Fusion proteins comprising anti-tigit antibodies and tgf-beta rs, pharmaceutical compositions thereof, and uses

Abstract

The present application belongs to the field of biology and medicine, and relates to a fusion protein comprising an anti-TIGIT antibody and a TGF-beta R, a pharmaceutical composition thereof and purposes. Specifically, the fusion protein of the present application comprises: a first protein functional region targeting TIGIT, and a second protein functional region having TGF-beta binding activity; wherein the first protein functional region is an anti-TIGIT antibody or an antigen binding fragment thereof; and the heavy chain variable region of the anti-TIGIT antibody comprises HCDR1-HCDR3 with the amino acid sequences shown in SEQ ID NOs: 3-5, respectively; and the light chain variable region thereof comprises LCDR1-LCDR3 with the amino acid sequences shown in SEQ ID NOs: 8-10, respectively. The fusion protein of the present application can simultaneously inhibit TIGIT and reduce the level of TGF-beta, and has good potential for preparing anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the fields of biology and medicine, and relates to a fusion protein comprising an anti-TIGIT antibody and TGF-βR, its pharmaceutical composition, and its uses. Background Technology

[0002] 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 classical immunoreceptor tyrosine inhibitory motif (ITIM) and immunoglobulin tyrosine tail (ITT) motifs. TIGIT is highly expressed in lymphocytes, especially 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).

[0003] 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 the killing effect of NK cells on cells that highly express 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). However, reports have found that blocking PD-1 and TIGIT simultaneously can enhance the killing effect of CD8+ T cells (Johnston RJ, Comps-Agrar L, Hackney J, et al. The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function[J]. Cancercell, 2014, 26(6):923-937).The latest research found that TIGIT, as an immune checkpoint of NK cells, can lead to NK cell exhaustion during tumor development. It has been shown that anti-TIGIT monoclonal antibodies can reverse NK cell exhaustion and have been 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-cell lymphoma, plasma cell carcinoma, etc. (Zhang Q, Bi J, Zheng X, et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity[J]. Nature immunology, 2018, 19(7):723-732).

[0004] Furthermore, reports indicate that TIGIT inhibitors, alone or in combination with PD-1 inhibitors plus CD96 inhibitors, can significantly reduce the growth of B16 melanoma in wild-type and CD155- / - mouse models (Li XY, Das I, Lepletier A, et al. Cd155 loss enhances tumor suppression via combined host and tumor-intrinsic mechanisms. J Clin Invest 2018; 128:2613-25). CD112R inhibitors, alone or in combination with TIGIT inhibitors and / or PD-1 inhibitors, can increase the ability of TILs to produce cytokines in ovarian tumors, endometrial tumors, and lung tumors (Whelan S, Ophir E, Kotturi MF, et al. PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+ T-cell Function. Cancer Immunol Res 2019; 7:257-68).

[0005] The transforming growth factor-β (TGF-β) superfamily is a diverse group of cytokines, further divided into subfamilies such as TGF-β, activins, inhibins, growth differentiation factor (GDF), glial-derived neurotrophic factor (GDNFs), Nodal, Lefty, and anti-Müllerian hormone (Table A). The proteins of the three TGF-β subfamilies are respectively designated as TGF-β1, TGF-β2, and TGF-β3, with TGF-β1 being the most highly expressed subtype. After TGF-β1, TGF-β2, and TGF-β3 bind to their receptors, they mediate a series of biological responses through Smad and non-Smad signaling pathways, including promoting epithelial-mesenchymal transitions (EMT), promoting tissue fibrosis, promoting angiogenesis, promoting tumor immune escape, and having both anti-cancer and cancer-promoting effects (J Massagué. TGFbeta in Cancer.[J].Cell,2008,134(2):215-230.).

[0006] TGF-β receptors (TGF-βR) are widely distributed on the surface of normal and tumor cells in humans, including three superfamilies of receptors. 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 written as TGF-βRIII) and Endoglin. Among them, TGF-βRⅠ, TGF-βRII, and TGF-βRIII can each bind to TGF-β1, TGF-β2, and TGF-β3 (Pawlak John B, Blobe Gerard C, TGF-β superfamily co-receptors in cancer. DevDyn, 2021). The TGF-β family and its receptors are shown in Table A (based on a review summary by Carl-Henrik Heldin1 and Aristidis Moustakas. Cold Spring HarbPerspect Biol. 2016.). TGF-βRII and TGF-βRII are serine / threonine protein kinase receptors. TGF-βRII is a core molecule in the TGF-β signaling pathway, binding with high affinity to TGF-β and subsequently forming a heterotetrameric receptor complex with the TGF-βRI dimer. TGF-βRII then phosphorylates and activates TGF-βRII via autophosphorylation. Activated TGF-βRII phosphorylates downstream Smad pathway-related proteins, regulating the transcription and translation of downstream target genes, thereby triggering disease-related biological responses. TGF-βRIII has a weaker affinity for TGF-β than TGF-βRII and TGF-βRII and lacks an intracellular segment, thus it cannot connect to downstream signaling pathways. Its function is to capture TGF-β and present it to TGF-βRII. Other TGF-β receptors can also bind to TGF-β (Sang Xiaohong et al., Research progress on small molecule inhibitors targeting TGF-β and receptors [J]. Acta Pharmaceutica Sinica, 2019(9).).

[0007] Table A: TGF-β family and receptors

[0008]

[0009]

[0010]

[0011] During tumor progression, tumor cells, mesenchymal fibroblasts, and other cells in the tumor microenvironment (TME) secrete large amounts of TGF-β, mediating immunosuppression. TGF-β inhibits the differentiation of naive T cells into Th1 cells that mediate anti-tumor activity, and T cells lacking TGF-βRII exhibit enhanced Th1 responses (Eduard, Batlle, Joan, et al. Transforming Growth Factor-β Signaling in Immunity and Cancer.[J].Immunity,2019.). The expression levels of granzyme A, granzyme B, perforin, interferon-gamma (IFN-γ), and FasL on effector T cells with tumor-killing activity decrease under the influence of TGF-β, leading to tumor cell immune escape. Tregs are key cells in tumor microenvironment-mediated tumor immunosuppression, inhibiting the function of effector T cells with tumor-killing activity. TGF-β produced by tumor cells induces a large number of Tregs in the tumor microenvironment, enhancing tolerance to tumor antigens and promoting tumor immune escape (Chen Dan, Ran Yan. Research progress on the regulation of tumor cells and tumor-associated immune cells by TGF-β [J]. Modern Medicine and Health, 2020, 36(09):1354-1358.). Currently, the indications for investigational antibody drugs targeting TGF-β molecules in the clinical stage include melanoma, renal cell carcinoma, breast cancer, cervical cancer, advanced non-small cell lung cancer, prostate cancer, pancreatic ductal adenocarcinoma, advanced solid tumors, and metastatic solid tumors.

[0012] Fc receptors are immunoglobulin family proteins expressed on the surface of specific immune cells, used to recognize antibody Fc regions and mediate immune responses. After the antibody Fab region recognizes an antigen, its antibody Fc region binds to the Fc receptor on immune cells (such as killer cells), initiating immune cell responses such as phagocytosis and ADCC.

[0013] Based on the type of antibody recognized by the Fc receptor and the different cells expressing it, Fc receptors are mainly divided into three types: FcγR, FcαR, and FcεR. FcγR can be further divided into four subtypes: FcγRI (also known as CD64), FcγRII (also known as CD32), FcγRIII (also known as CD16), and FcRn (also known as Neonatal Fc receptor). Among them, FcγRI, FcγRII, and FcγRIII are closely related to ADCC effects. FcγRIII is the most important molecule mediating ADCC, and it has two highly homologous subtypes, FcγRIIIa and FcγRIIIb, in different cell types. In the FcγRIIIa population, there are two subtypes: high-affinity FcγRIIIa caused by single nuclear stem polymorphism (SNP) sites, namely FcγRIIIa_V158 and low-affinity FcγRIIIa_F158. FcγRI has a high affinity for the Fc region of IgG and participates in the ADCC process. There are three subtypes of FcγRII: FcγRIIa, FcγRIIb, and FcγRIIc (also known as CD32a, CD32b, and CD32c, respectively). Among them, FcγRIIa has ADCC activity. There are two subtypes of FcγRIIa in the human population caused by single nucleotide mutations, namely FcγRIIa_H131 and FcγRIIa_R131. FcγRIIb is an inhibitory receptor. FcγRIIb is a typical inhibitory FcγR that can inhibit the nearby ITAM pathway. For example, after the immune complex binds to BCR, the Fc segment will bind to FcγRIIb on the same cell, negatively regulating B cell activation and reducing the secretion of antibodies and cytokines (Hogarth PM, Pietersz GA. 2012, NATURE REVIEWS DRUG DISCOVERY, 11(4):311-331).

[0014] Currently, there is a need to develop a drug that simultaneously inhibits the TIGIT and TGF-β signaling pathways. Summary of the Invention

[0015] Through in-depth research and creative labor, the inventors have obtained a fusion protein containing an anti-TIGIT antibody and TGF-βR. The inventors have surprisingly discovered that this fusion protein can effectively bind both TIGIT and TGF-β simultaneously, demonstrating its potential for preparing anti-tumor drugs. Therefore, the following invention is provided:

[0016] One aspect of the present invention relates to a fusion protein comprising:

[0017] Targeting the first protein functional region of TIGIT, and

[0018] A second protein functional region possessing TGF-β binding activity;

[0019] in,

[0020] The first protein functional region is an anti-TIGIT antibody or its antigen-binding fragment;

[0021] Furthermore, the anti-TIGIT antibody has a heavy chain variable region containing amino acid sequences as shown in SEQ ID NOs:3-5, namely HCDR1-HCDR3; and a light chain variable region containing amino acid sequences as shown in SEQ ID NOs:8-10, namely LCDR1-LCDR3.

[0022] In some embodiments of the present invention, the fusion protein wherein the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is selected from SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17; and

[0023] The amino acid sequence of the light chain variable region of the anti-TIGIT antibody is selected from SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:25.

[0024] In some embodiments of the present invention, the fusion protein, wherein...

[0025] The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6.

[0026] The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19.

[0027] The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19.

[0028] The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21.

[0029] The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:23.

[0030] The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21.

[0031] The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:23.

[0032] The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25; or

[0033] The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.

[0034] In some embodiments of the present invention, the fusion protein, wherein the anti-TIGIT antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, chimeric antibody or biantibody.

[0035] In some embodiments of the present invention, the fusion protein, wherein...

[0036] The anti-TIGIT antibody includes a non-CDR region, and the non-CDR region is derived from a species other than rodents, such as from human antibodies.

[0037] In some embodiments of the present invention, the fusion protein wherein the heavy chain constant region of the anti-TIGIT antibody is Ig gamma-1 chain C region (e.g., NCBI ACCESSION: P01857) or Ig gamma-4 chain C region (e.g., NCBI ACCESSION: P01861.1); and the light chain constant region is Ig kappa chain C region (e.g., NCBI ACCESSION: P01834).

[0038] In some embodiments of the present invention, the fusion protein has a stronger binding ability to the ligand CD155-hFc-Biotin than the control antibody RG6058(hG4).

[0039] In some embodiments of the present invention, the fusion protein is wherein the fusion protein is expressed at an EC50 concentration of less than 0.08 nM or less than 0.10 nM. 50 Combined with TIGIT-mFc; preferably, the EC 50 Measured by indirect ELISA method.

[0040] In some embodiments of the present invention, the fusion protein is wherein the fusion protein has an EC50 concentration of less than 0.3 nM, less than 0.4 nM, less than 0.5 nM, or less than 0.6 nM. 50 Combining TGF-β1, TGF-β2 and / or TGF-β3; preferably, the EC 50 Measured by indirect ELISA method.

[0041] In some embodiments of the present invention, the fusion protein, wherein the anti-TIGIT antibody is an antibody produced by the hybridoma cell line LT019, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:C2020208.

[0042] In some embodiments of the present invention, the fusion protein, wherein...

[0043] According to the EU numbering system, when the heavy chain constant region of the anti-TIGIT antibody is the heavy chain constant region of IgG1, the heavy chain constant region of the anti-TIGIT antibody has one of the following mutation combinations:

[0044] L234A and L235A;

[0045] L234A and G237A;

[0046] L235A and G237A; or

[0047] L234A, L235A, G237A.

[0048] In some embodiments of the present invention, the fusion protein, wherein...

[0049] According to the EU numbering system, when the heavy chain constant region of the anti-TIGIT antibody is the heavy chain constant region of IgG4, the heavy chain constant region of the anti-TIGIT antibody has one of the following combinations of mutations:

[0050] F234A and L235A;

[0051] F234A and G237A;

[0052] L235A and G237A; or

[0053] F234A, L235A, G237A.

[0054] In some embodiments of the present invention, the fusion protein, wherein...

[0055] The second protein functional region is the TGF-β receptor, its extracellular region, or a truncated fragment of the extracellular region of the TGF-β receptor with TGF-β receptor function;

[0056] Preferably, the TGF-β receptor is TGF-βRII, TGF-βRII, or TGF-βRIII, or the extracellular region of TGF-βRII, TGF-βRII, or TGF-βRIII, or a truncated fragment of the extracellular region of TGF-βRII, TGF-βRII, or TGF-βRIII;

[0057] Preferably, the amino acid sequence of the functional region of the second protein is as shown in any one of SEQ ID NOs:33-39.

[0058] In some embodiments of the present invention, the fusion protein:

[0059] The amino acid sequence of the heavy chain of the anti-TIGIT antibody is shown in SEQ ID NO:27 or SEQ ID NO:31, and the amino acid sequence of its light chain is shown in SEQ ID NO:29.

[0060] In some embodiments of the present invention, the fusion protein wherein the first protein functional region and the second protein functional region are directly connected or connected through a linker fragment.

[0061] In some embodiments of the present invention, the fusion protein is wherein the linker fragment is (GGGGS)n or (GGGGS)nG, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

[0062] In some embodiments of the present invention, the fusion protein wherein the first protein functional region and the second protein functional region are independently one, two, or more.

[0063] In some embodiments of the present invention, the fusion protein wherein the TGF-β receptor or an extracellular fragment thereof is linked to the C-terminus of the heavy chain of the anti-TIGIT antibody.

[0064] This invention relates to a fusion protein comprising:

[0065] Targeting the first protein functional region of TIGIT, and

[0066] A second protein functional region possessing TGF-β binding activity;

[0067] in,

[0068] The first protein has one functional region, and the second protein has two functional regions;

[0069] The first protein functional region is an anti-TIGIT antibody or its antigen-binding fragment, and the second protein functional region is a TGF-β receptor, its extracellular region, or a truncated fragment of the extracellular region of a TGF-β receptor with TGF-β receptor function.

[0070] The amino acid sequence of the heavy chain of the anti-TIGIT antibody is shown in SEQ ID NO:27 or SEQ ID NO:31, and the amino acid sequence of its light chain is shown in SEQ ID NO:29.

[0071] The amino acid sequence of the functional region of the second protein is shown in SEQ ID NO:33;

[0072] The second protein functional region is linked to the C-terminus of the heavy chain of the anti-TIGIT antibody via a linker fragment;

[0073] Preferably, the amino acid sequence of the linker fragment is as shown in SEQ ID NO:44.

[0074] In some embodiments of the present invention, the fusion protein is used to treat and / or prevent tumors;

[0075] Preferably, the tumor is selected from one or more of the following: 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-cell lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma.

[0076] Preferably, the non-small cell lung cancer is advanced non-small cell lung cancer.

[0077] In a broader sense, the fusion protein of this invention can also be called an antibody.

[0078] Another aspect of the invention relates to isolated nucleic acid molecules that encode the fusion protein described in any one of the invention.

[0079] Another aspect of the invention relates to a carrier comprising the isolated nucleic acid molecules of the invention.

[0080] Another aspect of the invention relates to a host cell comprising the isolated nucleic acid molecules of the invention, or the vector of the invention.

[0081] Another aspect of the present invention relates to a conjugate comprising a fusion protein portion and a conjugation portion, wherein the fusion protein portion is any one of the fusion proteins described in the present invention, and the conjugation portion is a detectable marker; preferably, the conjugation portion is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0082] In some embodiments of the present invention, the conjugate is used for the treatment and / or prevention of tumors;

[0083] Preferably, the tumor is selected from one or more of the following: 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-cell lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma.

[0084] Preferably, the non-small cell lung cancer is advanced non-small cell lung cancer.

[0085] Another aspect of the invention relates to the use of the fusion protein or conjugate of the invention in the preparation of a kit for detecting the presence or level of TIGIT and / or TGF-β in a sample.

[0086] Another aspect of the invention relates to a pharmaceutical composition comprising any one of the fusion proteins or conjugates of the invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0087] In some embodiments of the present invention, the pharmaceutical composition further comprises one or more tumor chemotherapy drugs;

[0088] Preferably, the tumor chemotherapy drug is a tyrosine kinase inhibitor; more preferably, the tumor chemotherapy drug is anlotinib or a pharmaceutically acceptable salt thereof (e.g., hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., methanesulfonate).

[0089] In some embodiments of the present invention, the pharmaceutical composition, wherein the unit dose of the pharmaceutical composition, calculated according to the mass of the fusion protein therein, is 100mg-1500mg, 200mg-1000mg, 200mg-800mg, 300mg-600mg, 400mg-500mg, or 450mg.

[0090] In some embodiments of the present invention, the pharmaceutical composition is an injection.

[0091] Another aspect of the invention relates to a combination product comprising individually packaged first and second products.

[0092] in,

[0093] The first product comprises the fusion protein, the conjugate, or the pharmaceutical composition described in any one of the present invention.

[0094] The second product comprises one or more tumor chemotherapy drugs; preferably, the tumor chemotherapy drug is a tyrosine kinase inhibitor; more preferably, the tumor chemotherapy drug is anlotinib or a pharmaceutically acceptable salt thereof (e.g., hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., methanesulfonate);

[0095] Preferably, the first product and the second product further comprise one or more pharmaceutically acceptable excipients.

[0096] Preferably, the combined product further includes a product instruction manual.

[0097] In some embodiments of the present invention, the combined product, wherein the unit dose of the first product, calculated according to the mass of the fusion protein therein, is 100mg-1500mg, 200mg-1000mg, 200mg-800mg, 300mg-600mg, 400mg-500mg, or 450mg.

[0098] In some embodiments of the present invention, the unit dose of the second product, calculated according to the mass of the active ingredient therein, is 0.1mg-100mg, 0.5mg-50mg, 1mg-20mg, 2mg-15mg, 4mg-12mg, or 8mg-12mg.

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

[0100] The first and second products are independently injectable formulations.

[0101] Another aspect of the present invention relates to the use of any fusion protein or conjugate of the present invention in the preparation of medicaments for treating and / or preventing tumors;

[0102] Preferably, the tumor is selected from one or more of the following: 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-cell lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma.

[0103] Preferably, the non-small cell lung cancer is advanced non-small cell lung cancer.

[0104] Another aspect of the invention relates to a method of treating and / or preventing tumors, comprising the step of administering to a subject in need an effective amount of any one of the fusion proteins or conjugates of the invention;

[0105] Preferably, the tumor is selected from one or more of the following: 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-cell lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma.

[0106] Preferably, the non-small cell lung cancer is advanced non-small cell lung cancer.

[0107] In some embodiments of the invention, the step of administering an effective amount of any one of the drug combinations of the invention to a subject in need is performed before or after surgical treatment, and / or before or after radiotherapy.

[0108] In some embodiments of the present invention, the method wherein...

[0109] The single-dose dose of the fusion protein of the present invention is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; or, the single-dose dose of the fusion protein 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.

[0110] Preferably, the drug is administered once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, or 3 weeks;

[0111] Preferably, the administration method is intravenous infusion or intravenous injection.

[0112] The variable regions of the light and heavy chains determine antigen binding; each chain's variable region contains three hypervariable regions, called complementarity-determining regions (CDRs), where the heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3, and the light chain (L) CDRs include LCDR1, LCDR2, and LCDR3. In this invention, CDRs are defined by the IMGT numbering system; please refer to Ehrenmann F, Kaas Q, Lefranc MP. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic Acids Research, 2009; 38(suppl_1):D301-D307.

[0113] Using techniques well-known to those skilled in the art, such as analyzing the amino acid sequence of the CDR of the following monoclonal antibody sequence according to the IMGT definition, the results are as follows:

[0114] HCDR and LCDR of murine TIGIT monoclonal antibodies

[0115] HCDR1:GHSFTSDYA(SEQ ID NO:3)

[0116] HCDR2: ISYSDST (SEQ ID NO:4)

[0117] HCDR3:ARLDYGNYGGAMDY(SEQ ID NO:5)

[0118] LCDR1: QHVSTA (SEQ ID NO:8)

[0119] LCDR2: SAS (SEQ ID NO:9)

[0120] LCDR3:QQHYITPWT(SEQ ID NO:10)

[0121] The three HCDRs and three LCDRs of the humanized TIGIT monoclonal antibody are the same as those of the mouse TIGIT monoclonal antibody.

[0122] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0123] As used in this article, the term EC 50 The half-maximal concentration (WMC) is the concentration that produces a 50% maximum effect.

[0124] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various regions or domains follows Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the definition of the IMGT numbering system, see Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0125] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0126] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975; 256(5517):495), but it can also be obtained using recombinant DNA technology (see US Patent 4,816,567).

[0127] 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 (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, 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 antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, Jones et al., Nature 1986; 321:522-525; Reichmann et al., Nature, 1988; 332:323-329; Presta, Curr. Op. Struct. Biol. 1992; 2:593-596; and Clark, Immunol. Today 2000; 21:397-402. In some cases, the antigen-binding fragment of the antibody is a diabetic, where V... H and V LThe domain is expressed on a single polypeptide chain, but the linker is too short to allow pairing between two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448 and Poljak RJ et al., Structure 1994; 2:1121-1123).

[0128] As described in this article, a fusion protein is a protein product co-expressed by two genes obtained through DNA recombination. Methods for producing and purifying antibody-antigen binding fragments are well-known in the art (e.g., Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15).

[0129] As used herein, the term "single-chain fragment variable (ScFv)" refers to an antibody heavy chain variable region (V) linked by linkers. H ) and antibody light chain variable region (V L The molecules of V. L and V H The domain enables the formation of monovalent molecules by linker pairing with single polypeptide chains (see, for example, Birdet et al., Science 1988; 242:423-426 and Huston et al., Proc. Natl. Acad. Sci. USA 1988; 85:5879-5883). Such scFv molecules can have a general structure: NH2-V L -Connection fragment-V H -COOH or NH2-V H -Connection fragment-V L-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al, Protein Eng. 1995; 8:725-731, Choi et al, Eur. J. Immunol. 2001; 31:94-106, Hu et al, Cancer Res. 1996; 56:3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293:41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1):51-59.

[0130] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0131] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0132] 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, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0133] In this invention, the term "ADCP" refers to antibody-dependent cellular phagoxytosis. The Fc fragment of an antibody bound to a cell surface antigen binds to the Fc receptor of a phagocytic cell, such as a macrophage, thereby mediating the phagocytosis of antibody-bound cells by the phagocyte.

[0134] In this invention, the term "ADCC" refers to antibody-dependent cell-mediated cytotoxicity. The Fab fragment of an antibody binds to antigenic epitopes on virus-infected cells or tumor cells, while its Fc fragment binds to Fc receptors (FcRs) on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells.

[0135] In this invention, the term "CDC" refers to complement-dependent cytotoxicity. When an antibody specifically binds to its corresponding antigen on the cell membrane surface, a complex is formed, activating the complement system. This, in turn, leads to the formation of a MAC on the target cell surface, resulting in target cell lysis. Complement can cause the lysis of various bacteria and other pathogenic cells, and is an important defense mechanism against pathogenic infection.

[0136] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0137] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have an equilibrium dissociation constant of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to an antigen (e.g., TIGIT protein). K can be measured using methods known to those skilled in the art. D For example, measurements can be taken using a Fortebio molecular interaction analyzer.

[0138] 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. Furthermore, in this 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.

[0139] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19). th (ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0140] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes 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 characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0141] In this invention, unless otherwise specified, the terms "first" (e.g., first protein functional region or first product) and "second" (e.g., second protein functional region or second product) are used for distinguishing reference or clarifying expression, and do not have a typical meaning of order.

[0142] Beneficial effects of the invention

[0143] The present invention achieves any one or more of the following technical effects (1)-(9):

[0144] (1) The fusion protein of the present invention (e.g., TF01 or TF02) can simultaneously inhibit TIGIT and reduce TGF-β levels.

[0145] (2) The fusion protein of the present invention (e.g., TF01 or TF02) can bind to TIGIT very specifically and can block the binding of TIGIT to CD155 very effectively, thereby specifically relieving the immunosuppression of the body by TIGIT.

[0146] (3) The fusion protein of the present invention (e.g., TF01 or TF02) can bind to TGF-β very specifically and can effectively block the binding of TGF-β to TGF-β receptor, specifically relieve the immunosuppression of the body by TGF-β and activate the immune response.

[0147] (4) The fusion protein of the present invention has good potential for preparing anti-tumor drugs.

[0148] (5) The first and second protein functional regions of the fusion protein of the present invention have a synergistic effect, which is better than using one of the protein functional regions alone.

[0149] (6) The fusion protein of the present invention, particularly TF01, completely eliminates its binding activity with Fc receptors FcγRI, FcγRIIb, FcγRIIa_H131 and / or FcγRIIa_R131, FcγRIIIa_V158 and / or FcγRIIIa_F158, thereby eliminating its ADCC or ADCP activity.

[0150] (7) The fusion protein of the present invention, particularly TF01, completely eliminates its binding activity with complement C1q, thereby completely eliminating its CDC activity.

[0151] (8) The fusion proteins of the present invention, especially TF01 and TF02, can effectively block the immunosuppression of immune cells induced by TIGIT and induce cells to secrete IL-2.

[0152] (9) The fusion protein of the present invention, especially TF01, can effectively block the immunosuppression of immune cells induced by TGF-β and induce cells to secrete INF-γ. Attached Figure Description

[0153] Figure 1 Results of the binding activity assay of TF01, TF02, 26B12H2L2 (hG4DM), RG6058 (hG4) with antigen TIGIT-mFc.

[0154] Figure 2 Results of the detection of the binding activity of TF01, TF02, TGF-βRII-mFc with TGF-β1.

[0155] Figure 3 Results of the detection of the binding activity of TF01, TF02, TGF-βRII-mFc with TGF-β2.

[0156] Figure 4 Results of the detection of the binding activity of TF01, TF02, TGF-βRII-mFc with TGF-β3.

[0157] Figure 5 The results of the assay of the activity of the fusion protein of anti-TIGIT antibody and TGF-βR competing with TGF-βRII-His-Biotin for binding to human TGF-β1.

[0158] Figure 6 The results of the assay of the activity of the fusion protein of anti-TIGIT antibody and TGF-βR competing with TGF-βRII-His-Biotin for binding to human TGF-β3.

[0159] Figure 7 The activity assay results showed that the fusion protein of anti-TIGIT antibody and TGF-βR competitively binds to human CD155-hFc-Biotin to human TIGIT-mFC.

[0160] Figure 8 Results of the assay for the binding activity of the anti-TIGIT antibody and TGF-βR fusion protein to TIGIT on the surface of the 293T-TIGIT membrane.

[0161] Figure 9 : TF01 competes with CD155 for binding to cell membrane surface antigen TIGIT detection results.

[0162] Figure 10 : TF01 competes with CD112 for binding to cell membrane surface antigen TIGIT detection results.

[0163] Figure 11 Results of the affinity constant detection between TF01 and FcγRI.

[0164] Figure 12 Results of the detection of the affinity constant between 26B12H2L2(hG1WT) and FcγRI. Figure 13 Results of the affinity constant detection between TF01 and FcγRIIIa_V158.

[0165] Figure 14 Figure 1: Detection results of affinity constants between 26B12H2L2(hG1WT) and FcγRIIIa_V158.

[0166] Figure 15 Results of the affinity constant detection between TF01 and FcγRIIIa_F158.

[0167] Figure 16 Results of the affinity constant detection between 26B12H2L2(hG1WT) and FcγRIIIa_F158.

[0168] Figure 17 Results of affinity constant detection between TF01 and FcγRIIa_H131.

[0169] Figure 18 Results of affinity constant detection between 26B12H2L2(hG1WT) and FcγRIIa_H131.

[0170] Figure 19 Results of affinity constant detection between TF01 and FcγRIIa_R131.

[0171] Figure 20 Results of affinity constant detection between 26B12H2L2(hG1WT) and FcγRIIa_R131.

[0172] Figure 21 Results of the affinity constant detection between TF01 and FcγRIIb.

[0173] Figure 22 Results of the affinity constant detection between 26B12H2L2(hG1WT) and FcγRIIb.

[0174] Figure 23 Results of the affinity constant test between TF01 and C1q.

[0175] Figure 24 Results of the affinity constant test between 26B12H2L2(hG1WT) and C1q.

[0176] Figure 25 ADCP effect results of RG6058(hG1WT), RG6058(hG4WT), 26B12H2L2(hG1WT), and TF01.

[0177] Figure 26 Evaluation results of the blocking activity of the anti-TIGIT antibody and TGF-βR fusion protein against IL-2 secretion in a co-culture system of CD112-Jurkat-TIGIT and THP-1 cells.

[0178] Figure 27 Evaluation results of the blocking activity of the anti-TIGIT antibody and TGF-βR fusion protein against CD155 in inhibiting IL-2 secretion in a co-culture system of Jurkat-TIGIT and THP-1 cells.

[0179] Figure 28 Evaluation results of the bioactivity of the anti-TIGIT antibody and TGF-βR fusion protein in promoting IL-2 secretion in the Jurkat-TIGIT and HT1080-aCD3scFv cell co-culture system.

[0180] Figure 29Evaluation results of the blocking activity of the anti-TIGIT antibody and TGF-βR fusion protein against IFN-γ secretion in the TGF-β1 inhibitory system during the secondary immune response to CMV antigen in PBMCs.

[0181] Figure 30 The efficacy of the anti-TIGIT antibody and TGF-βR fusion protein against the Scid Beige mouse MDA-MB-231 xenograft model was investigated. Results are expressed as mean ± standard error and analyzed by two-way ANOVA and Bonferroni test. Compared with the isotype control group, *p<0.05, **p<0.01, ***p<0.001.

[0182] Figure 31 Effect of the fusion protein of anti-TIGIT antibody and TGF-βR on body weight in Scid Beige mouse MDA-MB-231 xenograft model.

[0183] Biological materials involved in preservation:

[0184] The hybridoma cell line LT019 (TIGIT-26B12) was deposited at the China Center for Type Culture Collection (CCTCC) on October 23, 2020, with accession number CCTCC NO:C2020208. The deposit address is Wuhan University, Wuhan, China, 430072, China.

[0185] This invention relates to the following sequences 1-48:

[0186] 1. Amino acid sequence of 26B12VH

[0187] EVQLQESGPGLVKPSQSLSLTCTVTGHSFTSDYAWNWIRQFPGNRLEWMGYISYSDSTNYNPSLKSRISITRDTSKNQFFLQMNSVTTEDTATYYCARLDYGNYGGAMDYWGQGTSVTVSS(SEQ ID NO:1)

[0188] 2. Nucleic acid sequence of 26B12VH

[0189] GAGGTGCAGCTGCAGGAGTCTGGACCTGGCCTGGTGAAACCCTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCCACTCATTCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAGACTGGAGTGGATGGGCTACATAAGCTACAGTGATAGCACTAACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCTTGCAGATGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATTGGACTATGGTAACTACGGTGGGGCTATGGACTACTGGGGTCAAGGGACCTCAGTCACCGTCTCCTCA(SEQ ID NO: 2)

[0190] 3. HCDR1:

[0191] GHSFTSDYA(SEQ ID NO: 3)

[0192] 4. HCDR2:

[0193] ISYSDST(SEQ ID NO: 4)

[0194] 5. HCDR3:

[0195] ARLDYGNYGGAMDY(SEQ ID NO: 5)

[0196] 6. Amino acid sequence of 26B12VL

[0197] DIVLTQSHEFMSTSLRDRVSITCKSSQHVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVKAEDLAVYYCQQHYITPWTFGGGTKLEIK(SEQ ID NO: 6)

[0198] 7. Nucleic acid sequence of 26B12VL

[0199] GATATTGTGCTAACTCAGTCTCACGAATTCATGTCCACCTCATTACGAGACAGGGTCAGCATCACCTGCAAATCCAGTCAACATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATATTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATAAAA(SEQ ID NO:7)

[0200] 8. LCDR1:

[0201] QHVSTA(SEQ ID NO:8)

[0202] 9. LCDR2:

[0203] SAS(SEQ ID NO:9)

[0204] 10. LCDR3:

[0205] QQHYITPWT(SEQ ID NO:10)

[0206] 11. Amino acid sequence of 26B12H1VH

[0207] DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWNWIRQFPGKGLEWIGYISYSDSTNYNPSLKSRITISRDTSKNQFFLQLNSVTAADTATYYCARLDYGNYGGAMDYWGQGTSVTVSS(SEQ ID NO:11)

[0208] 12. Nucleic acid sequence of 26B12H1VH

[0209] GATGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTTCCCAGACCCTGTCTCTGACCTGTACAGTGTCTGGCCACAGCTTCACATCCGACTACGCCTGGAACTGGATCAGGCAGTTTCCAGGCAAGGGCCTGGAGTGGATCGGCTACATCTCTTATAGCGACTCCACCAACTATAATCCCTCTCTGAAGAGCCGGATCACCATCAGCAGAGATACATCCAAGAACCAGTTCTTTCTGCAGCTGAACAGCGTGACAGCCGCCGACACCGCCACATACTATTGCGCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTACTGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO: 12)

[0210] 13. Amino acid sequence of 26B12H2VH

[0211] DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWSWIRQPPGKGLEWIGYISYSDSTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARLDYGNYGGAMDYWGQGTSVTVSS (SEQ ID NO: 13)

[0212] 14. Nucleic acid sequence of 26B12H2VH

[0213] GATGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCAAGCCAGACCCTGTCCCTGACCTGTACAGTGTCCGGCCACTCTTTTACAAGCGACTACGCCTGGTCTTGGATCAGGCAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCTACATCTCCTATTCTGACAGCACCAACTATAATCCCTCCCTGAAGTCTCGGGTGACCATCTCTAGAGATACAAGCAAGAACCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGTGTACTATTGCGCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC(SEQ ID NO: 14)

[0214] 15. Amino acid sequence of 26B12H3VH

[0215] DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWSWIRQPPGKGLEWIGYISYSDSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDYGNYGGAMDYWGQGTSVTVSS(SEQ ID NO: 15)

[0216] 16. Nucleic acid sequence of 26B12H3VH

[0217] GATGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCAAGCCAGACCCTGTCCCTGACCTGTACAGTGTCCGGCCACTCTTTTACAAGCGACTACGCCTGGTCTTGGATCAGACAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCTACATCTCCTATTCTGACAGCACCAACTATAATCCCTCCCTGAAGTCTAGAGTGACCATCTCTGTGGATACAAGCAAGAACCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGTGTACTATTGCGCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 16)

[0218] 17. Amino acid sequence of 26B12H4VH

[0219] DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWNWIRQFPGKGLEWMGYISYSDSTNYNPSLKSRITISRDTSKNQFFLQLNSVTAADTATYYCARLDYGNYGGAMDYWGQGTSVTVSS (SEQ ID NO: 17)

[0220] 18. Nucleic acid sequence of 26B12H4VH

[0221] GATGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTTCCCAGACCCTGTCTCTGACCTGTACAGTGTCTGGCCACAGCTTCACATCCGACTACGCCTGGAACTGGATCAGGCAGTTTCCAGGCAAGGGCCTGGAGTGGATGGGCTACATCTCTTATAGCGACTCCACCAACTATAATCCCTCTCTGAAGAGCCGGATCACCATCAGCAGAGATACATCCAAGAACCAGTTCTTTCTGCAGCTGAACAGCGTGACAGCCGCCGACACCGCCACATACTATTGCGCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTACTGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC(SEQ ID NO: 18)

[0222] 19. Amino acid sequence of 26B12L1VL

[0223] DIQMTQSPKSLSTSVGDRVTITCRSSQHVSTAVAWYQQKPGKSPKLLIYSASYRYSGVPDRFSGSGSGTDFTFTISSVQPEDFATYYCQQHYITPWTFGGGTKLEIK(SEQ ID NO: 19)

[0224] 20. Nucleic acid sequence of 26B12L1VL

[0225] GACATCCAGATGACCCAGTCCCCTAAGTCCCTGTCTACAAGCGTGGGCGATCGGGTGACCATCACATGTAGAAGCTCCCAGCACGTGTCTACCGCAGTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTATTCCGCCTCTTACAGGTATTCCGGAGTGCCAGACCGGTTTAGCGGCTCCGGCTCTGGCACCGATTTCACCTTTACAATCTCTAGCGTGCAGCCAGAGGACTTCGCCACATACTATTGCCAGCAGCACTACATCACCCCATGGACCTTCGGCGGCGGCACAAAGCTGGAGATCAAG(SEQ ID NO: 20)

[0226] 21. Amino acid sequence of 26B12L2VL

[0227] DIQMTQSPSSLSASVGDRVTITCRSSQHVSTALAWYQQKPGKSPKLLIYSASSRYSGVPDRFSGSGSGTDFTFTISSLQPEDFATYYCQQHYITPWTFGGGTKLEIK(SEQ ID NO:21)

[0228] 22. Nucleic acid sequence of 26B12L2VL

[0229] GACATCCAGATGACCCAGTCCCCTAGCTCCCTGTCTGCCAGCGTGGGCGATAGGGTGACCATCACATGTAGATCTAGCCAGCACGTGTCTACAGCCCTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTACTCCGCCTCCTCTAGGTATTCTGGAGTGCCAGACCGGTTTTCCGGCTCTGGCAGCGGCACCGATTTCACCTTTACAATCAGCTCCCTGCAGCCAGAGGACTTCGCCACATACTATTGCCAGCAGCACTATATCACCCCATGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG(SEQ ID NO:22)

[0230] 23. Amino acid sequence of 26B12L3VL

[0231] DIQMTQSPSSLSASVGDRVTITCRASQHVSTALAWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYITPWTFGGGTKLEIK(SEQ ID NO:23)

[0232] 24. Nucleic acid sequence of 26B12L3VL

[0233] GACATCCAGATGACCCAGTCCCCTAGCTCCCTGAGCGCCTCCGTGGGCGATAGGGTGACCATCACATGTAGAGCCTCTCAGCACGTGAGCACAGCCCTGGCATGGTACCAGCAGAAGCCAGGCAAGGCCCCTAAGCTGCTGATCTATAGCGCCTCTAGCCTGCAGTCCGGAGTGCCATCTCGGTTCTCTGGCAGCGGCTCCGGAACCGACTTTACCCTGACAATCTCCTCTCTGCAGCCAGAGGATTTCGCCACATACTATTGCCAGCAGCACTACATCACCCCATGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG(SEQ ID NO:24)

[0234] 25. Amino acid sequence of 26B12L4VL

[0235] DIQMTQSPKSMSTSVGDRVTITCRSSQHVSTAVAWYQQKPGKSPKLLIYSASYRYSGVPDRFSGSGSGTDFTFTISSVQPEDFATYYCQQHYITPWTFGGGTKLEIK(SEQ ID NO:25)

[0236] 26. Nucleic acid sequence of 26B12L4VL

[0237] GACATCCAGATGACCCAGTCCCCTAAGTCCATGTCTACAAGCGTGGGCGACAGGGTGACCATCACATGTAGAAGCTCCCAGCACGTGTCTACCGCAGTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTATTCCGCCTCTTACAGGTATTCCGGAGTGCCAGACCGGTTTAGCGGCTCCGGCTCTGGCACCGATTTCACCTTTACAATCTCTAGCGTGCAGCCAGAGGACTTCGCCACATACTATTGCCAGCAGCACTACATCACCCCATGGACCTTCGGCGGCGGCACAAAGCTGGAGATCAAG(SEQ ID NO:26)

[0238] 27. Amino acid sequence of the heavy chain of 26B12H2L2(hG4DM)

[0239] DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWSWIRQPPGKGLEWIGYISYSDSTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARLDYGNYGGAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO: 27)

[0240] 28. Nucleic acid sequence of the heavy chain of 26B12H2L2(hG4DM)

[0241]

[0242] 29. Amino acid sequence of the 26B12H2L2(hG4DM) light chain

[0243] DIQMTQSPSSSLSASVGDRVTITCRSSQHVSTALAWYQQKPGKSPKLLIYSASSRYSGVPDRFSGSGSGTDFTFTISSLQPEDFATYYCQQHYITPWTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:29)

[0244] 30. Nucleic acid sequence of the 26B12H2L2(hG4DM) light chain

[0245] GACATCCAGATGACCCAGTCCCCTAGCTCCCTGTCTGCCAGCGTGGGCGATAGGGTGACCATCACATGTAGATCTAGCCAGCACGTGTCTACAGCCCTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTACTCCGCCTCCTCTAGGTATTCTGGAGTGCCAGACCGGTTTTCCGGCTCTGGCAGCGGCACCGATTTCACCTTTACAATCAGCTCCCTGCAGCCAGAGGACTTCGCCACATACTATTGCCAGCAGCACTATATCACCCCATGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCAGCCCCATCTGTCTTCATTTTTCCCCCTAGTGACGAGCAGCTGAAATCCGGAACAGCCTCTGTGGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAGGTGCAGTGGAAAGTCGATAACGCTCTGCAGAGTGGCAATTCACAGGAGAGCGTGACTGAACAGGACTCCAAGGATTCTACCTATAGTCTGAGCTCCACTCTGACCCTGTCCAAAGCAGATTACGAAAAGCACAAAGTGTATGCCTGTGAGGTCACCCACCAGGGGCTGAGTTCTCCAGTCACCAAATCCTTCAACAGAGGCGAATGT(SEQ ID NO: 30)

[0246] 31. Heavy chain amino acid sequence of 26B12H2L2 (hG1DM)

[0247] DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWSWIRQPPGKGLEWIGYISYSDSTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARLDYGNYGGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 31)

[0248] 32. 26B12H2L2(hG1DM) heavy chain nucleic acid sequence

[0249]

[0250] 33. Amino acid sequence of the extracellular region truncated fragment of TGF-βRII:

[0251] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ IDNO:33)

[0252] 34. Amino acid sequence of TGF-βRI: (Underlined portion is the extracellular region sequence)

[0253] MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATA LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHN SMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEL AAVIAGPVCFVCISLMLMVYICHNRTVIHHRVPNEEDPSLDRPFISEGTTLKDLIYDMTTSGSGSGLPLLVQRTIARTIVLQESIGKGRFGEVWRGKWRGEEVAVKIFSSREERSWFREAEIYQTVMLRHENILGFIAADNKDNGTWTQLWLVSDYHEHGSLFDYLNRYTVTVEGMIKLALSTASGLAHL HMEIVGTQGKPPAIARDLKSKNILVKKNGTCCIADLGLAVRHDSATDTIDIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLVFWEIARRCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEALRVMAKIMRECWYANGAARLTALRIKKTLSQLSQQEGIKM(SEQ ID NO:34)

[0254] 35. Amino acid sequence of the extracellular region of TGF-βRI:

[0255] LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEL(SEQ ID NO:35)

[0256] 36. Amino acid sequence of TGF-βRII: (Underlined portion is extracellular region sequence)

[0257] MGRGLLRGLWPLHIVLWTRIAS TIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCM SNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN DNIIFSEEYNTSNPDLLLVIFQ VTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK(SEQ ID NO: 36)

[0258] 37. Amino acid sequence of the extracellular region of TGF-βRⅡ:

[0259] TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ(SEQ ID NO: 37)

[0260] 38. Amino acid sequence of TGF-βRⅢ: (the underlined part is the extracellular region sequence)

[0261] MTSHYVIAIFALMSSCLATA GPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLN LRTAGQGPGQLQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSL TAETEERNFPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEG CVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKCKKSVNWVIKSFDVKGSLKIIA PNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNAEEMGDEEVHTIPP ELRILLDPGALPALQNPPIRGGEGQNGGLPFPFPDISRRVWNEEGEDGLPRPKDPVIPSIQLFPGLREPEEVQGSV DIALSVKCDNEKMIVAVEKDSFQASGYSGMDVTLLDPTCKAKMNGTHFVLESPLNGCGTRPRWSALDGVVYYNSIV IQVPALGDSSGWPDGYEDLESGDNGFPGDMDEGDASLFTRPEIVVFNCSLQQVRNPSSFQEQPHGNITFNMELYNT DLFLVPSQGVFSVPENGHVYVEVSVTKAEQELGFAIQTCFISPYSNPDRMSHYTIIENICPKDESVKFYSPKRVHF PIPQADMDKKRFSFVFKPVFNTSLLFLQCELTLCTKMEKHPQKLPKCVPPDEACTSLDASIIWAMMQNKKTFTKPL AVIHHEAESKEKGPSMKEPNPISPPIFHGLDTLTVMGIAFAAFVIGALLTGALWYIYSHTGETAGRQQVPTSPPASENSSAAHSIGSTQSTPCSSSSTA(SEQ ID NO: 38)

[0262] 39. Amino acid sequence of the extracellular region of TGF-βRⅢ:

[0263] GPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKCKKSVNWVIKSFDVKGSLKIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNAEEMGDEEVHTIPPELRILLDPGALPALQNPPIRGGEGQNGGLPFPFPDISRRVWNEEGEDGLPRPKDPVIPSIQLFPGLREPEEVQGSVDIALSVKCDNEKMIVAVEKDSFQASGYSGMDVTLLDPTCKAKMNGTHFVLESPLNGCGTRPRWSALDGVVYYNSIVIQVPALGDSSGWPDGYEDLESGDNGFPGDMDEGDASLFTRPEIVVFNCSLQQVRNPSSFQEQPHGNITFNMELYNTDLFLVPSQGVFSVPENGHVYVEVSVTKAEQELGFAIQTCFISPYSNPDRMSHYTIIENICPKDESVKFYSPKRVHFPIPQADMDKKRFSFVFKPVFNTSLLFLQCELTLCTKMEKHPQKLPKCVPPDEACTSLDASIIWAMMQNKKTFTKPLAVIHHEAESKEKGPSMKEPNPISPPIFHGLDTLTV(SEQ ID NO: 39)

[0264] 40. Amino acid sequence of the heavy chain of Tiragolumab:

[0265] EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 40)

[0266] 41. Amino acid sequence of the light chain of Tiragolumab:

[0267] DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 41)

[0268] 42. Amino acid sequence of the heavy chain variable region of RG6058 (hG4):

[0269] EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS(SEQ ID NO:42)

[0270] 43. Amino acid sequence of the variable region of the RG6058(hG4) light chain:

[0271] DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK(SEQ ID NO:43)

[0272] 44. The amino acid sequence of the linker fragment:

[0273] GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO:44)

[0274] 45. Anti-HEL & TGFβ antibody heavy chain amino acid sequence

[0275] EVQLEQSGAELMKPGASVKISCKATGYTFTTYWIEWIKQRPGHSLEWIGEILPGSDSTYYNEKVKGKVTFTADASSNTAYMQLSSLTSEDSAVYYCARGDGFYVYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO: 45)

[0276] 46. Amino acid sequence of anti-HEL&TGFβ antibody light chain

[0277] DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYTSQSMSGIPSRFSGSGSGTDFTLSINSVETEDFGVYFCQQSGSWPRTFGGGTKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 46)

[0278] 47. Heavy chain amino acid sequence of RG6058 (G1DM)

[0279] EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:47)

[0280] 48. Light chain amino acid sequence of RG6058 (G1DM)

[0281] DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:48) Detailed Implementation

[0282] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0283] BALB / c mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center.

[0284] C57 mice were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine.

[0285] MDA-MB-231 cells and U87-MG cells were purchased from ATCC.

[0286] The positive control antibody, Tiragolumab, has heavy chain and light chain amino acid sequences derived from the antibody described in WHO.Proposed INN:List 117.WHO Drug Information,31(2):p104,2017, and the sequences are identical to SEQ ID NOs:40-41, respectively.

[0287] The Tiragolumab described in this invention is the same antibody as RG6058 (hG1WT).

[0288] The positive control antibody RG6058 (hG4) has a sequence derived from the antibody described in the published patent CN108290946A. The amino acid sequences of its heavy chain variable region and light chain variable region are identical to those of SEQ ID NOs:42-43, respectively.

[0289] The positive control antibody RG6058 (hG1DM) was prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd., batch number 20180816. Its heavy chain amino acid sequence is shown in SEQ ID NOs:47, and its light chain amino acid sequence is shown in SEQ ID NOs:48.

[0290] In the following embodiments of the present invention, the isotype control antibody used is human anti-Hen Egg Lysozyme IgG (anti-HEL, i.e., human IgG, abbreviated as hIgG), whose variable region sequence is derived from Acierno et al.'s paper "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al. J Mol Biol. 2007; 374(1):130-46). The hIgG1DM and hIgG4WT used in the embodiments are isotype control antibodies of anti-HEL with hG1DM and hG4WT constant region sequences, which were prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0291] The anti-HEL & TGFβ antibody was constructed by fusing the C-terminus of the heavy chain of a human hG4DM,k subtype anti-HEL antibody with TGFβRII protein, and was prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd. Its heavy chain amino acid sequence is shown in SEQ ID NOs:45, and its light chain amino acid sequence is shown in SEQ ID NOs:46.

[0292] TIGIT-mFc protein (batch number 20171110), TGF-βRII-mFc (batch number 20200528), CD155-hFc-Biotin (batch number 20170721), CD155-mFc (batch number 20190726), and CD112-mFc (batch number 20190726) were all produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd. according to known sequences. Among them, mFc represents the Fc protein fragment of mouse IgG; hFc represents the Fc protein fragment of human IgG.

[0293] TGF-β1 protein was purchased from Sino Biologcal Inc., catalog number: LC13DE3108.

[0294] TGF-β2 protein was purchased from Peprotech, catalog number: 0420345E0620.

[0295] TGF-β3 protein was purchased from Peprotech, catalog number: 0713410A0219.

[0296] In the following embodiments of the present invention, the 293T-TIGIT cell line used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-TIGIT cell line was prepared by viral infection of HEK293T cells (Wuhan University). The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was plenti6.3 / V5-TIGITFL-BSD (where TIGIT, Genebank ID: NP_776160.2; the vector plenti6.3 / V5 TOPO was purchased from Invitrogen, product number: K531520).

[0297] In the following embodiments of the present invention, the CHO-K1-TIGIT cell line used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The CHO-K1-TIGIT cell line was prepared by viral infection of CHO-K1 cells (Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. JVirol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was plenti6.3 / V5-TIGITFL-BSD (where TIGIT, Genebank ID: NP_776160.2; the vector plenti6.3 / V5 TOPO was purchased from Invitrogen, product number: K531520).

[0298] In the following embodiments of the present invention, the Jurkat-TIGIT cell line used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The Jurkat-TIGIT cell line was prepared by viral infection of Jurkat cells (Chinese Academy of Sciences Cell Center). The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was plenti6.3 / V5-TIGITFL-BSD (where TIGIT, Genebank ID: NP_776160.2; the vector plenti6.3 / V5 TOPO was purchased from Invitrogen, product number: K531520).

[0299] In the following embodiments of the present invention, the HT1080-aCD3scFv cell line used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The HT1080-aCD3scFv cell line was prepared by viral infection of HT-1080 cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). The virus preparation used was 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was pCDH-aCD3scFv-Puro (wherein the aCD3scFv sequence was derived from the anti-human CD3 Mus musculus OKT3 hybridoma antibody; the vector pCDH-CMV-MCS-EF1-Puro was purchased from Youbao Biotechnology, product number: VT1480).

[0300] Example 1: Preparation of anti-TIGIT antibody

[0301] 1. Preparation of hybridoma cell line LT019

[0302] The antigen used to prepare the anti-TIGIT antibody was human TIGIT-mFc (TIGIT is Genbank ID: NP_776160.2). Spleen cells from immunized mice were fused with mouse myeloma cells to create hybridoma cells. Using human TIGIT-hFc as the antigen, the hybridoma cells were screened using an indirect ELISA method to obtain hybridoma cells capable of secreting antibodies that specifically bind to TIGIT. The selected hybridoma cells were then subjected to limiting dilution to obtain stable hybridoma cell lines. These hybridoma cell lines were named hybridoma cell line LT019, and the monoclonal antibody secreted by them was named 26B12.

[0303] The hybridoma cell line LT019 (also known as TIGIT-26B12) was deposited at the China Center for Type Culture Collection (CCTCC) on October 23, 2020, with accession number CCTCC NO: C2020208. The deposit address is Wuhan University, Wuhan, China, 430072, China.

[0304] 2. Preparation of anti-TIGIT antibody 26B12

[0305] The LT019 cell line prepared above was cultured in CD medium (Chemical Defined Medium, containing 1% penicillin and streptomycin) at 5% CO2 and 37°C. After 7 days, the cell culture supernatant was collected, centrifuged at high speed, filtered through a microporous membrane under vacuum, and purified using a HiTrap protein A HP column to obtain antibody 26B12.

[0306] Example 2: Sequence analysis of anti-TIGIT antibody 26B12

[0307] mRNA was extracted from the LT019 cell line cultured in Example 1 according to the method of the Total RNA Extraction Kit for Cultured Bacteria (Tiangen, catalog number DP430).

[0308] According to Invitrogen III. The First-Strand Synthesis System for RT-PCR kit instructions describe the synthesis of cDNA and its PCR amplification.

[0309] The PCR amplification products were directly cloned using TA. For specific procedures, please refer to the instructions for the pEASY-T1Cloning Kit (TransgenCT101).

[0310] The TA clone product was directly sequenced, and the sequencing results are as follows:

[0311] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, with a fragment length of 363 bp.

[0312] Its encoded amino acid sequence is shown in SEQ ID NO:1, and has a length of 121 amino acids.

[0313] The sequence of the heavy chain HCDR1 is shown in SEQ ID NO:3, the sequence of HCDR2 is shown in SEQ ID NO:4, and the sequence of HCDR3 is shown in SEQ ID NO:5.

[0314] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:7, and its length is 321bp.

[0315] Its encoded amino acid sequence is shown in SEQ ID NO:6, and has a length of 107 amino acids.

[0316] The sequence of the light chain LCDR1 is shown in SEQ ID NO:8, the sequence of LCDR2 is shown in SEQ ID NO:9, and the sequence of LCDR3 is shown in SEQ ID NO:10.

[0317] Example 3: Design and preparation of humanized and mutant antibodies against human TIGIT

[0318] 1. Light and heavy chain design of humanized anti-human TIGIT antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4.

[0319] Based on the three-dimensional crystal structure of human TIGIT protein and the sequence of antibody 26B12 obtained in Example 2, variable region sequences (antibody constant region sequences, from the NCBI database, heavy chain constant regions are all Iggamma-1 chain C region, ACCESSION: P01857; light chain constant regions are Ig kappa chain C region, ACCESSION: P01834) of antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L3, 26B12H1L4, and 26B12H4L4 were designed.

[0320] The designed variable region sequence is shown in Table 1 below.

[0321] Table 1: Variable region sequence of humanized antibodies against human TIGIT

[0322]

[0323] The above eight antibodies, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4, all have a heavy chain variable region nucleic acid sequence length of 363 bp, encoding an amino acid sequence length of 121 aa; and a light chain variable region nucleic acid sequence length of 321 bp, encoding an amino acid sequence length of 107 aa.

[0324] Furthermore, the above 8 antibodies share the same HCDR1-HCDR3 and LCDR1-LCDR3, as follows:

[0325] The sequence of HCDR1 is shown in SEQ ID NO:3, the sequence of HCDR2 is shown in SEQ ID NO:4, and the sequence of HCDR3 is shown in SEQ ID NO:5;

[0326] The sequence of LCDR1 is shown in SEQ ID NO:8, the sequence of LCDR2 is shown in SEQ ID NO:9, and the sequence of LCDR3 is shown in SEQ ID NO:10.

[0327] 2. Preparation of humanized antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4

[0328] The constant region of the heavy chain is the Iggamma-1 chain C region, ACCESSION: P01857; the constant region of the light chain is the Ig kappa chain C region, ACCESSION: P01834.

[0329] The cDNA and light chain cDNA of 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, 26B12H2L4, and 26B12H4L4 were cloned into the pUC57simple vector (provided by GenScript) to obtain:

[0330] pUC57simple-26B12H1, pUC57simple-26B12L1;

[0331] pUC57simple-26B12H4, pUC57simple-26B12L1;

[0332] pUC57simple-26B12H2, pUC57simple-26B12L2;

[0333] pUC57simple-26B12H3, pUC57simple-26B12L2;

[0334] pUC57simple-26B12H2, pUC57simple-26B12L3;

[0335] pUC57simple-26B12H3, pUC57simple-26B12L3;

[0336] pUC57simple-26B12H1, pUC57simple-26B12L4; and

[0337] pUC57simple-26B12H4, pUC57simple-26B12L4.

[0338] Following the standard techniques described in *Molecular Cloning: A Laboratory Manual (Second Edition)*, the full-length heavy and light chain genes synthesized by EcoRI and HindIII restriction enzyme digestion were subcloned into the expression vector pcDNA3.1 to obtain expression plasmids pcDNA3.1-26B12H1, pcDNA3.1-26B12L1, pcDNA3.1-26B12H4, pcDNA3.1-26B12H2, pcDNA3.1-26B12L2, pcDNA3.1-26B12H3, pcDNA3.1-26B12L3, and pcDNA3.1-26B12L4. Further sequencing analysis of the heavy / light chain genes in the recombinant expression plasmids was then performed. Subsequently, 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) containing the corresponding light and heavy chain recombinant plasmids were designed. pcDNA3.1-26B12H2 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H1 / pcDNA3.1-26B12L4, and pcDNA3.1-26B12H4 / pcDNA3.1-26B12L4 were co-transfected into 293F cells, and the culture medium was collected for purification. After sequencing verification, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected, and affinity purification was performed using a Protein A column to obtain humanized antibodies.

[0339] The humanized antibody 26B12H2L2 is also referred to as 26B12H2L2(hG1WT) in this invention.

[0340] 3. Design of humanized antibodies 26B12H2L2 (hG1DM) and 26B12H2L2 (hG4DM)

[0341] Based on 26B12H2L2, the inventors obtained the humanized antibody 26B12H2L2(hG1DM) with constant region mutations by introducing a point mutation (L234A) from leucine to alanine at position 234 (according to the EU numbering system, the same below) and a point mutation (L235A) from leucine to alanine at position 235 in the constant region of its heavy chain. The amino acid sequence of the heavy chain 26B12H2(hG1DM) is shown in SEQ ID NO:31; the amino acid sequence of the light chain is shown in SEQ ID NO:29.

[0342] Based on 26B12H2L2, the inventors kept the variable region of the antibody unchanged and used the Iggamma-4chain Cregion, ACCESSION: P01861.1 as the constant region of the heavy chain. They obtained the humanized antibody 26B12H2L2(hG4DM) with constant region mutations by introducing a point mutation from phenylalanine to alanine (F234A) at position 234 and a point mutation from leucine to alanine (L235A) at position 235 of the constant region of the heavy chain. The amino acid sequence of the heavy chain 26B12H2(hG4DM) is shown in SEQ ID NO:27; the amino acid sequence of the light chain is shown in SEQ ID NO:29.

[0343] The preparation of humanized antibodies 26B12H2L2 (hG1DM) and 26B12H2L2 (hG4DM) can be carried out by referring to the method in step 2 above.

[0344] Example 4: Sequence design and preparation of the fusion protein of anti-TIGIT antibody and TGF-βR

[0345] 1. Sequence Design

[0346] The composition of the anti-TIGIT antibody and TGF-βR fusion protein in this invention is shown in Table 2 below.

[0347] Table 2: Heavy and light chain composition of the fusion protein of anti-TIGIT antibody and TGF-βR

[0348]

[0349] The TGF-β receptor portion has two peptide chains, which are linked to the C-terminus of the heavy chain of the IgG portion via linker fragments.

[0350] 2. Antibody expression and purification

[0351] 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 plasmids pUC57simple-TF01H, pUC57simple-TF02H, pUC57simple-TF02L, and pUC57simple-TF02L, respectively.

[0352] Plasmids pUC57simple-TF01H / pUC57simple TF01L and pUC57simple-TF02H / pUC57simple TF02L were digested with HindIII and EcoRI, respectively. The heavy and light chains obtained by electrophoresis were subcloned into the pcDNA3.1 vector, 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 the sample was loaded onto a HiTrap MabSelect SuRe column. Proteins were eluted with Elution Buffer in one step, the target sample was recovered, and the medium was changed to PBS. The fusion proteins TF01 and TF02 of anti-TIGIT antibody and TGF-βR were thus prepared.

[0353] Example 5: ELISA method for determining the binding activity of TF01 and TF02 with antigen or TGF-β

[0354] 1. Indirect ELISA method was used to measure TF01, TF02, 26B12H2L2 (hG4DM), RG6058 (hG4) and antigen, respectively. Binding activity of TIGIT-mFc

[0355] The specific method is as follows:

[0356] The ELISA plate was coated with 2 μg / mL goat anti-mouse IgG Fc (Jackson, catalog number: 115-005-071) and incubated overnight at 4°C. The plate was then washed once with PBST and blocked for 2 hours at 37°C with 1% BSA in PBS solution. After blocking, the plate was washed three times with PBST, and 1 μg / mL TIGIT-mFc was added. The plate was incubated at 37°C for 30 minutes, washed three times with PBST, and then serially diluted antibody (antibody dilution gradient detailed in Table 3) was added. The plate was incubated at 37°C for 30 minutes, and then washed three times with PBST. After washing, HRP-labeled goat anti-human IgG (H+L) (Jackson, catalog number: 109-035-098) secondary antibody working solution diluted 1:5000 was added, and the plate was incubated at 37°C for 30 minutes. After incubation, wash the plate four times with PBST, then add TMB (Neogen, 308177) for 5 min in the dark, and finally add stop solution to terminate the reaction. Immediately place the plate in a microplate reader and read the OD values ​​of each well at 450 nm. Analyze the data using SoftMax Pro 6.2.1 software.

[0357] The test results are shown in Table 3 and Figure 1 As shown.

[0358] Table 3: ELISA detection of the binding of TF01, TF02, 26B12H2L2 (hG4DM), RG6058 (hG4) to TIGIT-mFc

[0359]

[0360]

[0361] As shown in the figure, TF01, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) can effectively bind to the antigen TIGIT-mFc in a dose-dependent manner, and the EC50 of each dose is [data missing]. 50 The values ​​are shown in Table 3. The binding efficiencies (EC50) of TF01, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) (as a control) were obtained by quantitative analysis of the absorbance of the bound antibodies and curve simulation calculation. 50 The values ​​are 0.067 nM, 0.091 nM, 0.046 nM, and 0.055 nM, respectively.

[0362] The above experimental results show that TF01, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) have effective binding activity to TIGIT-mFc.

[0363] 2. Indirect ELISA method was used to measure TF01, TF02, TGF-βRII-mFc and TGF-β1, TGF-β2, TGF-β3, respectively. Binding activity

[0364] The specific method is as follows:

[0365] TGF-β1, TGF-β2, and TGF-β3 were coated onto ELISA plates at concentrations of 1 μg / mL, 2 μg / mL, and 1 μg / mL, respectively, and incubated overnight at 4°C. The plates were then washed once with PBST, and blocked at 37°C for 2 hours using a 1% BSA-containing PBS solution as the blocking buffer. After blocking, the plates were washed three times with PBST. Serially diluted PBST solutions of antibody and TGF-βRII-mFc were added, followed by the addition of the target antibody and TGF-βRII-mFc. The plates were incubated at 37°C for 30 minutes, and then washed three times with PBST. After washing the plate, add HRP-labeled goat anti-human IgG (H+L) (Jackson, catalog number: 109-035-098) and HRP-labeled goat anti-mouse IgG Fc (Jackson, catalog number: 115-035-071) secondary antibody working solution diluted 1:5000, and then incubate at 37°C for 30 minutes. After incubation, wash the plate four times with PBST, then add TMB (Neogen, 308177) for color development in the dark for 5 minutes, and add stop solution to terminate the color development reaction. Immediately place the ELISA plate in a microplate reader and read the OD values ​​of each well at a wavelength of 450 nm. Analyze the data using SoftMax Pro 6.2.1 software.

[0366] The test results are shown in Tables 4, 5, and 6. Figure 2 , Figure 3 and Figure 4 As shown.

[0367] Table 4: ELISA detection of the binding of TF01, TF02, TGF-βRII-mFc to TGF-β1

[0368]

[0369] Table 5: ELISA detection of the binding of TF01, TF02, TGF-βRII-mFc to TGF-β2

[0370]

[0371]

[0372] Table 6: ELISA detection of the binding of TF01, TF02, TGF-βRII-mFc to TGF-β3

[0373]

[0374]

[0375] The binding efficiency (EC50) of TF01 with TGF-β1, TGF-β2, and TGF-β3 was obtained by quantitative absorbance analysis of the bound antibodies and curve simulation calculation. 50 The binding efficiencies (ECs) of TF02 with TGF-β1, TGF-β2, and TGF-β3 were 0.207 nM, 0.477 nM, and 0.215 nM, respectively. 50 The binding efficiencies (EC50) of TGF-βR2-mFc (as a positive control) to TGF-β1, TGF-β2, and TGF-β3 were 0.224 nM, 0.594 nM, and 0.234 nM, respectively. 50 The values ​​were 0.356 nM, 1.029 nM, and 0.402 nM, respectively.

[0376] The results showed that TF01 and TF02 exhibited effective binding activity to TGF-β1, TGF-β2, and TGF-β3 in a dose-dependent manner. Furthermore, TF01, TF02, and TGF-βRII-mFc all effectively bound to TGF-β1, TGF-β2, and TGF-β3, respectively, with TF01 and TF02 showing stronger binding affinity to these three molecule types than TGF-βRII-mFc.

[0377] Example 6: Competitive ELISA method to determine the competitive binding of TF01, TF02 and TGF-βRII-His-Biotin to human cells. TGF-β1 and TGF-β3 activity

[0378] Experimental Procedure: TGF-β1 and TGF-β3 were coated onto ELISA plates at 2 μg / mL and incubated overnight at 4°C. After incubation, the antigen-coated ELISA plates were washed once with PBST, followed by blocking with 1% BSA in PBS solution for 2 hours. After blocking, the plates were washed three times with PBST. Serially diluted antibodies and TGF-βRII-mFc were added to the ELISA plates. For TGF-β1 plates: after incubation at room temperature for 60 minutes, the plates were washed three times with PBST, and 0.01 μg / mL of TGF-βRII-His-Biotin was added and incubated at room temperature for 10 minutes. For TGF-β3 plates: after incubation at 37°C for 30 minutes, the plates were washed three times with PBST, and 0.01 μg / mL of TGF-βRII-His-Biotin was added and incubated at 37°C for 30 minutes. After incubation, the plates were washed three times with PBST. After washing the plate, add SA-HRP working solution diluted 1:4000 and incubate at 37°C for 30 minutes. After incubation, wash the plate four times with PBST, then add TMB (Neogen, 308177) for color development in the dark for 4 minutes. Stop the color development reaction by adding stop solution. Immediately place the plate in a microplate reader and read the OD values ​​of each well at 450 nm. Analyze the data using SoftMax Pro 6.2.1 software.

[0379] The results of the antibody's competitive activity against TGF-βRII-His-Biotin in binding to TGF-β1 and TGF-β3 are shown in Tables 7 and 8. Figure 5 , Figure 6 As shown. A curve was fitted with antibody concentration on the x-axis and absorbance value on the y-axis to calculate the EC50 of the antibody competing with TGF-βRII-His-Biotin for binding to TGF-β1 and TGF-β3. 50 The results are shown in Tables 7 and 8 below.

[0380] Table 7: Activity assay results of TF01, TF02 and TGF-βRII-His-Biotin competing for TGF-β1 binding.

[0381]

[0382] Table 8: Activity assay results of TF01, TF02 and TGF-βRII-His-Biotin competing for TGF-β3 binding.

[0383]

[0384] Quantitative absorbance analysis of the bound antibodies was performed, and curve simulation calculations were used to obtain the EC50 binding efficiency of TF01, TF02, TGF-βRII-mFc, and TGF-βRII-His-Biotin in competitive binding to TGF-β1. 50 The binding efficiencies (ECs) of TF01, TF02, TGF-βRII-mFc, and TGF-βRII-His-Biotin to TGF-β3 were 5.579 nM, 7.469 nM, and 6.475 nM, respectively. 50 The values ​​are 3.569 nM, 3.879 nM, and 2.808 nM, respectively.

[0385] The results showed that TF01 and TF02 could effectively bind to TGF-β1 and TGF-β3 in a dose-dependent manner. Furthermore, TF01 had a stronger ability to bind to TGF-β1 than TGF-βRII-His-Biotin. TF01's ability to competitively bind to TGF-β1 with TGF-βRII-His-Biotin was stronger than that with TGF-βRII-mFc.

[0386] Example 7: Competitive ELISA method for determining the competitive binding of TF01, TF02 and CD155-hFc-Biotin to human TIGIT- mFc activity

[0387] Human TIGIT-mFc (TIGIT Genbank ID: NP_776160.2) was coated onto an ELISA plate at a concentration of 2 μg / mL and incubated overnight at 4°C. After incubation, the plate was blocked with 1% BSA in PBS at 37°C for 2 hours. After blocking, the plate was washed three times and patted dry. The antibody was serially diluted to seven concentrations at a 1:3 ratio using 20.18 nM (final concentration 10.09 nM) as the starting concentration on a dilution plate, with a blank control included. An equal volume of 4 μg / mL (final concentration 2 μg / mL) of human CD155-hFc-Biotin solution was added, mixed, and incubated at room temperature for 20 minutes. The resulting mixture was then added to the coated ELISA plate and incubated at 37°C for 30 minutes. After incubation, wash the plate three times with PBST and blot dry. Add SA-HRP (KPL, 14-30-00) working solution and incubate at 37°C for 30 minutes. After incubation, wash the plate four times and blot dry. Then add TMB (Neogen, 308177) and develop the color in the dark for 5 minutes. Add stop solution to terminate the color development reaction. Immediately place the microplate in a microplate reader and read the OD values ​​of each well at 450 nm. Analyze the data using SoftMax Pro 6.2.1 software.

[0388] Test results as follows Figure 7As shown in Table 9, the OD values ​​for each dose are listed below. The binding EC values ​​were obtained by quantitatively analyzing the absorbance of the bound antibody and using curve simulation of antibody binding efficiency. 50 (Table 9)

[0389] Table 9: Activity assay results of TF01, TF02, 26B12H2L2 (hG4DM), RG6058 (hG4) and CD155-hFc-Biotin competing for binding with TIGIT-mFc

[0390]

[0391]

[0392] TF01, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) (as controls) blocked the binding of TIGIT-mFc to its ligand CD155-hFc-Biotin in EC2 cells. 50 The values ​​are 1.463 nM, 1.963 nM, 1.423 nM, and 1.675 nM, respectively.

[0393] The results showed that TF01, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) (as a control) could effectively block the binding of the antigen human CD155-hFc-Biotin to its receptor human TIGIT-mFc, and the blocking efficiency showed a dose-dependent relationship. TF01 had a stronger ability to block the binding of TIGIT-mFc to its ligand CD155-hFc-Biotin than the control antibody RG6058 (hG4).

[0394] Example 8: FACS detection of the binding activity of TF01 to TIGIT on the surface of 293T-TIGIT membrane

[0395] Collect 293T-TIGIT cells in logarithmic growth phase, at a ratio of 3 x 10⁻⁶. 5Cells were transferred per well to a clear conical 96-well plate. 1% PBSA was added, and the plate was centrifuged at 350g for 5 min, then the supernatant was discarded. 100 μL of antibody diluted with 1% PBSA (final concentrations of 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, 0.0041 nM, and 0.00041 nM) was added, and the mixture was gently incubated on ice for 1 h. 1% PBSA was added, and the plate was centrifuged at 350g for 5 min, then the supernatant was discarded. The cells were resuspended in 350-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog number: 109-095-098), and the mixture was incubated on ice in the dark for 0.5 h. 1% PBSA was added, and the plate was centrifuged at 350g for 5 min, then the supernatant was discarded. 400 μL of PBSA was added to resuspend the cell pellet, and the pellet was transferred to a flow cytometer for FACS analysis. Calibur detection.

[0396] The experimental results are shown in Table 10 and Figure 8 As shown, both TF01 and RG6058(hG4) (control antibody) can specifically bind to TIGIT on the surface of the 293T-TIGIT membrane.

[0397] Table 10: FACS detection of the binding activity of TF01 and RG6058 (hG4) to TIGIT on the surface of 293T-TIGIT cells.

[0398] 0.00041 8.83 9.07 0.0041 10.00 10.30 0.041 18.50 19.50 0.41 97.40 106.00 1.23 244.00 251.00 3.7 497.00 508.00 11.1 596.00 629.00 33.3 604.00 630.00 100 589.00 630.00 <![CDATA[EC 50 (nM)]]> 1.540 1.612 R2 0.9981 0.9981

[0399] Under the same experimental conditions, TF01 and RG6058(hG4) bound EC2 cells to 293T-TIGIT cells. 50 The values ​​are 1.540 nM and 1.612 nM, respectively.

[0400] The results showed that TF01 had effective activity in binding TIGIT on the surface of the 293T-TIGIT membrane in a dose-dependent manner, and its binding ability was stronger than that of the control antibody RG6058 (hG4).

[0401] Example 9: Competitive flow cytometry determination of TF01 competing with CD155 or CD112 for cell membrane surface antigens TIGIT

[0402] 293T-TIGIT cells were routinely digested, with 300,000 cells / well per sample, and centrifuged and washed. 100 μL of serially diluted antibody was added to each well, and the cells were incubated on ice for 30 min. 100 μL of LCD155 / CD112-mFc (prepared by Kangfang Biotechnology, batch number: 20190726 / 20190726) was added to each well, and the mixture was stirred to achieve a final concentration of 10 nM / 30 nM. The cells were incubated on ice for 1 hour. 1% PBSA was added, and the cells were centrifuged at 350g for 5 min, then the supernatant was discarded. 100 μL of APC Goat anti-mouse IgG antibody (Biolegend, catalog number: 405308) diluted 1:300 was added to each well. For blank samples, 100 μL of 1% PBSA was added. The mixture was stirred and incubated on ice in the dark for 30 min. After centrifugation, washing, and resuspending, the cells were transferred to flow cytometry tubes for analysis.

[0403] The results are as follows Figure 9 and Figure 10 As shown, EC 50 The values ​​are shown in Table 11. The competitive binding EC50 of TF01 was calculated using quantitative fluorescence analysis and curve fitting. 50 The values ​​are 1.2720 nM and 0.7445 nM, respectively.

[0404] Table 11: Fluorescence intensity analysis of TF01 competitively binding to 293T-TIGIT surface antigen by FACS

[0405]

[0406] The results showed that the TF01 antibody effectively blocked the binding of CD155 and / or CD112 to TIGIT on the surface of 293T-TIGIT host cells in a dose-dependent manner.

[0407] Example 10: Affinity detection of TF01 with Fc receptor FcγRI

[0408] The Fc receptor FcγRI (also known as CD64) can bind to the Fc terminus of IgG antibodies, participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic antibodies to bind to the Fc receptor affects their safety and efficacy. In this study, the affinity constant between TF01 and FcγRI was detected using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of the antibody.

[0409] The experimental method for detecting the affinity constants of corresponding antibodies with FcγRI using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was a solution of PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. FcγRI (purchased from Sinobio) solution at a concentration of 1 μg / mL was added to the HIS1K sensor and immobilized for 50 s to allow FcγRI to adhere to the sensor surface. The binding and dissociation parameters of the antibody with FcγRI were measured in buffer solutions at antibody concentrations ranging from 3.12 to 50 nM (two-fold serial dilution). The sample plate was vibrated at 1000 rpm, the detection temperature was 30 °C, and the frequency was 5.0 Hz. Data were analyzed using a 1:1 model to obtain the affinity constants.

[0410] The results of the affinity constant determination between FcγRI and TF01 are shown in Table 12 and Figure 11 - Figure 12 As shown.

[0411] Table 12: Kinetic parameters of TF01 binding to FcγRI

[0412]

[0413] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0414] The results showed that 26B12H2L2(hG1WT) could bind to FcγRI with an affinity constant of 6.51E-09M. TF01 did not bind to FcγRI or had an extremely low binding signal, so the results were not analyzed and no corresponding data were obtained.

[0415] The results showed that the binding activity of TF01 to FcγRI was effectively eliminated.

[0416] Example 11: Affinity determination of TF01 with Fc receptor FcγRIIIa and its subtypes

[0417] (1) Determination of the affinity constant between FcγRIIIa_V158 and TF01

[0418] The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) can bind to the Fc terminus of IgG antibodies, mediating ADCC effects. In this experiment, the affinity constant between TF01 and FcγRIIIa_V158 was measured using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of the antibody.

[0419] The experimental method for detecting the affinity constants of corresponding antibodies using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIIa_V158 was immobilized on the HIS1K sensor for 60 s. The sensor equilibrated in the buffer for 60 s. The immobilized FcγRIIIa_V158 bound to each antibody (concentration 31.25-500 nM, 2-fold dilution) for 60 s. The antibody dissociated in the buffer for 60 s. The sample plate vibrated at 1000 rpm, the detection temperature was 30℃, and the frequency was 5.0 Hz. Data were analyzed using a 1:1 model to obtain the affinity constants.

[0420] The results of the affinity constant determination between FcγRIIIa_V158 and TF01 are shown in Table 13 and Figure 13 - Figure 14 As shown.

[0421] Table 13: Kinetic parameters of TF01 combined with FcγRIIIa_V158

[0422]

[0423] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0424] The results showed that 26B12H2L2(hG1WT) could bind to FcγRIIIa_V158 with an affinity constant of 5.54E-08M. TF01 was not analyzed because it did not bind to FcγRIIIa_V158 or had an extremely low binding signal.

[0425] The results showed that the binding activity of TF01 to FcγR IIIa_V158 was effectively eliminated.

[0426] (2) Determination of the affinity constant between FcγRIIIa_F158 and TF01

[0427] The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) can bind to the Fc terminus of IgG antibodies, mediating ADCC. In this experiment, the affinity constant between TF01 and FcγRIIIa_F158 was detected using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of each antibody.

[0428] The experimental method for detecting the affinity constant of TF01 and FcγRIIIa_F158 using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 s. The sensor equilibrated in the buffer for 60 s. The immobilized FcγRIIIa_F158 bound to various antibodies (31.25-500 nM, 2-fold dilution) for 60 s. The antibodies dissociated in the buffer for 60 s. The sample plate was vibrated at 1000 rpm, the detection temperature was 30℃, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model to obtain the affinity constant.

[0429] The results of the affinity constant determination between FcγRIIIa_F158 and TF01 are shown in Table 14 and Figure 15 - Figure 16 As shown.

[0430] Table 14: Kinetic parameters of TF01FcγRIIIa_F158 bonding

[0431]

[0432] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0433] The results showed that 26B12H2L2(hG1WT) could bind to FcγRIIIa_F158 with an affinity constant of 9.97E-08M. TF01 did not bind to FcγRIIIa_F158 or the binding signal was extremely low. The results were not analyzed, so no corresponding data were obtained.

[0434] The results showed that the binding activity of TF01 to FcγRIIIa_F158 was effectively eliminated.

[0435] Example 12: Affinity determination of TF01 with Fc receptor FcγRIIa and its subtypes

[0436] (1) Determination of the affinity constant between FcγRIIa_H131 and TF01

[0437] The Fc receptor FcγRIIa_H131 (also known as CD32a_H131) can bind to the Fc terminus of IgG antibodies, participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic antibodies to bind to the Fc receptor affects their safety and efficacy. In this study, the affinity constant between TF01 and FcγRIIa_H131 was measured using a Fortebio Octet molecular interaction analyzer to evaluate the binding ability of each test antibody to the Fc receptor.

[0438] The experimental method for detecting the affinity constant of TF01 and FcγRIIa_H131 using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_H131 was immobilized on the NTA sensor at a height of approximately 1.0 nm. The sensor was equilibrated in the buffer for 60 s. The immobilized FcγRIIa_H131 then bound to various antibodies at concentrations ranging from 12.5 to 200 nM (two-fold serial dilution) for 60 s. The antibodies then dissociated in the buffer for 60 s. The sample plate was vibrated at 1000 rpm, the detection temperature was 30 °C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model to obtain the affinity constant.

[0439] The results of the affinity constant determination between FcγRIIa_H131 and TF01 are shown in Table 15 and Figure 17 - Figure 18 As shown.

[0440] Table 15: Kinetic parameters of TF01 and FcγRIIa_H131 binding

[0441]

[0442] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0443] The results showed that 26B12H2L2(hG1WT) could bind to FcγRIIa_H131 with an affinity constant of 3.90E-08M. TF01 did not bind to FcγRIIa_H131 or the binding signal was extremely low. The results were not analyzed, so no corresponding data were obtained.

[0444] The results showed that the binding activity of TF01 to FcγRIIa_H131 was effectively eliminated.

[0445] (2) Determination of the affinity constant between FcγRIIa_R131 and TF01

[0446] The Fc receptor FcγRIIa_R131 (also known as CD32a_R131) can bind to the Fc terminus of IgG antibodies, participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic antibodies to bind to the Fc receptor affects their safety and efficacy. In this study, the affinity constant between TF01 and FcγRIIa_R131 was measured using a Fortebio Octet molecular interaction analyzer to evaluate the binding ability of each test antibody to the Fc receptor.

[0447] The experimental method for detecting the affinity constant of TF01 and FcγRIIa_R131 using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_R131 was immobilized on the NTA sensor at a height of approximately 1.0 nm. The sensor was equilibrated in the buffer for 60 s. The immobilized FcγRIIa_R131 then bound to various antibodies at concentrations ranging from 12.5 to 200 nM (two-fold serial dilution) for 60 s. The antibodies then dissociated in the buffer for 60 s. The sample plate was vibrated at 1000 rpm, the detection temperature was 30 °C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model to obtain the affinity constant.

[0448] The results of the affinity constant determination between FcγRIIa_R131 and TF01 are shown in Table 16 and Figure 19 - Figure 20 As shown.

[0449] Table 16: Kinetic parameters of TF01 combined with FcγRIIa_R131

[0450]

[0451] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0452] The results showed that 26B12H2L2(hG1WT) could bind to FcγRIIa_H131 with an affinity constant of 3.22E-08M. TF01 did not bind to FcγRIIa_R131 or had an extremely low binding signal, so the results were not analyzed and no corresponding data were obtained.

[0453] The results showed that the binding activity of TF01 to FcγRIIa_R131 was effectively eliminated.

[0454] Example 13: Determination of the affinity constant between FcγRIIb and TF01

[0455] The Fc receptor FcγRIIb (also known as CD32b) can bind to the Fc terminus of IgG antibodies. In this experiment, the affinity constants of each test antibody with FcγRIIb were detected using a FortebioOctet molecular interaction analyzer to evaluate the binding ability of TF01 to the Fc receptor.

[0456] The experimental method for detecting the affinity constant of TF01 and FcγRIIb using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIb was immobilized on the NTA sensor at a height of approximately 1.0 nm. The sensor was equilibrated in the buffer for 60 s. hFCGR2B-his immobilized on the sensor then bound to various antibodies at concentrations ranging from 12.5 to 200 nM (two-fold serial dilution) for 60 s. The antibodies then dissociated in the buffer for 60 s. The sample plate was vibrated at 1000 rpm, the detection temperature was 30 °C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model to obtain the affinity constant.

[0457] The results of the affinity constant determination between FcγRIIb and TF01 are shown in Table 17 and Figure 21 - Figure 22 As shown.

[0458] Table 17: Kinetic parameters of TF01 binding with FcγRIIb

[0459]

[0460] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0461] The results showed that 26B12H2L2(hG1WT) could bind to FcγRIIb with an affinity constant of 4.12E-08M. TF01 did not bind to FcγRIIb or had an extremely low binding signal, so the results were not analyzed and no corresponding data were obtained.

[0462] The results showed that the binding activity of TF01 to FcγRIIb was effectively eliminated.

[0463] Example 14: Affinity determination of TF01 and C1q

[0464] Serum complement C1q can bind to the Fc terminus of IgG antibodies, mediating the CDC effect. The ability of therapeutic antibodies to bind to C1q affects the safety and efficacy of these antibodies. In this study, the affinity constant between TF01 and C1q was detected using a Fortebio Octet molecular interaction analyzer to evaluate the CDC activity of these antibodies.

[0465] The experimental method for detecting the affinity constant of the corresponding antibody with C1q using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 50 μg / mL antibody was immobilized on the FAB2G sensor at a height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 s. The antibody immobilized on the sensor then bound to the antigen C1q at a concentration of 0.625–10 nM (two-fold serial dilution) for 60 s. The antigen and antibody then dissociated in the buffer for 60 s. The sample plate was vibrated at 1000 rpm, the detection temperature was 30°C, and the detection frequency was 5.0 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant. Fortebio Data Acquisition 7.0 and Fortebio Data Analysis 7.0 were used for data acquisition and analysis.

[0466] The results of the affinity constant determination between TF01 and C1q are shown in Table 18 and Figure 23 - Figure 24 As shown.

[0467] Table 18: Kinetic parameters of TF01 and C1q bonding

[0468]

[0469] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0470] The results showed that 26B12H2L2(hG1WT) could bind to C1q with an affinity constant of 1.28E-09M. TF01 did not bind to C1q or had an extremely low binding signal, so the results were not analyzed and no corresponding data were obtained.

[0471] The results showed that the binding activity of TF01 to C1q was effectively eliminated.

[0472] Example 15: Antibody-mediated phagocytosis activity of TF01 against CHO-K1-TIGIT

[0473] To detect antibody-dependent cellular phagoxytosis (ADCP) activity, mouse macrophages were used as effector cells, and cell lines overexpressing TIGIT were used as target cells to detect their ADCP-mediated effect.

[0474] Take frozen MBMM (mouse bone marrow-derived macrophages) (derived from C57 mice), add DMEM + 10% FBS + 100 ng / mL M-CSF (macrophage colony-stimulating factor, Peprotech, catalog number: 315-02), centrifuge at 1200 x g for 5 min, collect cells, add DMEM + 10% FBS + 100 ng / mL M-CSF medium, and thaw overnight.

[0475] Collect target cells (CHO-K1-TIGIT), centrifuge at 170xg for 5 min, and wash once with PBS. Perform trypan blue counting. Dilute CFSE (5(6)-carboxydiacetic acid fluorescein succinimide ester, Biolegend, catalog number: 423801) to 2.5 μM with PBS. Resuspend the cells in an appropriate amount of diluted CFSE (staining density: 10 million cells / mL) and incubate for 20 min. Add 6 mL of DMEM complete medium (containing 10% FBS) to stop staining, centrifuge at 170xg for 5 min, discard the supernatant, and add 1 mL of... Incubate in DMEM complete medium for 10 min; dilute the antibody to the required concentration with DMEM complete medium and design an isotype control antibody; add target cells (150,000 / well) to a 96-well conical plate, add antibody (100 μL), mix well, incubate on ice for 40 min, centrifuge at 170 x g for 5 min and wash twice; collect macrophages (MBMM), centrifuge at 750 x g for 5 min, discard the supernatant and count, resuspend the cells in DMEM complete medium, adjust the macrophage concentration (50,000 / 100 μL), add to the 96-well conical plate containing target cells, resuspend and mix well, incubate at 37°C for 2 h; add 100 μL of 1% PBSA at room temperature to each well, centrifuge at 750 x g for 5 min and discard the supernatant; wash once with 200 μL of PBSA; add APC anti-mouse / human CD11b antibody (Biolegend, catalog number: 101212) (using PBSA) to each well at 100 μL / well. Add the 500-fold dilution to the corresponding sample, mix well, and incubate on ice for 40 min; add 100 μL of 1% PBSA to each well, centrifuge at 750 x g for 5 min, and discard the supernatant; wash each well once with 200 μL of PBSA; resuspend each tube with 200 μL of 1% PBSA and load onto the instrument. Macrophages in the instrument are APC-positive, and macrophages that phagocytose are double-positive for both APC and CFSE. The ratio of double-positive cells to APC-positive cells is used as the phagocytosis rate, and this is used to evaluate antibody-mediated ADCP activity. Calculate the ADCP activity of each group using the following formula, expressed as P%:

[0476]

[0477] The results are as follows Figure 25 As shown.

[0478] The results showed that RG6058(hG1WT), RG6058(hG4), and 26B12H2L2(hG1WT) had ADCP effects at the same concentration; the phagocytosis rate of TF01 at the same concentration was comparable to that of the isotype control antibody group, indicating that TF01 did not have ADCP effects.

[0479] The results showed that the amino acid mutations introduced by TF01 could effectively eliminate its ADCP effect.

[0480] Example 16: The inhibitory effect of the fusion protein of anti-TIGIT antibody and TGF-βR on CD112 / CD155 in a co-culture system Evaluation of IL-2 secretion blocking activity

[0481] 1. Evaluation of the inhibitory activity of the fusion protein of anti-TIGIT antibody and TGF-βR on IL-2 secretion in CD112-mediated Jurkat-TIGIT and THP-1 cell co-culture system.

[0482] 2 μg / mL anti-human CD3 antibody (prepared by Kangfang Biotechnology, batch number: 20170830) was used to coat 96-well plates (Corning, model: 3599) at 37℃ for 2 h; the coating solution was removed, and the plates were washed once with pre-cooled PBS (300 μL); Jurkat-TIGIT (constructed by Kangfang Biotechnology) cell counting was performed, and 50,000 cells were seeded into each well of the 96-well plate; the antibody was added according to the experimental design, and the plates were incubated at 37℃ for 30 min; CD112-hFc (prepared by Kangfang Biotechnology, batch number: 20180209) was added according to the experimental design; THP-1 (purchased from the Chinese Academy of Sciences, catalog number: 3131C0001000700057) cell counting was performed, and 50,000 cells were seeded into each well of the 96-well plate; the plates were incubated for 48 h; the culture supernatant was collected, and the IL-2 content was detected using an IL-2 ELISA kit (Dakewei, catalog number: 1110202).

[0483] The results are as follows Figure 26 As shown in the figure. The results showed that CD112 had a significant inhibitory effect on IL-2 secretion. Antibodies 26B12H2L2 (hG4DM), TF01, TF02, RG6058 (hG1DM), and RG6058 (hG4) could all effectively block the inhibition of IL-2 secretion by CD112 in the system, and the activities of TF01 and TF02 were stronger than those of the positive control antibodies RG6058 (hG1DM) and RG6058 (hG4).

[0484] 2. Evaluation of the inhibitory activity of the anti-TIGIT antibody and TGF-βR fusion protein on IL-2 secretion in CD155-mediated Jurkat-TIGIT and THP-1 cell co-culture system.

[0485] 2 μg / mL anti-human CD3 antibody (prepared by Kangfang Biotechnology, batch number: 20170830) was used to coat 96-well plates (Corning, model: 3599) at 37℃ for 2 h; the coating solution was removed, and the plates were washed once with pre-cooled PBS (300 μL); Jurkat-TIGIT (constructed by Kangfang Biotechnology) cell counting was performed, and 50,000 cells were seeded into each well of the 96-well plate; the antibody was added according to the experimental design, and the plates were incubated at 37℃ for 30 min; CD155-hFc (prepared by Kangfang Biotechnology, batch number: 20180209) was added according to the experimental design; THP-1 (purchased from the Chinese Academy of Sciences, catalog number: 3131C0001000700057) cell counting was performed, and 50,000 cells were seeded into each well of the 96-well plate; the plates were incubated for 48 h; the culture supernatant was collected, and the IL-2 content was detected using an IL-2 ELISA kit (Dakewei, catalog number: 1110202).

[0486] The results are as follows Figure 27 As shown in the figure. The results showed that CD155 had a significant inhibitory effect on IL-2 secretion. Antibodies TF01 and RG6058 (hG1DM) could both effectively block the inhibition of IL-2 secretion by CD155 in the system, and their activities were comparable.

[0487] Example 17: Fusion protein of anti-TIGIT antibody and TGF-βR in Jurkat-TIGIT and HT1080-aCD3scFv Evaluation of the bioactivity of IL-2 secretion-promoting agents in cell co-culture systems

[0488] Jurkat-TIGIT and HT1080-aCD3scFv cells (constructed by Kangfang Biotechnology) in logarithmic growth phase were collected and counted, with 50,000 Jurkat-TIGIT cells per well and 10,000 HT1080-aCD3scFv cells per well; diluted antibodies (antibody gradient 3nM, 30nM, 300nM) were added; soluble anti-human CD28 antibody (3μg / mL) (R&D, catalog number: MAB342-500) was added; the cells were incubated in an incubator for 48 h; the culture supernatant was collected, and the IL-2 content was detected using an IL-2 ELISA kit (Dakewei, catalog number: 1110202).

[0489] The results are as follows Figure 28 As shown.

[0490] The results showed that antibodies TF01, TF02 and RG6058(hG4) could effectively promote IL-2 secretion in the co-culture system of Jurkat-TIGIT and HT1080-scFv cells, and their activities were comparable.

[0491] Example 18: The fusion protein of anti-TIGIT antibody and TGF-βR during the secondary immune response of PBMCs to CMV antigen Evaluation of the blocking activity of TGF-β1 inhibitory system against IFN-γ secretion

[0492] Resuscitated PBMCs (from healthy donors) were seeded in complete culture medium (RPMI 1640 + 10% FBS) and incubated overnight. The following day, cells were collected, counted, and viability assessed. 200,000 cells / well were seeded into 96-well round-bottom plates (Corning, catalog number: 3799). Serially diluted antibodies, CMV (final concentration 0.02 μg / mL, Mabtech, catalog number: 3619-1), and TGF-β1 (final concentration 3 ng / mL, Genscript, catalog number: Z03411) were added according to the experimental design, to a final volume of 200 μL. (TGF-β1 and antibody were incubated at 37°C for 10 min). The plates were incubated for 4 days. After 4 days, the cell culture supernatant was collected, and the IFN-γ content was detected using an IFN-γ ELISA kit (Dakeway, catalog number: 1110002).

[0493] The results are as follows Figure 29 As shown in the figure. The results showed that CMV antigen could induce a secondary immune response in PBMCs, resulting in the secretion of IFN-γ. TGF-β1 had a significant inhibitory effect on IFN-γ secretion in the system. The combination of antibody TF01, anti-HEL&TGFβ, and anti-HEL&TGFβ+26B12H2L2(hG1DM) could effectively block the inhibition of IFN-γ secretion by TGF-β1, while 26B12H2L2(hG1DM) alone was ineffective. The blocking activity of TF01 was superior to that of the control antibody anti-HEL&TGFβ, and comparable to that of the combination of anti-HEL&TGFβ+26B12H2L2(hG1DM).

[0494] Example 19: The fusion protein of anti-TIGIT antibody and TGF-βR in a mouse tumor cell subcutaneous transplantation model Pharmacodynamic evaluation

[0495] To detect the in vivo tumor-suppressive activity of the anti-TIGIT antibody and TGF-βR fusion protein, MDA-MB-231 cells (purchased from ATCC) were first inoculated subcutaneously into the mammary fat pads of female Scid Bseige mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged 6.71-9.57 weeks. Seventeen days after inoculation, mice were randomly divided into three groups of eight mice each based on tumor volume. Each mouse received an intraperitoneal injection of human peripheral blood mononuclear cells (hPBMCs) and was administered the drug; the day of grouping was defined as day 0. Administration was done via intraperitoneal injection, once a week for a total of six administrations. hPBMCs were injected intraperitoneally on day 8 after grouping. The modeling process and specific administration methods are shown in Table 19. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated.

[0496] Table 19: Dosing regimens for treating the Scid Beige mouse MDA-MB-231 xenograft model with the fusion protein of anti-TIGIT antibody and TGF-βR.

[0497]

[0498] The results are as follows Figure 30 As shown in the figure. The results indicate that, compared with the isotype control antibody, the fusion protein TF01 of anti-TIGIT antibody and TGF-βR can effectively inhibit the growth of mouse tumors.

[0499] In addition, such as Figure 31 As shown, tumor-bearing mice tolerated the test drug TF01 well, and there was no effect on the body weight of tumor-bearing mice in any group.

[0500] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. A fusion protein having the following composition: Targeting the first protein functional region of TIGIT, and A second protein functional region possessing TGF-β binding activity; in, The first protein functional region is an anti-TIGIT antibody, and the anti-TIGIT antibody has a heavy chain variable region containing amino acid sequences as shown in SEQ ID NOs: 3-5, namely HCDR1-HCDR3; and a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 8, an amino acid sequence of LCDR2 with the amino acid sequence SAS, and an amino acid sequence of LCDR3 as shown in SEQ ID NO:

10. The second protein functional region is the extracellular region of TGF-βRII or a truncated fragment of the extracellular region of TGF-βRII with TGF-β receptor function; The first protein has one functional region, and the second protein has two functional regions; The first protein functional region and the second protein functional region are connected by a linker fragment; The extracellular region of the TGF-βRII or a truncated fragment of the extracellular region of the TGF-βRII with TGF-β receptor function is linked to the C-terminus of the heavy chain of the anti-TIGIT antibody.

2. The fusion protein according to claim 1, wherein, The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is selected from SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17; and The amino acid sequence of the light chain variable region of the anti-TIGIT antibody is selected from SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO:

25.

3. The fusion protein according to claim 1, wherein, The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

6. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

19. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

19. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

21. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

23. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

21. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

23. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25; or The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

25.

4. The fusion protein according to claim 1, wherein, The anti-TIGIT antibody is a humanized antibody or a chimeric antibody.

5. The fusion protein according to claim 1, wherein, The anti-TIGIT antibody includes a non-CDR region, and the non-CDR region is derived from a human antibody.

6. The fusion protein according to claim 1, wherein, The heavy chain constant region of the anti-TIGIT antibody is either the Ig gamma-1 chain C region or the Ig gamma-4 chain C region; the light chain constant region is the Ig kappa chain C region.

7. The fusion protein according to claim 6, wherein, The Ig gamma-4 chain C region is shown in NCBIACCESSION: P01857, and the Ig kappa chain C region is shown in NCBI ACCESSION: P01834.

8. The fusion protein according to claim 6, wherein, The Ig gamma-1 chain C region is shown in NCBIACCESSION: P01861.1, and the Ig kappa chain C region is shown in NCBI ACCESSION: P01834.

9. The fusion protein according to claim 1, wherein, The fusion protein binds more strongly to the ligand CD155-hFc-Biotin than the control antibody RG6058(hG4), which is a human IgG4 antibody against TIGIT. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 42, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

43.

10. The fusion protein according to claim 1, wherein, The fusion protein has an EC50 concentration of less than 0.08 nM. 50 Combined with TIGIT-mFc; the EC 50 Measured by indirect ELISA method.

11. The fusion protein according to claim 1, wherein, The fusion protein has an EC50 concentration of less than 0.10 nM. 50 Combined with TIGIT-mFc; the EC 50 Measured by indirect ELISA method.

12. The fusion protein according to claim 1, wherein, The fusion protein has an EC value of less than 0.3 nM. 50 Combined with TGF-β1, TGF-β2, or TGF-β3; the EC 50 Measured by indirect ELISA method.

13. The fusion protein according to claim 1, wherein, The fusion protein has an EC50 of less than 0.4 nM. 50 Combined with TGF-β1, TGF-β2, or TGF-β3; the EC 50 Measured by indirect ELISA method.

14. The fusion protein according to claim 1, wherein, The fusion protein has an EC50 concentration of less than 0.5 nM. 50 Combined with TGF-β1, TGF-β2, or TGF-β3; the EC 50 Measured by indirect ELISA method.

15. The fusion protein according to claim 1, wherein, The fusion protein has an EC50 of less than 0.6 nM. 50 Combined with TGF-β1, TGF-β2, or TGF-β3; the EC 50 Measured by indirect ELISA method.

16. The fusion protein according to claim 1, wherein, The anti-TIGIT antibody is an antibody produced by the hybridoma cell line LT019, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2020208.

17. The fusion protein according to any one of claims 1 to 16, wherein, According to the EU numbering system, when the heavy chain constant region of the anti-TIGIT antibody is the heavy chain constant region of IgG1, the heavy chain constant region of the anti-TIGIT antibody has one of the following mutation combinations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, G237A.

18. The fusion protein according to any one of claims 1 to 16, wherein, According to the EU numbering system, when the heavy chain constant region of the anti-TIGIT antibody is the heavy chain constant region of IgG4, the heavy chain constant region of the anti-TIGIT antibody has one of the following combinations of mutations: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A, G237A.

19. The fusion protein according to any one of claims 1 to 16, wherein, The amino acid sequence of the functional region of the second protein is shown in SEQ ID NO: 33 or SEQ ID NO:

37.

20. The fusion protein according to any one of claims 1 to 16, wherein, The amino acid sequence of the heavy chain of the anti-TIGIT antibody is shown in SEQ ID NO: 27 or SEQ ID NO: 31, and the amino acid sequence of its light chain is shown in SEQ ID NO:

29.

21. The fusion protein according to claim 1, wherein, The connection segment is (GGGGS)n or (GGGGS)nG, where n is a positive integer.

22. The fusion protein according to claim 1, wherein, n can be 1, 2, 3, 4, 5, or 6.

23. A fusion protein having the following composition: Targeting the first protein functional region of TIGIT, and A second protein functional region possessing TGF-β binding activity; in, The first protein has one functional region, and the second protein has two functional regions; The first protein functional region is an anti-TIGIT antibody or its antigen-binding fragment, and the second protein functional region is a TGF-β receptor, its extracellular region, or a truncated fragment of the extracellular region of a TGF-β receptor with TGF-β receptor function. The amino acid sequence of the heavy chain of the anti-TIGIT antibody is shown in SEQ ID NO: 27 or SEQ ID NO: 31, and the amino acid sequence of its light chain is shown in SEQ ID NO:

29. The amino acid sequence of the functional region of the second protein is shown in SEQ ID NO: 33; The second protein functional region is linked to the C-terminus of the heavy chain of the anti-TIGIT antibody via a linker fragment.

24. The fusion protein according to claim 23, wherein, The amino acid sequence of the linker fragment is shown in SEQ ID NO:

44.

25. An isolated nucleic acid molecule encoding the fusion protein of any one of claims 1 to 24.

26. A carrier comprising the isolated nucleic acid molecule of claim 25.

27. A host cell comprising the isolated nucleic acid molecule of claim 25, or the vector of claim 26.

28. A conjugate comprising a fusion protein portion and a conjugation portion, wherein, The fusion protein portion is the fusion protein according to any one of claims 1 to 24, and the coupling portion is a detectable marker.

29. The coupling according to claim 28, wherein, The coupling component is a radioactive isotope, a fluorescent substance, a colored substance, or an enzyme.

30. The coupling according to claim 28, wherein, The coupling part is a luminescent material.

31. Use of the fusion protein of any one of claims 1 to 24 or the conjugate of any one of claims 28 to 30 in the preparation of a kit for detecting the presence or level of TIGIT and / or TGF-β in a sample.

32. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 24 or the conjugate of any one of claims 28 to 30.

33. The pharmaceutical composition of claim 32, further comprising a pharmaceutically acceptable carrier and / or excipient.

34. The pharmaceutical composition according to claim 32 or 33, further comprising one or more tumor chemotherapy drugs.

35. The pharmaceutical composition according to claim 34, wherein, The tumor chemotherapy drug is a tyrosine kinase inhibitor.

36. The pharmaceutical composition according to claim 34, wherein, The tumor chemotherapy drug is anlotinib or its pharmaceutically acceptable salt.

37. The pharmaceutical composition according to claim 36, wherein, The medicinal salt is a hydrochloride salt.

38. The pharmaceutical composition according to claim 34, wherein, The tumor chemotherapy drug is lenvatinib or its pharmaceutically acceptable salt.

39. The pharmaceutical composition according to claim 38, wherein, The medicinal salt is a methanesulfonate.

40. The pharmaceutical composition according to claim 32 or 33, wherein, The unit dose of the pharmaceutical composition, calculated based on the mass of the fusion protein therein, is 100mg-1500mg.

41. The pharmaceutical composition according to claim 32 or 33, wherein, The unit dose of the pharmaceutical composition, calculated based on the mass of the fusion protein therein, is 200 mg to 1000 mg.

42. The pharmaceutical composition according to claim 32 or 33, wherein, The unit dose of the pharmaceutical composition, calculated based on the mass of the fusion protein therein, is 200 mg to 800 mg.

43. The pharmaceutical composition according to claim 32 or 33, wherein, The unit dose of the pharmaceutical composition, calculated based on the mass of the fusion protein therein, is 300mg-600mg.

44. The pharmaceutical composition according to claim 32 or 33, wherein, The unit dose of the pharmaceutical composition, calculated based on the mass of the fusion protein therein, is 400mg-500mg.

45. The pharmaceutical composition according to claim 32 or 33, wherein, The unit dose of the pharmaceutical composition, calculated based on the mass of the fusion protein therein, is 450 mg.

46. ​​A combination product comprising individually packaged first and second products, in, The first product comprises the fusion protein of any one of claims 1 to 24, the conjugate of any one of claims 28 to 30, or the pharmaceutical composition of any one of claims 32 to 45; The second product contains one or more chemotherapy drugs for cancer treatment.

47. The combined product according to claim 46, wherein, The second product contains a tumor chemotherapy drug that is a tyrosine kinase inhibitor.

48. The combined product according to claim 46, wherein, The second product contains anlotinib or its pharmaceutically acceptable salt as a tumor chemotherapy drug.

49. The combined product according to claim 48, wherein, The medicinal salt is a hydrochloride salt.

50. The combined product according to claim 46, wherein, The second product contains lenvatinib or its pharmaceutically acceptable salt as a tumor chemotherapy drug.

51. The combined product according to claim 50, wherein, The medicinal salt is a methanesulfonate.

52. The combined product according to any one of claims 46 to 51, wherein, Both the first product and the second product also independently contain one or more pharmaceutically acceptable excipients.

53. The combined product according to any one of claims 46 to 51, wherein, The combined product also includes a product instruction manual.

54. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the first product, calculated based on the mass of the fusion protein therein, is 100mg-1500mg.

55. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the first product, calculated based on the mass of the fusion protein therein, is 200mg-1000mg.

56. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the first product, calculated based on the mass of the fusion protein therein, is 200mg-800mg.

57. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the first product, calculated based on the mass of the fusion protein therein, is 300mg-600mg.

58. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the first product, calculated based on the mass of the fusion protein therein, is 400mg-500mg.

59. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the first product, calculated based on the mass of the fusion protein therein, is 450 mg.

60. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the second product, calculated based on the mass of its active ingredient, is 0.1 mg to 100 mg.

61. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the second product, calculated based on the mass of its active ingredient, is 0.5 mg to 50 mg.

62. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the second product, calculated based on the mass of its active ingredient, is 1mg-20mg.

63. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the second product, calculated based on the mass of its active ingredient, is 2mg-15mg.

64. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the second product, calculated based on the mass of its active ingredient, is 4mg-12mg.

65. The combined product according to any one of claims 46 to 51, wherein, The unit dose of the second product, calculated based on the mass of its active ingredient, is 8mg-12mg.

66. Use of the fusion protein according to any one of claims 1 to 24 or the conjugate according to any one of claims 28 to 30 in the preparation of a medicament for treating tumors; in, The tumor is selected from one or more of the following: 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-cell lymphoma, plasma cell carcinoma, renal cell carcinoma, and prostate cancer.

67. The use according to claim 66, wherein, The non-small cell lung cancer mentioned is advanced non-small cell lung cancer.

68. The use according to claim 66, wherein, The pancreatic cancer mentioned is pancreatic ductal adenocarcinoma.

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