Cell population as well as preparation method and application thereof

Abstract

The invention discloses a cell population as well as a preparation method and application thereof, and belongs to the technical field of biology. The cell population comprises engineered immune cells, the engineered immune cells comprise an exogenous nucleic acid, the insertion of the exogenous nucleic acid into a B2M gene fragment region causes the B2M gene to be disrupted, and the exogenous nucleic acid comprises a nucleic acid encoding a chimeric antigen receptor. The cell population destroys B2M by knocking nucleic acid encoding a chimeric antigen receptor (CAR) into a B2M locus, can be used for preparing a universal cell therapy product, and overcomes the problems that autologous T cell therapy is limited by the number and quality of T cells of a patient, and the preparation period is long.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a cell population, a preparation method and an application thereof. Background Art

[0002] In the field of cell therapy, especially in tumor treatment, cell immunotherapy has shown great potential. Among them, CAR-T (chimeric antigen receptor T cell) therapy, as an important cell immunotherapy, has achieved significant breakthroughs in the treatment of various malignant tumors.

[0003] The key to CAR-T therapy is the creation of CAR-T cells by knocking in genes carrying specific antigen receptors, enabling them to recognize and attack tumor cells. However, the CAR-T positivity rates of current CAR-T products derived through CAR-T editing technology are relatively low, which means that directly using CAR-T products with low CAR-positivity rates can reduce therapeutic efficacy. Therefore, how to improve the CAR-positivity rates of CAR-T products has become a pressing technical challenge for those skilled in the art.

[0004] In addition, the use of autologous T cell therapy is limited by the quantity and quality of the patient's T cells and has a long preparation cycle. Summary of the Invention

[0005] To address the low CAR positivity rate of the above-mentioned CAR-T products, the present invention provides an engineered immune cell that disrupts B2M by knocking a nucleic acid encoding a chimeric antigen receptor (CAR) into the B2M locus. This engineered immune cell can be used to prepare universal cell therapy products, overcoming the problems of autologous T cell therapy being limited by the number and quality of the patient's T cells and having a long preparation cycle.

[0006] On the one hand, the present invention provides a cell population, which comprises engineered immune cells, wherein the engineered immune cells comprise exogenous nucleic acid, wherein the exogenous nucleic acid is inserted into the B2M gene fragment region to destroy the B2M gene, and the exogenous nucleic acid comprises a nucleic acid encoding a chimeric antigen receptor; in the cell population, cells expressing the chimeric antigen receptor account for more than 60% of the total cell number.

[0007] For example: in the cell population, the cells expressing the chimeric antigen receptor account for more than 60.74%, more than 65%, more than 70%, more than 75%, more than 80%, more than 80.26%, more than 80.50%, more than 81.45%, more than 81.96%, more than 82.15%, more than 82.54%, more than 82.83%, more than 83.86%, more than 84%, more than 85%, more than 90%, more than 91.31%, more than 95%, more than 95.90%, more than 96.72%, more than 99.02%, and more than 99.51% of the total cell number.

[0008] In some embodiments, the exogenous nucleic acid further comprises a nucleic acid encoding a cytokine. Preferably, the cytokine is selected from at least one of IL15, IL-21, IL-9, IL-7, IL-2, IL-12 and IL-18; more preferably, the cytokine is selected from IL15 and / or IL-21.

[0009] In some embodiments, the nucleotide sequence of IL15 is SEQ ID NO:10, and the amino acid sequence is SEQ ID NO:11.

[0010] In some embodiments, the nucleotide sequence of IL-21 is SEQ ID NO: 26, and the amino acid sequence is SEQ ID NO: 27.

[0011] The above scheme knocks in cytokines while gene editing, and cultures the gene-edited cell population. Engineered immune cells expressing cytokines (such as CAR-T cells) will be cultured preferentially, and immune cell populations with a high CAR positivity rate will be obtained through self-enrichment. No subsequent purification or enrichment is required, which simplifies the production process and reduces production costs.

[0012] In some of the schemes, the exogenous nucleic acid is expressed by the endogenous promoter of the B2M gene or the exogenous MND promoter, preferably by the endogenous promoter of the B2M gene. The MND promoter, which is a promoter replaced by the dl587rev primer binding site in the negative regulatory region of the myeloproliferative sarcoma virus enhancer, can guide the sustained expression of the CAR polypeptide in resting, activated and amplified T cells, and such expression can effectively redirect engineered immune cells to induce cytotoxic activity against tumors or cancer cells. The expression of CAR in T cells driven by the MND promoter is comparable to that of the EF1α promoter, with little difference. The present invention has found through experiments that expression with the endogenous promoter of the B2M gene has a higher CAR positivity rate.

[0013] In some embodiments, the exogenous nucleic acid is inserted into the Chr15: 44711358-44718851 region;

[0014] Preferably, when the Cas12a editing system is selected to insert an exogenous nucleic acid, the exogenous nucleic acid is inserted into the Chr15:44715615-44715634 region;

[0015] When the Cas9 editing system is used to insert exogenous nucleic acid, the exogenous nucleic acid is inserted into the Chr15: 44715521-44715540 and / or Chr15: 44715516-44715535 region.

[0016] In some embodiments, the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence, and the extracellular domain recognizes and binds to the antigen;

[0017] The intracellular domain comprises an activation domain, or comprises a co-stimulatory domain and an activation domain.

[0018] In some embodiments, the extracellular domain comprises an antibody or antigen-binding fragment, such as a scFv, a single domain antibody, or a F(ab)'.

[0019] In some embodiments, the transmembrane domain is selected from the transmembrane domains of the following proteins: CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and / or CD154; preferably, it is the transmembrane domain of CD8.

[0020] In some embodiments, the activation domain comprises a functional signaling domain selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, FcεRIγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10 and / or DAP12; preferably, it is a functional signaling domain of CD3ζ.

[0021] In some embodiments, the costimulatory domain is selected from the costimulatory domains of the following proteins: 4-1BB, CD27, CD28, OX-40 and / or ICOS; preferably, it is the costimulatory domain of CD28 and / or 4-1BB.

[0022] In some embodiments, the chimeric antigen receptor further comprises a hinge region, which connects the extracellular domain and the transmembrane domain; preferably, the hinge region comprises the hinge region of CD8α or CD28, for example, the hinge region of CD8α.

[0023] In some embodiments, the chimeric antigen receptor further comprises a linker signal peptide connected to the extracellular domain. Preferably, the linker signal peptide is a signal peptide of CD8α.

[0024] In some embodiments, the extracellular domain targets a tumor antigen; preferably, the tumor antigen is selected from GPC3, CLDN18.2, GCC, EGFRvIII, ROR1, CLDN6, MSLN, ALPP, MUC1, LGR5, HER2, OR2H1, DLL-3, C-MET, glyco-cMET, glyco-LAMP1, CD123, CD33, CLL-1, CD70, CD38, FLT3, and GRP78. For example, the antigen-binding domain targeting the tumor antigen GPC3 can be GC33 scFv (nucleotide sequence of SEQ ID NO: 4) or YP7 scFv (sequence of SEQ ID NO: 29, amino acid sequence of SEQ ID NO: 30).

[0025] In some embodiments, the activation domain is a functional signal transduction domain of CD3ζ. Preferably, the activation domain comprises a nucleotide sequence as shown in SEQ ID NO: 9.

[0026] In some of these embodiments, the costimulatory domain is a CD28 costimulatory domain and / or a 4-1BB costimulatory domain. Preferably, the costimulatory domain comprises a nucleotide sequence as shown in SEQ ID NO: 7 and / or SEQ ID NO: 8.

[0027] In some embodiments, the transmembrane domain is the transmembrane domain of CD8. Preferably, the transmembrane domain comprises the nucleotide sequence shown in SEQ ID NO: 6.

[0028] In some embodiments, the hinge region is the hinge region of CD8α. Preferably, the hinge region comprises the nucleotide sequence shown in SEQ ID NO: 5.

[0029] In some embodiments, the linker signal peptide is a signal peptide of CD8α. Preferably, the linker signal peptide comprises a nucleotide sequence as shown in SEQ ID NO: 3.

[0030] It is understandable that the structure and specific sequence of the above-mentioned chimeric antigen receptor can be selected according to common knowledge in the art, and it does not affect the CAR positivity rate of gene editing.

[0031] In some embodiments, the nucleic acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 1 or a sequence with more than 90% homology to SEQ ID NO: 1, for example, more than 95% or more than 98%.

[0032] In some embodiments, the source cells of the engineered immune cells are selected from one or more of: T cells, NK cells, NKT cells, monocytes, mast cells, macrophages, dendritic cells, CIK cells and stem cell-derived immune effector cells;

[0033] More preferably, the source cells are T cells, NK cells or NKT cells; for example, T cells.

[0034] On the other hand, the present invention also discloses a method for preparing the above-mentioned engineered immune cells, comprising the following steps: delivering CRISPR / Cas nuclease, gRNA targeting the B2M gene, and a donor template to the immune cells, performing gene editing on the immune cells, and obtaining a cell population containing the engineered immune cells;

[0035] The donor template comprises a first homology arm sequence, a second homology arm sequence and the exogenous nucleic acid of the above-mentioned cell population, and the guide sequence of the gRNA binds to the Chr15:44711358-44718851 target region.

[0036] The above-mentioned gene editing system targeting the Chr15:44711358-44718851 region is a gene editing system with high knock-in efficiency obtained through screening, which can obtain positive CAR immune cells with high editing efficiency.

[0037] In some embodiments, the guide sequence of the gRNA is as shown in SEQ ID NO: 17 or has 1-5 mismatches with the sequence shown in SEQ ID NO: 17, and the CRISPR / Cas nuclease is Cpf1 Ultra; further, the first homology arm sequence is as shown in SEQ ID NO: 13 or a sequence with more than 90% homology to SEQ ID NO: 13, and the second homology arm sequence is selected as shown in SEQ ID NO: 14 or a sequence with more than 90% homology to SEQ ID NO: 14.

[0038] In some embodiments, the guide sequence of the gRNA is as shown in SEQ ID NO:22 or has 1-5 mismatches with the sequence shown in SEQ ID NO:22, and the CRISPR / Cas nuclease is Cas9; further, the first homology arm sequence is as shown in SEQ ID NO:24 or a sequence with more than 90% homology to SEQ ID NO:24, and the second homology arm sequence is selected as shown in SEQ ID NO:25 or a sequence with more than 90% homology to SEQ ID NO:25.

[0039] In some of these schemes, the gRNA can be modified using chemical modification methods well known to those skilled in the art, such as, in some embodiments, 2'-O-methyl (2'-O-Me) modification, 2'-fluoro (2'-F) modification. In some embodiments, the modification between the gRNA nucleotides comprises a phosphorothioate (PS) bond. In some schemes, the first three nucleotides at the 5' end and the last three nucleotides at the 3' end are modified. In some schemes, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are connected to a phosphorothioate (PS) bond.

[0040] In some of these schemes, gRNA can also be linked using modified ribonucleotides, deoxyribonucleotides, other synthetic bases, and synthetic backbones (such as peptide nucleic acid (PNA), locked nucleic acid (LNA), etc.). In some embodiments, the nucleic acid constituting the guide nucleotide sequence comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cyt ... 5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0041] In some of these schemes, the methods of delivering CRISPR / Cas nucleases and gRNA to the immune cells include: liposome delivery, lipid nanoparticle delivery, extracellular vesicle delivery, viral particle delivery or electroporation, preferably electroporation after forming RNP; the methods of delivering donor templates to the immune cells include: liposome delivery, lipid nanoparticle delivery, extracellular vesicle delivery, viral particle delivery, electroporation or minicircle DNA delivery, preferably through viral particle delivery or minicircle DNA delivery.

[0042] In some of the schemes, the method for preparing the cell population specifically comprises the following steps:

[0043] Gene editing: selecting a donor template containing the exogenous nucleic acid in the above-mentioned cell population, delivering CRISPR / Cas nuclease, gRNA targeting the B2M gene, and the donor template to the immune cells, and performing gene editing on the immune cells to obtain a cell population containing engineered immune cells;

[0044] Advantageous culture: The above cell population containing engineered immune cells is cultured to obtain.

[0045] In some embodiments, in the dominant culturing step, expression of the chimeric antigen receptor is detected in more than 80% of the cells cultured, preferably more than 90%, and more preferably more than 95%.

[0046] In some of these schemes, the culture medium used for cell culture is selected from T cell culture medium. Preferably, when the source cells of the engineered immune cells are T cells, the culture medium comprises the following components: T cell serum-free culture medium, 2% to 8% immune cell serum replacement by volume, and 100 to 1000 IU / ml of IL-2; when the source cells of the engineered immune cells are NKT cells, the culture medium comprises the following components: T cell serum-free culture medium, 2% to 8% immune cell serum replacement by volume, 100 to 1000 IU / ml of IL-2, and 5 to 30 ng / ml of IL-21.

[0047] In some embodiments, the cell culture conditions include a culture period of 5-25 days, preferably 5-15 days when the source cells are T cells, and preferably 5-25 days when the source cells are NKT cells. It is understood that the above cell culture conditions can be set in a conventional manner.

[0048] On the other hand, the present invention also provides a composition for gene editing, comprising a CRISPR / Cas nuclease and / or a nucleotide sequence encoding the CRISPR / Cas nuclease, and a gRNA, a nucleotide sequence encoding the gRNA, and / or a nucleotide composition comprising a nucleotide sequence encoding the gRNA.

[0049] The CRISPR / Cas nuclease is Cpf1 Ultra, and the guide sequence of the gRNA is as shown in SEQ ID NO: 17 or has 1-5 mismatches with the sequence shown in SEQ ID NO: 17;

[0050] or

[0051] The CRISPR / Cas nuclease is Cas9, and the guide sequence of the gRNA is as shown in SEQ ID NO: 22 or has 1-5 mismatches with the sequence shown in SEQ ID NO: 22.

[0052] In some embodiments, the composition further comprises a donor template comprising a first homology arm sequence, a second homology arm sequence, and the exogenous nucleic acid of the cell population described above;

[0053] When the guide sequence of the gRNA is as shown in SEQ ID NO: 17 or has 1-5 mismatches with the sequence shown in SEQ ID NO: 17, the first homology arm sequence is as shown in SEQ ID NO: 13 or a sequence with more than 90% homology to SEQ ID NO: 13, and the second homology arm sequence is selected as shown in SEQ ID NO: 14 or a sequence with more than 90% homology to SEQ ID NO: 14;

[0054] When the guide sequence of the gRNA is as shown in SEQ ID NO: 22 or has 1-5 mismatches with the sequence shown in SEQ ID NO: 22, the first homology arm sequence is as shown in SEQ ID NO: 24 or a sequence with more than 90% homology to SEQ ID NO: 24, and the second homology arm sequence is selected as shown in SEQ ID NO: 24 or a sequence with more than 90% homology to SEQ ID NO: 24.

[0055] On the other hand, the present invention also provides the use of the above-mentioned cell population, the above-mentioned preparation method, and the above-mentioned composition in the preparation of drugs for diagnosing, preventing and / or treating tumors.

[0056] In some of these regimens, the tumor includes a solid tumor and a non-solid tumor.

[0057] In some of the schemes, the solid tumor is selected from: liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, neurilemmoma, malignant fibrous histiocytoma and esophageal cancer; the non-solid tumor is selected from: B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

[0058] On the other hand, the present invention also provides a composition for diagnosing, preventing and / or treating tumors, comprising the above-mentioned cell population and / or the above-mentioned composition.

[0059] In some of these regimens, the tumor includes a solid tumor and a non-solid tumor.

[0060] In some of the schemes, the solid tumor is selected from: liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, neurilemmoma, malignant fibrous histiocytoma and esophageal cancer; the non-solid tumor is selected from: B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

[0061] On the other hand, the present invention also provides a method for diagnosing, preventing and / or treating tumors in patients in need thereof, characterized in that the method comprises administering an effective amount of the above-mentioned cell population and / or the above-mentioned composition to a patient in need thereof;

[0062] For example, the tumor may include solid tumors and non-solid tumors;

[0063] Preferably, the solid tumor is selected from the group consisting of liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, neurilemmoma, malignant fibrous histiocytoma and esophageal cancer; and the non-solid tumor is selected from the group consisting of B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

[0064] On the other hand, the present invention also provides a method for diagnosing, preventing and / or treating tumors in patients in need thereof, characterized in that the method comprises administering an effective amount of the above-mentioned cell population and / or the above-mentioned composition to a patient in need thereof;

[0065] For example, the tumor may include solid tumors and non-solid tumors;

[0066] Preferably, the solid tumor is selected from the group consisting of liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, neurilemmoma, malignant fibrous histiocytoma and esophageal cancer; and the non-solid tumor is selected from the group consisting of B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

[0067] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0068] The reagents and raw materials used in the present invention are commercially available.

[0069] The positive progress effect of the present invention is:

[0070] The cell population comprising engineered immune cells of the present invention destroys B2M by knocking nucleic acid encoding chimeric antigen receptor (CAR) into the B2M locus, and can be used to prepare universal cell therapy products, overcoming the problems that autologous T cell therapy is limited by the number and quality of the patient's T cells and has a long preparation cycle.

[0071] In addition, cytokines can be knocked in during gene editing, and the gene-edited cell population can be cultured. Engineered immune cells expressing cytokines (such as CAR-T cells) will be preferentially cultured, and immune cell populations with a high CAR positivity rate can be obtained through self-enrichment. No subsequent purification or enrichment is required, which simplifies the production process and reduces production costs.

[0072] At the same time, through the gene editing system with high knock-in efficiency obtained through screening, we can obtain positive CAR immune cells with high editing efficiency, and obtain cell therapy products with high CAR positivity rate, providing good quality assurance for the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is a graph showing the purity of T cells and B2M expression rate detected by flow cytometry after T cells were electroporated and cultured for 4 days in Example 2.

[0074] Figure 2 This is a graph showing the CAR positivity rate of T cells cultured for 4 days and 11 days after electroporation detected by flow cytometry in Example 2.

[0075] Figure 3 This is a flow cytometric analysis of B2M expression in iNKT cells cultured for 4 days after electroporation in Example 3.

[0076] Figure 4 This is a graph showing the purity, B2M expression rate, and CAR positivity of iNKT cells detected by flow cytometry after electroporation and culture for 4 days in Example 4.

[0077] Figure 5 This is a graph showing the purity of iNKT cells, B2M expression rate, and CAR positivity rate detected by flow cytometry after iNKT cells were electroporated and cultured for 18 days in Example 4.

[0078] Figure 6 This is a graph showing the B2M expression rate detected by flow cytometry in Example 5 after electroporation of T cells and culture for 4 days.

[0079] Figure 7 This is a graph showing the T cell purity, B2M expression rate, and CAR positivity rate detected by flow cytometry after T cell electroporation and culture for 4 days in Example 6. DETAILED DESCRIPTION

[0080] The term "chimeric antigen receptor" or "CAR (Chimeric Antigen Receptor)" used herein generally refers to a fusion protein comprising an extracellular domain and an intracellular domain that can bind to an antigen. CAR is a core component of a chimeric antigen receptor immune effector cell, which may include an antigen (e.g., tumor-associated antigen (TAA)) binding domain, a transmembrane domain, and an intracellular domain. In the present application, the chimeric antigen receptor can be combined with an immune effector cell receptor activation intracellular domain based on the antigen (e.g., GPC3) specificity of an antibody. Immune effector cells that are genetically modified to express CAR can specifically recognize and eliminate malignant cells that express the target antigen.

[0081] As used herein, the term "extracellular domain" generally refers to a domain that can specifically bind to an antigen, including but not limited to the extracellular domain of a single-chain antibody, an antibody or its antigen-binding fragment, a bound ligand, or a co-receptor. For example, the extracellular domain may comprise an antibody or its antigen-binding fragment that can specifically bind to an antigen expressed on a cell. The terms "binding domain," "extracellular domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" used in this application are used interchangeably and provide a domain or fragment of a CAR that has the ability to specifically bind to a target antigen (e.g., GPC3). The extracellular domain may be of natural, synthetic, semisynthetic, or recombinant origin. The "specific binding" generally refers to a measurable and reproducible interaction, such as binding between an antigen and an antibody, which can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to an antigen (which may be an epitope) is an antibody that binds to the antigen with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other antigens. As used herein, the term "epitope" and its grammatical alternatives may refer to a portion of an antigen that is recognized by an antibody, B cell, T cell, or engineered cell. For example, an epitope may be a tumor epitope or pathogen epitope recognized by a chimeric antigen receptor. Multiple epitopes within an antigen may also be recognized. Epitopes may also mutate.

[0082] The term "antibody" is used in the broadest sense herein to refer to a polypeptide or combination of polypeptides that comprises sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and various structures, as long as they exhibit the desired antigen-binding activity. In the present invention, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only complete antibodies but also antigen-binding fragments of antibodies. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which include mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art that can be used to transplant CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0083] "Antigen-binding fragments" herein do not possess the full structure of an intact antibody, but rather comprise only a portion or partial variant of an intact antibody that possesses the ability to bind to an antigen. Exemplarily, "antigen-binding fragments" herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0084] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0085] The term "transmembrane domain" as used herein refers to a structure that can anchor CAR to the plasma membrane of a cell, which is connected to the intracellular domain and plays a role in transmitting signals. The natural transmembrane portion of common cell adhesion molecules can be used for the "transmembrane domain" of CAR. For example, the natural transmembrane portion of CD8α or the natural transmembrane portion of CD28 can be used as the "transmembrane domain" in CAR.

[0086] As used herein, the term "activation domain" refers to a molecule on an immune effector cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

[0087] As used herein, the term "costimulatory domain" refers to a cognate binding partner on an immune effector cell that specifically binds a costimulatory ligand, thereby mediating a co-stimulatory response of the immune cell, such as, but not limited to, proliferation.

[0088] As used herein, the term "targeting" refers to specific binding, specifically to a non-random binding reaction between two molecules, such as an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 binds to the antigen with an affinity (KD) of M or less.

[0089] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0090] The term "tumor antigen" as used herein refers to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). TAA or TSA can be expressed on blood cancer cells. TAA or TSA can be expressed on solid tumor cells. The tumor antigen is selected from one or more of the following: CD19, CD133, CD123, CD20, CD22, CD30, CD33, CD171, CD80 / 86, CA125, C-met, L1CAM, EC, DLL3, CD99, GRP78, 5T4, CD138, CS-1 (also known as CD2 subclass 1, CRACC, SLAMF7, CD319 or 19A24), Glycan-3 (GPC3), Claudin 18.2 (Claudin 18.2), Glycan-4 (GPC4), Glycan-5 (GPC6), Glycan-7 (GPC8), Glycan-9 (GPC9), Glycan-10 (GPC11), Glycan-11 (GPC12), Glycan-10 (GPC13), Glycan-10 (GPC14), Glycan-10 (GPC15), Glycan-10 (GPC16), Glycan-10 (GPC17), Glycan-10 (GPC18), Glycan-10 (GPC19), Glycan-10 (GPC11), Glycan-10 (GPC11), Glycan-10 (GPC15), Glycan-10 (GPC16), Glycan-10 (GPC17), Glycan-10 (GPC19), Glycan-10 (GPC1 ... 18.2), guanylate cyclase C (GCC / GUCY2C), mesothelin (MSLN), epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), alpha-fetoprotein (AFP), tyrosine-protein kinase receptor UFO (AXL), death receptor 5 (DR5), NKG2D ligand, prostate stem cell antigen (PSCA), macrophage-stimulating protein receptor (MST1R), inhibitory leukocyte immunoglobulin-like receptor (LILRB4), C-type lectin-like molecule-1 (CLL-1 or CLECL1), epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation antigen (BCMA), Tn antigen (such as Tn Ag, GalNAcα-Ser / Thr), glyco-cMET, glyco-LAMP1, receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3);Tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, B7H3 (CD276), B7-H4, KIT (CD117), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), stage-specific embryonic antigen-4 (SSEA-4), folate receptor alpha (FR-α), receptor tyrosine-protein kinase ERBB2 (Her2 / neu), cell surface-associated mucin 1 (MUC1), cell surface-associated Mucin-16 (MUC16), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), mutant elongation factor 2 (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (e.g., proteasome, megalin) subunit type β 9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1;Sialyl Lewis adhesion molecule (sLe), transglutaminase 5 (TGS5), high molecular weight-melanoma associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tight junction protein 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5 member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), globoH The hexose moiety of glycoceramide (GloboH), breast differentiation antigen (NY-BR-1), urothelial differentiation-specific glycoprotein (uroplakin) 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenergic receptor β3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternative open reading frame protein (TARP), Wilm tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6 on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, p53, p53 mutant, prostate-specific protein (prostein), prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1);Rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), melanoma inhibitor of apoptosis protein (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytic leukemia virus oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), squamous cell carcinoma antigen 3 recognized by T cells (SART3), paired box protein Pax-5 (PAX5), pre-acrosomal protein binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchoring protein 4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, mutant heat shock protein 70-2 (mut hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of the IgA receptor (FCAR or CD890), leukocyte immunoglobulin-like receptor subfamily A, member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12, member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2), lymphocyte antigen 75 (LY75), placental alkaline phosphatase (ALPP), Fc receptor-like 5 (FCRL5) and / or immunoglobulin lambda-like polypeptide 1, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and olfactory receptor OR2H.

[0091] As used herein, the term "solid tumor" refers to a tumor selected from the group consisting of liver cancer, stomach cancer, lung cancer, breast cancer, colon cancer, renal cell carcinoma, non-small cell lung cancer, small intestine cancer, esophageal cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, liposarcoma, melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, neurilemmoma, malignant fibrous histiocytoma, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, spinal vertebral tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, and squamous cell carcinoma.

[0092] As used herein, the term "non-solid tumor" refers to a tumor selected from the group consisting of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, T-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, B lymphocytic neoplasm, and Waldenstrom macroglobulinemia.

[0093] As used herein, the term "glypican-3 (GPC3)" is a heparan sulfate (HS) glycoprotein, a member of the heparan sulfate proteoglycan family, anchored to the cell membrane surface via phosphatidylinositol (GPI). "GPC3" herein includes mature or immature, full-length, wild-type GPC3 proteins, as well as mutants (e.g., point mutations, insertion mutations, or deletion mutations), splice variants, orthologs, and fragments of the aforementioned GPC3. Exemplarily, "GPC3" herein can be derived from mammals, such as humans, primates, such as monkeys (e.g., rhesus monkeys and cynomolgus monkeys), and rodents, such as mice and rats.

[0094] The term "hinge region" as used herein can be considered as a part for providing flexibility to the extracellular antigen binding region, which is generally used to maintain further stable expression and activity of the chimeric antigen receptor in immune effector cells. The hinge region can be derived from the hinge region of CD8α or CD28 extracellular domain or IgG.

[0095] As used herein, the term "linker signal peptide" refers to a polypeptide attached to the N-terminus of a chimeric antigen receptor that may affect protein secretion efficiency. The linker signal peptide can be derived from a cell adhesion molecule, such as the signal peptide of CD8, or an immunoglobulin heavy chain signal peptide of an immune effector cell, such as a T cell or NK cell.

[0096] As used herein, the term "polypeptide" refers to a compound composed of amino acid residues covalently linked by peptide bonds. Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0097] As used herein, the term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence in biological processes, and the resulting biological properties. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is typically provided in sequence listings) and the non-coding strand (which serves as a template for transcription of the gene or cDNA) can be said to encode a protein or other product of the gene or cDNA.

[0098] The term "immune effector cells" as used herein refers to immune cells that can participate in the clearance of foreign antigens and perform effector functions in immune responses, and can be used in cell therapy as autologous cells or allogeneic cells for cell immunotherapy.

[0099] As used herein, the term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

[0100] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0101] Example 1

[0102] An engineered immune cell (CD3+ T cell) is prepared by the following method (using the Cpf1 system):

[0103] 1. Prepare T cells

[0104] PBMCs were resuscitated (purchased from Shanghai Heyousheng Biotechnology Co., Ltd.), CD3+ T cells were isolated using the EasySep™ Human T Cell Isolation Kit (purchased from Stem Cell Technology, Catalog No. 17951), and activated using T Cell TransAct™, human (purchased from Miltenyi Biotec, Catalog No. 130-128-758). T cells were then cultured in T cell medium composed of (hereinafter referred to as the following): Optivitro UniEx T Cell Serum-Free Medium (purchased from Ekosai, Catalog No. TE000-N052) + 5% by volume of CTS™ Immune Cell Serum Replacement (purchased from Gibco, Catalog No. A2596101) + 300 IU / ml IL-2 (purchased from Shuanglu Pharmaceutical, Xingjier® recombinant human interleukin-2 for injection).

[0105] 2. Prepare the donor template

[0106] AAV6 is a non-integrating virus that naturally tropes for leukocytes and is widely known as a highly efficient vector for delivering donor templates to T cells. This example, based on a third-generation CAR structure targeting GPC3, constructs AAV6 viruses loaded with four different donor templates (Source: Guangzhou Paizhen Biotechnology Co., Ltd.): AAV6 virus-1, AAV6 virus-2, AAV6 virus-3, and AAV6 virus-4.

[0107] AAV6 virus-1 contains an exogenous nucleic acid template sequence encoding CAR (chimeric antigen receptor), the coding sequence of CAR is SEQ ID NO: 1, the amino acid sequence of CAR is SEQ ID NO: 2, and it includes the following parts in sequence: CD8α signal peptide (sequence is SEQ ID NO: 3), GC33 scFv (i.e., the antigen binding domain targeting GPC3, coding sequence is SEQ ID NO: 4), CD8α hinge region (coding sequence is SEQ ID NO: 5), CD8α transmembrane region (coding sequence is SEQ ID NO: 6), CD28 costimulatory domain (coding sequence is SEQ ID NO: 7), 4-1BB costimulatory domain (i.e., CD137 costimulatory domain, coding sequence is SEQ ID NO: 8) and CD3ζ intracellular domain (coding sequence is SEQ ID NO: 9).

[0108] AAV6 virus-2 contains an exogenous nucleic acid template sequence encoding CAR and cytokine IL15. The CAR sequence is as described above, the coding sequence of IL15 is SEQ ID NO: 10, and the amino acid sequence of IL15 is SEQ ID NO: 11.

[0109] AAV6 virus-3 contains an exogenous nucleic acid template sequence encoding the MND promoter, CAR and cytokine IL15, wherein the CAR sequence and IL15 sequence are as described above, and the nucleotide sequence of the MND promoter is SEQ ID NO:12.

[0110] AAV6 virus-4 contains an exogenous nucleic acid template sequence encoding the MND promoter and CAR, wherein the CAR sequence is as described above, and the nucleotide sequence of the MND promoter is SEQ ID NO:12.

[0111] The above-mentioned exogenous nucleic acid is also connected to the first homology arm sequence and the second homology arm sequence (i.e., the left homology arm sequence and the right homology arm sequence) at both ends, respectively. The left homology arm sequence is shown in SEQ ID NO: 13, and the right homology arm sequence is shown in SEQ ID NO: 14.

[0112] Among them, the CAR provided by AAV6 virus-1 and the CAR-IL15 provided by AAV6 virus-2 are driven by the endogenous promoter of the knockout site B2M gene; the exogenous MND promoter is introduced between the CAR and IL15 of the CAR-IL15 provided by AAV6 virus-2 to drive the expression of IL15; in the structure of the MND promoter-CAR-IL15 provided by AAV6 virus-3, an exogenous MND promoter is introduced in front of the CAR to drive the expression of CAR and IL15, and the CAR coding sequence and IL15 coding sequence in the MND promoter-CAR-IL15 are connected by T2A (SEQ ID NO: 15); in the structure of the MND promoter-CAR provided by AAV6 virus-4, an exogenous MND promoter is introduced in front of the CAR to drive the expression of CAR.

[0113] That is, the AAV6 virus-1 donor template structure is: left homology arm sequence-CAR coding sequence-stop codon (TGA)-right homology arm sequence. The AAV6 virus-2 donor template structure is: left homology arm sequence-CAR coding sequence-stop codon (TGA)-MND promoter sequence-IL15 coding sequence-stop codon (TGA)-right homology arm sequence. The AAV6 virus-3 donor template structure is: left homology arm sequence-MND promoter sequence-CAR coding sequence-T2A sequence-IL15 coding sequence-stop codon (TGA)-right homology arm sequence. The AAV6 virus-4 donor template structure is: left homology arm sequence-MND promoter sequence-CAR coding sequence-stop codon (TGA)-right homology arm sequence.

[0114] 3. Prepare RNPs for the Cpf1 System

[0115] The RNP1 of the Cpf1 system was prepared by mixing gRNA1 (SEQ ID NO: 16, purchased from Gibco, Cat. No. C10010500CP; its guide sequence is SEQ ID NO: 17, binding to Chr15: 44715615-44715634) and Alt-R® As Cas12a (Cpf1) v3 (purchased from Integrated DNA Technologies, Cat. No. 1081069) in PBS. The concentration of gRNA1 in the RNP1 system was 10 μM, and the concentration of Alt-R® As Cas12a (Cpf1) v3 in the RNP system was 0.6 mg / mL.

[0116] 4. Cell transfection

[0117] On the third day after T cell activation, T cells were divided into five groups, namely, NC T cell group, endogenous promoter CAR T cells group, endogenous promoter CAR-IL15 T cells group, MND promoter CAR-IL15 T cells group and MND promoter CAR T cells group.

[0118] Among them, the NC T cell group did not receive any treatment. The endogenous promoter CAR T cell group, endogenous promoter CAR-IL15 T cell group, MND promoter CAR-IL15 T cell group, and MND promoter CAR T cell group were infected with AAV6 virus-1, AAV6 virus-2, AAV6 virus-3, and AAV6 virus-4, respectively, with an infection titer of 3×10 5GC / cell, 6 h after infection, centrifuge and remove the supernatant, and use the electroporation kit P3 Primary Cell 4D-Nucleofector™ XKit (purchased from Lonza, catalog number V4XP-3032) to electroporate the RNP obtained in step 3 into T cells. The electroporation program used is CA-137.

[0119] After electroporation of T cells, add 80 μL of T cell culture medium and transfer T cells to a cell culture plate to adjust the cell density to approximately 6 × 10 5 / ml and continue to culture.

[0120] Example 2

[0121] In this example, the engineered immune cell population prepared in Example 1 was tested for CAR positivity, and flow cytometry was performed on the T cells 4 and 11 days after electroporation.

[0122] 1. Methods

[0123] 1. CAR positive rate detection

[0124] The CAR positivity rate of T cells was analyzed by flow cytometry using a combination of FITC anti-human CD3 Antibody (purchased from Biolegend, Catalog No. 317306) and anti-G4Slinker antibody (name: Anti-(G4S)n(B02H1)mAb(APC), purchased from Shanghai Heyousheng, Catalog No. GS-ARAP100).

[0125] 2. B2M expression rate detection

[0126] The purity of T cells and the expression rate of B2M were analyzed by flow cytometry using FITC anti-human CD3 Antibody (purchased from Biolegend, Catalog No. 317306) and APC anti-human β2-microglobulin Antibody (purchased from Biolegend, Catalog No. 395712).

[0127] 2. Results

[0128] The results are as follows Figure 1 and Figure 2 As shown, Figure 1The figure shows the purity of T cells and the expression rate of B2M detected by flow cytometry after culturing T cells for 4 days after electroporation in Example 2. The purity of T cells in the NC T cell group, the endogenous promoter CAR T cell group, the endogenous promoter CAR-IL15 T cell group, the MND promoter CAR-IL15 T cell group, and the MND promoter CAR T cell group was very high, all above 98.5%. The B2M expression rate in the NC T cell group was 99.99%, and the knockout efficiency of B2M in the endogenous promoter CAR T cell group, the endogenous promoter CAR-IL15 T cell group, the MND promoter CAR-IL15 T cell group, and the MND promoter CAR T cell group was very high, above 95% (99.84%, 95.55%, 99.78%, and 98.24%, respectively). Figure 2 This is a graph of the CAR positivity rate detected by flow cytometry after T cells were electroporated for 4 days and 11 days in Example 2. It can be seen from the graph that after 4 days of culture, the CAR positivity rates of the NC T cell group, endogenous promoter CAR T cells group, endogenous promoter CAR-IL15 T cells group, MND promoter CAR-IL15 T cells group and MND promoter CAR T cells group were 0.39%, 84.00%, 88.38%, 44.81% and 49.94%, respectively; after 11 days of culture, the CAR positivity rates of the NC T cell group, endogenous promoter CAR T cells group, endogenous promoter CAR-IL15 T cells group, MND promoter CAR-IL15 T cells group and MND promoter CAR T cells group were 0.56%, 82.83%, 99.51%, 60.74% and 45.38%, respectively.

[0129] The results showed that there was no significant change in the CAR positivity rate of the endogenous promoter CAR T cells group after electroporation 4 days and 11 days after electroporation, and there was no significant change in the CAR positivity rate of the MND promoter CAR T cells group after 4 days and 11 days after electroporation. However, the CAR positivity rate of the endogenous promoter CAR-IL15 T cells group and the MND promoter CAR-IL15 T cells group after 11 days after electroporation was significantly improved compared with that after 4 days after electroporation, from 88.38% to 99.51% in the endogenous promoter CAR-IL15 T cells group and from 44.81% to 60.74% in the MND promoter CAR-IL15 T cells group. It can be seen that co-expression of the cytokine IL15 in CAR-T cells can make the CAR-T cells grow faster than T cells that have not been successfully transduced with CAR, giving CAR-T cells an advantage in culture and allowing self-enrichment to obtain CAR-T cells with a higher CAR positivity rate. Compared with the MND promoter-based CAR-IL15 T cell group and the MND promoter-based CAR T cell group, the endogenous promoter-based CAR T cell group and the endogenous promoter-based CAR-IL15 T cell group showed significantly higher CAR positivity rates at 4 and 11 days post-electroporation, respectively. This indicates that using the B2M endogenous promoter for CAR expression achieves higher insertion efficiency. In the endogenous promoter-based CAR-IL15 T cell group, CAR was expressed via the B2M endogenous promoter, with IL15 and CAR co-expressed. The CAR positivity rate was 99.51% at 11 days post-electroporation, indicating that CAR-T cell population proportions reached 99.51% through culture self-enrichment, achieving a surprising result.

[0130] Example 3

[0131] An engineered immune cell (iNKT cell-derived, B2M knockout) was prepared by the following method (using the Cpf1 system):

[0132] 1. Preparation of iNKT cells

[0133] PBMC cells were isolated from single-sample blood (purchased from Shanghai Heyousheng Company) using lymphocyte separation fluid (name: LymphoprepTM, purchased from Stemcell, product number 07811). PBMCs were then magnetically sorted to obtain iNKT cells (Anti-iNKT MicroBeads human, purchased from Miltenyi Biotec, catalog number 130-094-842). iNKT cells were resuspended and cultured in iNKT cell medium. The composition of the iNKT cell medium (the same below) is as follows: Optivitro UniEx T cell serum-free medium (purchased from Ekosai, catalog number TE000-N052) + 5% by volume of CTS™ immune cell serum replacement (purchased from Gibco, catalog number A2596101) + 300 IU / ml IL-2 (Xingjier® recombinant human interleukin-2 for injection purchased from Shuanglu Pharmaceuticals) + 10 ng / ml IL-21 (purchased from Beijing Tongli Haiyuan Biological, catalog number GMP-TL509). iNKT cells were activated with transact (T Cell TransAct, human, purchased from Miltenyi Biotec, Cat. No. 130-128-758) for 3 days, and then the transact stimulation was removed and cultured.

[0134] 2. Prepare RNPs for the Cpf1 System

[0135] RNP1 and RNP2 of the cpf1 system were prepared according to the RNP preparation method described in Example 1 and used to knock out the B2M locus. RNP1 used gRNA1 (SEQ ID NO: 16, with a guide sequence of SEQ ID NO: 17, binding to Chr15: 44715615-44715634); RNP2 used gRNA2 (SEQ ID NO: 18, with a guide sequence of SEQ ID NO: 19, binding to Chr15: 44711594-44711613).

[0136] 3. Cell transfection

[0137] On day 5 after iNKT cell activation, iNKT cells were divided into three groups: NC iNKT group, B2M gRNA1 iNKT group, and B2M gRNA2 iNKT group. The NC iNKT group did not receive any treatment. The B2M gRNA1 iNKT group and the B2M gRNA2 iNKT group were electroporated with RNP1 or RNP2 into iNKT cells using the P3 Primary Cell 4D-Nucleofector™ X Kit (purchased from Lonza, catalog number V4XP-3032). The electroporation protocol used was CA-137. After iNKT cell electroporation, 80 μL of iNKT cell culture medium was added, and the iNKT cells were transferred to a cell culture plate to adjust the cell density to 6 × 10 5 / ml and continue to culture.

[0138] 4. Editing efficiency detection

[0139] After iNKT cells were electroporated and cultured for 4 days, the B2M knockout efficiency was analyzed by flow cytometry staining with FITC anti-human β2-microglobulin Antibody (purchased from Biolegend, Cat. No. 395706).

[0140] The results are as follows Figure 3 As shown, Figure 3 Figure 3 shows flow cytometry analysis of B2M expression in iNKT cells cultured four days after electroporation. The results showed that the B2M knockout efficiency in the B2M gRNA1 iNKT group was 94%, while that in the B2M gRNA2 iNKT group was only 3.43%, indicating that gRNA1 is significantly more suitable for B2M knockout, disrupting B2M for the preparation of a universal cell therapy product and facilitating subsequent CAR insertion at the B2M site.

[0141] Example 4

[0142] An engineered immune cell (iNKT cell) is prepared by the following method (using the Cpf1 system):

[0143] 1. Preparation of iNKT cells

[0144] iNKT cells were prepared according to the method in Example 3.

[0145] 2. Cell transfection

[0146] On the 5th day after iNKT cell activation, iNKT cells were divided into two groups, namely, NC iNKT group and CAR iNKT group. The NCiNKT group did not receive any treatment, while the CAR iNKT group was infected with the AAV6 virus-2 in Example 1 with an infection titer of 3×105 GC / cells were infected for 6 h, and the supernatant was removed by centrifugation. The RNP1 described in Example 3 was electroporated into iNKT cells using the P3 Primary Cell 4D-Nucleofector™ X Kit (purchased from Lonza, catalog number V4XP-3032). The electroporation protocol used was CA-137.

[0147] After electroporation of iNKT cells, 80 μL of iNKT cell culture medium was added and iNKT cells were transferred to a cell culture plate to adjust the cell density to 6×10 5 / ml and continue to culture.

[0148] 3. Detection of iNKT cell purity, B2M knockout efficiency and CAR positivity rate

[0149] After electroporation of iNKT cells, the purity of iNKT cells, B2M knockout efficiency, and CAR positive rate were detected by flow cytometry on days 3 and 18 after culture. The detection method is as follows:

[0150] 1) The iNKT cell purity of CAR-iNKT cells was analyzed by flow cytometry using APC anti-human CD3 Antibody (purchased from Biolegend, Catalog No. 300412) and PE anti-human TCR Vα24-Jα18 (iNKT cell) Antibody (purchased from Biolegend, Catalog No. 342904).

[0151] 2) The B2M knockout efficiency of CAR-iNKT cells was analyzed by flow cytometry using APC anti-human β2-microglobulin Antibody (purchased from Biolegend, Catalog No. 395712) and PE anti-human TCR Vα24-Jα18 (iNKT cell) Antibody (purchased from Biolegend, Catalog No. 342904).

[0152] 3) The CAR positivity rate of CAR-iNKT cells was analyzed by flow cytometry using a combination of anti-G4Slinker antibody (Anti-(G4S)n(B02H1)mAb(APC), Catalog No. GS-ARAP100, purchased from Shanghai Heyousheng) and PE anti-human TCR Vα24-Jα18 (iNKT cell) Antibody (purchased from Biolegend, Catalog No. 342904).

[0153] The results are as follows Figure 4 and Figure 5As shown, Figure 4 This is a graph showing the purity, B2M expression rate, and CAR positivity of iNKT cells cultured for 4 days after electroporation in Example 4 using flow cytometry. Figure 5 This is a graph showing the purity of iNKT cells, B2M expression rate, and CAR positivity rate detected by flow cytometry after iNKT cells were electroporated and cultured for 18 days in Example 4.

[0154] The results showed that after culturing iNKT cells for 4 days after electroporation, the purity of NC iNKT group cells (CD3+iNKT+) in the CAR iNKT group was over 70%, the B2M site knockout efficiency (B2M-) was 95.74%, the CAR positive rate (CAR+) was 80.50%, and the positive rate of iNKT cells (iNKT+CAR+) was 67.38%, indicating that the insertion efficiency of CAR expression at the B2M site using gRNA1 was high. After 18 days of culture following electroporation, the purity of iNKT cells (CD3+iNKT+) in the CAR iNKT group was over 96%, the B2M knockout efficiency (B2M-) was 99.28%, the CAR positivity rate (CAR+) was 91.31%, and the positivity rate of iNKT cells (iNKT+CAR+) was 87.21%. This indicates that CAR-positive cells grow significantly faster than untransduced cells, giving CAR-positive cells an advantage during culture. Self-enrichment can increase the proportion of CAR-positive cells. Furthermore, the purity of iNKT cells (CD3+iNKT+) increased from 70.99% to 97.03%, demonstrating that self-enrichment can significantly improve iNKT cell purity. CAR-iNKT cells, obtained through self-enrichment through culture, can be used as cell therapy products with high CAR positivity, providing excellent quality assurance for therapeutic efficacy.

[0155] Example 5

[0156] An engineered immune cell (T cell-derived, B2M knockout) was prepared by the following method (using the Cas9 system):

[0157] 1. Prepare T cells

[0158] T cells were prepared and cultured according to the method in Example 1.

[0159] 2. Prepare RNPs for the SpCas9 System

[0160] The Cas9 system RNPs were prepared by mixing gRNA and SpCas9 Nuclease (purchased from Kaixia Biotechnology, Cat. No. Cas-EE109) in PBS for knockout of the B2M locus. The gRNA concentration in the RNP system was 10 μM, and the SpCas9 Nuclease concentration in the RNP system was 0.6 mg / mL.

[0161] In this example, three RNPs were used, namely RNP3, RNP4, and RNP5, differing only in that the gRNAs used were gRNA3, gRNA4, and gRNA5, respectively. gRNA3, gRNA4, and gRNA5 are all single-molecule gRNAs, each comprising the structure: 5'-[guide sequence]-[gRNA backbone sequence]-3'. The guide sequences of gRNA3, gRNA4, and gRNA5 are SEQ ID NO: 20 (binding to Chr15: 44715512-44715531), SEQ ID NO: 21 (binding to Chr15: 44715516-44715535), and SEQ ID NO: 22 (reverse complement to Chr15: 44715521-44715540), respectively, and the gRNA backbone sequence is SEQ ID NO: 23.

[0162] 3. Cell transfection

[0163] On the third day after T cell activation, T cells were divided into four groups: NC iNKT group, B2M gRNA3 T group, B2M gRNA4 T group, and B2M gRNA5 T group. The NC iNKT group did not receive any treatment. The B2M gRNA3 T group, B2M gRNA4 T group, and B2MgRNA5 T group were electroporated with RNP3, RNP4, or RNP5 into T cells using the P3 Primary Cell 4D-Nucleofector™ X Kit (purchased from Lonza, catalog number V4XP-3032). The electroporation protocol used was CA-137. After T cell electroporation, 80 μL of T cell culture medium was added, and the T cells were transferred to a cell culture plate to adjust the cell density to 6×10 5 / ml and continue to culture.

[0164] 4. Editing efficiency detection

[0165] Four days after electroporation, the B2M knockout efficiency was analyzed by flow cytometry staining with FITC anti-human β2-microglobulin Antibody (purchased from Biolegend, Cat. No. 395706).

[0166] The results are as follows Figure 6As shown, Figure 6 Figure 5 shows the flow cytometry analysis of B2M expression in T cells cultured for 4 days after electroporation in Example 5. The results showed that the B2M knockout efficiency was 12.60% in the B2M gRNA3 T group, 84.73% in the B2M gRNA4 T group, and 92.03% in the B2M gRNA5 T group, indicating that gRNA5 is significantly more suitable for B2M knockout in the Cas9 system, disrupting B2M for the preparation of universal cell therapy products and facilitating the subsequent insertion of CAR at the B2M site.

[0167] Example 6

[0168] An engineered immune cell (T cell) produced by the following method (using the Cas9 system):

[0169] 1. Prepare T cells

[0170] T cells were prepared according to the method of Example 1.

[0171] 2. Prepare the donor template

[0172] Based on the third-generation CAR structure, targeting GPC3, a donor template AAV virus-2' was constructed. Compared with the AAV6 virus-2 in Example 1, the only difference was that it was replaced with a homology arm suitable for the Cas9 system, wherein the left homology arm sequence is shown in SEQ ID NO: 24, and the right homology arm sequence is shown in SEQ ID NO: 25.

[0173] 3. Cell transfection

[0174] On the third day after T cell activation, the T cells were divided into two groups, namely, NC T cells group and CAR T cells group. The NCT cells group did not receive any treatment, while the CAR T cells group was infected with AAV virus-2' at an infection titer of 3×10 5 GC / cells were infected for 6 h, and the supernatant was removed by centrifugation. The RNP5 described in Example 5 was electroporated into T cells using the P3 Primary Cell 4D-Nucleofector™ XKit (purchased from Lonza, catalog number V4XP-3032). The electroporation procedure used was CA-137.

[0175] After electroporation of T cells, add 80 μL of T cell culture medium and transfer T cells to a cell culture plate to adjust the cell density to 6×10 5 / ml and continue to culture.

[0176] 4. T cell purity, B2M knockout efficiency and CAR positive rate detection

[0177] T cell purity, B2M knockout efficiency, and CAR positive rate were detected by flow cytometry 4 days after T cell electroporation. The detection method is as follows:

[0178] 1) The T cell purity and B2M knockout efficiency of CAR-T cells were analyzed by flow cytometry using FITC anti-human CD3 Antibody (purchased from Biolegend, Catalog No. 317306) and APC anti-human β2-microglobulin Antibody (purchased from Biolegend, Catalog No. 395712).

[0179] 2) The CAR positivity rate of T cells was analyzed by flow cytometry using a combination of FITC anti-human CD3 Antibody (purchased from Biolegend, Catalog No. 317306) and anti-G4Slinker antibody (Anti-(G4S)n(B02H1)mAb(APC), Catalog No. GS-ARAP100, purchased from Shanghai Heyousheng).

[0180] The results are as follows Figure 7 As shown, Figure 7 Figure 6 shows flow cytometry analysis of T cell purity, B2M expression rate, and CAR positivity after four days of culture following electroporation. The results show that after four days of culture, the CAR T cell group had a T cell (CD3+) purity of 99.09%, a B2M site knockout efficiency (B2M-) of 98.40%, and a CAR positivity rate (CAR+) of 81.96%. This indicates that gRNA5 insertion at the B2M site for CAR expression is highly efficient, facilitating the preparation of cell therapy products with high CAR positivity rates and providing excellent quality assurance for therapeutic efficacy.

[0181] Example 7

[0182] An engineered immune cell (using T cells as source cells) is prepared by the following method (using the Cpf1 system):

[0183] 1. Prepare T cells

[0184] T cells were prepared and cultured according to the method in Example 1.

[0185] 2. Prepare the donor template

[0186] Referring to the method of Example 1, two different AAV6 viruses were constructed, namely AAV6 virus-5 and AAV6 virus-6. The template nucleic acid sequences contained CAR-IL21 and CAR-IL15-IL21, respectively. The CAR structure and sequence were the same as in Example 1. The only difference between AAV6 virus-5 and AAV6 virus-2 was that the IL15 coding sequence was replaced with the IL21 coding sequence. The difference between AAV6 virus-6 and AAV6 virus-2 was that the IL15 coding sequence was replaced with a coding sequence for co-expression of IL15 and IL21. The coding sequence of IL21 is SEQ ID NO: 26, and the amino acid sequence of IL21 is SEQ ID NO: 27. The IL15 coding sequence and the IL21 coding sequence are connected by P2A (SEQ ID NO: 28).

[0187] 3. Cell transfection

[0188] On the third day after T cell activation, T cells were divided into five groups, namely, NC T cell group, CAR T cell group, CAR-IL15 T cell group, CAR-IL21 T cell group, and CAR-IL15-IL21 T cell group. The NC T cell group did not receive any treatment. The CAR T cell group, CAR-IL15 T cell group, CAR-IL21 T cell group, and CAR-IL15-IL21 T cell group were infected with AAV6 virus-1, AAV6 virus-2, AAV6 virus-5, and AAV6 virus-6, respectively, with an infection titer of 3×10 5 GC / cells were infected for 6 h, and the supernatant was removed by centrifugation. The RNP1 described in Example 1 was electroporated into T cells using the P3 Primary Cell 4D-Nucleofector™ X Kit (purchased from Lonza, catalog number V4XP-3032). The electroporation procedure used was CA-137.

[0189] After electroporation of T cells, add 80 μL of T cell culture medium and transfer T cells to a cell culture plate to adjust the cell density to 6×10 5 / ml and continue to culture.

[0190] 4. T cell purity, B2M knockout efficiency and CAR positive rate detection

[0191] Flow cytometry was performed on T cells 4 and 11 days after electroporation, using the same method as in Example 2. For the results, please refer to the table below. Table 1 shows the cell purity, B2M knockout efficiency, and CAR positivity data of flow cytometry analysis of T cells 4 and 11 days after electroporation in this example.

[0192] Table 1. T cell purity, B2M knockout efficiency, and CAR positive rate

[0193]

[0194] The results showed that co-expressing the cytokines IL15 or IL21 in CAR-T cells can increase the growth rate of CAR-T cells compared to non-CAR-transduced T cells, giving CAR-T cells an advantage in culture and allowing for self-enrichment of CAR-T cells with a higher CAR positivity rate. Furthermore, co-expressing both IL15 and IL21 in CAR-T cells can further accelerate the growth rate of CAR-T cells, giving them an advantage in culture and allowing for faster self-enrichment of CAR-T cells with a higher CAR positivity rate. This allows the resulting CAR-T cells to be used as high-CAR positivity cell therapy products, providing excellent quality assurance for therapeutic efficacy.

[0195] Example 8

[0196] An engineered immune cell (using T cells as source cells) is prepared by the following method (using the Cpf1 system):

[0197] 1. Prepare T cells

[0198] T cells were prepared and cultured according to the method in Example 1.

[0199] 2. Prepare the donor template

[0200] Referring to the method of Example 1, a new AAV6 virus, namely AAV6 virus-7, was constructed. The only difference between AAV6 virus-7 and AAV6 virus-2 was that the GC33 antibody in CAR was changed to the YP7 antibody (YP7 antibody coding sequence is SEQ ID NO: 29, amino acid sequence is SEQ ID NO: 30).

[0201] 3. Cell transfection

[0202] On the third day after T cell activation, T cells were divided into three groups, namely, NC T cell group, GC33-CAR-IL15 T cell group and YP7-CAR-IL15 T cell group. The NC T cell group did not receive any treatment. The GC33-CAR-IL15 T cell group and the YP7-CAR-IL15 T cell group were infected with AAV6 virus-2 and AAV6 virus-7, respectively, with an infection titer of 3×10 5GC / cells were infected for 6 h, and the supernatant was removed by centrifugation. The RNP of Example 1 was electroporated into T cells using the P3 Primary Cell 4D-Nucleofector™ X Kit (purchased from Lonza, catalog number V4XP-3032). The electroporation procedure used was CA-137.

[0203] After electroporation of T cells, add 80 μL of T cell culture medium and transfer T cells to a cell culture plate to adjust the cell density to 6×10 5 / ml and continue to culture.

[0204] 4. T cell purity, B2M knockout efficiency and CAR positive rate detection

[0205] Flow cytometry was performed on T cells 4 and 11 days after electroporation, using the same method as in Example 2. The results are shown in the table below. Table 2 shows the cell purity, B2M knockout efficiency, and CAR positivity data for flow cytometry analysis of T cells 4 and 11 days after electroporation.

[0206] Table 2. T cell purity, B2M knockout efficiency and CAR positive rate

[0207]

[0208] The results showed that co-expression of the cytokine IL15 in CAR-T cells and the use of GC33 or YP7 as the antibody in the CAR can make the CAR-T cells grow faster than T cells that are not transduced with CAR, allowing the CAR-T cells to gain an advantage in culture and self-enrichment to obtain CAR-T cells with a higher CAR positivity rate. The obtained CAR-T cells can be used as a high CAR positivity cell therapy product, providing a good quality guarantee for the treatment effect.

Claims

1. A cell population, characterized in that The cell population comprises engineered immune cells; The engineered immune cell comprises an exogenous nucleic acid, wherein the exogenous nucleic acid is inserted into the B2M gene segment region to disrupt the B2M gene, and the exogenous nucleic acid comprises a nucleic acid encoding a chimeric antigen receptor; In the cell population, cells expressing the chimeric antigen receptor account for more than 60% of the total cell number.

2. The cell population according to claim 1, wherein The exogenous nucleic acid further comprises a nucleic acid encoding a cytokine. Preferably, the cytokine is selected from at least one of IL15, IL-21, IL-9, IL-7, IL-2, IL-12 and IL-18; more preferably, the cytokine is selected from IL15 and / or IL-21.

3. The cell population according to claim 1, wherein The exogenous nucleic acid is expressed by the endogenous promoter of the B2M gene or the exogenous MND promoter, preferably by the endogenous promoter of the B2M gene.

4. The cell population according to claim 1, wherein The exogenous nucleic acid is inserted into the Chr15: 44711358-44718851 region; Preferably, when the Cas12a editing system is selected to insert an exogenous nucleic acid, the exogenous nucleic acid is inserted into the Chr15:44715615-44715634 region; When the Cas9 editing system is used to insert exogenous nucleic acid, the exogenous nucleic acid is inserted into the Chr15: 44715521-44715540 and / or Chr15: 44715516-44715535 region.

5. The cell population according to claim 1, wherein The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain connected in sequence; The extracellular domain recognizes and binds to the antigen; The intracellular domain comprises an activation domain, or comprises a co-stimulatory domain and an activation domain; Preferably, the extracellular domain comprises an antibody or antigen-binding fragment, such as scFv, single domain antibody or F(ab)'; And / or, the transmembrane domain is selected from the transmembrane domains of the following proteins: CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and / or CD154; preferably, the transmembrane domain of CD8; And / or, the activation domain comprises a functional signaling domain selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, FcεRIγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10 and / or DAP12; preferably, it is a functional signaling domain of CD3ζ; And / or, the costimulatory domain is selected from the costimulatory domains of the following proteins: 4-1BB, CD27, CD28, OX-40 and / or ICOS; preferably, the costimulatory domain of CD28 and / or 4-1BB.

6. The cell population according to claim 5, characterized in that The chimeric antigen receptor further comprises a hinge region, wherein the hinge region connects the extracellular domain and the transmembrane domain; preferably, the hinge region comprises the hinge region of CD8α or CD28, for example, the hinge region of CD8α; And / or, the chimeric antigen receptor further comprises a linker signal peptide connected to the extracellular domain, preferably, the linker signal peptide is a signal peptide of CD8α; And / or, the extracellular domain targets a tumor antigen; preferably, the tumor antigen is selected from GPC3, CLDN18.2, GCC, EGFRvIII, ROR1, CLDN6, MSLN, ALPP, MUC1, LGR5, HER2, OR2H1, DLL-3, C-MET, glyco-cMET, glyco-LAMP1, CD123, CD33, CLL-1, CD70, CD38, FLT3 and GRP78.

7. The cell population according to claim 6, characterized in that The activation domain is a functional signal transduction domain of CD3ζ. Preferably, the activation domain comprises the nucleotide sequence shown in SEQ ID NO: 9; And / or, the costimulatory domain is a CD28 costimulatory domain and / or a 4-1BB costimulatory domain, preferably, the costimulatory domain comprises the nucleotide sequence shown in SEQ ID NO: 7 and / or SEQ ID NO: 8; And / or, the transmembrane domain is the transmembrane domain of CD8, preferably, the transmembrane domain comprises the nucleotide sequence shown in SEQ ID NO: 6; And / or, the hinge region is the hinge region of CD8α, preferably, the hinge region comprises the nucleotide sequence shown in SEQ ID NO: 5; And / or, the connecting signal peptide is a signal peptide of CD8α. Preferably, the connecting signal peptide comprises a nucleotide sequence as shown in SEQ ID NO:

3.

8. The cell population according to claim 1, wherein The source cells of the engineered immune cells are selected from one or more of: T cells, NK cells, NKT cells, monocytes, mast cells, macrophages, dendritic cells, CIK cells and stem cell-derived immune effector cells; More preferably, the source cells are T cells, NK cells or NKT cells; for example, T cells.

9. The method for preparing a cell population according to any one of claims 1 to 8, characterized in that: The following steps are involved: delivering CRISPR / Cas nuclease, gRNA targeting the B2M gene, and a donor template to immune cells to perform gene editing on the immune cells to obtain a cell population containing engineered immune cells; The donor template comprises a first homology arm sequence, a second homology arm sequence and the exogenous nucleic acid of the cell population according to any one of claims 1 to 8, and the guide sequence of the gRNA binds to the Chr15:44711358-44718851 target region.

10. The method for preparing a cell population according to claim 9, wherein: The guide sequence of the gRNA is as shown in SEQ ID NO: 17 or has 1-5 mismatches with the sequence shown in SEQ ID NO: 17, and the CRISPR / Cas nuclease is Cpf1Ultra; further, the first homology arm sequence is as shown in SEQ ID NO: 13 or a sequence with greater than 90% homology to SEQ ID NO: 13, and the second homology arm sequence is as shown in SEQ ID NO: 14 or a sequence with greater than 90% homology to SEQ ID NO: 14; or The guide sequence of the gRNA is as shown in SEQ ID NO: 22 or has 1-5 mismatches with the sequence shown in SEQ ID NO: 22, and the CRISPR / Cas nuclease is Cas9; further, the first homology arm sequence is as shown in SEQ ID NO: 24 or a sequence with more than 90% homology to SEQ ID NO: 24, and the second homology arm sequence is selected as shown in SEQ ID NO: 25 or a sequence with more than 90% homology to SEQ ID NO:

25.

11. The method for preparing a cell population according to claim 9, wherein: Specifically include the following steps: Gene editing: selecting a donor template comprising the exogenous nucleic acid in the cell population of claim 9, delivering CRISPR / Cas nuclease, gRNA targeting the B2M gene, and the donor template to immune cells, and performing gene editing on the immune cells to obtain a cell population containing engineered immune cells; Advantageous culture: The above cell population containing engineered immune cells is cultured to obtain.

12. The preparation method according to claim 11, characterized in that At least one of the following conditions is met: (1) In the advantageous culture step, expression of the chimeric antigen receptor is detected in more than 80% of the cells, preferably more than 90%, and more preferably more than 95%; (2) The culture medium used for the cell culture is selected from T cell culture medium. Preferably, when the source cells of the engineered immune cells are T cells, the culture medium comprises the following components: T cell serum-free culture medium, 2% to 8% immune cell serum replacement by volume, and 100 to 1000 IU / mL of IL-2; when the source cells of the engineered immune cells are NKT cells, the culture medium comprises the following components: T cell serum-free culture medium, 2% to 8% immune cell serum replacement by volume, 100 to 1000 IU / ml of IL-2, and 5 to 30 ng / ml of IL-21; (3) The cell culture conditions include: a culture time of 5-25 days, preferably 5-15 days when the source cells are T cells, and preferably 5-25 days when the source cells are NKT cells.

13. A composition for gene editing, characterized in that comprising a CRISPR / Cas nuclease and / or a nucleotide sequence encoding the CRISPR / Cas nuclease, and a gRNA, a nucleotide sequence encoding the gRNA, and / or a nucleotide composition comprising a nucleotide sequence encoding the gRNA, The CRISPR / Cas nuclease is Cpf1 Ultra, and the guide sequence of the gRNA is as shown in SEQ ID NO: 17 or has 1-5 mismatches with the sequence shown in SEQ ID NO: 17; or The CRISPR / Cas nuclease is Cas9, and the guide sequence of the gRNA is as shown in SEQ ID NO: 22 or has 1-5 mismatches with the sequence shown in SEQ ID NO:

22.

14. The composition according to claim 13, characterized in that Also included is a donor template, wherein the donor template comprises a first homology arm sequence, a second homology arm sequence, and the exogenous nucleic acid of the cell population according to any one of claims 1 to 8; When the guide sequence of the gRNA is as shown in SEQ ID NO: 17 or has 1-5 mismatches with the sequence shown in SEQ ID NO: 17, the first homology arm sequence is as shown in SEQ ID NO: 13 or a sequence with more than 90% homology to SEQ ID NO: 13, and the second homology arm sequence is selected as shown in SEQ ID NO: 14 or a sequence with more than 90% homology to SEQ ID NO: 14; When the guide sequence of the gRNA is as shown in SEQ ID NO: 22 or has 1-5 mismatches with the sequence shown in SEQ ID NO: 22, the first homology arm sequence is as shown in SEQ ID NO: 24 or a sequence with more than 90% homology to SEQ ID NO: 24, and the second homology arm sequence is selected as shown in SEQ ID NO: 24 or a sequence with more than 90% homology to SEQ ID NO:

24.

15. Use of the cell population according to any one of claims 1 to 8, the preparation method according to any one of claims 9 to 12, or the composition according to any one of claims 13 to 14 in the preparation of a medicament for diagnosing, preventing and / or treating tumors; For example, the tumor may include solid tumors and non-solid tumors; Preferably, the solid tumor is selected from the group consisting of liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, neurilemmoma, malignant fibrous histiocytoma and esophageal cancer; and the non-solid tumor is selected from the group consisting of B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

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