RU2810952C2 - Pharmaceutical composition, including a ph20 variant of human hyaluronidase, and a medicinal product for subcutaneous administration - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents the use of PH20 variant containing one or more substitutions of amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T in wild-type PH20 having the amino acid sequence SEQ ID NO: 1, to obtain a pharmaceutical composition that is thermostable for a long time, including: (a) a medicinal product; and (b) the said PH20 variant, and where the medicinal product is not pembrolizumab.

EFFECT: invention makes it possible to obtain thermostable compositions containing PH20 variant.

26 cl, 20 dwg, 12 tbl, 28 ex

Description

Field of technology to which the invention relates

The present invention relates to a pharmaceutical composition comprising the PH20 variant of human hyaluronidase, which has increased enzymatic activity and thermal stability, and one or more drugs, as well as a method for treating a disease with their help.

The pharmaceutical composition according to the present invention can preferably be used for subcutaneous injection.

Prior Art

Drugs that need to be administered in high dose or multiple doses, especially antibody drugs and the like, are usually administered by intravenous injection, and such injection takes about 90 minutes or more, and intravenous injection must be accompanied by additional preparation procedure , thus creating inconvenience for the patient, doctors, and medical personnel, and additional costs arise. In contrast, subcutaneous injection has the advantage of allowing immediate administration, but the absorption rate is relatively slow compared with intravenous injection, and when the injection volume is 3-5 ml or more, it may cause swelling and pain at the injection site because absorption happens slowly. For this reason, subcutaneous injection of protein therapeutic agents is usually limited to the injection of a solution in a small amount of 2 ml or less. However, when hyaluronidase is administered subcutaneously (or subcutaneously) along with a therapeutic drug, the hyaluronic acid distributed in the extracellular matrix is hydrolyzed by hyaluronidase, and thus the viscosity of the subcutaneous area is reduced and the permeability to the substance is increased, and therefore a high dose or several doses of medication can be easily delivered to the body.

There are six types of hyaluronidase genes in humans: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1 and PH20/SPAM1. Hyal1 and Hyal2 are expressed in most tissues, and PH20/SPAM1 (hereafter referred to as PH20) is expressed in the sperm cell membrane and acrosomal membrane. HyalPS1 is not expressed because it is a pseudogene. PH20 is an enzyme (EC 3.2.1.35) that cleaves the β-1,4 bonds between N-acetylglucosamine and glucuronic acid, which are the sugars that make up hyaluronic acid. Human hyaluronidase PH20 has an optimal pH of 5.5, but shows some activity even at pH 7-8, whereas other human hyaluronidases, including Hyal1, have an optimal pH of 3-4 and very little activity at pH 7-8. pH value in subcutaneous areas in humans is about 7.4, which is practically neutral, and thus, among the various types of hyaluronidases, PH20 is widely used in clinical practice. Examples of clinical uses of PH20 include subcutaneous injection of an antibody therapeutic agent, use as an ocular relaxant and anesthetic additive in ophthalmic surgery, use to increase access of an anticancer therapeutic agent to tumor cells by hydrolyzing hyaluronic acid in the extracellular matrix of tumor cells, and use to accelerate resorption body fluids and blood that are excessively present in tissues.

Meanwhile, currently commercial PH20 is in a form extracted from the testes of cattle or sheep. Examples include Amphadase® (bovine hyaluronidase) and Vitrase® (ovine hyaluronidase).

Bovine testis hyaluronidase (BTH) is formed by removal of the signal peptide and the C-terminal 56 amino acids from wild-type bovine hyaluronidase PH20 by post-translational modification. BTH is also a glycoprotein and has a mannose content of 5% and a glucosamine content of 2.2%, based on total composition including amino acids (Borders and Raftery, 1968). When animal-derived hyaluronidase is reintroduced into the human body at high doses, a neutralizing antibody may be formed, and other animal-derived biomaterials contained as impurities in PH20 preparations may cause an allergic reaction. In particular, the use of PH20 extracted from bovine tissue is limited due to the risk of mad cow disease. To overcome these problems, studies have been conducted on recombinant human PH20 proteins.

It has been reported that recombinant human PH20 proteins have been expressed in yeast (P. pastoris), DS-2 insect cells, animal cells, etc. (Chen et al., 2016, Hofinger et al., 2007). Recombinant PH20 proteins produced in insect cells and yeast differ from human PH20 in terms of N-glycosylation patterns upon post-translational modification.

Among the hyaluronidases, the protein structures of Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidases (PDB ID: 1FCQ, 1FCU, 1FCV) were identified. Hyal1 consists of two domains, a catalytic domain and an EGF-like domain, and the catalytic domain has a (β/α) ₈ shape in which the alpha helix and beta strand, which characterize the secondary structure of the protein, are each repeated eight times (Chao et al., 2007). The EGF-like domain is completely conserved in variants in which the Hyal1 C terminus is differentially spliced. The amino acid sequences of Hyal1 and PH20 are 35.1% identical, and the tertiary structure of the PH20 protein has not yet been determined.

In a structure-function relationship study of the human PH20 protein, the C-terminal region of PH20 was found to be important for protein expression and enzymatic activity, and in particular, C-terminal termination at amino acids 477-483 was reported to be important for enzymatic expression and activity (Frost , 2007). The activity of the full-length PH20 protein (amino acids 1-509) or the pH20 variant having a truncated C-terminus at position 467 was only 10% or less of the activity of the pH20 variant having a truncated C-terminus at one site among positions 477-483 (Frost, 2007). Halozyme Therapeutics has developed rHuPH20 (amino acids 36-482), which is a recombinant protein where the C-terminus of the mature PH20 protein is cleaved at Y482 (Bookbinder et al., 2006; Frost, 2007).

Meanwhile, although research on the development of various subcutaneous injection therapeutics using human PH20 is ongoing, the problem of low stability of human PH20 itself still remains unresolved.

Based on this prior art, we have confirmed that human PH20 variants comprising one or more amino acid residue substitutions in the alpha helix 8 region (S347 to C381) and in the linker region (A333 to R346) between alpha helix 7 and alpha helix 8 in the amino acid sequence of wild type hyaluronidase PH20, and where some amino acids located at the N-terminus and/or C-terminus of PH20 are deleted, had very high enzymatic activity and thermostability, and therefore a patent application was filed (PCT/KR 2019/009215).

The authors of the present application have also confirmed that the PH20 variants in accordance with the present invention can be applied to pharmaceutical compositions or formulations including drugs, for example, antibody drugs, in particular, high-dose anti-HER2 antibodies or antibodies against immune checkpoints, and, Accordingly, the pharmaceutical compositions and formulations of the present invention comprising PH20 variants together with drugs such as anti-HER2 antibodies or anti-immune checkpoint antibodies can be used for subcutaneous injection and the activity of drugs such as antibody drugs and PH20 variants are very stable and can be stored for a long time, thus completing the present invention.

Summary of the invention

Technical problem solved by the invention

Thus, the present invention has been made in view of the above problems, and the object of the present invention is to provide a new pharmaceutical composition comprising a PH20 variant having enhanced enzymatic activity and heat stability, and a drug, wherein the heat stability and activity of the drug and the PH20 variant can be maintained for a long time. time; especially a pharmaceutical composition that can be used for subcutaneous injection.

Another object of the present invention is to provide a method of treating a disease, comprising administering a pharmaceutical composition in accordance with the present invention to a subject in need of treatment.

Technical solution

In accordance with the present invention, the above and other objects can be achieved by providing a pharmaceutical composition comprising (a) a drug and (b) a PH20 variant.

The PH20 variant contained in the pharmaceutical composition of the present invention may include one or more substitutions of amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T in the wild-type human PH20 protein having the amino acid sequence SEQ ID NO: 1, and may further include amino acid residue substitution(s) in one or more regions selected from the alpha helix 8 region (S347 to C381) and/or the linker region (A333 to R346) between alpha helix 7 and alpha helix 8, where certain amino acid residues located at the N-terminus and/or C-terminus are selectively removed.

The pharmaceutical composition according to the present invention may further include one or more additives selected from pharmaceutically acceptable additives, in particular a buffer, a stabilizer and a surfactant.

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

Description of drawings

The above and other objects, characteristics and other advantages of the present invention will be better understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Figure 1A illustrates chromatograms from size exclusion chromatography of trastuzumab in a stringent stability test at 45°C, and Figure 1B illustrates variation in monomeric protein purity of trastuzumab as a function of formulation in a stringent stability test at 45°C;

Figure 2 illustrates the results of measuring protein aggregation temperatures for compositions including trastuzumab and the new variant PH20 HP46;

Figure 3A is a weak cation exchange (WCX) chromatogram of trastuzumab in a stringent stability test at 45°C, Figure 3B illustrates changes (%) in the relative amounts of acidic variants in the compositions in a stringent stability test at 45°C, Figure 3C illustrates the changes (%) in the relative amounts of major peaks for the compositions in the 45°C severe stability test, and Figure 3D illustrates the changes (%) in the relative amounts of the major variants in the compositions in the 45°C severe stability test;

Figure 4 illustrates changes in trastuzumab monomeric protein purity in compositions 5-7 in a stringent stability test at 45°C;

Figure 5A illustrates the changes (%) in the relative amounts of acidic variants in compositions 5-7 in the stability test under severe conditions at 45°C, Figure 5B illustrates the changes (%) in the relative amounts of the main peaks in compositions 5, 6 and 7 in the stability test under severe conditions at 45°C, and Figure 5C illustrates the changes (%) in the relative amounts of the major variants in compositions 5, 6 and 7 in a stability test under severe conditions at 45°C;

Figure 6A illustrates the results of measuring the residual enzymatic activity of the Herceptin subcutaneous formulation (Herceptin SC), trastuzumab + PH20 wild type (HW2) and trastuzumab + PH20 variant HP46 on day 0 and day 1 in a severe stability test at 40°C. and Figure 6B illustrates the results of measuring the residual enzymatic activity of the Herceptin subcutaneous formulation, trastuzumab + PH20 wild type (HW2) and trastuzumab + PH20 variant HP46 on day 0 and day 1 in a severe stability test at 45°C;

Figure 7 illustrates the results of size exclusion chromatography analysis of compositions 8-10 in a stability test under severe conditions at 40°C for 14 days;

Figure 8A illustrates the results of analysis of changes in the size of protein particles of compositions 8-10 using DLS equipment, and Figure 8B illustrates the results of measuring protein aggregation temperatures;

Figure 9A illustrates the weak cation exchange (WCX) chromatogram of composition 8 in a severe stability test at 40°C, Figure 9B illustrates the changes (%) of the relative amounts of acid variants in compositions 8-10 in a severe stability test at 40°C C, Figure 9C illustrates the changes (%) in the relative amounts of major peaks for compositions 8-10 in the 40°C severe stability test, and Figure 9D illustrates the changes (%) in the relative amounts of the major variants in compositions 8-10 in the Stable under harsh conditions at 40°C;

Figure 10 illustrates the changes (%) in the relative enzymatic activity of compositions 8-10 in a severe stability test at 40°C;

Figure 11 illustrates changes in purity of trastuzumab monomers in compositions 11-13 in a severe stability test at 40°C;

Figure 12A illustrates the weak cation exchange (WCX) chromatogram of composition 11 in the 40°C severe stability test; Figure 12B illustrates the changes (%) in the relative amounts of acidic variants in compositions 11-13 in the 40°C severe stability test. C, Figure 12C illustrates the changes (%) in the relative amounts of major peaks in compositions 11-13 in the severe stability test at 40°C, and Figure 12D illustrates the changes (%) in the relative amounts of the major variants in compositions 11-13 in the test for Stable under harsh conditions at 40°C;

Figure 13 illustrates the changes (%) in the relative enzymatic activity of compositions 11-13 in a stability test under severe conditions at 40°C;

Figure 14 illustrates changes in the purity of rituximab monomers in compositions 14-16 in a severe stability test at 40°C;

Figure 15 illustrates changes in the relative enzymatic activity of compositions 14-16 in a severe stability test at 40°C;

Figure 16 illustrates changes in the relative enzymatic activity of compositions 17 and 18 in a severe stability test at 40°C;

Figure 17 illustrates the results of size exclusion chromatography analysis of compositions 19-22 at 40°C;

Figure 18 illustrates changes in the relative enzymatic activity of compositions 19-22 in a severe stability test at 40°C;

Figure 19 illustrates changes in enzymatic activity as a function of changes in pH for recombinant human PH20 and HP46; And

Figure 20 illustrates the results of a pharmacokinetic analysis of a Herceptin subcutaneous formulation (Herceptin SC) and a Herceptin biosimilar candidate (trastuzumab + HP46; Herceptin SC BS) in 9-week-old Sprague-Dawley rats where Herceptin and a Herceptin biosimilar candidate were administered at 18 mg/day. kg each, and the subcutaneous formulation contained 100 units of rHuPH20 and 100 units of HP46 (at pH 5.3).

Detailed Description and Exemplary Embodiments

Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as would be understood by one of ordinary skill in the art to which the invention relates. In general, the nomenclature used in this application is well known and widely used in the art.

An embodiment of the present invention relates to a pharmaceutical composition comprising (a) a drug and (b) a PH20 variant, and the pharmaceutical composition according to the present invention can be used for the prevention or treatment of a disease, and is preferably used for subcutaneous administration.

The human PH20 variant included in the pharmaceutical composition of the present invention has some amino acid residue substitutions in the region corresponding to the alpha helix region and/or linker region, preferably the alpha helix 8 region (S347 to C381), and/or linker region ( A333 to R346) between alpha helix 7 and alpha helix 8, more preferably the amino acid region between T341 and N363, and most preferably T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345- I361 or M345-N363 in the amino acid sequence of wild-type PH20 (having the amino acid sequence of SEQ ID NO: 1), preferably the mature wild-type PH20 protein (having the sequence consisting of L36-S490 in the amino acid sequence of SEQ ID NO: 1).

In the present invention, “wild-type mature PH20 protein” refers to a protein containing amino acid residues L36 to S490 of SEQ ID NO: 1, lacking M1 to T35, which form a signal peptide, and A491 to L509, which are not associated with the basic function of PH20 in the amino acid sequence of wild type PH20 having the sequence SEQ ID NO: 1.

Table 1. Amino acid sequence of wild type PH20 (SEQ ID NO: 1)

In particular, the PH20 variant or fragment thereof included in the pharmaceutical composition according to the present invention contains one or more mutations, preferably substitutions of amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T, and most preferably substituting one or more amino acid residues selected from the group consisting of L354I and N356E in wild-type PH20 having the sequence SEQ ID NO: 1.

In the present invention, the term "PH20 variant" is intended to include mutation of certain amino acid residues, preferably substitution of amino acid residues in the wild-type human PH20 sequence, as well as deletion of certain amino acid residues at the N-terminus and/or C-terminus together with such substitution of amino acid residues, and is used substantially in the same sense as the expression “PH20 variant or fragment thereof.”

The present inventors have tested new variants of PH20 or fragments thereof with increased enzymatic activity and thermostability compared to wild-type PH20, provided through previous studies based on experimental results in which enzymatic activity and protein aggregation temperature (Tagg) at neutral pH increase when The amino acid sequences of the alpha helix 8 region and the linker region between alpha helix 7 and alpha helix 8 of human PH20 are partially replaced by the amino acid sequences of the alpha helix 8 region and the linker region between alpha helix 7 and alpha helix 8 of Hyal1 with high hydrophilicity.

Accordingly, the PH20 variant included in the pharmaceutical composition of the present invention includes substitutions of one or more amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T, preferably substitutions of one or more amino acid residues selected from the group consisting of L354I and N356E, in the amino acid sequence of wild-type PH20 (having the amino acid sequence of SEQ ID NO: 1), preferably the mature wild-type PH20 protein (having the sequence consisting of L36-S490 in the amino acid sequence of SEQ ID NO: 1) ,

in which one or more amino acid residues are replaced in the region corresponding to the alpha helix region and/or linker region, preferably in the region of alpha helix 8 (S347 to C381) and/or linker region (A333 to R346) between the alpha helix 7 and alpha helix 8, more preferably in the amino acid region corresponding to T341-N363, T341-I361, L342-I361, S343-I361, I344-I361, M345-I361 or M345-N363.

Specifically, in the PH20 variant included in the pharmaceutical composition of the present invention, the alpha helix 8 region (S347 to C381) and/or the linker region (A333 to R346) of alpha helix 7 and alpha helix 8 of wild type PH20 , preferably mature wild-type PH20, may be replaced by several amino acid residues in the amino acid sequence corresponding to the Hyal1 region having the sequence SEQ ID NO: 51 (see Tables 2 and 3), but the present invention is not limited to this.

Table 2. Amino acid sequence of wild type Hyal1 (SEQ ID NO: 51).

Table 3. Comparison of alpha helices and amino acid sequences of PH20 and Hyal1 Alpha helix Amino acid sequence of PH20 Amino acid sequence of Hyal1 Alpha helix 1 From P56 to D65 N39 to G48 Alpha helix 3 From S119 to M135 S101 to I117 Alpha helix 4' K161 to N176 K144 to H159 Alpha helix 4 From S180 to R211 P163 to R194 Alpha helix 5 From F239 to S256 P222 to S239 Alpha helix 6 From A274 to D293 K257 to G277 Alpha helix 7 S317 to G332 P299 to G314 Alpha helix 8 S347 to C381 From T329 to C363

More specifically, the PH20 variant or fragment thereof included in the pharmaceutical composition of the present invention preferably includes a substitution of amino acid residue L354I and/or N356E in the amino acid sequence of wild-type PH20, preferably mature wild-type PH20,

and preferably further includes replacing an amino acid residue at one or more positions selected from T341 to N363, especially at one or more positions selected from the group consisting of T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361 and N363, but the present invention is not limited to these, and

more preferably, further includes one or more amino acid residue substitutions selected from the group consisting of T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T and N363G, but the present invention thereby not limited.

Preferably, the PH20 variant or fragment thereof included in the pharmaceutical composition of the present invention may include an amino acid residue substitution selected from M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T,

and may further include substitution of one or more amino acid residues selected from the group consisting of T341S, L342W, S343E, I344N and N363G, but the present invention is not limited to this.

More preferably, the PH20 variant or fragment thereof included in the pharmaceutical composition of the present invention may include, without limitation, any substitution selected from 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;

(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.

In the present specification, an expression that is described by a single letter amino acid residue code together with numbers, such as "S347", means the amino acid residue at the corresponding position in the amino acid sequence SEQ ID NO: 1.

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

The PH20 variant included in the pharmaceutical composition of the present invention is interpreted to include variants in which an amino acid residue at a particular position of the amino acid residue has undergone a conservative substitution.

As used herein, the term “conservative substitution” refers to modifications of a PH20 variant that involve replacing one or more amino acids with amino acids having similar biochemical properties that do not cause a loss of biological or biochemical function of the corresponding PH20 variant.

A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined and well 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 uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine), amino acids with non-polar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), amino acids with beta-branched side chains (for example, threonine, valine and isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan and histidine).

It is expected that the PH20 variant included in the pharmaceutical composition in accordance with the present description will still retain its activity despite the presence of conservative amino acid substitutions.

In addition, a PH20 variant or fragment thereof included in a pharmaceutical composition according to the present invention is interpreted to include PH20 variants or fragments thereof having substantially the same function and/or effect as the PH20 variants or fragments thereof according to the present invention, and having at least 80% or 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% amino acid sequence homology with the PH20 variant or fragment thereof according to the present invention.

The PH20 variants of the present invention have increased levels of expression in animal cells and increased levels of protein refolding, thereby increasing thermostability compared to the mature wild-type PH20 protein. In addition, the enzymatic activity of the PH20 variants was greater than or similar to that of mature wild-type PH20, despite increased thermostability.

In the meantime, it is known that when certain amino acids at the C-terminus of the mature wild-type PH20 protein, such as S490, are further cleaved off, enzymatic activity is reduced, but PH20 variants herein have shown increased thermostability, and increased or similar enzymatic activity at compared to wild-type mature PH20, even if the C-terminus of wild-type mature PH20 has an additional cleaved sequence. In addition, the PH20 variants retained their enzymatic activity when up to five amino acid residues were cleaved from the N-terminal amino acids, indicating that residues beginning with the P41 N-terminus played an important role in protein expression and enzymatic activity.

Accordingly, the PH20 variant included in the pharmaceutical composition of the present invention includes certain amino acid residue substitutions in the alpha helix 8 region (S347 to C381) and/or in the linker region (A333 to R346) between alpha helix 7 and alpha helix 8 of wild type PH20 and further includes deletions of certain amino acid residues at the C-terminus and/or N-terminus, but is not limited to this.

In one embodiment, the PH20 variant included in the pharmaceutical composition of the present invention may include certain deletions of amino acid residues at the N-terminus resulting from cleavage before an amino acid residue selected from the group consisting of M1-P42 at the N-terminus of the amino acid sequence SEQ ID NO: 1, preferably before amino acid residue L36, N37, F38, R39, A40, P41 or P42, and/or deletion of certain amino acid residues at the C-terminus by cleavage after an amino acid residue selected from the group consisting of V455-W509 at the C-terminus, preferably after an amino acid residue selected from the group consisting of V455-S490, and most preferably after the amino acid residue V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490.

The expression "cleavage before L36, N37, F38, R39, A40, P41 or P42 at the N-terminus" means, respectively, all amino acid residues from M1 to T35 immediately before L36, all amino acid residues from M1 to L36 immediately before N37, all amino acid residues M1 to N37 immediately before F38, all amino acid residues M1 to F38 immediately before R39, all amino acid residues M1 to R39 immediately before A40, all amino acid residues M1 to A40 immediately before P41, or all amino acid residues M1 to P41 immediately before P42 in the amino acid sequence SEQ ID NO: 1 is cleaved off and removed. The expression "cleavage before M1 at the N-terminus of SEQ ID NO: 1" means that no cleavage occurs at the N-terminus.

In addition, the expression "cleavage 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 C-terminus" means cleavage and removal of the amino acid residue following V455, C458, D461, C464, I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484 , P486, T488 or S490, respectively, in the sequence SEQ ID NO: 1. For example, a cleavage after S490 means a cleavage between S490 and A491.

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

Table 4. Amino acid sequences of PH20 variants according to the present invention, and their substitution/cleavage properties Name Sequence no. Replacement Subsequence HM1 5 12 amino acids are substituted M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G. HM2 6 7 amino acids replaced Y365F, I367L, L371S, A372G, K374L, M375L and V379A
NM3 7 19 amino acids are replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, N363G, Y365F, I367L, L371S, A372G, K374L, M375L V379A. HM4 8 17 amino acids are replaced by G340V, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T and N363G. NM6 9 11 amino acid residues are replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. HM7 10 16 amino acids are replaced by G340V, T341S L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. NM8 eleven 12 amino acids are replaced by I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. NM9 12 13 amino acids are substituted S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. NM10 13 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. NM11 14 13 amino acid residues are replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, Y365F and I367L. NM12 15 15 amino acid residues are replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, Y365F, I367L, L371S and A372G. NM13 16 11 amino acid residues were replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage was carried out before the F38 residue at the N-terminus. NM14 17 11 amino acid residues were replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage was carried out after the carboxyl group of I465. NM15 18 11 amino acid residues were replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage was carried out after the carboxyl group of F468. NM16 19 11 amino acid residues were replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage was carried out after the carboxyl group of P471. NM17 20 Amino acid residues L36-V47 are replaced by FRGPLLPNR, and 11 amino acids are replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. NM18 21 Amino acids L36-A52 are replaced by FRGPLLPNRPFTTV, and 11 amino acids are replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. NM19 22 14 amino acid residues were replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage was carried out before the F38 residue at the N-terminus and after the K470 residue at the C-con. this . NM20 23 14 amino acid residues were replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage was carried out before the F38 residue at the N-terminus and after the F468 residue at the C-con tse . NM21 24 15 amino acid residues are replaced by T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. HM24 25 The 11 amino acid residues are replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and the cleavage is carried out before the A40 residue at the N-terminus. NM25 26 11 amino acids are substituted M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, cleavage was carried out before the P42 residue at the N-terminus. NM29 27 14 amino acid residues were replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage was carried out before the L36 residue at the N-terminus and after the A467 residue at the C-con. this . NM30 28 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the L36 residue at the N-terminus and after the C464 residue at the C-con. this . NM31 29 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the L36 residue at the N-terminus and after the D461 residue at the C-con. tse . NM32 thirty 14 amino acid residues are replaced by L342W, S343E, i344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and i361T, and clearance was carried out in front on s- end. NM33 31 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the L36 residue at the N-terminus and after the V455 residue at the C-terminus this . NM34 32 15 amino acid residues are replaced by T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the K4 residue 70 on C -end. NM35 33 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the P472 residue at the C-con. this . NM36 34 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the M473 residue at the C-con. this . NM37 35 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue to N- and after the E474 residue to C-con tse. NM38 36 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the T475 residue at the C-terminal end this . NM39 37 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the E476 residue at the C-con. this . NM40 38 The 11 amino acid residues are replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and the cleavage is carried out before the N37 residue at the N-terminus. NM41 39 The 11 amino acid residues are replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and the cleavage is carried out before the R39 residue at the N-terminus. NM42 40 The 11 amino acid residues are replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and the cleavage is carried out before the P41 residue at the N-terminus. NM43 41 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the I465 residue at the C-con. this . HM44 42 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the D466 residue at the C-con. this . NM45 43 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, cleavage was carried out before the F38 residue at the N-terminus and after the A467 residue at the C-terminus . NM46 44 15 amino acid residues are replaced by T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the F4 residue 68 on C -end. NM47 45 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the P478 residue at the C-con. this . HM48 46 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, cleavage was carried out before the F38 residue at the N-terminus and after the I480 residue at the C-terminus . HM49 47 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the Y482 residue at the C-con. this . NM50 48 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the A484 residue at the C-con this . NM51 49 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the P486 residue at the C-con. this . NM52 50 14 amino acid residues are replaced by L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and cleavage is carried out before the F38 residue at the N-terminus and after the T488 residue at the C-con. this .

Meanwhile, previous studies have reported that the enzymatic activity of wild-type PH20 varies depending on the cleavage positions of amino acid residues located at the C-terminus. However, herein, the particular alpha helix that forms the secondary structure of PH20 has been replaced by the alpha helix of another human hyaluronidase, with the construction of PH20 variants having higher stability than wild type PH20, and in these variants the interaction between the substituted alpha helix domain and other secondary structures of PH20 show a different character from that of wild-type PH20, such that the variants have some or higher levels of enzymatic activity, regardless of the position of the C-terminal cleavage.

Additionally, herein, attempts have been made to increase the expression of the recombinant PH20 protein by using the signal peptide of other proteins exhibiting high levels of protein expression in animal cells instead of the original human PH20 signal peptide.

Thus, in another embodiment, the PH20 variant included in the pharmaceutical composition of the present invention may include at its N-terminus a signal peptide derived from human hyaluronidase-1 (Hyal1), human growth hormone or human serum albumin instead of the wild-type PH20 signal peptide type, which consists of M1-T35, and may preferably include, as shown in Table 5, a human growth hormone-derived signal peptide having the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA in accordance with SEQ ID NO: 2, human serum albumin-derived signal peptide , having the amino acid sequence MKWVTFISLLFLFSSAYS according to SEQ ID NO: 3, or a signal peptide derived from human Hyal1 having the amino acid sequence MAAHLLPICALFLTLLDMAQG according to SEQ ID NO: 4, but the present invention is not limited to this.

Table 5. Amino acid sequence of human growth hormone, human serum albumin, or human Hyal1 signal peptide Origin of signal peptide Amino acid sequence SEQ ID No.: Human growth hormone MATGSRTSLLLAFGLLCLPWLQEGSA 2 Human serum albumin MKWVTFISLLFLFSSAYS 3 Hyal1 person MAAHLLPICALFLTLLDMAQG 4

Among the PH20 variants included in the pharmaceutical composition of the present invention, the variant having a 6xHis tag attached to the C-terminus was designated as HM, and the variant without the 6xHis tag was designated as HP. In addition, the mature wild-type PH20 protein (L36-S490) with a 6xHis tag attached to its C-terminus was designated as WT, and the mature wild-type PH20 (L36-Y482) without the 6xHis tag and in which the C-terminus split off after Y482, was designated HW2.

HP46 (SEQ ID NO: 44) is a variant of human PH20 obtained by modeling the protein structure using Hyal1 (PDB ID: 2PE4) (Chao et al., 2007), which is a human hyaluronidase with a known tertiary protein structure, followed by by replacing the amino acid sequence of alpha helix 8 and the linker region between alpha helix 7 and alpha helix 8 with the amino acid sequence of Hyal1, and with cleavage of the N-terminus at F38 and cleavage of the C-terminus after F468. In particular, alpha helix 8 is located outside the tertiary structure of the PH20 protein and interacts less with neighboring alpha helices or beta strands than other alpha helices. Generally, enzymatic activity and thermostability have a trade-off relationship between them, and thus, the higher the thermostability of a protein, the lower the enzymatic activity, whereas when enzymatic activity increases due to improved flexibility of the protein structure, thermostability tends to decrease. However, the specific activity of HP46 measured by turbidimetric assay at pH 7.0 was approximately 46 U/μg, which is estimated to be approximately twice that of wild-type PH20, where the activity was approximately 23 U/μg.

The thermal stability of a protein can be assessed based on the melting temperature Tm, at which 50% of the protein's tertiary structure is denatured, and the aggregation temperature Tagg, at which aggregation between proteins occurs. In general, the aggregation temperature of a protein is usually lower than its melting temperature. Alpha helix 8 of Hyal1 exhibits greater hydrophilicity than alpha helix 8 of PH20. The substituted alpha helix 8 of Hyal1 increases the surface hydrophilicity of the HP46 protein, thereby causing an aggregation delay effect between proteins that occurs due to hydrophobic interactions, and thus the aggregation temperature is 51°C, which is observed to be an increase of 4 .5°C compared to the wild-type PH20 aggregation temperature of 46.5°C.

HP46 is a variant in which the amino acid residues in alpha helix 8 and the linker region between alpha helix 7 and alpha helix 8 are replaced, with T341 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 approximately 2-fold, and even in a gel substrate assay, the resulting variant was confirmed to hydrolyze 5-6 times more hyaluronic acid than wild type PH20. The substrate gel assay involves protein denaturation and refolding processes, which means that protein tertiary structure refolding and HP46 reduction are enhanced compared to wild-type PH20.

The amount of the PH20 variant in the pharmaceutical composition of the present invention is at least 50 U/ml, preferably in the range of 100 U/ml to 20,000 U/ml, more preferably in the range of about 150 U/ml to 18,000 U. /ml, even more preferably in the range from 1000 U/ml to 16000 U/ml, and most preferably in the range from 1500 U/ml to 12000 U/ml.

Examples of the drug included in the pharmaceutical composition according to the present invention include, but are not limited to, protein drugs, antibody drugs, small molecules, aptamers, RNA interference drugs, antisense oligonucleotides and cellular therapeutic agents such as chimeric antigen receptor (CAR)-T or CAR-natural killer (NK), and not only currently commercially available drugs can be used, but also drugs in clinical trials or in development.

As the drug, a protein drug or an antibody drug can preferably be used.

A “protein drug” included in the pharmaceutical composition according to the present invention is a drug that is composed of amino acids and thus exhibits a therapeutic or prophylactic effect through the activity of a protein, is a drug that is composed of a protein other than antibody drug, and may be selected from the group consisting of a cytokine, a therapeutic enzyme, a hormone, a soluble receptor and a fusion protein thereof, insulin or an analog thereof, bone morphogenetic protein (BMP), erythropoietin and a serum protein, but the present disclosure is not limited.

The cytokine 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.

The therapeutic enzyme may include, but is not limited to, β-glucocerebrosidase and agalsidase-β.

The soluble receptor included in the pharmaceutical composition of the present invention is an extracellular domain of the receptor, and its fusion protein is a protein in which an Fc region or the like from an antibody is fused to a soluble receptor. A soluble receptor is a soluble form of a receptor to which a disease-associated ligand binds, and examples thereof include a form in which the Fc region is fused to a soluble TNF-α receptor (eg, a product containing the ingredient etanercept, and similar forms), a form in in which the Fc region is fused to a soluble VEGF receptor (a product containing the ingredient alefacept and similar forms), a form in which the Fc region is fused to CTLA-4 (for example, a product containing the ingredient abatacept or belatacept and similar forms), a form in which in which the Fc region is fused to a soluble interleukin 1 receptor (for example, a product containing the ingredient rilonacept and similar forms), and a form in which the Fc region is fused to a soluble LFA3 receptor (for example, a product containing the ingredient alefacept and similar forms), but the present invention is not limited to this.

A hormone included in the pharmaceutical composition according to the present invention refers to a hormone administered to the body or an analogue thereof for the treatment or prevention of diseases caused by hormone deficiency and the like, and examples thereof include human growth hormone, estrogen and progesterone, but not limited to them.

The whey protein included in the pharmaceutical composition of the present invention is a protein present in plasma and includes both proteins extracted from plasma and produced recombinant proteins, and examples thereof may include fibrinogen, von Willebrand factor, albumin, thrombin, factor II (FII), factor V (FV), factor VII (FVII), factor IX (FIX), factor X (FX) and factor XI (FXI), but are not limited to.

The antibody drug included in the pharmaceutical composition according to the present invention may be a monoclonal antibody drug or a polyclonal antibody drug.

The monoclonal antibody drug of the present invention is a protein comprising a monoclonal antibody and a monoclonal antibody fragment that are capable of specifically binding to an antigen associated with a particular disease. A monoclonal antibody also includes a bispecific antibody, and a protein comprising a monoclonal antibody or fragment thereof conceptually includes an antibody-drug conjugate (ADC).

Examples of disease-specific antigen include 4-1BB, integrin, amyloid beta, angiopoietin (angiopoietin 1 or 2), angiopoietin analog 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, folate receptor, GD2, GM-CSF, HER2, HER3, interferon receptor, interferon gamma, IgE, IGF-1 receptor, interleukin 1, interleukin 2 receptor, interleukin 4 receptor, interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6 receptor, interleukin 7, interleukin 12/23, interleukin 13, interleukin 17A, interleukin 17A receptor, interleukin 31 receptor, interleukin 36 receptor, LAG3, LFA3, LAG3, LFA3, PVSK9, PD-1, PD-L1, RANK- L, SLAMF7, tissue factor, TNF, VEGF, VEGF receptor and von Willebrand factor (vWF), but the present invention is not limited to these.

The following are, without limitation, proteins, including monoclonal antibodies or fragments of monoclonal antibodies against antigens associated with a particular disease:

utomilumab as an antibody against 4-1BB;

natalizumab, etrolizumab, vedolizumab and bimagrumab as anti-integrin antibodies;

bapineizumab, crenezumab, solanezumab, aducanumab and gantenerumab as anti-amyloid beta antibodies;

anti-angiopoietin antibodies such as AMG780 against angiopoietin 1 and 2, MEDI 3617 and nesvacumab against angiopoietin 2 and vanucizumab, which is a bispecific antibody against angiopoietin 2 and VEGF;

evinacumab as an antibody against angiopoietin analogue 3;

tabalumab, lanalumab and belimumab as antibodies against B-cell activating factor (BAFF);

omburtamab as an anti-B7-H3 antibody;

ravulizumab and eculizumab as antibodies against complement 5;

mogamulizumab as an anti-CCR4 antibody;

telixizumab, teplizumab and muromonab as anti-CD3 antibodies, tebentafusp as a bispecific antibody against GP100 and CD3, blinatumomab as a bispecific antibody against CD19 and CD3, and REGN1979 as a bispecific antibody against CD20 and CD3;

ibalizumab and zanolimumab as anti-CD4 antibodies;

itolizumab as an anti-CD6 antibody;

efalizumab as an anti-CD11a antibody;

inebilizumab, tafasitamab and loncastuximab tesirine, which is an ADC, as anti-CD19 antibodies;

ocrelizumab, ublituximab, obinutuzumab, ofatumumab, rituximab, tositumomab and ibritumomab tiuxetan, which is an ADC, as anti-CD20 antibodies;

epratuzumab, inotuzumab ozogamicin, which is an ADC, and moxetumomab pasudotox as anti-CD22 antibodies;

brentuximab vedotin as an anti-CD30 ADC;

vadastuximab, talirine and gemtuzumab ozogamicin as anti-CD33 ADCs;

daratumumab and isatuximab as anti-CD38 antibodies;

alemtuzumab as an anti-CD52 antibody;

crizanlizumab as an anti-CD62 antibody;

polatuzumab vedotin as an ADC against CD79b;

galiximab as an anti-CD80 antibody;

eptinezumab, fremanezumab, galcanezumab and erenumab as anti-CGRP antibodies;

zolbetuximab as an anti-claudin-18 antibody;

lampalizumab as an antibody against complement factor D;

tremelimumab, zalifrelimab and ipilimumab as anti-CTLA4 antibodies;

rovalpituzumab tesirine as an anti-DLL3 ADC;

cetuximab, depatuxizumab, zalutumumab, necitumumab and panitumumab as anti-EGF receptor antibodies;

emicizumab as a bispecific antibody against coagulation factor IX and coagulation factor X, which are hemophilia factors;

nipocalimab and rozanolixizumab as anti-Fc receptor antibodies;

burosumab as an anti-FGF23 antibody;

farletuzumab as an anti-folate receptor antibody and mirvetuximab soravtansine as an anti-folate receptor ADC;

dinutuximab and naxitamab as antibodies against GD2;

otilimab as an anti-GM-CSF antibody;

margetuximab, pertuzumab and trastuzumab as anti-HER2 antibodies and trastuzumab deruxtecan, trastuzumab emtansine and trastuzumab duocarmazine as anti-HER2 ADCs;

patritumab as an anti-HER3 antibody;

anifrolumab as an anti-interferon receptor antibody;

emapalumab as an anti-interferon gamma antibody;

ligelizumab and omalizumab as anti-IgE antibodies;

dalotuzumab, figitumumab and teprotumumab as antibodies against the IGF-1 receptor;

gebokizumab and canakinumab as antibodies against interleukin 1;

daclizumab and basiliximab as antibodies against interleukin 2 receptor;

dupilumab as an anti-interleukin 4 receptor antibody;

mepolizumab and reslizumab as antibodies against interleukin 5;

benralizumab as an anti-interleukin 5 receptor antibody;

clazakizumab, olokizumab, sirukumab and siltuximab as antibodies against interleukin 6;

sarilumab, satralizumab, tocilizumab and REGN88 as anti-interleukin 6 receptor antibodies;

secukinumab as an anti-interleukin 7 antibody;

ustekinumab and briakinumab as antibodies against interleukin 12/23;

lebrikizumab and tralokinumab as antibodies against interleukin 13;

ixekizumab and bimekizumab as antibodies against interleukin 17A;

brodalumab as an anti-interleukin 17A receptor antibody;

brazicumab, guselkumab, risankizumab, tildrakizumab and mirikizumab as anti-interleukin 23 antibodies;

nemolizumab as an anti-interleukin 31 receptor antibody;

spesolimab as an antibody against interleukin 36 receptor;

relatlimab as an anti-LAG3 antibody;

narsoplimab as an anti-NASP2 antibody;

fasinumab and tanezumab as anti-NGF antibodies;

alirocumab, evolocumab and bococizumab as antibodies against PVSK9;

lambrolizumab, balstilimab, camrelizumab, cemiplimab, dostarlimab, prolgolimab, shintilimab, spartalizumab, tislelizumab, pembrolizumab and nivolumab as anti-PD-1 antibodies;

atezolizumab, avelumab, envafolimab and durvalumab as anti-PD-L1 antibodies and bintrafusp alfa as bispecific antibodies against TGF beta and PD-L1;

denosumab as an anti-RANK-L antibody;

elotuzumab as an anti-SLAMF7 antibody;

concizumab and marstacimab as antibodies against tissue factor;

antibodies against TNF, in particular TNFα, including infliximab, adalimumab, golimumab, the antibody fragment certolizumab pegol and ozoralizumab, which is a bispecific antibody against TNF and albumin;

anti-VEGF antibodies, including brolucizumab, ranibizumab, bevacizumab and faricimab, which are bispecific antibodies against VEGF and Ang2;

ramucirumab as an anti-VEGF receptor antibody; And

caplacizumab as an anti-vWF antibody.

Meanwhile, overexpression of human epidermal growth factor receptor 2 (HER2), which promotes cell division, is observed in approximately 20-25% of breast cancer patients, and breast cancers with overexpression of HER2 receptor progress rapidly, are aggressive and have poor response to chemotherapy compared to breast cancer with low HER2 expression and therefore has a poor prognosis. Trastuzumab, which is a monoclonal antibody drug targeting HER2, 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 US Food and Drug Administration (FDA) in 1998 for the treatment of breast cancer in the US, and in 2003 by the Korean Food and Drug Administration (KFDA). Since then, the efficacy of trastuzumab has also been recognized in HER2-overexpressing gastric cancer and has therefore been used as a therapeutic agent in gastric cancer.

Roche's formulation of Herceptin intravenous injection (commercial name: Herceptin) consists of 440 mg trastuzumab as the main ingredient, and lyophilized trastuzumab is mixed with saline and administered intravenously. On the other hand, the trastuzumab subcutaneous injection formulation (commercial name Herceptin SC) is a 5 ml liquid formulation and contains 600 mg (120 mg/ml) trastuzumab as the main ingredient, and includes 20 mM histidine (pH 5. 5), 210 mM trehalose, 10 mM methionine, 0.04% polysorbate 20 and 10,000 rHuPH20 units (2000 U/ml, 0.004%, 40 μg/ml).

The shelf life of subcutaneous Herceptin preparations is 21 months. The intravenous trastuzumab formulation is in lyophilized form and has a shelf life of 30 months, and the subcutaneous trastuzumab formulation is in liquid form and has a short shelf life of 21 months. For this reason, it may be determined that the stability of one or more of trastuzumab and recombinant human hyaluronidase PH20 in liquid formulations is limited.

In this context, in the present invention, taking into account the characteristics of the PH20 variant according to the present invention, wherein compared with the wild type human hyaluronidase PH20 and the recombinant human PH20 available from Halozyme, the PH20 variant not only has increased enzymatic activity, but also has a high measured aggregation temperature protein, thus demonstrating increased thermal stability, the shelf life of the subcutaneous injection preparation is set to a long period, preferably 21 months or more.

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

The polyclonal antibody included in the pharmaceutical composition according to the present invention is preferably a serum antibody extracted from serum, such as immunoglobulin, but is not limited to it.

In the case of a small molecule compound, any drug that requires rapid action for prevention or treatment can be used without restrictions. For example, morphine-based painkillers can be used (Thomas et al., 2009). Additionally, when used as a therapeutic agent for tissue necrosis caused by anticancer drugs, the small molecule compound can be used alone or in combination with antidotes such as vinca alkaloids and taxanes (Kreidieh et al., 2016).

The pharmaceutical composition according to the present invention may further include one or more components selected from the group consisting of a buffer, a stabilizer and a surfactant.

The buffer included in the composition in accordance with the present invention can be used without limitation, provided that it achieves a pH of 4 to 8, preferably 5 to 7, and the buffer is preferably one or more buffers selected from the group consisting from malate, formate, citrate, acetate, propionate, pyridine, piperazine, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), histidine, Tris, bis-Tris, phosphate, ethanolamine, carbonate, piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES), imidazole, bis-Tris propane, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)propanesulfonic acid) (MOPS), hydroxyethylpiperazine ethanesulfonic acid (HEPES), pyrophosphate and triethanolamine, more preferably a histidine buffer, for example, but not limited to, L-histidine/HCl.

The buffer concentration may range from 0.001 to 200 mM, preferably from 1 mM to 50 mM, more preferably from 5 mM to 40 mM, and most preferably from 10 mM to 30 mM.

The stabilizers in the composition of the present invention may be used without limitation as long as they are commonly used in the art to stabilize proteins, and preferably the stabilizers may be, for example, one or more stabilizers selected from the group consisting of carbohydrates, sugars or their hydrates, sugar alcohols or hydrates, and amino acids.

The carbohydrates, sugars or sugar alcohols used as a stabilizer may be one or more selected from the group consisting of trehalose or its hydrates, sucrose, saccharin, glycerol, erythritol, threitol, xylitol, arabitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, polyglycitol, cyclodextrin, hydroxylpropylcyclodextrin and glucose, but are not limited to.

The amino acid may be one or more amino acids selected from the group consisting of glutamine, glutamic acid, glycine, lysine, lysylysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine, threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolysine, histidine and alanine, but are not limited to.

The concentration of sugars or sugar alcohols used as a stabilizer in the pharmaceutical composition according to the present description may range from 0.001 mM to 500 mM, preferably from 100 mM to 300 mM, more preferably from 150 mM to 250 mM, and most preferably from 180 mM up to 230 mM and in particular may be around 210 mM.

Moreover, the concentration of amino acids used as a stabilizer in the pharmaceutical composition of the present invention may range from 1 mM to 100 mM, preferably from 3 mM to 30 mM, more preferably from 5 mM to 25 mM, and most preferably from 7 mM up to 20 mM, and in particular may be in the range from 8 mM to 15 mM.

The composition according to the invention may further include a surfactant.

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 ethers, e.g. polyoxyethylene monolauryl ether, alkylphenyl polyethylene ether [Triton-X ], and polyoxyethylene-polyoxypropylene copolymer [Poloxamer and Pluronic] and sodium dodecyl sulfate (SDS), but is not limited to.

More preferably, polysorbate may be used. The polysorbate may be, but is not limited to, polysorbate 20 or polysorbate 80.

The concentration of the nonionic surfactant in the pharmaceutical composition according to the present invention may range from 0.0000001% (w/v) to 0.5% (w/v), preferably from 0.000001% (w/v). /vol.) to 0.4% (wt./vol.), more preferably from 0.00001% (wt./vol.) to 0.3% (wt./vol.) and most preferably from 0.001% ( w/v) up to 0.2% (w/v).

In one embodiment, the pharmaceutical composition of the present invention may include 50-350 mg/ml of an antibody, for example, an anti-HER2 antibody or an anti-immune checkpoint antibody, a histidine buffer providing a pH of 5.5±2.0; 10-400 mM α,α-trehalose, 1-50 mM methionine and 0.0000001% (w/v) to 0.5% (w/v) polysorbate.

In a more specific embodiment, the pharmaceutical composition of the present invention may include 120 mg/ml anti-HER2 antibody or anti-immune checkpoint antibody, 20 mM histidine buffer that provides a pH of 5.5 ± 2.0; 210 mM α,α-trehalose, 10 mM methionine and 2000 U/ml variant PH20, and may further include from 0.005% (w/v) to 0.1% (w/v) polysorbate.

The pharmaceutical composition according to the present invention can be administered by intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration and the like, and subcutaneous administration is preferably carried out by subcutaneous injection; and it is most preferable to use the pharmaceutical composition as an injection preparation for subcutaneous administration.

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

The injectable formulation for subcutaneous injection can be provided in a form ready for injection without additional dilution process, and can be provided placed in a pre-filled syringe, glass ampoule or plastic container.

The present invention also relates to a method of treating a disease using a pharmaceutical composition or preparation in accordance with the present invention.

The disease that can be treated with the pharmaceutical composition or composition of the present invention is not particularly limited, and there is no limitation if it is a disease that can be treated with the drug used in combination with the PH20 variant of the present invention.

A disease that can be treated using a pharmaceutical composition or composition in accordance with the present description may be, but is not limited to, cancer or an autoimmune disease.

The cancer or carcinoma that can be treated with the pharmaceutical composition or formulation in accordance with the present invention is not particularly limited and includes both solid cancers and hematological malignancies. Examples of such cancers include skin cancer such as melanoma, liver cancer, hepatocellular carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer , colon cancer, cervical cancer, brain tumor, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cancer cervical, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and glioma. Preferably, the cancer that can be treated using the pharmaceutical composition or formulation of the present invention may be selected from the group consisting of, but not limited to, gastric cancer, colorectal cancer, breast cancer, lung cancer and kidney cancer.

Autoimmune diseases that can be treated with the pharmaceutical composition or formulation in accordance with the present invention include rheumatoid arthritis, asthma, psoriasis, multiple sclerosis, allergic rhinitis, Crohn's disease, ulcerative colitis, systemic lupus erythematous, type I diabetes, inflammatory bowel disease (IBD) and atopic dermatitis, but not limited to.

The present invention also provides a method of treating a disease, comprising using a pharmaceutical composition or formulation according to the present invention to a subject in need of treatment, and the present invention further provides using a pharmaceutical composition or composition according to the present invention for treating a disease.

Unless otherwise defined herein, technical terms and scientific terms used herein have meanings commonly understood by those skilled in the art. In addition, repeated descriptions of the same technical configuration and operation as in the prior art will be omitted.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are provided for illustrative purposes only, and those skilled in the art will appreciate that these examples should not be construed as limiting the scope of the present invention.

Examples

Example 1. Composition development

Four formulations of trastuzumab were prepared for subcutaneous administration, as shown in Table 6. Positions 1-4 contain a total of 120 mg/ml trastuzumab and consist of 20 mM histidine/histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine and PH20 variant. The difference between compositions 1-4 is the concentration of nonionic surfactant, where composition 1 contains 0% polysorbate 20, composition 2 contains 0.005% polysorbate 20, composition 3 contains 0.04% polysorbate 20, and composition 4 contains 0.1 % polysorbate 20.

Table 6. Composition of compositions Composition 1 Composition 2 Composition 3 Composition 4 Antibody Trastuzumab (120 mg/ml) Buffer 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 from SEQ ID NO: 44 (2,000 U/ml)

Example 2 Spectrophotometer Analysis

Compositions 1 to 4 were left for 14 days at 45°C, and changes in protein concentration were analyzed using a spectrophotometer manufactured by Beckman. Each sample was diluted with distilled water so that the sample concentration was 0.4 mg/mL, and then the protein absorbance was measured at 280 nm using a spectrophotometer. In a stability test under severe conditions, ie at 45°C for 14 days, there was no significant change in protein concentration in compositions 1-4. However, hyaluronidase activity decreased rapidly at 45°C, and thus the enzymatic activity was not measured in the present example (see FIG. 6).

Example 3. Study of the ratio of trastuzumab monomers in each composition using size exclusion chromatography

For size exclusion chromatography analysis, a Shimadzu Prominence HPLC system and a TSK-gel G3000SWXL column (7.8 × 300 mm, 5 μm) and a TSK precolumn (6.0 × 4.0 mm, 7 μm) were used. The mobile phase was 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride. The analysis was performed for 35 minutes in isocratic separation mode at a flow rate of 0.5 ml/min. The sample was diluted with the analytical solvent to give a final concentration of 10 mg/mL, and after 20 μL was injected into the HPLC column, the absorbance at 280 nm of the column eluate was recorded. The ratio of trastuzumab monomers in the HPLC chromatogram was calculated and plotted.

When subjected to gel chromatography analysis in a stability test under stringent conditions, i.e. at 45°C for 14 days, compositions 1-4 showed 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 (about 1.5%), and there was no significant difference between compositions. In conclusion, when analyzed by size exclusion chromatography in a stability test under stringent conditions, i.e. 45°C, there were no significant differences in stability profiles between formulations depending on the concentration of polysorbate 20 (0-0.1% (w/v)) (see Fig. 1).

Example 4: Measurement of protein aggregation temperature of compositions containing trastuzumab and HP46

Dynamic light scattering (DLS) is used to analyze the heat-related denaturation properties of proteins. In the present experiment, the change in the size of a protein molecule according to a change in temperature was measured and used to calculate the temperature of protein aggregation. For DLS analysis, a Zetasizer-nano-ZS instrument from Malvern and a quartz cuvette (ZEN2112) were used. During the assay, the temperature was increased from 25°C to 85°C at 1°C intervals, and the sample was diluted to 1 mg/mL using each formulation buffer, and then 150 μL of sample was added to the assay cuvette.

The aggregation temperature in composition 1, not containing polysorbate 20, was 74°C, and the aggregation temperature in compositions 2-4 was 76°C (see Fig. 2).

Example 5 WCX Analysis of Compositions Containing Trastuzumab and HP46

For chromatographic analysis of WCX, an HPLC system available from Shimadzu Prominence was used, and the columns used were a TSKgel CM-STAT column (4.6 x 100 mm, 7 µm), and a TSKgel CMSTAT pre-column (3.2 mm i.d. x 1. 5 cm), etc. Mobile phase A was 10 mM sodium phosphate (pH 7.5) and mobile phase B was 10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. The assay was carried out over 55 minutes with 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 so that the final concentration was 1.0 mg/ml, 80 μl of the sample was injected into the HPLC, and then the absorbance of the column eluate was recorded at 280 nm. The ratio of trastuzumab monomers in the HPLC chromatogram was calculated and plotted.

Formulations 1-4 showed a similar pattern of change when the WCX assay was performed in a stability test under stringent conditions, i.e. at 45°C for 14 days. Specific changes include an increase in the relative abundance of acidic variants (about 30% change over 14 days), a decrease in the relative abundance of the main peak (about 44% change over 14 days), and an increase in the relative abundance of basic variants (about 15% change over 14 days). , and there was no significant difference depending on the composition. In conclusion, in the WCX assay in the stability test under stringent conditions, i.e. at 45°C, protein stability according to polysorbate 20 (0-0.1%) was similar (see Figure 3).

Example 6. Composition development

Three types of subcutaneous trastuzumab formulations were prepared as described in Table 7. Formulations 5 to 7 generally included 120 mg/ml trastuzumab, 20 mM histidine/histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine and HP46. The difference between compositions 5-7 is the stabilizer ingredient 3: composition 5 - 0.04% polysorbate 20, composition 6 - 50 mM Lys-Lys, and composition 3 - glycine.

Table 7. Composition of compositions Composition 5 Composition 6 Composition 7 Antibody Trastuzumab (120 mg/ml) Buffer 20 mM histidine/histiine-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 from SEQ ID NO:44 (2,000 U/ml)

Example 7 Spectrophotometer Analysis

Compositions 5-7 were left for 14 days at 45°C, and changes in protein concentration were analyzed using a spectrophotometer manufactured by Beckman. Each sample was diluted with distilled water so that the sample concentration was 0.4 mg/mL, and then the protein absorbance was measured at 280 nm using a spectrophotometer. In a stability test under stringent conditions, ie at 45°C for 14 days, there was no significant change in protein concentration in compositions 5-7. However, hyaluronidase activity decreased rapidly at 45°C and therefore enzymatic activity was not measured in the present example (see FIG. 6).

Example 8. Study of the ratio of trastuzumab monomers in each composition using size exclusion chromatography

For size exclusion chromatography analysis, an HPLC system from Shimadzu Prominence was used, using a TSK-gel G3000SWXL column (7.8 × 300 mm, 5 μm) and a TSK precolumn (6.0 × 4.0 mm, 7 μm). The mobile phase was 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride. Isocratic separation mode was used at a flow rate of 0.5 ml/min for 35 minutes. The sample was diluted with the analytical solvent to give a final concentration of 10 mg/mL, and the absorbance was measured at 280 nm after 20 μL of the sample was injected into the HPLC column. The ratio of trastuzumab monomers in the HPLC chromatogram was calculated and plotted.

When gel chromatography analysis was performed in a stability test under stringent conditions, that is, at 45°C for 14 days, compositions 5-7 showed 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 (about 1.5%), and there was no significant difference depending on the composition. In conclusion, as a result of gel chromatography analysis in a stability test under stringent conditions, that is, at 45°C, similar protein stability was shown in compositions with 0.04% polysorbate 20, 50 mM Lys-Lys and 50 mM glycine. (see figure 4).

Example 9. WCX chromatographic analysis of compositions containing trastuzumab and HP46

For WCX chromatographic analysis, an HPLC system from Shimadzu Prominence was used, and the columns used were TSK-gel CM-STAT (4.6 x 100 mm, 7 μm), TSK-gel CMSTAT pre-column (3.2 mm i.d. x 1. 5 cm), etc. Mobile phase A was 10 mM sodium phosphate (pH 7.5) and mobile phase B was 10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. The analysis was performed for 55 minutes using a linear concentration gradient separation mode of 0-30% at a flow rate of 0.8 ml/min. The sample was diluted with mobile phase A so that the final concentration was 1.0 mg/ml, 80 μl of the sample was injected into the HPLC, and then the absorbance was recorded at 280 nm. The ratio of trastuzumab monomers in the HPLC chromatogram was calculated and plotted.

Formulations 5-7 showed a similar pattern of change when the WCX assay was performed in a stability test under stringent conditions, i.e. at 45°C for 14 days. Specific changes include an increase in the relative abundance of acidic variants (about 30% change over 14 days), a decrease in the relative abundance of the main peak (about 44% change over 14 days), and an increase in the relative abundance of basic variants. (change of approximately 15% over 14 days), and there was no significant difference depending on the composition. In conclusion, by performing the WCX assay in a stability test under stringent conditions, i.e. at 45°C, similar protein stability was shown in formulations with 0.04% polysorbate 20, 50 mM Lys-Lys and 50 mM glycine (see Table 1). Fig.5).

Example 10. Evaluation of the stability of HP46 according to temperatures of 40°C and 45°C in trastuzumab and HP46 subcutaneous formulations

To evaluate the stability of HP46 in subcutaneous trastuzumab formulations, trastuzumab (120 mg/mL) and PH20 (2000 U/mL) were mixed. At this time, the buffer used contained 20 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine and 0.04% polysorbate 20. The enzymatic activity of the control sample was measured on day 0, and the experimental samples were left at 40 °C or 45 °C for 1 day, and then the enzymatic activity of each sample was measured.

Herceptin subcutaneous formulations, trastuzumab + HW2 and trastuzumab + HP46 were each left at 40°C for 1 day and then hyaluronidase activity was measured, and the corresponding samples showed activity of 51%, 47% and 94%, indicating that HP46 had the greatest thermal stability at 40°C (see Fig. 6). Next, the drugs Herceptin SC, Trastuzumab + HW2 and Trastuzumab + HP46 were left at 45°C for 1 day, and then the hyaluronidase activity was measured, and as a result, Herceptin SC and Trastuzumab + HW2 showed no enzymatic activity, but enzymatic activity the drug trastuzumab + HP46 was maintained (see Fig. 6).

Example 11. Composition development

Three formulations of trastuzumab were prepared for subcutaneous administration as shown in Table 8. Formulations 8-10 generally contained 120 mg/ml trastuzumab, 20 mM histidine/histidine-HCl (pH 5.5); 210 mM trehalose, 10 mM methionine and PH20 option. The difference between formulations 8-10 is the concentration of nonionic surfactant, with formulation 8 containing 0% polysorbate 20, formulation 9 containing 0.005% polysorbate 20, and formulation 10 containing 0.04% polysorbate 20.

Table 8. Composition of compositions Composition 8 Composition 9 Composition 10 Antibody Trastuzumab (120 mg/ml) Polysorbate 20 0% 0.005% 0.04% Buffer 20 mM histidine/histiine-HCl Stabilizer 1 210 mM trehalose Stabilizer 2 10 mM methionine pH 5.5 Hyaluronidase HP46 from SEQ ID NO:44 (2,000 U/ml)

Example 12 Analysis Using a Spectrophotometer

Formulations 8 to 10 were left for 14 days at 40°C, and changes in protein concentration were analyzed using a spectrophotometer manufactured by Beckman. Each sample was diluted with distilled water so that the sample concentration was 0.4 mg/mL, and then the protein absorbance was measured at 280 nm using a spectrophotometer. In a stability test under severe conditions, i.e. at 40°C for 14 days, there was no significant change in protein concentration in compositions 8-10.

Example 13 Analysis of the ratio of trastuzumab monomers in each composition using size exclusion chromatography

For size exclusion chromatography analysis, an HPLC system available from Shimadzu Prominence was used, and the columns used were TSK-gel G3000SWXL (7.8 × 300 mm, 5 μm) and TSK precolumn (6.0 × 4.0 mm, 7 μm) . The mobile phase was 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride. The analysis was performed for 35 minutes in isocratic separation mode at a flow rate of 0.5 ml/min. The sample was diluted with the analytical solvent to give a final concentration of 10 mg/mL, and the absorbance was measured at 280 nm after 20 μL of the sample was injected into the HPLC column. The ratio of trastuzumab monomers in the HPLC chromatogram was calculated and plotted.

When gel chromatography analysis was performed in a stability test under stringent conditions, that is, at 40°C for 14 days, compositions 8-10 showed 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 less than 1.0%), and there was no significant difference between compositions. In conclusion, when analyzed by size exclusion chromatography in a stability test under stringent conditions, i.e. at 40°C, there were no significant differences in stability profiles between formulations depending on the concentration (0-0.04%) of polysorbate 20 (see Fig.7).

Example 14 Measurement of protein aggregation temperature for compositions containing trastuzumab and HP46

Dynamic light scattering (DLS) is used to analyze heat-related denaturation properties of proteins in the field of protein drugs. In the present experiment, the change in the size of a protein molecule according to a change in temperature was measured and used to calculate the temperature of protein aggregation. For DLS analysis, a Zetasizer-nano-ZS instrument from Malvern and a quartz cuvette (ZEN2112) were used. During the assay, the temperature was increased from 25°C to 85°C at 1°C intervals, and the sample was diluted to 1 mg/mL using each formulation buffer, and then 150 μL of sample was added to the assay cuvette.

The aggregation temperature of composition 8, which did not contain polysorbate 20, was 78.3°C, composition 9 showed an aggregation temperature of 77.3°C, and composition 10 showed an aggregation temperature of 77.7°C. Example 13 showed no change in the monomer ratio of the protein despite not containing polysorbate 20, and by comparing the case without polysorbate 20 with the case where it contained polysorbate 20, it was confirmed that there was no difference in aggregation between proteins. These results indicate that a minimum amount of polysorbate of 20 is not necessarily required for subcutaneous trastuzumab formulations (see FIG. 8).

Example 15: WCX chromatographic analysis of compositions containing trastuzumab and HP46

For WCX chromatographic analysis, an HPLC system from Shimadzu Prominence was used, and the columns used were TSKgel CM-STAT column (4.6 x 100 mm, 7 µm), TSKgel CMSTAT pre-column (3.2 mm i.d. x 1.5 cm) and so on. Mobile phase A is 10 mM sodium phosphate (pH 7.5) and mobile phase B is 10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. The assay was carried out over 55 minutes with 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 so that the final concentration was 1.0 mg/ml, 80 μl of the sample was injected into the HPLC, and then the absorbance of the column eluate was recorded at 280 nm. The ratio of trastuzumab monomers in the HPLC chromatogram was calculated and plotted.

Formulations 8 to 10 showed a similar pattern of change when the WCX assay was performed in a stability test under stringent conditions, i.e., 40°C for 14 days. Specific changes include an increase in the relative abundance of acidic variants (about 10% change over 14 days), a decrease in the relative abundance of the main peak (about 40% change over 14 days), and an increase in the relative abundance of basic variants (about 300% change over 14 days). and there was no significant difference depending on the composition. In conclusion, when WCX was analyzed in a stability test under stringent conditions, i.e. at 40°C, protein stability at different polysorbate 20 contents (0-0.04%) was similar (see FIG. 9).

Example 16. Analysis of enzymatic activity of compositions containing trastuzumab and HP46

Turbidimetric assay for measuring enzymatic activity is a method of measuring by absorption the degree to which an aggregate is formed by binding of residual hyaluronic acid to acidified albumin (BSA), and when hyaluronic acid is hydrolyzed by PH20, the degree of binding to albumin is reduced, resulting in a decrease in absorption. BTH (Sigma) as a standardized product was diluted to 1 U/m, 2 U/ml, 5 U/ml, 7.5 U/ml, 10 U/ml, 15 U/ml, 20 U ./ml, 30 U/ml, 50 U/ml and 60 U/ml in appropriate tubes. Purified PH20 variant samples were diluted with enzyme dilution buffer (20 mM Tris-HCl, pH 7.0, 77 mM NaCl, 0.01% (w/v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X in appropriate test tubes. In fresh tubes, a hyaluronidase solution with a concentration of 3 mg/ml was diluted 10 times to a concentration of 0.3 mg/ml, so that the volume of each tube was 180 μl. 60 μl of the sample containing hyaluronidase was added to the diluted hyaluronic acid solution, mixed with it and left to react at 37°C for 45 minutes. After completion of the reaction, 50 μL of reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance was measured at 600 nm using a spectrophotometer.

By performing an activity assay in a stability test under stringent conditions, i.e., at 40°C for 14 days, it was confirmed that the higher the concentration of polysorbate 20, the greater the decrease in activity over time (see FIG. 10).

Example 17. Composition development

Three formulations of trastuzumab were prepared for subcutaneous administration, as shown in Table 9. Formulations 11 to 13 generally contained 120 mg/ml trastuzumab, 20 mM histidine/histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine and variant PH20. The difference between formulations 11-13 is the concentration of nonionic surfactant, with formulation 11 containing 0% polysorbate 80, formulation 12 containing 0.005% polysorbate 80 and formulation 13 containing 0.04% polysorbate 80.

Table 9. Composition of compositions Composition 11 Composition 12 Composition 13 Antibody Trastuzumab (120 mg/ml) Polysorbate 80 0% 0.005% 0.04% Buffer 20 mM histidine/histiine-HCl Stabilizer 1 210 mM trehalose Stabilizer 2 10 mM methionine pH 5.5 Hyaluronidase HP46 from SEQ ID NO:44 (2,000 U/ml)

When gel chromatography analysis was performed in a stability test under stringent conditions, that is, at 40°C for 14 days, formulations 11 to 13 showed 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 less than 1.0%), and there was no significant difference between compositions. In conclusion, as a result of gel chromatography analysis in a stability test under stringent conditions, i.e. at 40°C, there was no significant difference in the stability profiles between the formulations depending on the concentration (0-0.04%) of polysorbate 80 (see Fig. 11).

Example 18 WCX chromatographic analysis of compositions containing trastuzumab and HP46

For WCX chromatographic analysis, HPLC system from Shimadzu Prominence, TSKgel CM-STAT column (4.6 x 100 mm, 7 µm) and TSKgel CMSTAT pre-column (3.2 mm i.d. x 1.5 cm), etc. were used. . Mobile phase A was 10 mM sodium phosphate (pH 7.5) and mobile phase B was 10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. The assay was carried out over 55 minutes with 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 so that the final concentration was 1.0 mg/ml, 80 μl of the sample was injected into the HPLC, and then the absorbance of the column eluate was recorded at 280 nm. The ratio of trastuzumab monomers in the HPLC chromatogram was calculated and plotted.

Formulations 11 to 13 showed a similar pattern of changes when the WCX assay was performed in a stability test under stringent conditions, i.e. at 40°C for 14 days. Specific changes include an increase in the relative abundance of acidic variants (about 10% change over 14 days), a decrease in the relative abundance of the main peak (about 40% change over 14 days), and an increase in the relative abundance of basic variants (about 300% change over 14 days). and there was no significant difference depending on the composition. In conclusion, in the WCX assay in the stability test under stringent conditions, i.e. at 40°C, protein stability as a function of polysorbate 80 content (0-0.04%) was similar (see FIG. 12).

Example 19. Analysis of enzymatic activity of compositions containing trastuzumab and HP46

Turbidimetric assay for measuring enzymatic activity is a method of measuring the extent of absorption, where aggregates are formed due to the binding of residual hyaluronic acid to acidified albumin (BSA), and when hyaluronic acid is hydrolyzed by PH20, the extent of binding to albumin is reduced, resulting in decreased absorption . BTH (Sigma) as a standardized product was diluted to 1 U/ml, 2 U/ml, 5 U/ml, 7.5 U/ml, 10 U/ml, 15 U/ml, 20 U ./ml, 30 U/ml, 50 U/ml and 60 U/ml, adding to the appropriate tubes. Purified protein samples were diluted with enzyme dilution buffer (20 mM Tris-HCl, pH 7.0, 77 mM NaCl, 0.01% (w/v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X in corresponding test tubes. In fresh tubes, a hyaluronidase solution with a concentration of 3 mg/ml was diluted 10 times to a concentration of 0.3 mg/ml, so that the volume of each tube was 180 μl. 60 μl of the sample containing hyaluronidase was added to the diluted hyaluronic acid solution, mixed with it and left to react at 37°C for 45 minutes. After completion of the reaction, 50 μL of reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance was measured at 600 nm using a spectrophotometer.

By performing an activity assay in a stability test under stringent conditions, i.e., at 40°C for 14 days, it was confirmed that the higher the concentration of polysorbate 80, the more pronounced the decrease in activity over time (see FIG. 13 ).

Example 20. Composition development

Three types of rituximab compositions were prepared as described in Table 10. Formulations 14 to 16 generally included 120 mg/ml rituximab, 20 mM histidine/histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine and a variant PH20. The difference between formulations 14-16 is the concentration of nonionic surfactant: formulation 14 is 0% polysorbate 80, formulation 15 is 0.005% polysorbate 80, and formulation 16 is 0.06% polysorbate 80.

Table 10. Composition of compositions Composition 14 Composition 15 Composition 16 Rituximab 120 mg/ml (±10) Polysorbate 80 0% 0.005% 0.06% Buffer 20 mM histidine/histiine-HCl Stabilizer 1 210 mM trehalose Stabilizer 2 10 mM methionine pH 5.5 Hyaluronidase HP46 from SEQ ID NO:44 (2,000 U/ml)

When gel chromatography analysis was carried out in a stability test under stringent conditions, i.e. at 40°C for 7 days, compositions 14-16 showed a similar pattern of changes. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (less than about 1.0%), and there was no significant difference between compositions. In conclusion, gel chromatography analysis in a stability test under stringent conditions, i.e. at 40°C, showed no significant differences in stability profiles between formulations depending on the concentration (0-0.06%) of polysorbate . 80 (see Fig. 14).

Example 21. Analysis of enzymatic activity of compositions containing rituximab and HP46

Turbidimetric assay for measuring enzymatic activity is a method of measuring by absorption the degree to which an aggregate is formed by binding of residual hyaluronic acid to acidified albumin (BSA), and when hyaluronic acid is hydrolyzed by PH20, the degree of binding to albumin is reduced, resulting in a decrease in absorption. BTH (Sigma) as a standardized product was diluted to 1 U/ml, 2 U/ml, 5 U/ml, 7.5 U/ml, 10 U/ml, 15 U/ml, 20 U ./ml, 30 U/ml, 50 U/ml and 60 U/ml in appropriate tubes. Purified protein samples were diluted with enzyme dilution buffer (20 mM Tris-HCl, pH 7.0, 77 mM NaCl, 0.01% (w/v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X in corresponding test tubes. In fresh tubes, a hyaluronidase solution with a concentration of 3 mg/ml was diluted 10 times to a concentration of 0.3 mg/ml, so that the volume of each tube was 180 μl. 60 μl of the sample containing hyaluronidase was added to the diluted hyaluronic acid solution, mixed with it and left to react at 37°C for 45 minutes. After completion of the reaction, 50 μL of reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance was measured at 600 nm using a spectrophotometer.

By performing an activity assay in a stability test under stringent conditions, i.e., 40°C for 7 days, it was confirmed that the higher the concentration of polysorbate 80, the greater the decrease in activity over time (see FIG. 15).

Example 22: Analysis of Enzyme Activity in Compositions of Commercial Products Not Containing Polysorbate

Two types of commercial formulations of rituximab were prepared as described in Table 11. Composition 17 is a commercial buffer for subcutaneous injection formulations, and Composition 18 is a commercial buffer for intravenous injection formulations. Formulations 17 and 18 contained the PH20 variant and rituximab at concentrations of 120 mg/mL and 100 mg/mL, respectively, but did not contain polysorbate 80, unlike commercial product formulations.

Table 11. Composition of compositions Composition 17 Composition 18 Rituximab 120 mg/ml 100 mg/ml Buffer 20 mM histidine/histidine-HCl 145 mM sodium citrate Stabilizer 1 210 mM trehalose 145 mM NaCl Stabilizer 2 10 mM methionine 10 mM methionine pH 5.5 6.5 Hyaluronidase HP46 from SEQ ID NO:44 (2,000 U/ml)

Turbidimetric assay for measuring enzymatic activity is a method of measuring by absorption the degree to which an aggregate is formed by binding of residual hyaluronic acid to acidified albumin (BSA), and when hyaluronic acid is hydrolyzed by PH20, the degree of binding to albumin is reduced, resulting in a decrease in absorption. BTH (Sigma) as a standardized product was diluted to 1 U/ml, 2 U/ml, 5 U/ml, 7.5 U/ml, 10 U/ml, 15 U/ml, 20 U ./ml, 30 U/ml, 50 U/ml and 60 U/ml in appropriate tubes. Purified protein samples were diluted with enzyme dilution buffer (20 mM Tris-HCl, pH 7.0, 77 mM NaCl, 0.01% (w/v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X in corresponding test tubes. In fresh tubes, a hyaluronidase solution with a concentration of 3 mg/ml was diluted 10 times to a concentration of 0.3 mg/ml, so that the volume of each tube was 180 μl. 60 μl of the sample containing hyaluronidase was added to the diluted hyaluronic acid solution, mixed with it and left to react at 37°C for 45 minutes. After completion of the reaction, 50 μL of reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance was measured at 600 nm using a spectrophotometer.

By performing an activity assay in a stability test under stringent conditions, i.e. at 40°C for 6 days, it was confirmed that high activity was maintained even in compositions not containing polysorbate 80, and in particular composition 18 remained highly active (see Fig. 16).

Example 23. Composition development

Four types of pembrolizumab compositions were prepared as described in Table 12. Formulations 19, 20 and 21 generally comprise 25 mg/ml pembrolizumab, 10 mM histidine (pH 5.5), 7% sucrose, 10 mM methionine and the PH20 option. The difference between formulations 19-21 is the concentration of nonionic surfactant: formulation 19 is 0% polysorbate 80, formulation 20 is 0.005% polysorbate 80 and formulation 21 is 0.02% polysorbate 80. Composition 22 contains 25 mg/ml pembrolizumab and consists of 10 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine, 0.02% polysorbate 80 and PH20 variant.

Table 12. Composition of compositions Composition 19 Composition 20 Composition 21 Composition 22 Antibody Pembrolizumab (25 mg/ml) Buffer 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 from SEQ ID NO:44 (2,000 U/ml)

Example 24 Spectrophotometer Analysis

Formulations 19, 20, 21 and 22 were left for 7 days at 40°C, and changes in protein concentration were analyzed using a spectrophotometer manufactured by Beckman. Each sample was diluted with distilled water so that the sample concentration was 0.4 mg/mL, and then the protein absorbance was measured at 280 nm using a spectrophotometer.

In a stability test under severe conditions, ie at 40°C for 7 days, there was no significant change in protein concentration in compositions 19-22.

Example 25 Study of the ratio of pembrolizumab monomers in each composition using size exclusion chromatography

For size exclusion chromatography analysis, an HPLC system from Shimadzu Prominence was used, and a TSK-gel G3000SWXL column (7.8 × 300 mm, 5 μm) and a TSK precolumn (6.0 × 4.0 mm, 7 μm) were used. The mobile phase was 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride. The analysis was performed for 35 minutes in isocratic separation mode at a flow rate of 0.5 ml/min. The sample was diluted with the analytical solvent to give a final concentration of 10 mg/mL, and after injecting 20 μL of the sample onto the HPLC column, the absorbance of the column eluate was measured at 280 nm. The ratio of pembrolizumab monomers in the HPLC chromatogram was calculated and plotted.

When gel chromatography analysis was performed in a stability test under stringent conditions, that is, at 40°C for 7 days, compositions 19, 20, 21 and 22 showed similar patterns of change. There was no significant difference between compositions in the change patterns of high molecular weight (HMW) and low molecular weight (LMW) degradation products. In conclusion, when analyzed by size exclusion chromatography in a stability test under stringent conditions, i.e. at 40°C, compositions 19, 20, 21 and 22 did not show any significant differences, and there was no difference depending on type of sugar (see Fig. 17). These results were consistent with the results for trastuzumab and rituximab in previous examples.

Example 26: Measurement of Enzymatic Activity of Compositions Containing Pembrolizumab and HP46

Turbidimetric assay for measuring enzymatic activity is a method of measuring by absorption the extent to which an aggregate is formed by binding of residual hyaluronic acid to acidified albumin (BSA), and when hyaluronic acid is hydrolyzed by PH20, the extent of binding to albumin is reduced, resulting in decreased absorption . BTH (Sigma) as a standardized product was diluted to 1 U/ml, 2 U/ml, 5 U/ml, 7.5 U/ml, 10 U/ml, 15 U/ml, 20 U ./ml, 30 U/ml, 50 U/ml and 60 U/ml in appropriate tubes. Purified protein samples were diluted with enzyme dilution buffer (20 mM Tris-HCl, pH 7.0, 77 mM NaCl, 0.01% (w/v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X in corresponding test tubes. In fresh tubes, a hyaluronidase solution with a concentration of 3 mg/ml was diluted 10 times to a concentration of 0.3 mg/ml, so that the volume of each tube was 180 μl. 60 μl of the sample containing the enzyme was added to the dilute hyaluronic acid solution, mixed with it and left to react at 37°C for 45 minutes. After completion of the reaction, 50 μL of reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance was measured at 600 nm using a spectrophotometer.

By performing an activity assay in a stability test under stringent conditions, i.e., at 40°C for 7 days, it was confirmed that as the concentration of polysorbate 80 increased, the decrease in activity over time was slightly increased. It was also confirmed that when the same amount of polysorbate 80 was included, the decrease in activity was less pronounced in the composition containing trehalose than in the composition containing sucrose (see Fig. 18).

Example 27: pH Activity Profiles of Wild Type HP46 and HW2

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

A total of three 96-well plates were prepared for the enzyme-substrate reaction and designated as A, B, and C, and the experiment was performed.

Hyaluronic acid buffer pH 4.0, 4.5 or 5.0 was prepared using 20 mM acetic acid and 70 mM NaCl, and hyaluronic acid solution pH 5.5; 6.0; 6.5; 7.0 or 8.0 were prepared using 20 mM sodium phosphate and 70 mM NaCl. 20 mg of hyaluronic acid was dissolved in 10 ml of each of the prepared hyaluronic acid buffers to prepare a final solution of hyaluronic acid substrate, which was then diluted with each pH prepared hyaluronic acid buffer to obtain 500 μl of a solution with a concentration of 0.1 mg/ml , 0.25 mg/ml, 0.45 mg/ml, or 0.7 mg/ml, and 100 μl of each solution were dispensed into each well of a 96-well plate designated as plate A. Hyaluronic acid buffers, diluted and prepared according to with concentration were used as calibration curves to measure the concentration of hyaluronic acid.

pH 4.0, pH 4.5, or pH 5.0 enzyme buffer was prepared using 20 mM acetic acid, 0.01% (w/v) BSA, 70 mM NaCl, and pH 5.5 enzyme buffer , pH 6.0, pH 6.5, pH 7.0, or pH 8.0 were prepared using 20 mM sodium phosphate, 0.01% (w/v) BSA, and 70 mM NaCl.

Wild-type HP46 and HW2 enzymes were diluted with pH-adjusted enzyme buffer to 10 U/mL, and 50 μL of the resulting solution was dispensed into each well of a 96-well plate designated as B.

50 μl of sample was transferred from each well of a 96-well plate, designated A, to each well of a 96-well plate, designated B, followed by the reaction in a shaking incubator at 37°C for 45 minutes. 15 minutes before the completion of the reaction, 200 μl of acidic albumin solution was added to each well of the 96-well plate, designated as C, and when the reaction of the enzyme substrate was completed, 40 μl of sample was transferred from each well of the 96-well plate, designated as B, to each well 96-well plate designated as C, followed by continuing the reaction for 20 minutes. After 20 minutes, the optical density at 600 nm was measured, the amount of hyaluronic acid remaining after reaction with the enzyme substrate was calculated, and the enzyme activity profiles were determined as a function of pH (see FIG. 19).

Example 28 Pharmacokinetics Analysis Using Herceptin Subcutaneous Composition and Trastuzumab and HP46 in Sprague-Dawley Rats

To test whether the subcutaneous formulation of trastuzumab and HP46 exhibited the same pharmacokinetic properties as the subcutaneous formulation of Herceptin, an experiment was conducted using 9-week-old Sprague-Dawley rats. The dose of Herceptin and trastuzumab administered was 18 mg/kg body weight of the rat, the amount of rHuPH20 included in the Herceptin subcutaneous formulation was 100 U, and the amount of HP46 was also 100 U. In the pharmacokinetic analysis, trastuzumab and HP46 showed the same area under the curve (AUC) as the Herceptin subcutaneous formulation (see FIG. 20).

Industrial applicability

The pharmaceutical composition of the present invention can be used for subcutaneous administration, and it is also very stable, and the activity of the PH20 variants together with a drug, preferably an antibody drug or the like, can be maintained for a long time. Thus, the pharmaceutical composition can help reduce not only the production cost of subcutaneous injection drugs but also medical costs, and is very effective in terms of convenience for patients.

Although preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will understand that various modifications, additions and substitutions are possible without departing from the scope and spirit of the invention as disclosed in the appended claims.

Links

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LIST OF SEQUENCES

<110> ALTEOGEN, INC.

<120> PHARMACEUTICAL COMPOSITION, INCLUDING PH20 OPTION

HUMAN HYALURONIDASES,

AND MEDICINE FOR SUBCUTTANEOUS ADMINISTRATION

<130> 4240-536

<150> PCT/KR2020/003975

<151> 2020-03-24

<150> KR 10-2019-0033880

<151> 2019-03-25

<160> 51

<170> Patent In version 3.5

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Met Gly Val Leu Lys Phe Lys His Ile Phe Phe Arg Ser Phe Val Lys

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Ser Ser Gly Val Ser Gln Ile Val Phe Thr Phe Leu Leu Ile Pro Cys

20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro

35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe

50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg

65 70 75 80

Ile Asn Ala Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu

85 90 95

Gly Tyr Tyr Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly

100 105 110

Gly Ile Pro Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys

115 120 125

Lys Asp Ile Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val

130 135 140

Ile Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro

145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn

165 170 175

Val Gln Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe

180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys

195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys

210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn

225 230 235 240

Val Glu Ile Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser

245 250 255

Thr Ala Leu Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val

260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val

275 280 285

Ser Lys Ile Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr

290 295 300

Arg Ile Val Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu

305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile

325 330 335

Val Ile Trp Gly Thr Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu

340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn

355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln

370 375 380

Gly Val Cys Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu

385 390 395 400

Asn Pro Asp Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr

405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys

420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp

435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys

450 455 460

Ile Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile

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Phe Tyr Asn Ala Ser Pro Ser Thr Leu Ser Ala Thr Met Phe Ile Val

485 490 495

Ser Ile Leu Phe Leu Ile Ile Ser Ser Val Ala Ser Leu

500 505

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Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

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Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala

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Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

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Tyr Ser

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Met Ala Ala His Leu Leu Pro Ile Cys Ala Leu Phe Leu Thr Leu Leu

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Asp Met Ala Gln Gly

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Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

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Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

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Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

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Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

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Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

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Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

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Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Gly Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

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Ala Ser Pro Ser Thr Leu Ser

450 455

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Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

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Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

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Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

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Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

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Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

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Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

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Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

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Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Phe Ile Leu Asn Val Thr Ser

325 330 335

Gly Ala Leu Leu Cys Ser Gln Ala Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

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Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

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Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

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Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Gly Pro Phe Ile Leu Asn Val Thr Ser

325 330 335

Gly Ala Leu Leu Cys Ser Gln Ala Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

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Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Val Ser Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Gly Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

<210> 9

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Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

<210> 10

<211> 455

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 10

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Val Ser Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

<210> 11

<211> 455

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 11

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

<210> 12

<211> 455

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 12

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

<210> 13

<211> 455

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 13

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

<210> 14

<211> 455

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 14

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Phe Ile Leu Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

<210> 15

<211> 455

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 15

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Phe Ile Leu Asn Val Thr Ser

325 330 335

Gly Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

<210> 16

<211> 453

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 16

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala Ser

435 440 445

Pro Ser Thr Leu Ser

450

<210> 17

<211> 430

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 17

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile

420 425 430

<210> 18

<211> 433

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 18

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe

<210> 19

<211> 436

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 19

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro

435

<210> 20

<211> 452

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 20

Phe Arg Gly Pro Leu Leu Pro Asn Arg Pro Phe Leu Trp Ala Trp Asn

1 5 10 15

Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp Met

20 25 30

Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly Gln

35 40 45

Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr Ile

50 55 60

Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys Ile

65 70 75 80

Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe Tyr

85 90 95

Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu Trp

100 105 110

Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys Asn

115 120 125

Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu Thr

130 135 140

Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys Asp

145 150 155 160

Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn His

165 170 175

Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr Lys

180 185 190

Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg Asn

195 200 205

Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro Ser

210 215 220

Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr Val

225 230 235 240

Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp Ala

245 250 255

Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr Asp

260 265 270

Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe Gly

275 280 285

Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr Leu

290 295 300

Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr Met

305 310 315 320

Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala Lys

325 330 335

Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg Lys

340 345 350

Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe Ala

355 360 365

Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro Thr

370 375 380

Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys Tyr

385 390 395 400

Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp Ala

405 410 415

Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu Lys

420 425 430

Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala Ser Pro

435 440 445

Ser Thr Leu Ser

450

<210> 21

<211> 452

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 21

Phe Arg Gly Pro Leu Leu Pro Asn Arg Pro Phe Thr Thr Val Trp Asn

1 5 10 15

Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp Met

20 25 30

Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly Gln

35 40 45

Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr Ile

50 55 60

Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys Ile

65 70 75 80

Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe Tyr

85 90 95

Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu Trp

100 105 110

Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys Asn

115 120 125

Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu Thr

130 135 140

Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys Asp

145 150 155 160

Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn His

165 170 175

Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr Lys

180 185 190

Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg Asn

195 200 205

Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro Ser

210 215 220

Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr Val

225 230 235 240

Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp Ala

245 250 255

Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr Asp

260 265 270

Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe Gly

275 280 285

Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr Leu

290 295 300

Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr Met

305 310 315 320

Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala Lys

325 330 335

Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg Lys

340 345 350

Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe Ala

355 360 365

Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro Thr

370 375 380

Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys Tyr

385 390 395 400

Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp Ala

405 410 415

Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu Lys

420 425 430

Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala Ser Pro

435 440 445

Ser Thr Leu Ser

450

<210> 22

<211> 433

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 22

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys

<210> 23

<211> 431

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 23

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe

420 425 430

<210> 24

<211> 455

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 24

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Ser Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

<210> 25

<211> 451

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 25

Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp Asn Ala

1 5 10 15

Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp Met Ser

20 25 30

Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly Gln Gly

35 40 45

Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr Ile Asp

50 55 60

Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys Ile Ser

65 70 75 80

Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe Tyr Met

85 90 95

Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu Trp Arg

100 105 110

Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys Asn Arg

115 120 125

Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu Thr Glu

130 135 140

Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys Asp Phe

145 150 155 160

Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn His Leu

165 170 175

Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr Lys Lys

180 185 190

Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg Asn Asp

195 200 205

Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro Ser Ile

210 215 220

Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr Val Arg

225 230 235 240

Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp Ala Lys

245 250 255

Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr Asp Gln

260 265 270

Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe Gly Glu

275 280 285

Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr Leu Ser

290 295 300

Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr Met Asp

305 310 315 320

Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala Lys Met

325 330 335

Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg Lys Asn

340 345 350

Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe Ala Ile

355 360 365

Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro Thr Leu

370 375 380

Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys Tyr Ser

385 390 395 400

Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp Ala Val

405 410 415

Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu Lys Pro

420 425 430

Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala Ser Pro Ser

435 440 445

Thr Leu Ser

450

<210> 26

<211> 449

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 26

Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp Asn Ala Pro Ser

1 5 10 15

Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp Met Ser Leu Phe

20 25 30

Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly Gln Gly Val Thr

35 40 45

Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr Ile Asp Ser Ile

50 55 60

Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys Ile Ser Leu Gln

65 70 75 80

Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe Tyr Met Pro Val

85 90 95

Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu Trp Arg Pro Thr

100 105 110

Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys Asn Arg Ser Ile

115 120 125

Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu Thr Glu Ala Thr

130 135 140

Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys Asp Phe Leu Val

145 150 155 160

Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn His Leu Trp Gly

165 170 175

Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr Lys Lys Pro Gly

180 185 190

Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg Asn Asp Asp Leu

195 200 205

Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro Ser Ile Tyr Leu

210 215 220

Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr Val Arg Asn Arg

225 230 235 240

Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp Ala Lys Ser Pro

245 250 255

Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr Asp Gln Val Leu

260 265 270

Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe Gly Glu Thr Val

275 280 285

Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr Leu Ser Ile Thr

290 295 300

Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr Met Asp Thr Thr

305 310 315 320

Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala Lys Met Cys Ser

325 330 335

Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg Lys Asn Trp Asn

340 345 350

Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe Ala Ile Gln Leu

355 360 365

Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro Thr Leu Glu Asp

370 375 380

Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys Tyr Ser Thr Leu

385 390 395 400

Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp Ala Val Asp Val

405 410 415

Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu Lys Pro Pro Met

420 425 430

Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala Ser Pro Ser Thr Leu

435 440 445

Ser

<210> 27

<211> 432

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 27

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

<210> 28

<211> 429

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 28

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys

420 425

<210> 29

<211> 426

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 29

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp

420 425

<210> 30

<211> 423

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 30

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys

420

<210> 31

<211> 420

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 31

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys

305 310 315 320

Glu Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val

420

<210> 32

<211> 433

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 32

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Ser

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys

<210> 33

<211> 435

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 33

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro

435

<210> 34

<211> 436

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 34

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met

435

<210> 35

<211> 437

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 35

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met Glu

435

<210> 36

<211> 438

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 36

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met Glu Thr

435

<210> 37

<211> 439

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 37

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met Glu Thr Glu

435

<210> 38

<211> 454

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 38

Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala

1 5 10 15

Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu

20 25 30

Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr

35 40 45

Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro

50 55 60

Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln

65 70 75 80

Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr

85 90 95

Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu

100 105 110

Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr

115 120 125

Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser

130 135 140

Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly

145 150 155 160

Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro

165 170 175

Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His

180 185 190

Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys

195 200 205

Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr

210 215 220

Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu

225 230 235 240

Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro

245 250 255

Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe

260 265 270

Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr

275 280 285

Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly

290 295 300

Thr Leu Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu

305 310 315 320

Tyr Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala

325 330 335

Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile

340 345 350

Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn

355 360 365

Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys

370 375 380

Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser

385 390 395 400

Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr

405 410 415

Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe

420 425 430

Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala

435 440 445

Ser Pro Ser Thr Leu Ser

450

<210> 39

<211> 452

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 39

Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp Asn

1 5 10 15

Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp Met

20 25 30

Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly Gln

35 40 45

Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr Ile

50 55 60

Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys Ile

65 70 75 80

Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe Tyr

85 90 95

Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu Trp

100 105 110

Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys Asn

115 120 125

Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu Thr

130 135 140

Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys Asp

145 150 155 160

Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn His

165 170 175

Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr Lys

180 185 190

Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg Asn

195 200 205

Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro Ser

210 215 220

Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr Val

225 230 235 240

Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp Ala

245 250 255

Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr Asp

260 265 270

Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe Gly

275 280 285

Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr Leu

290 295 300

Ser Ile Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr Met

305 310 315 320

Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala Lys

325 330 335

Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg Lys

340 345 350

Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe Ala

355 360 365

Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro Thr

370 375 380

Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys Tyr

385 390 395 400

Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp Ala

405 410 415

Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu Lys

420 425 430

Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala Ser Pro

435 440 445

Ser Thr Leu Ser

450

<210> 40

<211> 450

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 40

Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp Asn Ala Pro

1 5 10 15

Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp Met Ser Leu

20 25 30

Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly Gln Gly Val

35 40 45

Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr Ile Asp Ser

50 55 60

Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys Ile Ser Leu

65 70 75 80

Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe Tyr Met Pro

85 90 95

Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu Trp Arg Pro

100 105 110

Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys Asn Arg Ser

115 120 125

Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu Thr Glu Ala

130 135 140

Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys Asp Phe Leu

145 150 155 160

Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn His Leu Trp

165 170 175

Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr Lys Lys Pro

180 185 190

Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg Asn Asp Asp

195 200 205

Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro Ser Ile Tyr

210 215 220

Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr Val Arg Asn

225 230 235 240

Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp Ala Lys Ser

245 250 255

Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr Asp Gln Val

260 265 270

Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe Gly Glu Thr

275 280 285

Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr Leu Ser Ile

290 295 300

Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr Met Asp Thr

305 310 315 320

Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala Lys Met Cys

325 330 335

Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg Lys Asn Trp

340 345 350

Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe Ala Ile Gln

355 360 365

Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro Thr Leu Glu

370 375 380

Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys Tyr Ser Thr

385 390 395 400

Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp Ala Val Asp

405 410 415

Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu Lys Pro Pro

420 425 430

Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala Ser Pro Ser Thr

435 440 445

Leu Ser

450

<210> 41

<211> 428

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 41

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Ser

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile

420 425

<210> 42

<211> 429

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 42

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Ser

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp

420 425

<210> 43

<211> 430

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 43

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Ser

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

<210> 44

<211> 431

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 44

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Ser

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe

420 425 430

<210> 45

<211> 441

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 45

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met Glu Thr Glu Glu Pro

435 440

<210> 46

<211> 443

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 46

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile

435 440

<210> 47

<211> 445

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 47

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr

435 440 445

<210> 48

<211> 447

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 48

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala

435 440 445

<210> 49

<211> 449

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 49

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala Ser

435 440 445

Pro

<210> 50

<211> 451

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 50

Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp Ala Trp

1 5 10 15

Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro Leu Asp

20 25 30

Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala Thr Gly

35 40 45

Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr Pro Tyr

50 55 60

Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro Gln Lys

65 70 75 80

Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile Thr Phe

85 90 95

Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp Glu Glu

100 105 110

Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val Tyr Lys

115 120 125

Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu Ser Leu

130 135 140

Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala Gly Lys

145 150 155 160

Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg Pro Asn

165 170 175

His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His His Tyr

180 185 190

Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile Lys Arg

195 200 205

Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu Tyr Pro

210 215 220

Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr Leu Tyr

225 230 235 240

Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile Pro Asp

245 250 255

Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val Phe Thr

260 265 270

Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr Thr Phe

275 280 285

Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp Gly Thr

290 295 300

Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile Lys Glu Tyr

305 310 315 320

Met Asp Thr Thr Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu Ala Ala

325 330 335

Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys Ile Arg

340 345 350

Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp Asn Phe

355 360 365

Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly Lys Pro

370 375 380

Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys Ser Cys

385 390 395 400

Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp Thr Asp

405 410 415

Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala Phe Leu

420 425 430

Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn Ala Ser

435 440 445

Pro Ser Thr

450

<210> 51

<211> 435

<212>PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 51

Met Ala Ala His Leu Leu Pro Ile Cys Ala Leu Phe Leu Thr Leu Leu

1 5 10 15

Asp Met Ala Gln Gly Phe Arg Gly Pro Leu Leu Pro Asn Arg Pro Phe

20 25 30

Thr Thr Val Trp Asn Ala Asn Thr Gln Trp Cys Leu Glu Arg His Gly

35 40 45

Val Asp Val Asp Val Ser Val Phe Asp Val Val Ala Asn Pro Gly Gln

50 55 60

Thr Phe Arg Gly Pro Asp Met Thr Ile Phe Tyr Ser Ser Gln Leu Gly

65 70 75 80

Thr Tyr Pro Tyr Tyr Thr Pro Thr Gly Glu Pro Val Phe Gly Gly Leu

85 90 95

Pro Gln Asn Ala Ser Leu Ile Ala His Leu Ala Arg Thr Phe Gln Asp

100 105 110

Ile Leu Ala Ala Ile Pro Ala Pro Asp Phe Ser Gly Leu Ala Val Ile

115 120 125

Asp Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Thr Lys

130 135 140

Asp Ile Tyr Arg Gln Arg Ser Arg Ala Leu Val Gln Ala Gln His Pro

145 150 155 160

Asp Trp Pro Ala Pro Gln Val Glu Ala Val Ala Gln Asp Gln Phe Gln

165 170 175

Gly Ala Ala Arg Ala Trp Met Ala Gly Thr Leu Gln Leu Gly Arg Ala

180 185 190

Leu Arg Pro Arg Gly Leu Trp Gly Phe Tyr Gly Phe Pro Asp Cys Tyr

195 200 205

Asn Tyr Asp Phe Leu Ser Pro Asn Tyr Thr Gly Gln Cys Pro Ser Gly

210 215 220

Ile Arg Ala Gln Asn Asp Gln Leu Gly Trp Leu Trp Gly Gln Ser Arg

225 230 235 240

Ala Leu Tyr Pro Ser Ile Tyr Met Pro Ala Val Leu Glu Gly Thr Gly

245 250 255

Lys Ser Gln Met Tyr Val Gln His Arg Val Ala Glu Ala Phe Arg Val

260 265 270

Ala Val Ala Ala Gly Asp Pro Asn Leu Pro Val Leu Pro Tyr Val Gln

275 280 285

Ile Phe Tyr Asp Thr Thr Asn His Phe Leu Pro Leu Asp Glu Leu Glu

290 295 300

His Ser Leu Gly Glu Ser Ala Ala Gln Gly Ala Ala Gly Val Val Leu

305 310 315 320

Trp Val Ser Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile

325 330 335

Lys Glu Tyr Met Asp Thr Thr Leu Gly Pro Phe Ile Leu Asn Val Thr

340 345 350

Ser Gly Ala Leu Leu Cys Ser Gln Ala Leu Cys Ser Gly His Gly Arg

355 360 365

Cys Val Arg Arg Thr Ser His Pro Lys Ala Leu Leu Leu Leu Asn Pro

370 375 380

Ala Ser Phe Ser Ile Gln Leu Thr Pro Gly Gly Gly Pro Leu Ser Leu

385 390 395 400

Arg Gly Ala Leu Ser Leu Glu Asp Gln Ala Gln Met Ala Val Glu Phe

405 410 415

Lys Cys Arg Cys Tyr Pro Gly Trp Gln Ala Pro Trp Cys Glu Arg Lys

420 425 430

Ser Met Trp

435

<---

Claims (41)

1. Use of a PH20 variant containing one or more amino acid residue substitutions selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1 to produce a pharmaceutical composition that is thermostable for a long time, including: (a) a drug; and (b) the specified variant PH20, and where the drug is not pembrolizumab. 2. Use according to claim 1, where the PH20 variant contains one or more substitutions of amino acid residues selected from the group consisting of L354I and N356E. 3. Use of a PH20 variant containing substitution(s) of amino acid residues L354I and/or N356E in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, and further comprising substitution(s) of amino acid residues at one or additional positions selected from the group , consisting of T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361 and N363, in wild-type PH20 having the amino acid sequence SEQ ID NO: 1, to obtain thermostable for a long-term pharmaceutical composition comprising: (a) a drug; and (b) the specified PH20 option. 4. Use of a PH20 variant containing substitution(s) of amino acid residues L354I and/or N356E in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, and further comprising substitution(s) of amino acid residues at one or more positions selected from the group , consisting of T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T and N363G, in wild-type PH20 having the amino acid sequence SEQ ID NO: 1, to obtain thermostable for a long-term pharmaceutical composition comprising: (a) a drug; and (b) the specified PH20 option. 5. Use according to claim 3, where the PH20 variant contains substitutions of amino acid residues M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. 6. Use according to claim 3, wherein the PH20 variant contains one or more amino acid residue substitutions selected from the group consisting of T341S, L342W, S343E, I344N and N363G. 7. Use according to claim 4, where the PH20 variant contains substitutions of amino acid residues specified in 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; (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. 8. The use of a PH20 variant containing one or more substitutions of amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T, in wild type PH20 having the amino acid sequence SEQ ID NO: 1, where the signal peptide SEQ ID NO: 1 is replaced by a signal peptide derived from human hyaluronidase-1 (Hyal1), human growth hormone or human serum albumin at the N-terminus of the PH20 variant to provide a long-term thermostable pharmaceutical composition comprising: (a) a drug means; and (b) the specified PH20 option. 9. Use according to claim 8, wherein in embodiment PH20 one or more amino acid residues are removed by cleavage before amino acid residue L36, N37, F38, R39, A40, P41 or P42 at the N-terminus. 10. Use according to claim 8, wherein in embodiment PH20 one or more amino acid residues are removed by cleavage after an amino acid residue selected from the group consisting of V455-S490 at the C-terminus. 11. Use according to claim 10, where in the PH20 variant one or more amino acid residues are removed by cleavage after amino acid residue 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 C-terminus. 12. Use of a PH20 variant comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 50 to obtain a long-term thermostable pharmaceutical composition comprising: (a) a drug; and (b) the specified PH20 option. 13. Use according to claim 12, wherein the PH20 variant has the sequence SEQ ID NO: 44. 14. The use of claim 1, wherein the drug is a protein drug, antibody, small molecule, aptamer, RNAi, antisense or cellular therapeutic agent. 15. Use according to claim 14, wherein the drug is an antibody, a soluble receptor or an Fc fusion protein thereof. 16. The use of claim 15, wherein the antibody binds to one or more antigens selected from the group consisting of 4-1BB, integrin, amyloid beta, angiopoietin, angiopoietin analog 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, receptor EGF, hemophilia factor, Fc receptor, FGF23, folate receptor, GD2, GM-CSF, HER2, HER3, interferon receptor, interferon gamma, IgE, IGF-1 receptor, interleukin 1, interleukin 2 receptor, interleukin 4 receptor, interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6 receptor, interleukin 7, interleukin 12/23, interleukin 13, interleukin 17A, interleukin 17A receptor, interleukin 31 receptor, interleukin 36 receptor, LAG3, LFA3, NGF, PVSK9 PD-1, PD-L1, RANK-L, SLAMF7, tissue factor, TNF, VEGF and vWF. 17. Use according to claim 16, where the antibody is one or more antibodies selected from the group consisting of utomilumab, natalizumab, etrolizumab, vedolizumab, bimagrumab, bapineizumab, crenezumab, solanezumab, aducanumab, gantenerumab, AMI 361780, nesvacumab, vanucizumab, evinacumab, tabalumab, lanalumab, belimumab, omburtamab, ravulizumab, eculizumab, mogamulizumab, otelixizumab, teplizumab, muromonab, tebentafusp, blinatumomab, REGN1979, ibalizumab, zanolimumab, itolizumab, efalizumab, inebilizumab, tafa situmab, loncastuximab tesirine, ocrelizumab, ublituximab, obinutuzumab, ofatumumab , rituximab, tositumomab, ibritumomab tiuxetan, epratuzumab, inotuzumab ozogamicin, moxetumomab pasudotox, brentuximab vedotin, vadastuximab thalirin, gemtuzumab ozogamicin, daratumumab, isatuximab, alemtuzumab, crizanlizumab, polatuz umab vedotin, galiximab, eptinezumab, fremanezumab, galcanezumab, erenumab, zolbetuximab, lampalizumab, tremelimumab, zalifrelimab, ipilimumab, rovalpituzumab tesirina, cetuximab, depatuxizumab, zalutumumab, necitumumab, panitumumab, emicizumab, nipocalimab, rozanolixizumab, burosumab, farletuzumab, mirvetuximab soravtansine, dinutuximab, naxitamab, otilimab, margetuximab, pertuzumab, trastuzumab, trastuzumab derutexan, trastuzumab emtansine, trastuzumab duocarmazine, patritumab, anifrolumab, emapalumab, ligelizumab, omalizumab, dalotuzumab, figitumumab, teprotumumab, gebokizumab, canakinumab, daclizumab, basiliximab, dupilumab, mepolizumab, reslizumab, benralizumab, clazakizumab, olokizu maba, sirukumab, siltuximab, sarilumab, satralizumab, tocilizumab, REGN88 , secukinumab, ustekinumab, briakinumab, lebrikizumab, tralokinumab, ixekizumab, bimekizumab, brodalumab, brazikumab, guselkumab, risankizumab, tildrakizumab, mirikizumab, nemolizumab, spesolimab, relatlimab, narsoplimab, facinumab, tanezumab ba, alirocumab, evolocumab, bococizumab, lambrolizumab, balstilimab, camrelizumab , cemiplimab, dostarlimab, prolgolimab, sintilimab, spartalizumab, tislelizumab, pembrolizumab, nivolumab, atezolizumab, avelumab, envafolimab, durvalumab, bintrafuspa alfa, denosumab, elotuzumab, concizumab, marstacimab, infliximab, adalimumab, goal imumab, certolizumab pegola, ozoralizumab, brolucizumab, ranibizumab , bevacizumab, faricimab, ramucirumab and caplacizumab. 18. The use of claim 15, wherein the soluble receptor or soluble receptor included in the Fc fusion protein from the soluble receptor is selected from the group consisting of soluble TNF-α receptor, soluble VEGF receptor, CTLA-4, soluble interleukin 1 receptor and soluble receptor LFA3. 19. The use of claim 18, wherein the soluble receptor Fc fusion protein is selected from the group consisting of etanercept, aflibercept, abatacept, belatacept, rilonacept and alefacept. 20. Use according to claim 1, wherein the pharmaceutical composition further contains one or more components selected from the group consisting of a buffer, a stabilizer and a surfactant. 21. Use according to claim 20, where the buffer contains one or more substances selected from the group consisting of malate, formate, citrate, acetate, propionate, pyridine, piperazine, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid ( MES), histidine, Tris, bis-Tris, phosphate, ethanolamine, carbonate, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), imidazole, propane Bis-Tris, N,N-bis(2- hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)propanesulfonic acid (MOPS), hydroxyethyl-piperazineethanesulfonic acid (HEPES), pyrophosphate and triethanolamine; the stabilizer includes one or more substances selected from the group consisting of carbohydrates, sugars or their hydrates, sugar alcohols or their hydrates and amino acids; And the surfactant includes one or more nonionic surfactants selected from the group consisting of polyoxyethylene sorbitan fatty acid ester, polyethylene polypropylene glycol, polyoxyethylene stearate, polyoxyethylene alkyl ethers, polyoxyethylene-polyoxypropylene copolymer, and sodium dodecyl sulfate ( SDS). 22. Use according to claim 21, where the carbohydrates, sugars or sugar alcohols include one or more components selected from the group consisting of trehalose or its hydrates, sucrose, saccharin, glycerol, erythritol, threitol, xylitol, arabitol, ribitol, mannitol, sorbitol , galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, polyglycitol, cyclodextrin, hydroxylpropylcyclodextrin and glucose; And amino acid includes one or more amino acids selected from the group consisting of glutamine, glutamic acid, glycine, lysine, lysyl lysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine , threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolysine, histidine and alanine. 23. Use according to claim 20, where the pharmaceutical composition contains a histidine buffer providing a pH of 5.5 ± 2.0, trehalose and methionine. 24. Use according to claim 20, where the pharmaceutical composition contains a histidine buffer providing a pH of 5.5 ± 2.0, trehalose, methionine and polysorbate. 25. Application according to claim 24, where the pharmaceutical composition contains a histidine buffer providing a pH of 5.5±2.0, 10-400 mM α,α-trehalose, 1-50 mM methionine and from 0.0000001% (wt./ vol.) up to 0.5% (w/v) polysorbate. 26. Application of the PH20 option selected from the group consisting of: a PH20 variant comprising one or more substitutions of amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1; a PH20 variant comprising substitution(s) of amino acid residues L354I and/or N356E in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, and further comprising substitution(s) of amino acid residues at one or more positions selected from the group consisting of T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361 and N363, in wild-type PH20 having the amino acid sequence SEQ ID NO: 1; a PH20 variant comprising substitution(s) of amino acid residues L354I and/or N356E in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, and further comprising substitution(s) of amino acid residues at one or more selected positions from the group consisting of T341S , L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T and N363G, in wild-type PH20 having the amino acid sequence SEQ ID NO: 1; a PH20 variant comprising one or more amino acid residue substitutions selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, wherein the signal peptide is SEQ ID NO: 1 is replaced by a signal peptide derived from human hyaluronidase-1 (Hyal1), human growth hormone, or human serum albumin at the N terminus of the PH20 variant; And a PH20 variant comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 50, to obtain a composition for subcutaneous injection that is thermostable over a long period of time.