Pharmaceutical combinations and uses

Abstract

The application belongs to the field of biological medicine, and particularly relates to a drug combination and use. Specifically, the drug combination comprises a bispecific protein specifically binding to PD-1 and VEGFA, and a bispecific protein specifically binding to TIGIT and TGF-beta R. The drug combination of the application can effectively treat or prevent tumors, and has a good application prospect.

Description

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to CN application No. 202311192415.9, filed on September 14, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention pertains to the field of biomedicine, specifically relating to drug combinations and their uses. More 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 may be in the form of a pharmaceutical composition or a pharmaceutical product. Background Technology

[0004] The transmembrane receptor PD-1 (programmed cell death 1) is a member of the CD28 gene family and is expressed in activated T cells, B cells, and myeloid cells. PD-1 ligands PDL1 (Programmed cell death 1 ligand 1, also known as PD-L1) and PDL2 (Programmed cell death 1 ligand 2, also known as PD-L2) both belong to the B7 superfamily. PDL1 is expressed in various cell types, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is expressed only in antigen-presenting cells such as dendritic cells and macrophages.

[0005] PD-1 / PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection and tumor immune escape. The expression of PD-1 is mainly in immune cells such as T cells, while the ligand of PD-1, PDL1, is mainly highly expressed in many human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway can activate the suppressed T cells, and then attack the cancer cells. Blocking the PD-1 / PDL1 signaling can promote the proliferation of tumor antigen-specific T cells, play a role in killing tumor cells, and then inhibit the growth of local tumor (Julie R et al., 2012, N Engl J Med. 366:2455-2465). In addition, tumors with high expression of PDL1 are accompanied by very difficult to detectable cancer (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA 104:3360-5). An effective method is to regulate the expression of PD-1 by injecting anti-PD-1 antibody in vivo. Due to the broad spectrum of anti-tumor prospects and amazing efficacy of PD-1 antibody, it is generally believed that the antibody against PD-1 pathway will bring breakthrough progress in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet M.B., Parisi G., et al., 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 can promote the division and proliferation of endothelial cells, promote the formation of new blood vessels, and increase vascular permeability. It binds to the vascular endothelial growth factor receptor on the cell surface and exerts its function by activating the tyrosine kinase signal transduction pathway. In tumor tissue, tumor cells, tumor-infiltrating macrophages and mast cells can secrete high levels of VEGF to stimulate tumor vascular endothelial cells in a paracrine manner, promote endothelial cell proliferation and migration, induce angiogenesis, promote tumor sustained growth, increase vascular permeability, cause fibrin deposition in the surrounding tissue, promote monocyte, fibroblast endothelial cell invasion, and facilitate tumor matrix formation and tumor cell entry into new blood vessels, thus promoting tumor metastasis. Therefore, inhibiting tumor angiogenesis is considered to be one of the most promising methods for tumor treatment. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD and PIGF. The vascular endothelial growth factor receptor (VEGFR) includes VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4) and Neuropilin-1 (NRP-1). Among them, the first three receptors are similar in structure and belong to the tyrosine kinase superfamily, and are composed of three parts of extracellular region, transmembrane fragment and intracellular region. The extracellular region is composed of immunoglobulin-like domains, and the intracellular region belongs to the tyrosine kinase region. VEGFR1 and VEGFR2 are mainly located on the surface of vascular endothelial cells, and VEGFR3 is mainly located on the surface of lymphatic endothelial cells.

[0007] VEGF family molecules have different affinities for several receptors. VEGFA mainly binds to VEGFR1, VEGFR2 and NRP-1 to exert its function. VEGFR1 is the earliest discovered receptor. Under normal physiological conditions, the affinity of VEGFR1 to VEGFA is higher than that of VEGFR2 to VEGFA, but its intracellular tyrosine kinase 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, and has a high tyrosine kinase activity compared with VEGFR1. VEGFR2 mediates the proliferation, differentiation and other behaviors of vascular endothelial cells, and the formation process and permeability of blood vessels after binding with ligand VEGFA (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 62(3) (2007): 179-213.), and mediates the transcription and expression of intracellular related protein genes through the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway after VEGFA binds with VEGFR2, and promotes the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene, 18(13) (1999): 2221-2230.).

[0009] VEGFR3 belongs to one of the members of the tyrosine kinase family, and is mainly expressed in the vascular endothelial cells of the embryonic period and the lymphatic endothelial cells of the adult period. VEGFC and VEGFD bind with VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote the neogenesis of lymphatic vessels; NRP-1 is a transmembrane protein that cannot independently transduce biological signals, but can mediate signal transduction after forming a complex with VEGF tyrosine kinase receptor. (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5) (2016): 146-148).

[0010] VEGFA and VEGFR2 are mainly involved in the regulation of angiogenesis, and before and after VEGFA binds with VEGFR2, a cascade reaction of numerous intermediate signals in the upstream and downstream pathways is triggered, eventually changing the physiological functions of endothelial cells in different forms such as proliferation, survival, migration, increased permeability and infiltration into surrounding tissues (Dong Hongchao et al., Modern Oncology Medicine, 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 is composed of an extracellular immunoglobulin variable (IgV) domain, a type 1 transmembrane domain, and an intracellular domain with a classic immunoreceptor tyrosine-based 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 as well as natural killer cells (NK cells) (Yu X, Harden K, Gonzalez L C, 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 for TIGIT binding (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. TIGIT binding to ligands CD155 and CD112 in NK cells can inhibit the killing effect of NK cells on CD155 and CD112 high expression cells (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 when PD-1 and TIGIT are blocked at the same time, the killing effect of CD8 + T cells (Johnston R J, 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, TIGIT can cause NK cell exhaustion during tumor development, and it was proved that anti-TIGIT monoclonal antibody 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 tumor, multiple myeloma, non-Hodgkin's lymphoma, B lymphoma, 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 group of functionally diverse cytokines, which is further divided into TGF-β, Activins, inhibins, growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), Nodal, Lefty and anti-Müllerian hormone (Table A) subfamilies. The proteins of the three TGF-β subfamilies are also known as TGF-β1, TGF-β2, TGF-β3, among which TGF-β1 is the highest expressed subtype. After binding to receptors, TGF-β1, TGF-β2, TGF-β3 mediate a series of biological responses 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 cancer suppression and promotion, etc. (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 cells and tumor cells in humans, including three receptor superfamily, type I classic TGF-β receptor family includes ALK1-7, among which TGF-βR I (also written as TβRI) is also called ALK5; type II classic TGF-β receptor family includes TGF-βRII (also written as TβRII), ActRII, ActRIIB, AMHRII and BMPRII; type III TGF-β receptor superfamily includes Betaglycan (also known as TGF-βRIII) and Endoglin; among them, TGF-βR I, TGF-βRII, TGF-βRIII can each bind to TGF-β1, TGF-β2 and TGF-β3 (Pawlak John B, Blobe Gerard C, TGF-β superfamily co-receptors in cancer. Dev Dyn, 2021). The TGF-β family and receptors are shown in Table A (based on a review summary of Carl-Henrik Heldin 1 and Aristidis Moustakas. Cold Spring Harb Perspect Biol. 2016). TGF-βRI and TGF-βR II are serine / threonine protein kinase receptors. In the TGF-β signaling pathway, TGF-βRII is a key molecule for signal transduction, which can bind to TGF-β with high affinity and then form a heterotetrameric receptor complex with TGF-βRI dimer, phosphorylate and activate TGF-βR I through TGF-βR II autophosphorylation, and activate the downstream Smad pathway-related protein, regulate the transcription and translation of downstream target genes, and then trigger disease-related biological responses. The affinity of TGF-βRIII for TGF-β is weaker than that of TGF-βR I and TGF-βR II, and it has no intracellular segment and cannot reach the downstream signaling pathway, and its function is to capture TGF-β and present it to TGF-βRII. Other TGF-β receptors can also bind to TGF-β (Xiao-Hong Sang et al., Research Progress of Small Molecule Inhibitors Targeting TGF-β and Receptors [J]. Acta Pharmaceutica Sinica, 2019 (9)).

[0015] Many tumor patients still cannot control the disease for a long time after receiving surgery or chemotherapy drug treatment, and the 5-year survival rate is still very low. In summary, it has great clinical significance to develop more toxic and more effective treatment methods and combined drug treatment regimens. SUMMARY

[0016] The present inventors, through in-depth research and creative labor, combine the bispecific protein specifically binding to PD-1 and VEGFA with the bispecific protein specifically binding to TIGIT and TGF-βR, and surprisingly find that the combination has a pharmacological effect of effectively inhibiting tumor growth, and the effect is better than that of the bispecific protein specifically binding to PD-1 and VEGFA or the bispecific protein specifically binding to TIGIT and TGF-βR. Thus the following invention is provided:

[0017] One aspect of the present application relates to a pharmaceutical combination comprising an effective amount of a first bispecific protein and an effective amount of a second bispecific protein, wherein:

[0018] (1) the first bispecific protein comprises:

[0019] a first protein functional region specifically binding to PD-1, and

[0020] a second protein functional region specifically binding to VEGFA;

[0021] wherein,

[0022] the first protein functional region comprises a heavy chain variable region VH1 and a light chain variable region VL1, the VH1 comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2 and HCDR3 shown in SEQ ID NO: 3; the VL1 comprises LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5 and LCDR3 shown in SEQ ID NO: 6;

[0023] the second protein functional region comprises a heavy chain variable region VH2 and a light chain variable region VL2, the VH2 comprises HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8 and HCDR3 shown in SEQ ID NO: 9; the VL2 comprises LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11 and LCDR3 shown in SEQ ID NO: 12;

[0024] (2) the second bispecific protein comprises:

[0025] a third protein functional region specifically binding to TIGIT, and

[0026] a fourth protein functional region specifically binding to TGF-β;

[0027] wherein,

[0028] the third protein functional region comprises a heavy chain variable region VH3 and a light chain variable region VL3, the VH3 comprises a HCDR1 as shown in SEQ ID NO: 13, a HCDR2 as shown in SEQ ID NO: 14 and a HCDR3 as shown in SEQ ID NO: 15; the VL3 comprises a LCDR1 as shown in SEQ ID NO: 16, a LCDR2 as shown in SEQ ID NO: 17 and a LCDR3 as shown in SEQ ID NO: 18;

[0029] 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 a TGF-β receptor function or a variant of a TGF-β receptor extracellular region having a TGF-β receptor function.

[0030] In the present application, the bispecific protein can also be referred to as a bispecific molecule. The first bispecific protein can also be referred to as a first bispecific molecule. The second bispecific protein can also be referred to as a second bispecific molecule.

[0031] In some embodiments of the present application, the bispecific protein is a bispecific antibody. In some embodiments of the present application, the first bispecific protein is a bispecific antibody; preferably, an anti-PD-1-anti-VEGFA bispecific antibody.

[0032] In some embodiments of the present application, the bispecific protein is an antibody-receptor fusion protein. In some embodiments of the present application, the second bispecific protein is an antibody-receptor fusion protein; preferably, an anti-TIGIT antibody-TGF-βR fusion protein.

[0033] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0034] the amino acid sequence of the heavy chain variable region VH1 is selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 24; and

[0035] the amino acid sequence of the light chain variable region VL1 is selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 26 and SEQ ID NO: 35.

[0036] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0037] the amino acid sequence of the heavy chain variable region VH1 is as shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region VL1 is as shown in SEQ ID NO: 22;

[0038] the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO: 26;

[0039] the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO: 35;

[0040] the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO: 22;

[0041] the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO: 26;

[0042] the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO: 35.

[0043] In some embodiments of the application, the pharmaceutical combination, wherein,

[0044] the amino acid sequence of the heavy chain variable region VH2 is set forth in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region VL2 is set forth in SEQ ID NO: 33.

[0045] In some embodiments of the application, the pharmaceutical combination, wherein,

[0046] the amino acid sequence of the heavy chain variable region VH3 is selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, and SEQ ID NO: 56; and

[0047] the amino acid sequence of the light chain variable region VL3 is selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64.

[0048] In some embodiments of the application, the pharmaceutical combination, wherein,

[0049] the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 48;

[0050] the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 58;

[0051] the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 64;

[0052] the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 60;

[0053] the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 62;

[0054] the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 54, and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 60;

[0055] the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 54, and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 62;

[0056] the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 56, and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 58; or

[0057] the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 56, and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 64.

[0058] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0059] 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.

[0060] The variant of the extracellular region of TGF-beta receptor with TGF-beta receptor function refers to a protein with TGF-beta receptor function after one or several (e.g. 2, 3, 4, 5 or more than 5) amino acid substitutions, deletions and / or insertions to the extracellular region of TGF-beta receptor.

[0061] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0062] The first protein functional region is IgG, and the second protein functional region is single chain antibody; or

[0063] The first protein functional region is single chain antibody, and the second protein functional region is IgG.

[0064] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0065] The first protein functional region is IgG1, and the second protein functional region is single chain antibody; or

[0066] The first protein functional region is single chain antibody, and the second protein functional region is IgG1.

[0067] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0068] The third protein functional region is IgG or single chain antibody.

[0069] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0070] The third protein functional region is IgG4 or single chain antibody.

[0071] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0072] The IgG is IgG1, IgG2, IgG3 or IgG4.

[0073] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0074] According to the EU numbering system, the heavy chain constant region of IgG1 has one of the following four combinations of mutations:

[0075] L234A and L235A;

[0076] L234A and G237A;

[0077] L235A and G237A; or

[0078] L234A, L235A, and G237A.

[0079] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0080] According to the EU numbering system, the heavy chain constant region of IgG4 has one of the following four combinations of mutations:

[0081] F234A and L235A;

[0082] F234A and G237A;

[0083] L235A and G237A; or

[0084] F234A, L235A, and G237A.

[0085] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0086] the second protein functional region is IgG, and the amino acid sequence of its heavy chain is as set forth in SEQ ID NO: 38, SEQ ID NO: 40, or SEQ ID NO: 42, and the amino acid sequence of its light chain is as set forth in SEQ ID NO: 44.

[0087] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0088] the third protein functional region is IgG, and the amino acid sequence of its heavy chain is as set forth in SEQ ID NO: 66 or SEQ ID NO: 70, and the amino acid sequence of its light chain is as set forth in SEQ ID NO: 68.

[0089] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0090] the amino acid sequence of the fourth protein functional region is as set forth in any one of SEQ ID NO: 72 to SEQ ID NO: 78 and SEQ ID NO: 88.

[0091] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0092] the first protein functional region is directly connected to the second protein functional region or is connected through a linker;

[0093] the third protein functional region is directly connected to the fourth protein functional region or is connected through a linker; and / or

[0094] the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or is connected through a linker.

[0095] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0096] the linker is a polypeptide as set forth in SEQ ID NO: 91 or SEQ ID NO: 92, or a polypeptide obtained by concatenating a plurality of (e.g., 2, 3, 4, 5, or 6) polypeptides as set forth in SEQ ID NO: 91, or a polypeptide obtained by concatenating a plurality of (e.g., 2, 3, 4, or 5) polypeptides as set forth in SEQ ID NO: 91 and further concatenating a polypeptide as set forth in SEQ ID NO: 92;

[0097] Preferably, the amino acid sequence of the linker is independently selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 83.

[0098] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0099] 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;

[0100] Preferably, the first protein functional region is 2, and the second protein functional region is 1.

[0101] Preferably, the third protein functional region is 1, and the fourth protein functional region is 2.

[0102] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0103] the second protein functional region is 1 IgG, 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.

[0104] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0105] the second protein functional region is 1 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.

[0106] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0107] the first protein functional region is 1 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.

[0108] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0109] The first protein functional region is one IgG1, the second protein functional region is two single-chain antibodies, and the single-chain antibodies are respectively connected at the C-terminus or N-terminus of the two heavy chains of the first protein functional region.

[0110] In one or more embodiments of the present application, the bispecific protein is a bispecific antibody, which is in the form of IgG-scFv, i.e., Morrison mode.

[0111] In one or more embodiments of the present application, the bispecific antibody, wherein the single-chain antibody is connected at the C-terminus of the heavy chain of the immunoglobulin. Since the immunoglobulin has two heavy chains, one immunoglobulin molecule is connected with two single-chain antibody molecules. Preferably, the two single-chain antibody molecules are the same. Preferably, the single-chain antibody is connected with the C-terminus of the heavy chain of the immunoglobulin by forming an amide bond through the aforementioned linker.

[0112] In one or more embodiments of the present application, the constant region of the immunoglobulin is humanized, for example, the heavy chain constant region is Ig gamma-1 chain C region, such as ACCESSION: P01857 or Ig gamma-4 chain C region, such as ACCESSION: P01861.1; and the light chain constant region is Ig kappa chain C region, such as ACCESSION: P01834.

[0113] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0114] The third protein functional region is one IgG, the fourth protein functional region is two, and the fourth protein functional region is respectively connected at the C-terminus or N-terminus of the two heavy chains of the third protein functional region.

[0115] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0116] The third protein functional region is one IgG4, the fourth protein functional region is two, and the fourth protein functional region is respectively connected at the C-terminus or N-terminus of the two heavy chains of the third protein functional region. In some embodiments of the present application, the pharmaceutical combination, wherein,

[0117] The first bispecific protein comprises:

[0118] a first protein functional region specifically binding to PD-1, and

[0119] a second protein functional region specifically binding to VEGFA;

[0120] the first protein functional region is 2, and the second protein functional region is 1;

[0121] the second protein functional region is IgG, and the first protein functional region is single-chain antibody;

[0122] the amino acid sequence of the heavy chain of the IgG is shown as SEQ ID NO: 42, and the amino acid sequence of the light chain thereof is shown as SEQ ID NO: 44;

[0123] the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO: 24, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO: 35;

[0124] two single-chain antibodies are respectively connected at the C-terminus of two heavy chains of IgG;

[0125] the first protein functional region is connected with the second protein functional region through a first linker; and the heavy chain variable region of the single-chain antibody is connected with the light chain variable region of the single-chain antibody through a second linker; the first linker and the second linker are the same or different;

[0126] 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.

[0127] Preferably, the amino acid sequences of the first linker and the second linker are both shown as SEQ ID NO: 36.

[0128] In some embodiments of the present application, the pharmaceutical combination, wherein,

[0129] the second bispecific protein comprises:

[0130] a third protein functional region specifically binding to TIGIT, and

[0131] a fourth protein functional region specifically binding to TGF-β;

[0132] wherein,

[0133] the third protein functional region is 1, and the fourth protein functional region is 2;

[0134] the third protein functional region is IgG, and the fourth protein functional region is a truncated fragment of the extracellular region of TGF-β receptor having the function of TGF-β receptor;

[0135] the amino acid sequence of the heavy chain of the IgG is shown as SEQ ID NO: 66 or SEQ ID NO: 70, and the amino acid sequence of the light chain thereof is shown as SEQ ID NO: 68;

[0136] the amino acid sequence of the fourth protein functional region is shown as SEQ ID NO: 72 or SEQ ID NO: 88;

[0137] the fourth protein functional region is connected to the C-terminal of the two heavy chains of the IgG through a third linker;

[0138] Preferably, the amino acid sequence of the third linker is shown as SEQ ID NO: 83.

[0139] In some embodiments of the present application, the pharmaceutical 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 application, and the second bispecific protein is selected from the anti-TIGIT antibodies and the fusion proteins TF01, TF02 and TF01T of TGF-βR of the present application.

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

[0141] In some embodiments of the present application, the pharmaceutical combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (G4M) of the present application, and the second bispecific protein is selected from the anti-TIGIT antibodies and the fusion proteins TF01, TF02 and TF01T of TGF-βR of the present application.

[0142] In some embodiments of the present application, the pharmaceutical combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) of the present application, and the second bispecific protein is selected from the anti-TIGIT antibodies and the fusion proteins TF01, TF02 and TF01T of TGF-βR of the present application.

[0143] In some embodiments of the present application, the pharmaceutical combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) of the present application, and the second bispecific protein is the anti-TIGIT antibodies and the fusion proteins TF01T of TGF-βR of the present application.

[0144] In some embodiments of the present application, the pharmaceutical combination, wherein the first bispecific protein and the second bispecific protein are mixed together, is a pharmaceutical composition.

[0145] In some embodiments of the present application, the pharmaceutical combination, wherein the first bispecific protein and the second bispecific protein are in separate packages, is a combination product.

[0146] In some embodiments of the present application, the pharmaceutical 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), more preferably 2:1.

[0147] In some embodiments of the present application, the pharmaceutical combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) of the present application, the second bispecific protein is the fusion protein TF01T of the anti-TIGIT antibody and TGF-βR of the present application, 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), more preferably 2:1.

[0148] In some embodiments of the present application, the pharmaceutical combination, wherein the unit dose of the first bispecific protein and the second bispecific protein independently is 100mg-2500mg, 100mg-2000mg, 100mg-1500mg, 100mg-1200mg, 100mg-1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg or 450mg, calculated by the mass of the first bispecific protein or the second bispecific protein.

[0149] In some embodiments of the present application, the pharmaceutical combination, wherein the pharmaceutical combination further comprises an effective amount of a tumor chemotherapy drug, such as a platinum drug, an alkylating agent, an antimetabolite, an antitumor antibiotic, a plant anticancer drug, a hormone or an immunological agent.

[0150] In some embodiments of the present application, the pharmaceutical combination, wherein the pharmaceutical combination further comprises one or more pharmaceutically acceptable adjuvants; preferably, further comprises a product instruction.

[0151] In some embodiments of the present application, the pharmaceutical 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), more preferably 1:1.

[0152] In some embodiments of the present application, the pharmaceutical combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) of the present application, the second bispecific protein is the fusion protein TF01T of the anti-TIGIT antibody and TGF-βR of the present application, 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), more preferably 1:1.

[0153] In some embodiments of the present application, the first bispecific protein and the second bispecific protein are the active ingredients (APIs).

[0154] In some embodiments of the present application, the pharmaceutical combination consists of the first bispecific protein, the second bispecific protein, and the pharmaceutically acceptable excipient.

[0155] In some embodiments of the present application, the first bispecific protein, the second bispecific protein, and the tumor chemotherapy drug are the active ingredients (APIs).

[0156] In some embodiments of the present application, the pharmaceutical combination consists of the first bispecific protein, the second bispecific protein, the tumor chemotherapy drug, and the pharmaceutically acceptable excipient.

[0157] The pharmaceutical combination according to any one of the present application for use in the treatment or prevention of a tumor;

[0158] 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, renal 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 cancer, and pancreatic cancer.

[0159] Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0160] Preferably, the liver cancer is hepatocellular carcinoma.

[0161] Preferably, the renal tumor is renal cell carcinoma.

[0162] Preferably, the breast cancer is triple negative breast cancer.

[0163] Preferably, the urothelial cancer is bladder cancer.

[0164] Preferably, the lymphoma is non-Hodgkin lymphoma or B-cell lymphoma.

[0165] In another aspect of the application, a unit formulation, preferably for the treatment of a tumor, is provided, wherein the unit formulation comprises 1-10000 mg (preferably 1-1000 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 one of the aspects of the application and 1-10000 mg (preferably 10-1000 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 one of the aspects of the application, and optionally one or more chemotherapeutics according to the application (such as a platinum drug and / or a fluorouracil-based antineoplastic drug); wherein the first bispecific protein, the second bispecific protein and the chemotherapeutics are each separately packaged.

[0166] The present application relates to a method for the prevention or treatment of a cancer or a tumor, wherein one or more unit formulations according to the application are administered to a subject in need thereof, preferably the first bispecific protein, the second bispecific protein and the chemotherapeutics in the unit formulation are each administered separately.

[0167] In another aspect of the application, a single drug dosage unit, preferably for the treatment of a tumor, is provided, comprising 0.1-10000 mg (preferably 1-1000 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 one of the aspects of the application and 0.1-10000 mg (preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of the second bispecific protein according to any one of the aspects of the application.

[0168] In one or more embodiments of the application, wherein the first bispecific protein, the second bispecific protein and / or the chemotherapeutics are in a form suitable for intravenous injection or intravenous infusion, preferably in the form of a liquid formulation.

[0169] In one or more embodiments of the present application, wherein the single administration dose of the first bispecific protein of any one of the present application and / or the second bispecific protein of any one of the present application is 0.1-100 mg per kg of body weight, preferably 1-10 mg per kg of body weight; or the single administration dose of the first bispecific protein of any one of the present application and / or the second bispecific protein of any one of the present application is 10-1000 mg per subject, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg per subject,

[0170] Preferably, the administration is twice a day to about once 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.

[0171] Preferably, the administration is intravenous drip, intravenous injection, subcutaneous injection or intraperitoneal injection.

[0172] Another aspect of the present application relates to the use of the pharmaceutical combination of any one of the present application in the manufacture of a medicament for treating or preventing a tumor;

[0173] 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, renal 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.

[0174] Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0175] Preferably, the liver cancer is hepatocellular carcinoma.

[0176] Preferably, the renal tumor is renal cell carcinoma.

[0177] Preferably, the breast cancer is triple-negative breast cancer.

[0178] Preferably, the urothelial cancer is bladder cancer.

[0179] Preferably, the lymphoma is non-Hodgkin lymphoma or B-cell lymphoma.

[0180] Still another aspect of the present application relates to a method of treating or preventing a tumor, comprising the step of administering to a subject in need thereof an effective amount of the pharmaceutical combination of any one of the present application;

[0181] 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, renal 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.

[0182] Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0183] Preferably, the liver cancer is hepatocellular carcinoma.

[0184] Preferably, the renal tumor is renal cell carcinoma.

[0185] Preferably, the breast cancer is triple-negative breast cancer.

[0186] Preferably, the urothelial cancer is bladder cancer.

[0187] Preferably, the lymphoma is non-Hodgkin lymphoma or B-cell lymphoma.

[0188] In some embodiments of the present application, the method for treating or preventing tumor, wherein the first bispecific protein and the second bispecific protein are independently administered before or after surgery, and / or before or after radiotherapy.

[0189] In some embodiments of the present application, the method for treating or preventing tumor, wherein,

[0190] The single administration dose of the first bispecific protein and the second bispecific protein is independently 0.1-100 mg per kg of body weight, preferably 5-50 mg or 5-15 mg per kg of body weight.

[0191] The first bispecific protein and the second bispecific protein are independently administered once every half day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks.

[0192] And / or

[0193] The administration mode is intravenous drip, intravenous injection, subcutaneous injection, or intraperitoneal injection.

[0194] Antibody therapeutics, especially monoclonal antibodies (mAB), have achieved good effects in the treatment of various diseases. The traditional experimental method for obtaining these therapeutic antibodies is to immunize animals with antigens, and to obtain antibodies targeting antigens in the immunized animals, or to improve the affinity of those antibodies with low affinity to antigens by affinity maturation.

[0195] The variable regions of light and heavy chains determine antigen binding; the variable regions of each chain contain three regions of hypervariability, termed complementarity determining regions (CDRs) (CDRs of heavy chain (H) include HCDR1, HCDR2, HCDR3, CDRs of light chain (L) include LCDR1, LCDR2, LCDR3; which are designated by Rabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Vol. 1-3, NIH Publication 91-3242, Bethesda Md,

[0196] Preferably, the CDRs can also be defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc.“IMGT / 3Dstructure-DB and IMGT / Domain Gap Align: 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.

[0197] The amino acid sequences of the CDR regions of the monoclonal antibodies of items (1) to (5) below were analyzed by means of techniques well known to those skilled in the art, and the results were as follows:

[0198] (1) 14C12

[0199] The amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO: 20, and the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO: 22.

[0200] The amino acid sequences of the 3 CDR regions of the variable region of the heavy chain are as follows:

[0201] HCDR1: GFAFSSYD (SEQ ID NO: 1)

[0202] HCDR2: ISGGGRYT (SEQ ID NO: 2)

[0203] HCDR3: ANRYGEAWFAY (SEQ ID NO: 3)

[0204] The amino acid sequences of the 3 CDR regions of the variable region of the light chain are as follows:

[0205] LCDR1: QDINTY (SEQ ID NO:4)

[0206] LCDR2: RAN (SEQ ID NO:5)

[0207] LCDR3: LQYDEFPLT (SEQ ID NO:6)

[0208] (2) 14C12H1L1

[0209] The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 26.

[0210] The amino acid sequences of the 3 CDR regions of the heavy chain variable region are identical to 14C12.

[0211] The amino acid sequences of the 3 CDR regions of the light chain variable region are identical to 14C12.

[0212] (3) 14C12H1L1(M)

[0213] The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 35.

[0214] The amino acid sequences of the 3 CDR regions of the heavy chain variable region are identical to 14C12.

[0215] The amino acid sequences of the 3 CDR regions of the light chain variable region are identical to 14C12.

[0216] (4) Bevacizumab

[0217] The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 33;

[0218] The amino acid sequences of the 3 CDR regions of the heavy chain variable region are as follows:

[0219] HCDR1: GYTFTNYG (SEQ ID NO: 7)

[0220] HCDR2: INTYTGEP (SEQ ID NO: 8)

[0221] HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 9)

[0222] The amino acid sequences of the 3 CDR regions of the heavy chain variable region are as follows:

[0223] LCDR1: QDISNY (SEQ ID NO: 10)

[0224] LCDR2: FTS (SEQ ID NO: 11)

[0225] LCDR3: QQYSTVPWT (SEQ ID NO: 12)

[0226] (5) 26B12

[0227] The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 46, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 48.

[0228] The amino acid sequences of the 3 CDR regions of the heavy chain variable region thereof are as follows:

[0229] HCDR1: GHSFTSDYA (SEQ ID NO: 13)

[0230] HCDR2: ISYSDST (SEQ ID NO: 14)

[0231] HCDR3: ARLDYGNYGGAMDY (SEQ ID NO: 15)

[0232] The amino acid sequences of the 3 CDR regions of the light chain variable region thereof are as follows:

[0233] LCDR1: QHVSTA (SEQ ID NO: 16)

[0234] LCDR2: SAS (SEQ ID NO: 17)

[0235] LCDR3: QQHYITPWT (SEQ ID NO: 18)

[0236] The 3 HCDRs and 3 LCDRs of the humanized TIGIT mAb are the same as those of the murine TIGIT mAb.

[0237] In the present application, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art, unless otherwise specified. Also, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory operation steps used herein are the conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of the related terms are provided as follows.

[0238] As used herein, when referring to the amino acid sequence of PD-1 protein (NCBI GenBank: NP_005009.2), it includes the full length of PD-1 protein, or the extracellular fragment of PD-1, PD-1 ECD, or a fragment comprising PD-1 ECD; also includes fusion proteins of PD-1 ECD, such as fragments fused with Fc protein fragment of mouse or human IgG (mFc or hFc). However, one skilled in the art understands that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced in the amino acid sequence of PD-1 protein without affecting its biological function. Therefore, in the present application, the term "PD-1 protein" or "PD-1" shall include all such sequences, including natural or artificial variants thereof. Also, when describing sequence fragments of PD-1 protein, it also includes the corresponding sequence fragments in natural or artificial variants thereof.

[0239] 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, also includes fusion proteins of VEGFA, such as fragments fused with Fc protein fragment of mouse or human IgG (mFc or hFc). However, one skilled in the art understands that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced in the amino acid sequence of VEGFA protein without affecting its biological function. Therefore, in the present application, the term "VEGFA protein" or "VEGFA" shall include all such sequences, including natural or artificial variants thereof. Also, when describing sequence fragments of VEGFA protein, it also includes the corresponding sequence fragments in natural or artificial variants thereof.

[0240] 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 VEGFR2 protein, or the extracellular fragment of VEGFR2, VEGFR2-ECD, or a fragment comprising VEGFR2-ECD; also includes fusion proteins of VEGFR2-ECD, such as fragments fused with Fc protein fragment of mouse or human IgG (mFc or hFc). However, one skilled in the art understands that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced in the amino acid sequence of VEGFR2 protein without affecting its biological function. Therefore, in the present application, the term "VEGFR2 protein" or "VEGFR2" shall include all such sequences, including natural or artificial variants thereof. Also, when describing sequence fragments of VEGFR2 protein, it also includes the corresponding sequence fragments in natural or artificial variants thereof.

[0241] As used herein, the VEGFR is VEGFR1 and / or VEGFR2 if not otherwise specified; the specific protein sequence thereof is known in the art, and can refer to the sequence disclosed in the prior art or GenBank. For example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321); VEGFR2 (VEGFR2, NCBI Gene ID: 3791).

[0242] As used herein, when referring to the amino acid sequence of TIGIT (NCBI GenBank ID: NP_776160.2), it includes the full length of TIGIT protein, or the extracellular immunoglobulin variable region (IgV) domain or a fragment comprising the extracellular immunoglobulin variable region (IgV) domain; also includes the fusion protein of TIGIT, such as the fragment fused with the Fc protein fragment of mouse or human IgG (mFc or hFc). However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced in the amino acid sequence of TIGIT protein without affecting its biological function. Therefore, in the present application, the term "TIGIT protein" or "TIGIT" should include all such sequences, including the sequence shown and its natural or artificial variants. And when describing the sequence fragment of TIGIT protein, it not only includes the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variants.

[0243] As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can cause 50% of the maximum effect.

[0244] As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains (each pair has one "light" (L) chain and one "heavy" (H) chain). In general terms, the heavy chain can be understood as the polypeptide chain with larger molecular weight in the antibody, and the light chain refers to the polypeptide chain with smaller molecular weight in the antibody. The light chain can be classified as kappa and lambda light chain. The heavy chain can be generally classified as mu, delta, gamma, alpha or epsilon, and defines the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light chain and the heavy chain, the variable region and the constant region are connected by a J region of about 12 or more amino acids, and the heavy chain further contains a D region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region is composed of three domains (C H1 , C H2 and C H3It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L Each region or domain forms an antibody binding site. The allocation of amino acids to each region or domain follows the definitions 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 of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0245] Antigen-binding fragments of an antibody (e.g., the antibody fragments 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 fragmentation methods), and the antigen-binding fragments of the antibody can be screened for specificity in the same manner as those used for intact antibodies.

[0246] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0247] As used herein, the terms "monoclonal antibody" and "monoclonal" refer to one antibody or a fragment of an antibody from a population of highly homogenous antibody molecules, i.e., a population of identical antibody molecules except for possible naturally-occurring mutations that can arise during production. A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies are in contrast to monoclonal antibodies and generally comprise at least two or more different antibodies, which typically recognize different epitopes on an antigen. Monoclonal antibodies can typically be obtained using the hybridoma technology first described by Kohler et al. (Kohler et al., Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, e.g., U.S. Patent 4,816,567).

[0248] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment in which all or a portion of the CDR regions of a human immunoglobulin (recipient antibody) are replaced by CDR regions of a non-human antibody (donor antibody), where the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. Additionally, some of the amino acid residues in the framework regions (FRs) of the recipient antibody can be replaced by corresponding amino acid residues of the non-human antibody, or by amino acid residues of other antibodies, to further refine or optimize antibody performance. For further details of humanized antibodies, see, e.g., 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).

[0249] As used herein, the term "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. An "epitope" is also referred to in the art as an "antigenic determinant." An epitope or antigenic determinant typically consists of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and typically has specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, an epitope often comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be "linear" or "conformational." See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). In a linear epitope, all of the points of interaction between the protein and the interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction exist across amino acid residues of the protein that are separated from one another.

[0250] As used herein, the term "isolated" or "isolation" refers to a substance or component obtained by artificial means from its natural state. If a "isolated" substance or component occurs in nature, it can be that the natural environment of the substance has been changed, or the substance has been separated from the natural environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living organism is not isolated, but a highly pure version of the same polynucleotide or polypeptide that is separated from its natural state is isolated. The term "isolated" or "isolation" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0251] As used herein, the term "E. coli expression system" refers to an expression system consisting of an E. coli (strain) and a vector, wherein the E. coli (strain) is derived from a commercially available strain, such as, but not limited to: GI698, ER2566, BL21(DE3), B834(DE3), BLR(DE3).

[0252] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and directs the expression of elements carried in the genetic material of the vector 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 (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0253] 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 E. 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 fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0254] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or has specificity for) an antigen refers to an antibody that binds to the antigen with an affinity 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. In some embodiments of the application, the term "targeting" refers to specific binding. D ) to the antigen. In some embodiments of the application, the term "targeting" refers to specific binding.

[0255] As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Typically, an antibody binds to an antigen with an affinity of less than about 10 -5M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or smaller dissociation equilibrium constant (K D binds to an antigen, for example, as measured using surface plasmon resonance (SPR) in a BIACORE instrument, or by Fortebio Octet Molecular Interaction instrument.

[0256] As used herein, the terms "monoclonal antibody" and "monoclonal" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by their commonly known single and three letter codes. For example, alanine can be represented by A or Ala.

[0257] As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is compatible, in pharmacological and / or physiological terms, with the subject and 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 adjusting agents, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0258] As used herein, the term "adjuvant" refers to a non-specific immune enhancer that, when delivered into the body together with or prior to an antigen, can enhance the immune response of the body to the antigen or change the type of immune response. There are many kinds 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 parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments at present. Aluminum hydroxide adjuvant is more commonly used in clinical experiments.

[0259] As used herein, the term "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactically effective amount refers to an amount that is sufficient to prevent, deter, or delay the onset of a disease; a therapeutically effective amount refers to an amount that is sufficient to cure or at least partially arrest the disease and its complications in an already afflicted patient. Determining such effective amounts is well within the capability of those skilled in the art. For example, an amount effective for therapeutic uses will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., the age, weight, and sex of the patient, the mode of administration of the drug, and other therapeutics then being administered to the patient.

[0260] A "recurrent" cancer is a cancer that regrows, either at the original site or at a distant site, after an initial treatment, e.g., surgery. A "locally recurrent" cancer is a cancer that occurs at the same location as the previously treated cancer after treatment.

[0261] A "metastatic" cancer refers to a cancer that spreads from one part of the body, e.g., the lung, to another part of the body.

[0262] As used herein, the term "completely eliminate" means that no binding signal or very low binding signal is detected by the existing instrument equipment, e.g., Fortebio Octet Molecular Interaction Instrument. In one embodiment of the present application, no binding signal or very low binding signal means that the binding signal (i.e., Response value or Response) is lower than 0.1 nm.

[0263] In the present application, if not otherwise specified, the "first" (e.g., first bispecific antibody, first protein functional region, first linker, etc.), "second" (e.g., second bispecific antibody, second protein functional region, second linker, etc.), "third" (e.g., third protein functional region, third linker, etc.), and "fourth" (e.g., fourth protein functional region, fourth linker, etc.) are for the purpose of distinction in reference or clarity in expression, and do not have the typical meaning of order.

[0264] In the present application, if not otherwise specified, "about" or "approximately" means floating up and down within the range of 10%, 20%, or 30% of the modified numerical value or physical quantity, e.g., about 100 minutes or approximately 100 minutes can be 90 minutes-110 minutes, 80 minutes-120 minutes, or 70 minutes-130 minutes.

[0265] The entire contents of the prior application Chinese Patent Publication CN112830972A are hereby incorporated by reference into the present application.

[0266] Advantages of the Invention

[0267] The present application achieves one or more of the technical effects described in items (1) to (2) below:

[0268] (1) The pharmaceutical combination of the present application can effectively prevent or treat tumors.

[0269] (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 DRAWINGS

[0270] Figure 1 : Anti-TIGIT antibody and TGF-βR fusion protein combined with anti-PD-1-anti-VEGFA bispecific antibody promote INF-γ secretion in PBMC+CMV system.

[0271] Figure 2 : Anti-TIGIT antibody and TGF-βR fusion protein combined with anti-PD-1-anti-VEGFA bispecific antibody promote INF-γ secretion in PBMC+CMV system (+TGF-β1).

[0272] Figure 3 : Reporter gene system verifies the blocking activity of anti-TIGIT antibody and TGF-βR fusion protein combined with anti-PD-1-anti-VEGFA bispecific antibody.

[0273] Figure 4 : Reporter gene system (+VEGF) verifies the blocking activity of anti-TIGIT antibody and TGF-βR fusion protein combined with anti-PD-1-anti-VEGFA bispecific antibody.

[0274] Some sequences related to the present application are as follows:

[0275] 19. Nucleic acid sequence encoding 14C12 heavy chain variable region: (354bp)

[0276]

[0277]

[0278] 20. Amino acid sequence of 14C12 heavy chain variable region: (118aa)

[0279]

[0280] 21. Nucleic acid sequence encoding 14C12 light chain variable region: (321bp)

[0281]

[0282] 22. Amino acid sequence of 14C12 H1 L1 heavy chain variable region (107 aa)

[0283]

[0284] 23. Nucleic acid sequence encoding 14C12 H1 L1 heavy chain variable region (354 bp)

[0285]

[0286]

[0287] 24. Amino acid sequence of 14C12 H1 L1 heavy chain variable region (118 aa) V

[0288]

[0289] 25. Nucleic acid sequence encoding 14C12 H1 L1 light chain variable region (321 bp)

[0290]

[0291] 26. Amino acid sequence of 14C12 H1 L1 light chain variable region (107 aa)

[0292]

[0293] 27. Nucleic acid sequence encoding 14C12 H1 L1 heavy chain (14C12 H1) (1344 bp)

[0294]

[0295]

[0296] 28. Amino acid sequence of 14C12 H1 L1 heavy chain (14C12 H1) (448 aa)

[0297]

[0298] 29. Nucleic acid sequence encoding 14C12 H1 L1 light chain (14C12 L1) (642 bp)

[0299]

[0300]

[0301] ​30. Amino acid sequence of 14C12H1L1 light chain (14C12L1): (214 aa)

[0302]

[0303] 31. Amino acid sequence of Bevacizumab heavy chain variable region (Bevacizumab-Hv): (123 aa)

[0304]

[0305] 32. Nucleic acid sequence encoding Bevacizumab heavy chain variable region (Bevacizumab-Hv): (369 bp)

[0306]

[0307] 33. Amino acid sequence of Bevacizumab light chain variable region (Bevacizumab-Lv): (107 aa)

[0308]

[0309] 34. Nucleic acid sequence encoding Bevacizumab light chain variable region (Bevacizumab-Lv): (321 bp)

[0310]

[0311]

[0312] 35. Light chain variable region 14C12L1(M) of 14C12H1L1(M) V : (108 aa, amino acid sequence mutation sites made based on 14C12H1L1 are underlined)

[0313]

[0314] 36. Linker

[0315]

[0316] 37. Linker

[0317]

[0318] 38. Amino acid sequence of heavy chain of immunoglobulin portion in VP101 (hGlWT): (453 aa)

[0319]

[0320] 39. Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion in VP101 (hGl WT): (1359 bp)

[0321]

[0322]

[0323] 40. Amino acid sequence of the heavy chain of the immunoglobulin portion in VP101 (hG4 WT): (450 aa)

[0324]

[0325] 41. Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion in VP101 (hG4 WT): (1350 bp)

[0326]

[0327] 42. Amino acid sequence of the heavy chain of the immunoglobulin portion in VP101 (hGl DM): (453 aa, mutation sites underlined)

[0328]

[0329]

[0330] 43. Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion in VP101 (hGl DM): (1359 bp, mutation sites underlined)

[0331]

[0332] 44. Amino acid sequence of the light chain of the immunoglobulin portion in VP101 (hGl DM): (214 aa)

[0333]

[0334] 45. Nucleic acid sequence encoding the light chain of the immunoglobulin portion in VP101 (hGl DM): (642 bp)

[0335]

[0336] 46. Amino acid sequence of 26B12 VH

[0337]

[0338] 47. Nucleic acid sequence of 26B12 VH

[0339]

[0340]

[0341] 48. Amino acid sequence of 26B12 VL

[0342]

[0343] 49. Nucleic acid sequence of 26B12 VL

[0344]

[0345] 50. Amino acid sequence of 26B12 H1 VH

[0346]

[0347] 51. Nucleic acid sequence of 26B12 H1 VH

[0348]

[0349] 52. Amino acid sequence of 26B12 H2 VH

[0350]

[0351]

[0352] 53. Nucleic acid sequence of 26B12 H2 VH

[0353]

[0354] 54. Amino acid sequence of 26B12 H3 VH

[0355]

[0356] 55. Nucleic acid sequence of 26B12 H3 VH

[0357]

[0358] 56. Amino acid sequence of 26B12 H4 VH

[0359]

[0360] 57. Nucleic acid sequence of 26B12 H4 VH

[0361]

[0362]

[0363] 58. Amino acid sequence of 26B12 L1 VL

[0364]

[0365] 59. Amino acid sequence of 26B12 L1 VL

[0366]

[0367] 60. Amino acid sequence of 26B12 L2 VL

[0368]

[0369] 61. Nucleic acid sequence of 26B12 L2 VL

[0370]

[0371] 62. Amino acid sequence of 26B12 L3 VL

[0372]

[0373] 63. Nucleic acid sequence of 26B12 L3 VL

[0374]

[0375] 64. Amino acid sequence of 26B12 L4 VL

[0376]

[0377] 65. Nucleic acid sequence of 26B12 L4 VL

[0378]

[0379] 66. Amino acid sequence of 26B12 H2 L2 (hG4DM) heavy chain

[0380]

[0381]

[0382] 67. Nucleic acid sequence of 26B12 H2 L2 (hG4DM) heavy chain

[0383]

[0384] 68. Amino acid sequence of 26B12 H2 L2 (hG4DM) light chain

[0385]

[0386] 69. Nucleic acid sequence of 26B12 H2 L2 (hG4DM) light chain

[0387]

[0388] 70. 26B12 H2L2 (hGlDM) heavy chain amino acid sequence

[0389]

[0390] 71. 26B12 H2L2 (hGlDM) heavy chain nucleic acid sequence

[0391]

[0392]

[0393] 72. Amino acid sequence of TGF-βRII ectodomain fragment (truncated):

[0394]

[0395] 73. Amino acid sequence of TGF-βRI:

[0396]

[0397]

[0398] 74. Amino acid sequence of TGF-βRI ectodomain:

[0399]

[0400] 75. Amino acid sequence of TGF-βRII:

[0401]

[0402] 76. Amino acid sequence of TGF-βRII ectodomain:

[0403]

[0404] 77. Amino acid sequence of TGF-βRIII:

[0405]

[0406]

[0407] 78. Amino acid sequence of TGF-βRIII ectodomain:

[0408]

[0409] 79. Amino acid sequence of Tiragolumab heavy chain:

[0410]

[0411]

[0412] 80. Amino acid sequence of the light chain of tiragolumab:

[0413]

[0414] 81. Amino acid sequence of the heavy chain variable region of RG6058 (hG4):

[0415]

[0416] 82. Amino acid sequence of the light chain variable region of RG6058 (hG4):

[0417]

[0418] 83. Amino acid sequence of the linker:

[0419]

[0420] 84. Amino acid sequence of the heavy chain of the anti-HEL & TGFβ antibody

[0421]

[0422]

[0423] 85. Amino acid sequence of the light chain of the anti-HEL & TGFβ antibody

[0424]

[0425] 86. Amino acid sequence of the heavy chain of RG6058 (G1DM)

[0426]

[0427] 87. Amino acid sequence of the light chain of RG6058 (G1DM)

[0428]

[0429] 88. Amino acid sequence of the TGF-βRII extracellular domain fragment variant of TF01T:

[0430]

[0431] 89. Amino acid sequence of the hlgG4 WT heavy chain constant region

[0432]

[0433] 90. hlgGl WT heavy chain constant region amino acid sequence

[0434]

[0435] 91. Repeat units in linkers

[0436]

[0437] 92. Fragments in linkers

[0438] DETAILED DESCRIPTION

[0439] Embodiments of the present application will be described in detail with reference to the drawings, of which the following examples are provided by way of illustration only. As persons skilled in the art will appreciate, the following examples are provided for the purpose of illustration only and should not be construed in any way to limit the scope of the present application. Unless otherwise indicated, conventional methods or those modifications thereof known to those skilled in the art were employed in the examples. Unless otherwise indicated, the reagents or instruments used in the examples were commercially available and were used according to the manufacturer’s instructions.

[0440] The isotype control antibody is human anti-Hen Egg Lysozyme IgG (anti-HEL antibody, or human IgG, hlgG for short). The sequence of the variable region of the Fab F10.6.6 sequence is from Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1): 130-146.) Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies. The hlgGl DM used in the examples is an isotype control antibody with hG1 DM constant region sequence of anti-HEL, which is prepared in the laboratory of Zhongshan Kangfang Biomedicals Co., Ltd. Batch number: 20190520.

[0441] Jurkat-NFAT-PD1-hTIGIT is a cell line expressing human TIGIT based on Jurkat-NFAT-PD1 cells (PD1 Effector cells, Promega, Cat No: J112A) constructed by transfection in Zhongshan Kangfang Biomedicals Co., Ltd.

[0442] Preparation Example 1: Sequence design of antibody 14C12 against PD-1 and humanized antibody 14C12H1L1 thereof

[0443] The amino acid sequences of the heavy chain and the light chain of the antibody 14C12 against PD-1 and the humanized antibody 14C12H1L1 thereof, and the encoding nucleic acid sequences are the same as those of 14C12, 14C12H1L1 in Chinese Patent Publication CN112830972A respectively.

[0444] (1) The heavy chain variable region sequence and the light chain variable region sequence of 14C12

[0445] The nucleic acid sequence encoding the heavy chain variable region of 14C12: SEQ ID NO: 19;

[0446] The amino acid sequence of the heavy chain variable region of 14C12: SEQ ID NO: 20;

[0447] The nucleic acid sequence encoding the light chain variable region of 14C12: SEQ ID NO: 21;

[0448] The amino acid sequence of the light chain variable region of 14C12: SEQ ID NO: 22.

[0449] (2) The heavy chain variable region sequence and the light chain variable region sequence, the heavy chain sequence and the light chain sequence of the humanized monoclonal antibody 14C12H1L1

[0450] The nucleic acid sequence encoding the heavy chain variable region of 14C12H1L1: SEQ ID NO: 23;

[0451] The amino acid sequence of the heavy chain variable region of 14C12H1L1: SEQ ID NO: 24;

[0452] The nucleic acid sequence encoding the light chain variable region of 14C12H1L1: SEQ ID NO: 25;

[0453] The amino acid sequence of the light chain variable region of 14C12H1L1: SEQ ID NO: 26;

[0454] The nucleic acid sequence encoding the heavy chain (14C12H1) of 14C12H1L1: SEQ ID NO: 27;

[0455] The amino acid sequence of the heavy chain (14C12H1) of 14C12H1L1: SEQ ID NO: 28;

[0456] The nucleic acid sequence encoding the light chain (14C12L1) of 14C12H1L1: SEQ ID NO: 29;

[0457] The amino acid sequence of the light chain (14C12L1) of 14C12H1L1: SEQ ID NO: 30.

[0458] Preparation Example 2: Preparation of antibody Bevacizumab against VEGFA

[0459] The amino acid sequences of the heavy chain variable region and the light chain variable region of the marketed anti-VEGFA monoclonal antibody Avastin (Bevacizumab) are referred to the Chinese patent publication CN1259962A. The nucleic acid sequences encoding the heavy chain variable region and the light chain variable region are entrusted to the company Jin Sui to synthesize.

[0460] The amino acid sequence of the heavy chain variable region of Bevacizumab (Bevacizumab-Hv) is: SEQ ID NO: 31.

[0461] The nucleic acid sequence encoding the heavy chain variable region of Bevacizumab (Bevacizumab-Hv) is: SEQ ID NO: 32.

[0462] The amino acid sequence of the light chain variable region of Bevacizumab (Bevacizumab-Lv) is: SEQ ID NO: 33.

[0463] The nucleic acid sequence encoding the light chain variable region of Bevacizumab (Bevacizumab-Lv) is: SEQ ID NO: 34.

[0464] The heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857; and the light chain constant region is Ig kappa chain C region, ACCESSION: P01834.

[0465] The heavy chain cDNA and the light chain cDNA of Bevacizumab are respectively cloned into the pcDNA3.1 vector to obtain the recombinant expression plasmid of the antibody Bevacizumab. The recombinant plasmid is transfected into 293F cells. After the 293F cell culture solution is purified, detection is performed.

[0466] The anti-VEGFA monoclonal antibody Avastin (Bevacizumab) is prepared.

[0467] Preparation Example 3: Sequence design of mutant 14C12H1L1(M) of humanized antibody 14C12H1L1 against PD-1

[0468] Based on 14C12H1L1, individual amino acids in the skeleton region (light chain) are mutated to obtain 14C12H1L1(M).

[0469] The heavy chain variable region of 14C12H1L1(M) is 14C12H1(M) V which is the same as the heavy chain variable region 14C12H1 of 14C12H1L1, i.e., the amino acid sequence is shown as SEQ ID NO: 24.

[0470] 14C12H1L1(M) light chain variable region of 14C12H1L1(M) V : SEQ ID NO: 35.

[0471] Preparation Example 4: Sequence design of bispecific antibody

[0472] 1. Sequence design

[0473] The structural pattern of the bispecific antibody in the present application belongs to the Morrison pattern (IgG-scFv), that is, the scFv fragment of another antibody is connected at the C-terminus of both heavy chains of one IgG antibody, and the main composition design of the heavy chain and the light chain is as follows Table 1.

[0474] On the basis of the above Bevacizumab, the VP101 antibody above with the amino acid sequence of the heavy chain variable region and the light chain variable region of 14C12H1L1(M) as the ScFv fragment part is called VP101(M). Relative to 14C12H1L1, 14C12H1L1(M) effectively optimizes the structure of the bispecific antibody and improves its effectiveness.

[0475] Table 1: Composition design of heavy chain and light chain of VP101(M) and VP101(G4M)

[0476]

[0477] In the above Table 1:

[0478] (1) The right lower corner marked "V" refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. The corresponding heavy chain or light chain without the mark "V" is the full length containing the constant region. The amino acid sequence and the coding nucleic acid sequence of these variable regions or full lengths are all referred to the corresponding sequences recorded in the above preparation example.

[0479] (2) The amino acid sequence of Linker2 is SEQ ID NO: 36.

[0480] Alternatively, SEQ ID NO: 37 can be used as Linker1 to replace the aforementioned Linker2.

[0481] (3) Bevacizumab-H uses Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region.

[0482] (4) Bevacizumab-G4H uses Ig gamma-4 chain C region, ACCESSION: P01861.1 as the heavy chain constant region.

[0483] 2. Expression and purification of antibody VP101(M)

[0484] The heavy chain cDNA sequence and the light chain cDNA sequence of VP101(M) were cloned into pUC57simple (provided by Geneart) vector, respectively, to obtain pUC57simple-VP101H and pUC57simple-VP101L plasmids.

[0485] The plasmids pUC57simple-VP101H and pUC57simple-VP101L were digested (HindIII & EcoRI), respectively, and the obtained heavy chain and light chain were subcloned into pcDNA3.1 vector, respectively, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture solution was subjected to high-speed centrifugation, the supernatant was concentrated, and then loaded onto a HiTrap MabSelectSuRe column, and the protein was eluted with Elution Buffer to recover the target sample antibody VP101, and then the solution was changed to PBS.

[0486] 3. Detection of antibody VP101(M)

[0487] The purified sample was added to the reduced protein electrophoresis loading buffer and the non-reduced protein electrophoresis loading buffer, respectively, boiled, and then subjected to SDS-PAGE electrophoresis detection.

[0488] In order to distinguish from the mutated antibody in the following Preparation Example 5, VP101(M) is also referred to as VP101(hG1WT) in the present application. The above-mentioned VP101(M) is referred to as "wild type", which adopts Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region, and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region.

[0489] The amino acid sequence of the heavy chain of the immunoglobulin part in VP101(hG1WT) (Bevacizumab-H): SEQ ID NO: 38;

[0490] The nucleic acid sequence encoding the heavy chain of the immunoglobulin part in VP101(hG1WT): SEQ ID NO: 39.

[0491] To distinguish it from the mutated antibody in Example 5, VP101(G4M) is also referred to as VP101(hG4WT) in this invention. The aforementioned VP101(G4M) is considered "wild type," and it uses Ig gamma-4chain C region, ACCESSION: P01861.1 as the heavy chain constant region and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region.

[0492] Amino acid sequence of the heavy chain of the immunoglobulin moiety in VP101(hG4WT): SEQ ID NO:40;

[0493] Nucleic acid sequence of the heavy chain encoding the immunoglobulin portion of VP101(hG4WT): SEQ ID NO:41.

[0494] Preparation Example 5: Design of non-variable region amino acid mutation based on humanized bispecific antibody VP101 (hG1WT)

[0495] Based on the VP101(hG1WT) obtained in Preparation Example 5, the inventors obtained VP101(hG1DM) by introducing a point mutation (L234A) from leucine to alanine at position 234 and a point mutation (L235A) from leucine to alanine at position 235 of its heavy chain.

[0496] The amino acid sequence of the heavy chain of the immunoglobulin portion of VP101(hG1DM) (453aa, mutation sites are underlined): SEQ ID NO:42;

[0497] The nucleic acid sequence of the heavy chain encoding the immunoglobulin portion of VP101(hG1DM) (1359bp, mutation sites are underlined): SEQ ID NO:43.

[0498] The immunoglobulin moieties of VP101(hG1DM), VP101(hG1WT), and VP101(hG4WT) have the same light chain amino acid sequence, and their encoded nucleic acid sequences are also the same.

[0499] Amino acid sequence of the light chain of the immunoglobulin moiety in VP101(hG1DM): SEQ ID NO:44;

[0500] Nucleic acid sequence encoding the light chain of the immunoglobulin portion of VP101(hG1DM): SEQ ID NO:45.

[0501] Preparation Example 6: Preparation of anti-TIGIT antibody

[0502] 1. Preparation of hybridoma cell line LT019

[0503] The antigen used for preparing the anti-TIGIT antibody is human TIGIT-mFc (TIGIT is Genbank ID: NP_776160.2). The spleen cells of the immunized mice are fused with mouse myeloma cells to prepare hybridoma cells. The hybridoma cells are screened by indirect ELISA method using human TIGIT-hFc as the antigen, and the hybridoma cells capable of secreting antibodies specifically binding to TIGIT are obtained. The screened hybridoma cells are subjected to limited dilution to obtain stable hybridoma cell strains. The above hybridoma cell strains are named as hybridoma cell strain LT019, and the monoclonal antibodies secreted by the hybridoma cell strain are named as 26B12, respectively.

[0504] The hybridoma cell strain LT019 (also referred to as TIGIT-26B12) is preserved in China Center for Type Culture Collection (CCTCC) on October 23, 2020, with the preservation number of CCTCC NO: C2020208, and the address of the preservation is Wuhan, Wuhan University, China, with the postcode of 430072.

[0505] 2. Preparation of anti-TIGIT antibody 26B12

[0506] The LT019 cell strain prepared above is cultured in a CD medium (Chemical Defined Medium containing 1% penicillin-streptomycin) at 37°C in 5% CO2. After 7 days, the cell culture supernatant is collected, centrifuged at high speed, filtered by vacuum through a microporous filter, and purified by using a HiTrap protein A HP column to prepare the antibody 26B12.

[0507] Preparation Example 7: Sequence analysis of anti-TIGIT antibody 26B12

[0508] The mRNA is extracted from the LT019 cell strain cultured in Preparation Example 6 according to the method of the Bacterial Total RNA Extraction Kit for Cultured Cells (Tiangen, item number DP430).

[0509] The cDNA is synthesized and PCR amplification is performed according to the instructions of the Invitrogen III First-Strand Synthesis System for RT-PCR kit.

[0510] The PCR amplification product is directly subjected to TA cloning, and the specific operation is performed according to the instructions of the pEASY-T1 Cloning Kit (Transgen CT101) kit.

[0511] The product of TA cloning is directly subjected to sequencing, and the sequencing results are as follows:

[0512] The nucleic acid sequence of the heavy chain variable region is shown as SEQ ID NO: 47, and the fragment is 363 bp long.

[0513] The encoded amino acid sequence is shown as SEQ ID NO: 46, and the length is 121 amino acids.

[0514] The sequence of HCDR1 of the heavy chain is shown as SEQ ID NO: 13, the sequence of HCDR2 is shown as SEQ ID NO: 14, and the sequence of HCDR3 is shown as SEQ ID NO: 15.

[0515] The nucleic acid sequence of the light chain variable region is shown as SEQ ID NO: 49, and the length is 321 bp.

[0516] The encoded amino acid sequence is shown as SEQ ID NO: 48, and the length is 107 amino acids.

[0517] The sequence of LCDR1 of the light chain is shown as SEQ ID NO: 16, the sequence of LCDR2 is shown as SEQ ID NO: 17, and the sequence of LCDR3 is shown as SEQ ID NO: 18.

[0518] Preparation Example 8: Design and preparation of humanized antibody against human TIGIT and mutant antibody

[0519] 1. Design of the light chain and heavy chain of the humanized antibody 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 against human TIGIT

[0520] According to 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 are designed (the constant region sequence of the antibody, the constant region of the heavy chain is Ig gamma-1 chain C region; the constant region of the light chain is Ig kappa chain C region).

[0521] The designed variable region sequences are shown in Table 2 below.

[0522] Table 2: Variable region sequences of humanized antibodies against human TIGIT

[0523]

[0524] The nucleic acid sequences of the heavy chain variable regions of the above eight antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 are all 363 bp in length, and the amino acid sequences encoded by them are all 121 aa in length; the nucleic acid sequences of the light chain variable regions are all 321 bp in length, and the amino acid sequences encoded by them are all 107 aa in length.

[0525] And the above eight antibodies have the same HCDR1-HCDR3 and LCDR1-LCDR3, as follows:

[0526] The sequence of HCDR1 is shown as SEQ ID NO: 13, the sequence of HCDR2 is shown as SEQ ID NO: 14, and the sequence of HCDR3 is shown as SEQ ID NO: 15;

[0527] The sequence of LCDR1 is shown as SEQ ID NO: 16, the sequence of LCDR2 is shown as SEQ ID NO: 17, and the sequence of LCDR3 is shown as SEQ ID NO: 18.

[0528] 2. Preparation of humanized antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4

[0529] The heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region is Ig kappa chain C region, ACCESSION: P01834.

[0530] The 26B12H1L1 heavy chain cDNA and light chain cDNA, 26B12H4L1 heavy chain cDNA and light chain cDNA, 26B12H2L2 heavy chain cDNA and light chain cDNA, 26B12H3L2 heavy chain cDNA and light chain cDNA, 26B12H2L3 heavy chain cDNA and light chain cDNA, 26B12H3L3 heavy chain cDNA and light chain cDNA, 26B12H1L4 heavy chain cDNA and light chain cDNA, 26B12H2L4 heavy chain cDNA and light chain cDNA, and 26B12H4L4 heavy chain cDNA and light chain cDNA were respectively cloned into the pUC57simple (provided by Kingsrosy) vector to obtain:

[0531] pUC57simple-26B12H1, pUC57simple-26B12L1;

[0532] pUC57simple-26B12H4, pUC57simple-26B12L1;

[0533] pUC57simple-26B12H2, pUC57simple-26B12L2;

[0534] pUC57simple-26B12H3, pUC57simple-26B12L2;

[0535] pUC57simple-26B12H2, pUC57simple-26B12L3;

[0536] pUC57simple-26B12H3, pUC57simple-26B12L3;

[0537] pUC57simple-26B12H1, pUC57simple-26B12L4; and

[0538] pUC57simple-26B12H4, pUC57simple-26B12L4.

[0539] The synthesized heavy and light chain full-length genes were digested with EcoRI & HindIII, and were subcloned into the expression vector pcDNA3.1 by restriction enzyme (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 according to the standard techniques introduced in Molecular Cloning: A Laboratory Manual (2nd Edition), and the heavy and light chain genes of the recombinant expression plasmids were further analyzed by sequencing. Then, the light and heavy chain recombinant plasmids containing the corresponding genes (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 designed to be co-transfected into 293F cells, and the culture solution was collected and purified. After the correct sequences were verified by sequencing, the expression plasmids of endotoxin-free level were prepared, and the plasmids were transiently transfected into HEK293 cells to express the antibodies. After the cells were cultured for 7 days, the cell culture solution was collected, and the humanized antibodies were obtained by affinity purification using Protein A column.

[0540] The humanized antibody 26B12H2L2 is also referred to as 26B12H2L2 (hG1WT) in the present application.

[0541] 3. Design of humanized antibodies 26B12H2L2 (hG1DM) and 26B12H2L2 (hG4DM)

[0542] Based on 26B12H2L2, the present inventors introduced a point mutation of leucine to alanine at the 234th position (according to the EU numbering system, the same below) in the heavy chain constant region (L234A) and a point mutation of leucine to alanine at the 235th position (L235A) to obtain the humanized antibody 26B12H2L2 (hG1DM) with constant region mutation.

[0543] The heavy chain amino acid sequence of 26B12H2L2(hG1DM) is shown as SEQ ID NO: 70; the light chain amino acid sequence is shown as SEQ ID NO: 68.

[0544] Based on 26B12H2L2, the antibody variable region is unchanged, the humanized antibody 26B12H2L2(hG4DM) with constant region mutation is obtained by using Iggamma-4 chain C region, ACCESSION: P01861.1 as the heavy chain constant region, and introducing a point mutation of phenylalanine to alanine at the 234th site (F234A) and a point mutation of leucine to alanine at the 235th site (L235A) in the heavy chain constant region.

[0545] The heavy chain amino acid sequence of 26B12H2L2(hG4DM) is shown as SEQ ID NO: 66; the light chain amino acid sequence is shown as SEQ ID NO: 68.

[0546] The preparation of the humanized antibodies 26B12H2L2(hG1DM) and 26B12H2L2(hG4DM) can refer to the method in step 2 above.

[0547] Preparation Example 9: Sequence design and preparation of fusion protein of anti-TIGIT antibody and TGF-βR

[0548] 1. Sequence design

[0549] The composition of the anti-TIGIT antibody and TGF-βR fusion protein in the application is as follows in Table 3.

[0550] Table 3: Composition of heavy chain and light chain of anti-TIGIT antibody and TGF-βR fusion protein

[0551]

[0552] The TGF-β receptor part has two peptide chains respectively, which are connected to the C-terminal of the heavy chain of the IgG part through a linker.

[0553] 2. Expression and purification of antibodies

[0554] The heavy chain cDNA sequence and the light chain cDNA sequence of TF01 and TF02 are respectively cloned into the pUC57simple (provided by Kingsway) vector to obtain pUC57simple-TF01H, pUC57simple-TF02H and pUC57simple-TF02L, pUC57simple-TF02L plasmids.

[0555] The plasmids pUC57simple-TF01H / pUC57simple TF01L and pUC57simple-TF02H / pUC57simple TF02L were respectively digested (HindIII&EcoRI), and the recovered heavy chain and light chain were subcloned into pcDNA3.1 vector, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture solution was centrifuged at high speed, the supernatant was concentrated, and then loaded onto a HiTrap MabSelect SuRe column. The protein was eluted with Elution Buffer in one step, and the target sample was recovered and replaced with PBS. Thus, the fusion proteins TF01 and TF02 of anti-TIGIT antibody and TGF-βR were prepared.

[0556] Preparation Example 10: Sequence design and preparation of fusion protein (mutant) of anti-TIGIT antibody and TGF-βR

[0557] The TGF-βRII extracellular region fragment in the fusion protein TF01 of anti-TIGIT antibody and TGF-βR in the application was subjected to point mutation, the serine at position 8 of the TGF-βRII extracellular region fragment (SEQ ID NO: 72) was mutated to threonine (SEQ ID NO: 88), and a new antibody named TF01T was obtained. The preparation method is referred to Preparation Example 9.

[0558] Table 4: Composition of heavy chain and light chain of fusion protein (mutant) of anti-TIGIT antibody and TGF-βR

[0559]

[0560] Example 1: Anti-TIGIT antibody and TGF-βR fusion protein in combination with anti-PD-1-anti-VEGFA bispecific antibody Promoting INF-γ secretion

[0561] According to the separation liquid Ficoll-Paque TMNormal human PBMC (isolated from healthy human peripheral blood) were obtained according to the operating instructions of the Plus (Cytiva, Cat: 17-1440-02). The day before the experiment, the PBMC 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% carbon dioxide incubator. On the day of the experiment, the antibodies (VP101 (hG1DM) and TF01T in a molar ratio of 1:1), CMV (Mabtech, Cat: 3619-1) and TGF-β1 (Genscript, Cat: Z03411) diluted in complete medium were added to the 96-well round-bottom plates (Corning, Cat: 3799) in equal volumes according to the experimental design, and controls were designed, and incubated at 37°C for 10 min; the PBMC were collected, counted and the viability was measured, and 200,000 cells / well were inoculated in the 96-well round-bottom plates, and CMV was added at a final concentration of 0.2 μg / mL, and the plates were gently shaken to mix, and then incubated in the incubator for 4 days (the final volume of the experimental system was 200 μL). After 4 days, the cell culture supernatant was collected, and the concentration of IFN-γ was detected by ELISA kit (Dakewe, Cat: 1110002).

[0562] The results are shown in Figure 1 and Figure 2 .

[0563] The results show that VP101 (hG1DM) monotherapy group, TF01T monotherapy group and VP101 (hG1DM) combined with TF01T group can effectively promote the secretion of IFN-γ by PBMC under CMV stimulation, and the ability of VP101 (hG1DM) combined with TF01T group to promote the secretion of IFN-γ by PBMC is significantly stronger than that of each monotherapy group. After adding TGF-β1 which can inhibit the secretion of IFN-γ in the system, the secretion of IFN-γ by VP101 (hG1DM) combined with TF01T group is higher than that without TGF-β1, showing stronger immunobiological activity of promoting PBMC recall response to CMV.

[0564] Example 2: Reporter gene system verifies the blocking activity of anti-TIGIT antibody and TGF-βR fusion protein in combination with anti-PD-1-anti-VEGFA bispecific antibody Figure 3

[0565] PDL1 aAPC / CHO-K1 cells (purchased from Promega, item number: J108A) were resuspended with Ham’s F-12 + 10% FBS medium (Ham’s F-12, Gibco, item number: 11765070; FBS, fetal bovine serum, Excell Bio, item number: FSP500) and seeded at 40,000 cells per well per 100 μL in a 96-well flat-bottom black plate (Corning, model number: 3916) overnight. The next day, the antibodies and VEGF (self-made by Kangfang Biotechnology, batch number: 20210508) were diluted with assay medium (i.e. RPMI 1640 + 10% FBS; RPMI 1640, Gibco, item number: 22400-089; FBS, fetal bovine serum, Excell Bio, item number: FSP500) according to the experimental design and incubated at 37°C for 30 min. Jurkat-NFAT-PD1-hTIGIT cells (constructed by Kangfang Biotechnology) and A549 cells (purchased from the Cell Resource Center of Shanghai Life Sciences Research Institute of the Chinese Academy of Sciences, item number: SCSP-503) were collected separately, resuspended with 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 the antibody or the antibody and VEGF pre-incubation 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 per well per 20 μL were added, and 10,000 A549 cells per well per 20 μL were added. A human IgG1DM control group and a blank control group were set up, and the plate was incubated in an incubator for 6 h. The 96-well flat-bottom black plate was removed, equilibrated to room temperature, and 80 μL / well of Firefly Glo Luciferase Reporter Gene Assay Kit (Yeasen, item number: 11404ES80) was added. After incubation in the dark for 5 min, the RLU value was read.

[0566] The results are shown in Table 1. Figure 4The results show that VP101 (hG1DM) single drug group, TF01T single drug group and VP101 (hG1DM) combined with TF01T group can effectively play the neutralization effect to block the interaction of TIGIT / PVR and PD-1 / PD-L1, and the neutralization biological activity of VP101 (hG1DM) combined with TF01T to block TIGIT / PVR and PD-1 / PD-L1 is better than that of VP101 (hG1DM) single drug and TF01T single drug.

[0567] In addition, VEGF is additionally added to the reporter gene system for pretreatment, and the results are shown in FIG. 6. ​ The results show that, compared with the isotype control human IgG1DM, the VP101 (hG1DM) single drug group and the VP101 (hG1DM) combined with TF01T group can significantly improve the expression of luciferase; and the blocking activity of VP101 (hG1DM) combined with TF01T is higher than that of the VP101 (hG1DM) single drug group.

[0568] In summary, the neutralization biological activity of VP101 (hG1DM) combined with TF01T to block TIGIT / PVR and PD-1 / PD-L1 is better than that of VP101 (hG1DM) single drug and TF01T single drug, and in the system containing VEGF, VP101 (hG1DM) combined with TF01T shows stronger neutralization activity.

[0569] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details in accordance with all the teachings disclosed herein, and such changes are within the scope of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A pharmaceutical combination product 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: a first protein functional region that specifically binds to PD-1, and a second protein functional region that specifically binds to VEGFA; wherein, the first protein functional region comprises a heavy chain variable region VH1 and a light chain variable region VL1, the VH1 comprises a HCDR1 as shown in SEQ ID NO: 1, a HCDR2 as shown in SEQ ID NO: 2, and a HCDR3 as shown in SEQ ID NO: 3; the VL1 comprises a LCDR1 as shown in SEQ ID NO: 4, a LCDR2 as shown in SEQ ID NO: 5, and a 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, the VH2 comprises a HCDR1 as shown in SEQ ID NO: 7, a HCDR2 as shown in SEQ ID NO: 8, and a HCDR3 as shown in SEQ ID NO: 9; the VL2 comprises a LCDR1 as shown in SEQ ID NO: 10, a LCDR2 as shown in SEQ ID NO: 11, and a LCDR3 as shown in SEQ ID NO: 12; (2) the second bispecific protein comprises: a third protein functional region that specifically binds to TIGIT, and a fourth protein functional region that specifically binds to TGF-β; wherein, the third protein functional region comprises a heavy chain variable region VH3 and a light chain variable region VL3, the VH3 comprises a HCDR1 as shown in SEQ ID NO: 13, a HCDR2 as shown in SEQ ID NO: 14, and a HCDR3 as shown in SEQ ID NO: 15; the VL3 comprises a LCDR1 as shown in SEQ ID NO: 16, a LCDR2 as shown in SEQ ID NO: 17, and a LCDR3 as shown in SEQ ID NO: 18; the fourth protein functional region is an amino acid sequence as shown in SEQ ID NO: 72, SEQ ID NO: 76, or SEQ ID NO: 88; the second protein functional region is 1 IgG, the first protein functional region is two single chain antibodies, and the single chain antibodies are respectively connected at the C-terminus of the two heavy chains of the second protein functional region; the third protein functional region is 1 IgG, the fourth protein functional region is 2, and the fourth protein functional region is respectively connected at the C-terminus of the two heavy chains of the third protein functional region.

2. The pharmaceutical combination product 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 of claim 1, wherein, the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 20 and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO: 22; the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 20 and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO: 26; the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 20 and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO: 35; the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 24 and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO: 22; the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 24 and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO: 26; or the amino acid sequence of the heavy chain variable region VH1 is set forth in SEQ ID NO: 24 and the amino acid sequence of the light chain variable region VL1 is set forth in SEQ ID NO:

35.

4. The pharmaceutical combination of claim 1, wherein, the amino acid sequence of the heavy chain variable region VH2 is set forth in SEQ ID NO: 31 and the amino acid sequence of the light chain variable region VL2 is set forth in SEQ ID NO:

33.

5. The pharmaceutical combination of claim 1, wherein, the amino acid sequence of the heavy chain variable region VH3 is selected from the group consisting of 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 the group consisting of 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 of claim 1, wherein, the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 46 and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 48; the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 50 and the amino acid sequence of the light chain variable region VL3 is set forth in SEQ ID NO: 58; the amino acid sequence of the heavy chain variable region VH3 is set forth in SEQ ID NO: 50 and the amino acid sequence of the light chain variable region VL3 is set forth 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 product according to claim 1, wherein, The IgG may be IgG1, IgG2, IgG3, or IgG4.

8. The pharmaceutical combination product according to claim 7, 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.

9. The pharmaceutical combination product according to claim 7, 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.

10. The pharmaceutical combination product according to claim 1, wherein, The second protein's 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.

11. The pharmaceutical combination product according to claim 1, 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.

12. The pharmaceutical combination product according to claim 1, wherein, The first protein functional region is directly connected to the second protein functional region or connected through a connector; The third protein functional region is directly connected to the fourth protein functional region or connected through a connector; and / or The heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a linker.

13. The pharmaceutical combination of claim 12, wherein, the linker is a polypeptide as set forth in SEQ ID NO: 91 or SEQ ID NO: 92, or a polypeptide obtained by concatenating a plurality of polypeptides as set forth in SEQ ID NO:

91.

14. The pharmaceutical combination of claim 13, wherein, the plurality of polypeptides as set forth in SEQ ID NO: 91 refers to 2, 3, 4, 5, or 6 polypeptides as set forth in SEQ ID NO:

91.

15. The pharmaceutical combination of claim 12, wherein, the linker is a polypeptide obtained by concatenating a plurality of polypeptides as set forth in SEQ ID NO: 91 and further concatenating a polypeptide as set forth in SEQ ID NO:

92.

16. The pharmaceutical combination of claim 15, wherein, the plurality of polypeptides as set forth in SEQ ID NO: 91 refers to 2, 3, 4, or 5 polypeptides as set forth in SEQ ID NO:

91.

17. The pharmaceutical combination of claim 12, wherein, the amino acid sequence of the linker is independently selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO:

83.

18. The pharmaceutical combination of claim 1, wherein, the second protein functional region is 1 IgG1.

19. The pharmaceutical combination of claim 1, wherein, the third protein functional region is 1 IgG4.

20. The pharmaceutical combination of claim 1, wherein, the first bispecific protein comprises: a first protein functional region that specifically binds to PD-1, and a second protein functional region that specifically binds to VEGFA; the first protein functional region is 2, and 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 as set forth in SEQ ID NO: 42, and the amino acid sequence of the light chain thereof is as set forth in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single-chain antibody is as set forth in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region of the single-chain antibody is as set forth in SEQ ID NO: 35; two single-chain antibodies are respectively connected at the C-terminus of the two heavy chains of the IgG; the first protein functional region is connected to the second protein functional region through a first linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody through a second linker; the first linker and the second linker are the same or different.

21. The pharmaceutical combination of claim 20, wherein, the amino acid sequence of the first linker and the second linker is independently selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO:

37.

22. The pharmaceutical combination of claim 20, wherein, The amino acid sequence of the first linker and the second linker is as shown in SEQ ID NO:

36.

23. The pharmaceutical combination of any one of claims 1 to 22, wherein, the second bispecific protein comprises: a third protein functional region that specifically binds to TIGIT, and a fourth protein functional region that specifically binds to TGF-β; wherein, the third protein functional region is 1, and the fourth protein functional region is 2; the third protein functional region is IgG, and the fourth protein functional region is a truncated fragment of a TGF-β receptor extracellular region having TGF-β receptor function; the amino acid sequence of the heavy chain of the IgG is as shown in SEQ ID NO: 66 or SEQ ID NO: 70, and the amino acid sequence of the light chain thereof is as shown in SEQ ID NO: 68; the amino acid sequence of the fourth protein functional region is as shown in SEQ ID NO: 72 or SEQ ID NO: 88; the fourth protein functional region is connected at the C-terminus of the two heavy chains of the IgG via a third linker.

24. The pharmaceutical combination of claim 23, wherein, the amino acid sequence of the third linker is as shown in SEQ ID NO:

83.

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

26. The pharmaceutical combination of any one of claims 1 to 22, wherein, the first bispecific protein and the second bispecific protein are in independent packages from each other.

27. The pharmaceutical combination of any one of claims 1 to 22, wherein, the mass ratio of the first bispecific protein to the second bispecific protein is (10: 1) to (1:5).

28. The pharmaceutical combination of any one of claims 1 to 22, wherein, the mass ratio of the first bispecific protein to the second bispecific protein is (5: 1) to (1:3).

29. The pharmaceutical combination of any one of claims 1 to 22, wherein, the mass ratio of the first bispecific protein to the second bispecific protein is (3: 1) to (1:2).

30. The pharmaceutical combination of any one of claims 1 to 22, wherein, the mass ratio of the first bispecific protein to the second bispecific protein is 2:

1.

31. The pharmaceutical combination of any one of claims 1 to 22, wherein, the molar ratio of the first bispecific protein to the second bispecific protein is (10: 1) to (1:10).

32. The pharmaceutical combination of any one of claims 1 to 22, wherein, the molar ratio of the first bispecific protein to the second bispecific protein is (5: 1) to (1:5).

33. The pharmaceutical combination of any one of claims 1 to 22, wherein, the molar ratio of the first bispecific protein to the second bispecific protein is (3: 1) to (1:3).

34. The pharmaceutical combination of any one of claims 1 to 22, wherein, the molar ratio of the first bispecific protein to the second bispecific protein is (2: 1) to (1:2).

35. The pharmaceutical combination according to any one of claims 1 to 22, wherein, the molar ratio of the first bispecific protein to the second bispecific protein is 1:

1.

36. The pharmaceutical combination of any one of claims 1 to 22, wherein, the unit dose of the first bispecific protein and the second bispecific protein independently is 100 mg-2500 mg, calculated by the mass of the first bispecific protein or the second bispecific protein.

37. The pharmaceutical combination of any one of claims 1 to 22, wherein, the unit dose of the first bispecific protein and the second bispecific protein independently is 100 mg-2000 mg, calculated by the mass of the first bispecific protein or the second bispecific protein.

38. The pharmaceutical combination of any one of claims 1 to 22, wherein, The unit dose of the first bispecific protein and the second bispecific protein independently is 100 mg-1500 mg, calculated on the mass of the first bispecific protein or the second bispecific protein.

39. The pharmaceutical combination of any one of claims 1 to 22, wherein, The unit dose of the first bispecific protein and the second bispecific protein independently is 100 mg-1200 mg, calculated on the mass of the first bispecific protein or the second bispecific protein.

40. The pharmaceutical combination according to any one of claims 1 to 22, wherein, The unit dose of the first bispecific protein and the second bispecific protein independently is 100 mg-1000 mg, calculated on the mass of the first bispecific protein or the second bispecific protein.

41. The pharmaceutical combination according to any one of claims 1 to 22, wherein, The unit dose of the first bispecific protein and the second bispecific protein independently is 200 mg-800 mg, calculated on the mass of the first bispecific protein or the second bispecific protein.

42. The pharmaceutical combination according to any one of claims 1 to 22, wherein, The unit dose of the first bispecific protein and the second bispecific protein independently is 200 mg-500 mg, calculated on the mass of the first bispecific protein or the second bispecific protein.

43. The pharmaceutical combination of any one of claims 1 to 22, wherein, The unit dose of the first bispecific protein and the second bispecific protein independently is 300 mg-600 mg, calculated on the mass of the first bispecific protein or the second bispecific protein.

44. The pharmaceutical combination of any one of claims 1-22, wherein, The unit dose of the first bispecific protein and the second bispecific protein independently is 400 mg-500 mg, calculated on the mass of the first bispecific protein or the second bispecific protein.

45. The pharmaceutical combination according to any one of claims 1 to 22, wherein, The unit dose of the first bispecific protein and the second bispecific protein independently is 450 mg, calculated on the mass of the first bispecific protein or the second bispecific protein.

46. The pharmaceutical combination of any one of claims 1 to 22, wherein, The pharmaceutical combination product further comprises an effective amount of a tumor chemotherapeutic drug.

47. The pharmaceutical combination of any one of claims 1 to 22, wherein, The pharmaceutical combination product further comprises an effective amount of a platinum drug, an alkylating agent, an antimetabolite, an antitumor antibiotic, a plant anticancer drug, a hormone, or an immunological agent.

48. The pharmaceutical combination of any one of claims 1 to 22, wherein, The pharmaceutical combination product further comprises one or more pharmaceutically acceptable excipients.

49. The pharmaceutical combination product according to claim 48, further comprising a product insert.

50. Use of the pharmaceutical combination product according to any one of claims 1 to 49 for the manufacture of a medicament for the treatment or prevention of a tumor.

51. The use according to claim 50, wherein, the tumor is selected from one or more of colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal 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.

52. The use according to claim 51, wherein, the lung cancer is non-small cell lung cancer or small cell lung cancer.

53. The use according to claim 51, wherein, the liver cancer is hepatocellular carcinoma.

54. The use according to claim 51, wherein, the renal tumor is renal cell carcinoma.

55. The use according to claim 51, wherein, the breast cancer is triple-negative breast cancer.

56. The use of claim 51, wherein, the urothelial carcinoma is a bladder cancer.

57. The use of claim 51, wherein, the lymphoma is a non-Hodgkin lymphoma or a B-cell lymphoma.

Images