceptable base addition salts of compounds of the invention containing an acidic group are prepared by known methods from organic and inorganic bases and include, for example, nontoxic alkali metal and alkaline earth bases, such as calcium, sodium, potassium and ammonium hydroxide; and nontoxic organic bases such as triethylamine, butylamine, piperazine, and tri(hydroxymethyl)methylamine.
The compounds of the invention, as well as the pharmaceutically acceptable salts thereof, may be incorporated into convenient dosage forms, such as capsules, impregnated wafers, tablets or preferably injectable preparations. Solid or liquid pharmaceutically acceptable carriers may be employed.
Preferably, the compounds of the invention are administered systemically, e.g., by injection or infusion. Administration may be by any known route, preferably intravenous, subcutaneous, intramuscular, intrathecal, intracerebroventricular, or intraperitoneal. (Other routes are noted below) Injectables can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
To enhance delivery or immunogenic activity, the compound can be incorporated into liposomes using methods and compounds known in the art.
DNA immunogens are administered via gene gun, or by injection intramuscularly or subcutaneously as is well-known in the art.
The pharmaceutical preparations are made following conventional techniques of pharmaceutical chemistry. The pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth. The peptides are formulated using conventional pharmaceutically acceptable parenteral vehicles for administration by injection. These vehicles are nontoxic and therapeutic, and a number of formulations are set forth in Remington's Pharmaceutical Sciences, Gennaro, 18th ed., Mack Publishing Co., Easton, Pa. (1990)). Nonlimiting examples of excipients are water, saline, Ringer's solution, dextrose solution and Hank's balanced salt solution. Formulations according to the invention may also contain minor amounts of additives such as substances that maintain isotonicity, physiological pH, and stability. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension. Optionally, a suspension may contain stabilizers.
The polypeptides nucleic acids and other useful compositions of the invention are preferably formulated in purified form substantially free of aggregates and other protein materials, preferably at concentrations of about 1.0 ng/ml to 100 mg/ml.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.
The murine macrophage-derived J774A.1 and the Chinese hamster ovarian epithelial (CHO)-K1 cell lines were obtained from the ATCC (Manassas, Va.). J774A.1 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin. CHO-K1 were cultured in Ham's F-12 medium supplemented with 10% FBS, and 1% penicillin/streptomycin. Both cell-lines were maintained at 37° C. in a humidified 5% CO2 incubator.
Alanine-substitutions in LF were generated by introducing mutations into a B. anthracis LF expression vector pSJ115 (Park et al., supra) with the use of the Quickchange™ site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) following manufacturer's instructions except that primer extension was allowed to continue for 18 min and the deoxynucleotide triphosphate (dNTP) stocks were modified to reflect the high deoxyadenylate and deoxythymidylate content (70%) of LF the gene (Bragg et al., supra).
The primers used for site-directed mutagenesis are listed in Table 1 below:
Mutations were confirmed by DNA sequencing of the region containing the mutation. In addition, the genes encoding all LF that demonstrated reduced toxicity were sequenced in their entirety to confirm that only the desired mutations were present.
Mutagenized proteins, they were first transformed into the E. coli dcm−/dam− strain SCS110 to obtain unmethylated plasmid DNA which was then transformed into a non-toxigenic, sporulation-defective strain of B. anthracis, BH445 (Park et al., supra, as described by Quinn et al., supra).
To prepare crude preparations of secreted protein, a single colony of transformed cells was used to inoculate 5 ml FA medium (Singh et al., supra). Cultures were allowed to grow at 37° C. for 14-16 h. Culture supernatant (2 ml) was then concentrated using a centrifugal filter (Microcon 100K MWCO; Millipore) and protein was recovered in 40 μl buffer (20 mM Hepes, pH 7.5, 25 mM NaCl). The concentration of each protein was estimated by direct comparison to Coomassie Blue-stained BSA standards (0.5 and 2.0 mg/ml) after separation on 10-20% SDS-PAGE gels.
To make high-purity preparations of LF and PA, 50 ml cultures were used to inoculate 5 L of FA medium in a BioFlo 100 fermentor (New Brunswick Laboratories) at 37° C., pH 7.4, while sparging with air at 3 L/min and with agitation set to increase from 100 rpm to 400 rpm as level of dO2 dropped below 50%. After 17-18 h of growth, the cells were removed by centrifugation (3500 g for 30 min., 4° C.), and the supernatant was sterile-filtered and concentrated by tangential flow filtration using a Millipore prep/scale-TFF cartridge with 1 ft2 of 30-KDa MWCO polyethersulfone membrane, collecting the filtrate at approximately 50 ml/min under a 1 bar back-pressure.
Expressed protein was purified by ammonium sulfate fractionation and fast pressure liquid chromatography (FPLC) using phenyl sepharose and Q sepharose columns following the procedures of Park et al., supra. The concentration of each protein was estimated using the bicinchoninic acid method (Smith et al., supra) and by densitometric analyses of Coomassie Blue-stained polyacrylamide gels.
Recombinant human MEK1 protein was expressed in Spodoptera frugiperda (Sf9) cells that had been infected with baculovirus containing human MEK1 ligated into the pVL1393 vector backbone (pKM636). Protein was isolated from supernatants of lysed cells and was eluted over 10 column volumes in a linear gradient from 0-500 mM NaCl from a 20 ml Q-Sepharose column. The peak fractions containing MEK proteins were pooled and loaded directly onto a 10 ml Ni-NTA column. After washing the column with 30 mM imidazole, MEK was eluted with 100 mM imidazole. At this point, the eluate was adjusted to 3 μM EDTA, 3 mM MnCl2, and 2 mM dithiothreitol (DTT), and 25 units of protein phosphatase 1 (New England Biolabs, Beverly, Mass.) were added to the reaction which was allowed to incubate for 4 hrs at 30° C. Samples were then concentrated and applied to a 320 ml Sephacryl 200 column in a buffer of 25 mM HEPES (pH 8.0), 100 mM NaCl, 2 mM DTT and 10% glycerol.
ERK2 protein was expressed in E. coli and purified by FPLC as described earlier (Duesbery et al., supra; Chopra et al., supra). Active B-Raf (Δ1-415) was purchased from Upstate Biotechnology, Inc.
Cells were grown in 96-well microplates to 70% confluence. To induce lysis, cells were treated with culture medium containing LeTx [PA (0.1 μg/ml) plus LF (0.01-10,000 ng/ml)] and incubated for 3 h at 37° C. At the end of the experiment, cell viability was determined using the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, Wis.) according to the manufacturer's instructions. The concentration of LF required to cause a 50% maximal decrease in absorbance at 570 nm (the EC50) was determined by linear regression.
PA-binding and translocation assays were performed as described by Lacy et al. (supra) and quantitated using a Packard Tri-Carb 3100TR liquid scintillation counter.
To assay MEK cleavage in cells we made lysates of J774A.1 macrophages which had been incubated for 2 h with 0.1 μg/ml PA and 0.01 μg/ml LF or LF mutants. Lysates were separated by denaturing SDS-PAGE and immunoblotted with antibodies raised against the NH2- or COOH-termini of MEK2 (N-20 and C-16, 1:1000; Santa Cruz Biotechnology, Santa Cruz, Calif.). In vitro MEK cleavage assays were performed using immunoblotting with antibodies raised against MEK (anti-MEK1/2, 1:1000; Cell Signaling) as described earlier (Chopra et al., supra). Alternatively, MEK-cleavage was assayed indirectly by reacting a constant concentration of MEK with varying the amounts of LF, using MEK activity (i.e. ERK phosphorylation) as a readout for LF activity. Briefly, 0.35 μg MEK1 was added to 3 μl cleavage buffer (20 mM 3-(N-morpholino) propanesulfonic acid (pH 7.2), 25 mM β-glycerophosphate, 5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, 1 mM sodium orthovanadate, and 1 mM dithiothreitol) in the presence of varying amounts of LF or mutant LF (0.002 μg to 10 μg) and in a total volume of 10 μl. These cleavage reactions were incubated at 30° C. for 10 min. After cooling on ice for 2 min, 10 μl kinase buffer (0.5 mM ATP diluted 9:1 with [γ32P]ATP (Amersham; 10 mCi/ml, 3000 mCi/mmol)), 75 mM MgCl2, and 0.4 μg of ERK2] was added and samples were incubated for 10 min at 30° C. After cooling on ice for 2 min one volume of 2× SDS-buffer was added and samples were incubated in a boiling water bath for 3 min. Proteins were then separated by SDS polyacrylamide electrophoresis on 10% gels and ERK2 phosphorylation was quantitated using a Fuji FLA-5000 PhosphorImager.
B-Raf kinase assays were performed as described previously (Copra et al., supra) and quantitated using a Fuji FLA-5000 PhosphorImager. Results were normalized to phosphorylation in the absence of LF and compared using an unpaired Students' t-Test.
Since a number of the conserved residues in the LFIR are long-chain aliphatic residues, the present inventors conceived that a complementary region on LF would contain clustered aliphatic residues and would lie close to the groove into which the NH2-terminus of MEK fits. A surface plot of LF shows three distinct clusters of aliphatic residues meeting this requirement (
To test this, site-directed mutagenesis was employed to substitute alanine for each of these residues and then evaluated the effects of these mutations upon LF activity using a macrophage toxicity assay (Friedlander, supra). The average concentration of crude preparations of wild-type LF required to cause a 50% maximal decrease in absorbance/cell viability (the EC50) was 15.6±16.7 nM. Mutation of most of the aliphatic residues tested caused a less than a 5-fold reduction in toxicity (12.9 nM≦EC50≦84.3 nM;
To determine whether other residues in this region of LF played a role in LF-toxicity, alanine substitutions were made at surface-exposed residues which were in proximity to L514. Of these, L285, R290, Q297, E515, and K518 were judged to have a neutral or marginal role in toxicity (40 nM≦EC50≦65 nM;
LF is a Zn2+-metalloprotease which specifically cleaves the NH2-termini of mitogen-activated protein kinase kinases. To determine whether clustered residues in domain II are required for LF proteolytic activity we assayed MEK2 cleavage by immunoblotting in J774A.1 macrophages which had been treated for 2 h with PA (0.1 μg/ml) plus wild-type LF or LF containing alanine mutations (0.01 μg/ml) in this region. Of the proteins tested, only wild-type LF and LF containing alanine mutations which had a neutral or marginal effect on toxicity were able to cleave the NH2-terminus of MEK2 (
To test whether our mutant LF were able to bind PA and translocate across the membrane binding and translocation assays were done using [35S]-Met-labeled LF. In these assays LF and PA63 were allowed to bind ANTXR on CHO-K1 cells at 4° C., at which temperature endocytosis does not occur. LF containing an alanine substitution at LF (Y236A) was the negative control—it has was previously shown to be incapable of binding to PA.
After unbound protein was washed away, the cells were treated with low or neutral pH buffer. The low pH buffer mimics the endosomal environment and triggers PA63 pore formation and the subsequent translocation of LF to the cytosol. After this, cells were exposed to pronase to remove any surface-bound label, washed, lysed, and assayed for 35S content. As shown in
Since the preceding assays were performed with relatively crude preparations of protein, it remained a possibility that the results we observed were caused by the effects of contaminants upon mutant LF and not wild-type LF activity. To test this we purified wild-type LF and selected LF double mutants by FPLC and re-assessed the toxicity of these preparations using macrophage cytotoxicity assays. The EC50 of wild-type LF was 10.9±5.9 nM (
To this point, the analyses indicated that point mutations at clustered residues of domain II reduce the proteolytic activity of LF. To directly test this, the proteolytic activity of FPLC-purified wild-type and mutant LF was tested in vitro by immunoblotting. The control was an FPLC-purified preparation of LF harboring a point mutation in the Zn2+-binding domain (E687C), which has been previously characterized as being non-toxic Klimpel, K R et al. (1994) Mol Microbiol 13:1093-1100 and proteolytically inactive (Duesbery et al., 1998, supra)). Incubation of 0.2 μg wild-type LF, but not E687C, with 0.2 μg NH2-terminally His6-tagged MEK1 increased the electrophoretic mobility of MEK1, consistent with NH2-terminal proteolysis as described. Unexpectedly, none of the mutant LF showed reduced proteolytic activity towards MEK (
Modified cleavage assays were performed in the concentration of LF was varied in the presence of a fixed amount of MEK and the kinase activity of MEK towards ERK was used as an indirect, but quantifiable, measure of proteolysis. Wild-type LF caused a robust inhibition of MEK activity and resulted in a 50% suppression of ERK phosphorylation at a molar ratio (LF:MEK) of 0.5±0.3 (
An alternative explanation for the foregoing observations is that decreased LF toxicity may be caused by a loss of substrate affinity that is independent of proteolytic activity. In lieu of a direct assay of LF binding to MEK, the present inventor and colleagues previously demonstrated that LF could competitively inhibit B-Raf phosphorylation of MEK and that this inhibition was independent of its proteolytic activity. The interpretation of these results was that LF and B-Raf bound to adjacent or overlapping epitopes on MEK. To determine whether point mutations at clustered residues of domain II reduced the affinity of LF for MEK. in vitro B-Raf-mediated MEK phosphorylation was assayed in the presence of LF or LF mutants. As reported earlier, LF caused an approximately 35% inhibition of MEK phosphorylation by B-Raf and this effect was independent of LF proteolytic activity since E687C also inhibited MEK phosphorylation by B-Raf (
LF is the principal virulence factor of anthrax toxin (Cataldi, A et al. (1990) Mol. Microbiol. 4:1111-17; Pezard, C et al. (1991) Infec Immun 59:3472-77; Pezard, C et al. (1993) J. Gen. Microbiol. 139:2459-63). To date, its only identified substrates are members of the MEK family of protein kinases. Consequently, the interaction between LF and MEK is an important concern for understanding the pathogenesis of anthrax as well as in the design of targeted therapeutic agents. This studies described above were undertaken to identify regions of LF which are required for interaction with MEK. As a starting point, the present inventor reasoned that since a number of the conserved residues in the LFIR are long-chain aliphatic residues, any region of LF with which it associated would (i) contain a cluster of surface-exposed aliphatic residues and (ii) lie adjacent to the catalytic groove where the active site complex would form. Regardless of the physiological relevance of these assumptions, tests of this hypothesis led to the identification of a single residue (L514) in domain II which, when replaced by an alanine residue, resulted in a substantial reduction in LF toxicity. Further alanine-substitution in the vicinity of L514 identified four additional residues which also play a role in LF toxicity. Though separated in primary sequence, the tertiary structure of LF brings these five residues side-by-side in a focused region which lies at one end of the groove which forms between domains III and IV and contains the active site (see
What role does this region play in LF toxicity? One key observation is that although mutant LF (i.e. L514A and L514A/N516A) were incapable of cleaving MEK in cell-based assays, they did so in vitro. This indicates that these mutants are sensitive to the context in which they encounter their substrate MEKs. In cells, the spatial distribution and accessibility of MEKs are influenced by scaffolding proteins such as MP1 (Schaeffer, H J et al. (1998) Science 281:1668-1671) and JIP-1 (Whitmarsh, A J et al. (1998) Science 281:1671-74). In addition, cellular MEKs may be modified post-translationally (e.g. by phosphorylation) and can associate with their cognate MAPKs as well as other regulatory molecules such as B-Raf. Any of these factors may limit the ability of mutant LF to bind and cleave MEKs in cells. Indeed, while the MEK1 scaffolding protein MP1 can associate with both recombinant MEK1 and MEK2 in vitro, it can only bind MEK1 in cells (Schaeffer et al., supra). While the present invention is not intended to be bound by potential mechanism(s), the simplest interpretation of the present observations is that the region herein identified defines a site which is necessary for LF to associate into a productive complex with MEKs. Several observations support this conception: (i) the effect of the mutations was specific; only mutations in this region, but not in clusters II or III, decreased LF toxicity, (ii) decreased toxicity was accompanied by decreased proteolysis of MEK2 in cells, (iii) mutations in this region did not alter “other” functions: binding to PA or translocation of LF across the cell membrane, (iv) the LF mutants L514A and L514A/N516A possessed in vitro proteolytic activity which was comparable to that of wild-type LF, (v) the LF mutants K294A/L514A and R491A/L514A display reduced ability to competitively inhibit B-Raf phosphorylation of MEK, and (vi) the region identified on domain II is spatially distinct from the active site and thus is not likely to directly participate in substrate proteolysis. Because mutant LF not only retained the ability to bind PA and internalize into cells but also (in the case of L514A and L514A/N516A) possessed wild-type levels of proteolytic activity, an explanation that that the mutations introduce gross, structural changes in LF with nonspecific effects.
Understanding the precise role this region plays in promoting the association of LF into a productive complex with MEKs requires further research. This region may be required to direct LF to MEKs within cells. In this case, mutations in this region of domain II would reduce the ability of LF to associate with proteins which co-localize with MEKs. Alternatively, this region may play a direct role in binding MEKs. The latter possibility is supported by the observations that the LF mutants K294A/L514A and R491A/L514A display reduced ability to competitively inhibit B-Raf phosphorylation of MEK. Moreover, indirect evidence supports the conclusion view that LF and MEK interact at sites outside the active site. Using yeast two-hybrid analysis to identify binding partners of LF, Vitale et al. (supra) isolated cDNA encoding MEK2 which lacked the NH2-terminal cleavage site. In addition, the present inventor and colleagues earlier demonstrated the existence of a conserved region located in C-terminus of MEK1 which is required for LF-mediated proteolysis of MEKs (Chopra, A P et al., 2003, supra).
Recent publications have identified lead compounds which may be adapted for use as small molecule inhibitors of LF activity (Dell'Aica, I et al. (2004) EMBO Rep 5:418-22; Min, D H et al. (2004) Nat Biotechnol 22:717-23; Turk, B E et al. (2004) Nat Struct Mol Biol 11:60-6; Tonello, F et al. (2002) Nature 418:386). These molecules were initially identified by LF cleavage (proteolysis) assays that used optimized peptide substrates which mimic the NH2-terminal cleavage site on MEKs. However, as shown herein, sites outside the active site complex on both LF and MEK are required for efficient proteolysis of MEK.
Thus, according to the present invention, novel and more effective anthrax therapeutics are molecules that are targeted to the region of LF defined by these residues, and which are used either alone or in combination with those identified molecules which target LF's active site.
The references cited above are all incorporated by reference herein, whether specifically incorporated or not.
Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/35722 | 10/3/2005 | WO | 00 | 11/19/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60614555 | Oct 2004 | US |
