Fusion protein containing a TGF-βRII extracellular domain fragment, pharmaceutical composition thereof, and use thereof

Abstract

The present disclosure relates to the fields of biology and pharmacology, and relates to a fusion protein comprising a TGF-βRII extracellular region fragment, a pharmaceutical composition thereof, and uses thereof. Specifically, the fusion protein of the present disclosure comprises a first protein functional region that targets an immune checkpoint and a second protein functional region that has TGF-β binding activity, wherein the second protein functional region is a variant of the TGF-βRII extracellular region fragment, in which the first serine at the N-terminus of the TGF-βRII extracellular region fragment is replaced with alanine, glycine, or threonine, and / or a fragment comprising the amino acid sequence set forth in SEQ ID NO: 59 is deleted from the TGF-βRII extracellular region fragment. The fusion protein of the present disclosure can simultaneously inhibit TIGIT and reduce TGF-β levels, demonstrating excellent potential for the preparation of antitumor drugs.

Description

[Technical Field]

[0001] The present disclosure relates to the fields of biology and pharmacology, and to a fusion protein comprising a TGF-βRII extracellular domain fragment, a pharmaceutical composition thereof, and uses thereof. [Background technology]

[0002] The transforming growth factor beta (TGF-β) superfamily is a functionally diverse class of cytokines that are further classified into subfamilies, such as TGF-β, activins, inhibins, growth differentiation factors (GDFs), glial cell line-derived neurotrophic factors (GDNFs), nodal, lefty, and anti-Müllerian hormone (Table A). Three proteins in the TGF-β subfamily are known as TGF-β1, TGF-β2, and TGF-β3, respectively, with TGF-β1 being the most highly expressed subtype. Upon binding to their receptors, TGF-β1, TGF-β2, and TGF-β3 mediate various biological responses via Smad and non-Smad signaling pathways, including promoting epithelial-mesenchymal transition (EMT), tissue fibrosis, angiogenesis, tumor immune evasion, and dual tumor suppressor and promoter effects (J Massague. TGFbeta in Cancer [J]. Cell, 2008, 134(2):215-230).

[0003] TGF-β receptors (TGF-βR) are widely distributed on the surface of normal and tumor cells in humans and comprise three receptor superfamilies: the classical TGF-β type I receptor family includes ALK1-7, TGF-βRI (also known as TβRI), also known as ALK5; the classical TGF-β type II receptor family includes TGF-βRII (also known as TβRII), ActRII, ActRIIB, AMHRII, and BMPRII; and the TGF-β type III receptor superfamily includes betaglycan (also known as TGF-βRIII) and endoglin. TGF-βRI, TGF-βRII, and TGF-βRIII can bind to TGF-β1, TGF-β2, and TGF-β3, respectively (Pawlak John B, Blobe Gerard C, TGF-β superfamily co-receptors in cancer. DevDyn, 2021). The TGF-β family and receptors are listed in Table A (based on a review by Carl-Henrik Heldin1 and Aristidis Moustakas, Cold Spring Harb Perspective Biol. 2016). TGF-βRI and TGF-βRII are serine / threonine protein kinase receptors. TGF-βRII, a key signaling molecule in the TGF-β signaling pathway, binds to TGF-β with high affinity and can then form a heterotetrameric receptor complex with a TGF-βRI dimer. Autophosphorylation of TGF-βRII leads to further phosphorylation and activation of TGF-βRI. Activated TGF-βRI phosphorylates downstream Smad pathway-related proteins, regulating the transcription and translation of downstream target genes, thereby triggering disease-related biological responses. TGF-βRIII has weaker affinity for TGF-β than TGF-βRI and TGF-βRII and lacks an intracellular segment. Therefore, TGF-βRIII 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, XH et al., Advances in research on small-molecule inhibitors targeting TGF-β and receptors [J]. Journal of Pharmacy, 2019(9)).

[0004] [Table 1-1]

[0005] [Table 1-2]

[0006] During tumor progression, tumor cells, mesenchymal fibroblasts, and other cells secrete large amounts of TGF-β in the tumor microenvironment (TME), mediating immunosuppression. TGF-β inhibits the differentiation of naive T cells into Th1 cells, which mediate antitumor activity, and TGF-βRII-deficient T cells have enhanced Th1 responses (Eduard, Batlle, Joan, et al., Transforming Growth Factor-β Signaling in Immunity and Cancer [J]. Immunity, 2019). Granzyme A, granzyme B, perforin, gamma-interferon (IFN-γ), and FasL, which are tumor-killing effector T cells, have reduced expression levels under the influence of TGF-β, resulting in immune escape of tumor cells. Tregs are key cells in the tumor microenvironment (TME) that mediate tumor immunosuppression and can inhibit the function of tumor-killing effector T cells. TGF-β produced by tumor cells induces the emergence of large numbers of Tregs in the TME, enhances tumor antigen tolerance, and promotes tumor immune escape (Chen Dan, Ran Yan. Advances in research on the regulation of tumor cells and tumor-associated immune cells by TGF-β [J]. Modern Medicine & Health, 2020, 36(09):1354-1358). Indications for TGF-β molecular-targeting antibody-based investigational drugs currently undergoing clinical trials 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.

[0007] TIGIT (also known as T cell Ig and ITIM domains, WUCAM, Vstm3, or VSIG9) is a member of the poliovirus receptor (PVR) / nectin family. TIGIT consists of an extracellular immunoglobulin variable region (IgV) domain, a type I transmembrane domain, and an intracellular domain containing classical immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoglobulin tail tyrosine (ITT) motifs. TIGIT binds to T cells in lymphocytes, particularly effector CD4 + T cells and regulatory CD4 + T cells (Treg cells), follicular helper CD4 + T cells, and effector CD8 + It is highly expressed in T cells and natural killer (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).

[0008] CD155 (also known as PVR, Necl5, or Tage4), CD112 (also known as PVRL2 / Nectin2), and CD113 (also known as PVRL3) are ligands that TIGIT binds to (Martinet L, Smyth M J. Balancing natural killer cell activation through paired receptors [J]. Nature Reviews Immunology, 2015, 15(4):243-254). CD155 is a high-affinity ligand for TIGIT. In NK cells, TIGIT binding to its ligands CD155 and CD112 can inhibit the killing of NK cells against cells with high expression of these two proteins (Stanietsky N, Simic H, Arapovic J, et al., The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity [J]. Proceedings of the National Academy of Sciences, 2009, 106(42):17858-17863). It has also been reported that the killing effect of CD8+ T cells can be enhanced by simultaneous blockade of PD-1 and TIGIT (Johnston RJ, Comps-Agrar L, Hackney J, et al., The immunoreceptor TIGIT regulates anti-tumor and antiviral CD8+ T cell effector function [J]. Cancer Cell, 2014, 26(6):923-937).Recent studies have revealed that TIGIT, an NK cell immune checkpoint, can cause NK cell exhaustion during tumor development and demonstrated that anti-TIGIT monoclonal antibodies can reverse NK cell exhaustion and be used for immunotherapy of various tumors, such as non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, non-Hodgkin's lymphoma, B lymphoma, and plasma cell carcinoma (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).

[0009] Furthermore, it has been reported that TIGIT blockers, used alone or in combination with PD-1 and CD96 blockers, can significantly reduce B16 melanoma growth 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 blockers, used alone or in combination with TIGIT and / or PD-1 blockers, can enhance the ability of TILs to produce cytokines in ovarian, endometrial, and lung cancers (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). Summary of the Invention [Problem to be solved by the invention]

[0010] Currently, there remains a need to develop novel fusion proteins containing a TGF-β receptor, particularly a TGF-βRII extracellular domain fragment, for example, which fusion protein further contains an anti-TIGIT antibody. [Means for solving the problem]

[0011] overview Through thorough research and creative efforts, the present inventors have prepared a fusion protein comprising an anti-TIGIT antibody and TGF-βR, and surprisingly found that the fusion protein can effectively bind to TIGIT and TGF-β simultaneously, and even overcomes the drawback of being easily cleaved or degraded, demonstrating its potential for preparing antitumor drugs. This disclosure is described in detail below.

[0012] One aspect of the present disclosure is a first protein functional region that targets an immune checkpoint; and a second protein functional domain having TGF-β binding activity; In a fusion protein comprising The second protein functional domain is a variant of a TGF-βRII extracellular domain fragment, and the variant of the TGF-βRII extracellular domain fragment comprises: The first serine at the N-terminus of the TGF-βRII extracellular region fragment is replaced with alanine, glycine, or threonine, and / or a fragment containing the amino acid sequence set forth in SEQ ID NO: 59 is deleted from the TGF-βRII extracellular region fragment.

[0013] In some embodiments of the present disclosure, a fusion protein is provided in which the amino acid sequence of the TGF-βRII extracellular region fragment is set forth in SEQ ID NO: 33 or SEQ ID NO: 37.

[0014] In some embodiments of the present disclosure, a fusion protein is provided, in which the amino acid sequence of a deleted fragment comprising the amino acid sequence set forth in SEQ ID NO: 59 is set forth in any one of SEQ ID NOs: 59 to 65.

[0015] In some embodiments of the present disclosure, A fusion protein is provided, in which the amino acid sequence of the variant of the extracellular region fragment of TGF-βRII is set forth in any one of SEQ ID NOs: 49 to 58.

[0016] In some embodiments of the present disclosure, a fusion protein is provided wherein the immune checkpoint is selected from one or more of PD-1, PD-L1, CTLA-4, LAG3, and TIGIT.

[0017] In some embodiments of the present disclosure, a fusion protein is provided, wherein the first protein functional region is an anti-PD-1 antibody, or an antigen-binding fragment thereof.

[0018] In some embodiments of the present disclosure, a fusion protein is provided, wherein the first protein functional domain is an anti-PD-L1 antibody, or an antigen-binding fragment thereof.

[0019] In some embodiments of the present disclosure, a fusion protein is provided, wherein the first protein functional region is an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0020] In some embodiments of the present disclosure, a fusion protein is provided, wherein the first protein functional region is an anti-LAG3 antibody, or an antigen-binding fragment thereof.

[0021] In some embodiments of the present disclosure, a fusion protein is provided, wherein the first protein functional region is an anti-TIGIT antibody, or an antigen-binding fragment thereof.

[0022] In some embodiments of the present disclosure, A fusion protein is provided in which the anti-TIGIT antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 and a light chain variable region comprising LCDR1 to LCDR3, The amino acid sequence of HCDR1 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:5; The amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:9, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:10.

[0023] In some embodiments of the present disclosure, 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; Fusion proteins are provided in which 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 disclosure, The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 6; The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 19; The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 19; The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 21; The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 23; The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 21; The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 23; the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 11 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 25; or The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 25. Fusion proteins are provided.

[0025] In some embodiments of the present disclosure, a fusion protein is provided in which the anti-TIGIT antibody, or antigen-binding fragment thereof, is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain variable fragment, humanized antibody, chimeric antibody, and diabody.

[0026] In some embodiments of the present disclosure, fusion proteins are provided in which the anti-TIGIT antibody comprises a non-CDR region derived from a non-mouse species, eg, a human antibody.

[0027] In some embodiments of the present disclosure, a fusion protein is provided in which the heavy chain constant region of the anti-TIGIT antibody is an Ig gamma 1 chain C region or an Ig gamma 4 chain C region, and the light chain constant region is an Ig kappa chain C region.

[0028] In some embodiments of the present disclosure, fusion proteins are provided that have stronger binding ability for the ligand CD155-hFc-biotin than the control antibody RG6058 (hG4).

[0029] In some embodiments of the present disclosure, EC is preferably measured by indirect ELISA. 50 Measure an EC of less than 0.08 nM or less than 0.10 nM 50 Fusion proteins that bind to TIGIT-mFc at

[0030] In some embodiments of the present disclosure, EC is preferably measured by indirect ELISA. 50 to measure an EC of less than 0.3 nM, less than 0.4 nM, less than 0.5 nM, or less than 0.6 nM. 50 Fusion proteins are provided that bind to TGF-β1, TGF-β2, and / or TGF-β3 at 25°C.

[0031] In some embodiments of the present disclosure, a fusion protein is provided, wherein the anti-TIGIT antibody is an antibody produced by hybridoma cell line LT019, deposited at the China Center for Typical Type Culture Collection (CCTCC) under CCTCC number C2020208.

[0032] In some embodiments of the present disclosure, according to the EU numbering system: When the heavy chain constant region of the anti-TIGIT antibody is IgG1, the heavy chain constant region may have the following mutation combinations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A and When the heavy chain constant region of the anti-TIGIT antibody is IgG4, the heavy chain constant region may have the following mutation combinations: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A, and G237A A fusion protein is provided having one of:

[0033] In some embodiments of the present disclosure, a fusion protein is provided in which the amino acid sequence of the heavy chain of an anti-TIGIT antibody is set forth in SEQ ID NO:27 or SEQ ID NO:31, and the amino acid sequence of the light chain is set forth in SEQ ID NO:29.

[0034] In some embodiments of the present disclosure, a fusion protein is provided in which a first protein functional domain and a second protein functional domain are linked directly or via a linker fragment.

[0035] In some embodiments of the present disclosure, a fusion protein is provided in which the linker fragment is a polypeptide set forth in SEQ ID NO: 69 or SEQ ID NO: 70, or a polypeptide obtained by linking a plurality of (e.g., two, three, four, five, or six) polypeptides set forth in SEQ ID NO: 69, or a polypeptide obtained by linking a plurality of (e.g., two, three, four, or five) polypeptides set forth in SEQ ID NO: 69 and further linking the linked polypeptides with a polypeptide set forth in SEQ ID NO: 70, and preferably, the amino acid sequence of the linker fragment is set forth in SEQ ID NO: 44 or SEQ ID NO: 66.

[0036] In some embodiments of the present disclosure, fusion proteins are provided in which the first protein functional domain and the second protein functional domain are independently one, two or more in number.

[0037] In some embodiments of the present disclosure, a fusion protein is provided in which a second protein functional region is linked to the C-terminus of a first protein functional region.

[0038] In some embodiments of the present disclosure, a fusion protein is provided in which a second protein functional region is linked to the C-terminus of the heavy chain of an anti-TIGIT antibody.

[0039] The present disclosure provides: a first protein functional region that targets TIGIT; and a second protein functional domain having TGF-β binding activity; In a fusion protein comprising the number of first protein functional regions is one and the number of second protein functional regions is two; the first protein functional region is an anti-TIGIT antibody, or an antigen-binding fragment thereof, and the second protein functional region is a variant of a TGF-βRII extracellular domain fragment; The amino acid sequence of the heavy chain of the anti-TIGIT antibody is set forth in SEQ ID NO: 27 or SEQ ID NO: 31, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 29; The amino acid sequence of the variant of the TGF-βRII extracellular region fragment is set forth in any one of SEQ ID NOs: 49 to 58; the second protein functional region is linked to the C-terminus of the heavy chain of the anti-TIGIT antibody by a linker fragment; Preferably, the amino acid sequence of the linker fragment is set forth in SEQ ID NO:44 or SEQ ID NO:66.

[0040] Without being bound by theory, some TGF-βR fusion proteins are unstable and have problems with TGF-βR cleavage, resulting in incomplete fusion protein structure.This causes certain difficulties in the process and quality control of fusion protein production, thereby affecting the purity and uniformity of the quality of fusion proteins, and even causing problems in terms of drug safety and efficacy.In practical production and application, it is also necessary to develop more stable antibody / TGF-βR fusion proteins.

[0041] In some embodiments of the present disclosure, fusion proteins are provided in which variants of the TGF-βRII extracellular region fragment are less cleaved and / or degraded during recombinant expression than fusion proteins using a TGF-βRII extracellular region fragment (e.g., SEQ ID NO: 33 or SEQ ID NO: 37).

[0042] In some embodiments of the present disclosure, the fusion protein is used in the treatment and / or prevention of tumors; Preferably, the tumor is selected from one or more of 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 lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma; Preferably, the non-small cell lung cancer is advanced non-small cell lung cancer.

[0043] In a broader sense, the fusion proteins of the present disclosure may also be referred to as antibodies.

[0044] Another aspect of the present disclosure relates to an isolated nucleic acid molecule encoding a fusion protein according to any of the embodiments of the present disclosure.

[0045] Yet another aspect of the present disclosure pertains to vectors comprising the isolated nucleic acid molecules of the present disclosure.

[0046] Yet another aspect of the present disclosure pertains to a host cell comprising the isolated nucleic acid molecule of the present disclosure or the vector of the present disclosure.

[0047] Yet another aspect of the present disclosure relates to a conjugate comprising a fusion protein portion and a conjugated portion, wherein the fusion protein portion is a fusion protein according to any of the embodiments of the present disclosure, and the conjugated portion is a detectable label, preferably the conjugated portion is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0048] In some embodiments of the present disclosure, the conjugate is used in the treatment and / or prevention of tumors; Preferably, the tumor is selected from one or more of 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 lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma; Preferably, the non-small cell lung cancer is advanced non-small cell lung cancer.

[0049] Yet another aspect of the present disclosure relates to the use of a fusion protein of the present disclosure or a conjugate of the present disclosure in the preparation of a kit for detecting the presence or level of TIGIT and / or TGF-β in a sample.

[0050] Yet another aspect of the present disclosure relates to a pharmaceutical composition comprising a fusion protein according to any embodiment of the present disclosure or a conjugate of the present disclosure, optionally further comprising one or more pharmaceutically acceptable auxiliary materials (e.g., carriers and / or excipients), and optionally further comprising one or more anti-tumor chemotherapeutic agents.

[0051] In some embodiments of the present disclosure, a pharmaceutical composition is provided, wherein the unit dose of the pharmaceutical composition is 100 mg to 1500 mg, 200 mg to 1000 mg, 200 mg to 800 mg, 300 mg to 600 mg, 400 mg to 500 mg, or 450 mg, calculated based on the mass of the fusion protein in the pharmaceutical composition.

[0052] In some embodiments of the present disclosure, the pharmaceutical composition is an injectable solution.

[0053] In some embodiments of the present disclosure, a pharmaceutical composition is provided in which a fusion protein according to any embodiment of the present disclosure or a conjugate of the present disclosure is the only active ingredient. (Note: Examiners may request a definition of pharmaceutical composition. If the definition is "comprising a fusion protein as the only active ingredient, and an excipient," this is equivalent to the definition of semi-closed [compare "consisting of a fusion protein and an excipient"].)

[0054] In some embodiments of the present disclosure, a pharmaceutical composition consists of a fusion protein according to any embodiment of the present disclosure or a conjugate of the present disclosure, and one or more pharmaceutically acceptable auxiliary materials.

[0055] Yet another aspect of the present disclosure relates to the use of a fusion protein according to any embodiment of the present disclosure or a conjugate of the present disclosure in the preparation of a medicament for the treatment and / or prevention of a tumor, Preferably, the tumor is selected from one or more of 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 lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma; Preferably, the non-small cell lung cancer is advanced non-small cell lung cancer.

[0056] Yet another aspect of the present disclosure relates to a method for treating and / or preventing a tumor, comprising administering to a subject in need thereof an effective amount of a fusion protein according to any embodiment of the present disclosure or a conjugate of the present disclosure; Preferably, the tumor is selected from one or more of 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 lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma; Preferably, the non-small cell lung cancer is advanced non-small cell lung cancer.

[0057] In some embodiments of the present disclosure, methods are provided, wherein administering to a subject in need thereof an effective amount of a fusion protein according to any embodiment of the present disclosure or a conjugate of the present disclosure is before or after surgery and / or before or after radiation therapy.

[0058] In some embodiments of the present disclosure, The single administration dose of the fusion protein of the present disclosure is 0.1 to 100 mg, preferably 1 to 10 mg per kg body weight; or the single administration dose of the fusion protein of the present disclosure is 10 to 1000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg per subject; Preferably, administration occurs once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 10 days, once every week, once every 2 weeks, or once every 3 weeks; Preferably, the route of administration is intravenous infusion or intravenous injection. A method is provided.

[0059] It is noted that dosages can vary depending on the type and severity of the condition to be treated.Furthermore, those skilled in the art understand that for any particular patient, specific dosage regimens should be adjusted over time according to the patient's needs and the professional judgment of the physician, and the dosage ranges provided herein are for illustrative purposes only and do not limit the use or scope of the fusion protein or pharmaceutical composition of the present disclosure.

[0060] In the present disclosure, the subject can be a mammal, for example, a human.

[0061] Yet another aspect of the present disclosure relates to a variant of a TGF-βRII extracellular domain fragment, the variant comprising: the first serine at the N-terminus of the TGF-βRII extracellular domain fragment is substituted with alanine, glycine, or threonine; or a fragment comprising the amino acid sequence set forth in SEQ ID NO: 59 is deleted from the TGF-βRII extracellular domain fragment; Preferably, the amino acid sequence of the TGF-βRII extracellular region fragment is set forth in SEQ ID NO:33 or SEQ ID NO:37.

[0062] In some embodiments of the present disclosure, a variant of a TGF-βRII extracellular region fragment is provided, in which the amino acid sequence of a deletion fragment comprising the amino acid sequence set forth in SEQ ID NO: 59 is set forth in any one of SEQ ID NOs: 59 to 65.

[0063] Some embodiments of the present disclosure provide a variant of the extracellular region fragment of TGF-βRII, wherein the amino acid sequence of the variant of the extracellular region fragment of TGF-βRII is set forth in any one of SEQ ID NOs: 49 to 58.

[0064] The variable regions of the light and heavy chains determine antigen binding, and each chain variable region contains three hypervariable regions called complementarity-determining regions (CDRs). The heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3, while the light chain (L) CDRs include LCDR1, LCDR2, and LCDR3. In this disclosure, CDRs are defined according to 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.

[0065] The amino acid sequences of the CDRs of the following monoclonal antibodies were analyzed by technical means well known to those skilled in the art, for example, according to the IMGT definition, and the results are as follows:

[0066] HCDR and LCDR of mouse TIGIT monoclonal antibody HCDR1: GHSFTSDYA (SEQ ID NO: 3) HCDR2: ISYSDST (SEQ ID NO: 4) HCDR3: ARLDYGNYGGAMDY (SEQ ID NO: 5) LCDR1: QHVSTA (SEQ ID NO: 8) LCDR2:SAS (SEQ ID NO: 9) LCDR3: QQHYITPWT (SEQ ID NO: 10) The three HCDRs and three LCDRs of the humanized TIGIT monoclonal antibody are the same as those of the murine TIGIT monoclonal antibody.

[0067] In the present disclosure, unless otherwise defined, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the laboratory procedures used herein for cell culture, molecular genetics, nucleic acid chemistry and immunology are common methods widely used in the corresponding fields.Meanwhile, in order to facilitate understanding of the present disclosure, the definitions and explanations of relevant terms are provided below.

[0068] The term EC as used herein 50 refers to the concentration for 50% of the maximum effect, i.e., the concentration that can cause 50% of the maximum effect.

[0069] As used herein, the term "antibody" refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains (each pair containing one "light" (L) chain and one "heavy" (H) chain). Antibody light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon. Antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE. In light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, with heavy chains further containing a "D" region of about 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 region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), between which conserved regions called framework regions (FRs) are distributed. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antibody-binding site.The assignment of amino acids to regions or domains is based on the 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 definitions in 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.

[0070] The term "antibody" is not limited by any specific method for producing the antibody. For example, antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody may be of a different isotype, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM.

[0071] As used herein, the terms "mAb" and "monoclonal antibody" refer to antibodies or antibody fragments derived from a highly homogeneous population of antibodies, i.e., a population of antibody molecules that are identical except for natural mutations that may occur spontaneously. Monoclonal antibodies are highly specific to a single epitope of an antigen. In contrast to monoclonal antibodies, polyclonal antibodies generally contain at least two or more different antibodies that recognize different epitopes of an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al. (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975, 256(5517):495), but can also be obtained using recombinant DNA technology (see, e.g., U.S. Pat. No. 4,816,567).

[0072] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced with the CDR regions of a non-human antibody (donor antibody), which can be a non-human (e.g., mouse, rat, or rabbit) antibody having the expected specificity, affinity, or reactivity. Furthermore, to further improve or optimize antibody performance, some amino acid residues in the framework region (FR) of the receptor antibody may be further replaced with corresponding amino acid residues of a non-human antibody or with amino acid residues of other antibodies. For further details on humanized antibodies, see, e.g., 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 an antibody is a V H Domains and V LDiabodies are those in which two domains are expressed on a single polypeptide chain. However, the linker used is too short to allow the two domains on the same chain to pair. Therefore, these domains are forced to pair with complementary domains on the other chain, and two antigen binding sites are generated (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).

[0073] The fusion protein described herein is the protein product of the co-expression of two genes by DNA recombination.Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and are described, for example, in Chapters 5-8 and 15 of Antibodies: A Laboratory Manual, Cold Spring Harbor Press.

[0074] As used herein, the term "single-chain variable fragment (ScFv)" refers to a fragment of an antibody heavy chain variable region (V) linked via a linker. H ) and antibody light chain variable region (V L ) refers to a molecule containing V L Domains and V H The domains are paired to form monovalent molecules by linkers that allow the generation of a single polypeptide chain (see, e.g., Bird et al., Science, 1988, 242:423-426, and Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883). Such scFv molecules have the following general structure: NH2-V L -Linker fragment-V H -COOH, or NH2-V H -Linker fragment-V LThe linker may have -COOH. Suitable linkers in the prior art consist of repeated GGGGS (SEQ ID NO: 69) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 66) 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 the present disclosure are described in 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.

[0075] The term "isolated" as used herein refers to being obtained by artificial means from a natural state. When a certain "isolated" substance or component exists in nature, it may be altered in its natural environment, or it may be isolated from its natural environment, or both. For example, a specific non-isolated polynucleotide or polypeptide naturally occurs in a specific living animal, and the same polynucleotide or polypeptide, isolated from such a natural state with high purity, is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0076] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may further contain a replication origin site.

[0077] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell, such as E. coli or Bacillus subtilis, a fungal cell, such as a yeast cell or Aspergillus, an insect cell, such as an S2 Drosophila cell or an Sf9, or an animal cell, such as a fibroblast, a CHO cell, a GS cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK293 cell, or a human cell.

[0078] In this disclosure, the term "ADCP" refers to antibody-dependent cellular phagocytosis. The Fc fragment of an antibody that binds to a cell surface antigen binds to an Fc receptor on a phagocytic cell, such as a macrophage, which then mediates phagocytosis of the antibody-bound cell by the phagocyte.

[0079] In the present disclosure, the term "ADCC" refers to antibody-dependent cell-mediated cytotoxicity. The Fab fragment of an antibody binds to an epitope on a virus-infected cell or tumor cell, and the Fc fragment of the antibody binds to an Fc receptor (FcR) on the surface of a killer cell (NK cell, macrophage, etc.), mediating direct killing of the target cell by the killer cell.

[0080] In the present disclosure, the term "CDC" refers to complement-dependent cytotoxicity. When an antibody specifically binds to a corresponding antigen on the cell membrane surface, a complex is formed, activating the complement system, and then MAC is formed on the surface of the target cell, resulting in subsequent target cell lysis. Complement can cause cell lysis of various bacteria and other pathogenic organisms, and is an important defense mechanism against pathogenic organism infection.

[0081] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, e.g., a reaction between an antibody and its target antigen. In certain embodiments, an antibody that specifically binds to an antigen (or is specific for an antigen) is one in which the antibody binds to an antigen within about 10 -5 Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 Affinity (K D ) means that the antibody binds to the antigen.

[0082] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. A smaller dissociation equilibrium constant indicates stronger antibody-antigen binding and higher affinity between the antibody and the antigen. Generally, antibodies have a dissociation equilibrium constant of about 10 -5Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 The dissociation equilibrium constant (K D ) binds to an antigen (e.g., TIGIT protein). D can be determined using methods known to those skilled in the art, for example, using a ForteBio molecular interaction instrument.

[0083] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably, and the terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably. Moreover, as used herein, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0084] As used herein, the term "pharmaceutically acceptable auxiliary material" refers to a carrier and / or excipient that is pharmaceutically and / or physiologically compatible with the subject and active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH 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.

[0085] The term "effective amount" as used herein refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactically effective amount for a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., a tumor), and a therapeutically effective amount refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient suffering from the disease. Determining such an effective amount is undoubtedly within the capabilities of a person skilled in the art. For example, a therapeutically effective amount will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's overall condition, such as age, weight, and sex, the route of administration, and other treatments administered at the same time.

[0086] In this disclosure, the terms "first" (e.g., a first protein functional domain, or a first product) and "second" (e.g., a second protein functional domain, or a second product) are used for distinction or clarity in expression and do not have their typical sequential meaning, unless otherwise specified. [Effects of the Invention]

[0087] Beneficial Effects of the Present Disclosure The present disclosure achieves one or more of the following technical effects (1) to (8).

[0088] (1) The fusion protein of the present disclosure can simultaneously inhibit TIGIT and reduce TGF-β levels.

[0089] (2) The fusion protein of the present disclosure binds to TIGIT with high specificity and can very effectively block the binding of TIGIT to CD155, thereby specifically eliminating the immune suppression of an organism caused by TIGIT.

[0090] (3) The fusion protein of the present disclosure binds to TGF-β with high specificity and can very effectively block the binding of TGF-β to the TGF-β receptor, thereby specifically eliminating the immune suppression of the organism caused by TGF-β and activating the immune response.

[0091] (4) The fusion protein of the present disclosure has excellent potential for the preparation of antitumor drugs.

[0092] (5) The first protein functional domain and the second protein functional domain of the fusion protein of the present disclosure have a synergistic effect that is superior to the effect of using either of the protein functional domains alone, for example, the effect of using an anti-TIGIT antibody alone.

[0093] (6) The fusion protein of the present disclosure can effectively block TIGIT-induced immunosuppression of immune cells and induce IL-2 secretion in the cells.

[0094] (7) The fusion protein of the present disclosure can effectively block TGF-β-induced immunosuppression of immune cells and induce cellular INF-γ secretion.

[0095] (8) The fusion protein of the present disclosure overcomes the drawback of being easily cleaved or degraded. [Brief explanation of the drawings]

[0096] [Figure 1] FIG. 1 shows the binding activity of TFOl, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) to the antigen TIGIT-mFc. [Figure 2] FIG. 1 shows the binding activity of TFOl, TF02, and TGF-βRII-mFc to TGF-β1. [Figure 3] FIG. 1 shows the binding activity of TFOl, TF02, and TGF-βRII-mFc to TGF-β2. [Figure 4] FIG. 1 shows the binding activity of TFOl, TF02, and TGF-βRII-mFc to TGF-β3. [Figure 5] FIG. 10: Activity of anti-TIGIT antibody-TGF-βR fusion protein competing with TGF-βRII-His-biotin for binding to human TGF-β1. [Figure 6]FIG. 10. Activity of anti-TIGIT antibody-TGF-βR fusion protein competing with TGF-βRII-His-biotin for binding to human TGF-β3. [Figure 7] FIG. 10: Activity of anti-TIGIT antibody-TGF-βR fusion proteins competing with human CD155-hFc-biotin for binding to human TIGIT-mFC. [Figure 8] FIG. 1 shows the binding activity of anti-TIGIT antibody-TGF-βR fusion protein to TIGIT on the surface of 293T-TIGIT membrane. [Figure 9] FIG. 10: Competition between TFOl and CD155 for binding to the cell membrane surface antigen TIGIT. [Figure 10] FIG. 10: Competition between TFO1 and CD112 for binding to the cell membrane surface antigen TIGIT. [Figure 11] Affinity constant of TF01 for FcγRI. [Figure 12] FIG. 10 is a diagram of the affinity constant of 26B12H2L2 (hG1WT) for FcγRI. [Figure 13] A graph showing the affinity constant of TF01 for FcγRIIIa_V158. [Figure 14] FIG. 10 shows the affinity constant of 26B12H2L2 (hG1WT) for FcγRIIIa_V158. [Figure 15] A graph showing the affinity constant of TF01 for FcγRIIIa_F158. [Figure 16] FIG. 10 shows the affinity constant of 26B12H2L2 (hG1WT) for FcγRIIIa_F158. [Figure 17] A graph showing the affinity constant of TF01 for FcγRIIa_H131. [Figure 18] FIG. 10 shows the affinity constant of 26B12H2L2 (hG1WT) for FcγRIIa_H131. [Figure 19] A graph showing the affinity constant of TF01 for FcγRIIa_R131. [Figure 20]FIG. 10 shows the affinity constant of 26B12H2L2 (hG1WT) for FcγRIIa_R131. [Figure 21] Affinity constant of TF01 for FcγRIIb. [Figure 22] FIG. 10 is a graph showing the affinity constant of 26B12H2L2 (hG1WT) for FcγRIIb. [Figure 23] FIG. 10. Affinity constant of TF01 for C1q. [Figure 24] FIG. 10 is a diagram of the affinity constant of 26B12H2L2 (hG1WT) for C1q. [Figure 25] FIG. 1 shows the ADCP effects of RG6058(hG1WT), RG6058(hG4WT), 26B12H2L2(hG1WT), and TF01. [Figure 26] FIG. 10 shows the blocking activity of anti-TIGIT antibody-TGF-βR fusion protein on CD112-mediated inhibition of IL-2 secretion in a co-culture system of Jurkat-TIGIT cells and THP-1 cells. [Figure 27] FIG. 10 shows the blocking activity of anti-TIGIT antibody-TGF-βR fusion protein on CD155-mediated inhibition of IL-2 secretion in a co-culture system of Jurkat-TIGIT cells and THP-1 cells. [Figure 28] FIG. 1 shows the biological activity of anti-TIGIT antibody-TGF-βR fusion protein in promoting IL-2 secretion in a co-culture system of Jurkat-TIGIT cells and HT1080-aCD3scFv cells. [Figure 29] FIG. 1 shows the blocking activity of anti-TIGIT antibody-TGF-βR fusion protein on the inhibition of IFN-γ secretion by TGF-β1 during the secondary immune response of PBMCs to CMV antigen. [Figure 30]Effect of anti-TIGIT antibody-TGF-βR fusion protein in Scid Beige mouse MDA-MB-231 xenograft tumor model. Results are expressed as mean ± standard error and subjected to two-way ANOVA followed by Bonferroni test. *p<0.05, **p<0.01, ***p<0.001 compared to isotype control group. [Figure 31] FIG. 10 shows the effect of anti-TIGIT antibody-TGF-βR fusion protein on body weight in a Scid Beige mouse MDA-MB-231 xenograft tumor model. [Figure 32] FIG. 1 shows the biological activity of antibodies TF01A, TF01G, TF01T, TF01t31, and TF01t37 in promoting IL-2 secretion in a co-culture system of Jurkat-TIGIT cells and HT1080-aCD3scFv cells. [Figure 33] FIG. 1 shows the neutralizing effect of anti-TIGIT antibody-TGF-βR fusion protein on the inhibition of IL-4-induced TF-1 proliferation by TGF-β1. [Figure 34] FIG. 1 shows the neutralizing effect of anti-TIGIT antibody-TGF-βR fusion protein on the inhibition of IL-4-induced TF-1 proliferation by TGF-β3. [Figure 35] FIG. 1 shows the results of a reporter gene assay evaluating the blocking of the interaction between TGF-β1 and TGF-βR by an anti-TIGIT antibody-TGF-βR fusion protein. [Figure 36] FIG. 1 shows the results of a reporter gene assay evaluating the blocking of the interaction between TGF-β3 and TGF-βR by an anti-TIGIT antibody-TGF-βR fusion protein. [Figure 37] FIG. 1 shows the results of a reporter gene assay evaluating the blocking of the interaction between TIGIT and PVR by an anti-TIGIT antibody-TGF-βR fusion protein. [Figure 38] FIG. 1 is a diagram showing the molecular weight of the anti-TIGIT antibody-TGF-βR fusion protein TF01. [Figure 39]FIG. 1 is a diagram showing the molecular weight of the anti-TIGIT antibody-TGF-βR fusion protein TF01A. [Figure 40] FIG. 1 is a diagram showing the molecular weight of the anti-TIGIT antibody-TGF-βR fusion protein TF01G. [Figure 41] FIG. 1 is a diagram showing the molecular weight of the anti-TIGIT antibody-TGF-βR fusion protein TF01T. [Figure 42] FIG. 1 is a diagram of the molecular weight of the anti-TIGIT antibody-TGF-βR fusion protein TF01t31. [Figure 43] FIG. 1 is a diagram of the molecular weight of the anti-TIGIT antibody-TGF-βR fusion protein TF01t37. [Figure 44] FIG. 1 shows the results of SEC-HPLC analysis of the anti-TIGIT antibody-TGF-βR fusion protein TF01. [Figure 45] FIG. 1 shows the results of SEC-HPLC analysis of the anti-TIGIT antibody-TGF-βR fusion protein TF01T. [Figure 46] FIG. 1 shows the binding activity of anti-TIGIT antibody-TGF-βR fusion protein to TGF-β1. [Figure 47] FIG. 10 shows the quantification of the biological activity of anti-TIGIT antibody-TGF-βR fusion protein in promoting IFN-γ secretion using a mixed lymphocyte reaction. DETAILED DESCRIPTION OF THE INVENTION

[0097] Deposited biological material: The hybridoma cell line LT019 (TIGIT-26B12) was deposited at the China Center for Typical Culture Collection (CCTCC) on October 23, 2020. The CCTCC name was CCTCC number C2020208, and the deposit address was Wuhan University, Wuhan, China, postal code 430072.

[0098] Some sequences included in this disclosure are as follows: 1. Amino acid sequence of 26B12VH EVQLQESGPGLVKPSQSLSLTCTVTGHSFTSDYAWNWIRQFPGNRLEWMGYISYSDSTNYNPSLKSRISITRDTSKNQFFLQMNSVTTEDTATYYCARLDYGNYGGAMDYWGQGTSVTVSS (SEQ ID NO: 1) 2. Nucleotide sequence of 26B12VH GAGGTGCAGCTGCAGGAGTCTGGACCTGGCCTGGTGAAACCTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCCACTCATTCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAGACTGGAGTGGATGGGCTACATAAGCTACAGTGATAGCACTAACTACAACCC ATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCTTGCAGATGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATTGGACTATGGTAACTACGGTGGGGCTATGGACTACTGGGGTCAAGGGACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 2) 3. HCDR1: GHSFTSDYA (SEQ ID NO: 3) 4. HCDR2: ISYSDST (SEQ ID NO: 4) 5. HCDR3: ARLDYGNYGGAMDY (SEQ ID NO: 5) 6. Amino acid sequence of 26B12VL DIVLTQSHEFMSTSLRDRVSITCKSSQHVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVKAEDLAVYYCQQHYITPWTFGGGTKLEIK (SEQ ID NO: 6) 7. Nucleotide sequence of 26B12VL GATATTGTGCTAACTCAGTCTCACGAATTCATGTCCACCTCATTACGAGACAGGGTCAGCATCACCTGCAAATCCAGTCAACATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTA CACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATATTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATAAAA (SEQ ID NO: 7) 8. LCDR1: QHVSTA (SEQ ID NO: 8) 9. LCDR2: SAS (SEQ ID NO: 9) 10. LCDR3: QQHYITPWT (SEQ ID NO: 10) 11. Amino acid sequence of 26B12H1VH DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWNWIRQFPGKGLEWIGYISYSDSTNYNPSLKSRITISRDTSKNQFFLQLNSVTAADTATYYCARLDYGNYGGAMDYWGQGTSVTVSS (SEQ ID NO: 11) 12. Nucleotide sequence of 26B12H1VH GATGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTTCCCAGACCCTGTCTCTGACCTGTACAGTGTCTGGCCACAGCTTCACATCCGACTACGCCTGGAACTGGATCAGGCAGTTTCCAGGCAAGGGCCTGGAGTGGATCGGCTACATCTCTTATAGCGACTCCACCAACTATAATCCCTCTCTGAAGAGCCGGATCACCATCAGCAGAGATACATCCAAGAACCAGTTCTTTCTGCAGCTGAACAGCGTGACAGCCGCCGACACCGCCACATACTATTGCGCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTACTGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC(SEQ ID NO: 12) 13. Amino acid sequence of 26B12H2 VH DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWSWIRQPPGKGLEWIGYISYSDSTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARLDYGNYGGAMDYWGQGTSVTVSS(SEQ ID NO: 13) 14. Nucleotide sequence of 26B12H2 VH GATGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCAAGCCAGACCCTGTCCCTGACCTGTACAGTGTCCGGCCACTCTTTTACAAGCGACTACGCCTGGTCTTGGATCAGGCAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCTACATCTCCTATTCTGACAGCACCAACTATAATCCCTCCCTGAAGTCTCGGGTGACCATCTCTAGAGATACAAGCAAGAACCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGTGTACTATTGCGCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC(SEQ ID NO: 14) 15. Amino acid sequence of 26B12H3 VH DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWSWIRQPPGKGLEWIGYISYSDSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDYGNYGGAMDYWGQGTSVTVSS (SEQ ID NO: 15) 16. Nucleotide sequence of 26B12H3VH GATGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCAAGCCAGACCCTGTCCCTGACCTGTACAGTGTCCGGCCACTCTTTTACAAGCGACTACGCCTGGTCTTGGATCAGACAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCTACATCTCCTATTCTGACAGCACCAACTATAATCC CTCCCTGAAGTCTAGAGTGACCATCTCTGTGGATACAAGCAAGAACCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGTGTACTATTGCGCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 16) 17. Amino acid sequence of 26B12H4VH DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWNWIRQFPGKGLEWMGYISYSDSTNYNPSLKSRITISRDTSKNQFFLQLNSVTAADTATYYCARLDYGNYGGAMDYWGQGTSVTVSS (SEQ ID NO: 17) 18. Nucleotide sequence of 26B12H4VH GATGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTTCCCAGACCCTGTCTCTGACCTGTACAGTGTCTGGCCACAGCTTCACATCCGACTACGCCTGGAACTGGATCAGGCAGTTTCCAGGCAAGGGCCTGGAGTGGATGGGCTACATCTCTTATAGCGACTCCACCAACTATAATCC CTCTCTGAAGAGCCGGATCACCATCAGCAGAGATACATCCAAGAACCAGTTCTTTCTGCAGCTGAACAGCGTGACAGCCGCCGACACCGCCACATACTATTGCGCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTACTGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO: 18) 19. Amino acid sequence of 26B12L1VL DIQMTQSPKSLSTSVGDRVTITCRSSQHVSTAVAWYQQKPGKSPKLLIYSASYRYSGVPDRFSGSGSGTDFTFTISSVQPEDFATYYCQQHYITPWTFGGGTKLEIK (SEQ ID NO: 19) 20. Nucleotide sequence of 26B12L1VL GACATCCAGATGACCCAGTCCCCTAAGTCCCTGTCTACAAGCGTGGGCGATCGGGTGACCATCACATGTAGAAGCTCCCAGCACGTGTCTACCGCAGTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTATTCCGCCTCTTACAGGTA TTCCGGAGTGCCAGACCGGTTTAGCGGCTCCGGCTCTGGCACCGATTTCACCTTTACAATCTCTAGCGTGCAGCCAGAGGACTTCGCCACATACTATTGCCAGCAGACTACATCACCCCATGGACCTTCGGCGGCGGCACAAAGCTGGAGATCAAG (SEQ ID NO: 20) 21. Amino acid sequence of 26B12L2VL DIQMTQSPSSLSASVGDRVTITCRSSQHVSTALAWYQQKPGKSPKLLIYSASSRYSGVPDRFSGSGSGTDFTFTISSLQPEDFATYYCQQHYITPWTFGGGTKLEIK (SEQ ID NO: 21) 22. Nucleotide sequence of 26B12L2VL GACATCCAGATGACCCAGTCCCCTAGCTCCCTGTCTGCCAGCGTGGGCGATAGGGTGACCATCACATGTAGATCTAGCCAGCACGTGTCTACAGCCCTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTACTCCGCCTCCTCTAGGTA TTCTGGAGTGCCAGACCGGTTTTCCGGCTCTGGCAGCGGCACCGATTTCACCTTTACAATCAGCTCCCTGCAGCCAGAGGACTTCGCCACATACTATTGCCAGCAGCACTATATCACCCCATGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO: 22) 23. Amino acid sequence of 26B12L3VL DIQMTQSPSSLSASVGDRVTITCRASQHVSTALAWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYITPWTFGGGTKLEIK (SEQ ID NO: 23) 24. Nucleotide sequence of 26B12L3VL GACATCCAGATGACCCAGTCCCCTAGCTCCCTGAGCGCCTCCGTGGGCGATAGGGTGACCATCACATGTAGAGCCTCTCAGCACGTGAGCACAGCCCTGGCATGGTACCAGCAGAAGCCAGGCAAGGCCCCTAAGCTGCTGATCTATAGCGCCTCTAGCCTGCA GTCCGGAGTGCCATCTCGGTTCTCTGGCAGCGGCTCCGGAACCGACTTTACCCTGACAATCTCCTCTCTGCAGCCAGAGGATTTCGCCACATACTATTGCCAGCAGACTACATCACCCCATGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO: 24) 25. Amino acid sequence of 26B12L4VL DIQMTQSPKSMSTSVGDRVTITCRSSQHVSTAVAWYQQKPGKSPKLLIYSASYRYSGVPDRFSGSGSGTDFTFTISSVQPEDFATYYCQQHYITPWTFGGGTKLEIK (SEQ ID NO: 25) 26. Nucleotide sequence of 26B12L4VL GACATCCAGATGACCCAGTCCCCTAAGTCCATGTCTACAAGCGTGGGCGACAGGGTGACCATCACATGTAGAAGCTCCCAGCACGTGTCTACCGCAGTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTATTCCGCCTCTTACAGGTA TTCCGGAGTGCCAGACCGGTTTAGCGGCTCCGGCTCTGGCACCGATTTCACCTTTACAATCTCTAGCGTGCAGCCAGAGGACTTCGCCACATACTATTGCCAGCAGACTACATCACCCCATGGACCTTCGGCGGCGGCACAAAGCTGGAGATCAAG (SEQ ID NO: 26) 27. Amino acid sequence of the heavy chain of 26B12H2L2 (hG4DM) DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWSWIRQPPGKGLEWIGYISYSDSTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARLDYGNYGGAMDYWGQ GTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 27) 28. Nucleotide sequence of the heavy chain of 26B12H2L2 (hG4DM) 29. Amino acid sequence of the light chain of 26B12H2L2 (hG4DM) DIQMTQSPSSLSASVGDRVTITCRSSQHVSTALAWYQQKPGKSPKLLIYSASSRYSGVPDRFSGSGSGTDFTFTISSLQPEDFATYYCQQHYITPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29) 30. Nucleotide sequence of the light chain of 26B12H2L2 (hG4DM) (SEQ ID NO: 30) 31. Amino acid sequence of the heavy chain of 26B12H2L2 (hG1DM) DVQLQESGPGLVKPSQTLSLTCTVSGHSFTSDYAWSWIRQPPGKGLEWIGYISYSDSTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARLDYGNYGGAMDYWGQG TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31) 32. Nucleotide sequence of the heavy chain of 26B12H2L2 (hG1DM) 33. Amino acid sequence of TGF-βRII extracellular domain fragment (truncated): IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 33) 34. Amino acid sequence of TGF-βRI: (Extracellular domain sequence is underlined) MEAAVAAPPRRLLLLVLAAAAAAAALLPGATA LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEL AAVIAGPVCFVCISLMLMVYICHNRTVIHHRVPNEEDPSLDRPFISEGTTLKDLIYDMTTSGSGSGLPLLVQRTIARTIVLQESIGKGRFGEVWRGKWRGEEVAVKIFSSREERSWFREAEIYQTVMLRHENILGFIAADNKDNGTWTQLWLVSDYHEHGSLFDYLNRYTVTVEGMIKLALSTASGLAHLHMEIVGTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVRHDSATDTIDIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLVFWEIARRCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEALRVMAKIMRECWYANGAARLTALRIKKTLSQLSQQEGIKM (SEQ ID NO: 34) 35. Amino acid sequence of the extracellular domain of TGF-βRI: LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEL (SEQ ID NO: 35) 36. Amino acid sequence of TGF-βRII: (Extracellular domain sequence is underlined) MGRGLLRGLWPLHIVLWTRIAS TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK (SEQ ID NO: 36) 37. Amino acid sequence of the extracellular domain of TGF-βRII: TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO: 37) 38. Amino acid sequence of TGF-βRIII: (Extracellular domain sequence is underlined) MTSHYVIAIFALMSSCLATA GPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAE YLQPKAAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKCKKSVNWVIKSFDVKGSLKIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNAEEMGDEEVHTIPPELRILLDPGALPALQNPPIRGGEGQNGGLPF PFPDISRRVWNEEGEDGLPRPKDPVIPSIQLFPGLREPEEVQGSVDIALSVKCDNEKMIVAVEKDSFQASGYSGMDVTLLDPTCKAKMNGTHFVLESPLNGCGTRPRWSALDGVVYYNSIVIQVPALGDSSGWPDGYEDLESGDNGFPGDMDEGDASLFTRPEIVVFNCSLQQVRNPSSFQEQPHGNITFNM ELYNTDLFLVPSQGVFSVPENGHVYVEVSVTKAEQELGFAIQTCFISPYSNPDRMSHYTIIENICPKDESVKFYSPKRVHFPIPQADMDKKRFSFVFKPVFNTSLLFLQCELTLCTKMEKHPQKLPKCVPPDEACTSLDASIIWAMMQNKKTFTKPLAVIHHEAESKEKGPSMKEPNPISPPIFHGLDTLTV MGIAFAAFVIGALLTGALWYIYSHTGETAGRQQVPTSPPASENSSAAHSIGSTQSTPCSSSSTA (SEQ ID NO: 38) 39. Amino acid sequence of the extracellular domain of TGF-βRIII: (SEQ ID NO: 39) 40. Amino acid sequence of the heavy chain of tiragolumab: EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40) 41. Amino acid sequence of the light chain of tiragolumab: DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 41) 42. Amino acid sequence of the heavy chain variable region of RG6058 (hG4): EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO: 42) 43. Amino acid sequence of the light chain variable region of RG6058 (hG4): DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK (SEQ ID NO: 43) 44. Amino acid sequence of the linker fragment: GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 44) 45. Amino acid sequence of the heavy chain of anti-HEL&TGFβ antibody (SEQ ID NO: 45) 46. ​​Amino acid sequence of the light chain of anti-HEL&TGFβ antibody DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYTSQSMSGIPSRFSGSGSGTDFTLSINSVETEDFGVYFCQQSGSWPRTFGGGTKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 46) 47. Amino acid sequence of the heavy chain of RG6058(G1DM) EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47) 48. Amino acid sequence of the light chain of RG6058(G1DM) DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 48) 49. Amino acid sequence of TGF-βRII extracellular domain fragment variant TF01A: IPPHVQKAVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 49) 50. Amino acid sequence of TGF-βRII extracellular domain fragment variant TF01G: IPPHVQKGVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 50) 51. Amino acid sequence of the TGF-βRII extracellular domain fragment variant of TF01T: IPPHVQKTVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 51) 52. Amino acid sequence of the TGF-βRII extracellular domain fragment variant of antibody TF01t31: VNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 52) 53. Amino acid sequence of the TGF-βRII extracellular domain fragment variant of antibody TF01t37: VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 53) 54. TGF-βRII extracellular domain fragment variant NNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 54) 55. Variant of TGF-βRII extracellular domain fragment NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 55) 56. TGF-βRII extracellular domain fragment variant DMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 56) 57. TGF-βRII extracellular domain fragment variant MIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 57) 58. TGF-βRII extracellular domain fragment variant IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 58) 59. Deletion fragment in the extracellular domain of TGF-βRII IPPHVQKS (SEQ ID NO: 59) 60. Deletion fragment in the extracellular domain of TGF-βRII IPPHVQKSVNNDMI (SEQ ID NO: 60) 61. Deletion fragment in the extracellular domain of TGF-βRII IPPHVQKSV (SEQ ID NO: 61) 62. Deletion fragment in the extracellular domain of TGF-βRII IPPHVQKSVN (SEQ ID NO: 62) 63. Deletion fragment in the extracellular domain of TGF-βRII IPPHVQKSVNN (SEQ ID NO: 63) 64. Deletion fragment in the extracellular domain of TGF-βRII IPPHVQKSVNND (SEQ ID NO: 64) 65. Deletion fragment in the extracellular domain of TGF-βRII IPPHVQKSVNNDM (SEQ ID NO: 65) 66. Linker fragment GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 66) 67. Amino acid sequence of the heavy chain constant region of hIgG4WT ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 67) 68. Amino acid sequence of the heavy chain constant region of hIgG1WT ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 68) 69. Linker fragment GGGGS (SEQ ID NO: 69) 70. Linker fragment GGGGSG (SEQ ID NO: 70) 71. Amino acid sequence of the heavy chain of B12-hG1 QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTADTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDNYYMDV WGKGTTVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 71) 72. Amino acid sequence of the light chain of B12-hG1 EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 72) Detailed Description

[0023] The embodiments of the present disclosure will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are merely illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. Experimental procedures in the examples without specified conditions were carried out under conventional conditions or conditions recommended by the manufacturer. Any reagents or equipment used without a specified manufacturer were commercially available conventional products.

[0099] BALB / c mice were purchased from the Guangdong Provincial Medical Experimental Animal Center.

[0100] C57 mice were purchased from the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine.

[0101] MDA-MB-231 cells, U87-MG cells, and TF-1 cells were purchased from ATCC.

[0102] A549 was purchased from the Cell Resource Center of Shanghai Institutes for Life Sciences, Chinese Academy of Sciences.

[0103] The amino acid sequences of the heavy and light chains of the positive control antibody tiragolumab are derived from the antibodies described in WHO. Proposed INN: List 117. WHO Drug Information, 31(2):104, 2017, and the sequences are identical to SEQ ID NOs: 40 to 41, respectively.

[0104] Tiragolumab and RG6058 (hG1WT) are the same antibody in this disclosure.

[0105] The sequence of the positive control antibody RG6058 (hG4) is derived from the antibody described in the published Chinese Patent Application Publication No. 108290946, and the amino acid sequences of the heavy and light chain variable regions are identical to SEQ ID NOs: 42-43, respectively.

[0106] The positive control antibody RG6058 (hG1DM) was prepared in the Akeso Biopharma laboratory and had lot number 20180816. The amino acid sequence of its heavy chain is set forth in SEQ ID NO: 47, and the amino acid sequence of its light chain is set forth in SEQ ID NO: 48.

[0107] The variable region sequence of human anti-hen egg lysozyme IgG (anti-HEL, or human IgG, abbreviated as hIgG), an isotype control antibody used in the examples of the present disclosure, is described in Acierno et al., "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies," J Mol Biol., 2007, 374(1):130-46. hIgG1DM and hIgG4WT used in the examples are isotype control antibodies for anti-HEL that contain the constant region sequences of hG1DM and hG4WT, and were prepared in the laboratory of Akeso Biopharma.

[0108] The anti-HEL&TGFβ antibody was constructed by fusing TGFβRII protein to the C-terminus of the heavy chain of subtype k anti-HEL antibody (human hG4DM) and prepared in the laboratories of Akeso Biopharma, Inc. The amino acid sequence of the heavy chain is set forth in SEQ ID NO: 45, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 46.

[0109] TIGIT-mFc protein (Lot No. 20171110), TGF-βRII-mFc (Lot No. 20200528), CD155-hFc-biotin (Lot No. 20170721), CD155-mFc (Lot No. 20190726), and CD112-mFc (Lot No. 20190726) were all produced by Akeso Biopharma according to known sequences. mFc represents the Fc protein fragment of mouse IgG, and hFc represents the Fc protein fragment of human IgG.

[0110] TGF-β1 protein was purchased from Sino Biological Company, catalog number LC13DE3108.

[0111] TGF-β2 protein was purchased from Peprotech, catalog number 0420345E0620.

[0112] TGF-β3 protein was purchased from Peprotech, catalog number 0713410A0219.

[0113] In the following examples of this disclosure, the 293T-TIGIT cell line used was constructed by Akeso Biopharma. The 293T-TIGIT cell line was prepared by viral infection of HEK293T cells (Wuhan University). A third-generation lentivirus system was used for virus preparation. 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. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vector used was plenti6.3 / V5-TIGITFL-BSD (TIGIT, Genebank ID: NP_776160.2; the vector plenti6.3 / V5 TOPO was purchased from Invitrogen, product number K531520).

[0114] In the following examples of the present disclosure, the CHO-K1-TIGIT cell line used was constructed by Akeso Biopharma. The CHO-K1-TIGIT cell line was prepared by viral infection of CHO-K1 cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). A third-generation lentivirus system was used for viral preparation. 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. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vector used was plenti6.3 / V5-TIGITFL-BSD (TIGIT, Genebank ID: NP_776160.2; vector plenti6.3 / V5 TOPO was purchased from Invitrogen, product number K531520).

[0115] In the following examples of the present disclosure, the Jurkat-TIGIT cell line used was constructed by Akeso Biopharma. The Jurkat-TIGIT cell line was prepared by viral infection of Jurkat cells (Cell Center, Chinese Academy of Sciences). A third-generation lentivirus system was used for virus preparation. 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. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vector used was plenti6.3 / V5-TIGITFL-BSD (TIGIT, Genebank ID: NP_776160.2; the vector plenti6.3 / V5 TOPO was purchased from Invitrogen, product number K531520).

[0116] In the following examples of the present disclosure, the HT1080-aCD3scFv cell line used was constructed by Akeso Biopharma. The HT1080-aCD3scFv cell line was prepared by viral infection of HT-1080 cells (Cell Resources Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). A third-generation lentivirus system was used for viral preparation. 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. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vector used was pCDH-aCD3scFv-Puro (the aCD3scFv sequence was derived from the anti-human CD3 mouse (Mus musculus) OKT3 hybridoma antibody; the vector pCDH-CMV-MCS-EF1-Puro was purchased from Youbio, product number VT1480).

[0117] In the following examples of the present disclosure, the 293T-TGFβR2-Luc cell line used was constructed by Akeso Biopharma. The 293T-TGFβR2-Luc cell line was prepared by viral infection of HEK293T cells (Wuhan University). A third-generation lentivirus system was used for viral preparation. 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. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vectors used were pCDH-hTGFBR2FL-puro and pCDH-Smad3 / 4-Luc2P-Hygro (hTGFBR2FL Genebank ID: NP_003233.4; vector pCDH-CMV-MCS-EF1-Puro was purchased from Youbio, product number VT1480; pCDH-Smad3 / 4-Luc2P-Hygro was constructed by Akeso Biopharma).

[0118] In the following examples of the present disclosure, the Jurkat-TIGIT-NFAT-Luc cell line used was constructed by Akeso Biopharma. The Jurkat-TIGIT-NFAT-Luc cell line was prepared by viral infection of Jurkat cells (Cell Center, Chinese Academy of Sciences). A third-generation lentivirus system was used for viral preparation. 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. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vectors used were plenti6.3 / V5-TIGITFL-BSD and pCDH-NFAT-hygro (TIGIT, Genebank ID: NP_776160.2; vector plenti6.3 / V5 TOPO was purchased from Invitrogen, product number K531520; pCDH-NFAT-hygro was constructed by Akeso Biopharma).

[0119] In the following examples of the present disclosure, the CHO-aAPC-PDL1-PVR cell line used was constructed by Akeso Biopharma. The CHO-aAPC-PDL1-PVR cell line was prepared by viral infection of PD-L1 aAPC / CHO-K1 cells (purchased from Promega). A third-generation lentivirus system was used for viral preparation. 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. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vector used was pCDH-PVRFL-Puro (PVRFL Genebank ID: NP_006496.4; the vector pCDH-CMV-MCS-EF1-Puro was purchased from Youbio, product number VT1480).

[0120] Example 1: Preparation of anti-TIGIT antibodies 1. Preparation of hybridoma cell line LT019 The antigen used to prepare anti-TIGIT antibodies was human TIGIT-mFc (TIGIT had Genbank ID: NP_776160.2). Spleen cells from immunized mice were fused with mouse myeloma cells to prepare hybridoma cells. Using human TIGIT-hFc as the antigen, the hybridoma cells were screened by indirect ELISA to obtain hybridoma cells capable of secreting antibodies that specifically bind to TIGIT. The hybridoma cells obtained by screening were subjected to limiting dilution assay to obtain stable hybridoma cell lines. The hybridoma cell line described above was designated hybridoma cell line LT019, and the monoclonal antibody secreted by the cell line was designated 26B12.

[0121] The hybridoma cell line LT019 (also called TIGIT-26B12) was deposited at the China Center for Typical Culture Collection (CCTCC) on October 23, 2020. The CCTCC name was CCTCC number C2020208, and the deposit address was Wuhan University, Wuhan, China, postal code 430072.

[0122] 2. Preparation of anti-TIGIT antibody 26B12 The LTO19 cell line prepared above was cultured in a chemically defined medium (CD medium, containing 1% penicillin-streptomycin) at 37°C and 5% CO. After 7 days, the cell culture supernatant was collected, centrifuged at high speed, filtered through a microfiltration membrane, and purified using a HiTrap Protein A HP column to obtain antibody 26B12.

[0123] Example 2: Sequence analysis of anti-TIGIT antibody 26B12 mRNA was extracted from the LT019 cell line cultured in Example 1 according to the instructions for the RNAprep pure Cell / Bacteria kit (Tiangen, catalog number DP430).

[0124] cDNA was synthesized and amplified by PCR according to the instructions of Invitrogen's SuperScript® III First Strand Synthesis System Kit for RT-PCR.

[0125] The PCR amplification product was directly subjected to TA cloning according to the instructions attached to the pEASY-T1 cloning kit (Transgen CT101).

[0126] The TA cloning product was directly sequenced, and the sequencing results are as follows:

[0127] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO:2, and the fragment is 363 bp in length.

[0128] The amino acid sequence encoded by the fragment is set forth in SEQ ID NO: 1 and is 121 amino acids in length.

[0129] The sequence of heavy chain HCDR1 is set forth in SEQ ID NO:3, the sequence of HCDR2 is set forth in SEQ ID NO:4, and the sequence of HCDR3 is set forth in SEQ ID NO:5.

[0130] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO: 7 and is 321 bp in length.

[0131] The amino acid sequence encoded by the fragment is set forth in SEQ ID NO: 6 and is 107 amino acids in length.

[0132] The sequence of the light chain LCDR1 is set forth in SEQ ID NO:8, the sequence of LCDR2 is set forth in SEQ ID NO:9, and the sequence of LCDR3 is set forth in SEQ ID NO:10.

[0133] Example 3: Design and preparation of anti-human TIGIT humanized and mutant antibodies 1. Design of the Light and Heavy Chains of Anti-Human TIGIT Humanized Antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 Based on the three-dimensional crystal structure of human TIGIT protein and the sequence of antibody 26B12 obtained in Example 2, the variable region sequences of antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 were designed (antibody constant region sequences were from the NCBI database, the heavy chain constant region was Ig gamma 1 chain C region, accession number P01857, and the light chain constant region was Ig kappa chain C region, accession number P01834).

[0134] The designed variable region sequences are shown in Table 1 below.

[0135] [Table 2]

[0136] The nucleotide sequences of the heavy chain variable regions of the eight antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 are all 363 bp long and encode amino acid sequences of 121 aa in length, while the nucleotide sequences of the light chain variable regions are all 321 bp long and encode amino acid sequences of 107 aa in length.

[0137] Furthermore, the above eight antibodies have the same HCDR1 to HCDR3 and LCDR1 to LCDR3, the sequence of HCDR1 is set forth in SEQ ID NO: 3, the sequence of HCDR2 is set forth in SEQ ID NO: 4, and the sequence of HCDR3 is set forth in SEQ ID NO: 5; The sequence of LCDR1 is set forth in SEQ ID NO:8, the sequence of LCDR2 is set forth in SEQ ID NO:9, and the sequence of LCDR3 is set forth in SEQ ID NO:10.

[0138] 2. Preparation of humanized antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 The heavy chain constant regions were all Ig gamma 1 chain C regions (accession number P01857), and the light chain constant regions were all Ig kappa chain C regions (accession number P01834).

[0139] The heavy and light chain cDNAs of 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, 26B12H2L4, and 26B12H4L4 were cloned into pUC57simple (provided by GenScript) vectors to prepare the following heavy and light chain cDNAs: pUC57simple-26B12H1 and pUC57simple-26B12L1; pUC57simple-26B12H4 and pUC57simple-26B12L1; pUC57simple-26B12H2 and pUC57simple-26B12L2; pUC57simple-26B12H3 and pUC57simple-26B12L2; pUC57simple-26B12H2 and pUC57simple-26B12L3; pUC57simple-26B12H3 and pUC57simple-26B12L3; pUC57simple-26B12H1 and pUC57simple-26B12L4; and pUC57simple-26B12H4 and pUC57simple-26B12L4 were obtained.

[0140] According to the standard techniques described in Molecular Cloning: A Laboratory Manual (2nd edition), the full-length genes of heavy and light chains synthesized by EcoRI & HindIII digestion were subcloned into pcDNA3.1 expression vector by digestion with restriction enzymes EcoRI & HindIII 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, and the heavy / light chain genes of the recombinant expression plasmids were further sequenced. Subsequently, the intended gene combinations containing the corresponding light chain recombinant plasmids and heavy chain recombinant plasmids (pcDNA3.1-26B12H1 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H4 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H2 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H2 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H1 / pcDNA3.1-26B12L4, and pcDNA3.1-26B12H4 / pcDNA3.1-26B12L4) were separately co-transfected into 293F cells, and the culture medium was then collected and purified. After verifying the product by sequencing, an endotoxin-free expression plasmid was prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected and affinity-purified on a Protein A column to obtain the humanized antibody.

[0141] Humanized antibody 26B12H2L2 is also referred to herein as 26B12H2L2(hG1WT).

[0142] 3. Design of humanized antibodies 26B12H2L2(hG1DM) and 26B12H2L2(hG4DM) By introducing a point mutation from leucine to alanine (L234A) at position 234 (according to the EU numbering system; this also applies hereinafter) and a point mutation from leucine to alanine (L235A) at position 235 in the heavy chain constant region of 26B12H2L2, the present inventors obtained a humanized antibody containing mutations in its constant region, i.e., 26B12H2L2(hG1DM). The amino acid sequence of 26B12H2(hG1DM), which is the heavy chain of 26B12H2L2(hG1DM), is set forth in SEQ ID NO:31, and the amino acid sequence of its light chain is set forth in SEQ ID NO:29.

[0143] By leaving the variable regions of 26B12H2L2 unchanged and using the Ig gamma 4 chain C region as the heavy chain constant region of the antibody, and introducing a point mutation from phenylalanine to alanine at position 234 (F234A) and a point mutation from leucine to alanine at position 235 (L235A) in the heavy chain constant region, the inventors obtained a humanized antibody, 26B12H2L2(hG4DM), containing mutations in its constant region. The amino acid sequence of 26B12H2(hG4DM), the heavy chain of 26B12H2L2(hG4DM), is set forth in SEQ ID NO:27, and the amino acid sequence of its light chain is set forth in SEQ ID NO:29.

[0144] Humanized antibodies 26B12H2L2(hG1DM) and 26B12H2L2(hG4DM) can be prepared according to the method described in step 2 above.

[0145] Example 4: Sequence design and preparation of anti-TIGIT antibody-TGF-βR fusion protein 1. Sequence design The compositions of the anti-TIGIT antibody-TGF-βR fusion proteins of the present disclosure are shown in Table 2 below.

[0146] [Table 3]

[0147] The TGF-β receptor portion had two peptide chains, each of which was linked to the C-terminus of the heavy chain of the IgG portion via a linker fragment.

[0148] 2. Antibody Expression and Purification The heavy and light chain cDNA sequences of TFO1 and TF02 were cloned into the pUC57simple vector (provided by Genscript) to obtain the plasmids pUC57simple-TF01H and pUC57simple-TF02H, and pUC57simple-TF01L and pUC57simple-TF02L, respectively. The plasmids pUC57simple-TF01H / pUC57simple-TF01L and pUC57simple-TF02H / pUC57simple-TF02L were digested with HindIII and EcoRI. The electrophoretically isolated heavy and light chains were subcloned separately into the pcDNA3.1 vector, and the recombinant plasmids were extracted and cotransfected into 293F cells. After 7 days of cell culture, the culture solution was centrifuged at high speed. The supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. The proteins were eluted in one step with elution buffer. The target samples were isolated and buffer-exchanged into PBS. Thus, anti-TIGIT antibody-TGF-βR fusion proteins TFO1 and TF02 were obtained.

[0149] Example 5: Sequence design and preparation of anti-TIGIT antibody-TGF-βR fusion protein (mutant) The serine at position 8 of the TGF-βRII extracellular domain fragment (SEQ ID NO: 33) was mutated to alanine, glycine, or threonine to create a point mutation in the TGF-βRII extracellular domain fragment in TFO1, an anti-TIGIT antibody-TGF-βR fusion protein of the present disclosure. The resulting novel antibodies were designated TFO1A, TFO1G, and TFO1T, respectively. These antibodies were prepared in accordance with Example 4.

[0150] [Table 4]

[0151] Example 6: Sequence design and preparation of anti-TIGIT antibody-TGF-βR fusion protein (truncated) The TGF-βRII extracellular domain fragment in TFO1, an anti-TIGIT antibody-TGF-βR fusion protein of the present disclosure, was partially truncated by deletion of the sequence IPPHVQKS to obtain a fusion protein designated TFO1t31, or by deletion of the sequence IPPHVQKSVNNDMI to obtain a fusion protein designated TFO1t37. These fusion proteins were prepared according to Example 4.

[0152] [Table 5]

[0153] Example 7: Quantification of the binding activity of TFOl and TF02 to antigen or TGF-β by ELISA 1. Quantification of the binding activity of TF01, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) to the antigen TIGIT-mFc by indirect ELISA The specific method was as follows: An 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 ELISA plate was then washed once with PBST and blocked with a PBS solution containing 1% BSA (blocking solution) at 37°C for 2 hours. After blocking, the ELISA plate was washed three times with PBST. 1 μg / mL TIGIT-mFc was then added, and the plate was incubated at 37°C for 30 minutes and then washed three times with PBST. Antibodies serially diluted in PBST solution (the antibody dilution gradient is listed in Table 5) were added. The ELISA plate containing the test antibody was incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, a working solution of HRP-conjugated goat anti-human IgG (H+L) (Jackson, Cat. No. 109-035-098) secondary antibody diluted 1:5000 was added, and the plate was then incubated at 37°C for 30 minutes. After incubation, the plate was washed four times with PBST. Color development was then carried out in the dark for 5 minutes using TMB (Neogen, 308177), and the color reaction was stopped by adding stop solution. The ELISA plate was immediately placed in a microplate reader, and the OD of each well of the ELISA plate was measured at a wavelength of 450 nm. Data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0154] The results are shown in Table 5 and Figure 1.

[0155] [Table 6]

[0156] The figure shows that TFO1, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) effectively bind to the antigen TIGIT-mFc in a dose-dependent manner. EC 50The values ​​are shown in Table 3. Quantitative absorbance analysis of bound antibodies, curve fitting, and calculations determined the binding efficiencies EC of TFO1, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) (as a control). 50 The values ​​obtained were 0.067 nM, 0.091 nM, 0.046 nM, and 0.055 nM, respectively.

[0157] The results of the above experiments show that TFO1, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) had the activity of effectively binding to TIGIT-mFc.

[0158] 2. Quantification of the binding activity of TF01, TF02, and TGF-βRII-mFc to TGF-β1, TGF-β2, and TGF-β3 by indirect ELISA The specific method was as follows: ELISA plates were coated with 1 μg / mL TGF-β1, 2 μg / mL TGF-β2, and 1 μg / mL TGF-β3, respectively, and incubated overnight at 4°C. The antigen-coated ELISA plates were then washed once with PBST and blocked with PBS solution containing 1% BSA (blocking solution) at 37°C for 2 hours. After blocking, the ELISA plates were washed three times with PBST. Serially diluted antibodies and TGF-βRII-mFc in PBST solution were added. The ELISA plates containing the test antibodies and TGF-βRII-mFc were incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, a working solution of HRP-conjugated goat anti-human IgG (H+L) (Jackson, Catalog No. 109-035-098) and HRP-conjugated goat anti-mouse IgG Fc (Jackson, Catalog No. 115-035-071) secondary antibodies diluted at a ratio of 1:5000 was added, and the plate was then incubated at 37°C for 30 minutes. After incubation, the plate was washed four times with PBST. Color development was then carried out in the dark for 5 minutes using TMB (Neogen, 308177), and the color reaction was stopped by adding stop solution. The ELISA plate was immediately placed in a microplate reader, and the OD of each well of the ELISA plate was measured at a wavelength of 450 nm. Data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0159] The results are shown in Tables 6, 7, and 8, and in Figures 2, 3, and 4.

[0160] [Table 7]

[0161] [Table 8]

[0162] [Table 9]

[0163] Quantitative absorbance analysis of the bound antibody, curve fitting, and calculations determined the binding efficiency (EC) of TF01 to TGF-β1, TGF-β2, and TGF-β3. 50 The binding efficiencies EC of TF02 to TGF-β1, TGF-β2, and TGF-β3 were obtained as 0.207 nM, 0.477 nM, and 0.215 nM, respectively. 50 The values ​​were 0.224 nM, 0.594 nM, and 0.234 nM, respectively, which were the binding efficiencies EC of TGF-βR2-mFc (as a positive control) to TGF-β1, TGF-β2, and TGF-β3. 50 The values ​​were 0.356 nM, 1.029 nM, and 0.402 nM, respectively.

[0164] These results indicate that TFO1 and TF02 effectively bound to TGF-β1, TGF-β2, and TGF-β3 in a dose-dependent manner. These results further indicate that TFO1, TF02, and TGF-βRII-mFc all effectively bound to TGF-β1, TGF-β2, and TGF-β3, and that the binding capacities of TFO1 and TF02 to TGF-β1, TGF-β2, and TGF-β3 were higher than those of TGF-βRII-mFc.

[0165] Example 8: Quantification of the ability of TF01 and TF02 to compete with TGF-βRII-His-biotin for binding to human TGF-β1 and TGF-β3 by competitive ELISA Experimental steps: ELISA plates were coated with 2 μg / mL TGF-β1 and 2 μg / mL TGF-β3, respectively, and incubated overnight at 4°C. After incubation, the antigen-coated ELISA plates were rinsed once with PBST and blocked for 2 hours with PBS solution containing 1% BSA (ELISA plate blocking solution). After blocking, the ELISA plates were washed three times with PBST. Serially diluted antibodies and TGF-βRII-mFc in PBST solution were added to the ELISA plates. The TGF-β1 ELISA plates were incubated at room temperature for 60 minutes, then washed three times with PBST, and then 0.01 μg / mL TGF-βRII-His-biotin was added, followed by incubation at room temperature for 10 minutes. The TGF-β3 ELISA plate was incubated at 37°C for 30 minutes, washed three times with PBST, and then 0.01 μg / mL TGF-βRII-His-biotin was added, followed by incubation at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST. After washing, SA-HRP working solution diluted 1:4000 was added, and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed four times with PBST. Color development was then carried out in the dark for 4 minutes using TMB (Neogen, 308177), and the color reaction was stopped by adding stop solution. The ELISA plate was immediately placed in a microplate reader, and the OD of each well of the ELISA plate was measured at a wavelength of 450 nm. Data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0166] The activity of antibodies competing with TGF-βRII-His-biotin for binding to TGF-β1 and TGF-β3 is shown in Tables 9 and 10 and Figures 5 and 6. Curves of antibody concentration (abscissa) versus absorbance (ordinate) were fitted to determine the EC values ​​of antibodies competing with TGF-βRII-His-biotin for binding to TGF-β1 and TGF-β3. 50 The values ​​were calculated and the results are shown in Tables 9 and 10 below.

[0167] [Table 10]

[0168] [Table 11]

[0169] Quantitative absorbance analysis of bound antibodies, curve fitting, and calculations determined the binding efficiency EC of TF01, TF02, and TGF-βRII-mFc to compete with TGF-βRII-His-biotin for binding to TGF-β1. 50 The EC values ​​obtained were 5.579 nM, 7.469 nM, and 6.475 nM, respectively, which were the binding efficiencies EC of TF01, TF02, and TGF-βRII-mFc to compete with TGF-βRII-His-biotin for binding to TGF-β3. 50 The values ​​were 3.569 nM, 3.879 nM, and 2.808 nM, respectively.

[0170] These results indicate that TFO1 and TF02 effectively bound to TGF-β1 and TGF-β3 in a dose-dependent manner. Furthermore, the binding ability of TFO1 to TGF-β1 was higher than that of TGF-βRII-His-biotin. The ability of TFO1 to compete with TGF-βRII-His-biotin for binding to TGF-β1 was higher than that of TGF-βRII-mFc.

[0171] Example 9: Quantification of the ability of TF01 and TF02 to compete with CD155-hFc-biotin for binding to human TIGIT-mFc by competitive ELISA An ELISA plate was coated with 2 μg / mL human TIGIT-mFc (TIGIT Genbank ID: NP_776160.2) and incubated overnight at 4°C. After incubation, the ELISA plate was blocked with PBS containing 1% BSA for 2 hours at 37°C. After blocking, the plate was washed three times and tapped dry. The antibody was serially diluted to seven concentrations at a 1:3 ratio on a dilution plate, starting at an initial concentration of 20.18 nM (final concentration: 10.09 nM), and a blank control was set up. An equal volume of 4 μg / mL (final concentration: 2 μg / mL) human CD155-hFc-biotin solution was then added. After thorough mixing, the mixture was incubated at room temperature for 20 minutes. The reaction mixture was then added to the coated ELISA plate, and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST and tapped dry. SA-HRP (KPL, 14-30-00) working solution was added, and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed four times and gently tapped dry. Color development was then carried out in the dark for 5 minutes using TMB (Neogen, 308177), and the color reaction was stopped by adding stop solution. The ELISA plate was immediately placed in a microplate reader, and the OD of each well of the ELISA plate was measured at a wavelength of 450 nm. Data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0172] The results are shown in Figure 7. The OD values ​​at various doses are shown in Table 9. Quantitative absorbance analysis and curve fitting of the bound antibody determined the antibody binding efficiency, EC 50 The values ​​were obtained (Table 11).

[0173] [Table 12]

[0174] EC of TF01, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) (as a control) blocking binding of TIGIT-mFc to its ligand CD155-hFc-biotin 50 The values ​​were 1.463 nM, 1.963 nM, 1.423 nM, and 1.675 nM, respectively.

[0175] These results show that TFO1, TF02, 26B12H2L2 (hG4DM), and RG6058 (hG4) (as a control) effectively blocked the binding of the antigen, human CD155-hFc-biotin, to its receptor, human TIGIT-mFc, and the blocking efficiency was dose-dependent. The ability of TFO1 to block the binding of TIGIT-mFc to its ligand, CD155-hFc-biotin, was greater than that of the control antibody, RG6058 (hG4).

[0176] Example 10: Quantification of TFO1 binding activity to TIGIT on the surface of 293T-TIGIT membrane by FACS 293T-TIGIT cells in the logarithmic growth phase were harvested and plated in a clear 96-well V-bottom plate at 3 × 10 5 Cells were transferred at 100 μL per well. 1% PBSA was added, the plate was centrifuged at 350×g for 5 minutes, and the supernatant was removed. 100 μL of antibody diluted in 1% PBSA (final concentrations: 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. After gentle and thorough mixing, the plate was incubated on ice for 1 hour. 1% PBSA was added, the plate was centrifuged at 350×g for 5 minutes, and the supernatant was removed. A 350-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog number 109-095-098) was added to resuspend the cells. After thorough mixing, the plate was incubated on ice in the dark for 0.5 hours. 1% PBSA was added, the plate was centrifuged at 350 g for 5 minutes, and the supernatant was removed. 400 μL of PBSA was added to resuspend the cell pellet, and the suspension was transferred to a flow cytometry tube and analyzed by FACS Calibur.

[0177] The experimental results are shown in Table 12 and Figure 8. Both TFO1 and RG6058 (hG4) (control antibody) specifically bound to TIGIT on the surface of the 293T-TIGIT membrane.

[0178] [Table 13]

[0179] EC of binding of TFO1 and RG6058 (hG4) to 293T-TIGIT cells under the same experimental conditions. 50 The values ​​were 1.540 nM and 1.612 nM, respectively.

[0180] These results indicate that TFO1 effectively binds to TIGIT on the surface of 293T-TIGIT membranes in a dose-dependent manner, and its binding ability is higher than that of the control antibody RG6058 (hG4).

[0181] Example 11: Quantification of the ability of TFO1 to compete with CD155 or CD112 for binding to the cell membrane surface antigen TIGIT by competitive flow cytometry 293T-TIGIT cells were routinely lysed and 3 × 10 5 Cells were plated per well per sample and washed by centrifugation. Appropriate serially diluted antibodies were added at 100 μL per well, and the plate was incubated on ice for 30 minutes. CD155 / CD112-mFc (prepared by Akeso Biopharma, Lot No. 20190726 / Lot No. 20190726) was added at 100 μL per well to a final concentration of 10 nM / 30 nM and mixed thoroughly. The plate was incubated on ice for 1 hour. 1% PBSA was added, the plate was centrifuged at 350 g for 5 minutes, and the supernatant was removed. APC goat anti-mouse IgG antibody (Biolegend, Cat. No. 405308) diluted 1:300 was added at 100 μL per well, and 100 μL of 1% PBSA was added to the blank sample. After thorough mixing, the plates were incubated on ice in the dark for 30 minutes, washed by centrifugation, and the cells were resuspended, transferred to flow cytometry tubes, and analyzed using a flow cytometer.

[0182] The results are shown in Figures 9 and 10. 50 The values ​​are shown in Table 13. Fluorometric analysis and curve fitting determined the competitive binding EC 50 The values ​​were calculated to be 1.2720 nM and 0.7445 nM.

[0183] [Table 14]

[0184] These results indicate that antibody TFO1 effectively blocked the binding of CD155 and / or CD112 to TIGIT on the surface of 293T-TIGIT host cells in a dose-dependent manner.

[0185] Example 12: Quantification of the affinity of TF01 for the Fc receptor FcγRI The Fc receptor FcγRI (also known as CD64) can bind to the Fc fragment of IgG antibodies and plays a role in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of a therapeutic antibody to bind to an Fc receptor affects the safety and efficacy of the antibody. In this experiment, the affinity constant of TFO1 for FcγRI was determined using a Fortebio Octet molecular interaction instrument to evaluate the ADCC activity of the antibody.

[0186] The experimental method for determining the affinity constant of the corresponding antibody for FcγRI using the Fortebio Octet molecular interaction instrument is briefly described below. The sample dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA (pH 7.4). A 1 μg / mL solution of FcγRI (purchased from Sinobio) was added to the HIS1K sensor, and the system was incubated for 50 seconds to immobilize FcγRI on the sensor surface. Both the association and dissociation constants of the antibody for FcγRI were determined in buffer. The antibody concentrations ranged from 3.12 to 50 nM (2-fold serial dilution). The sample plate shaking speed was 1000 rpm, the temperature was 30 °C, and the frequency was 5.0 Hz. The data were analyzed by 1:1 model fitting to obtain the affinity constant.

[0187] The affinity constant between FcγRI and TF01 is shown in Table 14 and Figures 11 and 12.

[0188] [Table 15]

[0189] N / A means that there was no binding between the antibody and the antigen or the binding signal was so weak that the results were not analyzed and no corresponding data was obtained.

[0190] These results show that 26B12H2L2 (hG1WT) has an affinity constant of 6.51 × 10 -9 M showed binding to FcγRI, whereas there was no binding or the binding signal was very weak between TFO1 and FcγRI, so the results were not analyzed and no corresponding data were obtained.

[0191] These results indicate that the binding activity of TFO1 to FcγRI was effectively eliminated.

[0192] Example 13: Quantification of the affinity of TFO1 for the Fc receptor FcγRIIIa and its subtypes (1) Determination of the affinity constant between FcγRIIIa_V158 and TF01 The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) can bind to the Fc fragment of IgG antibodies and mediate ADCC. In this experiment, the affinity constant of TFO1 for FcγRIIIa_V158 was determined using a Fortebio Octet molecular interaction instrument to evaluate the ADCC activity of the antibody.

[0193] The experimental method for determining the affinity constants of the corresponding antibodies using the Fortebio Octet molecular interaction instrument is briefly described below. The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIIa_V158 was immobilized on the HIS1K sensor for 60 seconds, and the sensor was equilibrated in the buffer for 60 seconds. The FcγRIIIa_V158 immobilized on the sensor was bound to each antibody (antibody concentration: 31.25–500 nM, 2-fold dilution) for 60 seconds, and the antibody was dissociated in the buffer for 60 seconds. The sample plate shaking speed was 1000 rpm, the temperature was 30°C, and the frequency was 5.0 Hz. The data were analyzed by 1:1 model fitting to obtain the affinity constants.

[0194] The affinity constant between FcγRIIIa_V158 and TF01 is shown in Table 15 and Figures 13 and 14.

[0195] [Table 16]

[0196] N / A means that there was no binding between the antibody and the antigen or the binding signal was so weak that the results were not analyzed and no corresponding data was obtained.

[0197] These results indicate that 26B12H2L2 (hG1WT) has an affinity constant of 5.54 × 10 -8M showed binding to FcγRIIIa_V158, whereas there was no binding or the binding signal was extremely weak between TF01 and FcγRIIIa_V158, so the results were not analyzed.

[0198] These results indicate that the binding activity of TFO1 to FcγRIIIa_V158 was effectively eliminated.

[0199] (2) Determination of the affinity constant between FcγRIIIa_F158 and TF01 The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) can bind to the Fc fragment of IgG antibodies and mediate ADCC. In this experiment, the affinity constant of TFO1 for FcγRIIIa_F158 was determined using a Fortebio Octet molecular interaction instrument to evaluate the ADCC activity of the antibody.

[0200] The experimental procedure for determining the affinity constant of TFO1 for FcγRIIIa_F158 using a Fortebio Octet molecular interaction instrument is briefly described below. The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIIa_F158 was immobilized on a HIS1K sensor for 120 seconds, and the sensor was equilibrated in the buffer for 60 seconds. The FcγRIIIa_F158 immobilized on the sensor was bound to each antibody (antibody concentration: 31.25–500 nM, 2-fold dilution) for 60 seconds, and the antibody was dissociated in the buffer for 60 seconds. The sample plate shaking speed was 1000 rpm, the temperature was 30°C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model fitting to obtain the affinity constant.

[0201] The affinity constant between FcγRIIIa_F158 and TF01 is shown in Table 16 and Figures 15 to 16.

[0202] [Table 17]

[0203] N / A means that there was no binding between the antibody and the antigen or the binding signal was so weak that the results were not analyzed and no corresponding data was obtained.

[0204] These results show that 26B12H2L2 (hG1WT) has an affinity constant of 9.97 × 10 -8 M showed binding to FcγRIIIa_F158, whereas there was no binding or the binding signal was extremely weak between TF01 and FcγRIIIa_F158, so the results were not analyzed and no corresponding data were obtained.

[0205] These results indicate that the binding activity of TF01 to FcγRIIIa_F158 was effectively eliminated.

[0206] Example 14: Quantification of the affinity of TFO1 for the Fc receptor FcγRIIa and its subtypes (1) Determination of the affinity constant between FcγRIIa_H131 and TF01 The Fc receptor FcγRIIa_H131 (also known as CD32a_H131) can bind to the Fc fragment of IgG antibodies and plays a role in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of a therapeutic antibody to bind to an Fc receptor affects the safety and efficacy of the antibody. In this experiment, the affinity constant of TFO1 for FcγRIIa_H131 was determined using a Fortebio Octet molecular interaction instrument to evaluate the Fc receptor binding ability of the test antibody.

[0207] The experimental procedure for determining the affinity constant of TFO1 for FcγRIIa_H131 using the Fortebio Octet molecular interaction instrument is briefly described below. The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_H131 was immobilized on the NTA sensor at an immobilization height of approximately 1.0 nm, and the sensor was equilibrated in the buffer for 60 seconds. The FcγRIIa_H131 immobilized on the sensor was allowed to bind to each antibody (antibody concentration: 12.5–200 nM, 2-fold serial dilution) for 60 seconds, and the antibody was allowed to dissociate in the buffer for 60 seconds. The sample plate shaking speed was 1000 rpm, the temperature was 30°C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model fitting to obtain the affinity constant.

[0208] The affinity constant between FcγRIIa_H131 and TF01 is shown in Table 17 and Figures 17 to 18.

[0209] [Table 18]

[0210] N / A means that there was no binding between the antibody and the antigen or the binding signal was so weak that the results were not analyzed and no corresponding data was obtained.

[0211] These results show that 26B12H2L2 (hG1WT) has an affinity constant of 3.90 × 10 -8 M showed binding to FcγRIIa_H131, whereas there was no binding or the binding signal was extremely weak between TF01 and FcγRIIa_H131, so the results were not analyzed and no corresponding data were obtained.

[0212] These results indicate that the binding activity of TF01 to FcγRIIa_H131 was effectively eliminated.

[0213] (2) Determination of the affinity constant between FcγRIIa_R131 and TF01 The Fc receptor FcγRIIa_R131 (also known as CD32a_R131) can bind to the Fc fragment of IgG antibodies and plays a role in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of a therapeutic antibody to bind to an Fc receptor affects the safety and efficacy of the antibody. In this experiment, the affinity constant of TFO1 for FcγRIIa_R131 was determined using a Fortebio Octet molecular interaction instrument to evaluate the Fc receptor binding ability of the test antibody.

[0214] The experimental method for determining the affinity constant of TFO1 for FcγRIIa_R131 using a Fortebio Octet molecular interaction instrument is briefly described below. The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_R131 was immobilized on the NTA sensor at an immobilization height of approximately 1.0 nm, and the sensor was equilibrated in the buffer for 60 seconds. The FcγRIIa_R131 immobilized on the sensor was allowed to bind to each antibody (antibody concentration: 12.5–200 nM, 2-fold serial dilution) for 60 seconds, and the antibody was allowed to dissociate in the buffer for 60 seconds. The sample plate shaking speed was 1000 rpm, the temperature was 30°C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model fitting to obtain the affinity constant.

[0215] The affinity constant between FcγRIIa_R131 and TF01 is shown in Table 18 and Figures 19 and 20.

[0216] [Table 19]

[0217] N / A means that there was no binding between the antibody and the antigen or the binding signal was so weak that the results were not analyzed and no corresponding data was obtained.

[0218] These results show that 26B12H2L2 (hG1WT) has an affinity constant of 3.22 × 10 -8 M showed binding to FcγRIIa_H131, whereas there was no binding or the binding signal was extremely weak between TF01 and FcγRIIa_R131, so the results were not analyzed and no corresponding data were obtained.

[0219] These results indicate that the binding activity of TFO1 to FcγRIIa_R131 was effectively eliminated.

[0220] Example 15: Determination of the affinity constant between FcγRIIb and TF01 The Fc receptor FcγRIIb (also known as CD32b) can bind to the Fc fragment of IgG antibodies. In this experiment, to evaluate the Fc receptor binding ability of TFO1, the affinity constant of the test antibody for FcγRIIb was determined using a Fortebio Octet molecular interaction instrument.

[0221] The experimental procedure for determining the affinity constant of TFO1 for FcγRIIb using the Fortebio Octet molecular interaction instrument is briefly described below. The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIb was immobilized on the NTA sensor at an immobilization height of approximately 1.0 nm, and the sensor was equilibrated in the buffer for 60 seconds. The hFCGR2B-his immobilized on the sensor was allowed to bind to each antibody (antibody concentration: 12.5–200 nM, 2-fold serial dilution) for 60 seconds, and the antibody was allowed to dissociate in the buffer for 60 seconds. The sample plate shaking speed was 1000 rpm, the temperature was 30°C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model fitting to obtain the affinity constant.

[0222] The affinity constant between FcγRIIb and TF01 is shown in Table 19 and Figures 21 and 22.

[0223] [Table 20]

[0224] N / A means that there was no binding between the antibody and the antigen or the binding signal was so weak that the results were not analyzed and no corresponding data was obtained.

[0225] These results indicate that 26B12H2L2 (hG1WT) has an affinity constant of 4.12 × 10 -8 M showed binding to FcγRIIb, whereas there was no binding or the binding signal was extremely weak between TFO1 and FcγRIIb, so the results were not analyzed and no corresponding data were obtained.

[0226] These results indicate that the binding activity of TFO1 to FcγRIIb was effectively eliminated.

[0227] Example 16: Quantification of the affinity of TF01 for C1q Serum complement C1q can bind to the Fc fragment of IgG antibodies and mediate CDC. The ability of therapeutic antibodies to bind C1q affects the safety and efficacy of the antibody. In this experiment, to evaluate the CDC activity of these antibodies, the affinity constant of TFO1 for C1q was determined using a Fortebio Octet molecular interaction instrument.

[0228] The experimental method for determining the affinity constant of the corresponding antibody for C1q using the Fortebio Octet molecular interaction instrument is briefly described below. The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 50 μg / mL of antibody was immobilized on a FAB2G sensor at an immobilization height of approximately 2.0 nm, and the sensor was equilibrated in the buffer for 60 seconds. The antibody immobilized on the sensor was allowed to bind to the antigen C1q (antigen concentration: 0.625–10 nM, 2-fold serial dilution) for 60 seconds, and the antigen and antibody were allowed to dissociate from each other in the buffer for 60 seconds. The sample plate shaking speed was 1000 rpm, the temperature was 30°C, and the frequency was 5.0 Hz. The data were analyzed by 1:1 model fitting to obtain the affinity constant. The data acquisition software was Fortebio Data Acquisition 7.0 and the data analysis software was Fortebio Data Analysis 7.0.

[0229] The affinity constant of TF01 for C1q is shown in Table 20 and Figures 23-24.

[0230] [Table 21]

[0231] N / A means that there was no binding between the antibody and the antigen or the binding signal was so weak that the results were not analyzed and no corresponding data was obtained.

[0232] These results indicate that 26B12H2L2 (hG1WT) has an affinity constant of 1.28 × 10 -9 M showed binding to C1q, whereas there was no binding or the binding signal was very weak between TFO1 and C1q, so the results were not analyzed and no corresponding data were obtained.

[0233] These results indicate that the binding activity of TFO1 to C1q was effectively eliminated.

[0234] Example 17: Antibody-mediated phagocytic activity of TFO1 on CHO-K1-TIGIT cells The purpose of this example was to quantify antibody-dependent cellular phagocytosis (ADCP) activity. Mouse macrophages were used as effector cells and a TIGIT-overexpressing cell line was used as target cells to evaluate antibody-mediated ADCP activity.

[0235] Cryopreserved mouse bone marrow-derived macrophages (MBMM) (derived from C57 mice) were added to DMEM + 10% FBS + 100 ng / mL M-CSF (macrophage colony-stimulating factor, Peprotech, Cat. No. 315-02), and the mixture was centrifuged at 1200 × g for 5 minutes. Cells were collected, added to DMEM + 10% FBS + 100 ng / mL M-CSF medium, and thawed overnight.

[0236] Target cells (CHO-K1-TIGIT) were collected, centrifuged at 170 × g for 5 minutes, and washed once with PBS. Cells were counted using trypan blue. 5(6)-carboxyfluorescein N-succinimidyl ester (CFSE, Biolegend, Cat. No. 423801) was diluted to 2.5 μM in PBS, and the cells were resuspended using an appropriate volume of CFSE dilution (staining density: 1 × 10 7 cells / mL). The cell suspension was incubated in an incubator for 20 minutes, and 6 mL of DMEM complete medium (containing 10% FBS) was added to stop the staining. The mixture was centrifuged at 170 × g for 5 minutes, and the supernatant was discarded. 1 mL of DMEM complete medium was added, and the mixture was incubated in an incubator for 10 minutes. Antibodies were diluted to the desired concentration in DMEM complete medium, and isotype control antibodies were designed. Target cells (1.5 × 10 5Cells / well) were added to a 96-well V-bottom plate, and antibody (100 μL) was added. After thorough mixing, the plate was incubated on ice for 40 minutes and washed twice by centrifugation (170 × g, 5 minutes). Macrophages (MBMM) were collected and centrifuged at 750 × g for 5 minutes, and the supernatant was discarded. Cells were counted and resuspended in DMEM complete medium, and the macrophage concentration was adjusted (5 × 10 4 Cells / 100 μL). The cell suspension was added to a 96-well V-bottom plate containing target cells. After resuspension and thorough mixing, the plate was incubated in a 37°C incubator for 2 hours, and 100 μL of 1% PBSA (room temperature) was added to each well. The plate was centrifuged at 750 × g for 5 minutes, and the supernatant was discarded. The cells were washed once with 200 μL of PBSA. APC anti-mouse / human CD11b antibody (Biolegend, catalog no. 101212) (diluted 1:500 in PBSA) was added to the corresponding sample at 100 μL / well. After thorough mixing, the plate was incubated on ice for 40 minutes, and 100 μL of 1% PBSA was added to each well. The plate was centrifuged at 750 × g for 5 minutes, and the supernatant was discarded. The cells in each well were washed once with 200 μL of PBSA, and 200 μL of 1% PBSA was added to each tube to resuspend the cells. The cells were then analyzed using the device. In the analysis system, macrophages were positive for APC, and phagocytic macrophages were double positive for APC and CFSE. The phagocytic rate was expressed as the ratio of the number of double positive cells to the number of APC positive cells, and antibody-mediated ADCP activity was evaluated based on the phagocytic rate. The ADCP activity of each group, expressed as P%, was calculated using the following formula: P% = number of phagocytic macrophages / total number of macrophages × 100% was calculated using

[0237] The results are shown in Figure 25.

[0238] The results show that RG6058(hG1WT), RG6058(hG4), and 26B12H2L2(hG1WT) exhibited ADCP activity at the same concentrations. At the same concentrations, the phagocytosis rate of the TFO1 group was comparable to that of the isotype control antibody group, indicating that TFO1 does not exhibit ADCP activity.

[0239] These results indicate that the amino acid mutations introduced into TFO1 effectively eliminated its ADCP activity.

[0240] Example 18: Evaluation of the blocking activity of anti-TIGIT antibody-TGF-βR fusion protein on the inhibition of IL-2 secretion by CD112 / CD155 in a co-culture system 1. Evaluation of the blocking activity of anti-TIGIT antibody-TGF-βR fusion protein on CD112-mediated inhibition of IL-2 secretion in a co-culture system of Jurkat-TIGIT cells and THP-1 cells. A 96-well plate (Corning, Model No. 3599) was coated with 2 μg / mL of anti-human CD3 antibody (prepared by Akeso Biopharma, Lot No. 20170830) for 2 hours at 37°C. The coating solution was removed, and the plate was washed once with pre-chilled PBS (300 μL). Jurkat-TIGIT cells (prepared by Akeso Biopharma) were counted and plated at 5 × 10 cells per well into a 96-well plate. 4 Antibodies were added according to the experimental design, and the plates were incubated at 37°C for 30 minutes. CD112-hFc (prepared by Akeso Biopharma, lot number 20180209) was added according to the experimental design. THP-1 cells (obtained from the Chinese Academy of Sciences, catalog number 3131C0001000700057) were counted and plated at 5 x 10 cells / well in a 96-well plate. 4 Cells were seeded at 1000 x g / well. The plates were incubated in an incubator for 48 hours. The culture supernatants were collected and the IL-2 content was quantified using an IL-2 ELISA kit (Dakewe, Cat. No. 1110202).

[0241] The results are shown in Figure 26. The results demonstrate that CD112 exhibited a significant inhibitory effect on IL-2 secretion. Antibodies 26B12H2L2 (hG4DM), TFOl, TF02, RG6058 (hG1DM), and RG6058 (hG4) all effectively blocked CD112-mediated inhibition of IL-2 secretion in this system, with TFOl and TF02 being superior in activity to the positive control antibodies RG6058 (hG1DM) and RG6058 (hG4).

[0242] 2. Evaluation of the blocking activity of anti-TIGIT antibody-TGF-βR fusion protein in inhibiting IL-2 secretion by CD155 in a co-culture system of Jurkat-TIGIT cells and THP-1 cells. A 96-well plate (Corning, Model No. 3599) was coated with 2 μg / mL of anti-human CD3 antibody (prepared by Akeso Biopharma, Lot No. 20170830) for 2 hours at 37°C. The coating solution was removed, and the plate was washed once with pre-chilled PBS (300 μL). Jurkat-TIGIT cells (prepared by Akeso Biopharma) were counted and plated at 5 × 10 cells per well into a 96-well plate. 4 Antibodies were added according to the experimental design, and the plates were incubated at 37°C for 30 minutes. CD155-hFc (prepared by Akeso Biopharma, lot number 20180209) was added according to the experimental design. THP-1 cells (purchased from the Chinese Academy of Sciences, catalog number 3131C0001000700057) were counted and plated at 5 × 10 cells / well in a 96-well plate. 4 Cells were seeded at 1000 x g / well. The plates were incubated in an incubator for 48 hours. The culture supernatants were collected and the IL-2 content was quantified using an IL-2 ELISA kit (Dakewe, Cat. No. 1110202).

[0243] The results are shown in Figure 27. The results demonstrate that CD155 exerted a significant inhibitory effect on IL-2 secretion. Both antibodies TFOl and RG6058 (hG1DM) effectively blocked CD155 inhibition of IL-2 secretion in this system and were comparable in activity.

[0244] Example 19: Evaluation of the biological activity of anti-TIGIT antibody-TGF-βR fusion protein in promoting IL-2 secretion in a co-culture system of Jurkat-TIGIT cells and HT1080-aCD3scFv cells Jurkat-TIGIT cells (constructed by Akeso Biopharma) and HT1080-aCD3scFv cells (constructed by Akeso Biopharma) in the logarithmic growth phase were harvested and counted, and 5 × 10 cells were added to each well. 4 Jurkat-TIGIT cells, and 1 x 10 cells in each well 4 HT1080-aCD3scFv cells were plated. Diluted antibody (antibody gradient: 3 nM, 30 nM, and 300 nM) was added, followed by the addition of soluble anti-human CD28 antibody (3 μg / mL) (R&D, catalog no. MAB342-500). The plate was incubated in an incubator for 48 hours. The culture supernatant was collected, and the IL-2 content was quantified using an IL-2 ELISA kit (Dakewe, catalog no. 1110202).

[0245] The results are shown in Figure 28.

[0246] The results show that antibodies TFO1, TF02, and RG6058 (hG4) all effectively promoted IL-2 secretion in a co-culture system of Jurkat-TIGIT cells and HT1080-aCD3scFv cells, and their activities were comparable.

[0247] Example 20: Evaluation of the blocking activity of anti-TIGIT antibody-TGF-βR fusion protein on the inhibition of IFN-γ secretion by TGF-β1 during the secondary immune response of PBMCs to CMV antigens PBMCs (from healthy donors) were thawed and seeded in complete medium (RPMI1640 + 10% FBS) and incubated overnight in an incubator. The next day, cells were harvested, counted, analyzed for viability, and plated at 2 × 10 cells per well into 96-well round-bottom plates (Corning, Cat. No. 3799). 5Cells were seeded at 1000 x g / well. Meanwhile, serially diluted antibodies, CMV (final concentration: 0.02 μg / mL, Mabtech, Cat. No. 3619-1), and TGF-β1 (final concentration: 3 ng / mL, Genscript, Cat. No. Z03411) were added to a final volume of 200 μL according to the experimental design (TGF-β1 and antibodies were incubated at 37°C for 10 minutes). The plates were incubated in an incubator for 4 days. After 4 days, the cell culture supernatants were collected and the IFN-γ content was quantified using an IFN-γ ELISA kit (Dakewe, Cat. No. 1110002).

[0248] The results are shown in Figure 29. The results indicate that CMV antigen triggers a secondary immune response in PBMCs, resulting in IFN-γ secretion, and that TGF-β1 exhibited a significant inhibitory effect on IFN-γ secretion in this system. Antibody TFO1, anti-HEL & TGFβ, and the combination of anti-HEL & TGFβ + 26B12H2L2 (hG1DM) all effectively blocked TGF-β1-mediated inhibition of IFN-γ secretion in this system, whereas 26B12H2L2 (hG1DM) alone did not block the inhibition. The blocking activity of TFO1 was superior to that of the control antibody, anti-HEL & TGFβ, and comparable to that of the combination of anti-HEL & TGFβ + 26B12H2L2 (hG1DM).

[0249] Example 21: Pharmacodynamic evaluation of anti-TIGIT antibody-TGF-βR fusion protein in a mouse tumor cell subcutaneous xenograft model The purpose of this example was to quantify the in vivo antitumor activity of the anti-TIGIT antibody-TGF-βR fusion protein. First, MDA-MB-231 cells (obtained from ATCC) were subcutaneously inoculated into the mammary fat pad of 6.71- to 9.57-week-old female Scid Beige mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). On day 17 after inoculation, the mice were randomized into three groups of eight mice according to tumor volume. Each mouse was intraperitoneally injected with human peripheral blood mononuclear cells (hPBMCs). The day of administration was defined as D0. The administration route was intraperitoneal. Mice were administered once weekly for 6 weeks. hPBMCs were intraperitoneally injected on day 8 after grouping. The model and specific administration route are shown in Table 21. After administration, the length and width of the tumors in each group were measured, and tumor volume was calculated.

[0250] [Table 22]

[0251] The results are shown in Figure 30. The results show that, compared to an isotype control antibody, the anti-TIGIT antibody-TGF-βR fusion protein TFO1 effectively inhibited tumor growth in mice.

[0252] Furthermore, as shown in Figure 31, the tumor-bearing mice showed good resistance to the test drug TFOl, and each group had no effect on the body weight of the tumor-bearing mice.

[0253] Example 22: Evaluation of the biological activity of anti-TIGIT antibody-TGF-βR fusion protein in promoting IL-2 secretion in a co-culture system of Jurkat-TIGIT cells and HT1080-aCD3scFv cells Jurkat-TIGIT cells (constructed by Akeso Biopharma) and HT1080-aCD3scFv cells (constructed by Akeso Biopharma) in the logarithmic growth phase were harvested and counted, and 5 × 10 cells were added to each well. 4 Jurkat-TIGIT cells, and 1 x 10 cells in each well 4HT1080-aCD3scFv cells were plated. Diluted antibody (final antibody concentrations: 3 nM, 30 nM, and 300 nM) was added, followed by the addition of soluble anti-human CD28 antibody (3 μg / mL) (R&D, catalog number MAB342-500). The plate was incubated in an incubator for 48 hours. The culture supernatant was collected, and the IL-2 content was quantified using an IL-2 ELISA kit (Dakewe, catalog number 1110202).

[0254] The results are shown in Figure 32.

[0255] The results show that antibodies TF01A, TF01G, TF01T, TF01t31, and TF01t37 all effectively promoted IL-2 secretion in a co-culture system of Jurkat-TIGIT cells and HT1080-aCD3scFv cells, and their activity was comparable to that of TF01.

[0256] Example 23: Neutralizing effect of anti-TIGIT antibody-TGF-βR fusion protein on TGF-β inhibition of IL-4-induced TF-1 cell proliferation TF-1 cells (purchased from ATCC, catalog number CRL-2003) were harvested, centrifuged at 110 × g for 5 minutes, washed twice with assay medium (1640 + 10% FBS + 2.5 g / L glucose), resuspended, counted, and the cell suspension (2 × 10 4A 96-well round-bottom plate was prepared using IL-4 (final concentration: 0.1 nM) (Peprotech, catalog number 200-04), TGF-β1 (final concentration: 3 ng / mL) (Genscript, catalog number Z03411), TGF-β3 (final concentration: 3 ng / mL) (Peprotech, catalog number 100-36E), and antibodies (final concentrations: 1000 nM, 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM) were diluted according to the experimental design. Equal volumes of antibody and TGF-β1 / 3 were added to each well of an 8-tube strip to design isotype and blank controls. The strip was incubated at room temperature for 30 minutes. The corresponding concentrations of antibodies preincubated with TGF-β1 and TGF-β3 were then added to a 96-well plate at 50 μL / well, followed by 50 μL of 0.4 nM IL-4. After thorough mixing, the plate was incubated at 37°C in a 5% CO2 incubator for 72 hours. After 72 hours, CCK-8 (purchased from Dojindo Laboratories, Japan, catalog number CK04) was added at 20 μL / well, and the plate was incubated at 37°C in a 5% CO2 incubator for 4 hours (a color change was observed during incubation). After thorough mixing, 150 μL of the mixture was transferred to a clean 96-well flat-bottom plate, and the OD at 450 nm was measured using a microplate reader.

[0257] The results are shown in Figures 33 and 34.

[0258] The results show that antibodies TF01G, TF01T, TF01t31, and TF01 all effectively neutralized the inhibition of IL-4-induced TF-1 cell proliferation by TGF-β1 and TGF-β3, and their activities were comparable.

[0259] Example 24: Reporter gene experiments on anti-TIGIT antibody-TGF-βR fusion proteins blocking the interaction between TGF-β1 / 3 and TGF-βR According to the experimental design, antibodies and TGF-β1 / TGF-β3 (prepared at a concentration of 4 ng / mL; final concentration: 1 ng / mL) (TGF-β1 purchased from Genscript, catalog number Z03411; TGF-β3 purchased from Peprotech, catalog number 100-36E) were separately diluted in DMEM + 10% FBS medium. 25 μL of antibody and 25 μL of TGF-β1 / TGF-β3 were added to each well of a 96-well black plate to designate isotype and blank controls. The plate was incubated for 30 minutes in a cell incubator at 37°C and 5% CO2. 293T-TGFβR2-Luc cells (constructed by Akeso Biopharma) were harvested, resuspended in DMEM + 10% FBS medium, counted, and analyzed for viability. 5 × 10 4 50 μL of cells / cell suspension was added to each well of a 96-well plate. The plate was incubated overnight in a cell incubator at 37°C, 5% CO2, and 50 μL of Bright-Glo™ Luciferase Assay Reagent (Promega, Catalog No. E2620) was added. The plate was incubated at room temperature for 30 minutes, and RLU (relative light units) values ​​were read using an Envision multilabel microplate tester.

[0260] The results are shown in Table 22 and Figures 35 and 36.

[0261] [Table 23]

[0262] The results show that antibodies TF01G, TF01T, and TF01t31 all effectively blocked the interaction between TGF-β1 / 3 and TGF-βR2, and their activity was comparable to that of TF01.

[0263] Example 25: Reporter gene experiments on anti-TIGIT antibody-TGF-βR fusion proteins blocking the interaction between TIGIT and PVR CHO-aAPC-PDL1-PVR cells (constructed by Akeso Biopharma) were plated in a 96-well black flat-bottom plate (Corning, model no. 3916) at 2 × 10 4 The cells were seeded at 1 × 10 per well and cultured overnight (100 μL per well) in Ham's F-12 + 10% FBS medium. The next day, the medium was removed from the plate, and Jurkat-TIGIT-NFAT-Luc cells (constructed by Akeso Biopharma) were added at 1 × 10 per well. 5 Cells were added at 50 μL / well (medium: 1640 + 10% FBS). Antibodies (final concentrations: 0.037 nM, 0.37 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM, 300 nM, 1000 nM, or 3000 nM) were added at 30 μL / well, and isotype and negative controls were added in a final volume of 80 μL / well. The plates were incubated in an incubator for 4.5 hours, then removed and equilibrated to room temperature. Bright-Glo™ Luciferase Assay System (Promega, Cat. No. E2650) was added at 80 μL / well. The plates were incubated in the dark for 2 minutes before RLU readings.

[0264] The results are shown in Figure 37.

[0265] The results show that antibodies TF01G, TF01T, and TF01t31 all effectively blocked the interaction between TIGIT and PVR, activated the NFAT pathway in effector cells, and activated the expression of the luciferase reporter gene, and their activities were comparable to those of TF01, 26B12H2L2(hG1WT), and 26B12H2L2(hG4DM).

[0266] Example 26: Molecular weight analysis of anti-TIGIT antibody-TGF-βR fusion protein The antibody was diluted to 1 mg / mL in 50 mM ammonium bicarbonate (pH 7.8). A 50 mL aliquot of the diluted sample was cleaved with 2 μL of PNGase-F (purchased from NEB, catalog number P0705L) for 1 hour. A 10 μL aliquot of the cleaved sample was added to 40 μL of 6 M guanidine hydrochloride (purchased from Sigma, catalog number V900385-500G) and 5 μL of 0.5 M TCEP (tris(2-carboxyethyl)phosphine, purchased from Thermo Fisher, catalog number 77720). After thorough mixing, the mixture was denatured and reduced at room temperature for 30 minutes. Mass spectrometry analysis was performed after C4 column separation.

[0267] The results are shown in FIGS.

[0268] The results show that the heavy chain of antibody TF01 was cleaved, reducing its molecular weight from 65.7 KD to 51.1 KD, while the heavy chain of antibodies TF01A was slightly cleaved and the heavy chains of TF01G, TF01T, TF01t31, and TF01t37 were not cleaved.

[0269] Example 27: SEC-HPLC analysis of anti-TIGIT antibody-TGF-βR fusion protein Protein samples of TF01 and TF01T were analyzed by SEC-HPLC using a UV detector (detection wavelength: 280 nm; mobile phase: 25 mM disodium hydrogen phosphate dodecahydrate, 25 mM anhydrous sodium dihydrogen phosphate, 300 mM sodium chloride, pH 6.5; flow rate: 0.8 mL / min; injection volume: 100 μg; run time: 20 min isocratic elution).

[0270] The results are shown in Table 23 and Figures 44 and 45.

[0271] [Table 24]

[0272] These results show that TFO1 exhibited significant peaks characteristic of degradation fragments, whereas the engineered mutant TFO1T overcame the degradation problem, i.e., the peaks characteristic of degradation fragments disappeared and the purity of the main HPLC peak was significantly increased.

[0273] Example 28: Quantification of binding activity of anti-TIGIT antibody-TGF-βR fusion protein to TGF-β by ELISA ELISA plates were coated with 1 μg / mL TGF-β1 and incubated overnight at 4°C. The antigen-coated ELISA plates were then washed once with PBST and blocked with PBS containing 1% BSA (blocking solution) at 37°C for 2 hours. After blocking, the ELISA plates were washed three times with PBST. Serially diluted antibodies and TGF-βRII-mFc in PBST were added. The ELISA plates containing the test antibodies and TGF-βRII-mFc were incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, a working solution of HRP-labeled goat anti-human IgG (H+L) (Jackson, Cat. No. 109-035-098) and HRP-labeled goat anti-mouse IgG Fc (Jackson, Cat. No. 115-035-071) secondary antibodies diluted 1:5000 was added, and the plates were then incubated at 37°C for 30 minutes. After incubation, the plate was washed four times with PBST. Then, color development was performed using TMB (Neogen, 308177) in the dark for 5 minutes, and the color reaction was stopped by adding stop solution. The ELISA plate was immediately placed in a microplate reader, and the OD of each well of the ELISA plate was measured at a wavelength of 450 nm. Data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0274] The results are shown in the table.

[0275] [Table 25]

[0276] Quantitative absorbance analysis of bound antibodies, curve fitting, and calculations determined the binding efficiencies EC of TF01T, TF01, and TGF-βRII-mFc (as a positive control) for TGF-β1. 50 The values ​​obtained were 0.100 nM, 0.164 nM, and 0.271 nM, respectively.

[0277] The results are shown in Figure 46.

[0278] The results show that TFO1T and TFO1 had effective binding activity to TGF-β1 in a dose-dependent manner. The results further show that TFO1T, TFO1, and TGF-βRII-mFc all effectively bound to TGF-β1, and the binding ability of TFO1T and TFO1 to TGF-β1 was higher than that of TGF-βRII-mFc.

[0279] Example 29: Quantification of the biological activity of anti-TIGIT antibody-TGF-βR fusion proteins to stimulate IFN-γ secretion using a mixed lymphocyte reaction Normal human PBMCs (from healthy donors) were obtained by isolation using Ficoll-Paque™ Plus isolation solution according to the manufacturer's instructions and cryopreserved. After thawing and overnight culture, the suspended cells were removed and adherent cells were cultured in 1640 complete medium supplemented with 2000 U / mL GM-CSF (Peprotech, catalog number 300-03) and 2000 U / mL IL-4 (Peprotech, catalog number 200-04) at 37°C in a 5% CO2 incubator for 3 days. A half-medium change was performed, and 2000 U / mL GM-CSF, 50 ng / mL IFN-γ (Sinobiological, catalog number 11725-HNAS-100), and 100 ng / mL LPS (Sigma, catalog number L4391) were added. The mixture was incubated for 2 days in a 5% carbon dioxide incubator at 37° C. After 2 days, the induced DC cells were routinely collected and cryopreserved.

[0280] Two days before the experiment, PBMCs were thawed, and after 2 hours, SEB (Staphylococcus aureus enterotoxin antigen, purchased from Toxin Technology, catalog number BT202) (final concentration: 0.1 μg / mL) was added, and the cells were stimulated and induced for 2 days. On the day of the experiment, SEB-stimulated PBMCs were routinely collected, centrifuged at 250 × g for 5 minutes, and the supernatant was removed. The cells were washed once with assay medium (i.e., RPMI1640 + 10% FBS), then resuspended and counted. On the day of the experiment, DC cells (derived from PBMCs of a healthy donor) were thawed 2 hours before and cultured for 2 hours in an incubator at 37°C and 5% carbon dioxide. DCs were collected, centrifuged at 250 × g for 5 minutes, and the supernatant was removed. The cells were resuspended in assay medium and counted. DC cells (1 × 10 4 / well), PBMCs (1 × 10 5 / well), and A549 human lung cancer cells (purchased from the Cell Resource Center of Shanghai Institutes for Life Sciences, Chinese Academy of Sciences, catalog number SCSP-503) (2 × 10 4 40 μL / well of TGF-β1 (final concentration: 3 ng / mL) and antibody (final concentration: 3 nM, 30 nM, or 300 nM) were plated into a 96-well flat-bottom plate. According to the experimental design, TGF-β1 (final concentration: 3 ng / mL) and antibody (final concentration: 3 nM, 30 nM, or 300 nM) were added to the corresponding experimental group wells at 40 μL / well. After thorough mixing, the plate was incubated in an incubator for 5 days. After 5 days, the supernatant was collected and the IFN-γ content was quantified by ELISA.

[0281] The results are shown in Figure 47.

[0282] The results show that, compared with the isotype control B12-hG1, both antibodies TF01T and 26B12H2L2(hG1WT) induced PBMCs to secrete more IFN-γ and promoted IFN-γ secretion, with the activity of TF01T being greater than that of the anti-TIGIT monoclonal antibody 26B12H2L2(hG1WT).

[0283] Although the specific embodiments of the present disclosure have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to these details according to all the teachings disclosed, and all of these changes fall within the scope of protection of the present disclosure. The full scope of the present disclosure is provided by the appended claims and any equivalents thereof.

Claims

1. a first protein functional region that targets an immune checkpoint; and Second protein functional domain having TGF-β binding activity A fusion protein comprising: the second protein functional domain is a variant of a TGF-βRII extracellular domain fragment, and the variant of the TGF-βRII extracellular domain fragment comprises: A fusion protein in which the first serine at the N-terminus of the TGF-βRII extracellular region fragment is replaced with alanine, glycine, or threonine, and / or a fragment comprising the amino acid sequence set forth in SEQ ID NO:59 is deleted from the TGF-βRII extracellular region fragment.

2. The fusion protein of claim 1, wherein the amino acid sequence of the TGF-βRII extracellular region fragment is set forth in SEQ ID NO: 33 or SEQ ID NO:

37.

3. The fusion protein according to claim 1 or 2, wherein the amino acid sequence of the deletion fragment comprising the amino acid sequence set forth in SEQ ID NO: 59 is set forth in any one of SEQ ID NOs: 59 to 65.

4. The fusion protein according to any one of claims 1 to 3, wherein the amino acid sequence of the variant of the TGF-βRII extracellular region fragment is set forth in any one of SEQ ID NOs: 49 to 58.

5. The fusion protein of any one of claims 1 to 4, wherein the immune checkpoint is selected from one or more of PD-1, PD-L1, CTLA-4, LAG3, and TIGIT.

6. The fusion protein according to any one of claims 1 to 5, wherein the first protein functional domain is an anti-TIGIT antibody or an antigen-binding fragment thereof.

7. the anti-TIGIT antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 and a light chain variable region comprising LCDR1 to LCDR3; The amino acid sequence of HCDR1 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:5; The fusion protein of claim 6, wherein the amino acid sequence of LCDR1 is set forth in SEQ ID NO: 8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO: 9, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:

10.

8. 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; The fusion protein of claim 7, wherein 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.

9. the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 6; the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 19; the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 19; the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 21; the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 23; the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 21; the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 23; the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 11 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 25; or the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is set forth in SEQ ID NO: 25; A fusion protein according to claim 7 or 8.

10. The fusion protein of any one of claims 7 to 9, wherein the anti-TIGIT antibody, or the antigen-binding fragment thereof, is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, a complementarity-determining region fragment, a single-chain variable fragment, a humanized antibody, a chimeric antibody, and a diabody.

11. The fusion protein of any one of claims 7 to 10, wherein the anti-TIGIT antibody comprises non-CDR regions derived from a non-mouse species, such as a human antibody.

12. The fusion protein according to any one of claims 7 to 11, wherein the heavy chain constant region of the anti-TIGIT antibody is an Ig gamma 1 chain C region or an Ig gamma 4 chain C region, and the light chain constant region is an Ig kappa chain C region.

13. The fusion protein according to any one of claims 7 to 12, which has stronger binding ability to the ligand CD155-hFc-biotin than the control antibody RG6058 (hG4).

14. Preferably, EC is measured by indirect ELISA. ５０ and measuring an EC ５０ The fusion protein according to any one of claims 7 to 13, which binds to TIGIT-mFc at the nucleotide sequence nucleotide 111.

15. Preferably, EC is measured by indirect ELISA. ５０ and measuring an EC50 of less than 0.3 nM, less than 0.4 nM, less than 0.5 nM, or less than 0.6 nM. ５０ The fusion protein according to any one of claims 7 to 14, which binds to TGF-β1, TGF-β2, or TGF-β3 at the nucleotide sequence nucleotide 1 ...

16. The fusion protein according to any one of claims 7 to 15, wherein the anti-TIGIT antibody is an antibody produced by hybridoma cell line LT019, deposited at the China Center for Typical Culture Collection (CCTCC) under CCTCC number C2020208.

17. According to the EU numbering system, When the heavy chain constant region of the anti-TIGIT antibody is IgG1, the heavy chain constant region may have any of the following mutation combinations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A having one of When the heavy chain constant region of the anti-TIGIT antibody is IgG4, the heavy chain constant region may have any of the following mutation combinations: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A, and G237A having one of The fusion protein according to any one of claims 7 to 16.

18. The fusion protein according to any one of claims 7 to 17, wherein the amino acid sequence of the heavy chain of the anti-TIGIT antibody is set forth in SEQ ID NO: 27 or SEQ ID NO: 31, and the amino acid sequence of the light chain of the anti-TIGIT antibody is set forth in SEQ ID NO:

29.

19. The fusion protein according to any one of claims 1 to 18, wherein the first protein functional domain and the second protein functional domain are linked directly or via a linker fragment.

20. The linker fragment is a polypeptide set forth in SEQ ID NO: 69 or SEQ ID NO: 70, or a polypeptide obtained by linking a plurality of (e.g., 2, 3, 4, 5, or 6) polypeptides set forth in SEQ ID NO: 69, or a polypeptide obtained by linking a plurality of (e.g., 2, 3, 4, or 5) polypeptides set forth in SEQ ID NO: 69 and further linking the linked polypeptides with a polypeptide set forth in SEQ ID NO:

70.

20. The fusion protein of claim 19, wherein the amino acid sequence of the linker fragment is set forth in SEQ ID NO: 44 or SEQ ID NO:

66.

21. 21. The fusion protein of any one of claims 1 to 20, wherein the first protein functional domain and the second protein functional domain are independently one, two or more in number.

22. The fusion protein of any one of claims 1 to 21, wherein the second protein functional region is linked to the C-terminus of the first protein functional region.

23. The fusion protein of any one of claims 7 to 22, wherein the second protein functional region is linked to the C-terminus of the heavy chain of the anti-TIGIT antibody.

24. a first protein functional region that targets TIGIT; and Second protein functional domain having TGF-β binding activity A fusion protein comprising: the number of said first protein functional domains is one and the number of said second protein functional domains is two; the first protein functional region is an anti-TIGIT antibody or an antigen-binding fragment thereof, and the second protein functional region is a variant of a TGF-βRII extracellular domain fragment; the amino acid sequence of the heavy chain of the anti-TIGIT antibody is set forth in SEQ ID NO:27 or SEQ ID NO:31, and the amino acid sequence of the light chain of the anti-TIGIT antibody is set forth in SEQ ID NO:29; the amino acid sequence of the variant of the TGF-βRII extracellular domain fragment is set forth in any one of SEQ ID NOs: 49 to 58; the second protein functional region is linked to the C-terminus of the heavy chain of the anti-TIGIT antibody by a linker fragment; Preferably, the amino acid sequence of the linker fragment is set forth in SEQ ID NO:44 or SEQ ID NO:

66.

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

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

27. 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 which is a fusion protein according to any one of claims 1 to 24 and a conjugated portion which is a detectable label, preferably a radioisotope, a fluorescent substance, a colored substance or an enzyme.

29. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 24 or the conjugate of claim 28, optionally further comprising one or more pharmaceutically acceptable auxiliary materials, Optionally, the pharmaceutical composition further comprises one or more anti-tumor chemotherapeutic agents.

30. 30. The pharmaceutical composition of claim 29, wherein the unit dose of the pharmaceutical composition is 100 mg to 1500 mg, 200 mg to 1000 mg, 200 mg to 800 mg, 300 mg to 600 mg, 400 mg to 500 mg, or 450 mg, calculated based on the mass of the fusion protein in the pharmaceutical composition.

31. Use of a fusion protein according to any one of claims 1 to 24 or a conjugate according to claim 28 in the preparation of a medicament for the treatment and / or prevention of tumors, comprising: Preferably, the tumor is selected from one or more of 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 lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma; Preferably, the non-small cell lung cancer is advanced non-small cell lung cancer.

32. Preferably, the tumor is selected from one or more of 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 lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma; The fusion protein of any one of claims 1 to 24 or the conjugate of claim 28 for use in the treatment and / or prevention of tumors, preferably wherein said non-small cell lung cancer is advanced non-small cell lung cancer.

33. A method for treating or preventing a tumor, comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1 to 24 or the conjugate of claim 28, Preferably, the tumor is selected from one or more of 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 lymphoma, plasma cell carcinoma, renal cell carcinoma, prostate cancer, and pancreatic ductal adenocarcinoma; Preferably, the non-small cell lung cancer is advanced non-small cell lung cancer.

34. A variant of the TGF-βRII extracellular domain fragment, the first serine at the N-terminus of the TGF-βRII extracellular domain fragment is substituted with alanine, glycine, or threonine; or a fragment comprising the amino acid sequence set forth in SEQ ID NO:59 is deleted from the TGF-βRII extracellular domain fragment; Preferably, the amino acid sequence of the TGF-βRII extracellular region fragment is set forth in SEQ ID NO: 33 or SEQ ID NO:

37.

35. A variant of the TGF-βRII extracellular region fragment according to claim 34, wherein the amino acid sequence of the deletion fragment comprising the amino acid sequence set forth in SEQ ID NO: 59 is set forth in any one of SEQ ID NOs: 59 to 65.

36. The variant of the TGF-βRII extracellular region fragment according to claim 34 or 35, wherein the amino acid sequence of the variant of the TGF-βRII extracellular region fragment is set forth in any one of SEQ ID NOs: 49 to 58.

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