WO2025054946A1 - Tumor-infiltrating lymphocyte with knockout of roquin-1 and/or regnase-1 genes and use thereof - Google Patents

Abstract

Provided are a tumor-infiltrating lymphocyte with knockout of Roquin-1 and/or Regnase-1 genes and the use thereof. By means of knocking out the Roquin-1 and/or Regnase-1 genes in the tumor infiltrating lymphocyte, the proliferation ability and the anti-tumor ability of the tumor infiltrating lymphocyte are improved.

Description

Tumor infiltrating lymphocytes knocking out Roquin-1 and/or Regnase-1 genes and applications thereof Technical Field

The present invention relates to the field of cell therapy, and in particular to modified tumor infiltrating lymphocytes and applications thereof.

Background Art

Cell therapy is a hot area in tumor immunotherapy. Currently, there are many cell therapy drugs on the market, among which the most common product is CAR-T cells. In addition, TCR-T, NK, CAR-M and TIL are also products that many pharmaceutical companies and biotechnology companies are competing to develop. However, although CAR-T and TCR-T therapies have made some progress in clinical practice, their efficacy against solid tumors is still flawed, especially in overcoming the tumor microenvironment, promoting proliferation, reducing exhaustion, and enhancing the persistence of tumor killing. There are still great challenges.

Tumor infiltrating lymphocytes (TIL) therapy is a new type of cellular immunotherapy. This type of lymphocyte is generally obtained through two main steps. First, tumor tissue fragments obtained from patients are cultured in a medium containing high concentrations of interleukin-2 (IL-2) to allow T cells to crawl out of the tumor tissue, thereby obtaining a small amount of TIL; then co-cultured with human PBMC trophoblast cells to amplify TIL in large quantities, thereby obtaining enough TIL for clinical use. Because of their natural homing ability, these cells can infiltrate into tumor tissues very well.

Compared with CAR-T cells and TCR-T cells, which can only recognize a single or fixed two to three antigens by expressing specific antigen recognition domains, TIL can rely on T cell surface receptors to recognize multiple antigens at the same time, which can effectively overcome the problem of insufficient efficacy caused by tumor heterogeneity and reduce tumor antigen escape. In addition, CAR-T therapy and TCR-T therapy are generally obtained by separating T cells from the patient's peripheral blood and then obtaining modified CAR-T cells and TCR-T cells through viral transduction, while TIL is T cells directly isolated from tumor tissue. This group of T cells can well recognize tumor antigens and has better homing and infiltration capabilities. In terms of efficacy and safety, according to current clinical reports, CAR-T has poor effects on solid tumors, is prone to recurrence, and is prone to serious adverse events including cytokine release syndrome and neurotoxicity, while TIL has a better overall efficacy against solid tumors, and there are currently no reports of serious adverse events. Since most TILs are modified using non-viral vectors, the safety risks caused by viral vectors are low.

RNA binding proteins (RBPs) are a class of immunomodulators that have not been fully investigated. Specifically, Regnase-1 and Roquin-1 are RBPs that act as negative immunomodulators to coordinate the fine-tuning and restriction of inflammatory gene expression. Studies have shown that Regnase-1 knockout can enhance the function of mouse OT-1 cells, Pmel TCR-T cells, and CD19-CAR CD8+T cells (Wei et al. (2019) Nature. 576(7787):471-476). Knockout of Regnase-1, Roquin-1 and -2, or all three genes, increases the expression of IL-2 and interferon-γ (IFN-γ) mRNA in human Jurkat T cells stimulated by nonphysiological stimulation with PMA and ionomycin, thereby bypassing the TCR complex (Cui et al. (2017), Journal of Immunology, 199:4066-4077). The existing patent (WO2023070080A1) also involves knocking out Regnase-1 and/or Roquin-1 to enhance the activity of CAR-T cells, but does not involve the treatment of TIL cells.

The use of adoptive transfer of TILs to treat large, refractory tumors is an effective method for treating patients with poor prognosis (Gattinoni et al., Nat. Rev. Immunol., 2006, 6, 383-393). Successful immunotherapy requires large amounts of TILs, and commercialization requires robust and reliable methods. Therefore, there is still a need for improved TIL cell therapy in this field.

Summary of the invention

The present invention uses tumor infiltrating lymphocytes as a research model, obtains ovarian cancer samples from subject tissues, and obtains a certain amount of TILs through in vitro culture. CRISPR-Cas9 technology is then used to knock out the Roquin-1 and/or Regnase-1 genes in TILs. It is observed that knocking out the Roquin-1 and/or Regnase-1 genes can promote TIL proliferation and cytokine secretion, and significantly reduces dependence on IL-2 during in vitro expansion.

Furthermore, the present invention also proves that TIL with knockout of Roquin-1 and/or Regnase-1 gene has better killing effect on tumor cells through in vitro killing models of P815 cells and primary tumor cell lines. In addition, the inventors also found that knockout of Roquin-1 and/or Regnase-1 can promote the increase of central memory T cells (TCM) in TIL subpopulations, suggesting that the gene-modified TIL may have better anti-tumor persistence in vivo.

Therefore, the present invention aims to reveal a new TIL regulatory target combination of Roquin-1 and Regnase-1 genes. The modified TIL has better proliferation ability and anti-tumor activity, which can well solve the problems of easy exhaustion, difficulty in sustainability, and easy recurrence of CAR-T and TCR-T in the process of fighting solid tumors, and provide a better immunotherapy product for patients.

Specifically, the first aspect of the present invention provides a modified tumor infiltrating lymphocyte.

The modified TIL provided by the present invention does not contain the Roquin-1 gene and/or the Regnase-1 gene, or the biological functions of the Roquin-1 gene product and/or the Regnase-1 gene product of the modified TIL are reduced or eliminated.

The Roquin-1 gene is also called the Rc3h1 gene, and its gene ID in the human genome is 149041 (updated on August 18, 2023); the Regnase-1 gene is also called the Zc3h12a gene, and its gene ID in the human genome is 80149 (updated on August 18, 2023).

In some embodiments, the expression and/or function of the Roquin-1 and/or Regnase-1 genes or their gene products of the above-mentioned TILs are reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%, respectively, compared to unmodified or control TILs.

In some embodiments, the properties of the TILs are improved compared to unmodified or control TILs.

In some embodiments, the improved TIL properties mentioned above include one or more selected from the following groups: improved TIL cell proliferation ability, improved cytokine secretion ability, improved granzyme secretion ability, increased proportion of central memory T cells (Central Memory T cell, TCM), reduced IL-2 dependence, and improved tumor cell killing ability.

On the other hand, the present invention provides a method for preparing the modified TIL, comprising treating the Roquin-1 gene and the Regnase-1 gene in the TIL using gene editing technology, RNA interference technology, PROTAC technology, antibodies or small molecule inhibitors.

In some embodiments, the gene editing technology includes CRISPR/Cas technology, transcription activator-like effector nuclease (TALEN) technology, zinc finger nuclease (ZFN) technology, single or multi-base mutation, guide editing or site-directed knock-in.

In some embodiments, the Cas types include: Cas9, Cas12a, Cas3, Cas13, Cas14, Cas7, Cas8, Cas10 and Cas11.

In some embodiments, the Cas9 types include: SpCas9, SaCas9, SpCas9-HF, eSpCas9, xCas9, cpf1.

In some preferred embodiments, the Roquin-1 and/or Regnase-1 genes are disrupted and/or knocked out by CRISPR/Cas methods.

In some preferred embodiments, the Roquin-1 and/or Regnase-1 genes are disrupted and/or knocked out by CRISPR/Cas9 method.

In some embodiments, the CRISPR/Cas9 technology comprises introducing into the TILs a CRISPR/Cas9 system that contains both a single-stranded guide RNA (sgRNA) targeting a target gene and a Cas9 nuclease.

In some embodiments, the sgRNA includes an sgRNA targeting the Roquin-1 gene and an sgRNA targeting the Regnase-1 gene.

In some embodiments, the CRISPR/Cas9 technology can use sgRNA targeting the Roquin-1 gene and sgRNA targeting the Regnase-1 gene separately or simultaneously.

The present invention provides a guide RNA (gRNA) that directs a site-directed modification polypeptide to a specific target nucleic acid sequence. The gRNA comprises a nucleic acid targeting segment and a protein binding segment. The nucleic acid targeting segment of the gRNA comprises a nucleotide sequence that is complementary to a sequence in a target nucleic acid sequence. Therefore, the nucleic acid targeting segment of the gRNA interacts with the target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing), and the nucleotide sequence of the nucleic acid targeting segment determines the position in the target nucleic acid to which the gRNA will bind. The nucleic acid targeting segment of the gRNA can be modified (e.g., by genetic engineering) to hybridize with any desired sequence in the target nucleic acid sequence.

The protein binding segment of the guide RNA interacts with the site-directed modification polypeptide (e.g., Cas protein) to form a complex. The guide RNA guides the bound polypeptide to a specific nucleotide sequence in the target nucleic acid through the above-mentioned nucleic acid targeting segment. The protein binding segment of the guide RNA comprises two nucleotide fragments, which are complementary to each other and form a double-stranded RNA duplex.

In some embodiments, the gRNA comprises two separate RNA molecules. In such embodiments, each of the two RNA molecules comprises a segment of nucleotides that are complementary to each other, such that the complementary nucleotides of the two RNA molecules hybridize to form a double-stranded RNA duplex of the protein-binding segment. In some embodiments, the gRNA comprises a single-stranded RNA molecule (sgRNA), the ends of the sequences forming the complementary region in the above two RNA molecules being connected to form the sgRNA.

The specificity of the gRNA for the target locus is mediated by the sequence of the nucleic acid binding segment, which comprises 20 nucleotides that are complementary to the target nucleic acid sequence within the target locus.

In some embodiments, the present invention provides an sgRNA targeting the Roquin-1 gene, wherein the targeting sequence is CCTGAATAAACTCCACCGCA (SEQ ID NO: 1) or a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 1; the targeting sequence of the sgRNA targeting the Regnase-1 gene is GTGGACTTCTTCCGGAAGCT (SEQ ID NO: 2) or a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 2.

In some embodiments, the Cas9-sgRNA RNP complex or a vector containing Cas9 protein and sgRNA expression elements is introduced into the above-mentioned TIL by chemical transfection, electroporation or vector delivery.

The above-mentioned vector delivery method includes delivery using a viral vector, a virus-like vector or a non-viral vector, and the viral vector includes but is not limited to a viral vector based on: vaccinia virus, polio virus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviral vector (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus and mammary tumor virus), etc. Suitable non-viral vectors are selected from plasmids, transposons, lipid nanoparticles, liposomes, exosomes, attenuated bacteria or virus-like particles.

In some preferred embodiments, the Cas9-sgRNA RNP complex is introduced into the above-mentioned TIL by electroporation.

Another aspect of the present invention provides a method for producing the modified TIL, comprising the following steps:

(1) processing a tumor sample obtained from a subject into multiple tumor fragments to obtain a TIL population;

(2) amplifying the obtained TIL population in vitro in a culture medium containing T cell growth factors to obtain a Pre-rep TIL population;

(3) Treating the Pre-rep TIL population using the preparation method described in any of the above embodiments, and expanding the culture in a culture medium containing trophoblast cells, so that the expression and/or function of the Roquin-1 and/or Regnase-1 genes are reduced or eliminated.

In some embodiments, the T cell growth factor is selected from one or more of IL-2, IL-7, IL-15, and IL-21, preferably IL-2.

In some embodiments, the final concentration of the T cell growth factor in step (2) above is about 750-6000 IU/mL, preferably 6000 IU/mL.

In some embodiments, the final concentration of the T cell growth factor in step (3) above is about 750-6000 IU/mL, preferably 3000 IU/mL.

In some embodiments, the above-described TILs can be used for autologous TIL therapy of a subject.

Another aspect of the present invention provides a pharmaceutical composition comprising the TIL according to any one of the above embodiments or the TIL treated by the method according to any one of the above embodiments and optionally a pharmaceutically acceptable carrier.

Another aspect of the present invention provides use of the TIL described in any of the above embodiments, or the TIL treated by the method described in any of the above embodiments, or the above pharmaceutical composition in the preparation of a drug for preventing and/or treating tumors.

In some embodiments, the tumor is a solid tumor.

In some preferred embodiments, the tumor is selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.

Another aspect of the present invention provides a method for treating a tumor in a subject in need thereof, the method comprising administering to the subject the TIL described in any one of the above embodiments, or the TIL treated by the method described in any one of the above embodiments, or the above pharmaceutical composition.

In some embodiments, the tumor is a solid tumor.

In some preferred embodiments, the tumor is selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.

On the other hand, the present invention provides a method of reducing or eliminating the expression and/or function of Roquin-1 and/or Regnase-1 genes in TIL, wherein the method comprises improving the proliferation ability of TIL cells, improving the cytokine secretion ability, improving the granzyme secretion ability, increasing the proportion of central memory T cells (Central Memory T cell, TCM), reducing IL-2 dependence, and improving the tumor cell killing ability.

The experiments of the present invention have shown that the present invention improves the properties of TIL by knocking out the Roquin-1 and/or Regnase-1 genes in TIL, and has a series of advantages compared with unmodified TIL:

1) It can promote TIL cell proliferation, reduce TIL exhaustion in the body, and play a better anti-tumor effect;

2) It can reduce the dependence on IL-2 during TIL expansion, thereby reducing the use of IL-2 in clinical applications, thereby improving clinical safety and reducing costs;

3) It can promote the increase of TCM proportion in TIL subpopulation, thereby increasing the persistence of TIL in the body, reducing tumor recurrence, and overcoming the shortcomings of CAR-T and TCR-T therapies in the treatment of solid tumors;

4) It can promote TIL cells to secrete more cytokines and granzymes, and have better anti-tumor activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Detection of Roquin-1 and/or Regnase-1 gene knockout efficiency.

Figure 2: Roquin-1 and/or Regnase-1 gene knockout significantly promoted TIL proliferation. Data are expressed as mean±SEM, n≥2, two-way ANOVA, ns, p＞0.05; *, p<0.05; **, p<0.01.

Figure 3: Roquin-1 and/or Regnase-1 gene knockout significantly reduced the dependence of TIL expansion on IL-2. Data are expressed as mean±SEM, n≥2, two-way ANOVA, ns, p＞0.05; *, p<0.05; **, p<0.01.

Figure 4: When the effector-target ratio was 5:1, knockout of Roquin-1 and/or Regnase-1 significantly promoted the secretion of interferon-γ by TILs. Data are expressed as mean±SEM, n≥2, two-tailed unpaired T test, ns, p＞0.05; *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 5: When the effector-target ratio was 10:1, knockout of Roquin-1 and/or Regnase-1 significantly promoted TIL secretion of granzyme B. Data are expressed as mean±SEM, n≥4, one-way ANOVA, ns, p＞0.05; *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 6: At different effector-target ratios, knockout of Roquin-1 and/or Regnase-1 genes can promote the killing of P815 cells by TIL. Data are expressed as mean±SEM, n≥2, two-tailed paired T test, ns, p＞0.05; *, p<0.05.

Figure 7: Roquin-1 and/or Regnase-1 gene knockout promoted the killing ability of TIL against primary ovarian cancer cells at an effector-target ratio of 10:1. n≥3, two-way ANOVA, ns, p＞0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Figure 8: Roquin-1 and/or Regnase-1 gene knockout promotes the increase in the proportion of TCMs in TILs.

Figure 9: Roquin-1 and/or Regnase-1 gene knockout slightly increased the proportion of CD4+ T cells in TILs.

DETAILED DESCRIPTION

Although the present invention can be implemented in many different forms, what is disclosed here is its specific illustrative embodiment that verifies the principle of the present invention.It should be emphasized that the present invention is not limited to the specific embodiment illustrated.In addition, any section heading used herein is only for organizational purposes and is not to be interpreted as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, terms in the singular should include plural forms, and terms in the plural should include singular forms. More specifically, as used in this specification and the appended claims, unless the context otherwise clearly indicates, the singular forms "a", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and/or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include endpoints and all values between the endpoints.

Generally, terms relating to the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and techniques thereof, are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Abbas et al., Cellular and Molecular Immunology, 6th ed., W.B. Saunders Company (2010); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001). g Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The terms used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. In addition, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

definition

In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the term "tumor infiltrating lymphocytes" or "TIL" refers to a population of cells originally obtained as leukocytes that have left the subject's bloodstream and migrated to a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include primary TILs and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations that have been expanded or proliferated, including but not limited to bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

As used herein, the term "Roquin-1" refers to a gene or protein that can encode a protein containing a ring finger domain and a zinc finger domain, which controls the activation and differentiation of T cells by regulating gene expression and plays a key role in adaptive immune response. The NCBI gene ID of Roquin-1 is 149041. In the present invention, Roquin-1 can include unprocessed Roquin-1, any form of processed Roquin-1, a variant of Roquin-1, or a substance containing a functionally active fragment of Roquin-1.

As used herein, the term "Regnase-1" refers to a gene or protein that can encode a protein containing a zinc finger domain, which can mediate downstream signals and play a key role in the growth, development and differentiation of cells and tissues. The NCBI gene ID of Regnase-1 is 80149. In the present invention, Regnase-1 can include unprocessed Regnase-1, any form of processed Regnase-1, variants of Regnase-1, or substances containing functionally active fragments of Regnase-1.

As used herein, the term "TIL properties" refers to the properties of TIL cells that are improved after being modified by the preparation method of the present invention. Changes in TIL properties may include: increased TIL proliferation ability, increased TIL cell number, increased survival ability, improved T cell subset ratios, increased cytokine secretion ability, increased tumor cell killing ability, or any combination thereof. The changes of the present invention may be an increase or decrease.

As used herein, the term "TCR-T" refers to T cell receptor therapy.

As used herein, the term "CAR-T" refers to chimeric antigen receptor T cell therapy.

As used herein, the terms "peripheral blood mononuclear cells" and "PBMC" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMC is an antigen presenting cell.

As used herein, the term "RNA interference" or "RNAi" refers to RNA-dependent silencing of gene expression initiated by double-stranded RNA (dsRNA) molecules in the cytoplasm of cells. The dsRNA molecules reduce or inhibit the accumulation of transcripts of target nucleic acid sequences, thereby silencing the gene or reducing the expression of the gene.

As used herein, the term "ZFN" refers to zinc-finger nucleases, which consist of a DNA recognition domain and a non-specific endonuclease. The DNA recognition domain is composed of a series of Cys2-His2 zinc-finger proteins (zinc-fingers) in series (usually 3 to 4), each of which recognizes and binds to a specific triplet base. Researchers can target and locate different DNA sequences by processing and modifying the zinc-finger DNA binding domain of ZFN, so that ZFN can bind to the target sequence in a complex genome and perform specific cutting by the DNA cleavage domain. In addition, by combining zinc-finger nuclease technology with intracellular DNA repair mechanisms, researchers can also edit the genome in vivo freely. At present, ZFN technology has been widely used in targeted gene mutations in a large number of plants, fruit flies, zebrafish, frogs, mice/rat and cattle. New species with modified genetic backgrounds can be generated by artificially modifying genomic information. This technology is also of great value in the field of medicine, has potential significance for gene therapy of diseases, and has very broad application prospects.

As used herein, the term "TALEN" refers to transcription activator-like effector nucleases, a new gene editing tool. TALE protein is a natural protein derived from the plant pathogen Xanthomonas, which also contains a DNA binding domain. The DNA binding domain in TALE protein is composed of a series of 33 to 35 amino acid repeat domains, each of which can recognize a base. The DNA binding specificity of TALE nuclease is mainly determined by two highly variable amino acids, which scientists call repeat-variable di-residues (RVD). Like zinc finger domains, this TALE repeat module can also be connected in series to recognize a long string of DNA sequences. However, cloning such a large repeat coding sequence of the TALE protein DNA sequence recognition domain is also a considerable challenge. In order to solve this problem, scientists have come up with many solutions. Now there are several schemes that allow researchers to quickly assemble any combination of TALE protein DNA sequence recognition domains. There are many large-scale, systematic research projects using various assembly strategies that have shown that TALE repeat recognition modules can be assembled together to recognize any DNA sequence. Since the official invention of TALEN technology in 2010, many research groups around the world have verified the specific cutting activity of TALEN using multiple animal and plant systems such as in vitro cultured cells, yeast, Arabidopsis, rice, fruit flies and zebrafish.

As used herein, the term "CRISPR" is a bacterial immune system that has been transformed by scientists into the hottest gene editing tool in recent years. CRISPR (Clusters of Regularly Interspaced Short Palindromic Repeats) technology was jointly discovered by scientists from MIT and the University of California, Berkeley in 2012. It is a double-stranded DNA endonuclease tool mediated by RNA sequences. It consists of two parts, one of which is a sgRNA of about 100bp, which is used to target and recognize the target double-stranded DNA, and the other part is a 1369 amino acid Cas9 protein, which can bind to the sgRNA and has DNA enzyme activity. The artificially designed sgRNA and Cas9 protein form a complex that can specifically cut the target DNA. When the cutting causes mismatch repair, it causes gene frameshift and achieves the purpose of knockout. When a repair DNA sequence is added, a purposeful editing will be performed.

Preferably, the Roquin-1 and Regnase-1 genes are knocked out by TALEN, ZFN, RNAi or CRISPR/Cas gene editing system, wherein the CRISPR/Cas gene editing system includes CRISPR/Cas9, CRISPR/Cas12a, CRISPR/Cas13 and CRISPR/Cas14. In one embodiment of the present invention, TIL cells with knockout of Roquin-1 and Regnase-1 genes are successfully obtained using the CRISPR/Cas9 system, which has the advantages of being safer and more reliable than the prior art. Similarly, other gene editing technologies including TALEN, ZFN, and RNAi also have their advantages, and can also be used to knock out Roquin-1 and Regnase-1 genes in immune cells by such gene editing methods. For the CRISPR/Cas gene editing system, in addition to CRISPR/Cas9, similar CRISPR/Cas12a, CRISPR/Cas13 and CRISPR/Cas14 can also be used for gene editing to knock out Roquin-1 and Regnase-1 genes.

As used herein, the term “sgRNA”, or single-stranded guide RNA, is used to anchor targeted DNA in CRISPR-Cas9 technology.

In some embodiments, the sgRNA design follows the principle that the PAM sequence is NGG and close to the CDS region, and is designed using software such as http://crispr.mit.edu and http://zifit.partners.org/ZiFiT/. In some embodiments, the chemically modified sgRNA has (i) methylation modification at the head and tail 1-10 bases (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases); (ii) methylation modification and phosphorylation modification; (iii) other modifications that can stabilize the sgRNA. The synthetic modified sgRNA is provided by a professional supplier.

As used herein, the term "RNP" refers to the Cas9:sgRNA ribonucleoprotein (RNP) complex. The RNP method is to form an RNP complex with Cas9 protein and sgRNA in vitro, and then deliver the RNP into T cells by electroporation. Its advantages are: high knockout efficiency and low off-target rate (Cas9 protein and sgRNA will be completely degraded by T cells within 24 hours). Its disadvantages are: absolutely endotoxin-free Cas9 protein is not easy to obtain, and the requirements for reagents and instruments are relatively high.

In some embodiments, the Cas9-sgRNA RNP complex is introduced into the activated TIL cells by electroporation 2-5 days after TIL cell activation.

As used herein, "CD4+ cells" refer to CD4-positive cells, such as T cells. The terms "CD4+ cells" and "CD4-positive cells" can be used synonymously. These cells can be identified by methods known in the art, such as by staining the cells with fluorescently labeled antibodies against CD4 and using fluorescence-activated cell sorting. For example, existing data can demonstrate that an increase in the proportion of CD4+ cells can increase the ability of the cell population to secrete IFN-γ and/or TNF, and can increase the effect of the T cell population in promoting tumor suppression. For example, see Tay, R.E., Richardson, E.K. et al. (2020). Cancer Gene Therapy, 1-13. However, the art lacks a method for increasing the proportion of CD4+ cells, and the present application can provide a method for affecting the proportion of CD4+ cells.

As used herein, the term "central memory T cells" refers to T cells that have long-term memory and are able to accept antigen restimulation. Central memory T cells may have a phenotype of CD45RA-CCR7+, for example, central memory T cells may be identified by CD45RA- and CCR7+. Central memory T cells may have a stronger ability to resist tumor growth than ordinary T cells.

As used herein, the term "solid tumor" refers to an abnormal tissue mass that does not typically contain a cyst or fluid area. Solid tumors can be benign or malignant. The term solid tumor cancer refers to a malignant, neoplastic or cancerous solid tumor. Solid tumor cancers include but are not limited to melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and renal cancer. The tissue structure of a solid tumor includes interdependent tissue compartments, including substance (cancer cells) and supporting stromal cells (cancer cells are dispersed therein and can provide a supporting microenvironment).

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds as described herein that is sufficient to achieve the intended application, including but not limited to disease treatment. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo) or the subject and disease condition being treated (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the mode of administration. The term also applies to doses that will induce a specific response in target cells (e.g., a decrease in platelet adhesion and/or cell migration). The specific dose will depend on the specific compound selected, the administration regimen to be followed, whether the compound is administered in combination with other compounds, the time of administration, the administration tissue, and the physical delivery system that carries the compound.

As used herein, the terms "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or therapeutic in terms of partially or completely curing a disease and/or an adverse reaction caused by the disease. As used herein, "treatment" includes any treatment of a disease in mammals, particularly humans, including: (a) preventing the occurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development or progression; and (c) alleviating the disease, i.e., causing regression of the disease and/or alleviating one or more symptoms of the disease. "Treatment" is also meant to include the delivery of an agent to provide a pharmacological effect, even in the absence of a disease or condition. For example, "treatment" includes the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease, such as in the case of a vaccine.

As used herein, the term "subject" can be a human or non-human animal, preferably a human, suffering from, for example, cancer.

As used herein, the term "autologous" refers to any material, e.g., TILs, derived from the same individual that is later reintroduced into the individual, e.g., during treatment.

As used herein, the term "pharmaceutical composition" refers to a preparation that allows the biological activity of the active ingredient to be effective and may not contain additional components that are unacceptably toxic to the subject to which the preparation will be administered. Such preparations are sterile. "Pharmaceutically acceptable" excipients (carriers, additives) are those excipients that can be reasonably administered to a subject to provide an effective dose of the active ingredient used.

As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient and/or salt thereof is chemically and/or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th edition, Pennsylvania: Mack Publishing Company, 1995). The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active drug ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active drug ingredient, its use in the therapeutic compositions of the present invention is contemplated. Other active drug ingredients (e.g., other drugs) may also be incorporated into the compositions and methods.

Example

The invention generally described herein will be more easily understood by reference to the following examples, and those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be the scope of protection of the present invention. The following examples are provided by way of illustration and are not intended to limit the present invention. These examples are not intended to represent that the following experiments are all or only experiments performed.

Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

Example 1: Preparation of tumor infiltrating lymphocytes with knockout of Roquin-1 and/or Regnase-1 genes

Medium preparation

50 mL of cell culture medium was prepared as shown in the following table:

Table 1: Culture medium composition

Cryopreservation medium: 75% CS10 cell cryopreservation medium (Biolife solutions, 210102), 2% human serum albumin (Jetbelin, Switzerland, S20170005), 100 IU/mL recombinant human interleukin-2 injection (Shuanglu Pharmaceutical, S20040008), and normal saline.

Isolation and expansion of TILs

1. Tumor tissues were isolated from ovarian cancer patients; the tissues were sourced from Shanghai Renji Hospital or Shanghai First Hospital.

2. Wash the tumor tissue with PBS, then cut it into tissue blocks of approximately ^3x3x3mm3 with sterile scissors and place them in a 6-well plate containing an appropriate amount of TCM-60 culture medium, with 5 blocks in each well.

3. After 14 days of continuous culture, Pre-rep TIL was obtained and frozen by adding freezing medium.

Knockdown of Roquin-1 and/or Regnase-1 genes in TIL cells

1. Resuscitate Pre-rep TIL in BCM-2 medium and perform gene knockout after overnight recovery.

2. Resuspend the lyophilized sgRNA (sequence as shown in either SEQ ID NO: 1 or 2) in RNase-free/DNase-free pyrogen-free water at a final concentration of 100 μM (100 pmol/μL).

3. Mix Nucleofector ^TM solution and Supplement at a ratio of 4.5:1 to form Lonza electroporation Buffer P3 (Lonza, 4XP-3024), 100 μL per reaction. Incubate at room temperature.

4. Place 15.0 μL Lonza electroporation Buffer P3, 9.0 μL sgRNA and 10.0 μL Cas9 protein (Thermofisher, A36499) into a sterile, RNase/DNase-free centrifuge tube and incubate at room temperature for 10 minutes to form an RNP mixture.

5. Select the EH115 program for T cell electroporation in the Lonza 4D-Nucleofector ^™ X electroporation module.

6. Preheat T25 culture flasks, each containing 7.0 mL of TCM-60 medium. Take an appropriate amount of TCM-0 medium (800 μL per reaction) and pre-culture in a 37°C incubator containing 5% CO ₂ .

7. Centrifuge the revived Pre-rep TIL culture at 300xg for 8 minutes, discard the supernatant, and resuspend the revived TIL in DPBS buffer.

8. Count and collect cells ( ^5x106 cells per reaction), centrifuge the required number of cells at 300xg for 8 minutes, and discard the supernatant.

9. Resuspend the cells again with 6.0 mL of pre-warmed DPBS buffer, centrifuge at 300 x g for 8 minutes, and completely remove the supernatant.

10. Gently resuspend the cells in Lonza electroporation Buffer P3 (70 μL per reaction).

11. Carefully add 70 μL of cell suspension to the RNP mixture and mix by gently pipetting 2 to 3 times.

12. Transfer the mixture to the electrode cup. Be careful to avoid bubbles in the well to avoid sparks.

13. Gently tap the side of the electrode cup to ensure that the sample covers the bottom of the electrode cup. If the liquid does not cover the entire bottom of the test tube, an error will be reported during the electrotransfer process.

14. Place the electrode cup with lid into the 4D-Nucleofector ^TM X electroporation module and check the correct orientation of the electrode cup.

15. Start the Nucleofection ^™ program by pressing "Start" on the 4D-Nucleofector ^™ Core Unit display.

16. Immediately after the run is complete, carefully remove the electrode cup from the holder and add 800 μL of pre-warmed TCM-0 medium to each well, avoiding pipetting and mixing. Incubate in a 37°C incubator with 5% _CO2 for 1 hour.

17. After standing, transfer the cells to a T25 flask containing 7.0 mL of pre-warmed TCM-60 medium and culture in a 37°C incubator containing 5% _CO2 .

Harvesting and characterizing TIL cells with knockout of Roquin-1 and/or Regnase-1 genes

1. 48 hours after the Pre-rep TIL electroporation, ^5x104 cells were taken and mixed with trophoblast cells at a ratio of 1:200 in BCM-2 medium and cultured statically for 4 days.

2. After 4 days, TILs were rapidly expanded in BCM-3 medium and reached the culture endpoint after 9 days. During this period, the cells were resuspended every other day to record the cell density and the culture system was adjusted according to the cell density to ensure that the live cell density was (1-3) ^x106 /mL.

3. At the end of the culture, more than 1x10 ⁶ cells were taken to extract the genome, and PCR was performed using the sequencing primers shown in SEQ ID NO: 3 to SEQ ID NO: 6, and then the gene knockout efficiency was tested by first-generation sequencing. The sequencing experiment was completed by Genewise or GenScript Biotech Co., Ltd.

4. The remaining TILs at the end of the culture were frozen using freezing medium with a cell density of ² x 107 cells per tube. They were cooled at -80°C for 24 hours and then transferred to a liquid nitrogen tank.

The gene knockout results are shown in Figure 1, indicating that the sgRNA shown in the present invention has good knockout efficiency.

Table 2: sgRNA and sequencing primer sequences used in this example

Example 2: Knockout of Roquin-1 and/or Regnase-1 genes promotes TIL proliferation

The TILs in Example 1 were statically mixed and cultured with trophoblast cells in BCM-2 medium for 4 days, and rapidly expanded in BCM-3 medium. The culture endpoint was reached after 9 days. During this period, the cells were resuspended every other day to record the cell density and cell viability, and the culture system was adjusted according to the cell density to ensure that the viable cell density was (1 to 3) x 10 ^{6 cells} /mL, and the cell expansion multiple during the rapid expansion period was recorded.

The results of cell expansion times during rapid expansion are shown in Figure 2, where Ctrl represents control TIL without knockout. It can be seen that the cell expansion times of TIL cells with knockout of Roquin-1 and/or Regnase-1 genes were significantly higher than those of the control group, indicating that knockout of Roquin-1 and/or Regnase-1 genes can promote TIL proliferation.

Example 3: Knockout of Roquin-1 and/or Regnase-1 genes reduces the dependence of TIL expansion on IL-2

The TILs frozen at the end of the culture in Example 1 were revived and cultured for another 4 days using TCM-0 medium. During this period, the cells were resuspended every other day to record the cell density and cell viability, and the culture system was adjusted according to the cell density to ensure that the viable cell density was (1 to 3) x 10 ⁶ cells/mL, and the total number of cells was calculated.

The results are shown in Figure 3. It can be seen that in the absence of IL-2, the number of TIL cells with knockout of Roquin-1 and/or Regnase-1 genes was significantly higher than that of the control group, indicating that knockout of Roquin-1 and/or Regnase-1 genes can reduce the dependence of TIL expansion on IL-2.

Example 4: Knocking out Roquin-1 and/or Regnase-1 genes can promote TIL secretion of IFN-γ and granzyme B

After 24h of culturing the frozen TIL at the end point in Example 1, TIL cells were added to culture wells containing P815-GFP cells ( ^3x104 /well) according to an effect-target ratio of 20:1 or 10:1 or 5:1 or 1:1, wherein the culture medium of each well contained 0.5μg/mL anti-CD3 antibody (OKT3), and the co-incubation basal culture fluid was TCM-0, with the anti-CD3 group as the control. After 24h of co-incubation, the cell culture plate was taken out, centrifuged at 400xg for 5min, and the cell supernatant was collected, and the dilution was 100 times (for Granzyme B detection) and 20 times (for IFN-γ detection) respectively, and then detected according to the Human Granzyme B ELISA kit (Invitrogen, BMS2027-2) and Human IFN gamma ELISA kit (Sigma, RAB0222) kit operating instructions.

The results are shown in Figures 4 and 5. It can be seen that at an effector-target ratio of 5:1, the concentration of IFN-γ secreted by TILs with double knockout of Roquin-1 and Regnase-1 genes was significantly higher than that of the control group; at an effector-target ratio of 10:1, the concentration of granzyme B secreted by TILs with single knockout of Regnase-1 gene or double knockout of Roquin-1 and Regnase-1 genes was significantly higher than that of the control group, indicating that knockout of Roquin-1 and/or Regnase-1 genes can promote the secretion of IFN-γ and granzyme B by TILs.

Example 5: Knocking out Roquin-1 and/or Regnase-1 genes can promote the killing function of TIL on P815 cells

The TIL from sample RC22 was gene edited, rapidly amplified and frozen with reference to the method described in Example 1. After 24 hours of resuscitation of the TIL, TIL cells were added to culture wells containing P815-GFP cells (3x10 ⁴ cells/well) at an effector-target ratio of 20:1, 10:1 or 5:1, wherein the culture medium in each well contained 0.5 μg/mL anti-CD3 antibody (OKT3), and the co-incubation basal culture medium was TCM-0, with the anti-CD3 group as the control. After standing at room temperature for 20 minutes, it was placed in the Incucyte device, and the green fluorescence signal was collected every 4 hours for a total of 48 hours, and the statistical data was analyzed using the Incucyte Basic analysis analysis module.

The results are shown in Figure 6. It can be seen that the killing rate of TIL with Roquin-1 and/or Regnase-1 gene knockout on P815 cells is higher than that of the control group, indicating that knockout of Roquin-1 and/or Regnase-1 gene can promote the killing function of TIL on P815 cells.

Example 6: Knocking out Roquin-1 and/or Regnase-1 genes can promote the killing function of TIL on primary ovarian cancer tumor cells

The primary ovarian cancer cells from the sample RC22 were prestained with red fluorescent dye After labeling with Cytolight Rapid Red Dye (Sartorius Cat. No. 4706) at 7°C for 20 min, the cells were seeded in a 96-well plate at a density of 1×10 ⁴ cells/well for later use.

The TILs derived from the frozen sample RC22 in Example 5 were resuscitated, and the TILs were labeled using the SuperView ^™ 488Caspase-3 live cell assay kit (US Everbright, S6007L), and co-incubated with primary ovarian cancer tumor cells in TCM-0 medium at an effector-target ratio of 20:1, 10:1, or 5:1. After standing at room temperature for 20 minutes, the cells were placed in the Incucyte device, and green and red fluorescence signals were collected every 4 hours for a total of 48 hours. The statistical data were analyzed using the Incucyte Basic analysis analysis module.

The results are shown in Figure 7. It can be seen that the killing rate of primary tumor cells by TIL with Roquin-1 and/or Regnase-1 gene knockout was higher than that of the control group, indicating that knockout of Roquin-1 and/or Regnase-1 gene can promote the killing function of TIL on primary tumor cells.

Example 7: Knockout of Roquin-1 and/or Regnase-1 genes can promote the increase of TCM subsets of TIL central memory cells

[Corrected 26.10.2023 in accordance with Article 91]
TIL was gene edited and cultured for 48 hours according to the method described in Example 1 to obtain Edited Pre-rep TIL. 5x10 ⁴ Edited Pre-rep TIL were taken and mixed with trophoblast cells at a ratio of 1:200 in BCM-2 culture medium containing three different concentrations of IL-2 (750IU/mL, 1500IU/mL, 3000IU/mL) for amplification and culture for 4 days. After 7 days, it was replaced with BCM-3 culture medium containing three different concentrations of IL-2 (750IU/mL, 1500IU/mL, 3000IU/mL) and continued to be cultured for 9 days. During this period, the cells were resuspended every other day to record the cell density and cell viability. The culture system was adjusted according to the cell density to ensure that the live cell density was (1-3)x10 ⁶ /mL. 1x10 ⁶ cells were collected at the end of the culture, and the cells were washed with PBS containing 2% FBS, and then incubated with antibodies at 4°C for 30min. The antibodies are shown in Table 3. After cells were washed twice, the cells were analyzed using a Miltenyi MACS Quant Analyzer 16 fully automated multicolor flow cytometer.

Table 3: Flow cytometry antibody information

Figure 8 shows that under stimulation of different IL2 concentrations, knockout of Roquin-1 and/or Regnase-1 genes can increase the proportion of TCMs. This suggests that TILs with target knockout may have better persistence (Role of memory T cell subsets for adoptive immunotherapy, Dirk H Busch et al. 2016). TEFF represents terminal effector T cells, T denotes early T cells, TCM central memory T cells, and TEM effector memory T cells.

Example 8: Knocking out Roquin-1 and/or Regnase-1 genes can slightly increase the proportion of CD4+ cells in TILs

[Corrected 26.10.2023 in accordance with Article 91]
TIL was gene edited and cultured for 48 hours according to the method described in Example 1 to obtain Edited Pre-rep TIL. Take 5x10 ⁴ Edited Pre-rep TIL, mix with trophoblast cells at a ratio of 1:200, and expand and culture in BCM-2 complete culture medium for 4 days. After 7 days, use BCM-3 rapid culture medium to continue to culture TIL cells for 9 days. During this period, resuspend the cells every other day to record the cell density and cell viability. Adjust the culture system according to the cell density to ensure that the live cell density is (1-3) x10 ⁶ / mL. Collect 1x10 ⁶ cells at the end of the culture, wash the cells with PBS containing 2% FBS, and incubate the antibodies at 4°C for 30 minutes. The antibodies are shown in Table 3. After the cells were washed twice, they were detected using Miltenyi MACS Quant Analyzer 16 fully automatic multicolor flow cytometer.

Figure 9 shows that knockout of Roquin-1 and/or Regnase-1 genes can slightly increase the proportion of CD4+ cells in TILs. This suggests that TILs with target knockout may have stronger killing function (CD4+T cells in cancer, Daniel E. Speiser et al. 2023; Revisiting the role of CD4+T cells in cancer immunotherapy—new insights into old paradigms, Rong En Tay et al. 2020; Multiple roles for CD4+T cells in anti-tumor immune responses, Richa rd Kennedy et al.2008; Adoptive cell therapy with CD4+T helper 1cells and CD8+cytotoxic T cells enhances complete rejection of a n established tumour, leading to generation of endogenous memory responses to non-targeted tumour epitopes, Kunyu Li, et al. 2017).

It should be understood that although the present invention has been described exemplarily according to its preferred embodiments, it should not be limited to the above embodiments. For those skilled in the art, the present invention can have various modifications and variations. The selection and application of specific antibodies can be adjusted and changed accordingly according to specific needs. Therefore, for those skilled in the art, several simple substitutions can be made without departing from the concept and principle of the present invention, which should all be included in the scope of protection of the present invention.

Claims (33)

A modified tumor infiltrating lymphocyte (TIL), wherein the expression and/or function of the Roquin-1 gene or its gene product is reduced or eliminated. A modified tumor-infiltrating lymphocyte, wherein the expression and/or function of Roquin-1 and Regnase-1 genes or their gene products are reduced or eliminated. According to the modified TIL according to any one of claims 1 or 2, the expression and/or function of the Roquin-1 and/or Regnase-1 gene or its gene product of the TIL is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%, respectively, compared to the unmodified or control TIL. According to any one of claims 1 to 3, the properties of the TIL are improved compared to unmodified or control TIL. According to the modified TIL of claim 4, the improved TIL properties include one or more selected from the following groups: improved TIL cell proliferation ability, improved cytokine secretion ability, improved granzyme secretion ability, increased proportion of central memory T cells (Central Memory T cell, TCM), reduced IL-2 dependence, and improved tumor cell killing ability. A method for preparing the modified TIL according to any one of claims 1 to 5, wherein the Roquin-1 and/or Regnase-1 gene or its gene product is treated by one or more of the following methods: gene editing technology, RNA interference technology, PROTAC technology, antibodies or small molecule inhibitors. The preparation method according to claim 6, wherein the gene editing technology adopts one or more of the following methods: CRISPR/Cas technology, transcription activator-like effector nuclease (TALEN) technology, zinc finger nuclease (Zinc-finger nuclease, ZFN) technology, single or multi-base mutation, guide editing or site-directed knock-in. The CRISPR/Cas technology according to claim 7, wherein the Cas types include: Cas9, Cas12a, Cas3, Cas13, Cas14, Cas7, Cas8, Cas10 and Cas11. The CRISPR/Cas9 technology according to claim 8, wherein the Cas9 types include: SpCas9, SaCas9, SpCas9-HF, eSpCas9, xCas9 and cpf1. The preparation method according to any one of claims 6 to 9, wherein the Roquin-1 and/or Regnase-1 genes are disrupted and/or knocked out by CRISPR/Cas technology. The preparation method according to any one of claims 6 to 10, wherein the Roquin-1 and/or Regnase-1 genes are disrupted and/or knocked out by CRISPR/Cas9 technology. The preparation method according to any one of claims 6 to 11, wherein the CRISPR/Cas9 technology comprises introducing a CRISPR/Cas9 system containing both a single-stranded guide RNA (sgRNA) targeting a target gene and a Cas9 nuclease into the TIL according to any one of claims 1 to 5. The preparation method according to any one of claims 6 to 12, wherein the sgRNA comprises an sgRNA targeting the Roquin-1 gene and an sgRNA targeting the Regnase-1 gene. According to the preparation method according to any one of claims 6 to 13, the CRISPR/Cas9 technology can use sgRNA targeting the Roquin-1 gene and sgRNA targeting the Regnase-1 gene separately or simultaneously. According to the preparation method according to any one of claims 6-14, the targeting sequence in the sgRNA targeting the Roquin-1 gene is CCTGAATAAACTCCACCGCA (SEQ ID NO: 1) or a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 1; the targeting sequence of the sgRNA targeting the Regnase-1 gene is GTGGACTTCTTCCGGAAGCT (SEQ ID NO: 2) or a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 2. The preparation method according to any one of claims 6 to 15, wherein the method for introducing the CRISPR / Cas9 system into the TIL comprises introducing a Cas9-sgRNA ribonucleoprotein (RNP) complex or a vector containing a Cas9 protein and an sgRNA expression element into the TIL according to any one of claims 1 to 5 by chemical transfection, electroporation or vector delivery. The preparation method according to any one of claims 6-16, wherein the Cas9-sgRNA RNP complex is introduced into the TIL according to any one of claims 1-5 by electroporation. A method for producing the TIL according to any one of claims 1 to 5, comprising the following steps: (1) processing a tumor sample obtained from a subject into multiple tumor fragments to obtain a TIL population; (2) expanding the obtained TIL population in vitro in a culture medium containing T cell growth factors to obtain a Pre-rep TIL population; (3) Treating the Pre-REP TIL population using the preparation method described in any one of claims 6 to 17, and co-culturing them with trophoblast cells in a culture medium containing T cell growth factors, so that the expression and/or function of the Roquin-1 and/or Regnase-1 genes are reduced or eliminated. The preparation method according to claim 18, wherein the T cell growth factor is selected from one or more of IL-2, IL-7, IL-15, and IL-21. The preparation method according to any one of claims 18 or 19, wherein the T cell growth factor is IL-2. The preparation method according to claim 18, wherein the final concentration of the T cell growth factor in step (2) is about 750-6000 IU/mL. The preparation method according to claim 18, wherein the final concentration of the T cell growth factor in step (2) is 6000 IU/mL. The preparation method according to claim 18, wherein the final concentration of the T cell growth factor in step (3) is about 750-6000 IU/mL. The preparation method according to claim 18, wherein the final concentration of the T cell growth factor in step (3) is 3000 IU/mL. The preparation method according to claim 18, wherein the TIL is autologous TIL for the subject. A pharmaceutical composition comprising the TIL according to any one of claims 1 to 5 or the TIL treated by the method according to any one of claims 6 to 25 and optionally a pharmaceutically acceptable carrier. Use of the TIL according to any one of claims 1 to 5, or the TIL treated by the method according to any one of claims 6 to 25, or the pharmaceutical composition according to claim 26 in the preparation of a medicament for preventing and/or treating tumors. The use according to claim 27, wherein the tumor is a solid tumor. The use according to any one of claims 27 or 28, wherein the solid tumor is selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and renal cancer. A method for treating a tumor in a subject in need thereof, the method comprising administering to the subject the TIL according to any one of claims 1 to 5, or the TIL treated by the method according to any one of claims 6 to 25, or the pharmaceutical composition according to claim 26. The method of claim 30, wherein the tumor is a solid tumor. The method of claim 30 or 31, wherein the solid tumor is selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and renal cancer. A use of reducing or eliminating the expression and/or function of Roquin-1 and/or Regnase-1 genes in TIL, the use comprising improving TIL cell proliferation ability, improving cytokine secretion ability, improving granzyme secretion ability, improving the proportion of central memory T cells (TCM), reducing IL-2 dependence, and improving tumor cell killing ability; the TIL is the TIL according to any one of claims 1 to 5 or the TIL treated by the method according to any one of claims 6 to 25.