TIGIT antibody and preparation method thereof

Abstract

The invention provides a TIGIT antibody and a preparation method thereof. Specifically, the invention develops a novel chimeric antibody or a humanized cloned antibody targeting the TIGIT. The invention also discloses a method for preparing the monoclonal antibody. The monoclonal antibody disclosed by the invention can be combined with the TIGIT protein with high specificity, block the combination of the TIGIT protein and a ligand of the TIGIT protein, and can promote T cells to secrete IFN-gamma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a TIGIT antibody and a preparation method and application thereof. Background Art

[0002] The application of monoclonal antibodies is one of the most successful and revolutionary treatments for tumors in the past 20 years. Compared with traditional chemical drugs, antibody drugs have higher specificity and lower toxicity. Although monoclonal antibody drugs have achieved continuous success, they still face many challenges.

[0003] Immune checkpoints refer to some inhibitory signaling pathways in the immune system that prevent tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and participate in maintaining tolerance to self-antigens. Using the inhibitory signaling pathways of immune checkpoints to inhibit T cell activity is an important mechanism for tumors to evade immune killing. Blocking immune checkpoints is one of the many effective strategies for activating anti-tumor immunity.

[0004] Inhibitors of immune checkpoint proteins have the potential to treat various tumor types, such as metastatic melanoma, lung cancer, breast cancer, renal cell carcinoma, etc. Recent studies of cancer immunotherapy approaches have shown promising results, especially for metastatic cancer cases. In addition, cancer immunotherapy has great potential in the treatment of blood cancers, including Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, etc. The side effects caused by immune checkpoint inhibitors are negligible, reversible and controllable, and effective immune checkpoint inhibitors can significantly improve the overall survival of cancer patients. Immune checkpoint inhibitors can be used in combination with targeted therapy or conventional radiotherapy and chemotherapy, and this combination therapy is effective in treating many types of cancer.

[0005] TIGIT (Ig and ITIM domain T cell immunoreceptor), also known as VSVG3 or VSVG9 (V-Set and immunoglobulin domain protein 3 or 9), is a type I transmembrane protein that is one of the main immune checkpoint molecules. It contains an extracellular Ig-V-like domain, a transmembrane domain and an intracellular C-terminal domain. The intracellular C-terminal domain consists of an ITIM (immunoreceptor tyrosine-based inhibitory motif) and an ITT (immunoglobulin tail tyrosine-based) similar motif, both of which are highly conserved between humans and mice. TIGIT is expressed in activated T cells, as well as in NK cells, memory T cells, some Treg cells and Tfh (follicular helper T). There are currently two main ligands reported to bind to TIGIT, CD155 and CD112, which are expressed in APCs, T cells and various non-hematopoietic cells, especially overexpressed in various tumor cells, such as melanoma cells. TIGIT has a higher affinity for binding to CD155 than CD112. After TIGIT binds to its ligand, it can induce inhibitory signals in the downstream cells through the ITIM (immunoreceptor tyrosine inhibitory motif) at the intracellular end of TIGIT, inhibiting the activation and proliferation of T cells and NK cells, while reducing the production of cytokines. In addition, TIGTI is constitutively highly expressed on the surface of Treg cells, participating in the downregulation of the activity of Treg on other effector T cells, such as Th1 and Th17 cells.

[0006] In addition to their inhibitory receptor TIGIT, CD155 and CD112 also have an activating receptor, CD226. Similar to TIGIT, CD226 also activates downstream signaling pathways by binding to ligands CD155 or CD112. The difference is that CD226 transmits positive co-stimulatory signals, while TIGIT transmits inhibitory signals. Therefore, the immunosuppression mechanism of TIGIT is relatively complex. On the one hand, TIGIT inhibits the positive co-stimulatory signals downstream of CD226 by competing with CD226 for ligands. On the other hand, as mentioned above, TIGIT itself inhibits the TCR signaling pathway to regulate the intensity of T cell response to antigens. In addition, Treg downregulates the activity of other effector T cells, thereby making the body show tolerance to self-antigens or exogenous weak antigen recognition, which ultimately leads to a decrease in the body's ability to fight tumors.

[0007] Like several other typical immune checkpoint proteins (such as CTLA-4, PD-1), studies have shown that using anti-TIGIT antibodies to specifically block the interaction between TIGIT / CD155 and CD112 ligands can improve the body's recognition of tumor antigens and stimulate the proliferation of antigen-specific T cells, thereby activating the immune system and enhancing anti-tumor immune responses. It has been proven in multiple syngeneic mouse tumor models that TIGIT blockade promotes anti-tumor activity. Therefore, drug blockade of the TIGIT / CD155 and CD112 pathways can provide new treatments for various cancers and other immune diseases.

[0008] At present, the existing TIGIT antibodies in this field still have many shortcomings, such as a small therapeutic window, limited indications, and high treatment costs. Therefore, there is an urgent need to develop a TIGIT blocking antibody to treat various cancers. Summary of the invention

[0009] The object of the present invention is to provide a TIGIT antibody and a method for preparing the same.

[0010] In a first aspect of the present invention, a heavy chain variable region of an antibody is provided, wherein the heavy chain variable region has a complementary determining region CDR selected from the following group:

[0011] (1) VH-CDR1 shown in SEQ ID NO.1,

[0012] VH-CDR2 shown in SEQ ID NO.2 or SEQ ID NO.25, and

[0013] VH-CDR3 shown in SEQ ID NO.3; or

[0014] (2) VH-CDR1 shown in SEQ ID NO.1,

[0015] VH-CDR2 shown in SEQ ID NO.7 or SEQ ID NO.26, and

[0016] VH-CDR3 shown in SEQ ID NO.8;

[0017] Among them, any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain TIGIT binding affinity.

[0018] In another preferred embodiment, the heavy chain variable region has a complementarity determining region CDR selected from the following group:

[0019] (1) VH-CDR1 shown in SEQ ID NO.1,

[0020] VH-CDR2 shown in SEQ ID NO.2, and

[0021] VH-CDR3 shown in SEQ ID NO.3; or

[0022] VH-CDR1 shown in SEQ ID NO.1,

[0023] VH-CDR2 shown in SEQ ID NO.25, and

[0024] VH-CDR3 shown in SEQ ID NO.3; or

[0025] (2) VH-CDR1 shown in SEQ ID NO.1,

[0026] VH-CDR2 shown in SEQ ID NO.7, and

[0027] VH-CDR3 shown in SEQ ID NO.8; or

[0028] VH-CDR1 shown in SEQ ID NO.1,

[0029] VH-CDR2 shown in SEQ ID NO.26, and

[0030] VH-CDR3 shown in SEQ ID NO.8.

[0031] In another preferred example, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO.10, 12, 27 or 28.

[0032] In another preferred example, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 29, 31, 32, or 33.

[0033] In the second aspect of the present invention, a heavy chain of an antibody is provided, wherein the heavy chain has the heavy chain variable region as described in the first aspect of the present invention.

[0034] In another preferred embodiment, the heavy chain also includes a heavy chain constant region.

[0035] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0036] In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region.

[0037] In another preferred embodiment, the heavy chain of the antibody is shown in SEQ ID NO.19 or 21.

[0038] In a third aspect of the present invention, there is provided a light chain variable region of an antibody, wherein the light chain variable region has a complementary determining region CDR selected from the following group:

[0039] (1) VL-CDR1 shown in SEQ ID NO.4,

[0040] VL-CDR2 shown in SEQ ID NO.5, and

[0041] VL-CDR3 shown in SEQ ID NO.6; or

[0042] (2) VL-CDR1 shown in SEQ ID NO.4,

[0043] VL-CDR2 shown in SEQ ID NO.5, and

[0044] VL-CDR3 shown in SEQ ID NO.9;

[0045] Among them, any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain TIGIT binding affinity.

[0046] In another preferred embodiment, the light chain variable region has a complementarity determining region CDR selected from the following group:

[0047] VL-CDR1 shown in SEQ ID NO.4,

[0048] VL-CDR2 shown in SEQ ID NO.5, and

[0049] VL-CDR3 shown in SEQ ID NO.6; or

[0050] VL-CDR1 shown in SEQ ID NO.4,

[0051] VL-CDR2 shown in SEQ ID NO.5, and

[0052] VL-CDR3 shown in SEQ ID NO.9.

[0053] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.11 or 13.

[0054] In another preferred example, the light chain variable region has an amino acid sequence shown in SEQ ID NO.30, 34, 35 or 36.

[0055] The fourth aspect of the present invention provides a light chain of an antibody, wherein the light chain has the light chain variable region as described in the third aspect of the present invention.

[0056] In another preferred embodiment, the light chain also includes a light chain constant region.

[0057] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0058] In another preferred embodiment, the light chain of the antibody is shown as SEQ ID NO. 20 or 22.

[0059] In a fifth aspect of the present invention, an antibody is provided, wherein the antibody has:

[0060] (1) the heavy chain variable region according to the first aspect of the present invention; and / or

[0061] (2) the light chain variable region as described in the third aspect of the present invention;

[0062] Alternatively, the antibody has: the heavy chain as described in the second aspect of the present invention; and / or the light chain as described in the fourth aspect of the present invention,

[0063] Among them, any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain TIGIT binding affinity.

[0064] In another preferred embodiment, the amino acid sequence of any of the above-mentioned CDRs contains a derivative CDR sequence in which 1, 2 or 3 amino acids have been added, deleted, modified and / or substituted, and the derivative antibody composed of VH and VL containing the derivative CDR sequence can retain the affinity for binding to TIGIT.

[0065] In another preferred embodiment, the ratio of the affinity F1 of the derived antibody for TIGIT to the affinity F0 of the corresponding non-derivatized antibody for TIGIT (F1 / F0) is 0.5-2, preferably 0.7-1.5, and more preferably 0.8-1.2.

[0066] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-5 (such as 1-3, preferably 1-2, more preferably 1).

[0067] In another preferred embodiment, the derivative sequence that has been subjected to addition, deletion, modification and / or substitution of at least one amino acid and is able to retain TIGIT binding affinity is an amino acid sequence with a homology or sequence identity of at least 96%.

[0068] In another preferred embodiment, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0069] In another preferred embodiment, the heavy chain constant region is of human origin, and / or the light chain constant region is of human origin.

[0070] In another preferred example, the heavy chain variable region of the antibody further includes a human framework region, and / or the light chain variable region of the antibody further includes a human framework region.

[0071] In another preferred example, the heavy chain variable region of the antibody further includes a framework region of mouse origin, and / or the light chain variable region of the antibody further includes a framework region of mouse origin.

[0072] In another preferred embodiment, the antibody is selected from the following group: animal-derived antibody, chimeric antibody, humanized antibody, fully human antibody, or a combination thereof.

[0073] In another preferred embodiment, the ratio of the immunogenicity Z1 of the chimeric antibody in humans to the immunogenicity Z0 of the non-chimeric antibody (such as mouse antibody) in humans (Z1 / Z0) is 0-0.5, preferably 0-0.2, and more preferably 0-0.05 (such as 0.001-0.05).

[0074] In another preferred embodiment, the antibody is a partially or fully humanized, or fully human monoclonal antibody.

[0075] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0076] In another preferred embodiment, the antibody is a blocking antibody or a non-blocking antibody, preferably a blocking antibody.

[0077] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0078] In another preferred embodiment, the antibody is a bispecific antibody or a multispecific antibody.

[0079] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0080] In another preferred embodiment, the antibody has one or more characteristics selected from the following group:

[0081] (a) Promote NK cell cytotoxicity;

[0082] (b) blocking the binding of TIGIT protein to its ligand CD155;

[0083] (c) Inhibit tumor growth.

[0084] In another preferred embodiment, the antibody has a heavy chain variable region as described in the first aspect of the present invention; and a light chain variable region as described in the third aspect of the present invention; wherein the heavy chain variable region comprises a CDR selected from the following group:

[0085] (1) VH-CDR1 shown in SEQ ID NO.1,

[0086] VH-CDR2 shown in SEQ ID NO.2, and

[0087] VH-CDR3 shown in SEQ ID NO.3; and

[0088] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0089] VL-CDR1 shown in SEQ ID NO.4,

[0090] VL-CDR2 shown in SEQ ID NO.5, and

[0091] VL-CDR3 shown in SEQ ID NO.6; or

[0092] VH-CDR1 shown in SEQ ID NO.1,

[0093] VH-CDR2 shown in SEQ ID NO.25, and

[0094] VH-CDR3 shown in SEQ ID NO.3; and

[0095] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0096] VL-CDR1 shown in SEQ ID NO.4,

[0097] VL-CDR2 shown in SEQ ID NO.5, and

[0098] VL-CDR3 shown in SEQ ID NO.6; or

[0099] (2) The heavy chain variable region comprises a CDR selected from the following group:

[0100] VH-CDR1 shown in SEQ ID NO.1,

[0101] VH-CDR2 shown in SEQ ID NO.7, and

[0102] VH-CDR3 shown in SEQ ID NO.8; and

[0103] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0104] VL-CDR1 shown in SEQ ID NO.4,

[0105] VL-CDR2 shown in SEQ ID NO.5, and

[0106] VL-CDR3 shown in SEQ ID NO.9; or

[0107] The heavy chain variable region comprises a CDR selected from the group consisting of:

[0108] VH-CDR1 shown in SEQ ID NO.1,

[0109] VH-CDR2 shown in SEQ ID NO.26, and

[0110] VH-CDR3 shown in SEQ ID NO.8; and

[0111] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0112] VL-CDR1 shown in SEQ ID NO.4,

[0113] VL-CDR2 shown in SEQ ID NO.5, and

[0114] VL-CDR3 shown in SEQ ID NO.9.

[0115] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.10, 27, or 33; and / or the light chain variable region has the amino acid sequence shown in SEQ ID NO.11, 34, 35 or 36.

[0116] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.12, 28, 29, 31, 32; and / or the light chain variable region has the amino acid sequence shown in SEQ ID NO.13 or 30.

[0117] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.10; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.11.

[0118] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.33; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.34.

[0119] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.33; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.35.

[0120] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.33; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.36.

[0121] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.27; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.11.

[0122] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.12; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.13.

[0123] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.28; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.13.

[0124] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.29; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.30.

[0125] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.31; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.30.

[0126] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.32; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.30.

[0127] In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO.10, 27, or 33 in the sequence listing.

[0128] In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequences shown in SEQ ID NO.12, 28, 29, 31, and 32 in the sequence listing.

[0129] A sixth aspect of the present invention provides a recombinant protein, wherein the recombinant protein comprises:

[0130] (i) the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention; and

[0131] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0132] The seventh aspect of the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:

[0133] (1) the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention; and

[0134] (2) The recombinant protein according to the sixth aspect of the present invention.

[0135] The eighth aspect of the present invention provides a vector, wherein the vector contains the polynucleotide described in the seventh aspect of the present invention.

[0136] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0137] The ninth aspect of the present invention provides a genetically engineered host cell, wherein the host cell contains the vector described in the eighth aspect of the present invention or the polynucleotide described in the seventh aspect of the present invention is integrated into its genome.

[0138] The tenth aspect of the present invention provides an antibody conjugate, the antibody conjugate comprising:

[0139] (a) an antibody portion, the antibody portion being selected from the group consisting of a heavy chain variable region as described in the first aspect of the invention, a heavy chain as described in the second aspect of the invention, a light chain variable region as described in the third aspect of the invention, a light chain as described in the fourth aspect of the invention, or an antibody as described in the fifth aspect of the invention, or a combination thereof; and

[0140] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0141] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0142] In an eleventh aspect of the present invention, a CAR construct is provided, wherein the scFv segment of the monoclonal antibody antigen binding region of the CAR construct is a binding region that specifically binds to TIGIT,

[0143] Furthermore, the heavy chain variable region of the scFv comprises a CDR selected from the following group:

[0144] VH-CDR1 shown in SEQ ID NO.1,

[0145] VH-CDR2 shown in SEQ ID NO.2, and

[0146] VH-CDR3 shown in SEQ ID NO.3; and

[0147] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0148] VL-CDR1 shown in SEQ ID NO.4,

[0149] VL-CDR2 shown in SEQ ID NO.5, and

[0150] VL-CDR3 shown in SEQ ID NO.6;

[0151] or

[0152] The heavy chain variable region comprises a CDR selected from the group consisting of:

[0153] VH-CDR1 shown in SEQ ID NO.1,

[0154] VH-CDR2 shown in SEQ ID NO.7, and

[0155] VH-CDR3 shown in SEQ ID NO.8; and

[0156] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0157] VL-CDR1 shown in SEQ ID NO.4,

[0158] VL-CDR2 shown in SEQ ID NO.5, and

[0159] VL-CDR3 shown in SEQ ID NO.9.

[0160] The twelfth aspect of the present invention provides a recombinant immune cell, wherein the immune cell expresses an exogenous CAR construct as described in the eleventh aspect of the present invention.

[0161] In another preferred embodiment, the immune cells include NK cells and T cells.

[0162] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0163] The thirteenth aspect of the present invention provides a pharmaceutical composition, which contains:

[0164] (i) an active ingredient, wherein the active ingredient is selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the antibody conjugate as described in the tenth aspect of the present invention, the immune cell as described in the twelfth aspect of the present invention, or a combination thereof; and

[0165] (ii) a pharmaceutically acceptable carrier.

[0166] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0167] In another preferred embodiment, the pharmaceutical composition is an injection.

[0168] The fourteenth aspect of the present invention provides a use of an active ingredient, wherein the active ingredient is selected from the following group: the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the antibody conjugate as described in the tenth aspect of the present invention, the immune cell as described in the twelfth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a diagnostic reagent or kit; and / or (b) preparing a drug for preventing and / or treating diseases related to abnormal TIGIT expression or function.

[0169] In another preferred embodiment, the diagnostic reagent is a test piece or a test plate.

[0170] In another preferred embodiment, the disease associated with abnormal TIGIT expression or function is selected from the following group: cancer, tumor, and infectious disease.

[0171] In another preferred embodiment, the diagnostic reagent or kit is used for:

[0172] (1) detecting TIGIT protein in a sample; and / or

[0173] (2) detecting endogenous TIGIT protein in tumor cells; and / or

[0174] (3) Detect tumor cells expressing TIGIT protein;

[0175] The drug is used to prevent and / or treat diseases related to abnormal expression or function of TIGIT, and the diseases related to abnormal expression or function of TIGIT are cancer, tumor, and infectious diseases.

[0176] In another preferred embodiment, the tumor is selected from the group consisting of germ cell tumor, lymphoma, hematologic tumor, sarcoma, melanoma, lung cancer, breast cancer, and renal cell carcinoma.

[0177] In another preferred embodiment, the blood tumor is selected from the following group: Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma.

[0178] In another preferred embodiment, the antibody is in the form of an antibody-drug conjugate (ADC).

[0179] In another preferred embodiment, the diagnostic reagent or kit is used to diagnose TIGIT-related diseases.

[0180] In another preferred embodiment, the diagnostic reagent or kit is used to detect TIGIT protein in a sample.

[0181] In a fifteenth aspect of the present invention, a method for in vitro detection (including diagnostic or non-diagnostic) of TIGIT protein in a sample is provided, the method comprising the steps of:

[0182] (1) contacting the sample with the antibody according to the fifth aspect of the present invention in vitro;

[0183] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of TIGIT protein in the sample.

[0184] A sixteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

[0185] (a) culturing the host cell according to the ninth aspect of the present invention under conditions suitable for expression;

[0186] (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.

[0187] The seventeenth aspect of the present invention provides a drug combination comprising:

[0188] (i) a first active ingredient, the first active ingredient comprising the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the antibody conjugate according to the tenth aspect of the present invention, the immune cell according to the twelfth aspect of the present invention, or the pharmaceutical composition according to the thirteenth aspect of the present invention, or a combination thereof;

[0189] (ii) a second active ingredient, which comprises a second antibody, or a chemotherapeutic agent.

[0190] In another preferred embodiment, the second antibody is selected from the following group: CTLA4 antibody, PD-1 antibody, PD-L1 antibody.

[0191] In another preferred embodiment, the second antibody is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, lambrolizumab and atezolizumab.

[0192] In another preferred embodiment, the chemotherapeutic agent is selected from the following group: docetaxel, carboplatin, or a combination thereof.

[0193] The eighteenth aspect of the present invention provides the use of the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the antibody conjugate as described in the tenth aspect of the present invention, the immune cell as described in the twelfth aspect of the present invention, or the pharmaceutical composition as described in the thirteenth aspect of the present invention in combination with a second antibody or a chemotherapeutic agent in the preparation of a medicament for treating diseases related to abnormal TIGIT expression or function.

[0194] In another preferred embodiment, the second antibody is selected from the following group: CTLA4 antibody, PD-1 antibody, PD-L1 antibody.

[0195] The nineteenth aspect of the present invention provides a method for treating diseases related to abnormal TIGIT expression or function, by administering to a subject in need thereof an effective amount of the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the antibody conjugate as described in the tenth aspect of the present invention, the immune cell as described in the twelfth aspect of the present invention, or the pharmaceutical composition as described in the thirteenth aspect of the present invention, or a combination thereof.

[0196] In another preferred embodiment, the disease associated with abnormal TIGIT expression or function is cancer, tumor, or infectious disease.

[0197] In another preferred embodiment, the tumor is selected from the group consisting of germ cell tumor, lymphoma, hematologic tumor, sarcoma, melanoma, lung cancer, breast cancer, and renal cell carcinoma.

[0198] In another preferred embodiment, the blood tumor is selected from the following group: Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma.

[0199] In another preferred embodiment, the method further comprises administering to a subject in need thereof an inhibitor of one or more immune checkpoint receptors or ligands.

[0200] In another preferred embodiment, the one or more immune checkpoint receptors or ligands are selected from the following group: CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, BTLA, VISTA, CD96, A2aR, A2bR, CD39, CD73, IDO and TDO.

[0201] In another preferred embodiment, the inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, lambrolizumab and atezolizumab.

[0202] In another preferred embodiment, the method further comprises: administering a safe and effective amount of a second antibody to the subject before, during and / or after administering the first active ingredient.

[0203] In another preferred embodiment, the second antibody is selected from the following group: PD-1 antibody, CTLA4 antibody.

[0204] In another preferred embodiment, the second antibody is a PD-1 antibody.

[0205] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0206] Figure 1 The binding activity of TIGIT-hFc protein to biotin-labeled ligands CD155-hFc and CD112-hFc is shown.

[0207] Figure 2 Shown are the results of flow cytometry (FACS) detection of HEK293 cells transfected with TIGIT protein.

[0208] Figure 3The results of flow cytometry (FACS) of HEK293 cells transiently transfected with pCP-hTIGIT plasmid are shown. Purple is the blank control cell line that is not transfected with hTIGIT, and green is the HEK293 cells transiently transfected with hTIGIT. The figure shows that 78.1% of HEK293 cells highly express TIGIT.

[0209] Figure 4 The figure shows the enzyme-linked immunosorbent assay (ELISA) to detect the antibody titer in the mouse serum after immunization with TIGIT-hFC protein.

[0210] Figure 5 The reaction activity of the chimeric antibody TIGIT with the human TIGIT extracellular domain protein in the enzyme-linked immunosorbent assay was shown.

[0211] Figure 6 Flow cytometry (FACS) assay was performed to detect the binding of chimeric antibodies to human TIGIT-expressing cells.

[0212] Figure 7 Flow cytometry (FACS) assay was performed to detect the binding of the chimeric antibody to monkey TIGIT-expressing cells.

[0213] Figure 8 The inhibition of the binding of TIGIT protein to its receptor CD155 by the chimeric antibody TIGIT antibody is shown.

[0214] Fig. 9 The chimeric antibody TIGIT promoted the expression of NFAT downstream reporter genes when Jurkat-NFAT / hTIGIT and 293F-hCD155 cells were co-cultured.

[0215] Fig.10 It was shown that the chimeric antibody TIGIT antibody promoted the expression of NFAT downstream reporter genes when Jurkat-NFAT / hTIGIT and 293F-hCD112 cells were co-cultured.

[0216] Fig.11 The chimeric TIGIT antibody was shown to mediate the ADCC effect of NK92-CD16a 176 on HEK293-hTIGIT cells.

[0217] Fig.12 The chimeric TIGIT antibody mediated the killing effect of NK92-hTIGIT-hCD226 on HT1080 cells.

[0218] Fig.13 The reaction activity of HumAb053 humanized TIGIT antibody with human TIGIT extracellular domain protein in enzyme-linked immunosorbent assay was shown.

[0219] Fig.14 Flow cytometry (FACS) assay was used to detect the binding of HumAb053 humanized TIGIT antibody to human TIGIT-expressing cells.

[0220] Fig.15 Flow cytometry (FACS) assay was used to detect the binding of HumAb053 humanized TIGIT antibody to mouse TIGIT-expressing cells.

[0221] Fig.16 The figure shows the flow cytometry (FACS) assay to detect the binding of HumAb053 humanized TIGIT antibody to monkey and mouse TIGIT expressing cells.

[0222] Fig.17 Shown is the inhibition of the binding of TIGIT protein to its receptor CD155 by HumAb053 humanized TIGIT antibody.

[0223] Fig.18 It was shown that HumAb053 humanized TIGIT antibody promoted the expression of NFAT downstream reporter genes when Jurkat-NFAT / hTIGIT and 293F-hCD155 cells were co-cultured. DETAILED DESCRIPTION

[0224] After extensive and in-depth research, the inventors obtained a group of anti-TIGIT antibodies through a large number of screenings. Specifically, mice were immunized with recombinant protein hTIGIT-Fc, plasmid vector pCP-hTIGIT encoding TIGIT, and HEK293 hTIGIT cells to obtain antibodies with different sequences and high activity. The TIGIT antibody of the present invention has the characteristics of high affinity and high activity. In addition, the antibodies with different sequences obtained by the present invention can specifically bind to the TIGIT protein, and their binding activity is lower than nanomolar; in addition, by reversing the inhibition of TIGIT on T cell activation activity, T cells can be activated to secrete IFNγ. The present invention was completed on this basis.

[0225] the term

[0226] In the present invention, "VH" refers to the heavy chain variable region, and "VL" refers to the light chain variable region. "VH-CDR1" refers to the CDR1 of the heavy chain variable region; "VH-CDR2" refers to the CDR2 of the heavy chain variable region; "VH-CDR3" refers to the CDR3 of the heavy chain variable region. "VL-CDR1" refers to the CDR1 of the light chain variable region; "VL-CDR2" refers to the CDR2 of the light chain variable region; "VL-CDR3" refers to the CDR3 of the light chain variable region.

[0227] TIGIT

[0228] TIGIT (T cell Ig and ITIM domain, Ig and ITIM domain T cell immunoreceptor), also known as VSVG3 or VSVG9 (V-Set and immunoglobulin domain protein 3 or 9), is a type I transmembrane protein that is one of the main immune checkpoint molecules. It contains an extracellular Ig-V-like domain, a transmembrane domain, and an intracellular C-terminal domain. The intracellular C-terminal domain consists of an ITIM (immunoreceptor tyrosine inhibitory motif) and an ITT (immunoglobulin tail tyrosine) similar motif, both of which are highly conserved between humans and mice.

[0229] Antibody

[0230] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0231] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0232] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of the different classes of immunoglobulins are well known in the art.

[0233] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0234] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0235] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombinant techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are from different animal species, such as a chimeric antibody having a variable region from a monoclonal antibody from a mouse, and a constant region from a human immunoglobulin (see, for example, U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

[0236] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0237] In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0238] Table A

[0239]

[0240]

[0241] Anti-TIGIT antibodies

[0242] In the present invention, the antibody is an anti-TIGIT antibody. The present invention provides a high-specificity and high-affinity antibody against TIGIT, which comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) amino acid sequence, and the light chain comprises a light chain variable region (VL) amino acid sequence.

[0243] Preferably, the heavy chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0244] VH-CDR1 shown in SEQ ID NO.1,

[0245] VH-CDR2 shown in SEQ ID NO.2 or SEQ ID NO.25 or SEQ ID NO.26 or SEQ ID NO.7, and

[0246] VH-CDR3 shown in SEQ ID NO.3 or SEQ ID NO.8; and

[0247] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0248] VL-CDR1 shown in SEQ ID NO.4,

[0249] VL-CDR2 shown in SEQ ID NO.5, and

[0250] VL-CDR3 shown in SEQ ID NO.6 or SEQ ID NO.9;

[0251] Among them, any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain TIGIT binding affinity.

[0252] More preferably, the heavy chain variable region comprises a CDR selected from the group consisting of:

[0253] (1) VH-CDR1 shown in SEQ ID NO.1,

[0254] VH-CDR2 shown in SEQ ID NO.2, and

[0255] VH-CDR3 shown in SEQ ID NO.3; and

[0256] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0257] VL-CDR1 shown in SEQ ID NO.4,

[0258] VL-CDR2 shown in SEQ ID NO.5, and

[0259] VL-CDR3 shown in SEQ ID NO.6;

[0260] or

[0261] VH-CDR1 shown in SEQ ID NO.1,

[0262] VH-CDR2 shown in SEQ ID NO.25, and

[0263] VH-CDR3 shown in SEQ ID NO.3; and

[0264] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0265] VL-CDR1 shown in SEQ ID NO.4,

[0266] VL-CDR2 shown in SEQ ID NO.5, and

[0267] VL-CDR3 shown in SEQ ID NO.6; or

[0268] (2) The heavy chain variable region comprises a CDR selected from the following group:

[0269] VH-CDR1 shown in SEQ ID NO.1,

[0270] VH-CDR2 shown in SEQ ID NO.7, and

[0271] VH-CDR3 shown in SEQ ID NO.8; and

[0272] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0273] VL-CDR1 shown in SEQ ID NO.4,

[0274] VL-CDR2 shown in SEQ ID NO.5, and

[0275] VL-CDR3 shown in SEQ ID NO.9; or

[0276] The heavy chain variable region comprises a CDR selected from the group consisting of:

[0277] VH-CDR1 shown in SEQ ID NO.1,

[0278] VH-CDR2 shown in SEQ ID NO.26, and

[0279] VH-CDR3 shown in SEQ ID NO.8; and

[0280] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0281] VL-CDR1 shown in SEQ ID NO.4,

[0282] VL-CDR2 shown in SEQ ID NO.5, and

[0283] VL-CDR3 shown in SEQ ID NO. 9. Wherein, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain TIGIT binding affinity.

[0284] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0285] Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

[0286] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0287] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0288] Wherein, the animal is preferably a mammal, such as a mouse.

[0289] The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR-grafted and / or modified antibody targeting TIGIT (eg, human TIGIT).

[0290] In the above content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0291] In the above content of the present invention, more preferably, the number of the added, deleted, modified and / or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0292] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 10 or 12.

[0293] In another preferred example, the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:11 or 13.

[0294] In another preferred embodiment, the antibody targeting TIGIT is 14F9A4 (mAb015) or 51F11A7 (mAb053).

[0295] Recombinant protein

[0296] The present invention also provides a recombinant protein, which includes one or more of the heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2) and heavy chain CDR3 (VH-CDR3) of the TIGIT antibody, and / or one or more of the light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2) and light chain CDR3 (VL-CDR3) of the TIGIT antibody, including

[0297] VH-CDR1 shown in SEQ ID NO.1,

[0298] VH-CDR2 shown in SEQ ID NO.2 or SEQ ID NO.25 or SEQ ID NO.26 or SEQ ID NO.7, and

[0299] VH-CDR3 shown in SEQ ID NO.3 or SEQ ID NO.8; and

[0300] The light chain variable region has a complementarity determining region CDR selected from the group consisting of:

[0301] VL-CDR1 shown in SEQ ID NO.4,

[0302] VL-CDR2 shown in SEQ ID NO.5, and

[0303] VL-CDR3 shown in SEQ ID NO.6 or SEQ ID NO.9;

[0304] Among them, any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain TIGIT binding affinity.

[0305] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0306] In another preferred example, the recombinant protein described in the present invention includes the heavy chain variable region of the TIGIT antibody and the light chain variable region of the TIGIT antibody, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:10 or 12, and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:11 or 13.

[0307] Preferably, the recombinant protein further comprises an antibody heavy chain constant region and / or an antibody light chain constant region, wherein the antibody heavy chain constant region is conventional in the art, preferably a rat antibody heavy chain constant region or a human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat light chain antibody constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.

[0308] The recombinant protein is a conventional protein in the art, preferably, it is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single-chain antibody (scFv), a single-domain antibody (sdAb), and a single-domain antibody (Signle-domainantibody), as well as a monoclonal antibody or polyclonal antibody prepared from the above antibodies. The monoclonal antibody can be developed by a variety of approaches and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.

[0309] The full-length antibody protein is a conventional full-length antibody protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and light chain variable region of the protein and the human heavy chain constant region and the human light chain constant region constitute a full-length human antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.

[0310] The single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids.

[0311] The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, which includes a light chain variable region, a light chain constant region and an Fd segment of a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0312] The single-domain antibody is a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

[0313] The single-domain antibody is a conventional single-domain antibody in the art, which only includes the heavy chain variable region.

[0314] Wherein, the preparation method of the recombinant protein is a conventional preparation method in the art. The preparation method is preferably: obtaining by separation from an expression transformant that recombinantly expresses the protein or obtaining by artificially synthesizing a protein sequence. The separation from the expression transformant that recombinantly expresses the protein is preferably the following method: cloning a nucleic acid molecule encoding the protein and having a point mutation into a recombinant vector, transforming the obtained recombinant vector into a transformant, obtaining a recombinant expression transformant, and culturing the obtained recombinant expression transformant to obtain the recombinant protein.

[0315] Nucleic Acids

[0316] The present invention also provides a nucleic acid encoding the heavy chain variable region or light chain variable region of the above-mentioned antibody (eg, anti-TIGIT antibody) or recombinant protein or anti-TIGIT antibody.

[0317] The method for preparing the nucleic acid is a conventional method in the art, and preferably comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis.

[0318] Those skilled in the art know that the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately introduced with substitution, deletion, change, insertion or addition to provide a polynucleotide homologue. The polynucleotide homologue of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining the antibody activity.

[0319] Carrier

[0320] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0321] The recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors. The expression vector is any conventional vector in the art, as long as it can carry the aforementioned nucleic acid molecule. The vector preferably includes various plasmids, cosmids, phages or virus vectors, etc.

[0322] The present invention also provides a recombinant expression transformant comprising the recombinant expression vector.

[0323] Wherein, the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: the above-mentioned recombinant expression vector is transformed into a host cell to obtain it. The host cell is any conventional host cell in the art, as long as it can satisfy the above-mentioned recombinant expression vector to stably replicate itself, and the nucleic acid carried can be effectively expressed. Preferably, the host cell is E.coli TG1 or E.coli BL21 cell (expressing single-chain antibody or Fab antibody), or HEK293 or CHO cell (expressing full-length IgG antibody). The aforementioned recombinant expression plasmid is transformed into a host cell to obtain a preferred recombinant expression transformant of the present invention. Wherein the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0324] Antibody preparation

[0325] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0326] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0327] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0328] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0329] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0330] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.

[0331] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0332] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0333] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0334] Antibody-drug conjugates (ADCs)

[0335] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0336] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, and preferably chemically coupled. The effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0337] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent can be any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0338] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection agents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0339] Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs). Typically, ADCs contain a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidase. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze when the pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by proteases.

[0340] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved through the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (e.g., iodinated, brominated or chlorinated), benzyl halides (e.g., iodinated, brominated or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

[0341] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

[0342] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins, camptothecins, Duocarmycin / duocarmycins, etoposides, maytansines and maytansinoids (e.g. DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g. pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), and vinca alkaloids.

[0343] In the present invention, drug-linkers can be used to form ADCs in one simple step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker reacts with a drug to form an ADC.

[0344] Typically, the functional groups on the linker are selected to react specifically with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety can be used to react specifically with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that when a complementary pair of reactive functional groups is selected for selective reaction of the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0345] The present invention also provides a method for preparing ADC, which may further comprise: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0346] In certain embodiments, the methods of the invention include: combining the antibody with the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further include: combining the antibody-linker conjugate with the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0347] In some embodiments, the antibody drug conjugate ADC has the following molecular formula:

[0348]

[0349] in:

[0350] Ab is antibody,

[0351] LU is the connector;

[0352] D is for medicine;

[0353] And the subscript p is a value selected from 1 to 8.

[0354] application

[0355] The present invention also provides uses of the antibodies, antibody conjugates ADCs, recombinant proteins, and / or immune cells of the present invention, for example, for preparing diagnostic preparations or preparing drugs.

[0356] Preferably, the drug is a drug for preventing and / or treating diseases associated with abnormal TIGIT expression or function.

[0357] In the present invention, the disease associated with abnormal TIGIT expression or function is a conventional disease associated with abnormal TIGIT expression or function in the art. Preferably, the disease associated with abnormal TIGIT expression or function is cancer, tumor, infectious disease.

[0358] In the present invention, the cancer is a conventional cancer in the art, preferably a germ cell tumor, lymphoma, hematologic tumor, sarcoma, melanoma, lung cancer, breast cancer, renal cell carcinoma. In the present invention, the hematologic tumor is a conventional tumor disease in the art, such as leukemia, Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, preferably leukemia.

[0359] The uses of the antibodies, ADCs, recombinant proteins, and / or immune cells of the present invention include (but are not limited to):

[0360] (i) Diagnosis, prevention and / or treatment of tumor occurrence, growth and / or metastasis, including but not limited to: hematological tumors, melanomas, sarcomas, lymphomas, adenomas, blastomas.

[0361] (ii) diagnosis, prevention and / or treatment of cancer, including but not limited to: blastoma, lymphoma, hematologic malignancy, sarcoma, melanoma, lung cancer, breast cancer, renal cell carcinoma.

[0362] (iii) diagnosis, prevention and / or treatment of tumors, including but not limited to: melanoma, Hodgkin's and non-Hodgkin's lymphoma, glioblastoma, hepatoma, glioma, myeloma, Wilms' tumor, hematological tumor.

[0363] Test Uses and Kits

[0364] The antibodies or ADCs thereof of the present invention can be used in detection applications, for example, for detecting samples to provide diagnostic information.

[0365] In the present invention, the sample (specimen) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, a resection sample of a tumor, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.

[0366] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0367] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0368] Pharmaceutical composition

[0369] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding immune cells, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the disease to be treated.

[0370] The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration. The injection preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injections or infusions, etc.

[0371] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0372] The pharmaceutical composition described in the present invention is a pharmaceutical composition for preventing and / or treating diseases related to abnormal TIGIT expression or function.

[0373] The pharmaceutical composition of the present invention can be directly used to bind to TIGIT protein molecules, and thus can be used to prevent and treat diseases such as tumors.

[0374] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0375] In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including a pharmaceutically acceptable excipient, filler or diluent, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of a pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0376] In the present invention, preferably, the amount of the pharmaceutical composition administered is an effective amount, which is an amount that can alleviate or delay the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the above factors on an individual basis and using experiments that do not exceed routine.

[0377] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0378] The present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease associated with abnormal TIGIT expression or function. Preferably, the disease associated with abnormal TIGIT expression or function is cancer or tumor.

[0379] Method and composition for detecting TIGIT protein in samples

[0380] The present invention also provides a method for detecting TIGIT protein in a sample (for example, detecting cells overexpressing TIGIT), comprising the following steps: contacting the above-mentioned antibody with the sample to be tested in vitro, and detecting whether the above-mentioned antibody and the sample to be tested combine to form an antigen-antibody complex.

[0381] The meaning of overexpression is conventional in the art, referring to the overexpression of TIGIT protein RNA or protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing and changes in protein degradation), as well as local overexpression and increased functional activity (such as in the case of increased enzymatic hydrolysis of the substrate) due to changes in protein transport patterns (increased nuclear localization).

[0382] In the present invention, the above-mentioned detection method of whether the antigen-antibody complex is formed by binding is a conventional detection method in the art, preferably a flow cytometry (FACS) detection.

[0383] The present invention provides a composition for detecting TIGIT protein in a sample, which comprises the above-mentioned antibody, recombinant protein, antibody conjugate, immune cell, or a combination thereof as an active ingredient. Preferably, it also comprises a compound composed of the functional fragments of the above-mentioned antibody as an active ingredient.

[0384] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0385] The main advantages of the present invention include

[0386] (1) The variable region sequences of the antibodies of the present invention are significantly different from those of existing antibodies, with a sequence homology of <92%.

[0387] (2) The antibodies of the present invention have strong affinity and specifically bind to TIGIT protein, and their binding activity is lower than nanomolar.

[0388] (3) The antibody of the present invention reverses the inhibition of TIGIT on T cell activation activity, activates T cells to secrete IFNγ, and has good stimulating activity for T cell activation.

[0389] (4) The present invention uses hybridoma technology to obtain antibodies. Compared with antibodies obtained from phage libraries, the antibodies have high affinity and good sequence expression.

[0390] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0391] The experimental methods in the following examples without specifying specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions.

[0392] The room temperature described in the examples is the conventional room temperature in the art, generally 10-30°C.

[0393] If not otherwise specified, the PBS described in the examples is PBS phosphate buffer, pH 7.2.

[0394] The sequence involved in the present invention is as follows:

[0395] Table B

[0396]

[0397]

[0398]

[0399] Example 1 Preparation of TIGIT Antibody

[0400] (I) Preparation of immunogen

[0401] Prepare immunogens including extracellular TIGIT protein, TIGIT recombinant cell line, expression plasmid of TIGIT DNA vector, etc.

[0402] Immunogen 1), the amino acid sequence Met22-Pro141 of the extracellular region of human TIGIT protein (as shown in positions 22-141 of SEQ ID No. 14 in the sequence list. For its amino acid sequence information, see Uniprot database, number Q495A1) was cloned into the pCpC vector with human IgG Fc fragment (hFc) (purchased from Invitrogen, V044-50) and the plasmid was prepared according to the established standard molecular biology method. For specific methods, see Sambrook, J., Fritsch, EF, and Maniatis T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). HEK293 cells (purchased from Invitrogen) were transiently transfected (PEI, Polysciences) and FreeStyle TM 293 (Invitrogen) was expanded and cultured at 37°C. After 4 days, the cell culture fluid was collected, and the cell components were removed by centrifugation to obtain the culture supernatant containing the extracellular region of the TIGIT protein. The culture supernatant was loaded onto a protein A affinity chromatography column (Mabselect Sure, purchased from GE Healthcare), and the changes in the ultraviolet absorbance value (A280nm) were monitored by an ultraviolet (UV) detector. After loading, the protein A affinity chromatography column was washed with PBS phosphate buffer (pH7.2) until the ultraviolet absorbance value returned to the baseline, and then eluted with 0.1M glycine hydrochloride (pH2.5), and the hFc-tagged TIGIT protein (TIGIT-hFc) eluted from the protein A affinity chromatography column was collected and dialyzed with PBS phosphate buffer (pH7.2) in a 4°C refrigerator overnight. The dialyzed protein was sterile filtered at 0.22 microns and then aliquoted and stored at -80°C to obtain the purified immunogen 1. Immunogen 1 needs to undergo a series of quality control tests before use, such as testing its protein concentration, purity, molecular weight and biological activity.

[0403] The results are as follows Figure 1 As shown in Table 1. Table 1 shows that the binding of TIGIT to CD155 at the protein level changes with the concentration of CD155. The data in the table are OD 450nm value.

[0404] The biological activity of immunogen 1 was detected by enzyme-linked immunosorbent assay (ELISA), specifically:

[0405] The hFc-tagged TIGIT protein (TIGIT-hFc, i.e., immunogen 1) was diluted to 0.5 μg / mL with PBS, added to the ELISA microplate at 100 μl / well, and incubated overnight at 4°C. After blocking with ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4, the percentage is mass percentage) at 37°C for two hours, the gradient dilution of biotin-labeled CD155–hFc and CD112-hFc was added, and incubated at 37°C for 1 hour. The preparation method of biotin-labeled CD155-hFc and CD112-hFc is as follows: CD155-hFc and CD112-hFc are reacted with a biotinylation reagent. The preparation method of the CD155-hFc and CD112-hFc is the same as that of immunogen 1, wherein the amino acid sequence information of the CD155 extracellular region protein (Gly27-Asp343) is referred to the Uniprot database, number P15151; the amino acid sequence information of the CD112 extracellular region protein (Gln32-Leu360) is referred to the Uniprot database, number Q92692; the biotinylation reagent is purchased from Sigma, product number B3295; the operation steps of the reaction with the biotinylation reagent are referred to the instructions of the biotinylation reagent. Add streptavidin-labeled horseradish peroxidase (purchased from Sigma product number S2438), incubate at room temperature for 30 minutes, and then add 100 microliters / well TMB color development solution. After incubation at room temperature for 15 minutes, add 50 microliters of 1N hydrochloric acid to terminate the color development reaction, and read the OD with an ELISA plate reader. 450nm reading.

[0406] The specific activity results are shown in Table 1 and Figure 1 As shown, TIGIT has a stronger binding ability to CD155 and a weaker binding ability to CD112, which is basically consistent with the description in the literature.

[0407] Table 1 Binding activity of TIGIT-hFc protein with biotinylated ligands CD155-hFc and CD112-hFc

[0408]

[0409] Immunogen 2), the full-length amino acid sequence of human TIGIT was cloned into the pIRES vector (purchased from Clontech) and the plasmid was prepared. After plasmid transfection (transfection using X-treme GENE HP DNA Transfection Reagent, purchased from Roche, Cat#06 366 236 001, and operated according to the instructions) of HEK293 cell line and CHOK1 cell line (both purchased from Invitrogen), selective culture was carried out in DMEM medium containing 10% (w / w) FBS containing 0.5μg / ml puromycin for 2 weeks, and subcloned in 96-well culture plates by limiting dilution method and cultured at 37°C, 5% (v / v) CO2. After about 2 weeks, some monoclonal wells were selected and expanded into 6-well plates. The amplified clones were stained with known TIGIT antibodies and screened by flow cytometry. Select a cell line with good growth, high fluorescence intensity and monoclonality to continue to expand the culture and freeze it in liquid nitrogen to obtain immunogen 2.

[0410] The specific selection results are shown in Table 2 and Figure 2 As shown, the values ​​in Table 2 are the average fluorescence intensity, and the higher the value, the higher the expression of TIGIT protein. In Table 2, HEK293 cells are blank control cell lines that are not transfected with hTIGIT, and their average fluorescence intensity value is 4.2. The clone number selected as the immune cell is 4B10, and its average fluorescence intensity value is 520.3. Figure 2 This indicates that HEK293 cells have a higher level of TIGIT expression.

[0411] Table 2 Results of flow cytometry (FACS) screening of HEK293 cells transfected with TIGIT protein

[0412]

[0413] Immunogen 3), human TIGIT full-length amino acid sequence cDNA was cloned into pCP vector (pCP-hTIGIT) and coated onto 1.0um gold colloidal bullets, and immunized with Helios gene gun (Bio-rad). The detailed method was formulated according to the instructions of Helios gene gun. Figure 3 This indicates that HEK293 cells transiently transfected with pCP-hTIGIT can express higher levels of TIGIT.

[0414] (II) Preparation of hybridoma cells and antibody screening

[0415] The mice used for immunization were Balb / c and SJL mice.

[0416] A. Immunogen 1) Balb / c and SJL mice aged 6 to 8 weeks (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were used for immunization, and the mice were raised under SPF conditions. During the initial immunization, the immunogen h TIGIT-hFc protein was emulsified with Freund's complete adjuvant and injected intraperitoneally at 0.25 ml, that is, each mouse was injected with 50 micrograms of immunogen protein. During the booster immunization, the immunogen protein was emulsified with Freund's incomplete adjuvant and injected intraperitoneally at 0.25 ml, that is, each mouse was injected with 25 micrograms of immunogen protein. There was a 2-week interval between the initial immunization and the first booster immunization, and a 3-week interval between each subsequent booster immunization. Blood was collected one week after each booster immunization, and the antibody titer and specificity of the immunogen protein in the serum were detected by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS). The results are shown in the following table. Figure 4 As shown in Table 3. Table 3 shows that the sera of mice immunized with TIGIT-hFc have different degrees of binding to the immunogen, showing antigen-antibody reaction, with the highest dilution being around 10,000. The blank control is 1% (w / w) BSA, and the batch refers to the mouse sera on the seventh day after the second booster immunization. The data in the table are OD450nm values.

[0417] B. Immunogen 2) Balb / c and SJL mice aged 6 to 8 weeks (purchased from Shanghai Slake Experimental Animal Co., Ltd.) were used for immunization, and the mice were raised under SPF conditions. The obtained HEK293-h TIGIT stable cell line containing human TIGIT was expanded and cultured in a T-75 cell culture flask to 90% confluence, and the culture medium was aspirated. Wash twice with DMEM basal medium (purchased from Invitrogen), and then treat with enzyme-free cell dissociation solution (purchased from Invitrogen) at 37°C until the cells can fall off the wall of the culture dish, and collect the cells. Wash twice with DMEM basal medium, count the cells, and dilute the cells to 2╳10 with phosphate buffer (pH7.2) 7 Cells per milliliter. Each mouse was intraperitoneally injected with 0.5 ml of cell suspension each time it was immunized. The interval between the first and second immunizations was 2 weeks, and the interval between each subsequent immunization was 3 weeks. Except for the first immunization, blood was collected 1 week after each immunization, and the antibody titer and specificity in the serum were detected by enzyme-linked immunosorbent assay (ELISA).

[0418] C. Immunogen 3) Balb / c and SJL mice aged 6 to 8 weeks (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were used for immunization. The mice were raised under SPF conditions. All mice were immunized 3-4 times with a gene gun via the abdomen, 3-4 shots each time, and 1.0 microgram of cDNA per shot. The interval between the first immunization and the first booster immunization was 2 weeks, and the interval between each subsequent immunization was 3 weeks. Blood was collected 7 days after each booster immunization, and the antibody titer in the serum was detected by enzyme-linked immunosorbent assay (ELISA).

[0419] Usually, the ELISA titer of most mice can reach more than 1:1000 after 2-3 immunizations. Figure 3 This is the result of antibody titer detection using enzyme-linked immunosorbent assay (ELISA) for TIGIT-hFc protein immune serum.

[0420] Table 3 ELISA assay to detect serum antibody titers of mice after TIGIT-hFc protein immunization

[0421]

[0422] The results showed that after three immunizations with the immunogen, the ELISA titer of most mice could reach above 1:1000, indicating that the mice produced a good humoral immune response to the immunogen and their spleen cells could be used for hybridoma cell preparation.

[0423] Mice with titers that meet the requirements can be selected for cell fusion and hybridoma preparation. Before cell fusion, the mice are immunized for the last time with 50-100 micrograms of purified TIGIT-hFc injected intraperitoneally. The mice were killed 3-5 days later and spleen cells were collected. The cells were washed three times by centrifugation at 1000 rpm with DMEM basal medium, and then mixed with mouse myeloma cells SP2 / 0 (purchased from ATCC) at a ratio of 5:1 in the number of live cells, and cell fusion was performed using high-efficiency electrofusion or PEG method (see METHODS IN ENZYMOLOGY, VOL. 220). The fused cells were diluted into DMEM medium containing 20% ​​fetal bovine serum and 1╳HAT, and the percentages are mass percentages. Then, the cells were diluted at 1╳10 5 / 200 microliters per well was added to a 96-well cell culture plate and placed in a 5% CO2, 37°C incubator. The percentages are volume percentages. After 14 days, the supernatant of the cell fusion plate was screened by enzyme-linked immunosorbent assay (ELISA) and microplate cell assay (Acumen). After 14 days, the positive clones with OD450nm>1.0 in the enzyme-linked immunosorbent assay (ELISA) and average fluorescence intensity value>100 in the microplate cell assay (Acumen) were expanded to 24-well plates and cultured in a medium containing 10% (w / w) fetal bovine serum DMEM (Invitrogen) at 37°C and 5% (v / v) CO2. After 3 days of culture, the culture fluid of the expanded culture in the 24-well plate was centrifuged, the supernatant was collected, and the supernatant was subjected to antibody subtype analysis. After 14 days, the binding activity of the antibody to TIGIT protein and TIGIT-positive cells was determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS).

[0424] According to the results of 24-well plate screening, hybridoma cells with OD450nm>1.0 in enzyme-linked immunosorbent assay (ELISA) and average fluorescence intensity value>100 in flow cytometry (FACS) were selected as qualified positive clones. Selected hybridoma cells that meet the conditions were subcloned in 96-well plates by limiting dilution method and cultured in DMEM medium containing 10% (w / w) FBS (purchased from Invitrogen) at 37°C and 5% (v / v) CO2. Ten days after subcloning, preliminary screening was performed by enzyme-linked immunosorbent assay (ELISA) and microplate cell detection (Acumen), and positive monoclones were selected and expanded to 24-well plates for further culture. Three days later, flow cytometry (FACS) was used to determine the positive antigen binding to evaluate biological activity (the evaluation criteria were OD450nm>1.0 in enzyme-linked immunosorbent assay (ELISA) and average fluorescence intensity value>100 in flow cytometry (FACS)).

[0425] According to the test results of the 24-well plate samples, the positive clones were expanded and cultured in a DMEM medium (purchased from Invitrogen) containing 10% (w / w) FBS at 37° C., 5% (v / v) CO2, and the cells were suspended in a freezing solution [DMEM containing 20% ​​(w / w) FBS and 10% (w / w) DMSO] and frozen in liquid nitrogen according to a conventional method to obtain the hybridoma cells of the present invention, which can be used for subsequent antibody production, purification and amino acid sequence determination.

[0426] Example 2 Identification of mouse antibodies

[0427] (I) Detection of antibody binding to TIGIT protein by ELISA

[0428] The amino acid sequence Met22-Pro141 of the extracellular region of the human TIGIT protein described in the step of Example 1 (as shown in positions 22-141 in the sequence table SEQ ID No. 14) was cloned into the pCpC vector with a human IgG Fc fragment (hFc), and HEK293 cells were transfected. The cell culture fluid was collected and purified to obtain the human TIGIT protein with an hFc tag (herein referred to as hTIGIT-hFc protein). The purified human TIGIT extracellular region protein (hTIGIT-hFc) was diluted with PBS to a final concentration of 1.0 μg / ml, and then added to a 96-well ELISA plate at 100 μl per well. Seal with plastic film and incubate at 4°C overnight. The next day, wash the plate twice with a plate washing solution (PBS + 0.01% Tween20), and add a blocking solution (PBS + 0.01% Tween20+1% BSA) for 1-2 hours at room temperature. Pour off the blocking solution, add 50-100 μl of the antibody sample to be tested to each well, incubate at 37°C for 1-2 hours, and wash the plate 2-3 times with plate washing solution (PBS+0.01% Tween20). Add HRP (horseradish peroxidase) labeled secondary antibody, incubate at 37°C for 1-2 hours, and wash the plate 2-3 times with plate washing solution (PBS+0.01% Tween20). Add 100 μl of TMB substrate to each well, incubate at room temperature for 15-30 minutes, and then add 100 μl of stop solution (1.0N HCl) to each well. Read the A450nm value using an ELISA plate reader (TiterMax 384plus, Molecular Device).

[0429] The results are shown in Table 4, and the tested antibodies can bind well to the human TIGIT extracellular domain protein. Among them, the EC50 of clone number 14F9A4 (wherein VH is shown in SEQ ID NO.10, and VL is shown in SEQ ID NO.11) and 51F11A7 antibody (wherein VH is shown in SEQ ID NO.12, and VL is shown in SEQ ID NO.13) are 0.118nM and 0.0633nM respectively. The IgG control is mouse IgG, and the data in the table are Absorbance OD450nm values.

[0430] Table 4 Reactivity of mouse TIGIT antibody with human TIGIT extracellular domain protein in ELISA

[0431]

[0432] (II) Flow cytometry (FACS) to detect the binding of antibodies to TIGIT-expressing cells

[0433] The pIRES plasmid containing the full-length nucleotide sequence encoding human TIGIT described in step (i) immunogen 2 of Example 1, wherein the database accession number of the human TIGIT nucleotide sequence is NM_173799.4, was transfected into the CHOK1 cell line to obtain a CHOK1 stable cell line containing human TIGIT (herein referred to as the CHOK1-hTIGIT stable cell line). The pIRES plasmid containing the full-length gene of monkey TIGIT, wherein the database accession number of the monkey TIGIT nucleotide sequence is XM_015445425.1, was transfected into the CHOK1 cell line to obtain a CHOK1 stable cell line containing monkey TIGIT (herein referred to as the CHOK1-cTIGIT stable cell line). The pIRES plasmid carrying the full-length mouse TIGIT gene, in which the database accession number of the mouse TIGIT nucleotide sequence is NM_001146325.1, was transfected into the CHOK1 cell line containing mouse TIGIT in the CHOK1 stable cell line (herein referred to as the CHOK1-mTIGIT stable cell line). The CHOK1-hTIGIT stable cell line, the CHOK1-cTIGIT stable cell line and the CHOK1-mTIGIT stable cell line were expanded and cultured in a T-75 cell culture flask to 90% confluence, the culture medium was aspirated, and the cells were washed twice with HBSS buffer (Hanks Balanced Salt Solution, purchased from Invitrogen), and then treated and collected with enzyme-free cell dissociation solution (Versene solution, purchased from Life technology). The cells were washed twice with HBSS buffer, and after cell counting, the cells were diluted to 2×10 with HBSS buffer. 6 1% goat serum blocking solution was added per ml of cells, the percentages are weight percentages. Incubate on ice for 30 minutes, then wash twice with HBSS buffer by centrifugation. The collected cells were suspended to 2×10 in FACS buffer (HBSS containing 1% BSA, the percentages are weight percentages). 6 Cells / mL, 100 microliters per well were added to a 96-well FACS reaction plate, and 100 microliters of the purified TIGIT antibody test sample obtained in Example 2 was added to each well, and incubated on ice for 2 hours. Wash twice by centrifugation with FACS buffer, add 100 microliters of fluorescent (Alexa488) labeled secondary antibody (purchased from Invitrogen) per well, and incubate on ice for 1 hour. Wash three times by centrifugation with FACS buffer, add 100 microliters of fixative [4% (v / v) paraformaldehyde] per well to suspend the cells, and wash twice by centrifugation with FACS buffer after 10 minutes. Suspend the cells with 100 microliters of FACS buffer, and detect and analyze the results with FACS (FACS Calibur, purchased from BD).

[0434] The results are shown in Table 5, Table 6 and Table 7. The tested antibodies can bind to human or monkey TIGIT proteins on the cell surface, but not mouse TIGIT proteins. The EC50 of clone 14F9A4 and 51F11A7 antibodies binding to human TIGIT protein are 1.13nM and 0.81nM, respectively, and the EC50 of binding to monkey TIGIT protein are 0.80nM and 0.27nM, respectively. The IgG control is mouse IgG, and the data in the table are the average fluorescence intensity values ​​of the cell population measured by the average fluorescence intensity.

[0435] Table 5 Flow cytometry (FACS) assay to detect the binding of antibodies to human TIGIT expressing cells

[0436]

[0437] Table 6 Flow cytometry (FACS) assay to detect the binding of antibodies to monkey TIGIT expressing cells

[0438]

[0439]

[0440] Table 7 Flow cytometry (FACS) assay to detect the binding of antibodies to mouse TIGIT-expressing cells

[0441]

[0442] (III) Detection of TIGIT antibody blocking the binding of TIGIT protein to its ligand CD155

[0443] The receptor ligand binding test of TIGIT protein detects whether TIGIT antibody blocks the binding of TIGIT protein to its CD155. Since the interaction between TIGIT protein and another ligand CD112 is relatively weak and cannot be detected by enzyme-linked immunosorbent assay (ELISA), we only checked the ligand CD155 in the TIGIT antibody blocking experiment here.

[0444] TIGIT extracellular domain protein (TIGIT-hFc) was diluted with PBS to a final concentration of 1.0 μg / mL, and then added to a 96-well ELISA plate at 100 μl per well. Seal with plastic film and incubate overnight at 4°C. The next day, wash the plate twice with plate washing solution [PBS containing 0.01% (v / v) Tween20], add blocking solution [PBS containing 0.01% (v / v) Tween20 and 1% (w / w) BSA] and block at room temperature for 2 hours. Pour out the blocking solution, first add 50 μl of the purified TIGIT antibody sample obtained in Example 1 to each well, then add biotin-labeled CD155 extracellular domain protein (CD155-hFc), 100 microliters per well, mix well and incubate at 37°C. After 2 hours, wash the plate 3 times with plate washing solution [PBS containing 0.01% (v / v) Tween20]. Add 100 μl of HRP (horseradish peroxidase) labeled avidin diluent (purchased from Sigma) to each well, incubate at 37°C for 2 hours, and wash the plate 3 times with plate washing solution [PBS containing 0.01% (v / v) Tween20]. Add 100 μl of TMB substrate to each well, incubate at room temperature for 30 minutes, and then add 100 μl of stop solution (1.0N HCl) to each well. Read the A450nm value using an ELISA plate reader (SpectraMax 384plus, Molecular Device).

[0445] The results are shown in Table 8, indicating that the obtained antibodies can inhibit the binding of TIGIT protein to its ligand CD155 to varying degrees. The IgG control is mouse IgG, and the data in the table are inhibition rates (%).

[0446] Table 8 Inhibition of TIGIT antibody on the binding of TIGIT protein to its receptor CD155

[0447]

[0448]

[0449] (IV) NFAT reporter gene detection experiment

[0450] The purpose is to use luciferase reporter gene overexpression technology to study whether anti-TIGIT antibodies can bind to the immune checkpoint TIGIT and relieve the inhibition of the NFAT signaling pathway in the human T lymphocyte line (Jurkat) caused by the interaction between TIGIT and CD155 or CD112.

[0451] 1. Construction of Jurkat-NFAT / hTIGIT, 293F-hCD155 and 293F-hCD112 cell lines

[0452] The pLVX-IRES-hTIGIT.Puro plasmid containing the full-length nucleotide sequence encoding human TIGIT, wherein the database accession number of the human TIGIT nucleotide sequence is NM_173799.4, was packaged into a lentivirus and used to infect the Jurkat-NFAT cell line to obtain a Jurkat-NFAT stable cell line containing human TIGIT (referred to herein as the Jurkat-NFAT / hTIGIT stable cell line, clone number 2G7-1D8).

[0453] The pLVX-IRES-hCD155.Puro plasmid containing the full-length nucleotide sequence encoding human CD155, wherein the amino acid sequence information of CD155 is referred to the Uniprot database, number P15151, was transfected into the 293F cell line to obtain a 293F stable cell line containing human CD155 (herein referred to as 293F-hCD155 stable cell line, clone number 3G2).

[0454] The pLVX-IRES-hCD112.Puro plasmid containing the full-length nucleotide sequence encoding human CD112, wherein the amino acid sequence information of CD112 is referred to the Uniprot database, number Q92692, was transfected into the 293F cell line to obtain a 293F mixed cell line containing human CD112 (herein referred to as 293F-hCD112 mixed cell line, numbered 2F5 pool).

[0455] Jurkat-NFAT / hTIGIT stable cell line, 293F-hCD155 stable cell line and 293F-hCD112 mixed cell line were expanded and cultured in T-75 cell culture flasks to 90% confluence, the medium was aspirated, and the cells were washed twice with PBS buffer (purchased from Corning), and then lysed with enzyme-free cell dissociation solution (TrypLE TM Express Enzyme, purchased from Lifetechnology) and the cells were collected (Jurkat cells were suspension cells and no enzyme dissociation was required).

[0456] 2. NFAT reporter gene detection experiment

[0457] Jurkat-NFAT / hTIGIT cells were transferred to Viewplate-96 white plate (Perkin Elmer, Cat No. 6005181), 1*10E5 cells / well, 20ul RPMI-1640 medium (Gibco, Ref11875-093) to suspend the cells. 20ul RPMI-1640 medium diluted antibodies were added to the corresponding culture wells, 1:5 gradient dilution, a total of 8 concentration gradients, the highest concentration was 10ug / ml. Place in a cell culture incubator (37°C, 5% CO2 (v / v)) and incubate for 20min.

[0458] 20ul of 293F-hCD155 cells (2*10E4 cells / well) or 293F-hCD112 cells (6*10E4 cells / well) suspended in RPMI-1640 medium were added to each well and the cells were placed in a cell culture incubator (37°C, 5% CO2 (v / v)) and incubated for 20 minutes.

[0459] Add 20ul RPMI-1640 culture medium OKT3 (final concentration 2ug / ml) to each well. Place in a cell culture incubator (37°C, 5% CO2 (v / v)) and incubate for 5 hours. Add 80ul ONE-Glo reagent to each well, and complete the detection according to the experimental manual of ONE-Glo fluorescence detection system (Promega, Ref E6120).

[0460] In the Jurkat-NFAT / hTIGIT cell line constructed in step 1, the effect of the TIGIT antibody of the present invention on the secretion of the NFAT downstream reporter gene luciferase in the NFAT reporter gene detection experiment is detected. Among them, the purpose of co-culturing Jurkat-NFAT / hTIGIT and 293F-hCD155 or 293F-hCD112 is to allow the TIGIT receptor protein on the surface of Jurkat cells to bind to the CD155 or CD112 ligand protein on the surface of 293F cells, play an immunosuppressive role and inhibit the expression and secretion of the NFAT downstream reporter gene luciferase. The addition of TIGIT antibodies is to bind and inhibit the interaction between the TIGIT receptor and the CD155 or CD112 ligand, release the immunosuppressive signal, and activate the expression and secretion of the NFAT downstream reporter gene luciferase.

[0461] The results are shown in Tables 9 and 10, wherein the mIgG control is mouse IgG, and the data in the table are fluorescence intensity values ​​(Foldinduction compared to mIgG control).

[0462] The results showed that in the NFAT reporter gene detection experiment, the tested antibody could inhibit the interaction between TIGIT and CD155 or CD112, and enhance the expression and secretion of the NFAT downstream reporter gene luciferase in Jurkat-NFAT / hTIGIT cells.

[0463] Therefore, the anti-TIGIT antibody of the present invention can relieve the inhibition of the NFAT signaling pathway of the human T lymphocyte line (Jurkat) caused by the interaction between TIGIT and CD155 or CD112 by binding to the immune checkpoint TIGIT.

[0464] Table 9 TIGIT antibodies promote the expression of NFAT downstream reporter genes when Jurkat-NFAT / hTIGIT and 293F-hCD155 cells are co-cultured

[0465]

[0466] Table 10 TIGIT antibodies promote the expression of NFAT downstream reporter genes when Jurkat-NFAT / hTIGIT and 293F-hCD112 cells are co-cultured

[0467]

[0468] Example 3 Determination of amino acid sequences of light and heavy chain variable regions

[0469] Total RNA isolation: After the supernatant obtained from the subclone culture in Example 1 was tested for antigen binding (i.e., after the assay and activity determination in Example 2), 5×107 hybridoma cells were collected by centrifugation, 1 mL of Trizol was added, mixed and transferred to a 1.5 mL centrifuge tube, and allowed to stand at room temperature for 5 minutes. 0.2 mL of chloroform was added, shaken for 15 seconds, allowed to stand for 2 minutes, and centrifuged at 4°C, 12000g for 5 minutes, and the supernatant was transferred to a new 1.5 mL centrifuge tube. 0.5 mL of isopropanol was added, the liquid in the tube was gently mixed, allowed to stand at room temperature for 10 minutes, and then centrifuged at 4°C, 12000g for 15 minutes, and the supernatant was discarded. 1 mL of 75% ethanol (the percentage is the volume percentage) was added, the precipitate was gently washed, and the supernatant was discarded after centrifugation at 4°C, 12000g for 5 minutes. The precipitate was dried, and DEPC-treated H2O was added to dissolve (dissolved in a 55°C water bath for 10 minutes) to obtain total RNA.

[0470] Reverse transcription and PCR: Take 1 μg of total RNA, prepare 20 μl system, add reverse transcriptase and react at 42°C for 60 minutes, and terminate the reaction at 7°C for 10 minutes. Prepare 50 μl PCR system, including 1 μl cDNA, 25 pmol of each primer, 1 μl DNA polymerase and matching buffer system, 250 μmol dNTPs. Set the PCR program, pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 35 seconds, and after 35 cycles, extend at 72°C for 5 minutes to obtain PCR products. The kit used for reverse transcription is PrimeScript RT Master Mix, purchased from Takara, catalog number RR036; the kit used for PCR is Q5 super-fidelity enzyme, purchased from NEB, catalog number M0492.

[0471] Cloning and sequencing: Take 5 μl of PCR product for agarose gel electrophoresis detection, and purify the positive samples using a column recovery kit, where the recovery kit is Gel&PCR Clean-up, purchased from MACHEREY-NAGEL, item number 740609. Perform ligation reaction: 50ng sample, 50ng T vector, 0.5μl ligase, 1μl buffer, 10μl reaction system, react at 16℃ for half an hour to obtain ligation product, wherein the ligation kit is T4 DNA ligase, purchased from NEB, item number M0402; take 5μl ligation product and add it to 100μl competent cells (Ecos 101 competent cells, purchased from Yeastern, item number FYE607), ice bath for 5 minutes. Then heat shock in 42℃ water bath for 1 minute, put back on ice for 1 minute, add 650μl antibiotic-free SOC medium, recover at 37℃ shaker at 200RPM for 30 minutes, take out 200μl and spread on LB solid medium containing antibiotics and culture in 37℃ incubator overnight. The next day, use the primers M13F and M13R on the T vector to configure a 30μl PCR system for colony PCR. Use a pipette tip to dip the colony into the PCR reaction system and blow and aspirate, and aspirate 0.5μl and spot it on another LB solid culture dish containing 100nM ampicillin to preserve the strain. After the PCR reaction is completed, take out 5μl for agarose gel electrophoresis detection, and sequence the positive samples. The sequencing steps refer to Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991).

[0472] Example 4 Preparation of chimeric antibodies

[0473] (I) Plasmid construction and preparation:

[0474] Example 2 has obtained purified TIGIT antibodies from the culture supernatant of hybridoma cells, and the heavy chain variable region and light chain variable region sequences of TIGIT antibodies have been clarified based on the sequencing results of Example 3. The heavy chain variable region sequence of TIGIT antibodies was recombined into an expression vector containing a signal peptide and a human heavy chain antibody IgG1 constant region (where the expression vector was purchased from Invitrogen, and the recombination step was also completed by Shanghai Ruizhi Chemical), and the light chain variable region sequence of TIGIT antibodies was recombined into an expression vector containing a signal peptide and a human antibody light chain kappa constant region, and the recombinant plasmid was obtained and verified by sequencing (the sequencing method is the same as the sequencing method in Example 3). The recombinant plasmid with improved purity was extracted using an alkaline lysis kit (purchased from MACHEREY-NAGEL), with a mass of more than 500 μg, and filtered through a 0.22 μm filter membrane (purchased from Millopore) for transfection.

[0475] (II) Cell transfection:

[0476] CHO-S cells (purchased from Invitrogen) were cultured in medium CD FortiCHO (purchased from Invitrogen). The shaking incubator was set at 37°C, 120 RPM, and 8% CO2 (v / v) concentration.

[0477] Dissolve 0.1 mg DNA in 5 ml CD FortiCHO medium and mix well to obtain medium A. Mix 5 ml CD FortiCHO medium and 0.5 mg PEI (purchased from Sigma) to obtain medium B. Combine 5 ml medium A and 5 ml medium B, mix well, and let stand for 20 minutes to obtain mixed solution C. Slowly add 10 ml mixed solution C to 100 ml CD FortiCHO medium containing CHO-S cells until the cell density of CHO-S reaches 4.0×10 6 / mL, add while shaking to avoid excessive concentration of PEI, and place in a shaker for culture. On the second day, add peptone to a final concentration of 10g / L. On days 3 to 9, measure the antibody titer of the culture medium. On days 8 to 9, centrifuge (3500RPM, 30 minutes) to collect the supernatant, filter through a 0.22μm filter membrane, and obtain the filtered cell supernatant for purification.

[0478] (III) Antibody purification: For continuously produced endotoxin-free chromatography columns and Protein A fillers (purchased from GE), treat with 0.1M NaOH for 30 minutes or rinse with 5 column volumes of 0.5M NaOH. For column materials and chromatography columns that have not been used for a long time, soak them in 1M NaOH for at least 1 hour, rinse with endotoxin-free water until neutral, and wash the column materials with 10 column volumes of 1% (v / v) Triton×100. Use 5 column volumes of PBS (PBS phosphate buffer, pH7.2) for balance, apply the filtered cell supernatant obtained in step (ii) to the column, and collect the flow-through if necessary. After the column is applied, wash with 5 column volumes of PBS. Elute with 5 column volumes of 0.1M pH3.0 Glycine-HCl, collect the eluate, and neutralize with 0.5 column volumes of 1MTris-HCl (1.5M NaCl) at pH8.5 to harvest TIGIT chimeric antibodies. All the above solutions need to be newly prepared. After harvesting the TIGIT chimeric antibody, dialyze it in 1×PBS for 4 hours to avoid endotoxin contamination. After dialysis, the concentration was determined by spectrophotometry or a kit, the antibody purity was determined by HPLC-SEC, and the antibody endotoxin content was detected by an endotoxin detection kit (purchased from Lonza).

[0479] Example 5 Identification of chimeric antibodies

[0480] (I) Detection of antibody binding to TIGIT protein by ELISA

[0481] The results are shown in Table 11. Figure 5 As shown, the chimeric TIGIT antibody can bind to the human TIGIT extracellular domain protein. The activities of each antibody are comparable, indicating that the antibody has a strong ability to bind to TIGIT.

[0482] The EC50 values ​​of clone 14F9A4 (mAb015) and 51F11A7 (mAb053) antibodies binding to human TIGIT protein were 0.14 nM and 0.13 nM, respectively. The IgG control was human IgG1, and the data in the table are A450nm values.

[0483] Table 11 Reactivity of chimeric TIGIT antibodies with human TIGIT extracellular domain protein in ELISA

[0484]

[0485] (II) Flow cytometry (FACS) to detect the binding of antibodies to TIGIT-expressing cells

[0486] The results are shown in Tables 12 and 14. Figure 6 and Figure 7As shown, the chimeric TIGIT antibody can bind to human and monkey TIGIT proteins on the cell surface to varying degrees.

[0487] The EC50 values ​​of clone 14F9A4 and 51F11A7 antibodies binding to human TIGIT protein were 0.70 nM and 1.061 nM, respectively, and the EC50 values ​​of binding to monkey TIGIT protein were 1.593 nM and 1.164 nM, respectively. The IgG control was chimeric human IgG1, and the data in the table were the average fluorescence intensity values ​​of the cell population measured by the average fluorescence intensity.

[0488] Table 12 Flow cytometry (FACS) assay to detect the binding of antibodies to human TIGIT expressing cells

[0489]

[0490]

[0491] Table 13 Flow cytometry (FACS) assay to detect the binding of antibodies to monkey TIGIT expressing cells

[0492]

[0493] (III) Detection of TIGIT antibody blocking the binding of TIGIT protein to its ligand CD155

[0494] The results are shown in Table 14 Figure 8 As shown, it is shown that the chimeric TIGIT antibody can inhibit the binding of TIGIT protein to its ligand CD155 to varying degrees. The IgG control is human IgG1, and the data in the table are OD405.

[0495] Table 14 Inhibition of TIGIT antibody on the binding of TIGIT protein to its receptor CD155

[0496]

[0497] (IV) NFAT reporter gene detection experiment

[0498] The results are shown in Tables 15 and 16. Fig. 9 , Fig.10 As shown, the chimeric IgG1 control is chimeric human IgG1, and the data in the table are fluorescence intensity values.

[0499] The results showed that the chimeric TIGIT antibody could inhibit the interaction between TIGIT and CD155 or CD112 in the NFAT reporter gene detection experiment, thereby enhancing the expression and secretion of the NFAT downstream reporter gene luciferase in Jurkat-NFAT / hTIGIT cells.

[0500] Table 15 TIGIT antibodies promote the expression of NFAT downstream reporter genes when Jurkat-NFAT / hTIGIT and 293F-hCD155 cells are co-cultured

[0501]

[0502] Table 16 TIGIT antibodies promote the expression of NFAT downstream reporter genes when Jurkat-NFAT / hTIGIT and 293F-hCD112 cells are co-cultured

[0503]

[0504]

[0505] (5) TIGIT antibody-dependent cell-mediated cytotoxicity assay (ADCC)

[0506] The pIRES plasmid containing the full-length nucleotide sequence of human TIGIT described in Example 1 (immunogen preparation section) was transfected into HEK293 cells to obtain a HEK293 stable cell line expressing human TIGIT (herein referred to as HEK293-hTIGIT stable cell line). TM The target cells HEK293-hTIGIT stable cell line in the logarithmic growth phase were digested with 5% paraformaldehyde (Express Enzyme, purchased from Life Technology), washed twice with PBS phosphate buffer (purchased from Corning), and the cell density was adjusted to 1×10 6 cells / mL, 1 μL of DELFIABATDA reagent (purchased from Perkin Elmer) was added to each mL of cell suspension for labeling, and after incubation at 37°C, 5% CO2 in a cell culture incubator for 20 min, the cells were washed three times with 10 mL of PBS phosphate buffer, and resuspended in experimental buffer (RPMI Medium 1640, no Phenol Red + 2% FBS, purchased from Gibio) to 1×10 5 cells / mL, and the cell suspension was added to a 96-well V-bottom cell culture plate (purchased from Corning), with 100 μL per well (the number of cells per well was 1×10 4 ). Add 50 μl of the antibody diluted in the assay buffer to the corresponding culture wells, dilute 1:5, a total of 9 concentration gradients, and the highest concentration is 100 nM. Place the culture plate in a 37°C, 5% CO2 cell culture incubator and incubate for 20 minutes. Collect the effector cells NK92 (purchased from Huabo Bio) overexpressing CD16a 176V in the logarithmic growth phase, centrifuge and resuspend in the assay buffer to a density of 6×10 5cells / mL, and add 50 μL per well to the culture wells, with the ratio of effector cells to target cells being 3: 1. The culture plate was placed in a 37°C, 5% CO2 cell culture incubator and incubated for 2.5 hours.

[0507] At the same time, a blank solvent control group (background), a target cell spontaneous release control group (TS), an effector cell spontaneous release control group (ES), a target cell and effector cell mixed spontaneous release group ((T+E).S) (i.e., the effector cell and target cell release group when the antibody concentration is 0 nM), and a target cell maximum release group (TM) were set up. 10 microliters of lysis solution were added to each well of the maximum release control group, and the cells were placed in a 37°C, 5% CO2 cell culture incubator for 30 minutes and centrifuged at 500g for 5 minutes. 25 microliters of supernatant were taken and placed in a colorless transparent bottom 96-well detection plate (purchased from Perkin Elmer), 200 microliters of Europium Solution detection solution were added and mixed, and after shaking at 250 rpm for 15 minutes, the fluorescence signal value with an emission wavelength of 615 nm was detected on an EnVision 2105 detector (purchased from Perkin Elmer). The killing rate ADCC was calculated according to the formula = [(experimental group-target cell and effector cell mixed spontaneous release control group) / (target cell maximum release group-target cell spontaneous release control group)]×100%. The results are shown in Table 17 and Fig.11 shown.

[0508] in, Fig.11 It was shown that after the TIGIT antibody binds to human TIGIT on the surface of target cells through the Fab segment, its IgG1Fc segment can bind to CD16A on the surface of NK cells, thereby causing NK cells to produce a nonspecific killing effect on target cells, namely ADCC effect.

[0509] Table 17 TIGIT antibody-mediated ADCC effect of NK92-CD16a on HEK293-hTIGIT cells

[0510]

[0511]

[0512] (6) TIGIT antibody-mediated cytotoxicity experiments based on blocking TIGIT / CD155 signaling

[0513] TIGIT antibodies bind to TIGIT on the surface of NK cells, blocking its interaction with CD155 on the surface of target cells, causing CD155 to bind to the co-stimulatory molecule CD226 on the surface of NK cells, thereby enhancing the NK cell's ability to kill target cells.

[0514] The pLVX-IRES-hTIGIT.Puro (NM_173799.4) plasmid containing the full-length nucleotide sequence of human TIGIT and the pLVX-IRES-CD226.neo (Q15762) plasmid containing the full-length nucleotide sequence of human CD226 were packaged into lentivirus and sequentially infected NK92 cell lines to obtain NK92 stable cell lines expressing human TIGIT and CD226 (herein referred to as NK92-hTIGIT-hCD226 stable cell lines). The NK92-hTIGIT-hCD226 stable cell line was expanded and cultured, and the effector cells NK92-hTIGIT-hCD226 in the logarithmic growth phase were collected, centrifuged and resuspended in an experimental buffer (RPMI Medium 1640, noPhenol Red + 2% FBS, purchased from Gibco) to a density of 4×10 6 cells / mL, and add the cell suspension to a 96-well V-bottom cell culture plate (purchased from Corning) at 50 μl per well. Add 50 μl of the antibody diluted in the experimental buffer to the corresponding culture wells, 1:2 gradient dilution, a total of 5 concentration gradients, and the highest concentration is 50 μg / ml. Place the culture plate in a 37°C, 5% CO2 cell culture incubator and incubate for 20 minutes. Target cells HT1080 labeled with DELFIA BATDA reagent (labeling steps are the same as step 1), add the labeled target cells to the culture wells, 100 μl per well (the number of target cells per well is 1×10 4 ), the ratio of effector cells to target cells was 20:1. The culture plate was placed in a 37°C, 5% CO2 cell culture incubator and incubated for 2 hours. The control group was set up, tested, and calculated in the same way as in step 1. The results are shown in Table 18 and Fig.12 shown.

[0515] Table 18 TIGIT antibody-mediated killing effect of NK92-hTIGIT-hCD226 on HT1080 cells

[0516]

[0517] Example 6 Modification and preparation of hotspot mutation antibodies

[0518] 6.1 Hotspot modification and identification of antibody sequences

[0519] 6.1.1 Hotspot mutation design

[0520] Antibody mAb015 has two hot spots that need to be mutated, namely, aspartic acid-glycine DG at positions 55-56 of the heavy chain and asparagine-glycine NG at positions 61-62 of the heavy chain. Among them, aspartic acid-glycine DG at positions 55-56 mutates to glutamic acid-glycine EG, or mutates to serine-glycine SG, or mutates to aspartic acid-alanine DA. Asparagine-glycine NG mutates to asparagine-alanine NA, or mutates to glutamine-glycine QG, or mutates to serine-glycine SG.

[0521] Antibody mAb053 has two hot spots that need to be mutated, namely, aspartic acid-glycine DG at positions 55-56 of the heavy chain and asparagine-glycine NG at positions 61-62 of the heavy chain. Among them, aspartic acid-glycine DG at positions 55-56 mutates to glutamic acid-glycine EG, or mutates to serine-glycine SG, or mutates to aspartic acid-alanine DA, or mutates to glycine-glycine GG. Asparagine-glycine NG at positions 61-62 mutates to asparagine-alanine NA, or mutates to glutamine-glycine QG, or mutates to serine-glycine SG, or mutates to alanine-glycine AG, or mutates to asparagine-aspartic acid ND.

[0522] mAb015VH SEQ ID NO.23

[0523]

[0524] mAb015VL SEQ ID NO.11

[0525] QIVLSQSPAILSASPGEKVTMTC RASSSVSYMH WYQQKPGSSPKPWIY ATSNLAS GVPARFSGSGSGTSYSLTITRVEAEDAATYYC QQWSSNPPIVT FGSGTKLEIK

[0526] mAb053_VH SEQ ID NO.24

[0527] mAb053_VL SEQ ID NO.13

[0528] QIVLSQSPAILSASPGEKVTMTC RASSSVSYMH WYQQKPGSSPKPWIY ATSNLAS GVPTRFSGSGSGTSYSLTISRVEAEDAATYYC QLWSGNPPMLT FGAGTKLELK

[0529] 6.2 Preparation of hotspot mutation antibodies

[0530] 6.2.1 Plasmid construction and preparation

[0531] The hot spots of the antibody sequence are modified by PCR-mediated site-directed mutagenesis. The principle is: using a circular plasmid as a template, a pair of primers that completely match and introduce mutations are used for PCR amplification. Because the template is a plasmid extracted from Escherichia coli that is methylated and sensitive to DpnI, and the PCR product is an unmethylated open circular plasmid with a nick, the template can be eliminated by DpnI restriction digestion, and only the newly amplified mutant plasmid remains in the final product.

[0532] The specific operation method is to first find the codon corresponding to each amino acid according to the codon table. Then determine the nucleotide mutation site, for example, mutate aspartic acid N to asparagine D. The codon corresponding to aspartic acid is AAT, and the codon corresponding to asparagine is GAT or GAC. We choose to mutate AAT to GAT, and only need to mutate one site. Then find the mutation site and the base sequence near it, and then design the primers of the mutation sequence with the mutation site as the center, and perform PCR amplification with the wild-type antibody plasmid as the template. The product is digested with DpnI for 1-3 hours and then transformed into DH5α. After coating the plate, pick a single colony for culture and sequencing the next day to obtain the plasmid used for transfection after verification.

[0533] 6.2.2 Transfection and purification

[0534] One day before transfection, passage the Expi 293F cells to a density of 2.5-3×10 6 viable cells / mL. The cell density and viability were tested on the day of transfection, and the density reached 4.5-5.5×10 6 viable cells / mL, and when the viability is between 95-99%, dilute the cells to 3×10 6 viable cells / mL, gently mix the cells, add 3mL to each well of a 24-well culture plate, and discard the excess cells.

[0535] Prepare ExpiFectamine TM 293 / plasmid DNA mixture

[0536] a. Gently invert to mix ExpiFectamine TM 293Reagent 4-5 times

[0537] b.Opti-MEM TM I Reduced Serum Medium dilutes the plasmid DNA and mixes by rotating or inverting.

[0538] c.Opti-MEM TM I Reduced Serum MediumExpiFectamine TM Mix the 293 Reagent by rotating or inverting 2-3 times and incubate at room temperature for 5 minutes.

[0539] d. Dilute ExpiFectamine TM Add 293 Reagent to the diluted plasmid DNA, rotate or invert to mix 2-3 times, and incubate at room temperature for 10-20 minutes;

[0540] The incubated ExpiFectamine TM The 293 / plasmid DNA mixture was slowly added to the cells with shaking; the cells were cultured in a shaking incubator at 37°C.

[0541] Add ExpiFectamine after 18-22 hours TM 293Transfection Enhancer 1andExpiFectamine TM 293 Transfection Enhancer 2, no preheating required, just mix and add

[0542] 6-7 days after transfection, the 293F culture was centrifuged (30 minutes, 3500 rpm), and the supernatant was collected and filtered through a 0.22 uM filter. The filtered supernatant was purified by Protein A magnetic beads.

[0543] Equilibration buffer: PBS phosphate buffer, pH 7.2;

[0544] Elution buffer: 0.1 mol / L glycine-HCl, pH 3.0;

[0545] Neutralization buffer: 1mol / L Tris-HCl, 1.5mol / L NaCl, pH 8.5

[0546] The antibody concentration of the purified antibody was measured at A280 using NanoDrop (spectrophotometer), and the purity of the antibody was measured using SEC-HPLC. The results are shown in Table 19.

[0547] Table 19 Summary of production test results of hotspot mutation antibodies

[0548]

[0549]

[0550] 6.3 Identification of hotspot mutation antibodies

[0551] 6.3.1 Octet Affinity Screening for Hotspot Mutation Antibodies

[0552] The Octet red96 instrument (purchased from Fortiebio) was used for single-concentration affinity detection and sorting. The specific operations and methods were based on the instrument manual and the detailed methods provided by the manufacturer. Specifically: Affinity determination was performed using an anti-human antibody sensor (AHCsensor, purchased from Fortiebio). The hotspot mutation antibody was diluted and reacted with the AHC sensor; the sensor bound to the hotspot mutation antibody was incubated with 100nM human TIGIT ECD-His at 30°C for three minutes, and then incubated with PBS solution at 30°C for 5 minutes; the binding and dissociation of the antibody and TIGIT protein were detected by detecting the change in interference wavelength using the Octet instrument, and then used The User Software software was used to fit the dissociation constant and the binding constant. The affinity constant is the ratio of the dissociation constant to the binding constant. The results are shown in Table 20:

[0553] Table 20 Affinity results of hotspot mutation antibodies and TIGIT at single concentration

[0554]

[0555] 6.3.2 ELISA to detect the binding of hotspot mutation antibodies to TIGIT protein

[0556] Purified human TIGIT extracellular domain protein (hTIGIT-hFc) was diluted with PBS to a final concentration of 1.0 μg / ml, and then added to a 96-well ELISA plate at 100 μl per well. Seal with plastic film and incubate at 4°C overnight. The next day, wash the plate twice with plate washing solution (PBS + 0.01% Tween20), add blocking solution (PBS + 0.01% Tween20 + 1% BSA) and block at room temperature for 1-2 hours. Pour out the blocking solution, add 50-100 μl of the antibody sample to be tested to each well, incubate at 37°C for 1-2 hours, and wash the plate 2-3 times with plate washing solution (PBS + 0.01% Tween20). Add HRP (horseradish peroxidase) labeled secondary antibody, incubate at 37°C for 1-2 hours, and wash the plate 2-3 times with plate washing solution (PBS + 0.01% Tween20). Add 100 μl of TMB substrate to each well, incubate at room temperature for 15-30 minutes, and then add 100 μl of stop solution (1.0 N HCl) to each well. Read the A450nm value using an ELISA plate reader (TiterMax 384plus, Molecular Device). The results are shown in Table 21.

[0557] Table 21 Reactivity of hotspot mutation antibodies with human TIGIT extracellular domain protein in ELISA

[0558] mAbs EC50(nM) mAbs EC50(nM) hIgG1 12.94 mAb015-WT 0.18 mAb053-WT 0.21 mAb015-V1 0.16 mAb053-V1 0.23 mAb015-V2 0.17 mAb053-V2 0.18 mAb015-V3 0.18 mAb053-V3 0.18 mAb015-V4 0.21 mAb053-V4 0.15 mAb015-V5 0.27 mAb053-V5 0.17 mAb015-V6 0.24 mAb053-V6 0.24 mAb015-V7 0.32 mAb053-V7 0.22 mAb015-V8 0.29 mAb053-V8 0.22 mAb015-V9 0.21 mAb053-V9 2.11 mAb015-V10 0.3 mAb053-V10 0.23 mAb015-V11 0.23 mAb053-V11 0.17 mAb015-V12 1.24 mAb053-V12 0.27 mAb015-V13 0.28 mAb053-V13 0.25 mAb015-V14 0.25 mAb053-V14 0.21 mAb015-V15 0.28 mAb053-V15 0.7 mAb053-V16 0.21 mAb053-V17 0.53 mAb053-V18

[0559] In the binding assay, the thermal mutation antibodies showed different binding to human TIGIT, so mAb015-V15 and mAb053-V7 were selected as the thermal mutation modification results of these two antibodies.

[0560] mAb015-V15 VH SEQ ID NO.27

[0561]

[0562] mAb015-V15 VL SEQ ID NO.11

[0563] QIVLSQSPAILSASPGEKVTMTC RASSSVSYMH WYQQKPGSSPKPWIY ATSNLAS GVPARFSGSGSGTSYSLTITRVEAEDAATYYC QQWSSNPPIVT FGSGTKLEIK

[0564] mAb053-V7 VH SEQ ID NO.28

[0565]

[0566] mAb053-V7 VL SEQ ID NO.13

[0567] QIVLSQSPAILSASPGEKVTMTC RASSSVSYMH WYQQKPGSSPKPWIY ATSNLAS GVPTRFSGSGSGTSYSLTISRVEAEDAATYYC QLWSGNPPMLT FGAGTKLELK

[0568] Example 7 Preparation and identification of humanized antibodies

[0569] The mAb015-V15 and mAb053-V7 sequences obtained after hotspot removal of mAb015 and mAb053 antibodies were humanized.

[0570] 7.1mAb053-V7

[0571] 7.1.1 Design of mAb053-V7

[0572] Through sequence alignment (NCBI-Igblast), the germline gene sequences with the highest homology to the heavy chain variable region and light chain variable region of the candidate antibody were selected as the variable region transplantation framework: IGHV1-2*06 (66.3%) and IGKV3-11*01 (63.2%). After selecting the human antibody framework, homology modeling was used to predict the key amino acids that may determine the structure in the mouse antibody constant region, and back mutations were designed for the grafted framework region.

[0573] According to the above principles, 5 heavy chain variable region sequences (HumAb053 VH 2-g0, HumAb053 VH 2-g1, HumAb053 VH 2-g2, HumAb053 VH 2-g3, HumAb053 VH 2-g4) and 4 light chain variable region sequences (HumAb053 VL 2-g0, HumAb053 VL 2-g1, HumAb053 VL 2-g2, HumAb053 VL 2-g3) were designed, and then cross-combined for expression, for a total of 20 expression combinations, as shown in Table 22.

[0574] Table 22 mAb053 humanized antibody expression combination

[0575]

[0576] Vector construction: The amplification primers were synthesized by Genewiz, and then the variable regions of the light chain and heavy chain were amplified by PCR. A 50 μL reaction system was prepared, including 50-100 ng of the heavy chain variable region, 1 ul of the light chain variable region, 1 ul of the forward and reverse primers, 1 ul of pfxD enzyme (purchased from invitrogen, 12344-012), 5 ul of 10*pfx buffer (supplier same pfx same enzyme), and water was added to make up to 50 μL. The PCR program was set up, pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 68°C for 30 seconds, and an additional extension at 68°C for 10 minutes after 25 cycles to obtain the PCR product. Take 5 μL of the PCR product for agarose gel electrophoresis detection, and purify the positive samples using a recovery kit, where the recovery kit is PureLink Quick Gelextraction kit, purchased from Qiagen, catalog number 28706.

[0577] Preparation of humanized antibodies: Perform ligation reaction: insert 20-40ng, enzyme-cut expression vector 60-100ng, recombinase Exnase (purchased from Vazyme, item number C112-01 / 02) 1μL, buffer 2μL, reaction system 10μL, react at 37°C for half an hour to obtain the ligation product, i.e., the constructed recombinant vector. The buffer is the buffer purchased with the recombinase; the heavy chain variable region is cloned into the expression vector containing the signal peptide and the human antibody heavy chain IgG1 constant region (the expression vector is purchased from Invitrogen, and the recombination step is a conventional step), and the light chain variable region is cloned into the expression vector containing the signal peptide and the human antibody light chain kappa constant region (the expression vector is purchased from Invitrogen, and the recombination step is a conventional step). Add 10 μL of the ligation product to 100 μL of competent cells (Ecos 101 competent cells, purchased from Yeastern, catalog number FYE607), heat shock in a 42°C water bath for 60 seconds, put back on ice for 3 minutes, take out 80 μL and spread it on LB solid culture medium containing ampicillin, and incubate it in a 37°C incubator overnight. The next day, use the primers pEF1A and pSV40 on the expression vector to configure a 30 μL PCR system for colony PCR. The system for colony PCR is: 1 μL of each primer, 15 μL of PCR premix (purchased from Novoprotein), and make up to 30 μL. Use a pipette tip to dip the colony into the PCR reaction system and blow and aspirate, and aspirate 0.5 μL and spot it on another LB solid culture dish containing 100 μg / mL ampicillin to preserve the strain. After the PCR reaction is completed, take out 4.5 μL for agarose gel electrophoresis detection, and sequence the positive samples.

[0578] 7.1.2 Expression of mAb053-V7 humanized antibody

[0579] The expression vectors of the recombinant antibody heavy and light chains with the correct sequence were amplified and then transiently transfected with FreeStyle TM 293-F cells (purchased from Invitrogen) were used to produce antibodies. Humanized expression was performed using 24-well plates.

[0580] Identification of humanized antibodies: The Octet red96 instrument was used to detect the binding of antibodies to human TIGIT in the cell supernatant.

[0581] Table 23 Single concentration affinity results of humanized antibodies to TIGIT

[0582]

[0583] The heavy and light chain expression vectors of humanized antibodies HumAb053-2-16, HumAb053-2-17, HumAb053-2-18, HumAb053-2-19, and HumAb053-2-20, whose affinities differed by less than 3 times from mAb053-V7, were amplified and transiently transfected into 30 mL FreeStyleTM 293-F cells (purchased from Invitrogen) to produce antibodies. During transfection, the density of 293-F cells should be 1-1.2×10 6 / mL, 30mL cells require 30μg of the above-constructed recombinant vector and 70μg of transfection reagent polyethyleneimine (PEI). The recombinant vector and PEI were added to 2mL of culture medium respectively, allowed to stand at room temperature for 5 minutes, filtered with a 0.22μm filter membrane, and the mixture of the recombinant vector and PEI was allowed to stand at room temperature for 15 minutes. Then the above mixture was slowly added to the cells and cultured at 130rpm in a 37°C, 8% (v / v) CO2 incubator. The culture supernatant and cell pellet were taken every day to detect the expression of antibodies. After 5 days, the cell culture medium was centrifuged at 3000g for 30 minutes, the supernatant was collected, and filtered with a 0.22μm filter. The monoclonal antibodies in 200mL of clarified supernatant were purified with 1mL MabSelectTMSuReTMcolumn (purchased from GE Healthcare). MabSelectTMSuReTMcolumn was first equilibrated with equilibration buffer (PBS phosphate buffer, pH7.2), MabSelectTMSuReTMcolumn. After loading, wash the MabSelectTMSuReTMcolumn with equilibration buffer, the volume of which is 5 times the volume of the protein A column bed. Elute the monoclonal antibody bound to the MabSelectTMSuReTMcolumn with eluent (0.1M glycine-HCl buffer, pH 3.0). Collect the eluted antibodies and add 10% (v / v) 1.0MTris-HCl buffer to neutralize the pH. Then immediately dialyze overnight with PBS phosphate buffer. Collect the dialyzed monoclonal antibodies, aseptically filter them with a 0.22μm filter, and store them aseptically to obtain purified TIGIT humanized antibodies. The obtained antibodies were tested for protein concentration and purity.

[0584] The results are shown in Table 24 below, which show that the yield and purity analysis of the antibody were normal.

[0585] Table 24 Analysis of humanized antibody purification results

[0586] Protein Products Volume (mL) Concentration (mg / ml) Yield (mg) Purity (%; SEC) Buffer HumAb053-2-16 0.7 2.659 1.86 100 PBS pH7.4 HumAb053-2-17 0.7 3.914 2.74 100 PBS pH7.4 HumAb053-2-18 0.7 2.808 1.97 100 PBS pH7.4 HumAb053-2-19 0.7 4.301 3.01 100 PBS pH7.4 HumAb053-2-20 0.7 4.377 3.06 100 PBS pH7.4

[0587] 7.1.3 Activity identification of mAb053-V7 humanized antibody

[0588] The binding of antibody to hTIGIT-ECD-Fc was detected by ELISA. Fig.13 As shown. Flow cytometry (FACS) assay detected the antibody and hTIGIT ( Fig.14 ), mTIGIT( Fig.15 ), cynoTIGIT( Fig.16 ) expressing cells.

[0589] The results showed that the obtained antibodies could bind to human TIGIT and monkey TIGIT on the cell surface, but not to mouse TIGIT.

[0590] The affinity and activity of the humanized mAb053 antibody were evaluated, and the results showed that its function was close to that of mAb053-V7. The results are shown in Table 25.

[0591] Among them, the preferred mAb053 humanized antibodies are HumAb053-2-16, HumAb053-2-17, and HumAb053-2-18.

[0592] The data in Table 25 are the mean fluorescence intensity values ​​of the cell population measured by MFI.

[0593] Table 25 Humanized antibody activity identification results

[0594]

[0595]

[0596] HumAb053-2-16VH SEQ ID NO.29

[0597] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMN WVRQAPGQGLEWMG QIFPGDADTNYNAKFKG RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR YLSALDY WGQGTTVTVSS

[0598] HumAb053-2-16VL,HumAb053-2-17VL,HumAb053-2-18VL SEQ ID NO.30

[0599] EIVLTQSPATLSLSPGERATLSC RASSSVSYMH WYQQKPGQSPRPWIY ATSNLAS GVPARFSGSGSGTDYTLTISSLEPEDFAVYYC QLWSGNPPMLT FGGGTKVEIK

[0600] HumAb053-2-17VH SEQ ID NO.31

[0601] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMN WVRQAPGQGLEWMG QIFPGDADTNYNAKFKG RVTMTADKSISTAYMELSRLRSDDTAVYYCAR YLSALDY WGQGTTVTVSS

[0602] HumAb053-2-18VH SEQ ID NO.32

[0603] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMN WVRQAPGQGLEWMG QIFPGDADTNYNAKFKG RVTMTADKSISTAYMELSRLRSDDTAVYFCAR YLSALDY WGQGTTVTVSS

[0604] 7.2 Design, expression and identification of mAb015-V15

[0605] Through sequence alignment (NCBI-Igblast), the germline gene sequences with the highest homology to the heavy chain variable region and light chain variable region of the candidate antibody were selected as the variable region transplantation framework: IGHV5-51*01 (65.3%) and IGKV3-20*01 (61.5%). After selecting the human antibody framework, homology modeling was used to predict the key amino acids that may determine the structure in the mouse antibody constant region, and back mutations were designed for the grafted framework region.

[0606] According to the above principles, four heavy chain variable region sequences (mAb015_VH1, mAb015_VH2, mAb015_VH3, mAb015_VH4) and four light chain variable region sequences (mAb015_VL1, mAb015_VL2, mAb015_VL3, mAb015_VL4) were designed respectively, and then cross-combined for expression, for a total of 16 expression combinations, as shown in Table 26.

[0607] Table 26 mAb015 humanized antibody expression combination

[0608]

[0609]

[0610] Identification of humanized antibodies: BIAcore T200 was used to detect the binding of supernatant expressed in eukaryotic cells to TIGIT-hFc. The supernatant capture time was 120 s, the flow rate was 10 ul / min, the binding time of analyte 100 nM TIGIT-hFc was 180 s, the dissociation time was 600 s, and the flow rate was 30 ul / min. The chip was regenerated with 10 mM Glycine-HCl. Data were obtained by

[0611] The results of affinity data are shown in Table 27.

[0612] Table 27 mAb015 humanized antibody affinity data

[0613]

[0614] The results showed that the affinity and activity of the mAb015 humanized antibodies were evaluated, and the results showed that the affinities of Hu015_VH1+VL4, Hu015_VH1+VL3, Hu015_VH1+VL2, Hu015_VH2+VL3, Hu015_VH2+VL2, and Hu015_VH2+VL4 were close to those of mAb015-V15. The results are shown in Table 27.

[0615] The preferred mAb015-V15 humanized antibodies are Hu015_VH1+VL4, Hu015_VH1+VL3, and Hu015_VH1+VL2.

[0616] Hu015_VH1 SEQ ID NO.33

[0617] EVQLVQSGAEVKKPGESLKISCKGSGYAFS SYWMN WVRQMPGKGLEWMG QIFPGDADTKYSGKFKG QVTISADKSISTAYLQWSSLKASDTAMYYCAR YLGGLDY WGQGTLVTVSS

[0618] Hu015_VL4 SEQ ID NO.34

[0619] EIVLTQSPGTLSLSPGERATLSC RASSSVSYMH WYQQKPGQSPRPWIY ATSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC QQWSSNPPIVT FGGGTKVEIK

[0620] Hu015_VL3 SEQ ID NO.35

[0621] EIVLTQSPGTLSLSPGERATLSC RASSSVSYMH WYQQKPGQAPRPWIY ATSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC QQWSSNPPIVT FGGGTKVEIK

[0622] Hu015_VL2 SEQ ID NO.36

[0623] EIVLTQSPGTLSLSPGERATLSC RASSSVSYMH WYQQKPGQAPRPWIY ATSNLAS GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQWSSNPPIVT FGGGTKVEIK

[0624] discuss:

[0625] Those skilled in the art know that there are different methods for preparing antibodies, for example:

[0626] Antibodies can be obtained by immunizing wild-type mice, but mouse antibodies need to be humanized to obtain humanized antibodies. The disadvantage is that the modified antibodies may have stronger immunogenicity and the antibody structure may change, resulting in loss of activity or poor manufacturability.

[0627] Fully human antibodies can be obtained by immunizing fully human transgenic mice, but the quantity or affinity of the antibodies obtained will be poor;

[0628] The antibody library of immune mice can be constructed and the antibodies can be expressed and screened for activity using phage display technology, but this involves the random recombination of the heavy and light chains of the antibodies, which will result in poor productivity of the formed antibodies.

[0629] Antibodies can be expressed and screened for activity by constructing a human antibody library using phage display technology. However, since there is no immunization, the resulting antibodies will have poor affinity.

[0630] The present invention uses hybridoma technology to obtain antibodies, which have high antibody affinity and good sequence expression compared to antibodies obtained from phage libraries. In addition, the present invention obtains antibodies with different sequences that can specifically bind to TIGIT antibodies, and their binding activity is lower than nanomolar, thereby reversing TIGIT's inhibition of T cell activation activity, thereby activating T cells to secrete IFNγ.

[0631] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A heavy chain variable region of an antibody, characterized in that The heavy chain variable region has a complementarity determining region CDR selected from the group consisting of: (1) VH-CDR1 shown in SEQ ID NO.1, VH-CDR2 shown in SEQ ID NO.2 or SEQ ID NO.25, and VH-CDR3 shown in SEQ ID NO.3; or (2) VH-CDR1 shown in SEQ ID NO.1, VH-CDR2 shown in SEQ ID NO.7 or SEQ ID NO.26, and VH-CDR3 shown in SEQ ID NO.8; Among them, any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain TIGIT binding affinity.

2. A heavy chain of an antibody, characterized in that The heavy chain has the heavy chain variable region as described in claim 1.

3. A light chain variable region of an antibody, characterized in that: The light chain variable region has a complementarity determining region CDR selected from the group consisting of: (1) VL-CDR1 shown in SEQ ID NO.4, VL-CDR2 shown in SEQ ID NO.5, and VL-CDR3 shown in SEQ ID NO.6; or (2) VL-CDR1 shown in SEQ ID NO.4, VL-CDR2 shown in SEQ ID NO.5, and VL-CDR3 shown in SEQ ID NO.9; Among them, any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain TIGIT binding affinity.

4. A light chain of an antibody, characterized in that The light chain has the light chain variable region as described in claim 3.

5. An antibody, characterized in that The antibody has: (1) the heavy chain variable region of claim 1; and / or (2) the light chain variable region of claim 3; Alternatively, the antibody has: the heavy chain according to claim 2; and / or the light chain according to claim 4, Among them, any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain TIGIT binding affinity.

6. A recombinant protein, characterized in that The recombinant protein comprises: (i) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5; and (ii) optionally a tag sequence to facilitate expression and / or purification.

7. A polynucleotide, characterized in that The polynucleotide encodes a polypeptide selected from the group consisting of: (1) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5; and (2) The recombinant protein according to claim 6.

8. A carrier, characterized in that The vector contains the polynucleotide according to claim 7.

9. A genetically engineered host cell, characterized in that The host cell contains the vector of claim 8 or the polynucleotide of claim 7 is integrated into its genome.

10. An antibody conjugate, characterized in that: This antibody conjugate contains: (a) an antibody portion, the antibody portion being selected from the group consisting of: The heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, or a combination thereof; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

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