An anti-VEGF / PD-L1 bispecific antibody and its use

Abstract

The present invention relates to the use of an anti-PD-L1 / VEGF bispecific antibody. Specifically, the present invention provides an anti-PD-L1 / VEGF bispecific antibody, which has the function of blocking the binding of PD-L1 to the receptor PD-1 and the function of blocking the binding of VEGF to its receptor. The bispecific antibody can play a better tumor-killing effect by activating the immune system and blocking tumor angiogenesis at the same time. In addition, the present invention provides the amino acid sequence of the bispecific antibody, the corresponding expression vector and the host cell capable of expressing the bispecific antibody, as well as the bispecific antibody production method of the present invention.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an anti-VEGF / PD-L1 bispecific antibody and uses thereof. Background Art

[0002] Programmed death 1 ligand 1 (PD-L1), also known as CD274, is a member of the B7 family and a ligand for PD-1. PD-L1 is a type I transmembrane protein with a total of 290 amino acids, including 1 IgV-like region, 1 IgC-like region, 1 transmembrane hydrophobic region, and 1 intracellular region composed of 30 amino acids. Unlike other B7 family molecules, PD-L1 has the function of negatively regulating immune responses. Studies have found that PD-L1 is mainly expressed in activated T cells, B cells, macrophages, and dendritic cells. In addition to lymphocytes, PD-L1 is also expressed in endothelial cells of various other tissues such as thymus, heart, placenta, etc., as well as various non-lymphoid systems such as melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, head and neck cancer, etc. PD-L1 has a wide range of functions in regulating autoreactive T and B cells and immune tolerance, and plays a role in T and B cell responses in peripheral tissues. High expression of PD-L1 on tumor cells is associated with poor prognosis in cancer patients.

[0003] Vascular endothelial growth factor, also known as vascular permeability factor (VPF) or vasculotropin, was first isolated from tumor cells. It is a dimeric glycoprotein molecule that can bind to heparin and can specifically act on vascular endothelial cells. It is a mitogen specific for vascular endothelial cells. It has the effects of increasing the permeability of venules and small veins, promoting the division and proliferation of vascular endothelial cells, and inducing angiogenesis. VEGF is involved in the onset and progression of many angiogenesis-dependent diseases, including cancer, certain inflammatory diseases, and diabetic retinopathy. Therefore, VEGF is an important target in the study of anti-tumor drugs.

[0004] Most of the antibody drugs currently on the market are monoclonal antibodies. Therapeutic monoclonal antibodies have been used to treat cancer, autoimmune diseases, inflammation and other diseases, most of which are specific to one target. However, patients may develop drug resistance or no response to monoclonal antibody treatment. In addition, some diseases are affected by many factors in the body, including different signaling pathways, different cytokines and receptor regulatory mechanisms, etc. Single-target immunotherapy does not seem to be enough to destroy cancer cells. Therefore, it is necessary to combine different drugs or use a multi-target strategy of multi-specific antibodies to achieve this.

[0005] Although bifunctional antibodies are the direction of antibody drug research and development, they face many challenges, such as preclinical evaluation models, low expression levels, poor stability, complex processes, and large differences in quality control. Therefore, the research and development of bifunctional antibodies has always been difficult.

[0006] Therefore, there is an urgent need in the art to develop an anti-tumor bispecific antibody that has good specificity, good efficacy and is easy to prepare. Summary of the invention

[0007] The purpose of the present invention is to provide an anti-VEGF / PD-L1 bispecific antibody with good specificity, good efficacy and easy preparation.

[0008] In a first aspect of the present invention, a bispecific antibody is provided, comprising:

[0009] (a) an anti-PD-L1 single domain antibody; and

[0010] (b) Anti-VEGF antibodies or elements.

[0011] In another preferred embodiment, the bispecific antibody comprises 1-3 anti-PD-L1 single domain antibodies, preferably, comprises 1 or 2 anti-PD-L1 single domain antibodies.

[0012] In another preferred embodiment, the PD-L1 single domain antibody can block the interaction between PD-1 and PD-L1.

[0013] In another preferred embodiment, the bispecific antibody further comprises an Fc segment.

[0014] In another preferred embodiment, the Fc segment of the bispecific antibody is selected from the following group: human IgG domain, CH1+CL1 domain, or a combination thereof.

[0015] In another preferred embodiment, the human IgG domain is a modified mutant IgG domain, preferably a LALA mutant IgG domain.

[0016] In another preferred embodiment, the bispecific antibody comprises polypeptides having structures as shown in Formula I and Formula II.

[0017] A1-L1-CH-L2-B (Formula I)

[0018] A2-L3-CL (Formula II)

[0019] in,

[0020] A1 is the heavy chain variable region VH of the anti-VEGF antibody;

[0021] A2 is the light chain variable region VL of the anti-VEGF antibody;

[0022] B is an anti-PD-L1 single-domain antibody;

[0023] L1, L2 and L3 are each independently a no or a connection element;

[0024] CH is a human IgG heavy chain constant region CH (preferably a LALA mutant);

[0025] CL is the human kappa light chain constant region CL; and

[0026] “-” is a peptide bond;

[0027] And wherein, the polypeptide represented by formula I and the polypeptide represented by formula II form a heterodimer through disulfide bonds.

[0028] In another preferred embodiment, the bispecific antibody is a polypeptide having a structure as shown in Formula III or Formula IV,

[0029] A3-L4-Fc-L5-B (Formula III)

[0030] B-L6-Fc-L7-A3 (Formula IV)

[0031] in,

[0032] A3 is the domain that can bind to VEGF and block its activity;

[0033] B is an anti-PD-L1 single-domain antibody;

[0034] L4, L5, L6 and L7 are each independently a no or a connection element;

[0035] Fc is the Fc segment of human IgG (preferably a LALA mutant); and

[0036] “-” is a peptide bond.

[0037] In another preferred example, the anti-VEGF heavy chain variable region VH has an amino acid sequence of a heavy chain variable region derived from bevacizumab, and its amino acid sequence is shown in SEQ ID NO: 3, or has a sequence identity of ≥85% (preferably 90%, more preferably 95%) to the sequence shown in SEQ ID NO: 3.

[0038] In another preferred example, the anti-VEGF light chain variable region VL has an amino acid sequence of a light chain variable region derived from bevacizumab, and its amino acid sequence is as shown in SEQ ID NO: 9, or has a sequence identity of ≥85% (preferably 90%, more preferably 95%) to the sequence shown in SEQ ID NO: 9.

[0039] In another preferred example, the anti-VEGF heavy chain variable region VH has an amino acid sequence of a heavy chain variable region derived from Ranibizumab, and its amino acid sequence is shown in SEQ ID NO: 13, or has a sequence identity of ≥85% (preferably 90%, more preferably 95%) to the sequence shown in SEQ ID NO: 13.

[0040] In another preferred example, the anti-VEGF light chain variable region VL has an amino acid sequence of a light chain variable region derived from Ranibizumab, and its amino acid sequence is as shown in SEQ ID NO: 15, or has a sequence identity of ≥85% (preferably 90%, more preferably 95%) to the sequence shown in SEQ ID NO: 15.

[0041] In another preferred example, the amino acid sequence of the domain that can bind to VEGF and block its activity is as shown in SEQ ID NO: 17, or has a sequence identity of ≥ 85% (preferably 90%, more preferably 95%) to the sequence shown in SEQ ID NO: 17.

[0042] In another preferred example, the amino acid sequence of the anti-PD-L1 single domain antibody is as shown in SEQ ID NO: 5, or has a sequence identity of ≥85% (preferably 90%, more preferably 95%) to the sequence shown in SEQ ID NO: 5.

[0043] In another preferred example, the human IgG heavy chain constant region CH has a LALA mutation, and its amino acid sequence is as shown in SEQ ID NO:4, or has a sequence identity of ≥85% (preferably 90%, more preferably 95%) to the sequence shown in SEQ ID NO:4.

[0044] In another preferred example, the amino acid sequence of the human κ light chain constant region CL is as shown in SEQ ID NO: 10, or has a sequence identity of ≥85% (preferably 90%, more preferably 95%) to the sequence shown in SEQ ID NO: 10.

[0045] In another preferred example, the Fc segment of human IgG has a LALA mutation, and its amino acid sequence is as shown in SEQ ID NO: 18, or has a sequence identity of ≥85% (preferably 90%, more preferably 95%) to the sequence shown in SEQ ID NO: 18.

[0046] In another preferred embodiment, the sequence of the linker element is (G 4 S) n , wherein n is a positive integer (eg, 1, 2, 3, 4, 5 or 6), preferably, n is 2 or 4.

[0047] In another preferred example, the amino acid sequence of the linker element is as shown in SEQ ID NO: 6 or 7, or has a sequence identity of ≥ 85% (preferably 90%, more preferably 95%) to the sequence shown in SEQ ID NO: 6 or 7.

[0048] In another preferred embodiment, the bispecific antibody contains a polypeptide having a structure as shown in Formula I and Formula II, wherein the amino acid sequence of the polypeptide having a structure as shown in Formula I is shown in SEQ ID NO: 1, and the amino acid sequence of the polypeptide having a structure as shown in Formula II is shown in SEQ ID NO: 8. (i.e., Ava-2GS-NSD)

[0049] In another preferred embodiment, the bispecific antibody contains a polypeptide having a structure as shown in Formula I and Formula II, wherein the amino acid sequence of the polypeptide having a structure as shown in Formula I is shown in SEQ ID NO: 2, and the amino acid sequence of the polypeptide having a structure as shown in Formula II is shown in SEQ ID NO: 8. (i.e., Ava-4GS-NSD)

[0050] In another preferred embodiment, the bispecific antibody contains a polypeptide having a structure as shown in Formula I and Formula II, wherein the amino acid sequence of the polypeptide having a structure as shown in Formula I is shown in SEQ ID NO: 11, and the amino acid sequence of the polypeptide having a structure as shown in Formula II is shown in SEQ ID NO: 14. (i.e., Luc-2GS-NSD)

[0051] In another preferred embodiment, the bispecific antibody contains a polypeptide having a structure as shown in Formula I and Formula II, wherein the amino acid sequence of the polypeptide having a structure as shown in Formula I is shown in SEQ ID NO: 12, and the amino acid sequence of the polypeptide having a structure as shown in Formula II is shown in SEQ ID NO: 14. (i.e., Luc-4GS-NSD)

[0052] In another preferred embodiment, the bispecific antibody is a polypeptide having a structure as shown in Formula III, and its amino acid sequence is shown in SEQ ID NO: 16. (i.e., NSD-Elyea)

[0053] In another preferred embodiment, the bispecific antibody is a polypeptide having a structure as shown in Formula III, and its amino acid sequence is shown in SEQ ID NO: 19. (i.e., Elyea-NSD)

[0054] In another preferred embodiment, the bispecific antibody contains polypeptides of structures shown in Formula I and Formula II at the same time, and the polypeptide shown in Formula I and the polypeptide shown in Formula II form a heterodimer i through disulfide bonds;

[0055] Furthermore, the heterodimers i form homodimers ii through disulfide bonds between CH domains.

[0056] In another preferred embodiment, the bispecific antibody is a polypeptide having a structure as shown in Formula III or Formula IV, and the polypeptides having a structure as shown in Formula III or Formula IV form homodimers through disulfide bonds between Fc segments.

[0057] In the second aspect of the present invention, an isolated polynucleotide is provided, wherein the polynucleotide encodes the bispecific antibody according to the first aspect of the present invention.

[0058] In another preferred example, when the bispecific antibody contains polypeptides having structures as shown in Formula I and Formula II, in the polynucleotide, the ratio of the polynucleotide sequence encoding the polypeptide having the structure shown in Formula I to the polynucleotide sequence encoding the polypeptide having the structure shown in Formula II is 1:1.

[0059] In the third aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide as described in the second aspect of the present invention.

[0060] In another preferred embodiment, the vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof; preferably, the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0061] In a fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector according to the third aspect of the present invention, or the polynucleotide according to the second aspect of the present invention is integrated into its genome;

[0062] Alternatively, the host cell expresses the bispecific antibody as described in the first aspect of the present invention.

[0063] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0064] In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.

[0065] In a fifth aspect of the present invention, a method for producing the bispecific antibody according to the first aspect of the present invention is provided, comprising the steps of:

[0066] (a) culturing the host cell according to the fourth aspect of the present invention under suitable conditions to obtain a culture containing the bispecific antibody; and

[0067] (b) purifying and / or isolating the culture obtained in step (a) to obtain the bispecific antibody.

[0068] In another preferred embodiment, the purification can be performed by protein A affinity column purification and separation to obtain the target antibody.

[0069] In another preferred embodiment, the purity of the target antibody after purification and separation is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.

[0070] In a sixth aspect of the present invention, an immunoconjugate is provided, wherein the immunoconjugate comprises:

[0071] (a) the bispecific antibody according to the first aspect of the present invention; and

[0072] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0073] In another preferred embodiment, the radioactive nuclides include:

[0074] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0075] (ii) therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, or a combination thereof.

[0076] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0077] In another preferred embodiment, the drug is a cytotoxic drug.

[0078] In another preferred embodiment, the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0079] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0080] In another preferred embodiment, the toxin is selected from the following group:

[0081] auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotonin, calicheamicin, saponin, officinalis) inhibitors, glucocorticoids, or a combination thereof.

[0082] In another preferred embodiment, the coupling moiety is a detectable label.

[0083] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and chemotherapeutic agents (such as cisplatin).

[0084] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) bispecific antibody as described in the first aspect of the present invention.

[0085] In another preferred embodiment, the multivalency refers to a bispecific antibody as described in the first aspect of the present invention containing multiple repeats in the amino acid sequence of the immunoconjugate.

[0086] In the seventh aspect of the present invention, there is provided a use of the bispecific antibody as described in the first aspect of the present invention, or the immunoconjugate as described in the sixth aspect of the present invention, for preparing a medicament, a reagent, a detection plate or a kit; wherein the reagent, the detection plate or the kit is used for: detecting PD-L1 and / or VEGF in a sample; wherein the medicament is used for treating or preventing a tumor expressing PD-L1 (i.e., PD-L1 positive) or a tumor expressing VEGF.

[0087] In another preferred embodiment, the conjugated portion of the immunoconjugate is a diagnostic isotope.

[0088] In another preferred embodiment, the reagent is one or more reagents selected from the following group: isotope tracers, contrast agents, flow cytometry detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and imaging agents.

[0089] In another preferred embodiment, the reagent for detecting PD-L1 and / or VEGF in the sample is a contrast agent for (in vivo) detecting PD-L1 and / or VEGF molecules.

[0090] In another preferred embodiment, the detection is in vivo detection or in vitro detection.

[0091] In another preferred embodiment, the detection includes flow cytometry detection and cell immunofluorescence detection.

[0092] In another preferred embodiment, the agent is used to block the interaction between PD-1 and PD-L1, and simultaneously block the interaction between VEGF and VEGFR.

[0093] In another preferred embodiment, the tumor includes but is not limited to: acute myeloid leukemia, chronic myeloid leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, colon cancer, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor.

[0094] In an eighth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0095] (i) the bispecific antibody according to the first aspect of the present invention, or the immunoconjugate according to the sixth aspect of the present invention; and

[0096] (ii) a pharmaceutically acceptable carrier.

[0097] In another preferred embodiment, the conjugated portion of the immunoconjugate is a drug, a toxin, and / or a therapeutic isotope.

[0098] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating tumors, such as cytotoxic drugs.

[0099] In another preferred embodiment, the other drugs for treating tumors include paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, and pembrolizumab.

[0100] In another preferred embodiment, the drug is used to block the interaction between PD-1 and PD-L1, and simultaneously block the interaction between VEGF and VEGFR.

[0101] In another preferred embodiment, the pharmaceutical composition is used to block the PD-1 / PD-L1 and / or VEGF / VEGFR signaling pathways.

[0102] In another preferred embodiment, the pharmaceutical composition is used to treat tumors expressing PD-L1 protein (ie, PD-L1 positive) and / or expressing VEGF protein (ie, VEGF positive).

[0103] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0104] In another preferred embodiment, the pharmaceutical composition is used to prepare drugs for preventing and treating tumors.

[0105] In the ninth aspect of the present invention, one or more uses of the bispecific antibody according to the first aspect of the present invention selected from the following group are provided, including:

[0106] (i) for detecting human PD-L1 molecules and / or VEGF molecules; (ii) for flow cytometry detection; (iii) for cell immunofluorescence detection; (iv) for treating tumors; (v) for tumor diagnosis; (vi) for blocking the interaction between PD-1 and PD-L1; and (vii) for blocking the interaction between VEGF and VEGFR.

[0107] In another preferred embodiment, the tumor is a tumor that expresses PD-L1 protein (ie, PD-L1 positive) and / or expresses VEGF protein (ie, VEGF positive).

[0108] In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

[0109] In the tenth aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has: (i) the bispecific antibody as described in the first aspect of the present invention; and (ii) an optional tag sequence that assists expression and / or purification.

[0110] In another preferred embodiment, the tag sequence includes a 6His tag, a HA tag and an Fc tag.

[0111] In another preferred embodiment, the recombinant protein specifically binds to PD-L1 and / or VEGF.

[0112] In the eleventh aspect of the present invention, a method for detecting PD-L1 and / or VEGF in a sample is provided, the method comprising the steps of: (1) contacting the sample with the bispecific antibody as described in the first aspect of the present invention; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PD-L1 and / or VEGF in the sample.

[0113] In the twelfth aspect of the present invention, a method for treating a disease is provided, the method comprising: administering to a subject in need thereof the bispecific antibody as described in the first aspect of the present invention, the immunoconjugate as described in the sixth aspect of the present invention, or the pharmaceutical composition as described in the eighth aspect of the present invention.

[0114] In another preferred embodiment, the subject includes a mammal, preferably a human.

[0115] In the thirteenth aspect of the present invention, a PD-L1 and / or VEGF detection reagent is provided, wherein the detection reagent comprises the immunoconjugate as described in the sixth aspect of the present invention and a carrier acceptable in detection.

[0116] In another preferred embodiment, the conjugated portion of the immunoconjugate is a diagnostic isotope.

[0117] In another preferred embodiment, the test-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0118] In another preferred embodiment, the detection reagent is one or more reagents selected from the following group: isotope tracers, contrast agents, flow cytometry detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and imaging agents.

[0119] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0120] In another preferred embodiment, the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, buccal preparation, inhaler).

[0121] In the fourteenth aspect of the present invention, a kit for detecting PD-L1 and / or VEGF is provided, the kit containing the immunoconjugate as described in the sixth aspect of the present invention or the detection reagent as described in the thirteenth aspect of the present invention, and instructions.

[0122] In another preferred embodiment, the instructions state that the kit is used for non-invasively detecting the expression of PD-L1 and / or VEGF in a subject to be tested.

[0123] In another preferred embodiment, the kit is used for detecting tumors expressing PD-L1 protein (ie, PD-L1 positive) and / or expressing VEGF protein (ie, VEGF positive).

[0124] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 Schematic diagram of the bispecific antibody structure is shown.

[0126] Figure 2 It was shown that the bispecific antibody of the present invention can bind to the human PD-L1 protein on the cell surface.

[0127] Figure 3 It was shown that the specific antibody of the present invention can block the binding between VEGF protein and VEGFR protein.

[0128] Figure 4 It was shown that the bispecific antibodies of the present invention can effectively activate T cells.

[0129] FIG5 shows that the bispecific antibody of the present invention has good thermal stability, and the antigen binding activity of the accelerated stability sample is similar to that of the original sample.

[0130] Figure 6 It was shown that the bispecific antibody of the present invention can effectively inhibit tumor growth in mice. DETAILED DESCRIPTION

[0131] After extensive and in-depth research and a large number of screenings, the inventors have developed an anti-PD-L1 / VEGF bispecific antibody for the first time. The experimental results show that the bispecific antibody of the present invention can be highly specific to human PD-L1 protein, can inhibit the PD-1 / PD-L1 pathway on the basis of targeting and neutralizing VEGF in the tumor microenvironment, can restore the activity of T cells, enhance the immune response, and thus more effectively improve the effect of inhibiting the occurrence and development of tumors. On this basis, the present invention was completed.

[0132] the term

[0133] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0134] Bispecific Antibodies

[0135] As used herein, the terms "bispecific antibody of the present invention", "bispecific antibody of the present invention", and "anti-PD-L1 / VEGF bispecific antibody" have the same meanings and refer to bispecific antibodies that specifically recognize and bind to PD-L1 and VEGF.

[0136] The present invention provides an anti-PD-L1 / VEGF bispecific antibody, which comprises: an anti-PD-L1 single domain antibody and an anti-VEGF antibody or element.

[0137] Preferably, the bispecific antibody comprises a polypeptide having a structure as shown in Formula I and Formula II.

[0138] A1-L1-CH-L2-B (Formula I)

[0139] A2-L3-CL (Formula II)

[0140] in,

[0141] A1 is the heavy chain variable region VH of the anti-VEGF antibody;

[0142] A2 is the light chain variable region VL of the anti-VEGF antibody;

[0143] B is an anti-PD-L1 single-domain antibody;

[0144] L1, L2 and L3 are each independently a no or a connection element;

[0145] CH is a human IgG heavy chain constant region CH (preferably a LALA mutant);

[0146] CL is the human kappa light chain constant region CL; and

[0147] “-” is a peptide bond;

[0148] And wherein, the polypeptide represented by formula I and the polypeptide represented by formula II form a heterodimer through disulfide bonds.

[0149] Alternatively, the bispecific antibody is a polypeptide having a structure as shown in Formula III or Formula IV,

[0150] A3-L4-Fc-L5-B (Formula III)

[0151] B-L6-Fc-L7-A3 (Formula IV)

[0152] in,

[0153] A3 is the domain that can bind to VEGF and block its activity;

[0154] B is an anti-PD-L1 single-domain antibody;

[0155] L4, L5, L6 and L7 are each independently a no or a connection element;

[0156] Fc is the Fc segment of human IgG (preferably a LALA mutant); and

[0157] “-” is a peptide bond.

[0158] In one embodiment, the bispecific antibody contains polypeptides of structures shown in Formula I and Formula II at the same time, and the polypeptide shown in Formula I and the polypeptide shown in Formula II form a heterodimer i through disulfide bonds;

[0159] Furthermore, the heterodimers i form homodimers ii through disulfide bonds between CH domains.

[0160] In another embodiment, the bispecific antibody is a polypeptide having a structure as shown in Formula III or Formula IV, and the polypeptides having a structure as shown in Formula III or Formula IV form homodimers through disulfide bonds between Fc segments.

[0161] As used herein, the terms "single domain antibody", "nanobody VHH", and "nanobody" have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0162] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a -folded configuration, connected by three CDRs forming a connecting loop, and can form a partially folded structure in some cases. The CDRs in each chain are closely together through the FR region and together with the CDRs of another chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0163] As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between CDRs, i.e., refers to those parts of the light chain and heavy chain variable regions of immunoglobulins that are relatively conserved between different immunoglobulins in a single species. The light chain and heavy chain of an immunoglobulin each have four FRs, referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can therefore be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain can therefore be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, wherein the derivative of the human antibody FR is substantially identical to the naturally occurring human antibody FR, i.e., the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0164] Knowing the amino acid sequence of the CDRs, one skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0165] As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0166] As used herein, the term "affinity" is theoretically defined by the equilibrium association between a complete antibody and an antigen. The affinity of the dual antibody of the present invention can be evaluated or determined by a KD value (dissociation constant) (or other determination methods), such as bio-layer interferometry (BLI), using a FortebioRed96 instrument to measure and determine.

[0167] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the light and heavy chain domains to fold into an exchange dual variable region immunoglobulin.

[0168] As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments thereof of the present invention to form conjugates. The present invention also includes cell surface markers or antigens combined with the PD-L1 / VEGF bispecific antibodies or fragments thereof.

[0169] As used herein, the term "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0170] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

[0171] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0172] In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to PD-L1 and / or VEGF proteins, such as proteins or polypeptides having heavy chain variable regions. They may or may not contain an initial methionine.

[0173] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0174] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0175] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0176] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0177] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0178] The antibodies of the present invention refer to bispecific antibodies with PD-L1 and / or VEGF protein binding activity. The term also includes variant forms of polypeptides that have the same functions as the antibodies of the present invention and contain the same CDR regions. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and optimally 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, adding one or several amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0179] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0180] The present invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of single domain antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0181] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0182] Table A

[0183] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0184] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0185] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0186] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include a polynucleotide further including additional coding and / or non-coding sequences.

[0187] The present invention also relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0188] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0189] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0190] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0191] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0192] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0193] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl 2 Another method is to use MgCl 2 If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0194] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0195] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0196] The antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0197] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0198] Therapeutic agents that can be combined or coupled to the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0199] Pharmaceutical composition

[0200] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0201] The pharmaceutical composition of the present invention can be directly used to bind to PD-L1 and / or VEGF protein molecules, and thus can be used to treat tumors. In addition, other therapeutic agents can also be used simultaneously.

[0202] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned single-domain antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0203] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 10 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0204] Labeled Antibodies

[0205] In a preferred embodiment of the present invention, the antibody carries a detectable marker. More preferably, the marker is selected from the group consisting of an isotope, a colloidal gold marker, a colored marker or a fluorescent marker.

[0206] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the PD-L1 / VEGF bispecific antibody can be labeled with colloidal gold to obtain a colloidal gold-labeled antibody.

[0207] Detection Methods

[0208] The present invention also relates to a method for detecting PD-L1 and / or VEGF protein. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of PD-L1 and / or VEGF protein in the dissolved sample.

[0209] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage solution.

[0210] Reagent test kit

[0211] The present invention also provides a kit containing the antibody (or fragment thereof) or the detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

[0212] The present invention also provides a detection kit for detecting PD-L1 and / or VEGF levels, which includes antibodies that recognize PD-L1 and / or VEGF proteins, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0213] application

[0214] As described above, the single domain antibody of the present invention has a wide range of biological and clinical application values, and its application involves multiple fields such as diagnosis and treatment of diseases related to PD-L1 and / or VEGF, basic medical research, biological research, etc. A preferred application is for clinical diagnosis and targeted therapy of PD-L1 and / or VEGF, such as tumor treatment.

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

[0216] 1) The bispecific antibody of the present invention is highly specific to human PD-L1 protein with a correct spatial structure.

[0217] 2) The bispecific antibody of the present invention has strong affinity.

[0218] 3) The bispecific antibody of the present invention is easy to produce.

[0219] 4) Inhibiting the PD-1 / PD-L1 pathway based on the targeted neutralization of VEGF in the tumor microenvironment can restore the activity of T cells, enhance the immune response, and more effectively improve the effect of inhibiting tumor occurrence and development.

[0220] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0221] Summary of the sequence of the present invention

[0222] Table B: Sequences related to antibodies of the present invention

[0223]

[0224]

[0225]

[0226] Example 1: Cloning and expression of anti-VEGF / PD-L1 bispecific antibodies

[0227] 1.1 Antibody structure design

[0228] In this example, six anti-VEGF / PD-L1 bispecific antibodies were constructed, namely:

[0229] Ava-2GS-NSD / Ava-4GS-NSD: It is composed of two polypeptide chains, and its structural diagram is as follows Figure 1As shown in A, peptide chain #1 has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, which comprises a VH amino acid sequence (SEQ ID NO:3) derived from the anti-VEGF antibody bevacizumab (Patent No.: WO1998045332), and a CH amino acid sequence derived from human IgG1 (introducing LALA mutation to reduce Fc function, SEQ ID NO:4) is directly connected to the C-terminus of the VH amino acid sequence, and the N-terminus of the anti-PD-L1 nanobody C-Ye-18-5 (Patent Application No.: 2019108631090) (SEQ ID NO:5) is connected through 11 amino acid residues (G 4 S) 2 G (SEQ ID NO: 6) (Ava-2GS-NSD) or 21 amino acid residues (G 4 S) 4 A flexible peptide of G (SEQID NO:7) (Ava-4GS-NSD) is connected to the C-terminus of the heavy chain; peptide chain #2 has the amino acid sequence shown in SEQ ID NO:8, which comprises a VL amino acid sequence derived from the anti-VEGF antibody bevacizumab (Bevacizumab) (SEQ IDNO 9), and a human κ light chain constant region (CL) amino acid sequence (SEQ ID NO:10) at the C-terminus of the VL amino acid sequence.

[0230] Luc-2GS-NSD / Luc-4GS-NSD: It is composed of 2 polypeptide chains, and its structural diagram is as follows Figure 1 As shown in B, peptide chain #1 has an amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, which comprises a VH amino acid sequence (SEQ ID NO: 13) derived from the anti-VEGF antibody Ranibizumab (Patent No.: WO2018175752), and a human IgG1CH amino acid sequence (introducing LALA mutation to reduce Fc function, SEQ ID NO: 4) is directly connected to the C-terminus of the VH amino acid sequence, and the N-terminus of the anti-PD-L1 nanobody C-Ye-18-5 (SEQ ID NO: 5) is connected through 11 amino acid residues (G 4 S) 2 G (SEQ ID NO: 6) (Luc-2GS-NSD) or 21 amino acid residues (G 4 S) 4A flexible peptide of G (SEQ ID NO:7) (Luc-4GS-NSD) is connected to the C-terminus of the heavy chain; peptide chain #2 has the amino acid sequence shown in SEQ ID NO:14, which comprises a VL amino acid sequence derived from the anti-VEGF antibody Ranibizumab (Ranibizumab) (SEQ IDNO15), and a human κ light chain constant region (CL) amino acid sequence (SEQ ID NO:10) at the C-terminus of the VL amino acid sequence.

[0231] NSD-Elyea: It is composed of one polypeptide chain, and its structural diagram is as follows Figure 1 Therefore, the peptide chain has the amino acid sequence shown in SEQ ID NO: 16, which comprises an amino acid sequence of the VEGF binding region derived from the anti-VEGF fusion protein Aflibercept (patent number: US7070959) (SEQ ID NO: 17), and an amino acid sequence derived from human IgG1Fc (introducing LALA mutation to reduce Fc function, SEQ ID 18) is directly connected to the C-terminus of the amino acid sequence, and the N-terminus of the anti-PD-L1 nanobody C-Ye-18-5 is connected through 21 amino acid residues (G 4 S) 4 A flexible peptide of G (SEQ ID NO: 7) was attached to the C-terminus of the heavy chain.

[0232] Elyea-NSD: It is composed of 1 polypeptide chain, and its structural diagram is as follows Figure 1 Therefore, the peptide chain has the amino acid sequence shown in SEQ ID NO: 19, which comprises the anti-PD-L1 nanobody C-Ye-18-5 (SEQ ID NO: 5), in which the C-terminus of the nanobody sequence is directly connected to the amino acid sequence derived from human IgG1 Fc (introducing LALA mutation to reduce Fc function, SEQ ID NO: 4), and the N-terminus of the VEGF binding region amino acid sequence (SEQ ID NO: 17) derived from the anti-VEGF fusion protein Aflibercept is connected through 21 amino acid residues (G 4 S) 4 A flexible peptide of G (SEQ ID NO: 7) was attached to the C-terminus of the heavy chain.

[0233] 1.2 Gene cloning and protein preparation

[0234] The above gene fragments were constructed into pCDNA3.1 vector. TMExpression system kit (purchased from Thermo), the plasmid was transferred into Expi-CHO cells, the transfection method was in accordance with the product manual, and the supernatant was collected after 5 days of cell culture and the target protein was purified by protein A magnetic beads (purchased from GenScript) sorting method. The magnetic beads were resuspended in an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH7.4) (1-4 times the volume of magnetic beads) and added to the sample to be purified, incubated at room temperature for 1 hour, and gently shaken during the period. The sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed 3 times with binding buffer. Elution buffer (0.1M sodium citrate, pH3.2) was added according to 3-5 times the volume of the magnetic beads, and the elution buffer was shaken at room temperature for 5-10 minutes, and then placed back on the magnetic stand, the elution buffer was collected, and transferred to a collection tube with neutralization buffer (1M Tris, pH8.54) and mixed.

[0235] Example 2: Bispecific Antibody Affinity Determination

[0236] ForteBio affinity determination was performed according to existing methods (Estep, P et al., Solution-based high-throughput antibody-antigen affinity and epitope fractionation measurements, MAbs, 2013.5(2): p.270-8). Briefly, the sensor was equilibrated offline in the assay buffer for 30 min, then online for 60 s to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHC sensor. The sensor was then placed in 100 nM PD-L1 or VEGF antigen for 5 min, after which the sensor was transferred to PBS for dissociation for 5 min. A 1:1 binding model was used for kinetic analysis.

[0237] Table 1. Candidate molecules and PD-L1 binding affinities

[0238] serial number KD(M) Kon(1 / Ms) Koff(1 / s) Ava-2GS-NSD 8.48E-09 3.19E+05 2.70E-03 Luc-2GS-NSD 9.52E-09 2.81E+05 2.67E-03 Ava-4GS-NSD 7.12E-09 3.54E+05 2.52E-03 Luc-4GS-NSD 8.08E-09 3.21E+05 2.60E-03 NSD-Elyea 5.33E-09 3.22E+05 1.72E-03 Elyea-NSD 8.34E-09 3.39E+05 2.83E-03 C-Ye-18-5 8.75E-09 1.98E+05 1.73E-03

[0239] Table 2. Candidate molecules and VEGF binding affinity

[0240] serial number KD(M) Kon(1 / Ms) Koff(1 / s) Ava-2GS-NSD 1.38E-09 2.59E+05 3.58E-04 Luc-2GS-NSD 1.33E-09 1.77E+05 2.36E-04 Ava-4GS-NSD 1.51E-09 2.49E+05 3.75E-04 Luc-4GS-NSD 1.35E-09 1.82E+05 2.45E-04 NSD-Elyea 1.10E-09 6.48E+05 7.12E-04 Elyea-NSD 4.01E-10 9.14E+05 3.66E-04 Ranibizumab 2.29E-09 1.75E+05 4.00E-04 Bevacizumab 1.79E-09 1.71E+05 3.06E-04

[0241] In the assay experiment described above, all purified samples of the present invention have binding activity with PD-L1 and VEGF proteins, and the binding activity is similar to that of the control antibody C-Ye-18-5 (PD-L1 single domain antibody), Ranibizumab and Bevacizumab.

[0242] Example 3: Purified bispecific antibody binds to PD-L1

[0243] CHO cells overexpressing human PD-L1 (CHO-hPD-L1 cells) were generated by transfecting the pCHO1.0 vector (purchased from Invitrogen) cloned into the MCS human PD-L1 cDNA (purchased from Sino Biological). The cell density of the expanded cultured CHO-hPD-L1 cells was adjusted to 2×10 6 cells / ml, 100μl / well was added to a 96-well flow plate, centrifuged and set aside. The purified PD-L1 antibody was diluted with PBS, starting from 400nM and diluted 3 times for a total of 12 points. 100μl / well of the diluted sample was added to the 96-well flow plate with cells, incubated at 4°C for 30min, and washed twice with PBS. 100μl / well was added to sheep F(ab')2 anti-human IgG-Fc (PE) (purchased from Abcam) diluted 100 times with PBS, incubated at 4°C for 30min, and washed twice with PBS. 100μl / well was added to PBS to resuspend the cells, and the detection was performed on a CytoFlex (Bechman) flow cytometer and the corresponding MFI was calculated.

[0244] In the above method, the experimental results are as follows Figure 2 As shown, all the purified samples of the present invention have binding activity to CHO-hPD-L1 cells, and the binding activity of the purified samples is similar to that of the control antibody C-Ye-18-5.

[0245] Example 4: ELISA detection of VEGF / VEGFR protein level binding blocking experiment

[0246] Use ELISA coating solution to dilute human VEGFR protein to an appropriate concentration, add to ELISA plate, and coat overnight at 4°C. Block with 5% BSA at room temperature for 1 hour. Dilute the sample to be tested in a gradient manner and incubate with biotinylated human VEGF protein at room temperature for 1 hour. Add the incubated sample to the blocked ELISA plate and react at room temperature for 2 hours. Wash 3 times with PBS solution, add diluted streptavidin (HRP) and react at room temperature for 1 hour, wash 3 times with PBS solution, add ELISA colorimetric solution, let stand at room temperature for 3 minutes, add ELISA stop solution, and read the absorbance value at 450nm. The results are as follows Figure 3 As shown, Ava-2GS-NSD, Luc-2GS-NSD, Ava-4GS-NSD, Luc-4GS-NSD ( Figure 3 A) and NSD-Elyea, Elyea-NSD( Figure 3 B) Both can completely block the interaction between VEGF and VEGFR proteins.

[0247] Example 5: Mixed lymphocyte reaction experiment

[0248] In this example, the activity of bispecific antibodies in activating T cells was detected by mixed lymphocyte reaction (MLR) assay. The specific experimental method is as follows.

[0249] PBMC cells (purchased from SAILY BIO, SLB-HPB) were revived and centrifuged. PBMC were resuspended in 10 ml X-VIVO-15 medium (purchased from LONZA), cultured in a cell culture incubator at 37°C for 2 h, and non-adherent cells were removed by aspiration. 10 ml DC medium (X-VIVO-15 medium with 10 ng / ml GM-CSF (purchased from R&D), 20 ng / ml IL-4) was added, cultured for 3 days, 5 ml DC medium was added, and cultured until the 6th day, DC maturation medium (X-VIVO-15 medium with 1000 U / ml TNF-α (purchased from R&D), 10 ng / ml IL-6 (purchased from R&D), 5 ng / ml IL-1β (purchased from R&D), 1 μM PGE2 (purchased from Tocris)) was added, cultured for 2 days, mature DC cells were collected, and the cell density was adjusted to 2×10 with X-VIVO-15 medium. 5 cells / ml.

[0250] PBMC cells from another donor (purchased from SAILY BIO, SLB-HPB) were resuscitated, centrifuged, and resuspended in 10 ml X-VIVO-15 medium. + T cell sorting kit (purchased from Stemcell) enriched CD4 + T cells, X-VIVO-15 resuspended CD4 + T cells, adjust the cell density to 2 × 10 6 cells / ml. + T cells were mixed with the mature DC cells collected above at a ratio of 1:1, and 100 μl / well was added to a 96-well U-bottom plate.

[0251] The purified bispecific antibody samples were diluted with X-VIVO-15 medium, starting from 200 nM and diluted 3-fold for a total of 9 points. 100 μl / well was added to the mixed cell wells and cultured for 5 days. The supernatant was collected and IFN-γ ( Figure 4 A) and IL2( Figure 4 B) Expression level.

[0252] The results are as follows Figure 4As shown, Ava-2GS-NSD, Luc-2GS-NSD, Ava-4GS-NSD, Luc-4GS-NSD, NSD-Elyea, and Elyea-NSD all showed good biological activity in the MLR experiment, and the activation levels were similar to or better than the control antibody C-Ye-18-5.

[0253] Example 6: Accelerated stability test determination

[0254] 6.1 Accelerated stability sample purity determination

[0255] This experiment evaluated the long-term thermal stability of the bispecific antibody by detecting the changes in purity and biological activity after being placed at 40°C for 30 days. The purity of the target antibody after being placed at 40°C for 0, 14 and 30 days was determined using the SEC method. The experimental results are shown in Table 3. The purity of the bispecific antibodies Ava-2GS-NSD, Luc-2GS-NSD, NSD-Elyea, and Elyea-NSD did not change significantly.

[0256] Table 3: Monomer ratio of accelerated stability samples

[0257] Sample name Day 0 14 days 30 days Ava-2GS-NSD 98.9％ 98.1％ 97.0％ Luc-2GS-NSD 97.4％ 97.3％ 96.3％ NSD-Elyea 93.0％ 97.1％ 94.1％ Elyea-NSD 99.7％ 100.0％ 98.4％

[0258] 6.2 Accelerated stability sample activity determination

[0259] In this experiment, the binding of accelerated stability test samples and CHO-PDL1 cells was detected by FACS method, and the method was the same as Example 3. The binding of accelerated stability samples and VEGF was detected by ELISA method, and the method was as follows: VEGFA recombinant protein was diluted to 1μg / ml with ELISA coating solution, 100μl / well was added to the ELISA plate, and coated overnight at 4°C. Discard the coating solution, add 250μl / well PBST to wash 3 times, and block with 5% BSA at room temperature for 1 hour for use. Dilute the purified sample with 1% BSA gradient. Discard the blocking solution from the ELISA plate, add 100μl / well of the diluted sample to the blocked ELISA plate, and incubate at room temperature for 2 hours. Wash three times with 250 μl / well PBST, add 100 μl / well goat anti-human Fc-HRP diluted with 1% BSA, incubate at room temperature for 1 hour, wash three times with 250 μl / well PBST, add 100 μl / well ELISA colorimetric solution, leave at room temperature for 3 minutes, add 50 μl / well ELISA stop solution, and read the absorbance at 450 nm.

[0260] The experimental results are shown in Figure 5. The binding of the bispecific antibody to CHO-PDL1 cells ( Figure 5A ) and binding to VEGF protein ( Figure 5BThe results show that the bispecific antibody of the present invention has good thermal stability.

[0261] Example 7: Pharmacokinetic evaluation in rats

[0262] This experiment tested the pharmacokinetic properties of the anti-PD-L1 / VEGF bispecific antibody in rats. Six SD rats (half male and half female, 12 / 12 hours light / dark adjustment, temperature 24±2°C, humidity 40-70%, free access to water and diet) were purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd. On the day of the experiment, SD rats were injected with Ava-2GS-NSD or Elyea-NSD molecules in the tail vein at a dose of 10 mg / kg.

[0263] Blood collection time point: 3min, 4 hours, 10 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours, 672 hours after administration, blood was collected from the rat jugular vein. The whole blood sample was placed at 2-8°C for 30min, centrifuged at 12000rpm for 5min to collect serum, and the resulting serum was centrifuged at 2-8°C, 12000rpm for 5min, and stored at -80°C. ELISA was used to detect the molecular weight of free Ava-2GS-NSD or Elyea-NSD in serum. The results are shown in Table 4. The half-life of the free state molecule of Ava-2GS-NSD of the present invention in SD rats is about 320.6 hours, and the half-life of the free state molecule of Elyea-NSD in SD rats is about 38.1 hours.

[0264] Table 4 T1 / 2 of bispecific antibodies in SD rats

[0265] Test drug Dosage T1 / 2 Ava-2GS-NSD IV 320.6 hours Elyea-NSD IV 38.1 hours

[0266] Example 8: Study on tumor inhibitory activity of bispecific antibodies

[0267] In this experiment, the human colon cancer LOVO cell / NOG mouse injection human PBMC model was used to determine the anti-tumor effect of the bispecific antibody. Sufficient LOVO cells (purchased from Addexbio) were cultured and expanded in vitro, and the cells were collected after trypsin digestion, washed three times with PBS, and counted. The number of cells was 2×10 6 The number of cells / mouse was inoculated subcutaneously on the right abdomen of female 8-week-old NOG severely immunodeficient mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). The subcutaneous tumor formation of cells in NOG mice was observed daily. After 8 days of inoculation, 6×10 6PBMC. Three days after the injection of PBMC, the maximum width axis W and the maximum length axis L of the subcutaneous tumor on the right abdomen of each animal were measured with a vernier caliper, and the weight of each mouse was weighed with an electronic balance. The subcutaneous tumor volume on the right abdomen of each mouse was calculated according to the tumor volume T = 1 / 2 × W × W × L. Mice with too large and too small tumor volumes were eliminated, and NOG mice were divided into 4 groups according to the average tumor volume, with 6 mice in each group. The mice were divided into groups according to the following grouping and dosing schedule and injected with the corresponding doses of Ava-2GS-NSD.

[0268] Table 5 Experimental scheme for tumor inhibition activity of anti-PD-L1 / VEGF bispecific antibodies

[0269] Group Drug category Dosage Dosing frequency Group 1 PBS — 2 times a week, 6 times in total Group 2 Ava-2GS-NSD 24 mg / kg 2 times a week, 6 times in total Group 3 Ava-2GS-NSD 4.8mg / kg 2 times a week, 6 times in total Group 4 Ava-2GS-NSD 1mg / kg 2 times a week, 6 times in total

[0270] The tumor volume and body weight of mice were measured twice a week. The body weight and tumor volume of mice were measured for the last time 31 days after tumor cell inoculation, and the mice were euthanized. Compared with the PBS group, Ava-2GS-NSD could inhibit tumor growth in a dose-dependent manner, with TGIs of 1 mg / kg, 4.8 mg / kg, and 24 mg / kg being 16.7%, 44.9%, and 55.6%, respectively. Among them, the tumor volume of the 4.8 mg / kg and 24 mg / kg groups was significantly different from that of the PBS group. The results of the average tumor weight, as well as the trend of tumor growth inhibition rate and tumor volume were basically consistent ( Figure 6 A and Table 6).

[0271] Table 6 Effects of each treatment group on LOVO tumor volume and TGI in NOG mice subcutaneously inoculated

[0272]

[0273]

[0274] Note: Compared with the PBS group, "***" P<0.001; "**" P<0.01.

[0275] The status of mice in each group was observed daily and no abnormality was found; the mice were weighed twice a week, and the weight of mice in each group did not decrease significantly. At the end of the experiment, the weight of mice in each dose group did not change by more than 13% compared with the weight at the beginning of treatment, and the weight of mice in the PBS group also decreased by 6%. It is more likely that the GvHD caused by PBMC reconstruction in mice caused the weight of each group of mice to decrease in the later stage of the experiment (see Figure 6 B); At the end of the experiment, no obvious lesions were found in the liver, kidney, lung and other major organs of mice in each group after dissection, indicating that the drugs in each group had no obvious toxicity to mice at the dosage used in this experiment.

[0276] In summary, Ava-2GS-NSD can inhibit the growth of LOVO subcutaneous transplanted tumors in a dose-dependent manner, with an effective dose of 4.8 mg / kg; no obvious toxicity was observed in mice at three dosages (1 mg / kg, 4.8 mg / kg and 24 mg / kg).

[0277] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Biomis Biotechnology (Zhuhai) Co., Ltd. <120> An anti-VEGF / PD-L1 bispecific antibody and its use <130> P2020-1322 <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 578 <212> PRT <213> Artificial sequence <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu 450 455 460 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 465 470 475 480 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp 485 490 495 Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 500 505 510 Ser Ile Asn Ser Asp Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys 515 520 525 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 530 535 540 Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 545 550 555 560 Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr Val 565 570 575 Ser Ser <210> 2 <211> 588 <212> PRT <213> artificial sequence <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu Val Gln Leu Gln Glu Ser 465 470 475 480 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 485 490 495 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met Tyr Trp Leu Arg Gln 500 505 510 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Asn Ser Asp Ser 515 520 525 Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 530 535 540 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys 545 550 555 560 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Pro Gly Gly Tyr 565 570 575 Ala Lys Gly Gln Gly Thr Gln Val Thr Val Ser Ser 580 585 <210> 3 <211> 123 <212> PRT <213> artificial sequence <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 329 <212> PRT <213> Artificial sequence <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 5 <211> 115 <212> PRT <213> artificial sequence <400> 5 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Ser Asp Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <400> 6 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 <210> 7 <211> 21 <212> PRT <213> Artificial sequence <400> 7 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly 20 <210> 8 <211> 214 <212> PRT <213> Artificial sequence <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 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> 9 <211> 107 <212> PRT <213> artificial sequence <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> artificial sequence <400> 10 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 11 <211> 578 <212> PRT <213> artificial sequence <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu 450 455 460 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 465 470 475 480 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp 485 490 495 Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 500 505 510 Ser Ile Asn Ser Asp Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys 515 520 525 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 530 535 540 Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 545 550 555 560 Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr Val 565 570 575 Ser Ser <210> 12 <211> 588 <212> PRT <213> artificial sequence <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu Val Gln Leu Gln Glu Ser 465 470 475 480 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 485 490 495 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met Tyr Trp Leu Arg Gln 500 505 510 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Asn Ser Asp Ser 515 520 525 Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 530 535 540 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys 545 550 555 560 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Pro Gly Gly Tyr 565 570 575 Ala Lys Gly Gln Gly Thr Gln Val Thr Val Ser Ser 580 585 <210> 13 <211> 123 <212> PRT <213> artificial sequence <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 214 <212> PRT <213> artificial sequence <400> 14 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 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> 15 <211> 107 <212> PRT <213> artificial sequence <400> 15 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 16 <211> 567 <212> PRT <213> artificial sequence <400> 16 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Ser Asp Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 115 120 125 Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 130 135 140 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 145 150 155 160 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 165 170 175 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 180 185 190 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 195 200 205 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 210 215 220 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 225 230 235 240 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 245 250 255 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 260 265 270 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 275 280 285 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 290 295 300 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 305 310 315 320 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 325 330 335 Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 340 345 350 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Asp Thr Gly Arg Pro 355 360 365 Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu 370 375 380 Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr 385 390 395 400 Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys 405 410 415 Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr 420 425 430 Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His 435 440 445 Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile 450 455 460 Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu 465 470 475 480 Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile 485 490 495 Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu 500 505 510 Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe 515 520 525 Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu 530 535 540 Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr 545 550 555 560 Phe Val Arg Val His Glu Lys 565 <210> 17 <211> 205 <212> PRT <213> artificial sequence <400> 17 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 165 170 175 Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys 195 200 205 <210> 18 <211> 226 <212> PRT <213> artificial sequence <400> 18 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 19 <211> 567 <212> PRT <213> artificial sequence <400> 19 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 165 170 175 Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195 200 205 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 210 215 220 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 225 230 235 240 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 245 250 255 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 305 310 315 320 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 325 330 335 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 340 345 350 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 355 360 365 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 385 390 395 400 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 420 425 430 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 435 440 445 Gly Gly Ser Gly Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val 450 455 460 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 465 470 475 480 Phe Ser Ser Tyr Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly 485 490 495 Leu Glu Trp Val Ser Ser Ile Asn Ser Asp Ser Ser Ser Thr Tyr Tyr 500 505 510 Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 515 520 525 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala 530 535 540 Val Tyr Tyr Cys Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly 545 550 555 560 Thr Gln Val Thr Val Ser Ser 565 <210> 20 <211> 108 <212> PRT <213> Artificial Sequence <400> 20 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 100 105

Claims

1. A bispecific antibody, characterized in that: The bispecific antibody comprises: (a) anti-PD-L1 single domain antibody; and (b) Anti-VEGF antibodies; Among them, the amino acid sequence of the anti-PD-L1 single domain antibody is shown in SEQ ID NO:

5.

2. The bispecific antibody according to claim 1, characterized in that The bispecific antibody comprises 1-3 anti-PD-L1 single domain antibodies.

3. The bispecific antibody according to claim 1, characterized in that The bispecific antibody further comprises an Fc segment.

4. The bispecific antibody according to claim 3, characterized in that The Fc segment of the bispecific antibody is selected from the following group: a human IgG domain, a CH1+CL1 domain, or a combination thereof.

5. The bispecific antibody according to claim 1, characterized in that The bispecific antibody contains polypeptides with structures shown in Formula I and Formula II, A1-L1-CH-L2-B (Formula I) A2-L3-CL (Formula II) in, A1 is the heavy chain variable region VH of the anti-VEGF antibody; A2 is the light chain variable region VL of the anti-VEGF antibody; B is an anti-PD-L1 single-domain antibody; L1, L2 and L3 are each independently a no or a connection element; CH is the human IgG heavy chain constant region CH; CL is the human kappa light chain constant region CL; and "-" is a peptide bond; And wherein, the polypeptide represented by formula I and the polypeptide represented by formula II form a heterodimer through disulfide bonds.

6. The bispecific antibody according to claim 5, characterized in that The human IgG heavy chain constant region CH is a LALA mutant human IgG heavy chain constant region CH.

7. The bispecific antibody according to claim 1, characterized in that The bispecific antibody is a polypeptide having a structure as shown in Formula III or Formula IV, A3-L4-Fc-L5-B (Formula III) B-L6-Fc-L7-A3 (Formula IV) in, The amino acid sequence of A3 is shown in SEQ ID NO: 17; B is an anti-PD-L1 single-domain antibody; L4, L5, L6 and L7 are each independently a no or a connection element; Fc is the Fc segment of human IgG; and "-" is a peptide bond.

8. The bispecific antibody according to claim 7, characterized in that The human IgG Fc segment is a LALA mutant human IgG Fc segment.

9. The bispecific antibody according to claim 5, characterized in that The heavy chain variable region VH of the anti-VEGF antibody has an amino acid sequence of the heavy chain variable region derived from Bevacizumab, and its amino acid sequence is shown in SEQ ID NO:

3.

10. The bispecific antibody according to claim 5, characterized in that The light chain variable region VL of the anti-VEGF antibody has an amino acid sequence of a light chain variable region derived from bevacizumab, and its amino acid sequence is shown in SEQ ID NO:

9.

11. The bispecific antibody according to claim 5, characterized in that The heavy chain variable region VH of the anti-VEGF antibody has an amino acid sequence of the heavy chain variable region derived from Ranibizumab, and its amino acid sequence is shown in SEQ ID NO:

13.

12. The bispecific antibody according to claim 5, characterized in that The light chain variable region VL of the anti-VEGF antibody has an amino acid sequence of the light chain variable region derived from Ranibizumab, and its amino acid sequence is shown in SEQ ID NO:

15.

13. The bispecific antibody according to claim 5, characterized in that The human IgG heavy chain constant region CH has a LALA mutation, and its amino acid sequence is shown in SEQ ID NO:

4.

14. The bispecific antibody according to claim 5, characterized in that The amino acid sequence of the human κ light chain constant region CL is shown in SEQ ID NO:

10.

15. The bispecific antibody according to claim 7, characterized in that The Fc segment of the human IgG has a LALA mutation, and its amino acid sequence is shown in SEQ ID NO:

18.

16. The bispecific antibody according to claim 5 or 7, characterized in that: The sequence of the connecting element is (G4S)n, wherein n is a positive integer.

17. The bispecific antibody according to claim 16, wherein n is 1, 2, 3, 4, 5 or 6.

18. The bispecific antibody according to claim 5 or 7, characterized in that: The amino acid sequence of the connecting element is shown in SEQ ID NO: 6 or 7.

19. The bispecific antibody according to claim 5, characterized in that The bispecific antibody contains polypeptides having structures as shown in Formula I and Formula II, wherein the amino acid sequence of the polypeptide having the structure as shown in Formula I is shown in SEQ ID NO: 1, and the amino acid sequence of the polypeptide having the structure as shown in Formula II is shown in SEQ ID NO:

8.

20. The bispecific antibody according to claim 5, characterized in that The bispecific antibody contains polypeptides having structures as shown in Formula I and Formula II, wherein the amino acid sequence of the polypeptide having the structure as shown in Formula I is shown in SEQ ID NO: 2, and the amino acid sequence of the polypeptide having the structure as shown in Formula II is shown in SEQ ID NO:

8.

21. The bispecific antibody according to claim 5, characterized in that The bispecific antibody contains polypeptides having structures as shown in Formula I and Formula II, wherein the amino acid sequence of the polypeptide having the structure as shown in Formula I is shown in SEQ ID NO: 11, and the amino acid sequence of the polypeptide having the structure as shown in Formula II is shown in SEQ ID NO:

14.

22. The bispecific antibody according to claim 5, characterized in that The bispecific antibody contains polypeptides having structures as shown in Formula I and Formula II, wherein the amino acid sequence of the polypeptide having the structure as shown in Formula I is shown in SEQ ID NO: 12, and the amino acid sequence of the polypeptide having the structure as shown in Formula II is shown in SEQ ID NO:

14.

23. The bispecific antibody according to claim 7, characterized in that The bispecific antibody is a polypeptide having a structure as shown in Formula III, and its amino acid sequence is shown in SEQ ID NO:

16.

24. The bispecific antibody according to claim 7, wherein The bispecific antibody is a polypeptide having a structure as shown in Formula III, and its amino acid sequence is shown in SEQ ID NO:

19.

25. The bispecific antibody according to claim 5, characterized in that The bispecific antibody contains polypeptides of structures shown in Formula I and Formula II at the same time, and the polypeptide shown in Formula I and the polypeptide shown in Formula II form a heterodimer i through disulfide bonds; Furthermore, the heterodimers i form homodimers ii through disulfide bonds between CH domains.

26. The bispecific antibody according to claim 7, wherein The bispecific antibody is a polypeptide having a structure as shown in Formula III or Formula IV, and the polypeptides having a structure as shown in Formula III or Formula IV form homodimers through disulfide bonds between Fc segments.

27. An isolated polynucleotide, characterized in that The polynucleotide encodes the bispecific antibody according to claim 1.

28. The polynucleotide according to claim 27, wherein The polynucleotide encodes the bispecific antibody of claim 5, and in the polynucleotide, the ratio of the polynucleotide sequence encoding the structural polypeptide shown in Formula I to the polynucleotide sequence encoding the structural polypeptide shown in Formula II is 1:

1.

29. A carrier, characterized in that The vector contains the polynucleotide according to claim 27.

30. A host cell, characterized in that The host cell contains the vector of claim 29, or the polynucleotide of claim 27 is integrated into its genome; Alternatively, the host cell expresses the bispecific antibody according to claim 1.

31. The host cell according to claim 30, characterized in that The host cells include prokaryotic cells or eukaryotic cells.

32. A method for producing the bispecific antibody of claim 1, characterized in that: Includes steps: (a) culturing the host cell according to claim 30 under suitable conditions to obtain a culture containing the bispecific antibody; and (b) purifying and / or isolating the culture obtained in step (a) to obtain the bispecific antibody.

33. The method of claim 32, wherein: The purification can be performed by protein A affinity column purification to obtain the target antibody.

34. An immunoconjugate, characterized in that The immunoconjugate contains: (a) the bispecific antibody according to claim 1; and (b) a conjugation moiety which is a detectable label.

35. An immunoconjugate, characterized in that The immunoconjugate contains: (a) the bispecific antibody according to claim 1; and (b) a conjugated moiety selected from the group consisting of drugs, toxins, cytokines, or enzymes, gold nanoparticles or nanorods, nanomagnetic particles, viral coat proteins or VLPs, or a combination thereof.

36. The immunoconjugate of claim 34 or 35, wherein: The immunoconjugate contains: a multivalent bispecific antibody as claimed in claim 1, wherein the multivalency refers to the bispecific antibody as claimed in claim 1 containing multiple repeats in the amino acid sequence of the immunoconjugate.

37. Use of the bispecific antibody according to claim 1 or the immunoconjugate according to claim 34 or 35, characterized in that: Used to prepare reagents, detection plates or kits; wherein the reagents, detection plates or kits are used to: detect PD-L1 and / or VEGF in samples.

38. The use according to claim 37, characterized in that The reagent is one or more reagents selected from the following group: isotope tracers, contrast agents, flow detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and imaging agents.

39. The use according to claim 37, characterized in that The detection is in vivo detection or in vitro detection.

40. The use according to claim 37, characterized in that The detection includes flow cytometry and cell immunofluorescence detection.

41. Use of the bispecific antibody according to claim 1 or the immunoconjugate according to claim 34 or 35, characterized in that: For preparing a medicament, wherein the medicament is used to treat or prevent a tumor expressing PD-L1 or a tumor expressing VEGF, and the tumor is selected from the following group: multiple myelopathy, colorectal cancer, breast cancer, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, or a combination thereof.

42. The use according to claim 41, characterized in that The leukemia is acute myeloid leukemia and chronic myeloid leukemia, or The lymphoma is non-Hodgkin's lymphoma.

43. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) the bispecific antibody of claim 1, or the immunoconjugate of claim 34 or 35; and (ii) a pharmaceutically acceptable carrier.

44. The pharmaceutical composition according to claim 43, wherein The pharmaceutical composition also contains other drugs for treating tumors.

45. The pharmaceutical composition of claim 44, wherein The other drug for treating tumors is a cytotoxic drug.

46. ​​The pharmaceutical composition of claim 43, wherein The drug is used to block the interaction between PD-1 and PD-L1, and simultaneously block the interaction between VEGF and VEGFR.

47. The pharmaceutical composition of claim 43, wherein The pharmaceutical composition is used for preparing medicine for preventing and treating tumors.

48. Use of one or more of the bispecific antibodies of claim 1 selected from the group consisting of: The uses include: (i) for detecting human PD-L1 molecules and / or VEGF molecules; (ii) for flow cytometry; (iii) for cell immunofluorescence detection; (iv) for blocking the interaction between PD-1 and PD-L1; and (v) for blocking the interaction between VEGF and VEGFR; And the use is for non-diagnostic and non-therapeutic purposes.

49. A recombinant protein, characterized in that The recombinant protein has: (i) the bispecific antibody according to claim 1; and (ii) a tag sequence that facilitates expression and / or purification.

50. A method for detecting PD-L1 and / or VEGF in a sample for non-diagnostic and non-therapeutic purposes, characterized in that: The method comprises the steps of: (1) contacting a sample with the bispecific antibody according to claim 1; and (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PD-L1 and / or VEGF in the sample.

51. A PD-L1 and / or VEGF detection reagent, characterized in that: The detection reagent comprises the immunoconjugate as described in claim 34 or 35 and a carrier acceptable for detection.

52. A kit for detecting PD-L1 and / or VEGF, characterized in that: The kit contains the immunoconjugate as described in claim 34 or 35 or the detection reagent as described in claim 51, and instructions.

53. The kit of claim 52, wherein The instructions state that the kit is used for non-invasively detecting the expression of PD-L1 and / or VEGF in a subject to be tested.

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