A bispecific antibody targeting human claudin and human pdl1 protein and uses thereof

Abstract

The application discloses a bispecific antibody targeting human claudin and human PDL1 protein, characterized by comprising an anti-human claudin 18.2 antibody part and an anti-PD-L1 antibody part. The bispecific antibody can block the combination of PD-1 / PD-L1 while combining with human claudin 18.2 protein, and can play a role in activating NK cells to kill tumor cells in innate immunity and promoting the killing effect of killer T lymphocytes on tumors in acquired immunity. The bispecific antibody has better antitumor efficacy than the anti-claudin 18.2 antibody alone.

Description

TECHNICAL FIELD

[0001] The present application relates to a bispecific antibody targeting human claudin and human PDL1 protein and application thereof, and belongs to the field of biological medicine. BACKGROUND

[0002] Bispecific antibody (BsAb) is also called bifunctional antibody, which can recognize and bind two different antigens and epitopes at the same time, and block two different signal pathways to play its role. Compared with ordinary antibodies, BsAb increases one specific antigen binding site, and shows the following advantages in treatment:

[0003] Mediating immune cell killing of tumor: one of the important mechanisms of BsAb is to mediate immune cell killing. BsAb has two antigen binding arms, one of which binds to the target antigen, and the other binds to the marker antigen on the effector cell, which can activate the effector cell to target and kill tumor cells.

[0004] Dual-target signal blocking, playing unique or overlapping functions, effectively preventing drug resistance: simultaneous binding of dual targets and blocking of dual signal pathways is another important mechanism of BsAb. Receptor tyrosine kinase (RTKs) is the largest class of enzyme-linked receptors, which plays an important regulatory role in cell proliferation, such as Her family. RTKs are abnormally highly expressed on the surface of tumor cells, leading to malignant proliferation of tumor cells, and are therefore important targets for tumor treatment. Single-target monoclonal antibodies against RTKs have been widely used in tumor treatment, but tumor cells can escape immunity by switching signal pathways or activating intracellular signals through HER family members themselves or different members through homodimerization or heterodimerization. Therefore, using BsAb drugs to block two or more RTKs or their ligands at the same time can reduce tumor cell escape and improve treatment effect.

[0005] Stronger specificity, targeting and reduced off-target toxicity: using the characteristics of two antigen binding arms of BsAb that can bind different antigens, two antigen binding arms bind to two antigens on the surface of cancer cells, which can effectively enhance the binding specificity and targeting of antibodies to cancer cells, and reduce off-target side effects;

[0006] Effective reduction of treatment costs: taking BiTE as an example, compared with traditional antibodies, it has strong competitiveness in tissue penetration rate, tumor cell killing efficiency, off-target rate and clinical indications, and significant clinical advantages. Especially in the use of dosage, because its treatment effect can reach 100-1000 times of ordinary antibodies, the use dosage can be as low as 1 / 2000 of the original, which significantly reduces the cost of drug treatment. Compared with combination therapy, the cost of bispecific antibody is also much lower than that of two single drugs combined.

[0007] PD-1 (CD279) was first reported in 1992. The human PD-1 coding gene PDCD1 is located at 2q37.3, with a full length of 2097 bp, composed of 6 exons, and the translation product is a PD-1 precursor protein composed of 288 amino acids. After the removal of the signal peptide composed of the first 20 amino acids, the mature protein is obtained. PD-1 includes an extracellular immunoglobulin variable region IgV domain, a hydrophobic transmembrane domain and an intracellular domain, and the N-terminal ITIM motif of the intracellular tail domain contains 2 phosphorylation sites, and the C-terminal is an ITSM motif. PD-1 is a membrane protein, belongs to the CD28 immunoglobulin superfamily, mainly expressed on the surface of activated T cells, in addition to CD4 - CD8 - T cells, activated NK cells and monocytes have low abundance expression. PD-1 has two ligands, PD-L1 (CD274, B7-H1) and PD-L2 (CD273, B7-DC) of the B7 protein family, and the amino acid sequences of PD-L1 and PD-L2 are 40% identical. The main difference between the two is the different expression patterns. PD-L1 is constitutively expressed in APCs, non-hematopoietic cells (such as vascular endothelial cells, pancreatic islet cells) and immune-privileged sites (such as placenta, testis and eye), and inflammatory cytokines such as type I and type II interferons, TNF-α and VEGF can induce the expression of PD-L1. PD-L2 is only expressed in activated macrophages and dendritic cells. After PD-1 binds to PD-L1 on activated T cells, the ITSM motif of PD-1 is tyrosine phosphorylated, which in turn leads to the dephosphorylation of downstream protein kinases Syk and PI3K, inhibits the activation of downstream AKT, ERK and other pathways, and ultimately inhibits the transcription and translation of genes and cytokines required for T cell activation, and plays a role in negatively regulating T cell activity.

[0008] In tumor cells, tumor cells and tumor microenvironment up-regulate PD-L1 expression and interact with tumor-specific CD8 +PD-1 binding on the surface of T cells negatively regulates T cell activity and inhibits immune response. Tumor cells can up-regulate PD-L1 expression through the following four ways: 1. amplification of the gene encoding PD-L1 (9p24.1); 2. EGFR, MAPK, PI3K-Akt signaling pathway activation, HIF-1 transcription factor, etc. can up-regulate the expression of PD-L1 at the transcriptional level; 3. EB virus induction (EB virus positive gastric cancer and nasopharyngeal carcinoma show high expression of PD-L1); 4. epigenetic regulation. In the tumor microenvironment, the stimulation of inflammatory factors such as interferon-γ can also induce the expression of PD-L1 and PD-L2. Inflammatory factors can induce other cells in the tumor microenvironment, including macrophages, dendritic cells and stromal cells, to express PD-L1 and PD-L2, and tumor-infiltrating T cells that can recognize tumor antigens can secrete interferon-γ, thereby inducing up-regulation of PD-L1 expression. This process is called "adaptive immune resistance", and tumor cells can achieve self-protection through this mechanism. There is more and more evidence that tumors use PD-1-dependent immune suppression to evade immunity. High expression of PD-L1 and PD-L2 has been found in various solid tumors and hematological malignancies. In addition, there is a strong correlation between the expression of PD-Ls and the poor prognosis of tumor cells, which proves the existence of esophageal cancer, gastric cancer, renal cancer, ovarian cancer, bladder cancer, pancreatic cancer and melanoma, etc.

[0009] Claudin 18.2 has been increasingly valued as a tumor-specific antigen of gastrointestinal tumors, especially gastric cancer, in recent years.

[0010] Tight junctions (TJs) play a key role in the intercellular material flow, and also maintain cell polarity by blocking the radial diffusion of membrane proteins and membrane lipids, in addition, they are involved in recruiting signal molecules that regulate cell proliferation, differentiation and movement. Tight junctions are formed by claudins (CLDNs), and the claudin family consists of more than 20 protein molecules, all of which contain a four-transmembrane domain and similar amino acid sequences, but have certain specificity in tissue distribution. CLDNs play a key role in regulating the selective permeability of paracellular pathways, CLDN2 and CLDN15 are involved in forming cation channels and cation pores, and CLDN4 / 7 / 10 are involved in forming anion channels and pores. The differential expression of CLDN proteins is considered to be associated with various cancers. CLDN1 and CLDN7 are down-regulated in invasive breast cancer, prostate cancer and esophageal cancer, and CLDN3 / 4 is found to be up-regulated to varying degrees in various cancers such as cervical cancer, colon cancer, esophageal cancer and gastric cancer. Sahin et al. found that in normal tissues, the isoform 2 subtype of CLDN18 (Claudin18.2) is only expressed in the differentiated epidermal cells of the gastric mucosa, and is not expressed in the gastric stem cell area, but is abnormally highly expressed in primary gastric cancer and its metastases. There are also reports of high expression of Claudin18.2 in pancreatic cancer, esophageal cancer and lung cancer. Since Claudin18.2 is located on the cell membrane surface, its biological function and characteristics determine that it is an ideal therapeutic target, and in recent years, there have been monoclonal antibodies targeting this target. SUMMARY

[0011] The purpose of the present application is to provide a bispecific antibody targeting human claudin and human PDL1 protein and its application.

[0012] The present application adopts the following technical solutions:

[0013] A bispecific antibody targeting human claudin18.2 and human PDL1 protein, comprising:

[0014] An antibody part against human claudin18.2 and an anti-PD-L1 antibody part.

[0015] Further, the bispecific antibody targeting human claudin18.2 and human PDL1 protein of the present application has the sequence shown in SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66.

[0016] Further, the bispecific antibody of the present application targeting human claudin 18.2 and human PDL1 protein, the anti-human claudin 18.2 antibody part binds to the extracellular region of human claudin 18.2 protein, and the sequence of the anti-human claudin 18.2 antibody part is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34.

[0017] Further, the bispecific antibody of the present application targeting human claudin 18.2 and human PDL1 protein, the anti-PD-L1 antibody, and the sequence thereof is shown in SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.

[0018] The bispecific antibody of any one of the above, for use in the preparation of a medicament for treating cancer, infection or immunoregulatory disease.

[0019] The bispecific antibody of any one of the above, for use in the preparation of a medicament for inhibiting tumor growth.

[0020] Further, the cancer or tumor is selected from the group or site of colorectal, breast, ovarian, pancreatic, gastric, esophageal, prostate, kidney, cervix, bone marrow cancer, lymphoma, leukemia, thyroid, endometrial, uterus, bladder, neuroendocrine, head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

[0021] In particular, in a first aspect of the present application, there is provided a bispecific antibody targeting human claudin 18.2 and human PD-L1 protein, the bispecific antibody comprising:

[0022] an anti-human claudin 18.2 antibody moiety and an anti-PD-L1 antibody moiety.

[0023] In another preferred embodiment, the bispecific antibody has both binding activity to human claudin 18.2 and binding activity to human PD-L1 protein.

[0024] In another preferred embodiment, the complementarity determining regions (CDRs) of the anti-human claudin 18.2 antibody comprise:

[0025] HCDR1 as set forth in SEQ ID NO: 93, 75, 79, 83, or 87,

[0026] HCDR2 as set forth in SEQ ID NO: 94, 76, 80, 84, or 88, and

[0027] HCDR3 as set forth in SEQ ID NO: 95, 77, 81, 85, or 89; and

[0028] LCDR1 as set forth in SEQ ID NO: 90 or 72,

[0029] LCDR2 as set forth in SEQ ID NO: 91 or 73, and

[0030] LCDR3 as set forth in SEQ ID NO: 92, 74, 78, 82, or 86.

[0031] In another preferred embodiment, the three heavy chain CDRs and three light chain CDRs of the anti-human claudin 18.2 antibody are selected from the group consisting of:

[0032] (Z1) HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID No: 93, 94, 95; and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID No: 90, 91, 92;

[0033] (Z2) HCDR1, HCDR2, and HCDR3 as shown in SEQ ID Nos: 75, 76, 77; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID Nos: 72, 73, 74;

[0034] (Z3) HCDR1, HCDR2, and HCDR3 as shown in SEQ ID Nos: 79, 80, 81; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID Nos: 72, 73, 78;

[0035] (Z4) HCDR1, HCDR2, and HCDR3 as shown in SEQ ID Nos: 83, 84, 85; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID Nos: 72, 73, 82;

[0036] (Z5) HCDR1, HCDR2, and HCDR3 as shown in SEQ ID Nos: 87, 88, 89; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID Nos: 72, 73, 86.

[0037] In another preferred embodiment, the complementarity determining regions CDRs of the anti-human claudin 18.2 antibody comprise: HCDR1 as shown in SEQ ID NO: 93, HCDR2 as shown in SEQ ID NO: 94, and HCDR3 as shown in SEQ ID NO: 95, and LCDR1 as shown in SEQ ID NO: 90, LCDR2 as shown in SEQ ID NO: 91, and LCDR3 as shown in SEQ ID NO: 92.

[0038] In another preferred embodiment, the anti-PD-L1 antibody is a single domain antibody.

[0039] In another preferred embodiment, the three complementarity determining regions CDRs of the single domain antibody comprise: HCDR1 as shown in SEQ ID NO: 69, HCDR2 as shown in SEQ ID NO: 70 or 96, and HCDR3 as shown in SEQ ID NO: 71.

[0040] In another preferred embodiment, the three complementarity determining regions CDRs of the single domain antibody comprise: HCDR1 as shown in SEQ ID NO: 69, HCDR2 as shown in SEQ ID NO: 70, and HCDR3 as shown in SEQ ID NO: 71.

[0041] In another preferred embodiment, the three complementarity determining regions CDRs of the single-domain antibody comprise: HCDR1 of SEQ ID NO: 69, HCDR2 of SEQ ID NO: 96, and HCDR3 of SEQ ID NO: 71.

[0042] In another preferred embodiment, the bispecific antibody is a dimer composed of two monomers, the monomers having a structure of Formula I from N-terminus to C-terminus:

[0043]

[0044] wherein,

[0045] L1, L2, and L3 are each independently a bond or a linker element;

[0046] VH represents a heavy chain variable region of an anti-human claudin 18.2 antibody;

[0047] VL represents a light chain variable region of an anti-human claudin 18.2 antibody;

[0048] CH represents a heavy chain constant region of an anti-human claudin 18.2 antibody;

[0049] CL represents a light chain constant region of an anti-human claudin 18.2 antibody;

[0050] VHH represents an anti-PD-L1 single-domain antibody;

[0051] “-” represents a peptide bond;

[0052] “~” represents a disulfide bond or a covalent bond. In another preferred embodiment, L1 and L3 are each a bond (such as a peptide bond). In another preferred embodiment, the heavy chain variable region (VH) of the anti-human claudin 18.2 antibody comprises HCDR1 of SEQ ID NO: 93, 75, 79, 83, or 87, HCDR2 of SEQ ID NO: 94, 76, 80, 84, or 88, and HCDR3 of SEQ ID NO: 95, 77, 81, 85, or 89.

[0053] In another preferred embodiment, the heavy chain variable region (VH) of the anti-human claudin 18.2 antibody further comprises a humanized FR region.

[0054] In another preferred embodiment, the amino acid sequence of the heavy chain variable region (VH) of the anti-human claudin 18.2 antibody is set forth in SEQ ID NO: 31, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34.

[0055] In another preferred embodiment, the light chain variable region (VL) of the anti-human claudin 18.2 antibody further comprises a humanized FR region.

[0056] In another preferred embodiment, the light chain variable region (VL) of the anti-human claudin 18.2 antibody further comprises a humanized FR region.

[0057] In another preferred embodiment, the amino acid sequence of the light chain variable region (VL) of the anti-human claudin 18.2 antibody is set forth in SEQ ID NO: 29, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 28, or SEQ ID NO: 30.

[0058] In another preferred embodiment, the heavy chain constant region (CH) of the anti-human claudin 18.2 antibody is of human or murine origin.

[0059] In another preferred embodiment, the light chain constant region (CL) of the anti-human claudin 18.2 antibody is of human or murine origin.

[0060] In another preferred embodiment, the anti-PD-Ll single domain antibody (VHH) comprises a HCDR1 as set forth in SEQ ID NO: 69, a HCDR2 as set forth in SEQ ID NO: 70, and a HCDR3 as set forth in SEQ ID NO: 71.

[0061] In another preferred embodiment, the anti-PD-Ll single domain antibody (VHH) comprises a HCDR1 as set forth in SEQ ID NO: 69, a HCDR2 as set forth in SEQ ID NO: 96, and a HCDR3 as set forth in SEQ ID NO: 71.

[0062] In another preferred embodiment, the anti-PD-Ll single domain antibody (VHH) further comprises humanized FR regions.

[0063] In another preferred embodiment, the anti-PD-Ll single domain antibody (VHH) has an amino acid sequence as set forth in SEQ ID NO: 51, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.

[0064] In another preferred embodiment, the anti-PD-Ll single domain antibody (VHH) has an amino acid sequence as set forth in SEQ ID NO: 51, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.

[0065] In another preferred embodiment, the anti-PD-Ll single domain antibody (VHH) has an amino acid sequence as set forth in SEQ ID NO: 51, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.

[0066] In another preferred embodiment, the anti-PD-Ll single domain antibody (VHH) has an amino acid sequence as set forth in SEQ ID NO: 51, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.

[0067] In another preferred embodiment, the anti-PD-Ll single domain antibody (VHH) has an amino acid sequence as set forth in SEQ ID NO: 51, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.

[0068] In another preferred embodiment, the amino acid sequence of the VL of the bispecific antibody is as set forth in SEQ ID NO: 21, the amino acid sequence of the VH is as set forth in SEQ ID NO: 26, and the amino acid sequence of the anti-PD-L1 single domain antibody (VHH) is as set forth in SEQ ID NO: 51.

[0069] In another preferred embodiment, the amino acid sequence of the L chain (VL-L3-CL) of the bispecific antibody is as set forth in SEQ ID NO: 63, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 65; and the amino acid sequence of the H chain (VH-L1-CH-L2-VHH) of the bispecific antibody is as set forth in SEQ ID NO: 64, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 66.

[0070] In another preferred embodiment, the amino acid sequence of the L chain (VL-L3-CL) of the bispecific antibody is as set forth in SEQ ID NO: 63, and the amino acid sequence of the H chain (VH-L1-CH-L2-VHH) is as set forth in SEQ ID NO: 64.

[0071] In another preferred embodiment, the amino acid sequence of the L chain (VL-L3-CL) of the bispecific antibody is as set forth in SEQ ID NO: 59, and the amino acid sequence of the H chain (VH-L1-CH-L2-VHH) is as set forth in SEQ ID NO: 60.

[0072] In another preferred embodiment, the amino acid sequence of the L chain (VL-L3-CL) of the bispecific antibody is as set forth in SEQ ID NO: 61, and the amino acid sequence of the H chain (VH-L1-CH-L2-VHH) is as set forth in SEQ ID NO: 62.

[0073] In another preferred embodiment, the amino acid sequence of the L chain (VL-L3-CL) of the bispecific antibody is as set forth in SEQ ID NO: 65, and the amino acid sequence of the H chain (VH-L1-CH-L2-VHH) is as set forth in SEQ ID NO: 66.

[0074] In another preferred embodiment, the bispecific antibody is partially or fully humanized.

[0075] In a second aspect of the present application, there is provided an isolated polynucleotide encoding the bispecific antibody of the first aspect of the present application.

[0076] In another preferred embodiment, the polynucleotide comprises DNA, RNA or cDNA.

[0077] In a third aspect of the present application, a vector is provided, wherein the vector comprises the polynucleotide according to the second aspect of the present application.

[0078] In another preferred embodiment, the expression vector is selected from the group consisting of: a plasmid, a viral vector.

[0079] In another preferred embodiment, the expression vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as adenovirus, adeno-associated virus (AAV), retrovirus, or other vectors.

[0080] In a fourth aspect of the present application, a genetically engineered host cell is provided, wherein the host cell comprises the vector according to the third aspect of the present application, or the polynucleotide according to the second aspect of the present application is integrated into the genome of the host cell.

[0081] In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0082] In another preferred embodiment, the host cell is selected from the group consisting of: E. coli, a yeast cell, a mammalian cell.

[0083] In a fifth aspect of the present application, a method for preparing the bispecific antibody according to the first aspect of the present application is provided, comprising the steps of:

[0084] (i) culturing the host cell according to the fourth aspect of the present application under suitable conditions, thereby obtaining a mixture comprising the bispecific antibody according to the first aspect of the present application;

[0085] (ii) purifying and / or isolating the mixture obtained in step (i), thereby obtaining the bispecific antibody.

[0086] In another preferred embodiment, the purification can be achieved by protein A affinity column purification.

[0087] In another preferred embodiment, the purity of the target antibody after purification and isolation is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.

[0088] In a sixth aspect of the present application, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises:

[0089] (a) the bispecific antibody according to the first aspect of the present application or the conjugate according to the seventh aspect of the present application; and

[0090] (b) a pharmaceutically acceptable carrier.

[0091] In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating cancer (or tumor), such as chemotherapeutic drugs.

[0092] In another preferred embodiment, the pharmaceutical composition is used for blocking the interaction of PD-1 and PD-L1, while binding to claudin 18.2 protein.

[0093] In another preferred embodiment, the pharmaceutical composition is used for treating a cancer (or tumor) expressing claudin 18.2 protein (i.e., claudin 18.2 positive).

[0094] In another preferred embodiment, the pharmaceutical composition is in an injectable form.

[0095] In a seventh aspect of the present application, there is provided an immunoconjugate comprising:

[0096] (a) the bispecific antibody of the first aspect of the present application; and

[0097] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0098] In an eighth aspect of the present application, there is provided the use of the bispecific antibody of the first aspect of the present application in the manufacture of a medicament for treating a cancer (or tumor), an infection, or an immunoregulatory disease.

[0099] In a ninth aspect of the present application, there is provided the use of the bispecific antibody of the first aspect of the present application in the manufacture of a medicament for inhibiting tumor growth.

[0100] In another preferred embodiment, the cancer or tumor is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, renal cancer, cervical cancer, myeloid cancer, lymphatic cancer, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, desmoid tumor, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

[0101] The main benefits of the present application include: the present application provides a bispecific antibody targeting Claudin18.2 and PD-L1 simultaneously, which can target Claudin18.2 and PD-L1 with high efficiency. The present application can improve the treatment effect of tumors expressing claudin18.2. The bispecific antibody can block the binding of PD-1 / PD-L1 while binding to human claudin18.2 protein, which can activate NK cells to kill tumor cells in innate immunity and promote the killing effect of killer T lymphocytes on tumors in acquired immunity, thereby having a synergistic tumor killing effect. The bispecific antibody has better antitumor efficacy than the anti-claudin18.2 antibody alone. BRIEF DESCRIPTION OF DRAWINGS

[0102] Figure 1 The structure of the anti-CLDN18.2 / anti-PD-L1 bispecific antibody molecule is shown.

[0103] Figure 2 The results of ELISA detection of the first group of humanized anti-Claudin18.2 antibody binding to human Claudin18.2 activity are shown.

[0104] Figure 3 The results of ELISA detection of the second group of humanized anti-Claudin18.2 antibody binding to human Claudin18.2 activity are shown.

[0105] Figure 4 The results of ELISA detection of the third group of humanized anti-Claudin18.2 antibody binding to human Claudin18.2 activity are shown.

[0106] Figure 5 The results of ELISA detection of the fourth group of humanized Claudin18.2 antibody binding to human Claudin18.2 activity are shown.

[0107] Figure 6 The results of ELISA detection of the fifth group of humanized Claudin18.2 antibody binding to human Claudin18.2 activity are shown.

[0108] Figure 7 The results of FACS detection of the first group of humanized anti-Claudin18.2 antibody binding to human Claudin18.2 activity are shown.

[0109] Figure 8 The results of FACS detection of the second group of humanized anti-Claudin18.2 antibody binding to human Claudin18.2 activity are shown.

[0110] Figure 9Results of FACS assay for the third group of humanized anti-Claudin 18.2 antibodies binding to human Claudin 18.2 activity are shown.

[0111] Figure 10 Results of FACS assay for the fourth group of humanized anti-Claudin 18.2 antibodies binding to human Claudin 18.2 activity are shown.

[0112] Figure 11 Results of FACS assay for the fifth group of humanized anti-Claudin 18.2 antibodies binding to human Claudin 18.2 activity are shown.

[0113] Figure 12 Results of ADCC of humanized anti-Claudin 18.2 antibodies are shown.

[0114] Figure 13 Results of CDC of humanized anti-Claudin 18.2 antibodies are shown.

[0115] Figure 14 Results of in vivo efficacy of humanized anti-Claudin 18.2 antibodies in CB.17-SCID model of immunodeficient mice are shown.

[0116] Figure 15 Results of ELISA assay for humanized anti-PD-L1 single domain antibody binding to human PD-L1 activity are shown.

[0117] Figure 16 Results of ELISA assay for humanized anti-PD-L1 single domain antibody blocking activity are shown.

[0118] Figure 17 Results of expression and purification of the first anti-CLDN18.2 / anti-PD-L1 bispecific antibody are shown.

[0119] Figure 18 Results of expression and purification of the second anti-CLDN18.2 / anti-PD-L1 bispecific antibody are shown.

[0120] Figure 19 Results of expression and purification of the third anti-CLDN18.2 / anti-PD-L1 bispecific antibody are shown.

[0121] Figure 20 Results of expression and purification of the fourth anti-CLDN18.2 / anti-PD-L1 bispecific antibody are shown.

[0122] Figure 21 Results of ELISA assay for anti-CLDN18.2 / anti-PD-L1 bispecific antibody binding activity are shown.

[0123] Figure 22Results of ELISA detection of anti-CLDN18.2 / anti-PD-L1 bispecific antibody blocking PD-L1 / PD-1 binding activity are shown.

[0124] Figure 23 Results of ELISA detection of anti-CLDN18.2 / anti-PD-L1 bispecific antibody blocking PD-L1 / CD80 binding activity are shown.

[0125] Figure 24 Results of ELISA detection of anti-CLDN18.2 / anti-PD-L1 bispecific antibody binding to human CLDN18.2 activity are shown.

[0126] Figure 25 Results of mixed lymphocyte reaction (MLR) detection of anti-CLDN18.2 / anti-PD-L1 bispecific antibody PD-L1 functional activity (candidate molecule CHO14 stimulating T cell proliferation, enhancing IFN-gamma secretion) are shown.

[0127] Figure 26 Results of mixed lymphocyte reaction (MLR) detection of anti-CLDN18.2 / anti-PD-L1 bispecific antibody PD-L1 functional activity (candidate molecule CHO14 stimulating T cell proliferation, enhancing IL-2 secretion) are shown.

[0128] Figure 27 Results of PBMC-mediated cell killing experiment dependent on anti-CLDN18.2 / anti-PD-L1 bispecific antibody are shown.

[0129] Figure 28 Results of in vivo efficacy evaluation of anti-CLDN18.2 / anti-PD-L1 bispecific antibody in the immunotarget humanized transgenic mouse C57BL / 6-hPDL1 model MC38-hPDL1-mClaudin18.2 are shown.

[0130] Figure 29 Results of in vivo efficacy evaluation of anti-CLDN18.2 / anti-PD-L1 bispecific antibody in the immune system humanized mouse PBMCengrafted-NCG model HCC827-hClaudin18.2 are shown.

[0131] Figure 30 Results of in vivo synergistic efficacy evaluation of anti-CLDN18.2 / anti-PD-L1 bispecific antibody in the C57BL / 6 model MC38-hPDL1-mClaudin18.2 are shown.

[0132] Figure 31 Results of in vivo synergistic efficacy evaluation of anti-CLDN18.2 / anti-PD-L1 bispecific antibody in the C57BL / 6 model MC38-hPDL1-mClaudin18.2 are shown. Detailed Implementation

[0133] Through extensive and in-depth research and screening, the inventors obtained highly specific and high-affinity anti-CLDN18.2 antibodies and anti-PD-L1 single-domain antibodies. Based on these, they performed humanization and gene recombination, thereby obtaining bispecific antibodies that simultaneously target human Claudin18.2 and human PD-L1. In vitro experiments demonstrated that the bispecific antibodies of this invention can specifically bind to human Claudin18.2 and human PD-L1 molecules and kill recombinant Claudin 18.2-expressing and naturally expressed PD-L1-expressing human lung cancer cells. In vivo pharmacodynamic experiments demonstrated that the bispecific antibodies of this invention have a synergistic effect, exhibiting superior antitumor activity compared to Claudin18.2 monoclonal antibodies and PD-L1 monoclonal antibodies in humanized mouse models. Based on these findings, this invention was completed.

[0134] the term

[0135] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0136] As used herein, the terms “bispecific antibody of the present invention”, “bispecific antibody of the present invention”, and “anti-claudin18.2 / PD-L1 bispecific antibody” have the same meaning and refer to bispecific antibodies that specifically recognize and bind to claudin18.2 and PD-L1.

[0137] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light (L) and two identical heavy (H) chains. Each heavy chain is linked to a light chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains varies among the different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. There are two types of light chain, lambda (l) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activities of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains different sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable region (VH) and three constant regions (CH1, CH2, and CH3, collectively referred to as CH). Both the variable regions of the light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant domains of the light chain (CL) and the heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement fixation, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody depends on the structural complementarity between the antibody combining site and the antigenic determinant. The antibody combining site is composed of residues from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) influence the overall domain structure and, in turn, the combining site. Complementarity determining regions or CDRs refer to amino acid sequences that together define the specificity of the binding affinity and the native Fv region of a binding site of a native immunoglobulin. Each of the light and heavy chains of an immunoglobulin has three CDRs, designated LCDR1 (CDR1-L), LCDR2 (CDR2-L), LCDR3 (CDR3-L) and HCDR1 (CDR1-H), HCDR2 (CDR2-H), HCDR3 (CDR3-H), respectively. A conventional antibody antigen binding site thus includes six CDRs, comprising the CDR set from each of the heavy and light chain v regions.

[0138] As used herein, the terms "single domain antibody", "VHH", "nanobody" have the same meaning and refer to the variable region of the heavy chain of an antibody, which is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with full functionality. Usually, the variable region of the heavy chain of an antibody is cloned after the antibody naturally lacks the light chain and the constant region 1 (CH1) of the heavy chain, to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0139] As used herein, the term "variable" refers to certain portions of the variable region of an antibody that differ in sequence among antibodies and are responsible for the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the light chain and the heavy chain variable regions. The more conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, joined by three CDRs, which are generally termed FR1, FR2, FR3 and FR4 in the heavy chain and FR1, FR2, FR3 and FR4 in the light chain, that are arranged from amino-terminus to carboxy-terminus on the variable region. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation in antibody-dependent cellular cytotoxicity.

[0140] As used herein, the term "framework region" (FR) refers to the amino acid sequences that are interposed between the CDRs, i.e. to those parts of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins of a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can thus be represented as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain can thus be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FRs of the present application are human antibody FRs or derivatives thereof which are essentially identical to naturally occurring human antibody FRs, i.e. which have a sequence identity of 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0141] Knowing the amino acid sequences of the CDRs, the skilled person can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0142] As used herein, the term "humanized antibody" is an antibody molecule derived from a non-human source antibody capable of binding to an antigen of interest, having one or more complementarity determining regions (CDRs) of non-human origin and a framework region from a human immunoglobulin molecule. Framework residues in the human framework regions will typically be substituted by the corresponding residue from the CDR donor antibody, to alter, preferably to improve, antigen binding. These framework substitutions can be identified by, for example, modeling of the interactions of CDR and framework residues in binding the antigen, to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. Antibodies can be humanized using a variety of techniques known in the art, such as CDR-grafting (EP 239,400, PCT Publication WO 91 / 09967, U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106, EP 519,596, Padlan, Molecular Immunology 28(4 / 5):489-498 (1991), Studnicka et al., Protein Engineering 7(6):805-814 (1994), Roguska. et al., Proc. Natl. Sci. USA 91 :969-973 (1994)), and chain-shuffling (U.S. Pat. No. 5,565,332), all incorporated herein by reference.

[0143] As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or greater, specifically 90%, 95%, 97%, 99%, or 100% identical) to a framework region of a naturally occurring human antibody.

[0144] The present application includes not only intact antibodies, but also fragments of antibodies that are immunologically active or fusion proteins of antibodies with other sequences. Thus, the present application also includes fragments, derivatives, and analogs of the antibodies.

[0145] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present application. Polypeptide fragments, derivatives, or analogs of the present application can be (i) polypeptides having one or more conservative or non-conservative amino acid substitutions, preferably conservative amino acid substitutions, wherein such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) polypeptides having a substitution group at one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, for example, a polyethylene glycol, or (iv) polypeptides formed by adding additional amino acid sequences to the polypeptide, such as leader sequences or secretion sequences or sequences for purification of the polypeptide or prosequences, or fusion proteins with a 6His tag. These fragments, derivatives, and analogs are within the scope of one skilled in the art in light of the teachings herein.

[0146] The antibodies of the present application refer to diabodies having claudin 18.2 and PD-L1 protein binding activity. The term also includes variants of polypeptides having the same function as the antibodies of the present application, comprising the same CDR regions. These variants include, but are not limited to, deletion, insertion, and / or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (typically within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with similar or identical properties is often made without changing the function of the protein. For another example, addition of one or several amino acids at the C-terminus and / or N-terminus also often does not change the function of the protein.

[0147] The variants of the polypeptides include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the DNA encoding the antibodies of the present application under high or low stringency conditions, and polypeptides or proteins obtained using antisera against the antibodies of the present application.

[0148] wherein "conservative variants" refer to polypeptides having up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids replaced by similar or identical properties amino acids compared to the amino acid sequence of the antibodies of the present application (especially the framework regions replaced by similar or identical properties amino acids). These conservative variant polypeptides are preferably generated by amino acid replacements according to Table A.

[0149] Table A

[0150] Original residue Representative substitution Preferred substitution Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala Leu

[0151] The present application also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0152] The polynucleotides encoding the mature polypeptides of the present application include: a coding sequence encoding only the mature polypeptide; a coding sequence encoding the mature polypeptide and various additional coding sequences; a coding sequence encoding the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0153] The term "polynucleotide encoding a polypeptide" can be a polynucleotide that includes only the coding sequence for the polypeptide, or a polynucleotide that also includes additional coding and / or non-coding sequences.

[0154] The present application also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides that hybridize to the polynucleotides of the present application under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0155] The nucleotide full-length sequence of the antibodies of the present application or fragments thereof can generally be obtained by PCR amplification, recombination or artificial synthesis. One possible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is relatively short. Generally, a long fragment of the sequence can be obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0156] Once the relevant sequence is obtained, it can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules that exist in isolated form.

[0157] At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be obtained by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present application by chemical synthesis.

[0158] The present application also relates to vectors comprising the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express the protein.

[0159] The host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are bacterial cells of E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0160] The transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, competent host cells can be harvested during the exponential phase of growth, treated with CaCl2, and the steps used are well known in the art. Another method uses MgCl2. If desired, the transformation can also be performed by electroporation. When the host is a eukaryote, DNA transfection methods such as co-precipitation with calcium phosphate, conventional mechanical methods such as microinjection, electroporation, packaging in liposomes, etc. can be used.

[0161] The transformants obtained can be cultured using conventional methods to express the polypeptide encoded by the gene of the present application. Depending on the host cells used, the culture medium used in the culture can be selected from various conventional media. The culture is performed under conditions suitable for the growth of the host cells. When the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0162] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0163] The antibodies of the present application can be used alone, or in combination or conjugation with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties, or any combination of the above.

[0164] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing detectable products.

[0165] Therapeutic agents that can be combined or conjugated with the antibodies of the present application include, but are not limited to, 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or benzyl-hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0166] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates of drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules with the antibodies of the present application or fragments thereof.

[0167] Bispecific antibodies of the present application

[0168] The present application provides a bispecific antibody targeting human claudin 18.2 and human PD-L1 protein, which comprises an anti-human claudin 18.2 antibody portion and an anti-PD-L1 antibody portion.

[0169] (1) Anti-human claudin 18.2 antibody portion

[0170] The anti-human claudin 18.2 antibody portion in the bispecific antibody of the present application is obtained by humanizing the murine-derived anti-claudin 18.2 antibody. By comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, high-homology heavy and light chain variable region germline genes of QP190191, QP192193, QP199200, QP201202, QP207208, respectively, are selected as templates, and the CDRs of the murine-derived antibody are transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Some important amino acid residues are selected for back mutation combination.

[0171] The heavy chain variable region (VH) of the murine-derived anti-claudin 18.2 antibody has the following amino acid sequence, and the CDRs are shown as underlined portions in the sequence:

[0172] >QD208 SEQ ID NO. 46

[0173] EVQLQQSGPELVKPGASVKMSCKASGYTFT SYIMH WVKQKPGQGLEWIG YINPYNDGTKYNEKFKG KATLTSDKSSSTVYMELSSLTSEDSAVYCCAR LGFTTRNAMDY WGQGTSVTVSS

[0174] >QP191 SEQ ID NO. 38

[0175] EVKLVESGGGLVKPGGSLKLSCAASGFTFS NYAMS WVRQTPEKRLEWVA SIISGGRTYYLDSEKG RFTISRDNARNNLYLQMSSLRSEDTAMYYCTR IYYGNSFDY WGQGTTLTVSS

[0176] >QD193 SEQ ID NO. 40

[0177] QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIG QIYPGNGDTTYNGKFKG QATLTADKSSSTVYMQLSSLTSEDSAVYFCAR FVKGNAMDY WGQGTSVTVSS

[0178] >QD200 SEQ ID NO. 42

[0179] DVQLVESGGGLVQPGGSRKLSCAASGFTFS SFGMH WVRQAPEKGLEWVA YISSGSNSIYYVDTVKG RFTISRDNPKNTLFLQMTSLKSEDTAMYYCAR NAYYGNSFDY WGQGTTLTVSS

[0180] >QD202 SEQ ID NO. 44

[0181] EVQLQQSGPELVKPGASVKMSCKASGYTFT NYFVH WVKQKPGQGLEWIG YINPYNDDTKYNEKFKG KATLTSDKSSSTAYMDLSSLTSEDSAVYYC LSLRFFAY WGQGTLVTVSA

[0182] The light chain variable region of the murine-derived anti-claudin 18.2 antibody has the following amino acid sequence, respectively: CDRs are shown as underlined portions in the sequence:

[0183] >QD207 SEQ ID NO:45

[0184] DIVMTQSPSSLSVSAGEKVTMNC KSSQSLLNSGNQKNYLA WYQQKPGQPPKLLIY GASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNDHSYPFT FGSGTKLEIK

[0185] >QD190 SEQ ID NO:37

[0186] DIVMTQSPSSQTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTDFTLTISNMQAEDLAVYYC QNDYSYPFT FGSGTKLEIK

[0187] >QD192 SEQ ID NO:39

[0188] DIVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQNPGQPPKMLIY WASTRES GVPDRFTGSGSGIDFSLTISSVQAEDLALYYC QNAYSYPFT FGSGTKLEIK

[0189] >QD199 SEQ ID NO:41

[0190] DIVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTVFTLTISSVQAEDLAVYFC QNNYYYPLT FGAGTKLELK

[0191] >QD201 SEQ ID NO:43

[0192] DIVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQKPGQAPKLLIY WASTRES GVPDRFIGSGSGTDFTLTISHVQAEDLAVYFCQNDYSYPLT FGAGTNLELK

[0193] The CDR (underlined portion) regions of the light and heavy chain variable regions of the above-mentioned antibodies are listed in Table B.

[0194] Table B

[0195]

[0196]

[0197] (2) Anti-PD-L1 antibody moiety

[0198] The anti-human PD-L1 antibody moiety in the bispecific antibody of the present application is obtained by humanizing the anti-PD-L1 single-domain antibody (anti-PD-L1 nanobody). By comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy and light chain variable region germline genes with high homology to QP1162 and QP1166 are selected as templates, respectively, and the CDRs of the single-domain antibody are transplanted into the corresponding human template (such as IGHV3-23 germline and J-region IGHJ4*01) to form a variable region sequence with the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Some important amino acid residues are selected for back mutation combination.

[0199] The amino acid sequence of the anti-PD-L1 single-domain antibody is shown below, and the CDR is shown as the underlined portion in the sequence:

[0200] > QD1162 SEQ ID NO: 55

[0201] QVQLVESGGGSVQSGGSLRLSCAASGFTYG TYAMS WFRQAPGKEREGVA CIDIYGRASYTDPVKG RFTISQDNAKNTLYLQMNSLKPEDTAMYYCA ARDFGYCTASWVHEGFSRY WGQGTQVTVSS

[0202] > QD1166 SEQ ID NO: 56

[0203] QVQLVESGGDSVQPGGSLRLSCAASGFTYG TYAMS WFRQAPGKEREGVA CIDIYGRTSYTDPVKG RFTISQDNAKNTLYLQMNSLKPEDTAMYYCA ARDFGYCTASWVHEGFSRY WGQGTQVTVSS

[0204] The CDR (underlined) regions of the single-domain antibodies described above are summarized in the following table

[0205]

[0206] As used herein, the terms "anti-PD-L1 single-domain antibody" and "anti-PD-L1 nanobody" are used interchangeably and refer to an antibody that targets PD-L1 molecules, which is naturally devoid of light chain, containing only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.

[0207] As used herein, the term "affinity" is defined theoretically by the equilibrium association between the intact antibody and the antigen. The affinity of the bispecific antibodies of the present application can be assessed or determined by KD values (dissociation constant) (or other means of determination), for example, by Bio-layer interferometry BLI using a Fortebio Red96 instrument.

[0208] Pharmaceutical composition

[0209] The present application also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the antibody or active fragment thereof or fusion protein thereof described above, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably the pH is about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumorally, intraperitoneally, intravenously, or topically.

[0210] The pharmaceutical composition of the present application can be directly used to bind claudin 18.2 and / or PD-L1 protein molecules, and thus can be used for the treatment of tumors. In addition, it can also be used in combination with other therapeutic agents.

[0211] The pharmaceutical composition of the present application contains a safe and effective amount (e.g. 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned bispecific antibody (or conjugate thereof) of the present application and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration mode. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by a conventional method using physiological saline or an aqueous solution containing dextrose and other adjuvants. The pharmaceutical composition such as a needle, a solution is preferably manufactured under sterile conditions. The administration amount of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents.

[0212] When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also consider the administration route, patient health status and other factors, which are within the skill of a skilled physician.

[0213] Applications

[0214] The present application also provides the use of the antibody of the present application, which relates to the use in the preparation of a medicament for treating cancer (or tumor), infection or immunoregulatory disease. A preferred use is for treating cancer (or tumor).

[0215] The main advantages of the present application include:

[0216] (1) The present application provides a bispecific antibody targeting human Claudin 18.2 and human PD-L1 simultaneously, which can target human Claudin 18.2 and human PD-L1 with high efficiency.

[0217] (2) The bispecific antibody of the present application can improve the therapeutic effect of claudin 18.2-expressing tumors. The bispecific antibody can not only activate NK cells to kill tumor cells in innate immunity, but also promote the killing effect of killer T lymphocytes on tumors in acquired immunity, so the bispecific antibody has better antitumor effect than the anti-claudin 18.2 antibody alone.

[0218] (3) The bispecific antibody of the present application has a synergistic effect.

[0219] The technical solutions of the present application will be described in detail below in combination with specific embodiments.

[0220] The experimental methods in the following embodiments without specific conditions are carried out according to the conventional conditions, or the conditions suggested by the raw material or commercial manufacturers. Or the experimental methods recorded in the biotechnology textbooks such as Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory, Methods in Molecular Biology, Cell Biology, etc. The reagents without specific sources are the conventional reagents purchased through commercial channels.

[0221] Example 1: Humanization of anti-Claudin18.2 hybridoma monoclonal antibody

[0222] By comparing the IMGT human antibody heavy and light chain variable region gene database and MOE software, the high homology heavy and light chain variable region genes of QP190191, QP192193, QP199200, QP201202 and QP207208 were selected as templates, and the CDR of the mouse-derived antibody was transplanted into the corresponding human template to form the variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Some important amino acid residues were selected for back mutation combination. The amino acid residues are determined and annotated by Kabat numbering system.

[0223] 1. Humanized anti-Claudin18.2 antibody molecular cloning

[0224] The primer PCR was designed to build the VH / VK gene fragments of each humanized antibody, and then the expression vector pQD with signal peptide and constant region gene (CH1-FC / CL) fragments was subjected to homologous recombination to construct the full-length antibody expression vector VH-CH1-FC-pQD / VK-CL-pQD.

[0225] The VH / VK containing recombinant gene fragments were designed using online software DNAWorks (v3.2.2) (http: / / helixweb.nih.gov / dnaworks / ): 5'-30bp signal peptide + VH / VK + 30bp CH1 / CL-3'. The VH / VK containing recombinant gene fragments were obtained by two-step PCR amplification using the primers designed above according to the Primer STAR GXL DNA polymerase operation manual of TaKaRa Company. The expression vector pQD (with signal peptide and constant region gene (CH1-FC / CL) fragment) was constructed and cleaved using the restriction enzyme BsmBI, which was designed to construct the expression vector pQD (with signal peptide and constant region gene (CH1-FC / CL) fragment) based on the characteristics of the recognition sequence and cleavage site. The vector was cleaved with BsmBI and the gel was recovered for later use. The recombinant expression vector VH-CH1-FC-pQD / VK-CL-pQD was constructed. The VH / VK containing recombinant gene fragments and the BsmBI-cleaved expression vector pQD were added to DH5H competent cells at a ratio of 3:1, respectively, and incubated at 0°C for 30 min, 42°C for 90 s, 5 times the volume of LB medium was added, and incubated at 37°C for 45 min. LB-Amp plates were coated and incubated at 37°C overnight, and single colonies were selected for sequencing to obtain the desired clones.

[0226] The humanized design light and heavy chain variable region sequences and protein expression numbers are shown in the following table. In this table, all antibody light chains use kappa light chain constant region CL (SEQ ID NO: 67), and antibody heavy chains use human IgG1 constant region (SEQ ID NO: 68):

[0227] Table 1: Humanized design light and heavy chain variable region sequences and protein expression numbers

[0228]

[0229]

[0230]

[0231] The humanized design light and heavy chain variable region sequences and protein expression numbers are shown in the following table. In this table, all antibody light chains use kappa light chain constant region CL (SEQ ID NO: 67), and antibody heavy chains use human IgG1 constant region (SEQ ID NO: 68):

[0232] Table 2: Human-mouse chimeric antibodies and control antibodies (IMAB362 is a control antibody, corresponding protein number QP024025)

[0233] Hybridoma clone number Protein number Heavy chain plasmid number Sequence number Light chain plasmid number Sequence number 175D10 (IMAB362) QP024025 QD025 SEQ ID NO: 36 QD024 SEQ ID NO: 35 36A2 QP190191 QD191 SEQ ID NO: 38 QD190 SEQ ID NO: 37 40F1 QP192193 QD193 SEQ ID NO: 40 QD192 SEQ ID NO: 39 43C11 QP199200 QD200 SEQ ID NO: 42 QD199 SEQ ID NO: 41 44C6 QP20122 QD202 SEQ ID NO: 44 QD201 SEQ ID NO: 43 51A3 QP207208 QD208 SEQ ID NO: 46 QD207 SEQ ID NO: 45 phage screening QP11151116 QD1115 SEQ ID NO: 48 QD1116 SEQ ID NO: 47

[0234] 2. Humanized anti-Claudin 18.2 antibody protein expression

[0235] 293E cell culture density was maintained between 0.2-3x10 6 / ml, using maintenance stage medium (GIBCO Freestyle 293 expression medium) for culture, centrifugation and medium replacement for cells to be transfected the day before transfection, adjusting the cell density to 0.5-0.8x10 6 / ml. On the day of transfection, the 293E cell density was 1-1.5x10 6 / ml. Prepare plasmid and transfection reagent PEI, the amount of plasmid to be transfected is 100ug / 100ml cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI, and let stand for 15 min, not more than 20 min. Slowly add the plasmid and PEI mixture to the 293E cells, and place them in a 8% CO2, 120 rpm, 37°C shaker for culture. On the fifth day of transfection, collect the cell supernatant by centrifugation at 4700 rpm for 20 min.

[0236] 3. Humanized anti-Claudin 18.2 antibody protein purification

[0237] Protein A affinity chromatography purification

[0238] Equilibrate the column with equilibration buffer, at least 3 CV, actual volume 20 ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibration buffer, flow rate 1 ml / min; centrifuge the culture supernatant and pass it through the column, sample 40 ml, flow rate 0.33 ml / min; equilibrate the column with equilibration buffer, at least 3 CV, actual volume 20 ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibration buffer, flow rate 0.33 ml / min; pass the column with elution buffer, when UV280 rises to 15 mAU, start collecting the elution peak (PAC-EP), when UV280 falls to 15 mAU, stop collecting, flow rate 1 ml / min. After the sample collection is complete, adjust the PAC-EP to neutral with pH adjustment buffer.

[0239] 4. Cell-ELISA detection of humanized anti-Claudin 18.2 antibody binding to human Claudin 18.2 activity (CHOS-human CLDN18.2 cell-ELISA)

[0240] The human Claudin 18.2 protein sequence was transfected on CHO-S cells, and a CHOS-hu Claudin 18.2 cell strain stably expressing human Claudin 18.2 protein was constructed.

[0241] Detection reagent: skim milk powder (BD, 232100), PBS (Shenguo, B548117-0500); HRP-anti human IgG (H+L) (jackson, 109-035-088); TMB (Luoyang Baoao Experimental Material Center, C060201); Elisa plate (costa, 9018) 1x PBS buffer: weigh NaCl 8.00g, KCl 0.20g, Na2HPO4·12H2O 2.9g, KH2PO4 0.2g into 800mL ddH2O, dissolve thoroughly, then make up to 1L, adjust pH to 7.4, sterilize at high temperature for standby. Or buy commercial 10x, 20x PBS solution and dilute to 1x PBS buffer for use. Blocking solution: weigh 5g of milk powder into PBS, the blocking solution needs to be prepared and used immediately. Stop solution (1mol / L H2SO4): add 109mL of 98% concentrated H2SO4 slowly to 2000mL of ddH2O. TMB color development at 37°C for 10min, placed in a shaker (120rpm), 100ul / well;

[0242] Experimental steps: seed 2E5 / well of cells CHOS-CLD18.2-16-2 in a U-shaped plate, wash once with ice PBS, 1200rpm, centrifuge for 3min. VCD: 1.21E6, actually 165ul / well; after washing, add blocking solution 200ul / well, incubate on ice for 1h. After blocking, centrifuge at 1200rpm for 3min, then discard the supernatant, incubate CLD18.2 control antibody 14-1 and samples, dilute 12 gradients according to the dilution ratio of 100ug / ml 1:2, finally set a blank control, add 100ul / well according to 100ug / mL, 33.33333333ug / mL, 11.11111111ug / mL, 3.703703704ug / mL, 1.234567901ug / mL, 0.411522634ug / mL, 0.137174211ug / mL, 0.045724737ug / mL, 0.015241579ug / mL, 0.005080526ug / mL, 0.001694ug / mL, 0ug / mL, mix thoroughly, then incubate on ice for 2h, wash with ice PBS for 3 times; add enzyme-labeled antibody: incubate HRP-anti human IgG (H+L) antibody according to the dilution ratio of 1:10000, 100ul / well, mix thoroughly, then incubate on ice for 1h, wash with ice PBS for 3 times. Add substrate color developing solution: add substrate color developing solution TMB 100ul / well, place in a shaker, 200rpm, 35°C, avoid light, color develop for 10min.

[0243] Termination: After the color development is completed, the reaction is terminated by quickly adding a termination solution in an amount of 100 μL / well. Detection: After centrifugation at 3500 rpm for 5 min, 120 ul of supernatant is transferred to an Elisa plate, and the OD value at A450 nm is measured on an enzyme label instrument. The results are analyzed using graphpad prism software: as shown in Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 and Figure 6 .

[0244] The experimental results are shown in Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 and Figure 6 , which prove that the humanized anti-Claudin 18.2 antibodies of the present application all bind to CHOS-Claudin 18.2.

[0245] 5. FACS detects the activity of humanized anti-Claudin 18.2 antibody binding to human Claudin 18.2

[0246] Collect cells CHOS, CHOS-CLDN18.2, 2E5 / well, 1000r centrifuge for 5 min, 3% BSA / PBS buffer 200ul / well block for 60 min, 4℃. Antibody 20ug / ml initial concentration, 1:4 dilution, 4℃ incubate for 60 min. Wash with PBS for 2 times, incubate PE-anti-human FC (1:200) 50ul / well, 4℃ incubate for 30 min, wash with PBS for 3 times, resuspend with PBS, and the FACS results are shown in Table 3, Table 4, Table 5, Table 6, Table 7 and Figure 7 、 Figure 8 、 Figure 9 、 Figure 10 and Figure 11 .

[0247] The EC50 and Max values of Table 3 and Figure 7 prove that the humanized antibodies QP14361435, QP14371433, QP14371435 of the mouse-derived chimeric antibody QP190191 bind to CHOS-CLDN18.2 cells better than or comparable to the mouse-derived chimeric antibody QP190191; the EC50 and Max values of Table 7 and Figure 11 prove that the humanized antibodies QP14561454, QP14581453, QP14581454 of the mouse-derived chimeric antibody QP201202 bind to CHOS-CLDN18.2 cells comparable to the mouse-derived chimeric antibody QP201202; the EC50 and Max values of Table 6 and Figure 10The EC50and Max values of the humanized antibodies QP14451440, QP14441441, QP14451442 prove that the binding of the humanized antibodies to CHOS-CLDN18.2 cells is equivalent to that of the murine chimeric antibody QP192193; Table 5 and Figure 9 The EC50and Max values of the humanized antibodies QP14491448, QP14501446, QP14501448 prove that the binding of the humanized antibodies to CHOS-CLDN18.2 cells is equivalent to that of the murine chimeric antibody QP199200; Table 4 and Figure 8 The EC50and Max values of the humanized antibodies QP14631461, QP14641460, QP14641461, QP14641462, QP14651460 prove that the binding of the humanized antibodies to CHOS-CLDN18.2 cells is superior to or equivalent to that of the murine chimeric antibody QP207208.

[0248] Table 3: FACS detection of the binding activity of the first group of humanized anti-Claudin 18.2 antibodies to human Claudin 18.2 (mean fluorescence value MFI)

[0249]

[0250]

[0251] Table 4: FACS detection of the binding activity of the second group of humanized anti-Claudin 18.2 antibodies to human Claudin 18.2 (mean fluorescence value MFI)

[0252]

[0253] Table 5: FACS detection of the binding activity of the third group of humanized anti-Claudin 18.2 antibodies to human Claudin 18.2 (mean fluorescence value MFI)

[0254]

[0255]

[0256] Table 6: FACS detection of the binding activity of the fourth group of humanized anti-Claudin 18.2 antibodies to human Claudin 18.2-CHOS cells (mean fluorescence value MFI)

[0257]

[0258] Table 7: FACS detection of the fifth group of humanized anti-Claudin 18.2 antibody binding to human Claudin 18.2 activity results (mean fluorescence value MFI)

[0259]

[0260]

[0261] 6. Identification of humanized anti-Claudin 18.2 antibody binding Claudin 18.2 specificity

[0262] Flow cytometry fluorescence sorting technology FACS identifies the specificity of the humanized antibodies of the present application binding Claudin 18.2 does not bind Claudin 18.1. The human Claudin 18.1 protein sequence was transfected on CHO-S cells, and a CHOS-hu Claudin 18.1 cell strain stably expressing human Claudin 18.1 protein was constructed.

[0263] Cells CHOS, CHOS-CLDN18.2, CHOS-CLDN18.1, 2E5 / well, 1000r centrifugation for 5min, 3% BSA / PBS buffer 200ul / well blocking for 60min, 4℃. Antibody 20ug / ml initial concentration, 1:4 dilution, 4℃ incubation for 60min. PBS wash 2 times, incubate PE-anti-human FC (1:200) 50ul / well, 4℃ incubate for 30min, PBS wash 3 times, PBS resuspension, FACS reading mean fluorescence value as shown in Table 8, FACS results show that the humanized anti-Claudin 18.2 antibodies of the present application all bind to CHOS-Claudin 18.2 cells, and do not bind to CHOS-Claudin 18.1 and CHOS cells, proving that the humanized anti-Claudin 18.2 antibodies specifically bind to Claudin 18.2 and do not bind to Claudin 18.1.

[0264] Table 8: FACS detection of humanized anti-Claudin 18.2 antibody binding to CHOS, CHOS-CLDN18.1, CHOS-CLDN18.2, respectively, mean fluorescence value (MFI)

[0265]

[0266]

[0267] 7. ADCC effect of humanized anti-Claudin 18.2 antibody (antibody-dependent cell-mediated cytotoxicity)

[0268] Prepare target cells (HEK293-CLDN18.2): Digest HEK293-CLDN18.2 cells with trypsin at 1000 rpm for 5 min. Replace with fresh medium and plate in 96-well plates at 20,000 cells / well, incubate overnight at 37°C with 5% CO2. Prepare antibodies: Serially dilute antibodies 1:5 with medium to obtain 10 concentrations (80 μg / ml – 0.000512 μg / ml, 0 μg / ml). Discard the medium from the 96-well plates and add 70 μL / well of each diluted antibody concentration, with replicates for each concentration. Prepare PBMCs: Centrifuge the PBMCs recovered on day 1, resuspend in medium, and count. Add 70 μL / well of PBMCs to the wells above at a PBMC:Target cell ratio of 50:1, incubate at 37°C for 4 hours. Add 15 μL of lysis buffer (1% Triton-X100) to the Max lysis well and incubate at 37°C for 10 min.

[0269] Prepare LDH reagent: Centrifuge a 96-well plate at 200g for 5 minutes, and transfer 100µl of supernatant per well to a new clear 96-well plate. Take the LDH cytotoxicity assay kit (Cayman, 10008882-480well), prepare the reaction solution, add 100µl per well, and gently vortex at 37°C for 30 minutes. Read the absorbance at 490nm. Analyze the data according to the formula: Con(µg / ml)%Maximal signal = (Test-Control) / (Max-Control) – Con(0µg / ml)%Maximal signal.

[0270] Experimental results are as follows Figure 12 As shown in Tables 9 and 10, the humanized anti-Claudin 18.2 antibodies QP14331437, QP14401445, QP14611463, the fully human antibody QP11151116, and the control antibody QP024025 (IMAB362 analogue) of the present invention can all kill HEK293-Claudin 18.2 cells stably expressing Claudin 18.2 in a concentration-dependent PBMC-mediated manner.

[0271] Table 9: 490nm readings

[0272] conc. (ug / ml) 40 8 1.6 0.32 0.064 0.0128 0.00256 0.00051 0.0001 0 MAX QP14331437 1.012 1.005 1.012 0.956 0.856 0.752 0.596 0.561 0.55 0.56 1.21 QP14401445 0.98 0.919 0.856 0.907 0.861 0.726 0.573 0.557 0.463 0.527 1.17 QP14611463 0.844 0.947 0.903 0.908 0.835 0.678 0.567 0.516 0.513 0.522 1.144 QP11151116 1.176 0.927 0.885 0.863 0.771 0.752 0.58 0.521 0.521 0.508 1.321 QP024025 0.794 0.79 0.791 0.777 0.675 0.561 0.487 0.479 0.448 0.465 1.141 Target + Ab 0.488 0.418 0.358 0.361 0.272 0.253 0.274 0.261 0.274 0.279

[0273] Table 10: Kill Percentage

[0274] conc. (ug / ml) QP14331437 QP14401445 QP14611463 QP11151116 QP024025 40 52.32％ 47.80％ 28.62％ 75.45％ 21.57％ 8 53.76％ 42.72％ 46.32％ 43.75％ 26.16％ 1.6 56.36％ 37.77％ 43.37％ 41.22％ 30.02％ 0.32 49.58％ 43.72％ 43.84％ 38.46％ 28.17％ 0.064 41.55％ 42.09％ 39.27％ 32.36％ 21.98％ 0.0128 31.27％ 28.52％ 23.43％ 31.27％ 11.04％ 0.00256 13.30％ 10.80％ 10.15％ 11.56％ 1.49％ 0.000512 10.46％ 10.03％ 5.65％ 6.19％ 1.70％ 0.0001024 8.31％ -1.11％ 4.30％ 5.17％ -2.74％ 0 9.02％ 5.43％ 4.88％ 3.36％ -1.33％ EC50 0.0162 0.0082 0.01148 0.00716 0.01562

[0275] 8. The CDC effect (complement-dependent cytotoxicity) of humanized anti-Claudin18.2 antibody

[0276] Reagents: cells HEK293-hCLDN18.2-H11, complement: normal human serum complement (quidel, A113), antibody: QP024025 (IMAB362 analogue), QP14631461 from synthesis in this example.

[0277] Experimental procedure:

[0278] Plating cells / media: Set up culture media background, i.e. add media 40ul / well only, set up maximum LDH release, i.e. add 40ul target cells, 12ul lysis solution 45min before detection. Set up volume correction, i.e. add media 40ul / well, set up LDH positive control, i.e. 1ul positive control vortexed, 5000 fold (5mL) dilution in PBS+1% BSA. Can be diluted in 10 fold gradient. Set up experimental, i.e. add 40ul / well target cells. Add complement+antibody mix: add 20% complement (diluted in cell culture media), 40ul / well, both control and experimental groups. Incubate cells at 37°C, 5% CO2, 1h15min, add 12ul / well lysis solution to maximum release and volume correction groups. Continue incubation at 37°C for 45min, then detect LDH with kit, read OD490nm, should be done within 1h after adding stop solution. Calculate: experimental minus background control, maximum release minus volume correction, formula: Percent cytotoxicity=100*OD490(experimental LDH release) / OD490(maximum LDH release).

[0279] CDC results are shown in Figure 13 Figure 6, which shows that both the humanized anti-Claudin 18.2 antibody QP14611463 and the control antibody QP024025 (IMAB362 analogue) can kill HEK293-Claudin 18.2 cells stably expressing Claudin 18.2 in a concentration dependent manner.

[0280] 9. In vivo efficacy of humanized anti-Claudin 18.2 antibodies in the CB.17-SCID model of immunodeficient mice HEK293-hClaudin 18.2

[0281] Experimental method: The stable transfection cell line HEK293-hClaudin18.2 cells stably expressing human claudin18.2 (hClaudin18.2) in logarithmic growth phase were collected, and the cell concentration was adjusted to 5x10 7 / mL with PBS buffer, 0.1 mL (1:1 Matrigel) cell suspension was inoculated subcutaneously on the right flank of CB-17 SCID mice. The inoculated mice were observed and the tumor growth was monitored, and the mice were grouped and dosed on the day of inoculation.

[0282] Experimental results: As shown in Table 10 and Table 11. Figure 14

[0283] Table 11: Tumor volume

[0284]

[0285] The test molecules were humanized claudin18.2 antibodies QP14331437 and QP14611463, and the positive control antibody was QP024025 (IMAB362 analog). The dosing regimen was 10 mpk x 10, q2d, i.v. On day 37 after dosing, the average tumor volume of the PBS group (negative control group) reached 1091.34 mm3, the average tumor volume of the QP14331437 group was 260.65 mm 3 , TGI = 76.12%, the average tumor volume of the QP14611463 group was 225.01 mm 3 , TGI = 79.38%, the average tumor volume of the IMAB362 group was 324.19 mm 3 , TGI = 70.29%; there were statistically significant differences in tumor volume between the three groups and the PBS group (t-test, p < 0.01).

[0286] This experiment shows that in the HEK293-CLDN18.2 model, the humanized anti-claudin18.2 antibodies of the application all show a trend of better anti-tumor ability than the control antibody QP024025 (IMAB362 analog).

[0287] Example 2: Humanization of anti-PD-L1 single-domain antibody

[0288] By comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy and light chain variable region germline genes with high homology to QP1162 and QP1166 were selected as templates, and the CDRs of the single-domain antibodies were transplanted into the corresponding human templates IGHV3-23 germline and J-region IGHJ4 * ​In 01, the variable region sequence is formed in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Some important amino acid residues are selected for reverse mutation combination. The amino acid residues are determined and annotated by the Kabat numbering system.

[0289] 1. Humanized anti-PD-L1 antibody molecule clone

[0290] Design primers to construct each humanized antibody VH gene fragment, and then perform homologous recombination with the expression vector pQD containing the signal peptide and constant region gene (FC) fragment to construct the full-length antibody expression vector VH-FC-pQD.

[0291] Use online software DNAWorks (v3.2.2) (http: / / helixweb.nih.gov / dnaworks / ) to design multiple primers to synthesize VH / VK containing the required gene fragments for recombination: 5'-30bp signal peptide+VH+30bp FC-3'. According to the TaKaRa Primer STAR GXL DNA polymerase operation manual, use the above designed multiple primers to amplify the VH / VK containing the required gene fragments for recombination in two steps. Construct and enzyme cut the expression vector pQD containing the signal peptide and constant region gene (FC) fragment. Use restriction enzymes such as BsmBI to design and construct the expression vector pQD containing the signal peptide and constant region gene (FC) fragment based on the characteristics of different recognition sequences and enzyme cutting sites. Cut the vector with BsmBI and recover the gel for future use. Recombinantly construct the expression vector VH-FC-pQD. Add the VH containing the required gene fragments for recombination and the BsmBI enzyme cut recovery expression vector pQD (containing the signal peptide and constant region gene (FC) fragment) to the DH5H competent cells in a ratio of 3:1, ice bath at 0°C for 30 min, heat shock at 42°C for 90 s, add 5 times the volume of LB medium, incubate at 37°C for 45 min, spread on LB-Amp plates, and incubate at 37°C overnight. Pick single colonies for sequencing to obtain each desired clone.

[0292] The variable region sequences of each cloned humanized light and heavy chain and protein expression numbers are shown in the table below. In this table, the antibody is fused to the human IgG1-FC constant region at its C-terminus:

[0293] Table 12: QP1162 and QP1166 humanization design

[0294]

[0295]

[0296] The VHH of humanization is consistent with the length of the original camel single-domain antibody, both of which are 126 aa.

[0297] At the same time, the chimeric antibody containing human constant region and camel variable region and the control antibody QP11801181 (atezolimab or Tecentriq analogue) were designed and expressed as shown in the following table:

[0298] Table 13: Chimeric antibody and control antibody

[0299]

[0300] 2. Humanized anti-PD-L1 antibody protein expression

[0301] The 293E cell culture density was maintained between 0.2-3x10 6 / ml, and the maintenance stage medium (GIBCO Freestyle 293 expression medium) was used for culture. The cells to be transfected were centrifuged and replaced with fresh medium the day before transfection, and the cell density was adjusted to 0.5-0.8x106 / ml. On the day of transfection, the 293E cell density was 1-1.5x10 6 / ml. Prepare the plasmid and transfection reagent PEI, and the amount of plasmid required for transfection is 100ug / 100ml cells. The mass ratio of PEI to plasmid used is 2:1. Mix the plasmid and PEI, and let stand for 15 min, not more than 20 min. Slowly add the plasmid and PEI mixture to the 293E cells, and place them in a 8% CO2, 120 rpm, 37°C shaker for culture. On the fifth day of transfection, collect the cell supernatant by centrifuging at 4700 rpm for 20 min.

[0302] 3. Purification of humanized anti-PD-L1 antibody protein

[0303] Protein A affinity chromatography purification

[0304] Equilibrate the column with equilibration buffer, at least 3 CV, actual volume 20 ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibration buffer, flow rate 1 ml / min; centrifuge the culture supernatant and pass it through the column, sample 40 ml, flow rate 0.33 ml / min; equilibrate the column with equilibration buffer, at least 3 CV, actual volume 20 ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibration buffer, flow rate 0.33 ml / min; pass the column with elution buffer, when UV280 rises to 15 mAU, start collecting the elution peak (PAC-EP), when UV280 falls to 15 mAU, stop collecting, flow rate 1 ml / min. After the sample collection is complete, adjust the PAC-EP to neutral with pH adjusting solution.

[0305] 4. ELISA detection of humanized anti-PD-L1 antibody binding to human PD-L1 recombinant protein activity (Binding-ELISA)

[0306] Coating antibody QP1162 / QP320 / QP321 / QP322 / QP1166 / QP323 / QP324 / QP325 0.75ug / ml, QP11801181 1.5ug / ml 50ul / well, 4°C overnight. PBS 3 times. Blocking: 3% BSA 250ul / well, RT lh. Incubate 2ug / ml Biotin QP004.3 (biotin-PDL1-FC) 1:4 dilution different concentration, RT lh. PBST 3 times, PBS 3 times. Incubate secondary antibody: HRP-strepavidin (1:5000) 50ul / well, PBST 6 times, PBS 3 times. Color development: TMB 100ul / well, color development 10min. 2M H2SO4 50ul / well stop.

[0307] Results are shown in Table 13 and Figure 6. Figure 15 As shown in Table 14 and Figure 7, humanized antibody QP322 of Nanobody QP1162, humanized antibody QP325 of Nanobody QP1166 and humanized antibody QP323 of Nanobody QP320 can all bind to PD-L1 protein, which is equivalent to the original Nanobody.

[0308] Table 14: Results of ELISA detection of humanized anti-PD-L1 antibody binding to human PD-L1 activity

[0309]

[0310] 5. ELISA detection of humanized anti-PD-L1 antibody blocking activity (Blocking-ELISA)

[0311] Coating protein QP1138 (PD1-FC) 2ug / ml 50ul / well, 4°C overnight. PBS 3 times. Blocking: 3% BSA 250ul / well, RT lh. Prepare 2ug / ml Biotin QP004.3 (biotin-PDL1-FC) and different concentration QP112015ug / ml, QP11801181 30ug / ml, 1:3 dilution, mix equal volume, RT lh. PBST 3 times, PBS 3 times. Incubate secondary antibody: HRP-strepavidin (1:5000) 50ul / well, PBST 6 times, PBS 3 times. Color development: TMB 100ul / well, color development 10min. 2M H2SO4 50ul / well stop.

[0312] Results are shown in Table 15 and Figure 8. Figure 16and shown in Table 15, the humanized antibody QP322 of Nanobody QP1162, the humanized antibody QP325 of Nanobody QP1166 can block the binding of PD-L1 to PD-1 protein, which is equivalent to the humanized Nanobody before.

[0313] Table 15: Activity identification of humanized anti-PD-L1 single domain antibody (Blocking-ELISA)

[0314]

[0315] 6. SPR assay of humanized anti-PD-L1 antibody binding to human PD-L1 and monkey PD-L1 affinity

[0316] Surface plasmon resonance (SPR) assay of affinity

[0317] Determination of the affinity of the molecule to be tested to the protein human PD-L1 and cynoPD-L1 by Biacore T200 (GE)

[0318] The antigen information is as follows:

[0319] Table 16: Protein number

[0320] Protein Number Protein Description Catalog Number QPP09.1 PD-L1 Protein, Human, Recombinant (His Tag) SinoBiologic, 10084-H08H QPP10.1 PD-L1 Protein, Cynomolgus, Recombinant (His Tag) SinoBiologic, 90251-C08H

[0321] Table 17: SPR affinity results

[0322]

[0323]

[0324] The experimental results show that the SPR affinity results show that the humanized anti-PD-L1 antibodies QP322 and QP325 can bind to human PD-L1 protein and monkey PD-L1 protein. Among them, the humanized Nanobody QP322 has equivalent binding affinity to human and monkey PD-L1 protein as the camel Nanobody QP1162, and the humanization is successful.

[0325] Example 3: Anti-CLDN18.2 / anti-PD-L1 bispecific antibody

[0326] In this example, the humanized Nanobody QP322 is respectively connected with the heavy chain of anti-CLDN18.2 through the linker (G4S)4 to form a fusion protein, which is used to construct an anti-CLDN18.2 / anti-PD-L1 bispecific antibody.

[0327] The designed anti-CLDN18.2 / anti-PD-L1 bispecific antibody molecule has a form as shown in Figure 1 .

[0328] 1. Anti-CLDN18.2 / anti-PD-L1 bispecific antibody molecule clone

[0329] The primer PCR was designed to construct each humanized antibody VH gene fragment, which was then subjected to homologous recombination with the expression vector pQD (with signal peptide and constant region gene fragment) to construct the full-length antibody expression vector pQD.

[0330] The anti-CLDN18.2 / anti-PD-L1 bispecific antibody sequence and protein expression number are shown as follows:

[0331] Table 18: Anti-CLDN18.2 / anti-PD-L1 bispecific antibody sequence and protein expression number

[0332]

[0333]

[0334] 2. Anti-CLDN18.2 / anti-PD-L1 bispecific antibody protein expression

[0335] The 293E cell culture density was maintained between 0.2-3x106 / ml, and the maintenance stage medium (GIBCO Freestyle 293 expression medium) was used for culture. The cells were centrifuged and the medium was replaced the day before transfection, and the cell density was adjusted to 0.5-0.8x106 / ml. On the day of transfection, the 293E cell density was 1-1.5x106 / ml. The plasmid and transfection reagent PEI were prepared, and the amount of plasmid required for transfection was 100ug / 100ml of cells, and the mass ratio of PEI to plasmid was 2:1. The plasmid and PEI were mixed and allowed to stand for 15 min, not exceeding 20 min. The plasmid and PEI mixture was slowly added to the 293E cells, and the cells were cultured in a 8% CO2, 120 rpm, 37°C shaker. On the fifth day of transfection, the cells were collected by centrifugation at 4700 rpm for 20 min using a horizontal centrifuge.

[0336] 3. Anti-CLDN18.2 / anti-PD-L1 bispecific antibody expression and purification

[0337] Protein A affinity chromatography purification

[0338] Pass the equilibration solution through the column at a flow rate of at least 3 CV (20 ml), ensuring the pH and conductivity of the final effluent from the instrument are consistent with the equilibration solution. The flow rate is 1 ml / min. Pass the supernatant of the centrifuged culture solution through the column, loading 40 ml at a flow rate of 0.33 ml / min. Pass the equilibration solution through the column again at a flow rate of at least 3 CV (20 ml), ensuring the pH and conductivity of the final effluent from the instrument are consistent with the equilibration solution. The flow rate is 0.33 ml / min. Pass the elution solution through the column. Begin collecting the elution peak (PAC-EP) when UV280 rises to 15 mAU and stop collecting when UV280 falls to 15 mAU. The flow rate is 1 ml / min. After sample collection, adjust the PAC-EP to neutral using pH adjustment solution.

[0339] Four anti-CLDN18.2 / anti-PD-L1 bispecific antibodies were purified with Protein A, purified by SEC, concentrated, and then subjected to SEC testing to determine their purity. The results are summarized in the table below:

[0340] Table 19: SEC Purity

[0341]

[0342]

[0343] SEC purity was determined by HPLC. The four molecular spectra in the table are shown below: Figure 17 , Figure 18 , Figure 19 and Figure 20 As shown. QP3691433 Figure 17 QP3701440 purification as follows Figure 18 QP3711461 purification as follows Figure 19 QP3721116 purification as follows Figure 20 The results showed that the transient expression yield of the anti-Claudin 18.2 / PD-L1 bispecific antibody was good, and the SEC purity was excellent. The protein remained stable in physicochemical properties after concentration.

[0344] 4. ELISA detection of human PD-L1 binding activity of anti-CLDN18.2 / anti-PD-L1 bispecific antibody.

[0345] PD-L1 Binding ELISA:

[0346] Coating antibodies QP322 0.75ug / ml, QP11801181 1.5ug / ml, QP3691433, QP3701440, QP3711461, QP3721116 50ul / well, 4°C overnight. PBS 3 times. Blocking: 3% BSA 250ul / well, RT lh. Incubate 1ug / ml Biotin QP004.3 (biotin-PDL1-FC) 1:5 dilution different concentration, RT lh. PBST 6 times, PBS 3 times. Incubate secondary antibody: HRP-strepavidin (1:5000) 50ul / well, PBST 6 times, PBS 3 times. Color development: TMB 100ul / well, color development 10min. 2M H2SO4 50ul / well stop.

[0347] Results are shown in Table 20 and Figure 20. ELISA results showed that anti-Claudin 18.2 / PD-L1 bispecific antibodies QP3691433, QP3701440, QP3711461, QP3721116, in which the PD-L1 nanobody was fused at the C-terminus of the FC fragment, and the isotype control (QP322), in which the PD-L1 nanobody was fused at the N-terminus of the FC fragment, all bound to human PD-L1 protein with comparable EC50. Figure 21

[0348] Table 20: ELISA detection of anti-CLDN18.2 / anti-PD-L1 bispecific antibody molecules binding to human PD-L1 recombinant protein

[0349]

[0350]

[0351] 5. ELISA detection of anti-CLDN18.2 / anti-PD-L1 bispecific antibody molecules blocking PD-L1 / PD-1 binding activity (PD-L1 / PD-1 blocking)

[0352] PD-L1 / PD-1 blocking ELISA

[0353] ​Coating: Antibody QP1138 2ug / ml 50ul / well, 4C overnight. PBS 3 times. Blocking: 3% BSA 250ul / well, RT lh. Prepare 2ug / ml Biotin QP004.3 (biotin-PDL1-FC) and different concentrations of QP322 15ug / ml, QP1180 1181 30ug / ml, QP369 1433, QP370 1440, QP371 1461, QP372 1116 36ug / ml, 1:3 dilution, mix equal volume, RT lh. PBST 3 times, PBS 3 times. Incubate secondary antibody: HRP-strepavidin (1:5000) 50ul / well, PBST 3 times, PBS 3 times. Color development: TMB 100ul / well, color development 10min. 2M H2SO4 50ul / well stop.

[0354] Results are shown in Table 21. ELISA results showed that PD-L1 nanobody in anti-Claudin 18.2 / PD-L1 bispecific antibodies QP369 1433, QP370 1440, QP371 1461, QP372 1116 fused at C-terminus of FC and in isotype control (QP322) fused at N-terminus of FC, all could block the binding of human PD-L1 protein and PD-1 protein, with comparable IC50. Figure 22

[0355] Table 21: PD-L1 / PD-1 blocking ELISA results

[0356]

[0357] 6. ELISA detection of anti-CLDN18.2 / anti-PD-L1 bispecific antibodies blocking PD-L1 / CD80 binding activity (PD-L1 / CD80 blocking)

[0358] PD-L1 / CD80 blocking ELISA

[0359] Coat CD80-FC 4ug / ml, 4C overnight. PBS wash 3 times, 5% skim milk powder blocking, RT lh. Incubate final concentration 0.5ug / ml Biotin-QP004 + CHO14 (10ug / ml, 1:5 dilution), RT lh. PBST wash 5 times. HRP-Strepavidin (1:5000), PBST wash 6 times, PBS wash 3 times. TMB color development. Note: CHO14 protein is the protein obtained from CHO cell expression of QP371 1461. ​

[0360] Results are shown in Table 21 and Table 22. ELISA results show that the anti-Claudin 18.2 / PD-L1 bispecific antibody CHO14 (QP3711461) of the present application can block the binding of PD-L1 and CD80. Figure 23

[0361] Table 22: PD-L1 / CD80 blocking ELISA results

[0362]

[0363] 7. ELISA detection of anti-CLDN18.2 / anti-PD-L1 bispecific antibody binding CLDN18.2 activity

[0364] Detection reagent: skim milk powder (BD, 232100), PBS (Shenguo, B548117-0500); HRP-anti human IgG (H+L) (jackson, 109-035-088); TMB (Luoyang Baoao Tong Experimental Material Center, C060201); Elisa plate (costa, 9018) 1x PBS buffer: weigh NaCl 8.00 g, KCl 0.20 g, Na2HPO4·12H2O 2.9 g, KH2PO4 0.2 g into 800 mL ddH2O, dissolve thoroughly, make up to 1 L, adjust pH to 7.4, high temperature sterilization for standby. Or buy commercial 10x, 20x PBS solution dilute to 1x PBS buffer for use. Blocking solution: weigh 5 g of skim milk powder into PBS, the blocking solution needs to be prepared immediately. Stop solution (1 mol / L H2SO4): take 109 mL of 98% concentrated H2SO4 and slowly add it to 2000 mL of ddH2O. TMB 37°C color development for 10 min, placed in a shaker (120 rpm), 100ul / well;

[0365] ​Experimental steps: Seed 2E5 / well cells CHOS-CLD 18.2-16-2 in U-plate, ice PBS wash once, 1200 rpm, centrifugal 3 min; VCD: 1.21E6, actual 165ul / well; After washing, add blocking solution 200ul / well, incubate on ice for 1h. After blocking, centrifugal 3min at 1200rpm, discard the supernatant, incubate CHO14 samples, dilute 12 gradients according to the dilution ratio of 1:2, finally set a blank control, add 100ul / well according to 100ug / ml, 33.33333333ug / ml, 11.11111111ug / ml, 3.703703704ug / ml, 1.234567901ug / ml, 0.411522634ug / ml, 0.137174211ug / ml, 0.045724737ug / ml, 0.015241579ug / ml, 0.005080526ug / ml, 0.001694ug / ml, 0ug / ml, mix well, incubate on ice for 2h, ice PBS wash 3 times; Add enzyme-labeled antibody: incubate HRP-anti human IgG(H+L) antibody, dilute according to the dilution ratio of 1:10000, 100ul / well, mix well, incubate on ice for 1h, ice PBS wash 3 times. Add substrate developing solution: add substrate developing solution TMB 100ul / well, place in a shaker, 200rpm, 35C, avoid light, develop for 10min. Stop: after developing, quickly add stop solution 100ul / well to stop the reaction. Detection: after centrifugal 5min at 3500rpm, transfer 120ul supernatant to Elisa plate, measure the OD value of A450nm on the enzyme label instrument, use graphpad prism software to analyze the results.

[0366] The binding EC50 of CHO14 to CHOS-CLDN18.2 stable cell strain is 0.2653nM. It is proved that the candidate molecule

[0367] CHO14 binds to Claudin 18.2, and the results are shown in Figure 24

[0368] 8. Mixed lymphocyte reaction (MLR) detects anti-CLDN18.2 / anti-PD-L1 bispecific antibody PD-L1 functional activity

[0369] ​Mixed lymphocyte reaction (MLR) is to mix human T cells and allogeneic dendritic cells in a 96-well round-bottom plate at an appropriate ratio, and the lymphocytes are stimulated by alloantigen to activate, proliferate, and produce various cytokines. The PD-L1 antibody stimulates T cell proliferation and releases cytokines such as IL-2 / IFN-γ by blocking the immunosuppressive signal of PD-1 / PD-L1 binding in an antibody concentration-dependent manner. Using mixed lymphocyte reaction (MLR), whether the concentration of cytokines IFN-γ and IL-2 produced after T cell activation by anti-CLDN18.2 / anti-PD-L1 bispecific antibody has a significant antibody concentration-dependent effect is detected to prove the biological function of PD-L1 antibody in anti-CLDN18.2 / anti-PD-L1 bispecific antibody.

[0370] Preparation of DC (donor1) cells: resuscitate PBMC, use EasySep TM Human Monocyte Isolation Kit (Stemcell 19359) is used to separate monocytes, and rhGM-CSF (1000 U / ml) and rhIL4 (500 U / ml) are added to culture cells at 37°C for 6 days to induce iDC; semi-replace liquid every 2-3 days, while supplementing rhGM-CSF (1000 U / ml) and rhIL4 (500 U / ml); collect cells and centrifuge at 300x g for 5 min, resuspend with medium containing rhGM-CSF (1000 U / ml) and rhIL4 (500 U / ml), and add LPS (1 μg / ml) at the same time, continue to culture cells at 37°C for 1 day to induce mature DC; collect cells and count for standby. Preparation of T (donor2) cells: resuscitate PBMC, use EasySep TM Human CD4+T Cell Isolation Kit (Stemcell 17952) is used to separate CD4+T cells. Preparation of antibodies: dilute antibodies (initial concentration 10 ug / ml) with medium 1:5 gradient to 6 concentrations. Mix DC cells and T cells at a ratio of 1:10, add different concentrations of antibodies, and mix culture. On day 2, detect the expression of IL2 in the culture supernatant, and on day 5, detect the expression of IFNg in the culture supernatant.

[0371] In the mixed lymphocyte reaction experiment, the candidate molecule CHO14 has a significant antibody concentration-dependent effect on the concentration of cytokines IFNγ and IL-2 produced after T cell activation. It is proved that the biological function of PD-L1 antibody in candidate molecule CHO14, and CHO14 can significantly promote T cell proliferation and enhance the production of IL-2 and IFN-γ. As shown in Figure 25 and Figure 26 .

[0372] 9. PBMC-mediated cell killing assay dependent on anti-CLDN18.2 / anti-PD-L1 bispecific antibody

[0373] Prepare target cells (HCC827-CLDN18.2): Digest HCC827-CLDN18.2 cells with trypsin at 1000 rpm for 5 min. Replace with fresh medium and plate in 96-well plates at 20,000 cells / well, incubate overnight at 37°C with 5% CO2. Prepare antibodies: Serially dilute antibodies 1:5 (200 nM – 0.000512 nM, 0) to 10 concentrations using medium. Discard the medium from the target cells in the 96-well plates and add 70 μL / well of each diluted antibody concentration, with replicates for each concentration. Prepare PBMCs: Centrifuge the PBMCs recovered on day 1, resuspend in medium, and count. Add 70 μL / well of PBMCs to the above-mentioned wells at a PBMC:Target cell ratio of 50:1, incubate at 37°C for 4 hours. Add 15 μL of lysis buffer (1% Triton-X100) to the Max lysis well and incubate at 37°C for 10 min.

[0374] Prepare LDH reagents: Centrifuge a 96-well plate at 200g for 5 minutes, and transfer 100µl of supernatant per well to a new clear 96-well plate. Take the LDH cytotoxcity assay kit (Cayman, 10008882-480well), prepare the reaction solution, add 100µl per well, and gently vortex at 37°C for 30 minutes. Read the absorbance at 490nm. Analyze the data according to the formula: Con(µg / ml)%Maximal signal = (Test-Control) / (Max-Control) – Con(0µg / ml)%Maximal signal.

[0375] The results are as follows Figure 27 As shown in Tables 23 and 24, in the ADCC experiment, both the anti-Claudin 18.2 monoclonal antibody QP14611463 and the anti-CLDN18.2 / anti-PD-L1 bispecific antibody QP3711461 exhibited concentration-dependent PBMC-mediated killing of recombinant Claudin 18.2 and naturally PD-L1-expressing human lung cancer cells HCC827-CLDN18.2. The PBMC-mediated killing of human lung cancer cells HCC827-CLDN18.2 dependent on the bispecific antibody QP3711461 was superior to that mediated by the anti-Claudin 18.2 monoclonal antibody QP14611463.

[0376] Table 23: 490nm readings

[0377]

[0378] Table 24: Killing percentage

[0379]

[0380]

[0381] 10. In vivo efficacy evaluation of anti-CLDN18.2 / anti-PD-L1 bispecific antibody in C57BL / 6-hPDL1 model MC38-hPDL1-mClaudin18.2 of immunotargeting humanization transgenic mice

[0382] Experimental method: The logarithmic growth phase mouse colon cancer cells MC38-hPDL1 (Tg)-mClaudin18.2 (Tg) cells (which overexpress human PDL1 and mouse Claudin18.2, and knockout mouse PDL1) were removed from the culture medium and washed twice with PBS, then inoculated subcutaneously on the right flank of C57BL / 6-hPDL1 mice, with an inoculation amount of 5x10 5 / 100 μL / mouse. The mice were observed after inoculation and the growth of the tumors was monitored. On the 8th day after inoculation, when the average tumor volume reached 82.85 mm 3 , the mice were randomly divided into 4 groups, 9 mice in each group. The day of grouping was defined as D0 day, and the drug administration started on D0 day.

[0383] Experimental results: As shown in Table 25. Figure 28

[0384] Table 25: Tumor volume

[0385]

[0386] The test molecule was anti-CLDN18.2 / anti-PD-L1 bispecific antibody QP3711461, and the dosages were 1.5 mpk, 4 mpk, and 10 mpk, respectively, administered BIWx3, i.v. On the 28th day after administration, the average tumor volume of the PBS group (negative control group) reached 1033.97 mm 3 , the average tumor volume of the QP3711461 (1.5 mpk) group was 932.52 mm 3 , TGI = 12.19%, the average tumor volume of the QP3711461 (4 mpk) group was 360.92 mm 3 , TGI = 61.81%, and the average tumor volume of the QP3711461 (10 mpk) group was 294.50 mm 3 ​​​​​​​​​​​​​​​​, TGI = 69.53%; the tumor volumes of the 4 mpk group and the 10 mpk group were statistically significantly different from that of the PBS group (t test, p < 0.01).

[0387] This experiment shows that our anti-CLDN18.2 / anti-PD-L1 bispecific antibody exhibits superior anti-tumor ability in the MC38-hPDL1-mClaudin18.2 model of the immunotargeting humanized transgenic mouse.

[0388] 11. In vivo efficacy evaluation of anti-CLDN18.2 / anti-PD-L1 bispecific antibody in the PBMC engrafted-NCG model HCC827-hClaudin18.2 of the immune system humanized mouse

[0389] Experimental method: Take the logarithmic growth period human lung adenocarcinoma stable cell line HCC827-hClaudin18.2 cells (which naturally express human PDL1 and overexpress human Claudin18.2), remove the culture medium and wash twice with PBS, then inoculate subcutaneously on the right flank of NCG mice, inoculation amount: 5 x 10 6 / 100 μL / each. Observe the mice after inoculation and monitor the growth of the tumor, on the 7th day after inoculation, when the average tumor volume reaches about 150 mm 3 , inoculate human PBMC (5 x 10 6 / 100 μL / each). Randomly divide into 4 groups according to the tumor volume, 9 in each group. The day of grouping is defined as D0 day, and the drug administration starts on D0 day.

[0390] Experimental results: as shown in Figure 29 and Table 26.

[0391] Table 26: Tumor volume in the in vivo efficacy evaluation in the PBMC engrafted-NCG model HCC827-hClaudin18.2 of the immune system humanized mouse

[0392]

[0393] The test molecule is anti-CLDN18.2 / anti-PD-L1 bispecific antibody QP3711461, the dosages are 4 mpk and 10 mpk, BIW x 3, i.v. administration; the control antibody molecule is Tecentriq, the dosage is 5 mpk, BIW x 3, i.v. administration. On the 24th day after administration, the average tumor volume of the PBS group (negative control group) reached 1306.8 mm 3 , the average tumor volume of the QP1461371 (4 mpk) group was 258.51 mm 3, TGI = 80.22%, QP1461371 (10 mpk) group average tumor volume 104.81 mm 3 , TGI = 91.98%, Tecentriq (5 mpk) group average tumor volume 90.90 mm 3 , TGI = 93.04%; QP3711461 (4 mpk) group, QP3711461 (10 mpk) group and Tecentriq (5 mpk) group were statistically significantly different from the PBS group (t test, p < 0.01) in tumor volume.

[0394] This experiment shows that our anti-CLDN18.2 / anti-PD-L1 bispecific antibody also shows superior anti-tumor ability in the HCC827-hClaudin18.2 model of the humanized immune system mouse.

[0395] 12. In vivo synergistic efficacy evaluation of anti-CLDN18.2 / anti-PD-L1 bispecific antibody in C57BL / 6 model MC38-hPDL1-mClaudin18.2

[0396] Experimental method: Take the mouse colon cancer cells MC38-hPDL1 (Tg)-mClaudin18.2 (Tg) cells (which overexpress human PDL1 and mouse Claudin18.2, and knock out mouse PDL1) in the logarithmic growth phase, remove the culture medium and wash twice with PBS, then inoculate in the right flank of C57BL / 6 mice, inoculation amount: 5 x 10 5 / 100 μL / each. Observe the mice after inoculation and monitor the growth of the tumor, on the 7th day after inoculation, when the average tumor volume reaches about 90 mm 3 , randomly divide into 4 groups according to the tumor volume, 10 in each group. The day of grouping is defined as D0 day, and the drug administration starts on D0 day.

[0397] Experimental results are shown in Figure 30 and Table 27.

[0398] Table 27 Tumor volume

[0399]

[0400] The test molecules were anti-CLDN18.2 / anti-PD-L1 bispecific antibody QP3711461, mutant PD-L1 binding ability molecule QP30771461 (QP3711461-ΔPDL1 null), and mutant Claudin 18.2 binding ability molecule QP30891902 (QP3711461-ΔClaudin 18.2 null), and the administration dose was 5 mg / kg, Q2Dx6, ip. On the 13th day after administration, the average tumor volume of the PBS group (negative control group) reached 1262.27 mm 3 , the average tumor volume of the QP3711461 group was 532.87 mm 3 , TGI = 62.24%, the average tumor volume of the QP30771461 group was 1173.62 mm 3 , TGI = 7.59%, the average tumor volume of the QP30891902 group was 794.75 mm 3 , TGI = 39.63%; the tumor volume of the QP30771461 group was statistically significantly different from that of the PBS group (t test, p < 0.01), and the tumor volume of the mutant molecule QP30891902 group was statistically significantly different from that of the PBS group (t test, p < 0.05).

[0401] This experiment shows that the anti-CLDN18.2 / anti-PD-L1 bispecific antibody has a synergistic effect in the MC38-hPDL1-mClaudin18.2 model of C57BL / 6 mice, and exhibits more superior anti-tumor activity than Claudin18.2 monoclonal antibody and PD-L1 monoclonal antibody.

[0402] 13. Anti-CLDN18.2 / anti-PD-L1 bispecific antibody in vivo synergistic efficacy evaluation in C57BL / 6 model MC38-hPDL1-mClaudin18.2

[0403] Experimental method: The logarithmic growth phase mouse colon cancer cells MC38-hPDL1 (Tg)-mClaudin18.2 (Tg) cells (which overexpress human PDL1 and mouse Claudin18.2, and knock out mouse PDL1) were removed from the culture medium and washed twice with PBS, then inoculated subcutaneously on the right flank of C57BL / 6 mice, and the inoculation amount was 5x10 5 / 100 μL / each. The inoculated mice were observed and the tumor growth was monitored, and on the 6th day after inoculation, 15 mice were randomly divided into 3 groups according to the tumor volume. The day of grouping was defined as D0 day, and the administration was started on D0 day.

[0404] Experimental results are shown in Figure 31 and Table 28.

[0405] Table 28 Tumor volume

[0406]

[0407] The tested molecules were anti-CLDN18.2 / anti-PD-L1 bispecific antibody QP3711461, mutant PD-L1 binding ability molecule QP30771461 (QP3711461-ΔPDL1 null), and mutant Claudin 18.2 binding ability molecule QP30891902 (QP3711461-ΔClaudin 18.2 null), and the dosages were 5 mg / kg (QP3711461 group), 5 mg / kg+5 mg / kg (QP30771461+QP30891902 combined administration group), respectively, Q3Dx6, iv. administration. On day 19 after administration, the average tumor volume of the PBS group (negative control group) reached 838.76 mm 3 , the average tumor volume of the QP3711461 group was 313.63 mm 3 , TGI=67.15%, the average tumor volume of the QP30771461+QP30891902 combined administration group was 478.61 mm 3 , TGI=45.98%; the tumor volume of the QP3711461 group was statistically significantly different from that of the PBS group (t test, p<0.01), and the tumor volume of the QP30771461+QP30891902 combined administration group was statistically significantly different from that of the PBS group (t test, p<0.05).

[0408] This experiment showed that the anti-CLDN18.2 / anti-PD-L1 bispecific antibody had a synergistic effect in the MC38-hPDL1-mClaudin18.2 model of C57BL / 6 mice, and exhibited more superior anti-tumor activity than Claudin18.2 monoclonal antibody and PD-L1 monoclonal antibody (p=0.052).

[0409] All the documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. In addition, it should be understood that various modifications or changes can be made to the present application by those skilled in the art upon reading the above description of the present application, and these equivalent forms also fall within the scope defined by the claims attached hereto. SEQUENCE LISTING <110> ZHIYU BIOTECHNOLOGY (SHANGHAI) CO., LTD. <120> A bispecific antibody targeting human claudin and human PDL1 protein and application thereof <130> P2021-0670 <150> CN2020103446768 <151> 2020‑04‑27 <160> 96 <170> PatentIn version 3.5 <210> 1 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 1 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 2 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 2 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 3 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 3 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 4 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 4 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser He He Ser Gly Gly Arg Thr Tyr Tyr Leu Asp Ser Glu Lys 50 55 60 Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Arg He Tyr Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gin Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 5 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 5 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser He He Ser Gly Gly Arg Thr Tyr Tyr Leu Asp Ser Glu Lys 50 55 60 Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Arg He Tyr Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gin Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 6 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 6 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser He He Ser Gly Gly Arg Thr Tyr Tyr Leu Asp Ser Glu Lys 50 55 60 Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Arg He Tyr Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gin Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 7 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 7 Asp He Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr He Asn Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gin Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 8 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 8 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 9 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 9 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ile Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 10 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 10 Asp Ile Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gin Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Pro Pro Lys Met Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ile Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu lie 100 105 110 Lys <210> 11 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 11 Gln Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Gin lie Tyr Pro Gly Asn Gly Asp Thr Thr Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Val Lys Gly Asn Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 12 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 12 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 Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asn Gly Asp Thr Thr Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Val Lys Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 13 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 Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asn Gly Asp Thr Thr Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Phe Val Lys Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 14 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 14 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asn Tyr Tyr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 15 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 15 Asp Ile Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gin Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Phe Cys Gin Asn 85 90 95 Asn Tyr Tyr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 16 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 16 Asp Ile Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr lie Ser Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gin Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Pro Pro Lys Leu Leu lie Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Val Phe Thr Leu Thr 65 70 75 80 lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Phe Cys Gin Asn 85 90 95 Asn Tyr Tyr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu lie 100 105 110 Lys <210> 17 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 17 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr He Ser Ser Gly Ser Asn Ser He Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Ala Tyr Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 18 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 18 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Asn Ser Ile Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Ala Tyr Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 19 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 19 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 Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Asn Ser Ile Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Ala Tyr Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 20 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 20 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 21 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 21 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 22 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 22 Asp Ile Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gin Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gin Ala Glu Asp Val Ala Val Tyr Phe Cys Gin Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 23 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 23 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 24 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 24 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 Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Phe Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Ser Leu Arg Phe Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 25 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 25 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 Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Phe Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Ser Leu Arg Phe Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 26 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 26 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 Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Phe Val His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Ser Leu Arg Phe Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 27 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 27 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 Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Phe Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Ser Leu Arg Phe Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 28 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 28 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 29 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 29 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu lie Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Asn 85 90 95 Asp His Ser Tyr Pro Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu lie 100 105 110 Lys <210> 30 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 30 Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Pro Pro Lys Leu Leu lie Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 31 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 31 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 Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 32 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 32 Glu 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 Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 33 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 33 Glu 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 Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 34 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 34 Glu 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 Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ile Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Cys Cys 85 90 95 Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 35 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 35 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 36 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 36 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 37 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 37 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Gln Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Asn Met Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 38 <211> 117 <212> PRT <213> Mus musculus <400> 38 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Ile Ser Gly Gly Arg Thr Tyr Tyr Leu Asp Ser Glu Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Asn Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Thr 85 90 95 Arg Ile Tyr Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 39 <211> 113 <212> PRT <213> Mus musculus <400> 39 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gin Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Asn Pro Gly Gin 35 40 45 Pro Pro Lys Met Leu lie Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly lie Asp Phe Ser Leu Thr 65 70 75 80 lie Ser Ser Val Gin Ala Glu Asp Leu Ala Leu Tyr Tyr Cys Gin Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu lie 100 105 110 Lys <210> 40 <211> 118 <212> PRT <213> Mus musculus <400> 40 Gln Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp lie 35 40 45 Gly Gin lie Tyr Pro Gly Asn Gly Asp Thr Thr Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Gin Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Phe Val Lys Gly Asn Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 41 <211> 113 <212> PRT <213> Mus musculus <400> 41 Asp He Val Met Thr Gin Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gin Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Pro Pro Lys Leu Leu He Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Val Phe Thr Leu Thr 65 70 75 80 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Val Phe Thr Leu ThrIle Ser Ser Val Gin Ala Glu Asp Leu Ala Val Tyr Phe Cys Gin Asn 85 90 95 Asn Tyr Tyr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 42 <211> 119 <212> PRT <213> Mus musculus <400> 42 Asp Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gin Ala Pro Gin Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr He Ser Ser Gly Ser Asn Ser He Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe 65 70 75 80 Leu Gin Met Thr Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asn Ala Tyr Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gin Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 43 <211> 113 <212> PRT <213> Mus musculus <400> 43 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser His Val Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Asn Leu Glu Leu 100 105 110 Lys <210> 44 <211> 113 <212> PRT <213> Mus musculus <400> 44<400> 44 Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Phe Val His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu Glu Trp He 35 40 45 Gly Tyr He Asn Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Leu Ser Leu Arg Phe Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 <210> 45 <211> 113 <212> PRT <213> Mus musculus <400> 45 Asp He Val Met Thr Gin Ser Pro Ser Ser Leu Ser Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Asn Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Pro Pro Lys Leu Leu lie Tyr Gly Ala Ser Thr Arg Gin Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 lie Ser Ser Val Gin Ala Gin Asp Leu Ala Val Tyr Tyr Cys Gin Asn 85 90 95 Asp His Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Gin lie 100 105 110 Lys <210> 46 <211> 120 <212> PRT <213> Mus musculus <400> 46 Glu Val Gin Leu Gin Gin Ser Gly Pro Gin Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 lie Met His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu Gin Trp lie 35 40 45 Gly Tyr lie Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Cys Cys 85 90 95 Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 47 <211> 107 <212> PRT <213> Mus musculus <400> 47 Asp lie Gin Met Thr Gin Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Ser lie Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 48 <211> 120 <212> PRT <213> Mus musculus <400> 48 Glu Val Gln Leu Leu 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 Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Tyr Tyr Tyr Tyr Phe Trp Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 49 <211> 126 <212> PRT <213> Mus musculus <400> 49 Glu Val Gln Leu Leu 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 Thr Tyr Gly Thr Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe 100 105 110 Ser Arg Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 50 <211> 126 <212> PRT <213> Mus musculus <400> 50 Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Cys He Asp He Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys 50 55 60 Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe 100 105 110 Ser Arg Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 51 <211> 126 <212> PRT <213> Mus musculus <400> 51 Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr 20 25 30 Ala Met Ser Trp Phe Arg Gin Ala Pro Gly Lys Gly Arg Glu Gly Val 35 40 45 Ala Cys lie Asp lie Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Gin Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe 100 105 110 Ser Arg Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 52 <211> 126 <212> PRT <213> Mus musculus <400> 52 Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Cys lie Asp lie Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe 100 105 110 Ser Arg Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 53 <211> 126 <212> PRT <213> Mus musculus <400> 53 Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Cys lie Asp lie Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Gin Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe 100 105 110 Ser Arg Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 54 <211> 126 <212> PRT <213> Mus musculus <400> 54 Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr 20 25 30 Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe 100 105 110 Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 55 <211> 126 <212> PRT <213> Camelus <400> 55 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr 20 25 30 Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gin Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Gin Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe 100 105 110 Ser Arg Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 115 120 125 <210> 56 <211> 126 <212> PRT <213> Camelus <400> 56 Gln Val Gin Leu Val Gin Ser Gly Gly Asp Ser Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr 20 25 30 Ala Met Ser Trp Phe Arg Gin Ala Pro Gly Lys Gin Arg Gin Gin Val 35 40 45 Ala Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Gin Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Gin Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe 100 105 110 Ser Arg Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 115 120 125 <210> 57 <211> 214 <212> PRT <213> Homo sapiens <400> 57 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin 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 Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin 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 Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 58 <211> 448 <212> PRT <213> Homo sapiens <400> 58 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp He His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp He Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 59 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 59 Asp Ile Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gin Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 60 <211> 592 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 60 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ile Ser Gly Gly Arg Thr Tyr Tyr Leu Asp Ser Glu Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Arg He Tyr Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gin 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 Gin 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 Gin Thr Tyr He 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 Asp Glu Leu 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 Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 465 470 475 480 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly 485 490 495 Thr Tyr Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu 500 505 510 Gly Val Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro 515 520 525 Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Leu 530 535 540 Tyr Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr 545 550 555 560 Cys Ala Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu 565 570 575 Gly Phe Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 580 585 590 <210> 61 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 61 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 62 <211> 593 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 62 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 Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asn Gly Asp Thr Thr Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Phe Val Lys Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin 465 470 475 480 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr 485 490 495 Gly Thr Tyr Ala Met Ser Trp Phe Arg Gin Ala Pro Gly Lys Gly Arg 500 505 510 Glu Gly Val Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp 515 520 525 Pro Val Lys Gly Arg Phe Thr Ile Ser Gin Asp Asn Ser Lys Asn Thr 530 535 540 Leu Tyr Leu Gin Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr 545 550 555 560 Tyr Cys Ala Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His 565 570 575 Glu Gly Phe Ser Arg Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser 580 585 590 Ser <210> 63 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 63 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gin Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 64 <211> 595 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 64 Gln Val Gln Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ile Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Phe Thr Thr Arg Asn 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 Asp Glu Leu 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 Gin 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 Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460 Gly Gly Gly Gly Ser Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu 465 470 475 480 Val Gin Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 485 490 495 Thr Tyr Gly Thr Tyr Ala Met Ser Trp Phe Arg Gin Ala Pro Gly Lys 500 505 510 Gly Arg Glu Gly Val Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr 515 520 525 Thr Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Gin Asp Asn Ser Lys 530 535 540 Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Lys Ala Glu Asp Thr Ala 545 550 555 560 Val Tyr Tyr Cys Ala Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp 565 570 575 Val His Gin Gly Phe Ser Arg Tyr Trp Gin Gin Gly Thr Leu Val Thr 580 585 590 Val Ser Ser 595 <210> 65 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 65 Asp Ile Gin Met Thr Gin Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 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> 66 <211> 595 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide <400> 66 Glu Val Gln Leu Leu 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 Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Tyr Tyr Tyr Tyr Phe Trp Tyr 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 Gin 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 Gin Pro Arg Glu Pro Gin Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin 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 Gin 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 Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460 Gly Gly Gly Gly Ser Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu 465 470 475 480 Val Gin Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 485 490 495 Thr Tyr Gly Thr Tyr Ala Met Ser Trp Phe Arg Gin Ala Pro Gly Lys 500 505 510 Gly Arg Glu Gly Val Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr 515 520 525 Thr Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Gin Asp Asn Ser Lys 530 535 540 Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Lys Ala Glu Asp Thr Ala 545 550 555 560 Val Tyr Tyr Cys Ala Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp 565 570 575 Val His Glu Gly Phe Ser Arg Tyr Trp Gly Gin Gly Thr Leu Val Thr 580 585 590 Val Ser Ser 595 <210> 67 <211> 107 <212> PRT <213> Homo sapiens <400> 67 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 68 <211> 330 <212> PRT <213> Homo sapiens <400> 68 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 69 <211> 5 <212> PRT <213> Camelus <400> 69 Thr Tyr Ala Met Ser 1 5 <210> 70 <211> 16 <212> PRT <213> Camelus <400> 70 Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys Gly 1 5 10 15 <210> 71 <211> 19 <212> PRT <213> Camelus <400> 71 Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe 1 5 10 15 Ser Arg Tyr <210> 72 <211> 17 <212> PRT <213> Mus musculus <400> 72 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 73 <211> 7 <212> PRT <213> Mus musculus <400> 73 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 74 <211> 9 <212> PRT <213> Mus musculus <400> 74 Gln Asn Asp Tyr Ser Tyr Pro Phe Thr 1 5 <210> 75 <211> 5 <212> PRT <213> Mus musculus <400> 75 Asn Tyr Ala Met Ser 1 5 <210> 76 <211> 16 <212> PRT <213> Mus musculus <400> 76 Ser Ile Ile Ser Gly Gly Arg Thr Tyr Tyr Leu Asp Ser Glu Lys Gly 1 5 10 15 <210> 77 <211> 9 <212> PRT <213> Mus musculus <400> 77 Ile Tyr Tyr Gly Asn Ser Phe Asp Tyr 1 5 <210> 78 <211> 9 <212> PRT <213> Mus musculus <400> 78 Gln Asn Ala Tyr Ser Tyr Pro Phe Thr 1 5 <210> 79 <211> 5 <212> PRT <213> Mus musculus <400> 79 Ser Tyr Trp Met Asn 1 5 <210> 80 <211> 17 <212> PRT <213> Mus musculus <400> 80 Gln Ile Tyr Pro Gly Asn Gly Asp Thr Thr Tyr Asn Gly Lys Phe Lys 1 5 10 15 Gly <210> 81 <211> 9 <212> PRT <213> Mus musculus <400> 81 Phe Val Lys Gly Asn Ala Met Asp Tyr 1 5 <210> 82 <211> 9 <212> PRT <213> Mus musculus <400> 82 Gln Asn Asn Tyr Tyr Tyr Pro Leu Thr 1 5 <210> 83 <211> 5 <212> PRT <213> Mus musculus <400> 83 Ser Phe Gly Met His 1 5 <210> 84 <211> 17 <212> PRT <213> Mus musculus <400> 84 Tyr Ile Ser Ser Gly Ser Asn Ser Ile Tyr Tyr Val Asp Thr Val Lys 1 5 10 15 Gly <210> 85 <211> 10 <212> PRT <213> Mus musculus <400> 85 Asn Ala Tyr Tyr Gly Asn Ser Phe Asp Tyr 1 5 10 <210> 86 <211> 9 <212> PRT <213> Mus musculus <400> 86 Gln Asn Asp Tyr Ser Tyr Pro Leu Thr 1 5 <210> 87 <211> 5 <212> PRT <213> Mus musculus <400> 87 Asn Tyr Phe Val His 1 5 <210> 88 <211> 17 <212> PRT <213> Mus musculus <400> 88 Tyr Ile Asn Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 89 <211> 8 <212> PRT <213> Mus musculus <400> 89 Leu Ser Leu Arg Phe Phe Ala Tyr 1 5 <210> 90 <211> 17 <212> PRT <213> Mus musculus <400> 90 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 91 <211> 7 <212> PRT <213> Mus musculus <400> 91 Gly Ala Ser Thr Arg Glu Ser 1 5 <210> 92 <211> 9 <212> PRT <213> Mus musculus <400> 92 Gln Asn Asp His Ser Tyr Pro Phe Thr 1 5 <210> 93 <211> 5 <212> PRT <213> Mus musculus <400> 93 Ser Tyr Ile Met His 1 5 <210> 94 <211> 17 <212> PRT <213> Mus musculus <400> 94 Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 95 <211> 11 <212> PRT <213> Mus musculus <400> 95 Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr 1 5 10 <210> 96 <211> 16 <212> PRT <213> Camelus <400> 96 Cys Ile Asp Ile Tyr Gly Arg Thr Ser Tyr Thr Asp Pro Val Lys Gly 1 5 10 15

Claims

1. A bispecific antibody targeting human claudin 18.2 and human PD-L1 protein, characterized in that, The bispecific antibody comprises: an anti-human claudin 18.2 antibody portion and an anti-PD-L1 antibody portion; the 3 heavy chain CDRs and 3 light chain CDRs of the anti-human claudin 18.2 antibody are selected from any one of the following groups: (Z1) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 93, 94, 95; and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 90, 91, 92; (Z2) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 75, 76, 77; and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 72, 73, 74; (Z3) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 79, 80, 81; and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 72, 73, 78; (Z4) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 83, 84, 85; and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 72, 73, 82; (Z5) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 87, 88, 89; and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 72, 73, 86; The anti-PD-L1 antibody is a single domain antibody, and the 3 complementarity determining regions (CDRs) of the single domain antibody are: HCDR1 as shown in SEQ ID NO: 69, HCDR2 as shown in SEQ ID NO: 70 or 96, and HCDR3 as shown in SEQ ID NO:

71.

2. The bispecific antibody of claim 1, wherein The bispecific antibody is a dimer composed of two monomers, and the monomer has a structure as shown in Formula I from N-terminal to C-terminal: wherein, L1, L2 and L3 are each independently a bond or a linker element; VH represents a heavy chain variable region of an anti-human claudin 18.2 antibody; VL represents a light chain variable region of an anti-human claudin 18.2 antibody; CH represents a heavy chain constant region of an anti-human claudin 18.2 antibody; CL represents a light chain constant region of an anti-human claudin 18.2 antibody; VHH represents an anti-PD-L1 single domain antibody; "-" represents a peptide bond; "~" represents a disulfide bond or a covalent bond.

3. The bispecific antibody of claim 1 or 2, wherein The amino acid sequence of the heavy chain variable region of the anti-human claudin 18.2 antibody is as shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-human claudin 18.2 antibody is as shown in SEQ ID NO: 29, and the amino acid sequence of the anti-PD-L1 single domain antibody is as shown in SEQ ID NO:

51. or, the amino acid sequence of the heavy chain variable region of the anti-human claudin 18.2 antibody is shown as SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the anti-human claudin 18.2 antibody is shown as SEQ ID NO: 1, and the amino acid sequence of the anti-PD-L1 single domain antibody is shown as SEQ ID NO: 51; or, the amino acid sequence of the heavy chain variable region of the anti-human claudin 18.2 antibody is shown as SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the anti-human claudin 18.2 antibody is shown as SEQ ID NO: 8, and the amino acid sequence of the anti-PD-L1 single domain antibody is shown as SEQ ID NO: 51; or, the amino acid sequence of the heavy chain variable region of the anti-human claudin 18.2 antibody is shown as SEQ ID NO: 48, and the amino acid sequence of the light chain variable region of the anti-human claudin 18.2 antibody is shown as SEQ ID NO: 47, and the amino acid sequence of the anti-PD-L1 single domain antibody is shown as SEQ ID NO:

51.

4. The bispecific antibody of claim 3, wherein the amino acid sequence of the L chain of the bispecific antibody is shown as SEQ ID NO: 63, and the amino acid sequence of the H chain of the bispecific antibody is shown as SEQ ID NO: 64; or, the amino acid sequence of the L chain of the bispecific antibody is shown as SEQ ID NO: 59, and the amino acid sequence of the H chain of the bispecific antibody is shown as SEQ ID NO: 60; or, the amino acid sequence of the L chain of the bispecific antibody is shown as SEQ ID NO: 61, and the amino acid sequence of the H chain of the bispecific antibody is shown as SEQ ID NO: 62; or, the amino acid sequence of the L chain of the bispecific antibody is shown as SEQ ID NO: 65, and the amino acid sequence of the H chain of the bispecific antibody is shown as SEQ ID NO:

66.

5. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-4. The polynucleotide encodes the bispecific antibody of any one of claims 1-4.

6. A vector, characterized in that, The vector contains the polynucleotide of claim 5.

7. A genetically engineered host cell, characterized in that, The host cell contains the vector of claim 6, or the polynucleotide of claim 5 is integrated into the genome.

8. A method of making a bispecific antibody according to any one of claims 1 to 4, characterized in that, comprising the steps of: (i) culturing the host cell of claim 7 under suitable conditions, so as to obtain a mixture containing the bispecific antibody; (ii) purifying and / or isolating the mixture obtained in step (i), so as to obtain the bispecific antibody.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition contains: (a) the bispecific antibody of any one of claims 1-4; and (b) a pharmaceutically acceptable carrier.

10. An immunoconjugate, comprising, The immunoconjugate comprises: (a) the bispecific antibody of any one of claims 1-4; and (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

11. Use of the bispecific antibody according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment of a cancer or a tumor; said cancer or tumor is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, kidney cancer, cervical cancer, bone marrow cancer, lymphatic cancer, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, desmoid fibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

12. Use of the bispecific antibody according to any one of claims 1 to 4 for the manufacture of a medicament for the inhibition of tumor growth; said tumor is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, kidney cancer, cervical cancer, bone marrow cancer, lymphatic cancer, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, desmoid fibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

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