Pharmaceutical composition for subcutaneous injection comprising human hyaluronidase ph20 variant and drug

Abstract

This invention relates to a pharmaceutical composition comprising pembrolizumab and a PH20 variant. The PH20 variant comprises the amino acid sequence of any one of SEQ ID NO: 5 to SEQ ID NO: 50.

Description

[Technical Field]

[0001] This invention relates to a pharmaceutical composition comprising a human hyaluronidase PH20 variant having enhanced enzyme activity and thermal stability, and one or more drugs, and a method of using the composition to treat a disease.

[0002] The pharmaceutical composition according to the present invention is preferably used for subcutaneous injection. [Previous Technology]

[0003] Medications requiring high or multiple doses, especially antibody drugs, are generally administered intravenously. This type of injection takes approximately 90 minutes or longer and involves additional preparation procedures, making it inconvenient for patients, doctors, and healthcare personnel, and incurring additional costs. In contrast, subcutaneous injection offers the advantage of immediate administration, but its absorption rate is relatively lower than intravenous injection. Furthermore, when the injection volume is 3-5 ml or more, slow absorption can cause swelling and pain at the injection site. Therefore, subcutaneous injection of protein therapeutics is typically limited to small doses of 2 ml or less. However, when hyaluronidase is administered subcutaneously (or injected subcutaneously) along with the therapeutic drug, hyaluronic acid distributed in the extracellular matrix is ​​hydrolyzed by the action of hyaluronidase. This reduces viscosity and increases permeability in the subcutaneous area, allowing high or multiple doses of the drug to be easily delivered into the body.

[0004] There are six hyaluronidase genes in the human body: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20 / SPAM1. Hyal1 and Hyal2 are expressed in most tissues, while PH20 / SPAM1 (hereinafter referred to as PH20) is expressed in spermatocyte membranes and acrosomal membranes. HyalPS1 is a pseudogene and therefore not expressed. PH20 is an enzyme that cleaves the β-1,4 bond between N-acetylglucosamine and glucuronic acid (EC 3.2.1.35), which are the sugars that make up hyaluronic acid. The optimal pH for human hyaluronidase PH20 is 5.5, but it also exhibits some activity even at pH 7-8. Other human hyaluronidases, including Hyal1, have an optimal pH of 3-4 and exhibit relatively weak activity at pH 7-8. The pH value of the subcutaneous region in the human body is approximately 7.4, which is essentially neutral. Therefore, among various types of hyaluronidase, PH20 is widely used in clinical applications. Examples of clinical uses of PH20 include subcutaneous injection of antibody therapeutics, use as an eye relaxant and anesthetic additive in ophthalmic surgery, increasing the entry of anticancer therapeutics into tumor cells by hydrolyzing hyaluronic acid located in the extracellular matrix of tumor cells, and promoting the resorption of excess body fluids and blood in tissues.

[0005] Meanwhile, commercially available PH20 is currently in the form of extract from bovine or ovine testes. Examples include Amphadase® (bovine hyaluronidase) and Vitrase® (ovine hyaluronidase).

[0006] Bovine testicular hyaluronidase (BTH) is obtained by removing a signaling peptide and 56 amino acids from the carboxyl terminus of wild-type bovine PH20 during post-translational modification. BTH is also a glycoprotein, and based on its total amino acid composition, it contains 5% mannose and 2.2% glucosamine (Borders and Raftery, 1968). When animal-derived hyaluronidase is repeatedly administered to humans in high doses, neutralizing antibodies are produced, and other animal-derived biological materials besides PH20 may cause allergic reactions as impurities. In particular, the use of bovine PH20 is limited due to concerns about mad cow disease. To overcome these problems, research has been conducted on recombinant human PH20 protein.

[0007] Recombinant human PH20 protein has been reported to be expressed in yeast (P. pastoris), DS-2 insect cells, and animal cells (Chen et al., 2016, Hofinger et al., 2007). The N-glycosylation pattern of recombinant PH20 protein produced by insect cells and yeast differs from that of human PH20 during post-translational modification.

[0008] Among hyaluronidases, the protein structures of Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidase (PDB ID: 1FCQ, 1FCU, 1FCV) have been determined. Hyal1 consists of two domains: a catalytic domain and an EGF-like domain. The catalytic domain is in the form of (β / α)8, where the α-helix and β-strand are repeated eight times each (Chao et al., 2007), and the α-helix and β-strand represent the secondary structure of the protein. The EGF-like domain is completely preserved in various variants of Hyal1 where the C-terminus is cleaved differently. Hyal1 and PH20 share 35.1% amino acid sequence similarity. The tertiary structure of PH20 has not yet been discovered.

[0009] In studies on the structure / function relationship of human PH20, the carboxyl-terminal domain of PH20 has been found to be quite important for protein performance and enzyme activity. In particular, it has been reported that the C-terminus of amino acids 477-483 is quite important for enzyme performance and activity (Frost, 2007). The activity of full-length PH20 (amino acids 1-509) or PH20 variants truncated at position 467 is only 10% or less of the activity of PH20 variants truncated at one of the C-terminal positions 477 to 483 (Frost, 2007). Halozyme Therapeutics developed rHuPH20 (amino acids 36-482), which is a recombinant protein of mature PH20 cleaved at the Y482 C-terminus (Bookbinder et al., 2006; Frost, 2007).

[0010] Meanwhile, although research is underway to develop various therapeutic drugs using human PH20 via subcutaneous injection, the low stability of human PH20 itself remains unresolved.

[0011] In this technical context, the inventors of the present invention have demonstrated that human PH20 variants containing one or more amino acid residue substitutions have very high enzyme activity and thermal stability, and have therefore filed a patent application (PCT / KR 2019 / 009215), wherein one or more amino acid residues substitute the α-helix 8 domain (S347 to C381) and the linker domain (A333 to R346) between the α-helix 7 and the α-helix 8 in the amino acid sequence of wild-type hyaluronidase PH20, and some amino acids located at the amino terminus and / or carboxyl terminus of PH20 in human PH20 variants are cleaved. [Summary of the Invention]

[0012] The inventors of this invention have also confirmed that the PH20 variant according to this invention can be applied to pharmaceutical compositions or formulations containing drugs, such as antibody drugs, especially high-dose anti-HER2 antibodies or immune checkpoint antibodies. Therefore, pharmaceutical compositions and formulations containing the PH20 variant and drugs such as anti-HER2 antibodies or immune checkpoint antibodies according to this invention can be used for subcutaneous injection. The activity of the antibody drug and the PH20 variant is very stable and can be maintained for a long time, thus completing this invention.

[0013] The present invention was made in view of the aforementioned problems. One object of the present invention is to provide a novel pharmaceutical composition, particularly a pharmaceutical composition that can be used for subcutaneous injection, the pharmaceutical composition comprising a PH20 variant and a drug having enhanced enzyme activity and thermal stability, wherein the thermal stability and activity of the drug and the PH20 variant can be maintained for a long time.

[0014] Another object of the present invention is to provide a method for treating a disease, comprising administering a pharmaceutical composition according to the present invention to a subject in need of treatment.

[0015] According to the present invention, the above and other objectives can be achieved by a pharmaceutical composition comprising (a) a drug and (b) a PH20 variant.

[0016] The PH20 variant contained in the pharmaceutical composition according to the present invention comprises one or more amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T in wild-type human PH20 having the amino acid sequence of SEQ ID NO: 1, and may further comprise amino acid residue substitutions in one or more regions selected from the α-helix 8 domain (S347 to C381) and / or the linking domain between α-helix 7 and α-helix 8 (A333 to R346), wherein some amino acid residues located at the amino terminus and / or carboxyl terminus are selectively cleaved.

[0017] The pharmaceutical composition according to the present invention may further comprise one or more of pharmaceutically acceptable additives, especially buffers, stabilizers and surfactants.

[0018] The pharmaceutical composition according to the present invention can be used in the form of an injectable preparation for subcutaneous injection.

Implementation Method

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is that which is common and well-known in the art.

[0021] One embodiment of the present invention relates to a pharmaceutical composition comprising (a) a drug and (b) a PH20 variant. The pharmaceutical composition according to the present invention can be used for the prevention or treatment of diseases, and is preferably suitable for subcutaneous injection.

[0022] The PH20 variant contained in the pharmaceutical composition according to the present invention preferably has some amino acid residue substitutions in the amino acid sequence of wild-type PH20 (having the amino acid sequence of SEQ ID NO: 1), preferably in the amino acid sequence of mature wild-type PH20 (having the sequence consisting of L36 to S490 of the amino acid sequence of SEQ ID NO: 1), in the regions corresponding to the α-helix domain and / or its linking domain, preferably in the α-helix 8 domain (S347 to C381) and / or in the linking domain between α-helix 7 and α-helix 8 (A333 to R346), more preferably in the amino acid regions of T341 to N363, and most preferably in T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361 or M345 to N363.

[0023] In this invention, "mature wild-type PH20" refers to a protein containing amino acid residues L36 to S490 of SEQ ID NO: 1, which lacks M1 to T35, which form a message peptide in the amino acid sequence of wild-type PH20 having the sequence of SEQ ID NO: 1, and A491 to L509, which are not related to the substantial function of PH20.

[0024] Table 1, amino acid sequence of wild-type PH20 (SEQ ID NO: 1) MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKA KKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWN ESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLC QEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL

[0025] Specifically, the PH20 variant and fragment contained in the pharmaceutical composition according to the present invention contain one or more mutations in the wild-type PH20 having the sequence of SEQ ID NO: 1, preferably selected from the group consisting of amino acid residues of S343E, M345T, K349E, L353A, L354I, N356E and I361T, and most preferably selected from the group consisting of L354I and N356E.

[0026] In this invention, the term "PH20 variant" is intended to include mutations in some amino acid residues, preferably substitutions of amino acid residues in the wild-type human PH20 sequence, and deletions of some amino acid residues at the amino terminus and / or carboxyl terminus together with such substitutions, and is used in a substantially the same sense as "PH20 variant or fragment thereof".

[0027] The inventors of this invention have demonstrated, based on experimental results, that when the amino acid sequence portions of the α-helix 8 domain and the linker domain between the α-helix 7 and α-helix 8 of human PH20 are replaced by the amino acid sequence of the α-helix 8 domain and the linker domain between the α-helix 7 and α-helix 8 of the highly hydrophilic Hyal1, the enzyme activity and protein aggregation temperature (Tagg) increase at neutral pH. Through this prior research, novel PH20 variants and their fragments with enhanced enzyme activity and thermal stability compared to wild-type PH20 can be provided.

[0028] Therefore, the PH20 variant contained in the pharmaceutical composition according to the present invention preferably includes, in the amino acid sequence of the wild-type PH20 (having the amino acid sequence of SEQ ID NO: 1), or in the amino acid sequence of the mature wild-type PH20 (having the sequence consisting of L36 to S490 in the amino acid sequence of SEQ ID NO: 1), one or more amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T, preferably one or more amino acid residues selected from the group consisting of L354I and N356E.

[0029] Preferably, in the region corresponding to the α-helix domain and / or its linking domain, in the α-helix 8 domain (S347 to C381) and / or the linking domain between α-helix 7 and α-helix 8 (A333 to R346), more preferably in the amino acid region corresponding to T341 to N363, T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361 or M345 to N363, one or more amino acid residues are substituted.

[0030] Specifically, in the PH20 variant contained in the pharmaceutical composition according to the present invention, in wild-type PH20, preferably in mature wild-type PH20, the α-helix 8 domain (S347 to C381) and / or the linker domain (A333 to R346) between α-helix 7 and α-helix 8 may be replaced by some amino acid residues, which are some amino acid residues in the amino acid sequence of the corresponding region of Hyal1 having the sequence of SEQ ID NO: 51 (see Tables 2 and 3), but the present invention is not limited thereto.

[0031] Table 2, wild-type Hyal1 (SEQ ID NO: The amino acid sequence of 51) is MAAHLLPICALFLTLLDMAQGFRGPLLPNRPFTTVWNANTQWCLERHGVDVDVSVFDVVANPGQTFRGPDMTIFYSSQLGTYPYYTPTGEPVFGGLPQNASL IAHLARTFQDILAAIPAPDFSGLAVIDWEAWRPRWAFNWDTKDIYRQRSRALVQAQHPDWPAPQVEAVAQDQFQGAARAWMAGTLQLGRALRPRGLWGFYGFPDCYNYDFLS PNYTGQCPSGIRAQNDQLGWLWGQSRALYPSIYMPAVLEGTGKSQMYVQHRVAEAFRVAVAAGDPNLPVLPYVQIFYDTTNHFLPLDELEHSLGESAAQGAAGVVLWVSWE NTRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCSGHGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSLRGALSLEDQA​​QMAVEFKCRCYPGWQAPWCERKSMW

[0032] Table 3, Comparison of amino acid sequences between PH20 and Hyal1 α-helix amino acid sequence of PH20 The amino acid sequence of Hyal1 α-helix 1 P56 to D65 N39 to G48 α-helix 3 S119 to M135 S101 to I117 α-helix 4' K161 to N176 K144 to H159 α-helix 4 S180 to R211 P163 to R194 α-helix 5 F239 to S256 P222 to S239 α-helix 6 A274 to D293 K257 to G277 α-helix 7 S317 to G332 P299 to G314 α-helix 8 S347 to C381 T329 to C363

[0033] More specifically, the PH20 variant or fragment thereof included in the pharmaceutical composition according to the present invention preferably includes, in the amino acid sequence of wild-type PH20, and more preferably in the amino acid sequence of mature wild-type PH20, the substitution of amino acid residues L354I and / or N356E; and more preferably further includes the substitution of amino acid residues selected from one or more positions from T341 to N363, preferably selected from T341, L342, S343, I344, M345, S347, and M34. 8. One or more positions of the group consisting of K349, L352, L353, D355, E359, I361 and N363, but the invention is not limited thereto; and, more preferably, it includes substitution of one or more amino acid residues selected from the group consisting of T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T and N363G, but the invention is not limited thereto.

[0034] Preferably, the PH20 variant contained in the pharmaceutical composition according to the present invention may contain amino acid residue substitutions selected from M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; and may further contain one or more amino acid residue substitutions selected from the group consisting of T341S, L342W, S343E, I344N and N363G, but the present invention is not limited thereto.

[0035] More preferably, the PH20 variant or fragment thereof included in the pharmaceutical composition according to the present invention may include, but is not limited to, any substitution selected from the group consisting of: (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (d) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G; (e) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; and (f) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T.

[0036] In this invention, an amino acid residue is represented by a single letter code and a number, such as "S347", to indicate the position of the amino acid residue in the amino acid sequence of SEQ ID NO: 1.

[0037] For example, "S347" indicates that the amino acid residue at position 347 in the amino acid sequence of SEQ ID NO: 1 is serine. In addition, "S347T" indicates that the serine residue at position 347 in SEQ ID NO: 1 is replaced with threonine.

[0038] The PH20 variant contained in the pharmaceutical composition according to the present invention is interpreted as a variant in which an amino acid residue at a specific amino acid residue position is conservatively substituted.

[0039] As used herein, “conservative substitution” refers to the modification of the PH20 variant, which involves the substitution of one or more amino acids with similar biochemical properties, and does not result in the loss of the corresponding biological or biochemical function of the PH20 variant.

[0040] "Retained amino acid substitution" refers to an amino acid residue being replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined and are known in the art. 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 nonpolar side chains (e.g., glycine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0041] It is expected that the PH20 variant contained in the pharmaceutical composition according to the present invention will retain its activity despite having retained amino acid substitutions.

[0042] Furthermore, the PH20 variant or fragment contained in the pharmaceutical composition according to the present invention is interpreted as containing a PH20 variant or fragment that has substantially the same function and / or effect as the PH20 variant or fragment according to the present invention, and has at least 80% or 85% amino acid sequence homology with the PH20 variant or fragment according to the present invention, preferably at least 90%, more preferably at least 95%, and most preferably at least 99%.

[0043] The PH20 variant according to the present invention exhibits increased expression in animal cells and an increased protein folding rate, thereby possessing increased thermal stability compared to the mature wild-type PH20. Furthermore, despite the increased thermal stability, the enzyme activity of the PH20 variant still exceeds or is similar to that of the wild-type PH20.

[0044] It is known that when some amino acids, such as S490, at the carboxyl terminus of mature wild-type PH20 are additionally cleaved, enzyme activity decreases. However, the PH20 variant according to the present invention exhibits enhanced thermal stability and enhanced or similar enzyme activity compared to mature wild-type PH20, even when the carboxyl terminus of mature wild-type PH20 has an additionally cleaved sequence. Furthermore, the PH20 variant retains its enzyme activity even when up to five amino acid residues are cleaved from the amino terminus, indicating that residues from P41 onwards from the amino terminus play an important role in protein expression and enzyme activity.

[0045] Therefore, the PH20 variant contained in the pharmaceutical composition of the present invention contains some amino acid residue substitutions in the α-helix 8 domain (S347 to C381) and / or the linker domain (A333 to R346) between α-helix 7 and α-helix 8 of wild-type PH20, and further contains some amino acid residue deletions at the carboxyl terminus and / or amino terminus, but the present invention is not limited thereto.

[0046] In some embodiments, the PH20 variant included in the pharmaceutical composition according to the present invention may include the deletion of some amino acid residues at the amino terminus and / or the deletion of some amino acid residues at the carboxyl terminus, wherein the deletion of some amino acid residues at the amino terminus is derived from the amino acid residues selected from SEQ ID NO: The cleavage preceding the amino acid residues of the group consisting of M1 to P42 at the amino terminus of the amino acid sequence 1 is preferably preceding amino acid residues L36, N37, F38, R39, A40, P41, or P42. Some amino acid deletions at the carboxyl terminus originate from cleavage following the amino acid residues of the group consisting of V455 to W509 selected from the carboxyl terminus, preferably following the group consisting of V455 to S490, and most preferably following V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490.

[0047] The statement “removal before L36, N37, F38, R39, A40, P41, or P42 at the amino terminus” means that all amino acid residues from M1 to T35 immediately preceding L36, all amino acid residues from M1 to L36 immediately preceding N37, all amino acid residues from M1 to N37 immediately preceding F38, all amino acid residues from M1 to F38 immediately preceding R39, all amino acid residues from M1 to R39 immediately preceding A40, all amino acid residues from M1 to A40 immediately preceding P41, or all amino acid residues from M1 to P41 immediately preceding P42 are cleaved and removed. The statement “removal before M1 at the amino terminus of SEQ ID NO: 1” means that no removal occurs at the amino terminus.

[0048] Furthermore, the statement "removal after V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490 at the carboxyl terminus" respectively means cutting off and removing the SEQ ID NO: The amino acid residues following V455, C458, D461, C464, I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490 in the sequence 1. For example, a cleavage at S490 means a cleavage between S490 and A491.

[0049] Preferably, the human PH20 variant included in the pharmaceutical composition according to the present invention may have an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOS: 5 to 50, and more preferably has an amino acid sequence of SEQ ID NO: 44, but the present invention is not limited thereto. In the PH20 variant constructed in specific embodiments according to the present invention, the sequences of substituted or cleaved amino acids are shown in Table 4 below.

[0050] Table 4, amino acid sequences and substitution / removal characteristics of the PH20 variant according to the present invention. name Serial Number replace sequence HM1 5 Twelve amino acids were replaced with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T and N363G. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWE EWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPS IYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLGPYIINVTLAAKMC SQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM2 6 Seven amino acids were replaced with Y365F, I367L, L371S, A372G, K374L, M375L and V379A. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPFILNVTSGALLCSQALCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM3 7 19 amino acids were substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, N363G, Y365F, I367L, L371S, A372G, K374L, M375L and V379A. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM4 8 Seventeen amino acids were substituted with G340V, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWVSWENTRTKESCQAIKEYMDTTLGPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM6 9 11 amino acid residues were substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM7 10 Sixteen amino acids were substituted with G340V, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWVSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM8 11 Twelve amino acids were substituted with I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSNTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM9 12 13 amino acids were substituted as S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HMl0 13 14 amino acid residues were substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HMl1 14 13 amino acid residues were substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, Y365F and I367L. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPFILNVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM12 15 15 amino acid residues were substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, Y365F, I367L, L371S and A372G. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWE EWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPS IYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPFILNVTSGAKMC SQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM13 16 Eleven amino acid residues were substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were cleaved before the amino-terminal residue F38. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEW RPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSI YLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMC SQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM14 17 Eleven amino acids were replaced with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and the carboxyl group of I465 was cleaved. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLS WLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEY MDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCI HM15 18 Eleven amino acids were replaced with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and the carboxyl group of F468 was cleaved. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMA VIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSW LWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMD TTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF HM16 19 Eleven amino acids were replaced with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were cleaved after the carboxyl group at P471. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAV IDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLW NESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTL NPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKP HM17 20 Amino acids L36 to V47 were replaced with FRGPLLPNR, and 11 amino acids were replaced with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. FRGPLLPNRPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWR PTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIY LNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM18 twenty one Amino acids L36 to A52 were replaced with FRGPLLPNRPFTTV, and 11 amino acids were replaced with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. FRGPLLPNRPFTTVWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWR PTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIY LNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM19 twenty two Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue K470 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVI DWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLW NESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTT LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLK HM20 twenty three Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue F468 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAV IDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWL WNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDT TLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF HM21 twenty four Fifteen amino acid residues were replaced with T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWE EWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPS IYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMC SQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM24 25 Eleven amino acid residues were replaced with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were cleaved before the amino-terminal residue A40. APPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWR PTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIY LNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM25 26 Eleven amino acids were substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were cleaved before residue P42 at the amino terminus. PVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPT WARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYL NTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM29 27 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before the amino-terminal residue L36 and after the carboxyl-terminal residue A467. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMA VIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSW LWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMD TTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDA HM30 28 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and the residues before the amino terminus L36 and at the carboxyl terminus were also replaced with these residues. C464 was then removed. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDL SWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKE YMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVC HM31 29 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before the amino-terminal residue L36 and after the carboxyl-terminal residue D461. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLG MAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDD LSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAI KEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIAD HM32 30 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were cleaved before the amino-terminal residue L36 and after the carboxyl-terminal residue C458. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNL GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRN DDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQ AIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVC HM33 31 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before the amino-terminal residue L36 and after the carboxyl-terminal residue V455. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNL GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKES CQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAV HP34 32 Fifteen amino acid residues were replaced with T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were placed before residue F38 at the amino terminus and after residue K470 at the carboxyl terminus. Perform excision. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVI DWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLW NESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTT LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLK HM35 33 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue P472 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVI DWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWN ESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLN PYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPP HM36 34 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue M473 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVID WEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNE STALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNP YIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPM HM37 35 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue E474 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVID WEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNE STALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNP YIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPME HM38 36 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue T475 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVID WEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNES TALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPY IINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMET HM39 37 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue E476 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVID WEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNES TALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYI INVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETE HM40 38 Eleven amino acid residues were replaced with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before the amino-terminal residue N37. NFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEE WRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSI YLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMC SQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM41 39 Eleven amino acid residues were substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were cleaved before the amino-terminal residue R39. RAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWR PTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIY LNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM42 40 Eleven amino acid residues were replaced with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue P41 at the amino terminus. PPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRP TWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYL NTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM43 41 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue I465 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAV IDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSW LWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYM DTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCI HM44 42 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue D466 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAV IDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSW LWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYM DTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCID HM45 43 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue A467 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAV IDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWL WNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMD TTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDA HP46 44 Fifteen amino acid residues were replaced with T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue F468 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAV IDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWL WNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDT TLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF HM47 45 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue P478 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDW EEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNEST ALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYII NVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEP HM48 46 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue I480 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDW EEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTA LYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINV TLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQI HM49 47 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue Y482 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWE EWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTAL YPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVT LAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY HM50 48 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue A484 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWE EWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALY PSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLA AKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNA HM51 49 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue P486 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEE WRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYP SIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAA KMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASP HM52 50 Fourteen amino acid residues were replaced with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and were removed before residue F38 at the amino terminus and after residue T488 at the carboxyl terminus. FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEE WRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPS IYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKM CSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPST

[0051] Meanwhile, previous studies have reported that the enzymatic activity of wild-type PH20 varies depending on the cleavage position of the amino acid residue at the carboxyl terminus. However, in this invention, the specific α-helix that forms the secondary structure of PH20 is replaced by the α-helix of human hyaluronidase, thereby constructing a PH20 variant with higher stability than wild-type PH20. In these variants, the interaction between the replaced α-helix domain and other secondary structures of PH20 exhibits a pattern different from that of wild-type PH20, resulting in a variant with some degree or higher enzymatic activity regardless of the cleavage position at the carboxyl terminus.

[0052] Furthermore, in this invention, an attempt is made to increase the expression of the recombinant PH20 protein by replacing the original human PH20 signaling peptide with signaling peptides of other proteins that exhibit high protein expression in animal cells.

[0053] Therefore, in another embodiment, the PH20 variant contained in the pharmaceutical composition according to the present invention may contain a signaling peptide derived from human hyaluronidase-1 (Hyal1), human growth hormone or human serum albumin at its amino terminus, replacing the signaling peptide of wild-type PH20 composed of M1 to T35, and preferably may contain a signaling peptide derived from human growth hormone having the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA according to SEQ ID NO: 2, a signaling peptide derived from human serum albumin having the amino acid sequence MKWVTFISLLFLFSSAYS according to SEQ ID NO: 3, or a signaling peptide derived from human Hyal1 having the amino acid sequence MAAHLLPICALFLTLLDMAQG according to SEQ ID NO: 4, as shown in Table 5, but the present invention is not limited thereto.

[0054] Table 5, amino acid sequences of the signaling peptides of human growth hormone, human serum albumin or human Hyal1. Sources of messaging peptides amino acid sequence SEQ ID NO: Human growth hormone MATGSRTSLLLAFGLLCLPWLQEGSA 2 Human serum albumin MKWVTFISLLFLFSSAYS 3 Human Hyal1 MAAHLLPICALFLTLLDMAQG 4

[0055] Among the PH20 variants included in the pharmaceutical compositions according to the present invention, the variant having a histidine tag (6xHis-tag) attached to the carboxyl terminus is named HM, and the variant without the histidine tag is named HP. Furthermore, the mature wild-type PH20 (L36-S490) having a histidine tag attached to the carboxyl terminus is named WT, and the mature wild-type PH20 (L36 to Y482) without the histidine tag and whose carboxyl terminus is cleaved after Y482 is named HW2.

[0056] HP46 (SEQ ID NO: 44) is a variant of PH20 obtained by modeling the protein structure using Hyal1 (PDB ID: 2PE4), a human hyaluronidase with a known protein tertiary structure (Chao et al., 2007), followed by replacing the amino acid sequences of α-helix 8 and the linker domain between α-helix 7 and α-helix 8 with the amino acid sequence of Hyal1, and cleaving the amino terminus at F38 and the carboxyl terminus after F468. In particular, α-helix 8 is located outside the tertiary structure of PH20 and has less interaction with adjacent α-helices or β-strands compared to other α-helices. Generally, there is a trade-off relationship between enzyme activity and thermostability; therefore, the higher the thermostability of a protein, the lower its enzyme activity, and when enzyme activity increases due to improved protein structural flexibility, thermostability tends to decrease. However, the specific activity of HP46 measured by turbidity assay at pH 7 was approximately 46 units / µg, which was evaluated to be approximately twice the specific activity of wild-type PH20 (approximately 23 units / µg).

[0057] The thermal stability of proteins can be assessed based on the melting temperature (Tm) at which 50% of the protein's tertiary structure denatures and the aggregation temperature (Tagg) at which proteins aggregate. Generally, the aggregation temperature of proteins tends to be lower than their melting point. Compared to the α-helix 8 of PH20, the α-helix 8 of Hyal1 exhibits higher hydrophilicity. The substituted α-helix 8 of Hyal1 increases the surface hydrophilicity of HP46 proteins, thereby delaying the aggregation effect between proteins caused by hydrophobic interactions. Therefore, the aggregation temperature is 51°C, which is an increase of 4.5°C compared to the aggregation temperature of wild-type PH20 (46.5°C).

[0058] HP46 is a variant in which amino acid residues in α-helix 8 and the linker domain between α-helix 7 and α-helix 8 are substituted, with T341 being replaced by serine. When amino acid residue 341 is threonine, the enzyme activity is similar to that of wild-type PH20, but when substituted with serine, the enzyme activity increases by approximately 2-fold, and it has been confirmed that even in substrate gel assays, the resulting variant hydrolyzes 5 to 6 times more hyaluronic acid compared to wild-type PH20. Substrate gel assays involve protein denaturation and refolding processes, and represent an enhanced refolding and recovery of the protein tertiary structure of HP46 compared to wild-type PH20.

[0059] In the pharmaceutical composition according to the present invention, the amount of the PH20 variant is at least 50 units / mL, preferably in the range of 100 units / mL to 20,000 units / mL, more preferably in the range of about 150 units / mL to about 18,000 units / mL, even more preferably in the range of 1,000 units / mL to 16,000 units / mL, and most preferably in the range of 1,500 units / mL to 12,000 units / mL.

[0060] Examples of drugs included in the pharmaceutical compositions of the present invention include, but are not limited to, protein drugs, antibody drugs, small molecules, aptamers, RNAi, antisense RNA and cell therapy agents, such as chimeric antigen receptor T-cells (CAR-T) or chimeric antigen receptor natural killer cells (CAR-NK), which may use not only currently commercially available drugs, but also drugs in clinical trials or under development.

[0061] As a drug, protein drugs or antibody drugs are preferred.

[0062] The "protein drug" contained in the pharmaceutical composition of the present invention is a drug composed of amino acids, and thus exhibits the efficacy of treating or preventing diseases through the activity of proteins. It is a drug composed of proteins different from antibody drugs, and may be selected from the group consisting of free cytokines, therapeutic enzymes, hormones, soluble receptors and their fusion proteins, insulin or analogues thereof, bone morphogenetic protein (BMP), erythropoietin and serum-derived protein, but the present invention is not limited thereto.

[0063] The interleukins included in the pharmaceutical composition of the present invention may be selected from the group consisting of interferon, interleukin, colony-stimulating factor (CSF), tumor necrosis factor (TNF) and tissue growth factor (TGF), but the present invention is not limited thereto.

[0064] Therapeutic enzymes may include, but are not limited to, β-glucocerebrosidase and agalsidase β.

[0065] The extracellular domain of the soluble receptor contained in the pharmaceutical composition according to the present invention is fused to the protein of the soluble receptor via the Fc region of a fusion protein antibody or similar. The soluble receptor is a soluble form of a receptor bound to a disease-associated ligand, examples of which include forms with the Fc region fused to the TNF-α soluble receptor (e.g., products containing etanercept and similar forms), forms with the Fc region fused to the vascular endothelial growth factor (VEGF) soluble receptor (e.g., products containing alefacept and similar forms), forms with the Fc region fused to CTLA-4 (e.g., products containing abatacept or belatacept and similar forms), forms with the Fc region fused to the interleukin-1 soluble receptor (e.g., products containing rilonacept and similar forms), and forms with the Fc region fused to the LFA3 soluble receptor (e.g., products containing alefacept and similar forms), but the invention is not limited thereto.

[0066] The hormone contained in the pharmaceutical composition according to the present invention refers to a hormone or analogue thereof injected into the human body for the treatment or prevention of diseases caused by hormone deficiency, etc. Examples include, but are not limited to, human growth hormone, estrogen and progesterone.

[0067] The serum-derived proteins contained in the pharmaceutical compositions of the present invention are proteins present in plasma, and include proteins extracted from plasma and recombinant proteins generated therefrom. Examples may include, but are not limited to, fibrinogen, von Willebrand factor, albumin, thrombin, factor II (FII), factor V (FV), factor VII (FVII), factor IX (FIX), factor X (FX), and factor (FXI).

[0068] The antibody drug contained in the pharmaceutical composition according to the present invention may be a monoclonal antibody drug or a polyclonal antibody drug.

[0069] The monoclonal antibody drug according to the present invention comprises a protein containing a monoclonal antibody and a fragment of a monoclonal antibody, wherein the monoclonal antibody and the fragment of a monoclonal antibody can specifically bind to an antigen associated with a specific disease. The monoclonal antibody also includes a bispecific antibody, and the protein containing the monoclonal antibody or a fragment thereof conceptually includes an antibody-drug conjugate (ADC).

[0070] Examples of antigens associated with specific diseases include 4-1BB, integrin, amyloid beta, angiopoietin (angiopoietin 1 or 2), angiopoietin analogue 3, B-cell-activating factor (BAFF), B7-H3, complement 5, CCR4, CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD62, CD79b, CD80, CGRP, claudin-18, complement factor D, CTLA4, DLL3, EGF receptor, hemophilia factor, Fc receptor, FGF23, and folate receptor. Interleukin receptors, GD2, GM-CSF, HER2, HER3, interferon receptors, interferon-γ, IgE, IGF-1 receptors, interleukin 1, interleukin 2 receptors, interleukin 4 receptors, interleukin 5, interleukin 5 receptors, interleukin 6, interleukin 6 receptors, interleukin 7, interleukin 12 / 23, interleukin 13, interleukin 17A, interleukin 17 receptor A, interleukin 31 receptors, interleukin 36 receptors, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, RANK-L, SLAMF7, tissue factor, TNF, VEGF, VEGF receptors, and von Willebrand factor (vWF), but the invention is not limited thereto.

[0071] The following are, but not limited to, proteins containing monoclonal antibodies or monoclonal antibody fragments targeting specific diseases: utomilumab as an anti-4-1BB antibody; natalizumab, etrolizumab, vedolizumab, and bimagrumab as antibodies against integrins; bapineuzumab, crenezumab, solanezumab, aducanumab, and gantenerumab as antibodies against amyloid β; antibodies against angiotrophin, such as AMG780 and MEDI 3617 against angiotrophin 1 and 2, and nesvacumab against angiotrophin 2, and vanucizumab as a bispecific antibody against angiotrophin 2 and VEGF; Evinacumab as an antibody against angiotrophin analogue 3; tabalumab, lanalumab, and belimumab as antibodies against cellular activating factor (BAFF); omburtamab as an antibody against B7-H3. Ravulizumab and eculizumab are antibodies against complement 5; mogamulizumab is an antibody against CCR4; otelixizumab, teplizumab, and muromonab are antibodies against CD3; tebentafusp is a bispecific antibody against GP100 and CD3; blinatumomab is a bispecific antibody against CD19 and CD3; and REGN1979 is a bispecific antibody against CD20 and CD3; ibalizumab and zanolimumab are antibodies against CD4; itolizumab is an antibody against CD6; efalizumab is an antibody against CD11a; inebilizumab, tafasitamab, and loncastuximab; tesirine (an antibody-drug complex) is an antibody against CD19; ocrelizumab, ublituximab, obinutuzumab, ofatumumab, rituximab, tositumomab, and ibritumomab Tiuxetan (an antibody-drug complex) is an antibody targeting CD20; epratuzumab, inotuzumab ozogamicin (an antibody-drug complex), and moxetumomab pasudotox are antibodies targeting CD22; brentuximab vedotin is an antibody-drug complex targeting CD30.Vadastuximab, talirine, and gemtuzumab ozogamicin are antibody-drug complexes targeting CD33; daratumumab and isatuximab are antibody-drug complexes targeting CD38; alemtuzumab is an antibody targeting CD52; crizanlizumab is an antibody targeting CD62; polaruzumab vedotin is an antibody-drug complex targeting CD79b; galiximab is an antibody targeting CD80; eptinezumab, fremanezumab, galcanezumab, and erenumab are antibodies targeting CGRP; zolbetuximab is an antibody targeting necrin-18; lampalizumab is an antibody targeting complement factor D; tremelimumab, zalifrelimab, and ipilimumab are antibodies targeting CTLA4; and rovalpituzumab tesirine is an antibody-drug complex targeting DLL3. cetuximab, depatuxizumab, zalutumumab, necitumumab, and panitumumab are antibodies targeting the EGF receptor; emicizumab is a bispecific antibody targeting coagulation factor IX and factor X (which is hemophilia factor); nipocalimab and rozanolixizumab are antibodies targeting the Fc receptor; burosumab is an antibody targeting FGF23; farletuzumab is an antibody targeting the folate receptor, and mirvetuximab soravtansine is an antibody-drug complex targeting the folate receptor; dinutuximab and naxitamab are antibodies targeting GD2; otilimab is an antibody targeting GM-CSF; margetuximab, pertuzumab, and trastuzumab are antibodies targeting HER2, and trastuzumab deruxtecan, trastuzumab emtansine, and trastuzumab duocarmazine are antibody-drug complexes targeting HER2. Patritumab is an antibody targeting HER3; anifrolumab is an antibody targeting the interferon receptor; emapalumab is an antibody targeting interferon-gamma; ligelizumab and omalizumab are antibodies targeting IgE.Dalotuzumab, figitumumab, and teprotumumab are antibodies against the IGF-1 receptor; gebokizumab and canakinumab are antibodies against interleukin-1; daclizumab and basiliximab are antibodies against the interleukin-2 receptor; dupilumab is an antibody against the interleukin-4 receptor; mepolizumab and reslizumab are antibodies against interleukin-5; benralizumab is an antibody against the interleukin-5 receptor; clazakizumab, olokizumab, sirukumab, and siltuximab are antibodies against interleukin-6; sarilumab, satralizumab, tocilizumab, and REGN88 are antibodies against the interleukin-6 receptor; secukinumab is an antibody against interleukin-7; ustekinumab and briakinumab are antibodies against interleukin-12 / 23. Lebrikizumab and tralokinumab are antibodies against interleukin-13; ixekizumab and bimekizumab are antibodies against interleukin-17A; brodalumab is an antibody against interleukin-17 receptor A; brazikumab, guselkumab, risankizumab, tildrakizumab, and mirikizumab are antibodies against interleukin-23; nemolizumab is an antibody against interleukin-31 receptor; spesolimab is an antibody against interleukin-36 receptor; relatlimab is an antibody against LAG3; narsoplimab is an antibody against NSP2; fasinumab and tanezumab are antibodies against NGF; alirocumab, evolocumab, and bococizumab are antibodies against PVSK9. Lambrolizumab, balstilimab, camrelizumab, cemiplimab, dostarlimab, prolgolimab, shintilimab, spartalizumab, tislelizumab, pembrolizumab, and nivolumab are antibodies against PD-1; atezolizumab, avelumab, envafolimab, and durvalumab are antibodies against PD-L1; and bintrafusp alpha is a bispecific antibody against TGF beta and PD-L1.Denosumab is an antibody against RANK-L; elotuzumab is an antibody against SLAMF7; concizumab and marstacimab are antibodies against tissue factor; antibodies against TNF, especially TNFα, include infliximab, adalimumab, golimumab, the antibody fragment certolizumab pegol, and ozoralizumab (which is a bispecific antibody against TNF and albumin); antibodies against VEGF include brolucizumab, ranibizumab, bevacizumab, and faricimab (which is a bispecific antibody against VEGF and Ang2); ramucirumab is an antibody against the VEGF receptor; and caplacizumab is an antibody against vWF.

[0072] Simultaneously, overexpression of human epidermal growth factor receptor 2 (HER2) has been observed in approximately 20-25% of breast cancer patients. HER2-overexpressing breast cancer progresses rapidly, is aggressive, and exhibits a lower response to chemotherapy compared to HER2-low-expressing breast cancer, thus resulting in a poor prognosis. Trastuzumab is a monoclonal antibody targeting HER2. It specifically binds to HER2 on the surface of HER2-overexpressing cancer cells, inhibiting cell replication and proliferation signaling, thereby slowing tumor progression. Trastuzumab was approved by the United States Food and Drug Administration (FDA) for the treatment of breast cancer in the United States in 1998 and by the Korean Food and Drug Administration (KFDA) in 2003. Since then, the efficacy of trastuzumab has also been recognized in HER2-overexpressing gastric cancer, and it has been used as a therapeutic agent for gastric cancer.

[0073] Roche's Herceptin subcutaneous injection formulation (trade name: Herceptin) consists of 440 mg of trastuzumab as the main ingredient. The freeze-dried trastuzumab is mixed with normal saline and injected intravenously. On the other hand, the trastuzumab subcutaneous injection formulation (trade name: Herceptin SC) is a 5 ml liquid formulation containing 600 mg (120 mg / ml) of trastuzumab as the main ingredient, and contains 20 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine, 0.04% polysorbate 20 and 10,000 units of rHuPH20 (2,000 units / ml, 0.004%, 40 μg / ml) as additives.

[0074] The shelf life of the subcutaneous injection formulation of trastuzumab is 21 months. The intravenous injection formulation of trastuzumab is in a freeze-dried form and has a shelf life of 30 months, but the subcutaneous injection formulation of trastuzumab is in liquid form and has a shorter shelf life of 21 months. Therefore, it can be estimated that the stability of one or more trastuzumab formulations and recombinant human hyaluronidase PH20 in liquid formulations is limited.

[0075] In this document, in the present invention, from the viewpoint of the characteristics of the PH20 variant according to the present invention, compared with wild-type human hyaluronidase PH20 and recombinant human PH20 available from Halozyme, the PH20 variant not only has increased enzyme activity, but also has a high protein aggregation temperature, thus exhibiting improved thermal stability, and the shelf life of the subcutaneous injection formulation is set to be long-term, preferably 21 months or longer.

[0076] In the pharmaceutical composition according to the present invention, the content of the antibody drug may be in the range of 5 mg / mL to 500 mg / mL, more preferably 20 mg / mL to 200 mg / mL, more preferably 100 mg / mL to 150 mg / mL, most preferably 120 ± 18 mg / mL, for example, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL.

[0077] The multi-antibody contained in the pharmaceutical composition according to the present invention is preferably a serum antibody extracted from serum, such as an immunoglobulin, but is not limited thereto.

[0078] In the case of small molecule compounds, any drug requiring rapid effect for treatment or prevention can be used without restriction. For example, morphine-based painkillers can be used (Thomas et al., 2009). Furthermore, when used as a treatment for tissue necrosis caused by anticancer drugs, small molecule compounds can be used alone or in combination with antidotes (e.g., Vinca alkaloids and Taxanes) (Kreidieh et al., 2016).

[0079] The pharmaceutical composition according to the present invention may further comprise one or more of the group consisting of buffers, stabilizers and surfactants.

[0080] The buffer solution contained in the pharmaceutical composition of the present invention can be used without limitation, as long as it can achieve a pH of 4 to 8, preferably 5 to 7. The buffer solution is preferably selected from malate, formate, citrate, acetate, propionate, pyridine, piperazine, dimethyl arsenate, succinate, 2-(N-hydroxylinyl)ethanesulfonic acid (MES), histidine, tris(hydroxymethyl)aminomethane (Tris), bis(2-hydroxyethyl)aminotris(hydroxymethyl)aminomethane (bis-Tris), phosphate, ethanolamine, carbonate, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), imidazole, 1,3-bis(tris(hydroxymethyl)methylamino)propane (BIS-TRIS). The group consisting of one or more of propane, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-hydroxylinyl)propanesulfonic acid (MOPS), hydroxyethylpiperazine ethanesulfonic acid (HEPES), pyrophosphate and triethanolamine, preferably a histidine buffer, such as L-histidine / HCl, but not limited thereto.

[0081] The concentration of the buffer solution may be in the range of 0.001 mM to 200 mM, preferably 1 mM to 50 mM, more preferably 5 mM to 40 mM, and most preferably 10 mM to 30 mM.

[0082] In the pharmaceutical composition according to the invention, stabilizers may be used without limitation, as long as they are generally used in the art to stabilize proteins. Preferably, stabilizers may be, for example, one or more of the group consisting of carbohydrates, sugars or their hydrates, sugar alcohols or their hydrates, and amino acids.

[0083] The carbohydrate, sugar or sugar alcohol used as a stabilizer may be selected from one or more of the group consisting of trehalose or its hydrate, sucrose, saccharin, glycerol, butyltetraol, threitol, xylitol, arabinitol, ribitol, mannitol, sorbitol, galactitol, trehalitol, idoteol, inositol, heptamethasone, isomaltitol, maltitol, polyol, cyclodextrin, hydroxypropyl cyclodextrin and glucose, but is not limited thereto.

[0084] The amino acid may be one or more of the group consisting of glutamic acid, glutamic acid, glycine, lysine, lysilysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, aspartic acid, aspartic acid, ursine, isoleucine, taurine, theanine, threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolidine, histidine, and alanine, but is not limited thereto.

[0085] In the pharmaceutical composition according to the present invention, the concentration of sugar or sugar alcohol used as a stabilizer may be in the range of 0.001 mM to 500 mM, preferably 100 mM to 300 mM, more preferably 150 mM to 250 mM, most preferably 180 mM to 230 mM, and specifically, may be about 210 mM.

[0086] Furthermore, in the pharmaceutical composition according to the present invention, the amino acid used as a stabilizer may be in the range of 1 mM to 100 mM, preferably 3 mM to 30 mM, more preferably 5 mM to 25 mM, most preferably 7 mM to 20 mM, and specifically, may be in the range of 8 mM to 15 mM.

[0087] The pharmaceutical composition according to the present invention may further contain surfactants.

[0088] Preferably, the surfactant may be a nonionic surfactant, such as polyoxyethylene-sorbitan fatty acid ester (polysorbate or Tween), polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether (e.g., polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether [Triton-X] and polyoxyethylene-polyoxypropylene copolymer [Poloxamer and Pluronic]) and sodium dodecyl sulfonate (SDS), but is not limited thereto.

[0089] More preferably, polysorbate can be used. The polysorbate may be polysorbate 20 or polysorbate 80, but is not limited thereto.

[0090] In the pharmaceutical composition according to the present invention, the concentration of the nonionic surfactant may be in the range of 0.0000001% (w / v) (weight / volume) to 0.5% (w / v), more preferably 0.000001% (w / v) to 0.4% (w / v), more preferably 0.00001% (w / v) to 0.3% (w / v), and most preferably 0.001% (w / v) to 0.2% (w / v).

[0091] In one embodiment, the pharmaceutical composition according to the present invention may comprise 50-350 mg / mL of antibody (e.g., anti-HER2 antibody or immune checkpoint antibody), histidine buffer providing pH 5.5 ± 2.0, 10-400 mM α,α-trehalose, 1-50 mM methionine, and 0.0000001% (w / v) to 0.5% (w / v) of polysorbate.

[0092] In a more specific embodiment, the pharmaceutical composition according to the present invention may comprise 120 mg / mL of anti-HER2 antibody or immune checkpoint antibody, 20 mM amino acid buffer at pH 5.5 ± 2.0, 210 mM α,α-trehalose, 10 mM methionine and 2,000 units / mL of PH20 variant, and may further comprise 0.005% (w / v) to 0.1% (w / v) of polysorbate.

[0093] The pharmaceutical composition according to the present invention can be administered by means of: intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, local administration, intrapulmonary administration, rectal administration and the like. Subcutaneous administration is preferably carried out by subcutaneous injection, and the pharmaceutical composition is more preferably used as an injectable preparation for subcutaneous injection.

[0094] Therefore, another embodiment of the present invention provides a formulation comprising the pharmaceutical composition according to the present invention, preferably for subcutaneous injection.

[0095] Injectable formulations for subcutaneous injection may be provided in a ready-to-inject form that does not require an additional dilution process and may be supplied in prefilled syringes, glass ampoules or plastic containers.

[0096] The present invention also relates to a method of treating a disease, which uses a pharmaceutical composition or preparation according to the present invention.

[0097] The diseases that can be treated with the pharmaceutical composition or preparation according to the present invention are not particularly limited, as long as they are diseases that can be treated with a drug that can be combined with the PH20 variant according to the present invention.

[0098] Diseases that can be treated with the pharmaceutical composition or preparation according to the present invention may be cancer or autoimmune diseases, but are not limited thereto.

[0099] The cancers or malignant tumors that can be treated using the pharmaceutical compositions or preparations according to the present invention are not particularly limited, and include both solid cancers and blood cancers. Examples of these cancers include, but are not limited to, skin cancer (e.g., 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, cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma. Preferably, the cancers that can be treated with the pharmaceutical composition or preparation according to the present invention may be selected from the group consisting of gastric cancer, colorectal cancer, breast cancer, lung cancer and kidney cancer, but are not limited thereto.

[0100] Autoimmune diseases that can be treated with the pharmaceutical compositions or preparations according to the present invention include, but are not limited to, rheumatoid arthritis, asthma, psoriasis, multiple sclerosis, allergic rhinitis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, type 1 diabetes, inflammatory bowel disease (IBD), and atopic dermatitis.

[0101] The present invention also provides a method for treating a disease, comprising administering a pharmaceutical composition or preparation according to the present invention to a subject in need of treatment, and the present invention further provides the use of a pharmaceutical composition or preparation according to the present invention for treating a disease.

[0102] Unless otherwise defined, the technical and scientific terms used in this invention have the meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, repeated descriptions of the same technical configurations and operations as those in the prior art will be omitted.

[0103] The invention will be further described in detail below with reference to examples. These examples are provided for illustrative purposes only and will be apparent to those skilled in the art that they should not be construed as limiting the scope of the invention.

[0104] 〔Example〕

[0105] 〔Example 1, Formulation Development〕

[0106] As shown in Table 6, four subcutaneous trastuzumab formulations were prepared. Formulations 1 to 4 typically contain 120 mg / mL of trastuzumab and are composed of 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and a pH20 variant. The difference between formulations 1 to 4 lies in the concentration of the nonionic surfactant, where formulation 1: 0% polysorbate 20, formulation 2: 0.005% polysorbate 20, formulation 3: 0.04% polysorbate 20, and formulation 4: 0.1% polysorbate 20.

[0107] Table 6, Composition of the Formulation Formulation 1 Formulation 2 Formulation 3 Formulation 4 Antibody Trastuzumab (120 mg / mL) buffer solution 20 mM histidine / histidine-HCl Stabilizer 1 210 mM Trehalose Stabilizer 2 10 mM methionine Polysorbate 20 0% 0.005% 0.04% 0.1% Hyaluronidase HP46 (2,000 units / mL) of SEQ ID NO: 44

[0108] 〔Example 2, Measurement using a spectrophotometer〕

[0109] Formulations 1 through 4 were placed at 45°C for 14 days, and changes in protein concentration were analyzed using a Beckman spectrophotometer. Each sample was diluted with distilled water to a concentration of 0.4 mg / mL, and the absorbance of the protein at 280 nm was then measured using a spectrophotometer. In the stability test under harsh conditions (i.e., 14 days at 45°C), there were no significant changes in protein concentration for formulations 1 through 4. However, hyaluronidase activity decreased sharply at 45°C; therefore, enzyme activity was not measured in this example (see Figure 6).

[0110] [Example 3, using particle size sieving chromatography to study the monomer ratio of Trastuzumab in various formulations]

[0111] For size-exclusion chromatography analysis, an HPLC system provided by Shimadzu Prominence and a TSK-gel G3000SWXL (7.8 x 300 mm, 5 μm) and a TSK guard column (6.0 x 4.0 mm, 7 μm) were used. A 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used as the mobile phase. Analysis was performed for 35 minutes at a flow rate of 0.5 mL / min using isocranial separation mode. The sample was diluted with the analytical solvent to a final concentration of 10 mg / mL. After injecting 20 μL of sample into the HPLC column, the absorbance of the precipitate at 280 nm was recorded. The monomer proportion of Trastuzumab in the HPLC chromatogram was calculated and plotted.

[0112] When particle size distribution chromatography was performed in the stability test under harsh conditions (i.e., 14 days at 45°C), formulations 1 to 4 exhibited similar patterns of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (approximately 1.5%), and these differences were not significant across formulations. In conclusion, the results of particle size distribution chromatography analysis in the stability test under harsh conditions (i.e., at 45°C) showed no significant difference in stability among the formulations based on the concentration of polysorbate 20 (0–0.1% (w / v)) (see Figures 1A–1B).

[0113] 〔Example 4, Measuring the protein aggregation temperature of a formulation containing Trastuzumab and HP46〕

[0114] Dynamic light scattering (DLS) was used to analyze the denaturation properties of proteins caused by heat. In this experiment, the size changes of protein molecules according to temperature changes were measured and used to calculate the protein aggregation temperature. For the DLS analysis, a Zetasizer-nano-ZS instrument and a quartz cuvette (ZEN2112) provided by Malvern were used. During the analysis, the temperature was increased from 25°C to 85°C at 1°C intervals. The sample was diluted to 1 mg / mL using various formulation buffers, and then 150 µL of sample was added to the cuvette for analysis.

[0115] The aggregation temperature of formulation 1, which does not contain polysorbate 20, is 74°C, and the aggregation temperature of formulations 2 to 4 is 76°C (see Figures 2A and 2B).

[0116] 〔Example 5, Weak cation exchange chromatography determination of a formulation containing Trastuzumab and HP46〕

[0117] For weak cation exchange chromatography (WCX) analysis, an HPLC system provided by Shimadzu Prominence was used, with TSKgel CM-STAT columns (4.6 x 100 mm, 7 μm), TSKgel guard gel CMSTAT (3.2 mm x 1.5 cm inner diameter), and similar columns as the columns. Mobile phase A was 10 mM sodium phosphate (pH 7.5), and mobile phase B was 10 mM sodium phosphate containing 0.1 M sodium chloride (pH 7.2). Analysis was performed for 55 minutes at a linear concentration gradient of 0-30% mobile phase B at a flow rate of 0.8 mL / min. The sample was diluted with mobile phase A to a final concentration of 1.0 mg / mL, and 80 μL of the sample was injected into the HPLC. The absorbance of the column precipitate at 280 nm was then recorded. The monomeric proportion of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0118] When weak cation exchange chromatography analysis was performed in stability tests under harsh conditions (i.e., 14 days at 45°C), formulations 1 through 4 exhibited similar patterns of change. Specific changes included an increase in the relative content of acidic variants (approximately 30% change over 14 days), a decrease in the relative content of the main peak (approximately 44% change over 14 days), and an increase in the relative content of basic variants (approximately 15% change over 14 days), with no significant differences between formulations. In conclusion, the protein stability of polysorbate 20 (0–0.1%) was similar in weak cation exchange chromatography analysis under harsh conditions (i.e., 45°C) (see Figures 3A–3D).

[0119] 〔Example 6, Formulation Development〕

[0120] As shown in Table 7, three subcutaneous trastuzumab formulations were prepared. Formulations 5 to 7 typically contain 120 mg / mL of trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and HP46. The difference between formulations 5 to 7 lies in the composition of stabilizer 3, where formulation 5 contains 0.04% polysorbate 20, formulation 6 contains 50 mM Lys-Lys, and formulation 7 contains glycine.

[0121] Table 7, Composition of the Formulation Formulation 5 Formulation 6 Formulation 7 Antibody Trastuzumab (120 mg / mL) buffer solution 20 mM histidine / histidine-HCl Stabilizer 1 210 mM Trehalose Stabilizer 2 10 mM methionine Stabilizer 3 0.04% Polysorbate 20 50 mM Lys-Lys 50 mM glycine Hyaluronidase HP46 (2,000 units / mL) of SEQ ID NO: 44

[0122] 〔Example 7, Measurement using spectrophotometry〕

[0123] Formulations 5 through 7 were placed at 45°C for 14 days, and changes in protein concentration were analyzed using a Beckman spectrophotometer. Each sample was diluted with distilled water to a concentration of 0.4 mg / mL, and the absorbance of the protein at 280 nm was then measured using a spectrophotometer. In the stability test under harsh conditions (i.e., 14 days at 45°C), there were no significant changes in protein concentration for formulations 5 through 7. However, hyaluronidase activity decreased sharply at 45°C; therefore, enzyme activity was not measured in this example (see Figure 6).

[0124] [Example 8, using particle size sieving chromatography to study the monomer ratio of Trastuzumab in various formulations]

[0125] For particle size sieving chromatography analysis, an HPLC system provided by Shimadzu Prominence was used, with a TSK-gel G3000SWXL (7.8 x 300 mm, 5 μm) and a TSK guard column (6.0 x 4.0 mm, 7 μm) as the column. A 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used as the mobile phase. Isocratic separation mode was applied at a flow rate of 0.5 mL / min for 35 min. The sample was diluted with analytical solvent to a final concentration of 10 mg / mL, and the absorbance at 280 nm was measured after injecting 20 μL of sample into the HPLC column. The monomeric proportion of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0126] When particle size distribution chromatography was performed in stability tests under harsh conditions (i.e., 14 days at 45°C), formulations 5 through 7 exhibited similar patterns of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) impurities and a decrease in monomer content (approximately 1.5%), and these differences were not significant across formulations. In conclusion, particle size distribution chromatography analysis in stability tests under harsh conditions (i.e., at 45°C) showed similar protein stability in formulations containing 0.04% polysorbate 20, 50 mM Lys-Lys, and 50 mM glycine (see Figure 4).

[0127] 〔Example 9, Weak cation exchange chromatography analysis of a formulation containing Trastuzumab and HP46〕

[0128] For weak cation exchange chromatography analysis, an HPLC system provided by Shimadzu Prominence was used, with TSKgel CM-STAT (4.6 x 100 mm, 7 μm), TSKgel protective gel CMSTAT (3.2 mm x 1.5 cm inner diameter), and similar materials as columns. Mobile phase A was 10 mM sodium phosphate (pH 7.5), and mobile phase B was 10 mM sodium phosphate containing 0.1 M sodium chloride (pH 7.2). Analysis was performed for 55 minutes using a linear concentration gradient of 0-30% mobile phase B at a flow rate of 0.8 mL / min. The sample was diluted with mobile phase A to a final concentration of 1.0 mg / mL, and 80 μL of the sample was injected into the HPLC system. The absorbance at 280 nm was then recorded. The monomeric proportion of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0129] When weak cation exchange chromatography was performed in stability tests under harsh conditions (i.e., 14 days at 45°C), formulations 5 through 7 exhibited similar patterns of change. Specific changes included an increase in the relative content of the acidic variant (approximately 30% change over 14 days), a decrease in the relative content of the main peak (approximately 44% change over 14 days), and an increase in the relative content of the basic variant (approximately 15% change over 14 days), with no significant differences between formulations. In conclusion, the results of weak cation exchange chromatography analysis in stability tests under harsh conditions (i.e., 45°C) showed similar protein stability in formulations containing 0.04% polysorbate 20, 50 mM Lys-Lys, and 50 mM glycine (see Figures 5A-5C).

[0130] [Example 10, Stability assessment of HP46 in subcutaneous formulations of Trastuzumab and HP46 at 40°C and 45°C]

[0131] To evaluate the stability of HP46 in the subcutaneous injection formulation of trastuzumab, trastuzumab (120 mg / mL) was mixed with pH 20 (2000 units / mL). The buffer solution used contained 20 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine, and 0.04% polysorbate 20. Enzyme activity in the control group samples was measured on day 0, while the experimental group samples were incubated at 40°C or 45°C for 1 day, after which the enzyme activity of each sample was measured.

[0132] The various trastuzumab subcutaneous injection formulations (trastuzumab + HW2 and trastuzumab + HP46) were placed at 40°C for 1 day, and then the hyaluronidase activity was measured. The results showed that the activities were 51%, 47%, and 94%, respectively, indicating that HP46 had the best thermal stability at 40°C (see Figures 6A-6B). In addition, the trastuzumab subcutaneous injection formulations (trastuzumab + HW2 and trastuzumab + HP46) were placed at 45°C for 1 day, and then the hyaluronidase activity was measured. The results showed that the trastuzumab subcutaneous injection formulation and trastuzumab + HW2 did not have enzyme activity, but the enzyme activity of trastuzumab + HP46 was still present (see Figures 6-6B).

[0133] 〔Example 11, Formulation Development〕

[0134] As shown in Table 8, three subcutaneous trastuzumab formulations were prepared. Formulations 8 to 10 typically contain 120 mg / mL of trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and a pH20 variant. The difference between formulations 8 to 10 lies in the concentration of the nonionic surfactant, where formulation 8 contains 0% polysorbate 20, formulation 9 contains 0.005% polysorbate 20, and formulation 10 contains 0.04% polysorbate 20.

[0135] Table 8, Composition of the Formulation Formulation 8 Formulation 9 Formulation 10 Antibody Trastuzumab (120 mg / mL) Polysorbate 20 0% 0.005% 0.04% buffer solution 20 mM histidine / histidine-HCl Stabilizer 1 210 mM Trehalose Stabilizer 2 10 mM methionine pH 5.5 Hyaluronidase HP46 (2,000 units / mL) of SEQ ID NO: 44

[0136] 〔Example 12, Measurement using a spectrophotometer〕

[0137] Formulations 8 through 10 were placed at 40°C for 14 days, and changes in protein concentration were analyzed using a Beckman spectrophotometer. Each sample was diluted with distilled water to a concentration of 0.4 mg / mL, and the absorbance of the protein at 280 nm was then measured using a spectrophotometer. In the stability test under harsh conditions (i.e., 14 days at 40°C), there were no significant changes in protein concentration in formulations 8 through 10.

[0138] [Example 13, using particle size sieving chromatography to study the monomer ratio of Trastuzumab in various formulations]

[0139] For particle size sieving chromatography analysis, an HPLC system provided by Shimadzu Prominence was used, with a TSK-gel G3000SWXL (7.8 x 300 mm, 5 μm) and a TSK guard column (6.0 x 4.0 mm, 7 μm) as the column. A 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used as the mobile phase. Analysis was performed for 35 minutes at a flow rate of 0.5 mL / min using isocranial separation mode. The sample was diluted with analytical solvent to a final concentration of 10 mg / mL, and the absorbance at 280 nm was measured after injecting 20 μL of sample into the HPLC column. The monomeric proportion of Trastuzumab in the HPLC chromatogram was calculated and plotted.

[0140] When particle size distribution chromatography was performed in the stability test under harsh conditions (i.e., 14 days at 40°C), formulations 8 to 10 exhibited similar patterns of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (approximately below 1.0%), and these differences were not significant across formulations. In conclusion, the results of particle size distribution chromatography analysis in the stability test under harsh conditions (i.e., at 40°C) showed no significant difference in stability between formulations based on the concentration of polysorbate 20 (0–0.04%) (see Figure 7).

[0141] [Example 14, measuring the protein aggregation temperature of a formulation containing Trastuzumab and HP46]

[0142] In the field of protein drugs, dynamic light scattering (DLS) is used to analyze the denaturation properties of proteins caused by heat. In this experiment, the size change of protein molecules according to temperature changes was measured and used to calculate the protein aggregation temperature. For dynamic light scattering analysis, a Zetasizer-nano-ZS instrument and a quartz photocell (ZEN2112) provided by Malvern were used. During the analysis, the temperature was increased from 25°C to 85°C at 1°C intervals. The sample was diluted to 1 mg / mL using various formulation buffers, and then 150 µL of sample was added to the photocell for analysis.

[0143] Formulation 8, which does not contain polysorbate 20, exhibits an aggregation temperature of 78.3°C, formulation 9 exhibits an aggregation temperature of 77.3°C, and formulation 10 exhibits an aggregation temperature of 77.7°C. In Example 13, even without polysorbate 20, the monomer ratio of the protein did not change. Comparing the results with and without polysorbate 20 confirms that there is no difference in the aggregation of these proteins. These results indicate that for subcutaneous injection formulations of trastuzumab, it is not necessary to minimize the amount of polysorbate 20 (see Figures 8A-8B).

[0144] 〔Example 15, Weak cation exchange chromatography analysis of a formulation containing Trastuzumab and HP46〕

[0145] For weak cation exchange chromatography analysis, an HPLC system provided by Shimadzu Prominence was used, with TSKgel CM-STAT columns (4.6 x 100 mm, 7 μm), TSKgel protective gel CMSTAT (3.2 mm x 1.5 cm inner diameter), and similar columns as the columns. Mobile phase A was 10 mM sodium phosphate (pH 7.5), and mobile phase B was 10 mM sodium phosphate containing 0.1 M sodium chloride (pH 7.2). Analysis was performed for 55 minutes at a linear concentration gradient of 0-30% mobile phase B at a flow rate of 0.8 mL / min. The sample was diluted with mobile phase A to a final concentration of 1.0 mg / mL, and 80 μL of the sample was injected into the HPLC. The absorbance of the column precipitate at 280 nm was then recorded. The monomer proportion of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0146] When weak cation exchange chromatography analysis was performed in stability tests under harsh conditions (i.e., 14 days at 40°C), formulations 8 to 10 exhibited similar patterns of change. Specific changes included an increase in the relative content of acidic variants (approximately 10% change over 14 days), a decrease in the relative content of the main peak (approximately 40% change over 14 days), and an increase in the relative content of basic variants (approximately 300% change over 14 days), with no significant differences between formulations. In conclusion, the protein stability of polysorbate 20 (0–0.04%) was similar under weak cation exchange chromatography analysis in stability tests under harsh conditions (i.e., at 40°C) (see Figures 9A–9D).

[0147] 〔Example 16, Measuring the enzyme activity of a formulation containing Trastuzumab and HP46〕

[0148] The turbidimetric assay for measuring enzyme activity is a method that measures the degree of aggregation formed by the binding of residual hyaluronic acid and acidified albumin (BSA) by absorbance. When hyaluronic acid is hydrolyzed at pH 20, the amount bound to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma) as a standard was diluted to 1 unit / mL, 2 units / mL, 5 units / mL, 7.5 units / mL, 10 units / mL, 15 units / mL, 20 units / mL, 30 units / mL, 50 units / mL, and 60 units / mL, and prepared in individual tubes. Purified PH20 variant samples were diluted to 100X, 300X, 600X, 1200X, and 2400X concentrations using enzyme dilution buffer (20 mM Tris·HCl, pH 7.0, 77 mM sodium chloride, 0.01% (w / v) bovine serum albumin) and prepared in individual tubes. In clean tubes, a hyaluronidase solution with a concentration of 3 mg / mL was diluted 10-fold to a concentration of 0.3 mg / mL, resulting in a volume of 180 μL in each tube. 60 μL of the hyaluronidase-containing sample was added to the diluted hyaluronic acid solution and mixed, then reacted at 37°C for 45 minutes. After the reaction was complete, 50 μL of the reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well pan and shaken for 10 minutes. The absorbance at 600 nm was then measured using spectrophotometry.

[0149] The results of the activity analysis in the stability test under harsh conditions (i.e., 14 days at 40°C) confirmed that the higher the concentration of polysorbate 20, the greater the decrease in activity over time (see Figure 10).

[0150] 〔Example 17, Formulation Development〕

[0151] As shown in Table 9, three subcutaneous trastuzumab formulations were prepared. Formulations 11 to 13 typically contain 120 mg / mL of trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and a pH20 variant. The difference between formulations 11 to 13 lies in the concentration of the nonionic surfactant, where formulation 11: 0% polysorbate 80, formulation 12: 0.005% polysorbate 80, and formulation 13: 0.04% polysorbate 80.

[0152] Table 9, Composition of the Formulation Formulation 11 Formulation 12 Formulation 13 Antibody Trastuzumab (120 mg / mL) Polysorbate 80 0% 0.005% 0.04% buffer solution 20 mM histidine / histidine-HCl Stabilizer 1 210 mM Trehalose Stabilizer 2 10 mM methionine pH 5.5 Hyaluronidase HP46 (2,000 units / mL) of SEQ ID NO: 44

[0153] When particle size chromatography was performed in stability tests under harsh conditions (i.e., 14 days at 40°C), formulations 11 to 13 exhibited similar patterns of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (approximately below 1.0%), and these differences were not significant across formulations. In conclusion, the results of particle size chromatography analysis in stability tests under harsh conditions (i.e., at 40°C) showed no significant difference in stability between formulations based on the concentration of polysorbate 80 (0–0.04%) (see Figure 11).

[0154] 〔Example 18, Weak cation exchange chromatography analysis of a formulation containing Trastuzumab and HP46〕

[0155] For weak cation exchange chromatography analysis, an HPLC system provided by Shimadzu Prominence was used, with TSKgel CM-STAT columns (4.6 x 100 mm, 7 μm), TSKgel protective gel CMSTAT (3.2 mm x 1.5 cm inner diameter), and similar columns as the columns. Mobile phase A was 10 mM sodium phosphate (pH 7.5), and mobile phase B was 10 mM sodium phosphate containing 0.1 M sodium chloride (pH 7.2). Analysis was performed for 55 minutes at a linear concentration gradient of 0-30% mobile phase B at a flow rate of 0.8 mL / min. The sample was diluted with mobile phase A to a final concentration of 1.0 mg / mL, and 80 μL of the sample was injected into the HPLC. The absorbance of the column precipitate at 280 nm was then recorded. The monomer proportion of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0156] When weak cation exchange chromatography analysis was performed in stability tests under harsh conditions (i.e., 14 days at 40°C), formulations 11 to 13 exhibited similar patterns of change. Specific changes included an increase in the relative content of the acidic variant (approximately 10% change over 14 days), a decrease in the relative content of the main peak (approximately 40% change over 14 days), and an increase in the relative content of the basic variant (approximately 300% change over 14 days), with no significant differences between formulations. In conclusion, the protein stability of polysorbate 80 (0–0.04%) was similar under weak cation exchange chromatography analysis in stability tests under harsh conditions (i.e., at 40°C) (see Figures 12A–12D).

[0157] 〔Example 19, Measuring the enzyme activity of a formulation containing Trastuzumab and HP46〕

[0158] The turbidimetric assay for measuring enzyme activity is a method that measures the degree of aggregation formed by the binding of residual hyaluronic acid and acidified albumin (BSA) by absorbance. When hyaluronic acid is hydrolyzed at pH 20, the amount bound to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma) as a standard was diluted to 1 unit / mL, 2 units / mL, 5 units / mL, 7.5 units / mL, 10 units / mL, 15 units / mL, 20 units / mL, 30 units / mL, 50 units / mL, and 60 units / mL, and prepared in individual tubes. Purified protein samples were diluted to 100X, 300X, 600X, 1200X, and 2400X concentrations using enzyme dilution buffer (20 mM Tris·HCl, pH 7.0, 77 mM sodium chloride, 0.01% (w / v) bovine serum albumin) and prepared in individual tubes. In clean tubes, a hyaluronidase solution with a concentration of 3 mg / mL was diluted 10-fold to a concentration of 0.3 mg / mL, resulting in a volume of 180 μL in each tube. 60 μL of the hyaluronidase-containing sample was added to the diluted hyaluronic acid solution and mixed, then reacted at 37°C for 45 minutes. After the reaction was complete, 50 μL of the reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well pan and shaken for 10 minutes. The absorbance at 600 nm was then measured using spectrophotometry.

[0159] The results of the activity analysis in the stability test under harsh conditions (i.e., 14 days at 40°C) confirmed that the higher the concentration of polysorbate 80, the greater the decrease in activity over time (see Figure 13).

[0160] 〔Example 20, Formulation Development〕

[0161] As shown in Table 10, three rituximab formulations were prepared. Formulations 14 to 16 typically contain 120 mg / mL rituximab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and a pH20 variant. The difference between formulations 14 to 16 lies in the concentration of the nonionic surfactant, where formulation 14: 0% polysorbate 80, formulation 15: 0.005% polysorbate 80, and formulation 16: 0.06% polysorbate 80.

[0162] Table 10, Composition of the Formulation Formulation 14 Formulation 15 Formulation 16 Rituximab 120 mg / mL (± 10) PS 80 0% 0.005% 0.06% buffer solution 20 mM histidine / histidine-HCl Stabilizer 1 210 mM Trehalose Stabilizer 2 10 mM methionine pH 5.5 Hyaluronidase HP46 (2,000 units / mL) of SEQ ID NO: 44

[0163] When particle size chromatography was performed in stability tests under harsh conditions (i.e., 7 days at 40°C), formulations 14 to 16 exhibited similar patterns of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (approximately below 1.0%), and these differences were not significant across formulations. In conclusion, the results of particle size chromatography analysis in stability tests under harsh conditions (i.e., at 40°C) showed no significant difference in stability between formulations based on the concentration of polysorbate 80 (0–0.06%) (see Figure 14).

[0164] [Example 21, measuring the enzyme activity of a formulation containing Rituximab and HP46]

[0165] The turbidimetric assay for measuring enzyme activity is a method that measures the degree of aggregation formed by the binding of residual hyaluronic acid and acidified albumin (BSA) by absorbance. When hyaluronic acid is hydrolyzed at pH 20, the amount bound to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma) as a standard was diluted to 1 unit / mL, 2 units / mL, 5 units / mL, 7.5 units / mL, 10 units / mL, 15 units / mL, 20 units / mL, 30 units / mL, 50 units / mL, and 60 units / mL, and prepared in individual tubes. Purified protein samples were diluted to 100X, 300X, 600X, 1200X, and 2400X concentrations using enzyme dilution buffer (20 mM Tris·HCl, pH 7.0, 77 mM sodium chloride, 0.01% (w / v) bovine serum albumin) and prepared in individual tubes. In clean tubes, a hyaluronidase solution with a concentration of 3 mg / mL was diluted 10-fold to a concentration of 0.3 mg / mL, resulting in a volume of 180 μL in each tube. 60 μL of the hyaluronidase-containing sample was added to the diluted hyaluronic acid solution and mixed, then reacted at 37°C for 45 minutes. After the reaction was complete, 50 μL of the reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well pan and shaken for 10 minutes. The absorbance at 600 nm was then measured using spectrophotometry.

[0166] The results of the activity analysis in the stability test under harsh conditions (i.e., 7 days at 40°C) confirmed that the higher the concentration of polysorbate 80, the greater the decrease in activity over time (see Figure 15).

[0167] [Example 22, measuring the enzyme activity of a commercially available formulation that does not contain polysorbate]

[0168] As shown in Table 11, two commercially available rituximab formulations were prepared. Formulation 17 is a commercially available buffer solution for subcutaneous injection, and formulation 18 is a commercially available buffer solution for intravenous injection. Formulations 17 and 18 contain a PH20 variant and contain 120 mg / mL and 100 mg / mL of rituximab, respectively, but unlike commercially available products, they do not contain polysorbate 80.

[0169] Table 11, Composition of the Formulation Formulation 17 Formulation 18 Rituximab 120 mg / mL 100 mg / mL buffer solution 20 mM Histidine / Histidine-HCl 25 mM sodium citrate Stabilizer 1 210 mM Trehalose 145 mM sodium chloride Stabilizer 2 10 mM methionine 10 mM methionine pH 5.5 6.5 Hyaluronidase HP46 (2,000 units / mL) of SEQ ID NO: 44

[0170] The turbidimetric method for measuring enzyme activity is a method that measures the degree of aggregation formed by the binding of residual hyaluronic acid and acidified albumin (BSA) by absorbance. When hyaluronic acid is hydrolyzed at pH 20, the amount bound to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma) as a standard was diluted to 1 unit / mL, 2 units / mL, 5 units / mL, 7.5 units / mL, 10 units / mL, 15 units / mL, 20 units / mL, 30 units / mL, 50 units / mL, and 60 units / mL, and prepared in individual tubes. Purified protein samples were diluted with enzyme dilution buffer (20 mM Tris·HCl, pH 7.0, 77 mM sodium chloride, 0.01% (w / v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X, and prepared in individual tubes. In clean tubes, a hyaluronidase solution with a concentration of 3 mg / mL was diluted 10-fold to a concentration of 0.3 mg / mL, resulting in a tube volume of 180 μL. 60 μL of the hyaluronidase-containing sample was added to the diluted hyaluronic acid solution and mixed, then reacted at 37°C for 45 minutes. After the reaction was complete, 50 μL of the reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well pan and shaken for 10 minutes. The absorbance at 600 nm was then measured using spectrophotometry.

[0171] The results of the activity analysis in the stability test under harsh conditions (i.e., 6 days at 40°C) confirmed that high activity can be maintained even when polysorbate 80 is not contained in the formulation, especially formulation 18 maintains high activity (see Figure 16).

[0172] 〔Example 23: Formulation Development〕

[0173] As shown in Table 12, four pembrolizumab formulations were prepared. Formulations 19, 20, and 21 typically contain 25 mg / mL of pembrolizumab, 10 mM histidine (pH 5.5), 7% sucrose, 10 mM methionine, and a pH20 variant. The difference between formulations 19 and 21 lies in the concentration of the nonionic surfactant, where formulation 19 contains 0% polysorbate 80, formulation 20 contains 0.005% polysorbate 80, and formulation 21 contains 0.02% polysorbate 80. Formulation 22 contains 25 mg / mL of pembrolizumab and consists of 10 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine, 0.02% polysorbate 80, and a pH20 variant.

[0174] Table 12, Composition of the Formulation Formulation 19 Formulation 20 Formulation 21 Formulation 22 Antibody Pembrolizumab (25 mg / mL) buffer solution 10 mM histidine (pH 5.5) Stabilizer 1 7% sucrose 7% sucrose 7% sucrose 210 mM Trehalose Stabilizer 2 10 mM Methionine 10 mM Methionine 10 mM Methionine 10 mM Methionine Polysorbate 80 0% 0.005% 0.02% 0.02% Hyaluronidase HP46 (2,000 units / mL) of SEQ ID NO: 44

[0175] 〔Example 24, Measurement using a spectrophotometer〕

[0176] Formulations 19 to 22 were placed at 40°C for 7 days, and the changes in protein concentration were analyzed using a Beckman spectrophotometer. Each sample was diluted with distilled water to a concentration of 0.4 mg / mL, and the absorbance of the protein at 280 nm was then measured using a spectrophotometer.

[0177] In stability tests under harsh conditions (i.e., 7 days at 40°C), the protein concentrations of formulations 19 to 22 did not change significantly.

[0178] [Example 25, using particle size sieving chromatography to study the monomer ratio of Pembrolizumab in various formulations]

[0179] For particle size sieving chromatography analysis, an HPLC system provided by Shimadzu Prominence was used, with a TSK-gel G3000SWXL (7.8 x 300 mm, 5 μm) and a TSK guard column (6.0 x 4.0 mm, 7 μm) as the column. A 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used as the mobile phase. Analysis was performed for 35 minutes at a flow rate of 0.5 mL / min using isocranial separation mode. The sample was diluted with the analytical solvent to a final concentration of 10 mg / mL, and the absorbance at 280 nm was measured after injecting 20 μL of sample into the HPLC column. The monomeric proportion of pembrolizumab in the HPLC chromatogram was calculated and plotted.

[0180] When particle size distribution chromatography was performed in the stability test under harsh conditions (i.e., 7 days at 40°C), formulations 19 to 22 exhibited similar patterns of change. There were no significant differences in the degradation products at high molecular weight (HMW) and low molecular weight (LMW) levels, regardless of the formulation. In conclusion, the results of particle size distribution chromatography analysis in the stability test under harsh conditions (i.e., at 40°C) showed no significant differences between formulations 19 and 22, and these differences were not based on the type of sugar (see Figure 17). These results are consistent with those obtained with trastuzumab and rituximab as previously illustrated.

[0181] 〔Example 26, Measuring the enzyme activity of a formulation containing Pembrolizumab and HP46〕

[0182] The turbidimetric method for measuring enzyme activity is a method that measures the degree of aggregation formed by the binding of residual hyaluronic acid and acidified albumin (BSA) by absorbance. When hyaluronic acid is hydrolyzed at pH 20, the amount bound to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma) as a standard was diluted to 1 unit / mL, 2 units / mL, 5 units / mL, 7.5 units / mL, 10 units / mL, 15 units / mL, 20 units / mL, 30 units / mL, 50 units / mL, and 60 units / mL, and prepared in individual tubes. Purified protein samples were diluted with enzyme dilution buffer (20 mM Tris·HCl, pH 7.0, 77 mM sodium chloride, 0.01% (w / v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X, and prepared in individual tubes. In clean tubes, a hyaluronidase solution with a concentration of 3 mg / mL was diluted 10-fold to a concentration of 0.3 mg / mL, resulting in a tube volume of 180 μL. 60 μL of the enzyme-containing sample was added to the diluted hyaluronic acid solution and mixed, then reacted at 37°C for 45 minutes. After the reaction was complete, 50 μL of the reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well pan and shaken for 10 minutes. The absorbance at 600 nm was then measured using spectrophotometry.

[0183] The results of activity analysis in stability tests under harsh conditions (i.e., 7 days at 40°C) confirmed that the activity decreased significantly with increasing concentration of polysorbate 80. It was also confirmed that when the same amount of polysorbate 80 was contained, the decrease in activity in the trehalose-containing formulation was less than the decrease in activity in the sucrose-containing formulation (see Figure 18).

[0184] 〔Example 27, pH-activity diagram of HP46 and wild-type HW2〕

[0185] For the experiment used to confirm the pH-activity profiles of HP46 and wild-type HW2, a microturbidimetric assay method was used. For each pH value, hyaluronic acid buffer was prepared to dissolve the hyaluronic acid used as the substrate, and enzyme buffer was prepared to dilute the enzyme.

[0186] Prepare three 96-well discs for the reaction between the enzyme and the substrate, and label them as A, B and C, and conduct the experiment.

[0187] Hyaluronic acid buffer solutions with pH values ​​of 4.0, 4.5, or 5.0 were prepared using 20 mM acetic acid and 70 mM sodium chloride, and hyaluronic acid solutions with pH values ​​of 5.5, 6.0, 6.5, 7.0, or 8.0 were prepared using 20 mM sodium phosphate and 70 mM sodium chloride. 20 mg of hyaluronic acid was dissolved in 10 mL of each of the prepared hyaluronic acid buffer solutions to prepare the final hyaluronic acid substrate solution. This solution was then diluted with the respective hyaluronic acid buffer solutions prepared according to the pH values ​​to prepare 500 μL final solutions with concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.45 mg / mL, or 0.7 mg / mL. 100 μL of each solution was dispensed into each well of a 96-well disk labeled A. The concentration of hyaluronic acid was measured using the concentration-prepared and diluted hyaluronic acid buffer solutions as a calibration curve.

[0188] Enzyme buffers with pH 4.0, 4.5 or 5.0 were prepared using 20 mM acetic acid, 0.01% (w / v) BSA and 70 mM sodium chloride, and enzyme buffers with pH 5.5, 6.0, 6.5, 7.0 or 8.0 were prepared using 20 mM sodium phosphate, 0.01% (w / v) BSA and 70 mM sodium chloride.

[0189] HP46 and wild-type HW2 enzymes were prepared into 10 units / mL enzyme buffer solution according to pH value, and 50 μL of the final solution was dispensed into each well of a 96-well plate labeled B.

[0190] Transfer 50 μL of sample from each well of a 96-well plate labeled A to each well of a 96-well plate labeled B, and then react at 37°C with a shaking incubator for 45 minutes. 15 minutes before the reaction is complete, distribute 200 μL of acidic albumin solution into each well of a 96-well plate labeled C. When the enzyme acceptance reaction is complete, transfer 40 μL of sample from each well of a 96-well plate labeled B to each well of a 96-well plate labeled C, and then react for 20 minutes. After 20 minutes, measure the absorbance at 600 nm, calculate the amount of hyaluronic acid remaining after the enzyme acceptance reaction, and complete the enzyme activity graph based on pH (see Figure 19).

[0191] [Example 28, Pharmacokinetics of Henapine subcutaneous injection formulation with Trastuzumab and HP46 in Sprague-Dawley rats]

[0192] To test whether the subcutaneous formulations of trastuzumab and HP46 exhibited the same pharmacokinetic properties as the subcutaneous formulation of hyaluronic acid, 9-week-old Staunch II rats were used in the experiment. The dosage of hyaluronic acid and trastuzumab was 18 mg / kg (rice body weight), and the amount of rHuPH20 contained in the subcutaneous formulation of hyaluronic acid was 100 U, as was the amount of HP46. In the pharmacokinetic test, trastuzumab and HP46 exhibited the same area under the curve (AUC) as the subcutaneous formulation of hyaluronic acid (see Figure 20).

[0193] The pharmaceutical composition according to the present invention can be used for subcutaneous injection and is quite stable. The activity of the PH20 variant and the drug can be maintained for a long time, and the drug is preferably an antibody drug or analogue. Therefore, the pharmaceutical composition not only helps to reduce the cost of producing subcutaneous injection preparations, but also reduces medical costs, and is quite effective in terms of patient convenience.

[0194] Although preferred embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various modifications, additions and substitutions may be made without departing from the scope and spirit of the invention as disclosed in the claims.

[0195] 〔References〕 Bookbinder, L.H., Hofer, A., Haller, M.F., Zepeda, M.L., Keller, G.A., Lim, J.E., Edgington, T.S., Shepard, H.M., Patton, J.S., and Frost, G.I. (2006). A recombinant human enzyme for enhanced interstitial transport of therapeutics. J Control Release 114, 230-241. Borders jr., C.L. and Raftery, A. (1968) Purification and Partial Characterization of Testicular Hyaluronidase. J Biol Chem 243, 3756-3762 Chao, K.L., Muthukumar, L., and Herzberg, O. (2007). Structure of human hyaluronidase-1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesis. Biochemistry 46, 6911-6920. Chen, K.J., Sabrina, S., El-Safory, N.S., Lee, G.C., and Lee, C.K. (2016) Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris. J Biosci Bioeng. 122, 673-678 Frost, G.I. (2007). Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin Drug Deliv 4, 427-440 Hofinger, E.S., Bernhardt, G., and Buschauer, A. (2007) Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction. Glycobiology 17, 963-971 Kreidieh, F.Y., Moukadem, H.A., and Saghir, N.S.E. (2016) Overview, prevention and management of chemotherapy extravasation. World J Clin Oncol 7, 87-97. Thomas, J.R., Yocum, R.C., Haller, M.F., and Flament J. (2009) The INFUSE-Morphine IIB Study: Use of Recombinant Human Hyaluronidase (rHuPH20) to Enhance the Absorption of Subcutaneous Morphine in Healthy Volunteers. J Pain Symptom Manag 38, 673-682.

Brief Description of the Drawings

[0019] The above and other objects, features, and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings. Figure 1A shows a particle size sieving chromatography diagram of trastuzumab in a stability test under harsh conditions of 45°C. Figure 1B shows the change in purity of trastuzumab monomer protein in the formulation in a stability test under harsh conditions of 45°C. Figures 2A and 2B show the results of measuring the protein aggregation temperature of a formulation containing trastuzumab and the novel PH20 variant HP46. Figure 3A is a weak cation exchange (WCX) chromatography diagram of trastuzumab in a stability test under harsh conditions of 45°C. Figure 3B shows the change (%) in the relative amount of the acidic variant in the formulation in a stability test under harsh conditions of 45°C. Figure 3C shows the change (%) in the relative amount of the main peak in the formulation in a stability test under harsh conditions of 45°C. Figure 3D shows the change (%) in the relative amount of the basic variant in the formulation during stability testing under harsh conditions at 45°C. Figure 4 shows the change in purity of trastuzumab monomeric protein in formulations 5-7 during stability testing under harsh conditions at 45°C. Figure 5A shows the change (%) in the relative amount of the acidic variant in formulations 5-7 during stability testing under harsh conditions at 45°C. Figure 5B shows the change (%) in the relative amount of the main peak in formulations 5-7 during stability testing under harsh conditions at 45°C. Figure 5C shows the change (%) in the relative amount of the basic variant in formulations 5-7 during stability testing under harsh conditions at 45°C. Figure 6A shows the results of stability tests conducted at 40°C, measuring the residual enzyme activity of Herceptin subcutaneous injection (Herceptin SC), trastuzumab + wild-type PH20 (HW2), and trastuzumab + PH20 variant HP46 on days 0 and 1. Figure 6B shows the results of stability tests conducted at 45°C, measuring the residual enzyme activity of Herceptin subcutaneous injection, trastuzumab + wild-type PH20 (HW2), and trastuzumab + PH20 variant HP46 on days 0 and 1. Figure 7 shows the particle size sieving chromatography results of formulations 8-10 during a 14-day stability test at 40°C. Figure 8A shows the results of measuring changes in protein particle size using dynamic light scattering (DLS) equipment. Figure 8B shows the results of measuring protein aggregation temperature.Figure 9A shows the weak cation exchange (WCX) chromatography of formulation 8 under harsh conditions at 40°C. Figure 9B shows the relative amount (%) of acidic variants in formulations 8-10 under harsh conditions at 40°C. Figure 9C shows the relative amount (%) of the main peak in formulations 8-10 under harsh conditions at 40°C. Figure 9D shows the relative amount (%) of basic variants in formulations 8-10 under harsh conditions at 40°C. Figure 10 shows the relative enzyme activity (%) of formulations 8-10 under harsh conditions at 40°C. Figure 11 shows the change in purity of trastuzumab monomers in formulations 11-13 under harsh conditions at 40°C. Figure 12A shows the weak cation exchange (WCX) chromatography of formulation 11 in a stability test under harsh conditions at 40°C. Figure 12B shows the change (%) in the relative amount of acidic variants in formulations 11-13 in a stability test under harsh conditions at 40°C. Figure 12C shows the change (%) in the relative amount of the main peak in formulations 11-13 in a stability test under harsh conditions at 40°C. Figure 12D shows the change (%) in the relative amount of basic variants in formulations 11-13 in a stability test under harsh conditions at 40°C. Figure 13 shows the change (%) in the relative enzyme activity of formulations 11-13 in a stability test under harsh conditions at 40°C. Figure 14 shows the change in the purity of rituximab monomers in formulations 14-16 in a stability test under harsh conditions at 40°C. Figure 15 shows the change (%) of relative enzyme activity of formulations 14-16 in stability tests under harsh conditions at 40°C. Figure 16 shows the change of relative enzyme activity of formulations 17 and 18 in stability tests under harsh conditions at 40°C. Figure 17 shows the particle size distribution chromatography results of formulations 19-22 at 40°C. Figure 18 shows the change of relative enzyme activity of formulations 19-22 in stability tests under harsh conditions at 40°C. Figure 19 shows the change of enzyme activity of recombinant human PH20 and HP46 according to pH changes.Figure 20 shows the pharmacokinetic results of the subcutaneous product of Heamin (Heamin SC) and its biosimilar candidate (trastuzumab + HP46; Heamin SC BS) in 9-week-old Sprague-Dawley rats. Heamin and its biosimilar candidate were injected at a dose of 18 mg / kg per rat. The subcutaneous formulation contained 100 units of rHuPH20 and 100 units of HP46 (at pH 5.3). [Sequence List]

Claims

1. A pharmaceutical composition comprising: (a) pembrolizumab; and (b) a PH20 variant, the amino acid sequence of which is modified SEQ ID NO: 1, comprising: (i) amino acid residues of M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T of SEQ ID NO: 1, (ii) deletion of the amino terminus of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39 or M1 to A40 of SEQ ID NO: 1, and (iii) deletion of the carboxyl terminus, wherein the carboxyl terminus of the PH20 variant is selected from SEQ ID NO: It ends with any of I465 to S490 of 1.

2. The pharmaceutical composition as described in claim 1 further comprises one or more pharmaceutically acceptable additives.

3. The pharmaceutical composition as claimed in claim 2, wherein the one or more pharmaceutically acceptable additives comprise one or more of buffers, stabilizers, and surfactants.

4. A pharmaceutical composition comprising: (a) pembrolizumab; and (b) a PH20 variant, the amino acid sequence of which is modified SEQ ID NO: 1, comprising: (i) (A): substitution of amino acid residues of M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T in SEQ ID NO: 1; and (B): substitution of one or more amino acid residues selected from the group consisting of T341S, L342W, S343E, I344N and N363G; (ii) SEQ ID NO: The deletion of the amino terminus of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39, M1 to A40 or M1 to P41 of SEQ ID NO: 1; and (iii) the deletion of the carboxyl terminus, wherein the carboxyl terminus of the PH20 variant ends with any one of I465 to S490 selected from SEQ ID NO:

1.

5. The pharmaceutical composition as claimed in claim 4, wherein the amino acid residue substitutions of the PH20 variant are composed of amino acid residue substitutions from any of the following groups: (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; and (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T and N363G.

6. The pharmaceutical composition of claim 4, wherein the amino acid residue substitution of the PH20 variant is composed of amino acid residue substitutions from any of the following groups: (a) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; and (b) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T.

7. The pharmaceutical composition as described in claim 4 further comprises one or more pharmaceutically acceptable additives.

8. The pharmaceutical composition as claimed in claim 7, wherein the one or more pharmaceutically acceptable additives comprise one or more of a buffer, a stabilizer, and a surfactant.

9. A pharmaceutical composition comprising: (a) pembrolizumab; and (b) a PH20 variant, the amino acid sequence of which is a modified SEQ ID NO: 1, comprising: (i) amino acid residues of T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T of SEQ ID NO: 1; (ii) deletion of the amino terminus of amino acid residues M1 to T35, M1 to N37, M1 to F38, M1 to R39 or M1 to A40 of SEQ ID NO: 1; and (iii) deletion of the carboxyl terminus, wherein the carboxyl terminus of the PH20 variant is selected from SEQ ID NO:

1. NO: 1 ends with any of I465 to S490.

10. The pharmaceutical composition as described in claim 9 further comprises one or more pharmaceutically acceptable additives.

11. The pharmaceutical composition as claimed in claim 10, wherein the one or more pharmaceutically acceptable additives comprise one or more of a buffer, a stabilizer, and a surfactant.

12. The pharmaceutical composition according to any one of claims 4 to 8, wherein the deletion at the amino end is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39 or M1 to A40 of SEQ ID NO:

1.

13. The pharmaceutical composition of any one of claims 1 to 11, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO:

1.

14. The pharmaceutical composition of any one of claims 1 to 11, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO:

1.

15. The pharmaceutical composition of any one of claims 1 to 11, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35 of SEQ ID NO:

1.

16. The pharmaceutical composition of any one of claims 1 to 11, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to L36 of SEQ ID NO:

1.

17. The pharmaceutical composition according to any one of claims 1 to 8, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to N37 of SEQ ID NO:

1.

18. The pharmaceutical composition according to any one of claims 1 to 11, wherein the carboxyl terminus of the PH20 variant ends with an amino acid residue of SEQ ID NO: 1, namely I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490.

19. The pharmaceutical composition according to any one of claims 1 to 11, wherein the carboxyl terminus of the PH20 variant ends with an amino acid residue of SEQ ID NO: 1, namely I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, or Y482.

20. The pharmaceutical composition of any one of claims 1 to 11, wherein the carboxyl terminus of the PH20 variant ends with an amino acid residue I465, D466, A467, F468, K470 or P471 of SEQ ID NO:

1.

21. The pharmaceutical composition of any one of claims 1 to 11, wherein the carboxyl terminus of the PH20 variant ends with amino acid residue I465, F468 or P471 of SEQ ID NO:

1.

22. The pharmaceutical composition of any one of claims 1 to 8, wherein the carboxyl terminus of the PH20 variant ends with amino acid residue F468 of SEQ ID NO:

1.

23. The pharmaceutical composition according to any one of claims 1 to 11, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39 or M1 to A40 of SEQ ID NO: 1, and the carboxyl terminus of the PH20 variant ends with amino acid residues I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 of SEQ ID NO:

1.

24. The pharmaceutical composition according to any one of claims 1 to 8, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39 or M1 to A40 of SEQ ID NO: 1, and the carboxyl terminus of the PH20 variant ends with amino acid residues I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480 or Y482 of SEQ ID NO:

1.

25. The pharmaceutical composition according to any one of claims 1 to 8, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39 or M1 to A40 of SEQ ID NO: 1, and the carboxyl terminus of the PH20 variant ends with amino acid residues I465, D466, A467, F468, K470 or P471 of SEQ ID NO:

1.

26. The pharmaceutical composition according to any one of claims 1 to 8, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39 or M1 to A40 of SEQ ID NO: 1, and the carboxyl terminus of the PH20 variant ends with amino acid residues I465, F468 or P471 of SEQ ID NO:

1.

27. The pharmaceutical composition according to any one of claims 1 to 8, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39 or M1 to A40 of SEQ ID NO: 1, and the carboxyl terminus of the PH20 variant ends with amino acid residue F468 of SEQ ID NO:

1.

28. The pharmaceutical composition of claim 23, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO:

1.

29. The pharmaceutical composition of claim 23, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO:

1.

30. The pharmaceutical composition of claim 24, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO:

1.

31. The pharmaceutical composition of claim 24, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO:

1.

32. The pharmaceutical composition of claim 25, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO:

1.

33. The pharmaceutical composition of claim 25, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO:

1.

34. The pharmaceutical composition of claim 26, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO:

1.

35. The pharmaceutical composition of claim 26, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO:

1.

36. The pharmaceutical composition of claim 27, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO:

1.

37. The pharmaceutical composition of claim 27, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO:

1.

38. The pharmaceutical composition according to any one of claims 9 to 11, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO: 1, and the carboxyl terminus of the PH20 variant ends with amino acid residues I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 of SEQ ID NO:

1.

39. The pharmaceutical composition according to any one of claims 9 to 11, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO: 1, and the carboxyl terminus of the PH20 variant ends with amino acid residues I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480 or Y482 of SEQ ID NO:

1.

40. The pharmaceutical composition according to any one of claims 9 to 11, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO: 1, and the carboxyl terminus of the PH20 variant ends with amino acid residues I465, D466, A467, F468, K470 or P471 of SEQ ID NO:

1.

41. The pharmaceutical composition according to any one of claims 9 to 11, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO: 1, and the carboxyl terminus of the PH20 variant ends with amino acid residues I465, F468 or P471 of SEQ ID NO:

1.

42. The pharmaceutical composition according to any one of claims 9 to 11, wherein the deletion of the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38 or M1 to R39 of SEQ ID NO: 1, and the carboxyl terminus of the PH20 variant ends with amino acid residue F468 of SEQ ID NO:

1.

43. The pharmaceutical composition of claim 38, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO:

1.

44. The pharmaceutical composition of claim 39, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO:

1.

45. The pharmaceutical composition of claim 40, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO:

1.

46. ​​The pharmaceutical composition of claim 41, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO:

1.

47. The pharmaceutical composition of claim 42, wherein the deletion at the amino terminus is the deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37 or M1 to F38 of SEQ ID NO: 1.

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