Bispecific antibodies targeting PD1 and VEGF

Abstract

The present invention provides bispecific antibodies that target PD1 and VEGF. Bispecific antibodies capable of binding to a human VEGF protein and a human PD-1 protein are provided. These bispecific antibodies are effective in the treatment of cancer.

Description

[0001] This application is a divisional application of application number 202380083500.3, filed on December 8, 2023, entitled "Bispecific Antibody Targeting PD1 and VEGF". Background Technology

[0002] Vascular endothelial growth factor (VEGF) stimulates angiogenesis. VEGF participates in angiogenesis and vascularization. The normal functions of VEGF include generating new blood vessels during embryonic development, generating new blood vessels after injury, generating muscles after exercise, and generating new blood vessels to bypass blocked blood vessels.

[0003] VEGF may also contribute to disease progression. Solid tumors cannot grow beyond a certain size without sufficient blood supply. Cancers that express VEGF can grow and metastasize. Furthermore, overexpression of VEGF can lead to vascular diseases of the retina and other parts of the body. Inhibiting VEGF can be used to treat certain cancers and age-related macular degeneration.

[0004] Programmed cell death protein 1, also known as PD-1 and CD279 (differentiation cluster 279), is a protein on the surface of T cells and B cells. It plays a role in regulating the immune system's response to human cells by downregulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This may prevent autoimmune diseases, but it may also prevent the immune system from killing cancer cells.

[0005] PD-1 is an immune checkpoint that prevents autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressor T cells).

[0006] PD-L1 is a ligand of PD-1, which is highly expressed in various cancers, and therefore the role of PD-1 in cancer immune evasion is well-established. Monoclonal antibodies targeting PD-1 that enhance the immune system are being developed for cancer treatment. Inhibiting the interaction between PD-1 and PD-L1 can enhance T cell responses and mediate anti-tumor activity.

[0007] Bispecific antibodies targeting VEGF and PD-1 proteins have been proposed, but developing bispecific antibodies with good stability and activity has proven challenging. Summary of the Invention

[0008] This disclosure provides bispecific antibodies that bind to both VEGF and PD-1 proteins. As shown in the experimental examples, these bispecific antibodies exhibit high binding affinity to both proteins and effectively inhibit VEGF enzyme activity and block the binding of PD-1 to PD-L1, thereby leading to T cell activation.

[0009] One embodiment of this disclosure provides a bispecific antibody comprising an anti-VEGF moiety and an anti-PD-1 moiety, wherein the anti-VEGF moiety comprises an anti-VEGF antibody or antigen-binding fragment having binding specificity to human VEGF protein, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH), said heavy chain variable region comprising CDRH1 containing the amino acid sequence of SEQ ID NO:9, CDRH2 containing the amino acid sequence of SEQ ID NO:10, and CDRH3 containing the amino acid sequence of SEQ ID NO:11; and a light chain variable region (VL), said light chain variable region comprising CDRL1 containing the amino acid sequence of SEQ ID NO:12, CDRL2 containing the amino acid sequence of SEQ ID NO:13, and CDRL3 containing the amino acid sequence of SEQ ID NO:14, and wherein the anti-PD-1 moiety comprises an anti-PD-1 single-domain antibody having binding specificity to human PD-1 protein, said single-domain antibody comprising CDR1 containing the amino acid sequence of SEQ ID NO:5, CDR2 containing the amino acid sequence of SEQ ID NO:6, and CDRL3 containing the amino acid sequence of SEQ ID NO:14. CDR3 for the amino acid sequence of NO:7 or 8.

[0010] In some embodiments, the VH of the anti-VEGF antibody or antigen-binding fragment contains the amino acid sequence of SEQ ID NO:3, and the VL of the anti-VEGF antibody or antigen-binding fragment contains the amino acid sequence of SEQ ID NO:4.

[0011] 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 an LALA mutation.

[0012] In some implementations, the anti-PD-1 single-domain antibody comprises the amino acid sequence of SEQ ID NO:1 or 2.

[0013] In some embodiments, the anti-PD-1 single-domain antibody is located at the C-terminus of the anti-VEGF antibody or its antigen-binding fragment. 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 a single anti-PD-1 single-domain antibody on each peptide chain. In some embodiments, the bispecific antibody comprises two anti-PD-1 single-domain antibodies on each peptide chain. 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.

[0014] In one embodiment, a bispecific antibody is provided comprising the amino acid sequences of SEQ ID NO:16 and 17. In one embodiment, a bispecific antibody is provided comprising the amino acid sequences of SEQ ID NO:18 and 17. In one embodiment, a bispecific antibody is provided comprising the amino acid sequences of SEQ ID NO:19 and 17.

[0015] In one embodiment, a composition comprising the bispecific antibody of this disclosure and a pharmaceutically acceptable carrier is also provided. One or more polynucleotides encoding the heavy chain of the bispecific antibody of this disclosure are also provided. In one embodiment, the one or more polynucleotides encode the bispecific antibody.

[0016] Another embodiment provides cells comprising one or more polynucleotides of the present disclosure. Another embodiment provides a method of treating cancer in a patient in need, comprising administering a bispecific antibody to the patient. In some embodiments, 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. Attached Figure Description

[0017] Figure 1 A schematic diagram showing the anti-PD1-VEGF bispecific antibody.

[0018] Figure 2 The results showed that all tested anti-PD1-VEGF bispecific antibodies bound to human VEGF protein in a concentration-dependent manner.

[0019] Figure 3 The results showed that all tested anti-PD1-VEGF bispecific antibodies bound to the PD1 protein in humans and cynomolgus monkeys in a concentration-dependent manner.

[0020] Figure 4The results showed that all tested anti-PD1-VEGF bispecific antibodies bound to human PD1-overexpressing CHO-K1 cells in a concentration-dependent manner.

[0021] Figure 5 The results showed that all tested anti-PD1-VEGF bispecific antibodies blocked VEGF-mediated signaling in a concentration-dependent manner during VEGF reporter gene assays.

[0022] Figure 6 The results showed that all tested anti-PD1-VEGF bispecific antibodies blocked PD1-mediated signaling in a concentration-dependent manner in the PD1 reporter gene assay.

[0023] Figure 7A -B shows that the anti-PD1-VEGF bispecific antibody can stimulate the production of IFN-γ and IL-2 in primary CD4+ T cells in a classic mixed lymphocyte reaction.

[0024] Figure 8 The results showed that the anti-PD-1-VEGF bispecific antibody B12D1-9 inhibited VEGF-induced HUVEC cell proliferation in a concentration-dependent manner.

[0025] Figure 9 The results showed that the anti-PD-1-VEGF bispecific antibodies B12D1-6 and B12D1-9 exhibited strong anti-tumor efficacy in the HuH-7 xenograft model of PBMC humanized NOG mice. Detailed Implementation

[0026] 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 complete antibody or any antigen-binding fragment or single chain thereof. Therefore, the term “antibody” includes any molecule containing a protein or peptide that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. Examples of such molecules include, but are not limited to, complementarity-determining regions (CDRs) or ligand-binding portions of the heavy or light chain, variable regions of the heavy or light chain, constant regions of the heavy or light chain, framework (FR) regions or any portion thereof, or at least a portion of a binding protein.

[0027] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror-image aptamers, and biantibodies. The term "antibody fragment" also includes any synthetic or genetically modified protein that functions like an antibody by binding to a specific antigen to form a complex.

[0028] "Single-chain variable fragment" or "scFv" refers to the variable region (V) of the heavy chain of immunoglobulins. H ) and light chain variable region (V L A fusion protein of ( ). In some aspects, the regions are linked by short linker peptides of 10 to 25 amino acids. The linkers may be glycine-rich for flexibility and serine or threonine-rich for solubility, and may carry V H N-terminus and V L The C-terminus is linked, and vice versa. Despite the removal of the constant region and the introduction of a linker, this protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and described, for example, in U.S. Patent 5,892,019.

[0029] Single-domain antibodies (sdAbs or VHHs), also known as nanobodies, are antibody fragments composed of a single monomeric variable antibody domain. Like intact antibodies, they can selectively bind to specific antigens. Single-domain antibodies have a molecular weight of only 12-15 kDa, much smaller than ordinary antibodies (150-160 kDa).

[0030] "Specific binding" or "specific to" generally means that an antibody binds to an epitope through its antigen-binding domain, and that this binding requires some 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 is easier for the antibody to bind to an epitope through its antigen-binding domain than to a random, unrelated epitope. This article uses the term "specificity" to define the relative affinity of an antibody for a given epitope. For example, antibody "A" can be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" can be said to have higher specificity for binding to epitope "C" than for binding to a related epitope "D."

[0031] As used herein, the term "treat" or "treatment" refers to therapeutic treatment and protective or preventative measures aimed at preventing or slowing (alleviating) unwanted physiological changes or conditions, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, stabilization (i.e., no worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean extended survival compared to expected survival without treatment. Those who require treatment include those who already have a disease or condition, those who are susceptible to a disease or condition, and those who will take preventative measures against a disease or condition.

[0032] Anti-VEGF and anti-PD-1 bispecific antibody This disclosure provides a bispecific anti-VEGF and anti-PD-1 antibody with high affinity and inhibitory activity against both human VEGF and PD-1 proteins. The antibody binds efficiently to soluble and cell surface VEGF and PD-1, with binding levels comparable to their parent antibodies and to the benchmark anti-VEGF and anti-PD-1 bispecific antibody VP101 developed by Akeso. Surprisingly, however, one of the tested bispecific antibodies, B12D1-6, is significantly superior to VP101 (Example 5 and...). Figure 7A -B).

[0033] Structural diagram of bispecific antibodies is shown in Figure 1 In all bispecific antibodies, one (B12D1-9 and B12D1-10) or two (B12D1-6) anti-PD-1 nanobodies / VHHs (VH18 or VH8M1) are fused to the C-terminus of a full-size, LALA-mutated IgG1 antibody against bevacizumab. Therefore, B12D1-6 contains four anti-PD-1 VHHs, while B12D1-9 and B12D1-10 each contain only two anti-PD-1 VHHs.

[0034] According to one embodiment of this disclosure, a bispecific antibody with binding specificity to both human VEGF and PD-1 proteins is provided. In some embodiments, the bispecific antibody comprises an anti-VEGF moiety (or anti-VEGF unit) and an anti-PD-1 moiety (or anti-PD-1 unit). As used herein, the terms "miety" or "unit" refer to a portion of the bispecific antibody, which typically comprises one or both fragments having the desired binding activity. Each portion comprises one or more antibody or antigen-binding fragments.

[0035] In one embodiment, the bispecific antibody has an anti-VEGF moiety, which preferably comprises a Fab fragment (two VH / VL pairs), or further forms a full-size IgG antibody with an Fc fragment. In some embodiments, the Fc fragment includes an LALA mutation. The LALA mutation is a silent mutation in IgG Fc where leucine (L) residues at positions 234 and 235 (EU number) are mutated to alanine (A). The Fc fragment can be an IgG1, IgG2, IgG3, or IgG4 Fc fragment. In some embodiments, the Fc fragment also includes an N297A mutation at position 297 (EU number).

[0036] 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 embodiment, one (or each) anti-PD-1 VHH is located on the N-terminal side of the VH of the anti-VEGF moiety. In one embodiment, one (or each) anti-PD-1 VHH is located on the N-terminal side of the VL of the anti-VEGF moiety. In one embodiment, one (or each) anti-PD-1 VHH is located on the C-terminal side of the VL of the anti-VEGF moiety. In one embodiment, one (or each) anti-PD-1 VHH is located on the C-terminal side of the Fc chain (of the anti-VEGF antibody).

[0037] In some embodiments, the peptide chain of the bispecific antibody includes a single anti-PD-1 VHH. In some embodiments, the peptide chain of the bispecific antibody includes exactly two anti-PD-1 VHHs. In some embodiments, the peptide chain of the bispecific antibody includes exactly three anti-PD-1 VHHs.

[0038] In some embodiments, each heavy chain of the anti-VEGF antibody is fused to an anti-PD-1 VHH, for example, 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, for example, both at the N-terminus of the VH or the C-terminus of the Fc (in tandem). In some embodiments, each heavy chain of the anti-VEGF antibody is fused to a single 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.

[0039] In some embodiments, anti-PD-1 VHHs are 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 tandemly linked (e.g., B12D-6), they can be linked via a peptide linker. An exemplary linker is (G4S)4 (SEQ ID NO:15).

[0040] Exemplary anti-VEGF antibodies are described. In one embodiment, the anti-VEGF antibody includes the CDR region (SEQ ID NO: 9-14) of bevacizumab, which is shown in Table 1A. In some embodiments, the anti-VEGF portion includes the Fab fragment of bevacizumab (VH (SEQ ID NO: 3) and VL (SEQ ID NO: 4) sequences shown in Table 1A. Figure 1 middle).

[0041] Exemplary anti-PD-1 single-domain antibodies are also described. In one embodiment, the anti-PD-1 single-domain antibody includes three CDR regions of VH18 (SEQ ID NO: 5, 6, and 7). In one embodiment, the anti-PD-1 single-domain antibody includes three CDR regions of VH8M1 (SEQ ID NO: 5, 6, and 8). In some embodiments, the anti-PD-1 single-domain antibody includes the sequence of VH18 (SEQ ID NO: 1). In some embodiments, the anti-PD-1 single-domain antibody includes the sequence of VH8M1 (SEQ ID NO: 2).

[0042] Table 1B provides specific exemplary heavy and light chain sequences. 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.

[0043] 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.

[0044] 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.

[0045] In some embodiments, the antibody comprises an amino acid sequence or one or more portions that do not normally associate with the antibody. Exemplary modifications are described in more detail below. For example, the antibodies of this disclosure may comprise a flexible linker sequence or may be modified to add a functional portion (e.g., PEG, drug, toxin, or label).

[0046] The antibodies, variants, or derivatives thereof disclosed herein include modified derivatives, i.e., by covalently linking any type of molecule to the antibody such that the covalent link does not prevent the antibody from binding to the epitope. For example, but not limited to, antibodies can be modified by: glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization via known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Any of many chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, antibodies may contain one or more non-classical amino acids.

[0047] In some implementations, antibodies may be conjugated with therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceuticals, or PEG.

[0048] Antibodies may be conjugated or fused with therapeutic agents, which may include detectable markers such as radioactive markers, immunomodulators, hormones, enzymes, oligonucleotides, photosensitizing therapeutics or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancers, non-radioactive markers, combinations thereof, and other such agents known in the art.

[0049] Antibodies can be detectably labeled by conjugation to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding peptide is then determined by detecting the presence of light emitted during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, thermoacridonium ester, imidazole, acridineonium salts, and oxalates.

[0050] Polynucleotides encoding antibodies and methods for preparing antibodies This disclosure also provides isolated polynucleotide or nucleic acid molecules encoding antibodies, variants, or derivatives thereof. The polynucleotides of this 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 individual polynucleotide molecules. Furthermore, the polynucleotides of this 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 individual polynucleotide molecules.

[0051] Methods for manufacturing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptide disclosed herein are fully human. Fully human antibodies can be manufactured using techniques described in the art and as 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 attack, but with the endogenous locus disabled. Exemplary techniques that can be used to manufacture such antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140, ​​the entire contents of which are incorporated herein by reference.

[0052] Treatment As described herein, the antibodies, variants, or derivatives disclosed herein may be used in certain therapeutic and diagnostic methods.

[0053] Therefore, in some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method involves administering an effective amount of the disclosed antibody to the patient. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient overexpresses VEGF. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient overexpresses PD-1.

[0054] Non-limiting examples of cancer include 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.

[0055] This disclosure also provides cell therapy, and more specifically chimeric antigen receptor (CAR) T-cell therapy. Suitable T cells can be used, which are contacted with (or alternatively modified to express) the bispecific antibodies of this disclosure. Through such contact or modification, the T cells can be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer as disclosed herein. The T cells may be, for example, but not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0056] In some implementations, T cells are isolated from the cancer patient themselves. In other implementations, T cells are provided by a donor or cell bank. When T cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0057] The antibodies or variants or derivatives thereof disclosed herein may be used to treat, prevent, diagnose, and / or prognose other diseases or conditions associated with increased cell survival, including but not limited to the progression and / or metastasis of malignancies and related conditions, such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including medulloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia. Macroglobulinemia, heavy chain diseases, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, 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 gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, and Wilm's tumor. Tumors, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0058] The specific dosage and treatment regimen for any given patient will depend on a variety of factors, including the specific antibody, variant, or derivative thereof used, the patient's age, weight, general health condition, sex, and diet, as well as the timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of healthcare professionals regarding these factors is within the realm of ordinary skill in the art. The dosage 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 dosage used can be determined using pharmacological and pharmacokinetic principles well known in the art.

[0059] The methods of administration of antibodies, variants, or other similar substances include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides or compositions can be administered via any convenient route, such as by infusion or concentrated injection, absorption through the epithelial or mucosal skin layer (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides of this disclosure can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, percutaneously (e.g., via powder, ointment, drops, or transdermal patch), buccally, or as oral or nasal sprays.

[0060] As used in this article, “parenteral” refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0061] Administration can be systemic or local. Furthermore, it may be necessary to introduce the antibodies of this disclosure into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be achieved, for example, via an intraventricular catheter connected to a reservoir (e.g., an Ommaya reservoir). Lung administration may also be used, for example, by using an inhaler or nebulizer, and formulations with nebulizing agents.

[0062] It may be necessary to apply the antigen-binding peptides or compositions of this disclosure topically to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion during surgery, percutaneous application such as in conjunction with postoperative wound dressings, by injection, via catheter, via suppositories, or via implants that are porous, non-porous, or gel-like materials, including membranes such as salivary elastic membranes or fibers. Preferably, when applying the proteins (including antibodies) of this disclosure, care must be taken to use materials that do not absorb the proteins.

[0063] The amount of the disclosed antibody that can effectively treat, inhibit, and prevent inflammatory, immune, or malignant diseases, conditions, or disorders can be determined using standard clinical techniques. Additionally, in vitro assays may be optionally used to help identify the optimal dose range. The precise dose to be used in the formulation will also depend on the route of administration, the severity of the disease, condition, or disorder, and should be determined based on the physician's judgment and the individual patient's situation. The effective dose can be deduced from dose-response curves obtained from in vitro or animal model testing systems.

[0064] As a general recommendation, the dose of the antigen-binding peptide of this disclosure administered to patients is typically 0.1 mg / kg to 100 mg / kg patient body weight, 0.1 mg / kg to 20 mg / kg patient body weight, or 1 mg / kg to 10 mg / kg patient body weight. Generally, due to the immune response to foreign peptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower doses and lower frequencies of administration of human antibodies are usually possible. Furthermore, the dosage and frequency of administration of the antibodies of this disclosure can be reduced by modifying (e.g., lipidation) to enhance antibody uptake and tissue penetration (e.g., into the brain).

[0065] Methods for treating infectious or malignant diseases, ailments, or conditions (including the administration of antibodies, variants, or derivatives thereof disclosed herein) are typically tested in vitro and then in vivo in acceptable animal models to obtain the desired therapeutic or preventative activity before being used in humans. Suitable animal models (including transgenic animals) are well known to those skilled in the art. For example, in vitro assays demonstrating the therapeutic efficacy of the antigen-binding peptides described herein include the effect of the antigen-binding peptide on cell lines or patient tissue samples. The effect of the antigen-binding peptide on cell lines and / or tissue samples can be determined using techniques known to those skilled in the art, such as assays disclosed elsewhere herein. In vitro assays that can be used to determine whether to instruct the administration of a specific antigen-binding peptide according to this disclosure include in vitro cell culture assays in which patient tissue samples are grown in a culture and exposed to or otherwise administered the compound, and the effect of such compound on the tissue sample is observed.

[0066] Various delivery systems are known and can be used to administer the antibodies of this disclosure or the polynucleotides encoding the antibodies of this disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, and receptor-mediated endocytosis (see, for example, Wu and Wu, 1987). J. Biol. Chem (262:4429-4432), constructing nucleic acids as part of retroviral vectors or other vectors, etc.

[0067] In another embodiment, the compositions of this disclosure are administered in combination with an antitumor agent, antiviral agent, antibacterial agent, antibiotic, or antifungal agent. Any of these agents known in the art may be administered in the compositions of this disclosure.

[0068] In another embodiment, the compositions disclosed herein are administered in combination with a chemotherapeutic agent. Chemotherapy agents that can be administered in conjunction with the compositions disclosed herein include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); anti-estrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, fluorouridine, interferon alpha-2b, glutamate, plicamycin, mercaptopurine, and 6-thioguanine); and cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arbinoside, cyclophosphamide, estradiol mustard, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate). Sulfate); hormones (e.g., medroxyprogesterone, estradiol sodium phosphate, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol, chlorestradiol and testosterone); nitrogen mustard derivatives (e.g., mephalen, chlorambucil, nitrogen mustard and thiotepa); steroids and combinations (e.g., betamethasone sodium phosphate); and other chemotherapeutic agents (e.g., dacarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate and etoposide).

[0069] In another embodiment, the compositions of this disclosure are administered in combination with cytokines. Cytokines that can be administered with the compositions of this disclosure include, but are 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.

[0070] In other embodiments, the compositions disclosed herein are administered in combination with other treatment or preventative measures (e.g., radiotherapy).

[0071] Composition This disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0072] In the specific implementation plan, the term "pharmaceutical acceptable" means a substance approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia (US Pharmacopeia) or other recognized pharmacopoeia, for use in animals, and more specifically, for use in humans. Furthermore, a "pharmaceutical acceptable carrier" will typically be a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation adjuvant.

[0073] The term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; and agents for modulating tension, such as sodium chloride or dextrose. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated into suppositories with conventional binders and carriers (e.g., triglycerides). Oral formulations may include standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in a purified form, and an appropriate amount of carrier to provide a suitable form of administration to the patient. The formulation should be suitable for the mode of administration. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0074] In the embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to standard procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer and a local anesthetic (e.g., lidocaine) to reduce pain at the injection site. Typically, the components are provided separately or mixed together in unit doses as a dry lyophilized powder or anhydrous concentrate in, for example, an airtight container (e.g., an ampoule or capsule), indicating the amount of active agent. When the composition is intended for administration by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is intended for administration by injection, a sterile ampoule of water or saline for injection can be provided so that the components can be mixed prior to administration.

[0075] The compounds disclosed herein can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions (e.g., anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.) and those formed with cations (e.g., cations derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

[0076] Example Example 1: Generation of anti-PD1 / VEGF bispecific antibody Each of two anti-PD1 nanobodies, VH18 and VH8M1, was fused with the anti-VEGF antibody bevacizumab to generate an anti-PD1-VEGF bispecific antibody.

[0077] The bispecific antibody is a full-length IgG-VHH fusion. The anti-VEGF portion is the complete IgG portion, while the two anti-PD1 nanobodies are each positioned at the C-terminus of the IgG1 Fc fragment with the LALA mutation via a (G4S)4 linker (SEQ ID NO:15).

[0078] Three bispecific antibodies were prepared, including B12D1-6, B12D1-9, and B12D1-10. Their structural diagrams are illustrated in [the diagram]. Figure 1 In the figure, B12D1-9 and B12D1-10 each contain a single copy of an anti-PD1 nanobody (sdAb) at the C-terminus of each heavy chain. In contrast, B12D1-6 contains two anti-PD1 nanobodies tandemly. The linker between the two nanobodies in B12D1-6 is also (G4S)4 (SEQ ID NO:15).

[0079] Table 1 shows the antibody sequences.

[0080] Table 1. Antibody Sequences Table 1A. CDR Sequences Table 1B. Bispecific antibody sequences The resulting bispecific antibodies were transiently generated in CHO-K1 cells and used for in vitro characterization, including PD1 binding, VEGF binding, and cell-based functional assays.

[0081] Example 2: Antigen binding of anti-PD1-VEGF bispecific antibody This embodiment evaluates the antigen-binding activity of the bispecific antibody of Example 1 against PD1 and VEGF.

[0082] ELISA with VEGF To test the VEGF binding activity of the anti-PD1-VEGF bispecific antibody, microtiter plates were coated overnight at 4°C with 0.5 mg / ml human VEGF protein diluted in 100 ml of PBS, followed by blocking with 300 ml / well of 3% BSA. Three-fold dilutions of antibody, starting at 100 nM, were added to each well and incubated at 25°C for 1 hour. The plates were washed with ELISA wash buffer (1×DPBS containing 0.5% Tween-20) and then incubated at 25°C for 60 minutes with peroxidase-conjugated anti-human IgG secondary antibody. After washing, the plates were developed with TMB substrate and analyzed at OD 450 nm using a microplate reader. In this assay, the anti-PD1-VEGF antibody VP101, developed by Akeso and bevacizumab, was included as a reference antibody.

[0083] like Figure 2 As shown in Table 2, the anti-PD1-VEGF antibodies (B12D1-6, B12D1-9, and B12D1-10) bind efficiently to VEGF in a concentration-dependent manner. They exhibit binding activity comparable to the baseline VP101 and the parental antibody bevacizumab.

[0084] Table 2. Human VEGF ELISA binding Combined with PD1 ELISA To evaluate the PD1 binding activity of the bispecific antibodies, human PD1 and cynomolgus monkey PD1 diluted in PBS were coated onto microplates overnight at 4°C. Trifold dilutions of the test antibody, starting at 100 nM, were added to each well and incubated at 25°C for 1 hour. The plates were then washed with ELISA wash buffer (1×DPBS containing 0.5% Tween-20) and incubated with peroxidase-conjugated anti-human IgG secondary antibody at 25°C for 60 minutes. After four washes with ELISA wash buffer, the plates were tested using TMB-ELISA substrate solution, terminated with ELISA stop solution, and analyzed at OD 450 nm using a microplate reader. Pembrolizumab and the anti-PD1 parental antibody VH18 were used as reference antibodies in this study.

[0085] like Figure 3 As shown in Tables 3A-b, anti-PD1-VEGF antibodies (B12D1-6 and B12D1-9) bind to human PD1 in a concentration-dependent manner, EC2. 50 The potency (for B12D1-6) was 0.078 nM and (for B12D1-9) was 0.169 nM, comparable to the parental antibody and the benchmark VP101. Regarding cynomolgus monkey PD1 binding, B12D1-6 and B12D1-9 showed similar activity to VP101, but lower potency compared to the parental antibody and pembrolizumab.

[0086] Table 3A. Human PD1 ELISA binding Table 3B. Cynomolgus monkey PD1 ELISA binding PD1 binding based on cells To further determine the PD-1 binding activity of the bispecific antibodies, CHO-K1 cells overexpressing human PD-1 were incubated at 4°C for 30 min with different concentrations of anti-PD1-VEGF bispecific antibody and a reference antibody. The cells were then washed twice with FACS buffer and stained with PE-conjugated secondary antibodies at 4°C for 30 min. After washing twice with FACS buffer, the molecular weight fraction (MFI) of the PE was analyzed using a NovoCyte flow cytometer. In this study, baseline VP101, pembrolizumab, and anti-PD-1 parental nanobodies were used as reference antibodies.

[0087] like Figure 4 As shown in Table 4, 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 showed similar binding to EC1 cells as pembrolizumab.50 .

[0088] Table 4. Cell-based human PD1 binding Example 3: VEGF neutralizing activity in VEGF reporter gene assay To evaluate the ability of the anti-PD1-VEGF bispecific antibody to neutralize soluble VEGF, a VEGF reporter gene assay was used.

[0089] In this assay, HEK293 cells were modified to stably express VEGFR2 and possess an NK-kB luciferase reporter gene construct integrated into the genome. 10 ng / ml of soluble VEGF was incubated with the HEK293 reporter gene cells at 37°C for 6 hours in the presence of a continuous concentration of bispecific antibody or reference antibody. Then, the luciferase substrate was added, and the luminescence intensity was measured using a microplate reader.

[0090] like Figure 5 As shown in Table 5, the bispecific antibodies (B12D1-6, B12D1-9, and B12D1-10) effectively blocked VEGF function in a concentration-dependent manner, EC50. 50 The levels were 0.344 nM (for B12D1-6), 0.493 nM (for B12D1-9), and 0.567 nM (for B12D1-10), comparable to the baseline VP101 and the anti-VEGF parental antibody bevacizumab.

[0091] Table 5. VEGF reporter gene assay Example 4: PD1 blocking activity in PD1 reporter gene assay To evaluate the role of the anti-PD1-VEGF bispecific antibody in enhancing T cell activation, this example used a robust in vitro functional PD-1 reporter gene assay.

[0092] In short, the study aimed to simultaneously overexpress human PD1 and the luciferase reporter gene, both under the control of NFAT response elements, on Jurkat T cells, while overexpressing PDL1 and the antigen-independent TCR stimulant OKT3 on CHO-K1 cells. When these two cell types were co-cultured, negative signaling delivered on Jurkat cells via the PD1-PDL1 linker inhibited OKT3-mediated TCR activation and NFAT-driven luciferase gene expression.

[0093] like Figure 6As shown in Table 6, the addition of serially diluted anti-PD1-VEGF bispecific antibody effectively enhanced the luminescent signal in 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 benchmark VP101. B12D1-9 exhibited activity comparable to VP101.

[0094] Table 6. PD1 blocking activity Example 5: Stimulation of IFN-γ and IL-2 release by activated CD4+ T cells in a mixed lymphocyte response The effects of the anti-PD1-VEGF bispecific antibody on primary CD4+ T cells were further investigated using PBMCs from healthy donors.

[0095] In short, 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 at days 2 and 5. The concentrations of IL-2 (day 2) and IFN-γ (day 5) in the supernatant were measured using a standard ELISA kit.

[0096] like Figure 7A As shown in -B, anti-PD1-VEGF bispecific antibodies (B12D1-6 and B12D1-9) can stimulate the production of IFN-γ and IL-2 in primary CD4+ T cells in a concentration-dependent manner. Notably, B12D1-6 exhibits superior T cell activation potency compared to VP101 and B12D1-9.

[0097] Example 6: Inhibition of VEGF-induced proliferation of primary HUVEC cells To evaluate the inhibitory activity of the anti-PD1-VEGF bispecific antibody on the proliferation of VEGF-dependent primary HUVEC cells, HUVEC cells were incubated with different concentrations of test antibody or reference antibody in the presence of VEGF protein at 37°C in a 5% CO2 incubator for 5 days. After incubation, CellTiter-Glo was added to the mixture, and the luminescence intensity was then measured using a microplate reader.

[0098] like Figure 8 As shown, the anti-PD-1-VEGF bispecific antibody B12D1-9 inhibited VEGF-induced HUVEC cell proliferation in a concentration-dependent manner. The IC50 of B12D1-9... 50 The estimated value is 0.907 nM (Table 7). The potency is comparable to that of the reference antibody VP101 and bevacizumab.

[0099] Table 7. Inhibition of VEGF-induced cell proliferation Example 7: In vivo antitumor efficacy of anti-PD1-VEGF bispecific antibody in HuH-7 humanized NOG mouse model To evaluate the anti-tumor efficacy of the anti-PD1-VEGF bispecific antibody, a CDX tumor model of NOG mice transplanted with human PBMCs inoculated with HuH-7 tumor cells was used.

[0100] Subcutaneous implantation of 5×10⁶ PBMC-derived NOG mice 6 One HuH-7 cell. When the average tumor volume grows to 60 mm 3 Mice with tumors were randomly divided into four groups (N=6 / group) and administered either a mediator (DPBS) or test antibodies intraperitoneally. Test antibodies B12D1-6, B12D1-9, and VP101 were administered twice weekly at doses of 10 mg / kg, 8.77 mg / kg, and 10 mg / kg, respectively. These doses were equal and expressed in nmol / kg. Tumor volume was monitored three times weekly using a caliper during the experiment. Figure 9 As shown, all tested antibodies exhibited significant antitumor efficacy against HuH-7, and the efficacy of B12D1-6 and B12D1-9 was comparable to that of the reference antibody VP101.

[0101] The present invention also includes the following embodiments: 1. A bispecific antibody comprising an anti-VEGF moiety and an anti-PD-1 moiety. The anti-VEGF portion comprises an anti-VEGF antibody or antigen-binding fragment having binding specificity to human VEGF protein. The antibody or antigen-binding fragment comprises a heavy chain variable region (VH), which includes CDRH1 containing the amino acid sequence of SEQ ID NO:9, CDRH2 containing the amino acid sequence of SEQ ID NO:10, and CDRH3 containing the amino acid sequence of SEQ ID NO:11; and a light chain variable region (VL), which includes CDRL1 containing the amino acid sequence of SEQ ID NO:12, CDRL2 containing the amino acid sequence of SEQ ID NO:13, and CDRL3 containing the amino acid sequence of SEQ ID NO:14. The anti-PD-1 portion comprises an anti-PD-1 single-domain antibody that has binding specificity to human PD-1 protein, wherein the single-domain antibody comprises CDR1 containing the amino acid sequence of SEQ ID NO:5, CDR2 containing the amino acid sequence of SEQ ID NO:6, and CDR3 containing the amino acid sequence of SEQ ID NO:7 or 8.

[0102] 2. The bispecific antibody as described in Embodiment 1, wherein 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.

[0103] 3. The bispecific antibody as described in Embodiment 1 or 2, wherein the anti-VEGF antibody or antigen-binding fragment is a full-size Fab antibody.

[0104] 4. The bispecific antibody as described in Embodiment 3, wherein the full-size Fab antibody comprises an IgG Fc fragment.

[0105] 5. The bispecific antibody as described in Embodiment 4, wherein the IgG Fc fragment contains an LALA mutation.

[0106] 6. The bispecific antibody as described in any one of embodiments 1 to 5, wherein the anti-PD-1 single-domain antibody comprises the amino acid sequence of SEQ ID NO: 1 or 2.

[0107] 7. The bispecific antibody as described in any one of embodiments 1 to 6, wherein the anti-PD-1 single-domain antibody is located on the C-terminal side of the anti-VEGF antibody or its antigen-binding fragment.

[0108] 8. The bispecific antibody as described in Embodiment 7, wherein 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.

[0109] 9. The bispecific antibody as described in Embodiment 7 or 8, wherein each peptide chain of the bispecific antibody comprises a single anti-PD-1 single-domain antibody.

[0110] 10. The bispecific antibody as described in Embodiment 7 or 8, wherein each peptide chain of the bispecific antibody comprises two anti-PD-1 single-domain antibodies.

[0111] 11. The bispecific antibody as described in Embodiment 10, wherein the two anti-PD-1 single-domain antibodies on each peptide chain are linked by a (G4S)4 (SEQ ID NO:15) adapter.

[0112] 12. The bispecific antibody as described in Embodiment 1, comprising the amino acid sequences of SEQ ID NO:16 and 17.

[0113] 13. The bispecific antibody as described in Embodiment 1, comprising the amino acid sequences of SEQ ID NO:18 and 17.

[0114] 14. The bispecific antibody as described in Embodiment 1, comprising the amino acid sequences of SEQ ID NO:19 and 17.

[0115] 15. A composition comprising a bispecific antibody as described in any one of embodiments 1 to 14 and a pharmaceutically acceptable carrier.

[0116] 16. One or more polynucleotides encoding a heavy chain of a bispecific antibody as described in any one of embodiments 1 to 14.

[0117] 17. One or more polynucleotides as described in Embodiment 16, which encode the bispecific antibody.

[0118] 18. A cell comprising one or more polynucleotides as described in embodiment 16 or 17.

[0119] 19. A method of treating cancer in a patient in need, comprising administering to the patient a bispecific antibody as described in any one of embodiments 1 to 14.

[0120] 20. The method of embodiment 19, 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.

[0121] * * * The scope of this disclosure is not limited to the specific embodiments described, which are intended as a single illustration of individual aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. Those skilled in the art will appreciate that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations thereof, provided they fall within the scope of the appended claims and their equivalents.

[0122] All publications and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

1. A bispecific antibody comprising: (i) An anti-VEGF antibody or its antigen-binding fragment, comprising: (a) A Fab fragment, wherein the Fab fragment binds to human VEGF protein and comprises a heavy chain variable region (VH), the heavy chain variable region comprising CDRH1 containing the amino acid sequence of SEQ ID NO:9, CDRH2 containing the amino acid sequence of SEQ ID NO:10, and CDRH3 containing the amino acid sequence of SEQ ID NO:11; and a light chain variable region (VL), the light chain variable region comprising CDRL1 containing the amino acid sequence of SEQ ID NO:12, CDRL2 containing the amino acid sequence of SEQ ID NO:13, and CDRL3 containing the amino acid sequence of SEQ ID NO:14; and (b) IgG Fc fragment; (ii) A first anti-PD-1 single-domain antibody (sbAb), the first anti-PD-1 sbAb binding to human PD-1 protein and comprising CDR1 containing the amino acid sequence of SEQ ID NO:5, CDR2 containing the amino acid sequence of SEQ ID NO:6, and CDR3 containing the amino acid sequence of SEQ ID NO:7; and (iii) A second anti-PD-1 sbAb, which binds to human PD-1 protein and comprises CDR1 containing the amino acid sequence of SEQ ID NO:5, CDR2 containing the amino acid sequence of SEQ ID NO:6, and CDR3 containing the amino acid sequence of SEQ ID NO:

7. The first anti-PD-1 sbAb and the second anti-PD-1 sbAb are respectively fused with the anti-VEGF antibody or its antigen-binding fragment at the C-terminus of the IgG Fc fragment.

2. A bispecific antibody comprising: (i) An anti-VEGF antibody or its antigen-binding fragment, comprising: (a) A Fab fragment, wherein the Fab fragment binds to human VEGF protein and comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:3 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:4; and (b) IgG Fc fragment; (ii) A first anti-PD-1 sbAb, wherein the first anti-PD-1 sbAb binds to human PD-1 protein and contains the amino acid sequence of SEQ ID NO: 1; and (iii) A second anti-PD-1 sbAb, which binds to human PD-1 protein and contains the amino acid sequence of SEQ ID NO:

1. The first anti-PD-1 sbAb and the second anti-PD-1 sbAb are respectively fused with the anti-VEGF antibody or its antigen-binding fragment at the C-terminus of the IgG Fc fragment.

3. A bispecific antibody comprising: (i) An anti-VEGF antibody or its antigen-binding fragment, comprising: (a) A Fab fragment, wherein the Fab fragment binds to human VEGF protein and comprises a heavy chain variable region (VH) comprising CDRH1 containing the amino acid sequence of SEQ ID NO:9, CDRH2 containing the amino acid sequence of SEQ ID NO:10, and CDRH3 containing the amino acid sequence of SEQ ID NO:11; and a light chain variable region (VL) comprising CDRL1 containing the amino acid sequence of SEQ ID NO:12, CDRL2 containing the amino acid sequence of SEQ ID NO:13, and CDRL3 containing the amino acid sequence of SEQ ID NO:14; (b) IgG Fc fragment; (ii) A first anti-PD-1 sbAb, wherein the first anti-PD-1 sbAb binds to human PD-1 protein and comprises CDR1 containing the amino acid sequence of SEQ ID NO:5, CDR2 containing the amino acid sequence of SEQ ID NO:6 and CDR3 containing the amino acid sequence of SEQ ID NO:

7. (iii) A second anti-PD-1 sbAb, which binds to human PD-1 protein and comprises CDR1 containing the amino acid sequence of SEQ ID NO:5, CDR2 containing the amino acid sequence of SEQ ID NO:6, and CDR3 containing the amino acid sequence of SEQ ID NO:7; (iv) A third anti-PD-1 sbAb, said third anti-PD-1 sbAb binding to human PD-1 protein and comprising CDR1 containing the amino acid sequence of SEQ ID NO:5, CDR2 containing the amino acid sequence of SEQ ID NO:6, and CDR3 containing the amino acid sequence of SEQ ID NO:7; and (v) A fourth anti-PD-1 sbAb, said fourth anti-PD-1 sbAb binding to human PD-1 protein and comprising CDR1 containing the amino acid sequence of SEQ ID NO:5, CDR2 containing the amino acid sequence of SEQ ID NO:6, and CDR3 containing the amino acid sequence of SEQ ID NO:7, and The first anti-PD-1 sbAb and the second anti-PD-1 sbAb are respectively fused to the anti-VEGF antibody or its antigen-binding fragment at the C-terminus of the IgG Fc fragment, and The third anti-PD-1 sbAb is fused with the first anti-PD-1 sbAb at the C-terminus of the first anti-PD-1 sbAb, and The fourth anti-PD-1 sbAb is fused with the second anti-PD-1 sbAb at the C-terminus of the first anti-PD-1 sbAb.

4. A bispecific antibody comprising: (i) An anti-VEGF antibody or its antigen-binding fragment, comprising: (a) A Fab fragment, wherein the Fab fragment binds to human VEGF protein and comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:3 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:4; and (b) IgG Fc fragment; (ii) A first anti-PD-1 sbAb, wherein the first anti-PD-1 sbAb binds to human PD-1 protein and contains the amino acid sequence of SEQ ID NO: 1; (iii) A second anti-PD-1 sbAb, which binds to human PD-1 protein and contains the amino acid sequence of SEQ ID NO:1; (iv) A third anti-PD-1 sbAb, said third anti-PD-1 sbAb binding to human PD-1 protein and comprising the amino acid sequence of SEQ ID NO: 1; and (v) A fourth anti-PD-1 sbAb, said fourth anti-PD-1 sbAb binding to human PD-1 protein and containing the amino acid sequence of SEQ ID NO:1, and The first anti-PD-1 sbAb and the second anti-PD-1 sbAb are respectively fused to the anti-VEGF antibody or its antigen-binding fragment at the C-terminus of the IgG Fc fragment, and The third anti-PD-1 sbAb is fused with the first anti-PD-1 sbAb at the C-terminus of the first anti-PD-1 sbAb, and The fourth anti-PD-1 sbAb is fused with the second anti-PD-1 sbAb at the C-terminus of the second anti-PD-1 sbAb.

5. A bispecific antibody comprising: (i) An anti-VEGF antibody or its antigen-binding fragment, comprising: (a) A Fab fragment, wherein the Fab fragment binds to human VEGF protein and comprises a heavy chain variable region (VH), the heavy chain variable region comprising CDRH1 containing the amino acid sequence of SEQ ID NO:9, CDRH2 containing the amino acid sequence of SEQ ID NO:10, and CDRH3 containing the amino acid sequence of SEQ ID NO:11; and a light chain variable region (VL), the light chain variable region comprising CDRL1 containing the amino acid sequence of SEQ ID NO:12, CDRL2 containing the amino acid sequence of SEQ ID NO:13, and CDRL3 containing the amino acid sequence of SEQ ID NO:14; and (b) IgG Fc fragment; (ii) A first anti-PD-1 sbAb, wherein the first anti-PD-1 sbAb binds to human PD-1 protein and comprises CDR1 containing the amino acid sequence of SEQ ID NO:5, CDR2 containing the amino acid sequence of SEQ ID NO:6, and CDR3 containing the amino acid sequence of SEQ ID NO:8; and (iii) A second anti-PD-1 sbAb, which binds to human PD-1 protein and comprises CDR1 containing the amino acid sequence of SEQ ID NO:5, CDR2 containing the amino acid sequence of SEQ ID NO:6, and CDR3 containing the amino acid sequence of SEQ ID NO:

8. The first anti-PD-1 sbAb and the second anti-PD-1 sbAb are respectively fused with the anti-VEGF antibody or its antigen-binding fragment at the C-terminus of the IgG Fc fragment.

6. A bispecific antibody comprising: (i) An anti-VEGF antibody or its antigen-binding fragment, comprising: (a) A Fab fragment, wherein the Fab fragment binds to human VEGF protein and comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:3 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:4; and (b) IgG Fc fragment; (ii) A first anti-PD-1 sbAb, wherein the first anti-PD-1 sbAb binds to human PD-1 protein and contains the amino acid sequence of SEQ ID NO:2; and (iii) A second anti-PD-1 sbAb, which binds to human PD-1 protein and contains the amino acid sequence of SEQ ID NO:

2. The first anti-PD-1 sbAb and the second anti-PD-1 sbAb are respectively fused with the anti-VEGF antibody or its antigen-binding fragment at the C-terminus of the IgG Fc fragment.

7. The bispecific antibody of any one of claims 1, 3, and 5, wherein the VH of the Fab fragment of the anti-VEGF antibody comprises the amino acid sequence of SEQ ID NO:3, and the VL of the Fab fragment of the anti-VEGF antibody comprises the amino acid sequence of SEQ ID NO:

4.

8. The bispecific antibody of claim 1 or 3, wherein the first anti-PD-1 sbAb and / or the second anti-PD-1 sbAb comprises the amino acid sequence of SEQ ID NO:

1.

9. The bispecific antibody of claim 5, wherein the first anti-PD-1 sbAb, the second anti-PD-1 sbAb, the third anti-PD-1 sbAb and / or the fourth anti-PD-1 sbAb comprises the amino acid sequence of SEQ ID NO:

2.

10. The bispecific antibody according to any one of claims 1 to 9, wherein the IgG Fc fragment contains L234A and L235A amino acid substitutions compared to the wild-type IgG Fc fragment, according to EU designations.

11. The bispecific antibody according to any one of claims 1 to 10, wherein the first anti-PD-1 sbAb and / or the second anti-PD-1 sbAb are fused to the C-terminus of the IgG Fc fragment via a (G4S)4 (SEQ ID NO:15) linker.

12. The bispecific antibody according to any one of claims 3 to 4, 7 to 8, and 10 to 11, wherein the third anti-PD-1 sbAb is fused to the first anti-PD-1 sbAb at the C-terminus of the first anti-PD-1 sbAb via a (G4S)4 (SEQ ID NO:15) linker, and wherein the fourth anti-PD-1 sbAb is fused to the second anti-PD-1 sbAb at the C-terminus of the second anti-PD-1 sbAb via a (G4S)4 (SEQ ID NO:15) linker.

13. A bispecific antibody comprising an anti-VEGF moiety and an anti-PD-1 moiety, wherein the bispecific antibody comprises the amino acid sequences of SEQ ID NO:18 and 17.

14. A bispecific antibody comprising an anti-VEGF moiety and an anti-PD-1 moiety, wherein the bispecific antibody comprises the amino acid sequences of SEQ ID NO:16 and 17.

15. A bispecific antibody comprising an anti-VEGF moiety and an anti-PD-1 moiety, wherein the bispecific antibody comprises the amino acid sequences of SEQ ID NO:19 and 17.

16. A bispecific antibody comprising: (i) A first polypeptide containing the amino acid sequence of SEQ ID NO: 17; (ii) A second polypeptide comprising the amino acid sequence of SEQ ID NO: 18; (iii) A third polypeptide comprising the amino acid sequence of SEQ ID NO: 18; and (iv) A fourth polypeptide containing the amino acid sequence of SEQ ID NO:

17.

17. A bispecific antibody comprising: (i) A first polypeptide containing the amino acid sequence of SEQ ID NO: 17; (ii) A second polypeptide comprising 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.

18. A bispecific antibody comprising: (i) A first polypeptide containing the amino acid sequence of SEQ ID NO: 17; (ii) A second polypeptide comprising 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.

19. The bispecific antibody of any one of claims 16 to 18, wherein the first polypeptide and the second polypeptide are interconnected by a disulfide bond, the second polypeptide and the third polypeptide are interconnected by two disulfide bonds, and the third polypeptide and the fourth polypeptide are interconnected by a disulfide bond.

20. A composition comprising a bispecific antibody as described in any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. A polynucleotide encoding (i) the heavy chain of a bispecific antibody as described in any one of claims 1 to 19 or (ii) the heavy and light chains of a bispecific antibody as described in any one of claims 1 to 19.

22. A method for preparing a bispecific antibody as described in any one of claims 1 to 19, comprising expressing a polynucleotide encoding the heavy chain of the bispecific antibody and a polynucleotide encoding the light chain of the bispecific antibody in CHO cells.

23. A bispecific antibody prepared by the method of claim 22.

24. A cell comprising a heavy chain polynucleotide encoding a bispecific antibody as described in any one of claims 1 to 19 and a light chain polynucleotide encoding a bispecific antibody as described in any one of claims 1 to 19, wherein the cell is optionally a CHO-K1 cell.

25. The bispecific antibody according to any one of claims 1 to 19, wherein the bispecific antibody is produced in CHO-K1 cells.

26. The bispecific antibody according to any one of claims 1 to 19, wherein the bispecific antibody is produced by expressing a polynucleotide encoding the heavy chain of the bispecific antibody and a polynucleotide encoding the light chain of the bispecific antibody.

27. Use of the bispecific antibody according to any one of claims 1 to 19, 23 and 25 to 26 in the preparation of a medicament for treating cancer in patients in need.

28. The method of claim 27, 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.

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