Pharmaceutical combination and use

Abstract

Provided are a pharmaceutical combination and a use. The pharmaceutical combination contains a bispecific protein specifically binding to PD-1 and VEGFA, and a bispecific protein specifically binding to TIGIT and TGF-βR. The pharmaceutical combination can effectively treat or prevent tumors, and has good application prospects.

Description

Drug combinations and uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the application with CN application number 202311192415.9 and application date September 14, 2023, and claims its priority. The entire content of the CN application is hereby introduced as a whole into this application. Technical Field

[0003] The present invention relates to biopharmaceuticals and relates to drug combinations and their uses. Specifically, the drug combination comprises a bispecific protein that specifically binds to PD-1 and VEGFA, and a bispecific protein that specifically binds to TIGIT and TGF-βR. The drug combination can be in the form of a pharmaceutical composition or a combination product. Background Art

[0004] The transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 gene family and is expressed on activated T cells, B cells, and myeloid cells. PD-1's ligands, PDL1 (programmed cell death 1 ligand 1, also abbreviated as PD-L1) and PDL2 (programmed cell death 1 ligand 2, also abbreviated as PD-L2), both belong to the B7 superfamily. PDL1 is expressed on a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is expressed only on antigen-presenting cells such as dendritic cells and macrophages.

[0005] The PD-1 / PDL1 signaling pathway plays a crucial role in regulating immune tolerance, microbial infection, and tumor immune escape. PD-1 is primarily expressed in immune cells such as T cells, and its ligand, PDL1, is highly expressed in many human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway can activate suppressed T cells, allowing them to attack cancer cells. Blocking PD-1 / PDL1 signaling can promote the proliferation of tumor antigen-specific T cells, killing tumor cells and thereby inhibiting local tumor growth (Julie R et al., 2012, N Engl J Med. 366:2455-2465). Furthermore, tumors with high PDL1 expression are associated with cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA 104:3360-5). One effective approach is to modulate PD-1 expression through in vivo injection of anti-PD-1 antibodies. Due to the broad-spectrum anti-tumor potential and amazing efficacy of PD-1 antibodies, the industry generally believes that antibodies targeting the PD-1 pathway will bring breakthrough progress in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., 2015, Semin Oncol. 42(3): 466-473), leukemia and anemia (Held SA, Heine A, et al., 2013, Curr Cancer Drug Targets. 13(7): 768-74).

[0006] Vascular endothelial growth factor (VEGF) is a growth factor that promotes endothelial cell proliferation, angiogenesis, and vascular permeability. It binds to VEGF receptors on the cell surface and exerts its functions by activating tyrosine kinase signaling pathways. Within tumor tissue, tumor cells, tumor-invading macrophages, and mast cells secrete high levels of VEGF, which paracrine-stimulates tumor vascular endothelial cells, promoting endothelial cell proliferation and migration, inducing angiogenesis, and promoting sustained tumor growth. It also increases vascular permeability, triggers fibrin deposition in surrounding tissues, and promotes the infiltration of monocytes, fibroblasts, and endothelial cells. This facilitates tumor stroma formation and the entry of tumor cells into new blood vessels, promoting tumor metastasis. Therefore, inhibiting tumor angiogenesis is considered one of the most promising cancer treatments. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, and PIGF. Vascular endothelial growth factor receptors (VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4), and neuropilin-1 (NRP-1). The first three receptors are structurally similar, belonging to the tyrosine kinase superfamily. They are composed of an extracellular domain, a transmembrane segment, and an intracellular domain. The extracellular domain consists of an immunoglobulin-like domain, while the intracellular domain is a tyrosine kinase domain. VEGFR1 and VEGFR2 are primarily located on the surface of vascular endothelial cells, while VEGFR3 is primarily located on the surface of lymphatic endothelial cells.

[0007] VEGF family molecules have different affinities for several receptors. VEGFA mainly binds to VEGFR1, VEGFR2, and NRP-1 to exert its effects. VEGFR1 is the first receptor discovered. Under normal physiological conditions, the affinity of VEGFR1 for VEGFA is higher than that of VEGFR2 for VEGFA, but its intracellular tyrosinase activity is lower than that of VEGFR2 (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5) (2016): 146-148).

[0008] VEGFR2 is a major regulator of angiogenesis and construction. Compared with VEGFR1, VEGFR2 has a higher tyrosine kinase activity. After binding to its ligand VEGFA, VEGFR2 mediates the proliferation and differentiation of vascular endothelial cells, as well as the formation of blood vessels and vascular permeability (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 62(3)(2007):179-213.). After binding to VEGFR2, VEGFA mediates the transcriptional expression of intracellular related protein genes through the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway, promoting the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene, 18(13)(1999):2221-2230.).

[0009] VEGFR3 is a member of the tyrosine kinase family and is primarily expressed in vascular endothelial cells during the embryonic period and lymphatic endothelial cells during adulthood. VEGFC and VEGFD bind to VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote lymphangiogenesis. NRP-1 is a non-tyrosine kinase transmembrane protein that cannot independently transduce biological signals. It can only mediate signal transduction after forming a complex with the VEGF tyrosine kinase receptor. (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5)(2016): 146-148).

[0010] VEGFA and VEGFR2 are primarily involved in regulating angiogenesis. The binding of VEGFA to VEGFR2 triggers a cascade of numerous intermediate signals in upstream and downstream pathways, ultimately altering the physiological functions of endothelial cells in various ways, including proliferation, survival, migration, increased permeability, and infiltration into surrounding tissues (Dong Hongchao et al., Modern Oncology, Vol. 22, No. 9, September 2014, pp. 2231-3).

[0011] TIGIT (T cell Ig and ITIM domain, also known as WUCAM, Vstm3, VSIG9) is a member of the poliovirus receptor (PVR) / nectin family. TIGIT consists of an extracellular immunoglobulin variable region (IgV) domain, a type 1 transmembrane domain, and an intracellular domain with a classical immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoglobulin tyrosine tail (ITT) motif. TIGIT is highly expressed in lymphocytes, especially in effector and regulatory CD4 + T cells (Treg cells), follicular helper CD4 + T cells, effector CD8 +T cells and natural killer cells (NK cells) (Yu X, Harden K, Gonzalez LC, et al. The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells[J]. Nature immunology, 2009, 10(1): 48).

[0012] CD155 (also known as PVR, Necl5 or Tage4), CD112 (also known as PVRL2 / nectin 2) and CD113 (also known as PVRL3) are ligands bound by TIGIT (Martinet L, Smyth M J. Balancing natural killer cell activation through paired receptors [J]. Nature Reviews Immunology, 2015, 15 (4): 243-254.), among which CD155 is a high-affinity ligand for TIGIT. In NK cells, TIGIT binding ligands CD155 and CD112 can inhibit the killing effect of NK cells on cells with high expression of CD155 and CD112 (Stanietsky N, Simic H, Arapovic J, et al. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity [J]. Proceedings of the National Academy of Sciences, 2009, 106 (42): 17858-17863). It has been reported that blocking PD-1 and TIGIT simultaneously can enhance CD8 + The killing effect of T cells (Johnston RJ, Comps-Agrar L, Hackney J, et al. The immunoreceptor TIGIT regulates antitumor and antiviral CD8 +T cell effector function[J].Cancer cell,2014,26(6):923-937). In the latest research, it was found that TIGIT, as an immune checkpoint of NK cells, can lead to NK cell exhaustion during tumor development. It was also proved that anti-TIGIT monoclonal antibodies can reverse NK cell exhaustion and be used for immunotherapy of various tumors such as non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, non-Hodgkin's lymphoma, B lymphocytic neoplasm, plasma cell cancer, etc. (Zhang Q, Bi J, Zheng X, et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity[J].Nature immunology,2018,19(7):723-732).

[0013] The transforming growth factor-β (TGF-β) superfamily is a diverse class of cytokines, further divided into subfamilies including TGF-β, activins, inhibins, growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), Nodal, Lefty, and anti-Müllerian hormone (AMH) (Table A). The three TGF-β subfamily proteins are designated TGF-β1, TGF-β2, and TGF-β3, with TGF-β1 being the most highly expressed isoform. After binding to receptors, TGF-β1, TGF-β2, and TGF-β3 mediate a series of biological reactions through Smad and non-Smad signaling pathways, including promoting epithelial-mesenchymal transitions (EMT), promoting tissue fibrosis, promoting angiogenesis, promoting tumor immune escape, and having dual effects of tumor suppression and promotion (J Massagué. TGFbeta in Cancer. [J]. Cell, 2008, 134(2): 215-230.).

[0014] TGF-β receptors (TGF-βR) are widely distributed on the surface of normal human cells and tumor cells, including three receptor superfamilies. The type I classical TGF-β receptor family includes ALK1-7, among which TGF-βRI (also written as TβRI) is also called ALK5; the type II classical TGF-β receptor family includes TGF-βRII (also written as TβRII), ActRII, ActRIIB, AMHRII and BMPRII; the type III TGF-β receptor superfamily includes Betaglycan (also known as TGF-βRIII) and Endoglin; among them, TGF-βRI, TGF-βRII, and TGF-βRIII can each bind to TGF-β1, TGF-β2, and TGF-β3 (Pawlak John B, Blobe Gerard C, TGF-βsuperfamily co-receptors in cancer. DevDyn, 2021). The TGF-β family and receptors are shown in Table A (based on a review by Carl-Henrik Heldin1 and Aristidis Moustakas. Cold Spring Harb Perspect Biol. 2016). TGF-βRI and TGF-βRII are serine / threonine protein kinase receptors. TGF-βRII is a key signal transducer in the TGF-β signaling pathway. It binds to TGF-β with high affinity and then forms a heterotetrameric receptor complex with the TGF-βRI dimer. TGF-βRII then phosphorylates and activates TGF-βRI through autophosphorylation of TGF-βRII. Activated TGF-βRII phosphorylates downstream Smad pathway proteins, regulating the transcription and translation of downstream target genes and triggering disease-related biological responses. TGF-βRIII has a weaker affinity for TGF-β than TGF-βRI and TGF-βRII and lacks an intracellular domain, preventing it from accessing downstream signaling pathways. Its function is to capture TGF-β and present it to TGF-βRII. Other TGF-β receptors can also bind to TGF-β (Sang Xiaohong et al., Research progress on small molecule inhibitors targeting TGF-β and receptors [J]. Acta Pharmaceutica Sinica, 2019(9).).

[0015] Many cancer patients cannot achieve long-term disease control after surgery or chemotherapy, and their 5-year survival rate remains very low. In summary, the development of less toxic, more effective treatments and combined drug therapy regimens has great clinical significance.

[0016] Summary of the Invention

[0017] After in-depth research and creative work, the inventors combined a bispecific protein that specifically binds to PD-1 and VEGFA with a bispecific protein that specifically binds to TIGIT and TGF-βR. Surprisingly, they discovered that this combination has a pharmacological effect of effectively inhibiting tumor growth, and the effect is superior to that of a single bispecific protein that specifically binds to PD-1 and VEGFA or a single bispecific protein that specifically binds to TIGIT and TGF-βR. This provides the following invention:

[0018] One aspect of the present invention relates to a pharmaceutical combination comprising an effective amount of a first bispecific protein and an effective amount of a second bispecific protein, wherein:

[0019] (1) The first bispecific protein comprises:

[0020] specifically binds to the first protein domain of PD-1, and

[0021] Specifically binds to the second protein domain of VEGFA;

[0022] in,

[0023] The first protein functional region comprises a heavy chain variable region VH1 and a light chain variable region VL1, wherein the VH1 comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; the VL1 comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6;

[0024] The second protein functional region comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein the VH2 comprises the HCDR1 of SEQ ID NO: 7, the HCDR2 of SEQ ID NO: 8, and the HCDR3 of SEQ ID NO: 9; the VL2 comprises the LCDR1 of SEQ ID NO: 10, the LCDR2 of SEQ ID NO: 11, and the LCDR3 of SEQ ID NO: 12;

[0025] (2) The second bispecific protein comprises:

[0026] specifically binds to the third protein domain of TIGIT, and

[0027] Specifically binds to the fourth protein functional domain of TGF-β;

[0028] in,

[0029] The third protein functional region comprises a heavy chain variable region VH3 and a light chain variable region VL3, wherein the VH3 comprises the HCDR1 of SEQ ID NO: 13, the HCDR2 of SEQ ID NO: 14, and the HCDR3 of SEQ ID NO: 15; the VL3 comprises the LCDR1 of SEQ ID NO: 16, the LCDR2 of SEQ ID NO: 17, and the LCDR3 of SEQ ID NO: 18;

[0030] The fourth protein functional region is a TGF-β receptor, a TGF-β receptor extracellular region, a truncated fragment of the TGF-β receptor extracellular region having TGF-β receptor function, or a variant of the TGF-β receptor extracellular region having TGF-β receptor function.

[0031] In the present invention, a bispecific protein may also be referred to as a bispecific molecule. A first bispecific protein may also be referred to as a first bispecific molecule. A second bispecific protein may also be referred to as a second bispecific molecule.

[0032] In some embodiments of the present invention, the bispecific protein is a bispecific antibody. In some embodiments of the present invention, the first bispecific protein is a bispecific antibody; preferably, it is an anti-PD-1-anti-VEGFA bispecific antibody.

[0033] In some embodiments of the present invention, the bispecific protein is an antibody-receptor fusion protein. In some embodiments of the present invention, the second bispecific protein is an antibody-receptor fusion protein; preferably, it is an anti-TIGIT antibody-TGF-βR fusion protein.

[0034] In some embodiments of the present invention, the drug combination, wherein,

[0035] The amino acid sequence of the heavy chain variable region VH1 is selected from SEQ ID NO: 20 and SEQ ID NO: 24; and

[0036] The amino acid sequence of the light chain variable region VL1 is selected from SEQ ID NO: 22, SEQ ID NO: 26 and SEQ ID NO: 35.

[0037] In some embodiments of the present invention, the drug combination, wherein,

[0038] The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO: 22;

[0039] The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO: 26;

[0040] The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO: 35;

[0041] The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO: 22;

[0042] The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO: 26; or

[0043] The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO: 35.

[0044] In some embodiments of the present invention, the drug combination, wherein,

[0045] The amino acid sequence of the heavy chain variable region VH2 is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region VL2 is shown in SEQ ID NO: 33.

[0046] In some embodiments of the present invention, the drug combination, wherein,

[0047] The amino acid sequence of the heavy chain variable region VH3 is selected from SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56; and

[0048] The amino acid sequence of the light chain variable region VL3 is selected from SEQ ID NO:48, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62 and SEQ ID NO:64.

[0049] In some embodiments of the present invention, the drug combination, wherein,

[0050] The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO: 48;

[0051] The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO: 58;

[0052] The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO: 64;

[0053] The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO: 60;

[0054] The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO: 62;

[0055] The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO: 54, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO: 60;

[0056] The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO: 54, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO: 62;

[0057] The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO: 56, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO: 58; or

[0058] The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO: 56, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO: 64.

[0059] In some embodiments of the present invention, the drug combination, wherein,

[0060] The fourth protein functional region is TGF-βRⅠ, TGF-βRII, TGF-βRIII, the extracellular region of TGF-βRⅠ, the extracellular region of TGF-βRII, the extracellular region of TGF-βRIII, a truncated fragment of the extracellular region of TGF-βRⅠ having TGF-β receptor function, a truncated fragment of the extracellular region of TGF-βRII having TGF-β receptor function, a truncated fragment of the extracellular region of TGF-βRIII having TGF-β receptor function, or a variant of the extracellular region of TGF-β receptor having TGF-β receptor function.

[0061] The variant of the TGF-β receptor extracellular region having TGF-β receptor function refers to a protein that still has TGF-β receptor function after one or several (for example, 2, 3, 4, 5 or more) amino acids are replaced, deleted and / or inserted into the TGF-β receptor extracellular region.

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

[0063] The first protein functional region is IgG, and the second protein functional region is a single-chain antibody; or

[0064] The first protein functional region is a single-chain antibody, and the second protein functional region is IgG.

[0065] In some embodiments of the present invention, the drug combination, wherein,

[0066] The first protein functional region is IgG1, and the second protein functional region is a single-chain antibody; or

[0067] The first protein functional region is a single-chain antibody, and the second protein functional region is IgG1.

[0068] In some embodiments of the present invention, the drug combination, wherein,

[0069] The third protein functional region is IgG or single-chain antibody.

[0070] In some embodiments of the present invention, the drug combination, wherein,

[0071] The third protein functional region is IgG4 or a single-chain antibody.

[0072] In some embodiments of the present invention, the drug combination, wherein,

[0073] The IgG is IgG1, IgG2, IgG3 or IgG4.

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

[0075] According to the EU numbering system, the heavy chain constant region of IgG1 has one of the following four mutation combinations:

[0076] L234A and L235A;

[0077] L234A and G237A;

[0078] L235A and G237A; or

[0079] L234A, L235A and G237A.

[0080] In some embodiments of the present invention, the drug combination, wherein,

[0081] According to the EU numbering system, the heavy chain constant region of IgG4 has one of the following four mutation combinations:

[0082] F234A and L235A;

[0083] F234A and G237A;

[0084] L235A and G237A; or

[0085] F234A, L235A and G237A.

[0086] In some embodiments of the present invention, the drug combination, wherein,

[0087] The second protein functional region is IgG, and the amino acid sequence of its heavy chain is shown in SEQ ID NO: 38, SEQ ID NO: 40 or SEQ ID NO: 42, and the amino acid sequence of its light chain is shown in SEQ ID NO: 44.

[0088] In some embodiments of the present invention, the drug combination, wherein,

[0089] The third protein functional region is IgG, and the amino acid sequence of its heavy chain is shown in SEQ ID NO: 66 or SEQ ID NO: 70, and the amino acid sequence of its light chain is shown in SEQ ID NO: 68.

[0090] In some embodiments of the present invention, the drug combination, wherein,

[0091] The amino acid sequence of the fourth protein functional region is shown in any one of SEQ ID NO: 72 to SEQ ID NO: 78 and SEQ ID NO: 88.

[0092] In some embodiments of the present invention, the drug combination, wherein,

[0093] The first protein functional region is directly connected to the second protein functional region or is connected via a linker;

[0094] The third protein functional region is directly connected to the fourth protein functional region or is connected via a linker; and / or

[0095] The heavy chain variable region of the single-chain antibody is directly linked to the light chain variable region of the single-chain antibody or is linked via a linker.

[0096] In some embodiments of the present invention, the drug combination, wherein,

[0097] The linker is a polypeptide as set forth in SEQ ID NO:91 or SEQ ID NO:92, or a polypeptide obtained by concatenating multiple (e.g., 2, 3, 4, 5, or 6) polypeptides as set forth in SEQ ID NO:91, or a polypeptide obtained by concatenating multiple (e.g., 2, 3, 4, or 5) polypeptides as set forth in SEQ ID NO:91 and further concatenating the polypeptide as set forth in SEQ ID NO:92;

[0098] Preferably, the amino acid sequence of the linker is independently selected from SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 83.

[0099] In some embodiments of the present invention, the drug combination, wherein,

[0100] The first protein functional region, the second protein functional region, the third protein functional region and the fourth protein functional region are independently 1, 2 or more;

[0101] Preferably, the number of the first protein functional regions is 2, and the number of the second protein functional region is 1;

[0102] Preferably, the number of the third protein functional region is one, and the number of the fourth protein functional region is two.

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

[0104] The second protein functional region is an IgG, the first protein functional region is two single-chain antibodies, and the single-chain antibodies are respectively connected to the C-termini or N-termini of the two heavy chains of the second protein functional region.

[0105] In some embodiments of the present invention, the drug combination, wherein,

[0106] The second protein functional region is an IgG1, the first protein functional region is two single-chain antibodies, and the single-chain antibodies are respectively connected to the C-terminus or N-terminus of the two heavy chains of the second protein functional region.

[0107] In some embodiments of the present invention, the drug combination, wherein,

[0108] The first protein functional region is an IgG, the second protein functional region is two single-chain antibodies, and the single-chain antibodies are respectively connected to the C-terminus or N-terminus of the two heavy chains of the first protein functional region.

[0109] In some embodiments of the present invention, the drug combination, wherein,

[0110] The first protein functional region is an IgG1, the second protein functional region is two single-chain antibodies, and the single-chain antibodies are respectively connected to the C-terminus or N-terminus of the two heavy chains of the first protein functional region.

[0111] In one or more embodiments of the present invention, the bispecific protein is a bispecific antibody in the form of IgG-scFv, ie, Morrison format.

[0112] In one or more embodiments of the present invention, the bispecific antibody comprises a single-chain antibody linked to the C-terminus of an immunoglobulin heavy chain. Since immunoglobulins have two heavy chains, two single-chain antibody molecules are linked to one immunoglobulin molecule. Preferably, the two single-chain antibody molecules are identical. Preferably, the single-chain antibody is linked to the C-terminus of the immunoglobulin heavy chain via an amide bond formed by the aforementioned linker.

[0113] In one or more embodiments of the present invention, the constant region of the immunoglobulin is humanized, for example, the heavy chain constant region adopts the Ig gamma-1 chain C region, such as ACCESSION: P01857 or the Ig gamma-4 chain C region, such as ACCESSION: P01861.1; the light chain constant region adopts the Ig kappa chain C region, such as ACCESSION: P01834.

[0114] In some embodiments of the present invention, the drug combination, wherein,

[0115] The third protein functional region is one IgG, the fourth protein functional region is two, and the fourth protein functional region is respectively connected to the C-terminus or N-terminus of the two heavy chains of the third protein functional region.

[0116] In some embodiments of the present invention, the drug combination, wherein,

[0117] The third protein functional region is one IgG4, the fourth protein functional region is two, and the fourth protein functional region is connected to the C-terminus or N-terminus of the two heavy chains of the third protein functional region. In some embodiments of the present invention, the drug combination, wherein,

[0118] The first bispecific protein comprises:

[0119] specifically binds to the first protein domain of PD-1, and

[0120] Specifically binds to the second protein domain of VEGFA;

[0121] There are two first protein functional regions and one second protein functional region;

[0122] Wherein, the second protein functional region is IgG, and the first protein functional region is a single-chain antibody;

[0123] The amino acid sequence of the heavy chain of the IgG is shown in SEQ ID NO: 42, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 44;

[0124] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 35;

[0125] Two single-chain antibodies are connected to the C-termini of the two heavy chains of IgG;

[0126] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker are the same or different;

[0127] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO: 36 and SEQ ID NO: 37;

[0128] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO: 36.

[0129] In some embodiments of the present invention, the drug combination, wherein,

[0130] The second bispecific protein comprises:

[0131] specifically binds to the third protein domain of TIGIT, and

[0132] Specifically binds to the fourth protein functional domain of TGF-β;

[0133] in,

[0134] The number of the third protein functional region is one, and the number of the fourth protein functional region is two;

[0135] The third protein functional region is IgG, and the fourth protein functional region is a truncated fragment of the TGF-β receptor extracellular region having TGF-β receptor function;

[0136] The amino acid sequence of the IgG heavy chain is shown in SEQ ID NO: 66 or SEQ ID NO: 70, and the amino acid sequence of the light chain is shown in SEQ ID NO: 68;

[0137] The amino acid sequence of the fourth protein functional region is shown in SEQ ID NO: 72 or SEQ ID NO: 88;

[0138] The fourth protein functional region is connected to the C-termini of the two heavy chains of IgG through a third linker;

[0139] Preferably, the amino acid sequence of the third linker is as shown in SEQ ID NO:83.

[0140] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein is selected from the anti-PD-1-anti-VEGFA bispecific antibodies VP101(M), VP101(G4M) and VP101(hG1DM) of the present invention, and the second bispecific protein is selected from the anti-TIGIT antibody of the present invention and the TGF-βR fusion protein TF01, TF02 and TF01T.

[0141] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (M) of the present invention, and the second bispecific protein is selected from the anti-TIGIT antibody of the present invention and the TGF-βR fusion protein TF01, TF02 and TF01T.

[0142] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (G4M) of the present invention, and the second bispecific protein is selected from the anti-TIGIT antibody of the present invention and the TGF-βR fusion protein TF01, TF02 and TF01T.

[0143] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) of the present invention, and the second bispecific protein is selected from the anti-TIGIT antibody of the present invention and the TGF-βR fusion protein TF01, TF02 and TF01T.

[0144] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) of the present invention, and the second bispecific protein is the fusion protein TF01T of the anti-TIGIT antibody of the present invention and TGF-βR.

[0145] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein and the second bispecific protein are mixed together, is a pharmaceutical composition.

[0146] In some embodiments of the present invention, the pharmaceutical combination, wherein the first bispecific protein and the second bispecific protein are in separate packages, is a combination product.

[0147] In some embodiments of the present invention, the drug combination, wherein the mass ratio of the first bispecific protein to the second bispecific protein is (10:1) to (1:5), preferably (5:1) to (1:3) or (3:1) to (1:2), and more preferably 2:1.

[0148] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) of the present invention, and the second bispecific protein is the fusion protein TF01T of the anti-TIGIT antibody and TGF-βR of the present invention, and the mass ratio of the first bispecific protein to the second bispecific protein is (10:1) to (1:5), preferably (5:1) to (1:3) or (3:1) to (1:2), and more preferably 2:1.

[0149] In some embodiments of the present invention, the pharmaceutical combination, wherein the unit dose of the first bispecific protein and the second bispecific protein, calculated based on the mass of the first bispecific protein or the second bispecific protein, is independently 100 mg-2500 mg, 100 mg-2000 mg, 100 mg-1500 mg, 100 mg-1200 mg, 100 mg-1000 mg, 200 mg-800 mg, 200 mg-500 mg, 300 mg-600 mg, 400 mg-500 mg, or 450 mg.

[0150] In some embodiments of the present invention, the drug combination further comprises an effective amount of a tumor chemotherapy drug, such as a platinum drug, an alkylating agent, an antimetabolite, an anti-tumor antibiotic, a plant-based anticancer drug, a hormone or an immune preparation.

[0151] In some embodiments of the present invention, the pharmaceutical combination further comprises one or more pharmaceutically acceptable excipients; preferably, further comprises product instructions.

[0152] In some embodiments of the present invention, the drug combination, wherein the molar ratio of the first bispecific protein to the second bispecific protein is (10:1) to (1:10), preferably (5:1) to (1:5), (3:1) to (1:3) or (2:1) to (1:2), and more preferably 1:1.

[0153] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) of the present invention, and the second bispecific protein is the fusion protein TF01T of the anti-TIGIT antibody and TGF-βR of the present invention, and the molar ratio of the first bispecific protein to the second bispecific protein is (10:1) to (1:10), preferably (5:1) to (1:5), (3:1) to (1:3) or (2:1) to (1:2), and more preferably 1:1.

[0154] In some embodiments of the present invention, the first bispecific protein and the second bispecific protein are pharmaceutically active ingredients (APIs) or active ingredients.

[0155] In some embodiments of the present invention, the pharmaceutical combination consists of a first bispecific protein, a second bispecific protein, and pharmaceutically acceptable excipients.

[0156] In some embodiments of the invention, the first bispecific protein, the second bispecific protein, and the tumor chemotherapeutic drug are pharmaceutically active ingredients (APIs) or active ingredients.

[0157] In some embodiments of the present invention, the drug combination consists of a first bispecific protein, a second bispecific protein, a tumor chemotherapy drug, and pharmaceutically acceptable excipients.

[0158] The pharmaceutical combination according to any one of the present invention is used for treating or preventing tumors;

[0159] Preferably, the tumor is selected from one or more of colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, multiple myeloma, plasma cell carcinoma and pancreatic cancer;

[0160] Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer;

[0161] Preferably, the liver cancer is hepatocellular carcinoma;

[0162] Preferably, the kidney tumor is renal cell carcinoma;

[0163] Preferably, the breast cancer is triple-negative breast cancer;

[0164] Preferably, the urothelial cancer is bladder cancer;

[0165] Preferably, the lymphoma is non-Hodgkin's lymphoma or B lymphocytic neoplasm.

[0166] In another aspect of the present invention, a unit dosage form is provided, preferably for use in treating tumors, wherein the unit dosage form comprises 1-10,000 mg (preferably 1-1,000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg, or 100 mg) of the first bispecific protein according to any aspect of the present invention and 1-10,000 mg (preferably 10-1,000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, or 200 mg) of the second bispecific protein according to any aspect of the present invention, and optionally one or more chemotherapeutic agents according to the present invention (such as platinum drugs and / or fluorouracil anti-tumor drugs); wherein the first bispecific protein, the second bispecific protein, and the chemotherapeutic agent are packaged separately.

[0167] The present invention relates to a method for preventing or treating cancer or tumors, wherein one or more unit preparations of the present invention are administered to a subject in need thereof. Preferably, the first bispecific protein, the second bispecific protein, and the chemotherapeutic agent in the unit preparation are administered separately.

[0168] In another aspect of the present invention, a single pharmaceutical dosage unit is provided, preferably for the treatment of tumors, comprising 0.1-10,000 mg (preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of a first bispecific protein according to any one of the present invention and 0.1-10,000 mg (preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of a second bispecific protein according to any one of the present invention.

[0169] In one or more embodiments of the present invention, the first bispecific protein, the second bispecific protein and / or the chemotherapeutic drug are in a form suitable for intravenous injection or intravenous drip, preferably in the form of a liquid preparation.

[0170] In one or more embodiments of the present invention, the single dose of the first bispecific protein of any one of the present invention and / or the second bispecific protein of any one of the present invention is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; or the single dose of the first bispecific protein of any one of the present invention and / or the second bispecific protein of any one of the present invention is 10-1000 mg per subject, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg.

[0171] Preferably, the dosage is from twice a day to about every other day, or once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks;

[0172] Preferably, the administration method is intravenous drip, intravenous injection, subcutaneous injection or intraperitoneal injection.

[0173] Another aspect of the present invention relates to the use of any one of the drug combinations of the present invention in the preparation of a drug for treating or preventing tumors;

[0174] Preferably, the tumor is selected from one or more of colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, multiple myeloma, plasma cell carcinoma and pancreatic cancer;

[0175] Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer;

[0176] Preferably, the liver cancer is hepatocellular carcinoma;

[0177] Preferably, the kidney tumor is renal cell carcinoma;

[0178] Preferably, the breast cancer is triple-negative breast cancer;

[0179] Preferably, the urothelial cancer is bladder cancer;

[0180] Preferably, the lymphoma is non-Hodgkin's lymphoma or B lymphocytic neoplasm.

[0181] Another aspect of the present invention relates to a method for treating or preventing tumors, comprising the step of administering an effective amount of the pharmaceutical combination described in any one of the present invention to a subject in need thereof;

[0182] Preferably, the tumor is selected from one or more of colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, multiple myeloma, plasma cell carcinoma and pancreatic cancer;

[0183] Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer;

[0184] Preferably, the liver cancer is hepatocellular carcinoma;

[0185] Preferably, the kidney tumor is renal cell carcinoma;

[0186] Preferably, the breast cancer is triple-negative breast cancer;

[0187] Preferably, the urothelial cancer is bladder cancer;

[0188] Preferably, the lymphoma is non-Hodgkin's lymphoma or B lymphocytic neoplasm.

[0189] In some embodiments of the present invention, in the method for treating or preventing tumors, the first bispecific protein and the second bispecific protein are independently administered before or after surgery, and / or before or after radiotherapy.

[0190] In some embodiments of the present invention, the method for treating or preventing tumors, wherein:

[0191] The single administration dose of the first bispecific protein and the second bispecific protein is independently 0.1-100 mg / kg body weight, preferably 5-50 mg or 5-15 mg / kg body weight;

[0192] The first bispecific protein and the second bispecific protein are independently administered once every half a day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks;

[0193] and / or

[0194] The administration method is intravenous drip, intravenous injection, subcutaneous injection or intraperitoneal injection.

[0195] Antibody therapeutics, particularly monoclonal antibodies (mABs), have demonstrated promising therapeutic effects in the treatment of a wide range of diseases. Traditional experimental approaches to obtaining these therapeutic antibodies involve immunizing animals with antigens to generate antibodies that target the antigen, or improving antibodies with low affinity for the antigen through affinity maturation.

[0196] The variable regions of the light and heavy chains determine antigen binding; the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3, and the light chain (L) CDRs include LCDR1, LCDR2, and LCDR3; these were named by Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda, MD).

[0197] Preferably, CDRs may also be defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 38. suppl_1 (2009): D301-D307.

[0198] The amino acid sequences of the CDR regions of the monoclonal antibody sequences in items (1) to (5) below were analyzed using techniques well known to those skilled in the art. The results are as follows:

[0199] (1)14C12

[0200] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22.

[0201] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0202] HCDR1: GFAFSSYD (SEQ ID NO: 1)

[0203] HCDR2: ISGGGRYT (SEQ ID NO: 2)

[0204] HCDR3:ANRYGEAWFAY(SEQ ID NO:3)

[0205] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0206] LCDR1:QDINTY (SEQ ID NO:4)

[0207] LCDR2:RAN (SEQ ID NO:5)

[0208] LCDR3:LQYDEFPLT (SEQ ID NO:6)

[0209] (2)14C12H1L1

[0210] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26.

[0211] The amino acid sequences of the three CDR regions of its heavy chain variable region are identical to those of 14C12.

[0212] The amino acid sequences of the three CDR regions of its light chain variable region are identical to those of 14C12.

[0213] (3)14C12H1L1(M)

[0214] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 35.

[0215] The amino acid sequences of the three CDR regions of its heavy chain variable region are identical to those of 14C12.

[0216] The amino acid sequences of the three CDR regions of its light chain variable region are identical to those of 14C12.

[0217] (4) Bevacizumab

[0218] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33;

[0219] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0220] HCDR1: GYTFTNYG (SEQ ID NO: 7)

[0221] HCDR2: INTYTGEP (SEQ ID NO: 8)

[0222] HCDR3:AKYPHYYGSSHWYFDV(SEQ ID NO:9)

[0223] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0224] LCDR1:QDISNY (SEQ ID NO: 10)

[0225] LCDR2:FTS (SEQ ID NO: 11)

[0226] LCDR3:QQYSTVPWT (SEQ ID NO: 12)

[0227] (5)26B12

[0228] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:48.

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

[0230] HCDR1:GHSFTSDYA(SEQ ID NO:13)

[0231] HCDR2: ISYSDST (SEQ ID NO: 14)

[0232] HCDR3:ARLDYGNYGGAMDY(SEQ ID NO:15)

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

[0234] LCDR1: QHVSTA (SEQ ID NO: 16)

[0235] LCDR2: SAS (SEQ ID NO: 17)

[0236] LCDR3:QQHYITPWT(SEQ ID NO:18)

[0237] The three HCDRs and three LCDRs of the humanized TIGIT monoclonal antibody are the same as those of the mouse TIGIT monoclonal antibody.

[0238] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0239] As used herein, when referring to the amino acid sequence of the PD-1 protein (NCBI GenBank: NP_005009.2), it includes the full length of the PD-1 protein, or the extracellular fragment PD-1ECD or a fragment containing the PD-1ECD; it also includes a fusion protein of the PD-1ECD, such as a fragment fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally or artificially introduced into the amino acid sequence of the PD-1 protein without affecting its biological function. Therefore, in the present invention, the term "PD-1 protein" or "PD-1" should include all such sequences, including natural or artificial variants thereof. Furthermore, when describing a sequence fragment of the PD-1 protein, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0240] As used herein, when referring to the amino acid sequence of VEGFA protein (GenBank ID: NP_001165097.1), it includes the full length of VEGFA protein, and also includes fusion proteins of VEGFA, such as fragments fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions) may occur naturally or be artificially introduced into the amino acid sequence of VEGFA protein without affecting its biological function. Therefore, in the present invention, the term "VEGFA protein" or "VEGFA" should include all such sequences, including natural or artificial variants thereof. Furthermore, when describing a sequence fragment of VEGFA protein, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0241] As used herein, when referring to the amino acid sequence of VEGFR2 protein (also known as KDR, GenBank ID: NP_002244), it includes the full length of the VEGFR2 protein, or the extracellular fragment VEGFR2-ECD or a fragment comprising VEGFR2-ECD; it also includes a fusion protein of VEGFR2-ECD, such as a fragment fused to the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced into the amino acid sequence of the VEGFR2 protein without affecting its biological function. Therefore, in the present invention, the term "VEGFR2 protein" or "VEGFR2" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of the VEGFR2 protein, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0242] As used herein, unless otherwise specified, the VEGFRs are VEGFR1 and / or VEGFR2; their specific protein sequences are known in the art and can be found in existing literature or GenBank. For example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321) and VEGFR2 (VEGFR2, NCBI Gene ID: 3791).

[0243] As used herein, when referring to the amino acid sequence of TIGIT (NCBI GenBank ID: NP_776160.2), it includes the full length of the TIGIT protein, or the extracellular immunoglobulin variable region (IgV) domain or a fragment comprising the extracellular immunoglobulin variable region (IgV) domain; it also includes a fusion protein of TIGIT, such as a fragment fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the TIGIT protein without affecting its biological function. Therefore, in the present invention, the term "TIGIT protein" or "TIGIT" should include all such sequences, including the sequence shown and its natural or artificial variants. Furthermore, when describing a sequence fragment of the TIGIT protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.

[0244] As used herein, the term EC 50It refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0245] As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. In a general sense, the heavy chain can be understood as the polypeptide chain with the larger molecular weight in the antibody, and the light chain refers to the polypeptide chain with the smaller molecular weight in the antibody. Light chains can be classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are connected by a J region of about 12 or more amino acids, and the heavy chain also includes a D region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H1 、C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The V domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L) respectively form antibody binding sites. The allocation of amino acids to each region or domain follows the definition of Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The term "antibody" is not limited to any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0246] Antibody antigen-binding fragments (e.g., those described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and screened for specificity in the same manner as for intact antibodies.

[0247] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0248] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for possible spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and generally contain at least two or more different antibodies, which generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al. (Kohler et al., Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, for example, US Patent 4,816,567).

[0249] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced with amino acid residues of the corresponding non-human antibody, or with amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, Jones et al., Nature, 321: 522 525 (1986); Reichmann et al., Nature, 332: 323 329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593 596 (1992); and Clark, Immunol. Today 21: 397 402 (2000).

[0250] As used herein, the term "epitope" refers to the site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to as an "antigenic determinant" in the art. An epitope or antigenic determinant is typically composed of chemically active surface groups of a molecule, such as amino acids or carbohydrates or sugar side chains, and typically has specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be "linear" or "conformational." See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all interacting points between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interacting points exist across separate protein amino acid residues.

[0251] As used herein, the term "isolated" or "isolated" refers to something that is obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and a highly pure identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0252] As used herein, the term "E. coli expression system" refers to an expression system consisting of E. coli (strain) and a vector, wherein the E. coli (strain) is derived from commercially available strains, such as but not limited to: GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3).

[0253] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0254] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0255] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity of M or less (K D In some embodiments of the present invention, the term "targeting" refers to specific binding.

[0256] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind with a dissociation equilibrium constant of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less dissociation equilibrium constant (K D ) binding to the antigen, e.g., as measured using surface plasmon resonance (SPR) in a BIACORE instrument, or by a Fortebio Octet molecular interaction instrument.

[0257] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0258] As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0259] As used herein, the term "adjuvant" refers to a nonspecific immunopotentiator that, when delivered to the body together with an antigen or in advance, can enhance the body's immune response to the antigen or change the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0260] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactic effective amount is an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutic effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered treatments.

[0261] A "recurrent" cancer is one that grows back in the original site or at a distant site after responding to initial treatment (such as surgery). A "locally recurrent" cancer is one that appears after treatment in the same location as a previously treated cancer.

[0262] "Metastatic" cancer is cancer that has spread from one part of the body (such as the lungs) to another part of the body.

[0263] As used herein, the term "completely eliminated" refers to the absence of a binding signal or an extremely low binding signal as detected by existing instrumentation (e.g., the Fortebio Octet molecular interaction instrument). In one embodiment of the present invention, the absence of a binding signal or an extremely low binding signal refers to a binding signal (i.e., a response value or Response) of less than 0.1 nm.

[0264] In the present invention, unless otherwise specified, the “first” (e.g., a first bispecific antibody, a first protein functional region, a first linker, etc.), “second” (e.g., a second bispecific antibody, a second protein functional region, a second linker, etc.), “third” (e.g., a third protein functional region, a third linker, etc.) and “fourth” (e.g., a fourth protein functional region, a fourth linker, etc.) are for the purpose of distinguishing between references or clarifying the expression, and do not have a typical order meaning.

[0265] In the present invention, unless otherwise specified, "about" or "approximately" means fluctuating within a range of 10%, 20% or 30% of the modified numerical value or physical quantity, for example, about 100 minutes or about 100 minutes can be 90 minutes to 110 minutes, 80 minutes to 120 minutes or 70 minutes to 130 minutes.

[0266] The entire contents of the prior Chinese patent application CN112830972A are hereby incorporated into the present application by reference.

[0267] Advantageous Effects of the Invention

[0268] The present invention achieves one or more of the following technical effects (1) to (2):

[0269] (1) The drug combination of the present invention can effectively prevent or treat tumors.

[0270] (2) The combination of anti-TIGIT antibody-TGF-βR fusion protein and anti-PD-1-anti-VEGFA bispecific antibody has a pharmacological effect of effectively inhibiting tumor growth, and the effect is better than that of anti-PD-1-anti-VEGFA bispecific antibody alone or anti-TIGIT antibody-TGF-βR fusion protein alone, achieving a synergistic technical effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0271] Figure 1: The fusion protein of anti-TIGIT antibody and TGF-βR combined with anti-PD-1-anti-VEGFA bispecific antibody promotes INF-γ secretion in the PBMC+CMV system.

[0272] Figure 2: The fusion protein of anti-TIGIT antibody and TGF-βR combined with anti-PD-1-anti-VEGFA bispecific antibody promotes INF-γ secretion in the PBMC+CMV system (+TGF-β1).

[0273] Figure 3: Reporter gene system verifying the blocking activity of the fusion protein of anti-TIGIT antibody and TGF-βR combined with anti-PD-1-anti-VEGFA bispecific antibody.

[0274] Figure 4: Reporter gene system (+VEGF) verifies the blocking activity of the fusion protein of anti-TIGIT antibody and TGF-βR combined with anti-PD-1-anti-VEGFA bispecific antibody.

[0275] The partial sequences involved in the present invention are as follows:

[0276] 19. Nucleic acid sequence encoding the 14C12 heavy chain variable region: (354 bp)

[0277] 20. Amino acid sequence of the heavy chain variable region of 14C12: (118aa)

[0278] 21. Nucleic acid sequence encoding the 14C12 light chain variable region: (321 bp)

[0279] 22. 14C12 light chain variable region encoded amino acid sequence: (107aa)

[0280] 23. Nucleic acid sequence encoding the 14C12H1L1 heavy chain variable region: (354 bp)

[0281] 24. 14C12H1L1 heavy chain variable region 14C12H1(M) V Amino acid sequence: (118aa)

[0282] 25. Nucleic acid sequence encoding the light chain variable region of 14C12H1L1: (321 bp)

[0283] 26. Amino acid sequence of the light chain variable region of 14C12H1L1: (107aa)

[0284] 27. Nucleic acid sequence encoding 14C12H1L1 heavy chain (14C12H1): (1344 bp)

[0285] 28. Amino acid sequence of 14C12H1L1 heavy chain (14C12H1): (448aa)

[0286] 29. Nucleic acid sequence encoding 14C12H1L1 light chain (14C12L1): (642 bp)

[0287] 30. Amino acid sequence of 14C12H1L1 light chain (14C12L1): (214aa)

[0288] 31. Amino acid sequence of Bevacizumab heavy chain variable region (Bevacizumab-Hv): (123aa)

[0289] 32. Nucleic acid sequence encoding the Bevacizumab heavy chain variable region (Bevacizumab-Hv): (369 bp)

[0290] 33. Amino acid sequence of Bevacizumab light chain variable region (Bevacizumab-Lv): (107aa)

[0291] 34. Nucleic acid sequence encoding the Bevacizumab light chain variable region (Bevacizumab-Lv): (321 bp)

[0292] 35. Light chain variable region of 14C12H1L1(M) V :(108aa, the amino acid sequence mutation sites based on 14C12H1L1 are underlined)

[0293] 36. Connector

[0294] 37. Connector

[0295] 38. Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1WT): (453 aa)

[0296] 39. Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1WT): (1359 bp)

[0297] 40. Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG4WT): (450 aa)

[0298] 41. Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG4WT): (1350 bp)

[0299] 42. Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1DM): (453 aa, mutation sites are underlined)

[0300] 43. Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1DM): (1359 bp, mutation sites are underlined)

[0301] 44. The amino acid sequence of the light chain of the immunoglobulin portion of VP101 (hG1DM): (214 aa)

[0302] 45. Nucleic acid sequence encoding the light chain of the immunoglobulin portion of VP101 (hG1DM): (642 bp)

[0303] 46. ​​Amino acid sequence of 26B12VH

[0304] 47. Nucleic acid sequence of 26B12VH

[0305] 48. Amino acid sequence of 26B12VL

[0306] 49. Nucleic acid sequence of 26B12VL

[0307] 50. Amino acid sequence of 26B12H1VH

[0308] 51. Nucleic acid sequence of 26B12H1VH

[0309] 52. Amino acid sequence of 26B12H2VH

[0310] 53. Nucleic acid sequence of 26B12H2VH

[0311] 54. Amino acid sequence of 26B12H3VH

[0312] 55. Nucleic acid sequence of 26B12H3VH

[0313] 56. Amino acid sequence of 26B12H4VH

[0314] 57. Nucleic acid sequence of 26B12H4VH

[0315] 58. Amino acid sequence of 26B12L1VL

[0316] 59. Nucleic acid sequence of 26B12L1VL

[0317] 60. Amino acid sequence of 26B12L2VL

[0318] 61. Nucleic acid sequence of 26B12L2VL

[0319] 62. Amino acid sequence of 26B12L3VL

[0320] 63. Nucleic acid sequence of 26B12L3VL

[0321] 64. Amino acid sequence of 26B12L4VL

[0322] 65. Nucleic acid sequence of 26B12L4VL

[0323] 66. Amino acid sequence of the heavy chain of 26B12H2L2 (hG4DM)

[0324] 67. Nucleic acid sequence of 26B12H2L2 (hG4DM) heavy chain

[0325] 68. Amino acid sequence of the light chain of 26B12H2L2 (hG4DM)

[0326] 69. Nucleic acid sequence of 26B12H2L2 (hG4DM) light chain

[0327] 70. 26B12H2L2 (hG1DM) heavy chain amino acid sequence

[0328] 71. 26B12H2L2 (hG1DM) heavy chain nucleic acid sequence

[0329] 72. Amino acid sequence of TGF-βRII extracellular domain fragment (truncated):

[0330] 73.Amino acid sequence of TGF-βRI:

[0331] 74.Amino acid sequence of the extracellular region of TGF-βRI:

[0332] 75.Amino acid sequence of TGF-βRⅡ:

[0333] 76.Amino acid sequence of the extracellular region of TGF-βRⅡ:

[0334] 77. Amino acid sequence of TGF-βRⅢ:

[0335] 78.Amino acid sequence of the extracellular region of TGF-βRⅢ:

[0336] 79.Amino acid sequence of Tiragolumab heavy chain:

[0337] 80.Amino acid sequence of Tiragolumab light chain:

[0338] 81.Amino acid sequence of the heavy chain variable region of RG6058 (hG4):

[0339] 82. RG6058 (hG4) light chain variable region amino acid sequence:

[0340] 83.Amino acid sequence of the linker:

[0341] 84. Anti-HEL & TGFβ antibody heavy chain amino acid sequence

[0342] 85.Anti-HEL & TGFβ antibody light chain amino acid sequence

[0343] 86. Heavy chain amino acid sequence of RG6058 (G1DM)

[0344] 87. Light chain amino acid sequence of RG6058 (G1DM)

[0345] Amino acid sequence of the TGF-βRII extracellular domain variant of 88.TF01T:

[0346] 89.hIgG4WT heavy chain constant region amino acid sequence

[0347] 90.hIgG1WT heavy chain constant region amino acid sequence

[0348] 91. Repeating units in linkers

[0349] 92. Fragments in the linker DETAILED DESCRIPTION

[0350] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0351] The sequence of the isotype control antibody is human anti-hen egg lysozyme IgG (i.e., anti-HEL antibody, or human IgG, abbreviated as hIgG). The sequence is derived from the variable region sequence of the Fab F10.6.6 sequence in the Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1): 130-146). The hIgG1DM used in the examples is an isotype control antibody with the hG1DM constant region sequence for anti-HEL, prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd., batch number: 20190520.

[0352] Jurkat-NFAT-PD1-hTIGIT is a cell expressing human TIGIT constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. based on Jurkat-NFAT-PD1 cells (PD1 Effector cells, Promega, catalog number: J112A) through transfection.

[0353] Preparation Example 1: Sequence Design of Anti-PD-1 Antibody 14C12 and Its Humanized Antibody 14C12H1L1

[0354] The amino acid sequences of the heavy and light chains of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1, as well as the encoding nucleic acid sequences, are identical to those of 14C12 and 14C12H1L1 in Chinese Patent Publication CN112830972A, respectively.

[0355] (1) Heavy chain variable region sequence and light chain variable region sequence of 14C12

[0356] Nucleic acid sequence encoding the 14C12 heavy chain variable region: SEQ ID NO: 19;

[0357] Amino acid sequence of the 14C12 heavy chain variable region: SEQ ID NO: 20;

[0358] Nucleic acid sequence encoding the 14C12 light chain variable region: SEQ ID NO: 21;

[0359] The amino acid sequence encoded by the 14C12 light chain variable region is: SEQ ID NO: 22.

[0360] (2) Heavy chain variable region sequence, light chain variable region sequence, heavy chain sequence, and light chain sequence of humanized monoclonal antibody 14C12H1L1

[0361] Nucleic acid sequence encoding the 14C12H1L1 heavy chain variable region: SEQ ID NO: 23;

[0362] Amino acid sequence of the heavy chain variable region of 14C12H1L1: SEQ ID NO: 24;

[0363] Nucleic acid sequence encoding the light chain variable region of 14C12H1L1: SEQ ID NO: 25;

[0364] Amino acid sequence of the light chain variable region of 14C12H1L1: SEQ ID NO: 26;

[0365] Nucleic acid sequence encoding 14C12H1L1 heavy chain (14C12H1): SEQ ID NO: 27;

[0366] Amino acid sequence of 14C12H1L1 heavy chain (14C12H1): SEQ ID NO: 28;

[0367] Nucleic acid sequence encoding 14C12H1L1 light chain (14C12L1): SEQ ID NO: 29;

[0368] Amino acid sequence of 14C12H1L1 light chain (14C12L1): SEQ ID NO: 30.

[0369] Preparation Example 2: Preparation of anti-VEGFA antibody Bevacizumab

[0370] The amino acid sequences of the heavy and light chain variable regions of the marketed anti-VEGFA monoclonal antibody Avastin (Bevacizumab) were referenced in Chinese Patent Publication CN1259962A. The nucleic acid sequences encoding the heavy and light chain variable regions were synthesized by GenScript.

[0371] Amino acid sequence of Bevacizumab heavy chain variable region (Bevacizumab-Hv): SEQ ID NO: 31;

[0372] Nucleic acid sequence encoding the Bevacizumab heavy chain variable region (Bevacizumab-Hv): SEQ ID NO: 32;

[0373] Amino acid sequence of Bevacizumab light chain variable region (Bevacizumab-Lv): SEQ ID NO: 33;

[0374] Nucleic acid sequence encoding the Bevacizumab light chain variable region (Bevacizumab-Lv): SEQ ID NO: 34.

[0375] The heavy chain constant region all used the Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region all used the Ig kappa chain C region, ACCESSION: P01834.

[0376] The heavy and light chain cDNAs of bevacizumab were cloned into the pcDNA3.1 vector to generate recombinant expression plasmids for the antibody bevacizumab. The recombinant plasmids were transfected into 293F cells. The 293F cell culture fluid was purified and then tested.

[0377] The anti-VEGFA monoclonal antibody Avastin (Bevacizumab) was produced.

[0378] Preparation Example 3: Sequence Design of Mutant 14C12H1L1(M) of Anti-PD-1 Humanized Antibody 14C12H1L1

[0379] Based on 14C12H1L1, individual amino acids in its framework region (light chain) were mutated to obtain 14C12H1L1(M).

[0380] Heavy chain variable region of 14C12H1L1(M) V : Identical to the heavy chain variable region 14C12H1 of 14C12H1L1, i.e., the amino acid sequence is shown in SEQ ID NO: 24.

[0381] Light chain variable region of 14C12H1L1(M) V :SEQ ID NO:35.

[0382] Preparation Example 4: Sequence Design of Bispecific Antibodies

[0383] 1. Sequence Design

[0384] The structural model of the bispecific antibody in the present invention belongs to the Morrison model (IgG-scFv), that is, the C-termini of the two heavy chains of an IgG antibody are connected to the scFv fragment of another antibody, and the main components of the heavy chain and light chain are designed as shown in Table 1 below.

[0385] Based on the aforementioned bevacizumab, the VP101 antibody, named VP101(M), utilizes the amino acid sequences of the heavy and light chain variable regions of 14C12H1L1(M) as the ScFv fragment. Compared to 14C12H1L1, 14C12H1L1(M) effectively optimizes the bispecific antibody structure and enhances its effectiveness.

[0386] Table 1: Composition design of heavy and light chains of VP101(M) and VP101(G4M)

[0387] In Table 1 above:

[0388] (1) The "V" in the lower right corner refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. If "V" is not marked, the corresponding heavy chain or light chain is the full length including the constant region. The amino acid sequences of these variable regions or full lengths and their encoding nucleic acid sequences are referenced to the corresponding sequences described in the preparation examples above.

[0389] (2) The amino acid sequence of Linker2 is: SEQ ID NO: 36.

[0390] Alternatively, SEQ ID NO: 37 can be used as Linker 1 to replace the aforementioned Linker 2.

[0391] (3) Bevacizumab-H uses the Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region.

[0392] (4) Bevacizumab-G4H uses the Ig gamma-4 chain C region, ACCESSION: P01861.1 as the heavy chain constant region.

[0393] 2. Expression and Purification of Antibody VP101(M)

[0394] The heavy chain cDNA sequence and light chain cDNA sequence of VP101(M) were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-VP101H and pUC57simple-VP101L plasmids, respectively.

[0395] Plasmids pUC57simple-VP101H and pUC57simple-VP101L were digested with HindIII and EcoRI, and the heavy and light chains recovered by electrophoresis were subcloned into the pcDNA3.1 vector. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and then loaded onto a HiTrap MabSelect SuRe column. The protein was eluted in one step with Elution Buffer to recover the target antibody VP101, and the buffer was exchanged with PBS.

[0396] 3. Detection of Antibody VP101(M)

[0397] The purified samples were added into reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer respectively, boiled and then subjected to SDS-PAGE electrophoresis detection.

[0398] To distinguish it from the mutated antibody described in Preparation Example 5, VP101(M) is also referred to as VP101(hG1WT) in the present invention. This "wild-type" VP101(M) employs the Ig gamma-1 chain C region, ACCESSION: P01857, as the heavy chain constant region and the Ig kappa chain C region, ACCESSION: P01834, as the light chain constant region.

[0399] Amino acid sequence of the heavy chain (Bevacizumab-H) of the immunoglobulin portion of VP101 (hG1WT): SEQ ID NO: 38;

[0400] Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1WT): SEQ ID NO: 39.

[0401] To distinguish it from the mutated antibody described in Preparation Example 5, VP101(G4M) is also referred to as VP101(hG4WT) in the present invention. VP101(G4M) is considered "wild-type" and 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.

[0402] Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG4WT): SEQ ID NO: 40;

[0403] Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG4WT): SEQ ID NO: 41.

[0404] Preparation Example 5: Design of amino acid mutations in the non-variable region of the humanized bispecific antibody VP101 (hG1WT)

[0405] Based on VP101 (hG1WT) obtained in Preparation Example 5, the present inventors obtained VP101 (hG1DM) by introducing a point mutation from leucine to alanine (L234A) at position 234 of its heavy chain and a point mutation from leucine to alanine (L235A) at position 235.

[0406] Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1DM) (453 aa, mutation sites are underlined): SEQ ID NO: 42;

[0407] Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1DM) (1359 bp, mutation sites are underlined): SEQ ID NO: 43.

[0408] The light chain amino acid sequences of the immunoglobulin portions of VP101 (hG1DM), VP101 (hG1WT), and VP101 (hG4WT) are identical, as are their encoding nucleic acid sequences.

[0409] Amino acid sequence of the light chain of the immunoglobulin portion of VP101 (hG1DM): SEQ ID NO: 44;

[0410] Nucleic acid sequence encoding the light chain of the immunoglobulin portion of VP101 (hG1DM): SEQ ID NO: 45.

[0411] Preparation Example 6: Preparation of anti-TIGIT antibodies

[0412] 1. Preparation of Hybridoma Cell Line LT019

[0413] The antigen used to prepare anti-TIGIT antibodies is human TIGIT-mFc (TIGIT is Genbank ID: NP_776160.2). The spleen cells of immunized mice were fused with mouse myeloma cells to make hybridoma cells. Using human TIGIT-hFc as an antigen, the hybridoma cells were screened by indirect ELISA to obtain hybridoma cells that can secrete antibodies that specifically bind to TIGIT. The hybridoma cells obtained by screening were subjected to a limiting dilution method to obtain stable hybridoma cell lines. The above hybridoma cell lines were named hybridoma cell line LT019, and the monoclonal antibodies secreted by them were named 26B12.

[0414] The hybridoma cell line LT019 (also known as TIGIT-26B12) was deposited in the China Center for Type Culture Collection (CCTCC) on October 23, 2020, with the deposit number CCTCC NO: C2020208, and the deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0415] 2. Preparation of Anti-TIGIT Antibody 26B12

[0416] The LT019 cell line prepared above was cultured in CD medium (Chemical Defined Medium, containing 1% penicillin-streptomycin) at 5% CO₂ and 37°C. After 7 days, the cell culture supernatant was collected, centrifuged, vacuum filtered through a microporous filter, and purified using a HiTrap protein A HP column to produce the antibody 26B12.

[0417] Preparation Example 7: Sequence Analysis of Anti-TIGIT Antibody 26B12

[0418] According to the method of the bacterial total RNA extraction kit for cultured cells (Tiangen, catalog number DP430), mRNA was extracted from the LT019 cell line cultured in Preparation Example 6.

[0419] According to Invitrogen III First-Strand Synthesis System for RT-PCR Kit Instructions: Synthesize cDNA and perform PCR amplification.

[0420] The PCR amplified products were directly subjected to TA cloning. The specific operation was carried out according to the instructions of the pEASY-T1 Cloning Kit (Transgen CT101).

[0421] The TA cloned products were directly sequenced, and the sequencing results were as follows:

[0422] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 47, and the fragment is 363 bp long.

[0423] The amino acid sequence encoded by the gene is shown in SEQ ID NO: 46, which has a length of 121 amino acids.

[0424] The sequence of the heavy chain HCDR1 is shown in SEQ ID NO: 13, the sequence of HCDR2 is shown in SEQ ID NO: 14, and the sequence of HCDR3 is shown in SEQ ID NO: 15.

[0425] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 49, with a length of 321 bp.

[0426] The amino acid sequence encoded by the gene is shown in SEQ ID NO: 48, which has a length of 107 amino acids.

[0427] The sequence of the light chain LCDR1 is shown in SEQ ID NO: 16, the sequence of LCDR2 is shown in SEQ ID NO: 17, and the sequence of LCDR3 is shown in SEQ ID NO: 18.

[0428] Preparation Example 8: Design and Preparation of Humanized Antibodies and Mutated Antibodies Against Human TIGIT

[0429] 1. Light and heavy chain design of humanized anti-human TIGIT antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4

[0430] Based on the three-dimensional crystal structure of human TIGIT protein and the sequence of antibody 26B12 obtained in Preparation Example 7, the variable region sequences of antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 were designed (antibody constant region sequences, the heavy chain constant region all used the Ig gamma-1 chain C region; the light chain constant region was the Ig kappa chain C region).

[0431] The designed variable region sequences are shown in Table 2 below.

[0432] Table 2: Variable region sequences of humanized antibodies against human TIGIT

[0433] The above eight antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4, the nucleic acid sequence length of the heavy chain variable region is 363 bp, the length of the amino acid sequence encoded therein is 121 aa; the nucleic acid sequence length of the light chain variable region is 321 bp, the length of the amino acid sequence encoded therein is 107 aa.

[0434] And the above 8 antibodies have the same HCDR1-HCDR3 and LCDR1-LCDR3, as follows:

[0435] The sequence of HCDR1 is shown in SEQ ID NO: 13, the sequence of HCDR2 is shown in SEQ ID NO: 14, and the sequence of HCDR3 is shown in SEQ ID NO: 15;

[0436] The sequence of LCDR1 is shown in SEQ ID NO: 16, the sequence of LCDR2 is shown in SEQ ID NO: 17, and the sequence of LCDR3 is shown in SEQ ID NO: 18.

[0437] 2. Preparation of Humanized Antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4

[0438] The heavy chain constant region all used the Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region all used the Ig kappa chain C region, ACCESSION: P01834.

[0439] The heavy chain cDNA and light chain cDNA of 26B12H1L1, the heavy chain cDNA and light chain cDNA of 26B12H4L1, the heavy chain cDNA and light chain cDNA of 26B12H2L2, the heavy chain cDNA and light chain cDNA of 26B12H3L2, the heavy chain cDNA and light chain cDNA of 26B12H2L3, the heavy chain cDNA and light chain cDNA of 26B12H3L3, the heavy chain cDNA and light chain cDNA of 26B12H1L4, the heavy chain cDNA and light chain cDNA of 26B12H2L4, and the heavy chain cDNA and light chain cDNA of 26B12H4L4 were cloned into pUC57simple (provided by GenScript) vector to obtain the following:

[0440] pUC57simple-26B12H1, pUC57simple-26B12L1;

[0441] pUC57simple-26B12H4, pUC57simple-26B12L1;

[0442] pUC57simple-26B12H2, pUC57simple-26B12L2;

[0443] pUC57simple-26B12H3, pUC57simple-26B12L2;

[0444] pUC57simple-26B12H2, pUC57simple-26B12L3;

[0445] pUC57simple-26B12H3, pUC57simple-26B12L3;

[0446] pUC57simple-26B12H1, pUC57simple-26B12L4; and

[0447] pUC57simple-26B12H4, pUC57simple-26B12L4.

[0448] Referring to the standard techniques described in the Molecular Cloning Laboratory Manual (Second Edition), the full-length heavy and light chain genes synthesized by EcoRI & HindIII digestion were subcloned into the expression vector pcDNA3.1 by restriction enzyme digestion (EcoRI & HindIII) to obtain expression plasmids pcDNA3.1-26B12H1, pcDNA3.1-26B12L1, pcDNA3.1-26B12H4, pcDNA3.1-126B12H2, pcDNA3.1-26B12L2, pcDNA3.1-26B12H3, pcDNA3.1-26B12L3 and pcDNA3.1-26B12L4, and the heavy / light chain genes of the recombinant expression plasmids were further sequenced and analyzed. Subsequently, the recombinant plasmids containing the corresponding light and heavy chain gene combinations (pcDNA3.1-26B12H1 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H4 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H2 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L2 , pcDNA3.1-26B12H2 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H1 / pcDNA3.1-26B12L4, and pcDNA3.1-26B12H4 / pcDNA3.1-26B12L4) were co-transfected into 293F cells, and the culture medium was collected for purification. After sequencing verification, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected and affinity purified using a Protein A column to obtain the humanized antibody.

[0449] The humanized antibody 26B12H2L2 is also referred to as 26B12H2L2 (hG1WT) in the present invention.

[0450] 3. Design of humanized antibodies 26B12H2L2(hG1DM) and 26B12H2L2(hG4DM)

[0451] Based on 26B12H2L2, the present inventors introduced a leucine-to-alanine point mutation (L234A) at position 234 (according to the EU numbering system, the same below) and a leucine-to-alanine point mutation (L235A) at position 235 of the heavy chain constant region to obtain a humanized antibody with constant region mutations, 26B12H2L2(hG1DM). The heavy chain amino acid sequence of 26B12H2L2(hG1DM) is shown in SEQ ID NO:70; the light chain amino acid sequence is shown in SEQ ID NO:68.

[0452] The present inventors, based on 26B12H2L2, retained the variable region of the antibody and used Ig gamma-4 chain C region, ACCESSION: P01861.1, as the heavy chain constant region. They also introduced a phenylalanine-to-alanine point mutation (F234A) at position 234 of the heavy chain constant region, and a leucine-to-alanine point mutation (L235A) at position 235, to obtain a humanized antibody with constant region mutations, 26B12H2L2 (hG4DM). The heavy chain amino acid sequence of 26B12H2L2 (hG4DM) is shown in SEQ ID NO: 66; the light chain amino acid sequence is shown in SEQ ID NO: 68.

[0453] The preparation of humanized antibodies 26B12H2L2 (hG1DM) and 26B12H2L2 (hG4DM) can refer to the method in step 2 above.

[0454] Preparation Example 9: Sequence Design and Preparation of Fusion Protein of Anti-TIGIT Antibody and TGF-βR

[0455] 1. Sequence Design

[0456] The composition of the fusion protein of the anti-TIGIT antibody and TGF-βR in the present invention is shown in Table 3 below.

[0457] Table 3: Composition of heavy and light chains of anti-TIGIT antibody and TGF-βR fusion proteins

[0458] The TGF-β receptor portion has two peptide chains, each of which is connected to the C-terminus of the heavy chain of the IgG portion through a linker.

[0459] 2. Antibody Expression and Purification

[0460] The heavy chain cDNA sequences and light chain cDNA sequences of TF01 and TF02 were cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-TF01H, pUC57simple-TF02H, pUC57simple-TF02L, and pUC57simple-TF02L plasmids, respectively.

[0461] Plasmids pUC57simple-TF01H / pUC57simple TF01L and pUC57simple-TF02H / pUC57simple TF02L were digested with HindIII and EcoRI, respectively. The heavy and light chains were recovered by electrophoresis and subcloned into the pcDNA3.1 vector. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and then loaded onto a HiTrap MabSelect SuRe column. The protein was eluted in one step with Elution Buffer, and the target sample was recovered and exchanged with PBS. This produced the anti-TIGIT antibody and TGF-βR fusion proteins TF01 and TF02.

[0462] Preparation Example 10: Sequence Design and Preparation of Fusion Protein (Mutant) of Anti-TIGIT Antibody and TGF-βR

[0463] A point mutation was performed on the TGF-βRII extracellular region fragment in the fusion protein TF01 of the anti-TIGIT antibody and TGF-βR in the present invention, and the serine at position 8 of the TGF-βRII extracellular region fragment (SEQ ID NO: 72) was mutated to threonine (SEQ ID NO: 88), resulting in a new antibody named TF01T. The preparation method thereof is referred to Preparation Example 9.

[0464] Table 4: Composition of heavy and light chains of fusion proteins (mutants) of anti-TIGIT antibodies and TGF-βR

[0465] Example 1: Combination of anti-TIGIT antibody and TGF-βR fusion protein with anti-PD-1-anti-VEGFA bispecific antibody to promote INF-γ secretion

[0466] Ficoll-Paque separation medium TMNormal human PBMCs (isolated from healthy human peripheral blood) were isolated using the Cytiva Plus (Cytiva, Cat: 17-1440-02) operating instructions. The day before the experiment, PBMCs were resuscitated and cultured in complete medium (i.e., RPMI 1640 + 10% FBS; RPMI 1640, Gibco, Cat: 22400-089; FBS, fetal bovine serum, Excell Bio, Cat: FSP500) at 37°C in a 5% CO2 incubator. On the day of the experiment, antibodies (VP101 (hG1DM) to TF01T at a 1:1 molar ratio), CMV (Mabtech, Catalog No. 3619-1), and TGF-β1 (Genscript, Catalog No. Z03411) were serially diluted in complete medium. Equal volumes of TGF-β1 (final concentration of 3 ng / mL) and antibodies were added to 96-well round-bottom plates (Corning, Model 3799) according to the experimental design. A control group was also included and incubated at 37°C for 10 min. PBMCs were collected, counted, and viability was measured. 200,000 cells / well were seeded in 96-well round-bottom plates. CMV was added at a final concentration of 0.2 μg / mL, and the plates were gently tapped to mix. The cells were then cultured in an incubator for 4 days (final experimental volume, 200 μL). After 4 days, cell culture supernatants were collected and IFN-γ concentrations were measured using an ELISA kit (Dakoway, Catalog No. 1110002).

[0467] The results are shown in Figures 1 and 2.

[0468] The results showed that under CMV stimulation, VP101(hG1DM) alone, TF01T alone, and VP101(hG1DM) combined with TF01T all effectively promoted PBMC secretion of IFN-γ, and the VP101(hG1DM) combined with TF01T group was significantly more potent than any of the other single-drug groups. Adding TGF-β1, which inhibits IFN-γ secretion, to this system increased IFN-γ secretion in the VP101(hG1DM) combined with TF01T group compared to the group without TGF-β1, demonstrating a stronger immunogenic biological activity in promoting PBMC recall responses to CMV.

[0469] Example 2: Reporter gene system verification of the blocking activity of the anti-TIGIT antibody and TGF-βR fusion protein combined with the anti-PD-1-anti-VEGFA bispecific antibody

[0470] PDL1 aAPC / CHO-K1 cells (purchased from Promega, catalog number J108A) were resuspended in Ham's F-12 medium with 10% FBS (Ham's F-12, Gibco, catalog number 11765070; FBS, fetal bovine serum, Excell Bio, catalog number FSP500) and seeded at 40,000 cells / well / 100 μL in a 96-well flat-bottom black plate (Corning, model number 3916) for overnight culture. The next day, antibodies and VEGF (prepared by Kangfang Bio, batch number 20210508) were diluted in assay medium (RPMI 1640 with 10% FBS, Gibco, catalog number 22400-089; FBS, fetal bovine serum, Excell Bio, catalog number FSP500) according to the experimental design and preincubated at 37°C for 30 min. Jurkat-NFAT-PD1-hTIGIT cells (Zhongshan Kangfang Biotechnology) and A549 cells (purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Cat. No. SCSP-503) were collected and resuspended in assay medium, and the cell density was adjusted. The medium in the 96-well flat-bottom black plate was removed, and 40 μL / well of antibody or antibody and VEGF preincubation solution (wherein the molar ratio of VP101 (hG1DM) to TF01T was 1:1) was added according to the experimental design. 50,000 Jurkat-NFAT-PD1-hTIGIT cells / well / 20 μL and 10,000 A549 cells / well / 20 μL were added. Isotype control (human IgG1DM) and blank control groups were set up, and the cells were incubated in an incubator for 6 hours. A 96-well flat-bottom black plate was taken out and equilibrated to room temperature. 80 μL / well of Firefly Glo Luciferase Reporter Gene Assay Kit (Yeasen, Cat. No.: 11404ES80) was added. The plate was incubated in the dark for 5 min and then the RLU value was read.

[0471] The results are shown in Figure 3. In this reporter gene system, the interactions between TIGIT / PVR and PD-1 / PD-L1 can inhibit the activation of the NFAT signaling pathway. Therefore, blocking TIGIT / PVR and / or PD-1 / PD-L1 can activate the NFAT pathway in effector cells and activate the expression of the luciferase reporter gene. The higher the luciferase expression, the stronger the fluorescence intensity. The results show that the VP101(hG1DM) monotherapy group, the TF01T monotherapy group, and the VP101(hG1DM) combined with TF01T group can all effectively neutralize and block the TIGIT / PVR and PD-1 / PD-L1 interactions. In addition, the neutralizing biological activity of VP101(hG1DM) combined with TF01T in blocking TIGIT / PVR and PD-1 / PD-L1 is superior to that of VP101(hG1DM) monotherapy and TF01T monotherapy.

[0472] In addition, VEGF was added to this reporter gene system for pretreatment, and the results are shown in Figure 4. The results showed that compared with the isotype control human IgG1DM, both the VP101(hG1DM) monotherapy group and the VP101(hG1DM) combined with TF01T group significantly increased luciferase expression; and the blocking activity of VP101(hG1DM) combined with TF01T was higher than that of the VP101(hG1DM) monotherapy group.

[0473] In summary, the neutralizing biological activity of VP101(hG1DM) combined with TF01T in blocking TIGIT / PVR and PD-1 / PD-L1 is better than that of VP101(hG1DM) alone and TF01T alone, and in a system containing VEGF, VP101(hG1DM) combined with TF01T shows stronger neutralizing activity.

[0474] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A pharmaceutical combination comprising an effective amount of a first bispecific protein and an effective amount of a second bispecific protein, wherein: (1) The first bispecific protein comprises: specifically binds to the first protein domain of PD-1, and The second protein domain that specifically binds to VEGFA; in, The first protein functional region comprises a heavy chain variable region VH1 and a light chain variable region VL1, wherein the VH1 comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; the VL1 comprises LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6; The second protein functional region comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein the VH2 comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8, and HCDR3 as shown in SEQ ID NO:9; the VL2 comprises LCDR1 as shown in SEQ ID NO:10, LCDR2 as shown in SEQ ID NO:11, and LCDR3 as shown in SEQ ID NO:12; (2) The second bispecific protein comprises: specifically binds to the third protein domain of TIGIT, and Specifically binds to the fourth protein functional domain of TGF-β; in, The third protein functional region comprises a heavy chain variable region VH3 and a light chain variable region VL3, wherein the VH3 comprises HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 14, and HCDR3 as shown in SEQ ID NO: 15; the VL3 comprises LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 17, and LCDR3 as shown in SEQ ID NO: 18; The fourth protein functional region is a TGF-β receptor, a TGF-β receptor extracellular region, a truncated fragment of a TGF-β receptor extracellular region having TGF-β receptor function, or a variant of a TGF-β receptor extracellular region having TGF-β receptor function.

2. The pharmaceutical combination according to claim 1, wherein The amino acid sequence of the heavy chain variable region VH1 is selected from SEQ ID NO: 20 and SEQ ID NO: 24; and The amino acid sequence of the light chain variable region VL1 is selected from SEQ ID NO:22, SEQ ID NO:26 and SEQ ID NO:

35.

3. The pharmaceutical combination according to any one of claims 1 to 2, wherein: The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO: 22; The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO:20, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO:35; The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO: 22; The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO: 26; or The amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO:24, and the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO:

35.

4. The pharmaceutical combination according to any one of claims 1 to 3, wherein The amino acid sequence of the heavy chain variable region VH2 is shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region VL2 is shown in SEQ ID NO:

33.

5. The pharmaceutical combination according to any one of claims 1 to 4, wherein: The amino acid sequence of the heavy chain variable region VH3 is selected from SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:56; and The amino acid sequence of the light chain variable region VL3 is selected from SEQ ID NO:48, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62 and SEQ ID NO:

64.

6. The pharmaceutical combination according to any one of claims 1 to 5, wherein: The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO:48; The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO:50, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO:58; The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO:50, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO:64; The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO:52, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO:60; The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO:52, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO:62; The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO:54, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO:60; The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO:54, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO:62; The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO: 56, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO: 58; or The amino acid sequence of the heavy chain variable region VH3 is shown in SEQ ID NO:56, and the amino acid sequence of the light chain variable region VL3 is shown in SEQ ID NO:

64.

7. The pharmaceutical combination according to any one of claims 1 to 6, wherein: The fourth protein functional region is TGF-βRⅠ, TGF-βRII, TGF-βRIII, the extracellular region of TGF-βRⅠ, the extracellular region of TGF-βRII, the extracellular region of TGF-βRIII, a truncated fragment of the extracellular region of TGF-βRⅠ with TGF-β receptor function, a truncated fragment of the extracellular region of TGF-βRII with TGF-β receptor function, a truncated fragment of the extracellular region of TGF-βRIII with TGF-β receptor function, or a variant of the extracellular region of TGF-β receptor with TGF-β receptor function.

8. The pharmaceutical combination according to any one of claims 1 to 7, wherein: The first protein functional region is IgG, and the second protein functional region is a single-chain antibody; or The first protein functional region is a single-chain antibody, and the second protein functional region is IgG.

9. The pharmaceutical combination according to any one of claims 1 to 8, wherein: The third protein functional region is IgG or single-chain antibody.

10. The pharmaceutical combination according to any one of claims 8 to 9, wherein: The IgG is IgG1, IgG2, IgG3 or IgG4.

11. The pharmaceutical combination according to any one of claims 8 to 10, wherein: According to the EU numbering system, the heavy chain constant region of IgG1 has one of the following four mutation combinations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A.

12. The pharmaceutical combination according to any one of claims 8 to 10, wherein According to the EU numbering system, the heavy chain constant region of IgG4 has one of the following four mutation combinations: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A and G237A.

13. The pharmaceutical combination according to any one of claims 1 to 12, wherein: The second protein functional region is IgG, and the amino acid sequence of its heavy chain is shown in SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42, and the amino acid sequence of its light chain is shown in SEQ ID NO:

44.

14. The pharmaceutical combination according to any one of claims 1 to 13, wherein The third protein functional region is IgG, and the amino acid sequence of its heavy chain is shown in SEQ ID NO:66 or SEQ ID NO:70, and the amino acid sequence of its light chain is shown in SEQ ID NO:

68.

15. The pharmaceutical combination according to any one of claims 1 to 14, wherein: The amino acid sequence of the fourth protein functional region is shown in any one of SEQ ID NO:72 to SEQ ID NO:78 and SEQ ID NO:

88.

16. The pharmaceutical combination according to any one of claims 1 to 15, wherein: The first protein functional region is directly connected to the second protein functional region or connected via a linker; The third protein functional region is directly connected to the fourth protein functional region or is connected via a linker; and / or The heavy chain variable region of the single chain antibody is directly linked to the light chain variable region of the single chain antibody or is linked via a linker.

17. The pharmaceutical combination according to claim 16, wherein The linker is a polypeptide as shown in SEQ ID NO:91 or SEQ ID NO:92, or a polypeptide obtained by connecting multiple (e.g., 2, 3, 4, 5 or 6) polypeptides as shown in SEQ ID NO:91 in series, or a polypeptide obtained by connecting multiple (e.g., 2, 3, 4 or 5) polypeptides as shown in SEQ ID NO:91 in series and further connecting with a polypeptide as shown in SEQ ID NO:92 in series; Preferably, the amino acid sequence of the linker is independently selected from SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:

83.

18. The pharmaceutical combination according to any one of claims 1 to 17, wherein The first protein functional region, the second protein functional region, the third protein functional region and the fourth protein functional region are independently 1, 2 or more than 2; Preferably, the number of the first protein functional regions is 2, and the number of the second protein functional region is 1; Preferably, the number of the third protein functional region is one, and the number of the fourth protein functional region is two.

19. The pharmaceutical combination according to any one of claims 1 to 18, wherein The second protein functional region is one IgG (eg, IgG1), the first protein functional region is two single-chain antibodies, and the single-chain antibodies are respectively connected to the C-termini or N-termini of the two heavy chains of the second protein functional region.

20. The pharmaceutical combination according to any one of claims 1 to 19, wherein The third protein functional region is one IgG (eg, IgG4), the fourth protein functional region is two, and the fourth protein functional region is respectively connected to the C-terminus or N-terminus of the two heavy chains of the third protein functional region.

21. The pharmaceutical combination according to any one of claims 1 to 20, wherein: The first bispecific protein comprises: specifically binds to the first protein domain of PD-1, and The second protein domain that specifically binds to VEGFA; The number of the first protein functional regions is 2, and the number of the second protein functional region is 1; Wherein, the second protein functional region is IgG, and the first protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the IgG is shown in SEQ ID NO:42, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 35; Two single-chain antibodies are connected to the C-termini of the two heavy chains of IgG; The first protein functional region is connected to the second protein functional region via 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 via 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: 36 and SEQ ID NO: 37; Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO:

36.

22. The pharmaceutical combination according to any one of claims 1 to 21, wherein The second bispecific protein comprises: specifically binds to the third protein domain of TIGIT, and Specifically binds to the fourth protein functional domain of TGF-β; in, The third protein functional region is one, and the fourth protein functional region is two; The third protein functional region is IgG, and the fourth protein functional region is a truncated fragment of the TGF-β receptor extracellular region having TGF-β receptor function; The amino acid sequence of the heavy chain of the IgG is shown in SEQ ID NO:66 or SEQ ID NO:70, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO:68; The amino acid sequence of the fourth protein functional region is shown in SEQ ID NO:72 or SEQ ID NO:88; The fourth protein functional region is connected to the C-termini of the two heavy chains of IgG through a third linker; Preferably, the amino acid sequence of the third linker is as shown in SEQ ID NO:

83.

23. The pharmaceutical combination according to any one of claims 1 to 22, wherein: The first bispecific protein and the second bispecific protein are mixed together, and the drug combination is a pharmaceutical composition.

24. The pharmaceutical combination according to any one of claims 1 to 22, wherein: The first bispecific protein and the second bispecific protein are in separate packages, and the drug combination is a combination product.

25. The pharmaceutical combination according to any one of claims 1 to 24, wherein The mass ratio of the first bispecific protein to the second bispecific protein is (10:1) to (1:5), (5:1) to (1:3), (3:1) to (1:2) or 2:

1.

26. The pharmaceutical combination according to any one of claims 1 to 24, wherein The molar ratio of the first bispecific protein to the second bispecific protein is (10:1) to (1:10), (5:1) to (1:5), (3:1) to (1:3), (2:1) to (1:2) or 1:

1.

27. The pharmaceutical combination according to any one of claims 1 to 26, wherein Calculated according to the mass of the first bispecific protein or the second bispecific protein, the unit dose of the first bispecific protein and the second bispecific protein is independently 100 mg-2500 mg, 100 mg-2000 mg, 100 mg-1500 mg, 100 mg-1200 mg, 100 mg-1000 mg, 200 mg-800 mg, 200 mg-500 mg, 300 mg-600 mg, 400 mg-500 mg or 450 mg.

28. The pharmaceutical combination according to any one of claims 1 to 27, wherein The drug combination also contains an effective amount of tumor chemotherapy drugs, such as platinum drugs, alkylating agents, anti-metabolites, anti-tumor antibiotics, plant anticancer drugs, hormones or immune preparations.

29. The pharmaceutical combination according to any one of claims 1 to 28, wherein The pharmaceutical combination further comprises one or more pharmaceutically acceptable excipients; preferably, also comprises a product instruction sheet.

30. Use of the drug combination according to any one of claims 1 to 29 in the preparation of a drug for treating or preventing tumors; Preferably, the tumor is selected from one or more of colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, urothelial carcinoma, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, multiple myeloma, plasma cell cancer and pancreatic cancer; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the kidney tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer; Preferably, the urothelial cancer is bladder cancer; Preferably, the lymphoma is non-Hodgkin's lymphoma or B-lymphocytic neoplasm.

31. The pharmaceutical combination according to any one of claims 1 to 29, for use in treating or preventing tumors; Preferably, the tumor is selected from one or more of colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, urothelial carcinoma, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, multiple myeloma, plasma cell cancer and pancreatic cancer; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the kidney tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer; Preferably, the urothelial cancer is bladder cancer; Preferably, the lymphoma is non-Hodgkin's lymphoma or B-lymphocytic neoplasm.

32. A method for treating or preventing tumors, comprising the step of administering to a subject in need thereof an effective amount of the pharmaceutical combination of any one of claims 1 to 29; Preferably, the tumor is selected from one or more of colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, urothelial carcinoma, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, multiple myeloma, plasma cell cancer and pancreatic cancer; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the kidney tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer; Preferably, the urothelial cancer is bladder cancer; Preferably, the lymphoma is non-Hodgkin's lymphoma or B-lymphocytic neoplasm.

33. The method of claim 32, wherein: The first bispecific protein and the second bispecific protein are independently administered before or after surgery, and / or before or after radiotherapy.

34. The method according to any one of claims 32 to 33, wherein: The single administration dose of the first bispecific protein and the second bispecific protein is independently 0.1-100 mg, 5-50 mg or 5-15 mg per kilogram of body weight; The first bispecific protein and the second bispecific protein are independently administered once every half a day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks; and / or The administration method is intravenous drip, intravenous injection, subcutaneous injection or intraperitoneal injection.

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