Bispecific antibody targeting PD1 and VEGF

Abstract

Bispecific antibodies capable of binding to human VEGF protein and human PD-1 protein are provided. These bispecific antibodies are effective in treating cancer.

Description

[Background technology]

[0001] Vascular endothelial growth factor (VEGF) stimulates the formation of blood vessels. VEGF is involved in both vasculogenesis and angiogenesis. The normal function of VEGF is to create new blood vessels during embryonic development, after injury, in muscle after exercise, and to bypass blocked blood vessels.

[0002] VEGF may also contribute to the development of disease. Without a sufficient blood supply, solid tumors cannot grow beyond a limited size. Cancers that can express VEGF can grow and metastasize. Overexpression of VEGF can also cause vascular disease in the retina of the eye and other parts of the body. Inhibition of VEGF may be useful in the treatment of certain cancers and age-related macular degeneration.

[0003] Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a protein on the surface of T cells and B cells that is involved in controlling the immune system's response to the body's cells by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases but may also prevent the immune system from killing cancer cells.

[0004] PD-1 is an immune checkpoint that prevents autoimmunity through two mechanisms: first, PD-1 promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes; second, PD-1 reduces apoptosis of regulatory T cells (anti-inflammatory, suppressor T cells).

[0005] PD-1 ligand, PD-L1, is highly expressed in some cancers, and the role of PD1 in cancer immune evasion is well established. Monoclonal antibodies targeting PD-1 to enhance the immune system have been developed for cancer treatment. Blockade of the interaction between PD-1 and PD-L1 can enhance T cell responses and mediate antitumor activity.

[0006] Bispecific antibodies targeting both VEGF and PD-1 proteins have been proposed, but developing bispecific antibodies with good stability and activity has proven difficult. Summary of the Invention

[0007] The present disclosure provides bispecific antibodies that have binding specificity for both VEGF and PD-1 proteins. As shown in the experimental examples, these bispecific antibodies exhibit high binding affinity for both proteins and are effective in inhibiting VEGF enzymatic activity and blocking the binding of PD-1 to PD-L1, resulting in T cell activation.

[0008] One embodiment of the present disclosure provides a bispecific antibody comprising an anti-VEGF portion and an anti-PD-1 portion, wherein the anti-VEGF portion comprises an anti-VEGF antibody or antigen-binding fragment that has binding specificity for human VEGF protein, and the anti-VEGF antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:9, a CDRH2 comprising the amino acid sequence of SEQ ID NO:10, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:11, and a light chain variable region (VL) comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:12, a CDRL2 comprising the amino acid sequence of SEQ ID NO:13, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:14; and the anti-PD-1 portion comprises an anti-PD-1 single domain antibody that has binding specificity for human PD-1 protein, and the anti-PD-1 single domain antibody comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:5, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7 or 8.

[0009] In some embodiments, the VH of the anti-VEGF antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:3, and the VL of the anti-VEGF antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:4.

[0010] In some embodiments, the anti-VEGF antibody or antigen-binding fragment is a full-size Fab antibody. In some embodiments, the full-size Fab antibody comprises an IgG Fc fragment. In some embodiments, the IgG Fc fragment comprises a LALA mutation.

[0011] In some embodiments, the anti-PD-1 single domain antibody comprises the amino acid sequence of SEQ ID NO: 1 or 2.

[0012] In some embodiments, the anti-PD-1 single domain antibody is positioned C-terminally of the anti-VEGF antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 single domain antibody is fused to the C-terminus of the anti-VEGF antibody via a (G4S)4 (SEQ ID NO: 15) linker. In some embodiments, the bispecific antibody comprises one anti-PD-1 single domain antibody on each peptide chain of the bispecific antibody. In some embodiments, the bispecific antibody comprises two anti-PD-1 single domain antibodies on each peptide chain of the bispecific antibody. In some embodiments, the two anti-PD-1 single domain antibodies on each peptide chain are linked via a (G4S)4 (SEQ ID NO: 15) linker.

[0013] In one embodiment, a bispecific antibody is provided comprising the amino acid sequences of SEQ ID NOs: 16 and 17. In one embodiment, a bispecific antibody is provided comprising the amino acid sequences of SEQ ID NOs: 18 and 17. In one embodiment, a bispecific antibody is provided comprising the amino acid sequences of SEQ ID NOs: 19 and 17.

[0014] Also provided in one embodiment is a composition comprising a bispecific antibody of the present disclosure and a pharmaceutically acceptable carrier. Additionally provided is one or more polynucleotides encoding the heavy chain of the bispecific antibody of the present disclosure. In one embodiment, the one or more polynucleotides encode the bispecific antibody.

[0015]

[0010] Yet another embodiment provides a cell comprising one or more polynucleotides of the present disclosure. Another embodiment provides a method of treating cancer in a patient in need thereof, the method comprising administering to the patient the bispecific antibody. In some embodiments, the cancer is selected from the group consisting of bladder cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. [Brief explanation of the drawings]

[0016] [Figure 1] 1 shows a schematic diagram of an anti-PD1-VEGF bispecific antibody. [Figure 2] Figure 1 shows that all tested anti-PD1-VEGF bispecific antibodies bind to human VEGF protein in a concentration-dependent manner. [Figure 3] Figure 1 shows that all tested anti-PD1-VEGF bispecific antibodies bind to human PD1 protein and cynomolgus monkey PD1 protein in a concentration-dependent manner. [Figure 4] Figure 1 shows that all tested anti-PD1-VEGF bispecific antibodies bind to human PD1-overexpressing CHO-K1 cells in a concentration-dependent manner. [Figure 5] Figure 1 shows that all tested anti-PD1-VEGF bispecific antibodies concentration-dependently blocked VEGF-mediated signaling in a VEGF reporter gene assay. [Figure 6] Figure 1 shows that all tested anti-PD1-VEGF bispecific antibodies concentration-dependently blocked PD1-mediated signaling in a PD1 reporter gene assay. [Figure 7A] We show that the test anti-PD1-VEGF bispecific antibody can stimulate the production of IFN-γ in primary CD4+ T cells in a classical mixed lymphocyte reaction. [Figure 7B] We show that the test anti-PD1-VEGF bispecific antibody can stimulate IL-2 production in primary CD4+ T cells in a classical mixed lymphocyte reaction. [Figure 8]1 shows that the anti-PD1-VEGF bispecific antibody B12D1-9 inhibited VEGF-induced proliferation of HUVEC cells in a concentration-dependent manner. [Figure 9] 1 shows that the anti-PD1-VEGF bispecific antibodies B12D1-6 and B12D1-9 exhibited strong anti-tumor effects in the HuH-7 xenograft model in PBMC-humanized NOG mice. DETAILED DESCRIPTION OF THE INVENTION

[0017] definition As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody or any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule that comprises at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, a heavy or light chain complementarity-determining region (CDR) or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0018] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, or scFv. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0019] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and light chain (V LIn some embodiments, these regions are connected by a short linker peptide of 10 to about 25 amino acids. The linker may be rich in glycine to increase flexibility, or rich in serine or threonine to increase solubility, and the V H N-terminus of V L The scFv molecule can be linked to the C-terminus of the antibody or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. scFv molecules are well known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0020] A "single domain antibody" (sdAb or VHH), also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Like whole antibodies, single domain antibodies can selectively bind to specific antigens. With a molecular weight of only 12-15 kDa, single domain antibodies are much smaller than typical antibodies (150-160 kDa).

[0021] "Specifically binds" or "having specificity for" generally means that an antibody binds to an epitope via its antigen-binding domain, and that such binding requires a degree of complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope via its antigen-binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind epitope "C" with greater specificity than it does to the related epitope "D."

[0022] As used herein, the term "treat" or "treatment" refers to both therapeutic and prophylactic or preventative treatment, the purpose of which is to prevent or slow (alleviate) an undesired physiological change or disorder (such as the progression of cancer). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, diminishment of the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, remission or alleviation of the disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0023] Anti-VEGF anti-PD-1 bispecific antibody The present disclosure provides anti-VEGF, anti-PD-1 bispecific antibodies with high affinity and inhibitory activity for both human VEGF and human PD-1 proteins. These antibodies can effectively bind to both the soluble and cellular surfaces of VEGF and PD-1 at levels comparable to their parent antibodies and the benchmark anti-VEGF, anti-PD-1 bispecific antibody VP101 developed by Akeso. Surprisingly, however, one of the bispecific antibodies tested, B12D1-6, significantly outperformed VP101 (Example 5 and Figures 7A-B).

[0024] The structures of the bispecific antibodies are shown in Figure 1. In all bispecific antibodies, one (B12D1-9 and B12D1-10) or two (B12D1-6) anti-PD-1 nanobody / VHHs (VH18 or VH8M1) were fused to the C-terminus of an IgG1 antibody carrying the full-size LALA mutation of bevacizumab. Thus, B12D1-6 contained four anti-PD-1 VHHs, while B12D1-9 and B12D1-10 each contained only two anti-PD-1 VHHs.

[0025] According to one embodiment of the present disclosure, a bispecific antibody is provided that has binding specificity for both human VEGF protein and human PD-1 protein. In some embodiments, the bispecific antibody comprises an anti-VEGF portion (or anti-VEGF unit) and an anti-PD-1 portion (or anti-PD-1 unit). As used herein, the term "portion" or "unit" refers to a portion of a bispecific antibody, typically comprising one or two fragments having the desired binding activity. Each portion comprises one or more antibodies or antigen-binding fragments.

[0026] In one embodiment, the bispecific antibody has an anti-VEGF portion, which preferably comprises a Fab fragment (two VH / VL pairs) or further together with an Fc fragment to form a full-size IgG antibody. In some embodiments, the Fc fragment comprises a LALA mutation. The LALA mutation is a silencing mutation in the IgG Fc, in which the leucine (L) residues at positions 234 and 235 (EU numbering) are mutated to alanine (A). The Fc fragment can be an IgG1, IgG2, IgG3, or IgG4 Fc fragment. In some embodiments, the Fc fragment further comprises an N297A mutation at position 297 (EU numbering).

[0027] In some embodiments, the bispecific antibody has an anti-PD-1 moiety, or more specifically, one, two, three, four or more anti-PD-1 VHHs. In one example, one (or each) anti-PD-1 VHH is located N-terminal to the VH of the anti-VEGF moiety. In one example, one (or each) anti-PD-1 VHH is located N-terminal to the VL of the anti-VEGF moiety. In one example, one (or each) anti-PD-1 VHH is located C-terminal to the VL of the anti-VEGF moiety. In one example, one (or each) anti-PD-1 VHH is located C-terminal to the Fc chain (of an anti-VEGF antibody).

[0028] In some embodiments, one anti-PD-1 VHH is included in the peptide chain of the bispecific antibody. In some embodiments, exactly two anti-PD-1 VHHs are included in the peptide chain of the bispecific antibody. In some embodiments, exactly three anti-PD-1 VHHs are included in the peptide chain of the bispecific antibody.

[0029] In some embodiments, each heavy chain of the anti-VEGF antibody is fused to one anti-PD-1 VHH, such as at the N-terminus of the VH or the C-terminus of the Fc. In some embodiments, each heavy chain of the anti-VEGF antibody is fused to two anti-PD-1 VHHs, such as both at the N-terminus of the VH or the C-terminus of the Fc (linked). In some embodiments, each heavy chain of the anti-VEGF antibody is fused to one anti-PD-1 VHH at the N-terminus of the VH or to another anti-PD-1 VHH at the C-terminus of the Fc.

[0030] In some embodiments, the anti-PD-1 VHH is fused to the anti-VEGF moiety via a peptide linker. An exemplary linker is (G4S)4 (SEQ ID NO: 15). When two anti-PD-1 VHHs are linked (e.g., B12D-6), they can be linked via a peptide. An exemplary linker is (G4S)4 (SEQ ID NO: 15).

[0031] Exemplary anti-VEGF antibodies are described. In one embodiment, the anti-VEGF antibody comprises the CDR regions of bevacizumab (SEQ ID NOS: 9-14) shown in Table 1A. In some embodiments, the anti-VEGF portion comprises a Fab fragment of bevacizumab (the VH (SEQ ID NO: 3) and VL (SEQ ID NO: 4) sequences are shown in Figure 1).

[0032] Exemplary anti-PD-1 single domain antibodies are also described. In one embodiment, the anti-PD-1 single domain antibody comprises the three CDR regions of VH18 (SEQ ID NOs: 5, 6, and 7). In one embodiment, the anti-PD-1 single domain antibody comprises the three CDR regions of VH8M1 (SEQ ID NOs: 5, 6, and 8). In some embodiments, the anti-PD-1 single domain antibody comprises the sequence of VH18 (SEQ ID NO: 1). In some embodiments, the anti-PD-1 single domain antibody comprises the sequence of VH8M1 (SEQ ID NO: 2).

[0033] Specific examples of heavy and light chain sequences are shown in Table 1B. In one embodiment, the bispecific antibody comprises two heavy chains, each having the amino acid sequence of SEQ ID NO: 16, and two light chains, each having the amino acid sequence of SEQ ID NO: 17.

[0034] In one embodiment, the bispecific antibody comprises two heavy chains each having the amino acid sequence of SEQ ID NO: 18 and two light chains each having the amino acid sequence of SEQ ID NO: 17.

[0035] In one embodiment, the bispecific antibody comprises two heavy chains each having the amino acid sequence of SEQ ID NO: 19 and two light chains each having the amino acid sequence of SEQ ID NO: 17.

[0036] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibody of the present disclosure may comprise a flexible linker sequence or may be modified to attach a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0037] The antibodies, variants, or derivatives of the present disclosure include derivatives that are modified, i.e., modified by the covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, but not by limitation, antibodies can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, antibodies can contain one or more non-classical amino acids.

[0038] In some embodiments, the antibody may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0039] The antibody may be conjugated or fused to a therapeutic agent, which may include a detectable label such as a radiolabel, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or toxin, an ultrasound-enhancing agent, a non-radioactive label, combinations thereof, and other such agents known in the art.

[0040] An antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0041] Polynucleotides encoding antibodies and methods for preparing antibodies The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants, or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules.

[0042] Methods for producing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be produced using techniques described in the art and described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous gene locus has been disabled. Exemplary techniques that can be used to produce such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated by reference in their entireties.

[0043] Treatment method As described herein, the antibodies, variants or derivatives of the present disclosure may be used in certain therapeutic and diagnostic methods.

[0044] Thus, in some embodiments, methods of treating cancer in a patient in need thereof are provided. In one embodiment, the method involves administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one of the patient's cancer cells (e.g., stromal cells) overexpresses VEGF. In some embodiments, at least one of the patient's cancer cells (e.g., stromal cells) overexpresses PD-1.

[0045] Non-limiting examples of cancer include bladder cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0046] Cell therapy, and more specifically, chimeric antigen receptor (CAR) T cell therapy, is also provided in the present disclosure. Suitable T cells contacted with the bispecific antibody of the present disclosure (or alternatively engineered to express the bispecific antibody of the present disclosure) can be used. After such contact or engineering, the T cells can then be introduced into a cancer patient in need of treatment. The cancer patient can have any of the types of cancer disclosed herein. The T cells can be, for example, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

[0047] In some embodiments, the T cells are isolated from the cancer patient themselves. In some embodiments, the T cells are provided by a donor or from a cell bank. When the T cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0048] Additional diseases or conditions associated with increased cell viability that may be treated, prevented, diagnosed, and / or predicted with the antibodies or variants or derivatives thereof of the present disclosure include malignancies and leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroid leukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and, but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, spondyloma, angiosarcoma, endothelial tumors, and leukemias. These include, but are not limited to, progression and / or metastasis of related disorders such as solid tumors, including sarcomas and carcinomas such as sarcoma, lymphangiosarcoma, lymphangioendothelioma scleroma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, semimoma, embryonal tumor, Wells tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0049] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular antibody, variant, or derivative thereof used, the patient's age, weight, general health, sex, and diet, time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by a medical caregiver is within the skill of the art. The amount will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0050] Methods of administration of antibodies, variants, or derivatives include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral administration. The antigen-binding polypeptides or compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous layers (e.g., oral, rectal, and intestinal mucosa, etc.), or may be administered in conjunction with other biologically active agents. Thus, pharmaceutical compositions containing antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, drops, or transdermal patches), buccally, or as an oral or nasal spray.

[0051] As used herein, the term "parenteral" refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0052] Administration can be systemic or local. Furthermore, it may be desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer and formulation with an aerosolizing agent.

[0053] It may be desirable to administer an antigen-binding polypeptide or composition of the present disclosure locally to the area in need of treatment. This can be achieved, for example, but not limited to, by local infusion during surgery, by topical application, e.g., in combination with a wound dressing after surgery, by injection, by catheter, by suppository, or by implant, which is an implant of porous, non-porous, or gelatinous material, including membranes such as sialastic membranes, or fibers. Preferably, when administering proteins, including antibodies of the present disclosure, care must be taken to use materials to which the protein does not absorb.

[0054] The amount of an antibody of the present disclosure effective for treating, suppressing, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. In addition, in vitro assays may optionally be used to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease, disorder, or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0055] As a general proposition, the dosage of an antigen-binding polypeptide of the present disclosure administered to a patient is typically 0.1 mg / kg to 100 mg / kg of the patient's body weight, 0.1 mg / kg to 20 mg / kg of the patient's body weight, or 1 mg / kg to 10 mg / kg of the patient's body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to the foreign polypeptide. Therefore, lower dosages of human antibodies and less frequent administration are often possible. Furthermore, the dosage and frequency of administration of antibodies of the present disclosure can be reduced by enhancing antibody uptake (e.g., into the brain) and tissue penetration, for example, by modifications such as lipidation.

[0056] Methods for treating infectious or malignant diseases, conditions, or disorders involving the administration of an antibody, variant, or derivative thereof of the present disclosure are typically tested in vitro in an acceptable animal model, followed by in vivo testing, for the desired therapeutic or prophylactic activity prior to use in humans. Suitable animal models, including transgenic animals, are well known to those of skill in the art. For example, in vitro assays for demonstrating the therapeutic utility of the antigen-binding polypeptides described herein include the effect of the antigen-binding polypeptide on a cell line or a patient tissue sample. The effect of the antigen-binding polypeptide on a cell line and / or tissue sample can be determined using techniques well known to those of skill in the art, such as the assays disclosed elsewhere herein. According to the present disclosure, in vitro assays that can be used to determine whether administration of a particular antigen-binding polypeptide is indicated include in vitro cell culture assays in which a patient tissue sample is grown in culture and exposed to or otherwise administered a compound, and the effect of such compound on the tissue sample is observed.

[0057] Various delivery systems are known and can be used to administer an antibody of the present disclosure or a polynucleotide encoding an antibody of the present disclosure, including, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.

[0058] In further embodiments, the compositions of the present disclosure are administered in combination with an antitumor agent, an antiviral agent, an antibacterial or antibiotic agent, or an antifungal agent. Any of these agents known in the art can be administered in the compositions of the present disclosure.

[0059] In another embodiment, the compositions of the present disclosure are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the compositions of the present disclosure include antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin), antiestrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine), cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate). estramustine phosphate), hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone), nitrogen mustard derivatives (e.g., mephalen, colambucil, mechlorethamine (nitrogen mustard), and thiotepa), steroids and combinations (e.g., betamethasone sodium phosphate), and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0060] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines, including, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0061] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

[0062] composition The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0063] In specific embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, and more specifically, in humans, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia. Moreover, a "pharmaceutically acceptable carrier" will generally be a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid of any type.

[0064] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when pharmaceutical compositions are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates, if desired. Antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for adjusting tonicity such as sodium chloride or dextrose are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions may be formulated as suppositories, using traditional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with an appropriate amount of carrier so as to provide a proper dosage form for the patient. The formulation should be appropriate for the mode of administration. The formulation stock may be contained in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0065] In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in an airtightly sealed container such as an ampoule or sachet indicating the amount of active ingredient. When the composition is administered by infusion, it can be dispensed into an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0066] The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. [Example]

[0067] Example 1: Generation of anti-PD1 / VEGF bispecific antibodies Two anti-PD1 nanobodies, VH18 and VH8M1, were each fused to the anti-VEGF antibody bevacizumab to generate anti-PD1-VEGF bispecific antibodies.

[0068] The bispecific antibody was a full-length IgG-VHH fusion antibody: the anti-VEGF moiety was a complete IgG moiety, and two anti-PD1 nanobodies were attached to the C-terminal side of an IgG1 Fc fragment carrying the LALA mutation via a (G4S)4 linker (SEQ ID NO: 15).

[0069] Three bispecific antibodies were prepared, including B12D1-6, B12D1-9, and B12D1-10. Their structures are shown in Figure 1. As shown in the figure, B12D1-9 and B12D1-10 each contained a single copy of an anti-PD1 nanobody (sdAb) at the C-terminus of each heavy chain. In contrast, B12D1-6 contained two linked anti-PD1 nanobodies. The linker between the two nanobodies in B12D1-6 was also (G4S)4 (SEQ ID NO: 15).

[0070] The antibody sequences are shown in Table 1. [Table 1] [Table 2] [Table 3-1] [Table 3-2]

[0071] The resulting bispecific antibodies were transiently produced in CHO-K1 cells and subjected to in vitro characterization including PD1 binding, VEGF binding, and cell-based functional assays.

[0072] Example 2: Antigen binding of anti-PD1-VEGF bispecific antibodies In this example, the antigen-binding activity of the bispecific antibody of Example 1 against PD1 and VEGF was evaluated.

[0073] ELISA binding to VEGF To test the VEGF-binding activity of the anti-PD1-VEGF bispecific antibodies, microtiter plates were coated overnight at 4°C with 100 μl of 0.5 μg / ml human VEGF protein diluted in PBS and then blocked with 300 μl / well of 3% BSA. Three-fold dilutions of antibody starting at 100 nM were added to each well and incubated for 1 hour at 25°C. The plates were washed with ELISA wash buffer (1x DPBS containing 0.5% Tween-20) and then incubated with a peroxidase-conjugated anti-human IgG secondary antibody for 60 minutes at 25°C. After washing, the plates were developed with TMB substrate and analyzed by a microplate reader at OD 450 nm. The anti-PD1-VEGF antibody VP101 developed by Akeso and bevacizumab were included as reference antibodies in this assay.

[0074] As shown in Figure 2 and Table 2, the anti-PD1-VEGF antibodies (B12D1-6, B12D1-9, and B12D1-10) efficiently bound to VEGF in a concentration-dependent manner, and they showed binding activity comparable to that of the reference VP101 and the parent antibody bevacizumab. [Table 4]

[0075] ELISA binding to PD1 To evaluate the PD1-binding activity of the test bispecific antibodies, human PD1 and cynomolgus monkey PD1 diluted in PBS were each coated onto a microplate overnight at 4°C. Three-fold dilutions of the test antibody, starting at 100 nM, were added to each well and incubated at 25°C for 1 hour. The plate was then washed with ELISA wash buffer (1x DPBS containing 0.5% Tween-20) and incubated with a peroxidase-conjugated anti-human IgG secondary antibody for 60 minutes at 25°C. After washing four times with ELISA wash buffer, the plate was detected with TMB-ELISA substrate solution, then stopped with ELISA stop solution, and analyzed by a microplate reader at OD 450 nm. Pembrolizumab and the anti-PD1 parent antibody VH18 were included as reference antibodies in this study.

[0076] As shown in Figure 3 and Table 3A-b, the anti-PD1-VEGF antibodies (B12D1-6 and B12D1-9) had EC values ​​comparable to the parental antibody and the reference antibody VP101: 0.078 nM for B12D1-6 and 0.169 nM for B12D1-9. 50 B12D1-6 and B12D1-9 bound to human PD1 in a concentration-dependent manner at 100 ng / mL. With regard to cynomolgus monkey PD1 binding, B12D1-6 and B12D1-9 showed similar activity to VP101, but were less potent than the parent antibody and pembrolizumab. [Table 5] [Table 6]

[0077] Cell-based binding to PD1 To further determine the PD-1 binding activity of the bispecific antibodies, CHO-K1 cells overexpressing human PD-1 were incubated with different concentrations of the anti-PD1-VEGF bispecific antibody and reference antibody for 30 minutes at 4°C. The cells were then washed twice with FACS buffer and stained with a PE-conjugated secondary antibody for 30 minutes at 4°C. After washing twice with FACS buffer, the MFI of PE was analyzed using a NovoCyte flow cytometer. In this study, the reference antibodies included VP101, pembrolizumab, and the anti-PD-1 parent nanobody.

[0078] As shown in Figure 4 and Table 4, the anti-PD1-VEGF antibodies (B12D1-6, B12D1-9, and B12D1-10) bound to human PD-1-overexpressing CHO-K1 cells in a concentration-dependent manner. B12D1-6 had a binding EC 50 showed. [Table 7]

[0079] Example 3: VEGF neutralizing activity in a VEGF reporter assay To assess the ability of anti-PD1-VEGF bispecific antibodies to neutralize soluble VEGF, a VEGF reporter gene assay was used.

[0080] In this assay, HEK293 cells were engineered to stably express VEGFR2 and harbor an NK-kB luciferase reporter construct. Soluble VEGF at a concentration of 10 ng / ml was incubated with the HEK293 reporter cells in the presence of serial concentrations of bispecific or reference antibodies for 6 hours at 37°C. Luciferase substrate was then added, and luminescence intensity was measured using a microplate reader.

[0081] As shown in Figure 5 and Table 5, the bispecific antibodies (B12D1-6, B12D1-9, and B12D1-10) had EC values ​​comparable to those of the reference VP101 and the parent anti-VEGF antibody bevacizumab: 0.344 nM for B12D1-6, 0.493 nM for B12D1-9, and 0.567 nM for B12D1-10. 50 effectively blocked VEGF function in a concentration-dependent manner. [Table 8]

[0082] Example 4: PD1 blocking activity in PD1 reporter assay To assess the efficacy of anti-PD1-VEGF bispecific antibodies in enhancing T cell activation, a robust in vitro functional PD-1 reporter gene assay was used in this example.

[0083] Briefly, human PD1 and a luciferase reporter gene under the control of an NFAT response element were simultaneously overexpressed in Jurkat T cells, and PDL1 and the antigen-independent TCR stimulator OKT3 were overexpressed in CHO-K1 cells. When these two cell types were cocultured, negative signaling delivered to Jurkat cells by PD1-PDL1 ligation suppressed OKT3-mediated TCR activation and NFAT-driven luciferase gene expression.

[0084] As shown in Figure 6 and Table 6, the addition of serially diluted anti-PD1-VEGF bispecific antibodies effectively enhanced the luminescence signal from Jurkat-PD1 cells. The PD1-blocking activity of antibody B12D1-6 was similar to that of the commercially available antibody pembrolizumab and superior to that of the reference antibody VP101. B12D1-9 showed activity comparable to that of VP101. [Table 9]

[0085] Example 5: Stimulation of IFN-γ and IL-2 release by activated CD4+ T cells in a mixed lymphocyte reaction The effect of anti-PD1-VEGF bispecific antibodies on primary CD4+ T cells was further tested using PBMCs from healthy donors.

[0086] Briefly, human dendritic cells (DCs) were differentiated from CD14+ monocytes for 7 days. Purified CD4+ T cells isolated from another donor were co-cultured with DCs for 5 days in the presence of serially diluted test antibodies. Culture supernatants were collected on days 2 and 5, respectively. The concentrations of IL-2 (day 2) and IFN-γ (day 5) in the supernatants were measured using standard ELISA kits.

[0087] As shown in Figure 7A-B, the anti-PD1-VEGF bispecific antibodies (B12D1-6 and B12D1-9) were able to stimulate the production of IFN-γ and IL-2 in primary CD4+ T cells in a concentration-dependent manner. Notably, the T cell activation potency of B12D1-6 was superior to that of VP101 and B12D1-9.

[0088] Example 6: VEGF-induced inhibition of primary HUVEC cell proliferation To evaluate the activity of anti-PD1-VEGF bispecific antibodies to inhibit VEGF-dependent proliferation of primary HUVEC cells, HUVEC cells were incubated with different concentrations of test or reference antibodies in the presence of VEGF protein for 5 days at 37°C in a 5% CO2 incubator. After incubation, CellTiter-Glo was added to the mixture, and the luminescence intensity was then measured using a microplate reader.

[0089] As shown in Figure 8, the anti-PD1-VEGF bispecific antibody B12D1-9 inhibited VEGF-induced HUVEC cell proliferation in a concentration-dependent manner. The IC 50 was estimated to be 0.907 nM (Table 7). The potency is comparable to that of the reference antibodies VP101 and bevacizumab. [Table 10]

[0090] Example 7: In vivo anti-tumor efficacy of anti-PD1-VEGF bispecific antibodies in the HuH-7 humanized NOG mouse model To evaluate the antitumor efficacy of the anti-PD1-VEGF bispecific antibody, we used a CDX tumor model using human PBMC-engrafted NOG mice inoculated with HuH-7 tumor cells.

[0091] 5 x 10 human PBMCs into humanized NOG mice 6 HuH-7 cells were subcutaneously implanted. The average tumor volume was 60 mm 3 When tumors grew to 1000 mcg / kg, tumor-bearing mice were randomized into four groups (N=6 / group) and intraperitoneally administered vehicle (DPBS) or test antibodies. Test antibodies B12D1-6, B12D1-9, and VP101 were administered twice weekly at 10 mg / kg, 8.77 mg / kg, and 10 mg / kg, respectively. The doses were equal, less than nmol / kg. Tumor volume was monitored three times weekly by caliper measurement during the experimental period. As shown in Figure 9, all test antibodies had significant antitumor effects against HuH-7, and the efficacy of B12D1-6 and B12D1-9 was comparable to that of the reference antibody VP101. * * *

[0092] The present disclosure should not be limited in scope by the specific embodiments described, which are intended as single descriptions of individual aspects of the disclosure; any structures or methods that are functionally equivalent are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and structures of the present disclosure without departing from the spirit or scope of the disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure provided they fall within the scope of the appended claims and their equivalents.

[0093] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A bispecific antibody comprising an anti-VEGF moiety and an anti-PD-1 moiety, The bispecific antibody is a bispecific antibody containing the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO:

18.

2. (i) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 17, (ii) A second polypeptide containing the amino acid sequence of SEQ ID NO: 18, (iii) A third polypeptide comprising the amino acid sequence of Sequence ID No. 18, and (iv) A fourth polypeptide containing the amino acid sequence of SEQ ID NO: 17, A bispecific antibody containing [specific antibody].

3. The bispecific antibody according to claim 2, wherein the first polypeptide and the second polypeptide are linked to each other via disulfide bonds, the second polypeptide and the third polypeptide are linked to each other via two disulfide bonds, and the third polypeptide and the fourth polypeptide are linked to each other via disulfide bonds.

4. A composition comprising the bispecific antibody described in claim 1 and a pharmaceutically acceptable carrier.

5. (i) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 18 or (ii) the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO:

18.

6. (i) one or more polynucleotides encoding the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 18, or (ii) one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 17 and one or more polynucleotides encoding the amino acid sequence of SEQ ID NO:

18.

7. A pharmaceutical composition for treating cancer in patients requiring cancer treatment, comprising an effective amount of the bispecific antibody described in claim 1.

8. The pharmaceutical composition according to claim 7, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

9. The pharmaceutical composition according to claim 7, wherein the cancer is bladder cancer.

10. The pharmaceutical composition according to claim 7, wherein the cancer is breast cancer.

11. The pharmaceutical composition according to claim 7, wherein the cancer is colorectal cancer.

12. The pharmaceutical composition according to claim 7, wherein the cancer is head and neck cancer.

13. The pharmaceutical composition according to claim 7, wherein the cancer is kidney cancer.

14. The pharmaceutical composition according to claim 7, wherein the cancer is lung cancer.

15. The pharmaceutical composition according to claim 7, wherein the cancer is melanoma.

16. The cell according to claim 6, wherein the cell is a CHO-K1 cell.

17. A method for producing a bispecific antibody according to claim 1, comprising: (i) expressing one or more polynucleotides encoding the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 18 in CHO cells; or (ii) expressing one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 17 and one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 18 in CHO cells.

18. The bispecific antibody according to claim 1, wherein the bispecific antibody is produced in CHO-K1 cells.

19. The bispecific antibody according to claim 1, wherein the bispecific antibody is produced by (i) expressing one or more polynucleotides encoding the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 18 in CHO-K1 cells, or (ii) expressing one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 17 and one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 18 in CHO-K1 cells.

20. A bispecific antibody comprising an anti-VEGF moiety and an anti-PD-1 moiety, The bispecific antibody is a bispecific antibody containing the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO:

16.

21. (i) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 17, (ii) A second polypeptide containing the amino acid sequence of SEQ ID NO: 16, (iii) A third polypeptide comprising the amino acid sequence of SEQ ID NO: 16, and (iv) A fourth polypeptide containing the amino acid sequence of SEQ ID NO: 17, A bispecific antibody containing [specific antibody].

22. The bispecific antibody according to claim 21, wherein the first polypeptide and the second polypeptide are linked to each other via disulfide bonds, the second polypeptide and the third polypeptide are linked to each other via two disulfide bonds, and the third polypeptide and the fourth polypeptide are linked to each other via disulfide bonds.

23. A composition comprising the bispecific antibody described in claim 20 and a pharmaceutically acceptable carrier.

24. (i) the amino acid sequence of SEQ ID NO: 16 or (ii) a polynucleotide encoding the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO:

16.

25. (i) one or more polynucleotides encoding the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 16, or (ii) one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 17 and one or more polynucleotides encoding the amino acid sequence of SEQ ID NO:

16.

26. The cell according to claim 25, wherein the cell is a CHO-K1 cell.

27. A method for producing a bispecific antibody according to claim 20, comprising: (i) expressing one or more polynucleotides encoding the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 16 in CHO cells; or (ii) expressing one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 17 and one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 16 in CHO cells.

28. The bispecific antibody according to claim 20, wherein the bispecific antibody is produced in CHO-K1 cells.

29. The bispecific antibody according to claim 20, wherein the bispecific antibody is produced by (i) expressing one or more polynucleotides encoding the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 16 in CHO-K1 cells, or (ii) expressing one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 17 and one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 16 in CHO-K1 cells.

30. A pharmaceutical composition for treating cancer in patients requiring cancer treatment, comprising an effective amount of the bispecific antibody described in claim 20.

31. The pharmaceutical composition according to claim 30, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

32. The pharmaceutical composition according to claim 30, wherein the cancer is bladder cancer.

33. The pharmaceutical composition according to claim 30, wherein the cancer is breast cancer.

34. The pharmaceutical composition according to claim 30, wherein the cancer is colorectal cancer.

35. The pharmaceutical composition according to claim 30, wherein the cancer is head and neck cancer.

36. The pharmaceutical composition according to claim 30, wherein the cancer is kidney cancer.

37. The pharmaceutical composition according to claim 30, wherein the cancer is lung cancer.

38. The pharmaceutical composition according to claim 30, wherein the cancer is melanoma.

39. A bispecific antibody comprising an anti-VEGF moiety and an anti-PD-1 moiety, The bispecific antibody is a bispecific antibody containing the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO:

19.

40. (i) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 17, (ii) A second polypeptide containing the amino acid sequence of SEQ ID NO: 19, (iii) A third polypeptide comprising the amino acid sequence of SEQ ID NO: 19, and (iv) A fourth polypeptide containing the amino acid sequence of SEQ ID NO: 17, A bispecific antibody containing [specific antibody].

41. The bispecific antibody according to claim 40, wherein the first polypeptide and the second polypeptide are linked to each other via disulfide bonds, the second polypeptide and the third polypeptide are linked to each other via two disulfide bonds, and the third polypeptide and the fourth polypeptide are linked to each other via disulfide bonds.

42. A composition comprising the bispecific antibody described in claim 39 and a pharmaceutically acceptable carrier.

43. (i) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 19 or (ii) the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO:

19.

44. (i) one or more polynucleotides encoding the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 19, or (ii) one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 17 and one or more polynucleotides encoding the amino acid sequence of SEQ ID NO:

19.

45. The cell according to claim 44, wherein the cell is a CHO-K1 cell.

46. A method for producing a bispecific antibody according to claim 39, comprising: (i) expressing one or more polynucleotides encoding the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 19 in CHO cells; or (ii) expressing one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 17 and one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 19 in CHO cells.

47. The bispecific antibody according to claim 39, wherein the bispecific antibody is produced in CHO-K1 cells.

48. The bispecific antibody according to claim 39, wherein the bispecific antibody is produced by (i) expressing one or more polynucleotides encoding the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 19 in CHO-K1 cells, or (ii) expressing one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 17 and one or more polynucleotides encoding the amino acid sequence of SEQ ID NO: 19 in CHO-K1 cells.

49. A pharmaceutical composition for treating cancer in patients requiring cancer treatment, comprising an effective amount of the bispecific antibody described in claim 39.

50. The pharmaceutical composition according to claim 49, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

51. The pharmaceutical composition according to claim 49, wherein the cancer is bladder cancer.

52. The pharmaceutical composition according to claim 49, wherein the cancer is breast cancer.

53. The pharmaceutical composition according to claim 49, wherein the cancer is colorectal cancer.

54. The pharmaceutical composition according to claim 49, wherein the cancer is head and neck cancer.

55. The pharmaceutical composition according to claim 49, wherein the cancer is kidney cancer.

56. The pharmaceutical composition according to claim 49, wherein the cancer is lung cancer.

57. The pharmaceutical composition according to claim 49, wherein the cancer is melanoma.

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