Pharmaceutical composition and application

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a medicine composition and application. Specifically, the pharmaceutical composition comprises a bispecific protein which is specifically combined with PD-1 and VEGFA, and a bispecific protein which is specifically combined with TIGIT and TGF-beta R. The pharmaceutical composition disclosed by the invention can be used for effectively treating or preventing tumors and has a good application prospect.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

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

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

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

[0005] The PD-1 / PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune escape. PD-1 is mainly expressed in immune cells such as T cells, and the ligand PDL1 of PD-1 is highly expressed in many human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway can activate suppressed T cells and attack cancer cells. Blocking the PD-1 / PDL1 signal can promote the proliferation of tumor antigen-specific T cells, play a role in killing tumor cells, and inhibit local tumor growth (Julie R et al., 2012, N Engl J Med. 366: 2455-2465). In addition, tumors with high expression of PDL1 are accompanied by cancers that are difficult to detect (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 antibodies in vivo. Due to the broad-spectrum anti-tumor prospects and amazing efficacy of PD-1 antibodies, the industry generally believes that antibodies targeting the PD-1 pathway will bring breakthrough progress in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet 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 type of growth factor that can promote endothelial cell division and proliferation, promote the formation of new blood vessels, and increase vascular permeability. It binds to the vascular endothelial growth factor receptor on the cell surface and exerts its function by activating the tyrosine kinase signal transduction pathway. In tumor tissues, tumor cells, tumor-invading macrophages and mast cells can secrete high levels of VEGF, which stimulates tumor vascular endothelial cells in a paracrine form, promotes endothelial cell proliferation and migration, induces angiogenesis, promotes continuous tumor growth, and increases vascular permeability, causing fibrin deposition in surrounding tissues, promoting the infiltration of monocytes, fibroblasts and endothelial cells, which is beneficial to the formation of tumor matrix and the entry of tumor cells into new blood vessels, promoting tumor metastasis, and thus inhibiting tumor angiogenesis is considered to be one of the most promising tumor treatment methods. The VEGF family includes: VEGFA, VEGFB, VEGFC, VEGFD and PIGF. Vascular endothelial growth factor receptors (VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4) and Neuropilin-1 (NRP-1). Among them, the first three receptors are similar in structure and belong to the tyrosine kinase superfamily. They are composed of three parts: the extracellular region, the transmembrane segment and the 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 effects. VEGFR1 is the first receptor discovered. Under normal physiological conditions, the affinity of VEGFR1 to VEGFA is higher than that of VEGFR2 to VEGFA, but its intracellular tyrosinase activity is lower than that of VEGFR2 (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5)(2016): 146-148).

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

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

[0010] VEGFA and VEGFR2 are mainly involved in regulating angiogenesis. Before and after the binding of VEGFA and VEGFR2, many intermediate signals in the upstream and downstream pathways will be triggered to form a cascade reaction, which will eventually change the physiological function of endothelial cells in different forms such as proliferation, survival, migration, increased permeability and infiltration into surrounding tissues (Dong Hongchao et al., "Modern Oncology", Vol. 22, No. 9, September 2014, pp. 2231-3).

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

[0012] CD155 (also known as PVR, Necl5 or Tage4), CD112 (also known as PVRL2 / nectin 2) and CD113 (also known as PVRL3) are ligands that bind to TIGIT (Martinet L, Smyth M J. Balancing natural killer cell activation through paired receptors [J]. Nature Reviews Immunology, 2015, 15 (4): 243-254.), among which CD155 is a high-affinity ligand for TIGIT. In NK cells, TIGIT binding ligands CD155 and CD112 can inhibit NK cells from killing cells with high expression of CD155 and CD112 (Stanietsky N, Simic H, Arapovic J, et al. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity [J]. Proceedings of the National Academy of Sciences, 2009, 106 (42): 17858-17863). It has been reported that blocking PD-1 and TIGIT at the same time can enhance CD8 + The killing effect of T cells (Johnston RJ, Comps-Agrar L, Hackney J, et al. The immunoreceptor TIGITregulates antitumor and antiviral CD8 +T cell effector function[J].Cancer cell, 2014, 26(6):923-937). In the latest study, it was found that TIGIT, as an immune checkpoint of NK cells, can lead to 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 cancer, multiple myeloma, non-Hodgkin's lymphoma, B lymphocytic neoplasm, plasma cell cancer, etc. (Zhang Q, BiJ, 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 class of cytokines with diverse functions, which are divided into subfamilies such as TGF-β, activins, inhibins, growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), Nodal, Lefty, and anti-Müllerian hormone (Table A). The proteins of the three TGF-β subfamilies are called TGF-β1, TGF-β2, and TGF-β3, among which TGF-β1 is the most highly expressed subtype. After binding to receptors, TGF-β1, TGF-β2, and TGF-β3 mediate a series of biological reactions through Smad and non-Smad signaling pathways, including promoting epithelial-mesenchymal transitions (EMT), promoting tissue fibrosis, promoting angiogenesis, promoting tumor immune escape, and having dual effects of tumor suppression and promotion (J Massagué. TGFbeta in Cancer. [J]. Cell, 2008, 134 (2): 215-230.).

[0014] TGF-β receptor (TGF-βR) is widely distributed on the surface of normal human cells and tumor cells, including three receptor superfamilies. The type I classical TGF-β receptor family includes ALK1-7, among which TGF-βRⅠ (also written as TβRI) is also called ALK5; the type II classical TGF-β receptor family includes TGF-βRII (also written as TβRII), ActRII, ActRIIB, AMHRII and BMPRII; the type III TGF-β receptor superfamily includes Betaglycan (also known as TGF-βRIII) and Endoglin; among them, TGF-βRⅠ, TGF-βRII, and TGF-βRIII can each bind to TGF-β1, TGF-β2, and TGF-β3 (Pawlak John B, BlobeGerard C, TGF-βsuperfamily co-receptors in cancer. DevDyn, 2021). The TGF-β family and receptors are shown in Table A (based on the review summary of Carl-Henrik Heldin1 and Aristidis Moustakas. Cold Spring Harb Perspect Biol. 2016.). TGF-βRI and TGF-βRⅡ are serine / threonine protein kinase receptors. In the TGF-β signaling pathway, TGF-βRII is the core key molecule of signal transduction. It can bind to TGF-β with high affinity and then form a heterotetrameric receptor complex with TGF-βRI dimer. It phosphorylates and activates TGF-βRI through autophosphorylation of TGF-βRⅡ. The activated TGF-βRI phosphorylates downstream Smad pathway-related proteins, regulates the transcription and translation of downstream target genes, and then triggers disease-related biological responses. TGF-βRIII has a weaker affinity for TGF-β than TGF-βRI and TGF-βRⅡ, and does not have an intracellular segment, so it cannot be connected to the downstream signaling pathway. Its function is to capture TGF-β and present it to TGF-βRII. Other TGF-β receptors can also bind to TGF-β (Sang Xiaohong et al., Research progress of small molecule inhibitors targeting TGF-β and receptors [J]. Acta Pharmaceutica Sinica, 2019(9).).

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

[0016] After in-depth research and creative work, the inventors combined a bispecific protein that specifically binds to PD-1 and VEGFA with a bispecific protein that specifically binds to TIGIT and TGF-βR, and surprisingly found that the combination has a pharmacological effect of effectively inhibiting tumor growth, and the effect is better than a single bispecific protein that specifically binds to PD-1 and VEGFA or a single bispecific protein that specifically binds to TIGIT and TGF-βR. The following invention is provided:

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

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

[0019] specifically binds to the first protein domain of PD-1, and

[0020] The second protein domain that specifically binds to VEGFA;

[0021] in,

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

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

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

[0025] specifically binds to the third protein domain of TIGIT, and

[0026] Specifically binds to the fourth protein functional domain of TGF-β;

[0027] in,

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

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

[0030] In the present invention, a bispecific protein may also be referred to as a bispecific molecule. A first bispecific protein may also be referred to as a first bispecific molecule. A second bispecific protein may also be referred to as a second bispecific molecule.

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

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

[0033] In some embodiments of the present invention, the drug combination, wherein

[0034] The amino acid sequence of the heavy chain variable region VH1 is selected from 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 SEQ ID NO:22, SEQ ID NO:26 and SEQ ID NO:35.

[0036] In some embodiments of the present invention, the drug combination, wherein

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

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

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

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

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

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

[0043] In some embodiments of the present invention, the drug combination, wherein

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

[0045] In some embodiments of the present invention, the drug combination, wherein

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

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

[0048] In some embodiments of the present invention, the drug combination, wherein

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

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

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

[0052] 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;

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

[0054] 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;

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

[0056] 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

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

[0058] In some embodiments of the present invention, the drug 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 with TGF-β receptor function, a truncated fragment of the extracellular region of TGF-βRII with TGF-β receptor function, a truncated fragment of the extracellular region of TGF-βRIII with TGF-β receptor function, or a variant of the extracellular region of TGF-β receptor with TGF-β receptor function.

[0060] The variant of the TGF-β receptor extracellular region having TGF-β receptor function refers to a protein that still has TGF-β receptor function after one or several (e.g., 2, 3, 4, 5 or more) amino acids are replaced, deleted and / or inserted into the TGF-β receptor extracellular region.

[0061] In some embodiments of the present invention, the drug combination, wherein

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

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

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

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

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

[0067] In some embodiments of the present invention, the drug combination, wherein

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

[0069] In some embodiments of the present invention, the drug combination, wherein

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

[0071] In some embodiments of the present invention, the drug combination, wherein

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

[0073] In some embodiments of the present invention, the drug combination, wherein

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

[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 invention, the drug combination, wherein

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

[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 invention, the drug combination, wherein

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

[0087] In some embodiments of the present invention, the drug combination, wherein

[0088] 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.

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

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

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

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

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

[0094] The heavy chain variable region of the single chain antibody is directly linked to the light chain variable region of the single chain antibody or is linked via a linker.

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

[0096] The linker is a polypeptide as shown in SEQ ID NO:91 or SEQ ID NO:92, or a polypeptide obtained by connecting multiple (e.g., 2, 3, 4, 5 or 6) polypeptides as shown in SEQ ID NO:91 in series, or a polypeptide obtained by connecting multiple (e.g., 2, 3, 4 or 5) polypeptides as shown in SEQ ID NO:91 in series and further connecting with a polypeptide as shown in SEQ ID NO:92 in series;

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

[0098] In some embodiments of the present invention, the drug 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 number of the first protein functional regions is 2, and the number of the second protein functional region is 1;

[0101] Preferably, the number of the third protein functional region is one, and the number of the fourth protein functional region is two.

[0102] In some embodiments of the present invention, the drug combination, wherein

[0103] The second protein functional region is one IgG, the first protein functional region is two single-chain antibodies, and the single-chain antibodies are respectively connected to the C-termini or N-termini of the two heavy chains of the second protein functional region.

[0104] In some embodiments of the present invention, the drug combination, wherein

[0105] The second protein functional region is one IgG1, the first protein functional region is two single-chain antibodies, and the single-chain antibodies are respectively connected to the C-termini or N-termini of the two heavy chains of the second protein functional region.

[0106] In some embodiments of the present invention, the drug combination, wherein

[0107] The first protein functional region is one 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 invention, the drug 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 to the C-termini or N-termini of the two heavy chains of the first protein functional region.

[0110] In one or more embodiments of the present invention, the bispecific protein is a bispecific antibody in the form of IgG-scFv, namely Morrison model.

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

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

[0113] In some embodiments of the present invention, the drug 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 to the C-terminus or N-terminus of the two heavy chains of the third protein functional region.

[0115] In some embodiments of the present invention, the drug 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 connected to the C-terminus or N-terminus of the two heavy chains of the third protein functional region. In some embodiments of the present invention, the drug combination, wherein,

[0117] The first bispecific protein comprises:

[0118] specifically binds to the first protein domain of PD-1, and

[0119] The second protein domain that specifically binds to VEGFA;

[0120] The number of the first protein functional regions is 2, and the number of the second protein functional region is 1;

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

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

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

[0124] Two single-chain antibodies are connected to the C-termini of the two heavy chains of IgG;

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

[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 as shown in SEQ ID NO:36.

[0128] In some embodiments of the present invention, the drug combination, wherein

[0129] The second bispecific protein comprises:

[0130] specifically binds to the third protein domain of TIGIT, and

[0131] Specifically binds to the fourth protein functional domain of TGF-β;

[0132] in,

[0133] The third protein functional region is one, and the fourth protein functional region is two;

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

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

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

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

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

[0139] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein is selected from the anti-PD-1-anti-VEGFA bispecific antibodies VP101(M), VP101(G4M) and VP101(hG1DM) of the present invention, and the second bispecific protein is selected from the fusion proteins TF01, TF02 and TF01T of the anti-TIGIT antibody and TGF-βR of the present invention.

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

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

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

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

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

[0145] In some embodiments of the present invention, the drug 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 invention, the drug combination, wherein the mass ratio of the first bispecific protein to the second bispecific protein is (10:1) to (1:5), preferably (5:1) to (1:3) or (3:1) to (1:2), and more preferably 2:1.

[0147] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) of the present invention, and the second bispecific protein is the fusion protein TF01T of the anti-TIGIT antibody of the present invention and TGF-βR, and the mass ratio of the first bispecific protein to the second bispecific protein is (10:1) to (1:5), preferably (5:1) to (1:3) or (3:1) to (1:2), and more preferably 2:1.

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

[0149] In some embodiments of the present invention, the drug combination further comprises an effective amount of tumor chemotherapy drugs, such as platinum drugs, alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones or immune preparations.

[0150] In some embodiments of the present invention, the drug combination further comprises one or more pharmaceutically acceptable excipients; preferably, further comprises a product instruction sheet.

[0151] In some embodiments of the present invention, the drug combination, wherein the molar ratio of the first bispecific protein to the second bispecific protein is (10:1) to (1:10), preferably (5:1) to (1:5), (3:1) to (1:3) or (2:1) to (1:2), and more preferably 1:1.

[0152] In some embodiments of the present invention, the drug combination, wherein the first bispecific protein is the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) of the present invention, and the second bispecific protein is the fusion protein TF01T of the anti-TIGIT antibody of the present invention and TGF-βR, and the molar ratio of the first bispecific protein to the second bispecific protein is (10:1) to (1:10), preferably (5:1) to (1:5), (3:1) to (1:3) or (2:1) to (1:2), and more preferably 1:1.

[0153] In some embodiments of the invention, the first bispecific protein and the second bispecific protein are pharmaceutically effective ingredients (API) or active ingredients.

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

[0155] In some embodiments of the invention, the first bispecific protein, the second bispecific protein and the tumor chemotherapeutic drug are pharmaceutically effective ingredients (API) or active ingredients.

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

[0157] The pharmaceutical combination according to any one of the present invention is used for treating or preventing tumors;

[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, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, urothelial carcinoma, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, multiple myeloma, plasma cell cancer and pancreatic cancer;

[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 kidney 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's lymphoma or B-lymphocytic neoplasm.

[0165] In another aspect of the present invention, it relates to a unit preparation, preferably for treating tumors, wherein the unit preparation 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 aspect of the present invention 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 aspect of the present invention, and optionally one or more chemotherapeutic drugs according to the present invention (such as platinum drugs and / or fluorouracil anti-tumor drugs); wherein the first bispecific protein, the second bispecific protein and the chemotherapeutic drug are packaged separately.

[0166] The present invention relates to a method for preventing or treating cancer or tumors, wherein one or more unit preparations of the present invention are administered to a subject in need thereof, preferably, the first bispecific protein, the second bispecific protein and the chemotherapeutic drug in the unit preparation are administered separately.

[0167] In another aspect of the present invention, it relates to a single pharmaceutical dosage unit, preferably for treating tumors, 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 a first bispecific protein according to any one of the present invention 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 a second bispecific protein according to any one of the present invention.

[0168] In one or more embodiments of the present invention, the first bispecific protein, the second bispecific protein and / or the chemotherapeutic drug are in a form suitable for intravenous injection or intravenous drip, preferably in the form of a liquid preparation.

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

[0170] Preferably, the dosage is from twice a day to about once every other day, or once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks;

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

[0172] Another aspect of the present invention relates to the use of the drug combination described in any one of the present invention in the preparation of a drug for treating or preventing tumors;

[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, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, urothelial carcinoma, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, multiple myeloma, plasma cell cancer and pancreatic cancer;

[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 kidney 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's lymphoma or B-lymphocytic neoplasm.

[0180] Another aspect of the present invention relates to a method for treating or preventing tumors, comprising the step of administering an effective amount of the drug combination described in any one of the present invention to a subject in need;

[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, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, urothelial carcinoma, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, multiple myeloma, plasma cell cancer and pancreatic cancer;

[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 kidney 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's lymphoma or B-lymphocytic neoplasm.

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

[0189] In some embodiments of the present invention, the method for treating or preventing tumors, wherein:

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

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

[0192] and / or

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

[0194] Antibody therapeutics, especially monoclonal antibodies (mABs), have achieved good results in the treatment of many diseases. The traditional experimental method to obtain these therapeutic antibodies is to immunize animals with antigens, obtain antibodies targeting the antigen in the immunized animals, or improve antibodies with low affinity to the antigen through affinity maturation.

[0195] The variable regions of the light and heavy chains determine the binding of antigens; the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3; they were named by Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda Md),

[0196] Preferably, CDRs can also be defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSFand MhcSF." Nucleic acids research 38. suppl_1 (2009): D301-D307.

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

[0198] (1)14C12

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

[0200] The amino acid sequences of the three CDR regions of its heavy chain variable region 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 three CDR regions of its light chain variable region 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 shown in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:26.

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

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

[0212] (3)14C12H1L1(M)

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

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

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

[0216] (4) Bevacizumab

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

[0218] The amino acid sequences of the three CDR regions of its 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 three CDR regions of its 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 in SEQ ID NO:46, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:48.

[0228] The amino acid sequences of the three CDR regions of its heavy chain variable region 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 three CDR regions of its light chain variable region 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 three HCDRs and three LCDRs of the humanized TIGIT monoclonal antibody are the same as those of the mouse TIGIT monoclonal antibody.

[0237] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[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 PD-1ECD of PD-1 or a fragment containing PD-1ECD; it also includes a fusion protein of PD-1ECD, such as a fragment fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that in the amino acid sequence of PD-1 protein, mutations or variations (including but not limited to substitutions, deletions and / or additions) may be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "PD-1 protein" or "PD-1" shall include all such sequences, including natural or artificial variants thereof. Furthermore, when describing a sequence fragment of PD-1 protein, it also includes a corresponding sequence fragment in its natural or artificial variant.

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

[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 VEGFR2-ECD of VEGFR2 or a fragment comprising VEGFR2-ECD; it also includes the fusion protein of VEGFR2-ECD, such as a fragment fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions) may be naturally generated or artificially introduced into the amino acid sequence of VEGFR2 protein without affecting its biological function. Therefore, in the present invention, the term "VEGFR2 protein" or "VEGFR2" shall include all such sequences, including its natural or artificial variants. And, when describing the sequence fragment of VEGFR2 protein, it also includes the corresponding sequence fragment in its natural or artificial variant.

[0241] As used herein, unless otherwise specified, the VEGFR is VEGFR1 and / or VEGFR2; its specific protein sequence is a known sequence in the prior art, and can refer to the sequence disclosed in existing literature 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 the TIGIT protein, or the extracellular immunoglobulin variable region (IgV) domain or a fragment containing the extracellular immunoglobulin variable region (IgV) domain; it also includes a fusion protein of TIGIT, such as a fragment fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) may be naturally generated or artificially introduced into the amino acid sequence of the TIGIT protein without affecting its biological function. Therefore, in the present invention, the term "TIGIT protein" or "TIGIT" shall include all such sequences, including the sequence shown and its natural or artificial variants. Furthermore, when describing a sequence fragment of the TIGIT protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.

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

[0244] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. In a general sense, the heavy chain can be understood as the polypeptide chain with a larger molecular weight in the antibody, and the light chain refers to the polypeptide chain with a smaller molecular weight in the antibody. Light chains can be classified as κ and λ light chains. Heavy chains can generally be classified as μ, δ, γ, α or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are connected by a J region of about 12 or more amino acids, and the heavy chain also includes a D region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H1 , C H2 and C H3Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) respectively form antibody binding sites. The allocation of amino acids to each region or domain follows the definition of Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The term "antibody" is not limited by any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0245] Antibody antigen-binding fragments (e.g., those described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and screened for specificity in the same manner as for intact antibodies.

[0246] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0247] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least two or more different antibodies, which usually recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. (Kohler et al., Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, for example, US Patent 4,816,567).

[0248] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity. In addition, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, Jones et al., Nature, 321:522 525 (1986); Reichmann et al., Nature, 332:323 329 (1988); Presta, Curr. Op. Struct. Biol., 2:593 596 (1992); and Clark, Immunol. Today 21:397 402 (2000).

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

[0250] As used herein, the term "isolated" or "isolated" refers to that obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

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

[0252] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[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 Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0254] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less affinity (K D ) binds to the antigen. In some embodiments of the present invention, the term "targeting" refers to specific binding.

[0255] As used herein, the term “K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind to the antigen with a dissociation equilibrium constant of less than about 10 -5M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or less dissociation equilibrium constant (K D ) is bound to an antigen, e.g., as measured using surface plasmon resonance (SPR) in a BIACORE instrument, or by a FortebioOctet molecular interaction instrument.

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

[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 pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic 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 immunopotentiator that, when delivered into the body together with an antigen or in advance, can enhance the body's immune response to the antigen or change the type of immune response. There are many kinds of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvants are used more in clinical trials.

[0259] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, a disease prevention effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease; a disease treatment effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0260] A "recurrent" cancer is one that grows back in the original site or at a distant site after responding to initial treatment (such as surgery). A "locally recurrent" cancer is one that appears after treatment in the same location as a previously treated cancer.

[0261] "Metastatic" cancer is cancer that has spread from one part of the body (such as the lungs) to another part of the body.

[0262] As used herein, the term "completely eliminate" refers to the absence of binding signal or extremely low binding signal as detected by existing instrumentation (e.g., FortebioOctet molecular interaction instrument). In one embodiment of the present invention, the absence of binding signal or extremely low binding signal refers to a binding signal (i.e., response value or Response) lower than 0.1 nm.

[0263] In the present invention, unless otherwise specified, the “first” (e.g., a first bispecific antibody, a first protein functional region, a first linker, etc.), “second” (e.g., a second bispecific antibody, a second protein functional region, a second linker, etc.), “third” (e.g., a third protein functional region, a third linker, etc.) and “fourth” (e.g., a fourth protein functional region, a fourth linker, etc.) are for the purpose of distinguishing references or clarifying expressions, and do not have a typical order meaning.

[0264] In the present invention, unless otherwise specified, "about" or "approximately" means fluctuating within a range of 10%, 20% or 30% of the modified numerical value or physical quantity, for example, about 100 minutes or about 100 minutes can be 90 minutes-110 minutes, 80 minutes-120 minutes or 70 minutes-130 minutes.

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

[0266] Advantageous Effects of the Invention

[0267] The present invention achieves one or more of the following technical effects (1) to (2):

[0268] (1) The drug combination of the present invention 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 THE DRAWINGS

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

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

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

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

[0274] The partial sequence involved in the present invention is as follows:

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

[0276]

[0277]

[0278] 20.14C12 amino acid sequence of heavy chain variable region: (118aa)

[0279]

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

[0281]

[0282] 22.14C12 light chain variable region encoded amino acid sequence: (107aa)

[0283]

[0284] 23. Nucleic acid sequence encoding the heavy chain variable region of 14C12H1L1: (354 bp)

[0285]

[0286]

[0287] 24.14C12H1L1 Heavy chain variable region 14C12H1 (M) V Amino acid sequence: (118aa)

[0288]

[0289] 25. Nucleic acid sequence encoding the light chain variable region of 14C12H1L1: (321 bp)

[0290]

[0291] 26.14C12H1L1 light chain variable region amino acid sequence: (107aa)

[0292]

[0293] 27. Nucleic acid sequence encoding 14C12H1L1 heavy chain (14C12H1): (1344 bp)

[0294]

[0295]

[0296] 28. Amino acid sequence of 14C12H1L1 heavy chain (14C12H1): (448aa)

[0297]

[0298] 29. Nucleic acid sequence encoding 14C12H1L1 light chain (14C12L1): (642 bp)

[0299]

[0300]

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

[0302]

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

[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): (107aa)

[0308]

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

[0310]

[0311]

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

[0313]

[0314] 36. Connector

[0315]

[0316] 37. Connector

[0317]

[0318] 38. The amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1WT): (453aa)

[0319]

[0320] 39. Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1WT): (1359 bp)

[0321]

[0322]

[0323] 40. Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG4WT): (450 aa)

[0324]

[0325] 41. Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG4WT): (1350 bp)

[0326]

[0327] 42. The amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1DM): (453aa, the mutation site is underlined)

[0328]

[0329]

[0330] 43. Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1DM): (1359 bp, the mutation site is underlined)

[0331]

[0332] 44. The amino acid sequence of the light chain of the immunoglobulin portion of VP101 (hG1DM): (214aa)

[0333]

[0334] 45. Nucleic acid sequence encoding the light chain of the immunoglobulin portion of VP101 (hG1DM): (642 bp)

[0335]

[0336] Amino acid sequence of 46.26B12VH

[0337]

[0338] Nucleic acid sequence of 47.26B12VH

[0339]

[0340]

[0341] Amino acid sequence of 48.26B12VL

[0342]

[0343] Nucleic acid sequence of 49.26B12VL

[0344]

[0345] Amino acid sequence of 50.26B12H1VH

[0346]

[0347] Nucleic acid sequence of 51.26B12H1VH

[0348]

[0349] Amino acid sequence of 52.26B12H2VH

[0350]

[0351]

[0352] Nucleic acid sequence of 53.26B12H2VH

[0353]

[0354] Amino acid sequence of 54.26B12H3VH

[0355]

[0356] Nucleic acid sequence of 55.26B12H3VH

[0357]

[0358] Amino acid sequence of 56.26B12H4VH

[0359]

[0360] Nucleic acid sequence of 57.26B12H4VH

[0361]

[0362]

[0363] Amino acid sequence of 58.26B12L1VL

[0364]

[0365] Nucleic acid sequence of 59.26B12L1VL

[0366]

[0367] Amino acid sequence of 60.26B12L2VL

[0368]

[0369] Nucleotide sequence of 61.26B12L2VL

[0370]

[0371] Amino acid sequence of 62.26B12L3VL

[0372]

[0373] Nucleic acid sequence of 63.26B12L3VL

[0374]

[0375] Amino acid sequence of 64.26B12L4VL

[0376]

[0377] Nucleic acid sequence of 65.26B12L4VL

[0378]

[0379] Amino acid sequence of 66.26B12H2L2 (hG4DM) heavy chain

[0380]

[0381]

[0382] Nucleic acid sequence of 67.26B12H2L2 (hG4DM) heavy chain

[0383]

[0384] Amino acid sequence of 68.26B12H2L2 (hG4DM) light chain

[0385]

[0386] 69.26B12H2L2 (hG4DM) light chain nucleic acid sequence

[0387]

[0388] 70.26B12H2L2 (hG1DM) heavy chain amino acid sequence

[0389]

[0390] 71.26B12H2L2 (hG1DM) heavy chain nucleic acid sequence

[0391]

[0392]

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

[0394]

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

[0396]

[0397]

[0398] 74.Amino acid sequence of the extracellular region of TGF-βRⅠ:

[0399]

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

[0401]

[0402] 76.Amino acid sequence of the extracellular region of TGF-βRⅡ:

[0403]

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

[0405]

[0406]

[0407] 78.Amino acid sequence of the extracellular region of TGF-βRⅢ:

[0408]

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

[0410]

[0411]

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

[0413]

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

[0415]

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

[0417]

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

[0419]

[0420] 84. Anti-HEL & TGFβ antibody heavy chain amino acid sequence

[0421]

[0422]

[0423] 85. Anti-HEL & TGFβ antibody light chain amino acid sequence

[0424]

[0425] 86. Heavy chain amino acid sequence of RG6058 (G1DM)

[0426]

[0427] 87. Light chain amino acid sequence of RG6058 (G1DM)

[0428]

[0429] The amino acid sequence of the TGF-βRII extracellular region fragment variant of 88.TF01T is:

[0430]

[0431] 89.hIgG4WT heavy chain constant region amino acid sequence

[0432]

[0433] 90.hIgG1WT heavy chain constant region amino acid sequence

[0434]

[0435] 91. Repeating units in linkers

[0436]

[0437] 92. Fragments in the linker

[0438] DETAILED DESCRIPTION

[0439] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0440] The sequence of the isotype control antibody is human anti-hen egg lysosome antibody (human anti-Hen Egg Lysozyme IgG, i.e., anti-HEL antibody, or human IgG, referred to as hIgG) from the variable region sequence of the Fab F10.6.6 sequence in Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374 (1): 130-146.). The hIgG1DM used in the embodiment is the isotype control antibody of anti-HEL with the hG1DM constant region sequence, which was prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd., batch number: 20190520.

[0441] Jurkat-NFAT-PD1-hTIGIT is a cell expressing human TIGIT constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. based on Jurkat-NFAT-PD1 cells (PD1 Effector cells, Promega, catalog number: J112A) through transfection.

[0442] Preparation Example 1: Sequence Design of Anti-PD-1 Antibody 14C12 and Humanized Antibody 14C12H1L1

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

[0444] (1) Heavy chain variable region sequence and light chain variable region sequence of 14C12

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

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

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

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

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

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

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

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

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

[0454] Nucleic acid sequence encoding 14C12H1L1 heavy chain (14C12H1): SEQ ID NO: 27;

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

[0456] Nucleic acid sequence encoding 14C12H1L1 light chain (14C12L1): SEQ ID NO: 29;

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

[0458] Preparation Example 2: Preparation of anti-VEGFA antibody Bevacizumab

[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) refer to Chinese patent publication CN1259962A. The nucleic acid sequences encoding the heavy chain variable region and the light chain variable region were commissioned to be synthesized by GenScript.

[0460] Amino acid sequence of Bevacizumab heavy chain variable region (Bevacizumab-Hv): SEQ ID NO: 31;

[0461] Nucleic acid sequence encoding Bevacizumab heavy chain variable region (Bevacizumab-Hv): SEQ ID NO: 32;

[0462] Amino acid sequence of Bevacizumab light chain variable region (Bevacizumab-Lv): SEQ ID NO: 33;

[0463] Nucleic acid sequence encoding Bevacizumab light chain variable region (Bevacizumab-Lv): SEQ ID NO:34.

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

[0465] The heavy chain cDNA and light chain cDNA of Bevacizumab were cloned into the pcDNA3.1 vector to obtain the recombinant expression plasmid of the antibody Bevacizumab. The recombinant plasmid was transfected into 293F cells. The 293F cell culture fluid was purified and then tested.

[0466] The anti-VEGFA monoclonal antibody Avastin (Bevacizumab) was produced.

[0467] Preparation Example 3: Sequence Design of Mutant 14C12H1L1(M) of Anti-PD-1 Humanized Antibody 14C12H1L1

[0468] Based on 14C12H1L1, individual amino acids in its framework region (light chain) were mutated to obtain 14C12H1L1(M).

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

[0470] Light chain variable region of 14C12H1L1(M)14C12L1(M) V :SEQ ID NO:35.

[0471] Preparation Example 4: Sequence Design of Bispecific Antibodies

[0472] 1. Sequence Design

[0473] The structural model of the bispecific antibody in the present invention belongs to the Morrison model (IgG-scFv), that is, the C-termini of the two heavy chains of an IgG antibody are connected to the scFv fragment of another antibody, and the main components of the heavy chain and light chain are designed as shown in Table 1 below.

[0474] Based on the above-mentioned Bevacizumab, the VP101 antibody with the amino acid sequences of the heavy chain variable region and light chain variable region of the above-mentioned 14C12H1L1(M) as the ScFv fragment is called VP101(M). Compared with 14C12H1L1, 14C12H1L1(M) effectively optimizes the structure of the bispecific antibody and improves its effectiveness.

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

[0476]

[0477] In Table 1 above:

[0478] (1) The "V" in the lower right corner refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. If "V" is not marked, the corresponding heavy chain or light chain is the full length including the constant region. The amino acid sequences of these variable regions or full lengths and their encoding nucleic acid sequences are all referred to the corresponding sequences recorded in the above preparation examples.

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

[0480] Optionally, 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 light chain cDNA sequence of VP101(M) were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-VP101H and pUC57simple-VP101L plasmids, respectively.

[0485] Plasmids pUC57simple-VP101H and pUC57simple-VP101L were digested (HindIII & EcoRI), and the heavy and light chains were recovered by electrophoresis and subcloned into pcDNA3.1 vectors. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and then loaded onto the HiTrap MabSelectSuRe column. The protein was eluted in one step with Elution Buffer to recover the target sample antibody VP101, and the solution was changed to PBS.

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

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

[0488] In order to distinguish it from the mutated antibody in the subsequent Preparation Example 5, VP101(M) is also referred to as VP101(hG1WT) in the present invention. The above-mentioned VP101(M) is a "wild type" and uses 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 (Bevacizumab-H) of the immunoglobulin portion of VP101 (hG1WT): SEQ ID NO: 38;

[0490] Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1WT): SEQ ID NO:39.

[0491] In order to distinguish it from the mutated antibody in the subsequent Preparation Example 5, VP101 (G4M) is also referred to as VP101 (hG4WT) in the present invention. The above-mentioned VP101 (G4M) is a "wild type", which uses Ig gamma-4 chain 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] The amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG4WT): SEQ ID NO: 40;

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

[0494] Preparation Example 5: Design of amino acid mutations in the non-variable region of the humanized bispecific antibody VP101 (hG1WT)

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

[0496] Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1DM) (453 aa, the mutation site is underlined): SEQ ID NO: 42;

[0497] Nucleic acid sequence encoding the heavy chain of the immunoglobulin part in VP101 (hG1DM) (1359 bp, the mutation site is underlined): SEQ ID NO:43.

[0498] The light chain amino acid sequences of the immunoglobulin portions of VP101(hG1DM), VP101(hG1WT) and VP101(hG4WT) are identical, and their encoding nucleic acid sequences are also identical.

[0499] The amino acid sequence of the light chain of the immunoglobulin portion of 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 antibodies

[0502] 1. Preparation of hybridoma cell line LT019

[0503] The antigen used to prepare anti-TIGIT antibodies is human TIGIT-mFc (TIGIT is Genbank ID: NP_776160.2). The spleen cells of immunized mice were fused with mouse myeloma cells to make hybridoma cells. Using human TIGIT-hFc as an antigen, the hybridoma cells were screened by indirect ELISA to obtain hybridoma cells that can secrete antibodies that specifically bind to TIGIT. The hybridoma cells obtained by screening were subjected to limiting dilution to obtain stable hybridoma cell lines. The above hybridoma cell lines were named hybridoma cell lines LT019, and the monoclonal antibodies secreted by them were named 26B12.

[0504] The hybridoma cell line LT019 (also known as TIGIT-26B12) was deposited in the China Center for Type Culture Collection (CCTCC) on October 23, 2020, with the deposit number CCTCCNO: C2020208, and the deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0505] 2. Preparation of Anti-TIGIT Antibody 26B12

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

[0507] Preparation Example 7: Sequence Analysis of Anti-TIGIT Antibody 26B12

[0508] According to the method of the total RNA extraction kit for cultured cells and bacteria (Tiangen, catalog number DP430), mRNA was extracted from the LT019 cell line cultured in Preparation Example 6.

[0509] According to Invitrogen III First-Strand Synthesis Systemfor RT-PCR Kit Instructions Synthesize cDNA and perform PCR amplification.

[0510] The PCR amplification product was directly subjected to TA cloning. For specific operations, refer to the instructions of the pEASY-T1 Cloning Kit (Transgen CT101).

[0511] The products of TA cloning were directly sequenced, and the sequencing results were as follows:

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

[0513] The amino acid sequence encoded by it is shown in SEQ ID NO:46, with a length of 121 amino acids.

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

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

[0516] The amino acid sequence encoded by it is shown in SEQ ID NO: 48, with a length of 107 amino acids.

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

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

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

[0520] Based on the three-dimensional crystal structure of human TIGIT protein and the sequence of antibody 26B12 obtained in Preparation Example 7, the variable region sequences of antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 were designed (antibody constant region sequences, the heavy chain constant region all adopted Ig gamma-1 chain C region; the light chain constant region was 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 above 8 antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4, the length of the nucleic acid sequence of the heavy chain variable region is 363bp, and the length of the amino acid sequence encoded by them is 121aa; the length of the nucleic acid sequence of the light chain variable region is 321bp, and the length of the amino acid sequence encoded by them is 107aa.

[0525] And the above 8 antibodies have the same HCDR1-HCDR3 and LCDR1-LCDR3, as follows:

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

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

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

[0529] The heavy chain constant regions all used Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant regions all used Ig kappa chain C region, ACCESSION: P01834.

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

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

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

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

[0542] Based on 26B12H2L2, the inventors obtained the humanized antibody 26B12H2L2 (hG1DM) with constant region mutations by introducing a point mutation from leucine to alanine (L234A) at site 234 (according to the EU numbering system, the same below) and a point mutation from leucine to alanine (L235A) at site 235 of its heavy chain constant region.

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

[0544] Based on 26B12H2L2, the inventors kept the antibody variable region unchanged, used Iggamma-4chain Cregion, ACCESSION: P01861.1 as the heavy chain constant region, and introduced a point mutation from phenylalanine to alanine (F234A) at site 234 of the heavy chain constant region, and a point mutation from leucine to alanine (L235A) at site 235, thereby obtaining a humanized antibody 26B12H2L2 (hG4DM) with a constant region mutation.

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

[0546] The preparation of 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 fusion protein of the anti-TIGIT antibody and TGF-βR in the present invention is shown in Table 3 below.

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

[0551]

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

[0553] 2. Antibody Expression and Purification

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

[0555] Plasmids pUC57simple-TF01H / pUC57simple TF01L and pUC57simple-TF02H / pUC57simple TF02L were digested (HindIII & EcoRI), and the heavy and light chains recovered by electrophoresis were subcloned into pcDNA3.1 vectors, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and then loaded onto the HiTrap MabSelect SuRe column. The protein was eluted in one step with Elution Buffer, the target sample was recovered, and the solution was replaced with PBS. In this way, 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] A point mutation was performed on the TGF-βRII extracellular region fragment in the fusion protein TF01 of the anti-TIGIT antibody and TGF-βR in the present invention, and the serine at position 8 of the TGF-βRII extracellular region fragment (SEQ IDNO: 72) was mutated to threonine (SEQ IDNO: 88) to obtain a new antibody named TF01T. The preparation method thereof is referred to Preparation Example 9.

[0558] Table 4: Composition of heavy and light chains of fusion proteins (mutants) of anti-TIGIT antibodies and TGF-βR

[0559]

[0560] Example 1: Combination of anti-TIGIT antibody and TGF-βR fusion protein with anti-PD-1-anti-VEGFA bispecific antibody Promotes INF-γ secretion

[0561] Separation medium Ficoll-Paque TMNormal human PBMC (isolated from healthy human peripheral blood) was isolated according to the operating instructions of Cytiva Plus (Cytiva, Cat: 17-1440-02). One day before the experiment, 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, antibodies (wherein the molar ratio of VP101 (hG1DM) to TF01T was 1:1), CMV (Mabtech, Catalog No.: 3619-1) and TGF-β1 (Genscript, Catalog No.: Z03411) diluted in gradient with complete medium were used for grouping according to the experimental design. TGF-β1 (final concentration of 3 ng / mL) and antibodies were added in equal volumes to a 96-well round-bottom plate (Corning, Model: 3799), and a control group was designed. The cells were incubated at 37°C for 10 min. PBMCs were collected, counted and viability was measured. 200,000 cells / well were inoculated in a 96-well round-bottom plate, and CMV with a final concentration of 0.2 μg / mL was added. After gently tapping the edge of the plate to mix, the cells were placed in an 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 (Dakoway, Catalog No.: 1110002).

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

[0563] The results showed that under CMV stimulation, VP101(hG1DM) alone, TF01T alone, and VP101(hG1DM) plus TF01T could effectively promote PBMC to secrete IFN-γ, and the ability of VP101(hG1DM) plus TF01T to promote PBMC to secrete IFN-γ was significantly stronger than that of each single drug group. After adding TGF-β1, which can inhibit IFN-γ secretion in the system, the IFN-γ secretion of VP101(hG1DM) plus TF01T group was higher than that without TGF-β1, showing a stronger immunobiological activity of promoting PBMC to recall the response to CMV.

[0564] Example 2: Reporter gene system verification of anti-TIGIT antibody and TGF-βR fusion protein and anti-PD-1-anti-VEGFA Blocking activity of bispecific antibody combinations

[0565] PDL1 aAPC / CHO-K1 cells (purchased from Promega, catalog number: J108A) were resuspended in Ham's F-12 + 10% FBS medium (Ham's F-12, Gibco, catalog number: 11765070; FBS, fetal bovine serum, Excell Bio, catalog number: FSP500) and inoculated into a 96-well flat-bottom black plate (Corning, model: 3916) at 40,000 / well / 100 μL for overnight culture. The next day, according to the experimental design, antibodies and VEGF (Kangfang Bio's own, batch number: 20210508) were diluted in assay medium (i.e., RPMI 1640 + 10% FBS; RPMI 1640, Gibco, catalog number: 22400-089; FBS, fetal bovine serum, Excell Bio, catalog number: FSP500), and the antibodies and VEGF were pre-incubated at 37°C for 30 minutes. Jurkat-NFAT-PD1-hTIGIT cells (Zhongshan Kangfang Bioconstruction) and A549 cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number: SCSP-503) were collected separately, resuspended in analysis medium, and the cell density was adjusted. The medium in the 96-well flat-bottom black plate was removed, and 40 μL / well of antibody or antibody and VEGF preincubation solution (wherein the molar ratio of VP101 (hG1DM) to TF01T was 1:1) was added according to the experimental design, 50,000 Jurkat-NFAT-PD1-hTIGIT cells / well / 20 μL were added, and 10,000 A549 cells / well / 20 μL were added, and isotype control group (human IgG1DM) and blank control group were set up, and incubated in an incubator for 6 hours. The 96-well flat-bottom black plate was taken out and equilibrated to room temperature. 80 μL / well of FireflyGlo Luciferase Reporter Gene Assay Kit (Yeasen, Catalog No.: 11404ES80) was added. After incubation in the dark for 5 min, the RLU value was read.

[0566] The results are as follows Figure 3As shown. In this reporter gene system, the interaction between TIGIT / PVR and PD-1 / PD-L1 can inhibit the activation of the NFAT signaling pathway. Therefore, blocking TIGIT / PVR and (or) PD-1 / PD-L1 can activate the NFAT pathway of effector cells and activate the expression of the luciferase reporter gene. The higher the luciferase expression, the stronger the fluorescence intensity. The results showed that the VP101 (hG1DM) monotherapy group, the TF01T monotherapy group, and the VP101 (hG1DM) combined with TF01T group can effectively play a neutralizing role in blocking the interaction between TIGIT / PVR and PD-1 / PD-L1, and the neutralizing biological activity of VP101 (hG1DM) combined with TF01T in blocking TIGIT / PVR and PD-1 / PD-L1 is better than that of VP101 (hG1DM) monotherapy and TF01T monotherapy.

[0567] In addition, VEGF was added to the reporter gene system for pretreatment. Figure 4 The results showed that compared with the isotype control human IgG1DM, both the VP101(hG1DM) monotherapy group and the VP101(hG1DM) combined with TF01T group could significantly increase the expression of luciferase; and the blocking activity of VP101(hG1DM) combined with TF01T was higher than that of VP101(hG1DM) monotherapy group.

[0568] In summary, the neutralizing biological activity of VP101(hG1DM) combined with TF01T in blocking TIGIT / PVR and PD-1 / PD-L1 is better than that of VP101(hG1DM) alone and TF01T alone, and in a system containing VEGF, VP101(hG1DM) combined with TF01T exhibits stronger neutralizing activity.

[0569] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

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

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

35.

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

35.

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

33.

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

64.

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

64.

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

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

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

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

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

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

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

44.

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

68.

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

88.

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

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

83.

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

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

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

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

36.

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

83.

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

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

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

1.

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

1.

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

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

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

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

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