TIM3 protein mutant capable of enhancing T cell function and application of TIM3 protein mutant

Abstract

The invention relates to a TIM3 protein mutant capable of enhancing T cell functions and application of the TIM3 protein mutant. Specifically, the TIM3 protein mutant is subjected to amino acid residue mutation at one or two of the following sites corresponding to a wild type TIM3 protein: the Y265th site and the Y272th site. The mutant provided by the invention can break tumor microenvironment immunosuppression so as to enhance the tumor treatment effect of T cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and in particular to a TIM3 protein mutant capable of enhancing T cell function and application thereof. BACKGROUND

[0002] T cells play a central role in adaptive immune responses and are essential for pathogen clearance and tumor immune surveillance. However, under pathological conditions such as chronic infection and tumor microenvironment, T cells often enter a state of functional exhaustion, characterized by sustained decline in effector function, sustained high expression of inhibitory receptors, and reduced cell proliferation capacity. This exhausted state severely limits the efficacy of adoptive T cell therapy (such as CAR-T, TCR-T) and immunotherapy strategies such as immune checkpoint inhibitors.

[0003] TIM3 (T-cell immunoglobulin and mucin domain protein 3) is an inhibitory receptor on the surface of T cells. Under chronic inflammation and tumor antigen stimulation, the expression of TIM3 is significantly up-regulated, and the combination of TIM3 and its ligand Galectin-9 leads to T cell functional decline and apoptosis. On the other hand, TIM3 has a dual role in the regulation of T cell function. Unlike other classic inhibitory receptors such as PD-1, LAG-3, etc., the intracellular segment of TIM3 does not have a typical inhibitory signaling fragment. At the same time, TIM3 plays an important role in the rapid proliferation and differentiation of T cells under acute infection and other conditions. TIM3-deficient T cells cannot successfully complete the antigen activation process, but the specific molecular mechanism and molecular regulation process have not been reported.

[0004] Currently, anti-tumor immunotherapy strategies targeting TIM3 monoclonal antibodies have reached the clinical trial stage, but all TIM3 monoclonal antibody clinical trials currently underway have failed to achieve the desired efficacy. This may be related to the dual role of TIM3 in the regulation of T cell anti-tumor function. Simply blocking TIM3 may lead to antigen activation disorders in T cells in the tumor microenvironment, thereby leading to poor efficacy of TIM3 monoclonal antibodies.

[0005] Therefore, there is an urgent need in the art to develop more precise, long-acting, and fundamentally enhanced T cell function intervention strategies. SUMMARY

[0006] Based on the above-mentioned prior art limitations, the present application aims to provide a genetically engineered mutant of TIM3 protein located on the surface of immune cells (particularly T cells). The mutant is mutated by amino acid substitution at a specific site, thereby endowing the immune cells expressing the mutant with the ability of proliferation and the ability of secreting toxic cytokines. Compared with the wild-type TIM3 protein, the mutant provided by the present application can break the immune suppression of tumor microenvironment, thereby enhancing the therapeutic effect of immune cells on tumors, and providing a new molecular tool and treatment strategy for developing a new generation of engineered immune cells (such as CAR-T, TCR-T or tumor infiltrating lymphocytes) with stronger anti-tumor activity and persistence.

[0007] Solution for solving the problem In a first aspect, the present application provides a mutant of TIM3 protein, wherein the mutant of TIM3 protein is mutated at one or two of the following sites corresponding to the wild-type TIM3 protein: Y265 and Y272.

[0008] Preferably, the mutant of TIM3 protein is mutated at the Y265 and / or Y272 site corresponding to the wild-type TIM3 protein to D.

[0009] Preferably, the mutant of TIM3 protein is mutated at the Y265 and / or Y272 site corresponding to the wild-type TIM3 protein to D.

[0010] In a second aspect, the present application provides a polynucleotide encoding the mutant of TIM3 protein according to any one of the above-mentioned aspects.

[0011] In a third aspect, the present application provides an expression vector comprising the polynucleotide according to the above-mentioned aspects.

[0012] In a fourth aspect, the present application provides a cell comprising the expression vector according to the above-mentioned aspects, or the genome of the cell is integrated with the polynucleotide according to the above-mentioned aspects.

[0013] Preferably, the cell comprises phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils.

[0014] Preferably, the cell comprises monocytes, macrophages and / or dendritic cells.

[0015] Preferably, the cell comprises B cells, T cells, natural killer cells and / or natural killer T cells (NKT).

[0016] Preferably, the cell is a T cell, preferably the T cell is a CD8+ T cell.

[0017] Preferably, the cell is tumor infiltrating lymphocyte (TIL).

[0018] In a fifth aspect, the present application provides a pharmaceutical composition comprising the mutant of any one of the above, or the cell of any one of the above, and optionally, a pharmaceutically acceptable carrier.

[0019] In a sixth aspect, the present application provides use of the mutant of any one of the above, or the cell of any one of the above, or the pharmaceutical composition of the above, in the preparation of a medicament for preventing and / or treating tumor.

[0020] Effects of the application The present application constructs a mutant of TIM3 with remodeled function by site-directed mutagenesis of the 265th and 272nd amino acids of TIM3 protein. The mutant has significantly improved in vivo and in vitro proliferation potential of T cells, significantly increased expression of cytotoxic secretory factor IFNγ, and significantly enhanced antigen activation ability of T cells, thereby enhancing its anti-tumor function. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 FIG. 1 is a schematic diagram of point mutation recombinant TIM3 lentivirus, wherein TIM3-FL is a schematic diagram of structure without mutation, TIM3-2YD is TIM3 with the 265th and 272nd tyrosine mutated to aspartic acid, and TIM3-2YF is TIM3 with the 265th and 272nd tyrosine mutated to phenylalanine; Figure 2 FIG. 2 is a recombinant lentivirus vector containing TIM3 with the 265th and 272nd tyrosine mutated to aspartic acid; Figure 3 FIG. 3 is a recombinant lentivirus vector containing TIM3 with the 265th and 272nd tyrosine mutated to phenylalanine; Figure 4 FIG. 4 is a schematic diagram of the functional detection results of TIM3-2YD or TIM3-2YF gene modified T cells, wherein A is a result graph of Flag tag protein expression of PMBC cells transfected with different lentivirus vectors; B is a result graph of flow cytometry detection of IFN-γ secreted by PMBC cells containing different lentivirus vectors, and C is a result schematic diagram of the proliferation ability of PMBC cells transfected with different lentivirus vectors; Figure 5 FIG. 5 is a schematic diagram of the efficacy evaluation results of TIM3-2YD or TIM3-2YF gene modified TILs of human cervical cancer origin in a nude mouse xenograft model, wherein A is a flowchart of the nude mouse xenograft model experiment, B is a graph of the growth curve of the subcutaneous tumor of the nude mouse, wherein the arrow indicates the time point of TIL treatment, C is a statistical result of the weight of the subcutaneous tumor of the nude mouse, and D is a dissection result of the subcutaneous tumor of the nude mouse. DETAILED DESCRIPTION

[0022] In order to make the technical solutions and beneficial effects of the present application more obvious and easy to understand, the following will be described in detail by way of listing specific embodiments. The drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meaning as the technical and scientific terms in the technical field to which the present application belongs.

[0023] The present inventors have conducted extensive and in-depth research and found, through a large number of experimental studies, that site-directed mutation of T cell surface receptor TIM3 protein can effectively enhance the antigen activation ability of T cells, improve the expansion ability of T cells and promote the secretion of cytotoxic factors, and can obtain T cells with enhanced function, which have better survival ability and killing effect in the process of killing tumor cells. Based on this, the present application is completed.

[0024] T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) was first discovered in 2002 and is an immune regulatory protein in the TIM family. Members of the TIM family are encoded by three genes in humans (HAVCR1, HAVCR2, and TIMD4, which encode TIM1, TIM3, and TIM4, respectively). Among the TIM family, TIM3 is the most interesting because it is involved in the regulation of immune responses in autoimmune diseases and cancer. Initial studies suggested that it was a molecule expressed by CD4+ and CD8+ T cells that produced interferon gamma (IFNγ), but later studies showed that many other types of T cells (Treg cells), myeloid cells, natural killer cells (NK cells), macrophages, and mast cells can also express TIM3.

[0025] TIM3 is highly expressed in mature CD56(dim)CD16(+) NK cells and heterogeneously expressed in CD56(bright)CD16(-) NK cells. Studies have found that the expression of TIM3 can be induced on CD56(bright)CD16(-) NK cells after stimulation by cytokines such as IL-15, IL-12, and IL-18, which suggests that TIM3 can be a marker of NK cell maturation. High expression of TIM3 is also a marker of effector NK cells, indicating that these cells are producing IFNγ and are undergoing degranulation.

[0026] Overexpression of TIM3 on macrophages appears to attenuate the massive inflammatory response to zymosan, thus preventing adverse tissue disease. In line with this, TIM3 expression is upregulated in peripheral blood mononuclear cells of patients with acute sepsis, but is suppressed in patients with severe sepsis. TIM3 is also highly expressed on human peripheral blood mononuclear cells during pregnancy, where it appears to attenuate the response to TLR4 stimulation.

[0027] On mast cells, TIM3 is thought to have an activating function. TIM3 antibody enhances the secretion of IL-3, IL-4, IL-6 and IL-13 in mast cells after IgE sensitization and in vitro antigen-dependent activation. In addition, in vitro experiments show that both genetic deletion or siRNA knockdown of TIM3 significantly reduces IL-6 production after mast cell activation, and genetic deletion of TIM3 abrogates antigen-mediated glycolysis, an effect mediated by the FcεRII receptor.

[0028] Thus, TIM3 exerts different functions in different cells, and further studies are needed to decipher the role of TIM3 signaling in various cells and its impact.

[0029] In the present application, the creative finding is that site-directed mutation of TIM3 in T cells or TIL cells can effectively promote the proliferation of T cells and the expression of IFNγ factor, and has excellent anti-tumor effect.

[0030] As used herein, the term "tumor infiltrating lymphocytes" or "TILs" generally refers to a population of cells originally obtained as white blood cells, the cells of the present application have left the bloodstream of a subject and migrated into a tumor. TILs can include, but are not limited to, CD8 cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs can include primary TILs and secondary TILs. "Primary TILs" can be those TIL cells obtained from a tissue sample of a subject, and "secondary TILs" can be any TIL population that has been expanded or is expanded in the present application. In some embodiments, the tumor infiltrating lymphocytes of the present application can be unpurified or can be interpenetrating with tumor cells. In one embodiment, the TILs of the present application can refer to a population of TILs.

[0031] As used herein, the term "corresponding to" has the meaning generally understood by one of ordinary skill in the art. In particular, "corresponding to" means that after homology or sequence identity alignment of two sequences, a position in one sequence corresponds to a specified position in the other sequence. Thus, for example, "corresponding to wild type TIM3" means that an amino acid sequence is aligned with the amino acid sequence of wild type TIM3 and the position in the amino acid sequence that corresponds to wild type TIM3 is found.

[0032] The present application provides a TIM3 protein mutant, which has mutation of amino acid residue at one or two positions corresponding to the following positions of wild type TIM3 protein: position Y265, Y272.

[0033] In some embodiments, the TIM3 protein mutant has mutation of amino acid residue at position Y265 and / or Y272 of wild type TIM3 protein to D (Aspartic acid).

[0034] In some embodiments, the TIM3 protein mutant has mutation of Y (Tyrosine) at position 265 to D (Aspartic acid) of wild type TIM3 protein.

[0035] In some embodiments, the TIM3 protein mutant has mutation of Y (Tyrosine) at position 272 to D (Aspartic acid) of wild type TIM3 protein.

[0036] In some embodiments, the TIM3 protein mutant has mutation of amino acid residue at positions Y265D, Y272D of wild type TIM3 protein.

[0037] In this application, the amino acid sequence of wild type TIM3 protein is as SEQ ID NO. 1 (Accession No: NP_116171.3).

[0038] SEQ ID NO. 1 (TIM3-FL) MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP In some embodiments, the sequence of the TIM3 protein mutant is set forth in SEQ ID NO. 2.

[0039] SEQ ID NO. 2 (TIM3-2YD) MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIDTIEENVDEVEEPNEYYCYVSSRQQPSQPLGCRFAMP In some embodiments, the sequence of the TIM3 protein mutant is set forth in SEQ ID NO. 3.

[0040] SEQ ID NO. 3 (TIM3-2YF) MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIFTIEENVFEVEEPNEYYCYVSSRQQPSQPLGCRFAMP The second aspect of the present application provides a polynucleotide encoding the TIM3 protein mutant according to any one of the above.

[0041] The third aspect of the present application provides an expression vector comprising the polynucleotide according to the above.

[0042] In the present application, vectors derived from retroviruses such as lentivirus are suitable tools for achieving long-term gene transfer, because they allow long-term, stable integration of the transgene into the cell genome and replication with the replication of the daughter cell genome. Lentiviral vectors have advantages over vectors derived from onco-retroviruses such as murine leukemia viruses, because they can transduce non-proliferating cells, and have the advantage of low immunogenicity.

[0043] Generally, the nucleic acid sequence of the present application can be ligated downstream of a promoter and incorporated into an expression vector by conventional procedures. The vector can be integrated into and replicated with the eukaryotic cell genome. Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

[0044] The expression vectors of the present application can also be used in standard gene delivery protocols for nucleic acid immunization and gene therapy. Methods of gene delivery are known in the art. See, e.g., U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference in their entireties.

[0045] The polynucleotide sequence can be cloned into many types of vectors. For example, the nucleic acid sequence can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids, among others. Particular vectors of interest include expression vectors, replication vectors, and the like.

[0046] Further, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described in, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory, New York, 2001) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functional in an organism, a promoter sequence, convenient restriction sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0047] A number of viral-based systems have been developed and used for gene transduction of mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject in vivo or ex vivo. A number of retroviral systems are known in the art. In one embodiment, a lentiviral vector is used. A number of DNA viral systems are known in the art. In some embodiments, an adenoviral vector is used. A number of adenoviral vectors are known in the art.

[0048] The expression vector introduced into the cell can also include either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of the expressing cells from the population of transfected or infected cells by the viral vector. In other aspects, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker gene and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable marker genes include, for example, antibiotic resistance genes, such as neomycin and the like.

[0049] Methods of introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, e.g., mammalian (e.g., human T cells), bacterial, yeast, or insect cells, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0050] Physical methods of introducing polynucleotides into host cells include calcium phosphate precipitation, cationic complex transfection, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells that include vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory, New York, 2001). Preferred methods of introducing polynucleotides into host cells are liposome transfection and cationic complex polyethylenimine transfection.

[0051] Biological methods of introducing polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus, among others. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0052] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres (beads), and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0053] In preferred embodiments, the vector is a lentiviral vector.

[0054] It should be understood that, in the present application, in addition to using multiple lentiviruses for transduction, direct transfection of mRNA or plasmids, or methods such as expressing artificial transcription factors, can also be used to express TIM3 protein in immune cells such as T cells, TIL cells, and the like.

[0055] The fourth aspect of the present application provides a cell comprising the expression vector described above, or the genome of the cell is integrated with the polynucleotide described above.

[0056] In certain embodiments, the cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil, and / or a basophil.

[0057] In certain embodiments, the cell comprises a monocyte, a macrophage, and / or a dendritic cell.

[0058] In certain embodiments, the monocyte is a peripheral blood mononuclear cell (PBMC).

[0059] In certain embodiments, the cell comprises a B cell, a T cell, a natural killer cell, and / or a natural killer T cell (NKT).

[0060] In certain embodiments, the cell is a T cell.

[0061] In certain embodiments, the T cell is a CD8+ T cell.

[0062] In certain embodiments, the cell is a tumor infiltrating lymphocyte (TIL).

[0063] In certain embodiments, the TIL is a TIL derived from a tumor tissue, a tumor associated lymph node with or without tumor metastasis, a tumor metastasis lesion, a fragment of a paracancerous tissue, a pleural effusion, and / or an abdominal effusion and / or a TIL derived from a TIL recovered after cryopreservation. For example, the TIL of the present application can be obtained by processing a tumor tissue into tumor fragments.

[0064] In one aspect, the present application provides a method of preparing the above-mentioned cell, the method comprising: (1) providing a cell to be engineered; and (2) engineering the cell such that a TIM3 mutant is expressed in the cell, thereby obtaining the engineered immune cell as described in any of the above.

[0065] In certain embodiments, the cell to be engineered comprises a monocyte, a macrophage, and / or a dendritic cell.

[0066] In certain embodiments, the monocyte is a peripheral blood mononuclear cell (PBMC).

[0067] In certain embodiments, the cell to be engineered comprises a B cell, a T cell, a natural killer cell, and / or a natural killer T cell (NKT).

[0068] In certain embodiments, the cell to be engineered is a T cell.

[0069] In certain embodiments, the T cell is a CD8+ T cell.

[0070] In certain embodiments, the cell to be engineered comprises an allogeneic and autologous source.

[0071] In certain embodiments, the cell to be engineered is a tumor infiltrating lymphocyte (TIL).

[0072] In some embodiments, in step (2), the polynucleotide or vector described above is introduced into the cell so that the TIM3 mutant is expressed in the cell. Any means known in the art and conventional can be used to introduce the polynucleotide or vector described above into the cell in the preparation method, such as but not limited to liposome introduction, nanoparticle delivery, vector, transfection, heat shock, electroporation, transduction, gene gun or microinjection.

[0073] In some embodiments, the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and / or peritoneal effusion and not in vitro expanded are subjected to first stage in vitro expansion and second stage in vitro expansion, and the TIM3 mutant is expressed in the cells in the first stage expansion.

[0074] In some embodiments, the first stage in vitro expansion comprises preparing the TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, fragments of paracancerous tissue, pleural effusion and / or peritoneal effusion and not in vitro expanded into a single cell suspension, isolating, and culturing in human lymph culture medium containing 1000-2000 IU / mL of rhIL-2, then inoculating cells at 5E6 / well per well, and culturing in X-VIVO medium without IL-2, then transfecting the cells with the constructed recombinant lentivirus containing the TIM3 mutant sequence, adding rh-IL2 to the culture medium to a final concentration of 2000 IU / mL after a period of culture, and continuing to culture to obtain recombinant lentivirus modified TILs.

[0075] In some embodiments, the second stage in vitro expansion comprises co-culturing the X-ray irradiated feeder cells with the recombinant lentivirus modified TILs to obtain genetically engineered TIL cells.

[0076] In some embodiments, the intensity of the irradiation is 200-250 Gy, such as 210 Gy, 220 Gy, 230 Gy, 240 Gy, 250 Gy, etc.

[0077] In some embodiments, the ratio of feeder cells to recombinant lentivirus modified TILs is 1:30-50, such as 1:30, 1:35, 1:40, 1:45, 1:50.

[0078] In some embodiments, the co-culture medium is X-VIVO medium containing rhIL-2.

[0079] In certain embodiments, the rhIL-2 is at a final concentration of 3000-6000 IU / mL, e.g., 3000 IU / mL, 3500 IU / mL, 4000 IU / mL, 4500 IU / mL, 5000 IU / mL, 5500 IU / mL, 6000 IU / mL, etc. in X-VIVO medium.

[0080] In certain embodiments, the rhIL-2 is at a final concentration of 3000-6000 IU / mL, e.g., 3000 IU / mL, 3500 IU / mL, 4000 IU / mL, 4500 IU / mL, 5000 IU / mL, 5500 IU / mL, 6000 IU / mL, etc. in X-VIVO medium.

[0081] Methods of introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell, by any of a number of methods in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0082] In a fifth aspect, the present application provides a pharmaceutical composition comprising the mutant of any one of the above, or the cell of any one of the above, and a pharmaceutically acceptable carrier.

[0083] As used herein, "pharmaceutical composition" refers to a preparation of the mutant or cell of the present application with a medium generally accepted in the art for delivering a biologically active ingredient (the mutant or cell provided by the present application) to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate administration of the organism, to facilitate absorption of the active ingredient, and to exert a biological activity.

[0084] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent that is approved by a relevant government regulatory agency for use in humans or domestic animals.

[0085] In a sixth aspect, the present application provides use of the mutant of any one of the above, or the cell of any one of the above, in the preparation of a medicament for preventing and / or treating a tumor.

[0086] In certain embodiments, the tumor is a solid tumor or a hematological tumor; In certain embodiments, the solid tumor comprises gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.

[0087] In certain embodiments, the hematological tumor comprises acute myeloid leukemia (AML), pre-B lymphoblastic leukemia / lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, B lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B cell lymphoma, hairy cell leukemia, MALT type marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma / leukemia, pre-lymphoblastic lymphoma / leukemia, T lymphocytic leukemia, T granular lymphocytic leukemia, aggressive NK cell leukemia, mature T cell lymphoma / leukemia, extranodal nasal type NK / T cell lymphoma, enteropathy type T cell lymphoma, mycosis fungoides / sezary syndrome (MF / SS), peripheral T cell lymphoma, anaplastic large cell lymphoma, acute lymphatic leukemia (ALL), pre-T cell lymphoma / leukemia, Hodgkin's lymphoma, mast cell leukemia (MCL).

[0088] In a seventh aspect, the present application provides a method for treating cancer or tumor, comprising: administering to a subject in need thereof a safe and effective amount of the cell of any one of the above, or the pharmaceutical composition of the above.

[0089] The pharmaceutical composition of the present application can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease— although appropriate dosages can be determined by clinical trials.

[0090] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount", or "therapeutic amount", the precise amount of the composition of the present application to be administered can be determined by a physician with consideration of individual patient circumstances, and the age, weight, tumor size, extent of infection or metastasis, and individual 4 to 10 9 cells / kg body weight, preferably 10 5 to 10 6The T cell composition can be administered at a dose of 1 x 105cells / kg body weight, including all integer values within that range. The T cell composition can also be administered multiple times at these doses. The cells can be administered by infusion using techniques known in the art of immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by monitoring the patient for signs of disease and adjusting the treatment accordingly by one of skill in the medical arts.

[0091] Administration of the subject compositions can be carried out in any convenient manner, including by spray, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the compositions of the present application are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the compositions of the present application are preferably administered by i.v. injection. The compositions can be injected directly into a tumor, lymph node or site of infection.

[0092] An eighth aspect of the present application provides an in vitro method of enhancing the expansion capacity of immune cells and / or promoting the secretion of cytotoxic factors, comprising introducing into a cell a polynucleotide as described above.

[0093] In certain embodiments, the immune cell is as described in any of the above. For example, the immune cell is a T cell, and in particular and preferred embodiments, the immune cell is a TIL cell.

[0094] In certain embodiments, the cytotoxic factor is IFNγ.

[0095] Numerous specific embodiments of the present application have been described herein as examples. The skilled person will readily understand other advantages and benefits of the present application from the disclosure contained herein. The present application can also be embodied in other different specific embodiments or applications, and the various details of the application can be modified in light of different viewpoints and applications without departing from the spirit of the present application.

[0096] Before further describing the specific embodiments of the present application, it is to be understood that the scope of the protection of the present application is not limited to the specific details described below; it is also to be understood that the terminology used herein is for the purpose of describing the specific embodiments only and is not intended to limit the scope of the present application.

[0097] Unless otherwise indicated, the experimental methods, detection methods, preparation methods disclosed in the present application all employ conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields. These techniques are well described in the existing literature, see for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P. B. Becker, ed.) Humana Press, Totowa, 1999, etc.

[0098] In addition to the specific methods, devices, materials used in the examples, according to the mastery of the prior art by those skilled in the art and the description of the present application, any method, device and material of the prior art similar or equivalent to the method, device and material described in the examples of the present application can also be used to implement the present application. Unless otherwise specified, the parts and percentages are parts by weight and weight percentages.

[0099] The raw materials and reagents used in the present application are common reagents in the art, and can be purchased from commercial products or synthesized according to known methods, unless otherwise specified. Of course, it is also not excluded that the synthesis can be carried out according to the methods disclosed in the prior art.

[0100] Example 1 Preparation of recombinant lentivirus vector The above-mentioned gene fragments (as shown in Figure 1 The above-mentioned gene fragments (as shown in

[0101] After a period of culture, the virus supernatant after transfection was collected, and the titer of the lentivirus was adjusted to 5x10 7 TU, and were named TIM3-FL, TIM3-2YD (the tyrosine at positions 265 and 272 was mutated to aspartic acid, the plasmid map is shown in Figure 2 TIM3-2YF (the tyrosine at positions 265 and 272 was mutated to phenylalanine, the plasmid map is shown in Figure 3 and a blank vector control group was constructed.

[0102] Preparation and detection of enhanced T cells Fresh human PBMCs were collected and the concentration was adjusted to 3E6 / mL, and were labeled with CFSE. Human PBMCs were activated in a 24-well plate coated with human OKT3 for 12 hours (the culture system was 1640+10% FBS, and the volume was 1 mL). Then, the above-mentioned three kinds of lentiviruses and 8 μg / mL of Polybrene were added, and the final concentration of the lentivirus was 3x10 7 TU. Then, centrifugation was performed at 37°C and 500g. Then, the 24-well plate was left to stand for 12 hours, half of the liquid was replaced, and the final concentration of rhIL-2 in the culture system was 1000 IU / mL. Then, the culture was continued for 72 hours. At the end of the experiment, the expression level of the Flag-tagged protein gene was detected by WB after lentivirus transfection of human PBMCs. PBMCs were collected, and flow cytometry was used to detect IFN-γ (previously stimulated with BD Leukocyte Activation Cocktail for 6 hours). Flow cytometry was used to detect the fluorescence intensity of CFSE.

[0103] The detection results are shown in Figure 4 , wherein Figure 4 In FIG. A, the expression level of the Flag-tagged protein after different lentivirus treatments was detected by immunoblotting. It can be seen that the expression level of the Flag-tagged protein was significantly up-regulated. The IFN-γ cytokine secretion ability of the TIM3-2YD gene modified T cells was significantly improvedFigure 4 Medium B), and the proliferative capacity of T cells was also improved Figure 4 Medium C).

[0104] Example 3 Preparation and detection of enhanced TIL cells Isolation and purification of TIL: Collect cervical cancer tissue of patients (obtained by surgical resection or puncture biopsy), cut the tissue after removing necrotic tissue by physical method, digest using collagenase IV and hyaluronidase, prepare single cell suspension, then gradient density centrifugation (800g, 2 speed, 0 speed) is performed on the single cell suspension by lymphocyte separation medium. Then collect the middle density layer. Then wash the cells with 1640 medium containing 50 μg / mL gentamicin, and put the separated TIL into cell culture plate in human lymphocyte culture medium containing low concentration of rhIL-2 (2000 IU / mL) for 5-10 days to obtain purified CD3+ T lymphocytes.

[0105] Recombinant lentivirus modification of TIL cells: prepare blank vector control group (Vector-Control) and two point mutation recombinant TIM3 lentivirus (TIM3-2YD and TIM3-2YF) in advance using 293T cells, and the virus titer is 5x10 7 TU. Coat the low-adhesion 24-well plate in advance using human OKT3, and remove the OKT3 before cell plating. At 72 hours of TIL expansion, plate TIL cells at a density of 5E6 / well in a 24-well plate with a total volume of 0.5 mL, and culture in X-VIVO medium without IL-2 for 12 hours. Then add 0.5 mL of lentivirus to each well to be infected, and add 5 μg / mL of Polybrene. Then centrifuge the 24-well plate at 500g, 37°C for 1 hour. After centrifugation, stand the 24-well plate in the incubator for 10 hours. Then half-exchange the cells in the 24-well plate, and add rh-IL2 at a final concentration of 2000 IU / mL in the culture volume for 24 hours, then separate part of the cells for transfection efficiency detection.

[0106] Rapid expansion and in vitro culture of genetically engineered TIL: prepare Feeder cells (healthy human PBMC at a density of at least 3E7 / mL), irradiate the Feeder cells with an X-ray irradiator at 230 Gy, then mix the Feeder cells with the transfected TIL cells at a ratio of 1:40, and culture in human lymphocyte culture medium such as X-VIVO, etc., and add rhIL-2 at a final concentration of 6000 IU / mL, which can be cultured in culture bottles or bags, and replace or add human lymphocyte culture medium containing rhIL-2 as needed.

[0107] Animal experiment evaluation of TIL cell treatment efficacy: Three different lentivirus transfected TIL cells (2E5 / each) were reinfused into SiHa tumor-bearing (size about 50-70mm 3 ) nude mice (4 mice in each group, using mice injected with equal amount of PBS via tail vein as blank control group, see Figure 5 A) via tail vein. Then time-tumor growth curve was drawn, measured once every 3 days, and subcutaneous tumors were isolated for weight statistics and gross photography at the end of the experiment, see Figure 5 .

[0108] As can be seen from Figure 5 , the tumor growth rate of mice receiving TIL treatment was significantly slower than that of the blank control group. The mice receiving TIM3-2YD (tyrosine at positions 265 and 272 mutated to aspartic acid) mutant TIL treatment had significantly slower subcutaneous tumor growth rate and significantly lower tumor burden than the mice receiving empty vector control group and TIM3-2YF group TIL treatment.

[0109] It should be understood that the above examples are exemplary and are not intended to include all possible embodiments encompassed by the claims. Various modifications and changes can also be made on the basis of the above examples without departing from the scope of the disclosure. Similarly, any combination of the technical features of the above examples can also be made to form additional embodiments of the application that can not have been explicitly described. Therefore, the above examples only express several embodiments of the application and do not limit the protection scope of the patent.

Claims

1. A TIM3 protein mutant, characterized in that, The TIM3 protein mutant has a mutation in one or two amino acid residues at the following sites corresponding to the wild-type TIM3 protein: sites Y265 and Y272.

2. The TIM3 protein mutant according to claim 1, characterized in that, The TIM3 protein mutant has a D mutation at amino acid residues at positions Y265 and / or Y272 corresponding to the wild-type TIM3 protein.

3. The TIM3 protein mutant according to claim 1 or 2, characterized in that, The TIM3 protein mutants have the following amino acid residue mutations corresponding to the wild-type TIM3 protein: Y265D and Y272D.

4. A polynucleotide, characterized in that, The polynucleotide encodes the TIM3 protein mutant according to any one of claims 1 to 3.

5. An expression carrier, characterized in that, The expression vector comprises the polynucleotide of claim 4.

6. A cell, characterized in that, The cell contains the expression vector of claim 5, or the genome of the cell is integrated with the polynucleotide of claim 4.

7. The cell according to claim 6, characterized in that, The cells include phagocytes, lymphocytes, neutrophils, eosinophils and / or basophils; Preferably, the cells include monocytes, macrophages, and / or dendritic cells; Preferably, the cells include B cells, T cells, natural killer cells and / or natural killer T cells (NKT).

8. The cell according to claim 7, characterized in that, The cells are T cells, preferably CD8+ T cells; Preferably, the cells are tumor-infiltrating lymphocytes (TILs).

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mutant of any one of claims 1 to 3, or the cell of any one of claims 6 to 8, and optionally, a pharmaceutically acceptable carrier.

10. The use of the mutant according to any one of claims 1 to 3, or the cell according to any one of claims 6 to 8, or the pharmaceutical composition according to claim 9 in the preparation of a drug for the prevention and / or treatment of tumors.

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