阅读说明：本技术 一种白介素2突变体 (Interleukin 2 mutant ) 是由 胡辉 于 2020-05-14 设计创作，主要内容包括：本发明提供了一种白介素2突变体、包含该突变体的融合蛋白、抗体或缀合物以及包含所述突变体、融合蛋白、抗体或缀合物的药物组合物。与野生型白介素2相比,该突变体消除了与IL-2Rα的结合,降低了与IL-2Rβγ二聚体的结合,但保持了刺激肿瘤免疫细胞,包括但不限于T细胞和NK细胞的增殖。本发明的IL-2突变体消除了对高亲和力IL-2受体的亲和力,同时降低了对中等亲和力IL-2受体的亲和力。本发明的白介素2突变体更加适用于融合蛋白、抗体的缀合物。本发明的白介素2突变体相对于野生型白介素2的突变位点极少,所以潜在的免疫原性极低。同时没有利用天然IL-2进行免疫治疗产生的各种副作用。(The invention provides an interleukin 2 mutant, a fusion protein, an antibody or a conjugate containing the mutant and a pharmaceutical composition containing the mutant, the fusion protein, the antibody or the conjugate. Compared with wild type interleukin 2, the mutant eliminates the binding with IL-2R alpha, reduces the binding with IL-2R beta gamma dimer, but keeps stimulating the proliferation of tumor immune cells, including but not limited to T cells and NK cells. The IL-2 mutants of the invention eliminate the affinity for high affinity IL-2 receptors, while reducing the affinity for intermediate affinity IL-2 receptors. The interleukin 2 mutant of the invention is more suitable for conjugates of fusion protein and antibody. The interleukin 2 mutant of the present invention has very few mutation sites compared with the wild type interleukin 2, so the potential immunogenicity is very low. Meanwhile, various side effects generated by immunotherapy by utilizing the natural IL-2 are avoided.)

1. An interleukin 2 mutant, wherein the IL-2 mutant is mutated at an amino acid residue at one or two of the following positions corresponding to wild-type IL-2: 42. 44.

2. The IL-2 mutant protein according to claim 1, wherein the IL-2 mutant is mutated at an amino acid residue corresponding to position 42 of wild-type IL-2 selected from the group consisting of: F42N, F42A, F42G, F42Q, F42E, F42D, F42P, F42S, F42T, F42K, F42R, F42V; or

The IL-2 mutant is mutated at the 44 position corresponding to wild-type IL-2 with an amino acid residue selected from the group consisting of: F44T, F44A, F44G, F44Q, F44E, F44D, F44P, F44S, F44N, F44K, F44R, F44V;

preferably, the IL-2 mutant is mutated at the position corresponding to position 42 of the wild-type IL-2 with an amino acid residue selected from the group consisting of: F42N, F42A, F42G, F42P, F42S, F42T, F42E, F42D; or

The IL-2 mutant is mutated at the 44 position corresponding to wild-type IL-2 with an amino acid residue selected from the group consisting of: F44A, F44G, F44P, F44S, F44T, F44E, F44D.

3. The IL-2 mutant protein according to claim 1 or 2, wherein the IL-2 mutant is mutated at the following amino acid residues corresponding to wild-type IL-2: F42N, F44T.

4. A fusion protein or conjugate comprising the IL-2 mutant of any one of claims 1-3 and a non-IL-2 functional moiety.

5. A bispecific or trispecific antibody comprising an IL-2 mutant of any one of claims 1-3 and a non-IL-2 functional moiety.

6. A polynucleotide encoding the IL-2 mutant of any one of claims 1-3 or the fusion protein or conjugate of claim 4 or the bispecific or trispecific antibody of claim 5.

7. An expression vector comprising the polynucleotide of claim 6.

8. A host cell comprising the expression vector of claim 7 or having the polynucleotide of claim 6 integrated into the genome of the host cell.

9. A pharmaceutical composition comprising an IL-2 mutant of any one of claims 1-3 or a fusion protein or conjugate of claim 4 or a bispecific or trispecific antibody of claim 5, and a pharmaceutically acceptable excipient.

10. Use of an IL-2 mutant of any one of claims 1-3 or a fusion protein or conjugate of claim 4 or a bispecific or trispecific antibody of claim 5 in the manufacture of a medicament for treating a disease in an individual.

Technical Field

The present invention relates to the field of protein engineering. In particular, the present invention relates to novel interleukin-2 (IL-2) mutants that have eliminated binding to IL-2 ra, reduced binding to IL-2R β γ dimer, but retain the ability to stimulate proliferation of tumor immune cells, including but not limited to T cells and NK cells, as compared to the wild-type IL-2 original protein, and methods for making the same. The interleukin 2 mutant of the invention is more suitable for conjugates of fusion protein and antibody. Can be used for immunotherapy without the side effects of immunotherapy with native IL-2.

Background

Interleukin-2 (IL-2, Interleukin-2) is a type I cytokine, also known as T cell growth factor, produced by activated CD4+ and CD8+ T cells. Activated Dendritic Cells (DCs), Natural Killer (NK) cells and NKT cells can also produce IL-2. IL-2 is a multifunctional cytokine with a very important role in lymphocyte homeostasis.

IL-2 is able to increase NK cell activity and mediate activation-induced cell death. IL-2 can induce T cell expansion to enhance adoptive immunotherapy. IL-2 can promote the proliferation and development of regulatory T cells (Tregs) and maintain the homeostasis and the functions of the Tregs. Approved by the FDA for the treatment of melanoma and renal cell carcinoma in the 80 s of the 20 th century, but due to the short half-life of IL-2 in vivo, about 15min, about 60,000 IU/kg and 720,000IU/kg were required to be injected every 8h during the treatment, thus causing some adverse reactions. In addition, high doses of IL-2 can cause vascular (or capillary) leak syndrome (VLS) in patients, and low dose IL-2 regimens have been tested in patients to avoid VLS, however, at the cost of reduced treatment outcome.

IL-2 has three receptor subunits: IL-2R α, IL-2R β and IL-2R γ. Can be classified into receptors with different affinities according to the difference of binding subunits, and IL-2R alpha beta gamma is high affinity (Kd 10)^-11) IL-2R beta gamma is medium affinity (Kd 10)^-9) IL-2R α is low affinity (Kd 10)^-8) A receptor. Different types of receptors are expressed on different cell surfaces. The surface of regulatory T cells (Treg) expresses a high-affinity receptor (IL-2R alpha beta gamma), and is sensitive to low-dose IL-2; whereas effector T cells (Teff) express a moderate affinity receptor (IL-2R β γ) on their surface, sensitive to high doses of IL-2.

IL-2 is a multifunctional cytokine. The low dose of IL-2 can selectively stimulate the proliferation of Treg cells, and the treatment mechanism is that the surface of the Treg cells can express a high-affinity IL-2 receptor (IL-2R alpha beta gamma). In clinical experiments, low-dose IL-2 has good curative effect on treating self diseases such as Systemic Lupus Erythematosus (SLE), type I diabetes mellitus (T1D) and the like, and is expected to become a potential drug for treating the self immune diseases. Teff cells express moderate affinity receptors on their surface and are sensitive to high doses of IL-2. High doses of IL-2 induce the proliferation of Teff cells and are used for the treatment of cancer, AIDS, etc., but high doses of IL-2 produce a range of toxicities during the treatment, such as arrhythmia, dyspnea and hypoxia, pulmonary edema, etc. IL-2 is a known potent T cell and NK cell somatomedin, but its use is limited by the side effects described above.

Tumor immunity is a method for effectively treating tumors in recent years, and killing of tumor cells by in vivo T cells and NK cells is realized through PD-1 or PD-L1 inhibitors and other similar immunosuppressive agents such as CTLA-4, CD-47 antibodies and the like. In recent years, more and more bispecific antibodies are regarded as important in tumor immunity, for example, PD1 monoclonal antibody is selected by roche to couple IL-2 quadruple mutant (US20180326010) for tumor immunity. The IL-2 quadruple mutant (CN103492411A) mutates amino acids of three sites of F44A, Y45A and L72G in IL-2, reduces the affinity of IL-2 protein to a high-affinity IL-2 receptor and retains the affinity of the mutant IL-2 protein to a medium-affinity IL-2 receptor, but simultaneously reduces the biological activity of the obtained IL-2 mutant.

Therefore, there is a need in the art for the use of IL-2 for tumor immunization to stably produce IL-2 mutants that are effective in treating tumors.

Disclosure of Invention

The invention provides an interleukin 2 mutant protein, a fusion protein, an antibody or a conjugate containing the interleukin 2 mutant protein, and a pharmaceutical composition containing the interleukin 2 mutant protein, the fusion protein, the antibody or the conjugate. Compared with wild type interleukin 2, the interleukin 2 mutant of the invention eliminates the binding with IL-2R alpha, reduces the binding with IL-2R beta gamma dimer, still can retain the required biological activity, and stimulates the proliferation of tumor immune cells, including but not limited to T cells and NK cells. The interleukin 2 mutant protein is more suitable for conjugates of fusion protein and antibody. The interleukin 2 mutant protein of the invention has extremely low potential immunogenicity. The interleukin 2 mutant protein of the present invention can overcome the problems associated with IL-2 immunotherapy.

In a first aspect, the present invention provides an IL-2 mutant, which has a mutation in an amino acid residue such that the binding capacity of IL-2 to its receptor is altered as compared to wild-type IL-2; the IL-2 mutants have eliminated affinity for high affinity IL-2 receptors while reducing affinity for medium affinity IL-2 receptors.

In a preferred embodiment, the high affinity IL-2 receptor is a heterotrimeric form of the IL-2 receptor, consisting of a receptor alpha subunit, a receptor beta subunit, and a receptor gamma subunit; the medium affinity IL-2 receptor is a peptide that comprises only the IL-2 receptor beta subunit and the IL-2 receptor gamma subunit, and no IL-2 receptor alpha subunit.

In a preferred embodiment, the IL-2 mutant has a binding affinity for the high affinity IL-2 receptor that is reduced by 55% or more, more preferably 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, as compared to wild-type IL-2, most preferably the IL-2 mutant does not bind to the high affinity IL-2 receptor;

the binding affinity of the IL-2 mutant to the medium affinity IL-2 receptor is reduced by more than 10%, more preferably by more than 20%, more preferably by more than 30%, more preferably by more than 40%, more preferably by more than 50%, more than 60%, more preferably by more than 70%, more preferably by more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the binding affinity of the wild-type IL-2 to the medium affinity IL-2 receptor; most preferably, the IL-2 mutant has a reduced binding affinity for the medium affinity IL-2 receptor compared to the binding affinity of wild-type IL-2 for the medium affinity IL-2 receptor.

In a preferred embodiment, the IL-2 mutant retains the proliferative effect of activating tumor immune cells, including but not limited to T cells and NK cells.

In a specific embodiment, the IL-2 mutant has mutations in amino acid residues corresponding to positions 42 and 44 of wild-type IL-2.

In a specific embodiment, the IL-2 mutant has an amino acid residue mutation at one or both of the following positions corresponding to wild-type IL-2: 42 and 44.

In a preferred embodiment, the IL-2 mutant is mutated at the position corresponding to position 42 of the wild-type IL-2 with an amino acid residue selected from the group consisting of: F42N, F42A, F42G, F42P, F42S, F42T, F42E, F42D; or

The IL-2 mutant is mutated at the 44 position corresponding to wild-type IL-2 with an amino acid residue selected from the group consisting of: F44A, F44G, F44P, F44S, F44T, F44E, F44D.

In a preferred embodiment, the IL-2 mutant is mutated at one or two of the following amino acid residues corresponding to wild-type IL-2: F42N, F44T.

In a preferred embodiment, the IL-2 mutant has an elimination of the O sugar site.

In a preferred embodiment, the IL-2 mutant is mutated at position 3 corresponding to wild-type IL-2, thereby eliminating the O sugar site.

In a preferred embodiment, the IL-2 mutant is mutated at the following amino acid residue at position 3 corresponding to the wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P; preferably T3A.

In a preferred embodiment, the IL-2 mutant has a mutation at cys position 125: C125L, C125S, C125A; preferably C125S.

In a second aspect, the present invention provides an IL-2 mutant, wherein the amino acid residues of the IL-2 mutant are mutated as compared to wild-type IL-2; the IL-2 mutants have eliminated affinity for high affinity IL-2 receptors while reducing affinity for medium affinity IL-2 receptors.

In a preferred embodiment, the high affinity IL-2 receptor is a heterotrimeric form of the IL-2 receptor, consisting of a receptor alpha subunit, a receptor beta subunit, and a receptor gamma subunit; the medium affinity IL-2 receptor is a peptide that comprises only the IL-2 receptor beta subunit and the IL-2 receptor gamma subunit, and no IL-2 receptor alpha subunit.

In a preferred embodiment, the IL-2 mutant has a binding affinity for the high affinity IL-2 receptor that is reduced by 55% or more, more preferably 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, as compared to wild-type IL-2, most preferably the IL-2 mutant does not bind to the high affinity IL-2 receptor;

the binding affinity of the IL-2 mutant to the medium affinity IL-2 receptor is reduced by more than 10%, more preferably by more than 20%, more preferably by more than 30%, more preferably by more than 40%, more preferably by more than 50%, more than 60%, more preferably by more than 70%, more preferably by more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the binding affinity of the wild-type IL-2 to the medium affinity IL-2 receptor; most preferably, the IL-2 mutant has a reduced binding affinity for the medium affinity IL-2 receptor compared to the binding affinity of wild-type IL-2 for the medium affinity IL-2 receptor.

In a preferred embodiment, the IL-2 mutant retains the proliferative effect of activating tumor immune cells, including but not limited to T cells and NK cells.

In a specific embodiment, the amino acid residues of the IL-2 mutant are mutated as compared to the wild-type IL-2, thereby affecting binding to the IL-2 receptor.

In a specific embodiment, the IL-2 mutant has mutations in amino acid residues corresponding to positions 42 and 44 of wild-type IL-2.

In a specific embodiment, the IL-2 mutant has an amino acid residue mutation at one or more of the following positions corresponding to wild-type IL-2: 42 and 44.

In a preferred embodiment, the IL-2 mutant is mutated at position 42 corresponding to wild-type IL-2: F42N, F42A, F42G, F42Q, F42E, F42D, F42P, F42S, F42T, F42K, F42R, F42V.

In a preferred embodiment, the IL-2 mutant is mutated at position 44 corresponding to wild-type IL-2: F44T, F44A, F44G, F44Q, F44E, F44D, F44P, F44S, F44N, F44K, F44R, F44V.

In a preferred embodiment, the IL-2 mutant has an elimination of the O sugar site.

In a preferred embodiment, the IL-2 mutant is mutated at position 3 corresponding to wild-type IL-2, thereby eliminating the O sugar site.

In a preferred embodiment, the IL-2 mutant is mutated at the following amino acid residue at position 3 corresponding to the wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P; preferably T3A.

In a preferred embodiment, the IL-2 mutant has a mutation at cys position 125: C125L, C125S, C125A; preferably C125S. In a preferred embodiment, the non-IL-2 functional moiety is selected from the group consisting of:

in a third aspect, the invention provides a fusion protein or conjugate comprising an IL-2 mutant according to the first or second aspect and a non-IL-2 functional moiety.

In a preferred embodiment, the non-IL-2 functional moiety is selected from the group consisting of:

fc fragments, including but not limited to: fc fragments of human IgG1, IgG2, IgG3 and IgG4, and Fc fragment mutants with homology of more than 90%;

human Serum Albumin (HSA);

anti-HSA antibodies and fragments thereof;

anti-albumin polypeptides or antibodies;

transferrin;

a human chorionic gonadotrophin beta subunit Carboxy Terminal Peptide (CTP);

elastin-like polypeptides (ELP);

an antigen-binding moiety.

A cellular receptor or ligand.

In a preferred embodiment, the IL-2 mutant and the non-IL-2 functional moiety in the fusion protein may be linked directly or via a linker; the linker may be a repeat of AAA or GS, including but not limited to a repeat of G3S or a repeat of G4S; such as (G3S) 4.

In a preferred embodiment, the IL-2 mutant or fusion protein may be further modified to form a conjugate as follows:

polyethylene glycol modification (pegylation);

polysialation modification (PSA);

modifying saturated fatty acid;

hyaluronic acid modification (Hyaluronic acid, HA);

polyamino acid modification (proline-alamine-serine polymer, PASylation).

In a fourth aspect, the invention provides a bispecific or trispecific antibody comprising an IL-2 mutant of the first or second aspect and an antibody moiety.

In a preferred embodiment, the antibody moiety is selected from the group consisting of:

fc fragments, including but not limited to: fc fragments of human IgG1, IgG2, IgG3 and IgG4, and Fc fragment mutants with homology of more than 90%;

in a preferred embodiment, the antigen binding moiety is:

an antibody or active antibody fragment thereof;

fab molecules, scFv molecules and VHH molecules; or

In preferred embodiments, the IL-2 mutant in the bispecific or trispecific antibody may be linked directly to a non-IL-2 functional moiety, or may be linked by a linker; the linker may be a repeat of AAA or GS, including but not limited to a repeat of G3S or a repeat of G4S; such as (G3S) 4.

In a preferred embodiment, the IL-2 mutant, non-IL-2 functional moiety, may be linked to other cytokines or tumor markers to form a trispecific antibody.

In a fifth aspect, the present invention provides a polynucleotide encoding the IL-2 mutant of the first or second aspect, or the fusion protein or conjugate of the third aspect, or the bispecific or trispecific antibody of the fourth aspect.

In a sixth aspect, the present invention provides an expression vector comprising the polynucleotide of the fifth aspect.

In a seventh aspect, the present invention provides a host cell comprising an expression vector according to the sixth aspect, or having integrated into its genome a polynucleotide according to the fifth aspect.

In a preferred embodiment, the host cell is a eukaryotic cell; preferably yeast, insect cells, animal cells; more preferably animal cells; mammalian cells, such as chinese hamster ovary cells, are most preferred.

In an eighth aspect, the present invention provides a pharmaceutical composition comprising an IL-2 mutant of the first or second aspect, or a fusion protein or conjugate of the third aspect, or a bispecific or trispecific antibody of the fourth aspect, and a pharmaceutically acceptable excipient.

In a ninth aspect, the invention provides the use of an IL-2 mutant of the first or second aspect, or a fusion protein or conjugate of the third aspect, or a bispecific or trispecific antibody of the fourth aspect, in the manufacture of a medicament for the in vitro expansion of T lymphocytes, natural killer NK cells or for the treatment of a disease in an individual.

In a preferred embodiment, the disease is a disease for which IL-2 is used as an immunotherapy.

In preferred embodiments, the disease is cancer, an immune disease, Human Immunodeficiency Virus (HIV) infection, Hepatitis C Virus (HCV) infection, rheumatoid arthritis, atopic dermatitis, and the like.

In a preferred embodiment, the cancer, systemic lupus erythematosus, immune disease, diabetes, Human Immunodeficiency Virus (HIV) infection, Hepatitis C Virus (HCV) infection, rheumatoid arthritis, atopic dermatitis, and the like is treated by stimulating the immune system or by proliferating immune cells.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 SDS-PAGE of purified products of IL-2 mutant and IL-2 wild-type fusion proteins; in the figure, M: prestained Protein Ladder; 1: IL-2-HSA; 2: IL-2gm 17-HSA;

FIG. 2 shows the binding of IL-2 mutant, IL-2 wild type, to IL-2 Ra detected using Biacore, where the Rmax for IL-2gm17-HSA is 0 and the Rmax for IL-2-HSA is 140;

FIG. 3 shows the binding of IL-2 mutant, IL-2 wild type, and IL-2R β γ dimer as measured by Biacore; wherein the Rmax of IL-2gm17-HSA is 2.5 and the Rmax of IL-2-HSA is 17. As shown in fig. 3, IL-2gm17-HSA reduced affinity for IL-2R β γ dimer relative to IL-2-HSA;

FIG. 4 shows the stimulation of NK92 cell proliferation by interleukin-2 mutant and wild type IL-2;

FIG. 5 shows the proliferation of NK cells by IL-2gm17-HSA and wild-type IL-2-HSA;

FIG. 6 shows the proliferation of Treg cells by IL-2gm17-HSA and wild-type IL-2-HSA; and

FIGS. 7a-f show the sequences SEQ ID NO 1-6 used in the present invention.

Detailed Description

The inventors have conducted extensive and intensive studies and unexpectedly found that a novel IL-2 mutant modified by glycosylation after site-directed mutagenesis of an IL-2 polypeptide or a novel IL-2 mutant polypeptide not modified by glycosylation can eliminate the affinity of IL-2 protein for a high-affinity IL-2 receptor, reduce the affinity of the mutant IL-2 protein for a medium-affinity IL-2 receptor, retain the biological activity of IL-2, and stimulate the proliferation of tumor immune cells including but not limited to T cells and NK cells to achieve the purpose of treatment. The mutant is more suitable for the conjugate composition of fusion protein and antibody, and can achieve the effect of inhibiting or treating tumors by stimulating the proliferation of immune cells. The present invention has been completed based on this finding.

IL-2 mutants of the invention

In the present invention, the IL-2 polypeptide is subjected to amino acid residue changes by site-directed mutagenesis, thereby altering the binding capacity or affinity of IL-2 to its receptor, but retaining biological activity. The IL-2 mutant of the invention can better stimulate the proliferation of tumor immune cells, including but not limited to T cells and NK cells, and compared with wild type IL-2, the side effect is also obviously reduced, thereby realizing better treatment aim.

The IL-2 mutants of the invention are preferably expressed in eukaryotic cells and obtained by cell culture. Yeast, insect cells, animal cells can be selected, and transgenic animals can also be selected. In particular embodiments, the host cell is a eukaryotic cell; preferably yeast, insect cells, animal cells; the animal cell may be a mammalian cell, including but not limited to CHO cells, 293 cells, SP/20 cells, NS0 cells.

When yeast cells or insect cells are used as host cells, it is possible that the glycoforms of the IL-2 mutants obtained are non-human. Those skilled in the art will recognize that non-human glycoforms can be further modified into adult glycoforms.

In other embodiments, prokaryotic bacterial expression fermentation or in vitro cell-free synthesis may also be used to obtain IL-2 mutants.

With respect to the number of mutation sites introduced, amino acid residue mutation may occur at any one of two sites, positions 42 and 44, in the wild-type IL-2. Thus, the IL-2 mutants of the invention may be mutated at the amino acid residue corresponding to position 42 of the wild-type IL-2 protein: F42N, F42A, F42G, F42Q, F42E, F42D, F42P, F42S, F42T, F42K, F42R, F42V; and/or the following amino acid residue mutations occur at position 44 corresponding to the wild-type IL-2 protein: F44T, F44A, F44G, F44Q, F44E, F44D, F44P, F44S, F44N, F44K, F44R, F44V;

preferably, the IL-2 mutant is mutated at the position corresponding to position 42 of the wild-type IL-2 with an amino acid residue selected from the group consisting of: F42N, F42A, F42G, F42P, F42S, F42T, F42E, F42D; or

The IL-2 mutant is mutated at the 44 position corresponding to wild-type IL-2 with an amino acid residue selected from the group consisting of: F44A, F44G, F44P, F44S, F44T, F44E, F44D.

Based on the routine practice in the field, the original O-sugar site in the IL-2 polypeptide can be eliminated, the O sugar does not affect the IL-2 biological activity, the O sugar has complex structure and difficult analysis, and in order to reduce the complexity of production quality control, the glycosylation site can be eliminated by using genetic engineering mutation technology. Thus, the IL-2 mutants of the invention may be mutated at the 3-position corresponding to the wild-type IL-2 protein by the following amino acid residues: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P; preferably T3A. During purification and renaturation of IL-2 gene products, e.g.mismatching of disulfide bonds or intermolecular disulfide bond formation, reduces IL-2 activity. At present, point mutation is applied, namely cysteine at the position 125 is mutated into leucine or serine, so that only one disulfide bond can be formed, and the activity in the renaturation process of IL-2 is ensured. It has also been reported that a novel rIL-2 is produced by a protein engineering technique, in which cysteine at position 125 of an IL-2 molecule is changed to alanine, and the specific activity of the modified IL-2 is significantly increased as compared with that of the natural IL-2. Thus, the IL-2 mutants of the invention may be mutated at the amino acid residue corresponding to position 125 of the wild-type IL-2 protein: C125L, C125A, C125S; preferably C125S.

"corresponds to"

The term "corresponding to" as used herein has the meaning commonly understood by a person of ordinary skill in the art. Specifically, "corresponding to" means the position of one sequence corresponding to a specified position in the other sequence after alignment of the two sequences by homology or sequence identity. Thus, for example, reference to "corresponding to wild-type IL-2" means that an amino acid sequence is aligned with the amino acid sequence of wild-type IL-2 to find the position in the amino acid sequence that corresponds to wild-type IL-2.

Fusion proteins or conjugates of the invention

Based on the IL-2 mutants of the invention, the skilled worker knows that the IL-2 mutants of the invention can be made as fusion proteins or conjugates with other functional moieties of non-IL-2. In this context, a conjugate refers to a water-soluble polymer covalently linked to the residues of a mutant IL-2 polypeptide. In specific embodiments, the non-IL-2 functional moiety includes, but is not limited to: fc fragment, Human Serum Albumin (HSA), anti-HSA antibody or antibody fragment, transferrin, human chorionic gonadotropin beta subunit Carboxyl Terminal Peptide (CTP), elastin-like polypeptide (ELP), antigen binding portion, and cytokine, specifically interleukin, interferon, tumor necrosis factor superfamily, colony stimulating factor, chemotactic factor, growth factor, etc.

Based on the routine operation in this field, the technicians in this field knows how to obtain the IL-2 mutant fusion protein or conjugate. For example, the IL-2 mutants of the invention can be linked directly to other non-IL-2 functional moieties or can be linked via a linker. The linker may be a repeat of AAA or GS, including but not limited to a repeat of G3S or a repeat of G4S; such as (G3S) 4.

Further, the IL-2 mutant or fusion protein conjugate may be subjected to polyethylene glycol modification (pegylation), polysialylation modification (PSA), saturated fatty acid modification, Hyaluronic acid modification (HA), or polyamino acid modification (PAS) to form a conjugate.

The bispecific or trispecific antibodies of the invention

The development of disease is usually caused by multiple pathogenic factors, and simultaneous blockade of multiple targets may lead to better therapeutic effect, so bispecific antibodies (BsAb) are produced at the same time. Tumor immunotherapy is currently the new direction for treating tumors. Bispecific antibodies can bind to two different antigens, and thus have a broad development prospect in the field of tumor therapy. Bispecific antibodies were originally prepared using chemical coupling or hybridoma hybridization. The rapid development of recombinant DNA technology has revolutionized the structure of bispecific antibodies, mainly classified into two major classes, the IgG class containing Fc region and the non-IgG class without Fc region. The structure of the IgG type bispecific antibody is similar to that of a monoclonal antibody, the protein relative molecular mass is large, and the half-life period of plasma is long. The non-IgG type bispecific antibody has more diverse structural forms, smaller protein relative molecular mass, stronger tissue permeability and shorter plasma half-life.

Based on the IL-2 mutants of the invention, the skilled person knows that the IL-2 mutants of the invention can be covalently linked to antibody domains. In particular embodiments, the antibody domain includes, but is not limited to: IgG-type antibodies and non-IgG-type antibodies. In a preferred embodiment, the antibody domain may be an antibody or an active antibody fragment thereof, a Fab molecule, a scFv molecule and a VHH molecule, an immunoglobulin molecule, a receptor protein molecule or a ligand protein molecule; the immunoglobulin molecule may be an IgG molecule.

Based on the routine operation in this field, the technicians in this field knows how to obtain the IL-2 mutant bispecific antibody. For example, the IL-2 mutants of the invention can be linked directly to other non-IL-2 functional moieties or can be linked via a linker. The linker may be a repeat of AAA or GS, including but not limited to a repeat of G3S or a repeat of G4S; such as (G3S) 4.

Optionally, the mutant of the present invention may be coupled to an antibody against a T cell surface antigen, or may be coupled to an antibody against a tumor cell surface antigen. Preferably, the mutant of the present invention may be coupled to a T cell surface antigen antibody.

Optionally, the mutant of the present invention can be coupled to a T cell surface antigen antibody to form a bispecific antibody, and can also be coupled to a tumor cell surface antigen antibody to form a bispecific antibody. Optionally, the mutant of the present invention can be coupled to a T cell surface antigen antibody to form a trispecific antibody, or coupled to a tumor cell surface antigen antibody to form a trispecific antibody. Optionally, the mutant of the present invention can be coupled to a T cell surface antigen antibody or a tumor cell surface antigen antibody to form a trispecific antibody.

Pharmaceutical compositions of the invention and modes of administration thereof

On the basis of the IL-2 mutant, the invention also provides a pharmaceutical composition. In a specific embodiment, the pharmaceutical composition of the invention comprises an IL-2 mutant of the invention or a fusion protein or conjugate of claim 5 or a bispecific or trispecific antibody of claim 7 and optionally pharmaceutically acceptable excipients.

Optionally, the composition of the present invention further comprises a pharmaceutically acceptable excipient. If desired, pharmaceutically acceptable excipients may be added to the IL-2 mutant polypeptides, fusion proteins or conjugates, bispecific antibodies or trispecific antibodies of the invention to form a composition.

Uses and methods of use of the IL-2 mutants of the invention

As described above, the IL-2 mutants of the present invention are capable of eliminating the affinity of the IL-2 protein for the high affinity IL-2 receptor and reducing the affinity of the mutant IL-2 protein for the medium affinity IL-2 receptor while retaining the biological activity of IL-2, thereby better stimulating tumor immune cells, including but not limited to T cells and NK cells, to proliferate. Therefore, the IL-2 mutant, the fusion protein, the conjugate, the bispecific antibody or the trispecific antibody and the pharmaceutical composition can be prepared into corresponding medicaments. The medicament can be used for in vitro expansion of T lymphocytes, natural killer NK cells or treatment of diseases using IL-2 as immunotherapy. In specific embodiments, the disease is cancer; for example, there is a need for cancers that are treated by stimulating the immune system or by proliferating immune cells. In particular embodiments, the disease may be systemic lupus erythematosus, an immune disease, diabetes, Human Immunodeficiency Virus (HIV) infection, Hepatitis C Virus (HCV) infection, rheumatoid arthritis, atopic dermatitis, and the like.

The invention can also be used as a substitute for wild type IL-2 in the in vitro expanded cells in the treatment process of cells such as CAT-T, CAR-NK and the like.

The invention has the advantages that:

1. the IL-2 mutant protein eliminates the affinity with a high-affinity IL-2 receptor, reduces the affinity with a medium-affinity IL-2 receptor, and simultaneously retains the biological activity of IL-2;

2. compared with other IL-2 mutants in the prior art, the IL-2 mutant has few mutation sites, can achieve affinity reduction only by two mutation points, and keeps the effect of biological activity;

3. the IL-2 mutant protein avoids overlapping mutation to reduce specific activity, can affect the combination of two different receptor regions only through the mutation of two sites, and reduces potential immunogenicity;

4. the IL-2 mutant of the invention is convenient for production and quality control, generally does not need the process of in vitro modification, reduces steps and improves production efficiency;

5. the IL-2 mutants of the invention facilitate the formation of bifunctional or multifunctional fusion proteins or immunological compositions with other molecules;

6. the IL-2 mutants of the invention may be used in immunotherapy, but do not cause vascular (or capillary) leak syndrome (VLS) caused by native IL-2; and

the invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Examples

Example 1 Synthesis of mutant Interleukin-2 (IL-2) proteins

1. Gene synthesis

The nucleotide sequence encoding the amino acid sequence of a mutant of interleukin-2 (IL-2) protein was obtained by an automated gene synthesis method. In the examples, the terminal addition of the HSA tag to the gene fragment is advantageous for purification, and the tag is also a common means for prolonging the half-life of protein drugs. The gene fragment is flanked by single restriction enzyme cleavage sites. All gene synthesis sequences are designed with a 5' DNA sequence encoding a leader peptide that targets secretion of the protein in eukaryotic cells.

Number of mutations	Mutation site	Example mutant names	Protein tag
				3	3. 42 and 44	IL-2gm17(T3A,F42N,F44T)	HSA

2. Plasmid construction

The synthesized gene was subcloned into pTT5 plasmid using molecular biology reagents according to the manufacturer's instructions.

3. Expression of mutant Interleukin-2 (IL-2) proteins

IL-2 mutant molecules were named IL-2gm17-HSA (SEQ ID NO:1) and wild type IL-2-HSA (SEQ ID NO:2), and were constructed into eukaryotic expression vectors by molecular cloning to prepare IL-2gm17-HSA and IL-2-HSA expression vectors, respectively. Transient transfection expression of IL-2 mutant molecules was performed using 293E cells cultured in Freestyle medium. 24 hours before transfection, 0.5X 10 cells were inoculated into 1L cell culture flasks⁶293E cells/ml 150ml, 5% CO at 37 ℃₂Shaking and culturing at 120rpm in an incubator. 150 μ l of 293fectin was added to 2.85ml of OptiMEM at the time of transfection, and after thoroughly mixing, the mixture was placed in a chamberIncubation for 2 minutes at room temperature; at the same time, 150. mu.g of each plasmid for expression was diluted to 3ml using OptiMEM. The diluted transfection reagent and plasmid are mixed well, incubated at room temperature for 15 minutes, and then the mixture is added into the cells and mixed well. Shaking and culturing at 37 deg.C and 5% CO2 incubator at 120rpm for 7 days. The cell culture supernatant was collected and filtered through a 0.22 micron filter and then purified by Q-HP ion exchange chromatography (GE) using 20mM Tris 0-500mM NaCl, pH8.0, with samples collected in volume succession. Fractions collected were subjected to SDS-PAGE using 4-20% gradient gel (Kinsley), and pooled according to electrophoretic purity. The purity of the purified protein after the sample was checked by SDS-PAGE, and the results are shown in FIG. 1. The protein Marker in lane M, IL-2-HSA in lane 1, and IL-2gm17-HSA in lane 7.

Example 2 preparation of receptor proteins

To study the binding capacity of the IL-2 mutant molecules to IL-2R α receptor and IL-2R β γ heterodimerization receptor, human IL-2R α receptor and IL-2R β γ heterodimerization receptor proteins were prepared.

The human IL-2R alpha receptor is designed by linking the IL-2R alpha extracellular domain coding sequence with the 6 XHis Tag coding sequence (SEQ ID NO:3) and cloning into a eukaryotic expression vector. Transient transfection expression of IL-2R α receptor was performed using 293E cells cultured in Freestyle medium. 24 hours before transfection, 0.5X 10 cells were inoculated into 1L cell culture flasks⁶293E cells/ml 150ml, 5% CO at 37 ℃₂Shaking and culturing at 120rpm in an incubator. When in transfection, 150 mu l of 293fectin is firstly added into 2.85ml of OptiMEM, and after fully and uniformly mixing, the mixture is incubated for 2 minutes at room temperature; at the same time, 150. mu.g of the plasmid for expressing IL-2 R.alpha.receptor was diluted to 3ml using OptiMEM. Mixing the diluted transfection reagent and plasmid, incubating at room temperature for 15min, adding the mixture into cells, mixing, and adding 5% CO at 37 deg.C₂Shaking at 120rpm in incubator for 7 days. The cell culture supernatant was collected and filtered through a 0.22 μ M filter and then purified by using a Ni-NTA affinity column (GE) eluting with 20mM PB-0.5M NaCl-100mM imidazole. The protein was purified by SDS-PAGE using 4-20% gradient gel (Kinsley).

The design of the human IL-2R beta gamma heterodimerization receptor utilizes the 'Knobs into Holes' technology, connects the IL-2R beta extracellular domain coding sequence with the Fc fragment (SEQ ID NO:4) of the code 'Knobs', and clones into a eukaryotic expression vector; the IL-2R gamma extracellular domain coding sequence was ligated to the Fc fragment encoding "Holes" (SEQ ID NO:5) and cloned into a eukaryotic expression vector. Transient transfection expression of IL-2R β γ heterodimerization receptor was performed using 293E cells cultured in Freestyle medium. 24 hours before transfection, 0.5X 10 cells were inoculated into 1L cell culture flasks⁶Cells/ml 293E cells 150ml, 37 ℃, 5% CO2 incubator 120rpm shake culture. When in transfection, 150 mu l of 293fectin is firstly added into 2.85ml of OptiMEM, and after fully and uniformly mixing, the mixture is incubated for 2 minutes at room temperature; at the same time, 75. mu.g each of the plasmids for expressing IL-2 R.beta.gamma.heterodimerization receptor was diluted to 3ml using OptiMEM. Mixing the diluted transfection reagent and plasmid, incubating at room temperature for 15min, adding the mixture into cells, mixing, and adding 5% CO at 37 deg.C₂Shaking at 120rpm in incubator for 7 days. The cell culture supernatant was collected, filtered through a 0.22 μ M filter, and then purified using a MabSelect Sure affinity chromatography column (GE), eluting with 20mM sodium citrate, pH3.0, and pH adjusted to neutral with 1M Tris base. The protein was purified by SDS-PAGE using 4-20% gradient gel (Kinsley).

Example 3 affinity assay for detecting bound receptors Using biacore

To investigate the affinity of the IL-2 mutant for the receptor relative to wild type, the affinity of the IL-2 mutant molecule IL-2gm17-HSA and wild type IL-2-HSA for the human IL-2R α subunit was determined by Biacore 8k (ge) using recombinant monomeric IL-2R α subunits under the following conditions: human IL-2R α subunit was immobilized on CM5 chip (190 RU). IL-2gm17-HSA and IL-2-HSA were used as analytes in HBS-EP buffer at 25 ℃. For IL-2R α, the analyte concentration was 200nM down to 1.526nM (1:2 dilution) and the flow was 30 μ l/min (180 seconds on binding time, 300 seconds off time). For IL-2R α, regeneration was performed with 20mM NaOH,30ul/min for 10 seconds. For IL-2R α, 1:1 binding was used, RI ≠ 0, R max ═ global fit data.

As shown in FIG. 2, the Rmax of IL-2gm17-HSA was 0, and the Rmax of IL-2-HSA was 140. IL-2gm17-HSA had eliminated affinity for IL-2R α relative to IL-2-HSA.

The affinity of the IL-2 mutant molecule IL-2gm17-HSA and wild-type IL-2-HSA for the human IL-2R β γ heterodimer was determined by Biacore 8k (ge) using recombinant IL-2R β γ heterodimer under the following conditions: human hIL-2R β, γ ECD-N-hIgG1Fc was immobilized on Protein A chips (400 RU). IL-2gm17-HSA and IL-2-HSA were used as analytes in HBS-EP buffer at 25 ℃. For IL-2R β γ, the analyte concentration was 200nM down to 1.5625nM (1:2 dilution) with a flow of 30 μ l/min (180 seconds on binding time, 300 seconds off time). For IL-2R β γ, regeneration was performed with 10mM Glycine (pH1.5),30ul/min,30 sec. For IL-2R β γ, 1:1 binding was used, RI ≠ 0, R max ═ local fit data.

As shown in FIG. 3, the Rmax of IL-2gm17-HSA was 2.5 and the Rmax of IL-2-HSA was 17. As shown in FIG. 3, IL-2gm17-HSA has reduced affinity for IL-2R β γ dimer relative to IL-2-HSA. Thus, IL-2gm17-HSA abolished affinity for IL-2R α and reduced affinity for IL-2R β γ dimer relative to IL-2-HSA.

Example 4 cell proliferation assay Using NK92 cells

NK92 cell is an IL-2 dependent NK cell line derived from peripheral blood mononuclear cells of a 50 year old white male with aggressive non-Hodgkin's lymphoma. Part of the cells express CD25 on their cell surface. IL-2gm1-HSA (SEQ ID NO:6), which IL-2 mutant eliminates the affinity of interleukin 2 protein for high affinity IL-2 receptors while reducing the affinity for medium affinity IL-2 receptors. The inventors used NK92 cells to evaluate the activity of IL-2gm17-HSA and IL-2-HSA in a cell proliferation assay.

NK-92 cells in logarithmic growth phase were harvested, washed once with the basal medium MEM-alpha and co-cultured (5000 cells/well) with different concentrations of IL-2gm17-HSA, IL-2gm1-HSA and IL-2-HSA in the experimental medium MEM-alpha from Gibco (cat. No. 32561-. The full wavelength fluorescence was measured by end-point method using a microplate reader (from Molecular Devices, model I3x) with 100. mu.l of ATP detection substrate CellTiter-Glo (from Promega (cat. No. G7571)) per well.

The activity of IL-2gm17-HSA and IL-2-HSA was measured using a cell proliferation assay, and a summary of the results is shown in FIG. 4. All tested items induced NK92 cell growth in a dose-dependent manner. In the case of comparable cell proliferation fold, EC₅₀The larger, the less activity was demonstrated to stimulate the growth of NK 92. This change was due to CD25 binding influenced by its mutant protein, whereas the IL-2gm1-HSA mutant protein retained IL-2R signaling activation by the IL-2R β γ heterodimer, so that the cells were efficiently proliferated after increasing concentrations. The specific activity of IL-2gm1-HSA relative to IL-2-HSA for stimulating the proliferation of NK92 cells was 1.07%, demonstrating that IL-2gm1-HSA abolished the binding of CD25 to some NK92 cells expressing CD25 on their surface, with reduced stimulation due to the absence of IL-2R α β γ heterotrimer formation. IL-2gm1-HSA stimulated the NK92 cell proliferation effect about 100-fold lower relative to IL-2-HSA. The IL-2gm17-HSA mutant protein not only affected the binding to CD25, but also attenuated the activation of IL-2R signaling by IL-2R β γ heterodimer, so that the cells were efficiently proliferated after increasing the concentration. The specific activity of IL-2gm17-HSA on stimulating NK92 cell proliferation relative to IL-2-HSA was 0.0055%, demonstrating that IL-2gm17-HSA abolished binding to CD25 on NK92 cells partially expressing CD25 on their surface, and the absence of IL-2R α β γ heterotrimer also resulted in reduced stimulation. IL-2gm17-HSA stimulated the NK92 cell proliferation effect about 10000-fold lower relative to IL-2-HSA. It is shown that the IL-2gm17-HSA mutant protein reduces the activation of IL-2R signaling by IL-2R beta gamma heterodimer, so that the cells can be effectively proliferated after the concentration is increased, and the biological activity is kept.

Example 5 measurement of IL-2 mutants induced PBMC proliferation assay

Fresh blood samples of Chinese healthy people (n-2) were collected in heparin sodium tubes and PBMCs were isolated, resuspended in RPMI-1640 medium (containing 10% FBS) and plated in 48-well plates (1 x 10)⁶One/well), PBMC were stimulated with different concentrations of IL-2gm17-HSA, and wild-type IL-2-HSA at 37Co-culturing for 6 days in a 5% carbon dioxide incubator at the temperature of. FACS staining was performed with cell surface and intracellular marker antibodies to detect different cell populations, samples were obtained by a lsrfortessa cell analyzer.

NK cells are defined as CD3-/CD56+ and Treg cells as CD3+ CD4+ CD25+ Foxp3 +.

NK cell proliferation after 6 days incubation with different concentrations of IL-2gm17-HSA and wild type IL-2-HSA is shown in FIG. 5.

IL-2gm17-HSA showed slightly less stimulatory proliferation of NK cells relative to wild-type IL-2-HSA at concentrations of 0-20 nM; at 20-1000nM concentration, IL-2gm17-HSA showed a significantly increased stimulatory effect on NK cells relative to wild-type IL-2-HSA.

The proliferation of Treg cells after 6 days incubation with different concentrations of IL-2gm17-HSA and wild-type IL-2-HSA is shown in FIG. 6.

At the concentration of 0-20nM, the stimulation and proliferation effect of IL-2gm17-HSA on Treg cells is slightly reduced compared with that of wild type IL-2-HSA; at 20nM and 1000nM concentrations, IL-2gm17-HSA showed a significantly reduced proliferation-stimulating effect on Treg cells relative to wild-type IL-2-HSA.

In the experiment, IL-2gm17-HSA plays a remarkable role in stimulating the proliferation of NK cells and inhibiting the proliferation of Treg cells.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

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Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg

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Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

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Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys

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Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys

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Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val

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Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser

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Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly

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Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile

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Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu

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Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp

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Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro

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Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

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Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

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Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

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Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

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Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

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Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

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Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

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Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

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His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly

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Gly Ser Asp Asp Asp Asp Lys Ala Val Asn Gly Thr Ser Gln Phe Thr

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Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp

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Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg

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Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser

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Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr

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Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg

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Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg

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Leu Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu Thr His Arg

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Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg

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His Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu

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Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu

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Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys

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Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu

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Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr

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<400> 5

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Ala Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

20 25 30

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

35 40 45

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

50 55 60

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

65 70 75 80

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

85 90 95

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

100 105 110

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

115 120 125

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

130 135 140

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

145 150 155 160

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys

165 170 175

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

180 185 190

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

195 200 205

Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

210 215 220

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

225 230 235 240

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly

245 250 255

Gly Ser Asp Asp Asp Asp Lys Leu Asn Thr Thr Ile Leu Thr Pro Asn

260 265 270

Gly Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr

275 280 285

Asp Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe

290 295 300

Val Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu

305 310 315 320

Pro Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp

325 330 335

Asn Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile

340 345 350

Thr Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr

355 360 365

Phe Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr

370 375 380

Gln Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn

385 390 395 400

Leu Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn

405 410 415

Asn Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr

420 425 430

Asp Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys

435 440 445

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

450 455 460

Arg Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu

465 470 475 480

Trp Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro

485 490 495

Phe Leu Phe Ala Leu Glu Ala

500

<210> 6

<211> 721

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Asn Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr Ala Ala Ala Asp Ala His Lys Ser Glu Val Ala

130 135 140

His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu

145 150 155 160

Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

165 170 175

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp

180 185 190

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp

195 200 205

Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala

210 215 220

Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln

225 230 235 240

His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

245 250 255

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys

260 265 270

Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

275 280 285

Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys

290 295 300

Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu

305 310 315 320

Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys

325 330 335

Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val

340 345 350

Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser

355 360 365

Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly

370 375 380

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile

385 390 395 400

Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu

405 410 415

Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp

420 425 430

Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser

435 440 445

Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly

450 455 460

Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val

465 470 475 480

Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

485 490 495

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu

500 505 510

Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys

515 520 525

Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu

530 535 540

Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val

545 550 555 560

Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His

565 570 575

Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val

580 585 590

Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg

595 600 605

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe

610 615 620

Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala

625 630 635 640

Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu

645 650 655

Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys

660 665 670

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

675 680 685

Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe

690 695 700

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

705 710 715 720

Leu