Patent Application
20230250408
The present invention relates to novel human PH20 variants or fragments thereof having increased enzymatic activity and thermal stability compared to human hyaluronidase, which is an enzyme that hydrolyzes hyaluronic acid, and more particularly to PH20 variants or fragments thereof, which include one or more amino acid residue substitutions, deletions and/or insertions in hyaluronidase variants having the amino acid sequence of SEQ ID NO: 3, and optionally in which one or more amino acid residues are deleted from the N-terminus and/or C-terminus, a method for producing the same, and a pharmaceutical composition containing the same.
The human skin is composed of the epidermis, the dermis, and a subcutaneous fat layer, and there are six types of glycosaminoglycans in the skin. These glycosaminoglycans include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratin sulfate.
These glycosaminoglycans are composed of repeating disaccharide sugar units. The number of repeating disaccharide sugar units is different among glycosaminoglycans, but ranges from several hundreds to several thousands. Among the glycosaminoglycans, hyaluronic acid is present in the skin more than half of the amount in the body. Hyaluronic acid is synthesized by hyaluronan synthase present in the cell membrane, is present alone without binding to proteoglycans, and is the only glycosaminoglycan having no sulfate group. Other glycosaminoglycans bind to proteoglycans and have a sulfate group. Hyaluronic acid consists of glucuronic acid and N-acetylglucosamine, alternately linked via β-1,4 and β-1,3 bonds, and is composed of about 5,000 repeating units of these disaccharides. It is known that about one-third (5 g) of hyaluronic acid in the human body is degraded every day.
Hyaluronidases are enzymes that degrade hyaluronic acid present in the extracellular matrix. Six hyaluronidase genes are known in humans: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20/SPAM1. Human Hyal1 and Hyal2 are expressed in most tissues. PH20/SPAM1 (hereinafter referred to as PH20) is expressed in the sperm plasma membrane and the acrosomal membrane. However, HyalPS1 is not expressed, because it is a pseudogene. Hyaluronidases are divided, depending on the method by which hyaluronic acid is cleaved, into three types: enzymes (EC 3.2.1.35) that cleave β-1,4 bonds between N-acetylglucosamine and glucuronic acid by the use of H2O; enzymes (EC 3.2.1.36) that cleave β-1,3 bonds between N-acetylglucosamine and glucuronic acid by the use of H2O; and bacterial hyaluronidases (EC 4.2.99.1) that cleave β-1,4 bonds without using H2O.
The catalytic amino acids of Hyal1 are D129 and E131, which hydrolyze hyaluronic acid by substrate-assisted catalysis. Hyal1 exhibits optimum activity at an acidic pH of 3 to 4, and has no enzymatic activity at a pH of 4.5 or higher. In contrast to Hyal1, PH20 exhibits activity throughout a wide pH range of 3 to 8.
Arming et al. identified that the catalytic amino acids of PH20 are D111 and E113 (Arming et al., 1997). Arming et al. designated Leu as the first amino acid of the PH20, from which a signal peptide or the like is removed, and thus the catalytic amino acids of the PH20 containing the signal peptide correspond to D146 and E148, respectively.
Hyaluronidase hydrolyzes hyaluronic acid, thereby reducing the viscosity of hyaluronic acid in the extracellular matrix and increasing the permeability thereof into tissue (skin). The subcutaneous area of the skin has a neutral pH of about 7.0 to 7.5. Thus, among various types of hyaluronidases, PH20 is widely used in clinical practice (Bookbinder et al., 2006). In examples in which PH20 is used in clinical practice, PH20 is used as an eye relaxant and an anesthetic additive in ophthalmic surgery, and is also co-administered with an antibody therapeutic agent which is injected subcutaneously (Bookbinder et al., 2006). In addition, based on the property of hyaluronic acid, which is overexpressed in tumor cells, PH20 is used to hydrolyze hyaluronic acid in the extracellular matrix of tumor cells, thereby increasing the access of an anticancer therapeutic agent to the tumor cells. In addition, it is also used to promote resorption of body fluids and blood, which are excessively present in tissue.
PH20 was first identified in guinea pig sperm by Lathrop et al., and is also known to be expressed in sperm of different species. Human PH20 gene was cloned by Lin et al. and Gmachl et al. Human PH20 has the amino acid sequence of SEQ ID NO: 1, which consists of 509 amino acid residues, and exhibits 60% amino acid identity with guinea pig PH20 gene. Human PH20 enzyme is encoded from the SPAM1 (sperm adhesion molecule-1) gene, and Ser490 of PH20 is present in the form of being bound to glycosylphosphatidylinositol (GPI) on the surface of the sperm plasma membrane and in the acrosomal membrane. Sperm hydrolyzes hyaluronic acid using PH20 when it penetrates oocytes through the hyaluronan-rich cumulus layer of the oocytes. PH20 is present in an amount corresponding to 1% or less of the amount of proteins in sperm, and has six N-glycosylation sites (N82, N166, N235, N254, N368, and N393).
Currently commercially available PH20 is obtained by extraction from the testes of cattle or sheep. Examples thereof include Amphadase® (bovine hyaluronidase) and Vitrase® (sheep hyaluronidase).
Bovine testicular hyaluronidase (BTH) is obtained by removing a signal peptide and 56 amino acids on the C-terminal from bovine wild-type PH20 during post-translational modification. BTH is also a glycoprotein, and has a mannose content of 5% and a glucosamine content of 2.2% based on the total components including amino acids. When animal-derived hyaluronidase is repeatedly administered to the human body at a high dose, a neutralizing antibody can be produced. Since animal-derived hyaluronidase contains other biomaterials in addition to PH20, it may cause an allergic reaction when administered to the human body (Bookbinder et al., 2006). In particular, the production and the use of PH20 extracted from cattle can be limited due to concerns of mad cow disease. In order to overcome this problem, studies on the recombinant protein of human PH20 have been conducted.
Recombinant protein of human PH20 has been reported to be expressed in yeast (P. pastoris), DS-2 insect cells, and animal cells. The recombinant PH20 proteins produced in insect cells and yeast differ from human PH20 in terms of the pattern of N-glycosylation during post-translational modification.
Hyaluronidases, protein structures of which have been identified are Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidase (PDB ID: 1FCQ, 1FCU, 1FCV). Hyal1 is composed of two domains, a catalytic domain and an EGF-like domain. The catalytic domain is in the form of (β/α)8 in which an alpha-helix and a beta-strand, which characterize the secondary structure of the protein, are each repeated eight times (Chao et al., 2007). The EGF-like domain is completely conserved in variants in which the C-terminus of Hyal1 is spliced differently. The amino acid sequences of Hyal1 and PH20 are 35.1% identical, and the protein structure of PH20 has not yet been found.
A recombinant protein of human PH20 was developed by Halozyme Therapeutic, Inc. and has been sold under the trade name Hylenex® (Bookbinder et al., 2006; Frost, 2007).
When D146 and E148, which are the catalytic amino acids of PH20, were mutated to asparagine (D146N) and glutamine (E148Q), respectively, there was no enzymatic activity (Arming et al., 1997). In addition, when R246 of PH20 was substituted with glycine, the enzymatic activity was reduced by 90%, and when E319 was substituted with glutamine and R322 was substituted with threonine, the enzymatic activity disappeared. A variant in which 36 amino acids at the C-terminus of PH20 were removed (truncation of amino acids 474-509) exhibited a 75% reduction in enzymatic activity compared to wild-type PH20. This mutant was not secreted extracellularly, but remained in HeLa cells. A mutant in which C-terminal 134 amino acids were removed from PH20 had no enzymatic activity and was not secreted extracellularly. According to Frost et al., the C-terminal 477-483 region of PH20 is essential for soluble expression (Frost, 2007). The activity of full-length PH20 (1-509) or a PH20 variant having a C-terminus truncated at position 467 was merely 10% of a PH20 variant having a C-terminus truncated at one of positions 477 to 483 (Frost, 2007).
Recombinant PH20 is medically used as a carrier to promote subcutaneous delivery of pharmaceuticals, to reduce intraocular pressure in patients with ophthalmic diseases, to delay stenosis after surgery, as a dispersant to improve the activity of chemotherapeutic agents in diseases such as cancer, as an auxiliary therapeutic agent for surgery, and the like.
In particular, in the case of protein drugs, recently, high-dose products with high concentrations ranging from tens of mg to hundreds of mg per 1 mL have been developed, and thus the application of recombinant PH20 as a carrier to promote subcutaneous delivery of such protein drugs is increasing. Such protein drugs may have problems of low physical stability resulting from an increase in viscosity and aggregation of proteins due to the high concentration thereof. In addition, the aggregation of proteins is irreversible, and small amounts of proteins start to aggregate and aggregate form larger clumps (Schon et al., 2015). That is, recombinant PH20 administered in combination undergoes aggregation, thus reducing the stability of protein drugs.
Meanwhile, conventional recombinant PH20 is still insufficient from the aspects of thermal stability and expression level. Therefore, there is great demand in industry for a recombinant hyaluronidase having further improved biological and physico-chemical properties.
Therefore, the present invention has been made in view of the above problems, and it is one object of the present invention to provide a PH20 variant or fragment thereof which is improved in thermal stability, enzyme activity and expression level, compared to wild-type PH20, preferably mature wild-type PH20.
It is another object of the present invention to provide a composition for treating cancer containing the PH20 variant or fragment thereof and a method of treating cancer using the same.
In accordance with one aspect of the present invention, the above and other objects can be accomplished by the provision of a PH20 variant or fragment thereof comprising one or more amino acid residue substitutions, deletions and/or insertions in a hyaluronidase variant having the amino acid sequence of SEQ ID NO: 3, and in which one or more amino acid residues at the N-terminus or C-terminus are selectively deleted.
In accordance with another aspect of the present invention, there are provided a composition for treating cancer containing the PH20 variant or fragment thereof and a method of treating cancer using the same.
The PH20 variants or fragments thereof according to the present invention have increased protein expression levels and show an increase in protein aggregation temperature of 4-11.5° C. or so when expressed in CHO (ExpiCHO) cells so that they are efficiently produced and are imparted with higher thermal stability compared to the mature wild-type PH20.
Further, as the result of a substrate-gel assay, one of tests to measure the activity of hyaluronidase, the PH20 variants or fragments thereof according to the present invention have improved protein refolding so that they are re-natured faster than the mature wild-type PH20, and the original enzymatic activity is maintained regardless of the C-terminal cleavage position.
Furthermore, the PH20 variants or fragments thereof according to the present invention have low immunogenicity, so that they can be repeatedly administered to the human body.
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
More specifically,
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as appreciated by those skilled in the field to which the present invention pertains. In general, the nomenclature used herein is well-known in the art and is ordinarily used.
In the present invention, the position of the amino acid residue of each variant is referred from the amino acid sequence according to SEQ ID NO: 1, when described based on wild-type PH20, and the position of the amino acid residue of each variant is referred from the amino acid sequence according to SEQ ID NO: 3, when described based on the PH20 variant having SEQ ID NO: 3.
The present inventors found through previous research that a hyaluronidase PH20 variant, which includes one or more amino acid residue substitutions in the region corresponding to an alpha-helix region and/or a linker region thereof, preferably an alpha-helix 8 region (S347 to C381) and/or a linker region (A333 to R346) between alpha-helix 7 and alpha-helix 8, in wild-type PH20 having an amino acid sequence of SEQ ID NO: 1, preferably mature wild-type PH20, and optionally in which one or more of the N-terminal and/or C-terminal amino acid residues are selectively cleaved and deleted, exhibits superior efficacy compared to conventional wild-type PH20 or fragments thereof, and filed a patent application regarding this finding (see WO 2020/022791A).
As used herein, the term “mature wild-type PH20” means a protein consisting of amino acid residues L36 to Y482 or L36 to S490 of SEQ ID NO: 1, which lack M1 to T35, which form a signal peptide, and N483 to L509 or A491 to L509, which are not related to the substantial enzymatic function of PH20, in the amino acid sequence of SEQ ID NO: 1 of wild-type PH20.
Specifically, the present inventors found through previous research that, when amino acid sites corresponding to T341 to I361, which is a part of an alpha-helix 8 region (S347 to C381) and/or a linker region (A333 to R346) between alpha-helix 7 and alpha-helix 8, in wild-type PH20 having an amino acid sequence of SEQ ID NO: 1 is substituted with amino acid residues corresponding to wild-type Hyal1 having the sequence of SEQ ID NO: 2, the expression efficiency and enzymatic activity are improved, and fragments in which a part of the amino acid sequence at the N-terminus and C-terminus is deleted also exhibit superior expression efficiency and high enzymatic activity.
As a result of continuous research, the inventors found that a variant having the sequence of SEQ ID NO: 3, constructed by substituting the amino acid region corresponding to T341 to I361 of wild-type PH20 having the amino acid sequence of SEQ ID NO: 1 with the corresponding amino acid sequence of wild-type Hyal1 having the sequence of SEQ ID NO: 2, still exhibits excellent expression efficiency and high enzymatic activity, as well as remarkably improved protein aggregation temperature (Tagg) compared to the wild-type PH20, although it includes additional substitutions, deletions and/or insertions of amino acid residues, and further optionally includes deletions of one or more amino acid residues at the N-terminus and/or C-terminus. Based on this finding, the present invention has been completed.
The variant having the sequence of SEQ ID NO: 3 is constructed by substituting 15 amino acid residues, namely, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1.
In this regard, the PH20 variant or fragment thereof according to the present invention includes substitution, deletion and/or insertion of one or more amino acid residues in the PH20 variant having the amino acid sequence of SEQ ID NO: 3, and optionally includes deletion of one or more amino acid residues at the N-terminus and/or C-terminus.
As described above, the variant having the amino acid sequence of SEQ ID NO: 3 is a variant in which amino acid residues of T341 to I361 of wild-type PH20 are substituted with corresponding amino acid residues of wild-type Hyal1 (see Table 2). The variant having the amino acid sequence of SEQ ID NO: 3 or a fragment thereof including amino acid residue deletion at the N-terminus and C-terminus was identified as a variant having activity and stability superior to those of wild-type PH20 in previous research.
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Specifically, the PH20 variant or fragment thereof according to the present invention may include one or more mutations, preferably one or more amino acid residue substitutions, deletions and/or insertions in the amino acid sequence of SEQ ID NO: 3, and has a higher protein aggregation temperature (Tagg), which is an index indicating protein stability, than the wild-type PH20. In addition, the PH20 variant according to the present invention does not include the wild-type PH20 of SEQ ID NO: 1.
As used herein, the term “PH20 variant” is intended to include a variant having not only a mutation of one or more amino acid residues, preferably substitution, deletion and/or insertion of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 3, but also deletion of one or more amino acid residues at the N-terminus or C-terminus thereof together with the substitution, deletion and/or insertion of the amino acid residues, and is used with substantially the same meaning as the expression “PH20 variant or fragment thereof”.
Preferably, the PH20 variant according to the present invention includes amino acid residue substitution, insertion, and/or deletion at one or more positions selected from the group consisting of R39, D65 to L68, N82, T84, I102 to I105, T132 to Y134, N166, L179 to T182, T185 to K187, V241 to K244, N266 to Q269, P271, V272, K290 to P292, Q311 to K314, G340 to N363, L441, S442, D451 to D453, D461, V463 and D461 to V463 in a variant having the amino acid sequence of SEQ ID NO: 3, and has a higher protein aggregation temperature (Tagg) than that of wild-type PH20.
The PH20 variant according to the present invention may include a mutation at 20 or fewer, preferably 17 or fewer, more preferably 15 or fewer amino acid positions in the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.
More preferably, the PH20 variant or fragment thereof according to the present invention includes at least one amino acid residue substitution selected from the group consisting of R39K, D65A, E66A, P67A, L68A, N82A, T84N, I102A, D103A, S104A, S104N, I105A, I105Q, T132A, T132S, F133A, Y134A, N166A, N166K, L179A, L179S, L179I, L179F, S180T, S180A, L181A, L181M, T182A, T185A, E186A, E186D, K187A, V241A, E242A, I243A, K244A, N266A, T267A, Q268A, Q268D, Q268I, Q268N, Q269A, P271A, V272A, K290A, I291A, I291G, I291L, P292A, P292D, Q311A, V312A, L313A, L313P, L313M, K314A, G340Q, S341H, S341D, S341T, W342I, W342D, W342H, W342L, E343V, E343S, E343Y, E343Q, N344F, N344I, T345E, T345K, T345S, R346M, R346F, R346L, R346T, R346S, R346A, T347Q, T347E, T347V, T347W, T347H, T347S, K348Q, K348F, K348D, K348T, K348E, K348M, E349L, E349W, E349A, S350Q, S350I, S350D, S350T, S350E, S350N, Q352E, Q352G, Q352Y, Q352W, Q352T, A353E, A353Y, A353H, A353K, I354E, I354Q, I354S, I354V, I354A, I354N, I354T, I354R, I354W, I354L, K355Q, K355H, K355D, E356M, E356F, E356I, E356L, E356Q, E356V, E356D, Y357W, Y357F, M358V, M358R, M358Y, M358L, D359K, D359V, D359Y, D359Q, D359T, D359S, D359E, T360Y, T360R, T360L, T360D, T360S, T361M, T361E, T361H, T361L, T361D, T361I, L362A, N363M, N363E, L441A, S442A, D451A, D451S, T452A, T452D, T452H, T452K, T452G, T452P, T452M, T452F, D453A, D461R, D461A, G462A, V463Y and V463A in the variant having the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.
In the present invention, an expression described by a one-letter amino acid residue code together with numbers, such as “S341”, means the amino acid residue at each position in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
For example, “S341” means that the amino acid residue at position 341 in the amino acid sequence of SEQ ID NO: 3 is serine and “S341H” means that serine at position 341 of SEQ ID NO: 3 is substituted with histidine.
The PH20 variant or a fragment thereof according to the present invention is interpreted as including variants or fragments thereof in which an amino acid residue at a specific amino acid residue position is conservatively substituted.
As used herein, the term “conservative substitution” refers to modifications of a PH20 variant that involve the substitution of one or more amino acids with other amino acids having similar biochemical properties that do not result in loss of the biological or biochemical function of the PH20 variant.
The term “conservative amino acid substitution” refers to substitution of the amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined and are well known in the art to which the present invention pertains. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids with nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).
It is found that the PH20 variant or fragments thereof of the present invention retains the activity thereof despite having conservative amino acid substitutions.
In addition, the PH20 variant or fragment thereof according to the present invention is interpreted to include PH20 variants or fragments thereof having substantially the same function and/or effect as those/that of the PH20 variant or fragment thereof according to the present invention, and having amino acid sequence homology of at least 80% or 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% to the PH20 variant or fragment thereof according to the present invention.
The PH20 variants or fragments thereof according to the present invention have increased expression levels and protein refolding rate, and thereby have higher thermal stability than mature wild-type PH20. Furthermore, the enzymatic activity of the PH20 variants was greater than or similar to that of mature wild-type PH20 despite the increase in thermal stability.
Meanwhile, although the mature wild-type PH20 variant having cleavage at the C-terminal is known to have decreased enzymatic activity, the PH20 variants according to the present invention exhibit similar or increased enzymatic activity and expression efficiency, and high protein aggregation temperatures (Tagg) due to the more rapid protein refolding and thermal stability thereof, although one or more amino acid residues at the C-terminus are cleaved and deleted, and/or 1 to 7, preferably, 1 to 5 amino acid residues at the N-terminus are cleaved and deleted.
Accordingly, the PH20 variant or fragment thereof according to the present invention is characterized in that it includes one or more amino acid mutations, preferably one or more amino acid residue substitutions, deletions and/or insertions in the variant having the amino acid sequence of SEQ ID NO: 3 or the like, and one or more residues of N-terminus and/or C-terminus are additionally deleted, but is not limited thereto.
In one embodiment, the PH20 variant or fragment thereof according to the present invention may be one in which cleavage occurs before an amino acid residue selected from the group consisting of M1 to P42 from the N-terminus, preferably before an amino acid residue L36, N37, F38, R39, A40, P41, or P42 at the N-terminus, in the amino acid sequence of SEQ ID NO: 3, so that one or more amino acid residues from the N-terminus are deleted, and/or cleavage occurs after an amino acid residue selected from the group consisting of V455 to L509, preferably after an amino acid residue selected from the group consisting of V455 to S490, most preferably after an amino acid residue V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490 at the C-terminus, so that one or more amino acid residues at the C-terminus are deleted.
The expression “cleavage occurs before an amino acid residue selected from the group consisting of M1 to P42 at the N-terminus” means that a portion of amino acid residues immediately before the selected amino acid residue from among M1 to P42 at the N-terminus is cleaved and deleted. The expression “cleavage occurs before M1” means that no cleavage occurs at the N-terminus.
For example, the expression “cleavage occurs before an amino acid residue L36, N37, F38, R39, A40, P41, or P42” means that all amino acid residues from M1 to T35 immediately before L36, all amino acid residues from M1 to L36 immediately before N37, all amino acid residues from M1 to N37 immediately before F38, all amino acid residues from M1 to F38 immediately before R39, all amino acid residues from M1 to R39 immediately before A40, all amino acid residues from M1 to A40 immediately before P41, or all amino acid residues from M1 to P41 immediately before P42 in the amino acid sequence of SEQ ID NO: 3 according to the present invention are cleaved and removed.
In addition, the expression “cleavage occurs after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus” means that a portion of amino acid residues immediately before the selected amino acid residue from among M1 to P42 at the N-terminus is cleaved and deleted.
For example, the expression “cleavage occurs after an amino acid residue V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 at the C-terminus” means that an amino acid residue after the amino acid residue V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 in the amino acid sequence of SEQ ID NO: 3 according to the present invention is cleaved or all the amino acid residue(s) from the amino acid residue immediately after the selected amino acid residue to L509 is/are deleted.
Preferably, the novel PH20 variant or fragment thereof according to the present invention is characterized in that it includes an amino acid residue substitution, deletion or insertion at one or more positions in the variant having the amino acid sequence of SEQ ID NO: 3, a truncation before F38 at the N-terminus, and a truncation after F468 at the C-terminus.
More preferably, the novel PH20 variant or fragment thereof according to the present invention may include an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOS: 163 to 316, but is not limited thereto.
The sequences of the substituted or cleaved amino acids in the PH20 variant constructed in the specific embodiment according to the present invention are as shown in Table 6.
In addition, in the present invention, an attempt was made to increase the expression of a recombinant PH20 protein using other signal peptide of proteins highly expressed in animal cells, instead of using the original signal peptide of PH20.
Therefore, in another embodiment, the novel PH20 variant according to the present invention may be one in which the N-terminus further includes a human growth hormone signal peptide having an amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA of SEQ ID NO: 4, a human serum albumin signal peptide having an amino acid sequence MKWVTFISLLFLFSSAYS of SEQ ID NO: 5, or a human Hyal1 signal peptide having an amino acid sequence MAAHLLPICALFLTLLDMAQG of SEQ ID NO: 6 as shown in Table 3 below, instead of the signal peptide of wild-type PH20, which consists of M1 to T35, but is not limited thereto.
The expression “instead of the signal peptide of wild-type PH20, which consists of M1 to T35” means the case in which the signal peptide in the amino acid sequence of SEQ ID NO: 3 is partially or completely deleted, and thus does not perform the function thereof. In addition, the expression is meant to include the case in which a portion of the N-terminus is further deleted, for example, the case in which cleavage occurs before the N37, F38, R39, A40, P41 or P42 residue occurs so that an additional deletion of the N-terminus together with the deletion of the signal peptide of wild-type PH20 occurs.
In another aspect, the present invention is directed to a composition for treating cancer containing the novel PH20 variant or fragment thereof according to the present invention and a method for treating cancer using the same.
The cancers or carcinomas that can be treated by the novel PH20 variant or fragment thereof according to the present invention are not particularly limited, but include both solid cancers and blood cancers. The cancer may be selected from the group consisting of skin cancer such as melanoma, liver cancer, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, uterine cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, renal cancer, esophageal cancer, biliary tract cancer, testis cancer, rectal cancer, head and neck cancer, ureteral cancer, osteosarcoma, neurocytoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and neuroglioma, but is not limited thereto. Preferably, cancers that can be treated by the composition according to the present invention may be selected from the group consisting of colorectal cancer, breast cancer, lung cancer and renal cancer, but are not limited thereto.
The composition of the present invention may be a pharmaceutical composition. The pharmaceutical composition may further include a pharmaceutically acceptable composition. The composition may contain one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, which are typically used in the preparation of drugs, but is not limited thereto. In addition, the pharmaceutical composition may further contain one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions and preservatives, which are typically used in the preparation of drugs.
The pharmaceutical composition may be administered orally or parenterally. The parenteral administration is carried out by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, and the like. For oral administration, the active ingredient in the oral composition needs to be formulated into a coated dosage form or into a dosage form that can protect the active ingredient from disintegrating in the stomach, considering that peptides and proteins are digested in the stomach. Alternatively, the present composition may be administered via any device by which the active ingredient can move to the target cell of interest.
The pharmaceutical composition may be formulated in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, grains, powders, granules, tablets or capsules, and may additionally include dispersing or stabilizing agents for the purpose of formulation.
In particular, the composition for treating cancer according to the present invention may be used in combined therapy with other anticancer drugs.
An anticancer drug that can be used in combined therapy with the novel PH20 variant or fragment thereof according to the present invention is preferably a chemical anticancer drug, an antibody-based anticancer drug, a biological anticancer drug, an RNAi, or a cell therapeutic agent, but is not limited thereto.
Preferably, the anticancer drug that can be used in combined therapy with the novel PH20 variant or fragment thereof according to the present invention is preferably an immuno-oncologic agent, and more preferably an immune checkpoint inhibitor, but is not limited thereto.
In addition, the present invention is directed to a method for treating cancer using the novel PH20 variant or fragment in combination with other anticancer agents, particularly the anticancer agents described above.
In another aspect, the present invention is directed to a nucleic acid encoding the PH20 variant or fragment thereof.
The nucleic acids, as used herein, may be present in cells, in the cell lysate, or in the partially purified or substantially pure form. “Isolated” or “to be substantially pure”, when referring to nucleic acids, refer to those that have been purified and thus separated from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. The nucleic acids of the present invention may be DNA or RNA.
In still another aspect, the present invention is directed to a recombinant expression vector including the nucleic acid. For expression of the PH20 variant or fragment thereof according to the present invention, a DNA encoding the PH20 variant or fragment thereof can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the PH20 variant), and the DNA can be inserted into an expression vector such that it is “operatively linked” to transcriptional and translational control sequences.
As used herein, the term “operatively linked” is intended to mean that a gene encoding the PH20 variant or fragment thereof is ligated into a vector such that transcriptional and translational control sequences serve intended functions thereof of regulating the transcription and translation of the gene encoding the PH20 variant or fragment thereof. The expression vector and expression control sequences are chosen to be compatible with the expression host cell that is used. The genes encoding the PH20 are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites on a fragment of the gene encoding the PH20 variant or fragment thereof and a vector, or blunt-end ligation if no restriction enzyme sites are present).
In addition, the recombinant expression vectors carry regulatory sequences that control the expression of a gene encoding the PH20 variant or fragment thereof in the host cell. The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the genes encoding the PH20 variant or fragment thereof. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the desired level of expression of the protein, etc.
In yet another aspect, the present invention is directed to a host cell including the nucleic acid or the vector. The host cell according to the present invention is preferably selected from the group consisting of animal cells, plant cells, yeasts, E. coli., and insect cells, but is not limited thereto.
Specifically, the host cell according to the present invention include prokaryotic cells such as E. coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., fungi such as Aspergillus sp., yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa, and eukaryotic cells such as lower eukaryotic cells, and higher other eukaryotic cells such as insect cells.
In addition, the host cells that can be used in the present invention may be derived from plants or mammals. Preferably, examples of the host cells include, but are not limited to, monkey kidney cells (C0S7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cells, HuT 78 cells and HEK293 cells. More preferably, CHO cells may be used.
The nucleic acid or the vector is transfected into a host cell. Transfection can be performed using various techniques that are generally used to introduce foreign nucleic acid (DNA or RNA) into prokaryotic or eukaryotic cells, for example, electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection. In order to express the PH20 variant or fragment thereof of the present invention, various combinations of recombinant expression vectors and host cells can be employed. The preferred expression vector for eukaryotic cells includes gene expression regulatory sequences derived from, but not limited to, SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. Expression vectors that can be used for bacterial hosts include bacterial plasmids such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and derivatives thereof, obtained from E. coli; a plasmid having broad host range, such as RP4; phage DNAs exemplified by various phage lambda derivatives, such as, λgt10, λgt11 and NM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phage. An expression vector available for yeast cells may be a 2-μm plasmid and derivatives thereof. Expression vectors for insect cells include pVL941.
In another aspect, the present invention is directed to a method for producing a PH20 variant or fragment thereof, the method including culturing the host cell and expressing the PH20 variant or fragment thereof according to the present invention.
When a recombinant expression vector capable of expressing the PH20 variant or fragment thereof is introduced into mammalian host cells, the PH20 variant or fragment thereof can be produced by culturing the host cells for a period of time such that the PH20 variant or fragment thereof is expressed in the host cells, preferably a period of time such that the PH20 variant is secreted into the medium during culture of the host cells.
In an alternative embodiment, the expressed PH20 variant or fragment thereof can be isolated and purified from the host cells. Isolation or purification of the PH20 variant or fragment thereof can be performed by conventional isolation/purification methods (e.g., chromatography) that are used for proteins. The chromatography may include a combination of one or more selected from affinity chromatography, ion exchange chromatography, and hydrophobic chromatography, but is not limited thereto. In addition to the chromatography, a combination of filtration, ultrafiltration, salting out, dialysis, and the like may be used.
In order to confirm the industrial applicability of the enzyme, it is necessary to analyze the catalytic reaction rate of the enzyme. Types of enzymatic reactions include an enzyme reaction with an active site with fixed reactivity and an enzyme reaction with several active sites with various reactivity. It is known that the catalytic reaction rate of enzymes having an active site with fixed reactivity, such as hyaluronidase, follows the Michaelis-Menten rate formula.
The Michaelis-Menten's enzyme kinetic is premised on the assumption of an enzymatic reaction as a two-step reaction system including a reversible reaction step in which Complex [ES] of Enzyme (E)-Substrate (S) is formed and an irreversible reaction step in which the ES complex is dissociated to yield Product (P). In this case, kf, kr and kcat are the rate constants of the reaction in each direction (Alan Fersht (1977) Enzyme structure and mechanism).
The enzymatic reaction assumes that the process of reacting the enzyme with the substrate to produce the ES complex rapidly reaches equilibrium, or may be considered to be a pseudo-steady state assuming that d[ES]/dt≈0 is satisfied by sufficiently lowering the concentration of the enzyme by performing a reaction that maintains a sufficiently high substrate concentration. Since the rate equations assuming fast equilibrium or pseudo-steady state are derived in the same manner, a pseudo-steady state in which the substrate concentration is initially higher than the enzyme concentration is assumed in most experiments.
When conditions such as “the amount of enzyme is constant before and after the reaction” and “when a chemical reaction reaches chemical equilibrium, the reaction rate at which the product is obtained is equal to the rate at which the product is decomposed again” are used under such an assumption, the reaction rate of the final product may be expressed by the following Michaelis-Menten rate formula. In this case, KM=(kr+kcat)/kf, and Vmax=kcat [E]0.
The Lineweaver-Burk equation is used to experimentally analyze the enzyme reaction rate using the Michaelis-Menten rate formula. This equation shows the relationship between the reciprocal 1/V of the experimentally measured reaction rate with the reciprocal 1/[S] of the given substrate concentration in the experiment. Statistical verification that this equation is a linear equation demonstrates that the enzyme reaction is a reaction following Michaelis-Menten's rate formula, and KM and Vmax can be calculated using this equation.
Enzymes that catalyze a chemical reaction have a transition state after binding to a substrate at an active site, and the activation energy for reaching the transition state having high energy is lowered through multiple bonds with the substrate. The equilibrium constant for reaching this transition state is proportional to kcat/KM. Here, 1/KM is an index that combines the degree to which an enzyme-substrate complex is produced by bonding the enzyme to the substrate with the degree to which the enzyme-substrate complex is maintained without being decomposed, and kcat is the equilibrium constant at which a product is obtained from the enzyme-substrate complex. Therefore, kcat/KM can be said to be an indicator of how much product can be obtained from the substrate and the enzyme, that is, the catalytic efficiency of the enzyme.
The industrial availability of hyaluronidase is proportional to the catalytic efficiency thereof. In particular, when the enzyme is injected subcutaneously along with a polymeric pharmacologically active substance such as a monoclonal antibody, the catalytic efficiency of hyaluronidase plays an important role. In the case where the variant according to the present invention has higher kcat/KM than the wild-type PH20, when the hyaluronidase combined with the polymeric pharmacologically active substance is administered subcutaneously, hyaluronic acid present therein is rapidly decomposed and thus a superior effect of rapidly dispersing the pharmacologically active substance can be obtained. In addition, when the variant according to the present invention has a larger kcat than the wild-type PH20, the maximum reaction rate Vmax increases at the same enzyme concentration, thereby providing excellent effects of decomposing a greater amount of hyaluronic acid during the same period of time and dispersing the pharmacologically active substance in a wider region.
Therefore, in order to confirm the enzymatic properties of the PH20 variant according to the present invention, the enzyme reaction rate of each variant was analyzed, and V. (maximum enzyme reaction rate), KM (substrate concentration under 50% Vmax condition), kcat (substrate conversion rate), and kcat/KM (enzyme catalyst efficiency) thereof were compared in Example 4. The results described above demonstrate that the PH20 variant according to the present invention is superior to wild-type PH20.
Hereinafter, the present invention will be described in more detail with reference to examples. However, it will be obvious to those skilled in the art that these examples are provided only for illustration of the present invention and should not be construed as limiting the scope of the present invention.
For construction of PH20 variants, cDNA (clone ID: hMU002604) of wild-type PH20 was purchased from the Korean Human Gene Bank. Wild-type PH20 encodes amino acids from L36 to S490. The PH20 gene was amplified by polymerase chain reaction (hereinafter referred to as PCR) and inserted into the XhoI and NotI restriction enzyme sites of a pcDNA3.4-TOPO vector. For expression in ExpiCHO cells, the signal peptide of human growth hormone, human serum hormone or human Hyal1 was used as a signal peptide instead of the original signal peptide of PH20. For protein purification using a HisTrap column, the DNA sequence of a His-tag was located at the 3′-end of the PH20 cDNA. The amino acid substitution of PH20 variants was performed using PCR, and the amino acid substitution was confirmed through DNA sequencing.
The list of primers used in cloning of the PH20 variants are summarized in Table 4 below, and the specific sequences of the primers are summarized in Table 5 below.
After finding a PH20 variant with increased enzymatic activity and thermal stability, the His-tag-free cDNA of the PH20 variant was also constructed.
The PH20 variant was constructed as follows using cDNA of the PH20 variant.
The expression of the variant was performed using the ExpiCHO expression system. When the cell density of ExpiCHO cells reached 6×106/mL, a plasmid including the wild-type or variant PH20 cDNA inserted in the pcDNA3.4-TOPO vector was transfected into the ExpiCHO cells using ExpiFectamine CHO reagent. As a cell culture medium, ExpiCHO expression medium (100 to 500 mL) was used. After transfection, the ExpiCHO cells were cultured with shaking at 130 rpm for a total of 6 days, during which the cells were cultured at 37° C. for 1 day and were further cultured at a lower temperature of 32° C. for 5 days. After completion of the culture, the cell supernatant was collected by centrifugation at 10,000 rpm for 30 min.
The recombinant proteins of the C-terminal His-tag-attached wild-type PH20 and variant PH20, produced in the ExpiCHO cells, were purified through three-step column chromatography using an AKTA prime system (GE Healthcare Systems), and the three-step column chromatography was performed using a HisTrap HP column-Q Sepharose column-phenyl HP column, and a Q Sepharose column-HisTrap HP column-butyl HP column, respectively, depending on the variant.
The purification using the HisTrap HP column, the Q Sepharose column and the phenyl HP column was performed as follows. For protein purification using the HisTrap column, buffer A (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl, 0.5 M imidazole) were prepared. The protein was bound to the HisTrap column, and the column was flushed with 5 column volumes (CV) of buffer A to remove non-specifically bound proteins. It was confirmed that the conductivity was maintained at a constant level, and then the column was flushed with 5 CV of 20% buffer B to elute the protein. The eluted protein was dialyzed with dialysis buffer (20 mM sodium phosphate, pH 7.5, 50 mM NaCl). For protein purification using the Q Sepharose column, buffer A (20 mM sodium phosphate, pH 7.5) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl) were prepared. The protein was bound to the Q Sepharose column, and the column was flushed with 5 CV of buffer A to remove nonspecifically bound proteins, and then was flushed with 5 CV of buffer B at a concentration gradient of 0 to 100% to elute the protein. For protein purification using the phenyl HP column, buffer A (20 mM sodium phosphate, pH 7.0, 1.5 M (NH4)2SO4) and buffer B (20 mM sodium phosphate, pH 7.0) were prepared. The protein was bound to the phenyl column, and the column was flushed with 5 CV of buffer A to remove nonspecifically bound proteins, and then was flushed with 5 CV of buffer B at a concentration gradient of 0 to 100% to elute the protein.
The purification using the Q Sepharose column, the HisTrap HP column and the butyl HP column was performed as follows. For protein purification using the Q Sepharose column, buffer A (20 mM NaPi, 15 mM NaCl, pH 8.0) and buffer B (20 mM NaPi, 500 mM NaCl, pH 8.0) were prepared. To adjust the pH and conductivity of the culture solution to be the same as Buffer A, the pH was titrated to 8 using 1 M Tris buffer, and the conductivity was adjusted to 5 mS/cm or less by adding water (PW) thereto. Then, the culture solution was filtered through a membrane having 0.22-μm pores therein. The protein was bound to the Q Sepharose column, and the column was flushed with 5 CV of buffer A to remove nonspecifically bound proteins and was then flushed with 5 CV of buffer B to elute the target protein. For protein purification using the HisTrap HP column, buffer A (20 mM NaPi, 500 mM NaCl, pH 7.5) and buffer B (20 mM NaPi, 500 mM NaCl, 500 mM Imidazole, pH 7.5) were prepared. The protein sample was bound to the HisTrap HP column, the column was flushed with 10 CV of 7% buffer B to remove non-specifically bound proteins, and the column was then flushed with 3 CV of 40% buffer B to elute the protein. For protein purification using the butyl HP column, buffer A (20 mM NaPi, 1.5 M Ammonium sulfate, pH 7.0) and buffer B (20 mM NaPi, pH 7.0) were prepared. A 3 M ammonium sulfate solution and a protein sample to be loaded onto the column were mixed in a ratio of 1:1 and then the resulting mixture was filtered through a membrane having 0.22-μm pores therein. The protein sample was bound to the butyl HP column, and the column was flushed with 5 CV of buffer A to remove impurities. Then, the target protein was eluted with a linear concentration gradient of 0-100% buffer B, and was dialyzed using a dialysis buffer (20 mM NaPi, 100 mM NaCl, pH 7.0). The variant according to the present invention was purified by the method suggested in the present invention, 10% SDS-PAGE analysis was performed on each purified product, and the results are shown in
The enzymatic activities of wild-type PH20 and variant PH20 were measured by turbidimetric assay.
The turbidimetric assay is a method of measuring the absorbance in the precipitate that is produced when hyaluronic acid is mixed with albumin (BSA). When hyaluronic acid is hydrolyzed by PH20, the absorbance of the precipitate that is produced upon mixing with albumin decreases. The turbidimetric assay is generally performed as follows. Hyaluronidase PH20 (Sigma) was diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50 and 60 units/mL and prepared in each tube. The purified protein sample was dissolved in an enzyme diluent buffer (20 mM Tris·HCl, pH 7.0, 77 mM NaCl, 0.01% (w/v) bovine serum albumin), diluted to 100×, 300×, 600×, 1200× and 2400×, and prepared in respective tubes. In fresh tubes, the hyaluronic acid solution having a concentration of 3 mg/mL was diluted 10-fold to a concentration of 0.3 mg/mL so that the volume in each tube became 180 μL. 60 μL of enzyme was added to and mixed with the diluted hyaluronic acid solution and allowed to react at 37° C. for 45 minutes. After completion of the reaction, 50 μL of the reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance was measured at 600 nm using a spectrophotometer.
Methods of measuring the thermal stability of the protein include a method of measuring the aggregation temperature by dynamic light scattering (DLS), a method of measuring the melting temperature (Tm) in real-time PCR using Sypro-Orange dye, and a method of measuring the enzymatic activity after allowing the protein to stand at a predetermined temperature for a predetermined time, etc. In the method of measuring the aggregation temperature by DLS, the aggregation of molecules is measured using light scattering, and thus the sensitivity is high and the aggregation temperature is generally lower than the melting temperature of the protein. Since each variant is prepared as a solution of the same concentration of 0.2 mg/mL and is then measured, the physical properties of each variant can be compared using the resulting value as the aggregation temperature (Philo, J. S. (2009) Cur. Pharm. Biotech. 10, 359-372).
The amino acid sequences of the PH20 variants constructed by substitution or cleavage of the amino acids from the PH20 variant having the sequence of SEQ ID NO: 3 in the present invention are shown in Table 6 below.
In the present invention, the experiment was conducted on a variant in which six histidines were added for protein purification at the C-terminus in the sequence shown in Table 6. It was found that this addition to the C-terminus did not affect the enzyme activity or protein stability. The variant according to the present invention was named as a combination of HM and serial number, and the variants according to Example 3 were named “Hyal2-variant”, “Hyal3-variant”, and “Hyal4-variant”.
WHLHD
YLVDTLNPYIINVTLAAKMCSQV
E
IKEYMDTTLNPYIINVTLAAKMCSQVLC
A
LDMSLFSFIGSPRINATGQGVTIFYVDRL
A
TQQSPVAATLYVRNRVREAIRVSKIPDA
A
TEATEKAKQEFEKAGKDFLVETIKLGKL
A
TEATDKAKQEFEKAGKDFLVETIKLGK
M
TEATDKAKQEFEKAGKDFLVETIKLGK
A
TEATAKAKQEFEKAGKDFLVETIKLGK
A
TEATDKAKQEFEKAGKDFLVETIKLGK
A
TEATDKAKQEFEKAGKDFLVETIKLGK
A
TEATAKAKQEFEKAGKDFLVETIKLGK
A
TEATAKAKQEFEKAGKDFLVETIKLGK
Further study on the structure and function of the protein was conducted through research on variants including the cleavage at the N-terminal and C-terminal based on the amino acid sequence of SEQ ID NO: 3. As a result of the expression amount and activity analysis of the prepared variant, the aggregation temperature is shown in Table 7.
Expression level and specific activity were analyzed by the turbidimetric assay described in Example 1. The results of the assay are shown. At this time, activity in the culture solution exceeding 300 unit/mL was marked as “>LOQ”, and activity after purification exceeding 15 unit/μg was marked as “>LOQ” based on the limit of quantification (LOQ) set for each of the activity in the culture solution and the activity after purification. In the opposite case, the inequality sign was changed. The expression level and limits of quantification (LOQ) of the activity analysis, and test results based thereon are shown in Table 7. The aggregation temperature of wild-type PH20 (L36-Y482) of SEQ ID NO: 1 is 46.5° C., and the aggregation temperature of the PH20 variant (F38-F468) of SEQ ID NO: 3 is 51° C.
As can be seen from Table 7 above, among the variants having the amino acid sequence of SEQ ID NO: 3, a total of 133 types of variants having one amino acid residue substitution, namely, HM63, HM64, HM65, HM66, HM67, HM69, HM70, HM71, HM72, HM73, HM74, HM75, HM76, HM77, HM78, HM79, HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM90, HM91, HM92, HM93, HM94, HM95, HM97, HM98, HM99, HM100, HM101, HM102, HM103, HM104, HM105, HM106, HM107, HM110, HM111, HM112, HM114, HM115, HM116, HM117, HM118, HM121, HM125, HM126, HM130, HM131, HM132, HM133, HM134, HM135, HM136, HM138, HM139, HM140, HM141, HM142, HM143, HM144, HM145, HM152, HM153, HM154, HM155, HM156, HM157, HM158, HM159, HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168, HM169, HM170, HM171, HM172, HM173, HM174, HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185, HM186, HM190, HM191, HM192, HM193, HM194, HM195, HM196, HM197, HM198, HM199, HM203, HM204, HM205, HM208, HM210, HM211, HM212, HM213, HM214, HM216, HM217, HM218, HM219, HM220, HM231, HM232, HM233, HM234, HM235, HM243, HM245 and HM246, were variants that still maintain the activity in the purified fraction obtained after purification and have an aggregation temperature of 48 to 58° C. and thus exhibit excellent thermal stability. Thereamong, a total of 65 types of variants, namely HM63, HM64, HM65, HM66, HM67, HM69, HM70, HM71, HM72, HM73, HM74, HM75, HM76, HM77, HM78, HM79, HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM90, HM91, HM92, HM93, HM94, HM95, HM98, HM99, HM100, HM101, HM102, HM103, HM104, HM105, HM106, HM107, HM110, HM111, HM112, HM114, HM115, HM116, HM117, HM118, HM121, HM125, HM126, HM130, HM131, HM132, HM133, HM134, HM135, HM136, HM138, HM139, HM140, HM141, HM142, HM143, HM144 and HM145, are variants that are mutated at one of substitution sites in the sequence of SEQ ID NO: 3 from PH20, and have an aggregation temperature of 48° C. to 58° C. There among, a total of 68 types of variants, namely HM97, HM152, HM153, HM154, HM155, HM156, HM157, HM158, HM159, HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168, HM169, HM170, HM171, HM172, HM173, HM174, HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185, HM186, HM190, HM191, HM192, HM193, HM194, HM195, HM196, HM197, HM198, HM199, HM203, HM204, HM205, HM208, HM210, HM211, HM212, HM213, HM214, HM216, HM217, HM218, HM219, HM220, HM231, HM232, HM233, HM234, HM235, HM243, HM245 and HM246, are variants that are mutated at one position of sites other than the substitution sites in SEQ ID NO: 3 from PH20, and have an aggregation temperature of 48° C. to 56° C.
As a result, it can be seen that the variant having substitution at one position from SEQ ID NO: 3 has a higher aggregation temperature than that of wild-type PH20 (L36-Y482) of SEQ ID NO: 1, regardless of the substitution position. However, among them, HM174, HM208, HM210 and HM211 were found to have lower activity in the culture solution than 300 unit/mL, which is LOQ, but have higher activity after purification than 15 unit/μg, which is LOQ. In this case, it is considered that the characteristics of the variant itself cannot be accurately analyzed when the activity of the variant is measured only in the culture solution.
In addition, as shown in Table 7 above, among the variants having the amino acid sequence of SEQ ID NO: 3, HM146, HM147, HM149, HM262 and HM263 retain the same mutations as the variant having the amino acid sequence of SEQ ID NO: 3, that is, substitution of amino acid residues, but further include cleavage at the N-terminus and the C-terminus, which means that the expression and activity of proteins in the variants having the amino acid sequence of SEQ ID NO: 3 are not affected by further cleavage at the N-terminus and the C-terminus. These variants have an aggregation temperature of 49° C. to 53° C., which is not very different from that of the variant of SEQ ID NO: 3, which means that the physical properties of the variants were also unaffected by further cleavage at the N-terminus and the C-terminus.
In addition, in the variants having the amino acid sequence of SEQ ID NO: 3, a total of 13 types of variants, namely HM96, HM150, HM254, HM261, HM266, HM268, HM271, HM275, HM276, HM279, HM280, HM287 and HM288, which are variants including one or more amino acid substitutions and cleavages among those listed in Table 7 above, successfully expressed proteins, further retained enzyme activity, and had an aggregation temperature of 48° C. to 59° C. This means that even in the case of such multiple substitutions, the activity and physical properties of proteins were maintained. However, multiple substitutions exhibited unpredictable enzymatic activity and aggregation temperature which are unpredictable only with the combination of characteristics that are obtained in each single substitution constituting the same.
The amino acid sequences of Hyal2 (TTSTETCQYLKDYLTRL), Hyal3 (SSSEEECWHLHDYLVDT), and Hyal4 (TASKANCTKVKQFVSSD), which are the corresponding parts of hyaluronidases present in humans, other than Hyal1, in place of M345 to I361 sites in the amino acid sequence of the wild-type PH20 of SEQ ID NO: 1, were introduced into the M345 to I361 sites to investigate how the stability of proteins changes.
The variants constructed by substituting the M345 to I361 sites of mature wild-type PH20 (L36-S490) with corresponding sequences of Hyal2, Hyal3, and Hyal4, are referred to as “Hyal2-variant”, “Hyal3-variant” and “Hyal4-variant”, respectively.
The Hyal2-variant, Hyal3-variant and Hyal4-variant were constructed, and then the thermal stability of these variants was analyzed (see
In addition, in order to confirm whether or not these variants were expressed in ExpiCHO cell culture, the variants were purified by the same method using a HisTrap column, and the expression levels of proteins were compared by SDS-PAGE analysis. The result showed that the expression level of the Hyal3-variant was the highest, followed by the Hyal2-variant and Hyal4-variant in descending order.
SDS-PAGE analysis was performed to confirm the thermal stability of the variants according to the present invention. The purified wild-type PH20 of SEQ ID NO: 1 (L36-Y482) and the purified protein of SEQ ID NO: 3 (F38-F468) of the PH20 variants according to the present invention were stored at 42° C. for 7 days, followed by 10% SDS-PAGE analysis under reducing and non-reducing conditions (
As a result, wild-type PH20 (L36-Y482) was observed to aggregate (Lane G in
In order to analyze the enzyme kinetics of the variants according to the present invention, the enzyme activity was measured by the Morgan-Elson method (Takahashi, T. et al (2003) Anal. Biochem. 322:257-263). The Morgan-Elson method is a colorimetric method that assays red substances (at 545 nm) produced by the reaction of the reducing end of N-acetyl-D-glucosamine (GlcNAc) produced upon hydrolysis of hyaluronic acid by hyaluronidase with para-dimethylaminobenzaldehyde (DMAB), which is an Ehrlich's Reagent. N-acetyl-D-glucosamine (GlcNAc, Sigma) diluted to 0.25, 0.50, 0.75, 1.00 or 1.25 mM in dilution buffer solution (0.1 M NaPi, 0.1 M NaCl, 1.5 mM saccharic acid 1,4-lactone, pH 5.35) was reduced by treatment with tetraborate in each test tube, and then DMAB was added to induce colorimetric reaction. After the reaction, absorbance was measured at 545 nm to create a standard reaction curve for GlcNAc. Hyaluronic acid as a substrate was diluted to 0.54, 0.65, 0.87, 1.23 or 2.17 μM in a dilution buffer solution in each test tube, and hyaluronidase was added thereto, followed by reaction at 37° C. for 5 minutes and heating at 100° C. for 5 minutes to terminate the enzyme reaction. The resultant sample after enzyme reaction was reduced by treatment with tetraborate, and DMAB was added to induce colorimetric reaction. After the reaction, absorbance was measured at 545 nm, and enzyme activity was measured using the standard reaction curve of GlcNAc above. The enzyme kinetics of the wild-type PH20 of SEQ ID NO: 1 and the PH20 variant according to the present invention were analyzed using this method. As a result, the linearity of the Lineweaver-Burk curve was detected, which means that the PH20 variant according to the present invention follows the Michaelis-Menten enzyme kinetics equation.
Table 8 shows Vmax (maximum enzyme reaction rate), KM (50% substrate concentration), kcat (substrate conversion rate), and kcat/KM (enzyme catalyst efficiency) obtained as the result of analysis of enzyme kinetics regarding wild-type PH20 (L36-Y482) of SEQ ID NO: 1, variant PH20 (F38-F468) of SEQ ID NO: 3, HM261, and HM268. It can be seen that, as the value of KM decreases, the substrate-binding capacity of the enzyme increases, and as the value of kcat increases, the substrate conversion rate of the enzyme increases, so kcat/KM (enzyme catalyst efficiency) of each PH20 variant is higher than that of the wild-type PH20. In addition, the kcat of each of SEQ ID NO: 3, HM261 and HM268 is greater than that of the wild-type PH20 of SEQ ID NO: 1, and thus the substrate conversion rate of the enzyme is larger than that of the wild-type PH20 of SEQ ID NO: 1, so the industrial availability of each PH20 variant is greater than that of the wild-type PH20.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0009046 | Jan 2020 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/000943 | 1/25/2021 | WO |
