WO2021136263A1 - 一种经修饰的免疫效应细胞及其制备方法 - Google Patents

一种经修饰的免疫效应细胞及其制备方法 Download PDF

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WO2021136263A1
WO2021136263A1 PCT/CN2020/140799 CN2020140799W WO2021136263A1 WO 2021136263 A1 WO2021136263 A1 WO 2021136263A1 CN 2020140799 W CN2020140799 W CN 2020140799W WO 2021136263 A1 WO2021136263 A1 WO 2021136263A1
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Prior art keywords
gene
immune effector
hla
cells
expression
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PCT/CN2020/140799
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English (en)
French (fr)
Chinese (zh)
Inventor
尚小云
蒋海娟
王丹
沈慧
马丽
辛雨
徐凡丽
李甲璐
马少文
赵丹
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Jiangsu T Maximum Biotech Co Ltd
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Jiangsu T Maximum Biotech Co Ltd
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Priority to KR1020227026014A priority Critical patent/KR20220124197A/ko
Priority to CA3163304A priority patent/CA3163304A1/en
Priority to AU2020418199A priority patent/AU2020418199B2/en
Priority to JP2022540754A priority patent/JP7670358B2/ja
Priority to CN202080090688.0A priority patent/CN115003802B/zh
Priority to EP20911252.3A priority patent/EP4086342A4/en
Publication of WO2021136263A1 publication Critical patent/WO2021136263A1/zh
Priority to US17/563,804 priority patent/US20220193135A1/en
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • This application relates to the field of biomedicine, in particular to a modified immune effector cell and a preparation method thereof.
  • Anti-tumor immunotherapy can cause a long-lasting and strong response in a variety of malignant tumors, can be used to treat many different types of cancer, and shows a wide range of potential.
  • Current anti-tumor immunotherapy mainly includes two types of immunotherapy: immune cell-targeted monoclonal antibody (mAb) therapy and adoptive cell therapy (ACT).
  • mAb monoclonal antibody
  • ACT adoptive cell therapy
  • ACT refers to the reinfusion of autologous or allogeneic lymphocytes stimulated and expanded in vitro into the human body to achieve the anti-tumor effect, but this therapy is only effective in patients with consistent MHC polymorphisms.
  • CAR-T is a new and effective MHC-independent adoptive cell therapy.
  • CAR also known as chimeric antigen receptor
  • CAR is an artificial receptor that mimics the function of TCR. It can specifically recognize antigens on the surface of tumor cells to target killing. Dead tumor cells.
  • the timely and successful production and infusion of autologous CAR T cells is the biggest obstacle to the implementation of effective CAR T cell therapy.
  • the patient was treated with chemotherapy before, and chemotherapy drugs will cause the patient's T cells to expand in vitro or reduce their function, resulting in failure to produce A sufficient quantity of CAR-T cell products, or poor quality.
  • autologous CAR-T is mostly used in ALL or CLL patients, and its application in solid tumor patients faces great challenges. For example, the heterogeneity of tumor antigens limits the application of autologous CAR-T.
  • the present application provides a modified immune effector cell, wherein the expression and/or activity of the TRAC gene and the HLA-A gene are down-regulated compared with the expression and/or activity of the corresponding gene in the corresponding cell without the modification ,
  • the expression and/or activity of the B2M gene is not down-regulated, and the expression and/or activity of the CIITA gene is not down-regulated.
  • the modification causes the expression and/or activity of two genes to be down-regulated, wherein the two genes consist of the TRAC gene and the HLA-A gene.
  • the expression and/or activity of the TRAC gene and HLA-A gene are down-regulated, the expression and/or activity of the B2M gene is not down-regulated, and the expression and/or activity of the CIITA gene is not down-regulated compared with the corresponding wild-type cell. /Or the activity is not down-regulated.
  • the expression and/or activity of two genes are down-regulated compared to corresponding wild-type cells, wherein the two genes consist of the TRAC gene and the HLA-A gene.
  • the immune effector cells include T cells.
  • the down-regulation of the expression level and/or activity of the gene includes down-regulating the expression and/or activity of the nucleic acid molecule encoding the gene; and/or down-regulating the expression and/or expression of the protein product encoded by the gene / Or the activity is down-regulated.
  • the modification includes: gene mutation and/or gene silencing.
  • the modification includes administering one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, and CRISPR/Cas9 system to the immune effector cell.
  • the modification includes administering the CRISPR/Cas9 system to the immune effector cells.
  • the alleles of the HLA-A gene are selected from the following group: A*02, A*11, A*24, A*30, A*33, A*03, A*01 and A*26.
  • the expression level and/or activity of at most 2 alleles in the HLA-A gene are down-regulated.
  • the expression level and/or activity of one allele in the HLA-A gene is down-regulated.
  • the modification includes administering to the immune effector cell an sgRNA targeting the exon portion of the HLA-A gene.
  • the sgRNA targeted to the exon part of the HLA-A gene comprises the nucleotide sequence shown in any one of SEQ ID Nos. 16-54 and 91-92.
  • the modification further comprises administering sgRNA targeting the exon part of the TRAC gene to the immune effector cell.
  • the sgRNA targeted to the exon part of the TRAC gene comprises the nucleotide sequence shown in any one of SEQ ID No. 1-15.
  • the antisense RNA comprises the nucleotide sequence shown in any one of SEQ ID No. 93-96.
  • the modification further comprises administering a Cas protein to the cell.
  • the Cas protein includes Cas9 protein.
  • the immune effector cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR), which includes an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory structure, and a primary signaling structure area.
  • CAR chimeric antigen receptor
  • the antigen binding domain specifically binds to tumor antigens.
  • the tumor antigen is selected from the following group: CD19, CD133, CD123, CD22, CD30, CD171, CA125, C-met, L1CAM, EC, DLL3, CD99, CS1, 5T4, CD138, CS- 1 (also known as CD2 subclass 1, CRACC, SLAMF7, CD319 or 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), nerve Ganglioside G2 (GD2), Ganglioside GD3, TNF receptor family member B cell maturation antigen (BCMA), Tn antigen (such as Tn'Ag, GalNAc ⁇ -Ser/Thr), prostate specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72), CD38,
  • the antigen-binding domain is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, single domain antibodies, and antigen-binding fragments thereof.
  • the antigen binding domain targets solid tumors.
  • the solid tumor is selected from the following group: lung cancer, breast cancer, colon cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, esophageal cancer, bone cancer, pancreatic cancer, skin cancer, Head or neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumor, bladder cancer, kidney or ureter cancer, renal pelvis cancer , Central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem glioma, pituitary adenoma,
  • CNS
  • the antigen binding domain targets non-solid tumors.
  • the non-solid tumor is 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 myelogenous leukemia (CML), acute myeloid leukemia (AML), B-cell young lymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse Large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative disease, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma , Myelodysplastic and myelodysplastic syndrome, Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoi
  • the transmembrane domain comprises a protein derived from a protein selected from the group consisting of CD28, CD3e, CD27, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2.
  • the costimulatory domain comprises a costimulatory domain selected from the following proteins: CD137, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7 , CD160 (BY55), LIGHT, NKG2C, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2 CD18, ITGB7, TNFR2, TRANCE/RANKL, DNAM1
  • the primary intracellular signaling domain comprises a functional signaling domain selected from the following proteins: CD3 ⁇ , FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ (Fc ⁇ R1b), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, DAP10 and/or DAP12.
  • a functional signaling domain selected from the following proteins: CD3 ⁇ , FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ (Fc ⁇ R1b), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, DAP10 and/or DAP12.
  • the hinge region connects the antigen binding domain and the transmembrane domain, and the hinge region comprises a hinge region derived from a protein selected from the group consisting of: human Ig (immunoglobulin) hinge Region, GS linker, KIR2DS2 hinge region or CD8a hinge region.
  • This application also provides a method for preparing the modified immune effector cell described in this application, which comprises the following steps: down-regulating the expression and/or activity of the corresponding gene in the corresponding cell without the modification.
  • Expression and/or activity of TRAC gene and HLA-A gene in immune effector cells Does not down-regulate the expression and/or activity of the B2M gene, and does not down-regulate the expression and/or activity of the CIITA gene.
  • the modification causes the expression and/or activity of two genes to be down-regulated, wherein the two genes consist of the TRAC gene and the HLA-A gene.
  • the expression and/or activity of the TRAC gene and HLA-A gene are down-regulated, the expression and/or activity of the B2M gene is not down-regulated, and the expression and/or activity of the CIITA gene is not down-regulated compared with the corresponding wild-type cell. /Or the activity is not down-regulated.
  • the expression and/or activity of two genes are down-regulated compared to corresponding wild-type cells, wherein the two genes consist of the TRAC gene and the HLA-A gene.
  • the down-regulating the expression level and/or activity of the gene includes down-regulating the expression and/or activity of the nucleic acid molecule encoding the gene; and/or down-regulating the expression and/or the protein product encoded by the gene Or the activity is down-regulated.
  • the modification includes: gene mutation and/or gene silencing.
  • the modification includes administering one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, and CRISPR/Cas9 system to the immune effector cell.
  • the modification includes administering the CRISPR/Cas9 system to the immune effector cells.
  • the modification includes administering to the immune effector cell an sgRNA targeting the exon portion of the HLA-A gene.
  • the sgRNA targeted to the exon part of the HLA-A gene comprises the nucleotide sequence shown in any one of SEQ ID Nos. 16-54 and 91-92.
  • the modification includes administering to the immune effector cell an sgRNA targeting the exon portion of the TRAC gene.
  • the sgRNA targeted to the exon part of the TRAC gene comprises the nucleotide sequence shown in any one of SEQ ID No. 1-15.
  • the antisense RNA comprises the nucleotide sequence shown in any one of SEQ ID No. 93-96.
  • the modification further comprises administering a Cas protein to the cell.
  • the Cas protein includes Cas9 protein.
  • the application also provides a composition, which includes the modified immune effector cell described in the application and a pharmaceutically acceptable carrier.
  • the composition includes a cell population, wherein the cell population includes the modified immune effector cells described herein.
  • This application also provides the application of the modified immune effector cells described in this application in the preparation of CAR-T cells.
  • the application also provides the application of the modified immune effector cells described in the application in the preparation of medicines, and the medicines are used for allogeneic therapy.
  • the application also provides a method of allogeneic treatment, the method comprising administering the modified immune effector cells described in the application to a patient or a subject.
  • the present application also provides the modified immune effector cells described in the present application, which are used in allogeneic therapy.
  • This application also provides the application of the modified immune effector cells described in this application in the preparation of medicines for the treatment of tumors.
  • the application also provides a method of treating tumors, the method comprising administering the modified immune effector cells described in the application to a patient or a subject.
  • the application also provides the modified immune effector cells, which are used to treat tumors.
  • the tumor includes solid tumors and non-solid tumors.
  • the tumor is selected from the following group: lung cancer, breast cancer, colon cancer, renal cell cancer, liver cancer, non-small cell lung cancer, small bowel cancer, esophageal cancer, bone cancer, pancreatic cancer, skin cancer, head Or neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Huo Chikin’s disease, non-Hodgkin’s lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumor, bladder cancer, kidney or ureter cancer, renal pelvis cancer, Central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi
  • CNS
  • FIG 1 shows the results of Sanger sequencing of the TRAC gene in this application after Sg9RNA editing.
  • Figure 2 shows the results of TA clone detection after Sg9RNA editing of the TRAC gene in this application.
  • FIG. 3 shows the results of flow cytometric detection of the TRAC gene in this application after Sg9RNA editing.
  • FIG. 4 shows the results of Sanger sequencing of the HLA-A02 gene in this application after Sg2RNA editing.
  • FIG. 5 shows the results of Sanger sequencing of the HLA-A02 gene in this application after Sg5RNA editing.
  • Figure 6 shows the results of Sanger sequencing of the HLA-A11 gene in this application after Sg10-3RNA editing.
  • Figure 7 shows the results of Sanger sequencing of the HLA-A11 gene in this application after Sg21RNA editing.
  • Figures 8A-8B show the results of simultaneous knockout of HLA-A02 and TRAC in the modified immune effector cells of the present application.
  • FIGS 9A-9B show the protein levels of HLA-A02 and TRAC in the modified immune effector cells of the present application.
  • Figures 10A-10D show the mRNA levels of TRAC, HLA-A, B2M and CIITA in the modified immune effector cells of the present application.
  • Figures 11A-11B show the protein levels of B2M and CIITA in the modified immune effector cells of the present application.
  • Figures 12A-12D show the protein levels of TRAC, HLA-A, B2M and CIITA in the modified immune effector cells of the present application.
  • Figures 13A-13B show the knockout of TRAC and HLA-A mRNA levels in the modified immune effector cells of the present application.
  • Figures 14A-14B show the protein levels of CD69 and CD137 in the modified immune effector cells of the present application.
  • Figure 15 shows the co-culture of the modified immune effector cells of the present application and NK cells.
  • Figure 16 shows the level of IFN- ⁇ expressed by the modified immune effector cells of the present application.
  • Figures 17A-17D show the protein levels of TRAC, HLA-A, B2M and CIITA in the modified immune effector cells of the present application.
  • Figure 18 shows the infection efficiency of the modified immune effector cells of the present application on CAR.
  • Figure 19 shows the expansion factor of the modified immune effector cells of the present application.
  • Figure 20 shows the killing effect of the modified immune effector cells of the present application on CD19-positive target cells.
  • Figure 21 shows the dosing regimen for administering the modified immune effector cells of the present application.
  • Figure 22 shows the killing effect of the modified immune effector cells of the present application on tumors in mice.
  • immune effector cells generally refers to immune cells that participate in immune responses and perform effector functions.
  • the exercise of effector functions may include removing foreign body antigens or promoting immune effector responses.
  • Immune effector cells may include plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.
  • the term "modification” generally refers to changing the state or structure of a cell and/or a change in the state or structure of a cell.
  • the change is usually compared with the state or structure of the cell without the modification.
  • the change may include a change in the expression level or function of the endogenous gene, for example, the expression level of the endogenous gene in the cell is down-regulated by genetic engineering means, Up-regulation or non-expression, the genetic engineering means may include homologous recombination, CRISPR/Cas9 system gene editing, etc.; the changes may also include changes in cell protein expression, structure or function, for example, through the expression level of the endogenous gene or
  • the change in the expression of the corresponding protein, the change in structure or function achieved by the change in function such as the change in protein expression, the change in structure or function achieved by regulating protein translation and post-translational modification; the change may also include the introduction of foreign sources Genes, expression of foreign proteins, etc.
  • T cell receptor alpha con-stant T cell receptor alpha con-stant
  • T cell receptor T cell receptor
  • TCR T cell receptor
  • MHC major histocompatibility complex
  • TCR usually consists of two different protein chains (ie heterodimers). In humans, the TCR in most T cells consists of an ⁇ chain and a ⁇ chain (encoded by TRA and TRB, respectively). This type of T cell is called ⁇ ⁇ T cell. In a small number of T cells, TCR consists of a ⁇ chain.
  • ⁇ T cell this type of T cell is called ⁇ T cell.
  • ⁇ T cells account for about 95% of the total number of T cells, and ⁇ T cells account for about 5% of the total number of T cells. This ratio varies during ontogeny and disease states (such as leukemia), and varies between species. different.
  • Each chain that composes the TCR contains variable and constant regions.
  • the gene encoding the ⁇ chain (TRA, such as the information shown in HGNC: 12027) is located on chromosome 14 and consists of multiple gene fragments, including variable Segment (V), connecting segment (J) and constant region (C).
  • TRAC gene usually refers to the gene sequence encoding the constant region (C) of the alpha chain of the T cell receptor (for example, the information shown in HGNC: 12029), which is located in Chromosome 14 (14q11.2; 14: 22,547,505-22,552,131).
  • C constant region
  • HGNC 12029
  • one of the variable segment (V) genes encoding the N segment antigen recognition domain is rearranged with one of the connecting segment (J) to produce a functional V region exon, which is transcribed and spliced with
  • the constant regions (C) are connected to form the T cell receptor alpha chain coding sequence.
  • HLA-A generally refers to a type of human leukocyte antigen (human leukocyte antigens) polypeptide chain, which is encoded by the HLA-A gene located on human chromosome 6p21.3 (for example, the information shown in HGNC:4931).
  • HLA-A is one of the three main types of peptides that make up class I MHC molecules on the surface of human cells.
  • Others include HLA-B and HLA-C.
  • the heterodimer composed of the ⁇ chain encoded by the HLA-A gene and the ⁇ chain ( ⁇ 2-microglobulin) encoded by the B2M gene is the HLA-A MHC I molecule.
  • the ⁇ chain encoded by the HLA-A gene may include an ⁇ 1 domain, an ⁇ 2 domain, an ⁇ 3 domain, a transmembrane region, and a cytoplasmic region, wherein the ⁇ 1 domain and the ⁇ 2 domain can be combined with the peptide fragments so that the MHC I molecules (such as HLA-A) present the peptides to cells of the immune system.
  • the alpha chain of the MHC I molecule is polymorphic, and its primary structure has many changes. As of December 2013, there are 2432 known HLA-A alleles. , Encoding 1740 active proteins and 117 invalid proteins.
  • HLA-A alleles may include those included in IMGT/HLA database version 3.38.0 (https://www.ebi.ac.uk/ipd/imgt/hla/) and named by the WHO HLA Factor Nomenclature Committee The sequence information of the different HLA-A alleles.
  • B2M usually refers to ⁇ 2 microglobulin ( ⁇ 2-microglobulin), which is one of the components of MHC class I molecules.
  • ⁇ 2 microglobulin also called ⁇ chain
  • B2M is usually expressed in all nucleated cells.
  • ⁇ 2 microglobulin is encoded by the B2M gene located at 15q21.1 (for example, the information shown in HGNC:914).
  • CIITA generally refers to the transactivator of the major histocompatibility complex (MHCII) of class II.
  • the transactivator may be a protein with an acidic transcription activation domain, 4 LRR (leucine-rich repeat sequences) and a GTP binding domain.
  • the protein can be located in the cell nucleus, as a positive regulator of major histocompatibility complex (MHCII) gene transcription, and is called the "master control factor" for the expression of these genes.
  • the protein can also bind to GTP and use the binding with GTP to transport itself to the nucleus, where it usually acts in a co-activator-like manner through acetyltransferase (AT) activity.
  • the protein is encoded by a gene located at 16p13.13 (for example, the information shown in HGNC:7067), which can generate several transcript variants encoding different isoforms.
  • wild-type cell generally refers to a naturally-occurring or naturally-derived cell.
  • T cell generally refers to thymus-derived cells that participate in various cell-mediated immune responses.
  • nucleic acid or “polynucleotide” or “nucleic acid molecule” generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers in single-stranded or double-stranded form. Unless specifically limited, the term may include nucleic acids containing analogs of natural nucleotides that have binding properties similar to the reference nucleic acid (for example, sequence information is shown) and in a manner similar to naturally occurring nucleotides. metabolism. Unless otherwise specified, the sequence of a nucleic acid may include its variants modified in a conservative manner, such as degenerate codon substitutions, alleles, orthologs, SNPs and complementary sequences, as well as the sequences explicitly indicated.
  • expression generally refers to the transcription and/or translation of a specific nucleotide sequence.
  • the term "gene mutation” generally refers to a change in the base pair composition or arrangement sequence of a gene in the structure. For example, point mutations caused by a single base change, or deletions, duplications, and insertions of multiple bases.
  • gene silencing generally refers to preventing the expression of certain genes through regulatory mechanisms.
  • TGS transcriptional gene silencing
  • post-transcriptional gene silencing post-transcriptional gene silencing
  • PTGS post-transcriptional gene silencing
  • Gene silencing is generally considered to be a gene knockdown mechanism, and common methods for silencing genes can be RNAi.
  • endogenous refers to any substance derived from or produced inside an organism, cell, tissue, or system.
  • exogenous refers to any substance introduced from or produced outside an organism, cell, tissue, or system.
  • antisense RNA generally refers to a single-stranded RNA complementary to the transcription product mRNA (messenger RNA). Antisense RNA can inhibit gene expression by binding to mRNA. For example, the binding of antisense RNA to target mRNA causes the double-stranded RNA molecule to increase its sensitivity to RNase III and cause it to degrade; for example, antisense RNA binds to the upstream non-coding region of mRNA, thereby directly inhibiting the translation of target mRNA .
  • siRNA generally refers to the abbreviation of Small interfering RNA (small interfering RNA) or short in-terfering RNA (short interfering RNA).
  • siRNA is a type of double-stranded non-coding RNA molecule with a length of about 18-28 base pairs, which can cause degradation of mRNA through complementary binding with mRNA, thereby interfering with the expression of specific genes.
  • siRNA may be a product obtained by treating long double-stranded RNA or shRNA with Dicer enzyme.
  • siRNA enters the cell to form an RNA-induced silencing complex (RISC) with other proteins, the sense strand is degraded, and the antisense strand can be combined with a complementary targeting sequence to achieve gene silencing.
  • RISC RNA-induced silencing complex
  • shRNA usually refers to the abbreviation of short hairpin RNA, that is, “short hairpin RNA”.
  • shRNA usually includes two short inverted repeats, separated by a loop sequence in the middle, forming a hairpin structure.
  • 5-6 T bases can be included as the transcription terminator of RNA polymerase III.
  • shRNA can enter the cell via a viral vector or plasmid, and be transcribed under the action of polymerase II or polymerase III.
  • the transcription product is exported from the nucleus (usually via Exportin 5) and then processed by Dicer for delivery.
  • RISC the sense strand is degraded, and the antisense strand can be combined with the complementary targeting sequence to achieve gene silencing.
  • CRISPR/Cas system generally refers to a group of molecules including RNA-guided nucleases or other effector molecules and gRNA molecules, which can direct and implement RNA-guided nucleases or other effector molecules.
  • the nucleic acid is modified at the target sequence, for example to cause degradation of the target sequence.
  • the CRISPR system comprises gRNA and Cas protein, for example, Cas9 protein.
  • a system containing Cas9 or a functional mutant thereof is referred to as the "Cas9 system” or "CRISPR/Cas9 system” in this application.
  • gRNA molecules and Cas molecules can be complexed to form a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • gRNA molecule or “guide RNA”, “guide RNA”, “guide RNA”, “guide RNA molecule”, and “gRNA” are used interchangeably, and generally refer to the ability to promote specific guidance RNA guidance Nuclease or other effector molecules (generally complexed with gRNA molecules) to nucleic acid molecules on the target sequence.
  • the hybridization of a part of the gRNA with DNA for example, through the gRNA steering domain
  • the binding of a part of the gRNA molecule to RNA-guided nucleases or other effector molecules for example, at least through gRNAtracr
  • the gRNA molecule is composed of a single continuous polynucleotide molecule, referred to herein as a "single guide RNA” or “sgRNA” or the like.
  • the gRNA molecule is composed of multiple (for example, two) polynucleotide molecules that can associate themselves (generally by hybridization), and are referred to herein as “dual guide RNA” or “dgRNA” or the like.
  • Cas protein generally refers to the enzyme responsible for cutting DNA in the CRISPR/Cas system. It can include enzymes from type I, II, and III CRISPR/Cas systems. For example, Cas3, Cas9, Cas10.
  • Cas9 protein generally refers to the enzyme from the bacterial type II CRISPR/Cas system responsible for cutting DNA. Cas9 can include wild-type proteins and functional mutants thereof.
  • locus is also called a gene locus or locus, which refers to a fixed position on a chromosome, such as where a certain gene is located.
  • the arrangement position of the locus in the genome is called a genetic map.
  • CAR chimeric antigen receptor
  • TAA tumor associated antigen
  • ITAM immunoreceptor tyrosine-based activation motifs
  • binding domain generally refers to (specifically) binding to a given target epitope or a given target site on a target molecule (for example an antigen), or to the given target epitope or A given target site interacts, or recognizes the given target epitope or the domain of the given target site.
  • the term "specific binding” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which can determine the target in the presence of a heterogeneous population of molecules (including biomolecules) The presence.
  • an antibody that specifically binds a target is an antibody that binds to the target with greater affinity, affinity, easier, and/or longer duration than it binds to other targets.
  • the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include but does not require exclusive binding.
  • transmembrane domain generally refers to a polypeptide or protein that is encoded by at least one exon including an extracellular region, a transmembrane region, and an intracellular region at the DNA level.
  • the transmembrane domain generally contains three different structural regions: the N-terminal extracellular region, the conserved transmembrane stretch region in the middle, and the C-terminal cytoplasmic region.
  • the transmembrane domain may also contain intracellular or cytoplasmic regions.
  • the term "hinge region” generally refers to a region between the binding domain and the transmembrane domain in the CAR structure.
  • the hinge region is usually derived from the IgG family, such as IgG1 and IgG4, and some are derived from IgD and CD8.
  • the hinge region has a certain degree of flexibility, which affects the space constraint between the CAR molecule and its specific target, and then affects CAR T Contact between cells and tumor cells.
  • costimulatory usually refers to the source of lymphocyte activation of the second signal, usually by the surface costimulatory molecules of immune cells involved in adaptive immunity (T cell/B cell or antigen presenting cell/T cell) And its receptor interaction.
  • T cell/B cell or antigen presenting cell/T cell adaptive immunity
  • costimulatory molecules of immune cells involved in adaptive immunity T cell/B cell or antigen presenting cell/T cell
  • the complete activation of T cells depends on the effects of dual signals and cytokines.
  • the first signal of T cell activation comes from the specific binding of its receptor TCR and antigen, that is, T cell recognition of the antigen
  • the second signal of T cell activation comes from costimulatory molecules, that is, the costimulatory molecules of antigen-presenting cells and the surface of T cells The interaction of the corresponding receptors.
  • costimulatory domain generally refers to the intracellular part of the corresponding receptor of the costimulatory molecule, which can transmit a costimulatory signal (also referred to as a second signal).
  • a costimulatory signal also referred to as a second signal.
  • CD137 or other costimulatory molecule receptors
  • costimulatory domain from CD137 can be activated after the extracellular binding domain in the CAR structure binds to the corresponding antigen, and transduce costimulatory signals.
  • the term "primary signal transduction domain” generally refers to an amino acid sequence capable of generating signals that promote the immune effector function of CAR-containing cells, such as CAR-T cells.
  • immune effector functions in, for example, CAR-T cells may include cytolytic activity and auxiliary activity, including secretion of cytokines.
  • the primary signaling domain transduces effector function signals and directs cells to perform specialized functions.
  • the entire primary signaling domain can be used, in many cases, it is not necessary to use the entire chain. In terms of using truncated portions of the primary signaling domain, such truncated portions can be used to replace the complete chain as long as it can transduce effector function signals.
  • the term primary signaling domain is therefore intended to include any truncated portion of the intracellular signaling domain sufficient to transduce effector function signals.
  • tumor antigen generally refers to molecules (such as proteins, sugars, or lipids) that are expressed intact or as fragments on the surface of tumor cells and can be used to preferentially guide drugs to tumor cells.
  • the tumor antigen may be a marker expressed by both normal cells and cancer cells, for example, a lineage marker, for example, CD19 on B cells.
  • the tumor antigen may be a cell surface molecule that is overexpressed in tumor cells, for example, a 1-fold over-expression, a 2-fold over-expression, and a 3-fold or more over-expression compared to normal cells.
  • the cell surface molecules are abnormally expressed in tumor cells, for example, molecules that contain deletions, additions, or mutations compared to corresponding molecules expressed on normal cells.
  • tumor antigens may be exclusively expressed on the surface of tumor cells intact or as fragments (e.g., MHC/peptide), and are not synthesized or expressed on the surface of normal cells.
  • the CAR in the present application may include an antigen-binding domain that binds to a peptide presented by MHC, such as an antibody or antibody fragment.
  • MHC major histocompatibility complex
  • TCR T cell receptors
  • the MHC class I complex is constitutively expressed by all nucleated cells.
  • Virus-specific and/or tumor-specific peptide/MHC complexes can be used for unique classes of cell surface targets for immunotherapy. (See, for example, Sastry et al., J Virol.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are the same, except for possible naturally occurring mutations and/or post-translational modifications (such as isomerization, amidation) that may be present in very small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Unlike typical polyclonal antibody preparations that contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized from hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous antibody population and should not be interpreted as requiring the production of antibodies by any specific method.
  • polyclonal antibody generally refers to a composition of different antibody molecules.
  • Polyclonal antibodies can bind or react with multiple different specific epitopes on the same or different antigens.
  • the antigen-specific variability of a polyclonal antibody is located in the variable regions of the individual antibodies constituting the polyclonal antibody, for example, in the complementarity determining regions (CDR) 1, CDR2, and CDR3 regions.
  • CDR complementarity determining regions
  • polyclonal antibodies can be prepared by immunizing animals with the target sFGFR or part thereof.
  • polyclonal antibodies can be prepared by mixing multiple monoclonal antibodies having the specificity of the desired target sFGFR.
  • human antibody generally refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences.
  • Human antibodies are well known in the prior art (see, for example, van Dijk, M.A. and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). It is also possible to generate human antibodies in transgenic animals (e.g., mice) that, after immunization, can generate a complete repertoire or selection of human antibodies without endogenous immunoglobulin production (e.g., Jakobovits, A., etc., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A.
  • Human antibodies can also be generated in phage display libraries (e.g. Hoogenboom, HR and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, etc., J. Mol. Biol. 222 (1991) 581-597).
  • the term "human antibody” can also include antibodies modified in the constant region.
  • humanized antibody generally refers to an antibody containing heavy and light chain variable region sequences from non-human species (such as mouse), but at least a portion of the VH and/or VL sequences have been The changes are similar to human germline variable sequences.
  • the term “humanized antibody” is a framework that can immunospecifically bind to a related antigen and includes a framework (FR) region substantially having the amino acid sequence of a human antibody and a complementarity determining region (CDR) substantially having the amino acid sequence of a non-human antibody.
  • FR framework
  • CDR complementarity determining region
  • substantially in the context of CDR means that the amino acid sequence of the CDR is at least 80%, for example at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR.
  • a humanized antibody basically contains at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin
  • the CDR regions and all or substantially all of the framework regions are framework regions with consensus sequences of human immunoglobulins.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin.
  • single domain antibody generally refers to a type of antibody that lacks the light chain of the antibody but contains the variable region of the heavy chain. Because of its small molecular weight, it is also called Nanobody. Single-domain antibodies were first found in camelid animals, and similar antibodies were also found in chondrophytes such as nurse sharks, great star sharks, and rays. For example, antibodies that lack both the light chain of traditional antibodies and the CH1 region of the heavy chain constant region are called heavy chain antibodies (HcAb). HcAbs are commonly found in various camelid animals.
  • a heavy chain antibody called Ig new antigen receptor, or IgNAR for short is composed of two identical heavy chains, and the heavy chain contains 5 constant regions and 1 variable region.
  • the variable region of a heavy chain antibody is only composed of the variable region of an antibody heavy chain. Similar to the Fab of a traditional antibody, this variable region can specifically bind to an antigen, so the heavy chain antibody can perform the same function as a traditional antibody.
  • tumor generally refers to neoplasms or solid lesions formed by abnormal cell growth.
  • the tumor can be a solid tumor or a non-solid tumor.
  • a tangible mass that can be detected by clinical examinations such as X-ray film, CT scan, B-ultrasound, or palpation can be called a solid tumor.
  • X-ray, CT scan, B-ultrasound and palpation cannot Tumors seen or palpated, such as leukemia, can be called non-solid tumors.
  • CD namely cluster of differentiation, also called cluster of differentiation
  • CD molecules have many uses, and are usually used as important receptors or ligands for cells. Some CDs can participate in the signal cascade of cells to change the behavior of cells. Some CD proteins have nothing to do with cell signal transduction, but have other functions, such as cell adhesion. As of April 21, 2016, the total number of CD molecules in humans is 371.
  • the term "pharmaceutically acceptable carrier” generally refers to a pharmaceutically acceptable substance, composition or vehicle involved in carrying, storing, transporting, or administering cell preparations.
  • a pharmaceutically acceptable carrier may include a pharmaceutically acceptable salt, wherein the term “pharmaceutically acceptable salt” includes a salt of the active compound prepared using a relatively non-toxic acid or base, depending on the characteristics of the cells described in the present application , Such as sodium chloride.
  • Pharmaceutically acceptable carriers may also include organic acids (such as lactic acid), biologically active substances (such as polypeptides, antibodies, etc.), antibiotics (such as penicillin, streptomycin), and the like.
  • Pharmaceutically acceptable carriers may also include hydrogels, such as hydrogels containing polyacrylamide.
  • Pharmaceutically acceptable carriers may include storage solutions, cryopreservation solutions, injection solutions, etc. that can be used for cells. Generally speaking, the pharmaceutically acceptable carrier can maintain the activity of the cells it carries and does not hinder its therapeutic efficacy; the pharmaceutically acceptable carrier can also help the storage, transportation, and cell recovery of the cells. Proliferation, migration, and suitable for clinical applications.
  • composition generally refers to a composition suitable for administration to patients, human patients.
  • the composition described in this application may comprise the immune effector cells described in this application, and optionally a pharmaceutically acceptable carrier.
  • the acceptable ingredients of the composition are non-toxic to the recipient at the dosage and concentration used.
  • the compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • allogeneic therapy generally refers to a treatment method in which organs, tissues, cells, etc. not derived from the subject or patient are administered to the subject or patient to achieve the purpose of treatment.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a modified immune effector cell, wherein the expression and/or the expression and/or the activity of the TRAC gene and the HLA-A gene are compared with the expression and/or activity of the corresponding gene in the corresponding cell without the modification.
  • the activity is down-regulated, the expression and/or activity of the B2M gene is not down-regulated, and the expression and/or activity of the CIITA gene is not down-regulated.
  • the present application also provides a method for preparing the modified immune effector cell described in the present application, which includes the following steps: and the expression and/or activity of the corresponding gene in the corresponding cell without the modification.
  • the modification causes the expression and/or activity of two genes to be down-regulated, wherein the two genes are composed of the TRAC gene and the HLA-A gene.
  • the immune effector cells may include plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells and/or bone marrow-derived phagocytes.
  • NK natural killer
  • NKT natural killer T
  • the plasma cells refer to effector B cells/antibody secreting cells, which may include primitive plasma cells, young plasma cells, Russell bodies, Dutcher bodies, flame cells, and the like.
  • the B cell refers to all B cell types except plasma cells, for example, pre-B cells, immature B cells, mature B cells, activated B cells, and the like.
  • the T cells may include: helper T cells (Th), which can assist humoral immunity and cellular immunity; suppressor T cells (Ts) ), the suppressor T cells can inhibit cellular immunity and humoral immunity; effector T cells (Te), which can release lymphokines; cytotoxic T cells (Tc), so The cytotoxic T cells can kill target cells; Delayed type hypersensitivity T cells (Td), which can participate in the effect of type IV allergies; Amplified T cells (Ta) The amplified T cells (Ta) can act on Th and Ts to expand the immune effect; primitive or natural T cells (Virgin or Natural T cells) can differentiate into effector T cells or after contact with antigens. Memory T cell; Memory T cell (Memory T cell, Tm), the memory T cell can memory specific antigen stimulation.
  • Th helper T cells
  • Ts suppressor T cells
  • Tc cytotoxic T cells
  • Tc cytotoxic T cells
  • Ta The amplified T cells (Ta) can act on Th and Ts to
  • the cytotoxic T cell may have a cell surface marker CD8+.
  • the helper T cell may have a cell surface marker CD4+.
  • the corresponding cells without the modification may include wild-type cells and/or artificially modified cells.
  • the wild-type cells may include naturally-occurring or naturally-derived cells, such as plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells and/or cells isolated from the human body.
  • the human body is a healthy human body.
  • the healthy human body may include those without tumors or immune system related diseases or disorders.
  • the artificial modification may be an artificial modification that does not use the TRAC gene, HLA-A gene, B2M gene, and CIITA gene as the recognition site/target.
  • the artificial modification does not affect the TRAC gene, HLA gene.
  • -A gene, B2M gene and CIITA gene expression and/or activity said not affecting the expression and/or activity of TRAC gene, HLA-A gene, B2M gene and CIITA gene refers to the separation from the human body or from the Compared with corresponding cells obtained from human precursor cells or pluripotent cells that have not been further artificially modified, the expression and/or activity of TRAC gene, HLA-A gene, B2M gene and CIITA gene remain unchanged. The expression and/or activity is not completely the same.
  • the mRNA quantitative analysis of the TRAC gene, HLA-A gene, B2M gene, and CIITA gene in the two is not significant.
  • Sexual difference P>0.05
  • using conventional technical means in the field to detect, compared with the two TRAC gene, HLA-A gene, B2M gene and CIITA gene corresponding polypeptide/protein quantitative analysis does not have significant Sexual difference (P>0.05).
  • the artificially modified cells may include CAR-T cells; for example, the CAR-T cells may include CAR-T cells whose CAR molecules contain binding domains targeting the following molecules: CD19, PSCA, CD123, CD20, CEA , FAP, CD133, EGFR, EGFRVIII, BCMA, PSMA, Her2, CA125, EphA2, C-met, L1CAM, VEGFR, CS1, ROR1, EC, NY-ESO-1, MUC1, LewisY, GPC3, GD2, DLL3, CD99 , 5T4, CD22, CD30, CD33, CD138 and/or CD171.
  • TRAC gene HLA-A gene, B2M gene and CIITA gene
  • the expression and/or activity of the TRAC gene and the HLA-A gene are down-regulated, the expression and/or activity of the B2M gene is not down-regulated, and the expression and/or the CIITA gene The activity was not down-regulated.
  • the expression and/or activity of two genes are down-regulated compared with the corresponding wild-type cells, wherein the two genes are composed of TRAC gene and HLA-A gene.
  • the down-regulating the expression level and/or activity of the gene includes down-regulating the expression and/or activity of the nucleic acid molecule encoding the gene; and/or the expression and/or the protein product encoded by the gene Activity is down-regulated.
  • the protein product may include a polypeptide.
  • the expression and/or activity of the TRAC gene and HLA-A gene are down-regulated or the down-regulation of the expression and/or activity of the TRAC gene and HLA-A gene in the immune effector cell may include and Compared with the modified corresponding cell, the modification changes the nucleotide sequence structure of the TRAC gene and the HLA-A gene.
  • the nucleotide sequence may include a coding region or a non-coding region, such as a cis-regulatory element sequence, an exon sequence, and the cis-regulatory element sequence may include a promoter.
  • the changes may include partial or complete deletion of sequences, insertion of foreign fragments, base site mutations, etc.
  • the inserted foreign fragments may replace or destroy the sequence structure of the TRAC gene and the HLA-A gene, so that it cannot be normal.
  • the base site mutations may include frameshift mutations, missense mutations, nonsense mutations, and the like.
  • the alteration may include chemical group modification of the nucleotide sequence, such as methylation and the like.
  • the changes in the structure of the nucleotide sequence can be detected by gene sequencing, such as Sanger sequencing, sulfite sequencing, and the like.
  • the expression and/or activity of the TRAC gene and HLA-A gene are down-regulated or the down-regulation of the expression and/or activity of the TRAC gene and HLA-A gene in the immune effector cell can also include the Compared with the modified corresponding cells, the modification reduces the mNNA content of the TRAC gene and the HLA-A gene.
  • the mRNA content can be obtained by experimental methods and biostatistics methods well known to those skilled in the art, such as molecular probe in situ hybridization, real-time fluorescent quantitative PCR (qPCR, RT-PCR), and the real-time fluorescent quantitative PCR may include SYBR Green method, TaqMan method, two-hybrid probe method, molecular beacon method.
  • the detection result of the mRNA content allows unavoidable errors in experiments or statistics, and the errors may be well-known in the art.
  • the error may be in the range of ⁇ 10%, for example, ⁇ 8%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%.
  • the mNNA content is reduced by at least 30%, such as 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, for example, mNNA of TRAC gene and HLA-A gene cannot be detected in the modified immune effector cells.
  • the expression and/or activity of the TRAC gene and HLA-A gene are down-regulated or the down-regulation of the expression and/or activity of the TRAC gene and HLA-A gene in the immune effector cell can also include the Compared with the modified corresponding cells, the modification reduces the content of polypeptides expressed by the TRAC gene and the HLA-A gene.
  • the polypeptide refers to a polypeptide that has the same structure and function as the polypeptide produced by the expression of the TRAC gene and the HLA-A gene in the corresponding cell without the modification, and is produced by changes in the nucleotide sequence of the TRAC gene and the HLA-A gene Peptides with changed functions and structures are not in the comparison.
  • the polypeptide content can be obtained by experimental methods and biostatistical methods known to those skilled in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), cell immunofluorescence staining, Western blotting (Western blotting, WB).
  • ELISA enzyme-linked immunosorbent assay
  • WB Western blotting
  • the detection result of the polypeptide content allows unavoidable errors in experiments or statistics, and the errors may be well-known in the art.
  • the error may be in the range of ⁇ 10%, for example, ⁇ 8%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%.
  • the polypeptide content is reduced by at least 30%, such as 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, for example, the polypeptides expressed by the TRAC gene and the HLA-A gene cannot be detected in the modified immune effector cells.
  • the expression and/or activity of the TRAC gene and HLA-A gene are down-regulated or the down-regulation of the expression and/or activity of the TRAC gene and HLA-A gene in the immune effector cell may refer to knockout Or knock down the expression of the TRAC gene and HLA-A gene or perform other operations that disrupt the function of the TRAC protein and HLA-A protein.
  • the expression and/or activity of the B2M gene is not down-regulated, and the expression and/or activity of the CIITA gene is not down-regulated, or the expression and/or activity of the B2M gene is not down-regulated, and the CIITA is not down-regulated
  • the expression and/or activity of the gene may include that the mNNA content of the B2M gene and the CIITA gene does not decrease compared with the corresponding cell without the modification.
  • the mRNA content can be obtained by experimental methods and biostatistics methods well known to those skilled in the art, such as molecular probe in situ hybridization, real-time fluorescent quantitative PCR (qPCR, RT-PCR), and the real-time fluorescent quantitative PCR may include SYBR Green method, TaqMan method, two-hybrid probe method, molecular beacon method.
  • qPCR real-time fluorescent quantitative PCR
  • RT-PCR real-time fluorescent quantitative PCR
  • the detection result of the mRNA content allows unavoidable errors in experiments or statistics, and the errors may be well-known in the art.
  • the error may be in the range of ⁇ 10%, for example, ⁇ 8%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%.
  • the non-decreasing of the mNNA content may include an increase or no change in the content, and the constant may include that the corresponding mRNA quantitative analysis of the B2M gene and the CIITA gene does not have a significant difference between the two compared (P>0.05).
  • the expression and/or activity of the B2M gene is not down-regulated, and the expression and/or activity of the CIITA gene is not down-regulated, or the expression and/or activity of the B2M gene is not down-regulated, and the CIITA is not down-regulated
  • the expression and/or activity of the gene may include that the content of the polypeptide expressed by the B2M gene and the CIITA gene does not decrease compared with the corresponding cell without the modification.
  • the polypeptide refers to a polypeptide that has the same structure and function as the polypeptide produced by the expression of the B2M gene and the CIITA gene in the corresponding cell without the modification.
  • the polypeptide content can be obtained by experimental methods and biostatistical methods known to those skilled in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), cell immunofluorescence staining, Western blotting (Western blotting, WB).
  • ELISA enzyme-linked immunosorbent assay
  • WB Western blotting
  • the detection result of the polypeptide content allows unavoidable errors in experiments or statistics, and the errors may be well-known in the art.
  • the error may be in the range of ⁇ 10%, for example, ⁇ 8%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%.
  • the non-decreasing of the content of the polypeptide may include an increase or no change in the content, and the change may include that the quantitative analysis of the corresponding polypeptides of the B2M gene and the CIITA gene does not have a significant difference between the two compared (P>0.05).
  • the expression and/or activity of the B2M gene is not down-regulated, and the expression and/or activity of the CIITA gene is not down-regulated, or the expression and/or activity of the B2M gene is not down-regulated, and the CIITA is not down-regulated
  • the expression and/or activity of the gene may include not performing artificial intervention on the corresponding cell without the modification for the mRNA and/or polypeptide encoded by the CIITA gene and the B2M gene, the CIITA gene and the B2M gene, for example, the artificial intervention
  • the intervention may include the introduction of nucleotide molecules or other compounds that can target the CIITA gene and the B2M gene or the mRNA molecules encoded by the CIITA gene and the B2M gene or their encoded mRNA molecules and change their structure or content into the corresponding cells without the modification.
  • the expression and/or activity of the B2M gene is not down-regulated, and the expression and/or activity of the CIITA gene is not down-regulated, or the expression and/or activity of the B2M gene is not down-regulated, and the CIITA is not down-regulated
  • the expression and/or activity of the gene may include that the nucleotide sequence structure of the B2M gene and the CIITA gene has not changed compared with the corresponding cell without the modification, and the nucleotide sequence structure has not changed.
  • Gene sequencing tests such as Sanger sequencing, sulfite sequencing, etc.
  • the unaltered structure of the nucleotide sequence may include no artificial changes, and may also include naturally occurring changes that do not affect its function.
  • the expression and/or activity of the B2M gene is not down-regulated, and the expression and/or activity of the CIITA gene is not down-regulated, or the expression and/or activity of the B2M gene is not down-regulated, and the CIITA is not down-regulated
  • the expression and/or activity of the gene may mean that no modification is performed targeting the B2M gene or the protein encoded by the CIITA gene or the protein encoded by the CIITA gene.
  • the expression level and/or activity of at most two alleles in the HLA-A gene are down-regulated.
  • the two alleles may be a pair of alleles of the HLA-A gene in the immune effector cell; for example, the expression level and/or activity of one allele in the HLA-A gene Down.
  • the TRAC gene may include the gene shown in HGNC:12029 and all allele types thereof.
  • the TRAC gene and all allele types thereof may include the TRAC gene type that can be present in the immune effector cell.
  • the TRAC gene may include the nucleotide sequence shown in SEQ ID No. 55.
  • the TRAC gene may include a nucleotide sequence derived from a human and having 80% or more homology with the nucleotide sequence shown in SEQ ID No. 55, for example, 85% or more, 90% or Above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or above.
  • the HLA-A gene may include the gene shown in HGNC:4931 and all allele types thereof.
  • the HLA-A gene may include all HLAs named by the WHO HLA Factor Nomenclature Committee included in IMGT/HLA database version 3.38.0 (https://www.ebi.ac.uk/ipd/imgt/hla/) -A allele type, the HLA-A allele type and its sequence information disclosed in IMGT/HLA database version 3.38.0 are incorporated herein by reference.
  • the HLA-A allele may include A*02, A*24, A*01, A*03, A*32, A*11, A*26, A*68, A*23, A* 29. Any one or more of A*31, A*33, A*25, A*43, A*74, A*30, A*69.
  • the HLA-A allele may include A*02:01, A*03:01, A*01:01, A*24:02, A*68:01, A*11:01, A* Any one of 31:01:02, A*29:02, A*32:01, A*26, A*23:01, A*30:02, A*25:01, A*33:03 Or multiple.
  • the HLA-A allele may include A*02:01:01, A*01:01:01, A*03:01:01, A*24:02:01, A*11:01: 01, A*32:01:01, A*29:02:01, A*31:01:02, A*23:01:01, A*26:01:01, A*68:01:02, A*30:01:01, A*68:02:01, A*25:01:01, A*68:01:01,
  • the HLA-A allele may include A*02, A*30, A*03, A*01, A*24, A*32, A*68, A*11, A*26, A* 23. Any one or more of A*31 and A*25.
  • the HLA-A allele may include A*02:01, A*03:01, A*24:02, A*01:01, A*11:01, A*26:01, A* Any one or more of 25:01, A*68:01, A*32:01, A*31:01.
  • the HLA-A allele may include A*24, A*33, A*02, A*11, A*26, A*31, A*01, A*24:02, A*02: 01, A*33:03, A*11:01, A*26:01, A*02:06, A*31:01:02, A*26:03, A*26:02, A*02: Any one or more of 07, A*01:01, A*02:10, A*03:01.
  • the HLA-A allele may include A*02, A*24, A*33, A*11, A*26, A*31, A*30, A*03, A*01, A* 32, A*29, A*68, A*23, A*25, A*34, A*36, A*43, A*66, A*74.
  • the HLA-A allele may include A*24:02, A*33:03, A*02:01, A*11:01, A*02:01, A*31:01, A* 26:01, A*02:07, A*30:01, A*26:02, A*01:01,
  • the HLA-A allele may include A*02:01, A*11:01, A*24:02, A*30:01, A*26:01, A*23:01, A* 02:07, A*02:06, A*03:01, A*01:01, A*31:01:02, A*33:03, A*32:01, A*68:01, A* Any one or more of 02:03 and A*02:05.
  • the HLA-A allele may include A*03:01, A*02:01, A*23:01, A*01:01, A*30:02, A*30:01, A* 33:03, A*29:02, A*74:01, A*36:01, A*24:02, A*02:02, A*68:01, A*68:02, A*34: Any one or more of 02, A*66:02, A*31:01:02, A*32:01, A*02:05, A*66:01, A*26:01.
  • the HLA-A allele may include any one or more of A*02, A*11, A*24, A*30, A*33, A*03, A*01, A*26 Kind.
  • the HLA-A allele may include A*11:01, A*24:02, A*02:01, A*02:07, A*33:03, A*02:06, and A* Any one or more of 30:01.
  • the HLA-A alleles may include HLA-A*02:01:01:01, HLA-A*11:01:01:01, HLA-A*24:02:01, HLA-A* 30:01:01:01, HLA-A*33:01:01:01, HLA-A*03:01:01:01, HLA-A*01:01:01:01, HLA-A*26: Any one or more of 01:01:01.
  • the HLA-A gene may include a nucleic acid sequence as shown in any one of SEQ ID NOs. 56-63.
  • the HLA-A gene may include a nucleotide sequence derived from a human and having 80% or more homology with the nucleic acid sequence shown in any one of SEQ ID NO. 56-63, for example, 85% Or above, 90% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or above.
  • the modification may include gene knockout and/or gene silencing.
  • the modification may include deletion of all or part of a gene, gene mutation and/or gene silencing.
  • the gene knockout may include deletion of all or part of the gene, gene mutation, and the like.
  • the gene may include the HLA-A gene and/or the TRAC gene.
  • the modification may include knocking out any one of the two TRAC alleles and knocking out any one of the two HLA-A alleles in the immune cell.
  • the modification may include that two TRAC alleles in the immune cell are knocked out and any one of the two HLA-A alleles is knocked out.
  • the modification may include any one of the two TRAC alleles being knocked out and the two HLA-A alleles being knocked out.
  • the modification may include two TRAC alleles being knocked out and two HLA-A alleles being knocked out.
  • the deletion of the partial fragment of the gene may include the deletion of ⁇ 1 exon sequence.
  • the genetic mutation may include a change in base pair composition or arrangement order, which usually can lead to, for example, missense mutations, frameshift mutations, and/or nonsense mutations.
  • the missense mutation usually refers to a change in a certain base pair, which changes a codon encoding one amino acid to a codon encoding another amino acid, thereby changing the corresponding amino acid constituting the protein.
  • the frameshift mutation usually refers to the insertion or deletion of one or several bases that are not an integer multiple of 3 in the DNA strand, resulting in a change in the coding codon.
  • the nonsense mutation usually means that a certain base pair change causes a codon encoding an amino acid to become a terminator, and protein synthesis is terminated early when the site is reached.
  • the change in the composition or arrangement order of the base pairs may include a single nucleotide or base change (also called a point mutation) and/or a polynucleotide or base change.
  • the single nucleotide or base change may include the replacement of one base or nucleotide by another base or nucleotide, and the insertion or deletion of one base.
  • the polynucleotide or base change may include the loss of a base sequence, the insertion of a base sequence, and/or the rearrangement of a base sequence.
  • the base sequence may be a part of any exon/or intron in the gene.
  • the rearrangement may include repetition, inversion, translocation, etc. of the base sequence.
  • the gene silencing may include transcriptional gene silenc-ing (TGS) and/or post-transcriptional gene silen-cing (PTGS).
  • TGS transcriptional gene silenc-ing
  • PTGS post-transcriptional gene silen-cing
  • the silencing at the transcriptional level may include methylation of DNA molecules to inhibit DNA, such as promoter sequence methylation; the post-transcriptional gene silencing may include the specific intervention of the target RNA after the gene is transcribed. Changes in gene expression. For example, RNA interference (RNAi) reduces mRNA levels.
  • RNA interference RNA interference
  • the modification may include a technique or method that uses homologous recombination to replace an exogenous nucleotide sequence with an endogenous normal gene, thereby inactivating the endogenous normal gene.
  • the exogenous nucleotide sequence may be known.
  • the exogenous nucleotide sequence may be a partial fragment of the endogenous normal gene; for example, the exogenous nucleotide sequence may include a homology arm 1 from 5'to 3'in turn.
  • the nucleotide sequence to be inserted may include a reporter gene, a non-coding sequence, or a variant sequence of an endogenous normal gene.
  • the modification may include administering one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, and CRISPR/Cas9 system to the immune effector cell.
  • the antisense RNA may be a single-stranded RNA complementary to the transcription product mRNA (messenger RNA).
  • the complementarity is that at least 60% of the nucleic acid sequence of the antisense RNA is complementary to the mRNA, such as at least 70%, such as at least 80%, such as at least 90%, such as 100% complementary.
  • the binding of the antisense RNA to the target mRNA causes the sensitivity of the double-stranded RNA molecule to RNase III to increase, thereby degrading it.
  • the antisense RNA binds to the upstream non-coding region of the mRNA, thereby inhibiting the translation of the target mRNA.
  • the antisense RNA is artificially prepared.
  • the antisense RNA cannot form a short hairpin structure.
  • the antisense RNA may include the nucleotide sequence described in any one of SEQ ID NO: 93-96.
  • the siRNA may be a type of double-stranded non-coding RNA molecule with a length of about 18-28 base pairs.
  • the siRNA can cause degradation of mRNA through complementary binding to mRNA.
  • the length of the siRNA may be 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 base pairs.
  • the siRNA is artificially prepared.
  • the siRNA is obtained by treating long double-stranded RNA or shRNA in a cell with Dicer enzyme.
  • the shRNA refers to a type of RNA that can form a short hairpin structure.
  • the shRNA may include two short inverted repeats, and a loop sequence located between the two short inverted repeats.
  • At least 18 consecutive nucleic acid sequences in the shRNA are capable of complementary binding to the target mRNA, such as at least 19, such as at least 20, such as at least 21, such as at least 22, such as at least 23, such as at least 24 , Such as at least 25, such as at least 26.
  • the shRNA does not include the sgRNA described in this application.
  • the modification may include administering the CRISPR/Cas system to the immune effector cells.
  • the CRISPR/Cas system may include guide RNA (gRNA) and Cas enzyme.
  • the gRNA may include crRNA and tracrRNA.
  • the gRNA can be called dgRNA (dual molecule gRNA), when the crRNA and the tracrRNA are located in the same nucleotide molecule
  • the gRNA may be referred to as sgRNA (single molecule gRNA).
  • the CRISPR/Cas system may include gRNA nucleic acid sequence and Cas protein.
  • the RNP complex formed by gRNA nucleic acid sequence and Cas protein may include gRNA nucleic acid sequence and Cas protein.
  • the CRISPR/Cas system may also include a nucleic acid sequence encoding the gRNA and a nucleic acid sequence encoding the Cas protein.
  • the nucleic acid sequence encoding the gRNA and the nucleic acid sequence encoding the Cas protein may be placed in a plasmid, virus (for example, adenovirus, lentivirus, retrovirus) and other vectors commonly used in the field.
  • virus for example, adenovirus, lentivirus, retrovirus
  • the nucleic acid sequence encoding the gRNA and the nucleic acid sequence encoding the Cas protein may be located in the same vector or in different vectors.
  • the nucleic acid sequence encoding tracrRNA and the sequence encoding crRNA may be located in the same vector or in different vectors.
  • the promoters used to drive the expression of each coding sequence may be the same or different.
  • the CRISPR/Cas system may include more than one guide RNA.
  • Each guide RNA can contain different targeting sequences, allowing the CRISPR/Cas system to cleave more than one target sequence.
  • one or more guide RNAs can have the same or different characteristics, such as activity or stability in the CRISPR/Cas complex.
  • each guide RNA can be encoded on the same or different vectors.
  • the promoters used to drive the expression of more than one guide RNA can be the same or different.
  • the guide RNA includes a guide RNA that targets the HLA-A gene and the TRAC gene.
  • the modification also includes the application of Cas enzyme to the cell.
  • the Cas enzyme may include a Cas protein and a nucleic acid sequence encoding the Cas protein.
  • the Cas protein may include at least one domain that interacts with a guide RNA (gRNA); for example, the Cas protein can be guided to a target sequence by the guide RNA; for example, the guide RNA interacts with the Cas protein and the target sequence.
  • gRNA guide RNA
  • the guide RNA provides specificity for targeted cleavage
  • the Cas protein can be universal, pairing with different guide RNAs to cut different target sequences; for example,
  • the Cas protein can cut single-stranded or double-stranded DNA; for example, the Cas protein can cut RNA; for example, the Cas protein can cut RNA/DNA; for example, the Cas protein includes at least one DNA binding domain and At least one nuclease domain; for example, the nuclease domain can be heterologous to the DNA binding domain; for example, the nuclease activity can be changed by modifying the Cas protein; for example, the Cas protein can be used for binding and Regulate the expression or activity of DNA; for example, the Cas protein may be a Cas nuclease.
  • the CRISPR/Cas system may include type 1 or type 2 system components, including ribonucleic acid protein complexes (see, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov Et al., Molecular Cell, 60: 385-397 (2015).
  • the type 2 CRISPR/Cas system has a single-protein effector.
  • Cas proteins of type II, V, and VI can be single-protein, RNA guide nuclease, in this application It is referred to as "Type 2 Cas nucleases.”
  • Type 2 Cas nucleases can include Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins.
  • Cas9 or Cpf1 protein can include RuvC Like nuclease domain or HNH-like nuclease domain, the Cpf1 sequence in Zetsche is introduced in this application in its entirety.
  • the Cas protein can be from the type II CRISPR/Cas system (ie the Cas9 protein of the CRISPR/Cas9 system) or the type V CRISPR/Cas system (eg the Cpf1 protein).
  • the Cas protein may be from a type 2 CRISPR/Cas system, such as Cas9 protein or Cpf1 protein.
  • Class 2 Cas nuclease family proteins are enzymes with DNA endonuclease activity, which can be guided to cleave desired nucleic acid targets by designing appropriate guide RNAs described in this application.
  • the components of the Type 2 CRISPR/Cas system can be from a type IIA, IIB, IIC, V or VI system.
  • the Cas9 protein or its orthologs can be derived from the following exemplary species: Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus (Staphylococcus) aureus), Listeria in-nocua, Lactobacillus gasseri, Pasteurella novicida, Wolinella succinogenes, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum , Nocardiopsis rougevillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum
  • Lyngbya sp. Microcoleuschthonoplastes, Oscillatoria sp., Thermosiphoafricanus, Neisseriacinereal , Campylobacter lari or Corynebacterium diphtheria.
  • the Cas9 protein may be derived from Streptococcus pyogenes; for example, the Cas9 protein may be derived from Streptococcus thermophilus; for example, the Cas9 protein may be derived from Staphylococcus aureus; for example, the Cpf1 protein may be derived from Tulare Pasteurella spp Porphyromonas macacae.
  • the Cpf1 protein may be from the genus Aminococcus or Lachnospiraceae.
  • the Cas9 protein may contain 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the Cas9 protein of Streptococcus pyogenes. % Homology of amino acid sequence.
  • the Cas9 protein may include the amino acid sequence shown in SEQ ID NO:65.
  • the exemplary nucleic acid sequence encoding the Cas9 protein is described in the following documents: Cong et al., SCIENCE 2013, 399(6121): 819-823; Wang et al., CELL 2013, 153(4): 910-918; Mali et al. , SCIENCE 2013,399(6121):823-826; Jinek et al.,SCIENCE 2012,337(6096):816-821.
  • nucleotide sequence encoding the Cas9 protein is shown in SEQ ID NO: 64.
  • the Cas9 protein may be modified.
  • the modification may include amino acid substitutions, and other polypeptide fragments to form a fusion protein.
  • the other polypeptide fragments may include PEST sequences, ubiquitin, polyubiquitin, and nuclear localization signal (NLS).
  • the crRNA may include a targeting sequence, and the gRNA may target any target sequence through the targeting sequence of the crRNA.
  • the degree of complementarity between the targeting sequence and the target sequence on the target nucleic acid molecule may be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%.
  • the targeting sequence and the target sequence on the target nucleic acid molecule may be 100% complementary.
  • the targeting sequence and the target sequence on the target nucleic acid molecule may contain at least one mismatch.
  • the targeting sequence and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches.
  • the targeting sequence and the target sequence on the target nucleic acid molecule may contain 1-6 mismatches.
  • the targeting sequence and the target sequence on the target nucleic acid molecule may contain 5 or 6 mismatches.
  • the targeting sequence and the target sequence on the target nucleic acid molecule do not contain mismatches.
  • the length of the targeting sequence may depend on the CRISPR/Cas system and components used. For example, different Cas proteins from different bacterial species have different optimal targeting sequence lengths.
  • the targeting sequence may include lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 nucleotides.
  • the targeting sequence may comprise 18-24 nucleotides in length.
  • the targeting sequence may comprise 19-21 nucleotides in length.
  • the targeting sequence may comprise 20 nucleotides in length.
  • the crRNA may also include a crRNA flagpole sequence
  • the crRNA flagpole sequence may include any sequence that is complementary to tracrRNA and is sufficient to promote the formation of the CRISPR/Cas complex.
  • the flagpole sequence may include all or part of a naturally occurring crRNA sequence (also referred to as a "marker” or "handle") that is complementary to a tracrRNA in the same CRISPR/Cas system.
  • the flagpole sequence may include all or part of the repetitive sequence from the naturally occurring CRISPR/Cas system.
  • the flagpole sequence may include a truncated or modified marker or handle sequence.
  • the part of the tracrRNA that is complementary to the crRNA flagpole sequence may be referred to as a tracrRNA flagpole sequence.
  • the degree of complementarity between the tracrRNA and the flagpole portion that hybridizes to the tracrRNA along the shorter of the two sequences can be about 40%, 50%, 60%, 70%, 80% or more.
  • the tracrRNA and the flagpole part that hybridizes with tracrRNA is not 100% complementary along the shorter of the two sequences.
  • the length of the crRNA flagpole sequence can depend on the CRISPR/Cas system or tracrRNA used.
  • the crRNA flagpole sequence can comprise 10-50 nucleotides or more than 50 nucleotides in length.
  • the crRNA flagpole sequence can contain 15-40 nucleotides in length.
  • the crRNA flagpole sequence can contain 20-30 nucleotides in length.
  • the crRNA flagpole sequence can contain 22 nucleotides in length.
  • the length of the crRNA flagpole sequence can have no upper limit.
  • the tracrRNA may include all or part of the wild-type tracrRNA sequence from the naturally-occurring CRISPR/Cas system.
  • tracrRNA may comprise truncated or modified variants of wild-type tracrRNA.
  • the length of tracrRNA can depend on the CRISPR/Cas system used.
  • tracr RNA can contain lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70 , 80, 90, 100 or more than 100 nucleotides.
  • tracr is at least 20 nucleotides in length.
  • tracrRNA is at least 40 nucleotides in length.
  • tracrRNA may contain secondary structures, such as one or more hairpin or stem-loop structures, or one or more raised structures.
  • the guide RNA may include two RNA molecules, referred to herein as "dual guide RNA" or "dgRNA".
  • dgRNA may include a first RNA molecule containing crRNA and a second RNA molecule containing tracrRNA. The first and second RNA molecules can form an RNA duplex through the base pairing between the flagpole sequence on crRNA and tracrRNA.
  • the first RNA molecule from 5'to 3' may include a targeting sequence complementary to the target sequence, a crRNA flagpole sequence.
  • the second RNA molecule from 5'to 3' may include a tracrRNA flagpole sequence complementary to a crRNA flagpole sequence, a nuclease binding sequence, for example, the nuclease binding sequence may be a Cas nuclease, for example, Cas9.
  • the guide RNA may include a single RNA molecule, referred to as "single molecule gRNA" or "sgRNA".
  • sgRNA may include tracrRNA, crRNA covalently linked to tracrRNA.
  • crRNA and tracrRNA can be covalently linked via a linker nucleic acid sequence.
  • a single-molecule gRNA may include a stem-loop structure formed by base pairing between the flagpole sequences on crRNA and tracrRNA.
  • sgRNA is "Cas9sgRNA” that can mediate DNA cleavage by Cas9 protein.
  • sgRNA is "Cpf1sgRNA” that can mediate DNA cleavage by Cpf1 protein.
  • a single molecule of gRNA or sgRNA can include crRNA, loop, and tracrRNA from 5'to 3'.
  • the crRNA from 5'to 3' may include a targeting sequence complementary to the target sequence, and a crRNA flagpole sequence.
  • the tracrRNA from 5'to 3' may include a tracrRNA flagpole sequence complementary to a crRNA flagpole sequence, a nuclease binding sequence, for example, the nuclease binding sequence may be a Cas nuclease, for example, Cas9.
  • a single molecule of gRNA or sgRNA from 5'to 3' may include a targeting sequence complementary to the target sequence, a crRNA flagpole sequence, a loop, a tracrRNA flagpole sequence complementary to a crRNA flagpole sequence, and a nuclease binding sequence.
  • the gRNA may also contain modified nucleosides or nucleotides.
  • changing e.g., replacing) one or more non-linked phosphate oxygen and/or one or more of the main chain phosphodiester bond connected to phosphate oxygen; e.g., changing, e.g., replacing) the component of ribose, for example, replacing the ribose 2'hydroxyl; for example, replacing the phosphate moiety with a dephosphorylated linker; for example, modifying or replacing a naturally occurring nucleobase; for example, replacing or modifying the phosphoribose backbone; for example, modifying the 3'end or 5'end of the oligonucleotide,
  • removal, modification or replacement of terminal phosphate groups or conjugation moieties, capping or linker for example, 3'or 5'cap modification may include sugar and/or backbone modification); for example, modification or replacement of sugar.
  • introducing the modified nucleoside or nucleotide increases the stability to nucleases.
  • introducing the modified nucleoside or nucleotide reduces the innate immune response.
  • the innate immune response includes a cellular response to foreign nucleic acids (including single-stranded nucleic acids), and may involve the expression and release of cytokines (especially interferons) and the induction of cell death.
  • the modification may include administering sgRNA targeting the exon portion of the HLA-A gene to the immune effector cell.
  • the sgRNA targeted to the exon part of the HLA-A gene may comprise the nucleotide sequence shown in any one of SEQ ID Nos. 16-54 and 91-92.
  • the sgRNA targeting the exon part of the HLA-A gene may comprise a nucleus that is at least 70% homologous to the nucleotide sequence shown in SEQ ID No. 16-54, 91-92.
  • the nucleotide sequence for example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%.
  • the modification may include administering sgRNA targeting the exon portion of the TRAC gene to the immune effector cell.
  • the sgRNA targeting the exon part of the TRAC gene may comprise the nucleotide sequence shown in any one of SEQ ID No. 1-15.
  • the sgRNA targeting the exon part of the TRAC gene may comprise a nucleotide sequence that is at least 70% homologous to the nucleotide sequence shown in any one of SEQ ID No. 1-15, for example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%.
  • the immune effector cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR), and the CAR includes an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory structure, and a primary signal transduction domain.
  • CAR chimeric antigen receptor
  • the antigen binding domain specifically binds to tumor antigens.
  • the tumor antigen is selected from the following group: CD19, CD123, CD22, CD30, CD171, CA125, C-met, L1CAM, EC, DLL3, CD99, 5T4, CD138, CS-1 (also known as CD2 subclass 1 , CRACC, SLAMF7, CD319 or 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), nerve Ganglioside GD3, TNF receptor family member B cell maturation antigen (BCMA), Tn antigen (such as Tn Ag, GalNAc ⁇ -Ser/Thr), prostate specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan Body 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72
  • the antigen-binding domain may include an antibody or antigen-binding fragment thereof that specifically binds to the tumor antigen.
  • the antibodies or antigen-binding fragments thereof that specifically bind to GPC3 described in this application may include, but are not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, Diabodies, triabodies, tetrabodies, Fv fragments, scFv fragments, Fab fragments, Fab' fragments, F(ab')2 fragments and camel single domain antibodies.
  • the antibody may be a humanized antibody. It may be a framework (FR) region that immunospecifically binds to a relevant antigen (e.g., human CD19, BCMA, or GPC3) and contains substantially the amino acid sequence of a human antibody and a complementarity determining region that substantially has the amino acid sequence of a non-human antibody ( CDR) antibodies or variants, derivatives, analogs or fragments thereof.
  • a relevant antigen e.g., human CD19, BCMA, or GPC3
  • CDR non-human antibody
  • the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
  • the single chain antibody is scFv.
  • the antigen binding domain targets solid tumors.
  • the solid tumor is selected from the following group: liver cancer, gastric cancer, lung cancer, breast cancer, colon cancer, rectal cancer, renal cell cancer, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, Pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer , Central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal
  • the antigen binding domain targets non-solid tumors.
  • the non-solid tumor is selected from the following group: 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 myelogenous leukemia (CML), acute myeloid leukemia (AML), B-cell young lymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma , Follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disease, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and Myelodysplastic syndrome, non-Hodgkin’s lympho
  • the transmembrane domain may comprise a transmembrane domain selected from the following proteins: CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the costimulatory domain may comprise a costimulatory domain selected from the following proteins: CD137, CD28, 4-1BB, OX-40 and ICOS.
  • the intracellular signaling domain may include a signaling domain derived from CD3 ⁇ .
  • the hinge region connects the antigen binding domain and the transmembrane domain, and the hinge region includes a hinge region derived from a protein selected from the group consisting of IgG1, IgG4, IgD, and CD8.
  • the application also provides a composition, which includes the modified immune effector cell described in the application and a pharmaceutically acceptable carrier.
  • composition includes a cell population, wherein the cell population includes the modified immune effector cells described in this application.
  • the ratio of the number of modified immune effector cells to the total number of cells in the cell population is at least 0.001%, at least 0.01%, at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15 %, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 53%, at least 55%, at least 58%, at least 60%, at least 63%, At least 65%, at least 68%, at least 70%, at least 73%, at least 75%, at least 78%, at least 80%, at least 83%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
  • the cell population may include the modified immune effector cells and corresponding immune effector cells that have not been modified.
  • the modified immune effector cell may include a cell in which any one of two TRAC alleles is knocked out and any one of two HLA-A alleles is knocked out, two TRAC alleles Cells that have been knocked out and either of the two HLA-A alleles have been knocked out, cells in which either of the two TRAC alleles have been knocked out and the two HLA-A alleles have been knocked out, A cell in which two TRAC alleles have been knocked out and two HLA-A alleles have been knocked out.
  • the cell population may be a cell population obtained by genetically engineering the immune effector cell population, and the genetic engineering method may include administering the immune effector cell population described in this application to the immune effector cell population.
  • Antisense RNA, the siRNA, the shRNA and/or the CRISPR/Cas9 system may include the sgRNA targeting the exon portion of the HLA-A gene, the sgRNA targeting the exon portion of the TRAC gene, and the Cas9 protein.
  • the cell population may include a cell population obtained by editing the immune effector cell population by the CRISPR/Cas9 system, and the editing efficiency of the editing is at least 30%, at least 35%, at least 40%, at least 45 %, at least 50%, at least 53%, at least 55%, at least 58%, at least 60%, at least 63%, at least 65%, at least 68%, at least 70%, at least 73%, at least 75%, at least 78%, At least 80%, at least 83%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 %, at least 96%, at least 97%, at least 98%, at least 99%.
  • the editing efficiency can be obtained by Sanger sequencing, TA cloning sequencing, and flow cytometry.
  • the cell population may include the cell population of the immune effector cells obtained by administering the antisense RNA, the siRNA, and the shRNA described in this application, and the cell population is different from the cell population before the administration.
  • mRNA expression is reduced by at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 53%, at least 55%, at least 58%, at least 60%, at least 63%, at least 65%, at least 68%, at least 70%, at least 73%, at least 75%, at least 78%, at least 80%, at least 83%, at least 85%, at least 86% , At least 87%, at least 88%, at least 89%, at least 90%.
  • the cell population may include the cell population of the immune effector cells obtained by administering the antisense RNA, the siRNA, and the shRNA described in the present application, and the cell population is different from the cell population before the administration.
  • protein expression is reduced by at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 53%, at least 55%, at least 58%, at least 60%, at least 63%, at least 65%, at least 68%, at least 70%, at least 73%, at least 75%, at least 78%, at least 80%, at least 83%, at least 85%, at least 86% , At least 87%, at least 88%, at least 89%, at least 90%.
  • the acceptable ingredients of the composition are non-toxic to the recipient at the dosage and concentration used.
  • the pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are compatible with the immune effector cells, and are generally safe, non-toxic, and neither biologically Nor is it undesirable in other respects.
  • the pharmaceutically acceptable carrier may include storage solution at 2°C-8°C, cryopreservation solution, injection solution and the like.
  • the carrier may include the following components: adenosine, sodium chloride, albumin, interleukin-15, angiotensin-II, short peptides and polypeptides from the serum-free culture medium of human umbilical cord mesenchymal stem cells Compound etc.
  • the carrier may also include Normosol R (Abbott), Plasma-Lyte A (Baxter) injection, 5% dextrose water or Ringer's lactate solution.
  • the carrier may also include glycerol or DMSO.
  • the composition may comprise parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or direct injection into tissues.
  • the composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition may be administered without interruption.
  • the uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient's body, as described in WO2015/036583.
  • This application also provides the application of the modified immune effector cells described in this application in the preparation of CAR-T cells.
  • the application also provides the application of the modified immune effector cells described in the application in the preparation of medicines, and the medicines are used for allogeneic therapy.
  • This application also provides the application of the modified immune effector cells described in this application in the preparation of medicines for the treatment of tumors.
  • the tumor includes solid tumors and non-solid tumors.
  • the types of the solid tumor and the non-solid tumor are as described above.
  • the following examples are only to illustrate the modified immune effector cells, preparation methods and uses of the present application, and are not used to limit the scope of the present application.
  • the examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells.
  • Such methods are well known to those of ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold spring Harbor Labora-tory Press.
  • sgRNAs composed of 20 nucleotide sequences may appear repeatedly in the genome, use the website http://crispr.cos.uni-heidelberg.de to come. Carry out the design and evaluation of sgRNA, paste the exon sequence to the website, and design the sgRNA on the website and perform predictive evaluation. The higher the score in the evaluation, the higher the editing efficiency and the lower risk of off-target. Select the sgRNA with a higher score for the experiment.
  • the sgRNA targeting the TRAC gene is shown in SEQ ID No. 1-15
  • the sgRNA targeting the HLA-A02 gene is shown in SEQ ID No.
  • the sgRNA targeting the HLA-A11 gene is shown in SEQ ID No. 38.
  • the sgRNA targeting the HLA-A24 gene is shown in SEQ ID No. 47-54, synthesized by GenScript Biotechnology.
  • Peripheral blood was collected from healthy donors and diluted 1:1 with PBS buffer.
  • a new 50ml centrifuge tube first add the diluted cell separation solution (Ficoll) of 1/3 of the blood volume, and then add the blood cell diluent very slowly along the tube wall, and centrifuge at 800g at room temperature for 20 minutes (the centrifuge is set to increase speed 1, decrease speed 0).
  • the liquid in the centrifuge tube is divided into PBS and serum layer, white blood cell layer, lymphocyte separation liquid, and red blood cell layer from top to bottom.
  • Remove the PBS and serum layer move the white blood cell layer to a new 50ml centrifuge tube, add PBS to 40ml to wash the cells, and centrifuge at 450g for 10min. After centrifugation, the supernatant was discarded to obtain peripheral blood mononuclear cells. After the cells are resuspended, the cells are counted.
  • the EasySep TM human T cell sorting kit (purchased from StemCell Technologies, catalog number: 17951) was used to extract T cells from peripheral blood mononuclear cells (PBMC). Adjust the density of PBMC to 5 ⁇ 107 cells/ml, and add PBS buffer in the range of 0.25-2ml; first add cocktail and mix and then add isolation cocktail according to 50 ⁇ l/ml.
  • PBMC peripheral blood mononuclear cells
  • the electroporation kit (purchased from LONZA, article number V4XXP-3024) was used to transfer the RNP complex into the activated T cells prepared in Example 2 by electroporation.
  • Pre-warm the medium (X-VIVO15 medium + 10% FBS + IL2 (200U/ml) + IL7 (10ng/ml) + IL15 (5ng/ml)) in the well plate 30 minutes in advance.
  • HLA-A02 Sg5 shown in SEQ ID No. 17
  • SEQ ID No.18 or HLA-A11 sg21 (shown in SEQ ID No.91) or HLA-A11 Rsg2 (shown in SEQ ID No.92)
  • 10 ⁇ g Cas9 protein purchased from thermo, catalog number A36499
  • Count the cells take 3 ⁇ 5 ⁇ 10 4 cells, centrifuge at 2000r/min for 5min, try to remove the supernatant, then add 20 ⁇ l DE lysis buffer to each tube, add to the PCR tube after cell lysis, and transfer to the PCR machine after transient centrifugation.
  • Machine conditions 65°C for 30min, 4°C for 30s, 95°C for 2min, 16°C infinite.
  • Use the primer pair TRAC-For/TRAC-Rev, or HLA-A For/HLA-A Rev and use the cleavage product as a template for PCR.
  • the PCR primer sequence is shown in SEQ ID NO.66-81, and the PCR product is sent to Jin Weizhi Perform Sanger sequencing. After getting the sanger sequencing results, use the EditR editor in the website: https://moriaritylab.shinyapps.io/editr_v10/ to predict where the editing will occur and the editing efficiency.
  • the three detection results of TRAC single gene knockout are shown in Figures 1 to 3, and the calculation results of knockout efficiency are shown in Table 1.
  • the three detection methods are basically the same, and subsequent experiments only use the Sanger sequencing method to detect editing efficiency.
  • the electroporation kit (purchased from LONZA, article number: V4XXP-3024) was used to transfer the RNP complex into the activated T cells prepared in Example 2 by electroporation. Pre-warm the medium (X-VIVO15 medium + 10% FBS + IL2 (200U/ml) + IL7 (10ng/ml) + IL15 (5ng/ml)) in the well plate 30 minutes in advance.
  • RNP complex 20 ⁇ g TRAC sgRNA (TRAC Sg9), 20 ⁇ g HLA-A sgRNA (HLA-A02 Sg2 or HLA-A02 Sg5 or HLA-A11 sg21 or target HLA-A*24:02:01, HLA- A*30:01:01:01, HLA-A*33:01:01:01, HLA-A*03:01:01:01, HLA-A*01:01:01:01 or HLA-A* 26:01:01:01:01 sgRNA) was added to the PCR tube (no RNA), and then 10 ⁇ g Cas9 protein (purchased from thermo, catalog number A36499) was added to each of them, mixed gently, and incubated at room temperature for 12min.
  • Example 2 Count the activated T cells cultured in Example 2, centrifuge at 300g for 8min to discard the supernatant, add PBS to resuspend the cells, aspirate 1E7 cells and centrifuge again at 300g for 8min, discard the supernatant and resuspend the cells with 100 ⁇ l of the prepared electroporation buffer .
  • Add the pre-warmed culture medium to the electrospindle then transfer the cells into the pre-heated culture medium in the well plate with a matching pipette, and then place it in a 37°C, 5% CO 2 incubator for culture.
  • the double gene knockout efficiency can be detected by sequencing, and TRAC-negative and HLA-A-negative T cells whose double-gene knockout efficiency is not less than 80% can be obtained.
  • the result is shown in Figure 8-9.
  • Figure 8A shows the results of using HLA-A02 Sg5 to knock out HLA-A02, the upper row shows the results of the control group (that is, without using HLA-A02 Sg5 for knockout); the next row shows the simultaneous knockout of HLA -The results of A02 and TRAC;
  • Figure 8B shows the results of knocking out TRAC with TRAC Sg9, and the upper line shows the results of the control group (that is, the results of TRAC Sg9 are not used for knockout); the next line shows the simultaneous knockout Results of HLA-A02 and TRAC.
  • Example 2 (1) Using the activated T cells prepared in Example 2, they were divided into two groups, one group was used as a control, and the other group was prepared according to the method of Example 5 with double knockout T cells of the TRAC gene and HLA-A gene. Sanger sequencing was performed in step (1) of Example 4. According to the sequencing results, cells with TRAC and HLA-A double gene knockout were obtained. The prepared double gene knockout T cells are incubated with the corresponding TRAC and HLA-A antibodies, and the double gene knockout cell line can be obtained by flow sorting or magnetic bead sorting.
  • RNA extraction kit purchased from QIAGEN, article number: 74004
  • reverse transcription kit purchased from Applied Biosystems, article number: 4368814
  • cDNA was used as a template for quantitative PCR detection.
  • Figure 10-11 The result is shown in Figure 10-11.
  • Figure 10 shows the mRNA level measurement of gene expression
  • Figures 10A-10D show the mRNA levels of TRAC, HLA-A, B2M and CIITA respectively
  • WT refers to the situation without any knock-out treatment
  • double-knock group Refers to the result of T cell knockout of TRAC gene and HLA-A gene
  • Figure 11 shows the protein level determination of gene expression, in which Figures 11A-11B show the protein expression levels of B2M and CIITA respectively
  • NEG refers to the negative control
  • WT refers to the condition without any knock-out treatment
  • TRAC+HLA-A Double knockout refers to the result of double knockout of TRAC gene and HLA-A gene.
  • Example 7 Preparation of TRAC gene, HLA-A/B2M gene and CIITA gene knockout T cells and verify the expression changes of the corresponding three genes
  • TRAC, HLA-A and CIITA genes in T cells with TRAC, HLA-A and CIITA knockout was down-regulated; compared with control cells, TRAC, B2M and CIITA were knocked out
  • the protein expression of TRAC, HLA-A and CIITA genes in T cells was down-regulated.
  • FIGS 12A-12D show the knockout of TRAC, HLA-A and B2M protein levels in sequence.
  • WT refers to the situation without any knockout treatment
  • TRAC+HLA-A double knockout refers to the results of T cells with double knockout of TRAC gene and HLA-A gene
  • TRAC+HLA-A+CIITA triple knockout refers to TRAC
  • TRAC+B2M+CIITA triple-knockout refers to the results of B2M, CIITA, and TRAC three-gene knock-out T cells
  • TRAC+HLA-A knockdown refers to Example 9
  • Figure 12D shows the knockout of CIITA protein levels.
  • A is adenylate
  • T is thymidylate
  • R is adenylate or guanylate (purine)
  • Y is thymidylate or cytidine Acids (pyrimidines)
  • N is adenylate, thymidylate, guanylate or cytidine acid; compare the homology of selected sequences to avoid significant homology between antisense RNA and other genes or sequences , Resulting in off-target effects.
  • the homology analysis uses NCBI B
  • the designed antisense RNA sequence includes HLA-A-homo-551 (which includes the nucleotide sequence shown in SEQ ID NO.93); HLA-A-homo-NEG (which includes the nucleotide sequence shown in SEQ ID NO.94) Nucleotide sequence); TRAC-homo-375 (it includes the nucleotide sequence shown in SEQ ID NO. 95); TRAC-homo-NEG (it includes the nucleotide sequence shown in SEQ ID NO. 96).
  • the antisense RNA designed in Example 8 was used for double gene knockdown.
  • the company prepares a lentivirus (Zima) with TRAC gene and HLA-A gene antisense RNA sequence.
  • CD3 + T cells were prepared according to the method of Example 2 (D0 day), and activated with CD3/CD28 antibody magnetic beads, which will carry the antisense RNA sequences of the TRAC gene and the HLA-A gene (SEQ ID NO. 95 and SEQ ID NO.
  • the activated T cells were transfected with the lentivirus of 93) (D1 day), the lentiviral vector was washed off on D2, and the culture was continued to D5. Collect T cells cultured to D5 days, and detect gene knockdown efficiency by quantitative PCR or Western Blot.
  • T cells with TRAC gene and HLA-A gene knockdown can be obtained through flow sorting or magnetic bead sorting.
  • the results showed that the mRNA and protein expression levels of TRAC and HLA-A in the TRAC and HLA-A gene knockdown group were down-regulated.
  • Figures 13A-13B show the knockout of TRAC and HLA-A mRNA levels in sequence.
  • WT refers to the situation without any knock-out treatment
  • TRAC+HLA-A double knock refers to the result of double knockout of TRAC gene and HLA-A gene.
  • the knockout levels of TRAC and HLA-A protein levels can be seen in the results in FIG. 12.
  • Example 10 The difference between different T cell activities
  • TRAC+HLA-A double knockout refers to the results of T cells with double knockout of TRAC gene and HLA-A gene
  • TRAC+HLA-A+CIITA triple knockout refers to TRAC
  • TRAC+B2M+CIITA triple-knockout refers to the results of B2M, CIITA, and TRAC three-gene knock-out T cells
  • TRAC+HLA-A knockdown refers to Example 9 The prepared TRAC gene and HLA-A gene knockdown T cell result.
  • T cells without gene knockout, double gene knockout, three gene knockout, and double gene knockdown in Examples 2, 5, 7 and 9 were labeled with CFSE (invitrogen, C34554), and the cell count was 1 respectively.
  • CFSE invitrogen, C34554
  • *10 6 cells were co-cultured with NK cells (NK92MI) at a ratio of 1:1. After 24 hours, the co-cultured groups of cells were collected, and the ratio of CFSE-positive cells in the mixed cells was detected by flow cytometry.
  • NK+T refers to the situation where NK cells are co-cultured with T cells that have not undergone any knock-out treatment
  • NK+TRAC+HLA-A knockdown refers to NK cells with the TRAC gene and HLA-A gene prepared in Example 9
  • the results of knockdown T cells are co-cultured
  • NK+TRAC+HLA-A double knocking refers to the co-cultivation of NK cells and T cells with double knockout of TRAC gene and HLA-A gene
  • NK+TRAC+HLA -A+CIITA triple knocking refers to the situation where NK cells are co-cultured with TRAC, HLA-A and CIITA knockout T cells
  • NK+TRAC+B2M+CIITA triple knocking refers to the situation where NK cells are combined with B2M, CIITA and
  • Example 12 The difference between different T cell allogeneic immune rejection reactions
  • Peripheral blood from Donor 1 was used to prepare T cells without gene knockout, double gene knockout, three gene knockout, and double gene knockdown in Examples 2, 5, 7, and 9.
  • CD3 + T cells were prepared from peripheral blood from donor 2. Each group of cells prepared from the peripheral blood of Donor 1 was mixed with the peripheral blood of Donor 2 in equal proportions with the CD3 + T cells prepared in Example 2. After 24 hours, the expression level of IFN- ⁇ in the cell mixed system was measured. The results showed that the expression level of IFN- ⁇ in the double-knockout T cell group was lower than that in the triple-knockout T cell group.
  • TRAC+HLA-A double knock refers to the results of T cells with double knockout of TRAC gene and HLA-A gene
  • TRAC+HLA-A+CIITA Triple knockout refers to the results of TRAC, HLA-A and CIITA three gene knockout T cells
  • TRAC+B2M+CIITA triple knockout refers to the results of B2M, CIITA and TRAC three gene knockout T cells
  • TRAC+HLA-A knockout Low refers to the result of knockdown of TRAC gene and HLA-A gene prepared in Example 9.
  • Example 13 Preparation of CAR-T cells with double knockout of TRAC gene and HLA-A gene, CAR-T cells with triple knockout of TRAC gene, HLA-A gene and CIITA gene, and TRAC gene, B2M gene and CIITA gene Knockout CAR-T cells
  • Example 2 (1) Obtain CD3 + T cells (day D0) according to the method of Example 2, and activate them with CD3/CD28 antibody magnetic beads. After activation, perform lentiviral vectors (including CD19-CAR, CD20-CAR or BCMA-CAR) on day D1. Transfection with other lentivirus), wash off the lentiviral vector on D2, sort CAR-positive T cells on D3 and continue to culture until D5.
  • lentiviral vectors including CD19-CAR, CD20-CAR or BCMA-CAR
  • TRAC gene and HLA-A gene double gene knockout cells Take CAR-T cells on D5 days as initial cells, prepare TRAC gene and HLA-A gene double gene knockout cells according to the method in Example 5 and Example 7, respectively, TRAC gene, HLA-A gene CAR-T cells with CIITA gene, TRAC gene, B2M gene and CIITA gene knockout.
  • the above-mentioned double gene knockout and triple gene knockout CAR-T cells can be obtained by flow cytometry detection, and the yield of double gene knockout CAR-T cells is higher than that of triple gene knockout CAR-T cells.
  • Figures 17A-17D show the knockout of TRAC, HLA-A and B2M protein levels in sequence.
  • Figure 17D shows the knockout of CIITA protein levels.
  • WT refers to the condition without any knockout treatment
  • TRAC+HLA-A double knockout refers to the results of CAR-T cells with double knockout of TRAC gene and HLA-A gene
  • TRAC+HLA-A+CIITA triple knockout The results of CAR-T cells with TRAC, HLA-A and CIITA knockouts
  • TRAC+B2M+CIITA knockout refers to the results of CAR-T cells with B2M, CIITA and TRAC knockouts.
  • the transfection efficiency of CD19CAR is shown in Figures 18A-18B.
  • CAR30%+ represents the transfection efficiency of CD19 CAR.
  • Figure 19 shows the fold expansion of different cells. Among them, CAR-T cells with double gene knockout of TRAC gene and HLA-A gene had the highest amplification factor.
  • Figure 20 shows the killing effect on CD19 target cells Raji-Luciferase, among which CAR-T cells with double knockout of TRAC gene and HLA-A gene have the most significant killing effect.
  • each E/T ratio is the result corresponding to the legend A-D from left to right.
  • mice were injected intravenously with tumor cells. After the tumor was successfully established, the mice were infused with CAR-T cells with double gene knockout of TRAC gene and HLA-A gene, CAR-T cells with triple gene knockout or without gene knockout. T cells to monitor the tumor volume in mice.
  • Tumor growth rate was significantly slowed in mice with double-gene knockout CAR-T cells.
  • Figure 21-22 shows the way of administration to mice, i.v. means intravenous injection, CAR-T cells represent double-knockout CAR-T cells expressing CD19 CAR, and triple-knockout CAR-T cells.
  • Figure 20 shows the volume of tumors in mice after the administration of CAR-T cells. Among them, Figure 20 shows from left to right the three genes of CD19 CAR-T cells, TRAC, HLA-A, and CIITA that have been treated with physiological saline, unmodified T cells, TRAC gene and HLA-A gene double gene knockout, respectively.
  • mice The volume of tumors in mice after knockout of CD19 CAR-T cells, B2M, CIITA, and TRAC CD19 CAR-T cells. It was found that the mice that were injected with CAR-T cells with double knockout of TRAC gene and HLA-A gene, the tumor growth rate was significantly slowed down.

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EP4388105A4 (en) * 2021-08-16 2026-02-11 Beam Therapeutics Inc Persistent Allogeneic Modified Immune Cells and Their Methods of Use
WO2023164688A1 (en) * 2022-02-28 2023-08-31 Kite Pharma, Inc. Allogeneic therapeutic cells
JP2025507619A (ja) * 2022-02-28 2025-03-21 カイト ファーマ インコーポレイテッド 治療用同種異系細胞

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US20220193135A1 (en) 2022-06-23
CN115003802B (zh) 2024-09-03
CN113061580B (zh) 2022-09-06
CN115003802A (zh) 2022-09-02
JP2023508740A (ja) 2023-03-03
CA3163304A1 (en) 2021-07-08
JP7670358B2 (ja) 2025-04-30
EP4086342A4 (en) 2024-05-15
CN113061580A (zh) 2021-07-02
AU2020418199B2 (en) 2024-06-13
KR20220124197A (ko) 2022-09-13

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