Medical application of anti-PD-1-anti-VEGFA (vascular endothelial growth factor A) bispecific antibody

Abstract

The invention belongs to the field of tumor treatment and molecular immunology, and relates to medical application of an anti-VEGFA-anti-PD-1 bispecific antibody. Specifically, the invention relates to application of an anti-VEGFA-anti-PD-1 bispecific antibody or a pharmaceutical composition of the anti-VEGFA-anti-PD-1 bispecific antibody and chemotherapeutic drugs in preparation of drugs for treating non-small cell lung cancer brain metastasis. The bispecific antibody can effectively treat non-small cell lung cancer brain metastasis, is high in safety and has a good application prospect.

Description

[0001] Cross - reference to related applications

[0002] This application is based on and claims priority to an application with a CN application number of 202410108261.9 and a filing date of January 25, 2024. The entire content of this CN application is hereby incorporated herein by reference in its entirety. Technical field

[0003] The present invention belongs to the fields of tumor treatment and immunobiology, and relates to the medical use of an anti - PD - 1 - anti - VEGFA bispecific antibody. 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 drug in the preparation of a drug for treating brain metastases of non - small cell lung cancer. Background art

[0004] Tumors, especially malignant tumors, are diseases that seriously endanger human health in the world today and rank second among the causes of death from various diseases. Moreover, in recent years, their incidence has shown an obvious upward trend. The treatment effect of malignant tumors is poor, the late metastasis rate is high, and the prognosis is mostly not good. Although the current conventional treatment methods used clinically, such as radiotherapy, chemotherapy, and surgical treatment, have alleviated the pain to a great extent and extended the survival time, these methods all have great limitations and it is difficult to further improve their curative effects.

[0005] Vascular endothelial growth factor (VEGF) is a class of growth factors that can promote endothelial cell division and proliferation, promote the formation of new blood vessels, and increase vascular permeability. It binds to the vascular endothelial growth factor receptor on the cell surface and exerts its function by activating the tyrosine kinase signal transduction pathway. In tumor tissues, tumor cells, infiltrating macrophages and mast cells can secrete high levels of VEGF, which stimulates tumor vascular endothelial cells in a paracrine manner, promotes endothelial cell proliferation and migration, induces blood vessel formation, promotes continuous tumor growth, and increases vascular permeability, causing fibrin deposition in the surrounding tissues, promoting the infiltration of monocytes, fibroblasts and endothelial cells, facilitating tumor stroma formation and tumor cell entry into new blood vessels, and promoting tumor metastasis. Therefore, inhibiting tumor angiogenesis is considered to be one of the most promising tumor treatment methods at present. The VEGF family includes: VEGFA, VEGFB, VEGFC, VEGFD and PIGF. Vascular endothelial growth factor receptors (VEGFR) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4) and Neuropilin-1 (NRP-1). Among them, the first three receptors are similar in structure and all belong to the tyrosine kinase superfamily. They are all composed of three parts: an extracellular domain, a transmembrane segment and an intracellular domain. The extracellular domain is composed of immunoglobulin-like domains, and the intracellular domain belongs to the tyrosine kinase region. VEGFR1 and VEGFR2 are mainly located on the surface of vascular endothelial cells, and VEGFR3 is mainly 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 function. VEGFR1 is the earliest discovered receptor. Under normal physiological conditions, the affinity of VEGFR1 for VEGFA is higher than that of VEGFR2 for VEGFA, but its intracellular tyrosine kinase activity is lower than that of VEGFR2 (Ma Li, Chinese Journal of Birth Health & Heredity, 24(5)(2016): 146-148).

[0007] VEGFR2 is a major regulator of angiogenesis and vascular construction, and has high tyrosine kinase activity compared with VEGFR1. After VEGFR2 binds to the ligand VEGFA, it mediates the behaviors of vascular endothelial cells such as proliferation and differentiation, as well as the processes of blood vessel formation and vascular permeability (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 62(3)(2007): 179-213.). After VEGFA binds to VEGFR2, it mediates the transcriptional expression of related intracellular 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 belongs to one of the members of the tyrosine kinase family, and is mainly expressed in vascular endothelial cells during embryonic development and lymphatic endothelial cells in adulthood. VEGFC and VEGFD bind to VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells, promoting the neovascularization of lymphatic vessels; NRP-1 is a non-tyrosine kinase transmembrane protein that cannot independently transduce biological signals, and can mediate signal transduction only after forming a complex with the VEGF tyrosine kinase receptor. (Ma Li, Chinese Journal of Birth Health & Heredity, 24(5)(2016): 146-148).

[0009] VEGFA and VEGFR2 mainly participate in the regulation of angiogenesis. Before and after VEGFA binds to VEGFR2, it will trigger a cascade reaction of many intermediate signals in the upstream and downstream pathways, and finally change its physiological functions in different forms such as endothelial cell proliferation, survival, migration, increased permeability, and infiltration into surrounding tissues (Dong Hongchao et al., Journal of Modern Oncology, Vol. 22, No. 9, September 2014, pp. 2231-2233).

[0010] Currently, there are already a variety of humanized monoclonal antibodies targeting human VEGF, especially VEGFA, such as Bevacizumab, which was successively approved by the US 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 1 (PD-1), also known as CD279, is a type I transmembrane glycoprotein membrane surface receptor belonging to the CD28 immunoglobulin superfamily, which 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 cell membranes of a variety of cells, including non-hematopoietic system cells and various tumor cells. PD-L1 is mainly expressed on T cells, B cells, DCs, microvascular endothelial cells and various tumor cells. PD-L2 is only expressed on antigen-presenting cells such as dendritic cells and macrophages. The interaction between PD-1 and its ligands can inhibit lymphocyte activation, inhibit T cell proliferation and the secretion of cytokines such as IL-2 and IFN-γ.

[0012] Numerous studies have shown that the tumor microenvironment can protect tumor cells from being destroyed by immune cells. The expression of PD-1 on infiltrating lymphocytes in the tumor microenvironment is upregulated, and various primary tumor tissues are positive for PD-L1 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 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 cell infiltration into tumor tissues, upregulate anti-tumor immune effector factors such as IL-2, IFN-γ, granzyme B and perforin, thereby effectively inhibiting tumor growth.

[0013] In addition, anti-PD-1 antibodies can also be used to treat chronic viral infections. Chronic viral infections are often accompanied by the loss of function and reduction in the number of virus-specific effector T cells. By injecting PD-1 antibodies, the interaction between PD-1 and PD-L1 can be blocked, thereby 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, it is generally believed in the industry 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 M.B., 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 highly-instability (MSI-H) or mismatch repair deficiency (dMMR) tumors (multiple anti-PD-1 antibody drugs have been approved by the FDA, etc. for the treatment of tumors with MSI-H / dMMR characteristics).

[0015] Bispecific antibodies, also known as bispecific antibodies (Bispecific Antibody), are specific drugs that target two different antigens simultaneously and can be produced by immunoselection and purification. In addition, they can also be obtained through genetic engineering, which has corresponding flexibility in aspects such as binding site optimization, consideration of synthetic forms, and yield, so it has certain advantages. Currently, more than 45 forms have been proven to exist (Müller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24: 89-98). A variety of bispecific antibodies developed so far are in the IgG-ScFv form, namely the Morrison mode (Coloma M.J., Morrison S.L. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol., 1997; 15: 159-163). Due to this form similar to the naturally occurring IgG form, its advantages in antibody engineering, expression, and purification have been proven to be an ideal form of bispecific antibodies (Miller B.R., Demarest S.J., 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 indicate a worse prognosis. Tumor brain metastases can involve various intracranial tissues, including brain parenchyma, meninges, nerves, and capillaries, etc., and can be accompanied by epilepsy, cognitive decline, sensory or motor dysfunction, and cranial nerve lesions, severely reducing the quality of life of patients. The brain has a unique blood-brain barrier (BBB), which is composed of non-fenestrated endothelial cells tightly connected, and the end-feet 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 expression of endothelial cell adhesion molecules in the blood-brain barrier is low, hindering the excessive entry of peripheral immune cells into the central nervous system; on the other hand, conventional chemotherapy drugs and targeted drugs are also difficult to cross the blood-brain barrier and enter the intracranial to play a role (Daneman R, Zhou L, Agalliu D, et al. The mouse bloodbrain barriertranscriptome: a new resource for understanding the development and functionof 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, enabling tumor cells from the blood to invade the intracranial, thus leading to the occurrence of brain metastatic cancer.

[0017] A variety of peripheral immune cells, including lymphocytes and neutrophils, as well as various immune factors, are involved in the process of brain metastatic cancer, and these together constitute the immune microenvironment of the metastatic focus. When brain metastasis occurs, the activation of microglia can upregulate the expression of interleukin (IL), 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 highly express IL-1β, which further activates the Notch signaling pathway, and 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 brain metastasis in patients with advanced melanoma[J]. Cancer Rep (Hoboken), 2021, 4(6):e1419).

[0018] Lung cancer is one of the common malignant tumors globally, and non-small cell lung cancer (NSCLC) is the most common type of lung cancer. 10%-15% of NSCLC patients have brain metastases at the time of initial diagnosis, causing various neurological symptoms, and 24%-44% of advanced NSCLC patients develop brain metastases. The median survival of patients with NSCLC brain metastases after treatment at initial diagnosis is only 4-6 months. The vast majority of NSCLC patients with brain metastases are no longer suitable for surgical treatment at the time of diagnosis. Chemotherapy such as antifolate agents combined with platinum is still the main treatment for advanced NSCLC at present. However, due to the obstruction of the blood-brain barrier, the efficacy of the vast majority of chemotherapy drugs and targeted TKI drugs for brain metastases is limited. The third-generation TKI drugs have strong anti-tumor activity and a high passing rate through the blood-brain barrier, and often have better efficacy. However, these drugs are only applicable to some patients with common classical driver gene mutations with no obvious symptoms, and their application is also limited by drug resistance and safety.

[0019] Currently, immunotherapy is also used clinically to treat NSCLC brain metastases. Some studies have shown that immune checkpoint inhibitors (ICIs) can promote the recruitment of T cells (especially CD8 + T cells) from outside the skull into the skull, enabling 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 U S 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 that of positive patients (12.2%). It can be seen that the efficacy of ICIs alone is not ideal for the overall 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 combined with chemotherapy or multiple immune checkpoint inhibitors are also used to treat lung cancer brain metastases, but this will increase the incidence of immune-related adverse events (irAEs) (Yang Shanru, Tai Risheng, Wang Geng, Qu Shutao, Lei Lei, Li Na. Problems brought by immune checkpoint inhibitor therapy - immune-related adverse events irAEs[J]. Chinese Journal of Immunology, 2022, 38(16):2026-2030).

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

[0021] Through in-depth research and creative work, the inventor of the present invention has found that the anti-PD-1-anti-VEGFA bispecific antibody involved in the present invention, with or without chemotherapy, can effectively treat or prevent brain metastases of non-small cell lung cancer and has good safety at the same time.

[0022] Thus, the following invention is provided:

[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 metastases of non-small cell lung cancer, wherein,

[0024] the bispecific antibody comprises:

[0025] a first protein functional region targeting VEGFA, and

[0026] a second protein functional region targeting PD-1;

[0027] wherein,

[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 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;

[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 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.

[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, for the use described above,

[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 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.

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

[0036] 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)

[0038] 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)

[0040] 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)

[0042] 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)

[0044] 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)

[0046] 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.

[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 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.

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

[0051] 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)

[0053] 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)

[0055] 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)

[0057] 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)

[0059] 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)

[0061] 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.

[0062] In some embodiments of the present invention, for the use as described above, wherein,

[0063] The immunoglobulin is of human IgG1 subtype.

[0064] In some embodiments of the present invention, for the use as described above, 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, if not otherwise specified, the letter before the site represents the amino acid before mutation, and the letter after the site represents the amino acid after mutation.

[0072] In some embodiments of the present invention, for the use as described above, 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, for the use as described above, wherein,

[0075] 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;

[0076] 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

[0077] 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.

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

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

[0080] For the immunoglobulin, its heavy chain constant region is human Ig gamma-1 chain C region or human Ig gamma-4 chain C region, and its light chain constant region is human Ig kappa chain C region.

[0081] In some embodiments of the present invention, the constant regions of the immunoglobulin are 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 as 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 as 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 as shown in SEQ ID NO: 42.

[0086] In some embodiments of the present invention, for the use described above, the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin. Since an immunoglobulin consists 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, for the use described above, there are two single-chain antibodies, and one end of each single-chain antibody is respectively linked to the C-terminus or N-terminus of the two heavy chains of the immunoglobulin.

[0088] In some embodiments of the present invention, the V of the single-chain antibody H and V L There is a disulfide bond between them. Methods for introducing a disulfide bond between the V H and V L of the antibody are well known in the art. For example, see U.S. Patent US5,747,654; 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; Webberet.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, Cancer Res.54(1994)2714-2718, which are incorporated herein by reference.

[0089] In some embodiments of the present invention, for the use described above,

[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 formed by tandemly connecting multiple (such as 2, 3, 4, 5 or 6) polypeptides represented by SEQ ID NO: 43.

[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 than 2.

[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-terminals of the two heavy chains of the immunoglobulin.

[0097] In some embodiments of the present invention, the use, 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);

[0098] Preferably, the amino acid sequence of the single-chain antibody is 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 immunoglobulin against VEGFA, and the second protein functional region is a single-chain antibody against PD-1;

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

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

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

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

[0104] The bispecific antibody includes:

[0105] The first protein functional region targeting VEGFA, and

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

[0107] There is 1 first protein functional region and 2 second protein functional regions;

[0108] Among them, 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 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;

[0110] 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, SEQ ID NO:7 or SEQ ID NO:11;

[0111] The single-chain antibodies are respectively linked to the C termini of the two heavy chains of the immunoglobulin;

[0112] 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;

[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, for the use described above, wherein,

[0116] The bispecific antibody comprises:

[0117] The first protein functional region targeting VEGFA, and

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

[0119] There is 1 first protein functional region and 2 second protein functional regions;

[0120] Among them, 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 its light chain variable region 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 linked to the C-terminals of the two heavy chains of the immunoglobulin;

[0124] 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;

[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 shown in SEQ ID NO: 18.

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

[0128] The bispecific antibody comprises:

[0129] A first protein functional region targeting VEGFA, and

[0130] A second protein functional region targeting PD-1;

[0131] There is 1 first protein functional region and 2 second protein functional regions;

[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 its light chain is shown in SEQ ID NO: 26;

[0134] 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;

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

[0136] 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;

[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] A first protein functional region targeting VEGFA, and

[0142] A second protein functional region targeting PD-1;

[0143] There is 1 first protein functional region and 2 second protein functional regions;

[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 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;

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

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

[0148] The first protein functional region is linked to the second protein functional region through 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] A first protein functional region targeting VEGFA, and

[0153] A second protein functional region targeting PD-1;

[0154] There is 1 first protein functional region and 2 second protein functional regions;

[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 as SEQ ID NO:24, SEQ ID NO:20 or SEQ ID NO:22, and the amino acid sequence of its light chain is shown as SEQ ID NO:26;

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

[0158] The single-chain antibodies are respectively connected to the C-terminals of the two heavy chains of the immunoglobulin;

[0159] The first protein functional region is connected to the second protein functional region through 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, wherein the bispecific antibody is the sole 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 chemotherapeutic 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 chemotherapeutic 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, for the use described above, 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, for the use described above, 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, for the use described above, 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.

[0168] In some embodiments of the present invention, for the use described above, 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, for the use described above, the administration method of the bispecific antibody is intravenous drip, intravenous injection or intraperitoneal injection.

[0170] In some embodiments of the present invention, for the use described above, the combination is:

[0171] The bispecific antibody as described in any one of the present invention, pemetrexed and carboplatin; or

[0172] The bispecific antibody as described in any one of the present invention, paclitaxel and carboplatin.

[0173] In some embodiments of the present invention, for the use described above, 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, for the use described above, 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, for the use described above, 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, for the use described above, it is characterized by one or more of the following (1) to (3):

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

[0184] (2) The single-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, 2 weeks, 3 weeks, or 4 weeks;

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

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

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

[0188] Wherein, 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 field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, 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 injection solution. For example, a sterile injection solution can be prepared by incorporating the required dose of the bispecific antibody of the present invention in a suitable solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, a sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for ease of storage and use. Such a sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0192] In addition, the bispecific antibody of the present invention can be present in the drug in unit dose form for ease of administration. 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 in a liquid (e.g., injection) dosage form, it can contain the bispecific antibody of the present invention at 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.

[0193] The bispecific antibody or drug of the present invention can be administered by any suitable method known in the art, including but not limited to oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracisternal, inguinal, intravesical, local (e.g., powder, ointment or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection). Those skilled in the art will understand that the route and / or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the bispecific antibody or pharmaceutical composition of the present invention is administered by intravenous infusion or injection.

[0194] The bispecific antibody or drug provided by the present invention can be used alone or in combination, and can also 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 the administration of the bispecific antibody of the present invention or the pharmaceutical composition of the present invention.

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

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

[0197] In some embodiments of the present invention, the administration of the chemotherapy drug is simultaneous with, before, or after the administration of the bispecific antibody.

[0198] Typical non-limiting ranges for a therapeutically or prophylactically effective amount of the bispecific antibody of the present invention are 0.02 - 50 mg / kg, such as 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 condition to be treated. In addition, those skilled in the art will understand that for any particular patient, the specific dosing regimen should be adjusted over time according to the patient's needs and the professional evaluation of the physician; the dosage ranges given herein are for illustrative purposes only and do not limit the use or scope of the pharmaceutical compositions of the present invention.

[0199] In the present invention, the subject can be a mammal, such as a human.

[0200] In one or more embodiments of the present invention, the single-dose administration dosage of the anti-PD-1 - anti-VEGFA bispecific antibody is 0.1 - 100 mg per kilogram of body weight, preferably 1 - 50 mg (such as 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 administration dosage of the anti-PD-1 - anti-VEGFA bispecific antibody is 10 - 5000 mg per subject (such as approximately 100 mg, approximately 150 mg, approximately 200 mg, approximately 250 mg, approximately 300 mg, approximately 350 mg, approximately 400 mg, approximately 450 mg, approximately 500 mg, approximately 600 mg, approximately 700 mg, approximately 800 mg, approximately 900 mg, approximately 1000 mg, approximately 1200 mg, approximately 1500 mg, approximately 1800 mg, approximately 2000 mg, approximately 2500 mg or approximately 3000 mg), preferably 100 - 2500 mg, 500 - 2500 mg, 500 - 2000 mg, 500 - 2000 mg or 500 mg - 1500 mg;

[0201] Preferably, administration is 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;

[0202] Preferably, the administration method is intravenous drip, intravenous injection or intraperitoneal injection.

[0203] In some embodiments, the administration of the anti-PD-1-anti-VEGFA bispecific antibody is carried out in a cycle of 2 weeks (14 days) or 3 weeks (21 days), and preferably the anti-PD-1-anti-VEGFA bispecific antibody is intravenously administered on the first day (D1) of each cycle. For example, the anti-PD-1-anti-VEGFA bispecific antibody is administered at a frequency of once every two weeks (q2w) or once every three weeks (q3w).

[0204] In some embodiments of the present invention, 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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 ) of each subject, 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 , or 500 - 600 mg / m 2 .

[0209] In one or more embodiments of the present invention, the single-dose of pemetrexed is based on the body surface area (m2 ) 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 、 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.

[0210] 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.

[0211] 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 - 175 mg / m 2 、 or 131 - 175 mg / m 2 each time; preferably, the paclitaxel is administered once every 1 week, every 2 weeks, every 3 weeks or every 4 weeks.

[0212] In some embodiments, the non - small cell lung cancer is squamous cell non - small cell lung cancer (also known as squamous cell carcinoma, squamous carcinoma, squamous non - small cell lung cancer or squamous epithelial cell carcinoma). In some embodiments, the non - small cell lung cancer is non - small cell lung cancer with a histological predominance of squamous cell carcinoma. 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.

[0213] In one or more embodiments of the present invention, the non - small cell lung cancer patient has PD - L1 TPS≥50%, or 1% < PD - L1 TPS≥49%, or PD - L1 TPS≤1%.

[0214] Antibody - based therapeutic drugs, especially monoclonal antibodies, have achieved good therapeutic effects in the treatment of various diseases. The traditional experimental methods for obtaining these therapeutic antibodies are to immunize animals with antigens to obtain antibodies targeting the antigens in the immunized animals, or to improve the antibodies with low affinity for antigens through affinity maturation methods.

[0215] The variable regions of the light and heavy chains determine antigen binding; each variable region of the chain contains three hypervariable regions, called complementarity-determining regions (CDRs). The CDRs of the heavy chain (H Chain) include HCDR1, HCDR2, HCDR3, and the CDRs of the light chain (L Chain) include LCDR1, LCDR2, LCDR3; they were named by Kabat et al., see Bethesda M.d., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication (1-3) 1991: 91-3242.

[0216] Preferably, the CDRs can 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.

[0217] By means well-known to those skilled in the art, for example, by analyzing the amino acid sequences of the CDR regions of the monoclonal antibody sequences in items (1)-(3) below according to the IMGT definition through the VBASE2 database, the results are as follows:

[0218] (1) Bevacizumab

[0219] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 3.

[0220] The amino acid sequences of the 3 CDR regions of its heavy chain variable region are as follows:

[0221] HCDR1: GYTFTNYG (SEQ ID NO: 28)

[0222] HCDR2: INTYTGEP (SEQ ID NO: 29)

[0223] HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 30)

[0224] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0225] LCDR1: QDISNY (SEQ ID NO: 31)

[0226] LCDR2: FTS (SEQ ID NO: 32)

[0227] LCDR3: QQYSTVPWT (SEQ ID NO: 33)

[0228] (2) 14C12, 14C12H1L1 or 14C12H1L1(M)

[0229] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0230] HCDR1: GFAFSSYD (SEQ ID NO: 34)

[0231] HCDR2: ISGGGRYT (SEQ ID NO: 35)

[0232] HCDR3: ANRYGEAWFAY (SEQ ID NO: 36)

[0233] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0234] LCDR1: QDINTY (SEQ ID NO: 37)

[0235] LCDR2: RAN (SEQ ID NO: 38)

[0236] LCDR3: LQYDEFPLT (SEQ ID NO: 39)

[0237] (3) VP101(hG1WT) or VP101(hG1DM)

[0238] The amino acid sequences of the nine CDR regions of its heavy chain are as follows:

[0239] HCDR1: GYTFTNYG (SEQ ID NO: 28)

[0240] HCDR2: INTYTGEP (SEQ ID NO: 29)

[0241] HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 30)

[0242] HCDR4: GFAFSSYD (SEQ ID NO: 34)

[0243] HCDR5: ISGGGRYT (SEQ ID NO:35)

[0244] HCDR6: ANRYGEAWFAY (SEQ ID NO:36)

[0245] HCDR7: QDINTY (SEQ ID NO:37)

[0246] HCDR8: RAN (SEQ ID NO:38)

[0247] HCDR9: LQYDEFPLT (SEQ ID NO:39)

[0248] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0249] LCDR1: QDISNY (SEQ ID NO:31)

[0250] LCDR2: FTS (SEQ ID NO:32)

[0251] LCDR3: QQYSTVPWT (SEQ ID NO:33).

[0252] The antibody VP101 (hG1DM) of the present invention introduces amino acid mutations in the non-variable region of VP101 (hG1WT). Amino acid mutations are introduced at positions 234 and 235 according to the EU numbering system:

[0253] By introducing a point mutation of leucine to alanine (L234A) at position 234 and a point mutation of leucine to alanine (L235A) at position 235 in its heavy chain hinge region, VP101 (hG1DM) is obtained.

[0254] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are all conventional steps widely used in the corresponding fields. At the same time, to better understand the present invention, the definitions and explanations of related terms are provided below.

[0255] As used herein, when referring to the amino acid sequence of the VEGFA protein (GenBank ID: NP_001165097.1), it includes the full length of the 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, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can occur naturally or be introduced artificially in the amino acid sequence of the VEGFA protein without affecting its biological function. Therefore, in the present invention, the term "VEGFA protein" shall include all such sequences, including its natural or artificial variants. And when describing a sequence fragment of the VEGFA protein, it also includes the corresponding sequence fragment in its natural or artificial variants.

[0256] As used herein, when referring to the amino acid sequence of the 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 containing VEGFR2-ECD; it also includes fusion proteins of VEGFR2-ECD, such as fragments 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 occur naturally or be introduced artificially in the amino acid sequence of the VEGFR2 protein without affecting its biological function. Therefore, in the present invention, the term "VEGFR2 protein" shall include all such sequences, including its natural or artificial variants. And when describing a sequence fragment of the VEGFR2 protein, it also includes the corresponding sequence fragment in its natural or artificial variants.

[0257] As used herein, if not otherwise specified, the VEGFR is VEGFR1 and / or VEGFR2; its specific protein sequence is a known sequence in the prior art, and reference can be made to the sequences disclosed in the existing literature or GenBank. For example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321); VEGFR2 (VEGFR2, NCBI Gene ID: 3791).

[0258] As used herein, when referring to the amino acid sequence of the 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 of PD-1, PD-1ECD, or a fragment containing PD-1ECD; it also includes fusion proteins of PD-1ECD, such as fragments 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) may occur naturally or be introduced artificially in 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 their natural or artificial variants. And when describing a sequence fragment of the PD-1 protein, it also includes the corresponding sequence fragment in its natural or artificial variants.

[0259] As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximal effect.

[0260] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Generally speaking, the heavy chain can be understood as the polypeptide chain with a larger molecular weight in the antibody, and the light chain is the polypeptide chain with a smaller molecular weight in the antibody. The light chain can be classified into κ and λ light chains. The heavy chain is usually classified into μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE respectively. Within the light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region consists of 3 domains (C H1 , C H2 , and C H3 ). Each light chain consists of a light chain variable region (V L ) and a light chain constant region (C L ). The light chain constant region consists of one domain C L . The constant region of the antibody can mediate 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. V H and V LThe region can also be subdivided into regions with high variability (called complementarity determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V H and V L ) of each heavy chain / light chain pair respectively form the antibody binding site. The assignment 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 the definitions of Chothia & Lesk J. Mol. Biol. 196 (1987): 901-917; Chothia et al. Nature 342 (1989): 878-883 or 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. In particular, the heavy chain can also contain more than three CDRs, such as 6, 9, or 12. For example, in the bispecific antibodies of the present invention, the heavy chain can be the C-terminus of the heavy chain of an IgG antibody linked to the ScFv of another antibody, in which case the heavy chain contains 9 CDRs. The term "antibody" is not restricted by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0261] 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 the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as the "antigen-binding portion". See generally, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989)). Antigen-binding fragments of antibodies can be generated by recombinant DNA techniques 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 complementarity-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 on the polypeptide.

[0262] As used herein, the term "Fd fragment" means an antibody fragment consisting of the V H and C H1 domains; the term "Fv fragment" means an antibody fragment consisting of the V L and V H domains of a single arm of an antibody; the term "dAb fragment" means an antibody fragment consisting of the V H domain (Ward et al., Nature 341 (1989):544-546); the term "Fab fragment" means an antibody fragment consisting of the V L 、V H 、C L and C H1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region.

[0263] In some cases, an antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), in which the V L and V H domains are paired by a linker that enables them to be produced 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 -linker-V H -COOH or NH2-V H -linker-V L-COOH. Suitable prior art linkers consist of the repeating GGGGS (SEQ ID NO:43) amino acid sequence 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).

[0264] In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a bivalent antibody, in which the V H and V L domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains on the same chain, thus forcing the domains to pair with the complementary domain on the other chain and generating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90 (1993):6444-6448, and Poljak R. J. et al., Structure 2 (1994):1121-1123).

[0265] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0266] As used herein, unless the context clearly dictates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0267] As used herein, the terms "monoclonal antibody" and "mAb" refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, i.e., a group of identical antibody molecules except for possible naturally-occurring mutations. A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies, in contrast to monoclonal antibodies, generally contain at least two or more different antibodies that usually recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained by the hybridoma technique first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained by recombinant DNA techniques (see, e.g., U.S. Patent 4,816,567).

[0268] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain or / and heavy chain is derived from one antibody (which may be from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or / and heavy chain is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), but in any case retains the binding activity to the target antigen (U.S. Patent 4,816,567; Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 (1984): 6851-6855).

[0269] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment in which all or part of the CDR regions of a human immunoglobulin (recipient antibody) have been replaced with the CDR regions of a non-human antibody (donor antibody), where the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework regions (FR) of the recipient antibody can also be replaced with the amino acid residues of the corresponding non-human antibody or with the amino acid residues of another antibody to further improve or optimize the properties of the antibody. For more details on humanized antibodies, see, e.g., 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.

[0270] As used herein, the term "epitope" refers to the site on an antigen that is specifically bound by an immunoglobulin or antibody. An "epitope" is also referred to in the art as an "antigenic determinant". Epitopes or antigenic determinants typically consist of the chemically active surface groups of a molecule such as amino acids or carbohydrates or sugar side chains and usually have specific three-dimensional structural features as well as specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which may be "linear" or "conformational". See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all points of interaction between a protein and an interacting molecule (such as an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction exist across protein amino acid residues that are separated from one another.

[0271] As used herein, the term "isolated" or "separated" means obtained by artificial means from its natural state. If a "separated" substance or component occurs in nature, it may be that its natural environment has changed, or the substance has been separated from its natural environment, or both. For example, a certain polynucleotide or polypeptide that naturally exists in an in vivo animal in an unseparated state, and the highly purified same polynucleotide or polypeptide separated from this natural state is referred to as isolated. The term "isolated" or "separated" does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0272] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, enabling the genetic elements 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, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector can contain multiple elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can also contain an origin of replication.

[0273] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, 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 Drosophila S2 cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0274] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) refers to an antibody that binds to the antigen with an affinity (K -5 ) of less than about 10 -6 M, for example less than about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, or 10 D M or less. In some embodiments of the present invention, the term "targeting" refers to specific binding.

[0275] 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 an antibody and an 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, an antibody binds to the antigen with an affinity of less than about 10 -5M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or a dissociation equilibrium constant (K D ) that binds to an antigen, for example, as determined using surface plasmon resonance (SPR) in a BIACORE instrument or a Fortebio molecular interaction instrument.

[0276] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0277] 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 regulators, surfactants, adjuvants, ionic strength enhancers. For example, pH regulators include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.

[0278] In some embodiments, the anti-PD-1 - anti-VEGFA bispecific antibody or an antigen-binding fragment thereof can be formulated with one or more pharmaceutically acceptable carriers to form a suitable pharmaceutical composition.

[0279] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (such as a disease related to the binding of PD-1 and PD-L1 or a disease such as a tumor related to overexpression of VEGF) is an amount sufficient to prevent, inhibit, or delay the occurrence of the disease (such as a disease related to the binding of PD-1 and PD-L1 or a disease such as a tumor related to overexpression of VEGF); an effective amount for treating a disease is an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the general condition of the patient such as age, weight, and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0280] Advantages of the Invention

[0281] The present invention achieves any one or more of the following technical effects (1) to (4):

[0282] (1) The bispecific antibody in the present invention can effectively treat brain metastases of non-small cell lung cancer.

[0283] (2) When the bispecific antibody of the present invention is used to treat brain metastases of non-small cell lung cancer, the median progression-free survival of the patient is relatively long, and can even reach 19.3 months.

[0284] (3) When the bispecific antibody of the present invention is used to treat brain metastases of non-small cell lung cancer, the onset of action is fast, and partial remission is achieved in some patients 6 weeks after drug administration.

[0285] (4) The bispecific antibody in the present invention has good safety in the treatment of cancer, especially brain metastases of non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0286] Figure 1 : Head magnetic resonance imaging of a patient with brain metastases of non-small cell lung cancer 12 weeks after receiving the bispecific antibody VP101 (hG1DM) of anti-PD-1 - anti-VEGFA in combination with a chemotherapeutic drug. The position marked with a cross in a white circle at the baseline level is the tumor focus.

[0287] Figure 2 : Head magnetic resonance imaging of a patient with brain metastases of non-small cell lung cancer 42 weeks after receiving the bispecific antibody VP101 (hG1DM) of anti-PD-1 - anti-VEGFA alone.

[0288] Figure 3 : Intracranial remission rate (RANO criteria) of the bispecific antibody VP101 (hG1DM) of anti-PD-1 - anti-VEGFA in the treatment of brain metastases of non-small cell lung cancer.

[0289] Figure 4 : Median progression-free survival of the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) in patients with brain metastases from non-small cell lung cancer.

[0290] Some sequences involved in the present invention are shown in Table A below.

[0291] Table A

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308] Detailed implementation mode

[0309] The embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in the art (for example, referring to "Molecular Cloning: A Laboratory Manual", Third Edition, J. Sambrook et al., translated by Huang Peitang et al., Science Press) or according to the product instructions are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through market purchase.

[0310] Preparation Example 1: Preparation of the anti-VEGFA antibody Bevacizumab

[0311] The amino acid sequences of the heavy chain variable region and the light chain variable region of the marketed anti-VEGFA monoclonal antibody Avastin (Bevacizumab) refer to Chinese Patent Publication CN1259962A. The nucleic acid sequences encoding the heavy chain variable region and the light chain variable region were synthesized by GenScript Corporation.

[0312] The amino acid sequence of the Bevacizumab heavy chain variable region (Bevacizumab-VH) is shown in SEQ ID NO:1.

[0313] The nucleic acid sequence encoding the Bevacizumab heavy chain variable region is shown in SEQ ID NO:2.

[0314] The amino acid sequence of the Bevacizumab light chain variable region (Bevacizumab-VL) is shown in SEQ ID NO:3.

[0315] The nucleic acid sequence encoding the Bevacizumab light chain variable region is shown in SEQ ID NO:4.

[0316] The heavy chain constant region all adopts Ig gamma-1 chain C region, ACCESSION:P01857; the light chain constant region all adopts Ig kappa chain C region, ACCESSION:P01834.

[0317] The heavy chain cDNA and the light chain cDNA of Bevacizumab were respectively cloned into the pcDNA3.1 vector to obtain the recombinant expression plasmid of the antibody Bevacizumab. The recombinant plasmid was transfected into 293F cells. The culture solution of 293F cells was purified and then detected.

[0318] The anti-VEGFA monoclonal antibody Avastin (Bevacizumab) was prepared.

[0319] Preparation Example 2: Sequences of the anti-PD-1 antibody 14C12, its humanized antibody 14C12H1L1 and mutant 14C12H1L1(M) Design

[0320] 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 the same as those of 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A, respectively.

[0321] (1) Heavy chain variable region sequence and light chain variable region sequence of 14C12

[0322] The amino acid sequence of the heavy chain variable region of 14C12 is shown in SEQ ID NO:5.

[0323] The nucleic acid sequence encoding the heavy chain variable region of 14C12 is shown in SEQ ID NO:6.

[0324] The amino acid sequence of the light chain variable region of 14C12 is shown in SEQ ID NO:7.

[0325] The nucleic acid sequence encoding the light chain variable region of 14C12 is shown in SEQ ID NO:8.

[0326] (2) Heavy chain variable region sequence, light chain variable region sequence, heavy chain sequence and light chain sequence of the humanized monoclonal antibody 14C12H1L1

[0327] The amino acid sequence of the heavy chain variable region of 14C12H1L1 is shown in SEQ ID NO:9.

[0328] The nucleic acid sequence encoding the heavy chain variable region of 14C12H1L1 is shown in SEQ ID NO:10.

[0329] The amino acid sequence of the light chain variable region of 14C12H1L1 is shown in SEQ ID NO:11.

[0330] The nucleic acid sequence encoding the light chain variable region of 14C12H1L1 is shown in SEQ ID NO:12.

[0331] The amino acid sequence of the heavy chain (14C12H1) of 14C12H1L1 is shown in SEQ ID NO:13.

[0332] The nucleic acid sequence encoding the heavy chain (14C12H1) of 14C12H1L1 is shown in SEQ ID NO:14.

[0333] The amino acid sequence of the light chain (14C12L1) of 14C12H1L1 is shown in SEQ ID NO:15.

[0334] The nucleic acid sequence encoding the light chain (14C12L1) of 14C12H1L1 is shown in SEQ ID NO:16.

[0335] (3) The heavy chain variable region sequence and light chain variable region sequence of 14C12H1L1(M)

[0336] Individual amino acids in the framework region (light chain) of 14C12H1L1 were mutated to obtain 14C12H1L1(M).

[0337] The amino acid sequence of the light chain variable region 14C12L1(M) of 14C12H1L1(M) is shown in SEQ ID NO:17.

[0338] The amino acid sequence of the heavy chain variable region 14C12H1(M) of 14C12H1L1(M) is the same as that of the heavy chain variable region 14C12H1 of 14C12H1L1, that is, as shown in SEQ ID NO:9.

[0339] Preparation Example 3: Sequence design of bispecific antibody

[0340] 1. Sequence design

[0341] The structural pattern of the bispecific antibody in the present invention belongs to the Morrison pattern (IgG-scFv), that is, an scFv fragment of another antibody is connected to the C-terminus of each of the two heavy chains of an IgG antibody. The main composition design of its heavy chain and light chain is as shown in Table 1 below.

[0342] Based on the above Bevacizumab, the VP101 antibody with the amino acid sequences of the heavy chain variable region and light chain variable region of the above 14C12H1L1(M) as the ScFv fragment part is called VP101(M). Compared with 14C12H1L1, 14C12H1L1(M) effectively optimizes the structure of the bispecific antibody and improves its effectiveness.

[0343] Table 1: Composition design of the heavy chain and light chain of VP101(M) and VP101(G4M)

[0344]

[0345] In the above Table 1:

[0346] (1) Those marked with "V" in the lower right corner refer to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. Those without the "V" mark, the corresponding heavy chain or light chain is the full length including the constant region. The amino acid sequences and their encoding nucleic acid sequences of these variable regions or full lengths are all referred to the corresponding sequences recorded in the above Preparation Examples.

[0347] (2) The amino acid sequence of Linker1 is as shown in SEQ ID NO:18.

[0348] Optionally, the amino acid sequence of Linker 2 is as shown in SEQ ID NO:19. Linker 2 can replace the aforementioned Linker 1.

[0349] (3) Bevacizumab-H uses the Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region.

[0350] (4) Bevacizumab-G4H uses the Ig gamma-4 chain C region, ACCESSION: P01861.1 as the heavy chain constant region.

[0351] 2. Expression and purification of antibody VP101(M)

[0352] The cDNA sequences of the heavy chain and light chain of VP101(M) were respectively cloned into the pUC57simple vector (provided by GenScript Corporation) to obtain the plasmids pUC57simple-VP101H and pUC57simple-VP101L.

[0353] The plasmids pUC57simple-VP101H and pUC57simple-VP101L were respectively digested with enzymes (HindIII & EcoRI), and the heavy and light chains obtained by electrophoresis recovery 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, concentrated the supernatant and loaded onto a HiTrap MabSelect SuRe column, and the protein was eluted in one step with Elution Buffer and the target sample antibody VP101 was recovered, and the buffer was exchanged to PBS.

[0354] 3. Detection of antibody VP101(M)

[0355] The purified samples were respectively added to the reducing protein electrophoresis loading buffer and non-reducing protein electrophoresis loading buffer, and after boiling, SDS-PAGE electrophoresis was performed for detection.

[0356] In order to distinguish from the mutated antibody in Preparation Example 4, in the present invention, VP101(M) is also referred to as VP101(hG1WT). The above-mentioned VP101(M) as the "wild type" uses 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.

[0357] The amino acid sequence of the heavy chain of the immunoglobulin portion in VP101(hG1WT) is shown in SEQ ID NO:20.

[0358] The nucleic acid sequence encoding the heavy chain of the immunoglobulin portion in VP101(hG1WT) is shown in SEQ ID NO:21.

[0359] In order to distinguish it from the mutated antibody in Preparation Example 4, in the present invention, VP101(G4M) is also referred to as VP101(hG4WT). The above-mentioned VP101(G4M) is used as the "wild type", which uses 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.

[0360] The amino acid sequence of the heavy chain of the immunoglobulin portion in VP101(hG4WT) is shown in SEQ ID NO:22.

[0361] The nucleic acid sequence encoding the heavy chain of the immunoglobulin portion in VP101(hG4WT) is shown in SEQ ID NO:23.

[0362] Preparation Example 4: Amino acid mutation design of the non-variable region based on the humanized bispecific antibody VP101(hG1WT)

[0363] Based on VP101(hG1WT) obtained in Preparation Example 3, the present inventors introduced a point mutation of leucine to alanine (L234A) at the 234th site of its heavy chain and a point mutation of leucine to alanine (L235A) at the 235th site, and obtained VP101(hG1DM).

[0364] The amino acid sequence of the heavy chain of the immunoglobulin portion in VP101(hG1DM) is shown in SEQ ID NO:24.

[0365] The nucleic acid sequence encoding the heavy chain of the immunoglobulin portion in VP101(hG1DM) is shown in SEQ ID NO:25.

[0366] The amino acid sequences of the light chains of the immunoglobulin portions of VP101(hG1DM), VP101(hG1WT) and VP101(hG4WT) are the same, and their encoding nucleic acid sequences are also the same.

[0367] The amino acid sequence of the light chain of the immunoglobulin portion in VP101(hG1DM) is shown in SEQ ID NO:26.

[0368] The nucleic acid sequence encoding the light chain of the immunoglobulin portion in VP101 (hG1DM) is shown in SEQ ID NO: 27.

[0369] The amino acid sequence of "heavy chain of 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.

[0370] Among them, the amino acid sequence of the scFv is shown in SEQ ID NO: 45.

[0371] Example 1: Anti-PD-1 - anti-VEGFA bispecific antibody VP101 (hG1DM) Effectively treat non-small cell lung cancer Patients with brain metastases

[0372] Advanced NSCLC patients were from the AK112-201 (NCT04736823) and AK112-202 (NCT04900363) trials, had not received systemic cancer treatment, and had a performance status score of 0 - 1 and an age range of 18 - 75 years old, and were respectively eligible for the use of VP101 (hG1DM) in combination with chemotherapy or the use of VP101 (hG1DM) alone. The research code of VP101 (hG1DM) is AK112.

[0373] Each three - week period is a dosing cycle. On the first day of each cycle, drug treatment is given in sequence: first, AK112 is given by intravenous injection, then pemetrexed or paclitaxel is given by intravenous injection, and finally carboplatin is given by intravenous injection.

[0374] The dosing regimen of the trial numbered AK112-201 is as follows:

[0375] Patients with non - squamous non - small cell lung cancer were given VP101 (hG1DM) (20 mg / kg, Q3W) in combination with pemetrexed (500 mg / m 2 ) and carboplatin (AUC5 min*mg / mL, Q3W) for a total of 4 cycles. If the disease did not progress, VP101 (hG1DM) (20 mg / kg, Q3W) in combination with pemetrexed (500 mg / m 2 ) was given for maintenance treatment for 2 years;

[0376] Patients with squamous non - small cell lung cancer were given VP101 (hG1DM) (20 mg / kg, Q3W) in combination with paclitaxel (175 mg / m 2) And treated with carboplatin (AUC 5 min*mg / mL, Q3W) for a total of 4 cycles. If the disease does not progress, maintenance treatment with VP101 (hG1DM) (20 mg / kg, Q3W) is given for 2 years.

[0377] The dosing regimen of the trial numbered AK112-202 is as follows:

[0378] Patients were treated with VP101 (hG1DM) (20 mg / kg, Q3W) for a total of 4 cycles. If the disease does not progress, maintenance treatment with VP101 (hG1DM) (20 mg / kg, Q3W) is given for 2 years.

[0379] Initial examinations for brain metastases include cranial MRI or head CT scans to determine brain metastases. Brain metastases patients at baseline are eligible if they have no symptoms caused by brain metastases or are clinically stable for at least 2 weeks after treatment. Brain metastases patients who have received radiotherapy or are allergic and unable to receive subsequent magnetic resonance imaging cannot participate in the evaluation of the intracranial activity of the anti-PD-1-anti-VEGFA bispecific antibody. For brain metastases patients at the baseline level, brain magnetic resonance imaging is performed every 6 weeks during the treatment process. In addition to evaluating the response of extracranial target lesions according to the RECIST 1.1 criteria, brain metastases are continuously evaluated by 2 independent neuroradiologists (from the United States and China) according to the Response Assessment in Neuro-Oncology (RANO) criteria.

[0380] A total of 35 patients met the criteria for inclusion in this analysis: 28 patients were from the AK112-201 trial and 7 patients were from the AK112-202 trial. The median age of these patients was 60 years (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 patients, and 46% of the patients (46% of 35) had positive PD-L1 expression (PD-L1 TPS ≥ 1%).

[0381] The results showed that a total of 12 patients with brain metastases from non-small cell lung cancer (34%) achieved remission of intracranial tumor foci, and 8 of them achieved complete remission of intracranial tumor foci. Among these 12 patients with brain metastases from non-small cell lung cancer, 11 patients (39%) received combination therapy of VP101 (hG1DM) and chemotherapy, and 1 patient (14%) received monotherapy with VP101 (hG1DM). Among the 8 patients who achieved complete remission of intracranial tumor foci, 7 patients (25% of the total number of patients in the AK112-201 trial) received combination therapy of VP101 (hG1DM) and chemotherapy, and 1 patient (14% of the total number of patients in the AK112-202 trial) received monotherapy with VP101 (hG1DM), as Figure 3 shown. The median progression-free survival of patients with brain metastases in the two cohorts was 19.3 months (as Figure 4 shown), and there was no intracranial hemorrhage within 3 months after treatment of patients with brain metastases. Some patients achieved partial remission at the 6th week of drug administration. As an example, the results of one patient with brain metastases from non-small cell lung cancer who received combination therapy of VP101 (hG1DM) and chemotherapy are shown in Table 2 and Figure 1 shown, and the results showed that the brain metastases tumor foci of this non-small cell lung cancer patient were effectively relieved. In addition, the results of another patient with brain metastases from non-small cell lung cancer who received monotherapy with VP101 (hG1DM) are shown in Figure 2 shown, and the results showed that the brain metastases tumor foci of this non-small cell lung cancer patient were also effectively relieved.

[0382] Table 2: Evaluation results of intracranial tumor foci of Figure 1 the same patient with brain metastases from non-small cell lung cancer

[0383] Tumor assessment Week 6 Week 12 Week 18 Overall (RANO-BM) Partial response Partial response Partial response

[0384] Example 2: In vivo safety analysis of the anti-PD-1 - anti-VEGFA bispecific antibody

[0385] 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 adverse events frequently reported in clinical studies of bevacizumab. The inventors analyzed the incidence of these adverse events (≥ grade 3) in the clinical study of the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM). A total of 648 patients from 7 clinical trials (NCT04047290, NCT04597541, NCT05116007, NCT04736823, NCT04900363, NCT04870177, and NCT0518471) were included in the analysis dataset. The experimental results are shown in Table 3 and Table 4.

[0386] As shown in Table 3, the proportions of patients in the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) group who developed bleeding, hypertension, proteinuria, and gastrointestinal perforation greater than grade 3 were 0.5%, 4.6%, 1.1%, and 0.3%, respectively, which were lower than those in the bevacizumab group (the data of bevacizumab were from the drug label).

[0387] 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 of the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) was numerically lower compared to the published safety data of nivolumab (the data of nivolumab were from the drug label).

[0388] These data indicate that the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) has good in vivo safety.

[0389] Table 3: Incidence of bleeding, hypertension, proteinuria, and gastrointestinal perforation in clinical trials

[0390]

[0391] Table 4: Incidence of irAEs in clinical trials

[0392]

[0393] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that, based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are within the protection 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 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.

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 linking 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 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 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, the single-chain antibody against PD-1 is two molecules, 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 IgG-scFv form; Preferably, the bispecific antibody is a tetravalent bispecific antibody in the IgG-scFv form.

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; 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 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 antibody is 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 antibody is 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; (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 shown in SEQ ID NOs:45 - 48; 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; 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 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; 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 mode of the bispecific antibody is intravenous drip, intravenous injection or intraperitoneal injection.

18. The 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 administered according to the body surface area (m 2 ) of each subject 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, every 2 weeks, every 3 weeks or every 4 weeks; (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, 2 weeks, 3 weeks or 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 metastases are selected from one or more of squamous cell non-small cell lung cancer brain metastases, non-squamous cell non-small cell lung cancer brain metastases and lung adenocarcinoma brain metastases; Preferably, for patients with non-small cell lung cancer brain metastases, PD-L1 TPS≥50%, or 1%<PD-L1 TPS≥49%, or PD-L1 TPS≤1%.

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