Pharmaceutical use of Anti-PD-1-Anti-vegfa bispecific antibody

Abstract

The present invention relates to the field of tumor therapy and molecular immunology, relates to a pharmaceutical use of an anti-PD-1-anti-VEGFA bispecific antibody, and in particular, to a use of the anti-PD-1-anti-VEGFA bispecific antibody or a pharmaceutical combination of said antibody and a chemotherapy drug in the preparation of a drug for treating non-small cell lung cancer with brain metastases. The bispecific antibody can effectively treat non-small cell lung cancer with brain metastases, has high safety, and has good prospects of application.

Description

Medical uses of anti-PD-1-anti-VEGFA bispecific antibodies

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the application with CN application number 202410108261.9 and application date January 25, 2024, and claims its priority. The entire content of the CN application is hereby introduced as a whole into this application. Technical Field

[0003] The present invention belongs to the fields of tumor therapy and immunobiology, and relates to the medical use of anti-PD-1-anti-VEGFA bispecific antibodies. Specifically, the present invention relates to the use of an anti-human PD-1-anti-human VEGFA bispecific antibody or a drug combination thereof with a chemotherapeutic agent in the preparation of a medicament for treating brain metastases of non-small cell lung cancer. Background Art

[0004] Tumors, especially malignant tumors, are a serious threat to human health worldwide, ranking second among all causes of death from various diseases. Furthermore, their incidence has shown a significant upward trend in recent years. Malignant tumors are poorly treated, have a high rate of late-stage metastasis, and often have a poor prognosis. While conventional treatments currently used in clinical practice, such as radiotherapy, chemotherapy, and surgery, have significantly alleviated pain and prolonged survival, these methods all have significant limitations, making further improvement in their efficacy difficult.

[0005] Vascular endothelial growth factor (VEGF) is a growth factor that promotes endothelial cell proliferation, angiogenesis, and vascular permeability. It binds to VEGF receptors on the cell surface and exerts its functions by activating tyrosine kinase signaling pathways. Within tumor tissue, tumor cells, tumor-invading macrophages, and mast cells secrete high levels of VEGF, which paracrine-stimulates tumor vascular endothelial cells, promoting endothelial cell proliferation and migration, inducing angiogenesis, and promoting sustained tumor growth. It also increases vascular permeability, triggers fibrin deposition in surrounding tissues, and promotes the infiltration of monocytes, fibroblasts, and endothelial cells. This facilitates tumor stroma formation and the entry of tumor cells into new blood vessels, promoting tumor metastasis. Therefore, inhibiting tumor angiogenesis is considered one of the most promising cancer treatments. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, and PIGF. Vascular endothelial growth factor receptors (VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4), and neuropilin-1 (NRP-1). The first three receptors are structurally similar, belonging to the tyrosine kinase superfamily. They are composed of an extracellular domain, a transmembrane segment, and an intracellular domain. The extracellular domain consists of an immunoglobulin-like domain, while the intracellular domain is a tyrosine kinase domain. VEGFR1 and VEGFR2 are primarily located on the surface of vascular endothelial cells, while VEGFR3 is primarily located on the surface of lymphatic endothelial cells.

[0006] VEGF family molecules have different affinities for several receptors. VEGFA mainly binds to VEGFR1, VEGFR2, and NRP-1 to exert its effects. VEGFR1 is the first receptor discovered. Under normal physiological conditions, the affinity of VEGFR1 for VEGFA is higher than that of VEGFR2 for VEGFA, but its intracellular tyrosinase activity is lower than that of VEGFR2 (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5) (2016): 146-148).

[0007] VEGFR2 is a major regulator of angiogenesis and construction. Compared with VEGFR1, VEGFR2 has a higher tyrosine kinase activity. After binding to its ligand VEGFA, VEGFR2 mediates the proliferation and differentiation of vascular endothelial cells, as well as the formation of blood vessels and vascular permeability (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 62(3)(2007):179-213.). After binding to VEGFR2, VEGFA mediates the transcriptional expression of intracellular related protein genes through the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway, promoting the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene, 18(13)(1999):2221-2230.).

[0008] VEGFR3 is a member of the tyrosine kinase family and is primarily expressed in vascular endothelial cells during the embryonic period and lymphatic endothelial cells during adulthood. VEGFC and VEGFD bind to VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote lymphangiogenesis. NRP-1 is a non-tyrosine kinase transmembrane protein that cannot independently transduce biological signals. It can only mediate signal transduction after forming a complex with the VEGF tyrosine kinase receptor. (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5)(2016): 146-148).

[0009] VEGFA and VEGFR2 are mainly involved in regulating angiogenesis. Before and after the binding of VEGFA and VEGFR2, they will trigger a cascade reaction of numerous intermediate signals in the upstream and downstream pathways, ultimately changing the physiological functions of endothelial cells in different forms such as proliferation, survival, migration, increased permeability and infiltration into surrounding tissues (Dong Hongchao et al., "Modern Oncology", Vol. 22, No. 9, September 2014, pp. 2231-3).

[0010] Currently, there are many humanized monoclonal antibodies targeting human VEGF, especially VEGFA, such as Bevacizumab, which was approved by the U.S. Food and Drug Administration in 2004 for the treatment of various tumors such as non-small cell lung cancer, renal cell carcinoma, cervical cancer, and metastatic colorectal cancer.

[0011] Programmed cell death protein (PD-1), also known as CD279, is a type I transmembrane glycoprotein surface receptor belonging to the CD28 immunoglobulin superfamily. It is ubiquitously expressed on T cells, B cells, and myeloid cells. PD-1 has two natural ligands, PD-L1 and PD-L2. Both PD-L1 and PD-L2 belong to the B7 superfamily and are constitutively or inducibly expressed on the surface membranes of various cells, including non-hematopoietic cells and various tumor cells. PD-L1 is primarily expressed on T cells, B cells, dendritic cells, microvascular endothelial cells, and various tumor cells. PD-L2 is exclusively expressed on antigen-presenting cells such as dendritic cells and macrophages. The interaction between PD-1 and its ligands inhibits lymphocyte activation, T cell proliferation, and the secretion of cytokines such as IL-2 and IFN-γ.

[0012] A large number of studies have shown that the tumor microenvironment can protect tumor cells from destruction by immune cells, and the expression of PD-1 in infiltrating lymphocytes in the tumor microenvironment is upregulated, and a variety of primary tumor tissues are PD-L1 positive in immunohistochemical analysis, such as lung cancer, liver cancer, ovarian cancer, skin cancer, colon cancer, glioma, etc. At the same time, the expression of PD-L1 in tumors is significantly correlated with the poor prognosis of cancer patients. Blocking the interaction between PD-1 and its ligands can promote tumor-specific T cell immunity and improve the efficiency of immune clearance of tumor cells. A large number of clinical trials have shown that antibodies targeting PD-1 or PD-L1 can promote CD8 + T cells infiltrate into tumor tissues and upregulate anti-tumor immune effector factors such as IL-2, IFN-γ, granzyme B and perforin, thereby effectively inhibiting tumor growth.

[0013] Furthermore, anti-PD-1 antibodies can be used to treat chronic viral infections. Chronic viral infections are often accompanied by a loss of function and a decrease in the number of virus-specific effector T cells. Injection of PD-1 antibodies can block the interaction between PD-1 and PD-L1, effectively inhibiting the exhaustion of effector T cells in chronic viral infections.

[0014] Due to the broad-spectrum anti-tumor prospects and amazing efficacy of PD-1 antibodies, the industry generally believes that antibodies targeting the PD-1 pathway will bring breakthrough progress in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., Anti-PD-1 Therapy in Melanoma. Semin Oncol. Jun; 42(3)(2015): 466-473), lymphoma and anemia (Held SA, Heine A, et al., Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets. Sep; 13(7)(2013): 768-74), microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) tumors (many anti-PD-1 antibody drugs have been approved by the FDA and others for the treatment of tumors with MSI-H / dMMR characteristics).

[0015] Bifunctional antibodies, also known as bispecific antibodies, are specific drugs that simultaneously target two different antigens. They can be produced through immunoassay purification. Alternatively, they can be obtained through genetic engineering, which offers advantages in terms of flexibility in optimizing binding sites, considering synthetic formats, and achieving high yields. Currently, over 45 bispecific antibodies have been identified (Müller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24:89-98). Many bispecific antibodies currently developed are in the IgG-ScFv format, also known as the Morrison model (Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol., 1997; 15: 159-163). Due to its similarity to naturally occurring IgG, this format has advantages in antibody engineering, expression, and purification and has been proven to be an ideal form of bifunctional antibodies (Miller BR, Demarest SJ, et al., Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010; 23: 549-57; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways. MAbs 2011; 3: 299-309).

[0016] Brain metastases (BM) in patients with malignant tumors often portend a worse prognosis. Tumor brain metastases can involve multiple intracranial tissues, including brain parenchyma, spinal meninges, nerves, and capillaries, and can be accompanied by epilepsy, cognitive impairment, sensory or motor dysfunction, and cranial neuropathies, which seriously reduce the patient's quality of life. The brain has a unique blood-brain barrier (BBB), which is composed of tightly connected non-fenestrated endothelial cells. The foot-end processes of pericytes and astrocytes surround these non-fenestrated endothelial cells, providing support for the blood-brain barrier and maintaining its integrity (Desland FA, Hormigo A. The CNS and the brain tumor microenvironment: implications for glioblastoma immunotherapy [J]. Int J Mol Sci, 2020, 21(19): 7358). On the one hand, the low expression of endothelial cell adhesion molecules in the blood-brain barrier hinders the excessive entry of peripheral immune cells into the central nervous system; on the other hand, conventional chemotherapy drugs and targeted drugs also have difficulty crossing the blood-brain barrier and entering the brain to exert their effects (Daneman R, Zhou L, Agalliu D, et al. The mouse bloodbrain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells [J]. PLoS One, 2010, 5(10): e13741). However, the blood-brain barrier is often damaged to a certain extent at the lesion site, allowing blood-derived tumor cells to invade the brain, thereby leading to the occurrence of brain metastasis.

[0017] A variety of peripheral immune cells including lymphocytes and neutrophils, as well as various immune factors, are involved in the development of brain metastatic cancer, which together constitute the immune microenvironment of metastatic lesions. When brain metastasis occurs, microglial activation can upregulate interleukin (IL) expression, and IL-6 can exert an immunosuppressive effect on effector T cells (Lin YJ, Wei KC, Chen PY, et al. Roles of neutrophils in glioma and brain metastases[J]. Front Immunol, 2021, 12: 701383); the interaction between tumor cells and astrocytes can lead to high expression of IL-1β, thereby further activating the Notch signaling pathway. The activation of this pathway can increase the stemness of tumor stem cells and drive their proliferation and differentiation in the tumor microenvironment (Wang XC, Haaland B, Hu-Lieskovan S, et al. First line immunotherapy extends brain metastasis-free survival, improves overall survival, and reduces the incidence of brainmetastasis in patients with advanced melanoma[J]. CancerRep(Hoboken), 2021, 4(6): e1419).

[0018] Lung cancer is one of the most common malignancies worldwide, with non-small cell lung cancer (NSCLC) being the most common type of lung cancer. 10%-15% of NSCLC patients develop brain metastases at initial diagnosis, causing various neurological symptoms, and 24%-44% of patients with advanced NSCLC develop brain metastases. The median survival of patients with brain metastases from NSCLC who are initially diagnosed and treated is only 4-6 months. The vast majority of patients with NSCLC brain metastases are no longer suitable for surgical treatment at the time of diagnosis. Chemotherapy, such as antifolate agents combined with platinum, remains the mainstay of treatment for advanced NSCLC. However, due to the obstruction of the blood-brain barrier, the efficacy of most chemotherapy drugs and targeted TKIs for brain metastases is limited. Third-generation TKIs have strong anti-tumor activity and a high blood-brain barrier passage rate, often resulting in better efficacy. However, these drugs are only suitable for patients with some common classic driver gene mutations who do not have obvious symptoms. Their application is also limited by drug resistance and safety.

[0019] Currently, immunotherapy is also used in clinical practice to treat NSCLC brain metastasis. Studies have shown that immune checkpoint inhibitors (ICIs) can promote the activation of T cells (especially CD8 + T cells) are recruited from outside the skull to inside the skull, allowing more T cells to enter the brain to kill tumors (Taggart D, Andreou T, Scott KJ, Williams J, Rippaus N, Brownlie RJ, Ilett EJ, Salmond RJ, Melcher A, Lorger M. Anti-PD-1 / anti-CTLA-4 efficacy in melanoma brain metastases depends on extracranial disease and augmentation of CD8+T cell trafficking. Proc Natl Acad Sci US A. 2018 Feb 13; 115(7): E1540-E1549.). A study on durvalumab showed that among 25 patients with EGFR-positive / ALKs-positive brain metastases, the objective response rate of patients with negative driver genes was 16.4%, which was higher than the 12.2% of patients with positive drivers. This shows that the efficacy of ICIs alone is not ideal for patients with EGFR mutations (Garassino MC, Cho BC, Kim JH, et al. Durvalumab as third-line or later treatment for advanced non-small-cell lung cancer (ATLANTIC): an open-label, single-arm, phase 2 study [J]. Lancet Oncol, 2018, 19(4): 521-536). In addition, immune checkpoint inhibitors are combined with chemotherapy or multiple immune checkpoint inhibitors are used in combination to treat brain metastases of lung cancer, but the incidence of adverse immune reactions (irAEs) will be increased (Yang Shanru, Tai Risheng, Wang Geng, Qu Shutao, Lei Lei, Li Na. Problems caused by immune checkpoint inhibitor treatment-immune-related adverse reactions irAEs [J]. Chinese Journal of Immunology, 2022, 38(16): 2026-2030).

[0020] In summary, developing new drugs or treatments to treat brain metastases has great clinical significance. Summary of the Invention

[0021] After in-depth research and creative work, the inventors discovered that the anti-PD-1-anti-VEGFA bispecific antibody of the present invention, combined with or without chemotherapy, can effectively treat or prevent brain metastasis of non-small cell lung cancer while having good safety.

[0022] This provides the following invention:

[0023] One aspect of the present invention relates to the use of a bispecific antibody in the preparation of a medicament for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0024] The bispecific antibody comprises:

[0025] Targeting the first protein domain of VEGFA, and

[0026] Targeting the second protein functional region of PD-1;

[0027] in,

[0028] The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the immunoglobulin comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; and the single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively;

[0029] or,

[0030] The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; and, the immunoglobulin comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively.

[0031] In the present invention, unless otherwise specified, the bispecific antibody is an anti-VEGFA-anti-PD-1 bispecific antibody, particularly an anti-human VEGFA-anti-human PD-1 bispecific antibody.

[0032] In some embodiments of the present invention, the use, wherein,

[0033] The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17.

[0034] In some embodiments of the present invention, the use, wherein the bispecific antibody is selected from any one of the following (1)-(6):

[0035] (1)

[0036] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7;

[0037] (2)

[0038] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11;

[0039] (3)

[0040] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17;

[0041] (4)

[0042] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7;

[0043] (5)

[0044] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11; and

[0045] (6)

[0046] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17.

[0047] In some embodiments of the present invention, the use, wherein,

[0048] The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17.

[0049] In some embodiments of the present invention, the use, wherein the bispecific antibody is selected from any one of the following (7)-(12):

[0050] (7)

[0051] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0052] (8)

[0053] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0054] (9)

[0055] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 17; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0056] (10)

[0057] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0058] (11)

[0059] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3; and

[0060] (12)

[0061] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:17; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3.

[0062] In some embodiments of the present invention, the use, wherein,

[0063] The immunoglobulin is of human IgG1 subtype.

[0064] In some embodiments of the present invention, the use, wherein,

[0065] The immunoglobulin is of human IgG1 subtype;

[0066] Wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises one of the following mutation combinations:

[0067] L234A and L235A;

[0068] L234A and G237A;

[0069] L235A and G237A; or

[0070] L234A, L235A, G237A.

[0071] In the present invention, unless otherwise specified, the letters before the site represent the amino acid before mutation, and the letters after the site represent the amino acid after mutation.

[0072] In some embodiments of the present invention, the use, wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin further has one or more mutations selected from the following:

[0073] N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A, and K320A.

[0074] In some embodiments of the present invention, the use, wherein,

[0075] The amino acid sequence of the immunoglobulin heavy chain is shown in SEQ ID NO: 24, and the amino acid sequence of the immunoglobulin light chain is shown in SEQ ID NO: 26;

[0076] The amino acid sequence of the immunoglobulin heavy chain is shown in SEQ ID NO: 20, and the amino acid sequence of the immunoglobulin light chain is shown in SEQ ID NO: 26; or

[0077] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26.

[0078] In one or more embodiments of the present invention, the bispecific antibody is in the form of IgG-scFv, ie, Morrison format.

[0079] In one or more embodiments of the present invention, the use, wherein,

[0080] The immunoglobulin has a heavy chain constant region that is human Ig gamma-1 chain C region or human Ig gamma-4 chain C region, and a light chain constant region that is human Ig kappa chain C region.

[0081] In some embodiments of the present invention, the constant region of the immunoglobulin is humanized, for example, the heavy chain constant region adopts Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834; or

[0082] The heavy chain constant region of the immunoglobulin adopts Ig gamma-4 chain C region, ACCESSION: P01861.1; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834.

[0083] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region Ig gamma-1 chain C region (ACCESSION: P01857) is shown in SEQ ID NO: 40.

[0084] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region Ig gamma-4 chain C region (ACCESSION: P01861.1) is shown in SEQ ID NO: 41.

[0085] In some embodiments of the present invention, the amino acid sequence of the light chain constant region Ig kappa chain C region (ACCESSION: P01834) is shown in SEQ ID NO: 42.

[0086] In some embodiments of the present invention, the use described herein is characterized in that the single-chain antibody is linked to the C-terminus of an immunoglobulin heavy chain. Since immunoglobulins are composed of two heavy chains, two single-chain antibody molecules are linked to one immunoglobulin molecule. Preferably, the two single-chain antibody molecules are identical.

[0087] In some embodiments of the present invention, the use is described, wherein the number of the single-chain antibodies is two, and one end of each single-chain antibody is connected to the C-terminus or N-terminus of two heavy chains of immunoglobulin.

[0088] In some embodiments of the present invention, the V H With V L There are disulfide bonds between the V H and V LMethods for introducing disulfide bonds between proteins are well known in the art, for example, see U.S. Pat. No. 5,747,654; Rajagopal et al., Prot. Engin. 10 (1997) 1453-1459; Reiter et al., Nat. Biotechnol. 14 (1996) 1239-1245; Reiter et al., Protein Engineering 8 (1995) 1323-1331; Webber et al., Molecular Immunology 32 (1995) 249-258; Reiter et al., Immunity 2 (1995) 281-287; Reiter et al., JBC 269 (1994) 18327-18331; Reiter et al., Inter. J. of Cancer 58 (1994) 142-149; or Reiter et al., J. Biol. Am. Am. Am. Am. Prot. Am. et.al, Cancer Res. 54 (1994) 2714-2718; which is incorporated herein by reference.

[0089] In some embodiments of the present invention, the use, wherein,

[0090] The first protein functional region is directly connected to the second protein functional region or connected through a linker; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a linker.

[0091] In some embodiments of the present invention, the use, wherein,

[0092] The linker is a polypeptide represented by SEQ ID NO: 43 (GGGGS), or a polypeptide composed of multiple (eg, 2, 3, 4, 5 or 6) polypeptides represented by SEQ ID NO: 43 connected in series.

[0093] In some embodiments of the present invention, the use, wherein,

[0094] The first protein functional region and the second protein functional region are independently 1, 2 or more.

[0095] In some embodiments of the present invention, the use, wherein,

[0096] There are two single-chain antibodies, which are respectively connected to the C-termini of two heavy chains of immunoglobulin.

[0097] In some embodiments of the present invention, the use, wherein the single-chain antibody is independently linked in sequence: heavy chain variable region-linker-light chain variable region (VH-linker-VL), or light chain variable region-linker-heavy chain variable region (VL-linker-VH);

[0098] Preferably, the amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs: 45-48.

[0099] In some embodiments of the present invention, the use, wherein the first protein functional region is an anti-VEGFA immunoglobulin, and the second protein functional region is an anti-PD-1 single-chain antibody;

[0100] Preferably, the anti-PD-1 single-chain antibody is two molecules, which are respectively connected to the C-termini of the two heavy chains of the anti-VEGFA immunoglobulin;

[0101] Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv;

[0102] Preferably, the bispecific antibody is a tetravalent bispecific antibody in the IgG-scFv format.

[0103] In some embodiments of the present invention, the use, wherein,

[0104] The bispecific antibody comprises:

[0105] Targeting the first protein domain of VEGFA, and

[0106] Targeting the second protein functional region of PD-1;

[0107] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0108] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0109] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0110] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17, SEQ ID NO: 7 or SEQ ID NO: 11;

[0111] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0112] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0113] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0114] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0115] In some embodiments of the present invention, the use, wherein,

[0116] The bispecific antibody comprises:

[0117] Targeting the first protein domain of VEGFA, and

[0118] Targeting the second protein functional region of PD-1;

[0119] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0120] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0121] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0122] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17, SEQ ID NO: 7 or SEQ ID NO: 11;

[0123] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0124] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0125] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0126] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0127] In some embodiments of the present invention, the use, wherein,

[0128] The bispecific antibody comprises:

[0129] Targeting the first protein domain of VEGFA, and

[0130] Targeting the second protein functional region of PD-1;

[0131] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0132] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0133] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, SEQ ID NO: 20 or SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0134] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17;

[0135] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0136] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0137] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0138] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0139] In some embodiments of the present invention, the use, wherein,

[0140] The bispecific antibody comprises:

[0141] Targeting the first protein domain of VEGFA, and

[0142] Targeting the second protein functional region of PD-1;

[0143] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0144] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0145] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0146] The amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs: 45-48;

[0147] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0148] The first protein functional region and the second protein functional region are connected via a first linker;

[0149] Preferably, the first linker is selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0150] In some embodiments of the present invention, the use, wherein,

[0151] The bispecific antibody comprises:

[0152] Targeting the first protein domain of VEGFA, and

[0153] Targeting the second protein functional region of PD-1;

[0154] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0155] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0156] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, SEQ ID NO: 20 or SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0157] The amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs: 45-48;

[0158] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0159] The first protein functional region and the second protein functional region are connected via a first linker;

[0160] Preferably, the first linker is selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0161] In some embodiments of the present invention, the use is characterized in that the bispecific antibody is the only active ingredient.

[0162] In some embodiments of the present invention, the use is the use of the combination of the bispecific antibody and at least one chemotherapy drug in the preparation of a drug for treating or preventing brain metastasis of non-small cell lung cancer.

[0163] In some embodiments of the present invention, the use, wherein,

[0164] The chemotherapy drug is one or more selected from tyrosine kinase inhibitors, platinum drugs (such as cisplatin or carboplatin), pemetrexed, paclitaxel, docetaxel, temozolomide and fotemustine.

[0165] In some embodiments of the present invention, the use, wherein the unit dose of the bispecific antibody is 100 mg-4000 mg, 200 mg-3000 mg, 200 mg-2000 mg, 300 mg-2000 mg, 400 mg-2000 mg, 500 mg-2000 mg, 500 mg-1000 mg, 500 mg-1500 mg, 600 mg-2000 mg, 800 mg-2000 mg, 1000 mg-2000 mg or 1500 mg-2000 mg.

[0166] In some embodiments of the present invention, the use, wherein the unit dose of the bispecific antibody is 100 mg-1000 mg, 200 mg-800 mg, 200 mg-500 mg, 300 mg-600 mg, 400 mg-500 mg or 450 mg.

[0167] In some embodiments of the present invention, the use, wherein the single administration dose of the bispecific antibody is 1-50 mg, 10 mg-50 mg, 10 mg-40 mg, 10 mg-30 mg, 10 mg-20 mg, 10 mg-15 mg, 15 mg-30 mg or 15 mg-20 mg per kilogram of body weight.

[0168] In some embodiments of the present invention, the use, wherein the bispecific antibody is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks;

[0169] In some embodiments of the present invention, in the use, the bispecific antibody is administered by intravenous drip, intravenous injection or intraperitoneal injection.

[0170] In some embodiments of the present invention, the use, wherein the combination is:

[0171] The bispecific antibody according to any one of the present invention, pemetrexed, and carboplatin; or

[0172] The bispecific antibody according to any one of the present invention, paclitaxel and carboplatin.

[0173] In some embodiments of the present invention, the use, wherein the combination is:

[0174] bispecific antibody VP101 (hG1DM), pemetrexed, and carboplatin; or

[0175] Bispecific antibody VP101 (hG1DM), paclitaxel, and carboplatin.

[0176] In some embodiments of the present invention, the use, wherein the combination is:

[0177] bispecific antibody VP101 (hG1WT), pemetrexed, and carboplatin; or

[0178] Bispecific antibody VP101 (hG1WT), paclitaxel, and carboplatin.

[0179] In some embodiments of the present invention, the use, wherein the combination is:

[0180] bispecific antibody VP101 (hG4WT), pemetrexed, and carboplatin; or

[0181] Bispecific antibody VP101 (hG4WT), paclitaxel, and carboplatin.

[0182] In some embodiments of the present invention, the use is characterized by one or more of the following (1) to (3):

[0183] (1) The single dose of pemetrexed is based on the body surface area (m 2 )10-1500mg / m 2 100-1000mg / m 2 200-900mg / m 2 300-800mg / m 2 400-700mg / m 2 500-600mg / m 2; Preferably, the pemetrexed is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks;

[0184] (2) The single dose of carboplatin is independently AUC 2-6min*mg / mL or AUC 2.5-5min*mg / mL; preferably, the carboplatin is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks;

[0185] (3) The single dose of paclitaxel is based on the body surface area (m 2 )67.5-175mg / m 2 87.5-175mg / m 2 or 131-175 mg / m 2 ; Preferably, the paclitaxel is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks.

[0186] In some embodiments of the present invention, the use, wherein the non-small cell lung cancer brain metastasis is selected from one or more of squamous cell non-small cell lung cancer brain metastasis, non-squamous cell non-small cell lung cancer brain metastasis and lung adenocarcinoma brain metastasis.

[0187] In some embodiments of the present invention, the use is described, wherein the PD-L1 TPS of patients with brain metastases from non-small cell lung cancer is ≥50%, or 1% < PD-L1 TPS ≥49%, or PD-L1 TPS ≤1%.

[0188] TPS is the abbreviation of tumor cell proportion score.

[0189] In one or more embodiments of the present invention, the bispecific antibody is a humanized antibody.

[0190] In one or more embodiments of the present invention, the bispecific antibody is VP101 (hG1WT), VP101 (hG4WT) or VP101 (hG1DM).

[0191] The drug can be formulated into any dosage form known in the pharmaceutical art, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the bispecific antibody of the present invention into an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use.

[0192] In addition, the bispecific antibodies of the present invention can be present in medicine in unit dosage form, so that it is easy to use. In certain embodiments, the unit dose is at least 1 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. In the case where the pharmaceutical composition is a liquid (e.g., injection) dosage form, it can include a concentration of at least 0.1 mg / ml, such as at least 0.25 mg / ml, at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml, or at least 100 mg / ml of the bispecific antibodies of the present invention.

[0193] The bispecific antibodies or drugs of the present invention can be administered by any suitable method known in the art, including but not limited to, oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, preferred routes of administration / modes are parenteral (e.g., intravenous, subcutaneous, intraperitoneal or intramuscular). Technicians will appreciate that routes of administration and / or modes will vary depending on the intended purpose. In a preferred embodiment, the bispecific antibodies or pharmaceutical compositions of the present invention are administered by intravenous infusion or injection.

[0194] The bispecific antibodies or drugs provided by the present invention can be used alone or in combination, or can be used in combination with another pharmaceutically active agent (e.g., a tumor chemotherapy drug). Such another pharmaceutically active agent can be administered before, simultaneously with, or after administration of the bispecific antibody or pharmaceutical composition of the present invention.

[0195] In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be a single dose, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the treatment situation.

[0196] In one or more embodiments of the present invention, one treatment cycle is every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks. In some embodiments, one treatment cycle is every 3 weeks.

[0197] Another aspect of the present invention relates to a bispecific antibody for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0198] The bispecific antibody comprises:

[0199] Targeting the first protein domain of VEGFA, and

[0200] Targeting the second protein functional region of PD-1;

[0201] in,

[0202] The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the immunoglobulin comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; and the single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively;

[0203] or,

[0204] The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; and, the immunoglobulin comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively.

[0205] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0206] The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17.

[0207] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein the bispecific antibody is selected from any one of the following (1)-(6):

[0208] (1)

[0209] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7;

[0210] (2)

[0211] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11;

[0212] (3)

[0213] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17;

[0214] (4)

[0215] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7;

[0216] (5)

[0217] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11; and

[0218] (6)

[0219] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17.

[0220] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0221] The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17.

[0222] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein the bispecific antibody is selected from any one of the following (7)-(12):

[0223] (7)

[0224] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0225] (8)

[0226] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0227] (9)

[0228] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 17; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0229] (10)

[0230] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0231] (11)

[0232] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3; and

[0233] (12)

[0234] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:17; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3.

[0235] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0236] The immunoglobulin is of human IgG1 subtype;

[0237] Wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises one of the following mutation combinations:

[0238] L234A and L235A; or

[0239] L234A, L235A, G237A.

[0240] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0241] The amino acid sequence of the immunoglobulin heavy chain is shown in SEQ ID NO: 24, and the amino acid sequence of the immunoglobulin light chain is shown in SEQ ID NO: 26;

[0242] The amino acid sequence of the immunoglobulin heavy chain is shown in SEQ ID NO: 20, and the amino acid sequence of the immunoglobulin light chain is shown in SEQ ID NO: 26; or

[0243] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26.

[0244] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0245] The first protein functional region is directly connected to the second protein functional region or connected through a linker; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a linker.

[0246] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0247] The linker is a polypeptide shown in SEQ ID NO: 43, or a polypeptide consisting of multiple (eg, 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO: 43 connected in series.

[0248] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0249] The first protein functional region and the second protein functional region are independently 1, 2 or more.

[0250] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0251] There are two single-chain antibodies, which are respectively connected to the C-termini of two heavy chains of immunoglobulin.

[0252] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein the single-chain antibody is independently linked in sequence: heavy chain variable region-linker-light chain variable region (VH-linker-VL), or light chain variable region-linker-heavy chain variable region (VL-linker-VH);

[0253] Preferably, the amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs: 45-48.

[0254] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein the first protein functional region is an anti-VEGFA immunoglobulin, and the second protein functional region is an anti-PD-1 single-chain antibody;

[0255] Preferably, the anti-PD-1 single-chain antibody is two molecules, which are respectively connected to the C-termini of the two heavy chains of the anti-VEGFA immunoglobulin;

[0256] Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv;

[0257] Preferably, the bispecific antibody is a tetravalent bispecific antibody in the IgG-scFv format.

[0258] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0259] The bispecific antibody comprises:

[0260] Targeting the first protein domain of VEGFA, and

[0261] Targeting the second protein functional region of PD-1;

[0262] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0263] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0264] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0265] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17, SEQ ID NO: 7 or SEQ ID NO: 11;

[0266] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0267] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0268] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0269] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0270] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0271] The bispecific antibody comprises:

[0272] Targeting the first protein domain of VEGFA, and

[0273] Targeting the second protein functional region of PD-1;

[0274] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0275] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0276] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0277] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17, SEQ ID NO: 7 or SEQ ID NO: 11;

[0278] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0279] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0280] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0281] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0282] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0283] The bispecific antibody comprises:

[0284] Targeting the first protein domain of VEGFA, and

[0285] Targeting the second protein functional region of PD-1;

[0286] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0287] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0288] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, SEQ ID NO: 20 or SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0289] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17;

[0290] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0291] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0292] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0293] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0294] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0295] The bispecific antibody comprises:

[0296] Targeting the first protein domain of VEGFA, and

[0297] Targeting the second protein functional region of PD-1;

[0298] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0299] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0300] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0301] The amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs: 45-48;

[0302] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0303] The first protein functional region and the second protein functional region are connected via a first linker;

[0304] Preferably, the first linker is selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0305] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0306] The bispecific antibody comprises:

[0307] Targeting the first protein domain of VEGFA, and

[0308] Targeting the second protein functional region of PD-1;

[0309] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0310] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0311] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, SEQ ID NO: 20 or SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0312] The amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs: 45-48;

[0313] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0314] The first protein functional region and the second protein functional region are connected via a first linker;

[0315] Preferably, the first linker is selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0316] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein the bispecific antibody is administered in combination with at least one chemotherapeutic drug.

[0317] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein the bispecific antibody is administered in combination with at least one chemotherapeutic drug.

[0318] The chemotherapy drug is one or more selected from tyrosine kinase inhibitors, platinum drugs (such as cisplatin or carboplatin), pemetrexed, paclitaxel, docetaxel, temozolomide and fotemustine.

[0319] In some embodiments of the present invention, the bispecific antibody is characterized by one or more of the following (1) to (4):

[0320] (1) The unit dose of the bispecific antibody is 100 mg-4000 mg, 200 mg-3000 mg, 200 mg-2000 mg, 300 mg-2000 mg, 400 mg-2000 mg, 500 mg-2000 mg, or 1000 mg-2000 mg;

[0321] (2) The single dose of the bispecific antibody is 1-50 mg, 10 mg-50 mg, 10 mg-40 mg, 10 mg-30 mg, 10 mg-20 mg, 10 mg-15 mg, 15 mg-30 mg, or 15 mg-20 mg per kilogram of body weight;

[0322] (3) the bispecific antibody is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks;

[0323] (4) The bispecific antibody is administered by intravenous drip, intravenous injection or intraperitoneal injection.

[0324] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein:

[0325] The bispecific antibody is administered in combination with pemetrexed and carboplatin; or

[0326] The bispecific antibody was administered in combination with paclitaxel and carboplatin.

[0327] In some embodiments of the present invention, the bispecific antibody is used to treat or prevent brain metastasis of non-small cell lung cancer, wherein the bispecific antibody is administered in combination with pemetrexed and carboplatin, or the bispecific antibody is administered in combination with paclitaxel and carboplatin, and is characterized by one or more of the following (1) to (3):

[0328] (1) The single dose of pemetrexed is based on the body surface area (m 2 )10-1500mg / m 2 100-1000mg / m 2 200-900mg / m 2 300-800mg / m 2 400-700mg / m 2 500-600mg / m 2 ; Preferably, the pemetrexed is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks;

[0329] (2) The single dose of carboplatin is independently AUC 2-6min*mg / mL or AUC 2.5-5min*mg / mL; preferably, the carboplatin is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks;

[0330] (3) The single dose of paclitaxel is based on the body surface area (m 2 )67.5-175mg / m 2 87.5-175mg / m 2 or 131-175 mg / m 2 ; Preferably, the paclitaxel is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks.

[0331] In some embodiments of the present invention, the bispecific antibody, wherein the non-small cell lung cancer brain metastasis is selected from one or more of squamous cell non-small cell lung cancer brain metastasis, non-squamous cell non-small cell lung cancer brain metastasis and lung adenocarcinoma brain metastasis;

[0332] Preferably, the PD-L1 TPS of patients with non-small cell lung cancer brain metastasis is ≥50%, or 1% < PD-L1 TPS ≥49%, or PD-L1 TPS ≤1%.

[0333] Another aspect of the present invention relates to a method for treating or preventing brain metastasis of non-small cell lung cancer, comprising the step of administering to a subject in need thereof an effective amount of a bispecific antibody, wherein the bispecific antibody comprises:

[0334] Targeting the first protein domain of VEGFA, and

[0335] Targeting the second protein functional region of PD-1;

[0336] in,

[0337] The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the immunoglobulin comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; and the single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively;

[0338] or,

[0339] The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; and, the immunoglobulin comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively.

[0340] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0341] The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17.

[0342] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein the bispecific antibody is selected from any one of the following (1)-(6):

[0343] (1)

[0344] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7;

[0345] (2)

[0346] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11;

[0347] (3)

[0348] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17;

[0349] (4)

[0350] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7;

[0351] (5)

[0352] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11; and

[0353] (6)

[0354] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17.

[0355] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0356] The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17.

[0357] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein the bispecific antibody is selected from any one of the following (7)-(12):

[0358] (7)

[0359] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0360] (8)

[0361] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0362] (9)

[0363] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 17; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0364] (10)

[0365] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0366] (11)

[0367] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3; and

[0368] (12)

[0369] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:17; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3.

[0370] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0371] The immunoglobulin is of human IgG1 subtype.

[0372] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0373] The immunoglobulin is of human IgG1 subtype;

[0374] Wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises one of the following mutation combinations:

[0375] L234A and L235A;

[0376] L234A and G237A;

[0377] L235A and G237A; or

[0378] L234A, L235A, G237A.

[0379] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein the heavy chain constant region of the immunoglobulin further has one or more mutations selected from the following according to the EU numbering system:

[0380] N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A, and K320A.

[0381] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0382] The amino acid sequence of the immunoglobulin heavy chain is shown in SEQ ID NO: 24, and the amino acid sequence of the immunoglobulin light chain is shown in SEQ ID NO: 26;

[0383] The amino acid sequence of the immunoglobulin heavy chain is shown in SEQ ID NO: 20, and the amino acid sequence of the immunoglobulin light chain is shown in SEQ ID NO: 26; or

[0384] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26.

[0385] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0386] The first protein functional region is directly connected to the second protein functional region or connected through a linker; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a linker.

[0387] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0388] The linker is a polypeptide represented by SEQ ID NO: 43 (GGGGS), or a polypeptide composed of multiple (eg, 2, 3, 4, 5 or 6) polypeptides represented by SEQ ID NO: 43 connected in series.

[0389] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0390] The first protein functional region and the second protein functional region are independently 1, 2 or more.

[0391] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0392] There are two single-chain antibodies, which are respectively connected to the C-termini of two heavy chains of immunoglobulin.

[0393] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein the single-chain antibody is independently linked in sequence: heavy chain variable region-linker-light chain variable region (VH-linker-VL), or light chain variable region-linker-heavy chain variable region (VL-linker-VH);

[0394] Preferably, the amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs: 45-48.

[0395] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein the first protein functional region is an anti-VEGFA immunoglobulin, and the second protein functional region is an anti-PD-1 single-chain antibody;

[0396] Preferably, the anti-PD-1 single-chain antibody is two molecules, which are respectively connected to the C-termini of the two heavy chains of the anti-VEGFA immunoglobulin;

[0397] Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv;

[0398] Preferably, the bispecific antibody is a tetravalent bispecific antibody in the IgG-scFv format.

[0399] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0400] The bispecific antibody comprises:

[0401] Targeting the first protein domain of VEGFA, and

[0402] Targeting the second protein functional region of PD-1;

[0403] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0404] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0405] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0406] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17, SEQ ID NO: 7 or SEQ ID NO: 11;

[0407] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0408] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0409] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0410] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0411] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0412] The bispecific antibody comprises:

[0413] Targeting the first protein domain of VEGFA, and

[0414] Targeting the second protein functional region of PD-1;

[0415] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0416] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0417] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0418] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17, SEQ ID NO: 7 or SEQ ID NO: 11;

[0419] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0420] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0421] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0422] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0423] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0424] The bispecific antibody comprises:

[0425] Targeting the first protein domain of VEGFA, and

[0426] Targeting the second protein functional region of PD-1;

[0427] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0428] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0429] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, SEQ ID NO: 20 or SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0430] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17;

[0431] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0432] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0433] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0434] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0435] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0436] The bispecific antibody comprises:

[0437] Targeting the first protein domain of VEGFA, and

[0438] Targeting the second protein functional region of PD-1;

[0439] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0440] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0441] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0442] The amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs: 45-48;

[0443] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0444] The first protein functional region and the second protein functional region are connected via a first linker;

[0445] Preferably, the first linker is selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0446] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0447] The bispecific antibody comprises:

[0448] Targeting the first protein domain of VEGFA, and

[0449] Targeting the second protein functional region of PD-1;

[0450] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0451] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0452] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, SEQ ID NO: 20 or SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0453] The amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs: 45-48;

[0454] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0455] The first protein functional region and the second protein functional region are connected via a first linker;

[0456] Preferably, the first linker is selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0457] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein the bispecific antibody is the only active ingredient.

[0458] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer further comprises the step of administering an effective amount of at least one chemotherapeutic drug to a subject in need thereof.

[0459] In some embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0460] The chemotherapy drug is one or more selected from tyrosine kinase inhibitors, platinum drugs (such as cisplatin or carboplatin), pemetrexed, paclitaxel, docetaxel, temozolomide and fotemustine.

[0461] In some embodiments of the present invention, in the method for treating or preventing brain metastasis of non-small cell lung cancer, the chemotherapy drug is administered simultaneously with, before, or after the administration of the bispecific antibody.

[0462] A typical non-limiting range of a therapeutically or prophylactically effective amount of the bispecific antibodies of the present invention is 0.02-50 mg / kg, for example 0.1-50 mg / kg, 0.1-25 mg / kg, or 1-10 mg / kg. It should be noted that the dosage may vary depending on the type and severity of the symptoms to be treated. In addition, those skilled in the art understand that for any particular patient, the specific dosing regimen should be adjusted over time based on the patient's needs and the doctor's professional evaluation; the dosage ranges given here are for illustrative purposes only and do not limit the use or scope of the pharmaceutical compositions of the present invention.

[0463] In the present invention, the subject may be a mammal, such as a human.

[0464] In one or more embodiments of the present invention, the method for treating or preventing brain metastasis of non-small cell lung cancer, wherein:

[0465] The single dose of the anti-PD-1-anti-VEGFA bispecific antibody is 0.1-100 mg per kg body weight, preferably 1-50 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 10 mg-50 mg, 10 mg-40 mg, 10 mg-30 mg, 10 mg-20 mg, 10 mg-15 mg, 15 mg-30 mg, 15 mg-20 mg, 12 mg, 15 mg, 20 mg, 25 mg or 30 mg); alternatively, the single dose of the anti-PD-1-anti-VEGFA bispecific antibody is 10-5000 mg per subject. mg (e.g., about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg about 1500 mg, about 1800 mg, about 2000 mg, about 2500 mg or about 3000 mg), preferably 100-2500 mg, 500-2500 mg, 500-2000 mg, 500-2000 mg or 500 mg-1500 mg;

[0466] Preferably, the drug is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every 1 week, every 2 weeks, every 3 weeks or every 4 weeks;

[0467] Preferably, the administration method is intravenous drip, intravenous injection or intraperitoneal injection.

[0468] In some embodiments, the anti-PD-1-anti-VEGFA bispecific antibody is administered in a cycle of 2 weeks (14 days) or 3 weeks (21 days), and the anti-PD-1-anti-VEGFA bispecific antibody is preferably administered intravenously on the first day (D1) of each cycle. For example, the anti-PD-1-anti-VEGFA bispecific antibody is administered once every two weeks (q2w) or once every three weeks (q3w).

[0469] In some embodiments of the present invention, in the method for treating or preventing brain metastasis of non-small cell lung cancer, the unit dose of the bispecific antibody is 100 mg-4000 mg, 200 mg-3000 mg, 200 mg-2000 mg, 300 mg-2000 mg, 400 mg-2000 mg, 500 mg-2000 mg, 500 mg-1000 mg, 500 mg-1500 mg, 600 mg-2000 mg, 800 mg-2000 mg, 1000 mg-2000 mg or 1500 mg-2000 mg.

[0470] In one or more embodiments of the present invention, the unit dose of the bispecific antibody is 100 mg-1000 mg, 200 mg-800 mg, 200 mg-500 mg, 300 mg-600 mg, 400 mg-500 mg or 450 mg.

[0471] In one or more embodiments of the present invention, the unit dose of the tumor chemotherapy drug is 0.1 mg-100 mg, 0.5 mg-50 mg, 0.5 mg-10 mg, 1 mg-10 mg, 2 mg-8 mg or 1 mg-5 mg.

[0472] In one or more embodiments of the present invention, the unit dose of the tumor chemotherapy drug is 1 mg-20 mg, 2 mg-15 mg, 4 mg-12 mg or 8 mg-12 mg.

[0473] In one or more embodiments of the present invention, the single dose of the tumor chemotherapy drug is based on the body surface area (m 2 )10-1500mg / m 2 100-1000mg / m 2 200-900mg / m 2 300-800mg / m 2 400-700mg / m 2 , or 500-600 mg / m 2 .

[0474] In one or more embodiments of the present invention, the single dose of pemetrexed is based on the body surface area (m 2 )10-1500mg / m 2 100-1000mg / m 2 200-900mg / m 2 300-800mg / m 2 400-700mg / m 2 , or 500-600 mg / m 2 ; Preferably, the pemetrexed is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks.

[0475] In one or more embodiments of the present invention, the carboplatin is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks. In some embodiments, the carboplatin is administered at a dose of AUC 2-6 min*mg / mL or AUC 2.5-5 min*mg / mL each time; preferably, the carboplatin is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks.

[0476] In one or more embodiments of the present invention, the paclitaxel is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks. In some embodiments, the paclitaxel is administered at a dose of 67.5-175 mg / m 2 87.5-175mg / m 2 , or 131-175 mg / m 2 preferably, the paclitaxel is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks.

[0477] In some embodiments, the non-small cell lung cancer is squamous cell non-small cell lung cancer (also known as squamous cell carcinoma, squamous cell carcinoma, squamous non-small cell lung cancer or squamous epithelial cell carcinoma). In some embodiments, the non-small cell lung cancer is a non-small cell lung cancer with squamous cell carcinoma as the predominant histology. In some embodiments, the non-small cell lung cancer is non-squamous cell non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is adenocarcinoma (also known as lung adenocarcinoma). Preferably, the non-small cell lung cancer is locally advanced, recurrent and / or metastatic non-small cell lung cancer.

[0478] In one or more embodiments of the present invention, the non-small cell lung cancer patient has a PD-L1 TPS ≥ 50%, or 1% < PD-L1 TPS ≥ 49%, or PD-L1 TPS ≤ 1%.

[0479] Another aspect of the present invention relates to a method for preparing a drug for treating or preventing brain metastasis of non-small cell lung cancer, which uses a bispecific antibody, wherein:

[0480] The bispecific antibody comprises:

[0481] Targeting the first protein domain of VEGFA, and

[0482] Targeting the second protein functional region of PD-1;

[0483] in,

[0484] The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the immunoglobulin comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; and the single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively;

[0485] or,

[0486] The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; and, the immunoglobulin comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively.

[0487] In some embodiments of the present invention, the preparation method, wherein,

[0488] The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17.

[0489] In some embodiments of the present invention, the preparation method, wherein the bispecific antibody is selected from any one of the following (1)-(6):

[0490] (1)

[0491] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7;

[0492] (2)

[0493] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11;

[0494] (3)

[0495] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17;

[0496] (4)

[0497] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7;

[0498] (5)

[0499] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11; and

[0500] (6)

[0501] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17.

[0502] In some embodiments of the present invention, the preparation method, wherein,

[0503] The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17.

[0504] In some embodiments of the present invention, the preparation method, wherein the bispecific antibody is selected from any one of the following (7)-(12):

[0505] (7)

[0506] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0507] (8)

[0508] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0509] (9)

[0510] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 17; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0511] (10)

[0512] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0513] (11)

[0514] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3; and

[0515] (12)

[0516] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:17; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3.

[0517] In some embodiments of the present invention, the preparation method, wherein,

[0518] The immunoglobulin is of human IgG1 subtype;

[0519] Wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises one of the following mutation combinations:

[0520] L234A and L235A; or

[0521] L234A, L235A, G237A.

[0522] In some embodiments of the present invention, the preparation method, wherein,

[0523] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0524] The amino acid sequence of the immunoglobulin heavy chain is shown in SEQ ID NO: 20, and the amino acid sequence of the immunoglobulin light chain is shown in SEQ ID NO: 26; or

[0525] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26.

[0526] In some embodiments of the present invention, the preparation method, wherein,

[0527] The first protein functional region is directly connected to the second protein functional region or connected through a linker; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a linker.

[0528] In some embodiments of the present invention, the preparation method, wherein,

[0529] The linker is a polypeptide shown in SEQ ID NO: 43, or a polypeptide consisting of multiple (eg, 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO: 43 connected in series.

[0530] In some embodiments of the present invention, the preparation method, wherein,

[0531] The first protein functional region and the second protein functional region are independently 1, 2 or more.

[0532] In some embodiments of the present invention, the preparation method, wherein,

[0533] There are two single-chain antibodies, which are respectively connected to the C-termini of two heavy chains of immunoglobulin.

[0534] In some embodiments of the present invention, the preparation method, wherein the single-chain antibody is independently linked in sequence: heavy chain variable region-linker-light chain variable region (VH-linker-VL), or light chain variable region-linker-heavy chain variable region (VL-linker-VH);

[0535] Preferably, the amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs: 45-48.

[0536] In some embodiments of the present invention, the preparation method, wherein the first protein functional region is an anti-VEGFA immunoglobulin, and the second protein functional region is an anti-PD-1 single-chain antibody;

[0537] Preferably, the anti-PD-1 single-chain antibody is two molecules, which are respectively connected to the C-termini of the two heavy chains of the anti-VEGFA immunoglobulin;

[0538] Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv;

[0539] Preferably, the bispecific antibody is a tetravalent bispecific antibody in the IgG-scFv format.

[0540] In some embodiments of the present invention, the preparation method, wherein,

[0541] The bispecific antibody comprises:

[0542] Targeting the first protein domain of VEGFA, and

[0543] Targeting the second protein functional region of PD-1;

[0544] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0545] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0546] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0547] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17, SEQ ID NO: 7 or SEQ ID NO: 11;

[0548] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0549] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0550] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0551] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0552] In some embodiments of the present invention, the preparation method, wherein,

[0553] The bispecific antibody comprises:

[0554] Targeting the first protein domain of VEGFA, and

[0555] Targeting the second protein functional region of PD-1;

[0556] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0557] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0558] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0559] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17, SEQ ID NO: 7 or SEQ ID NO: 11;

[0560] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0561] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0562] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0563] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0564] In some embodiments of the present invention, the preparation method, wherein,

[0565] The bispecific antibody comprises:

[0566] Targeting the first protein domain of VEGFA, and

[0567] Targeting the second protein functional region of PD-1;

[0568] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0569] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0570] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, SEQ ID NO: 20 or SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0571] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17;

[0572] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0573] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0574] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0575] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0576] In some embodiments of the present invention, the preparation method, wherein,

[0577] The bispecific antibody comprises:

[0578] Targeting the first protein domain of VEGFA, and

[0579] Targeting the second protein functional region of PD-1;

[0580] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0581] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0582] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0583] The amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs: 45-48;

[0584] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0585] The first protein functional region and the second protein functional region are connected via a first linker;

[0586] Preferably, the first linker is selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0587] In some embodiments of the present invention, the preparation method, wherein,

[0588] The bispecific antibody comprises:

[0589] Targeting the first protein domain of VEGFA, and

[0590] Targeting the second protein functional region of PD-1;

[0591] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0592] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0593] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, SEQ ID NO: 20 or SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0594] The amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs: 45-48;

[0595] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0596] The first protein functional region and the second protein functional region are connected via a first linker;

[0597] Preferably, the first linker is selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0598] In some embodiments of the present invention, in the preparation method, the bispecific antibody is administered in combination with at least one chemotherapeutic drug.

[0599] In some embodiments of the present invention, the preparation method, wherein,

[0600] The chemotherapy drug is one or more selected from tyrosine kinase inhibitors, platinum drugs (such as cisplatin or carboplatin), pemetrexed, paclitaxel, docetaxel, temozolomide and fotemustine.

[0601] In some embodiments of the present invention, the preparation method is characterized by one or more of the following (1) to (4):

[0602] (1) The unit dose of the bispecific antibody is 100 mg-4000 mg, 200 mg-3000 mg, 200 mg-2000 mg, 300 mg-2000 mg, 400 mg-2000 mg, 500 mg-2000 mg, or 1000 mg-2000 mg;

[0603] (2) The single dose of the bispecific antibody is 1-50 mg, 10 mg-50 mg, 10 mg-40 mg, 10 mg-30 mg, 10 mg-20 mg, 10 mg-15 mg, 15 mg-30 mg, or 15 mg-20 mg per kilogram of body weight;

[0604] (3) the bispecific antibody is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks;

[0605] (4) The bispecific antibody is administered by intravenous drip, intravenous injection or intraperitoneal injection.

[0606] In some embodiments of the present invention, the preparation method, wherein:

[0607] The bispecific antibody is administered in combination with pemetrexed and carboplatin; or

[0608] The bispecific antibody was administered in combination with paclitaxel and carboplatin.

[0609] In some embodiments of the present invention, the preparation method is characterized by one or more of the following (1) to (3):

[0610] (1) The single dose of pemetrexed is based on the body surface area (m 2 )10-1500mg / m 2 100-1000mg / m 2 200-900mg / m 2 300-800mg / m 2 400-700mg / m 2 500-600mg / m 2 ; Preferably, the pemetrexed is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks;

[0611] (2) The single dose of carboplatin is independently AUC 2-6min*mg / mL or AUC 2.5-5min*mg / mL; preferably, the carboplatin is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks;

[0612] (3) The single dose of paclitaxel is based on the body surface area (m 2 )67.5-175mg / m 2 87.5-175mg / m 2 or 131-175 mg / m 2 ; Preferably, the paclitaxel is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks.

[0613] In some embodiments of the present invention, the preparation method, wherein the non-small cell lung cancer brain metastasis is selected from one or more of squamous cell non-small cell lung cancer brain metastasis, non-squamous cell non-small cell lung cancer brain metastasis and lung adenocarcinoma brain metastasis;

[0614] Preferably, in patients with non-small cell lung cancer brain metastases, PD-L1 TPS ≥ 50%, or 1% < PD-L1 TPS ≥ 49%, or PD-L1 TPS ≤ 1%.

[0615] Another aspect of the present invention relates to a pharmaceutical composition for treating or preventing brain metastasis of non-small cell lung cancer, comprising a bispecific antibody, wherein:

[0616] The bispecific antibody comprises:

[0617] Targeting the first protein domain of VEGFA, and

[0618] Targeting the second protein functional region of PD-1;

[0619] in,

[0620] The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the immunoglobulin comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; and the single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively;

[0621] or,

[0622] The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and the light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; and, the immunoglobulin comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively.

[0623] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0624] The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17.

[0625] In some embodiments of the present invention, the pharmaceutical composition, wherein the bispecific antibody is selected from any one of the following (1)-(6):

[0626] (1)

[0627] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7;

[0628] (2)

[0629] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11;

[0630] (3)

[0631] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17;

[0632] (4)

[0633] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7;

[0634] (5)

[0635] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11; and

[0636] (6)

[0637] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17.

[0638] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0639] The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17.

[0640] In some embodiments of the present invention, the pharmaceutical composition, wherein the bispecific antibody is selected from any one of the following (7)-(12):

[0641] (7)

[0642] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0643] (8)

[0644] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0645] (9)

[0646] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 17; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0647] (10)

[0648] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3;

[0649] (11)

[0650] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 3; and

[0651] (12)

[0652] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:17; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3.

[0653] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0654] The immunoglobulin is of human IgG1 subtype;

[0655] Wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises one of the following mutation combinations:

[0656] L234A and L235A; or

[0657] L234A, L235A, G237A.

[0658] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0659] The amino acid sequence of the immunoglobulin heavy chain is shown in SEQ ID NO: 24, and the amino acid sequence of the immunoglobulin light chain is shown in SEQ ID NO: 26;

[0660] The amino acid sequence of the immunoglobulin heavy chain is shown in SEQ ID NO: 20, and the amino acid sequence of the immunoglobulin light chain is shown in SEQ ID NO: 26; or

[0661] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26.

[0662] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0663] The first protein functional region is directly connected to the second protein functional region or connected through a linker; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a linker.

[0664] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0665] The linker is a polypeptide shown in SEQ ID NO: 43, or a polypeptide consisting of multiple (eg, 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO: 43 connected in series.

[0666] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0667] The first protein functional region and the second protein functional region are independently 1, 2 or more.

[0668] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0669] There are two single-chain antibodies, which are respectively connected to the C-termini of two heavy chains of immunoglobulin.

[0670] In some embodiments of the present invention, the pharmaceutical composition, wherein the single-chain antibody is independently linked in sequence: heavy chain variable region-linker-light chain variable region (VH-linker-VL), or light chain variable region-linker-heavy chain variable region (VL-linker-VH);

[0671] Preferably, the amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs: 45-48.

[0672] In some embodiments of the present invention, the pharmaceutical composition, wherein the first protein functional region is an anti-VEGFA immunoglobulin, and the second protein functional region is an anti-PD-1 single-chain antibody;

[0673] Preferably, the anti-PD-1 single-chain antibody is two molecules, which are respectively connected to the C-termini of the two heavy chains of the anti-VEGFA immunoglobulin;

[0674] Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv;

[0675] Preferably, the bispecific antibody is a tetravalent bispecific antibody in the IgG-scFv format.

[0676] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0677] The bispecific antibody comprises:

[0678] Targeting the first protein domain of VEGFA, and

[0679] Targeting the second protein functional region of PD-1;

[0680] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0681] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0682] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0683] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17, SEQ ID NO: 7 or SEQ ID NO: 11;

[0684] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0685] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0686] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0687] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0688] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0689] The bispecific antibody comprises:

[0690] Targeting the first protein domain of VEGFA, and

[0691] Targeting the second protein functional region of PD-1;

[0692] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0693] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0694] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0695] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17, SEQ ID NO: 7 or SEQ ID NO: 11;

[0696] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0697] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0698] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0699] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0700] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0701] The bispecific antibody comprises:

[0702] Targeting the first protein domain of VEGFA, and

[0703] Targeting the second protein functional region of PD-1;

[0704] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0705] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0706] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, SEQ ID NO: 20 or SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0707] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17;

[0708] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0709] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0710] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0711] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 18.

[0712] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0713] The bispecific antibody comprises:

[0714] Targeting the first protein domain of VEGFA, and

[0715] Targeting the second protein functional region of PD-1;

[0716] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0717] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0718] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 3;

[0719] The amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs: 45-48;

[0720] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0721] The first protein functional region and the second protein functional region are connected via a first linker;

[0722] Preferably, the first linker is selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0723] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0724] The bispecific antibody comprises:

[0725] Targeting the first protein domain of VEGFA, and

[0726] Targeting the second protein functional region of PD-1;

[0727] The number of the first protein functional region is one, and the number of the second protein functional region is two;

[0728] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0729] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, SEQ ID NO: 20 or SEQ ID NO: 22, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26;

[0730] The amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs: 45-48;

[0731] The single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin;

[0732] The first protein functional region and the second protein functional region are connected via a first linker;

[0733] Preferably, the first linker is selected from SEQ ID NO: 18 and SEQ ID NO: 19.

[0734] In some embodiments of the present invention, the pharmaceutical composition comprises the bispecific antibody as the only active ingredient.

[0735] In some embodiments of the present invention, the pharmaceutical composition, wherein the bispecific antibody is administered in combination with at least one chemotherapeutic drug.

[0736] In some embodiments of the present invention, the pharmaceutical composition, wherein the active ingredients are the bispecific antibody and at least one chemotherapeutic drug.

[0737] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0738] The chemotherapy drug is one or more selected from tyrosine kinase inhibitors, platinum drugs (such as cisplatin or carboplatin), pemetrexed, paclitaxel, docetaxel, temozolomide and fotemustine.

[0739] In some embodiments of the present invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0740] In some embodiments of the present invention, the pharmaceutical composition is composed of an active ingredient and one or more pharmaceutically acceptable excipients.

[0741] In some embodiments of the present invention, the pharmaceutical composition is composed of the bispecific antibody according to any one of the present invention and one or more pharmaceutically acceptable excipients.

[0742] In some embodiments of the present invention, the pharmaceutical composition is composed of the bispecific antibody according to any one of the present invention, one or more chemotherapeutic drugs, and one or more pharmaceutically acceptable excipients.

[0743] In some embodiments of the present invention, the pharmaceutical composition is characterized by one or more of the following (1) to (4):

[0744] (1) The unit dose of the bispecific antibody is 100 mg-4000 mg, 200 mg-3000 mg, 200 mg-2000 mg, 300 mg-2000 mg, 400 mg-2000 mg, 500 mg-2000 mg, or 1000 mg-2000 mg;

[0745] (2) The single dose of the bispecific antibody is 1-50 mg, 10 mg-50 mg, 10 mg-40 mg, 10 mg-30 mg, 10 mg-20 mg, 10 mg-15 mg, 15 mg-30 mg, or 15 mg-20 mg per kilogram of body weight;

[0746] (3) the bispecific antibody is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks;

[0747] (4) The bispecific antibody is administered by intravenous drip, intravenous injection or intraperitoneal injection.

[0748] In some embodiments of the present invention, the pharmaceutical composition comprises:

[0749] The bispecific antibody, pemetrexed and carboplatin are administered in combination; or

[0750] The bispecific antibody, paclitaxel, and carboplatin are administered in combination.

[0751] In some embodiments of the present invention, the pharmaceutical composition is characterized by one or more of the following (1) to (3):

[0752] (1) The single dose of pemetrexed is based on the body surface area (m 2 )10-1500mg / m 2 100-1000mg / m 2 200-900mg / m 2 300-800mg / m 2 400-700mg / m 2 500-600mg / m 2 ; Preferably, the pemetrexed is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks;

[0753] (2) The single dose of carboplatin is independently AUC 2-6min*mg / mL or AUC 2.5-5min*mg / mL; preferably, the carboplatin is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks;

[0754] (3) The single dose of paclitaxel is based on the body surface area (m 2 )67.5-175mg / m 2 87.5-175mg / m 2 or 131-175 mg / m 2 ; Preferably, the paclitaxel is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks.

[0755] In some embodiments of the present invention, the pharmaceutical composition, wherein the non-small cell lung cancer brain metastasis is selected from one or more of squamous cell non-small cell lung cancer brain metastasis, non-squamous cell non-small cell lung cancer brain metastasis and lung adenocarcinoma brain metastasis;

[0756] Preferably, the PD-L1 TPS of patients with non-small cell lung cancer brain metastasis is ≥50%, or 1% < PD-L1 TPS ≥49%, or PD-L1 TPS ≤1%.

[0757] Antibody therapeutics, particularly monoclonal antibodies, have demonstrated promising therapeutic effects in the treatment of a wide range of diseases. Traditional experimental approaches to obtaining these therapeutic antibodies involve immunizing animals with antigens to generate antibodies that target the antigen, or improving antibodies with low affinity for the antigen through affinity maturation.

[0758] The variable regions of the light and heavy chains determine antigen binding; each chain's variable region contains three hypervariable regions, called complementarity-determining regions (CDRs). The heavy chain (H chain) CDRs consist of HCDR1, HCDR2, and HCDR3, while the light chain (L chain) CDRs consist of LCDR1, LCDR2, and LCDR3. These terms were first used by Kabat et al. (Bethesda MD, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication (1-3) 1991:91-3242).

[0759] Preferably, CDRs may also be defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 38. suppl_1 (2009): D301-D307.

[0760] The amino acid sequences of the CDR regions of the monoclonal antibody sequences in items (1) to (3) below were analyzed using techniques well known to those skilled in the art, such as using the VBASE2 database according to the IMGT definition. The results are as follows:

[0761] (1) Bevacizumab

[0762] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3.

[0763] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0764] HCDR1: GYTFTNYG (SEQ ID NO: 28)

[0765] HCDR2: INTYTGEP (SEQ ID NO: 29)

[0766] HCDR3:AKYPHYYGSSHWYFDV(SEQ ID NO:30)

[0767] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0768] LCDR1:QDISNY (SEQ ID NO: 31)

[0769] LCDR2:FTS (SEQ ID NO:32)

[0770] LCDR3:QQYSTVPWT (SEQ ID NO: 33)

[0771] (2) 14C12, 14C12H1L1, or 14C12H1L1(M)

[0772] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0773] HCDR1: GFAFSSYD (SEQ ID NO: 34)

[0774] HCDR2: ISGGGRYT (SEQ ID NO: 35)

[0775] HCDR3:ANRYGEAWFAY(SEQ ID NO:36)

[0776] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0777] LCDR1:QDINTY (SEQ ID NO: 37)

[0778] LCDR2:RAN (SEQ ID NO:38)

[0779] LCDR3:LQYDEFPLT (SEQ ID NO:39)

[0780] (3) VP101(hG1WT) or VP101(hG1DM)

[0781] The amino acid sequences of the 9 CDR regions of its heavy chain are as follows:

[0782] HCDR1: GYTFTNYG (SEQ ID NO: 28)

[0783] HCDR2: INTYTGEP (SEQ ID NO: 29)

[0784] HCDR3:AKYPHYYGSSHWYFDV(SEQ ID NO:30)

[0785] HCDR4: GFAFSSYD (SEQ ID NO: 34)

[0786] HCDR5: ISGGGRYT (SEQ ID NO: 35)

[0787] HCDR6:ANRYGEAWFAY(SEQ ID NO:36)

[0788] HCDR7: QDINTY (SEQ ID NO: 37)

[0789] HCDR8:RAN (SEQ ID NO:38)

[0790] HCDR9: LQYDEFPLT (SEQ ID NO: 39)

[0791] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0792] LCDR1:QDISNY (SEQ ID NO: 31)

[0793] LCDR2:FTS (SEQ ID NO:32)

[0794] LCDR3: QQYSTVPWT (SEQ ID NO: 33).

[0795] The antibody VP101 (hG1DM) of the present invention introduces amino acid mutations in the non-variable region of VP101 (hG1WT). According to the EU numbering system, amino acid mutations are introduced at positions 234 and 235:

[0796] VP101 (hG1DM) was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A) and a point mutation from leucine to alanine at position 235 (L235A) in the hinge region of its heavy chain.

[0797] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0798] As used herein, when referring to the amino acid sequence of VEGFA protein (GenBank ID: NP_001165097.1), it includes the full length of VEGFA protein, and also includes fusion proteins of VEGFA, such as fragments fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions) may occur naturally or be artificially introduced into the amino acid sequence of VEGFA protein without affecting its biological function. Therefore, in the present invention, the term "VEGFA protein" should include all such sequences, including natural or artificial variants thereof. Furthermore, when describing a sequence fragment of VEGFA protein, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0799] As used herein, when referring to the amino acid sequence of VEGFR2 protein (also known as KDR, GenBank ID: NP_002244), it includes the full length of the VEGFR2 protein, or the extracellular fragment VEGFR2-ECD of VEGFR2 or a fragment comprising VEGFR2-ECD; it also includes a fusion protein of VEGFR2-ECD, for example, a fragment fused to the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced into the amino acid sequence of the VEGFR2 protein without affecting its biological function. Therefore, in the present invention, the term "VEGFR2 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of the VEGFR2 protein, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0800] As used herein, unless otherwise specified, the VEGFRs are VEGFR1 and / or VEGFR2; their specific protein sequences are known in the art and can be found in existing literature or GenBank. For example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321) and VEGFR2 (VEGFR2, NCBI Gene ID: 3791).

[0801] As used herein, when referring to the amino acid sequence of PD-1 protein (Programmed cell death protein 1, NCBI GenBank: NM_005018), it includes the full length of the PD-1 protein, or the extracellular fragment PD-1ECD or a fragment containing PD-1ECD; it also includes a fusion protein of PD-1ECD, such as a fragment fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced into the amino acid sequence of the PD-1 protein without affecting its biological function. Therefore, in the present invention, the term "PD-1 protein" should include all such sequences, including natural or artificial variants thereof. Furthermore, when describing a sequence fragment of the PD-1 protein, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0802] As used herein, the term EC 50 It refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0803] As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. In a general sense, heavy chains can be understood as polypeptide chains with larger molecular weights in antibodies, and light chains refer to polypeptide chains with smaller molecular weights in antibodies. Light chains can be classified as kappa and lambda light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H1 、C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V LThe V domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) respectively form an antibody binding site. The allocation of amino acids to each region or domain can follow the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196 (1987): 901-917; Chothia et al. Nature 342 (1989): 878-883 or the IMGT numbering system definition, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 38. suppl_1 (2009): D301-D307. definition. In particular, the heavy chain can also include more than 3 CDRs, such as 6, 9, or 12. For example, in the bispecific antibodies of the present invention, the heavy chain can be an ScFv of another antibody connected to the C-terminus of the heavy chain of an IgG antibody, in which case the heavy chain contains 9 CDRs. The term "antibody" is not limited by any specific method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0804] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989). Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides having at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0805] As used herein, the term "Fd fragment" means a fragment consisting of H and C H1 The term "Fv fragment" refers to an antibody fragment consisting of a single arm of the V domain. L and V H The term "dAb fragment" refers to an antibody fragment consisting of a V H The term "Fab fragment" refers to an antibody fragment consisting of V L 、V H 、C L and C H1 The term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

[0806] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), in which V L and V H The domains are paired to form monovalent molecules through linkers that enable them to be generated as a single polypeptide chain (see, e.g., Bird et al., Science 242 (1988): 423-426 and Huston et al., Proc. Natl. Acad. Sci. USA 85 (1988): 5879-5883). Such scFv molecules can have the general structure: NH2-V L -Connector-V H -COOH or NH2-V H -Connector-V L-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 43) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 18) can be used, but variants thereof can also be used (Holliger et al., Proc. Natl. Acad. Sci. USA 90 (1993): 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al., Protein Eng. 8 (1995): 725-731, Choi et al., Eur. J. Immunol. 31 (2001): 94-106, Hu et al., Cancer Res. 56 (1996): 3055-3061, Kipriyanov et al., J. Mol. Biol. 293 (1999): 41-56 and Roovers et al., Cancer Immunol. (2001).

[0807] In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a bivalent antibody in which V H and V L The domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90 (1993): 6444-6448, and Poljak RJ et al., Structure 2 (1994): 1121-1123).

[0808] Antibody antigen-binding fragments (e.g., those described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and screened for specificity in the same manner as for intact antibodies.

[0809] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0810] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for possible spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies, and these different antibodies typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, for example, US Patent 4,816,567).

[0811] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (US Patent 4,816,567 Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 (1984): 6851-6855).

[0812] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced with amino acid residues of the corresponding non-human antibody, or with amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, Jones et al., Nature, 321 (1986): 522-525; Reichmann et al., Nature, (1988) 332: 323-329; Presta, Curr. Op. Struct. Biol., 2 (1992): 593-596; and Clark, Immunol. Today 21 (2000): 397-402.

[0813] As used herein, the term "epitope" refers to the site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to as an "antigenic determinant" in the art. An epitope or antigenic determinant is typically composed of chemically active surface groups of a molecule, such as amino acids or carbohydrates or sugar side chains, and typically has specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be "linear" or "conformational." See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all interacting points between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interacting points exist across separate protein amino acid residues.

[0814] As used herein, the term "isolated" or "isolated" refers to something that is obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and a highly pure identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0815] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0816] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0817] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity of M or less (K D In some embodiments of the present invention, the term "targeting" refers to specific binding.

[0818] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind with a dissociation equilibrium constant of less than about 10 -5M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less dissociation equilibrium constant (K D ) binds to the antigen, e.g., as determined using surface plasmon resonance (SPR) in a BIACORE instrument or in a Fortebio molecular interaction instrument.

[0819] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0820] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0821] In some embodiments, the anti-PD-1-anti-VEGFA bispecific antibody or antigen-binding fragment thereof can be formulated with one or more pharmaceutically acceptable carriers to prepare a suitable pharmaceutical composition.

[0822] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a disease associated with the binding of PD-1 to PD-L1 or excessive expression of VEGF, such as a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a disease associated with the binding of PD-1 to PD-L1 or excessive expression of VEGF, such as a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0823] Advantageous Effects of the Invention

[0824] The present invention achieves any one or more of the following technical effects (1) to (4):

[0825] (1) The bispecific antibody of the present invention can effectively treat brain metastases of non-small cell lung cancer.

[0826] (2) The bispecific antibody of the present invention is used to treat brain metastases of non-small cell lung cancer, and the median progression-free survival of patients is longer, even reaching 19.3 months.

[0827] (3) The bispecific antibody of the present invention is effective in treating brain metastases of non-small cell lung cancer quickly, and some patients achieved partial remission after 6 weeks of administration.

[0828] (4) The bispecific antibody of the present invention has good safety in the treatment of cancer, especially brain metastasis of non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0829] Figure 1: MRI of the head of a patient with brain metastases from non-small cell lung cancer who received the anti-PD-1 and anti-VEGFA bispecific antibody VP101 (hG1DM) combined with chemotherapy 12 weeks after treatment. The white circles and x-marked areas at baseline indicate tumor lesions.

[0830] Figure 2: MRI of the head in a patient with brain metastases from non-small cell lung cancer who received monotherapy with the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) 42 weeks after treatment.

[0831] Figure 3: Intracranial response rate (RANO criteria) of the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) in the treatment of brain metastases from non-small cell lung cancer.

[0832] Figure 4: Median progression-free survival in patients with brain metastases from non-small cell lung cancer treated with the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM).

[0833] The partial sequences involved in the present invention are shown in Table A below.

[0834] Table A DETAILED DESCRIPTION

[0835] The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will appreciate that the following examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific techniques or conditions not specified in the examples are based on the techniques or conditions described in the literature in this area (e.g., with reference to "Molecular Cloning Experiment Guide" by J. Sambrook et al., translated by Huang Peitang et al., 3rd edition, Science Press) or according to product specifications. Reagents or instruments not specified by manufacturer are all conventional products that can be purchased commercially.

[0836] Preparation Example 1: Preparation of anti-VEGFA antibody Bevacizumab

[0837] The amino acid sequences of the heavy and light chain variable regions of the marketed anti-VEGFA monoclonal antibody Avastin (Bevacizumab) were referenced in Chinese Patent Publication CN1259962A. The nucleic acid sequences encoding the heavy and light chain variable regions were synthesized by GenScript.

[0838] The amino acid sequence of the Bevacizumab heavy chain variable region (Bevacizumab-VH) is shown in SEQ ID NO: 1.

[0839] The nucleic acid sequence encoding the Bevacizumab heavy chain variable region is shown in SEQ ID NO: 2.

[0840] The amino acid sequence of the Bevacizumab light chain variable region (Bevacizumab-VL) is shown in SEQ ID NO: 3.

[0841] The nucleic acid sequence encoding the Bevacizumab light chain variable region is shown in SEQ ID NO: 4.

[0842] The heavy chain constant region all used the Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region all used the Ig kappa chain C region, ACCESSION: P01834.

[0843] The heavy and light chain cDNAs of bevacizumab were cloned into the pcDNA3.1 vector to generate recombinant expression plasmids for the antibody bevacizumab. The recombinant plasmids were transfected into 293F cells. The 293F cell culture fluid was purified and then tested.

[0844] The anti-VEGFA monoclonal antibody Avastin (Bevacizumab) was produced.

[0845] Preparation Example 2: Sequence Design of Anti-PD-1 Antibody 14C12, Its Humanized Antibody 14C12H1L1, and Mutant 14C12H1L1(M)

[0846] The amino acid sequences of the heavy and light chains of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1, as well as the encoding nucleic acid sequences, are identical to those of 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A, respectively.

[0847] (1) Heavy chain variable region sequence and light chain variable region sequence of 14C12

[0848] The amino acid sequence of the 14C12 heavy chain variable region is shown in SEQ ID NO:5.

[0849] The nucleic acid sequence encoding the 14C12 heavy chain variable region is shown in SEQ ID NO:6.

[0850] The amino acid sequence of the light chain variable region of 14C12 is shown in SEQ ID NO:7.

[0851] The nucleic acid sequence encoding the light chain variable region of 14C12 is shown in SEQ ID NO:8.

[0852] (2) Heavy chain variable region sequence, light chain variable region sequence, heavy chain sequence, and light chain sequence of humanized monoclonal antibody 14C12H1L1

[0853] The amino acid sequence of the heavy chain variable region of 14C12H1L1 is shown in SEQ ID NO:9.

[0854] The nucleic acid sequence encoding the heavy chain variable region of 14C12H1L1 is shown in SEQ ID NO:10.

[0855] The amino acid sequence of the light chain variable region of 14C12H1L1 is shown in SEQ ID NO:11.

[0856] The nucleic acid sequence encoding the light chain variable region of 14C12H1L1 is shown in SEQ ID NO:12.

[0857] The amino acid sequence of the 14C12H1L1 heavy chain (14C12H1) is shown in SEQ ID NO:13.

[0858] The nucleic acid sequence encoding the 14C12H1L1 heavy chain (14C12H1) is shown in SEQ ID NO:14.

[0859] The amino acid sequence of the 14C12H1L1 light chain (14C12L1) is shown in SEQ ID NO:15.

[0860] The nucleic acid sequence encoding the 14C12H1L1 light chain (14C12L1) is shown in SEQ ID NO:16.

[0861] (3) Heavy chain variable region sequence and light chain variable region sequence of 14C12H1L1(M)

[0862] Based on 14C12H1L1, individual amino acids in its framework region (light chain) were mutated to obtain 14C12H1L1(M).

[0863] The amino acid sequence of the light chain variable region 14C12L1(M) of 14C12H1L1(M) is shown in SEQ ID NO:17.

[0864] The amino acid sequence of the heavy chain variable region 14C12H1(M) of 14C12H1L1 is identical to that of the heavy chain variable region 14C12H1 of 14C12H1L1, that is, as shown in SEQ ID NO:9.

[0865] Preparation Example 3: Sequence Design of Bispecific Antibodies

[0866] 1. Sequence Design

[0867] The structural model of the bispecific antibody in the present invention belongs to the Morrison model (IgG-scFv), that is, the C-termini of the two heavy chains of an IgG antibody are connected to the scFv fragment of another antibody, and the main components of the heavy chain and light chain are designed as shown in Table 1 below.

[0868] Based on the aforementioned bevacizumab, the VP101 antibody, named VP101(M), utilizes the amino acid sequences of the heavy and light chain variable regions of 14C12H1L1(M) as the ScFv fragment. Compared to 14C12H1L1, 14C12H1L1(M) effectively optimizes the bispecific antibody structure and enhances its effectiveness.

[0869] Table 1: Composition design of heavy and light chains of VP101(M) and VP101(G4M)

[0870] In Table 1 above:

[0871] (1) The "V" in the lower right corner refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. If "V" is not marked, the corresponding heavy chain or light chain is the full length including the constant region. The amino acid sequences of these variable regions or full lengths and their encoding nucleic acid sequences are referenced to the corresponding sequences described in the preparation examples above.

[0872] (2) The amino acid sequence of Linker1 is shown in SEQ ID NO: 18.

[0873] Optionally, the amino acid sequence of Linker 2 is shown in SEQ ID NO: 19. Linker 2 can replace the aforementioned Linker 1.

[0874] (3) Bevacizumab-H uses the Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region.

[0875] (4) Bevacizumab-G4H uses Ig gamma-4 chain C region, ACCESSION: P01861.1 as the heavy chain constant region.

[0876] 2. Expression and Purification of Antibody VP101(M)

[0877] The heavy chain cDNA sequence and light chain cDNA sequence of VP101(M) were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-VP101H and pUC57simple-VP101L plasmids, respectively.

[0878] Plasmids pUC57simple-VP101H and pUC57simple-VP101L were digested with HindIII and EcoRI, and the heavy and light chains recovered by electrophoresis were subcloned into the pcDNA3.1 vector. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and then loaded onto a HiTrap MabSelect SuRe column. The protein was eluted in one step with Elution Buffer to recover the target antibody VP101, and the buffer was exchanged with PBS.

[0879] 3. Detection of Antibody VP101(M)

[0880] The purified samples were added into reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer respectively, boiled and then subjected to SDS-PAGE electrophoresis detection.

[0881] To distinguish it from the mutated antibody described in Preparation Example 4, VP101(M) is also referred to herein as VP101(hG1WT). This "wild-type" VP101(M) employs the Ig gamma-1 chain C region, ACCESSION: P01857, as the heavy chain constant region and the Ig kappa chain C region, ACCESSION: P01834, as the light chain constant region.

[0882] The amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1WT) is shown in SEQ ID NO: 20.

[0883] The nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1WT) is shown in SEQ ID NO: 21.

[0884] To distinguish it from the mutated antibody described in Preparation Example 4, VP101(G4M) is also referred to as VP101(hG4WT) in the present invention. VP101(G4M) is considered "wild-type" and utilizes the Ig gamma-4 chain C region, ACCESSION: P01861.1, as the heavy chain constant region and the Ig kappa chain C region, ACCESSION: P01834, as the light chain constant region.

[0885] The amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG4WT) is shown in SEQ ID NO: 22.

[0886] The nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG4WT) is shown in SEQ ID NO: 23.

[0887] Preparation Example 4: Design of amino acid mutations in the non-variable region of the humanized bispecific antibody VP101 (hG1WT)

[0888] Based on VP101 (hG1WT) obtained in Preparation Example 3, the inventors obtained VP101 (hG1DM) by introducing a point mutation from leucine to alanine (L234A) at position 234 of its heavy chain and a point mutation from leucine to alanine (L235A) at position 235.

[0889] The amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1DM) is shown in SEQ ID NO: 24.

[0890] The nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1DM) is shown in SEQ ID NO: 25.

[0891] The light chain amino acid sequences of the immunoglobulin portions of VP101 (hG1DM), VP101 (hG1WT), and VP101 (hG4WT) are identical, as are their encoding nucleic acid sequences.

[0892] The amino acid sequence of the light chain of the immunoglobulin portion of VP101 (hG1DM) is shown in SEQ ID NO: 26.

[0893] The nucleic acid sequence encoding the light chain of the immunoglobulin portion of VP101 (hG1DM) is shown in SEQ ID NO: 27.

[0894] The amino acid sequence of the “heavy chain of the immunoglobulin portion + linker + scFv portion” (equivalent to the heavy chain of the bispecific antibody) in the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) is shown in SEQ ID NO: 44.

[0895] The amino acid sequence of scFv is shown in SEQ ID NO: 45.

[0896] Example 1: Anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) effectively treats patients with brain metastases from non-small cell lung cancer

[0897] Patients with advanced NSCLC from the AK112-201 (NCT04736823) and AK112-202 (NCT04900363) trials, aged 18-75 years, had not received prior systemic cancer therapy and had a performance status of 0-1. They were eligible for either VP101(hG1DM) combined with chemotherapy or VP101(hG1DM) alone. VP101(hG1DM) is being developed under the code name AK112.

[0898] Each three-week dosing cycle was administered sequentially on the first day of each cycle: AK112 was administered intravenously first, followed by pemetrexed or paclitaxel, and finally carboplatin was administered intravenously.

[0899] The dosing regimen for the trial numbered AK112-201 is as follows:

[0900] Patients with non-squamous non-small cell lung cancer were treated with VP101 (hG1DM) (20 mg / kg, Q3W) combined with pemetrexed (500 mg / m 2 ) and carboplatin (AUC5 min*mg / mL, Q3W) for 4 cycles. If the disease does not progress, VP101(hG1DM) (20 mg / kg, Q3W) combined with pemetrexed (500 mg / m 2 ) Maintenance treatment for 2 years;

[0901] Patients with squamous non-small cell lung cancer were treated with VP101 (hG1DM) (20 mg / kg, Q3W) combined with paclitaxel (175 mg / m 2 ) and carboplatin (AUC5 min*mg / mL, Q3W) for a total of 4 cycles. If the disease does not progress, VP101(hG1DM) (20 mg / kg, Q3W) is given as maintenance treatment for 2 years.

[0902] The dosing regimen for the trial numbered AK112-202 is as follows:

[0903] Patients were treated with VP101 (hG1DM) (20 mg / kg, Q3W) for 4 cycles. If the disease did not progress, VP101 (hG1DM) (20 mg / kg, Q3W) was given as maintenance treatment for 2 years.

[0904] Initial workup for brain metastases included cranial MRI or head CT to identify brain metastases. Patients with brain metastases at baseline were eligible if they had no symptoms caused by brain metastases or were clinically stable for at least 2 weeks after treatment. Patients with brain metastases who had received radiotherapy or were unable to undergo follow-up MRI due to allergies were not eligible to participate in the evaluation of the intracranial activity of the anti-PD-1-anti-VEGFA bispecific antibody. For patients with brain metastases at baseline, brain MRI was performed every 6 weeks during treatment. In addition to evaluating the response of extracranial target lesions according to RECIST 1.1 criteria, brain metastases were serially assessed by two independent neuroradiologists (from the United States and China) according to the Response Assessment in Neuro-Oncology (RANO) criteria.

[0905] A total of 35 patients met the criteria for inclusion in this analysis: 28 patients from the AK112-201 trial and 7 patients from the AK112-202 trial. The median age of these patients was 60 years (range, 42-69 years), 77% of the 35 patients were male, 26 patients (74.3%) had non-small cell lung adenocarcinoma, 5 patients (14.3%) had squamous non-small cell lung cancer, 4 patients (11.4%) had other types of NSCLC, and 46% of the patients (46% of the 35 patients) were PD-L1 positive (PD-L1 TPS ≥ 1%).

[0906] Results showed that a total of 12 patients (34%) with NSCLC brain metastases achieved a response to their intracranial tumors, including 8 patients with complete responses. Of these 12 patients with NSCLC brain metastases, 11 (39%) received VP101 (hG1DM) combined with chemotherapy, and 1 (14%) received VP101 (hG1DM) monotherapy. Of the 8 patients who achieved complete responses, 7 (25% of the total patients in the AK112-201 trial) received VP101 (hG1DM) combined with chemotherapy, and 1 (14% of the total patients in the AK112-202 trial) received VP101 (hG1DM) monotherapy, as shown in Figure 3. The median progression-free survival for patients with brain metastases in both cohorts was 19.3 months (Figure 4), and no patients with brain metastases experienced intracranial hemorrhage within 3 months of treatment. Some patients achieved partial responses as early as week 6 of dosing. As an example, the results for one patient with brain metastases from non-small cell lung cancer who received VP101 (hG1DM) combined with chemotherapy are shown in Table 2 and Figure 1, demonstrating effective remission of the brain metastases. Furthermore, the results for another patient with brain metastases from non-small cell lung cancer who received VP101 (hG1DM) monotherapy are shown in Figure 2, demonstrating similar effective remission of the brain metastases.

[0907] Table 2: Intracranial tumor lesion assessment results for the same patient with non-small cell lung cancer brain metastasis as in Figure 1

[0908] Example 2: In vivo safety analysis of anti-PD-1-anti-VEGFA bispecific antibodies

[0909] The safety and efficacy of the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) are currently being evaluated in multiple clinical trials. Bleeding, hypertension, proteinuria, and gastrointestinal perforation are frequently reported adverse events in bevacizumab clinical studies. The present inventors analyzed the incidence of these adverse events (≥ grade 3) in the clinical studies of the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM). 648 patients from 7 clinical trials (NCT04047290, NCT04597541, NCT05116007, NCT04736823, NCT04900363, NCT04870177, and NCT0518471) were included in the analysis data set. The experimental results are shown in Tables 3 and 4.

[0910] As shown in Table 3, the incidence of bleeding greater than grade 3, hypertension, proteinuria, and gastrointestinal perforation in the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) group was 0.5%, 4.6%, 1.1%, and 0.3%, respectively, which were lower than those in the bevacizumab group (the data for bevacizumab were derived from the drug instructions).

[0911] In addition, as shown in Table 4, the analysis of immune-related adverse events (irAEs) in these trials found that the number of safety events for the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) was numerically lower than the published safety data of nivolumab (data for nivolumab were derived from the drug instructions).

[0912] These data indicate that the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) has a good in vivo safety profile.

[0913] Table 3: Incidence of bleeding, hypertension, proteinuria, and gastrointestinal perforation in clinical trials

[0914] Table 4: Incidence of irAEs in clinical trials

[0915] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of a bispecific antibody in the preparation of a medicament for treating or preventing brain metastases of non-small cell lung cancer, wherein, the bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the immunoglobulin comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs: 28-30 respectively, and the light-chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 31-33 respectively; and, the single-chain antibody comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs: 34-36 respectively, and the light-chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 37-39 respectively; or, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the single-chain antibody comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs: 28-30 respectively, and the light-chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 31-33 respectively; and, the immunoglobulin comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs: 34-36 respectively, and its light-chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 37-39 respectively.

2. The use according to claim 1, wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light-chain variable region of the single-chain antibody is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO:

17.

3. Use according to any one of claims 1 to 2, wherein, The bispecific antibody is selected from any one of the following (1)-(6): (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 7; (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 11; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 17; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 7; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 11; and (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:

17.

4. The use according to claim 1, wherein, The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; and, the amino acid sequence of the heavy-chain variable region of the immunoglobulin is selected from SEQ ID NO:5 and SEQ ID NO:9, and the amino acid sequence of the light-chain variable region of the immunoglobulin is selected from SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:

17.

5. The use according to any one of claims 1 or 4, wherein, The bispecific antibody is selected from any one of (7)-(12) below: (7) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO:5, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO:7; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; (8) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO:5, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO:11; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; (9) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO:5, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO:17; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; (10) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO:9, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO:7; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; (11) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO:9, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO:11; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; and (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:17; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:

3.

6. The use according to any one of claims 1 to 5, wherein the immunoglobulin is of human IgG1 subtype; wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises one of the following mutation combinations: L234A and L235A; or L234A, L235A, G237A.

7. The use according to any one of claims 1 to 3 and 6, wherein the amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:24, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; the amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:20, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; or the amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:22, and the amino acid sequence of its light chain is as shown in SEQ ID NO:

26.

8. The use according to any one of claims 1 to 7, wherein the first protein functional region is directly connected to the second protein functional region or connected by a linker; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected by a linker.

9. The use according to claim 8, wherein the linker is the polypeptide shown in SEQ ID NO:43, or a polypeptide formed by tandemly connecting multiple (such as 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO:

43.

10. The use according to any one of claims 1 to 9, wherein the first protein functional region and the second protein functional region are independently 1, 2 or more than 2.

11. The use according to any one of claims 1 to 10, wherein there are two single-chain antibodies, which are respectively connected to the C-terminals of the two heavy chains of the immunoglobulin.

12. The use according to any one of claims 1 to 11, wherein The single-chain antibody is independently: heavy chain variable region-linker-light chain variable region (VH-linker-VL), or light chain variable region-linker-heavy chain variable region (VL-linker-VH); Preferably, the amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs:45-48.

13. Use according to any one of claims 1 to 12, wherein The first protein functional region is an immunoglobulin against VEGFA, and the second protein functional region is a single-chain antibody against PD-1; Preferably, there are two molecules of the single-chain antibody against PD-1, which are respectively connected to the C-terminals of the two heavy chains of the immunoglobulin against VEGFA; Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv; Preferably, the bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv.

14. Use according to any one of claims 1 to 13, wherein The bispecific antibody is selected from any one of the following (13)-(16): (13) The bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:1, and the amino acid sequence of its light chain variable region is as shown in SEQ ID NO:3; The amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:9 or SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:17, SEQ ID NO:7 or SEQ ID NO:11; The single-chain antibodies are respectively linked to the C termini of the two heavy chains of the immunoglobulin; The first protein functional region is linked to the second protein functional region through a first linker; and the heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody through a second linker; the first linker and the second linker are the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:18 and SEQ ID NO:19; Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO:18; (14) The bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:24, SEQ ID NO:20 or SEQ ID NO:22, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; The amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:17; The single-chain antibodies are respectively linked to the C termini of the two heavy chains of the immunoglobulin; The first protein functional region is linked to the second protein functional region through a first linker; and the heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody through a second linker; the first linker and the second linker are the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:18 and SEQ ID NO:19; Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO:18; (15) The bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:1, and the amino acid sequence of its light chain variable region is as shown in SEQ ID NO:3; The amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs: 45 - 48; The single-chain antibodies are respectively connected to the C termini of the two heavy chains of the immunoglobulin; The first protein functional region is connected to the second protein functional region through a first linker; Preferably, the first linker is selected from SEQ ID NO:18 and SEQ ID NO:19; (16) The bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:24, SEQ ID NO:20 or SEQ ID NO:22, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; The amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs: 45 - 48; The single-chain antibodies are respectively connected to the C termini of the two heavy chains of the immunoglobulin; The first protein functional region is connected to the second protein functional region through a first linker; Preferably, the first linker is selected from SEQ ID NO:18 and SEQ ID NO:

19.

15. Use according to any one of claims 1 to 14, which is the use of the combination of the bispecific antibody and at least one chemotherapeutic drug in the preparation of a drug for treating or preventing brain metastases of non-small cell lung cancer.

16. Use according to claim 15, wherein the chemotherapeutic drug is one or more selected from tyrosine kinase inhibitors, platinum drugs (such as cisplatin or carboplatin), pemetrexed, paclitaxel, docetaxel, temozolomide and fotemustine.

17. The use according to any one of claims 1 to 16, characterized in that One or more of the following (1) to (4): (1) The unit dose of the bispecific antibody is 100 mg - 4000 mg, 200 mg - 3000 mg, 200 mg - 2000 mg, 300 mg - 2000 mg, 400 mg - 2000 mg, 500 mg - 2000 mg, or 1000 mg - 2000 mg; (2) The single-dose administration dose of the bispecific antibody is 1 - 50 mg, 10 mg - 50 mg, 10 mg - 40 mg, 10 mg - 30 mg, 10 mg - 20 mg, 10 mg - 15 mg, 15 mg - 30 mg, or 15 mg - 20 mg per kilogram of body weight; (3) The bispecific antibody is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks; (4) The administration method of the bispecific antibody is intravenous drip, intravenous injection, or intraperitoneal injection.

18. Use according to any one of claims 1 to 17, wherein, The combination is: The bispecific antibody, pemetrexed, and carboplatin; or The bispecific antibody, paclitaxel, and carboplatin.

19. The use according to claim 18, wherein One or more of the following (1) to (3): (1) The single-dose of pemetrexed is calculated according to the body surface area of each subject (m 2 ) at 10 - 1500 mg / m 2 , 100 - 1000 mg / m 2 , 200 - 900 mg / m 2 , 300 - 800 mg / m 2 , 400 - 700 mg / m 2 , 500 - 600 mg / m 2 ; preferably, the pemetrexed is administered once every 1 week, 2 weeks, 3 weeks or 4 weeks; (2) The single-dose administration dose of the carboplatin is independently AUC 2 - 6 min*mg / mL or AUC 2.5 - 5 min*mg / mL; preferably, the carboplatin is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks; (3) The single-dose of paclitaxel is based on the body surface area of each subject (m 2 ) 67.5-175 mg / m 2 , 87.5-175 mg / m 2 or 131-175 mg / m 2 ; Preferably, the paclitaxel is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks.

20. Use according to any one of claims 1 to 19, wherein The non-small cell lung cancer brain metastasis is selected from one or more of squamous cell non-small cell lung cancer brain metastasis, non-squamous cell non-small cell lung cancer brain metastasis, and lung adenocarcinoma brain metastasis; Preferably, for patients with non-small cell lung cancer brain metastasis, PD-L1 TPS ≥ 50%, or 1% < PD-L1 TPS ≥ 49%, or PD-L1 TPS ≤ 1%.

21. A bispecific antibody for treating or preventing non-small cell lung cancer brain metastasis, where the bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; wherein the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the immunoglobulin comprises a heavy-chain variable region and a light-chain variable region, where the heavy-chain variable region comprises HCDR1 - HCDR3 with amino acid sequences shown in SEQ ID NOs: 28 - 30 respectively, and the light-chain variable region comprises LCDR1 - LCDR3 with amino acid sequences shown in SEQ ID NOs: 31 - 33 respectively; and, the single-chain antibody comprises a heavy-chain variable region and a light-chain variable region, where the heavy-chain variable region comprises HCDR1 - HCDR3 with amino acid sequences shown in SEQ ID NOs: 34 - 36 respectively, and the light-chain variable region comprises LCDR1 - LCDR3 with amino acid sequences shown in SEQ ID NOs: 37 - 39 respectively; Or, The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the single-chain antibody comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs: 28-30 respectively, and the light-chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 31-33 respectively; and, the immunoglobulin comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs: 34-36 respectively and its light-chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 37-39 respectively.

22. The bispecific antibody according to claim 21, wherein The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light-chain variable region of the single-chain antibody is selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO:

17.

23. The bispecific antibody according to any one of claims 21 to 22, wherein The bispecific antibody is selected from any one of the following (1)-(6): (1) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO: 7; (2) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO: 11; (3) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO: 17; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 7; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 11; and (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:

17.

24. The bispecific antibody according to claim 21, wherein, The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO:

17.

25. The bispecific antibody according to any one of claims 21 or 24, wherein, The bispecific antibody is selected from any one of the following (7)-(12): (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 7; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 3; (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 11; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 3; (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:17; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:7; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:11; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; and (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:17; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:

3.

26. The bispecific antibody according to any one of claims 21 to 25, wherein, The immunoglobulin is of human IgG1 subtype; Wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises one of the following mutation combinations: L234A and L235A; or L234A, L235A, G237A.

27. The bispecific antibody according to any one of claims 21 to 23 and 26, wherein, The amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:24, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; The amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:20, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; or The amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:22, and the amino acid sequence of its light chain is as shown in SEQ ID NO:

26.

28. The bispecific antibody according to any one of claims 21 to 27, wherein, The first protein functional region is directly connected to the second protein functional region or connected through a linker; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a linker.

29. The bispecific antibody according to claim 28, wherein the linker is the polypeptide shown in SEQ ID NO:43, or a polypeptide formed by tandem connection of multiple (such as 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO:

43.

30. The bispecific antibody according to any one of claims 21 to 29, wherein the first protein functional region and the second protein functional region are independently 1, 2 or more than 2.

31. The bispecific antibody according to any one of claims 21 to 30, wherein the single-chain antibody is two, and is respectively connected to the C termini of the two heavy chains of the immunoglobulin.

32. The bispecific antibody according to any one of claims 21 to 31, wherein, The single-chain antibody is independently connected in sequence: heavy chain variable region-linker-light chain variable region (VH-linker-VL), or light chain variable region-linker-heavy chain variable region (VL-linker-VH); Preferably, the amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NOs:45-48.

33. The bispecific antibody according to any one of claims 21 to 32, wherein, The first protein functional region is an immunoglobulin against VEGFA, and the second protein functional region is a single-chain antibody against PD-1; Preferably, the single-chain antibody against PD-1 is two molecules, and is respectively connected to the C termini of the two heavy chains of the immunoglobulin against VEGFA; Preferably, the bispecific antibody is a bispecific antibody in the IgG-scFv form; Preferably, the bispecific antibody is a tetravalent bispecific antibody in the IgG-scFv form.

34. The bispecific antibody according to any one of claims 21 to 33, which is selected from any one of the following (13)-(16): (13) The bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; The first protein functional region is 1, and the second protein functional region is 2; wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:1, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:9 or SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:17, SEQ ID NO:7 or SEQ ID NO:11; the single-chain antibody is respectively connected to the C termini of the two heavy chains of the immunoglobulin. The first protein functional region is linked to the second protein functional region by a first linker; and the heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody by a second linker; the first linker and the second linker are the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:18 and SEQ ID NO:19; Preferably, the amino acid sequences of both the first linker and the second linker are as shown in SEQ ID NO:18; (14) The bispecific antibody comprises: A first protein functional region targeting VEGFA, and A second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:24, SEQ ID NO:20 or SEQ ID NO:22, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; The amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:17; The single-chain antibody is respectively linked to the C termini of the two heavy chains of the immunoglobulin; The first protein functional region is linked to the second protein functional region by a first linker; and the heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody by a second linker; the first linker and the second linker are the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:18 and SEQ ID NO:19; Preferably, the amino acid sequences of both the first linker and the second linker are as shown in SEQ ID NO:18; (15) The bispecific antibody comprises: A first protein functional region targeting VEGFA, and A second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:1, and the amino acid sequence of its light chain variable region is as shown in SEQ ID NO:3; The amino acid sequence of the single-chain antibody is any one of the sequences in SEQ ID NOs:45 - 48; The single-chain antibody is respectively linked to the C termini of the two heavy chains of the immunoglobulin; The first protein functional region is linked to the second protein functional region by a first linker; Preferably, the first linker is selected from SEQ ID NO:18 and SEQ ID NO:19; (16) The bispecific antibody comprises: A first protein functional region targeting VEGFA, and A second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Among them, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:24, SEQ ID NO:20 or SEQ ID NO:22, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; The amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs: 45-48; The single-chain antibodies are respectively connected to the C-terminals of the two heavy chains of the immunoglobulin; The first protein functional region is connected to the second protein functional region through a first linker; Preferably, the first linker is selected from SEQ ID NO:18 and SEQ ID NO:

19.

35. The bispecific antibody according to any one of claims 21 to 34, wherein, The bispecific antibody is administered in combination with at least one chemotherapeutic drug.

36. The bispecific antibody according to claim 35, wherein, The chemotherapeutic drug is one or more selected from tyrosine kinase inhibitors, platinum drugs (such as cisplatin or carboplatin), pemetrexed, paclitaxel, docetaxel, temozolomide, and fotemustine.

37. The bispecific antibody according to any one of claims 21 to 36, characterized in that One or more of the following (1) to (4): (1) The unit dose of the bispecific antibody is 100 mg - 4000 mg, 200 mg - 3000 mg, 200 mg - 2000 mg, 300 mg - 2000 mg, 400 mg - 2000 mg, 500 mg - 2000 mg, or 1000 mg - 2000 mg; (2) The single-dose administration dose of the bispecific antibody is 1 - 50 mg, 10 mg - 50 mg, 10 mg - 40 mg, 10 mg - 30 mg, 10 mg - 20 mg, 10 mg - 15 mg, 15 mg - 30 mg, or 15 mg - 20 mg per kilogram of body weight; (3) The bispecific antibody is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks; (4) The administration method of the bispecific antibody is intravenous drip, intravenous injection, or intraperitoneal injection.

38. The bispecific antibody according to any one of claims 21 to 37, wherein: The bispecific antibody is administered in combination with pemetrexed and carboplatin; or The bispecific antibody is administered in combination with paclitaxel and carboplatin.

39. The bispecific antibody according to claim 38, wherein One or more of the following (1) to (3): (1) The single-dose of pemetrexed is based on the body surface area of each subject (m 2 ) 10 - 1500 mg / m 2 , 100 - 1000 mg / m 2 , 200 - 900 mg / m 2 , 300 - 800 mg / m 2 , 400 - 700 mg / m 2 , 500 - 600 mg / m 2 ; Preferably, the pemetrexed is administered once every 1 week, 2 weeks, 3 weeks or 4 weeks; (2) The single-dose administration dose of carboplatin is independently AUC 2 - 6 min*mg / mL or AUC 2.5 - 5 min*mg / mL; preferably, carboplatin is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks; (3) The single-dose of paclitaxel is based on the body surface area of each subject (m 2 ) 67.5 - 175 mg / m 2 , 87.5 - 175 mg / m 2 or 131 - 175 mg / m 2 ; Preferably, the paclitaxel is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks.

40. The bispecific antibody according to any one of claims 21 to 39, wherein The non-small cell lung cancer brain metastasis is one or more selected from squamous cell non-small cell lung cancer brain metastasis, non-squamous cell non-small cell lung cancer brain metastasis, and lung adenocarcinoma brain metastasis; Preferably, for patients with non-small cell lung cancer brain metastasis, PD-L1 TPS ≥ 50%, or 1% < PD-L1 TPS ≥ 49%, or PD-L1 TPS ≤ 1%.

41. A method for treating or preventing brain metastases of non-small cell lung cancer, comprising the step of administering an effective amount of a bispecific antibody to a subject in need thereof, wherein, the bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the immunoglobulin comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs: 28-30 respectively, and the light-chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 31-33 respectively; and, the single-chain antibody comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs: 34-36 respectively, and the light-chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 37-39 respectively; or, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the single-chain antibody comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs: 28-30 respectively, and the light-chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 31-33 respectively; and, the immunoglobulin comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1-HCDR3 with amino acid sequences shown in SEQ ID NOs: 34-36 respectively, and its light-chain variable region comprises LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 37-39 respectively.

42. The method according to claim 41, wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; the amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light-chain variable region of the single-chain antibody is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO:

17.

43. The method according to any one of claims 41 to 42, wherein, The bispecific antibody is selected from any one of the following (1)-(6): (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 7; (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 11; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 17; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 7; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 11; and (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:

17.

44. According to the method of claim 41, wherein, The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; and, the amino acid sequence of the heavy-chain variable region of the immunoglobulin is selected from SEQ ID NO:5 and SEQ ID NO:9, and the amino acid sequence of the light-chain variable region of the immunoglobulin is selected from SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:

17.

45. The method according to any one of claims 41 or 44, wherein, The bispecific antibody is selected from any one of the following (7)-(12): (7) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO:5, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO:7; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; (8) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO:5, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO:11; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; (9) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO:5, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO:17; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; (10) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO:9, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO:7; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; (11) The amino acid sequence of the heavy-chain variable region of the immunoglobulin is as shown in SEQ ID NO:9, and the amino acid sequence of the light-chain variable region of the immunoglobulin is as shown in SEQ ID NO:11; and, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is as shown in SEQ ID NO:3; and (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:17; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:

3.

46. The method according to any one of claims 41 to 45, wherein the immunoglobulin is of human IgG1 subtype; wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises one of the following mutation combinations: L234A and L235A; or L234A, L235A, G237A.

47. The method according to any one of claims 41 to 43 and 46, wherein the amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:24, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; the amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:20, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; or the amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:22, and the amino acid sequence of its light chain is as shown in SEQ ID NO:

26.

48. The method according to any one of claims 41 to 47, wherein the first protein functional region is directly connected to the second protein functional region or connected by a linker; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected by a linker.

49. The method according to claim 48, wherein the linker is the polypeptide shown in SEQ ID NO:43, or a polypeptide formed by tandemly connecting multiple (such as 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO:

43.

50. The method according to any one of claims 41 to 49, wherein the first protein functional region and the second protein functional region are independently 1, 2 or more than 2.

51. The method according to any one of claims 41 to 50, wherein there are two single-chain antibodies, which are respectively connected to the C-terminals of the two heavy chains of the immunoglobulin.

52. The method according to any one of claims 41 to 51, wherein, The single-chain antibody is independently: heavy chain variable region-linker-light chain variable region (VH-linker-VL), or light chain variable region-linker-heavy chain variable region (VL-linker-VH) connected in sequence; Preferably, the amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs:45-48.

53. The method according to any one of claims 41 to 52, wherein The first protein functional region is an immunoglobulin against VEGFA, and the second protein functional region is a single-chain antibody against PD-1; Preferably, there are two molecules of the single-chain antibody against PD-1, which are respectively connected to the C-terminals of the two heavy chains of the immunoglobulin against VEGFA; Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv; Preferably, the bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv.

54. The method according to any one of claims 41 to 53, wherein, The bispecific antibody is selected from any one of the following (13)-(16): (13) The bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:1, and the amino acid sequence of its light chain variable region is as shown in SEQ ID NO:3; The amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:9 or SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:17, SEQ ID NO:7 or SEQ ID NO:11; The single-chain antibodies are respectively connected to the C-terminals of the two heavy chains of the immunoglobulin; The first protein functional region is connected to the second protein functional region through a first linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody through a second linker; the first linker and the second linker are the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:18 and SEQ ID NO:19; Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO:18; (14) The bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:24, SEQ ID NO:20 or SEQ ID NO:22, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; The amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:17; The single-chain antibodies are respectively connected to the C-terminals of the two heavy chains of the immunoglobulin; The first protein functional region is connected to the second protein functional region through a first linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody through a second linker; the first linker and the second linker are the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:18 and SEQ ID NO:19; Preferably, the amino acid sequences of both the first linker and the second linker are as shown in SEQ ID NO:18; (15) The bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:1, and the amino acid sequence of its light chain variable region is as shown in SEQ ID NO:3; The amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs: 45-48; The single-chain antibodies are respectively connected to the C-termini of the two heavy chains of the immunoglobulin; The first protein functional region is connected to the second protein functional region through a first linker; Preferably, the first linker is selected from SEQ ID NO:18 and SEQ ID NO:19; (16) The bispecific antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO:24, SEQ ID NO:20 or SEQ ID NO:22, and the amino acid sequence of its light chain is as shown in SEQ ID NO:26; The amino acid sequence of the single-chain antibody is as shown in any one of SEQ ID NOs: 45-48; The single-chain antibodies are respectively connected to the C-termini of the two heavy chains of the immunoglobulin; The first protein functional region is connected to the second protein functional region through a first linker; Preferably, the first linker is selected from SEQ ID NO:18 and SEQ ID NO:

19.

55. The method according to any one of claims 41 to 54, which is the co-administration of the bispecific antibody and at least one chemotherapeutic drug.

56. The method according to claim 55, wherein the chemotherapeutic drug is one or more selected from tyrosine kinase inhibitors, platinum drugs (such as cisplatin or carboplatin), pemetrexed, paclitaxel, docetaxel, temozolomide, and fotemustine.

57. The method according to any one of claims 41 to 56, characterized in that One or more of the following (1) to (4): (1) The unit dose of the bispecific antibody is 100 mg - 4000 mg, 200 mg - 3000 mg, 200 mg - 2000 mg, 300 mg - 2000 mg, 400 mg - 2000 mg, 500 mg - 2000 mg, or 1000 mg - 2000 mg; (2) The single-dose administration of the bispecific antibody is 1-50 mg, 10 mg-50 mg, 10 mg-40 mg, 10 mg-30 mg, 10 mg-20 mg, 10 mg-15 mg, 15 mg-30 mg or 15 mg-20 mg per kilogram of body weight; (3) The bispecific antibody is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks; (4) The administration method of the bispecific antibody is intravenous drip, intravenous injection or intraperitoneal injection.

58. According to the method described in any one of claims 41 to 57, wherein: The bispecific antibody is administered in combination with pemetrexed and carboplatin; or The bispecific antibody is administered in combination with paclitaxel and carboplatin.

59. The method according to claim 58, wherein One or more of the following (1) to (3): (1) The single-dose of pemetrexed is calculated according to the body surface area of each subject (m 2 ) 10 - 1500 mg / m 2 , 100 - 1000 mg / m 2 , 200 - 900 mg / m 2 , 300 - 800 mg / m 2 , 400 - 700 mg / m 2 , 500 - 600 mg / m 2 ; Preferably, the pemetrexed is administered once every 1 week, 2 weeks, 3 weeks or 4 weeks; (2) The single-dose administration of carboplatin is independently AUC 2-6 min*mg / mL or AUC 2.5-5 min*mg / mL; preferably, carboplatin is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks; (3) The single-dose of paclitaxel is based on the body surface area of each subject (m 2 ) 67.5 - 175 mg / m 2 , 87.5 - 175 mg / m 2 or 131 - 175 mg / m 2 ; Preferably, the paclitaxel is administered once every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks.

60. A method according to any one of claims 41 to 59, wherein, The brain metastasis of non-small cell lung cancer is selected from one or more of squamous cell non-small cell lung cancer brain metastasis, non-squamous cell non-small cell lung cancer brain metastasis and lung adenocarcinoma brain metastasis; Preferably, for patients with brain metastasis of non-small cell lung cancer, PD-L1 TPS≥50%, or 1%<PD-L1 TPS≥49%, or PD-L1 TPS≤1%.

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