A bispecific antibody against pd1 x pdl1

Abstract

Provided are a bispecific antibody against PD1xPDL1, a preparation method and use thereof, and experimental results show that the bispecific antibody can better maintain the activity of each single antibody, can specifically bind to two target points of PD-1 and PD-L1 at the same time, and has good physicochemical properties.

Description

Technical Field

[0001] This invention relates to the field of antibodies, specifically to a bispecific antibody against PD1×PDL1, its preparation method, and its uses. Background Technology

[0002] Human programmed cell death receptor-1 (PD-1) is a type I membrane protein with 288 amino acids and is one of the known major immune checkpoints (Blank et al., 2005, Cancer Immunotherapy, 54:307-314). PD-1 is expressed on activated T lymphocytes. Its binding to ligands PD-L1 (programmed cell death-ligand 1) and PD-L2 (programmed cell death-ligand 2) can inhibit T lymphocyte activity and related in vivo cellular immune responses. PD-L2 is mainly expressed on macrophages and dendritic cells, while PD-L1 is widely expressed on B and T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, and heart tissue cells. Numerous studies have shown that the interaction between PD-1 and PD-L1 is not only essential for maintaining the balance of the immune system in the body, but also a major mechanism and cause for PD-L1-positive tumor cells to circumvent immune surveillance. By blocking the negative regulation of the PD-1 / PD-L1 signaling pathway by cancer cells, the immune system can be activated, promoting T cell-related tumor-specific cellular immune responses, thus opening the door to a new cancer treatment method—tumor immunotherapy.

[0003] PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily associated with CD28 and CTLA-4. Research shows that PD-1 negatively regulates antigen receptor signal transduction when it binds to its ligands (PD-L1 and / or PD-L2). The structure of mouse PD-1 and the co-crystallization structure of mouse PD-1 and human PD-L1 have been elucidated (Zhang, X. et al., Immunity 20:337-347 (2004); Lin et al., Proc. Natl. Acad. Sci. USA 105:3011-6 (2008)). PD-1 and similar family members are type I transmembrane glycoproteins containing a variable (V-type) Ig domain responsible for ligand binding and a cytoplasmic tail region responsible for binding signal transduction molecules. The PD-1 cytoplasmic tail region contains two tyrosine-based signal transduction motifs: ITIM (immunoreceptor tyrosine inhibition motif) and ITSM (immunoreceptor tyrosine switching motif).

[0004] PD-1 plays a crucial role in the immune evasion mechanisms of tumors. Tumor immunotherapy, which utilizes the body's own immune system to fight cancer, is a groundbreaking cancer treatment method. However, the tumor microenvironment can protect tumor cells from effective immune destruction; therefore, disrupting the tumor microenvironment has become a key focus of anti-tumor research. Existing research has identified the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by IFN-γ in most PD-L1-negative tumor cell lines), and is presumed to be an important target mediating tumor immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. USA 99: 12293-12297 (2002); Strome SE et al., Cancer Res., 63: 6501-6505 (2003)). Immunohistochemical evaluation of biopsies has revealed the expression of PD-1 (on tumor-infiltrating lymphocytes) and / or PD-L1 on tumor cells in many primary human tumors. Such cancers include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma, pancreatic cancer, and head and neck tumors. Therefore, blocking the interaction between PD-1 and PD-L1 can enhance the immune activity of tumor-specific T cells, helping the immune system to clear tumor cells. Consequently, PD-1 and PD-L1 have become popular targets for developing tumor immunotherapy drugs.

[0005] Bispecific antibodies are antibody molecules that can simultaneously and specifically bind to two antigens or two epitopes. Based on symmetry, bispecific antibodies can be classified into structurally symmetrical and asymmetrical molecules. Based on the number of binding sites, bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules. Bispecific antibodies are gradually becoming a new class of therapeutic antibodies, which can be used to treat various inflammatory diseases, cancer, and other diseases. Summary of the Invention

[0006] This invention provides a bispecific antibody against PD1×PDL1.

[0007] Therefore, the first objective of this invention is to provide a bispecific antibody against PD1×PDL1.

[0008] A second object of the present invention is to provide an isolated nucleotide encoding the bispecific antibody.

[0009] A third objective of the present invention is to provide an expression vector containing the nucleotides.

[0010] A fourth object of the present invention is to provide a host cell comprising the expression vector.

[0011] A fifth object of the present invention is to provide a method for preparing the bispecific antibody.

[0012] A sixth object of the present invention is to provide a pharmaceutical composition comprising the bispecific antibody.

[0013] A seventh object of the present invention is to provide the use of the bispecific antibody or the pharmaceutical composition thereon in the preparation of a medicament for treating cancer.

[0014] An eighth object of the present invention is to provide a method for treating cancer using the bispecific antibody or the pharmaceutical composition.

[0015] To achieve the above objectives, the present invention provides the following technical solution:

[0016] The first aspect of the present invention provides a bispecific antibody against PD1×PDL1, the bispecific antibody comprising two polypeptide chains and two light chains:

[0017] a) Each polypeptide chain, from its N-terminus to its C-terminus, sequentially comprises VH-PDL1—CH1—CH2—CH3—linker2—VL-PD1—linker1—VH-PD1 or VH-PDL1—CH1—CH2—CH3—linker2—VH-PD1—linker1—VL-PD1 or VL-PD1—linker1—VH-PD1—linker2—VH-PDL1—CH1—CH2—CH3 or VH-PD1—linker1—VL-PD1—linker2—VH-PDL1—CH1—CH2—CH3, and each light chain, from its N-terminus to its C-terminus, sequentially comprises VL-PDL1—CL; or

[0018] b) Each of the polypeptide chains, from the N-terminus to the C-terminus, sequentially comprises VH-PD1—CH1—CH2—CH3—linker2—VL-PDL1—linker1—VH-PDL1 or VH-PD1—CH1—CH2—CH3—linker2—VH-PDL1—linker1—VL-PDL1 or VL-PDL1—linker1—VH-PDL1—linker2—VH-PD1—CH1—CH2—CH3 or VH-PDL1—linker1—VL-PDL1—linker2—VH-PD1—CH1—CH2—CH3, and each of the light chains, from the N-terminus to the C-terminus, sequentially comprises VL-PD1—CL.

[0019] Wherein, VH-PDL1 is a heavy chain variable region bound to PD-L1, CH1-CH2-CH3 is a heavy chain constant region, VL-PD1 is a light chain variable region bound to PD-1, VH-PD1 is a heavy chain variable region bound to PD-1, VL-PDL1 is a light chain variable region bound to PD-L1, and CL is a light chain constant region.

[0020] In the bispecific antibody against PD1×PDL1, VH-PDL1 forms an antigen-binding site that specifically binds to PD-L1 with VL-PDL1, and VL-PD1 forms an antigen-binding site that specifically binds to PD-1 with VH-PD1.

[0021] According to a preferred embodiment of the present invention, the anti-PD1×PDL1 bispecific antibody further includes one or more of the following:

[0022] 1) The VH-PDL1 contains the heavy chain CDR with an amino acid sequence as shown in SEQ ID NO: 1-3;

[0023] 2) The VL-PDL1 contains the light chain CDR with an amino acid sequence as shown in SEQ ID NO: 4-6;

[0024] 3) The VH-PD1 contains the heavy chain CDR with an amino acid sequence as shown in SEQ ID NO: 7-9;

[0025] 4) The VL-PD1 contains a light chain CDR with an amino acid sequence as shown in SEQ ID NO: 10-12.

[0026] In one embodiment, the VH-PDL1 comprises H-CDR1 with the amino acid sequence shown in SEQ ID NO: 1.

[0027] In one embodiment, the VH-PDL1 comprises H-CDR2 with the amino acid sequence shown in SEQ ID NO: 2.

[0028] In one embodiment, the VH-PDL1 comprises the amino acid sequence H-CDR3 as shown in SEQ ID NO: 3.

[0029] In one embodiment, the VL-PDL1 comprises L-CDR1 with the amino acid sequence shown in SEQ ID NO: 4.

[0030] In one embodiment, the VL-PDL1 comprises the amino acid sequence L-CDR2 as shown in SEQ ID NO: 5.

[0031] In one embodiment, the VL-PDL1 comprises L-CDR3 with the amino acid sequence shown in SEQ ID NO: 6.

[0032] In one embodiment, the VH-PD1 comprises H-CDR1 with the amino acid sequence shown in SEQ ID NO: 7.

[0033] In one embodiment, the VH-PD1 comprises the amino acid sequence H-CDR2 as shown in SEQ ID NO: 8.

[0034] In one embodiment, the VH-PD1 comprises H-CDR3 with the amino acid sequence shown in SEQ ID NO: 9.

[0035] In one embodiment, the VL-PD1 comprises L-CDR1 with the amino acid sequence shown in SEQ ID NO: 10.

[0036] In one embodiment, the VL-PD1 comprises the amino acid sequence L-CDR2 as shown in SEQ ID NO: 11.

[0037] In one embodiment, the VL-PD1 comprises the amino acid sequence L-CDR3 as shown in SEQ ID NO: 12.

[0038] According to a preferred embodiment of the present invention, the VH-PDL1 has an amino acid sequence as shown in SEQ ID NO: 13, and / or the VL-PDL1 has an amino acid sequence as shown in SEQ ID NO: 14, and / or the VH-PD1 has an amino acid sequence as shown in SEQ ID NO: 15, and / or the VL-PD1 has an amino acid sequence as shown in SEQ ID NO: 16.

[0039] Linker2 consists of 3 G4S, and linker1 consists of 4 G4S.

[0040] According to the present invention, the heavy chain constant region includes the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and the light chain constant region includes the κ or λ light chain constant region.

[0041] According to a preferred embodiment of the present invention, the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 22 or SEQ ID NO: 23 or SEQ ID NO: 24, and / or, the light chain has an amino acid sequence as shown in SEQ ID NO: 18; or the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 25 or SEQ ID NO: 26 or SEQ ID NO: 27 or SEQ ID NO: 28, and / or, the light chain has an amino acid sequence as shown in SEQ ID NO: 21.

[0042] A second aspect of the invention provides an isolated nucleotide encoding the bispecific antibody.

[0043] The method for preparing the nucleotides described in this invention is a conventional method in the art, and preferably includes the following preparation method: obtaining the nucleotides encoding the above-mentioned monoclonal antibody by gene cloning technology such as PCR, or obtaining the nucleotides encoding the above-mentioned monoclonal antibody by artificial full-sequence synthesis.

[0044] Those skilled in the art will understand that the nucleotide sequence encoding the amino acid sequence of the aforementioned bispecific antibody can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homologue. The polynucleotide homologue of this invention can be prepared by substituting, deleting, or adding one or more bases encoding the bispecific antibody gene within the range of maintaining antibody activity.

[0045] A third aspect of the present invention provides an expression vector containing the nucleotides described above.

[0046] The expression vectors mentioned above are conventional expression vectors in the art, referring to expression vectors containing appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and / or sequences, and other appropriate sequences. The expression vectors can be viruses or plasmids, such as appropriate bacteriophages or phage particles. For more technical details, please refer to, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd edition, ColdSpring Harbor Laboratory Press, 1989. Many known techniques and protocols for nucleic acid manipulation can be found in *Current Protocols in Molecular Biology*, 2nd edition, edited by Ausubel et al.

[0047] The expression vector of the present invention is preferably pTT5, pDR1, pcDNA3.1, pcDNA3.4, pDHFF, pGM-CSF or pCHO1.0, and more preferably pcDNA3.4 or pTT5.

[0048] A fourth aspect of the present invention provides a host cell containing the expression vector described above.

[0049] The host cells described in this invention are any conventional host cells in the art, as long as they can stably and spontaneously replicate the recombinant expression vector and effectively express the carried nucleotides. The host cells include prokaryotic expression cells and eukaryotic expression cells. Specifically, the host cells are, for example: COS, CHO (Chinese Hamster Ovary), NSO, sf9, sf21, DH5α, BL21(DE3), E. coli TG1, BL21(DE3) cells, or CHO-K1 cells, 293E cells, or TG1. The host cells are more preferably CHO cells and / or 293E cells.

[0050] A fifth aspect of the present invention provides a method for preparing the bispecific antibody, the method comprising the following steps:

[0051] (a) Under expression conditions, host cells as described above are cultured to express the bispecific antibody;

[0052] (b) Isolate and purify the bispecific antibody described in (a).

[0053] The host cell culture method and the isolation and purification method of the anti-PD1×PDL1 bispecific antibody described in this invention are conventional methods in the art. For specific operating procedures, please refer to the relevant cell culture technique manual and bispecific antibody isolation and purification technique manual. The method for preparing the anti-PD1×PDL1 bispecific antibody disclosed in this invention includes: culturing the aforementioned host cells under expression conditions to express the anti-PD1×PDL1 bispecific antibody; and isolating and purifying the anti-PD1×PDL1 bispecific antibody. Using the above method, the recombinant protein can be purified into a substantially homogeneous substance, for example, appearing as a single band on SDS-PAGE electrophoresis.

[0054] The anti-PD1×PDL1 bispecific antibody disclosed in this invention can be separated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer or pH adjustment can be used to elute the bispecific antibody bound to the affinity column. The inventors of this invention conducted detection experiments on the obtained anti-PD1×PDL1 bispecific antibody. The experimental results show that this bispecific antibody can bind well to PD-1 and PDL-1, exhibiting high affinity.

[0055] A sixth aspect of the present invention provides a pharmaceutical composition comprising a bispecific antibody as described above and a pharmaceutically acceptable carrier.

[0056] The anti-PD1×PDL1 bispecific antibody provided by this invention can be combined with a pharmaceutically acceptable carrier to form a drug composition, thereby achieving a more stable therapeutic effect. The drug composition has a specific formulation that ensures the conformational integrity of the amino acid core sequence of the anti-PD1×PDL1 bispecific antibody disclosed in this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation).

[0057] A seventh aspect of the invention provides the use of the bispecific antibody or pharmaceutical composition as described above in the preparation of a medicament for treating cancer.

[0058] According to the present invention, the cancer is selected from one or more of melanoma, kidney cancer, lung cancer, liver cancer, stomach cancer, lymphoma, breast cancer, colorectal cancer, leukemia, prostate cancer, bone marrow cancer and other proliferative malignant diseases.

[0059] The anti-PD1×PDL1 bispecific antibody described in this invention can be used alone or in combination with other anti-tumor drugs.

[0060] The cancer treatment drug referred to in this invention refers to a drug that inhibits and / or treats tumors, and may include delaying the development of tumor-related symptoms and / or reducing the severity of these symptoms, further including alleviating existing tumor-related symptoms and preventing the occurrence of other symptoms, and also including reducing or preventing tumor metastasis, etc.

[0061] When the bispecific antibody against PD1×PDL1 and its pharmaceutical composition in this invention are administered to animals, including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The results of animal experiments and various other factors can be referenced, but the total dosage should not exceed a certain range.

[0062] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0063] An eighth aspect of the invention provides a method for treating cancer, comprising administering to a subject in need a bispecific antibody as described above or a pharmaceutical composition as described above.

[0064] According to the present invention, the cancer is selected from one or more of melanoma, kidney cancer, lung cancer, liver cancer, stomach cancer, lymphoma, breast cancer, colorectal cancer, leukemia, prostate cancer, bone marrow cancer and other proliferative malignant diseases.

[0065] When the anti-PD1×PDL1 bispecific antibody and its pharmaceutical composition are administered to subjects, the dosage must be a therapeutically effective amount. The therapeutically effective amount refers to an amount that is effective in treating cancer. Specifically, when the anti-PD1×PDL1 bispecific antibody and its pharmaceutical composition are administered to subjects, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. This can be referenced based on animal experiment results and various other factors, but the total dosage must not exceed a certain range.

[0066] The beneficial effects of the present invention are as follows: The present invention provides a bispecific antibody against PD1×PDL1. Experimental results show that the bispecific antibody can maintain the activity of each monoclonal antibody well and can simultaneously and specifically bind to two targets, PD-1 and PD-L1, and has good physicochemical properties. Attached Figure Description

[0067] Figure 1 This is a schematic diagram of the structure of the anti-PD1×PDL1 dual antibody of the present invention.

[0068] Figure 2A This is the HPLC-SEC detection chromatogram of the anti-PD1×PDL1 bispecific antibody of the present invention.

[0069] Figure 2B The results of SDS-PAGE analysis of the anti-PD1×PDL1 bispecific antibody of the present invention are shown.

[0070] Figure 3A The binding results of the anti-PD1×PDL1 bispecific antibody of the present invention to PD-1 were detected by ELISA.

[0071] Figure 3B The binding results of the anti-PD1×PDL1 bispecific antibody of the present invention to PD-L1 were detected by ELISA.

[0072] Figure 4 The binding results of the anti-PD1×PDL1 bispecific antibody of the present invention to both PD-1 and PD-L1 were detected by bispecific ELISA.

[0073] Figure 5AThe binding results of the anti-PD1×PDL1 bispecific antibody of the present invention to PD-1 / CHO cells were detected by FACS.

[0074] Figure 5B The binding results of the anti-PD1×PDL1 bispecific antibody of the present invention to N87-PDL1 cells were detected by FACS.

[0075] Figure 6 The results show the activity of PD1 / PD-L1 in cells blocked by the anti-PD1×PDL1 dual antibody of the present invention.

[0076] Figure 7A The results show that anti-PD1×PDL1 rev2 blocks the activity of the PD1 / PD-L1 signaling pathway on cells.

[0077] Figure 7B The results represent the effects of anti-PD1×PDL1 rev3, rev4, and rev5 on the activity of the PD1 / PD-L1 signaling pathway in cells.

[0078] Figure 7C The results represent the effects of anti-PD1×PDL1 rev6, rev7, and rev8 on the activity of the PD1 / PD-L1 signaling pathway in cells. Detailed Implementation

[0079] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region including a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 isotypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0080] In this invention, the term "bispecific antibody (Biantibody)" refers to an antibody molecule that can simultaneously and specifically bind to two antigens (targets) or two epitopes.

[0081] In this invention, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a substantially homogeneous population, meaning that the individual antibodies in this population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody formulations (which are typically mixtures of different antibodies targeting different antigenic determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring any special method to produce the antibody.

[0082] In this invention, the terms "Fab" and "Fc" refer to the ability of papain to cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of its light chain. The Fc fragment, or crystallizable fragment, consists of the antibody's CH2 and CH3 domains. The Fc fragment lacks antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells.

[0083] In this invention, the term "scFv" refers to a single-chain antibody fragment (scFv), which is composed of the variable regions of the antibody heavy chain and the variable regions of the light chain linked by a short peptide (linker) of 15 to 25 amino acids.

[0084] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the heavy and light chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0085] In this invention, the terms "antibody," "binding," and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11The antibody binds to the antigen with an equilibrium dissociation constant (KD) of M or smaller. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of antibody-antigen binding.

[0086] In this invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitopes of this invention are regions of an antigen that are bound to antibodies.

[0087] In this invention, the term "expression vector" can refer to pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, and other vectors used in mammalian expression systems. The expression vector includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0088] In this invention, the term "host cell" refers to a cell suitable for expressing the above-mentioned expression vector. It can be a eukaryotic cell, such as a mammalian or insect host cell culture system, which can be used for the expression of the fusion protein of this invention. CHO (Chinese Hamster Ovary), HEK293, COS, BHK, and derived cells of the above cells can all be used in this invention.

[0089] In this invention, the term "pharmaceutical composition" refers to the bispecific antibody of this invention that can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical formulation composition to exert its therapeutic effect more stably. These formulations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation).

[0090] The experimental materials used in the following examples are described below:

[0091] pcDNA TM 3.4 vector: purchased from Thermo Fisher, part number A14697;

[0092] CHO cells: purchased from Thermo Fisher Scientific, catalog number A29133;

[0093] 293E cells: from NRC biotechnology Research Institute;

[0094] PD-1 / PD-L1 Blockade Bioassay, Propagation model: purchased from Promega, part number J1252;

[0095] Human gastric cancer cell line NCI-N87: purchased from the American Type Culture Collection (ATCC);

[0096] SD rats: purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0097] The experimental reagents used in the following examples are described below:

[0098] Anti-PD-L1 monoclonal antibody: prepared according to the sequence in PCT / CN2020 / 090442;

[0099] Anti-PD-1 monoclonal antibody: prepared according to the sequence in WO2018 / 137576;

[0100] HRP-labeled mouse anti-human Fab antibody: purchased from Sigma, catalog number A0293;

[0101] HRP-labeled anti-6×His antibody: purchased from abcam, catalog number ab178563;

[0102] Goat anti-human IgG-FITC: purchased from Sigma, product number F4143;

[0103] PBS: Purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B548117;

[0104] PBST: PBS + 0.05% Tween 20;

[0105] BSA: Purchased from Sangon Biotech (Shanghai) Co., Ltd., item number A60332;

[0106] FBS: Purchased from Gibco, item number 10099;

[0107] TMB: Purchased from BD Company, item number 555214;

[0108] Bio-Glo Luciferase Assay System: Purchased from Promega, product number G7940.

[0109] The experimental instruments used in the following examples are described below:

[0110] PCR instrument: purchased from BioRad, model number C1000 Touch Thermal Cycler;

[0111] HiTrap MabSelectSuRe column: purchased from GE, part number 11-0034-95;

[0112] Beckman Coulter CytoFLEX flow cytometer: purchased from Beckman Coulter;

[0113] SpectraMax i3x microplate reader: purchased from Molecular Devices.

[0114] The following examples and experimental cases are further illustrative of the present invention and should not be construed as limiting the invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press.

[0115] Example 1: Construction of anti-PD1×PDL1 bispecific antibody molecule

[0116] This invention constructs an anti-PD1×PDL1 bispecific antibody, named anti-PD1×PDL1 BsAb, by tandemly linking the scFv (VL-linker1-VH, linker1 consisting of 4 GGGGS) of the anti-human PD-1 monoclonal antibody mAb1-25-Hu (sequence from WO2018 / 137576) to the C-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody M8 (sequence from PCT / CN2020 / 090442) via linker2 (3 GGGGS). The structure is as follows. Figure 1 As shown.

[0117] This invention constructs an anti-PD1×PDL1 bispecific antibody, named anti-PD1×PDL1 Rev2, by tandem linker 2 (3 GGGGS) of anti-human PD-1 monoclonal antibody mAb1-25-Hu's scFv2 (VH-linker1-VL, linker1 is 4 GGGGS) with linker 2 (3 GGGGS).

[0118] This invention constructs an anti-PD1×PDL1 bispecific antibody, named anti-PD1×PDL1 Rev3, by tandem linker 2 (3 GGGGS) of anti-human PD-1 monoclonal antibody mAb1-25-Hu's scFv2 (VH-linker1-VL, linker1 is 4 GGGGS) with linker 2 (3 GGGGS).

[0119] This invention constructs an anti-PD1×PDL1 bispecific antibody, named anti-PD1×PDL1 Rev4, by tandem linker 2 (3 GGGGS) of the scFv (VL-linker1-VH) of anti-human PD-1 monoclonal antibody mAb1-25-Hu with linker 2 (4 GGGGS) on the N-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody M8.

[0120] This invention constructs an anti-PD1×PDL1 bispecific antibody, named anti-PD1×PDL1 Rev5, by tandemly linking the scFv3 (VL-linker1-VH, linker1 consisting of 4 GGGGS) of anti-human PD-L1 monoclonal antibody M8 to the C-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody mAb1-25-Hu via linker2 (consisting of 3 GGGGS).

[0121] This invention constructs an anti-PD1×PDL1 bispecific antibody, named anti-PD1×PDL1 Rev6, by tandemly linking the scFv4 (VH-linker1-VL, linker1 consisting of 4 GGGGS) of anti-human PD-L1 monoclonal antibody M8 to the C-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody mAb1-25-Hu via linker2 (consisting of 3 GGGGS).

[0122] This invention constructs an anti-PD1×PDL1 bispecific antibody, named anti-PD1×PDL1 Rev7, by tandemly linking the scFv4 (VH-linker1-VL, linker1 consisting of 4 GGGGS) of anti-human PD-L1 monoclonal antibody M8 to the N-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody mAb1-25-Hu via linker2 (consisting of 3 GGGGS).

[0123] This invention constructs an anti-PD1×PDL1 bispecific antibody, named anti-PD1×PDL1 Rev8, by tandemly linking the scFv3 (VL-linker1-VH, linker1 consisting of 4 GGGGS) of anti-human PD-L1 monoclonal antibody M8 to the N-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody mAb1-25-Hu via linker2 (consisting of 3 GGGGS).

[0124] Bispecific antibodies and their corresponding monoclonal antibody heavy and light chain expression vectors were obtained through gene synthesis and conventional molecular cloning methods. The corresponding amino acid sequences are shown in Table 1, where CDR is encoded according to Kabat rules.

[0125] Table 1. Sequence information of the antibodies of the present invention

[0126] SEQ ID NO: sequence name 1 Amino acid sequence of the heavy chain complementarity-determining region H-CDR1 of the anti-PD-L1 monoclonal antibody

[0127] 2 The amino acid sequence of the heavy chain complementarity-determining region H-CDR2 of the anti-PD-L1 monoclonal antibody 3 Amino acid sequence of the heavy chain complementarity-determining region H-CDR3 of the anti-PD-L1 monoclonal antibody 4 Amino acid sequence of L-CDR1, the light chain complementarity-determining region of the anti-PD-L1 monoclonal antibody 5 The amino acid sequence of the light chain complementarity-determining region L-CDR2 of the anti-PD-L1 monoclonal antibody 6 Amino acid sequence of L-CDR3, the light chain complementarity-determining region of the anti-PD-L1 monoclonal antibody 7 Amino acid sequence of the heavy chain complementarity-determining region H-CDR1 of the anti-PD-1 monoclonal antibody 8 The amino acid sequence of the heavy chain complementarity-determining region H-CDR2 of the anti-PD-1 monoclonal antibody 9 The amino acid sequence of the heavy chain complementarity-determining region H-CDR3 of the anti-PD-1 monoclonal antibody 10 Amino acid sequence of L-CDR1, the light chain complementarity-determining region of the anti-PD-1 monoclonal antibody 11 Amino acid sequence of the light chain complementarity-determining region L-CDR2 of the anti-PD-1 monoclonal antibody 12 The amino acid sequence of the light chain complementarity-determining region L-CDR3 of the anti-PD-1 monoclonal antibody 13 The amino acid sequence of the heavy chain variable region of anti-PD-L1 monoclonal antibody 14 The amino acid sequence of the light chain variable region of anti-PD-L1 monoclonal antibody 15 The amino acid sequence of the heavy chain variable region of anti-PD-1 monoclonal antibody 16 The amino acid sequence of the light chain variable region of anti-PD-1 monoclonal antibody 17 The amino acid sequence of the polypeptide chain of anti-PD1×PDL1 BsAb 18 Amino acid sequence of the light chain of anti-PD-L1 monoclonal antibody 19 Amino acid sequence of the heavy chain of anti-PD-L1 monoclonal antibody 20 Amino acid sequence of the heavy chain of anti-PD-1 monoclonal antibody 21 Amino acid sequence of the light chain of anti-PD-1 monoclonal antibody 22 The amino acid sequence of the polypeptide chain of anti-PD1×PDL1 Rev2 23 The amino acid sequence of the polypeptide chain of anti-PD1×PDL1 Rev3 24 The amino acid sequence of the polypeptide chain of anti-PD1×PDL1 Rev4 25 The amino acid sequence of the polypeptide chain of anti-PD1×PDL1 Rev5 26 The amino acid sequence of the polypeptide chain of anti-PD1×PDL1 Rev6 27 The amino acid sequence of the polypeptide chain of anti-PD1×PDL1 Rev7 28 The amino acid sequence of the polypeptide chain of anti-PD1×PDL1 Rev8

[0128] Example 2 Expression and purification of anti-PD1×PDL1 bispecific antibody

[0129] The two polypeptide chains and light chain DNA fragments of the anti-PD1×PDL1 bispecific antibody anti-PD1×PDL1 BsAb were subcloned into the pcDN3.4 vector, and the recombinant plasmid was extracted and co-transfected into CHO cells and / or 293E cells. After culturing the cells for 5-7 days, the culture medium was centrifuged at high speed, filtered through a microporous membrane, and loaded onto a HiTrap MabSelectSuRe column. The protein was eluted with elution buffer containing 100mM citric acid at pH 3.5, and dialyzed to PBS at pH 7.4.

[0130] The purified protein was detected by HPLC. The HPLC-SEC chromatogram of the anti-PD1×PDL1 bispecific antibody BsAb is shown below. Figure 2A As shown, the purity of the bispecific antibody monomer reached over 96%. SDS-PAGE results are as follows. Figure 2B As shown, lanes 1 and 2 represent reduced and non-reduced SDS-PAGE of anti-PD1×PDL1 bispecific antibodies, while lanes 3 and 4 represent reduced and non-reduced SDS-PAGE of anti-PD-L1 monoclonal antibodies. The theoretical molecular weight of the bispecific antibodies is 197 kDa.

[0131] Example 3: Detection of the affinity of anti-PD1×PDL1 double antibody for antigen using enzyme-linked immunosorbent assay (ELISA).

[0132] 3.1 Affinity test with PD-1 antigen

[0133] To detect the affinity of anti-PD1×PDL1 bispecific antibody for PD-1 antigen, PD1-ECD-hFc protein (synthesized from the extracellular domain gene of UniProt (sequence number Q15116) with a signal peptide sequence added to its N-terminus and hFc added to its C-terminus, constructed into expression vectors pcDNA3.4 or pTT5 via EcoRI and HindIII restriction sites, transfected into HEK-293E cells for expression and purification) was diluted to 200 ng / ml in PBS buffer at pH 7.4. 100 μL / well was then added to an ELISA plate and incubated overnight at 4°C. The plate was washed twice the next day with PBST. Each well was then blocked with PBST + 1% BSA and incubated at 37°C for 1 h. The plate was washed twice with PBST. Then, the antibody to be tested, serially diluted with PBS + 1% BSA, was added. Anti-PD-1 monoclonal antibody was used as a positive control, starting at 200 nM and serially diluted 3-fold 12 times. Incubate at 37℃ for 1 hour; wash twice with PBST, add HRP-labeled mouse anti-human Fab secondary antibody, and incubate at 37℃ for another 40 minutes; wash three times with PBST and blot dry, add 100 μL TMB to each well, and incubate at room temperature (20±5℃) in the dark for 5 minutes; add 50 μL 2M H2SO4 stop solution to each well to terminate the substrate reaction, read the OD value at 450 nm using a microplate reader, and perform data analysis using GraphPad Prism, plotting and calculating EC50. 50 .

[0134] Experimental results are as follows Figure 3A As shown, anti-PD-1 monoclonal antibody and anti-PD1×PDL1 bispecific antibody (anti-PD1×PDL1 BsAb) bind to PD-1 antigen in EC2. 50 The values ​​are 0.29 nM and 0.30 nM, respectively, indicating that their affinity is comparable.

[0135] 3.2 Affinity detection with PD-L1 antigen

[0136] To test the affinity of anti-PD1×PDL1 bispecific antibodies for PD-L1 antigen, the PD-L1 extracellular domain gene of PDL1-ECD-His protein (based on the sequence provided by NCBI (NCBI accession number NP_054862.1)) was synthesized using PBS buffer at pH 7.4. A signal peptide sequence was added to its N-terminus, and a 6×His tag was added to its C-terminus. The protein was then constructed into expression vectors pcDNA3.4 or pTT5 via EcoRI and HindIII restriction enzyme sites, respectively. (The antibody was obtained by transfecting HEK-293E cells for expression and purification) was diluted to 1000 ng / ml, and then 100 μL / well was added to the ELISA plate; incubated overnight at 4°C; washed twice with PBST the next day; PBST + 1% BSA was added to each well for blocking, and the plate was blocked at 37°C for 1 h; washed twice with PBST; then the antibody to be tested was added in serially diluted with PBS + 1% BSA, with anti-PD-L1 monoclonal antibody as a positive control, starting at a concentration of 200 nM, and serially diluted 3-fold 12 times. Incubate at 37℃ for 1 hour; wash twice with PBST, add HRP-labeled mouse anti-human Fab secondary antibody, and incubate at 37℃ for another 40 minutes; wash three times with PBST and blot dry, add 100 μL TMB to each well, and incubate at room temperature (20±5℃) in the dark for 5 minutes; add 50 μL 2M H2SO4 stop solution to each well to terminate the substrate reaction, read the OD value at 450 nm using a microplate reader, and perform data analysis using GraphPad Prism, plotting and calculating EC50. 50 .

[0137] Experimental results are as follows Figure 3B As shown, anti-PD-L1 monoclonal antibodies and anti-PD1×PDL1 bispecific antibodies (anti-PD1×PDL1 BsAb) bind to PD-L1 antigen in EC. 50 The values ​​are 0.27 nM and 0.29 nM, respectively, indicating comparable affinity.

[0138] Example 4: Detection of the ability of anti-PD1×PDL1 bispecific ELISA to bind two antigens simultaneously.

[0139] To detect the ability of anti-PD1×PDL1 bispecific antibodies to simultaneously bind to PD-1 and PD-L1 antigens, PD1-ECD-hFc protein was diluted to 200 ng / ml with PBS buffer at pH 7.4, and 100 μL / well was added to each ELISA plate. The plate was incubated overnight at 4°C. The next day, the plate was washed twice with PBST. Each well was then blocked with PBST + 1% BSA and incubated at 37°C for 1 h. The plate was washed twice with PBST. Then, the antibody to be tested, serially diluted with PBS + 1% BSA, was added, starting at 100 nM and serially diluted 3-fold 12 times. The plate was incubated at 37°C for 1 h. After washing twice with PBST, 100 μL / well of 1000 ng / ml PDL1-ECD-His diluted with PBS at pH 7.4 was added to each ELISA plate. Incubate at 37℃ for 1 hour; wash twice with PBST, add secondary antibody HRP-anti-His, and incubate at 37℃ for another 40 minutes; wash three times with PBST and blot dry, add 100 μL TMB to each well, and incubate at room temperature (20±5℃) in the dark for 5 minutes; add 50 μL 2MH2SO4 stop solution to each well to terminate the substrate reaction, read the OD value at 450 nm using a microplate reader, and perform data analysis using GraphPad Prism, plotting and calculating EC50. 50 .

[0140] Experimental results are as follows Figure 4 As shown, the EC50 of the anti-PD1×PDL1 bispecific antibody anti-PD1×PDL1 BsAb 50 The value was 0.36 nM, while anti-PD-1 monoclonal antibody and anti-PD-L1 monoclonal antibody did not have the ability to bind to both antigens simultaneously.

[0141] Example 5: Detection of the binding affinity of anti-PD1×PDL1 bispecific antibodies to target cells

[0142] CHO cells stably transfected with PD-1 on their cell surface were used as target cells. The binding affinity of the anti-PD1×PDL1 bispecific antibody to these cells was determined by flow cytometry. Cells were washed three times with PBS containing 0.5% BSA, centrifuged at 300g for 5 minutes each time, and the supernatant was discarded. Cells were resuspended in PBS containing 0.5% BSA at a cell density of 1×10⁻⁶ cells / mL. 6Cells / mL, 100 μL / well added to 96-well plates. Starting with 200 nM anti-PD1×PDL1 bispecific antibody and positive control anti-PD-1 monoclonal antibody, serially diluted 11 times, 100 μL / well added to each well of the 96-well plate, and incubated at 4°C for 1 h. Cells were washed twice with PBS to remove unbound antibodies. Then, 100 μL of goat anti-human IgG-FITC was added, and the plate was incubated at 4°C for 30 min. After centrifugation at 300g for 5 min, cells were washed twice with PBS to remove unbound secondary antibody. Finally, cells were resuspended in 200 μL of PBS, and the binding affinity of the bispecific antibody to the cells was determined using a Beckman Coulter CytoFLEX flow cytometer. The obtained data were analyzed using GraphPad Prism software. Experimental results are as follows: Figure 5A As shown, the EC50 of anti-PD-1 monoclonal antibody and anti-PD1×PDL1 bispecific antibody anti-PD1×PDL1 BsAb 50 The values ​​are 0.64 nM and 1.43 nM, respectively, indicating that their affinity is comparable.

[0143] N87-PDL1 is a stable cell line constructed in our laboratory by transfecting NCI-N87 cells with PD-L1 using a lentiviral transfection method. N87-PDL1 cells in the logarithmic growth phase were digested with trypsin, washed three times with PBS containing 0.5% BSA, and centrifuged at 300g for 5 minutes each time, discarding the supernatant. The cells were resuspended in PBS with 0.5% BSA at a cell density of 1×10⁻⁶ cells / mL. 6 Cells / mL, 100 μL / well added to a 96-well plate. Anti-PD1×PDL1 bispecific antibody and positive control anti-PD-L1 monoclonal antibody were diluted to 120 nM, serially diluted 11 times, and 100 μL / well added to each well of the 96-well plate, then mixed thoroughly with N87-PDL1 cells. The remaining procedures were the same as above. Experimental results are as follows. Figure 5B As shown, ECG of anti-PD-L1 monoclonal antibody 50 The EC50 of the anti-PD1×PDL1 bispecific antibody BsAb is 0.11 nM. 50 The affinity is 0.15 nM, and the two have similar affinity.

[0144] Example 6: Anti-PD1×PDL1 dual anti-PD1 / PD-L1 activity blocking PD-1 / PD-L1 at the cellular level

[0145] This experiment used Promega's PD-1 / PD-L1 Blockade Bioassay, Propagation model, and methods.

[0146] Stable PD-L1 aAPC / CHO-K1 cell lines in logarithmic growth phase were trypsinized into single cells and transferred to white-bottomed 96-well plates (100 μL / well, 40,000 cells / well). The plates were incubated overnight at 37°C with 5% CO2. Anti-PD1×PDL1 bispecific antibody, anti-PD-L1 monoclonal antibody, and anti-PD-1 monoclonal antibody were diluted to 2× working solution concentrations, starting at 100 nM and increasing in 3-fold increments. Cell densities ranging from 1.4 to 2×10⁻⁶ cells / well were collected. 6 PD1 effector cells with a cell viability of over 95% were digested with trypsin to form 1.25 × 10⁻⁶ cells / mL. 6 Single-cell suspensions of cells / ml were prepared. PD-L1 aAPC / CHO-K1 cells cultured the previous day were collected, the supernatant was discarded, and 40 μl of serially diluted working solution of the antibody to be tested was added; then an equal volume of PD1 effector cells was added. The mixture was incubated at 37°C with 5% CO2 for 6 hours. 80 μl of Bio-Glo assay reagent was added to each well. After incubation at room temperature for 10 minutes, luminescence was read using a Spectramax i3. Data analysis, plotting, and IC50 calculation were performed using GraphPad Prism. 50 .

[0147] Experimental results are as follows Figure 6 As shown, the IC50 values ​​of anti-PD1×PDL1 BsAb, anti-PD-L1 monoclonal antibody, and anti-PD-1 monoclonal antibody are... 50 The IC50 values ​​for the bispecific antibody anti-PD1×PDL1 BsAb and the anti-PD-L1 monoclonal antibody were 0.24 nM, 0.42 nM, and 1.60 nM, respectively. 50 The IC50 of anti-PD-1 monoclonal antibodies is close to that of anti-PD-1 monoclonal antibodies. 50 It's on the larger side, but its high platform is a bit high.

[0148] Anti-PD1×PDL1 rev2, rev3, rev4, rev5, rev6, rev7, rev8 blocked the activity of the PD-1 / PD-L1 signaling pathway on cells, and the results were as follows: Figure 7A , 7B As shown in Figure 7C, anti-PD1×PDL1 rev2 was significantly weaker than the positive control anti-PD-1 monoclonal antibody; the activities of anti-PD1×PDL1 rev3 and rev6 were comparable to the positive control anti-PD-L1 monoclonal antibody; the activities of anti-PD1×PDL1 rev4 and rev5 were slightly weaker than the positive control anti-PD-L1 monoclonal antibody; and the activities of anti-PD1×PDL1 rev7 and rev8 were significantly weaker than the positive control anti-PD-L1 monoclonal antibody.

[0149] Example 7: Pharmacokinetic Study of Anti-PD1×PDL1 Bispecific Antibody

[0150] Four SD rats, each weighing approximately 200g, were used in each group. Each rat was injected with 2mg of the antibody prepared in Example 2 via tail vein injection. Blood samples were collected at the following time points after administration: 0h, 3h, 24h, 48h, 96h, 168h, 336h, and 504h. Blood was collected from the orbital cavity, and after natural clotting, the blood was centrifuged at 8000rpm / min to obtain serum.

[0151] The serum drug concentration of anti-PD1×PDL1 bispecific antibody BsAb was detected using the following method:

[0152] 1) Protein A coating of ELISA plates to detect the antibody Fab fragment. Coat with protein A at 100 ng / well, incubate overnight at 4°C; wash twice with PBST the next day, then block with PBS + 2% BSA at 37°C for 2 hours. Wash twice with PBST. Anti-PD1×PDL1 bispecific antibody standards were added to 1000 ng / mL, serially diluted 2-fold in 12 increments. Rat serum samples were diluted 1000-4000 times. Both groups of samples were added to the blocked ELISA plate and incubated for 1 hour; after washing twice with PBST, HRP-labeled mouse anti-human Fab antibody was added, and the plate was incubated at 37°C for 30 minutes; after washing three times with PBST, residual droplets were patted dry on absorbent paper, 100 μL of TMB was added to each well, and the plate was incubated at room temperature (20±5°C) in the dark for 5 minutes; 50 μL of 2M H2SO4 stop solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm using a microplate reader. The calculated half-life is 275.6 hours.

[0153] 2) Coat ELISA plates with PD1-ECD-hFc, 20 ng / well. The remaining steps are the same as above. The calculated half-life is 303.5 hours.

[0154] 3) Coat the ELISA plate with PDL1-ECD-hFc, 100 ng / well. The remaining steps are the same as above. The calculated half-life is 307 hours.

[0155] As shown in Table 2-4, the calculated half-life results from the three sets of ELISA results are similar, all around 300 hours, indicating that the analysis data are reliable.

[0156] Table 2. Results of protein A plate ELISA

[0157] experimental group Half-life (hours) Maximum concentration (μg / mL) 1 247.50433 170 2 316.48482 166 3 273.29592 150 4 265.20196 164

[0158] Table 3. Results of PD1-ECD-hFc plate ELISA

[0159] experimental group Half-life (hours) Maximum concentration (μg / mL) 1 299.46777 196 2 348.73796 202 3 295.3837 170 4 270.42874 174

[0160] Table 4. Results of PDL1-ECD-hFc plate ELISA

[0161] experimental group Half-life (hours) Maximum concentration (μg / mL) 1 280.15463 166 2 336 152 3 320.97912 146

[0162] 4 291.3996 166

[0163] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention. sequence list <110> 3SBio (Shanghai) Co., Ltd. <120> A bispecific antibody against PD1×PDL1 <150> 2020104876195 <151> 2020-06-02 <160> 28 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <400> 1 Ser Tyr Gly Val His 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <400> 2 Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <400> 3 Gln Leu Gly Leu Arg Ala Met Asp Tyr 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <400> 4 Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <400> 5 Tyr Ala Ser Gln Ser Phe Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <400> 6 Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <400> 7 Ser Tyr Asp Met Ser 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <400> 8 Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val Lys 1 5 10 15 Gly <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <400> 9 Pro Tyr Gly Gly Tyr Phe Asp Val 1 5 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <400> 10 Arg Ala Ser Gln Ser Ile Ser Asn Phe Leu His 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <400> 11 Tyr Ala Ser Gln Ser Ile Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Gln Gln Ser Asn Ser Trp Pro His Thr 1 5 <210> 13 <211> 117 <212> PRT <213> Artificial Sequence <400> 13 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 15 <211> 117 <212> PRT <213> Artificial Sequence <400> 15 Glu Val Lys 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 Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence <400> 16 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 706 <212> PRT Artificial Sequence <400> 17 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile 450 455 460 Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg 465 470 475 480 Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe Leu His 485 490 495 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Lys Tyr 500 505 510 Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly 515 520 525 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp 530 535 540 Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His Thr Phe 545 550 555 560 Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly 565 570 575 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys 580 585 590 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 595 600 605 Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser 610 615 620 Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ala Thr Ile 625 630 635 640 Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg 645 650 655 Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr Leu Gln Met 660 665 670 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Ser Pro 675 680 685 Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 690 695 700 Ser Ser 705 <210> 18 <211> 214 <212> PRT <213> Artificial Sequence <400> 18 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 19 <211> 447 <212> PRT <213> Artificial Sequence <400> 19 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 20 <211> 444 <212> PRT <213> Artificial Sequence <400> 20 Glu Val Lys 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 Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 21 <211> 214 <212> PRT <213> Artificial Sequence <400> 21 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His 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> 22 <211> 706 <212> PRT <213> Artificial Sequence <400> 22 Glu Val Lys 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 Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser 130 135 140 Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys 145 150 155 160 Arg Ala Ser Gln Ser Ile Ser Asn Phe Leu His Trp Tyr Gln Gln Lys 165 170 175 Pro Gly Gln Ala Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile 180 185 190 Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205 Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe 210 215 220 Cys Gln Gln Ser Asn Ser Trp Pro His Thr Phe Gly Gln Gly Thr Lys 225 230 235 240 Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys 260 265 270 Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu 275 280 285 Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu 290 295 300 Glu Trp Ile Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro 305 310 315 320 Ser Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln 325 330 335 Val Ser Phe Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr 340 345 350 Tyr Cys Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln 355 360 365 Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 370 375 380 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 385 390 395 400 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 405 410 415 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 420 425 430 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 435 440 445 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 450 455 460 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 465 470 475 480 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 485 490 495 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 500 505 510 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 515 520 525 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 530 535 540 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 545 550 555 560 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 565 570 575 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 580 585 590 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 595 600 605 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 610 615 620 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 625 630 635 640 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 645 650 655 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 660 665 670 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 675 680 685 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 690 695 700 Gly Lys 705 <210> 23 <211> 706 <212> PRT <213> Artificial Sequence <400> 23 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 450 455 460 Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 465 470 475 480 Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Asp Met 485 490 495 Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ala Thr 500 505 510 Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val Lys Gly 515 520 525 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr Leu Gln 530 535 540 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Ser 545 550 555 560 Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr 565 570 575 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 580 585 590 Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala 595 600 605 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala 610 615 620 Ser Gln Ser Ile Ser Asn Phe Leu His Trp Tyr Gln Gln Lys Pro Gly 625 630 635 640 Gln Ala Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly 645 650 655 Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 660 665 670 Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln 675 680 685 Gln Ser Asn Ser Trp Pro His Thr Phe Gly Gln Gly Thr Lys Val Glu 690 695 700 Ile Lys 705 <210> 24 <211> 706 <212> PRT <213> Artificial Sequence <400> 24 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 115 120 125 Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Asp 145 150 155 160 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ala 165 170 175 Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr Leu 195 200 205 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala 210 215 220 Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys 260 265 270 Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu 275 280 285 Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu 290 295 300 Glu Trp Ile Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro 305 310 315 320 Ser Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln 325 330 335 Val Ser Phe Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr 340 345 350 Tyr Cys Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln 355 360 365 Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 370 375 380 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 385 390 395 400 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 405 410 415 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 420 425 430 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 435 440 445 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 450 455 460 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 465 470 475 480 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 485 490 495 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 500 505 510 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 515 520 525 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 530 535 540 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 545 550 555 560 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 565 570 575 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 580 585 590 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 595 600 605 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 610 615 620 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 625 630 635 640 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 645 650 655 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 660 665 670 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 675 680 685 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 690 695 700 Gly Lys 705 <210> 25 <211> 703 <212> PRT <213> Artificial Sequence <400> 25 Glu Val Lys 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 Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly 435 440 445 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr 450 455 460 Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile 465 470 475 480 Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln 485 490 495 Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln 500 505 510 Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 515 520 525 Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr 530 535 540 Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly 545 550 555 560 Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln 580 585 590 Ser Gly Gly Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys 595 600 605 Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg 610 615 620 Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu Ile Trp Ser Gly 625 630 635 640 Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser 645 650 655 Arg Asp Thr Ser Lys Asn Gln Val Ser Phe Lys Ile Ser Ser Leu Thr 660 665 670 Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Gly Leu Arg 675 680 685 Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 690 695 700 <210> 26 <211> 703 <212> PRT <213> Artificial Sequence <400> 26 Glu Val Lys 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 Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly 435 440 445 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln 450 455 460 Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr 465 470 475 480 Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val 485 490 495 Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu Ile Trp Ser 500 505 510 Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile 515 520 525 Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe Lys Ile Ser Ser Leu 530 535 540 Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Gly Leu 545 550 555 560 Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 580 585 590 Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser 595 600 605 Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 610 615 620 Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro 625 630 635 640 Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser 645 650 655 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 660 665 670 Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 675 680 685 Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 690 695 700 <210> 27 <211> 703 <212> PRT <213> Artificial Sequence <400> 27 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser 130 135 140 Pro Asp Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys 165 170 175 Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe 180 185 190 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205 Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys 225 230 235 240 Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 260 265 270 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe 275 280 285 Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu 290 295 300 Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro 305 310 315 320 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 325 330 335 Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 340 345 350 Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln 355 360 365 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 370 375 380 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 385 390 395 400 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 405 410 415 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 420 425 430 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 435 440 445 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 450 455 460 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 465 470 475 480 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 485 490 495 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 500 505 510 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 515 520 525 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 530 535 540 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 545 550 555 560 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 565 570 575 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 580 585 590 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 595 600 605 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 610 615 620 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 625 630 635 640 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 645 650 655 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 660 665 670 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 675 680 685 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 690 695 700 <210> 28 <211> 703 <212> PRT <213> Artificial Sequence <400> 28 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln Ser 130 135 140 Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly 145 150 155 160 Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 165 170 175 Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser 180 185 190 Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe Lys 195 200 205 Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 225 230 235 240 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 260 265 270 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe 275 280 285 Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu 290 295 300 Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro 305 310 315 320 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 325 330 335 Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 340 345 350 Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln 355 360 365 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 370 375 380 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 385 390 395 400 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 405 410 415 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 420 425 430 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 435 440 445 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 450 455 460 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 465 470 475 480 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 485 490 495 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 500 505 510 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 515 520 525 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 530 535 540 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 545 550 555 560 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 565 570 575 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 580 585 590 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 595 600 605 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 610 615 620 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 625 630 635 640 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 645 650 655 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 660 665 670 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 675 680 685 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 690 695 700

Claims

1. A bispecific antibody against PD1×PDL1, characterized in that, The bispecific antibody comprises two polypeptide chains and two light chains: Each polypeptide chain, from its N-terminus to its C-terminus, sequentially comprises VH-PDL1—CH1—CH2—CH3—linker2—VL-PD1—linker1—VH-PD1 or VH-PDL1—CH1—CH2—CH3—linker2—VH-PD1—linker1—VL-PD1 or VL-PD1—linker1—VH-PD1—linker2—VH-PDL1—CH1—CH2—CH3 or VH-PD1—linker1—VL-PD1—linker2—VH-PDL1—CH1—CH2—CH3, and each light chain, from its N-terminus to its C-terminus, sequentially comprises VL-PDL1—CL; or Each polypeptide chain, from its N-terminus to its C-terminus, sequentially comprises VH-PD1—CH1—CH2—CH3—linker2—VL-PDL1—linker1—VH-PDL1 or VH-PD1—CH1—CH2—CH3—linker2—VH-PDL1—linker1—VL-PDL1 or VL-PDL1—linker1—VH-PDL1—linker2—VH-PD1—CH1—CH2—CH3 or VH-PDL1—linker1—VL-PDL1—linker2—VH-PD1—CH1—CH2—CH3, and each light chain, from its N-terminus to its C-terminus, sequentially comprises VL-PD1—CL. Wherein, VH-PDL1 is a heavy chain variable region that binds PD-L1, CH1-CH2-CH3 is a heavy chain constant region, VL-PD1 is a light chain variable region that binds PD-1, VH-PD1 is a heavy chain variable region that binds PD-1, VL-PDL1 is a light chain variable region that binds PD-L1, and CL is a light chain constant region; the amino acid sequences of the heavy chain CDR1-3 of VH-PDL1 are shown in SEQ ID NO: 1-3; the amino acid sequences of the light chain CDR1-3 of VL-PDL1 are shown in SEQ ID NO: 4-6; the amino acid sequences of the heavy chain CDR1-3 of VH-PD1 are shown in SEQ ID NO: 7-9; and the amino acid sequences of the light chain CDR1-3 of VL-PD1 are shown in SEQ ID NO: 10-12.

2. The bispecific antibody against PD1×PDL1 according to claim 1, characterized in that, The VH-PDL1 forms a specific antigen-binding site for PD-L1 with the VL-PDL1, and the VL-PD1 forms a specific antigen-binding site for PD-1 with the VH-PD1.

3. The bispecific antibody against PD1×PDL1 according to claim 1, characterized in that, The amino acid sequence of the VH-PDL1 is shown in SEQ ID NO: 13, and / or the amino acid sequence of the VL-PDL1 is shown in SEQ ID NO: 14, and / or the amino acid sequence of the VH-PD1 is shown in SEQ ID NO: 15, and / or the amino acid sequence of the VL-PD1 is shown in SEQ ID NO:

16.

4. The bispecific antibody against PD1×PDL1 according to claim 1, characterized in that, Linker2 consists of 3 G4S, and linker1 consists of 4 G4S.

5. The bispecific antibody against PD1×PDL1 according to claim 1, characterized in that, The heavy chain constant region includes the IgG1, IgG2, IgG3, and IgG4 heavy chain constant regions, and the light chain constant region includes the κ or λ light chain constant region.

6. The bispecific antibody against PD1×PDL1 according to claim 1, characterized in that, The amino acid sequence of the polypeptide chain is as shown in SEQ ID NO: 17 or SEQ ID NO: 22 or SEQ ID NO: 23 or SEQ ID NO: 24, and / or, the amino acid sequence of the light chain is as shown in SEQ ID NO: 18; or the amino acid sequence of the polypeptide chain is as shown in SEQ ID NO: 25 or SEQ ID NO: 26 or SEQ ID NO: 27 or SEQ ID NO: 28, and / or, the amino acid sequence of the light chain is as shown in SEQ ID NO:

21.

7. An isolated polynucleotide, characterized in that, The nucleotide encodes the bispecific antibody as described in any one of claims 1-6.

8. An expression carrier, characterized in that, The expression vector comprises the polynucleotide as described in claim 7.

9. A host cell, characterized in that, The host cell includes the expression vector as described in claim 8.

10. The method for preparing the bispecific antibody according to any one of claims 1-6, characterized in that, The preparation method includes the following steps: a) Under expression conditions, the host cells as described in claim 9 are cultured to express the bispecific antibody; b) Isolate and purify the bispecific antibody obtained in a).

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the bispecific antibody as described in any one of claims 1-6 and a pharmaceutically acceptable carrier.

12. Use of the bispecific antibody of any one of claims 1-6 or the pharmaceutical composition of claim 11 in the preparation of a medicament for treating cancer, wherein the cancer is selected from melanoma, renal cell carcinoma, lung cancer, liver cancer, gastric cancer, lymphoma, breast cancer, colorectal cancer, leukemia, prostate cancer, and bone marrow cancer.

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