Bispecific antibodies against pdl1 x kdr

Abstract

The application provides an anti-PDL1xKDR bispecific antibody. Experimental results show that the bispecific antibody can better maintain the activity of each single antibody, can specifically combine with two target points of PD-L1 and KDR at the same time, and has good physical and chemical properties.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more specifically, this invention discloses a bispecific antibody against PDL1×KDR. Background Technology

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

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

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

[0005] The protein encoded by KDR is also named vascular endothelial growth factor receptor-2 (VEGFR-2), a crucial receptor for angiogenesis signal transduction. VEGFR2 is primarily expressed in vascular endothelial cells, especially tumor endothelial cells, and mainly binds to VEGF-C, D, and A, promoting angiogenesis. The specific binding of VEGF to the extracellular region of VEGFR2 can activate multiple downstream signaling pathways, including MAPK, PI3K, PKC, and FAK, participating in endothelial cell budding, migration, vascular permeability, and tumor cell survival. VEGFR-2 is closely related to various diseases such as cancer, psoriasis, rheumatoid arthritis, and diabetic retinopathy. It plays a significant role, particularly in tumor growth, metastasis, and multidrug resistance. Therefore, VEGFR-2 has become an ideal target for treating these diseases, especially cancer. Given the rich vascularity of tumors, clinical trial data indicate that targeting VEGFR-2 is crucial for inhibiting tumor angiogenesis. However, the effects of anti-VEGFR-2 drugs used alone are often very short-lived, which may be closely related to excessive vascular trimming and extreme hypoxia. A 2018 study revealed the synergistic relationship between vascular normalization and the immune response. The tumor microenvironment contains a large number of immunosuppressive cells and dysfunctional effector T cells. The combined use of immune checkpoint inhibitors and anti-angiogenic drugs can significantly prolong the therapeutic window for tumor vascular normalization. Vascular normalization weakens the immunosuppressive process in the tumor microenvironment, increases T cell infiltration, and ultimately promotes tumor regression.

[0006] Bispecific antibodies are gradually becoming a new class of therapeutic antibodies that can be used to treat various inflammatory diseases, cancer, and other diseases.

[0007] There is an urgent need in this field to develop bispecific antibodies with excellent activity for the treatment of diseases such as cancer. Summary of the Invention

[0008] This invention provides a bispecific antibody against PDL1×KDR and its application.

[0009] Therefore, the first objective of this invention is to provide a bispecific antibody against PDL1×KDR.

[0010] A second object of the present invention is to provide an isolated nucleotide encoding the aforementioned bispecific antibody.

[0011] A third objective of this invention is to provide an expression vector comprising the aforementioned nucleotides.

[0012] A fourth object of the present invention is to provide a host cell comprising the expression vector described above.

[0013] The fifth object of the present invention is to provide a method for preparing the aforementioned bispecific antibody.

[0014] A sixth object of the present invention is to provide a pharmaceutical composition comprising the aforementioned bispecific antibody.

[0015] A seventh object of the present invention is to provide the use of the bispecific antibody or the pharmaceutical composition thereof in the preparation of a medicament for treating cancer.

[0016] An eighth object of the present invention is to provide a method for treating cancer using the bispecific antibody or the pharmaceutical composition described herein.

[0017] To achieve the above objectives, the present invention provides the following technical solution:

[0018] A first aspect of the present invention provides a bispecific antibody against PDL1×KDR, comprising two polypeptide chains and two light chains selected from the group consisting of:

[0019] (a) The polypeptide chain comprises VH-PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH-KDR-linker1-VL-KDR from the N-terminus to the C-terminus, and the light chain comprises VL-PDL1-CL from the N-terminus to the C-terminus; or

[0020] (b) the polypeptide chain comprises VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the light chain comprises VL-KDR-CL from the N-terminus to the C-terminus; or

[0021] (c) The polypeptide chain comprises VH-KDR-linker1-VL-KDR-linker2-VH-PDL1-CH1-CH2-CH3 or VL-KDR-linker1-VH-KDR-linker2-VH-PDL1-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the light chain comprises VL-PDL1-CL from the N-terminus to the C-terminus; or

[0022] (d) The polypeptide chain comprises VH-KDR-CH1-CH2-CH3-linker2-VL-PDL1-linker1-VH-PDL1 or VH-KDR-CH1-CH2-CH3-linker2-VH-PDL1-linker1-VL-PDL1 from the N-terminus to the C-terminus, and the light chain comprises VL-KDR-CL from the N-terminus to the C-terminus; or

[0023] (e) The polypeptide chain contains VH-PDL1-linker1-VL-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the light chain contains VL-KDR-CL from the N-terminus to the C-terminus.

[0024] Wherein, VH-PDL1 is a heavy chain variable region that incorporates PD-L1.

[0025] The VL-PDL1 mentioned above is a light chain variable region that incorporates PD-L1.

[0026] The VH-KDR mentioned above is a heavy chain variable region combined with KDR.

[0027] The VL-KDR mentioned above is a light chain variable region combined with KDR.

[0028] Linker1 and linker2 are each independently a flexible peptide linker.

[0029] CH1-CH2-CH3 is the heavy chain constant region, CL is the light chain constant region, VH-PDL1 and VL-PDL1 form an antigen binding site that specifically binds to PD-L1, and VH-KDR and VL-KDR form an antigen binding site that specifically binds to KDR.

[0030] In another preferred embodiment, the bispecific antibody has the activity of simultaneously binding to KDR and binding to PD-L1.

[0031] In another preferred embodiment, the flexible peptide linker comprises 6-30 amino acids, more preferably 10-25 amino acids.

[0032] In another preferred embodiment, the flexible peptide linker comprises 2-6 G4S and / or G3S.

[0033] In another preferred embodiment, the Linker1 consists of 3-5 G4S.

[0034] In another preferred embodiment, the Linker2 is 2-4 G4S.

[0035] In another preferred embodiment, linker1 consists of 4 G4S and linker2 consists of 3 G4S.

[0036] In another preferred embodiment, the anti-PDL1×KDR bispecific antibody comprises two polypeptide chains and two light chains, wherein:

[0037] (a) The polypeptide chain comprises VH-PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH-KDR-linker1-VL-KDR from the N-terminus to the C-terminus, and the light chain comprises VL-PDL1-CL from the N-terminus to the C-terminus; or

[0038] (b) The polypeptide chain contains VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the light chain contains VL-KDR-CL from the N-terminus to the C-terminus.

[0039] Wherein, VH-PDL1 is a heavy chain variable region that incorporates PD-L1.

[0040] The VL-PDL1 mentioned above is a light chain variable region that incorporates PD-L1.

[0041] The VH-KDR mentioned above is a heavy chain variable region combined with KDR.

[0042] The VL-KDR mentioned above is a light chain variable region combined with KDR.

[0043] Linker1 and linker2 are each independently flexible peptide linkers; CH1-CH2-CH3 is the heavy chain constant region, CL is the light chain constant region, VH-PDL1 and VL-PDL1 form a specific antigen binding site for PD-L1, and VH-KDR and VL-KDR form a specific antigen binding site for KDR.

[0044] In another preferred embodiment, the anti-PDL1×KDR bispecific antibody comprises two polypeptide chains and two light chains, wherein:

[0045] (a) The polypeptide chain comprises VH-PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH-KDR-linker1-VL-KDR from the N-terminus to the C-terminus, and the light chain comprises VL-PDL1-CL from the N-terminus to the C-terminus; or

[0046] (b) The polypeptide chain contains VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the light chain contains VL-KDR-CL from the N-terminus to the C-terminus.

[0047] Wherein, VH-PDL1 is a heavy chain variable region that incorporates PD-L1.

[0048] The VL-PDL1 mentioned above is a light chain variable region that incorporates PD-L1.

[0049] The VH-KDR mentioned above is a heavy chain variable region combined with KDR.

[0050] The VL-KDR mentioned above is a light chain variable region combined with KDR.

[0051] The linker1 consists of 4 G4S molecules, the linker2 consists of 3 G4S molecules, the CH1-CH2-CH3 region is the heavy chain constant region, the CL region is the light chain constant region, the VH-PDL1 and the VL-PDL1 form a specific antigen binding site for PD-L1, and the VH-KDR and the VL-KDR form a specific antigen binding site for KDR.

[0052] According to a preferred embodiment of the present invention, the VH-PDL1 contains a heavy chain CDR with amino acid sequences as shown in SEQ ID NO: 1-3, the VL-PDL1 contains a light chain CDR with amino acid sequences as shown in SEQ ID NO: 4-6, the VH-KDR contains a heavy chain CDR with amino acid sequences as shown in SEQ ID NO: 7-9, and the VL-KDR contains a light chain CDR with amino acid sequences as shown in SEQ ID NO: 10-12.

[0053] According to a preferred embodiment of the present invention, the VH-PDL1 has the amino acid sequence shown in SEQ ID NO: 13, the VL-PDL1 has the amino acid sequence shown in SEQ ID NO: 14, the VH-KDR has the amino acid sequence shown in SEQ ID NO: 15, and the VL-KDR has the amino acid sequence shown in SEQ ID NO: 16.

[0054] According to a preferred embodiment of the present invention, the heavy chain constant region includes the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and the light chain constant region includes the κ or λ light chain constant region.

[0055] According to a preferred embodiment of the present invention, the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24 or SEQ ID NO: 25, and the light chain has an amino acid sequence as shown in SEQ ID NO: 19; or the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, and the light chain has an amino acid sequence as shown in SEQ ID NO: 21.

[0056] A second aspect of the invention provides an isolated nucleotide encoding the bispecific antibody.

[0057] A third aspect of the present invention provides an expression vector containing the nucleotides described above.

[0058] A fourth aspect of the present invention provides a host cell containing the expression vector described above.

[0059] A fifth aspect of the present invention provides a method for preparing the aforementioned bispecific antibody, the method comprising the following steps:

[0060] (a) Under expression conditions, host cells as described above are cultured to express the bispecific antibody described above;

[0061] (b) Isolate and purify the bispecific antibody described in (a).

[0062] A sixth aspect of the present invention provides a pharmaceutical composition comprising a bispecific antibody as described above and a pharmaceutically acceptable carrier.

[0063] In another preferred embodiment, the pharmaceutical composition further contains an antitumor agent.

[0064] In another preferred embodiment, the pharmaceutical composition is a unit dosage form.

[0065] In another preferred embodiment, the antitumor agent may be present separately from the bispecific antibody in a separate package, or the antitumor agent may be conjugated to the bispecific antibody.

[0066] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form.

[0067] In another preferred embodiment, the parenteral dosage form includes intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavitary injection.

[0068] A seventh aspect of the invention provides the use of the bispecific antibody or pharmaceutical composition as described above in the preparation of a medicament for treating cancer.

[0069] According to the present invention, the cancer is selected from the group consisting of: colorectal cancer, non-small cell lung cancer, gastric cancer, gastroesophageal junction adenocarcinoma, melanoma, lung cancer, liver cancer, lymphoma, leukemia, prostate cancer, bone marrow cancer and other proliferative malignant diseases.

[0070] An eighth aspect of the invention provides a method for treating cancer, comprising administering to a subject in need a bispecific antibody as described above, or an immunoconjugate thereof, or a pharmaceutical composition as described above.

[0071] According to the present invention, the cancer is selected from the group consisting of: colorectal cancer, non-small cell lung cancer, gastric cancer, gastroesophageal junction adenocarcinoma, melanoma, lung cancer, liver cancer, lymphoma, leukemia, prostate cancer, bone marrow cancer and other proliferative malignant diseases.

[0072] A ninth aspect of the present invention provides an immunoconjugate comprising:

[0073] (a) a bispecific antibody as described in the first aspect of the present invention; and

[0074] (b) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0075] In another preferred embodiment, the conjugate is partially selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.) capable of producing detectable products.

[0076] In another preferred embodiment, the immunoconjugate includes an antibody-drug conjugate (ADC).

[0077] In another preferred embodiment, the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors.

[0078] Beneficial effects: This invention provides a bispecific antibody against PD-L1×KDR. Experimental results show that the bispecific antibody can maintain the activity of each monoclonal antibody well and can simultaneously and specifically bind to both PD-L1 and KDR targets, exhibiting good physicochemical properties. Attached Figure Description

[0079] Figure 1 is a schematic diagram of the structure of the anti-PDL1×KDR dual antibody molecule of the present invention, wherein, Figure 1AThe anti-KDR scFv is connected in series at the C-terminus of the anti-PDL1 mAb. Figure 1B The anti-PDL1 scFv is connected in series at the N end of the anti-KDR mAb.

[0080] Figure 2 shows the HPLC detection chromatogram and SDS-PAGE detection results of the anti-PDL1×KDR bispecific antibody. Figure 2A This is the HPLC detection chromatogram. Figure 2B This is the result of SDS-PAGE testing.

[0081] Figure 3 shows the ELISA results of binding of the anti-PDL1×KDR bispecific antibody to PD-L1 and KDR, respectively. Figure 3A For the binding result with PD-L1, Figure 3B The result is the combination with KDR.

[0082] Figure 4 This study describes the results of bispecific ELISA detection of the binding of anti-PDL1×KDR bispecific antibody to both PD-L1 and KDR.

[0083] Figure 5 The binding results of anti-PDL1×KDR bispecific antibody to N87-PDL1 cells were detected by FACS.

[0084] Figure 6 The results show that the activity of PD1 / PD-L1 on cells was blocked by the anti-PDL1×KDR dual antibody.

[0085] Figure 7 The results show that the activity of KDR binding to VEGF on cells was blocked by the anti-PDL1×KDR dual antibody.

[0086] Figure 8 To block the cellular activity of PD1 / PD-L1 with anti-PDL1×KDR bispecific antibodies (anti-PDL1×KDR rev3, rev4, rev5, rev6, rev7).

[0087] Figure 9 shows the activity of anti-PDL1×KDR bispecific antibodies (anti-PDL1×KDR rev3, rev4, rev5, rev6, rev7) in blocking the binding of KDR to VEGF on cells; among which... Figure 9A The blocking activity of anti-PDL1×KDR rev3,rev4; Figure 9B The blocking activity of anti-PDL1×KDR rev5, rev6; Figure 9C This represents the blocking activity of anti-PDL1×KDR rev7. Detailed Implementation

[0088] Through extensive and in-depth research and screening, the inventors have obtained a bispecific antibody composed of an anti-KDR antibody and an anti-PD-L1 antibody. This bispecific antibody is a homodimer. The bispecific antibody of this invention not only retains the activity of both the anti-KDR and anti-PD-L1 antibodies but can also bind to both KDR and PD-L1 simultaneously. This bispecific antibody can be developed into a highly effective anti-tumor drug. Based on this, the inventors completed this invention.

[0089] the term

[0090] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region including a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 isotypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0091] In this invention, the term "bispecific antibody (or bispecific antibody)" refers to an antibody molecule that can simultaneously and specifically bind to two antigens (targets) or two epitopes. Based on symmetry, bispecific antibodies can be classified into structurally symmetrical and asymmetrical molecules. Based on the number of binding sites, bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules.

[0092] In this invention, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a substantially homogeneous population, meaning that the individual antibodies in this population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody formulations (which are typically mixtures of different antibodies targeting different antigenic determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring any special method to produce the antibody.

[0093] In this invention, the terms "Fab" and "Fc" refer to the ability of papain to cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of its light chain. The Fc fragment, or crystallizable fragment, consists of the antibody's CH2 and CH3 domains. The Fc fragment lacks antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells.

[0094] In this invention, the term "scFv" refers to a single-chain antibody fragment (scFv), which is composed of the variable regions of the antibody heavy chain and the variable regions of the light chain, typically linked by a short peptide (linker) of 15 to 25 amino acids.

[0095] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the heavy and light chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0096] As used herein, the term "frame region" (FR) refers to the amino acid sequence inserted between CDRs, specifically those portions of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins within a single species. Each immunoglobulin light and heavy chain has four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can be represented as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain 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, that is, the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0097] Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the frame regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0098] As used herein, the term "human frame region" is a frame region that is substantially identical (approximately 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to the frame region of a naturally occurring human antibody.

[0099] As used herein, the term "linker" refers to an insertion into an immunoglobulin domain that provides sufficient mobility for the light and heavy chains to fold into one or more amino acid residues of an exchangeable dual variable region immunoglobulin. In this invention, preferred linkers are Linker1 and Linker2, wherein Linker1 links the VH and VL of a single-chain antibody (scFv), while Linker2 is used to link the scFv to the heavy chain of another antibody.

[0100] Suitable examples of linkers include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in the linker can vary depending on the type of secondary structural element that needs to be achieved in the linker.

[0101] Bispecific antibodies

[0102] The bispecific antibody of the present invention is an anti-PDL1×EGFR bispecific antibody, comprising an anti-PDL1 antibody portion and an anti-EGFR antibody portion.

[0103] Preferably, the sequence of the anti-PD-L1 antibody of the present invention is as described in patent application PCT / CN2020 / 090442. Those skilled in the art can also modify or alter the anti-PD-L1 antibody of the present invention using techniques well known in the art, such as adding, deleting, and / or substituting one or more amino acid residues, thereby further increasing the affinity or structural stability of anti-PD-L1, and obtaining the modified or altered results by conventional assay methods.

[0104] In this invention, "a conserved variant of the bispecific antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the bispecific antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0105] Table A

[0106] The initial residues Representative substitution Preferred replacement 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

[0107] Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0108] In this invention, the terms "antibody," "binding," and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The antibody binds to the antigen with an equilibrium dissociation constant (KD) of M or smaller. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of antibody-antigen binding.

[0109] In this invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitopes of this invention are regions of an antigen that are bound to antibodies.

[0110] The bispecific antibody of the present invention can be used alone or in combination or conjugated with a detectable marker (for diagnostic purposes), a therapeutic agent, or any combination of the above substances.

[0111] Nucleic acid encoding and expression vector

[0112] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0113] In this invention, the term "expression vector" refers to a vector carrying an expression cassette for expressing a specific target protein or other substance, such as plasmids, viral vectors (e.g., adenovirus, retrovirus), bacteriophages, yeast plasmids, or other vectors. Representative examples include, but are not limited to, pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, and other vectors used in mammalian expression systems. The expression vector includes a fusion DNA sequence linked to suitable transcriptional and translational regulatory sequences.

[0114] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0115] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0116] In this invention, the term "host cell" refers to a cell suitable for expressing the above-mentioned expression vector. It can be a eukaryotic cell, such as a mammalian or insect host cell culture system, which can be used for the expression of the fusion protein of this invention. CHO (Chinese Hamster Ovary), HEK293, COS, BHK, and derived cells of the above cells can all be used in this invention.

[0117] Pharmaceutical Compositions and Applications

[0118] This invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection. In this invention, the term "pharmaceutical composition" refers to a pharmaceutical formulation composition in which the bispecific antibody of this invention, together with a pharmaceutically acceptable carrier, can be formed to exert its therapeutic effect more stably. These formulations ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation). The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described bispecific antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. 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. Furthermore, the bispecific antibody of the present invention can also be used with other therapeutic agents.

[0119] When using a pharmaceutical composition, a safe and effective amount of the bispecific antibody or its immunoconjugate is administered to a mammal. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0120] The experimental materials used in the following examples are described below:

[0121] pcDNA TM 3.4 vector: purchased from Thermo Fisher, part number A14697;

[0122] CHO cells: purchased from Thermo Fisher Scientific, catalog number A29133;

[0123] 293E cells: from NRC biotechnology Research Institute;

[0124] Human gastric cancer cell line NCI-N87: purchased from the American Type Culture Collection (ATCC);

[0125] PD-1 / PD-L1 Blockade Bioassay, Propagation model: purchased from Promega, product number J1252;

[0126] VEGF Bioassay, Propagation Model: Purchased from Promega, product number GA1082.

[0127] The experimental reagents used in the following examples are described below:

[0128] Anti-PD-L1 monoclonal antibody: prepared according to the sequence in PCT / CN2020 / 090442;

[0129] Anti-KDR monoclonal antibody: The CDR region sequence is derived from clone number 3A10 in Dan Lu et al. (Dan Lu et al. Tailoring in VitroSelection for a Picomolar Affinity Human Antibody Directed against Vascular Endothelial Growth Factor Receptor 2 for Enhanced Neutralizing Activity. The Journal of Biological Chemistry, 2003, 278:43496-43507.), while other framework regions were obtained by our company after mutation.

[0130] HRP-labeled goat anti-human Fc antibody: purchased from Sigma, catalog number A0170;

[0131] FITC-labeled goat anti-human Fc antibody: purchased from Sigma, catalog number F9512;

[0132] HRP-labeled mouse anti-human Fab antibody: purchased from Sigma, catalog number A0293;

[0133] HRP-labeled anti-6×His antibody: purchased from abcam, catalog number ab178563;

[0134] Goat anti-human IgG-FITC: purchased from Sigma, product number F4143;

[0135] PBS: Purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B548117;

[0136] PBST: PBS + 0.05% Tween 20;

[0137] BSA: Purchased from Sangon Biotech (Shanghai) Co., Ltd., item number A60332;

[0138] FBS: Purchased from Gibco, item number 10099;

[0139] TMB: Purchased from BD Company, item number 555214;

[0140] Bio-Glo Luciferase Assay System: Purchased from Promega, product number G7940;

[0141] Solution: Purchased from Sigma, catalog number A6964-100mL.

[0142] The experimental instruments used in the following examples are described below:

[0143] PCR instrument: purchased from BioRad, model number C1000 Touch Thermal Cycler;

[0144] HiTrap MabSelectSuRe column: purchased from GE, part number 11-0034-95;

[0145] Beckman Coulter CytoFLEX flow cytometer: purchased from Beckman Coulter;

[0146] SpectraMax i3x microplate reader: purchased from Molecular Devices.

[0147] The following examples and experimental cases are further illustrative of the invention and should not be construed as limiting the invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0148] Example 1: Construction of anti-PDL1×KDR bispecific antibody molecule

[0149] This invention constructs an anti-PDL1×KDR bispecific antibody, named anti-PDL1×KDRBsAb1, by tandemly linking the scFv1 (VL-linker1-VH) of an anti-human KDR monoclonal antibody to the C-terminus of the heavy chain of an anti-human PD-L1 monoclonal antibody via linker2. The structure is as follows: Figure 1A As shown (the VL of scFv1 is linked to the C-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody via linker2).

[0150] This invention utilizes the scFv2 (VH-linker1-VL) of an anti-human KDR monoclonal antibody, cascaded to the C-terminus of the heavy chain of an anti-human PD-L1 monoclonal antibody via linker 2, to construct an anti-PDL1×KDR bispecific antibody, named anti-PDL1×KDRBsAb2. The structure is as follows: Figure 1A As shown (the VH of scFv2 is linked to the C-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody via linker2).

[0151] This invention utilizes the scFv3 (VL-linker1-VH) of an anti-human PD-L1 monoclonal antibody, tandemly linked to the N-terminus of the heavy chain of an anti-human KDR monoclonal antibody via linker2, to construct an anti-PDL1×KDR bispecific antibody, named anti-PDL1×KDR BsAb3. The structure is as follows: Figure 1B As shown (the VH of scFv3 is linked to the N-terminus of the heavy chain of the anti-human KDR monoclonal antibody via linker2).

[0152] This invention constructs an anti-PDL1×KDR bispecific antibody by tandemly linking the scFv2 (VH-linker1-VL) of the anti-human KDR monoclonal antibody to the N-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody via linker2, and naming it anti-PDL1×KDRRev3 (the VL of scFv2 is linked to the N-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody via linker2).

[0153] This invention constructs an anti-PDL1×KDR bispecific antibody by tandemly linking the scFv1 (VL-linker1-VH) of the anti-human KDR monoclonal antibody to the N-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody via linker2, and naming it anti-PDL1×KDRRev4 (the VH of scFv1 is linked to the N-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody via linker2).

[0154] This invention constructs an anti-PDL1×KDR bispecific antibody by tandemly linking the scFv3 (VL-linker1-VH) of the anti-human PD-L1 monoclonal antibody to the C-terminus of the heavy chain of the anti-human KDR monoclonal antibody via linker2. The antibody is named anti-PDL1×KDR Rev5 (the VL of scFv3 is linked to the C-terminus of the heavy chain of the anti-human KDR monoclonal antibody via linker2).

[0155] This invention constructs an anti-PDL1×KDR bispecific antibody by tandemly linking the scFv4 (VH-linker1-VL) of the anti-human PD-L1 monoclonal antibody to the C-terminus of the heavy chain of the anti-human KDR monoclonal antibody via linker2. The antibody is named anti-PDL1×KDR Rev6 (the VH of scFv4 is linked to the C-terminus of the heavy chain of the anti-human KDR monoclonal antibody via linker2).

[0156] This invention constructs an anti-PDL1×KDR bispecific antibody by tandemly linking the scFv4 (VH-linker1-VL) of the anti-human PD-L1 monoclonal antibody to the N-terminus of the heavy chain of the anti-human KDR monoclonal antibody via linker2. The antibody is named anti-PDL1×KDR Rev7 (the VL of scFv4 is linked to the N-terminus of the heavy chain of the anti-human KDR monoclonal antibody via linker2).

[0157] Among them, linker1 has 4 GGGGS, linker2 has 3 GGGGS, the CDR region sequence of the anti-human KDR monoclonal antibody comes from the sequence of clone number 3A10 in Dan Lu et al. (Dan Lu et al. Tailoring in Vitro Selection for a Picomolar Affinity Human Antibody Directed against Vascular Endothelial Growth Factor Receptor 2 for Enhanced Neutralizing Activity. The Journal of Biological Chemistry, 2003, 278: 43496-43507.), the other framework regions were obtained by our company after mutation, and the sequence of the anti-human PD-L1 monoclonal antibody M8 comes from PCT / CN2020 / 090442.

[0158] The heavy and light chain expression vectors of each bispecific antibody and its corresponding monoclonal antibody were obtained through gene synthesis and conventional molecular cloning methods. The corresponding amino acid sequences are shown in Table 1, where CDR is encoded according to the Kabat rule.

[0159] Table 1. Sequence information of the antibodies of the present invention

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167] Note: In the table above, the polypeptide chain of the anti-PDL1×KDR bispecific antibody refers to the heavy chain of the bispecific antibody (formed by scFv linked to the heavy chain end of the anti-human KDR monoclonal antibody through a linker), and the light chain of the bispecific antibody is not included.

[0168] Example 2 Expression and purification of anti-PDL1×KDR bispecific antibody

[0169] The polypeptide chain and light chain DNA fragments of the anti-PDL1×KDR bispecific antibody were subcloned into the pcDN3.4 vector, respectively. The recombinant plasmid was extracted and co-transfected into CHO cells and / or 293E cells. After culturing the cells for 5-7 days, the culture medium was centrifuged at high speed, filtered through a microporous membrane under vacuum, and then loaded onto a HiTrap MabSelectSuRe column. The protein was eluted with elution buffer containing 100 mM citric acid at pH 3.5. The target sample was recovered and dialyzed to PBS at pH 7.4.

[0170] The purified protein was detected by HPLC. The HPLC chromatogram of the anti-PDL1×KDR bispecific antibody is shown below. Figure 2A As shown, the monomer purity of the anti-PDL1×KDR BsAb1 bispecific antibody reached over 96%. The spectra of the other two bispecific antibodies (anti-PDL1×KDR BsAb2, BsAb3) were similar, with monomer purity also exceeding 96%. SDS-PAGE results are shown below. Figure 2B As shown, lanes 1 and 2 represent reduced and non-reduced SDS-PAGE of anti-PDL1×KDR BsAb1, lanes 3 and 4 represent reduced and non-reduced SDS-PAGE of anti-PDL1 monoclonal antibody, lanes 5 and 6 represent reduced and non-reduced SDS-PAGE of anti-PDL1×KDR BsAb2, and lanes 7 and 8 represent reduced and non-reduced SDS-PAGE of anti-PDL1×KDR BsAb3. The theoretical molecular weight of anti-PDL1×KDR BsAb1 and anti-PDL1×KDR BsAb2 is 197 KD, and the theoretical molecular weight of anti-PDL1×KDR BsAb3 is 196 KD.

[0171] Example 3: Determination of the affinity of anti-PDL1×KDR double antibody for antigen by enzyme-linked immunosorbent assay (ELISA)

[0172] 3.1 Affinity detection with PD-L1 antigen

[0173] To detect the affinity of anti-PDL1×KDR bispecific antibody for PD-L1 antigen, the PDL1-ECD-His protein (synthesized from the sequence provided by NCBI (NCBI accession number NP_054862.1), the extracellular domain gene of PD-L1 was modified by adding a signal peptide sequence to its N-terminus and a 6×His tag to its C-terminus, constructed into expression vectors through EcoRI and HindIII restriction sites, transfected into HEK-293E cells for expression, and purified) was diluted to 2000 ng / ml in PBS buffer at pH 7.4. 100 μl / well was then added to an ELISA plate; the plate was incubated overnight at 4°C; the plate was washed twice with PBST the next day; each well was blocked with PBST + 1% BSA and incubated at 37°C for 1 h; the plate was washed twice with PBST; then, the antibody to be tested was added in serially diluted PBS + 1% BSA, with anti-PD-L1 monoclonal antibody as a positive control, starting at 300 nM and serially diluted 3-fold 12 times. Incubate at 37℃ for 1 hour; wash twice with PBST, add HRP-labeled goat anti-human Fc antibody, and incubate at 37℃ for another 40 minutes; wash three times with PBST and blot dry, add 100 μl TMB to each well, and incubate at room temperature (20±5℃) in the dark for 5 minutes; add 50 μl 2M H2SO4 stop solution to each well to terminate the substrate reaction, read the OD value at 450 nm using a microplate reader, and perform data analysis using GraphPad Prism, plotting and calculating EC50. 50 The experimental results are as follows: Figure 3A As shown, the ECGs of anti-PD-L1 monoclonal antibodies, anti-PD-L1×KDR BsAb1, anti-PD-L1×KDR BsAb2, and anti-PD-L1×KDR BsAb3... 50 The concentrations were 0.13 nM, 0.13 nM, 0.14 nM, and 0.15 nM, respectively. The affinity of the three bispecific antibodies was comparable to that of the monoclonal antibody. The anti-PD-L1 monoclonal antibody had a slightly higher plateau, possibly due to the use of anti-Fc as the secondary antibody. Experiments have shown that the ELISA results for this sample differ slightly depending on the secondary antibody used. It can be concluded that the affinity of the bispecific antibodies is not weaker than that of the monoclonal antibody.

[0174] 3.2 Affinity detection with KDR antigen

[0175] To detect the affinity of anti-PDL1×KDR bispecific antibody for KDR antigen, the KDR-ECD-His protein (synthesized from the sequence provided by UniProt (sequence number P35968), with a signal peptide sequence added to its N-terminus and a 6×His tag added to its C-terminus, constructed into an expression vector via EcoRI and HindIII restriction sites, transfected into HEK-293E cells for expression and purification) was diluted to 2000 ng / ml in PBS buffer at pH 7.4. 100 μl / well was then added to an ELISA plate and incubated overnight at 4°C. The plate was washed twice with PBST the following day. Each well was then blocked with PBST + 1% BSA and incubated at 37°C for 1 h. The plate was washed twice with PBST. Then, the antibody to be tested, serially diluted with PBS + 1% BSA, was added. The anti-KDR monoclonal antibody served as a positive control, with an initial concentration of 300 nM and 12 serial 3-fold dilutions. Incubate at 37℃ for 1 hour; wash twice with PBST, add HRP-anti-Fab antibody, and incubate at 37℃ for another 40 minutes; wash three times with PBST and blot dry, add 100 μl TMB to each well, and incubate at room temperature (20±5℃) in the dark for 5 minutes; add 50 μl 2M H2SO4 stop solution to each well to terminate the substrate reaction, read the OD value at 450 nm using a microplate reader, and perform data analysis using GraphPad Prism, plotting and calculating EC50. 50 The experimental results are as follows: Figure 3B As shown, the ECGs of anti-KDR monoclonal antibodies, anti-PDL1×KDR BsAb1, anti-PDL1×KDR BsAb2, and anti-PDL1×KDR BsAb3... 50 The specific concentrations (0.26 nM, 0.23 nM, 0.23 nM, and 0.41 nM) showed that the affinities of anti-PDL1×KDR BsAb1 and anti-PDL1×KDR BsAb2 were slightly stronger than those of the monoclonal antibody. This may be due to the use of anti-Fab as the secondary antibody. Experiments have shown that the ELISA results for this sample differ slightly depending on the secondary antibody used. Therefore, it can be considered that the affinity of the dual antibody is not weaker than that of the monoclonal antibody.

[0176] Example 4: Detection of the ability of anti-PDL1×KDR bispecific antibody to bind to two antigens simultaneously using a bispecific ELISA.

[0177] To detect the ability of anti-PDL1×KDR bispecific antibodies to bind to both KDR and PD-L1 antigens simultaneously, PDL1-ECD-hFc protein (with the C-terminus of PDL1-ECD-His protein replaced by an hFc tag) was diluted to 1 μg / ml in PBS buffer (pH 7.4), and 100 μl / well was added to each ELISA plate. The plate was incubated overnight at 4°C. The next day, the plate was washed twice with PBST. Each well was then blocked with PBST + 1% BSA and incubated at 37°C for 1 h. The plate was washed twice with PBST. Then, the antibody to be tested, serially diluted with PBS + 1% BSA, was added, starting at 12 nM and serially diluted 3-fold eight times. The plate was incubated at 37°C for 1 h. After washing twice with PBST, 100 μl / well of 1 μg / ml KDR-ECD-His antigen diluted in PBS (pH 7.4) was added to each ELISA plate. Incubate at 37℃ for 1 hour; wash twice with PBST, add secondary antibody HRP-anti-His, and incubate at 37℃ for another 40 minutes; wash three times with PBST and blot dry, add 100 μl TMB to each well, and incubate at room temperature (20±5℃) in the dark for 5 minutes; add 50 μl 2M H2SO4 stop solution to each well to terminate the substrate reaction, read the OD value at 450 nm using a microplate reader, and perform data analysis using GraphPad Prism, plotting and calculating EC50. 50 The experimental results are as follows: Figure 4 As shown, the EC50 values ​​of anti-PDL1×KDR BsAb1, anti-PDL1×KDR BsAb2, and anti-PDL1×KDR BsAb3 were 0.13 nM, 0.14 nM, and 0.20 nM, respectively. Among them, anti-PDL1×KDR BsAb3 was slightly weaker than the other two bispecific antibodies, while the monoclonal antibody did not have the ability to bind to both antigens simultaneously.

[0178] Example 5: FACS detection of the binding affinity of anti-PDL1×KDR bispecific antibody to target cells

[0179] N87-PDL1 is a stable cell line constructed in our laboratory by transfecting NCI-N87 cells with PD-L1 using a lentiviral transfection method. N87-PDL1 cells in logarithmic growth phase were digested with trypsin, washed three times with PBS containing 0.5% BSA, and centrifuged at 300g for 5 minutes each time, discarding the supernatant. The cells were resuspended in PBS with 0.5% BSA at a cell density of 1×10⁻⁶ cells / mL. 6Cells / mL, 100 μL / well added to 96-well plates. Anti-PDL1×KDR bispecific antibody and positive control anti-PD-L1 monoclonal antibody were diluted to 120 nM, serially diluted 11 times, and 100 μL / well added to each well of the 96-well plate, mixed thoroughly with N87-PDL1 cells. Incubated at 4°C for 1 h. Cells were washed twice with PBS to remove unbound antibody. Then, 100 μL / well of FITC-labeled goat anti-human Fc antibody was added, and incubated at 4°C for 30 min. Centrifuged at 300g for 5 min, and washed twice with PBS to remove unbound secondary antibody. Finally, cells were resuspended in 200 μL PBS, and the binding affinity of the bispecific antibody to the cells was determined using a Beckman Coulter CytoFLEX flow cytometer. The obtained data were analyzed using GraphPadPrism software.

[0180] Experimental results are as follows Figure 5 As shown, ECG of anti-PD-L1 monoclonal antibody 50 EC50 of anti-PDL1×KDR BsAb1 is 0.13 nM. 50 EC50 of anti-PDL1×KDR BsAb2 is 0.15 nM. 50 EC50 of anti-PDL1×KDR BsAb3 is 0.15 nM. 50 The affinity was 0.21 nM, and the three bispecific antibodies had comparable affinity to the positive control anti-PD-L1 monoclonal antibody.

[0181] Example 6: Anti-PDL1×KDR dual antibody blocks the cellular activity of PD1 / PD-L1.

[0182] This experiment used Promega's PD-1 / PD-L1 Blockade Bioassay, Propagation model, and methods.

[0183] Log-phase PD-L1 aAPC / CHO-K1 cells were trypsinized into single cells and transferred to white-bottomed 96-well plates (100 μL / well, 40,000 cells / well). The plates were incubated overnight at 37°C with 5% CO2. Anti-PDL1×KDR bispecific antibody and anti-PD-L1 monoclonal antibody were diluted to 2× working solution concentration, starting at 66 nM and increasing in 3-fold increments. Cell densities ranging from 1.4 to 2×10⁻⁶ cells / well were collected. 6 PD1 effector cells with a cell viability of over 95% were digested with trypsin to form 1.25 × 10⁻⁶ cells / mL. 6Single-cell suspension at 1 / ml. Take PD-L1aAPC / CHO-K1 cells cultured the previous day, discard the supernatant, and add 40 μl of serially diluted penicillin / PD-L1 monoclonal antibody working solution; then add an equal volume of PD1 effector cells. Incubate at 37°C, 5% CO2 for 6 hours. Add 80 μl of Bio-Glo assay reagent to each well. After incubation at room temperature for 10 minutes, read luminescence using Spectramax i3. Seal the bottom of the plate with an opaque membrane before reading. Perform data analysis, plotting, and calculating IC50 using GraphPad Prism. 50 .

[0184] Experimental results are as follows Figure 6 As shown, the IC50 of anti-PD-L1 monoclonal antibody 50 The concentration was 0.23 nM. The IC50 values ​​of the three bispecific antibodies, anti-PDL1×KDR BsAb1, anti-PDL1×KDR BsAb2, and anti-PDL1×KDR BsAb3, were 0.23 nM. 50 The concentrations were 0.27 nM, 0.22 nM, and 0.30 nM, respectively, which are comparable to those of monoclonal antibodies.

[0185] Anti-PDL1×KDR Rev3, Rev4, Rev5, Rev6, and Rev7 blocked the activity of the PD1 / PD-L1 signaling pathway on cells, and the results were as follows: Figure 8 As shown, anti-PDL1×KDR rev5 was significantly worse than the positive control anti-PD-L1 monoclonal antibody; anti-PDL1×KDR rev6 was slightly worse than the positive control anti-PD-L1 monoclonal antibody; the activities of anti-PDL1×KDR rev3, rev4, and rev7 were not significantly different from those of the positive control anti-PD-L1 monoclonal antibody.

[0186] Example 7: Anti-PDL1×KDR dual antibody blocks the activity of KDR binding to VEGF on cells.

[0187] This experiment uses Promega's VEGF Bioassay, Propagation Model, and methods.

[0188] KDR / NFAT-RE HEK293 cells express KDR on their surface. When VEGF binds to KDR on the cell surface, the signal is transduced into the cell, and a fluorescent reporter gene is expressed, resulting in a detectable biofluorescent signal. When an anti-KDR antibody is added to block the binding of VEGF to KDR on the cell surface, the fluorescence signal weakens, showing a dose-response relationship with the concentration of the KDR antibody within a certain range.

[0189] Take one bottle of KDR / NFAT-RE HEK293 cells in logarithmic growth phase cultured at T75, wash once with D-PBS, and then add 3 mL of D-PBS. The solution was digested at 37°C for approximately 2 minutes, then an equal volume of assay buffer (DMED + 10% FBS) was added for neutralization, and the cells were dispersed by pipetting. The cells were centrifuged at 200g for 5 minutes. Cells were counted using trypan blue assay, and then the cell density was adjusted to 1.6 × 10⁻⁶ cells / min. 6 VEGF was diluted with assay buffer (DMED + 10% FBS) to a 3× dilution of 60 ng / mL, resulting in a working concentration of 20 ng / mL. 25 μL / well was added to each well of a 96-well white translucent plate. The test antibody and positive control were diluted with assay buffer (DMED + 10% FBS) to a 3× dilution of 1000 nM, and then serially diluted fourfold, with 25 μL / well added to each well of the 96-well white translucent plate. The plates were incubated at 37°C with 5% CO2 for 6 hours. After incubation, 75 μL / well of Bio-Glo assay reagent was added to each well, and the plates were incubated at room temperature for 10 minutes. Luminescence was then read using a Spectramax i3. The bottom of the plates was sealed with an opaque membrane before reading. Data analysis, plotting, and IC50 calculations were performed using GraphPad Prism. 50 .

[0190] Experimental results are as follows Figure 7 As shown, the IC of anti-KDR monoclonal antibody 50 The concentration was 3.10 nM. The IC50 values ​​of the three bispecific antibodies, anti-PDL1×KDR BsAb1, anti-PDL1×KDR BsAb2, and anti-PDL1×KDR BsAb3, were 3.10 nM. 50 The activity levels were 13.47 nM, 6.09 nM, and 4.35 nM, respectively, with anti-PDL1×KDR BsAb1 being slightly weaker than anti-PDL1×KDR BsAb2 and anti-PDL1×KDR BsAb3. The activity differences between the three groups of bispecific antibodies and monoclonal antibodies were not significant.

[0191] Anti-PDL1×KDR Rev3, Rev4, Rev5, Rev6, and Rev7 blocked the activity of KDR binding to VEGF on cells, and the results were as follows: Figure 9A , 9B As shown in Figures 9C, the activities of anti-PDL1×KDR rev3, rev4, rev5, rev6, and rev7 were not significantly different from those of the positive control anti-KDR monoclonal antibody.

[0192] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> 3SBio (Shanghai) Co., Ltd. <120> A bispecific antibody against PDL1×KDR <130> P2021-1249 <150> 2020104875313 <151> 2020-06-02 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain complementarity-determining region H-CDR1 of the anti-PD-L1 monoclonal antibody <400> 1 Ser Tyr Gly Val His 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the heavy chain complementarity-determining region H-CDR2 of the anti-PD-L1 monoclonal antibody <400> 2 Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the heavy chain complementarity-determining region H-CDR3 of the anti-PD-L1 monoclonal antibody <400> 3 Gln Leu Gly Leu Arg Ala Met Asp Tyr 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of L-CDR1, the light chain complementarity-determining region of anti-PD-L1 monoclonal antibody <400> 4 Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the light chain complementarity-determining region L-CDR2 of the anti-PD-L1 monoclonal antibody <400> 5 Tyr Ala Ser Gln Ser Phe Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of L-CDR3, the light chain complementarity-determining region of the anti-PD-L1 monoclonal antibody <400> 6 Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain complementarity-determining region H-CDR1 of the anti-KDR monoclonal antibody <400> 7 Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn 1 5 10 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the heavy chain complementarity-determining region H-CDR2 of the anti-KDR monoclonal antibody <400> 8 Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the heavy chain complementarity-determining region H-CDR3 of the anti-KDR monoclonal antibody <400> 9 Val Thr Asp Ala Phe Asp Ile 1 5 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of L-CDR1, the light chain complementarity-determining region of the anti-KDR monoclonal antibody <400> 10 Thr Gly Ser His Ser Asn Phe Gly Ala Gly Thr Asp Val His 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the light chain complementarity-determining region L-CDR2 of the anti-KDR monoclonal antibody <400> 11 Gly Asp Ser Asn Arg Pro Ser 1 5 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the light chain complementarity-determining region L-CDR3 of the anti-KDR monoclonal antibody <400> 12 Gln Ser Tyr Asp Tyr Gly Leu Arg Gly Trp Val 1 5 10 <210> 13 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the heavy chain variable region of anti-PD-L1 monoclonal antibody <400> 13 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the light chain variable region of anti-PD-L1 monoclonal antibody <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 15 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the heavy chain variable region of anti-KDR monoclonal antibody <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 16 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the light chain variable region of anti-KDR monoclonal antibody <400> 16 Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val 1 5 10 15 Thr Ile Ser Cys Thr Gly Ser His Ser Asn Phe Gly Ala Gly Thr Asp 20 25 30 Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 35 40 45 His Gly Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala 65 70 75 80 Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Tyr Gly Leu Arg 85 90 95 Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 17 <211> 707 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the polypeptide chain of anti-PDL1×KDR BsAb1 <400> 17 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Leu 450 455 460 Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val Thr Ile 465 470 475 480 Ser Cys Thr Gly Ser His Ser Asn Phe Gly Ala Gly Thr Asp Val His 485 490 495 Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile His Gly 500 505 510 Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys 515 520 525 Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp 530 535 540 Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Tyr Gly Leu Arg Gly Trp 545 550 555 560 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 580 585 590 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser 595 600 605 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser 610 615 620 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 625 630 635 640 Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 645 650 655 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 660 665 670 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 675 680 685 Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu Val Thr 690 695 700 Val Ser Ser 705 <210> 18 <211> 707 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the polypeptide chain of anti-PDL1×KDR BsAb2 <400> 18 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 450 455 460 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu 465 470 475 480 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Met 485 490 495 Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser 500 505 510 Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys Gly 515 520 525 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln 530 535 540 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 545 550 555 560 Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val 565 570 575 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 580 585 590 Ser Gly Gly Gly Gly Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly 595 600 605 Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser His Ser Asn 610 615 620 Phe Gly Ala Gly Thr Asp Val His Trp Tyr Gln His Leu Pro Gly Thr 625 630 635 640 Ala Pro Lys Leu Leu Ile His Gly Asp Ser Asn Arg Pro Ser Gly Val 645 650 655 Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala 660 665 670 Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser 675 680 685 Tyr Asp Tyr Gly Leu Arg Gly Trp Val Phe Gly Gly Gly Thr Lys Leu 690 695 700 Thr Val Leu 705 <210> 19 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the light chain of anti-PD-L1 monoclonal antibody <400> 19 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 20 <211> 705 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the polypeptide chain of anti-PDL1×KDR BsAb3 <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln Ser 130 135 140 Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly 145 150 155 160 Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 165 170 175 Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser 180 185 190 Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe Lys 195 200 205 Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 225 230 235 240 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 260 265 270 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 275 280 285 Ser Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 290 295 300 Glu Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala 305 310 315 320 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 325 330 335 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 340 345 350 Tyr Tyr Cys Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly 355 360 365 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 370 375 380 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 385 390 395 400 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 405 410 415 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 420 425 430 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 435 440 445 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 450 455 460 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 465 470 475 480 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 485 490 495 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 500 505 510 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 515 520 525 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 530 535 540 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 545 550 555 560 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 565 570 575 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 580 585 590 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 595 600 605 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 610 615 620 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 625 630 635 640 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 645 650 655 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 660 665 670 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 675 680 685 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 700 Lys 705 <210> 21 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the light chain of anti-KDR monoclonal antibody <400> 21 Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val 1 5 10 15 Thr Ile Ser Cys Thr Gly Ser His Ser Asn Phe Gly Ala Gly Thr Asp 20 25 30 Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 35 40 45 His Gly Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala 65 70 75 80 Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Tyr Gly Leu Arg 85 90 95 Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser<e 210 215 <210> 22 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain of anti-PD-L1 monoclonal antibody <400> 22 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 23 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain of anti-KDR monoclonal antibody <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 24 <211> 707 <212> PRT <213> Amino acid sequence of the polypeptide chain of anti-PDL1×KDR Rev3 <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Val Leu Thr Gln Pro Pro Ser Val 130 135 140 Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser His 145 150 155 160 Ser Asn Phe Gly Ala Gly Thr Asp Val His Trp Tyr Gln His Leu Pro 165 170 175 Gly Thr Ala Pro Lys Leu Leu Ile His Gly Asp Ser Asn Arg Pro Ser 180 185 190 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 195 200 205 Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 210 215 220 Gln Ser Tyr Asp Tyr Gly Leu Arg Gly Trp Val Phe Gly Gly Gly Thr 225 230 235 240 Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 245 250 255 Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val 260 265 270 Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser 275 280 285 Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly 290 295 300 Leu Glu Trp Ile Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn 305 310 315 320 Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn 325 330 335 Gln Val Ser Phe Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val 340 345 350 Tyr Tyr Cys Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly 355 360 365 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 370 375 380 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 385 390 395 400 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 405 410 415 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 420 425 430 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 435 440 445 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 450 455 460 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 465 470 475 480 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 485 490 495 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 500 505 510 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 515 520 525 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 530 535 540 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 545 550 555 560 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 565 570 575 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 580 585 590 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 595 600 605 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 610 615 620 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 625 630 635 640 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 645 650 655 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 660 665 670 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 675 680 685 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 690 695 700 Pro Gly Lys 705 <210> 25 <211> 707 <212> PRT <213> Amino acid sequence of the polypeptide chain of anti-PDL1×KDR Rev4 <400> 25 Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val 1 5 10 15 Thr Ile Ser Cys Thr Gly Ser His Ser Asn Phe Gly Ala Gly Thr Asp 20 25 30 Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 35 40 45 His Gly Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala 65 70 75 80 Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Tyr Gly Leu Arg 85 90 95 Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly 130 135 140 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 145 150 155 160 Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 165 170 175 Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser 180 185 190 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu 195 200 205 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 210 215 220 Cys Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu 225 230 235 240 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 245 250 255 Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val 260 265 270 Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser 275 280 285 Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly 290 295 300 Leu Glu Trp Ile Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn 305 310 315 320 Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn 325 330 335 Gln Val Ser Phe Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val 340 345 350 Tyr Tyr Cys Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly 355 360 365 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 370 375 380 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 385 390 395 400 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 405 410 415 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 420 425 430 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 435 440 445 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 450 455 460 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 465 470 475 480 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 485 490 495 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 500 505 510 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 515 520 525 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 530 535 540 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 545 550 555 560 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 565 570 575 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 580 585 590 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 595 600 605 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 610 615 620 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 625 630 635 640 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 645 650 655 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 660 665 670 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 675 680 685 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 690 695 700 Pro Gly Lys 705 <210> 26 <211> 705 <212> PRT <213> Amino acid sequence of the polypeptide chain of anti-PDL1×KDR Rev5 <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val 450 455 460 Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys Glu Lys Val 465 470 475 480 Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp 485 490 495 Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala 500 505 510 Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 515 520 525 Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala 530 535 540 Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly 545 550 555 560 Ala Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly 565 570 575 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu 580 585 590 Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu 595 600 605 Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val His Trp 610 615 620 Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu Ile Trp 625 630 635 640 Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr 645 650 655 Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe Lys Ile Ser Ser 660 665 670 Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Gly 675 680 685 Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 690 695 700 Ser 705 <210> 27 <211> 705 <212> PRT <213> Amino acid sequence of the polypeptide chain of anti-PDL1×KDR Rev6 <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln 450 455 460 Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln Ser Leu Ser 465 470 475 480 Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val His 485 490 495 Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu Ile 500 505 510 Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu 515 520 525 Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe Lys Ile Ser 530 535 540 Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu 545 550 555 560 Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 565 570 575 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 580 585 590 Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Asp Phe 595 600 605 Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Arg Ala Ser 610 615 620 Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys Pro Asp Gln 625 630 635 640 Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val 645 650 655 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 660 665 670 Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 675 680 685 Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 690 695 700 Lys 705 <210> 28 <211> 705 <212> PRT <213> Amino acid sequence of the polypeptide chain of anti-PDL1×KDR Rev7 <400> 28 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser 130 135 140 Pro Asp Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys 165 170 175 Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe 180 185 190 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205 Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys 225 230 235 240 Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 260 265 270 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 275 280 285 Ser Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 290 295 300 Glu Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala 305 310 315 320 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 325 330 335 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 340 345 350 Tyr Tyr Cys Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly 355 360 365 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 370 375 380 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 385 390 395 400 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 405 410 415 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 420 425 430 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 435 440 445 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 450 455 460 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 465 470 475 480 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 485 490 495 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 500 505 510 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 515 520 525 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 530 535 540 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 545 550 555 560 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 565 570 575 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 580 585 590 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 595 600 605 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 610 615 620 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 625 630 635 640 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 645 650 655 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 660 665 670 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 675 680 685 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 700 Lys 705

Claims

1. A bispecific antibody against PDL1×KDR, characterized in that, It comprises two polypeptide chains and two light chains selected from the group consisting of: (a) The polypeptide chain comprises VH-PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH-KDR-linker1-VL-KDR from the N-terminus to the C-terminus, and the light chain comprises VL-PDL1-CL from the N-terminus to the C-terminus; or (b) The polypeptide chain comprises VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the light chain comprises VL-KDR-CL from the N-terminus to the C-terminus. The VH-PDL1 is a heavy chain variable region that binds to PD-L1. The VH-PDL1 has three complementarity-determining regions (CDRs), and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 1-3. The VL-PDL1 is a light chain variable region that binds to PD-L1. The VL-PDL1 has three complementarity-determining regions (CDRs), wherein the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 4-6. The VH-KDR is a heavy chain variable region that binds to KDR. The VH-KDR has three complementarity-determining regions (CDRs), wherein the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7-9. The VL-KDR is a light chain variable region that binds to KDR. The VL-KDR has three complementarity-determining regions (CDRs), wherein the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 10-12. Linker1 and linker2 are each independently flexible peptide linkers, CH1-CH2-CH3 is a heavy chain constant region, CL is a light chain constant region, VH-PDL1 and VL-PDL1 form an antigen-binding site that specifically binds to PD-L1, and VH-KDR and VL-KDR form an antigen-binding site that specifically binds to KDR.

2. The bispecific antibody against PDL1×KDR as described in claim 1, characterized in that, It comprises two polypeptide chains and two light chains, wherein: (a) the polypeptide chains contain VH-PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH-KDR-linker1-VL-KDR from the N-terminus to the C-terminus, and the light chains contain VL-PDL1-CL from the N-terminus to the C-terminus; or (b) The polypeptide chain comprises VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the light chain comprises VL-KDR-CL from the N-terminus to the C-terminus. Wherein, VH-PDL1 is a heavy chain variable region that incorporates PD-L1. The VL-PDL1 mentioned above is a light chain variable region that incorporates PD-L1. The VH-KDR mentioned above is a heavy chain variable region combined with KDR. The VL-KDR mentioned above is a light chain variable region combined with KDR. The linker1 consists of 4 G4S molecules, the linker2 consists of 3 G4S molecules, the CH1-CH2-CH3 region is the heavy chain constant region, the CL region is the light chain constant region, the VH-PDL1 and the VL-PDL1 form a specific antigen binding site for PD-L1, and the VH-KDR and the VL-KDR form a specific antigen binding site for KDR.

3. The bispecific antibody as described in claim 1 or 2, characterized in that, The VH-PDL1 has the amino acid sequence shown in SEQ ID NO: 13, the VL-PDL1 has the amino acid sequence shown in SEQ ID NO: 14, the VH-KDR has the amino acid sequence shown in SEQ ID NO: 15, and the VL-KDR has the amino acid sequence shown in SEQ ID NO:

16.

4. The bispecific antibody as described in claim 1 or 2, characterized in that, The polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24 or SEQ ID NO: 25, and the light chain has an amino acid sequence as shown in SEQ ID NO: 19; or the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, and the light chain has an amino acid sequence as shown in SEQ ID NO:

21.

5. The bispecific antibody as described in claim 1 or 2, characterized in that, The heavy chain constant region includes the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and the light chain constant region includes the κ or λ light chain constant region.

6. An isolated nucleotide, characterized in that, The nucleotides encode the bispecific antibody as described in claim 1 or 2.

7. An expression carrier, characterized in that, The expression vector contains the nucleotides as described in claim 6.

8. A host cell, characterized in that, The host cell contains the expression vector as described in claim 7.

9. The method for preparing the bispecific antibody as described in claim 1 or 2, characterized in that, The method includes the following steps: (a) Under expression conditions, host cells as described in claim 8 are cultured to express the bispecific antibody; (b) Isolate and purify the bispecific antibody described in (a).

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the bispecific antibody as described in claim 1 or 2 and a pharmaceutically acceptable carrier.

11. Use of the bispecific antibody of claim 1 or 2 or the pharmaceutical composition of claim 10 in the preparation of a medicament for treating cancer, wherein the cancer is selected from the group consisting of: colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma, melanoma, lung cancer, liver cancer, and prostate cancer.

12. The use as described in claim 11, wherein the lung cancer is non-small cell lung cancer.

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