CN113754775A

CN113754775A - Bispecific antibody for resisting PD-L1 and HER2

Info

Publication number: CN113754775A
Application number: CN202010487636.9A
Authority: CN
Inventors: 朱祯平; 黄浩旻; 张学赛; 李晴柔
Original assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Current assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date: 2020-06-02
Filing date: 2020-06-02
Publication date: 2021-12-07
Also published as: CN115605513A; CN115605513B; WO2021244328A1; TW202146457A

Abstract

The invention provides a bispecific antibody against PD-L1 and HER 2. The bispecific antibody of the invention can simultaneously and specifically bind to two targets of PD-L1 and HER2, and has similar or even better biological activity than the monoclonal antibody.

Description

Bispecific antibody for resisting PD-L1 and HER2

Technical Field

The invention relates to the field of antibodies, and more particularly discloses a bispecific antibody against PD-L1 and HER 2.

Background

Human programmed cell death receptor-1 (PD-1) is a 288 amino acid type I membrane protein and is one of the known major Immune checkpoints (Immune Checkpoint) (Blank et al,2005, Cancer Immunotherapy,54: 307-) -314. PD-1 is expressed in activated T lymphocytes, and binding of the ligands PD-L1 (programmed cell death receptor-Ligand 1) and PD-L2 (programmed cell death receptor-Ligand 2) can inhibit the activity of T lymphocytes and the related in vivo cellular immune response. PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. Numerous studies have shown that the interaction of PD-1 and PD-L1 is not only necessary to maintain immune system balance in vivo, but also a major mechanism and cause PD-L1 expressing positive tumor cells to circumvent immune surveillance. By blocking the negative regulation and control of cancer cells to PD-1/PD-L1 signal channels, the immune system is activated, and the tumor specific cellular immune response related to T cells can be promoted, thereby opening the door of a new tumor treatment method, namely tumor immunotherapy.

PD-1 (encoded by gene Pdcd 1) is an immunoglobulin superfamily member that is associated with CD28 and CTLA-4. The results of the study show that PD-1 negatively regulates antigen receptor signaling when bound to its ligand (PD-L1 and/or PD-L2). The murine PD-1 structure and the cocrystal structure of murine PD-1 and human PD-L1 have been clarified (Zhang, X. et al, Immunity 20: 337-347 (2004); Lin et al, Proc. Natl. Acad. Sci. USA 105: 3011-6 (2008)). PD-1 and similar family members are type I transmembrane glycoproteins that contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail responsible for binding to a signaling molecule. The PD-1 cytoplasmic tail contains two tyrosine-based signaling motifs, the ITIM (immunoreceptor tyrosine inhibition motif) and the ITSM (immunoreceptor tyrosine transduction motif).

PD-1 plays an important role in the immune evasion mechanism of tumors. Tumor immunotherapy, namely, cancer resistance by using the immune system of the human body, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction, so how to break the tumor microenvironment becomes the key point of anti-tumor research. The role of PD-1 in the tumor microenvironment has been determined by prior work: PD-L1 is expressed in a number of mouse and human tumors (and can be induced by IFN-. gamma. in most PD-L1 negative tumor cell lines) and is presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al, Proc. Natl. Acad. Sci. U.S.A.99: 12293-12297 (2002); Strome S.E. et al, Cancer Res., 63: 6501-6505 (2003)). Biopsy evaluation by immunohistochemistry has revealed expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells in many primary tumors in humans. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, stomach cancer, oral squamous cell carcinoma, urothelial cell carcinoma, and pancreatic cancer, as well as head and neck tumors, among others. Therefore, the blocking of the interaction of PD-1/PD-L1 can improve the immunocompetence of tumor specific T cells and is beneficial to the immune system to eliminate tumor cells, so that PD-L1 becomes a hot target for developing tumor immunotherapy drugs.

HER2/neu (human epidermal growth factor receptor 2), also known as erbB2, has tyrosine protein kinase activity and is a member of the human epidermal growth factor receptor family and is expressed at low levels in only a few normal tissues of adults. However, studies show that HER2 is overexpressed in various tumors, such as the overexpression is existed in about 30% of breast cancer patients and 16% of gastric cancer patients, and the overexpression of HER2 in tumors can remarkably promote the neogenesis of tumor vessels, the growth of tumors and enhance the invasion and metastasis capacity of tumors, and is an important indicator for poor prognosis of patients of the kind. Thus, as early as 1998, the first monoclonal antibody drug Herceptin (Herceptin, Trastuzumab/Trastuzumab, Genentech/Roche) targeting HER2 was approved by the FDA for marketing and use in the treatment of HER 2-overexpressed breast and gastric cancers.

Bispecific antibodies refer to antibody molecules that specifically bind to two antigens or two epitopes simultaneously. Bispecific antibodies can be divided into structurally symmetric and asymmetric molecules according to symmetry. Bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules, depending on the number of binding sites. Bispecific antibodies are evolving into a new class of therapeutic antibodies that can be used to treat a variety of inflammatory, cancer, and other diseases.

Disclosure of Invention

The invention provides a bispecific antibody against PD-L1 and HER 2.

Accordingly, it is a first object of the present invention to provide a bispecific antibody against PD-L1 and HER 2.

It is a second object of the present invention to provide an isolated nucleic acid encoding said bispecific antibody.

The third purpose of the invention is to provide an expression vector containing the nucleotide.

The fourth object of the present invention is to provide a host cell comprising said expression vector.

The fifth object of the present invention is to provide a method for producing the bispecific antibody.

It is a sixth object of the present invention to provide a pharmaceutical composition comprising said bispecific antibody.

The seventh object of the present invention is to provide the use of said bispecific antibody or said pharmaceutical composition for the preparation of a medicament for the treatment of cancer.

An eighth object of the present invention is to provide the bispecific antibody or the pharmaceutical composition for use in a method for treating cancer.

In order to achieve the purpose, the invention provides the following technical scheme:

a first aspect of the invention provides a bispecific antibody against PD-L1 and HER2, comprising two polypeptide chains and two light chains, wherein:

(a) the polypeptide chain comprises VH-PDL1-CH1-CH2-CH3-L1-VH-HER2-L2-VL-HER2 or VH-HER2-L2-VL-HER2-L1-VH-PDL1-CH1-CH2-CH3 from N terminal to C terminal, and the light chain comprises VL-PDL1-CL from N terminal to C terminal; or

(b) Said polypeptide chain comprises, from N-terminus to C-terminus, VH-HER2-CH1-CH2-CH3-L1-VH-PDL1-L2-VL-PDL1 or VH-PDL1-L2-VL-PDL1-L1-VH-HER2-CH1-CH2-CH3, and said light chain comprises, from N-terminus to C-terminus, VL-HER 2-CL;

wherein VH-PDL1 is a heavy chain variable region binding to PD-L1, VL-PDL1 is a light chain variable region binding to PD-L1, VH-HER2 is a heavy chain variable region binding to HER2, VL-HER2 is a light chain variable region binding to HER2, and L1 and L2 are (G)₄S) x, x is 3, 4, 5 or 6, CH1-CH2-CH3 is a heavy chain constant region, CL is a light chain constant region, VH-PDL1 forms an antigen binding site with specificity binding to PD-L1 with VL-PDL1, and VH-HER2 forms an antigen binding site with specificity binding to HER2 with VL-HER 2.

According to the preferred embodiment of the present invention, L1 is (G)₄S)₃L2 is (G)₄S)₄。

According to a preferred embodiment of the invention, said VH-PDL1 comprises an amino acid sequence as set forth in SEQ ID NO: 1-3, and VL-PDL1 comprises an amino acid sequence set forth in SEQ ID NO: 4-6, and VH-HER2 comprising an amino acid sequence set forth in SEQ ID NO: 7-9 or SEQ ID NO: 13-15, and VL-HER2 comprising an amino acid sequence set forth in SEQ ID NO: 10-12 or SEQ ID NO: 16-18.

According to a preferred embodiment of the invention, said VH-PDL1 has the amino acid sequence as shown in SEQ ID NO: 19, and VL-PDL1 having the amino acid sequence shown in SEQ ID NO: 20, and the VH-HER2 has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 23, and VL-HER2 having the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 24.

According to a preferred embodiment of the invention, said polypeptide chain has the amino acid sequence as shown in SEQ ID NO: 31 or SEQ ID NO: 33, and the light chain has an amino acid sequence shown as SEQ ID NO: 26; or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 34, and the light chain has an amino acid sequence shown as SEQ ID NO: 28; or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 36, and the light chain has an amino acid sequence shown as SEQ ID NO: 30.

According to the invention, the heavy chain constant region comprises an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region and the light chain constant region comprises a kappa or lambda light chain constant region.

In a second aspect, the invention provides an isolated nucleotide encoding said bispecific antibody.

In a third aspect, the invention provides an expression vector, wherein the expression vector contains the nucleotide as described above.

In a fourth aspect, the present invention provides a host cell comprising an expression vector as described above.

In a fifth aspect, the present invention provides a method for preparing the bispecific antibody, the method comprising the steps of:

(a) culturing a host cell as described above under expression conditions, thereby expressing the bispecific antibody;

(b) isolating and purifying the bispecific antibody of (a).

A sixth aspect of the invention provides a pharmaceutical composition comprising a bispecific antibody as described above and a pharmaceutically acceptable carrier.

A seventh aspect of the invention provides the use of a bispecific antibody as described above or a pharmaceutical composition as described above for the manufacture of a medicament for the treatment of cancer.

According to the invention, the cancer is selected from the group consisting of: melanoma, renal cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases, and the like.

An eighth aspect of the invention provides a method of treating cancer comprising administering to a subject in need thereof a bispecific antibody as described above or a pharmaceutical composition as described above.

Has the advantages that: the invention provides an anti-PD-L1 and anti-HER 2 bispecific antibody, and in vitro and in vivo experimental results show that the bispecific antibody can be simultaneously and specifically combined with two targets of PD-L1 and HER2, and has biological activity similar to that of a monoclonal antibody or even better.

Drawings

FIG. 1 shows the results of an ELISA assay for the affinity of bispecific antibodies to PDL 1-ECD-his.

FIG. 2 shows the results of determining the affinity of bispecific antibodies to HER2-ECD-his by ELISA, wherein FIG. 2A shows the bispecific antibody with M8 and 607 combined and FIG. 2B shows the bispecific antibody with M8 and 612 combined.

FIG. 3 is the result of the competitive inhibition of PD-1 binding to PD-L1 by the bispecific antibody.

FIG. 4 shows the results of the bispecific antibody binding activity assay for the PD-1 and PD-L1-highly expressing cell lines.

Fig. 5 shows the results of the activity assay of the bispecific antibody in inhibiting BT474 cell proliferation.

Figure 6 is the results of the activity assay of bispecific antibodies to inhibit N87 cell proliferation.

Fig. 7 shows the results of the activity assay of the bispecific antibody in inhibiting BT474 cell proliferation.

Figure 8 is the results of the activity assay of bispecific antibodies to inhibit N87 cell proliferation.

Detailed Description

In the present invention, the terms "Antibody (abbreviated Ab)" and "Immunoglobulin G (abbreviated IgG)" are heterotetrameric proteins of about 150000 daltons having the same structural features, which consist of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes (isotypes). Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a constant region consisting of three domains, CH1, CH2, and CH 3. Each light chain has a variable region (VL) at one end and a constant region at the other end, the light chain constant region comprising a domain CL; the constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC) and the like. Heavy chain constant regions include IgG1, IgG2, IgG3, IgG4 subtypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by interpoly disulfide bonds formed between the hinge regions.

In the present invention, the term "bispecific antibody (diabody)" refers to an antibody molecule capable of specifically binding to two antigens (targets) or two epitopes simultaneously.

In the present invention, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically a mixture of different antibodies with epitopes against different antigens), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

In the present invention, the terms "Fab" and "Fc" mean that papain cleaves an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of the antibody. The Fc fragment, i.e., the crystallizable fragment (Fc), consists of the CH2 and CH3 domains of the antibody. The Fc region has no antigen binding activity and is the site of antibody interaction with effector molecules or cells.

In the present invention, the term "scFv" is a single chain antibody (scFv), and is formed by connecting an antibody heavy chain variable region and an antibody light chain variable region via a short peptide (linker) of 15 to 25 amino acids.

In the present invention, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy chain variable region and the light chain variable region. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)).

In the present invention, the terms "anti", "binding", "specific binding" refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. Typically, the antibody is present in an amount less than about 10^-7M, e.g. less than about 10^-8M、10^-9M、10^-10M、10^-11M or less binds the antigen with an equilibrium dissociation constant (KD). In the present invention, the term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity of an antibody to an antigen is determined in a BIACORE instrument using Surface Plasmon Resonance (SPR for short) or the relative affinity of the binding of an antibody to an antigen is determined using ELISA.

In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is a region of an antigen to which an antibody binds.

In the present invention, the term "expression vector" may be pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, etc., and other vectors used in mammalian expression systems, etc., and the expression vector includes a fusion DNA sequence to which appropriate transcription and translation regulatory sequences are ligated.

In the present invention, the term "host cell" refers to a cell suitable for expressing the above expression vector, and may be a eukaryotic cell, such as mammalian or insect host cell culture system, which can be used for the expression of the fusion protein of the present invention, and CHO (Chinese Hamster Ovary), HEK293, COS, BHK and derived cells of the above cell can be suitable for the present invention.

In the present invention, the term "pharmaceutical composition" means that the bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition for more stable therapeutic effect, which can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, while protecting the multifunctional group of the protein from degradation (including but not limited to aggregation, deamination or oxidation).

The following examples and experimental examples are intended to further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., Fritsch, e.f. and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

Example 1 construction of anti-PD-L1 and HER2 bispecific antibodies

The structural general formula of the bispecific antibody is as follows:

1)Ab-L1-scFv；

2)scFv-L1-Ab；

wherein Ab is an antibody comprising two heavy chains and two light chains;

the scFv is a single-chain antibody, is formed by connecting a heavy chain variable region and a light chain variable region of another antibody through a connecting sequence L2, and is respectively connected to the N tail end or the C tail end of two heavy chains of the Ab through a connecting sequence L1;

l1 and L2 are joining sequences (G)₄S)x，x may be 3, 4, 5 or 6.

The bispecific antibody is constructed by using the combination of an anti-human PD-L1 antibody M8 (the sequence is derived from PCT/CN2020/090442), an anti-human HER2 antibody 607 (the sequence is derived from US5821337 and is prepared according to the trastuzumab sequence) and an anti-human HER2 antibody 612 (the sequence is derived from WO2020/025013A1), and the specific structures are shown in Table 1.

TABLE 1 Structure of bispecific antibody of the present invention

Wherein PDL1-HC represents the heavy chain of M8, PDL1-LC represents the light chain of M8, 607-VH represents the heavy chain variable region of 607, 607-VL represents the light chain variable region of 607, and the rest are similar.

The bispecific antibody constructed according to the present invention has the relevant sequence information as shown in table 2, wherein the CDRs are encoded according to the Kabat rules.

TABLE 2 sequence information of the antibodies of the invention

Constructing a pTT5 expression vector (purchased from NRC biotechnology Research Institute) through EcoRI and HindIII enzyme cutting sites by a gene synthesis and a conventional molecular cloning method, obtaining an expression vector of a heavy chain and a light chain of each bispecific antibody and a corresponding monoclonal antibody, co-transfecting HEK F cells (purchased from Thermo Fisher, a product number A14527) with the expression vector of each heavy chain sequence and the corresponding light chain expression vector, expressing and purifying to obtain each bispecific antibody PDL1H-607scFv, 607H-PDL1scFv, 607scFv-PDL1H, PDL1 scFv-36607 1scFv-612H and 612H-PDL1scFv and corresponding monoclonal antibodies M8, 607 and 612.

Example 2 determination of affinity of bispecific antibodies targeting HER2 and PD-L1 by ELISA method

PDL1-ECD-his (NCBI accession number NP-054862.1) and HER2-ECD-his protein (NCBI accession number NP-004439.2) were prepared as follows: PD-L1 and HER2 ectodomain genes are respectively synthesized according to the sequences provided by NCBI, a signal peptide sequence is added at the N end of the ectodomain genes, a 6 XHis tag is added at the C end of the ectodomain genes, the ectodomain genes are respectively constructed into pTT5 expression vectors through EcoRI and HindIII enzyme cutting sites, and HEK-293F cells are transfected for expression and purified to obtain the recombinant plasmid.

PDL1-ECD-his or HER2-ECD-his proteins were diluted and coated on 96-well ELISA plates, 0.05. mu.g/well, overnight at 4 ℃, washed 3 times with PBST (PBS containing 0.05% Tween20), formulated with PBS as 2% BSA, 200. mu.L/well, blocked at room temperature for 2H, washed 2 times with PBST, diluted with PBST as 1% BSA to various concentrations and added to ELISA wells, 100. mu.L/well, set 3 multiple wells per concentration, incubated at room temperature for 1H, washed 3 times with PBST, diluted with PBST as 1% HRP at appropriate ratio (secondary antibody-anti human IgG, available from Lipore, Cathap 101, Cathap P), added to PBST as well as 100. mu.L/well, added with TMST as wash solution at room temperature for 3 times, added to PBST as 3 times at room temperature, 100 μ L/well, color development to desired color with 2M H₂SO₄The color reaction was stopped, 50. mu.L/well, each reaction solution was shaken well and OD450nm was measured with a microplate reader, the data was analyzed, and EC was calculated₅₀。

Experimental results As shown in FIGS. 1 and 2, respectively, the affinity (EC) of each bispecific antibody for PDL1-ECD-his and HER2-ECD-his₅₀) As shown in Table 3 and Table 4, respectively, it can be seen that the affinity of each bispecific antibody to PDL1-ECD-his is comparable to that of M8, all at the same level, and PDL1scFv-607H is slightly superior. While the affinity for HER2-ECD-his, in the bispecific antibody combination of 607 and M8, was superior to 607scFv-PDL1H and PDL1H-607scFv, comparable to that of 607 monoclonal antibody, and PDL1scFv-607H and 607H-PDL1scFv were relatively weak; at 612 with M8, both PDL1scFv-612H and 612H-PDL1scFv had comparable affinities for HER2-ECD-his, slightly weaker than 612 mAb.

TABLE 3 EC of each bispecific antibody against PDL1-ECD-his₅₀

TABLE 4 EC of each bispecific antibody against HER2-ECD-his₅₀

Example 3 bispecific antibody competitive inhibition of PD-1 binding Activity assay with PD-L1

PDL1-ECD-Fc protein (prepared as above except that the C-terminal tag was replaced with human Fc sequence) was diluted and coated on a 96-well ELISA plate, 0.2. mu.g/well, coated overnight at 4 ℃, PBST (PBS containing 0.05% Tween20) washed 3 times, 2% BSA was prepared in PBS, 200. mu.L/well, blocked for 2H at room temperature, after PBST was washed 2 times, 500ng/ml biotin-labeled PD1-Fc (i.e., PD1-Fc-biotin, accession number NP-005009.2 at NCBI, extracellular domain gene of PD-1 was obtained from NCBI registration information, cloned and prepared in the same manner as 1-ECD-Fc) solution PDL1H-607, 607H-PDL1, 607-PDL-1H, PDL 1-607H, PDL 1-

scFv

612H, 612H-PDL-1-8 was diluted to different concentrations and added to 100. mu.L/well in ELISA, setting 3 multiple wells for each concentration, incubating at room temperature for 1h, washing with PBST for 3 times, diluting a secondary antibody (HRP-labeled streptavidin purchased from Sigma, cat # S4672-5MG) with 1% BSA prepared from PBST in a proper ratio, adding ELISA wells at 100. mu.L/well, incubating at room temperature for 1h, washing with PBST for 3 times, adding TMB color developing solution at 100. mu.L/well, developing to a desired color, and washing with 2M H₂SO₄The color reaction was stopped, 50. mu.L/well, each reaction solution was shaken well and OD450nm was measured with a microplate reader, the data was analyzed, and EC was calculated₅₀。

The results are shown in FIG. 3, where each bispecific antibody competitively inhibits the binding of PD-1 to PD-L1Force (IC)₅₀) As shown in Table 5, it can be seen that each bispecific antibody competes for inhibition of PD-1 as compared to PD-L1 activity.

TABLE 5 IC of competitive inhibition of PD-1 binding to PD-L1 for each bispecific antibody₅₀

Example 4 bispecific antibody inhibits the binding Activity of PD-1 and PD-L1 high expressing cell lines

PD-1 high expression cell line PD-1Effect Cells and PD-L1 high expression cell line PD-L1 aAPC/CHO-K1 Cells were purchased from Promega corporation (cat # J1252).

1) PDL1-aAPC/CHO-K1 cells in logarithmic growth phase were trypsinized, centrifuged, counted, resuspended in 10% FBS Ham/F12 medium (purchased from Thermo Fisher, cat # 11765054), plated at 40000 cells per well at 100. mu.L/well, plated into white-plate clear-bottom 96-well plates in CO₂The cell culture chamber was grown overnight at 37 ℃.

2) M8, 607H-PDL1scFv, PDL1scFv-607H, PDL1H-607scFv, 607scFv-PDL1H, PDL1scFv-612H, 612H-PDL1scFv were prepared in 1640 medium (purchased from Thermo Fisher, Cat. No. 61870036) containing 1% FBS at a maximum concentration of 200nM, respectively, and diluted in 3-fold gradient (working concentration maximum dose of 100nM) while diluting PD-1Effect Cells to 1.25X 10 Cells in 1% FBS 1640 medium⁶one/mL.

3) The supernatant of the 96-well plate cultured with PDL1-aAPC/CHO-K1 cells was discarded, 40. mu.L of diluted PD-1Effect Cell suspension and 40. mu.L of diluted antibody were added, the plate was gently shaken, and the cells were further cultured in a Cell incubator at 37 ℃ for 6 hours.

4) Bio-Glo Luciferase Assay Reagent (from Promega, cat #: g7941) Incubate at room temperature for 10 min.

5) The luminescence intensity was measured in a multifunctional microplate reader and the data was analyzed by GraphPad Prism 6.

The results are shown in FIG. 4, and each bispecific antibody inhibits high expression of PD-1 and PD-L1IC of binding Activity of cell lines₅₀As shown in Table 6, it can be seen that PDL1H-607scFv, 607scFv-PDL1H are relatively superior in activity, and PDL1scFv-612H and PDL1scFv-607H are inferior.

TABLE 6 IC of each bispecific antibody inhibiting the binding of PD-1 and PD-L1 high expressing cell lines₅₀

Example 5 determination of cell proliferation inhibitory Activity of bispecific antibody against HER2 high expressing cell line

1) BT474 and N87 cells in logarithmic growth phase were trypsinized, counted, resuspended in complete medium, i.e., 1640 medium containing 10% FBS (purchased from Thermo Fisher, cat # 61870036), and seeded at 150 μ L/well into 96-well cell culture plates, breast cancer cells BT474 (purchased from chinese academy of sciences cell bank, cat #: TCHu143) 10000 cells per well, gastric cancer cell N87 (purchased from chinese academy of sciences cell bank, catalog No.: SCSP-534) in 8000 per well in CO₂The cells were incubated overnight at 37 ℃.

2) Diluting the antibody to be tested with complete culture medium according to 3-fold gradient, continuously diluting each group with 9 gradient, wherein the highest concentration of each antibody is 400nM (final working concentration is 100nM), adding 50 μ L of the above antibody to a 96-well cell culture plate, the final volume of each well is 200 μ L, setting the group without administration as negative control, continuously culturing in a cell culture box at 37 ℃ for 5d, and making 2 duplicate wells in each concentration.

3) The cell culture supernatant was discarded and 100. mu.L/well of CCK8 reaction solution (1: 10-fold dilution, purchased from Dojindo, cat # CK04), incubated at 37 ℃.

4) After the cell culture wells were developed to the desired depth, their absorbance was measured at a wavelength of 450nm in a multifunctional microplate reader and the data was analyzed by GraphPad Prism 6.

5) The cell survival rate and the growth inhibition rate were calculated according to the following formulas:

survival rate was (OD dose-OD blank)/(OD control-OD blank) × 100%. Growth inhibition rate is 1-survival rate.

The results of the experiments are shown in fig. 5, 6, 7 and 8, respectively, and the IC of each bispecific antibody for inhibiting proliferation of HER 2-highly expressed cell lines BT474 and N87₅₀As shown in Table 7, Table 8, Table 9, and Table 10, respectively, it can be seen that PDL1scFv-607H, 607H-PDL1scFv are more excellent in the inhibitory activity against BT474 cell proliferation for the double antibody combination of 607 and M8; PDL1H-607scFv was the least active against N87 cell proliferation inhibition, although IC of PDL1scFv-607H, 607H-PDL1scFv₅₀Comparable, but at high concentrations PDL1scFv-607H had the strongest maximal killing effect on N87 cells. For the diabody combination of 612 and M8, PDL1scFv-612H and 612H-PDL1scFv were comparable in cell proliferation inhibitory activity.

TABLE 7 IC inhibition of BT474 cell proliferation by bispecific antibodies₅₀

TABLE 8 IC inhibition of N87 cell proliferation by bispecific antibodies₅₀

TABLE 9 IC inhibition of BT474 cell proliferation by bispecific antibodies₅₀

TABLE 10 IC inhibition of N87 cell proliferation by bispecific antibodies₅₀

Sequence listing

<110> Sansheng Guojian pharmaceutical industry (Shanghai) GmbH

<120> a bispecific antibody against PD-L1 and HER2

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ser Tyr Gly Val His

1 5

<210> 2

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gln Leu Gly Leu Arg Ala Met Asp Tyr

1 5

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Tyr Ala Ser Gln Ser Phe Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Gln Ser Asn Ser Trp Pro Leu Thr

1 5

<210> 7

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Asp Thr Tyr Ile His

1 5

<210> 8

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 9

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr

1 5 10

<210> 10

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala

1 5 10

<210> 11

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> 12

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gln Gln His Tyr Thr Thr Pro Pro Thr

1 5

<210> 13

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Asp Tyr Ala Ile His

1 5

<210> 14

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 15

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Arg Asp Gly Gly Thr Ile Asn Tyr

1 5

<210> 16

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 17

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 18

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Ser Gln Ser Thr His Ile Pro Trp Thr

1 5

<210> 19

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 20

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

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

1 5 10 15

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

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala

65 70 75 80

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

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 21

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 24

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 25

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

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

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 26

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

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

1 5 10 15

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

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala

65 70 75 80

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

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

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

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 27

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

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

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

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

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

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

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

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

435 440 445

Gly Lys

450

<210> 28

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

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

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 29

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

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

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 30

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

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

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 31

<211> 709

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

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

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

450 455 460

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

465 470 475 480

Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile

485 490 495

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg

500 505 510

Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly

515 520 525

Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln

530 535 540

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg

545 550 555 560

Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

565 570 575

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln

595 600 605

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

610 615 620

Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln

625 630 635 640

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu

645 650 655

Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp

660 665 670

Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

675 680 685

Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr

690 695 700

Lys Val Glu Ile Lys

705

<210> 32

<211> 709

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

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

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

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

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

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

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

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

435 440 445

Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser

465 470 475 480

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

485 490 495

Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

500 505 510

Ile Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu

515 520 525

Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser

530 535 540

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

545 550 555 560

Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr

565 570 575

Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln

595 600 605

Ser Pro Asp Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr

610 615 620

Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln

625 630 635 640

Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser

645 650 655

Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

660 665 670

Phe Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr

675 680 685

Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr

690 695 700

Lys Leu Glu Ile Lys

705

<210> 33

<211> 709

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

130 135 140

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

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

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser

180 185 190

Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

245 250 255

Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly

260 265 270

Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly

275 280 285

Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly

290 295 300

Lys Gly Leu Glu Trp Ile Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp

305 310 315 320

Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser

325 330 335

Lys Asn Gln Val Ser Phe Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr

340 345 350

Ala Val Tyr Tyr Cys Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr

355 360 365

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly

370 375 380

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

385 390 395 400

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

405 410 415

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

420 425 430

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

435 440 445

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

450 455 460

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

465 470 475 480

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

485 490 495

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

500 505 510

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

515 520 525

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

530 535 540

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

545 550 555 560

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

565 570 575

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

580 585 590

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

595 600 605

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

610 615 620

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

625 630 635 640

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

645 650 655

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

660 665 670

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

675 680 685

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

690 695 700

Leu Ser Pro Gly Lys

705

<210> 34

<211> 709

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser

130 135 140

Pro Asp Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys

145 150 155 160

Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys

165 170 175

Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe

180 185 190

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr

210 215 220

Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

260 265 270

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile

275 280 285

Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

290 295 300

Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala

305 310 315 320

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

325 330 335

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

340 345 350

Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr

355 360 365

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

370 375 380

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

385 390 395 400

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

405 410 415

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

420 425 430

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

435 440 445

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

450 455 460

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

465 470 475 480

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

485 490 495

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

500 505 510

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

515 520 525

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

530 535 540

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

545 550 555 560

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

565 570 575

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

580 585 590

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

595 600 605

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

610 615 620

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

625 630 635 640

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

645 650 655

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

660 665 670

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

675 680 685

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

690 695 700

Leu Ser Pro Gly Lys

705

<210> 35

<211> 706

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser

130 135 140

Pro Asp Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys

145 150 155 160

Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys

165 170 175

Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe

180 185 190

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr

210 215 220

Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

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

260 265 270

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe

275 280 285

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

290 295 300

Glu Trp Ile Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn

305 310 315 320

Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser

325 330 335

Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val

340 345 350

Tyr Tyr Cys Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln

355 360 365

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

370 375 380

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

385 390 395 400

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

405 410 415

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

420 425 430

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

435 440 445

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

450 455 460

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

465 470 475 480

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

485 490 495

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

500 505 510

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

515 520 525

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

530 535 540

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

545 550 555 560

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

565 570 575

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

580 585 590

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

595 600 605

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

610 615 620

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

625 630 635 640

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

645 650 655

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

660 665 670

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

675 680 685

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

690 695 700

Gly Lys

705

<210> 36

<211> 706

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

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

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

450 455 460

Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln Ser Leu

465 470 475 480

Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val

485 490 495

His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu

500 505 510

Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser Arg

515 520 525

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

530 535 540

Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln

545 550 555 560

Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

565 570 575

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

580 585 590

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Asp

595 600 605

Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Arg Ala

610 615 620

Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys Pro Asp

625 630 635 640

Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe Ser Gly

645 650 655

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

660 665 670

Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln

675 680 685

Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

690 695 700

Ile Lys

705

Claims

1. Bispecific antibody against PD-L1 and HER2, characterized in that it comprises two polypeptide chains and two light chains, wherein:

wherein VH-PDL1 is a heavy chain variable region binding to PD-L1, VL-PDL1 is a light chain variable region binding to PD-L1, VH-HER2 is a heavy chain variable region binding to HER2, VL-HER2 is a light chain variable region binding to HER2, and L1 and L2 are (G)₄S) x, x is 3, 4, 5 or 6, CH1-CH2-CH3 is a heavy chain constant region, CL is a light chain constant region, VH-PDL1 and VL-PDL1 form an antigen binding site specifically binding to PD-L1, VH-HER2 forms an antigen binding site with VL-HER2 that specifically binds HER 2.

2. The bispecific antibody of claim 1, wherein L1 is (G)₄S)₃L2 is (G)₄S)₄。

3. The bispecific antibody of claim 1, wherein said VH-PDL1 comprises an amino acid sequence set forth in SEQ ID NO: 1-3, and VL-PDL1 comprises an amino acid sequence set forth in SEQ ID NO: 4-6, and VH-HER2 comprising an amino acid sequence set forth in SEQ ID NO: 7-9 or SEQ ID NO: 13-15, and VL-HER2 comprising an amino acid sequence set forth in SEQ ID NO: 10-12 or SEQ ID NO: 16-18.

4. The bispecific antibody of claim 3, wherein said VH-PDL1 has an amino acid sequence as set forth in SEQ ID NO: 19, and VL-PDL1 having the amino acid sequence shown in SEQ ID NO: 20, and the VH-HER2 has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 23, and VL-HER2 having the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 24.

5. The bispecific antibody of claim 4, wherein said polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 31 or SEQ ID NO: 33, and the light chain has an amino acid sequence shown as SEQ ID NO: 26; or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 34, and the light chain has an amino acid sequence shown as SEQ ID NO: 28; or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 36, and the light chain has an amino acid sequence shown as SEQ ID NO: 30.

6. The bispecific antibody of claim 1, wherein the heavy chain constant region comprises an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region and the light chain constant region comprises a kappa or lambda light chain constant region.

7. An isolated nucleotide encoding the bispecific antibody of any one of claims 1 to 6.

8. An expression vector comprising the nucleotide of claim 7.

9. A host cell comprising the expression vector of claim 8.

10. The method of making a bispecific antibody of any one of claims 1-6, comprising the steps of:

(a) culturing the host cell of claim 9 under expression conditions, thereby expressing the bispecific antibody;

(b) isolating and purifying the bispecific antibody of (a).

11. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-6 and a pharmaceutically acceptable carrier.

12. Use of the bispecific antibody of any one of claims 1-6 or the pharmaceutical composition of claim 11 in the manufacture of a medicament for the treatment of cancer.

13. The use of claim 12, wherein the cancer is selected from the group consisting of: melanoma, renal cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.

14. A method of treating cancer, comprising administering to a subject in need thereof the bispecific antibody of any one of claims 1-6 or the pharmaceutical composition of claim 11.

15. The method of claim 14, wherein the cancer is selected from the group consisting of: melanoma, renal cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.