A tim3 protein mutant capable of enhancing t cell function and application thereof

Abstract

The present application relates to a TIM3 protein mutant capable of enhancing T cell function and application thereof. Specifically, the TIM3 protein mutant is mutated at one or two of the following sites corresponding to the wild-type TIM3 protein: Y265 and Y272. The mutant provided by the present application can break the immune suppression of the tumor microenvironment, thereby enhancing the efficacy of T cell therapy for tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically a TIM3 protein mutant that can enhance T cell function and its application. Background Technology

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

[0003] TIM3 (T-cell immunoglobulin and mucin domain protein 3) is an inhibitory receptor on the surface of T cells. Its expression is significantly upregulated under chronic inflammation and tumor antigen stimulation, and it binds to its ligand Galectin-9, leading to T cell dysfunction and apoptosis. On the other hand, TIM3 has a dual role in regulating T cell function. Unlike other classic inhibitory receptors such as PD-1 and LAG-3, the intracellular domain of TIM3 lacks typical inhibitory signal transduction fragments. Simultaneously, TIM3 plays a crucial role in the rapid proliferation and differentiation of T cells under conditions such as acute infection. TIM3-deficient T cells cannot successfully complete the antigen activation process, but the specific molecular mechanisms and regulatory processes involved are currently unknown.

[0004] Currently, anti-tumor immunotherapy strategies targeting TIM3 monoclonal antibodies have reached the clinical trial stage, but all existing TIM3 monoclonal antibody clinical trials have failed to achieve the expected efficacy. This may be related to the dual nature of TIM3's regulation of T cell anti-tumor function; simply blocking TIM3 may lead to impaired antigen activation of T cells in the tumor microenvironment, thus resulting in poor efficacy of TIM3 monoclonal antibodies.

[0005] Therefore, there is an urgent need in this field to develop new intervention strategies that are more precise, long-lasting, and can fundamentally enhance T cell function. Summary of the Invention

[0006] Based on the aforementioned limitations of the prior art, this invention aims to provide a genetically engineered TIM3 protein mutant located on the surface of immune cells (especially T cells). This mutant, through amino acid substitution (mutation) at specific sites, endows immune cells expressing this mutant with proliferative capacity and the ability to secrete toxic cytokines. Compared to wild-type TIM3 protein, the mutant provided by this invention can break the immunosuppression of the tumor microenvironment, thereby enhancing the efficacy of immune cell therapy for tumors. This provides a new molecular tool and therapeutic 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 durability.

[0007] Solution for solving the problem In a first aspect, the present invention provides a TIM3 protein mutant, wherein the TIM3 protein mutant has a mutation of amino acid residues at one or two sites corresponding to the wild-type TIM3 protein: sites Y265 and Y272.

[0008] Preferably, 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.

[0009] Preferably, the TIM3 protein mutant has the following amino acid residue mutations corresponding to the wild-type TIM3 protein: Y265D and Y272D.

[0010] In a second aspect, the present invention provides a polynucleotide that encodes a TIM3 protein mutant as described in any of the preceding claims.

[0011] Thirdly, the present invention provides an expression vector comprising the polynucleotides described above.

[0012] Fourthly, the present invention provides a cell comprising the expression vector described above, or the genome of the cell having the polynucleotides described above integrated.

[0013] Preferably, the cells include phagocytes, lymphocytes, neutrophils, eosinophils, and / or basophils.

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

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

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

[0017] Preferably, the cells are tumor-infiltrating lymphocytes (TILs).

[0018] Fifthly, the present invention provides a pharmaceutical composition comprising any of the mutants described above, or any of the cells described above, and optionally, a pharmaceutically acceptable carrier.

[0019] In a sixth aspect, the present invention provides the use of any of the mutants described above, or any of the cells described above, or any of the pharmaceutical compositions described above in the preparation of drugs for the prevention and / or treatment of tumors.

[0020] The effects of the invention This invention constructs a functionally remodeled TIM3 mutant by site-directed mutation of amino acids 265 and 272 of the TIM3 protein. The mutant exhibits significantly enhanced T cell proliferation potential in vitro and in vivo, significantly increased expression of the cytotoxic secretory factor IFNγ, and significantly enhanced T cell antigen activation capacity, thereby strengthening its anti-tumor function. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of a point-mutated recombinant TIM3 lentivirus. TIM3-FL is the structural diagram without mutation, TIM3-2YD is the TIM3 structure with tyrosine at positions 265 and 272 mutated to aspartic acid, and TIM3-2YF is the TIM3 structure with tyrosine at positions 265 and 272 mutated to phenylalanine. Figure 2 It is a recombinant lentiviral vector containing tyrosine mutations at positions 265 and 272 of TIM3 to aspartic acid; Figure 3 It is a recombinant lentiviral vector containing tyrosine mutations at positions 265 and 272 of TIM3, which are replaced with phenylalanine. Figure 4 The diagram shows the functional detection results of T cells modified with TIM3-2YD or TIM3-2YF genes. In this diagram, A shows the expression results of Flag tag protein in PMBC cells transfected with different lentiviral vectors; B shows the results of flow cytometry detection of IFN-γ secretion by PMBC cells containing different lentiviral vectors; and C shows the proliferation capacity of PMBC cells transfected with different lentiviral vectors. Figure 5 This diagram illustrates the efficacy evaluation results of TILs modified with the TIM3-2YD or TIM3-2YF gene, derived from human cervical cancer, in a nude mouse xenograft model. In the diagram, A is a flowchart of the experimental process for the nude mouse xenograft model; B is a growth curve of subcutaneous tumors in nude mice, where the arrows indicate the time points when TIL treatment was administered; C is the statistical results of subcutaneous tumor weight in nude mice; and D is the anatomical results of subcutaneous tumors in nude mice. Detailed Implementation

[0022] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0023] Through extensive and in-depth research and numerous experimental studies, the inventors discovered that site-directed mutation of the T cell surface receptor TIM3 protein can effectively enhance the antigen activation ability of T cells, improve T cell proliferation capacity, and promote the secretion of cytotoxic factors. Furthermore, it yields functionally enhanced T cells with better survivability and killing effect during tumor cell destruction. Based on this, the present invention was completed.

[0024] Protein 3 (TIM3), containing T cell immunoglobulin and mucin domains, was first discovered in 2002 and is an immunomodulatory protein in the TIM family. Members of the TIM family are encoded by three human genes (HAVCR1, HAVCR2, and TIMD4, which encode TIM1, TIM3, and TIM4, respectively). Among the TIM family, TIM3 has received the most attention because it is involved in the regulation of immune responses in autoimmunity and cancer. Initial studies suggested it was primarily expressed by CD4+ and CD8+ T cells that produce interferon-γ (IFNγ), but later research has shown 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 exhibits heterogeneous expression in CD56(bright)CD16(-) NK cells. Studies have found that stimulation with cytokines such as IL-15, IL-12, and IL-18 induces TIM3 expression in CD56(bright)CD16(-) NK cells, suggesting that TIM3 may be a marker of NK cell maturation. High TIM3 expression is also a marker of effector NK cells, indicating that these cells are producing IFNγ and undergoing degranulation.

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

[0027] In mast cells, TIM3 is believed 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. Furthermore, in vitro experiments showed that TIM3 gene deletion or siRNA knockdown significantly reduced IL-6 production after mast cell activation, and TIM3 gene deletion eliminated antigen-mediated glycolysis, a process mediated by the FcεRII receptor.

[0028] Therefore, TIM3 plays different functions in different cells, and further research is needed to decipher the role and impact of TIM3 signaling in various cells.

[0029] In this invention, it was creatively discovered that site-directed mutation of TIM3 in T cells or TIL cells can effectively promote T cell proliferation and IFNγ expression, resulting in excellent anti-tumor effects.

[0030] As used herein, the term "tumor-infiltrating lymphocytes" or "TILs" generally refers to a cell population initially obtained as leukocytes, whose cells have left the subject's bloodstream and migrated into the tumor. TILs may 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 may include primary TILs and secondary TILs. "Primary TILs" can be those TIL cells obtained from a subject's tissue sample, and "secondary TILs" can be any TIL population that has been expanded or amplified in this invention. In some embodiments, the tumor-infiltrating lymphocytes of this invention may be unisolated and unpurified, or may be interinfiltrating with tumor cells. In one embodiment, the TILs of this invention may refer to a population of TILs.

[0031] As used herein, the term "corresponds to" has the meaning commonly understood by one of ordinary skill in the art. Specifically, "corresponds to" means that, after a homology or sequence identity alignment, one sequence corresponds to a specified position in another sequence. Thus, for example, "corresponds to wild-type TIM3" means that an amino acid sequence is aligned with the amino acid sequence of wild-type TIM3, and a site on that amino acid sequence that corresponds to wild-type TIM3 is found.

[0032] The present invention provides a TIM3 protein mutant, wherein the TIM3 protein mutant has a mutation of amino acid residues at one or two sites corresponding to the wild-type TIM3 protein: sites Y265 and Y272.

[0033] In some embodiments, the TIM3 protein mutant is mutated to D (aspartic acid) at the Y265 and / or Y272 sites corresponding to the wild-type TIM3 protein.

[0034] In some embodiments, the TIM3 protein mutant is mutated from Y (tyrosine) to D (aspartic acid) at position 265 corresponding to the wild-type TIM3 protein.

[0035] In some embodiments, the TIM3 protein mutant is mutated from Y (tyrosine) to D (aspartic acid) at position 272 corresponding to the wild-type TIM3 protein.

[0036] In some embodiments, the TIM3 protein mutant has mutations in the following amino acid residues corresponding to the wild-type TIM3 protein: Y265D and Y272D.

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

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

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

[0040] SEQ ID NO.3 (TIM3-2YF) MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPR MLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIFTIEENVFEVEEPNEYYCYVSSRQQPSQPLGCRFAMP A second aspect of the present invention provides a polynucleotide encoding a TIM3 protein mutant as described in any of the preceding claims.

[0041] A third aspect of the present invention provides an expression vector comprising the polynucleotides described above.

[0042] In this invention, vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow transgenes to be integrated stably and permanently into the cellular genome and replicate along with the daughter cell genome. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells and have the advantage of low immunogenicity.

[0043] Typically, the nucleic acid sequence of this invention can be ligated downstream of a promoter using standard procedures and incorporated into an expression vector. This vector can then be integrated into the eukaryotic cell genome and replicated thereafter. A typical cloning vector contains transcription and translation terminators, an initial sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0044] The expression vector of the present invention 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, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety.

[0045] Polynucleotide sequences 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, phage particles, phage derivatives, animal viruses, and entrapments. Specific vectors of interest include expression vectors, replication vectors, etc.

[0046] Furthermore, the expression vector can be provided to cells 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, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication that functions in the organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

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

[0048] Expression vectors introduced into cells may also contain either or both of optional marker genes or reporter genes to facilitate the identification and selection of expressing cells from transfected or infected cell populations via viral vectors. Alternatively, optional markers may be carried on a separate DNA segment and used in co-transfection procedures. Both the optional marker gene and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful optional marker genes include, for example, antibiotic resistance genes such as neomycin.

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

[0050] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, cationic complex transfection, lipid transfection, particle bombardment, microinjection, electroporation, and so on. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. For example, *Molecular Cloning: A Laboratory Manual* (Sambrook et al., ColdSpring Harbor Laboratory, New York, 2001). Preferred methods for introducing polynucleotides into host cells are liposome transfection and cationic complex polyethyleneimine transfection.

[0051] Biological methods for 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 cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, 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 macromolecular complexes, nanocapsules, and microspheres (beads); and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in both in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

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

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

[0055] A fourth aspect of the present invention provides a cell comprising the expression vector described above, or the genome of the cell having the polynucleotides described above integrated.

[0056] In some embodiments, the cells include phagocytes, lymphocytes, neutrophils, eosinophils, and / or basophils.

[0057] In some embodiments, the cells include monocytes, macrophages, and / or dendritic cells.

[0058] In some embodiments, the mononuclear cells are peripheral blood mononuclear cells (PBMCs).

[0059] In some embodiments, the cells include B cells, T cells, natural killer cells, and / or natural killer T cells (NKT).

[0060] In some embodiments, the cells are T cells.

[0061] In some embodiments, the T cells are CD8+ T cells.

[0062] In some embodiments, the cells are tumor-infiltrating lymphocytes (TILs).

[0063] In some embodiments, the TIL is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, fragments of adjacent normal tissue, pleural effusion and / or ascites, and / or TIL derived from cryopreservation followed by thawing. For example, the TIL of the present invention can be obtained by processing tumor tissue into tumor fragments.

[0064] On one hand, the present invention provides a method for preparing the cells described above, the method comprising: (1) Provide cells to be modified; and (2) The cells are modified to express the TIM3 mutant, thereby obtaining engineered immune cells as described in any of the above.

[0065] In some embodiments, the cells to be modified include monocytes, macrophages, and / or dendritic cells.

[0066] In some embodiments, the mononuclear cells are peripheral blood mononuclear cells (PBMCs).

[0067] In some embodiments, the cells to be modified include B cells, T cells, natural killer cells, and / or natural killer T cells (NKT).

[0068] In some embodiments, the cells to be modified are T cells.

[0069] In some embodiments, the T cells are CD8+ T cells.

[0070] In some embodiments, the cells to be modified include allogeneic and autologous sources.

[0071] In some embodiments, the cells to be modified are tumor-infiltrating lymphocytes (TILs).

[0072] In some embodiments, in step (2), the polynucleotide or vector described above is introduced into the cells to express the TIM3 mutant. The preparation method may employ any conventional method known in the art to introduce the polynucleotide or vector into the cells, including, but not limited to, liposome delivery, nanoparticle delivery, vector, transfection, heat shock, electrotransfection, transduction, gene gun, or microinjection.

[0073] In some implementations, TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, fragments of adjacent tissue, pleural effusion and / or ascites, and which have not been amplified in vitro, undergo a first-stage in vitro amplification and a second-stage in vitro amplification, wherein the TIM3 mutant is expressed in the cells during the first-stage amplification.

[0074] In some embodiments, the first stage of in vitro expansion includes preparing a single-cell suspension of TILs derived from tumor tissue, tumor-associated lymphoid nodes with or without tumor metastasis, metastatic lesions, fragments of adjacent normal tissue, pleural effusion, and / or ascites that have not been expanded in vitro. After separation, the TILs are cultured in human lymphocyte culture medium containing 1000-2000 IU / mL rhIL-2. Cells are then seeded at a rate of 5E6 cells / well and cultured in X-VIVO medium without IL-2. Subsequently, the cells are transfected with a recombinant lentivirus containing the TIM3 mutant sequence. After a period of culture, rh-IL2 is added to the culture medium to a final concentration of 2000 IU / mL, and the cells are cultured for a further period of time to obtain recombinant lentivirus-modified TILs.

[0075] In some implementations, the second stage of in vitro expansion includes co-culturing X-ray-irradiated feeder cells with TILs modified by recombinant lentivirus 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 implementations, the ratio of feeder cells to recombinant lentivirus-modified TILs is 1:30 to 50, for example, 1:30, 1:35, 1:40, 1:45, or 1:50.

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

[0079] In some embodiments, the final concentration of rhIL-2 in X-VIVO medium is 3000-6000 IU / mL, such as 3000 IU / mL, 3500 IU / mL, 4000 IU / mL, 4500 IU / mL, 5000 IU / mL, 5500 IU / mL, 6000 IU / mL, etc.

[0080] In some embodiments, the final concentration of rhIL-2 in X-VIVO medium is 3000-6000 IU / mL, such as 3000 IU / mL, 3500 IU / mL, 4000 IU / mL, 4500 IU / mL, 5000 IU / mL, 5500 IU / mL, 6000 IU / mL, etc.

[0081] Methods for introducing genes into cells and expressing genes into cells are known in the art. Within the scope of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0082] A fifth aspect of the present invention provides a pharmaceutical composition comprising any of the mutants described above, or any of the cells described above, and a pharmaceutically acceptable carrier.

[0083] As used herein, "pharmaceutical composition" refers to a formulation of the mutant or cell of the present invention with a medium generally accepted in the art for delivering a bioactive ingredient (the mutant or cell provided by the present invention) to a mammal (e.g., a human). This medium includes pharmaceutically acceptable carriers. The purpose of the pharmaceutical composition is to facilitate administration to the organism, thereby promoting the absorption of the active ingredient and the exertion of its bioactivity.

[0084] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, gliding agent, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that is permitted by the relevant governmental regulatory authority to be acceptable for human or animal use.

[0085] The sixth aspect of the present invention provides the use of the mutants described in any of the above claims, or the cells described above, in the preparation of drugs for the prevention and / or treatment of tumors.

[0086] In some embodiments, the tumor is a solid tumor or a hematoma; In some embodiments, the solid tumor includes gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or combinations thereof.

[0087] In some embodiments, the hematologic malignancies include: 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 lymphoma / leukemia, and pre-lymphoblastic lymphoma. Leukemia, T-cell lymphocytic leukemia, T-granular lymphocytic leukemia, aggressive NK cell leukemia, mature T-cell lymphoma / leukemia, extranodal nasal NK / T-cell lymphoma, enteropathic T-cell lymphoma, mycosis fungoides / sezary syndrome (MF / SS), peripheral T-cell lymphoma, anaplastic large cell lymphoma, acute lymphoblastic leukemia (ALL), pro-T-cell lymphoma / leukemia, Hodgkin's lymphoma, mast cell leukemia (MCL).

[0088] The seventh aspect of the present invention provides a method for treating cancer or tumor, comprising: administering a safe and effective amount of any of the cells described above, or the pharmaceutical composition described above, to a subject requiring treatment.

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

[0090] When referring to "immunologically effective amount," "antitumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that: pharmaceutical compositions comprising T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6The T-cell composition can be administered at a dose of cells per kg of body weight (including all integer values ​​within those ranges). These doses can also be administered multiple times. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

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

[0092] The eighth aspect of the present invention provides an in vitro method for enhancing the proliferative capacity of immune cells and / or promoting the secretion of cytotoxic factors, comprising introducing the aforementioned polynucleotides into cells.

[0093] In some embodiments, the immune cells are as described in any of the above embodiments. For example, the immune cells are T cells, and in a particular and preferred embodiment, the immune cells are TIL cells.

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

[0095] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0096] Before further describing specific embodiments of the present invention, 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0097] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sam Brook 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; *theseries METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATINSTRUCTURE 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, equipment, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described, used, and materials in the embodiments of this invention can be used to implement this invention. Unless otherwise stated, parts and percentages are parts by weight and weight percentages.

[0099] The raw materials and reagents used in this invention are all common reagents in the art. Unless otherwise specified, they can be purchased commercially or synthesized according to known methods. Of course, it is not excluded that they can be synthesized according to methods disclosed in the prior art.

[0100] Example 1: Preparation of Recombinant Lentiviral Vector Separately, the above-mentioned target gene fragments (such as...) Figure 1 The plasmids were constructed in the lentiviral expression vector pLV-CMV-Myc-EF1-Puro. Each vector plasmid was then mixed with the lentiviral packaging plasmids psPAX2 and pMD2.G in a ratio of 3:1:4. HEK293T cells with a growth density of 70-80% were seeded in 10cm culture dishes. The plasmids (6-8μg) were transfected into the cells using Lipo8000 transfection reagent (20-24μL) according to the instructions. The cells were then cultured to prepare the virus.

[0101] After culturing for a period of time, the supernatant of the transfected virus was collected, and the lentivirus titer was adjusted to 5 × 10⁻⁶. 7 TU, named TIM3-FL and TIM3-2YD respectively (tyrosine residues at positions 265 and 272 are point-mutated to aspartic acid, plasmid maps are shown below). Figure 2 As shown), TIM3-2YF (tyrosine residues at positions 265 and 272 are point-mutated to phenylalanine; plasmid map as shown). Figure 3 As shown in the figure (control plasmid), a blank vector control group was also constructed.

[0102] Example 2: Preparation and Detection of Enhanced T Cells Fresh human PBMCs were collected and adjusted to a concentration of 3E6 / mL, labeled with CFSE, and activated for 12 hours in 24-well plates pre-coated with human OKT3 (culture system: 1640 + 10% FBS, volume: 1 mL). Subsequently, the three lentiviruses mentioned above and 8 μg / mL of Polybrene were added, with a final lentivirus concentration of 3 × 10⁻⁶. 7 TU. The culture was then centrifuged at 37°C and 500g. After incubation in 24-well plates for 12 hours, a half-medium change was performed, and rhIL-2 was added to the culture system to a final concentration of 1000 IU / mL. The plates were then incubated for 72 hours. At the end of the experiment, human PBMCs were transfected with lentivirus, and the expression level of the Flag tag protein gene was detected by Western blotting. PBMCs were harvested and IFN-γ (pre-stimulated with BDLenoblastocyte Activation Cocktail for 6 hours) was detected by flow cytometry. The fluorescence intensity of CFSE was also detected by flow cytometry.

[0103] Test results are shown Figure 4 ,in, Figure 4 In Figure A, the expression levels of Flag tag protein after different lentiviral treatments were detected by immunoblotting, showing a significant upregulation of Flag tag protein expression. The IFN-γ cytokine secretion capacity of T cells modified with the TIM3-2YD gene was significantly enhanced. Figure 4 The proliferation capacity of B cells and T cells was also improved. Figure 4 (C)

[0104] Example 3: Preparation and detection of enhanced TIL cells Isolation and purification of TILs: Cervical cancer tissue was collected from patients (obtained through surgical resection or needle biopsy). Necrotic tissue was removed using physical methods, and the tissue was minced and digested with collagenase IV and hyaluronidase to prepare a single-cell suspension. The single-cell suspension was then subjected to gradient density centrifugation (800g, acceleration 2, deceleration 0) using lymphocyte separation medium. The intermediate density layer was then collected. The cells were then washed with 1640 medium containing 50 μg / mL gentamicin. The isolated TILs were placed in cell culture plates and cultured in human lymphocyte culture medium containing a low concentration of rhIL-2 (2000 IU / mL) for 5-10 days to obtain purified CD3+ T lymphocytes.

[0105] Recombinant lentiviral modification of TIL cells: A blank vector control group and two point-mutated recombinant TIM3 lentiviruses (TIM3-2YD and TIM3-2YF) were prepared in advance using 293T cells, with a viral titer of 5 × 10⁻⁶. 7 TU. Low-adhesion 24-well plates were pre-coated with human OKT3, which was removed before cell seeding. At 72 hours of TIL amplification, TIL cells were seeded into 24-well plates at a density of 5E6 cells / well (total volume 0.5 mL) and cultured in IL-2-free X-VIVO medium for 12 hours. Subsequently, 0.5 mL of lentivirus and 5 μg / mL of Polybrene were added to each well to be infected. The 24-well plates were then centrifuged at 500g, 37°C for 1 hour. After centrifugation, the 24-well plates were incubated for 10 hours. The medium in the 24-well plates was then partially changed, and rh-IL2 was added to the culture volume to a final concentration of 2000 IU / mL. After culturing for 24 hours, a portion of the cells was isolated for transfection efficiency analysis.

[0106] Rapid expansion and in vitro culture of genetically engineered TILs: Feeder cells (healthy human PBMCs with a density of at least 3E7 cells / mL) were prepared and irradiated with 230 Gy using an X-ray irradiator. Then, the Feeder cells and transfected TIL cells were mixed at a ratio of 1:40 and cultured in human lymphocyte culture medium such as X-VIVO, with the addition of rhIL-2 to a final concentration of 6000 IU / mL. This process can be carried out in culture flasks or culture bags, with the addition or replacement of rhIL-2-containing human lymphocyte culture medium as needed.

[0107] Animal experiments evaluating the efficacy of TIL cell therapy: TIL cells transfected with three different lentiviruses (2E5 / animal) were reinfused via tail vein into SiHa tumor-bearing cells (approximately 50-70 mm in size). 3 In nude mice (4 mice per group, with mice injected with an equal volume of PBS via tail vein as a blank control group, see below) Figure 5 (A). A time-tumor growth curve was then plotted, with measurements taken every 3 days. At the end of the experiment, subcutaneous tumors were dissected for weight analysis and gross imaging. The results are shown in […]. Figure 5 .

[0108] Depend on Figure 5 It can be seen that the tumor growth rate of mice treated with TIL was significantly slowed compared with the blank control group. Mice treated with TIL of the TIM3-2YD mutant (tyrosine point mutation at positions 265 and 272 to aspartic acid) showed significantly slower subcutaneous tumor growth and significantly reduced tumor burden compared with mice treated with TIL of the empty vector control group and the TIM3-2YF group.

[0109] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A TIM3 protein mutant, characterized in that, The TIM3 protein mutant has the following amino acid residue mutations corresponding to the wild-type TIM3 protein: Y265D and Y272D. The sequence of the TIM3 protein mutant is shown in SEQ ID NO.

2.

2. A polynucleotide, characterized in that, The polynucleotide encodes the TIM3 protein mutant of claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the polynucleotide of claim 2.

4. A cell, characterized in that, The cell contains the expression vector of claim 3, or the genome of the cell is integrated with the polynucleotide of claim 2.

5. The cell according to claim 4, characterized in that, The cells include phagocytes, lymphocytes, eosinophils, and / or basophils.

6. The cell according to claim 5, characterized in that, The cells include neutrophils, monocytes, macrophages, and / or dendritic cells.

7. The cell according to claim 5, characterized in that, The cells include B cells, T cells, and natural killer cells.

8. The cell according to claim 7, characterized in that, The cells in question are T cells.

9. The cell according to claim 8, characterized in that, The T cells mentioned are CD8+ T cells.

10. The cell according to claim 7, characterized in that, The cells in question are natural killer T cells.

11. The cell according to claim 5, characterized in that, The cells in question are tumor-infiltrating lymphocytes.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mutant of claim 1, or the cell of any one of claims 4 to 11, and optionally, a pharmaceutically acceptable carrier.

13. The use of the mutant of claim 1, or the cell of any one of claims 4 to 11, or the pharmaceutical composition of claim 12 in the preparation of a medicament for the prevention and / or treatment of cervical cancer.

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