Anti-TIM-3 monoclonal antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an anti-TIM-3 monoclonal antibody as well as a preparation method and application thereof. According to the present invention, the blocking anti-TIM-3 monoclonal antibody is successfully prepared, the binding and blocking characteristics and the in vitro and in vivo anti-tumor effects of the blocking anti-TIM-3 monoclonal antibody are represented in detail, and the anti-TIM-3 monoclonal antibody 15E10 shows strong anti-tumor activity, and comprises a heavy chain amino acid sequence and a light chain amino acid sequence selected from SEQ ID NO: 15-16. Therefore, the compound is expected to become a promising candidate drug for cancer immunotherapy, has the potential of preventing and treating cancers, and also has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-TIM-3 monoclonal antibody and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In the past decade, tumor immunotherapy has flourished. Tumor immunotherapy treats cancer by activating the patient's own immune system. It is another emerging field of tumor treatment after surgery, radiotherapy and chemotherapy. Tumor immunotherapy has developed rapidly, and its early representative treatment options include lymphokine activated killer cell (LAK) and cytokine activated killer cell (CIK) therapy. The second-generation immunotherapy gives CIK cells a certain specificity by infusing dendritic cells (DC), which is called DC-CIK therapy. The third-generation immunotherapy uses gene editing technology to transform the surface receptors of human T cells into CARs that can recognize specific tumor antigens, enabling them to attack tumor cells in a targeted manner. With the development of immunotherapy, the fourth-generation immunotherapy represented by immune checkpoint inhibitors and tumor vaccine technology has emerged.

[0004] Immune checkpoint blockade (ICB) therapy is one of the most promising emerging therapeutic strategies in the field of tumor immunotherapy. Immune checkpoints are key proteins and signaling pathways that regulate and balance the immune system. They regulate the activity and function of immune cells through interactions between ligands and receptors. Tumor cells can use immune checkpoints as an immune escape mechanism to evade immune system attacks by inhibiting the activity of immune cells. Immune checkpoint inhibitors can relieve this immunosuppressive effect and restore anti-tumor immune responses, thereby achieving the effect of tumor inhibition. Therefore, immune checkpoints can be used as an important target for tumor immunotherapy, and the research and development of immune checkpoint antibodies has important clinical significance and prospects. The FDA has approved a variety of immune checkpoint antibodies for the treatment of different types of tumors. In 2018, James P. Allison and Tasuku Honjo were awarded the Nobel Prize in Physiology or Medicine for their breakthrough discovery of immune checkpoint inhibitors, which is a milestone in the research of immune checkpoint inhibitors. At present, antibodies targeting the PD-1-PD-L1 pathway, such as Nivolumab and Pembrolizumab, have been widely used in the clinical treatment of various cancers and have achieved significant clinical effects. Antibodies targeting CTLA-4, such as Ipilimumab, have also been approved for the treatment of cancers such as melanoma.

[0005] Although ICB therapy has shown strong anti-tumor effects in clinical practice, some patients still have primary, adaptive resistance or immune-related adverse reactions. So far, therapeutic TIM-3 antibodies have not obtained effective anti-tumor activity in clinical trials, and the development of TIM-3McAb is still in the phase III clinical stage. Some TIM-3McAbs have been discontinued due to no significant efficacy or strong side effects, such as BGB-A425. Therefore, it is urgent to explore new immune checkpoint inhibitors to improve the therapeutic effect of current ICB therapy. Among them, T cell immunoglobulin mucin domainmolecule 3 (TIM-3) is a new immune checkpoint molecule that can be expressed in a variety of immune cells such as T cells, NK cells, monocytes and macrophages, tumor-associated dendritic cells, and can also be expressed in non-immune cells such as tumor cells and endothelial cells to exert its immunosuppressive effect. In addition, compared with other immune checkpoints, TIM-3 can bind to its four ligands to regulate a variety of immune cells in the tumor microenvironment (TME). Notably, the failure of programmed cell death protein 1 (PD-1) blockade and the development of adaptive resistance are associated with the upregulation of TIM-3, suggesting that dual blockade of immune checkpoints may play a synergistic role in anti-tumor immunotherapy.

[0006] In summary, blocking TIM-3 can play a key role in anti-PD-1 combined blocking therapy and salvage therapy after anti-PD-1 treatment failure, and is expected to break through the current dilemma of ICB therapy. However, the research and development of TIM-3 monoclonal antibodies (McAb) is not yet mature, and phase III clinical research is still ongoing. Some TIM-3McAbs have been terminated due to failure to show significant efficacy or serious side effects. Therefore, developing more effective functional TIM-3 antibodies and broadening the types and number of immune checkpoint inhibitors will lay the foundation for exploring more effective TIM-3 antibody drug-related combined treatment options. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides anti-TIM-3 monoclonal antibodies and preparation methods and applications thereof. Specifically, the present invention successfully prepared blocking anti-human TIM-3McAbs and characterized their binding and blocking properties, in vitro and in vivo anti-tumor effects in detail, so that they are expected to become promising candidate drugs for cancer immunotherapy. Based on the above research results, the present invention is completed.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect of the present invention, an anti-TIM-3 monoclonal antibody is provided, which comprises a complementary determining region (CDR) sequence selected from the following: SEQ ID NO: 2, 4, 6, 9, 11 and 13, or comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the complementary determining region sequence, respectively.

[0010] Preferably, for the anti-TIM-3 monoclonal antibody described above, the complementary determining region comprises a heavy chain complementary determining region and a light chain complementary determining region;

[0011] Wherein, the heavy chain complementarity determining region is:

[0012] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:2, a CDR2 having an amino acid sequence as shown in SEQ ID NO:4, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:6;

[0013] Wherein, the light chain complementary determining region is:

[0014] It comprises a CDR1 having an amino acid sequence as shown in SEQ ID NO:9, a CDR2 having an amino acid sequence as shown in SEQ ID NO:11, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:13.

[0015] Further, the anti-TIM-3 monoclonal antibody further comprises a framework region (FR) sequence selected from the following: SEQ ID NO: 1, 3, 5, 7, 8, 10, 12 and 14, or comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the framework region sequence, respectively.

[0016] Preferably, for the anti-TIM-3 monoclonal antibody described above, wherein the complementarity determining region comprises a heavy chain framework region and a light chain framework region;

[0017] Wherein, the heavy chain framework region is:

[0018] comprising FR1 of the amino acid sequence shown in SEQ ID NO: 1, FR2 of the amino acid sequence shown in SEQ ID NO: 3, FR3 of the amino acid sequence shown in SEQ ID NO: 5, and FR4 of the amino acid sequence shown in SEQ ID NO: 7;

[0019] Wherein, the light chain framework region is:

[0020] It comprises FR1 of the amino acid sequence shown in SEQ ID NO:8, FR2 of the amino acid sequence shown in SEQ ID NO:10, FR3 of the amino acid sequence shown in SEQ ID NO:12, and FR4 of the amino acid sequence shown in SEQ ID NO:14.

[0021] For the above-mentioned anti-TIM-3 monoclonal antibody, it comprises a heavy chain amino acid sequence and a light chain amino acid sequence selected from the following: SEQ ID NO: 15-16, or comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the heavy chain amino acid sequence and the light chain amino acid sequence, respectively.

[0022] Furthermore, the anti-TIM-3 monoclonal antibody may be a recombinant antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody or an antigen-binding fragment of these antibodies, which is not specifically limited herein.

[0023] Wherein, the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab') 2 fragment, Fv fragment or scFv fragment.

[0024] The second aspect of the present invention provides an isolated polynucleotide, which can encode the above-mentioned anti-TIM-3 monoclonal antibody.

[0025] The third aspect of the present invention provides an expression vector comprising the isolated polynucleotide described above.

[0026] The expression vector can be selected from: DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof; preferably, the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0027] A fourth aspect of the present invention provides a host cell comprising the above-mentioned expression vector, or comprising the above-mentioned polynucleotide integrated into the host cell genome; or, the host cell expresses the above-mentioned anti-TIM-3 monoclonal antibody.

[0028] The host cells include cells of prokaryotic and eukaryotic origin. Among them, prokaryotic organisms are preferably Escherichia coli, and eukaryotic organisms include yeast and mammalian cells, wherein mammalian cells include CHO, NS0, HEK293, PERC6, etc., preferably CHO cells.

[0029] A fifth aspect of the present invention provides a method for preparing an anti-TIM-3 monoclonal antibody, comprising the steps of:

[0030] (I) culturing the host cell under conditions suitable for producing anti-TIM-3 monoclonal antibodies to obtain a culture containing the anti-TIM-3 monoclonal antibodies; and,

[0031] (II) isolating or recovering the anti-TIM-3 monoclonal antibody from the culture.

[0032] The sixth aspect of the present invention provides an immunoconjugate, which includes the anti-TIM-3 monoclonal antibody of the present invention and a coupling portion, wherein the coupling portion is a detectable marker, a drug, a toxin, a cytokine, a viral coat protein or a VLP, etc.

[0033] Wherein, the detectable marker includes a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

[0034] The drug can be a cytotoxic drug, such as an anti-tubulin drug, a DNA minor groove binder, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite drug, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, and the like.

[0035] The toxins may be auristatins, chlortetracycline, maytansinoids, ricin, ethidium bromide, mitomycin, diphtheria toxin, abrin, gelonin, metotoxin, restrictocin, phenomycin, curcin, crotonin, calicheamicin, glucocorticoids, and the like.

[0036] The immunoconjugate contains a multivalent (eg, bivalent) anti-TIM-3 monoclonal antibody as described in the present invention. The multivalent refers to an anti-TIM-3 monoclonal antibody as described in the present invention that contains multiple repetitions in the amino acid sequence of the immunoconjugate.

[0037] The seventh aspect of the present invention provides a kit, which includes the above-mentioned anti-TIM-3 monoclonal antibody and / or the above-mentioned immunoconjugate; the kit can be used to detect the presence or level of TIM-3 in a sample. Preferably, the kit also includes a second antibody that specifically recognizes the bifunctional integrated antibody; optionally, the second antibody also includes a detectable marker, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

[0038] In one or more specific embodiments of the present invention, the kit further comprises instructions, according to which the kit can be used to non-invasively detect the expression of TIM-3 in the subject to be tested. It is understood that the above kit can be used for tumor detection, monitoring, diagnosis and prognosis.

[0039] An eighth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned anti-TIM-3 monoclonal antibody or the above-mentioned immunoconjugate.

[0040] Optionally, it also includes a pharmaceutically acceptable carrier and / or excipient. Moreover, according to the usual method, it can be made into a dosage form in the form of an oral preparation, an external preparation, a suppository, and a sterile injection solution such as a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, a spray, etc.

[0041] The non-pharmaceutical active ingredients such as carriers and / or excipients that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0042] In one or more specific embodiments of the present invention, the pharmaceutical composition of the present invention can be applied to the body by known means. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. It can be optionally applied via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such application can be carried out via single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be applied in the present invention can vary depending on various factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0043] In one or more specific embodiments of the present invention, the subject of administration of the pharmaceutical composition can be human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc. Among them, human is the most preferred.

[0044] The ninth aspect of the present invention provides the use of the above-mentioned anti-TIM-3 monoclonal antibody, immunoconjugate or pharmaceutical composition in any one or more of the following:

[0045] (A) preparing tumor detection, monitoring, diagnosis and / or prognosis products;

[0046] (B) Preparation of products for tumor prevention and / or treatment.

[0047] Tumor is used in the present invention as known to those skilled in the art. Benign tumors are defined as excessive proliferation of cells that are unable to form aggressive, metastatic tumors in the body. In contrast, malignant tumors are defined as cells with multiple cellular and biochemical abnormalities that are capable of forming systemic diseases (such as forming tumor metastases in distant organs).

[0048] In one or more specific embodiments of the present invention, medicine of the present invention can be used for treating malignant tumor.The example of malignant tumor of available drug therapy of the present invention includes solid tumor and hematologic tumor.Solid tumor can be for example the tumor of breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine gland (such as thyroid and adrenal cortex), esophagus, endometrium, germ cell, head and neck, kidney, liver, lung, larynx, tongue and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva.Malignant tumor includes hereditary cancer, for example squamous cell carcinoma, melanoma, retinoblastoma and Wilms' tumor etc.In addition, malignant tumor includes primary tumor in described organ and corresponding secondary tumor (tumor metastasis) in distal organ. Hematological tumors can be, for example, aggressive and indolent forms of leukemia and lymphoma, i.e., non-Hodgkin's disease, chronic and acute myeloid leukemia (CML / AML), acute lymphocytic leukemia (ALL), Hodgkin's disease, multiple myeloma, and T-cell lymphoma. Also included are myelodysplastic syndromes, plasmacytomas, tumoroid syndromes, and cancers of unknown primary site and AIDS-related malignancies.

[0049] More specifically, the product has any one or more of the following functions:

[0050] 1) Detecting the level of TIM-3 in the sample;

[0051] 2) regulating (e.g., downregulating) TIM-3 activity or level;

[0052] 3) blocking the binding of TIM-3 to its ligand;

[0053] 4) Enhance the function of T cells;

[0054] 5) Enhance the function of NK cells;

[0055] The ligands include: Gal-9, CEACAM-1, PtdSer;

[0056] The enhancement of T cell function includes: enhancing the ability of T cells to produce IL-2 and IFN-γ; promoting the proliferation of T cells;

[0057] The enhancing the function of NK cells includes: promoting the killing activity of NK cells.

[0058] The tenth aspect of the present invention provides a method for treating a disease, comprising: administering a therapeutically effective dose of the above-mentioned anti-TIM-3 monoclonal antibody, conjugate or pharmaceutical composition to the subject.

[0059] The disease may be a tumor.

[0060] The subject refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of active compounds or agents, including the compounds of the present invention, which can cause the biological or medical response of the tissue system, animal or human sought by the researcher, veterinarian, doctor or other medical personnel, including the alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the optimal dosage and interval of the active ingredients of the present invention are determined by their properties and external conditions such as the form, route and site of administration and the specific mammal being treated, and this optimal dosage can be determined using conventional techniques. It must also be recognized that the optimal course of treatment, that is, the daily dosage of the compound over a specified period of time, can be determined by methods known in the art.

[0061] One or more beneficial technical effects of the above technical solution:

[0062] The above technical scheme obtains anti-TIM-3 monoclonal antibodies through sample screening, which have high binding activity and have good anti-tumor activity as verified by experiments. Therefore, it is expected to become a promising candidate drug for cancer immunotherapy and has the potential to be used in the prevention and treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0064] Figure 1 are DNA sequences before and after codon optimization in the embodiments of the present invention, wherein A is the DNA sequence before optimization and B is the DNA sequence after optimization.

[0065] Figure 2 The hTIM-3-GST recombinant protein was identified by SDS-PAGE in the example of the present invention, wherein lane 1 is HAVR2 / GST: 5 μl, about 0.4 mg / ml; lane 2 is BSA 10 μg; lane 3 is BSA 5 μg; and lane 4 is BSA 2 μg.

[0066] Figure 3 The ELISA method for identifying the antibody titer in serum in the embodiment of the present invention is shown in Figure 1. A is the ELISA detection of HAVR2 / GST after the second immunization; and B is the ELISA detection of HAVR2 / GST after the third immunization.

[0067] Figure 4 The figure shows the effect of the supernatants of 15 hybridoma cells on the killing efficiency of NK cells in the examples of the present invention.

[0068] Figure 5 This is the SDS-PAGE verification and subtype identification of TIM-3McAb in the examples of the present invention.

[0069] Figure 6 It is the expression level of TIM-3 in THP-1 cells after PMA stimulation in the embodiment of the present invention.

[0070] Figure 7 This is the immunofluorescence assay of the specific binding of TIM-3McAb to TIM-3 molecules on the surface of THP-1 cells in the examples of the present invention.

[0071] Figure 8 ELISA is used to determine the binding of TIM-3McAb to TIM-3 protein in the embodiment of the present invention; wherein A is a detection diagram of the binding of different TIM-3McAb to TIM-3 protein; and B is the affinity constant of different TIM-3McAb to hTIM-3 protein.

[0072] Fig. 9 ELISA detection of the effect of TIM-3McAb in blocking the binding of TIM-3 to Gal-9 in the embodiment of the present invention; wherein A is a graph showing the competitive binding of a fixed concentration of Gal-9 protein with the TIM-3McAb to be tested to hTIM-3 protein; B is a graph showing the competitive binding of a different concentration gradient of Gal-9 protein with the TIM-3McAb to be tested to hTIM-3 protein.

[0073] Fig.10 The figure shows the effect of TIM-3McAb prepared in the embodiment of the present invention on blocking the binding of TIM-3 to CEACAM-1 by flow cytometry; wherein A is the expression efficiency of CEACAM-1 in the Jurkat cell line stably expressing CEACAM-1 detected by flow cytometry; B is a flow cytometric graph of TIM-3McAb blocking the binding of TIM-3 to CEACAM-1, and C is a statistical analysis graph.

[0074] Fig.11 The figure shows the effect of TIM-3McAb prepared in the embodiment of the present invention on blocking the binding of TIM-3 to PtdSer by flow cytometry; wherein A is the PtdSer expression efficiency of Jurkat cells after 30 minutes of UV radiation treatment detected by flow cytometry; B is a flow cytometric graph of TIM-3McAb blocking the binding of TIM-3 to PtdSer, and C is a statistical analysis graph.

[0075] Fig.12In the embodiment of the present invention, TIM-3McAb increases the levels of IL-2 and IFN-γ in activated T cells; wherein, AC are the effects of TIM-3McAb on T cell expression of cytokines Il2 (A), Ifng (B) and IFN-γ (C) in T cell systems activated by anti-CD3 antibodies and anti-CD28 antibodies, respectively; DF are the effects of TIM-3McAb on T cell expression of cytokines Il2 (D), Ifng (E) and IFN-γ (F) in T cell systems activated by SEB, respectively.

[0076] Fig.13 The level of IL-2 and IFN-γ secreted by T cells in the mixed lymphocyte reaction system enhanced by TIM-3McAb in the embodiment of the present invention; wherein A is the CSFE detected by flow cytometry + IL-2 + CD8 + T cell percentage; B is a statistical analysis chart; C is the detection of CSFE by flow cytometry + IFN-γ + CD8 + T cell percentage; D is the statistical analysis chart.

[0077] Fig.14 : TIM-3McAb promotes T cell proliferation in the embodiment of the present invention; wherein A is a microscopic examination of TIM-3McAb enhancing the proliferation level of T cells, B is the detection of CFSE attenuation using flow cytometry; C is a statistical analysis graph.

[0078] Fig.15 In the embodiment of the present invention, TIM-3McAb improves the in vitro killing efficiency of NK cells; wherein A is the determination of 7-AAD by flow cytometry + CFSE + A is the cell ratio diagram; B is the statistical analysis diagram.

[0079] Fig.16 In the examples of the present invention, TIM-3McAb 15E10 dose-dependently enhanced the level of IFN-γ secretion by T cells in vitro.

[0080] Fig.17 1 is a graph showing that TIM-3McAb 15E10 in the embodiment of the present invention enhances the killing activity of NK cells in a dose-dependent manner; wherein A is a graph showing the killing effect of 15E10 on improving NK cells in vitro determined by flow cytometry based on the YAC-1-NK cell killing system; and B is a statistical analysis graph.

[0081] Fig.18The figure shows that TIM-3McAb enhances the anti-tumor activity of hTIM-3 mice in the embodiment of the present invention; wherein A is the experimental design diagram, B is the actual diagram of mouse tumor, C is the statistical analysis diagram of mouse tumor volume change; D is the statistical analysis diagram of mouse tumor weight change; and E is the mouse weight change diagram.

[0082] Fig.19 The TIM-3McAb in the embodiment of the present invention improves the function of hTIM-3 tumor-bearing mouse T cells; wherein A is 15E10 to enhance TNF-α + CD8 + T cell ratio result diagram; B is 15E10 enhanced IFN-γ + CD8 + T cell ratio results.

[0083] Fig. 20 In the embodiment of the present invention, TIM-3McAb improves the function of NK cells in hTIM-3 tumor-bearing mice; wherein A is 15E10 to enhance TNF-α + NKp46 + Cell ratio results; B is 15E10 enhanced IFN-γ + NKp46 + Cell ratio results. DETAILED DESCRIPTION

[0084] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0085] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0086] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0087] The term "antibody" or "immunoglobulin" is used as a general term to include full-length antibodies, their individual chains, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as VHH domains or VH / VL domains, respectively) whether referring to heavy chain antibodies or conventional four-chain antibodies herein. In addition, the term "sequence" used herein (such as in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0088] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0089] The term "domain" (of a polypeptide or protein) refers to a folded protein structure that is able to maintain its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed or transferred to other proteins without loss of the function of the rest of the protein and / or the domain.

[0090] The term "single (structural) domain antibody (VHH)" refers to an immunoglobulin domain comprising four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Therefore, the general structure or sequence of a single domain antibody (VHH) can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single domain antibody (VHH) confers specificity to an antigen by having an antigen binding site.

[0091] The antibody amino acid sequence of the anti-TIM-3 monoclonal antibody 15E10 was analyzed by technical means well known to those skilled in the art, and the results are as follows:

[0092] 15E10 (IgG2a, kappa)

[0093] IgG2a:

[0094] FR-H1:EVKLVESGGGLVQPGGSLRLSCATSGFTFT(SEQ ID NO.1)

[0095] <h2 style=";text-align:left;direction:ltr">CDR-H1:DYYMS(SEQ ID NO.2)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0096] <h2 style=";text-align:left;direction:ltr"> FR-H2:WVRQPPGKALEWLG(SEQ ID NO.3)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0097] <h2 style=";text-align:left;direction:ltr"> CDR-H2:FIRNKANGYTTEYSASVKG(SEQ ID NO.4)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0098] <h2 style=";text-align:left;direction:ltr"> FR-H3:RFTISRDNSQSILYLQMNTLRAEDSATYYCAR(SEQ ID NO.5)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0099] <h2 style=";text-align:left;direction:ltr"> CDR-H3:DMDY(SEQ ID NO.6)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0100] <h2 style=";text-align:left;direction:ltr"> FR-H4:WGQGTTLTVSS(SEQ ID NO.7)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0101] <h2 style=";text-align:left;direction:ltr"> kappa:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0102] <h2 style=";text-align:left;direction:ltr"> FR-L1:NIMMTQSPSSLAVSAGEKVTMSC(SEQ ID NO.8)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0103] <h2 style=";text-align:left;direction:ltr"> CDR-L1:KSSQSVLYSSNQKNYLA(SEQ ID NO.9)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0104] <h2 style=";text-align:left;direction:ltr"> FR-L2:WYQQKPGQSPKLLIY(SEQ ID NO.10)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0105] <h2 style=";text-align:left;direction:ltr"> CDR-L2:WASTRES(SEQ ID NO.11)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0106] <h2 style=";text-align:left;direction:ltr"> FR-L3:GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC(SEQ ID NO.12)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0107] <h2 style=";text-align:left;direction:ltr"> CDR-L3:HQYLSSWT(SEQ ID NO.13)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0108] <h2 style=";text-align:left;direction:ltr"> FR-L4:FGGGTKLEIK(SEQ ID NO.14)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0109] <h2 style=";text-align:left;direction:ltr"> IgG2a:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0110] MKLWLNWIFLVTLLNGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTEYSASVKG RFTISRDNSQSILYLQMNTLRAEDSATYYCARDMDYWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPV(SEQ ID NO.15)

[0111] Kappa:

[0112] MESQTQVFLSLLLWVSGTCGNIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRF TGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK(SEQ ID NO.16)

[0113] The term "humanized antibody" refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin of a non-human species, wherein the rest of the immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only a complementarity determining region (CDR) transplanted to an appropriate framework region in a variable domain. The antigen binding site may be wild type, or modified by one or more amino acid substitutions, such as modification to be closer to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences (e.g., humanized single domain antibodies containing all three CDRs from alpacas). Other forms have one or more CDRs that have been altered relative to the original antibody.

[0114] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence identity" indicates the percentage of amino acids substituted with the same amino acid. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.

[0115] The term "affinity" is theoretically defined by the equilibrium association between intact antibody and antigen. Affinity herein can be measured by K DThe dissociation constant (or other determination method) is evaluated or determined, for example, by bio-layer interferometry (BLI) using a FortebioRed96 instrument.

[0116] An "effective amount" of an agent is that amount necessary to effect a physiological change in the cell or tissue to which it is administered.

[0117] A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes or prevents the adverse effects of a disease.

[0118] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.

[0119] The term "pharmaceutical composition" refers to a preparation which is in such form that the biological activity of the active ingredients contained therein is effective, and which contains no other ingredients which are unacceptably toxic to a subject to which the composition would be administered.

[0120] The term "treatment / prevention" refers to an attempt to alter the natural course of a disease in a treated individual, and may be a clinical intervention performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the formation of a disease or slow the progression of a condition.

[0121] The present invention is further explained by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present examples all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related conventional techniques in the field of the present technology. These technologies have been fully described in the existing literature.

[0122] Example

[0123] 1. Research Methods

[0124] 1.1 Preliminary screening of TIM-3 McAb

[0125] The NK-92 cell-K562 cell killing system was used to determine the effect of hybridoma supernatant on the killing ability of NK-92 cells, and the supernatant of 16 positive cells after fusion screening was preliminarily screened. The steps are as follows.

[0126] (1) Use NK-92 cells as effector cells. Take NK-92 cells, centrifuge at 800 rpm for 3 minutes, discard the supernatant, and resuspend in culture medium. Add TIM-3McAb at a concentration of 5 μg / mL (Mouse-IgG Isotype Control as a negative control) and incubate at 37°C for 2 hours.

[0127] (2) Use Fluc-K562 cells containing the firefly luciferase (Fluc) reporter gene as target cells, resuspend them in culture medium, and count them.

[0128] (3) According to the effector-target ratio, NK-92 cells were co-cultured with Fluc-K562 cells and incubated in a 37°C incubator for 6 h.

[0129] (4) Observe the status of the co-cultured cells under a microscope, add cell lysis buffer, shake on a shaker at room temperature for 10 min, and place the well plate in a -80 refrigerator to freeze and thaw the cells 1 to 3 times.

[0130] (5) After taking the cell culture plate out of the -80°C ultra-low temperature freezer, wait for it to return to room temperature, blow the cell lysate evenly, and transfer it to the ELISA plate.

[0131] (6) Add Luciferase substrate and use a microplate luminometer to detect the Luciferase value of the lysed cells in each well.

[0132] (7) The killing efficiency of NK-92 cells in different groups was calculated using the calculation formula: Killing rate of group A (%) = (average maximum release pore value - detection value of group A) / maximum release pore value × 100%. 1.2 SDS-PAGE identification of purified TIM-3McAb

[0133] (1) The purified TIM-3McAb was mixed with an equal amount of 2×SDS sample buffer, incubated at 100°C in a water bath for 3 min, and 10 μL of sample was added to each well.

[0134] (2) Electrophoresis: The concentration of the concentrated gel was 3.5% and the current intensity was 20 mA.

[0135] The separation gel concentration was 7.5% and the current intensity was 30 mA (non-reducing).

[0136] The concentration of the concentrated gel was 5% and the current intensity was 18 mA.

[0137] The separation gel concentration was 15% and the current intensity was 24 mA (reducing).

[0138] After about 3 hours of electrophoresis, when the bromophenol blue indicator contained in the sample migrates to about 3 mm from the bottom edge, the electrophoresis stops and the gel is peeled off. After staining with Coomassie brilliant blue methanol water for about 1 to 2 hours, the gel is destained until the protein bands are clear. After destaining, the gel is imaged and scanned for storage.

[0139] 1.3 Determination of TIM-3McAb cell binding activity

[0140] 1.3.1PMA promotes high expression of TIM-3 in THP-1 cells

[0141] According to 2×10 per well 6 THP-1 cells were seeded on a cell slide in a 24-well culture plate at a concentration of 10 cells / well. 50 ng / mL PMA was added to the THP-1 cell culture system and incubated at 37°C and 5% CO 2 After culturing in a cell culture incubator for 24 h, the cell adhesion status was observed under a microscope.

[55] Take the above about 5×10 5 Place cells in a centrifuge tube and centrifuge at 800 rpm for 5 min. Discard the supernatant and resuspend the cell pellet in PBS. Label with flow cytometry antibody APC-anti-human TIM-3 and incubate at 4°C in the dark for 30 min. After the antibody incubation, add 1 mL PBS to each tube, centrifuge at 3000 rpm for 3 min, discard the supernatant, resuspend in 200 μL PBS, and use flow cytometry to determine the expression level of TIM-3 in THP-1 cells.

[0142] 1.3.2 Immunofluorescence assay of TIM-3McAb cell binding activity

[0143] (1) The culture medium of THP-1 cells induced to adhere with phorbol myfismte acetate (PMA) in the above 24-well plate was discarded, and 1 mL of 5 μg / mL TIM-3McAb (Mouse-IgG Isotype Control as a negative control and commercial TIM-3 antibody MAB005 as a positive control) diluted in PBS was slowly added to each well and incubated at 4°C for 1 h.

[0144] (2) After incubation, the cells in the 24-well culture plate were washed three times with PBS, and 1 mL of 4% paraformaldehyde was added to each well for fixation for 15 min.

[0145] (3) After fixation, discard the paraformaldehyde and add PBS to the well plate and wash three times.

[0146] (4) Add goat serum to the cell slides in the 24-well plate and block at room temperature for 30 minutes. After blocking, discard the goat serum and add PBS to the plate for washing three times.

[0147] (5) Add FITC-anti Mouse IgG to the cell slides, incubate at 37°C for 30 min, and wash three times with PBST, each time for 3 min.

[0148] (6) Add 4',6-diamidino-2-phenylindole (DAPI) fluorescent dye to the cell slide, incubate at room temperature in the dark for 5 min, and wash three times with PBST.

[0149] (7) Seal the slides with a sealing solution containing an anti-fluorescence quencher, and then observe and collect images under a fluorescence microscope.

[0150] 1.4 ELISA to determine the affinity of TIM-3 monoclonal antibody

[0151] The binding affinity of TIM-3 antibodies to human TIM-3 protein was measured by ELISA.

[0152] (1) Coating: Dilute TIM-3 protein with PBS to a final concentration of 2 μg / mL, add 100 μL per well to the ELISA plate, seal the plate and place at 4°C for 24 h.

[0153] (2) Washing: discard the coating solution, use 0.05% PBST to wash the unbound coating solution in the ELISA plate, add 200 μL of PBST to each well of the ELISA plate, and wash by manual shaking for 3 times.

[0154] (3) Blocking: Use 1% bovine serum albumin (BSA) as the blocking solution, add 300 μL per well to the ELISA plate, and place at room temperature for 1 hour.

[0155] (4) The purified antibody to be tested was diluted with 1% BSA in a series of concentrations (100, 50, 10, 5, 1, 0.1, 0.01, 0.001 μg / mL), and 100 μL per well was added to the ELISA plate and incubated at 37° C. for 1 h.

[0156] (5) Washing: Use 0.05% PBST for washing. Add 200 μL of PBST to each well of the ELISA plate and wash by manual shaking for 3 times.

[0157] (6) Add enzyme-labeled secondary antibody: HRP-Goat anti-Mouse IgG was diluted with 1% BSA and 100 μL was added to each well of the ELISA plate. The reaction was incubated at 37°C for 1 h.

[0158] (7) Washing: Use 0.05% PBST for washing. Add 200 μL of PBST to each well of the ELISA plate and wash by manual shaking for 4 times.

[0159] (8) Color development: Add 100 μL of substrate solution to each well of the ELISA plate, react for 30 min at room temperature in the dark, and then add stop solution to terminate the reaction.

[0160] (9) Use an ELISA reader to measure the OD value at 450 nm and 630 nm and record the calculation.

[0161] 1.5 Determination of the blocking effect of TIM-3 monoclonal antibodies on the binding of TIM-3 to its ligand

[0162] 1.5.1 ELISA to detect the blocking effect of TIM-3McAb on the binding of TIM-3 to Gal-9

[0163] (1) Dilute TIM-3 protein to 1 μg / mL with PBS, add 100 μL per well to the ELISA plate, and place at 4°C for 24 h.

[0164] (2) Discard the coating solution and use 0.05% PBST to wash the unbound coating solution in the ELISA plate. Add 200 μL of PBST to each well of the ELISA plate and wash by manual shaking for 3 times.

[0165] (3) Use 1% BSA as blocking solution, add 300 μL per well to the ELISA plate and leave at room temperature for 1 hour.

[0166] (4) Wash with 0.05% PBST. Add 200 μL of PBST to each well of the ELISA plate and wash with manual shaking for 3 times.

[0167] (5) Gal-9 protein diluted to different concentrations (0, 5, 10, 50, 100, 500 μg / mL) was incubated with the TIM-3 antibody to be tested at 37°C for 1 h.

[0168] (6) Add 100 μL of the above mixture to each well of the ELISA plate and react at 37°C for 1 h.

[0169] (7) Wash with 0.05% PBST. Add 200 μL of PBST to each well of the ELISA plate and wash by manual shaking for 3 times.

[0170] (8) HRP-Goat anti-Mouse IgG was diluted with 1% BSA and 100 μL was added to each well of the ELISA plate and reacted at 37°C for 1 h.

[0171] (9) Wash the ELISA plate five times with PBST, add substrate reaction solution, incubate at room temperature in the dark for 30 min, and add stop solution.

[0172] (10) Use an ELISA reader to measure the OD value at 450 nm and 630 nm.

[0173] 1.5.2 Flow cytometry detection of the blocking effect of TIM-3McAb on the binding of TIM-3 to CEACAM-1

[0174] (1) Flow cytometry to detect the expression efficiency of CEACAM-1 on Jurkat-CEACAM-1 cells. Jurkat-CEACAM-1 cells were placed in a centrifuge tube and centrifuged at 800 rpm for 5 min. The supernatant was discarded and the cell pellet was resuspended in PBS. The cells were labeled with flow cytometry antibody BV421-anti-human CD66 and incubated at 4°C in the dark for 30 min. After the antibody incubation, 1 mL of PBS was added to each tube and centrifuged at 3000 rpm for 3 min. The supernatant was discarded and the pellet was resuspended in 200 μL of PBS. The pellet was filtered through a 200-mesh nylon mesh and then tested on a flow cytometer.

[0175] (2) Incubate TIM-3 protein at a concentration of 10 μg / mL with TIM-3McAb at a concentration of 10 μg / mL (Mouse-IgGIsotype Control as a negative control) on ice for 20 minutes. Incubate the above co-incubated mixture with Jurkat-CEACAM-1 cells on ice for 30 minutes. Label CEACAM-1 with flow cytometry antibody BV421-anti-human CD66 and label TIM-3 with APC-anti-human TIM-3, and incubate at 4°C in the dark for 30 minutes. After the antibody incubation, add 1 mL of PBS to each tube, centrifuge at 3000 rpm for 3 minutes, discard the supernatant, resuspend in 200 μL PBS, filter, and detect CEACAM-1 by flow cytometry. + TIM-3 + Fluorescence intensity of cells.

[0176] 1.5.3 Flow cytometry detection of the blocking effect of TIM-3McAb on the binding of TIM-3 to PtdSer

[0177] (1) Flow cytometry was used to detect the expression efficiency of PtdSer on Jurkat cells after UV irradiation. Jurkat cells were treated with UV irradiation for 30 min to induce cell apoptosis, and untreated Jurkat cells were used as negative control. FITC-Annexin V staining was performed using Annexin V binding buffer, and cell apoptosis was detected by flow cytometry, which was used for subsequent experiments.

[0178] (2) TIM-3 protein was pre-incubated with TIM-3McAb or Mouse-IgG Isotype Control (as a negative control) on ice for 30 min. Then, Jurkat cells induced by UV radiation to undergo apoptosis were incubated with the above pre-incubated mixture on ice for 30 min. TIM-3 was labeled with APC-anti-human TIM-3, and apoptotic cells were labeled with FITC-Annexin V staining using Annexin V binding buffer. Annexin V was analyzed by flow cytometry. + TIM-3 + The proportion of positive cells.

[0179] 1.6 Effect of TIM-3 mAb on T cell cytokine secretion levels

[0180] 1.6.1 Extraction of human PBMC

[0181] Extract PBMC using density gradient centrifugation. Draw 5-10 mL of peripheral blood from healthy volunteers, add the same volume of PBS and mix thoroughly. Prepare a new centrifuge tube, add the same volume of Ficoll as the peripheral blood, and then slowly add the peripheral blood and PBS mixture to the surface of Ficoll. Centrifuge the above mixture at 2000 rpm for 20 minutes, aspirate the middle white film layer cells, add 2-3 times the volume of PBS, centrifuge at 1500 rpm for 5 minutes, resuspend with culture medium, and count.

[0182] 1.6.2 SEB activated human PBMC

[0183] The superantigen Staphylococcal enterotoxin B (SEB) was used to stimulate human PBMC and activate T cells to measure the effect of TIM-3 antibody on T cell activation. The extracted PBMCs were inoculated in a 24-well plate, SEB was added at a concentration of 100 ng / mL, and incubated with TIM-3McAb at a concentration of 10 μg / mL (Mouse-IgG IsotypeControl as a negative control) for 5 days. After collecting the cells, centrifugation was performed, and the supernatant and cell pellet were taken to analyze the expression levels of IFN-γ and IL-2 by ELISA and qRT-PCR, respectively.

[0184] 1.6.3 Anti-CD3 / CD28 antibody activation of human PBMC

[0185] After mixing anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (5 μg / mL), 500 μL per well was added to a 24-well plate and incubated at 4°C overnight. The next day, the antibody was discarded, human PBMCs were added, and the plate was incubated at 37°C and 5% CO.2 Cultured in a cell culture incubator, the culture medium was replaced after 72 hours. TIM-3McAb (Mouse-IgG Isotype Control as a negative control) was added at a concentration of 10 μg / mL and incubated for 5 days. After collecting the cells, centrifugation was performed, and the supernatant and cell pellet were taken to analyze the expression levels of IFN-γ and IL-2 by ELISA and qRT-PCR, respectively.

[0186] 1.6.4 ELISA analysis

[0187] (1) Dilute the Capture Antibody with 1× Coating Buffer and add 100 μL per well to a 96-well plate. Seal the plate and incubate overnight at 4°C.

[0188] (2) Wash away unbound antibodies using wash buffer. Add 300 μL of wash buffer to each well and wash four times. After each wash, invert the plate and tap it firmly on absorbent paper to remove residual buffer.

[0189] (3) Add 200 μL of 1× Assay Diluent A to each well and incubate at room temperature for 1 h.

[0190] (4) Wash the plate 4 times with wash buffer.

[0191] (5) Dilute the standard or sample to the target concentration using 1× Assay Diluent A, add 100 μL to each well of the plate, and incubate at room temperature for 2 h.

[0192] (6) Wash the plate 4 times with wash buffer.

[0193] (7) Add 100 μL of 1× Detection Antibody solution to each well and incubate at room temperature for 1 h.

[0194] (8) Wash the plate 4 times with wash buffer.

[0195] (9) Add 100 μL of 1× Avidin-HRP solution to each well and incubate at room temperature for 30 min.

[0196] (10) Wash the plate 5 times with washing buffer. Add 100 μL of substrate solution to each well and incubate in the dark for 15 min.

[0197] (11) Add 100 μL of stop solution to each well and measure the absorbance at 450 nm using a microplate reader within 15 min.

[0198] 1.6.5 qRT-PCR analysis

[0199] (1) RNA extraction

[0200] Take the cell pellet, add 200μL TRIzol, mix and lyse at room temperature for 15min. Add 200μL chloroform, mix thoroughly, let stand at room temperature for 10min, and centrifuge at 4℃, 12000rpm for 15min. After centrifugation, the sample is divided into three layers, the upper colorless aqueous phase is the RNA layer, the middle is cloudy, and the bottom is the organic phase. Pipette the upper aqueous phase and transfer it to a 1.5mL RNase-free EP tube, add an equal volume of isopropanol, invert and mix, let stand at room temperature for 10min, centrifuge at 4℃, 12000rpm for 15min, discard the supernatant, and obtain the white precipitate at the bottom. Add 1mL75% diethylpro carbonate (DEPC)-ethanol, centrifuge at 4℃, 12000rpm for 5min, discard the supernatant, add 40mL DEPC water to dissolve, and store at -80℃ for use.

[0201] (2) Reverse transcription

[0202] Use Thermo's reverse transcription kit, take 1mg RNA as template, 4μL 5×Reaction Buffer, 1μL Oligo dT, 2μL 10mM dNTPs, 1μL RNase inhibitor and 1μL Reverse trancriptase, RNase Free water, the above system is 20μL. Then, place it in a 42℃ water bath for 1h and a 72℃ water bath for 5min to terminate the reaction. The product is cDNA. Add 80μL double distilled water (DDW) to the cDNA sample and mix well to obtain the cDNA working solution for subsequent qRT-PCR experiments.

[0203] (3) qPCR

[0204] The qPCR reaction system was as follows: 10 μL SYBR Green qPCR Master Mix, 0.5 μL Primer F (10 μM), 0.5 μL Primer R (10 μM), and 9 μL cDNA working solution.

[0205] 1.7 Mixed lymphoid reaction

[0206] (1) The spleens of 7- to 8-week-old male BALB / c-hTIM-3 mice and C57BL / 6 wild-type mice were obtained and prepared into spleen single cell suspensions.

[0207] (2) The spleen single cell suspension of BALB / c-hTIM-3 mice was centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 5 μM CFSE working solution. The cells were placed at 37°C and 5% CO2 Incubate in a cell culture incubator for 15 min, centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend in PBS as the reaction cell.

[0208] (3) Treat spleen single cells from C57BL / 6 wild-type mice with Mitomycin C at a concentration of 25 μg / mL and incubate at 37°C in the dark for 30 min. Centrifuge at 800 rpm for 5 min, discard the supernatant, resuspend in PBS, wash twice, and resuspend the cell pellet in complete medium to serve as stimulator cells.

[0209] (4) The spleen single cells of the above BALB / c-hTIM-3 mice and C57BL / 6 wild-type mice were transferred to a 24-well plate for co-incubation, and TIM-3McAb at a concentration of 10 μg / mL (Mouse-IgG Isotype Control was used as a negative control) was added and incubated at 37°C and 5% CO. 2 The cells were cultured in a cell culture incubator for 5 days.

[0210] (5) Collect cells and detect the proliferation level of CFSE-labeled cells by flow cytometry.

[0211] 1.8 Flow cytometry to detect the effect of TIM-3 monoclonal antibody on NK cell killing ability

[0212] (1) Centrifuge the YAC-1 cells at 800 rpm for 5 min, discard the supernatant, and resuspend the YAC-1 cells in 5 μM CFSE working solution diluted with PBS. Incubate the YAC-1 cells in a 37°C cell culture incubator for 15 min.

[0213] (2) The incubated YAC-1 cells were centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS.

[0214] (3) The above-mentioned CFSE-labeled YAC-1 cells were seeded in a 96-well plate as target cells.

[0215] (4) The spleen of hTIM-3 mice was harvested and prepared into a spleen single cell suspension.

[0216] (5) The hTIM-3 mouse spleen single cell suspension prepared above was incubated with 5 μg / mL TIM-3 monoclonal antibody (Mouse-IgG Isotype Control was used as a negative control) at 37°C for 2 h.

[0217] (6) After the co-incubation, the cells were inoculated into the 96-well plate containing YAC-1 cells at different effector-target ratios of 10:1, 20:1, and 50:1, and incubated in a cell culture incubator at 37°C for 6 h.

[0218] (7) The cells were transferred to a flow cytometry tube and centrifuged at 800 rpm for 5 min. The culture medium was discarded and the cells were resuspended in PBS buffer containing the nucleic acid dye 7-AAD. After incubation at 4°C in the dark for 5 min, CFSE was detected by flow cytometry. + 7-AAD + The proportion of cells.

[0219] 1.9 In vivo experiments in mice to detect the anti-tumor activity of TIM-3McAb

[0220] 1.9.1 H22 Cell Expansion

[0221] H22 cells were taken out of the -80℃ ultra-low temperature freezer and revived in a 37℃ water bath. After complete dissolution, 500μL was aseptically taken out in the clean bench and injected into the peritoneal cavity of BALB / c mice by intraperitoneal injection. After 5 to 7 days, the abdomen of the mouse was significantly swollen, and ascites tumors had formed. The mice were killed by cervical dislocation, the peritoneal fluid was aspirated, and the obtained cells were centrifuged at 1200rpm for 5min. The cells were resuspended in PBS and counted.

[0222] 1.9.2H22 Subcutaneous Tumor Formation and Drug Injection

[0223] In this study, 7- to 8-week-old male BALB / c-TIM-3-HU mice with humanized TIM-3 were used as research subjects. Each mouse was injected subcutaneously at the thigh root with approximately 2×10 6 H22 cells. The experimental mice were randomly divided into 2 groups, with 4 mice in each group. Tumor-bearing mice were injected intraperitoneally on the 7th, 10th, 13th and 16th days respectively. The experimental group mice were injected with TIM-3McAb (10 mg / kg) each time, and the control group mice were injected with Mouse-IgG Isotype Control (10 mg / kg) each time. The tumor size was recorded every 3 days, and the survival status of the mice was observed and the survival period was recorded. The tumor volume calculation formula is as follows: 4π / 3×(tumorlength / 2)×(tumor width / 2) 2 On the 18th day, the mice were euthanized by cervical dislocation, and the spleen and tumor of the mice were removed, photographed and weighed, and single cells in the spleen and mononuclear cells in the tumor tissue were extracted.

[0224] The steps for extracting mononuclear cells from tumor tissue are as follows:

[0225] (1) Cut the excised mouse tumor tissue into small pieces and digest it with collagenase II at 37°C for 1 h;

[0226] (2) Take out and shake every 15 minutes;

[0227] (3) Centrifuge at 600 rpm for 2 min, remove large precipitates and take the supernatant;

[0228] (4) Centrifuge at 1000 rpm for 8 min and discard the supernatant;

[0229] (5) Resuspend the pellet with 8 to 10 mL of red blood cell lysis buffer, lyse on ice for 8 min, centrifuge at 1200 rpm for 8 min, discard the supernatant, and retain the cell pellet;

[0230] (6) Resuspend the cell pellet in 40% percoll solution and centrifuge at 1200 rpm for 5 min to obtain mouse tumor tissue mononuclear cells.

[0231] 1.9.3 Detection of cell surface molecules by flow cytometry

[0232] The mouse tumor tissue mononuclear cells and spleen mononuclear cells obtained above were resuspended in PBS and counted. 6 Transfer cells to flow tubes. Add the corresponding flow antibody mixed solution to the above cell suspension and incubate at 4°C in the dark for 30 minutes. Add 1 mL PBS to each tube, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, resuspend the cell pellet in 200 μL PBS per tube, filter through a 200-mesh nylon mesh, and detect on a flow cytometer.

[0233] 1.9.4 Flow cytometry to detect intracellular molecules

[0234] (1) The mouse tumor tissue mononuclear cells and spleen mononuclear cells obtained above were resuspended in RPMI1640 medium containing PMA (concentration 50 ng / mL) and Lonomycin (concentration 1 μg / mL) and counted.

[0235] (2) The above cells were cultured at 2×10 6 Cells were seeded into 24-well plates and incubated at 37°C with 5% CO 2 Incubate in cell culture incubator for 2 h.

[0236] (3) Add 1 μL BFA to each well and incubate at 37°C and 5% CO 2 Incubate in a cell culture incubator for 4 h.

[0237] (4) After incubation, the cells were transferred to a flow cytometry tube, PBS was added, and the tube was centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and 100 μL of IC Fixation Buffer was added to each tube of cell pellet to resuspend it and incubate at 4°C for 20 min.

[0238] (5) Centrifuge at 1000 rpm for 3 min, discard the supernatant, add the flow cytometry antibody mixture prepared in Permeabilization Buffer, and incubate at 4°C in the dark for 30 min.

[0239] (6) After the antibody incubation, add 1 mL of PBS to each tube, centrifuge at 1000 rpm for 3 min, resuspend in 200 μL of PBS, filter through a 200-mesh nylon mesh, and detect on a flow cytometer.

[0240] 1.10 Statistical analysis

[0241] In this study, the data were processed using GraphPad Prism 8.0 software. The experiments were repeated three times in each group and analyzed using unpaired student's t test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no statistical significance.

[0242] 2. Research Results

[0243] 2.1 Preparation of TIM-3McAb

[0244] The hTIM-3 extracellular segment recombinant protein was obtained by prokaryotic expression, mice were immunized and serum antibodies were detected, and hybridoma cell lines producing TIM-3McAb were prepared and screened using hybridoma technology (commissioned by Beijing Aibo Biotechnology Co., Ltd.).

[0245] 2.2 Construction of recombinant plasmid pGEX6P1-hTIM-3

[0246] Referring to the gene sequence of hTIM-3 in GenBank, the gene was codon optimized according to the codon preference of Escherichia coli without changing the amino acid sequence. For example, the rare codons CGG, AGG, AGA and CGA encoding arginine were replaced with CGC or CGT, and the rare codons CCC and CCA encoding proline were replaced with CCG. Before codon optimization ( Figure 1 A) Figure 1 B) The gene sequence is as follows Figure 1 The human TIM-3 extracellular domain gene fragment synthesized after the above codon optimization was cloned into the pGEX-6P-1 vector to construct the recombinant plasmid pGEX-6P-1-hTIM-3.

[0247] 2.3 SDS-PAGE analysis of hTIM-3-GST recombinant protein

[0248] Next, SDS-PAGE was used to further verify the hTIM-3 extracellular domain recombinant protein. Among them, the BSA control sample was used as a molecular weight standard and loading control. According to the NCBI protein database, the size of the TIM-3 extracellular segment protein is about 23kDa, and the size of GST is about 26KDa. The results of the SDS-PAGE experiment are as follows Figure 2 As shown, the molecular weight of the sample protein is about 49 kDa, which confirms that the hTIM-3-GST recombinant protein band size we purified is correct and is indeed the desired hTIM-3 extracellular domain recombinant protein.

[0249] 2.4ELISA to identify the titer of antibodies in serum

[0250] The hTIM-3 recombinant protein purified above was used as an antigen to immunize mice three times, and the serum collected after the second and third immunizations was graded diluted, and the antibody titer in the serum was detected by ELISA. Figure 3 The experimental results showed that after three immunizations, the serum antibody titer of experimental mouse 1 reached more than 1 / 16000, experimental mouse 2 reached more than 1 / 8000, experimental mouse 3 reached more than 1 / 32000, experimental mouse 4 reached more than 1 / 16000, and experimental mouse 5 reached more than 1 / 8000, indicating that the immunization was successful and the next step of monoclonal antibody preparation could be carried out.

[0251] 2.5 Screening and purification of TIM-3 McAb

[0252] 2.5.1 Preliminary screening of TIM-3 McAb

[0253] The spleen cells of mice immunized with hTIM-3 recombinant protein were separated and fused with mouse myeloma cells SP2 / 0. Monoclonal antibodies were prepared by hybridoma technology. ELISA detection and identification were performed to obtain 15 hybridoma cell lines that stably secreted anti-hTIM-3 protein monoclonal antibodies. Next, the supernatants of the above 15 hybridoma cells were screened using the NK-92 cell-K562 cell killing system. Based on the killing activity of NK-92 cells, hybridoma cell lines that can promote the killing function of NK cells were screened. The killing efficiency was calculated according to the corresponding formula, and the results are as follows: Figure 4 As shown. Compared with the IgG control group, except for 16H9, the supernatants of the other 16 hybridoma cells can effectively enhance the in vitro killing effect of NK cells. Among them, the supernatants of the seven hybridoma cells, 1C4, 3A4, 10F1, 10H3, 15E10, 14G6, and 10B10, significantly enhanced the in vitro killing effect of NK cells. Five hybridoma cells, 1C4, 3A4, 10F1, 10H3, and 15E10, were selected for antibody purification.

[0254] 2.5.2 SDS-PAGE verification of purified TIM-3 McAb

[0255] The purified 1C4, 3A4, 10F1, 10H3, and 15E10 TIM-3McAbs were analyzed by SDS-PAGE. The experimental results are shown in Figure 5 As shown in A. Reducing SDS-PAGE detected that the above five strains of TIM-3McAb all contained a heavy chain of about 55 kDa and a light chain of about 25 kDa, confirming that the prepared antibodies were indeed correct, and further sequencing was performed to obtain their subtype information ( Figure 5 B).

[0256] 2.6 Cell binding activity and affinity of TIM-3McAb

[0257] 2.6.1 TIM-3McAb binds to the THP-1 cell surface

[0258] Next, the binding activity of the purified 1C4, 3A4, 10F1, 10H3, and 15E10 TIM-3McAbs to the TIM-3 molecules on the surface of THP-1 cells was determined by immunofluorescence. First, THP-1 cells were stimulated with PMA to highly express TIM-3. Flow cytometry results showed that after 50 ng / mL PMA continuously stimulated THP-1 cells for 24 hours, the expression level of TIM-3 in THP-1 cells could reach 50.4%.

[0259] The results of immunofluorescence detection showed that compared with the IgG isotype control, 1C4, 3A4, 10F1, 10H3, 15E10 and commercial mouse anti-human TIM-3McAb (MAB2365) could specifically bind to the TIM-3 molecules on the cell surface.

[0260] 2.6.2 Affinity of TIM-3 McAb

[0261] In order to evaluate the binding of the prepared anti-human TIM-3 McAb to human TIM-3 protein, the affinity constants of 1C4, 3A4, 10F1, 10H3, and 15E10 to hTIM-3 protein were determined by ELISA. Figure 8 As shown in A and B, the affinity constants of 1C4, 3A4, 10F1, 10H3, and 15E10 for hTIM-3 protein are 7.93E-8, ​​6.1E-9, 1.54E-8, 6.7E-9, and 3.9E-9, respectively. All five TIM-3McAbs have high affinity for human TIM-3 protein.

[0262] 2.7 Blocking effect of TIM-3McAb on the binding of TIM-3 to its ligand

[0263] 2.7.1 TIM-3McAb blocks TIM-3 binding to Gal-9

[0264] In order to verify the blocking effect of the prepared TIM-3McAb on the binding of TIM-3 to its ligand, the competitive binding ELISA method was used to analyze whether the prepared TIM-3McAb could block the interaction between TIM-3 and its ligand Gal-9. The plate was coated with hTIM-3 protein, and a fixed concentration of Gal-9 protein or a gradient dilution of 0, 5, 10, 50, 100 and 500 μg / mL of Gal-9 protein was made to compete with the tested TIM-3McAb for binding to hTIM-3 protein. IgG was used as a negative control. The results showed that compared with IgG, except for 10F1, 1C4, 3A4, 10H3 and 15E10 could compete with Gal-9 protein for binding to hTIM-3 protein, and inhibit the binding of TIM-3 to Gal-9 to a certain extent ( Fig. 9 A, B). 2.7.2 TIM-3McAb blocks the binding of TIM-3 to CEACAM-1

[0265] To verify the blocking effect of the TIM-3McAb we prepared on the binding of TIM-3 to its ligand CEACAM-1, flow cytometry was used to analyze the ability of the prepared TIM-3McAb to block the interaction between TIM-3 and its ligand CEACAM-1. First, flow cytometry was used to detect the expression efficiency of CEACAM-1 in the Jurkat cell line stably expressing CEACAM-1, which was about 97.4% ( Fig.10 A). Next, 10 μg / mL of TIM-3 protein was co-incubated with the TIM-3McAb or IgG control to be tested, and then co-incubated with Jurkat cells overexpressing CEACAM-1 on ice, and CEACAM-1 was detected by flow cytometry. + TIM-3 + The fluorescence intensity represents the binding efficiency of TIM-3 and CEACAM-1. Fig.10 The experimental results of B and C showed that the binding efficiency of TIM-3 and CEACAM-1 in the IgG control group was about 35.7%, while the binding efficiency of TIM-3 and CEACAM-1 in the 1C4, 3A4, 10F1, 10H3 and 15E10 groups was 28.9%, 28.9%, 28.9%, 28.9% and 28.9%, respectively ( Fig.10 B). This indicates that antibodies 1C4, 3A4, 10F1, 10H3 and 15E10 can block the binding of TIM-3 to CEACAM-1 to varying degrees ( Fig.10 C).

[0266] 2.7.3 TIM-3McAb blocks TIM-3 binding to PtdSer

[0267] PtdSer is a phospholipid abundant in the plasma membrane of eukaryotic cells. Flipping enzymes usually keep PtdSer on the inner side of the cell membrane of living cells. After apoptosis, PtdSer is flipped and exposed to the cell surface under the action of disrupting enzymes. PtdSer acts as an "eat me" signal for dead cells. Among them, TIM-3 is one of the PtdSer-specific receptors, which can mediate the phagocytic effect of phagocytes on apoptotic cells. In addition, Annexin V has a high affinity for PtdSer, and Annexin V labeled with fluorescein can be used to detect early apoptotic cells. Based on this, this project used flow cytometry to analyze the ability of the prepared TIM-3McAb to block the interaction between TIM-3 and its ligand PtdSer.

[0268] First, Jurkat cells were induced to apoptosis by UV irradiation for 45 min, and Jurkat cells without irradiation were used as negative control. After FITC-Annexin V flow cytometry staining, the PtdSer level of Jurkat cells was determined by flow cytometry. Fig.11 A shows that the PtdSer expression efficiency of Jurkat cells reached 89% after 30 min of UV radiation treatment. Next, TIM-3 protein was co-incubated with the TIM-3McAb or IgG control to be tested, and then co-incubated with Jurkat cells expressing PtdSer on ice, and Annexin V was analyzed by flow cytometry. + TIM-3 + Positive cells represent the binding efficiency of TIM-3 and PtdSer. The results showed that the binding efficiency of TIM-3 and PtdSer on the surface of apoptotic cells in the IgG control group was about 10.3%, while the binding efficiency of TIM-3 and PtdSer in the 1C4, 3A4, 10F1, 10H3 and 15E10 groups was 1.53%, 6.73%, 5.01%, 2.79% and 3.01%, respectively. Fig.10 B). The above results show that compared with the IgG control group, antibodies 1C4, 3A4, 10F1, 10H3 and 15E10 can block the binding of TIM-3 to PtdSer on the surface of apoptotic cells to a certain extent ( Fig.10 C).

[0269] 2.8TIM-3McAb enhances T cell function

[0270] 2.8.1TIM-3McAb enhances the ability of T cells to produce IL-2 and IFN-γ

[0271] Human peripheral blood mononuclear cells were stimulated with anti-CD3 antibody / anti-CD28 antibody and SEB, respectively. After 72 hours of culture, the levels of IFN-γ and IL-2 in the supernatant were detected by qRT-PCR and ELISA. The experimental results showed that in the T cell system activated by anti-CD3 antibody and anti-CD28 antibody, 1C4, 10F1, 10H3 and 15E10 could increase the level of cytokine Il2 expressed by T cells to varying degrees compared with the IgG control group ( Fig.12 A), antibodies 1C4, 3A4, 10F1, 10H3 and 15E10 can increase the level of cytokine IFN-γ produced by T cells to varying degrees ( Fig.12 B, C). Consistently, in the T cell system activated by SEB, 1C4, 3A4, 10F1, 10H3 and 15E10 could increase the levels of cytokines IL-2 and IFN-γ produced by T cells to varying degrees compared with the IgG control group ( Fig.12 DF).

[0272] In addition, the effect of the prepared TIM-3McAb on the secretion of IL-2 and IFN-γ by T cells in vitro was further verified by the mixed lymphocyte reaction system. Splenic mononuclear cells from C57BL / 6 wild-type mice were treated with Mitomycin C and co-cultured with CSFE-labeled spleen mononuclear cells from BALB / c-hTIM-3 mice. Flow cytometry was used to detect CSFE + IL-2 + CD8 + T cells and IFN-γ + CD8 + The results showed that compared with the IgG control group, antibodies 1C4, 3A4, 10F1, 10H3 and 15E10 could enhance the secretion of IL-2 by T cells ( Fig.13 A, B) and IFN-γ capacity ( Fig.13 C, D).

[0273] 2.8.2 TIM-3McAb promotes T cell proliferation

[0274] The effect of the prepared TIM-3McAb on T cell proliferation in vitro was further verified by a mixed lymphocyte reaction system. Splenic mononuclear cells of C57BL / 6 wild-type mice were treated with Mitomycin C and mixed with spleen mononuclear cells of BALB / c-hTIM-3 mice, and each TIM-3McAb and IgG antibody were added for co-incubation. The growth status of each group of cells was observed under a microscope, such as Fig.14As shown in A, compared with the IgG control group, antibodies 1C4, 3A4, 10F1, 10H3 and 15E10 can enhance the proliferation level of T cells, and microscopic observation shows that the cell colonies are enlarged and / or increased. In addition, spleen mononuclear cells of BALB / c-hTIM-3 mice were labeled with CFSE, and the attenuation of CFSE was detected by flow cytometry, as shown in Figure 2. Fig.14 B and Fig.14 As shown in C, antibodies 1C4, 3A4, 10F1, 10H3, and 15E10 significantly promoted T cell proliferation compared with the IgG control group.

[0275] 2.9TIM-3McAb enhances the killing effect of NK cells

[0276] Next, the effect of TIM-3McAb on the level of NK cell killing in vitro was further determined. CFSE-labeled YAC-1 cells were mixed with mouse spleen cell single cell suspension at three specific effector-target ratios of 10:1, 20:1, and 50:1, and co-incubated with each TIM-3McAb and IgG. 7-AAD was measured by flow cytometry. + CFSE + The experimental results of flow cytometry are as follows: Fig.15 A and Fig.15 As shown in Figure B, as the effector-target ratio increases, the NK cell killing efficiency also increases, and at the three effector-target ratios of 10:1, 20:1 and 50:1, antibodies 1C4, 3A4, 10F1, 10H3 and 15E10 can significantly improve the NK cell killing efficiency.

[0277] 2.10 15E10 can enhance the function of T cells and NK cells in a dose-dependent manner

[0278] Since the prepared 1C4, 3A4, 10F1, 10H3 and 15E10 antibodies all showed certain activity in assisting T cell activation, proliferation and promoting NK cell killing in in vitro experiments, we selected a relatively stable antibody 15E10, and further used the anti-CD3 / anti-CD28 antibody activation system to determine whether its effect on T cells was dose-dependent. The experimental results are shown in Figure A. Compared with the Mock group without anti-CD3 / anti-CD28 antibody activation, the IFN-γ level of the IgG isotype control group activated by anti-CD3 / anti-CD28 antibodies was significantly increased. Compared with the IgG isotype control group, 15E10 can enhance the level of IFN-γ secretion by T cells in vitro in a dose-dependent manner.

[0279] In addition, the YAC-1-NK cell killing system was used to determine whether 15E10 could enhance the killing effect of NK cells in vitro in an antibody-dependent manner. Fig.17 As shown in A and B, compared with the IgG isotype control group, 15E10 at three concentrations of 2 μg / mL, 4 μg / mL and 6 μg / mL at three effector-target ratios of 10:1, 20:1 and 50:1 can dose-dependently enhance the killing efficiency of NK cells in vitro.

[0280] 2.11TIM-3McAb enhances the antitumor activity of hTIM-3 tumor-bearing mice

[0281] Since 15E10 showed good functional activity in vitro and could increase the level of IFN-γ secreted by T cells and enhance the killing function of NK cells in a dose-dependent manner, we further clarified the anti-tumor activity of 15E10 monoclonal antibody in vivo. First, H22 cells were subcutaneously inoculated into male BALB / c-hTIM-3 mice aged 7 to 8 weeks with humanized TIM-3. After tumor formation, they were divided into groups and treated. The first group of mice was intraperitoneally injected with IgG isotype control antibodies, and the second group of mice was intraperitoneally injected with 15E10 (10 mg / kg, once every two days, for a total of four injections). The experimental design is as follows: Fig.18 As shown in A. Tumor size and mouse weight were measured every three days using a vernier caliper and a pan balance, and the data were recorded. The experimental results showed that compared with the IgG isotype control group, 15E10 could significantly slow down the growth rate of mouse tumors, and the volume of mouse tumors ( Fig.18 B, C) and tumor weight ( Fig.18 D) was significantly lower than that of the IgG isotype control group. In addition, there was no significant difference in the body weight of mice between the IgG isotype control group and the TIM-3McAb group, and TIM-3McAb had no significant effect on the body weight of mice ( Fig.18 E).

[0282] 2.12TIM-3McAb improves T cell function in hTIM-3 tumor-bearing mice

[0283] To further explore the effect of 15E10 on the immune cell function of tumor-bearing mice in vivo, mononuclear cells infiltrating tumor tissues and spleen mononuclear cells were extracted from mice, and the effect of TIM-3 monoclonal antibody 15E10 on CD8 + The results showed that 15E10 significantly enhanced the expression of TNF-α + CD8 + T cell ratio and IFN-γ + CD8 + The proportion of T cells ( Fig.19 AB).

[0284] 2.13TIM-3McAb improves NK cell function in hTIM-3 tumor-bearing mice

[0285] Mononuclear cells from tumor tissues and spleen mononuclear cells were extracted from tumor-bearing mice, and flow cytometry was used to determine the effect of 15E10 treatment on the killing function of NK cells in mice. The results showed that 15E10 significantly enhanced the expression of TNF-α + NKp46 + The proportion of cells and IFN-γ + NKp46 + The proportion of cells ( Fig. 20 AB).

[0286] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An anti-TIM-3 monoclonal antibody, characterized in that: It comprises a complementarity determining region sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 9, 11 and 13, or an amino acid sequence having at least 80% identity with the complementarity determining region sequence, respectively.

2. The anti-TIM-3 monoclonal antibody according to claim 1, characterized in that The complementary determining regions include a heavy chain complementary determining region and a light chain complementary determining region; The heavy chain complementarity determining region is: comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:2, a CDR2 having an amino acid sequence as shown in SEQ ID NO:4, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:6; The light chain complementarity determining region is: comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:9, a CDR2 having an amino acid sequence as shown in SEQ ID NO:11, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:13; Further, the anti-TIM-3 monoclonal antibody further comprises a framework region sequence selected from the following: SEQ ID NO: 1, 3, 5, 7, 8, 10, 12 and 14, or comprises an amino acid sequence having at least 80% identity with the framework region sequence, respectively; Further, for the anti-TIM-3 monoclonal antibody, wherein the complementarity determining region comprises a heavy chain framework region and a light chain framework region; Wherein, the heavy chain framework region is: comprising FR1 of the amino acid sequence shown in SEQ ID NO: 1, FR2 of the amino acid sequence shown in SEQ ID NO: 3, FR3 of the amino acid sequence shown in SEQ ID NO: 5, and FR4 of the amino acid sequence shown in SEQ ID NO: 7; Wherein, the light chain framework region is: It comprises FR1 of the amino acid sequence shown in SEQ ID NO:8, FR2 of the amino acid sequence shown in SEQ ID NO:10, FR3 of the amino acid sequence shown in SEQ ID NO:12, and FR4 of the amino acid sequence shown in SEQ ID NO:

14. For the above-mentioned anti-TIM-3 monoclonal antibody, it comprises a heavy chain amino acid sequence and a light chain amino acid sequence selected from the following: SEQ ID NO: 15-16, or comprises an amino acid sequence that has at least 80% identity with the heavy chain amino acid sequence and the light chain amino acid sequence, respectively.

3. An isolated polynucleotide, characterized in that The polynucleotide can encode the anti-TIM-3 monoclonal antibody according to claim 1 or 2.

4. An expression vector, characterized in that: It comprises the isolated polynucleotide according to claim 3.

5. A host cell, characterized in that It comprises the expression vector of claim 4, or comprises the polynucleotide of claim 3 integrated into the genome of a host cell; or, the host cell expresses the anti-TIM-3 monoclonal antibody of claim 1 or 2.

6. A method for preparing an anti-TIM-3 monoclonal antibody, characterized in that: Includes steps: (I) culturing the host cell according to claim 1 under conditions suitable for producing anti-TIM-3 monoclonal antibodies, thereby obtaining a culture containing the anti-TIM-3 monoclonal antibodies; and, (II) isolating or recovering the anti-TIM-3 monoclonal antibody from the culture.

7. An immunoconjugate, characterized in that: It comprises the anti-TIM-3 monoclonal antibody according to claim 1 or 2, and a coupling portion, wherein the coupling portion is a detectable marker, a drug, a toxin, a cytokine, a viral coat protein or a VLP.

8. A kit, characterized in that: It comprises the anti-TIM-3 monoclonal antibody according to claim 1 or 2, and / or the immunoconjugate according to claim 7; the kit is used to detect the presence or level of TIM-3 in a sample.

9. A pharmaceutical composition, characterized in that It includes the anti-TIM-3 monoclonal antibody according to claim 1 or 2, or the immunoconjugate according to claim 7.

10. Use of the anti-TIM-3 monoclonal antibody according to claim 1 or 2, the immunoconjugate according to claim 7, or the pharmaceutical composition according to claim 9 in any one or more of the following: (A) preparing tumor detection, monitoring, diagnosis and / or prognosis products; (B) preparing products for tumor prevention and / or treatment; Furthermore, the product has any one or more of the following functions: 1) Detecting the level of TIM-3 in the sample; 2) regulating TIM-3 activity or level; 3) blocking the binding of TIM-3 to its ligand; 4) Enhance the function of T cells; 5) Enhance the function of NK cells; Further, the ligands include Gal-9, CEACAM-1, and PtdSer; Furthermore, the enhancement of T cell function includes: enhancing the ability of T cells to produce IL-2 and IFN-γ; promoting the proliferation of T cells; Furthermore, the enhancement of NK cell function includes: promoting the killing activity of NK cells.

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