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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gene
immune effector
hla
cells
expression
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PCT/CN2020/140799
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English (en)
French (fr)
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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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江苏茂行科技有限公司
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Priority to JP2022540754A priority Critical patent/JP2023508740A/ja
Priority to AU2020418199A priority patent/AU2020418199A1/en
Priority to EP20911252.3A priority patent/EP4086342A4/en
Priority to KR1020227026014A priority patent/KR20220124197A/ko
Priority to CN202080090688.0A priority patent/CN115003802A/zh
Priority to CA3163304A priority patent/CA3163304A1/en
Publication of WO2021136263A1 publication Critical patent/WO2021136263A1/zh
Priority to US17/563,804 priority patent/US20220193135A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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.

Abstract

提供了一种经修饰的免疫效应细胞，其中与未经所述修饰的相应细胞中相应基因的表达和/或活性相比，TRAC基因和HLA-A基因的表达和/或活性被下调，B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调。提供制备所述的经修饰的免疫效应细胞的方法。

Description

一种经修饰的免疫效应细胞及其制备方法 技术领域
本申请涉及生物医药领域，具体的涉及一种经修饰的免疫效应细胞及其制备方法。
背景技术
抗肿瘤免疫疗法在多种恶性肿瘤中能够引起持久和强烈的反应，可以用于治疗许多不同类型的癌症，显示出广泛的潜力。当前抗肿瘤免疫疗法主要包括两种类型的免疫疗法：免疫细胞靶向单克隆抗体(mAb)疗法和过继性细胞疗法(ACT)。其中，ACT是指依赖在体外刺激和扩增的自体或者异体淋巴细胞回输到人体中，以达到抗肿瘤的效果，但是该疗法仅在MHC多态一致的患者中疗效相对好。CAR-T是新型有效的不依赖MHC的过继性细胞疗法，CAR又称嵌合抗原受体，是模拟TCR功能的人工受体，它能够特异性的识别肿瘤细胞表面的抗原，从而靶向杀死肿瘤细胞。但是及时并成功制造和输注自体CAR T细胞是实施有效CAR T细胞疗法的最大障碍，例如病人之前采用化疗方案治疗，化疗药物会引起病人T细胞在体外扩增困难或功能降低，导致不能产生足够数量的CAR-T细胞产品，或质量较差。另外目前自体CAR-T多用于ALL或CLL患者，在实体瘤患者中的应用面临很大的挑战，例如肿瘤抗原的异质性限制了自体CAR-T的应用。为了增加CAR的灵活性和扩大抗原识别的范围，通用型CAR-T应运而生。通用型CAR T细胞的主要设计原则是从同种异体健康供体产生肿瘤抗原特异性T细胞，通过该方式获得的CAR T细胞扩增效率和活力增强，然而异体T细胞的内源性TCR可识别受体的同种异体抗原，会导致移植物抗宿主病(GVHD)，同时HLA在异体T细胞表面的表达会导致宿主免疫系统的快速排斥(HVGR)。因此，同种异体细胞治疗引起免疫排斥反应的问题尚需进一步解决。
发明内容
本申请提供了一种经修饰的免疫效应细胞，其中与未经所述修饰的相应细胞中相应基因的表达和/或活性相比，TRAC基因和HLA-A基因的表达和/或活性被下调，B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调。
在某些实施方式中，所述修饰使得两种基因的表达和/或活性被下调，其中所述两种基因 由TRAC基因和HLA-A基因组成。
在某些实施方式中，与相应的野生型细胞相比，TRAC基因和HLA-A基因的表达和/或活性被下调，B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调。
在某些实施方式中，与相应的野生型细胞相比，两种基因的表达和/或活性被下调，其中所述两种基因由TRAC基因和HLA-A基因组成。
在某些实施方式中，所述免疫效应细胞包括T细胞。
在某些实施方式中，所述基因的表达水平和/或活性被下调包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
在某些实施方式中，所述修饰包括：基因突变和/或基因沉默。
在某些实施方式中，所述修饰包括向所述免疫效应细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和CRISPR/Cas9系统。
在某些实施方式中，所述修饰包括向所述免疫效应细胞施用CRISPR/Cas9系统。
在某些实施方式中，所述HLA-A基因的等位基因选自以下组：A*02，A*11，A*24，A*30，A*33，A*03，A*01和A*26。
在某些实施方式中，所述HLA-A基因中的至多2个等位基因的表达水平和/或活性下调。
在某些实施方式中，所述HLA-A基因中的1个等位基因的表达水平和/或活性下调。
在某些实施方式中，所述修饰包括向所述免疫效应细胞施用靶向所述HLA-A基因外显子部分的sgRNA。
在某些实施方式中，所述靶向所述HLA-A基因外显子部分的sgRNA包含SEQ ID No.16-54、91-92中任一项所示的核苷酸序列。
在某些实施方式中，所述修饰还包括向所述免疫效应细胞施用靶向所述TRAC基因外显子部分的sgRNA。
在某些实施方式中，所述靶向所述TRAC基因外显子部分的sgRNA包含SEQ ID No.1-15中任一项所示的核苷酸序列。
在某些实施方式中，所述反义RNA包含SEQ ID No.93-96中任一项所示的核苷酸序列。
在某些实施方式中，所述修饰还包括向所述细胞施用Cas蛋白。
在某些实施方式中，Cas蛋白包括Cas9蛋白。
在某些实施方式中，所述免疫效应细胞包含编码嵌合抗原受体(CAR)的核酸，所述CAR包括抗原结合结构域、铰链区、跨膜结构域、共刺激结构和初级信号传导结构域。
在某些实施方式中，所述抗原结合结构域特异性地结合肿瘤抗原。
在某些实施方式中，所述肿瘤抗原选自以下组：CD19、CD133、CD123、CD22、CD30、 CD171、CA125、C-met、L1CAM、EC、DLL3、CD99、CS1、5T4、CD138、CS-1(也称作CD2亚类1、CRACC、SLAMF7、CD319或19A24)、C型凝集素样分子-1(CLL-1或CLECL1)、CD33、表皮生长因子受体变体III(EGFRvIII)、神经节苷脂G2(GD2)、神经节苷脂GD3、TNF受体家族成员B细胞成熟抗原(BCMA)、Tn抗原(例如Tn Ag、GalNAcα-Ser/Thr)、前列腺特异性膜抗原(PSMA)；受体酪氨酸激酶样孤儿受体1(ROR1)、Fms样酪氨酸激酶3(FLT3)；肿瘤相关糖蛋白72(TAG72)、CD38、CD44v6、癌胚抗原(CEA)、上皮细胞黏附分子(EPCAM)、B7H3(CD276)、KIT(CD117)、白介素-13受体亚基α-2(IL-13Ra2或CD213A2)、间皮素、白介素11受体α(IL-11Ra)、前列腺干细胞抗原(PSCA)、蛋白酶丝氨酸21、血管内皮生长因子受体2(VEGFR2)、Lewis(Y)抗原、CD24、血小板衍生生长因子受体β(PDGFR-β)、阶段特异性胚胎抗原-4(SSEA-4)、CD20、叶酸受体α、受体酪氨酸-蛋白激酶ERBB2(Her2/neu)、细胞表面相关黏蛋白1(MUC1)、表皮生长因子受体(EGFR)、神经细胞黏附分子(NCAM)、Prostase、前列腺酸性磷酸酶(PAP)、突变的延伸因子2(ELF2M)、肝配蛋白B2、成纤维细胞活化蛋白α(FAP)、胰岛素样生长因子1受体(IGF-I受体)、碳酸酐酶IX(CAIX)、蛋白酶体(例如蛋白酶体、巨蛋白因子)亚基Β型9(LMP2)、糖蛋白100(gp100)、由断点簇集区(BCR)和Abelson鼠白血病病毒癌基因同源物1(Abl)组成的癌基因融合蛋白(bcr-abl)、酪氨酸酶、肝配蛋白A型受体2(EphA2)、岩藻糖基GM1；唾液酰Lewis黏附分子(sLe)、转谷氨酰胺酶5(TGS5)、高分子量-黑素瘤相关抗原(HMWMAA)、o-乙酰基-GD2神经节苷脂(OAcGD2)、叶酸受体β、肿瘤内皮标志物1(TEM1/CD248)、肿瘤内皮标志物7相关(TEM7R)、紧密连接蛋白6(CLDN6)、促甲状腺激素受体(TSHR)、G蛋白偶联受体C类第5群成员D(GPRC5D)、染色体X可读框61(CXORF61)、CD97、CD179a、间变性淋巴瘤激酶(ALK)、聚唾液酸、胎盘特异性1(PLAC1)、globoH glycoceramide的己糖部分(GloboH)、乳腺分化抗原(NY-BR-1)、尿路上皮分化特异糖蛋白(uroplakin)2(UPK2)、甲型肝炎病毒细胞受体1(HAVCR1)、肾上腺素受体β3(ADRB3)、泛连接蛋白(pannexin)3(PANX3)、G蛋白偶联受体20(GPR20)、淋巴细胞抗原6复合体基因座K9(LY6K)、嗅觉受体51E2(OR51E2)、TCRγ可变可读框蛋白(TARP)、Wilm肿瘤蛋白(WT1)；癌症/睾丸抗原1(NY-ESO-1)、癌症/睾丸抗原2(LAGE-1a)、黑素瘤相关抗原1(MAGE-A1)、位于第12p号染色体上的ETS转位变体基因6(ETV6-AML)、精子蛋白17(SPA17)、X抗原家族成员1A(XAGE1)、血管生成素结合性细胞表面受体2(Tie 2)、黑素瘤癌症睾丸抗原-1(MAD-CT-1)、黑素瘤癌睾丸抗原-2(MAD-CT-2)、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋白(prostein)、前列腺癌肿瘤抗原-1(PCTA-1或半乳糖凝集素8)、T细胞识别的黑素瘤抗原1(MelanA或MART1)；大鼠肉瘤(Ras)突变体、人端粒酶逆转录酶(hTERT)、肉瘤易位 断点、黑素瘤凋亡抑制蛋白(ML-IAP)、ERG(跨膜蛋白酶、丝氨酸2(TMPRSS2)ETS融合基因)、N-乙酰葡糖胺基转移酶V(NA17)、配对的框蛋白Pax-3(PAX3)、雄激素受体、细胞周期蛋白B1、v-myc鸟髓细胞增多症病毒癌基因神经母细胞瘤衍生的同源物(MYCN)、Ras同源物家族成员C(RhoC)、酪氨酸酶相关蛋白2(TRP-2)、细胞色素P450 1B1(CYP1B1)、T细胞识别的鳞状细胞癌抗原3(SART3)、配对的框蛋白Pax-5(PAX5)、前顶体蛋白结合蛋白sp32(OY-TES1)、淋巴细胞特异性蛋白质酪氨酸激酶(LCK)、A激酶锚定蛋白4(AKAP-4)、滑膜肉瘤X断点2(SSX2)、高级糖基化终末产物的受体(RAGE-1)、豆荚蛋白(legumain)、人乳头瘤病毒E6(HPV E6)、人乳头瘤病毒E7(HPV E7)、肠羧基酯酶、突变的热休克蛋白70-2(mut hsp70-2)、CD79a、CD79b、CD72、白细胞相关免疫球蛋白样受体1(LAIR1)、IgA受体的Fc片段(FCAR或CD890)、白细胞免疫球蛋白样受体亚家族A成员2(LILRA2)、CD300分子样家族成员f(CD300LF)、C型凝集素结构域家族12成员A(CLEC12A)、骨髓间质细胞抗原2(BST2)、含有EGF样模块的黏蛋白样激素受体样2(EMR2)、淋巴细胞抗原75(LY75)、磷脂酰基醇蛋白聚糖-3(GPC3)、Fc受体样5(FCRL5)和/或免疫球蛋白λ样多肽1。
在某些实施方式中，所述抗原结合结构域选自以下组：单克隆抗体、多克隆抗体、人抗体、人源化抗体、单域抗体和其抗原结合片段。
在某些实施方式中，所述抗原结合结构域靶向实体瘤。
在某些实施方式中，所述实体瘤选自以下组：肺癌、乳腺癌、结肠癌、肾细胞癌、肝癌、非小细胞肺癌、小肠癌、食道癌、骨癌、胰腺癌、皮肤癌、头或颈癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌、直肠癌、肛区癌、胃癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、霍奇金病、非霍奇金淋巴瘤、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、儿童实体瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管生成、脊枢椎肿瘤、脑干胶质瘤、垂体腺瘤、Kaposi肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤。
在某些实施方式中，所述抗原结合结构域靶向非实体瘤。
在某些实施方式中，所述非实体瘤选自以下组：慢性淋巴细胞白血病(CLL)、急性白血病、急性淋巴样白血病(ALL)、B细胞急性淋巴样白血病(B-ALL)、T细胞急性淋巴样白血病(T-ALL)、慢性髓性白血病(CML)、急性髓性白血病(AML)、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树状细胞肿瘤、Burkitt淋巴瘤、弥散性大B细胞淋巴瘤、滤泡淋巴瘤、多毛细胞白血病、小细胞或大细胞滤泡淋巴瘤、恶性淋巴细胞增生性疾病、MALT淋巴瘤、套细胞淋巴瘤、边缘区淋巴瘤、多发性骨髓瘤、脊髓发育不良和脊髓发育不良综合征、霍奇金淋巴瘤、 浆母细胞淋巴瘤、浆细胞样树状细胞肿瘤、B淋巴细胞瘤、Waldenstrom巨球蛋白血症。
在某些实施方式中，所述跨膜结构域包含源自选自下述蛋白：CD28、CD3e、CD27、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2。
在某些实施方式中，所述共刺激结构域包含选自下述蛋白的共刺激结构域：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、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、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(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和/或NKG2D。
在某些实施方式中，所述初级胞内信号传导结构域包含选自下述蛋白的功能性信号传导结构域：CD3ζ、FcRγ(FCER1G)、FcγRIIa、FcRβ(FcεR1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10和/或DAP12。
在某些实施方式中，所述铰链区连接所述抗原结合结构域和所述跨膜结构域，所述铰链区包含源自选自下述蛋白的铰链区：人Ig(免疫球蛋白)铰链区、GS接头、KIR2DS2铰链区或CD8a铰链区。
本申请还提供了制备本申请所述的经修饰的免疫效应细胞的方法，其包括以下的步骤：与未经所述修饰的相应细胞中相应基因的表达和/或活性相比，下调所述免疫效应细胞中TRAC基因和HLA-A基因的表达和/或活性。不下调B2M基因的表达和/或活性，且不下调CIITA基因的表达和/或活性。
在某些实施方式中，所述修饰使得两种基因的表达和/或活性被下调，其中所述两种基因由TRAC基因和HLA-A基因组成。
在某些实施方式中，与相应的野生型细胞相比，TRAC基因和HLA-A基因的表达和/或活性被下调，B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调。
在某些实施方式中，与相应的野生型细胞相比，两种基因的表达和/或活性被下调，其中 所述两种基因由TRAC基因和HLA-A基因组成。
在某些实施方式中，所述下调基因的表达水平和/或活性包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
在某些实施方式中，所述修饰包括：基因突变和/或基因沉默。
在某些实施方式中，所述修饰包括向所述免疫效应细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和CRISPR/Cas9系统。
在某些实施方式中，所述修饰包括向所述免疫效应细胞施用CRISPR/Cas9系统。
在某些实施方式中，所述修饰包括向所述免疫效应细胞施用靶向所述HLA-A基因外显子部分的sgRNA。
在某些实施方式中，所述靶向所述HLA-A基因外显子部分的sgRNA包含SEQ ID No.16-54、91-92中任一项所示的核苷酸序列。
在某些实施方式中，所述修饰包括向所述免疫效应细胞施用靶向所述TRAC基因外显子部分的sgRNA。
在某些实施方式中，所述靶向所述TRAC基因外显子部分的sgRNA包含SEQ ID No.1-15中任一项所示的核苷酸序列。
在某些实施方式中，所述反义RNA包含SEQ ID No.93-96中任一项所示的核苷酸序列。
在某些实施方式中，所述修饰还包括向所述细胞施用Cas蛋白。
在某些实施方式中，Cas蛋白包括Cas9蛋白。
本申请还提供了一种组合物，其包括本申请所述的经修饰的免疫效应细胞和药学上可接受的载体。
在某些实施方式中，所述组合物包含细胞群，其中所述细胞群包含本申请所述的经修饰的免疫效应细胞。
本申请还提供了本申请所述的经修饰的免疫效应细胞在制备CAR-T细胞中的应用。
本申请还提供了本申请所述的经修饰的免疫效应细胞在制备药物中的应用，所述药物用于异体治疗。
本申请还提供了一种异体治疗的方法，所述方法包括向患者或受试者施用本申请所述的经修饰的免疫效应细胞。
本申请还提供了本申请所述的经修饰的免疫效应细胞，其用于异体治疗。
本申请还提供了本申请所述的经修饰的免疫效应细胞在制备药物中的应用，所述药物用于治疗肿瘤。
本申请还提供了一种治疗肿瘤的方法，所述方法包括向患者或受试者施用本申请所述的 经修饰的免疫效应细胞。
本申请还提供了所述的经修饰的免疫效应细胞，其用于治疗肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和非实体瘤。
在某些实施方式中，所述肿瘤选自以下组：肺癌、乳腺癌、结肠癌、肾细胞癌、肝癌、非小细胞肺癌、小肠癌、食道癌、骨癌、胰腺癌、皮肤癌、头或颈癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌、直肠癌、肛区癌、胃癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、霍奇金病、非霍奇金淋巴瘤、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、儿童实体瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管生成、脊枢椎肿瘤、脑干胶质瘤、垂体腺瘤、Kaposi肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。
下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请中TRAC基因在Sg9RNA编辑后Sanger测序的结果。
图2显示的是本申请中TRAC基因在Sg9RNA编辑后TA克隆检测的结果。
图3显示的是本申请中TRAC基因在Sg9RNA编辑后流式细胞检测的结果。
图4显示的是本申请中HLA-A02基因在Sg2RNA编辑后Sanger测序的结果。
图5显示的是本申请中HLA-A02基因在Sg5RNA编辑后Sanger测序的结果。
图6显示的是本申请中HLA-A11基因在Sg10-3RNA编辑后Sanger测序的结果。
图7显示的是本申请中HLA-A11基因在Sg21RNA编辑后Sanger测序的结果。
图8A-8B显示的是本申请的经修饰的免疫效应细胞中同时敲除HLA-A02和TRAC的结果。
图9A-9B显示的是本申请的经修饰的免疫效应细胞中HLA-A02和TRAC的蛋白水平。
图10A-10D显示的是本申请的经修饰的免疫效应细胞中TRAC、HLA-A、B2M和CIITA的mRNA水平。
图11A-11B显示的是本申请的经修饰的免疫效应细胞中B2M和CIITA的蛋白水平。
图12A-12D显示的是本申请的经修饰的免疫效应细胞中TRAC、HLA-A、B2M和CIITA的蛋白水平。
图13A-13B显示的是本申请的经修饰的免疫效应细胞中TRAC和HLA-A mRNA水平的敲除情况。
图14A-14B显示的是本申请的经修饰的免疫效应细胞中CD69和CD137的蛋白水平。
图15显示的是本申请的经修饰的免疫效应细胞与NK细胞共培养的情况。
图16显示的是本申请的经修饰的免疫效应细胞表达IFN-γ的水平。
图17A-17D显示的是本申请的经修饰的免疫效应细胞中TRAC、HLA-A、B2M和CIITA的蛋白水平。
图18显示的是本申请的经修饰的免疫效应细胞对CAR的感染效率。
图19显示的是本申请的经修饰的免疫效应细胞的扩增倍数。
图20显示的是本申请的经修饰的免疫效应细胞对CD19阳性靶细胞的杀伤效果。
图21显示的是施用本申请的经修饰的免疫效应细胞的给药方案。
图22显示的是本申请的经修饰的免疫效应细胞对小鼠体内肿瘤的杀伤效果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
以下对本申请做进一步描述：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本申请中，术语“免疫效应细胞”通常是指参与免疫应答，行使效应功能的免疫细胞。例如所述行使效应功能可以包括清除异物抗原或促进免疫效应子应答等。免疫效应细胞可以包括浆细胞、T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和骨髓源性吞噬细胞。
在本申请中，术语“修饰”通常是指改变细胞的状态或结构和/或细胞的状态或结构的改变。 所述改变通常是与相应未经所述修饰的细胞的状态或结构相比，所述改变可以包括内源基因表达水平或功能的变化，例如通过基因工程手段使得细胞内源基因表达水平下调、上调或不表达，所述基因工程手段可以包括同源重组、CRISPR/Cas9系统基因编辑等；所述改变还可以包括细胞蛋白质表达、结构或功能的变化，例如通过所述内源基因表达水平或功能的变化而实现的相应蛋白质表达的变化、结构或功能的变化，例如通过调节蛋白质翻译、翻译后修饰而实现的蛋白质表达的变化、结构或功能的变化；所述改变还可以包括引入外源基因、表达外源蛋白质等。
在本申请中，术语“TRAC”通常是指T细胞受体α链恒定区(T cell receptor alpha con-stant)。T细胞受体(TCR)通常是指位于T细胞表面的特异性受体，能够识别与主要组织相容性复合物(MHC)分子结合的抗原。TCR通常由两条不同的蛋白质链组成(即异源二聚体)。在人类中，多数T细胞中的TCR由一条α链和一个β链(分别由TRA和TRB编码)组成，这一类T细胞被称为αβT细胞，少数的的T细胞中，TCR由γ链和δ链(分别由TRG和TRD编码)组成，这一类T细胞被称为γδT细胞。通常情况下，αβT细胞约占T细胞总数的95％，γδT细胞约占T细胞总数的5％，该比率在个体发育过程中和患病状态(例如白血病)中发生变化，物种之间也有所不同。组成TCR的每一条链都含有可变区与恒定区，在人类中，编码α链的基因(TRA，例如HGNC:12027所示的信息)位于14号染色体，由多基因片段构成，包括可变段(V)、连接段(J)以及恒定区(C)，TRAC基因通常是指编码T细胞受体α链恒定区(C)的基因序列(例如HGNC:12029所示的信息)，其位于14号染色体(14q11.2；14：22,547,505-22,552,131)。通常编码N段抗原识别域的可变段(V)基因中的1个与连接段(J)中的一个重排产生一个功能性V区外显子，该外显子被转录并通过剪接与恒定区(C)连接，从而形成T细胞受体α链编码序列。
在本申请中，“HLA-A”通常是指一类人类白细胞抗原(human leukocyte antigens)多肽链，由位于人类染色体6p21.3的HLA-A基因(例如HGNC:4931所示的信息)编码。HLA-A是构成人类细胞表面I类MHC分子的三种主要多肽类型之一，其他还包括HLA-B和HLA-C。由HLA-A基因编码的α链和B2M基因编码的β链(β2-微球蛋白)组成的异二聚体即为HLA-A类MHC I分子。所述由HLA-A基因编码的α链可以包含α1结构域、α2结构域域、α3结构域、跨膜区以及胞质区，其中α1结构域、α2结构域可以与肽段结合从而由MHC I分子(例如HLA-A类)将所述肽段呈递给免疫系细胞。在人类中，与大多数哺乳动物相似，MHC I分子的α链为多态性的，其一级结构有较多变化，截至2013年12月，共有2432个已知的HLA-A等位基因，编码1740个活性蛋白和117个无效蛋白。在本申请中，HLA-A等位基因可以 包括IMGT/HLA数据库3.38.0版(https://www.ebi.ac.uk/ipd/imgt/hla/)收录的由WHO HLA因子命名委员会命名的不同HLA-A等位基因的序列信息。
在本申请中，术语“B2M”通常是指是β2微球蛋白(β2-microglobulin)，是MHC I类分子的组成部分之一。β2微球蛋白(也称为β链)可以与HLA编码的α链组成MHC I类的分子。B2M通常在所有有核的细胞中都有表达。在人类中，β2微球蛋白由位于15q21.1的B2M基因(例如HGNC:914所示的信息)所编码。
在本申请中，术语“CIITA”通常是指Ⅱ类主要组织相容性复合体(MHCⅡ)的反式激活因子。所述反式激活因子可以是具有酸性转录激活结构域、4个LRR(富含亮氨酸的重复序列)和GTP结合结构域的蛋白质。所述蛋白质可位于细胞核中，作为II类主要组织相容性复合体(MHCⅡ)基因转录的正向调节剂，被称为表达这些基因的“主控制因子”。该蛋白质还可结合GTP，并利用与GTP结合来使其自身转运到细胞核中，在细胞核中，其通常使通过乙酰转移酶(AT)活性以类似共激活剂的方式起作用。在人类中，所述蛋白质由位于16p13.13的基因(例如HGNC:7067所示的信息)编码，能够产生几种编码不同同工型的转录物变体。
在本申请中，术语“野生型细胞”通常是指自然存在的或者自然来源的细胞。
在本申请中，术语“T细胞”通常是指胸腺衍生的细胞，其参与各种细胞介导的免疫反应。
在本申请中，术语“核酸”或“多核苷酸”或“核酸分子”通常时指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其单链形式或双链形式的聚合物。除非特别限定，否则该术语可以包括含天然核苷酸的类似物的核酸，所述核酸具有与参考核酸(例如示出了序列信息)相似的结合特性并且按照与天然存在核苷酸相似的方式代谢。除非另外说明，核酸的序列可以包括其保守方式修饰的变体，例如简并密码子置换、等位基因、直向同源物、SNP和互补序列，以及明确指出的序列。
在本申请中，术语“表达”通常是指特定核苷酸序列的转录和/或翻译。
在本申请中，术语“基因突变”通常是指基因在结构上发生的碱基对组成或排列顺序的改变。例如单个碱基改变所引起的点突变，或多个碱基的缺失、重复和插入等。
在本申请中，术语“基因沉默”通常是指通过调节机制阻止某些基因的表达。主要可以包括两种：一种是由于DNA甲基化、异染色质化以及位置效应等因素引起的转录水平上的基因沉默(transcriptional gene silencing,TGS)，另一种是转录后基因沉默(post-transcriptional gene silencing,PTGS)，即在基因转录后的水平上通过对靶标RNA进行特异性干预而影响基因的表达。通常情况下当基因沉默时，相应基因表达下调/减少。而当基因被敲除时则通常表现为不表达，例如在细胞中，某种特定基因的所有等位基因均被敲除后则表现为该基因的表达消失。 基因沉默通常被认为是一种基因敲低机制，通用于沉默基因的方法可以如RNAi等。
在本申请中，术语“内源”指来自生物、细胞、组织或系统或在其内部产生的任何物质。
在本申请中，术语“外源”指从生物、细胞、组织或系统外部引入或在其外部产生的任何物质。
在本申请中，术语“反义RNA”通常是指一种与转录产物mRNA(信使RNA)互补的单链RNA。反义RNA可通过与mRNA的结合抑制基因的表达。例如，反义RNA与靶mRNA结合后引起该双链RNA分子对RNA酶Ⅲ的敏感性增加，使其降解；例如，反义RNA与mRNA的上游非编码区结合，从而直接抑制靶mRNA的翻译。
在本申请中，术语“siRNA”通常是指Small interfering RNA(小干扰RNA)或short in-terfering RNA(短干扰RNA)的缩写。siRNA是一类双链非编码RNA分子，长度约为18-28个碱基对，可通过与mRNA的互补结合引起mRNA的降解从而干扰特定基因的表达。在某些实施方式中，siRNA可以是长双链RNA或shRNA经Dicer酶处理得到的产物。在某些实施方式中，siRNA进入细胞与其他蛋白质形成RNA诱导沉默复合体(RISC)，有义链发生降解，反义链可与互补的靶向序列结合，从而实现基因沉默。
在本申请中，术语“shRNA”通常是指short hairpin RNA的缩写，即“短发夹RNA”。shRNA通常包括两个短反向重复序列，中间由一茎环(loop)序列分隔，组成发夹结构。通常还可以包括5-6个T碱基作为RNA聚合酶Ⅲ的转录终止子。在某些实施方式中，shRNA可经由病毒载体或质粒进入细胞中，在聚合酶Ⅱ或聚合酶Ⅲ的作用下进行转录，转录产物自细胞核输出(通常可经由Exportin 5)后经Dicer处理后输送至RISC，有义链发生降解，反义链可与互补的靶向序列结合，从而实现基因沉默。
在本申请中，术语“CRISPR/Cas系统”通常是指包含RNA引导的核酸酶或其他效应分子和gRNA分子的一组分子，所述分子能够指引和实现由RNA引导的核酸酶或其他效应分子在靶序列处修饰核酸，例如引起靶序列降解。在某些实施方式中，CRISPR系统包含gRNA和Cas蛋白，例如，Cas9蛋白。包含Cas9或其功能性突变体的系统在本申请中称作“Cas9系统”或“CRISPR/Cas9系统”。在某些实施方式中，gRNA分子和Cas分子可以复合，以形成核糖核蛋白(RNP)复合体。
在本申请中，术语“gRNA分子”或“向导RNA”、“指导RNA”、“指引RNA”、“向导RNA分子”、“gRNA”可互换使用，通常是指能够促进特异性指引RNA引导的核酸酶或其他效应分子(一般与gRNA分子复合)至靶序列上的核酸分子。在某些实施方案中，通过gRNA的一部分与DNA(例如，通过gRNA导引结构域)杂交并且通过gRNA分子的一部分与RNA指 导的核酸酶或其他效应分子结合(例如，至少通过gRNAtracr)实现所述引导。在某些实施方案中，gRNA分子由单一的连续多核苷酸分子组成，在本文中称作“单一向导RNA”或“sgRNA”等。在其他实施方案中，gRNA分子由本身能够缔合(一般通过杂交)的多个(例如二个)多核苷酸分子组成，在本文中称作“双重向导RNA”或“dgRNA”等。
在本申请中，术语“Cas蛋白”通常是指CRISPR/Cas系统中负责剪切DNA的酶。可以包括来自Ⅰ、Ⅱ、Ⅲ型CRISPR/Cas系统的酶。例如，Cas3、Cas9、Cas10。
在本申请中，术语“Cas9蛋白”通常是指负责剪切DNA的来自细菌II型CRISPR/Cas系统的酶。Cas9可以包括野生型蛋白及其有功能性突变体。
在本申请中，“等位基因”通常是指基因座上的基因序列可能具有的不同变化的形式。基因座也称作基因位点或位点，是指染色体上的固定位置，例如某个基因所在。基因座在基因组中的排列位置称为基因图谱(genetic map)。
在本申请中，术语“嵌合抗原受体(CAR)”通常指识别肿瘤相关抗原(tumor associated antigen，TAA)的抗体的抗原结合区或其他靶分子的结合片段与胞内信号域“免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motifs,ITAM,通常可以为CD3ζ或FcεRIγ)”融合形成的抗原受体。例如，CAR的基本结构可以包括一个肿瘤相关抗原(tumor-associated antigen,TAA)或其他靶分子的抗原结合结构域(通常来源于单克隆抗体抗原结合区域的scFv)，一个胞外铰链区(Hinge area)，一个跨膜结构域(Transmembrane region)和一个胞内免疫受体酪氨酸活化基序(Immunoreceptor tyrosine-based activation mo-tif,ITAM)。
在本申请中，术语“结合结构域”通常是指(特异性)结合到靶分子(例如抗原)上的给定靶表位或给定靶位点，或与所述给定靶表位或给定靶位点相互作用，或识别所述给定靶表位或给定靶位点的结构域。
在本申请中，术语“特异性结合”通常指可测量的和可再现的相互作用，比如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况可决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在某些实施方式中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在另一个实施方式中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“跨膜结构域”通常指多肽或蛋白质，所述多肽或蛋白质在DNA水平上由至少一个包含胞外区、跨膜区和胞内区的外显子编码。跨膜结构域一般包含三个不同的结构区：N末端胞外区、中间保守的跨膜伸展区、以及C末端胞质区。跨膜结构域可能还包含 胞内区或胞质区。
在本申请中，术语“铰链区”通常指在CAR结构中位于结合结构域和跨膜结构域之间的一段区域。铰链区通常来源于IgG家族，例如IgG1和IgG4，还有些来源于IgD和CD8，通常铰链区具有一定程度的灵活性，从而影响CAR分子与其特异性靶点之间的空间约束，进而影响CAR T细胞与肿瘤细胞之间的接触。
在本申请中，术语“共刺激”通常指淋巴细胞激活第二信号的来源，通常由参与适应性免疫的免疫细胞(T细胞/B细胞间或抗原提呈细胞/T细胞间)表面共刺激分子及其受体相互作用而产生。例如，T细胞的完全活化有赖于双信号和细胞因子的作用。T细胞活化的第一信号来自其受体TCR与抗原的特异性结合，即T细胞对抗原识别；T细胞活化的第二信号来自共刺激分子，即抗原呈递细胞的共刺激分子与T细胞表面的相应受体的相互作用。
在本申请中，术语“共刺激结构域”通常指共刺激分子的相应受体胞内部分，所述胞内部分能够传导共刺激信号(也称作第二信号)。例如，在CAR-T细胞中，来自于CD137(或其他共刺激分子受体)的共刺激结构域可以在CAR结构中胞外的结合结构域与相应抗原结合后被活化，转导共刺激信号。
在本申请中，术语“初级信号传导结构域”通常指细胞内能够产生促进含有CAR的细胞例如CAR-T细胞的免疫效应子功能的信号的氨基酸序列。在例如CAR-T细胞中免疫效应子功能的实例可以包括细胞裂解活性和辅助活性，包括细胞因子的分泌。在某些实施方式中，初级信号传导结构域转导效应子功能信号并指导细胞进行特化功能。虽然可以使用整个初级信号传导结构域，但在许多情况下，不必使用整个链。就使用初级信号传导结构域的截短部分而言，此类截短部分可用于代替完整链，只要其能够转导效应子功能信号即可。术语初级信号传导结构域因此意在包括足以转导效应子功能信号的胞内信号传导结构域的任一截短部分。
在本申请中，术语“肿瘤抗原”通常是指在肿瘤细胞表面上完整或作为片段表达并且可用于偏好性导引药物至肿瘤细胞的分子(例如蛋白质、糖或脂质)。在某些实施方案中，肿瘤抗原可以是正常细胞和癌细胞二者表达的标志物，例如，谱系标志物，例如，B细胞上的CD19。在某些实施方案中，肿瘤抗原可以是在肿瘤细胞中过量表达的细胞表面分子，例如，与正常细胞相比，1倍过量表达、2倍过量表达，3倍或更多倍过量表达。在某些实施方式中，所述细胞表面分子在肿瘤细胞中不正常地表达，例如，与正常细胞上表达的相应分子相比含有缺失、添加或突变的分子。在某些实施方案中，肿瘤抗原可以排他地在肿瘤细胞表面上完整或作为片段(例如，MHC/肽)表达，并且在正常细胞的表面上不合成或不表达。在某些实施方式中，本申请中的CAR可以包括包含与MHC呈递的肽结合的抗原结合结构域，例如抗体或 抗体片段。通常，衍生自内源蛋白的肽与主要组织相容性复合体(MHC)I类分子结合，并且由CD8+T淋巴细胞上的T细胞受体(TCR)识别。MHC I类复合体由全部有核细胞组成型表达。病毒特异性和/或肿瘤特异性肽/MHC复合体可以用于免疫疗法的独特类别细胞表面靶。(参见例如Sastry等，J Virol.2011 85(5):1935-1942；Sergeeva等，Blood，2011 117(16):4262-4272；Verma等，J Immunol 2010 184(4):2156-2165；Willemsen等，Gene Ther 2001 8(21):1601-1608；Dao等，Sci Transl Med 2013 5(176):176ra33；Tassev等，Cancer Gene Ther 2012 19(2):84-100)。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体。即构成群体的各个抗体相同，除了可能以极小量存在的可能的天然存在突变和/或翻译后修饰(例如异构化、酰胺化)之外。单克隆抗体是高度特异性的，针对单一抗原性位点。与典型的包含针对不同决定簇(表位)的不同抗体的多克隆抗体制备物不同，每种单克隆抗体针对抗原上的单一决定簇。在它们的特异性以外，单克隆抗体的优势在于它们是由杂交瘤培养物合成的，未受到其它免疫球蛋白的污染。修饰语“单克隆”指示抗体从基本上同质的抗体群获得的特征，不应解释为要求通过任何特定方法来生成抗体。
在本申请中，术语“多克隆抗体”通常是指不同的抗体分子的组合物。多克隆抗体能够与同一或不同抗原上的多个不同特异性抗原决定簇结合或反应。多克隆抗体的抗原特异性的变异度位于构成多克隆抗体的单个抗体的可变区中，例如，位于互补决定区(CDR)1、CDR2和CDR3区。例如，多克隆抗体可以通过用靶sFGFR或其部分免疫动物来制备。例如，多克隆抗体可通过将具有所需靶sFGFR特异性的多个单克隆抗体混合来制备。
在本申请中，术语“人抗体”通常是指具有自人种系免疫球蛋白序列衍生的可变和恒定区的抗体。人抗体是现有技术中公知的(例如参见van Dijk，M.A.和van de Winkel，J.G.，Curr.Opin.Chem.Biol.5(2001)368-374)。还可以在转基因动物(例如小鼠)中生成人抗体，所述转基因动物在免疫后能够在没有内源免疫球蛋白生成的情况中生成人抗体的完整全集或选集(例如Jakobovits，A.等，Proc.Natl.Acad.Sci.USA 90(1993)2551-2555；Jakobovits，A.等，Nature362(1993)255-258；Brueggemann，M.等，YearImmunol.7(1993)33-40)。还可以在噬菌体展示文库中生成人抗体(例如Hoogenboom，H.R.和Winter，G.，J.Mol.Biol.227(1992)381-388；Marks，J.D.等，J.Mol.Biol.222(1991)581-597)。术语“人抗体”还可以包括在恒定区中进行修饰的抗体。
在本申请中，术语“人源化抗体”通常是指包含来自非人物种(例如小鼠)的重链和轻链可变区序列的抗体，但其中至少一部分VH和/或VL序列已被改变为类似于人种系可变序列。例如，术语“人源化抗体”为可以免疫特异性结合至相关抗原且包含基本上具有人类抗体的氨 基酸序列的框架(FR)区及基本上具有非人类抗体的氨基酸序列的互补决定区(CDR)的抗体或其变异体、衍生物、类似物或片段。术语“基本上”在CDR的情况下是指CDR的氨基酸序列与非人类抗体CDR的氨基酸序列至少80％、例如至少85％、至少90％、至少95％、至少98％或至少99％同一。人源化抗体基本上包含至少一个且通常两个可变域(Fab、Fab’、F(ab’)2、FabC、Fv)，其中所有或基本上所有CDR区对应于非人类免疫球蛋白的CDR区且所有或基本上所有框架区为具有人类免疫球蛋白共有序列的框架区。在某些实施方式中，人源化抗体还可包含至少一部分免疫球蛋白恒定区(Fc)，通常为人类免疫球蛋白的恒定区。
在本申请中，术语“单域抗体”通常是指缺失抗体轻链而含有重链可变区的一类抗体。因其分子量小，也被称为纳米抗体(Nanobody)。单域抗体最早在骆驼科动物中被发现，之后在护士鲨、大星鲨和鳐鱼等软骨鱼纲动物中也发现了类似的抗体。例如，既缺失传统抗体的轻链也缺少重链恒定区的CH1区域的抗体，被称为重链抗体(Heavy chain antibody，HcAb)，HcAb普遍存在于各种骆驼科动物中。例如，被称为新抗原受体(Ig new antigen receptor)的重链抗体，简称为IgNAR，这类抗体由两个相同的重链组成，重链包含5个恒定区和1个可变区。重链抗体的可变区仅由抗体重链的可变区组成，与传统抗体的Fab类似，该可变区可以与抗原特异性结合，因此重链抗体可以发挥和传统抗体一样的功能。
在本申请中，术语“肿瘤”通常指由异常细胞生长形成的赘生物或实体病变。在本申请中，肿瘤可以是实体瘤或非实体瘤。在某些实施方式中，可通过临床检查如x线摄片、CT扫描，B超、或触诊扪及到的有形肿块可称为实体瘤，X线、CT扫描，B超及触诊无法看到或扪及到的肿瘤例如白血病可称为非实体瘤。
在本申请中，术语“CD”，即分化簇(Cluster of differentiation)，也称作分化群，通常指的是用来识别用作免疫抗原标识的细胞表面分子。CD分子有许多用途，通常用作细胞的重要受体或配体。部分CD可参于细胞的讯号级联从而此改变细胞的行为，有些CD蛋白则与细胞信号传导无关，但有着其他功能，例如细胞黏附。截至2016年4月21日人类的CD分子总数为371，例如本申请中作为跨膜结构域来源的CD28、CD3e、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154，作为共刺激结构域来源的CD137、CD28、CD134(OX-40)和CD278(ICOS)。
在本申请中，术语“药学上可接受的载体”通常是指药学上可接受的涉及携带、储存、转运或施用细胞制剂的物质、组合物或媒介物。例如液体、半固体或固体填充剂，稀释剂，等渗剂，溶剂或包囊材料。药学上可接受的载体可以包括药学上可接受的盐，其中术语“药学上可接受的盐”包括使用相对无毒性的酸或碱制备的活性化合物的盐，取决于本申请所述细胞的 特性，例如氯化钠。药学上可接受的载体还可以包括有机酸(例如乳酸)、生物活性物质(例如多肽、抗体等)以及抗生素(例如青霉素、链霉素)等。药学上可接受的载体还可以包括水凝胶，例如含有聚丙烯酰胺的水凝胶。药学上可接受的载体可以包括可用于细胞的储存液、冻存液、注射液等。一般来说，所述药学上可接受的载体能够维持其所承载的细胞的活性并且不妨碍其治疗功效；所述药学上可接受的载体还可以有助于细胞的储存、转运，以及细胞的增殖、迁移，并且适用于临床应用。
在本申请中，术语“组合物”通常指涉及适合施用于患者、人患者的组合物。例如，本申请所述的组合物，其可以包含本申请所述的免疫效应细胞，以及任选地药学上可接受的载体。在某些实施方式中，组合物的可接受成分在所用剂量和浓度下对接受者无毒。本申请的组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“异体治疗”通常是指向受试者或患者施用非来自于该受试者或患者的器官、组织、细胞等以达到治疗目的的治疗方式。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
一方面，本申请提供了一种经修饰的免疫效应细胞，其中与未经所述修饰的相应细胞中相应基因的表达和/或活性相比，TRAC基因和HLA-A基因的表达和/或活性被下调，B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调。
另一方面，本申请还提供了一种制备本申请所述的经修饰的免疫效应细胞的方法，其包括以下的步骤：与未经所述修饰的相应细胞中相应基因的表达和/或活性相比，下调所述免疫效应细胞中TRAC基因和HLA-A基因的表达和/或活性。不下调B2M基因的表达和/或活性，且不下调CIITA基因的表达和/或活性。
在本申请中，所述修饰使得两种基因的表达和/或活性被下调，其中所述两种基因由TRAC基因和HLA-A基因组成。
免疫效应细胞
在本审请中，所述免疫效应细胞可以包括浆细胞、T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和/或骨髓源性吞噬细胞。
在本申请中，所述浆细胞是指效应B细胞/抗体分泌细胞，可以包括原始浆细胞、幼浆细胞、Russell小体、Dutcher小体和火焰状细胞等。
在本申请中，所述B细胞是指除浆细胞之外的所有B细胞类型，例如，前B细胞、不成熟B细胞、成熟B细胞、活化B细胞等。
在本申请中，所述T细胞可以包括：辅助性T细胞(Helper T cells,Th)，所述辅助性T细胞T细胞能够协助体液免疫和细胞免疫；抑制性T细胞(Suppressor T cells,Ts)，所述抑制性T细胞能够抑制细胞免疫及体液免疫；效应T细胞(Effector T cells,Te)，所述效应T细胞能够释放淋巴因子；细胞毒性T细胞(Cytotoxic T cells,Tc)，所述细胞毒性T细胞能够杀伤靶细胞；迟发性变态反应T细胞(Delayed type hypersensitivityT cells,Td)，所述迟发性变态反应T细胞能够参与Ⅳ型变态反应的作用；放大T细胞(Ta)，所述放大T细胞(Ta)能够作用于Th和Ts，扩大免疫效果；原始的或天然T细胞(Virgin or Natural T cells)原始的或天然T细胞能够和抗原接触后分化成效应T细胞或记忆T细胞；记忆T细胞(Memory T cell,Tm)，所述记忆T细胞能够记忆特异性抗原刺激。
例如，所述细胞毒性T细胞可以具有细胞表面标志CD8+。
例如，所述辅助性T细胞可以具有细胞表面标志CD4+。
在本申请中，所述未经所述修饰的相应细胞可以包括野生型细胞和/或经过人工改造的细胞。所述野生型细胞可以包括自然存在的或自然来源的细胞，例如分离自人体的浆细胞、T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和/或骨髓源性吞噬细胞，或者由分离自人体的前体细胞或多潜能细胞诱导分化得到免疫效应细胞；所述经过人工改造的细胞可以是分离自人体或由分离自人体的前体细胞/多潜能细胞诱导分化得到，之后进一步经过人工改造的浆细胞、T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和/或骨髓源性吞噬细胞。例如，所述人体为健康人体。例如，所述健康人体可以包括未患有肿瘤或免疫系统相关疾病或病症的。
在本申请中，所述人工改造可以是不以TRAC基因、HLA-A基因、B2M基因和CIITA基因为识别位点/靶点进行的人工改造，例如，所述人工改造不影响TRAC基因、HLA-A基因、B2M基因和CIITA基因的表达和/或活性，所述不影响TRAC基因、HLA-A基因、B2M基因和CIITA基因的表达和/或活性是指与所述分离自人体或由分离自人体的前体细胞或多潜能细胞诱导分化得到的、未进一步经过人工改造的相应细胞相比，TRAC基因、HLA-A基因、B2M基因和CIITA基因的表达和/或活性不变，所述表达和/或活性不变并非完全一致，例如，利用所属领域常规技术手段进行检测，相比的两者中所述TRAC基因、HLA-A基因、B2M基因和CIITA基因的mRNA定量分析不具有显著性差异(P>0.05)，例如，利用所属领域常规技术手段进行检测，相比的两者中所述TRAC基因、HLA-A基因、B2M基因和CIITA 基因的相应多肽/蛋白定量分析不具有显著性差异(P>0.05)。例如所述人工改造的细胞可以包括CAR-T细胞；例如所述CAR-T细胞可以包括其CAR分子含有靶向如下分子的结合结构域的CAR-T细胞：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和/或CD171。CD19、CD133、CD123、CD22、CD30、CD171、CA125、C-met、L1CAM、EC、DLL3、CD99、CS1、5T4、CD138、CS-1(也称作CD2亚类1、CRACC、SLAMF7、CD319或19A24)、C型凝集素样分子-1(CLL-1或CLECL1)、CD33、表皮生长因子受体变体III(EGFRvIII)、神经节苷脂G2(GD2)、神经节苷脂GD3、TNF受体家族成员B细胞成熟抗原(BCMA)、Tn抗原(例如Tn Ag、GalNAcα-Ser/Thr)、前列腺特异性膜抗原(PSMA)；受体酪氨酸激酶样孤儿受体1(ROR1)、Fms样酪氨酸激酶3(FLT3)；肿瘤相关糖蛋白72(TAG72)、CD38、CD44v6、癌胚抗原(CEA)、上皮细胞黏附分子(EPCAM)、B7H3(CD276)、KIT(CD117)、白介素-13受体亚基α-2(IL-13Ra2或CD213A2)、间皮素、白介素11受体α(IL-11Ra)、前列腺干细胞抗原(PSCA)、蛋白酶丝氨酸21、血管内皮生长因子受体2(VEGFR2)、Lewis(Y)抗原、CD24、血小板衍生生长因子受体β(PDGFR-β)、阶段特异性胚胎抗原-4(SSEA-4)、CD20、叶酸受体α、受体酪氨酸-蛋白激酶ERBB2(Her2/neu)、细胞表面相关黏蛋白1(MUC1)、表皮生长因子受体(EGFR)、神经细胞黏附分子(NCAM)、Prostase、前列腺酸性磷酸酶(PAP)、突变的延伸因子2(ELF2M)、肝配蛋白B2、成纤维细胞活化蛋白α(FAP)、胰岛素样生长因子1受体(IGF-I受体)、碳酸酐酶IX(CAIX)、蛋白酶体(例如蛋白酶体、巨蛋白因子)亚基Β型9(LMP2)、糖蛋白100(gp100)、由断点簇集区(BCR)和Abelson鼠白血病病毒癌基因同源物1(Abl)组成的癌基因融合蛋白(bcr-abl)、酪氨酸酶、肝配蛋白A型受体2(EphA2)、岩藻糖基GM1；唾液酰Lewis黏附分子(sLe)、转谷氨酰胺酶5(TGS5)、高分子量-黑素瘤相关抗原(HMWMAA)、o-乙酰基-GD2神经节苷脂(OAcGD2)、叶酸受体β、肿瘤内皮标志物1(TEM1/CD248)、肿瘤内皮标志物7相关(TEM7R)、紧密连接蛋白6(CLDN6)、促甲状腺激素受体(TSHR)、G蛋白偶联受体C类第5群成员D(GPRC5D)、染色体X可读框61(CXORF61)、CD97、CD179a、间变性淋巴瘤激酶(ALK)、聚唾液酸、胎盘特异性1(PLAC1)、globoH glycoceramide的己糖部分(GloboH)、乳腺分化抗原(NY-BR-1)、尿路上皮分化特异糖蛋白(uroplakin)2(UPK2)、甲型肝炎病毒细胞受体1(HAVCR1)、肾上腺素受体β3(ADRB3)、泛连接蛋白(pannexin)3(PANX3)、G蛋白偶联受体20(GPR20)、淋巴细胞抗原6复合体基因座K9(LY6K)、嗅觉受体51E2(OR51E2)、TCRγ可变可读框蛋 白(TARP)、Wilm肿瘤蛋白(WT1)；癌症/睾丸抗原1(NY-ESO-1)、癌症/睾丸抗原2(LAGE-1a)、黑素瘤相关抗原1(MAGE-A1)、位于第12p号染色体上的ETS转位变体基因6(ETV6-AML)、精子蛋白17(SPA17)、X抗原家族成员1A(XAGE1)、血管生成素结合性细胞表面受体2(Tie 2)、黑素瘤癌症睾丸抗原-1(MAD-CT-1)、黑素瘤癌睾丸抗原-2(MAD-CT-2)、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋白(prostein)、前列腺癌肿瘤抗原-1(PCTA-1或半乳糖凝集素8)、T细胞识别的黑素瘤抗原1(MelanA或MART1)；大鼠肉瘤(Ras)突变体、人端粒酶逆转录酶(hTERT)、肉瘤易位断点、黑素瘤凋亡抑制蛋白(ML-IAP)、ERG(跨膜蛋白酶、丝氨酸2(TMPRSS2)ETS融合基因)、N-乙酰葡糖胺基转移酶V(NA17)、配对的框蛋白Pax-3(PAX3)、雄激素受体、细胞周期蛋白B1、v-myc鸟髓细胞增多症病毒癌基因神经母细胞瘤衍生的同源物(MYCN)、Ras同源物家族成员C(RhoC)、酪氨酸酶相关蛋白2(TRP-2)、细胞色素P450 1B1(CYP1B1)、T细胞识别的鳞状细胞癌抗原3(SART3)、配对的框蛋白Pax-5(PAX5)、前顶体蛋白结合蛋白sp32(OY-TES1)、淋巴细胞特异性蛋白质酪氨酸激酶(LCK)、A激酶锚定蛋白4(AKAP-4)、滑膜肉瘤X断点2(SSX2)、高级糖基化终末产物的受体(RAGE-1)、豆荚蛋白(legumain)、人乳头瘤病毒E6(HPV E6)、人乳头瘤病毒E7(HPV E7)、肠羧基酯酶、突变的热休克蛋白70-2(mut hsp70-2)、CD79a、CD79b、CD72、白细胞相关免疫球蛋白样受体1(LAIR1)、IgA受体的Fc片段(FCAR或CD890)、白细胞免疫球蛋白样受体亚家族A成员2(LILRA2)、CD300分子样家族成员f(CD300LF)、C型凝集素结构域家族12成员A(CLEC12A)、骨髓间质细胞抗原2(BST2)、含有EGF样模块的黏蛋白样激素受体样2(EMR2)、淋巴细胞抗原75(LY75)、磷脂酰基醇蛋白聚糖-3(GPC3)、Fc受体样5(FCRL5)和/或免疫球蛋白λ样多肽1(IGLL1)。
TRAC基因，HLA-A基因，B2M基因和CIITA基因
在本申请中，与相应的野生型细胞相比，TRAC基因和HLA-A基因的表达和/或活性被下调，B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调。
在本申请中，与相应的野生型细胞相比，两种基因的表达和/或活性被下调，其中所述两种基因由TRAC基因和HLA-A基因组成。
在本申请中，其中所述下调基因的表达水平和/或活性包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
例如，所述蛋白产物可以包括多肽。
在本申请中，所述TRAC基因和HLA-A基因的表达和/或活性被下调或所述下调所述免疫效应细胞中TRAC基因和HLA-A基因的表达和/或活性可以包括与未经所述修饰的相应细 胞相比，所述修饰改变了所述TRAC基因和HLA-A基因的核苷酸序列结构。所述核苷酸序列可以包括编码区或非编码区，例如顺式调控元件序列、外显子序列，所述顺式调控元件序列可以包括启动子。所述改变可以包括部分或全部序列缺失、插入外源片段、碱基位点突变等，插入的外源片段可以是取代或破坏了TRAC基因和HLA-A基因的序列结构，使其不能够正常翻译；所述碱基位点突变可以包括移码突变、错义突变、无义突变等。所述改变改变可以包括核苷酸序列的化学基团修饰，例如甲基化等。所核苷酸序列结构的改变可通过基因测序检测，例如桑格测序、亚硫酸盐测序等。
在本申请中，所述TRAC基因和HLA-A基因的表达和/或活性被下调或所述下调所述免疫效应细胞中TRAC基因和HLA-A基因的表达和/或活性还可以包括与未经所述修饰的相应细胞相比，所述修饰使得所述TRAC基因和HLA-A基因的mNNA含量下降。所述mRNA含量可以通过所属领域技术人员所公知的实验方法及生物统计学方法获得，例如分子探针原位杂交、实时荧光定量PCR(qPCR、RT-PCR),所述实时荧光定量PCR可以包括SYBR Green法、TaqMan法、双杂交探针法、分子信标法。所述mRNA含量检测结果允许实验或统计中不可避免地误差，所述误差可以是所属领域公知的。所述误差可以在±10％的范围内，例如±8％，±6％，±5％，±4％，±3％范围内。所述mNNA含量下降至少为30％，例如35％，40％，45％，50％，55％，60％，65％，70％，75％，80％，85％，90％，95％，99％，例如，在所述经修饰的免疫效应细胞中检测不到TRAC基因和HLA-A基因的mNNA。
在本申请中，所述TRAC基因和HLA-A基因的表达和/或活性被下调或所述下调所述免疫效应细胞中TRAC基因和HLA-A基因的表达和/或活性还可以包括与未经所述修饰的相应细胞相比，所述修饰使得所述TRAC基因和HLA-A基因表达的多肽含量下降。所述多肽是指与未经所述修饰的相应细胞中TRAC基因和HLA-A基因表达产生的多肽结构、功能一致的多肽，而由TRAC基因和HLA-A基因核苷酸序列变化而产生的功能、结构变化了的多肽不在比较范围内。所述多肽含量可以通过所属领域技术人员所公知的实验方法及生物统计学方法获得，例如流式细胞分析、酶联免疫吸附法(ELISA)、细胞免疫荧光染色法、免疫印迹法(Western blotting，WB)。所述多肽含量检测结果允许实验或统计中不可避免地误差，所述误差可以是所属领域公知的。所述误差可以在±10％的范围内，例如±8％，±6％，±5％，±4％，±3％范围内。所述多肽含量下降至少为30％，例如35％，40％，45％，50％，55％，60％，65％，70％，75％，80％，85％，90％，95％，99％，例如，在所述经修饰的免疫效应细胞中检测不到TRAC基因和HLA-A基因表达的多肽。
在本申请中，所述TRAC基因和HLA-A基因的表达和/或活性被下调或所述下调所述免 疫效应细胞中TRAC基因和HLA-A基因的表达和/或活性可以是指敲除或敲低所述TRAC基因和HLA-A基因的表达或者进行其他破坏TRAC蛋白和HLA-A蛋白功能的操作。
在本申请中，所述B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调，或者所述不下调B2M基因的表达和/或活性，且不下调CIITA基因的表达和/或活性可以包括与未经所述修饰的相应细胞相比，所述B2M基因和CIITA基因的mNNA含量不下降。所述mRNA含量可以通过所属领域技术人员所公知的实验方法及生物统计学方法获得，例如分子探针原位杂交、实时荧光定量PCR(qPCR、RT-PCR),所述实时荧光定量PCR可以包括SYBR Green法、TaqMan法、双杂交探针法、分子信标法。所述mRNA含量检测结果允许实验或统计中不可避免地误差，所述误差可以是所属领域公知的。所述误差可以在±10％的范围内，例如±8％，±6％，±5％，±4％，±3％范围内。所述mNNA含量不下降可以包括含量上升或不变，所述不变可以包括相比的两者中所述B2M基因和CIITA基因的相应mRNA定量分析不具有显著性差异(P>0.05)。
在本申请中，所述B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调，或者所述不下调B2M基因的表达和/或活性，且不下调CIITA基因的表达和/或活性可以包括与未经所述修饰的相应细胞相比，所述B2M基因和CIITA基因表达的多肽含量不下降。所述多肽是指与未经所述修饰的相应细胞中B2M基因和CIITA基因表达产生的多肽结构、功能一致的多肽。所述多肽含量可以通过所属领域技术人员所公知的实验方法及生物统计学方法获得，例如流式细胞分析、酶联免疫吸附法(ELISA)、细胞免疫荧光染色法、免疫印迹法(Western blotting，WB)。所述多肽含量检测结果允许实验或统计中不可避免地误差，所述误差可以是所属领域公知的。所述误差可以在±10％的范围内，例如±8％，±6％，±5％，±4％，±3％范围内。所述多肽含量不下降可以包括含量上升或不变，所述不变可以包括相比的两者中所述B2M基因和CIITA基因的相应多肽定量分析不具有显著性差异(P>0.05)。
在本申请中，所述B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调，或者所述不下调B2M基因的表达和/或活性，且不下调CIITA基因的表达和/或活性可以包括没有对所述未经所述修饰的相应细胞进行针对CIITA基因和B2M基因、CIITA基因和B2M基因编码的mRNA和/或多肽进行人工干预，例如，所述人工干预可以包括向所述未经所述修饰的相应细胞中引入能够靶向CIITA基因和B2M基因或其编码的mRNA分子并改变其结构或含量的核苷酸分子或其他化合物。
在本申请中，所述B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调，或者所述不下调B2M基因的表达和/或活性，且不下调CIITA基因的表达和/或活 性可以包括与未经所述修饰的相应细胞相比，所述B2M基因和所述CIITA基因的核苷酸序列结构没有改变，所述核苷酸序列结构没有改变可通过基因测序检测，例如桑格测序、亚硫酸盐测序等。所述核苷酸序列结构没有改变可以包括没有进行人为的改变，还可以包括自然出现的、不影响其功能的改变。
在本申请中，所述B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调，或者所述不下调B2M基因的表达和/或活性，且不下调CIITA基因的表达和/或活性可以是指不进行以B2M基因或其编码的蛋白、CIITA基因或其编码的蛋白为靶点的修饰。
在本申请中，所述HLA-A基因中的至多2个等位基因的表达水平和/或活性下调。例如，所述2个等位基因可以是所述免疫效应细胞中HLA-A基因的一对等位基因；例如，所述HLA-A基因中的1个等位基因的表达水平和/或活性下调。
在本申请中，所述TRAC基因可以包括如HGNC:12029所示的基因及其所有等位基因类型。例如，所述TRAC基因及其所有等位基因类型可以包括能够存在于所述免疫效应细胞中的TRAC基因类型。例如，所述TRAC基因可以包括如SEQ ID No.55所示的核苷酸序列。
例如，所述TRAC基因可以包括来自于人类的并且与SEQ ID No.55所示的核苷酸序列具有80％或以上同源性的核苷酸序列，例如，85％或以上，90％或以上，95％或以上，96％或以上，97％或以上，98％或以上，99％或以上。
在本申请中，所述HLA-A基因可以包括如HGNC:4931所示的基因及其所有等位基因类型。例如，所述HLA-A基因可以包括IMGT/HLA数据库3.38.0版(https://www.ebi.ac.uk/ipd/imgt/hla/)收录的由WHO HLA因子命名委员会命名的所有HLA-A等位基因类型，IMGT/HLA数据库3.38.0版公开的HLA-A等位基因类型及其序列信息通过引用方式并入本文。
例如，所述HLA-A等位基因可以包括A*02、A*24、A*01、A*03、A*32、A*11、A*26、A*68、A*23、A*29、A*31、A*33、A*25、A*43、A*74、A*30、A*69中的任意一种或多种。
例如，所述HLA-A等位基因可以包括A*02:01、A*03:01、A*01:01、A*24:02、A*68:01、A*11:01、A*31:01:02、A*29:02、A*32:01、A*26、A*23:01、A*30:02、A*25:01、A*33:03中的任意一种或多种。
例如，所述HLA-A等位基因可以包括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、
A*02:05:01中的任意一种或多种。
例如，所述HLA-A等位基因可以包括A*02、A*30、A*03、A*01、A*24、A*32、A*68、A*11、A*26、A*23、A*31、A*25中的任意一种或多种。
例如，所述HLA-A等位基因可以包括A*02:01、A*03:01、A*24:02、A*01:01、A*11:01、A*26:01、A*25:01、A*68:01、A*32:01、A*31:01中的任意一种或多种。
例如，所述HLA-A等位基因可以包括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*02:07、A*01:01、A*02:10、A*03:01中的任意一种或多种。
例如，所述HLA-A等位基因可以包括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。
例如，所述HLA-A等位基因可以包括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、
A*03:01、A*30:04、A*26:03中的任意一种或多种。
例如，所述HLA-A等位基因可以包括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*02:03、A*02:05中的任意一种或多种。
例如，所述HLA-A等位基因可以包括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*34:02、A*66:02、A*31:01:02、A*32:01、A*02:05、A*66:01、A*26:01中的任意一种或多种。
例如，所述HLA-A等位基因可以包括A*02、A*11、A*24、A*30、A*33、A*03、A*01、A*26中的任意一种或多种。
例如，所述HLA-A等位基因可以包括A*11:01、A*24:02、A*02:01、A*02:07、A*33:03、A*02:06和A*30:01中的任意一种或多种。
例如，所述HLA-A等位基因可以包括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*26:01:01:01中的任意一种或多种。
例如，所述HLA-A基因可以包括如SEQ ID NO.56-63中任一项所示的核酸序列。
例如，所述HLA-A基因可以包括来自于人类的并且与SEQ ID NO.56-63中任一项所示的核酸序列具有80％或以上同源性的核苷酸序列，例如，85％或以上，90％或以上，95％或以 上，96％或以上，97％或以上，98％或以上，99％或以上。
修饰
在本申请中，所述修饰可以包括基因敲除和/或基因沉默。
例如，所述修饰可以包括基因全部或部分片段缺失、基因突变和/或基因沉默。
例如，所述基因敲除可以包括基因全部或部分片段缺失、基因突变等。
例如，所述基因可以包括所述HLA-A基因和/或所述TRAC基因。
例如，所述修饰可以包括所述免疫细胞中两个TRAC等位基因中的任意一个被敲除并且两个HLA-A等位基因中的任意一个被敲除。
例如，所述修饰可以包括所述免疫细胞中两个TRAC等位基因被敲除并且两个HLA-A等位基因中的任意一个被敲除。
例如，所述修饰可以包括两个TRAC等位基因中的任意一个被敲除并且两个HLA-A等位基因被敲除。
例如，所述修饰可以包括两个TRAC等位基因被敲除并且两个HLA-A等位基因被敲除。
例如，所述基因部分片段缺失可以包括≥1个外显子序列的缺失。
例如，所述基因突变可以包括碱基对组成或排列顺序的改变，所述改变通常能够导致例如错义突变、移码突变和/或无义突变。所述错义突变通常是指由于某个碱基对的改变，使编码一种氨基酸的密码子变成编码另外一种氨基酸的密码子，从而使构成蛋白质的相应氨基酸发生变化。所述移码突变通常是指在DNA链上，一个或几个非3的整数倍的碱基的插入或缺失从而导致编码密码子的变化。所述无义突变通常是指某个碱基对的改变使一个编码氨基酸的密码子变成终止子，蛋白质合成进行到该位点时提前终止。
例如，所述碱基对组成或排列顺序的改变可以包括单核苷酸或碱基的变化(也称作点突变)和/或多核苷酸或碱基的变化。例如，所述单核苷酸或碱基的变化可以包括一种碱基或核苷酸被另一种碱基或核苷酸所替换、一个碱基的插入或缺失。例如，所述多核苷酸或碱基的变化可以包括丢失一段碱基序列、插入一段碱基序列和/或一段碱基序列发生重排。所述一段碱基序列可以是基因中任意一个外显子/或内含子的一部分。所述重排可以包括所述一段碱基序列的重复、倒位、易位等。
例如，所述基因沉默可以包括转录水平上的基因沉默(tran-scriptional gene silenc-ing,TGS)和/或转录后基因沉默(post-transcriptional gene silen-cing,PTGS)。所述转录水平上的沉默可以包括DNA分子甲基化从而抑制DNA，例如启动子序列甲基化；所述转录后基因沉默可以包括所述基因转录后通过对靶标RNA进行特异性干预而导致的基因表达变化。例如通过RNA 干扰(RNAi)降低mRNA水平。
在本申请中，所述修饰可以包括利用同源重组将外源核苷酸序列替换掉内源的正常基因，从而使所述内源的正常基因失活的技术或方法。所述外源核苷酸序列可以是已知的。例如，所述外源核苷酸序列可以是所述内源的正常基因的部分片段；例如，所述外源核苷酸序列自5’至3’可以依次包括同源臂1、待插入的核苷酸序列、同源臂2。所述待插入的核苷酸序列可以包括报告基因、非编码序列或者内源的正常基因的变异序列。
在本申请中，所述修饰可以包括向所述免疫效应细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和CRISPR/Cas9系统。
在本申请中，所述反义RNA可以是一种与转录产物mRNA(信使RNA)互补的单链RNA。
例如，所述互补为所述反义RNA至少60％的核酸序列与所述mRNA互补，例如至少70％，例如至少80％，例如至少90％，例如100％互补。
例如，所述反义RNA与靶mRNA结合后引起该双链RNA分子对RNA酶Ⅲ的敏感性增加，从而使其降解。
例如，所述反义RNA与mRNA的上游非编码区结合，从而抑制靶mRNA的翻译。
例如，所述反义RNA是人工制备的。
例如，所述反义RNA不能够形成短发夹结构。
在本申请中，所述反义RNA可以包含SEQ ID NO：93-96中任一项所述的核苷酸序列。
在本申请中，所述siRNA可以是一类长度约为18-28个碱基对的双链非编码RNA分子。
例如，所述siRNA可以通过与mRNA的互补结合引起mRNA的降解。
例如，所述siRNA的长度可以是18、19、20、21、22、23、24、25、26、27、28个碱基对。
例如，所述siRNA是人工制备的。
例如，所述siRNA是由细胞内长双链RNA或shRNA经Dicer酶处理得到的。
在本申请中，所述shRNA是指一类能够形成短发夹结构的RNA。
例如，所述shRNA可以包括两个短反向重复序列，以及位于所述两个短反向重复序列之间的茎环(loop)序列。
例如，所述shRNA中至少有连续的18个核酸序列能够与靶mRNA互补结合，例如至少19个、例如至少20个、例如至少21个、例如至少22个、例如至少23个、例如至少24个、例如至少25个、例如至少26个。
例如，所述shRNA不包括本申请所述的sgRNA。
在本申请中，所述修饰可以包括向所述免疫效应细胞施用CRISPR/Cas系统。
CRISPR/Cas系统
在本申请中，所述CRISPR/Cas系统可以包含向导RNA(gRNA)和Cas酶。所述gRNA可以包括crRNA和tracrRNA。当所述crRNA和所述tracrRNA分别位于两条不同的核苷酸分子中时，所述gRNA可称为dgRNA(双分子gRNA)，当所述crRNA和所述tracrRNA位于同一条核苷酸分子中时，所述gRNA可称为sgRNA(单分子gRNA)。
在本申请中，所述CRISPR/Cas系统可以包括gRNA核酸序列和Cas蛋白。例如gRNA核酸序列与Cas蛋白形成的RNP复合体。
在本申请中，所述CRISPR/Cas系统还可以包括编码所述gRNA的核酸序列和编码Cas蛋白的核酸序列，编码所述gRNA的核酸序列和编码Cas蛋白的核酸序列可以置于质粒、病毒(例如腺病毒、慢病毒、反转录病毒)等所属领域常规使用的载体中。例如，所述编码所述gRNA的核酸序列和编码Cas蛋白的核酸序列可以位于相同的载体中，或者位于不同的载体中。例如，编码tracrRNA的核酸序列和编码crRNA的序列可以位于相同的载体中也可以位于不同的载体中。用于驱动各个编码序列表达的启动子可以是相同的或不同的。
在本申请中，所述CRISPR/Cas系统可以包含超过一个向导RNA。每个向导RNA可以包含不同的靶向序列，使得CRISPR/Cas系统切割超过一个靶序列。例如，一个或多个向导RNA可以具有相同或不同的特性，如活性或在CRISPR/Cas复合物中的稳定性。在使用超过一个向导RNA时，每个向导RNA可以在相同或不同的载体上编码。用于驱动所诉超过一个向导RNA表达的启动子可以是相同的或不同的。例如，所述向导RNA包括靶向HLA-A基因、靶向TRAC基因的向导RNA。
在本申请中，所述修饰还包括向所述细胞施用Cas酶。所述Cas酶可以包括Cas蛋白、编码Cas蛋白的核酸序列。
例如，所述Cas蛋白可以包含至少一个与向导RNA(gRNA)相互作用的结构域；例如，所述Cas蛋白可由向导RNA指导至靶序列；例如，所述向导RNA与Cas蛋白以及靶序列相互作用，使其指导Cas蛋白与靶序列的结合；例如，所述向导RNA为靶向切割提供特异性，所述Cas蛋白可以是通用的，与不同向导RNA配对来切割不同的靶序列；例如，所述Cas蛋白可以切割单链或双链DNA；例如，所述Cas蛋白可以切割RNA；例如，所述Cas蛋白可使RNA/DNA产生切口；例如，所述Cas蛋白包含至少一个DNA结合结构域和至少一个核酸酶结构域；例如，所述核酸酶结构域对DNA结合结构域而言可以是异源的；例如，可以通 过修饰Cas蛋白改变核酸酶活性；例如，所述Cas蛋白可用于结合和调节DNA的表达或活性；例如，所述Cas蛋白可以是Cas核酸酶。
在本申请中，所述CRISPR/Cas系统可以包含1类或2类系统成分，包括核糖核酸蛋白质复合物(参见例如Makarova等，Nat Rev Microbiol,13(11):722-36(2015)；Shmakov等，Molecular Cell,60:385-397(2015)。其中，2类CRISPR/Cas系统具有单蛋白质效应物。II、V和VI型Cas蛋白可以是单蛋白、RNA向导核酸酶，在本申请中称为“2类Cas核酸酶”。2类Cas核酸酶可以包括Cas9、Cpf1、C2c1、C2c2和C2c3蛋白。Cas9或Cpf1蛋白(Zetsche等,Cell,163:1-13(2015))可以包含RuvC样核酸酶结构域或HNH样核酸酶结构域，Zetsche中的Cpf1序列以其整体引入本申请。
例如，Cas蛋白可以来自II型CRISPR/Cas系统(即CRISPR/Cas9系统的Cas9蛋白)或V型CRISPR/Cas系统(例如Cpf1蛋白质)。例如，所述Cas蛋白可以来自2类CRISPR/Cas系统，例如Cas9蛋白或Cpf1蛋白。2类Cas核酸酶家族蛋白质是具有DNA内切核酸酶活性的酶，可通过设计本申请中进一步描述的适当的向导RNA来指导它们切割所希望的核酸靶标。
例如，所述2类CRISPR/Cas系统成分可以来自IIA型、IIB型、IIC型、V型或VI型系统。包括Cas9及其直向同源物。Cas9蛋白或其直向同源物可以来自如下示例性物种：化脓性链球菌(Streptococcus pyogenes)、嗜热链球菌(Streptococcus thermophilus)、链球菌属物种(Streptococcus sp.)、金黄色葡萄球菌(Staphylococcus aureus)、无害利斯特氏菌(Listeria in-nocua)、戈氏乳杆菌(Lactobacillus gasseri)、新凶手巴斯德氏菌(Francisella novicida)、产琥珀酸沃林氏菌(Wolinella succinogenes)、脑膜炎奈氏球菌(Neisseria meningitidis)、空肠弯曲杆菌(Campylobacter jejuni)、多杀巴斯德氏菌(Pasteurella multocida)、产琥珀酸丝状杆菌(Fibrobacter succinogene)、深红红螺菌(Rhodospirillum rubrum)、达松维尔拟诺卡氏菌(Nocardiopsis dassonvillei)、始旋链霉菌(Streptomycespristinaespiralis)、绿色产色链霉菌(Streptomyces viridochromogenes)、玫瑰链孢囊菌(Streptosporangium roseum)、酸热芽孢杆菌(Alicyclobacillusacidocaldarius)、假真菌样芽孢杆菌(Bacillus pseudomycoides)、德氏杆菌(Lactobacillusdelbrueckii)、唾液乳杆菌(Lactobacillus salivarius)、布氏乳杆菌(Lactobacillusbuchneri)、齿垢密螺旋体(Treponema denticola)、海洋微颤菌(Microscilla mari-na)、伯克氏菌目细菌(Burkholderiales bacterium)、Polaromonas naphthalenivorans、极地单胞菌属物种(Polaromonas sp.)、蓝丝菌属物种(Cyanothecesp.)、铜绿微囊蓝细菌(Microcystis ae-ruginosa)、聚球蓝细菌属物种(Synechococcussp.)、阿拉伯糖醋盐杆菌(Acetohalobium arabati-cum)、肉毒梭状芽孢杆菌(Clostridium botulinum)、艰难梭菌(Clostridium difficile)、海杆菌属物种 (Marinobacter sp.)、嗜盐亚硝化球菌(Nitrosococcus halophilus)、多变鱼腥蓝细菌(Anabaena variabilis)、产泡沫节球蓝细菌(Nodularia spumigena)、念珠蓝细菌属物种(Nostoc sp.)、最大节螺菌(Arthrospiramaxima)、盘状节螺蓝细菌(Arthrospira platensis)、节螺蓝细菌属物种(Arthrospirasp.)、鞘丝蓝细菌属物种(Lyngbya sp.)、土质体微鞘蓝细菌(Microcoleuschthonoplastes)、颤蓝细菌属物种(Oscillatoria sp.)、非洲栖热腔菌(Thermosipho africanus)、灰烬奈氏球菌(Neisseriacinereal)、红嘴鸥弯曲杆菌(Campylobacter lari)或白喉棒杆菌(Corynebacterium diphtheria)。
例如，所述Cas9蛋白可以来自化脓性链球菌；例如，所述Cas9蛋白可以来自嗜热链球菌；例如，所述Cas9蛋白可以来自金黄色葡萄球菌；例如，所述Cpf1蛋白可以来自土拉热巴斯德氏菌(Francisella tularensis)、氨基酸球菌属(Acidaminococcus)、挑剔真杆菌(Eubacterium eligens)、稻田氏钩端螺旋体(Leptospira inadai)、解糖胨拟杆菌(Prevotella dis-iens)或猕猴卟啉单胞菌(Porphyromonasmacacae)。例如，所述Cpf1蛋白可以来自氨基酸球菌属或毛螺菌科(Lachnospiraceae)。
例如，所述Cas9蛋白可以包含与化脓性链球菌Cas9具有60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％，或99％同源性的氨基酸序列。
例如，所述Cas9蛋白可以包含如SEQ ID NO：65所示的氨基酸序列。
例如，所述编码Cas9蛋白的示例性核酸序列如以下文献中描述：Cong等，SCIENCE 2013,399(6121):819-823；Wang等，CELL 2013,153(4):910-918；Mali等，SCIENCE 2013,399(6121):823-826；Jinek等，SCIENCE 2012,337(6096):816-821。
例如，所述编码Cas9蛋白的核苷酸序列如SEQ ID NO：64所示。
例如，所述Cas9蛋白可以是经过修饰的。例如，所述修饰可以包括氨基酸取代、与其他多肽片段组成融合蛋白。所述其他多肽片段可以包括PEST序列、泛蛋白、多聚泛蛋白、核定位信号(NLS)。
在本申请中，所述crRNA可以包含靶向序列，所述gRNA可以通过crRNA的靶向序列靶向任何目的序列。例如，所述靶向序列和靶核酸分子上的靶序列之间的互补程度可以是约60％、65％、70％、75％、80％、85％、90％、95％、97％、98％、99％或100％。例如，所述靶向序列和靶核酸分子上的靶序列可以100％互补。例如，所述靶向序列和靶核酸分子上的靶序列可以包含至少一个错配。例如，所述靶向序列和靶核酸分子上的靶序列可以包含1、2、3、4、5、6、7、8、9或10个错配。例如，所述靶向序列和靶核酸分子上的靶序列可以包含1-6个错配。例如，所述靶向序列和靶核酸分子上的靶序列可以包含5或6个错配。例如， 所述靶向序列和靶核酸分子上的靶序列不包含错配。
在本申请中，所述靶向序列的长度可取决于所使用的CRISPR/Cas系统和成分。例如，来自不同细菌物种的不同Cas蛋白质具有不同的最优靶向序列长度。此外，所述靶向序列可以包含长度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或超过50个核苷酸。例如，所述靶向序列可以包含长度18-24个核苷酸。例如，所述靶向序列可以包含长度19-21个核苷酸。例如，所述靶向序列可以包含长度20个核苷酸。
在本申请中，所述crRNA还可以包括crRNA旗杆序列，所述crRNA旗杆序列可以包含能够与tracrRNA的互补从而足以促进CRISPR/Cas复合物形成的任何序列。例如，所述旗杆序列可以包含与同一CRISPR/Cas系统中的tracrRNA互补的天然存在的crRNA的全部或部分序列(也称为“标记”或“把手”)。例如，旗杆序列可以包含来自天然存在的CRISPR/Cas系统的全部或部分重复序列。例如，旗杆序列可以包含截短或修饰的标记或把手序列。所述tracrRNA中与所述crRNA旗杆序列互补的部分可以称为tracrRNA旗杆序列。例如，tracrRNA和与tracrRNA杂交的旗杆部分之间沿两个序列中较短者的互补程度可以是约40％、50％、60％、70％、80％或更高。例如，tracrRNA和与tracr RNA杂交的旗杆部分之间沿两个序列中较短者的并非100％互补。crRNA旗杆序列的长度可取决于所使用的CRISPR/Cas系统或tracrRNA。例如，crRNA旗杆序列可以包含长度10-50个核苷酸或超过50个核苷酸。例如，crRNA旗杆序列可以包含长度15-40个核苷酸。例如，crRNA旗杆序列可以包含长度20-30个核苷酸。例如，crRNA旗杆序列可以包含长度22个核苷酸。在使用双向导RNA时，crRNA旗杆序列的长度可以没有上限。
在本申请中，所述tracrRNA可以包含来自天然存在的CRISPR/Cas系统的全部或部分野生型tracrRNA序列。例如，tracrRNA可以包含野生型tracrRNA的截短或修饰变体。tracrRNA的长度可取决于所使用的CRISPR/Cas系统。例如，tracr RNA可以包含长度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或超过100个核苷酸。例如，tracr长度为至少20个核苷酸。例如，tracrRNA长度为至少40个核苷酸。例如，tracrRNA可以包含二级结构，例如一个或多个发夹或茎-环结构、或一个或多个凸起结构。
在本申请中，所述向导RNA可以包含两个RNA分子，在本文中称为“双向导RNA”或“dgRNA”。例如，dgRNA可以包含含有crRNA的第一RNA分子和含有tracrRNA的第二RNA分子。该第一和第二RNA分子可通过crRNA和tracr RNA上的旗杆序列之间的碱基配对形 成RNA双链体。
例如，第一RNA分子从5'至3'可以包含与靶序列互补的靶向序列、crRNA旗杆序列。第二RNA分子从5'至3'可以包含与crRNA旗杆序列互补的tracrRNA旗杆序列、核酸酶结合序列，例如，所述核酸酶结合序列可以Cas核酸酶，例如，Cas9。
在本申请中，所述向导RNA可以包含单个RNA分子，称为“单分子gRNA”或“sgRNA”。例如，sgRNA可以包含tracrRNA、与tracrRNA共价连接的crRNA。例如，crRNA和tracrRNA可以通过接头核酸序列共价连接。例如，单分子gRNA可以包含通过crRNA和tracr RNA上的旗杆序列间的碱基配对形成的茎-环结构。例如，sgRNA是能够介导通过Cas9蛋白质进行DNA切割的“Cas9sgRNA”。例如，sgRNA是能够介导通过Cpf1蛋白质进行DNA切割的“Cpf1sgRNA”。
例如，单分子gRNA或sgRNA从5'至3'可以包含crRNA、环、和tracrRNA。crRNA从5'至3'可以包含与靶序列互补的靶向序列、crRNA旗杆序列。tracrRNA从5'至3'可以包含与crRNA旗杆序列互补的tracrRNA旗杆序列、核酸酶结合序列，例如，所述核酸酶结合序列可以Cas核酸酶，例如，Cas9。
例如，单分子gRNA或sgRNA从5'至3'可以包含与靶序列互补的靶向序列、crRNA旗杆序列、环、crRNA旗杆序列互补的tracrRNA旗杆序列、核酸酶结合序列。
在本申请中，所述gRNA中还可以包含修饰的核苷或核苷酸。例如，改变(例如替换)一个或两个非连接磷酸氧和/或主链磷酸二酯键中的一个或多个连接磷酸氧；例如，改变例如替换)核糖的组分，例如替换核糖上的2’羟基；例如用脱磷酸接头取代磷酸部分；例如修饰或替换天然存在的核碱基；例如，替换或修饰磷酸核糖主链；例如，修饰寡核苷酸的3’端或5’端，例如去除、修饰或替换末端磷酸基团或缀合部分，加帽或接头(例如3’或5’帽修饰可以包含糖和/或主链修饰)；例如，修饰或替换糖。
例如，引入所述修饰的核苷或核苷酸增加对核酸酶的稳定性。例如，引入所述修饰的核苷或核苷酸减少先天免疫反应。所述先天免疫反应包括对外源核酸(包括单链核酸)的细胞反应，可以涉及细胞因子表达和释放(尤其是干扰素)及细胞死亡的诱导。
例如，所述修饰可以包括向所述免疫效应细胞施用靶向所述HLA-A基因外显子部分的sgRNA。
例如，所述靶向所述HLA-A基因外显子部分的sgRNA可以包含SEQ ID No.16-54、91-92中任一项所示的核苷酸序列。
例如，所述靶向所述HLA-A基因外显子部分的sgRNA可以包含与SEQ ID No.16-54、 91-92中任一项所示的核苷酸序列至少70％同源的核苷酸序列，例如至少75％、80％、85％、90％、95％、96％、97％、98％、99％。
例如，所述修饰可以包括向所述免疫效应细胞施用靶向所述TRAC基因外显子部分的sgRNA。
例如，所述靶向所述TRAC基因外显子部分的sgRNA可以包含SEQ ID No.1-15中任一项所示的核苷酸序列。
例如，所述靶向所述TRAC基因外显子部分的sgRNA可以包含与SEQ ID No.1-15中任一项所示的核苷酸序列至少70％同源的核苷酸序列，例如至少75％、80％、85％、90％、95％、96％、97％、98％、99％。
嵌合抗原受体(CAR)
在本申请中，所述免疫效应细胞包含编码嵌合抗原受体(CAR)的核酸，所述CAR包括抗原结合结构域、铰链区、跨膜结构域、共刺激结构和初级信号传导结构域。
例如，所述抗原结合结构域特异性地结合肿瘤抗原。例如，所述肿瘤抗原选自以下组：CD19、CD123、CD22、CD30、CD171、CA125、C-met、L1CAM、EC、DLL3、CD99、5T4、CD138、CS-1(也称作CD2亚类1、CRACC、SLAMF7、CD319或19A24)、C型凝集素样分子-1(CLL-1或CLECL1)、CD33、表皮生长因子受体变体III(EGFRvIII)、神经节苷脂G2(GD2)、神经节苷脂GD3、TNF受体家族成员B细胞成熟抗原(BCMA)、Tn抗原(例如Tn Ag、GalNAcα-Ser/Thr)、前列腺特异性膜抗原(PSMA)；受体酪氨酸激酶样孤儿受体1(ROR1)、Fms样酪氨酸激酶3(FLT3)；肿瘤相关糖蛋白72(TAG72)、CD38、CD44v6、癌胚抗原(CEA)、上皮细胞黏附分子(EPCAM)、B7H3(CD276)、KIT(CD117)、白介素-13受体亚基α-2(IL-13Ra2或CD213A2)、间皮素、白介素11受体α(IL-11Ra)、前列腺干细胞抗原(PSCA)、蛋白酶丝氨酸21、血管内皮生长因子受体2(VEGFR2)、Lewis(Y)抗原、CD24、血小板衍生生长因子受体β(PDGFR-β)、阶段特异性胚胎抗原-4(SSEA-4)、CD20、叶酸受体α、受体酪氨酸-蛋白激酶ERBB2(Her2/neu)、细胞表面相关黏蛋白1(MUC1)、表皮生长因子受体(EGFR)、神经细胞黏附分子(NCAM)、Prostase、前列腺酸性磷酸酶(PAP)、突变的延伸因子2(ELF2M)、肝配蛋白B2、成纤维细胞活化蛋白α(FAP)、胰岛素样生长因子1受体(IGF-I受体)、碳酸酐酶IX(CAIX)、蛋白酶体(例如蛋白酶体、巨蛋白因子)亚基Β型9(LMP2)、糖蛋白100(gp100)、由断点簇集区(BCR)和Abelson鼠白血病病毒癌基因同源物1(Abl)组成的癌基因融合蛋白(bcr-abl)、酪氨酸酶、肝配蛋白A型受体2(EphA2)、岩藻糖基GM1；唾液酰Lewis黏附分子(sLe)、转谷氨酰胺酶5(TGS5)、高分子量-黑素瘤相 关抗原(HMWMAA)、o-乙酰基-GD2神经节苷脂(OAcGD2)、叶酸受体β、肿瘤内皮标志物1(TEM1/CD248)、肿瘤内皮标志物7相关(TEM7R)、紧密连接蛋白6(CLDN6)、促甲状腺激素受体(TSHR)、G蛋白偶联受体C类第5群成员D(GPRC5D)、染色体X可读框61(CXORF61)、CD97、CD179a、间变性淋巴瘤激酶(ALK)、聚唾液酸、胎盘特异性1(PLAC1)、globoH glycoceramide的己糖部分(GloboH)、乳腺分化抗原(NY-BR-1)、尿路上皮分化特异糖蛋白(uroplakin)2(UPK2)、甲型肝炎病毒细胞受体1(HAVCR1)、肾上腺素受体β3(ADRB3)、泛连接蛋白(pannexin)3(PANX3)、G蛋白偶联受体20(GPR20)、淋巴细胞抗原6复合体基因座K9(LY6K)、嗅觉受体51E2(OR51E2)、TCRγ可变可读框蛋白(TARP)、Wilm肿瘤蛋白(WT1)；癌症/睾丸抗原1(NY-ESO-1)、癌症/睾丸抗原2(LAGE-1a)、黑素瘤相关抗原1(MAGE-A1)、位于第12p号染色体上的ETS转位变体基因6(ETV6-AML)、精子蛋白17(SPA17)、X抗原家族成员1A(XAGE1)、血管生成素结合性细胞表面受体2(Tie 2)、黑素瘤癌症睾丸抗原-1(MAD-CT-1)、黑素瘤癌睾丸抗原-2(MAD-CT-2)、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋白(prostein)、前列腺癌肿瘤抗原-1(PCTA-1或半乳糖凝集素8)、T细胞识别的黑素瘤抗原1(MelanA或MART1)；大鼠肉瘤(Ras)突变体、人端粒酶逆转录酶(hTERT)、肉瘤易位断点、黑素瘤凋亡抑制蛋白(ML-IAP)、ERG(跨膜蛋白酶、丝氨酸2(TMPRSS2)ETS融合基因)、N-乙酰葡糖胺基转移酶V(NA17)、配对的框蛋白Pax-3(PAX3)、雄激素受体、细胞周期蛋白B1、v-myc鸟髓细胞增多症病毒癌基因神经母细胞瘤衍生的同源物(MYCN)、Ras同源物家族成员C(RhoC)、酪氨酸酶相关蛋白2(TRP-2)、细胞色素P450 1B1(CYP1B1)、T细胞识别的鳞状细胞癌抗原3(SART3)、配对的框蛋白Pax-5(PAX5)、前顶体蛋白结合蛋白sp32(OY-TES1)、淋巴细胞特异性蛋白质酪氨酸激酶(LCK)、A激酶锚定蛋白4(AKAP-4)、滑膜肉瘤X断点2(SSX2)、高级糖基化终末产物的受体(RAGE-1)、豆荚蛋白(legumain)、人乳头瘤病毒E6(HPV E6)、人乳头瘤病毒E7(HPV E7)、肠羧基酯酶、突变的热休克蛋白70-2(mut hsp70-2)、CD79a、CD79b、CD72、白细胞相关免疫球蛋白样受体1(LAIR1)、IgA受体的Fc片段(FCAR或CD890)、白细胞免疫球蛋白样受体亚家族A成员2(LILRA2)、CD300分子样家族成员f(CD300LF)、C型凝集素结构域家族12成员A(CLEC12A)、骨髓间质细胞抗原2(BST2)、含有EGF样模块的黏蛋白样激素受体样2(EMR2)、淋巴细胞抗原75(LY75)、磷脂酰基醇蛋白聚糖-3(GPC3)、Fc受体样5(FCRL5)和/或免疫球蛋白λ样多肽1(IGLL1)。
在本申请中，所述抗原结合结构域可以包括特异性结合所述肿瘤抗原的抗体或其抗原结合片段。例如，本申请所述的特异性结合GPC3抗体或其抗原结合片段可以包括但不限于重 组抗体、单克隆抗体、人抗体、人源化抗体、嵌合抗体、双特异性抗体、单链抗体、双抗体、三抗体、四抗体、Fv片段、scFv片段、Fab片段、Fab'片段、F(ab')2片段和骆驼单结构域抗体。
例如，所述抗体可以为人源化抗体。其可以为免疫特异性结合至相关抗原(例如人类CD19、BCMA或GPC3)且包含基本上具有人类抗体的氨基酸序列的框架(FR)区及基本上具有非人类抗体的氨基酸序列的互补决定区(CDR)的抗体或其变异体、衍生物、类似物或片段。此处的“基本上”在CDR的情况下是指CDR的氨基酸序列与非人类抗体CDR的氨基酸序列至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一。
例如，所述抗原结合片段可以包括Fab，Fab’，F(ab)2、Fv片段、F(ab’)2，scFv，di-scFv和/或dAb。
例如，所述单链抗体为scFv。
例如，所述抗原结合结构域靶向实体瘤。例如，所述实体瘤选自以下组：肝癌、胃癌、肺癌、乳腺癌、结肠癌、直肠癌、肾细胞癌、肝癌、非小细胞肺癌、小肠癌、食道癌、黑素瘤、骨癌、胰腺癌、皮肤癌、头或颈癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌、直肠癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、霍奇金病、非霍奇金淋巴瘤、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、儿童实体瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管生成、脊枢椎肿瘤、脑干胶质瘤、垂体腺瘤、Kaposi肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤以及所述癌症的转移病灶。
例如，所述抗原结合结构域靶向非实体瘤。例如，所述非实体瘤选自以下组：慢性淋巴细胞白血病(CLL)、急性白血病、急性淋巴样白血病(ALL)、B细胞急性淋巴样白血病(B-ALL)、T细胞急性淋巴样白血病(T-ALL)、慢性髓性白血病(CML)、急性髓样白血病(AML)、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树状细胞肿瘤、Burkitt淋巴瘤、弥散性大B细胞淋巴瘤、滤泡淋巴瘤、多毛细胞白血病、小细胞或大细胞滤泡淋巴瘤、恶性淋巴细胞增生性疾病、MALT淋巴瘤、套细胞淋巴瘤、边缘区淋巴瘤、多发性骨髓瘤、脊髓发育不良和脊髓发育不良综合征、非霍奇金淋巴瘤、霍奇金淋巴瘤、浆母细胞淋巴瘤、浆细胞样树状细胞肿瘤和Waldenstrom巨球蛋白血症。
在本申请中，所述跨膜结构域可以包含选自下述蛋白的跨膜结构域：CD28、CD3e、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
在本申请中，所述共刺激结构域可以包含选自下述蛋白的共刺激结构域：CD137、CD28、4-1BB、OX-40和ICOS。
在本申请中，所述胞内信号传导结构域可以包含源自CD3ζ的信号传导结构域。
在本申请中，所述铰链区连接所述抗原结合结构域和所述跨膜结构域，所述铰链区包含源自选自下述蛋白的铰链区：IgG1、IgG4、IgD、CD8。
组合物、应用
本申请还提供了一种组合物，其包括本申请所述的经修饰的免疫效应细胞和药学上可接受的载体。
例如，所述组合物包含细胞群，其中所述细胞群包含本申请中所述的经修饰的免疫效应细胞。
例如，所述经修饰的免疫效应细胞的个数与所述细胞群中细胞总数的比例为至少0.001％，至少0.01％，至少0.1％，至少1％，至少5％，至少10％，至少15％，至少20％，至少25％，至少30％，至少35％，至少40％，至少45％，至少50％，至少53％，至少55％，至少58％，至少60％，至少63％，至少65％，至少68％，至少70％，至少73％，至少75％，至少78％，至少80％，至少83％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或100％。
例如，所述细胞群中可以包括所述经修饰的免疫效应细胞和未经所述修饰的相应免疫效应细胞。
例如，所述经修饰的免疫效应细胞可以包括两个TRAC等位基因中的任意一个被敲除并且两个HLA-A等位基因中的任意一个被敲除的细胞、两个TRAC等位基因被敲除并且两个HLA-A等位基因中的任意一个被敲除的细胞、两个TRAC等位基因中的任意一个被敲除并且两个HLA-A等位基因被敲除的细胞、两个TRAC等位基因被敲除并且两个HLA-A等位基因被敲除的细胞。
例如，所述细胞群可以是所述免疫效应细胞的细胞群经基因工程手段处理后所得到的细胞群，所述基因工程手段处理可以包括向所述免疫效应细胞的细胞群施用本申请所述反义RNA、所述siRNA、所述shRNA和/或所述CRISPR/Cas9系统。例如，所述CRISPR/Cas9系统可以包括所述靶向HLA-A基因外显子部分的sgRNA、所述靶向所述TRAC基因外显子部分的sgRNA和所述Cas9蛋白。
例如，所述细胞群可以包括所述免疫效应细胞的细胞群经CRISPR/Cas9系统编辑后所得 到的细胞群，所述编辑的编辑效率为至少30％，至少35％，至少40％，至少45％，至少50％，至少53％，至少55％，至少58％，至少60％，至少63％，至少65％，至少68％，至少70％，至少73％，至少75％，至少78％，至少80％，至少83％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％。
例如，所述编辑效率可以通过Sanger测序、TA克隆测序、流式细胞计数获得。
例如，所述细胞群可以包括所述免疫效应细胞的细胞群经施用本申请所述反义RNA、所述siRNA、所述shRNA后所得到的细胞群，所述细胞群与所述施用前的相应免疫效应细胞的细胞群相比，mRNA表达降低至少10％，至少20％，至少30％，至少35％，至少40％，至少45％，至少50％，至少53％，至少55％，至少58％，至少60％，至少63％，至少65％，至少68％，至少70％，至少73％，至少75％，至少78％，至少80％，至少83％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％。
例如，所述细胞群可以包括所述免疫效应细胞的细胞群经施用本申请所述反义RNA、所述siRNA、所述shRNA后所得到的细胞群，所述细胞群与所述施用前的相应免疫效应细胞的细胞群相比，蛋白表达降低至少10％，至少20％，至少30％，至少35％，至少40％，至少45％，至少50％，至少53％，至少55％，至少58％，至少60％，至少63％，至少65％，至少68％，至少70％，至少73％，至少75％，至少78％，至少80％，至少83％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％。
例如，所述组合物的可接受成分在所用剂量和浓度下对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
例如，所述药学上可接受的载体可以包括与所述免疫效应细胞相容的任何和所有的溶剂、分散介质、等渗剂和吸收延迟剂，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。例如，所述药学上可接受的载体可以包括2℃-8℃储存液、冻存液、注射液等。例如所述载体可以包括如下成分：腺苷、氯化钠、白蛋白、白细胞介素-15、血管紧张素-II、来自人脐带间充质干细胞的无血清培养液中的短肽和多肽类化合物等。例如，所述载体还可以包括Normosol R(Abbott)、Plasma-Lyte A(Baxter)注射液、5％葡萄糖水或林格氏乳酸盐溶液。例如，所述载体还可以包括甘油或DMSO。
例如，所述组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述组合物可以通过输注或注射施用于患者或者受试者。在某些实施方式中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉 内、局部或真皮内施用。在某些实施方式中，所述药物组合物可以不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵系统来实现，以测量流入患者体内的治疗剂，如WO2015/036583所述。
本申请还提供了本申请所述的经修饰的免疫效应细胞在制备CAR-T细胞中的应用。
本申请还提供了本申请所述的经修饰的免疫效应细胞在制备药物中的应用，所述药物用于异体治疗。
本申请还提供了本申请所述的经修饰的免疫效应细胞在制备药物中的应用，所述药物用于治疗肿瘤。
例如，所述肿瘤包括实体瘤和非实体瘤。所述实体瘤、所述非实体瘤的种类如前文所述。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的经修饰的免疫效应细胞、制备方法和用途等，而不用于限制本申请发明的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Labora-tory Press。
实施例
实施例1 设计导向RNA
通过网站https://www.ncbi.nlm.nih.gov/，查找并下载相应基因序列(如SEQ ID No.55-63所示)，使用SnapGene软件打开基因序列，可在目的基因的不同外显子上设计sgRNA。在本实施例中采用的CRISPR/Cas9系统的sgRNA非限制性的设计原则为：5’-NNN(20)-NGG-3’，NGG被称为原间隔子相邻基序(PAM)，其中，N表示A、T、C或G。由于在同一外显子上可以设计出较多sgRNA，并且由20个核苷酸序列组成的sgRNA可能会在基因组中重复出现，所以利用网站http://crispr.cos.uni-heidelberg.de来进行sgRNA的设计与评估，将外显子序列粘贴至该网站，网站设计出sgRNA并进行预测评估，在评估中得分越高，则说明可能存在较高的编辑效率和较低的脱靶风险，从中选择得分较高的sgRNA进行试验。靶向TRAC基因的sgRNA如SEQ ID No.1-15所示，靶向HLA-A02基因的sgRNA如SEQ ID No.16-37所示，靶向HLA-A11基因的sgRNA如SEQ ID No.38-46所示，靶向HLA-A24基因的sgRNA如SEQ ID No.47-54所示，由金斯瑞生物科技公司合成。
实施例2：CD3 +T细胞制备
(1)从外周血中分离PBMC
从健康捐献者中采集外周血，用PBS缓冲液按照1：1进行外周血稀释。在新的50ml离心管中先加入稀释后血量1/3的细胞分离液(Ficoll)，然后沿着管壁非常缓慢加入血细胞稀释液，800g常温离心20min(离心机设置升速1、降速0)。离心后离心管中的液体自上而下分为PBS与血清层、白细胞层、淋巴细胞分离液、红细胞层。去除PBS与血清层，将白细胞层移至新的50ml离心管中，加入PBS至40ml清洗细胞，450g离心10min。离心后弃上清，即得到外周血单个核细胞。细胞重悬后进行细胞计数。
(2)复苏冻存的健康人PBMC
将冻存的健康人PBMC细胞在37℃水浴锅中进行复苏，完全融化后将细胞吸到含有10ml含10％FBS的X-VIVO15培养基(购自LONZA)的15ml离心管中，400g离心8min；去上清，加入2ml X-VIVO15培养基(含10％FBS和终浓度100μg/ml的DNase I)室温孵育15min，孵育的过程中不断振荡；将孵育结束后的溶液用40μm的滤网进行过滤，并吸取10ml PBS缓冲液重悬底部的细胞再加到滤网上，过虑后400g离心8min，离心后弃上清，细胞重悬后进行细胞计数。
(3)CD3 +T细胞分选
使用EasySep TM人T细胞分选试剂盒(购自StemCell Technologies，货号：17951)提取外周血单个核细胞(PBMC)中的T细胞。将PBMC密度调整至5×107细胞/ml，PBS缓冲液的添加范围在0.25-2ml；先加cocktail混匀再按照50μl/ml加入isolation cocktail，混匀后室温放置5min；将RapidSpheres用旋涡振荡仪涡旋30s后加按照40μl/ml加入至细胞中混匀；补加缓冲液至2.5ml的倍数，上下轻轻吹打2-3次；按照每管2.5ml分别加到冻存管中，将冻存管置于磁力架上，室温放置3min；轻轻打开冻存管盖，小心持两边拿起磁力架，倒置保持2-3s，将细胞液一次性倒入新的离心管中；用10-20ml缓冲液(视细胞量)重悬细胞后，300g离心10min，弃掉上清，得到CD3 +T细胞。
(4)T细胞激活
激活试剂按培养基:Transact＝99:1体积比进行配置，培养基为X-VIVO15培养基(含5％FBS、200U/ml IL2、10ng/ml IL7和5ng/ml IL15)、Transact购自美天旎。T细胞按每1×106个细胞用1ml激活试剂(含有10μl Transact)充分重悬后，放置于37℃、5％CO2培养箱中孵育3天。
实施例3.制备单基因敲除的T细胞
使用电转试剂盒(购自LONZA，货号V4XXP-3024)通过电转方式将RNP复合体转入实施例2制备的激活后T细胞。提前30min在孔板中预热培养基(X-VIVO15培养基+10％FBS+IL2(200U/ml)+IL7(10n g/ml)+IL15(5ng/ml))。电转缓冲液按照Nucleofector Solution：Supplement＝82:18进行配置。准备RNP复合体：TRAC的sgRNA序列为sg9(如SEQ ID No.1所示)，HLA-A的sgRNA序列为HLA-A02 Sg2(如SEQ ID No.17所示)或HLA-A02 Sg5(如SEQ ID No.18所示)或HLA-A11 sg21(如SEQ ID No.91所示)或HLA-A11 Rsg2(如SEQ ID No.92所示)，先将20μg sgRNA加入到PCR管(无RNA酶)中，再加入10μg Cas9蛋白(购自thermo，货号A36499)，轻轻混匀后，室温孵育12min。将实施例2培养的激活后T细胞进行计数，300g离心8min弃上清，加入PBS重悬细胞，吸取1E7个细胞重新300g离心8min，弃上清后用100μl配置好的电转缓冲液重悬细胞。将孵育好的RNP复合体加入上述细胞悬液中，轻柔混匀，然后将混合物轻轻地转移到电转杯中。将电转杯放在Lonza-4D电转仪上，选用EO-115电转程序进行电转。往电转杯中加入预热的培养基，然后用配套吸管将细胞转入孔板中预热的培养基中，然后放置于37℃、5％CO 2培养箱中培养。
实施例4.基因敲除效率检测方法的比较
(1)Sanger测序检测
细胞计数，取3～5×10 4细胞，2000r/min离心5min，尽量去干净上清，然后每管加20μl DE裂解液，细胞裂解后加到PCR管中，瞬时离心后上PCR仪，上机条件：65℃30min，4℃30s、95℃2min、16℃无限。使用引物对TRAC-For/TRAC-Rev，或HLA-A For/HLA-A Rev，将裂解产物作为模板进行PCR，PCR引物序列如SEQ ID NO.66-81所示，将PCR产物交送金唯智进行Sanger测序。得到sanger测序结果后，使用网站：https://moriaritylab.shinyapps.io/editr_v10/中的EditR编辑器预测编辑发生的位置以及编辑效率。
(2)TA克隆测序检测
利用AxyPrepTM PCR产物清洁试剂盒(购自AXYGEN)，将PCR产物纯化，随后用试剂盒(DNA A-Tailing Kit，购自TaKaRa)将纯化后的PCR产物加粘性末端，通过DNA Ligation Kit Ver2.1(购自TaKaRa)将产物连接至T载体(pMDTM19-T Vector Cloning Kit购自TaKaRa)，连接产物转化感受态细胞(DH5alpha)，然后涂布于含氨苄抗性的LB板，在37℃培养箱培养约12小时后挑单菌落，并将单菌落菌液交由金维智进行测序。敲除效率＝突变克隆数/总克 隆数。
(3)流式检测细胞计数
取10E5至10E8细胞，2000rpm离心5min，去上清，然后每管加100μl的PBS缓冲液重悬细胞，再加入anti-human AB TCR-APC(购自eBioscience)抗体5μl，HLA-A02 Monoclonal Antibody(BB7.2)，APC，eBioscince TM(购自invitrogen)抗体5μl，混合均匀后于室温孵育10min。2000rpm离心5min后再以PBS缓冲液洗2遍，重悬细胞并通过BD FACSAria流式细胞仪进行检测，可得细胞表面TCR、HLA-A02表达阳性率。敲除效率＝(A-B)/A×100％；A为对照组表达阳性率；B为敲除组表达阳性率。
TRAC单基因敲除的三种检测结果如图1至图3所示，敲除效率计算结果如表1所示，三种检测方法基本相同，后续试验仅采用Sanger测序方法检测编辑效率。
表1基因敲除效率检测方法结果
Figure PCTCN2020140799-appb-000001
针对HLA-A02基因编辑的Sanger测序方法结果如图4-5所示，编辑效率均为90％；针对HLA-A11基因编辑的Sanger测序方法结果如图6-7所示。
实施例5.制备TRAC基因和HLA-A基因双基因敲除的T细胞
使用电转试剂盒(购自LONZA，货号：V4XXP-3024)通过电转方式将RNP复合体转入实施例2制备的激活后T细胞。提前30min在孔板中预热培养基(X-VIVO15培养基+10％FBS+IL2(200U/ml)+IL7(10ng/ml)+IL15(5ng/ml))。电转缓冲液按照Nucleofector Solution：Supplement＝82:18进行配置。准备RNP复合体：分别将20μg TRAC sgRNA(TRAC Sg9)，20μg HLA-A sgRNA(HLA-A02 Sg2或HLA-A02 Sg5或HLA-A11 sg21或者靶向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或HLA-A*26:01:01:01的sgRNA)加入到PCR管(无RNA)中，再各自加入10μg Cas9蛋白(购自thermo，货号A36499)，轻轻混匀后，室温孵育12min。将实施例2培养的激活后T细胞进行计数，300g离心8min弃上清，加入PBS重悬细胞，吸取1E7个细胞重新300g离心8min，弃上清后用100μl配置好的电转缓冲液重悬细胞。将孵育好的TRAC 与HLA-A的RNP复合体加入上述细胞悬液中，轻柔混匀，然后将混合物轻轻地转移到电转杯中。将电转杯放在Lonza-4D电转仪上，选用EO-115电转程序进行电转。往电转杯中加入预热的培养基，然后用配套吸管将细胞转入孔板中预热的培养基中，然后放置于37℃、5％CO 2培养箱中培养。
通过测序检测双基因敲除效率，可得到双基因敲除效率均不低于80％的TRAC阴性、HLA-A阴性的T细胞。结果如图8-9所示。其中图8A显示的是利用HLA-A02 Sg5敲除HLA-A02的结果，其中上一行显示的是对照组结果(即没有使用HLA-A02 Sg5进行敲除)；下一行显示的是同时敲除HLA-A02和TRAC的结果；其中图8B显示的是利用TRAC Sg9敲除TRAC的结果，其中上一行显示的是对照组结果(即没有使用TRAC Sg9进行敲除)；下一行显示的是同时敲除HLA-A02和TRAC的结果。图9A-9B显示了敲除HLA-A02和TRAC蛋白水平的敲除情况，其中NEG指阴性对照，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指同时敲除HLA-A02和TRAC的结果。
实施例6.双基因敲除的T细胞中TRAC基因，HLA-A基因，B2M基因和CIITA基因与相应细胞中的相应基因的表达区别
(1)利用实施例2制备的激活后T细胞，分为两组，一组作为对照，另一组按照实施例5的方法制备TRAC基因和HLA-A基因双基因敲除的T细胞，按照实施例4步骤(1)的方式进行Sanger测序。依据测序结果获得TRAC和HLA-A双基因敲除的细胞。将制备的双基因敲除T细胞与相应TRAC和HLA-A抗体孵育，通过流式分选或磁珠分选，可以得到双基因敲除的细胞株。
(2)检测双基因敲除的T细胞与对照组相比mRNA表达水平的变化。使用RNA提取试剂盒(购自QIAGEN，货号：74004)提取RNA，使用逆转录试剂盒(购自Applied Biosystems，货号：4368814)对RNA进行逆转录得到cDNA，以cDNA为模板进行定量PCR检测。
(3)检测双基因敲除的T细胞与对照组相比蛋白表达水平的变化。使用全蛋白提取试剂(购自Thermo Scientific，货号：87787)提取蛋白，通过Western Blot方法或流式方法检测蛋白表达水平，使用的抗体分别为TRAC抗体(购自eBioscience货号：17-9986-42)、HLA-A抗体(购自Merck货号：17-9876-41)、B2M抗体(购自Invitrogen货号：A15770)和CIITA抗体(购自OriGene货号：CF812200)。
Sanger测序检测双基因敲除的T细胞中TRAC和/或HLA-A基因的核苷酸序列相对于对照组发生了变化；定量PCR显示双基因敲除的T细胞中TRAC和/或HLA-A基因mRNA表达量下调，而B2M和/或CIITA基因的mRNA表达量没有下调。FACS和Western Blot结果 显示双基因敲除的T细胞中蛋白表量下调，B2M和/或CIITA蛋白表达量没有下调。
结果如图10-11所示。其中，图10显示的是基因表达的mRNA水平测定，其中图10A-10D分别显示了TRAC、HLA-A、B2M和CIITA的mRNA水平；其中WT指没有经任何敲除处理的情况，双敲组指TRAC基因和HLA-A基因双基因敲除的T细胞的结果。图11显示的是基因表达的蛋白水平测定，其中图11A-11B分别显示了B2M和CIITA的蛋白表达水平；其中NEG指阴性对照，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的T细胞的结果。
实施例7.制备TRAC基因、HLA-A/B2M基因和CIITA基因三基因敲除的T细胞并验证其中相应三种基因的表达变化
(1)按照实施例6步骤(1)的方式准备对照组和TRAC基因、HLA-A基因和CIITA基因三基因敲除的细胞，以及TRAC基因、B2M基因和CIITA基因三基因敲除的细胞。
(2)按照实施例6步骤(3)的方式通过FACS和Western Blot方法检测蛋白表达水平变化。
相对于对照组细胞，TRAC、HLA-A和CIITA三基因敲除的T细胞中TRAC、HLA-A和CIITA基因的蛋白表达量下调；相对于对照组细胞，TRAC、B2M和CIITA三基因敲除的T细胞中TRAC、HLA-A和CIITA基因的蛋白表达量下调。
(3)使用TRAC(购自eBioscience，货号：17-9986-42)、HLA-A(购自Merck，货号：17-9876-41)、B2M(购自：Invitrogen，货号：A15770)抗体通过流式细胞术检测实施例6中的双基因敲除细胞和本实施例中的两种三基因敲除细胞的敲除效率，结果显示在单细胞水平同时实现多基因敲除的效率，双基因敲除明显高于三基因敲除。
结果如图12A-12D所示。其中图12A-12C依次为TRAC、HLA-A和B2M蛋白质水平的敲除情况。其中，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的T细胞的结果；TRAC+HLA-A+CIITA三敲指TRAC、HLA-A和CIITA三基因敲除的T细胞的结果；其中TRAC+B2M+CIITA三敲指B2M、CIITA和TRAC三基因敲除的T细胞的结果；TRAC+HLA-A敲低指实施例9制备的TRAC基因和HLA-A基因敲低的T细胞的结果。图12D显示了CIITA蛋白质水平的敲除情况。
图12的结果显示，和WT对照组相比，TRAC、HLA-A、CIITA和B2M的蛋白质水平下调。同时，与TRAC+HLA-A+CIITA三敲或TRAC+B2M+CIITA三敲相比，TRAC+HLA-A双敲的敲除效率更高。
实施例8 设计反义RNA序列
通过数据库https://www.ncbi.nlm.nih.gov/或www.ensembl.org/，获得相应基因(TRAC基因和HLA-A基因)的转录RNA序列(如SEQ ID NO.82-90所示)，参考如下原则设计siRNA：
尽量避免起始密码子下游50-100个核苷酸与终止密码子上游100个核苷酸的序列；选择长度小于30个核苷酸的序列；避免4个或以上的连续相同碱基；避免内含子区域；避免重复序列；避免单核苷酸多态性(SNP)位点；序列GC含量30％-60％之间，优先选择序列模式AA(N<sub>19)、NA(N<sub>21)或NAR(N<sub>17)YNN，A为腺苷酸；T为胸腺苷酸；R为腺苷酸或鸟苷酸(嘌呤类)；Y为胸腺苷酸或胞苷酸(嘧啶类)；N为腺苷酸、胸腺苷酸、鸟苷酸或胞苷酸；对选择的序列进行同源性比较分析，避免反义RNA与其他基因或序列具有显著的同源性，由此造成脱靶效应。同源性分析利用NCBI Blast tool:Nucleotide-nucleotide BLAST(blastn)，UCSC Blat tool或Ensembl Blast进行。
设计获得的反义RNA序列包括HLA-A-homo-551(其包含SEQ ID NO.93所示的核苷酸序列)；HLA-A-homo-NEG(其包含SEQ ID NO.94所示的核苷酸序列)；TRAC-homo-375(其包含SEQ ID NO.95所示的核苷酸序列)；TRAC-homo-NEG(其包含SEQ ID NO.96所示的核苷酸序列)。
实施例9 制备TRAC基因和HLA-A基因敲低的T细胞
利用通过实施例8设计的反义RNA进行双基因敲低。公司制备TRAC基因和HLA-A基因反义RNA序列的慢病毒(吉玛)。按照实施例2的方式制备CD3 +T细胞(D0天)，并用CD3/CD28抗体磁珠激活，将携带TRAC基因和HLA-A基因的反义RNA序列(SEQ ID NO.95和SEQ ID NO.93)的慢病毒转染激活的T细胞(D1天)，D2天洗去慢病毒载体，继续培养至D5天。收集培养至D5天的T细胞，通过定量PCR或Western Blot等方法对基因敲低效率进行检测。对获得的T细胞进行相应TRAC和HLA-A抗体标记，通过流式分选或磁珠分选的方式可以得到TRAC基因和HLA-A基因敲低的T细胞。结果显示，TRAC和HLA-A基因敲低组中TRAC和HLA-A的mRNA和蛋白表达水平均下调。其中，图13A-13B依次为TRAC和HLA-A mRNA水平的敲除情况。其中，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的T细胞的结果。其中，为TRAC和HLA-A蛋白质水平的敲除水平可以参见图12的结果。
实施例10 不同T细胞活性的区别
制备实施例2、5、7和9中的无基因敲除、双基因敲除、三种基因敲除和双基因敲低的 T细胞，比较几种T细胞活性各组细胞计数并分别各取1*10 6细胞接种24孔板中，每孔加入PHA(0.3μg/ml)(离子霉素+)或5ng/ml的PMA和50ng/ml的ionomycin于细胞中，继续培养5小时后，使用CD69(早期活化)(购自BD Biosciences，货号：FN50)、CD137(偏晚期)(购自BD Biosciences，货号：4B4-1)抗体，流式检测细胞的活化状态。结果表明，双基因敲除、双基因敲低的T细胞活性优于三基因敲除的T细胞。
CD69和CD137的蛋白质水平表达情况分别参见图14A-14B。其中，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的T细胞的结果；TRAC+HLA-A+CIITA三敲指TRAC、HLA-A和CIITA三基因敲除的T细胞的结果；其中TRAC+B2M+CIITA三敲指B2M、CIITA和TRAC三基因敲除的T细胞的结果；TRAC+HLA-A敲低指实施例9制备的TRAC基因和HLA-A基因敲低的T细胞的结果。
实施例11 不同T细胞对异体NK细胞反应性的区别
对实施例2、5、7和9中的无基因敲除、双基因敲除、三种基因敲除和双基因敲低的T细胞进行CFSE(invitrogen，C34554)标记，细胞计数，分别取1*10 6细胞并以1：1比例与NK细胞(NK92MI)进行共培养，24小时后收集共培养的各组细胞，流式细胞检测混合细胞中CFSE阳性细胞的比率。
结果表明，NK细胞对双基因敲除、双基因敲低的T细胞的杀伤毒性低于三基因敲除的T细胞。结果如图15所示。其中，NK+T指将NK细胞与没有经任何敲除处理的T细胞共培养的情况；NK+TRAC+HLA-A敲低指将NK细胞与实施例9制备的TRAC基因和HLA-A基因敲低的T细胞的结果共培养的情况；NK+TRAC+HLA-A双敲指将NK细胞与TRAC基因和HLA-A基因双基因敲除的T细胞共培养的情况；NK+TRAC+HLA-A+CIITA三敲指将NK细胞与TRAC、HLA-A和CIITA三基因敲除的T细胞共培养的情况；NK+TRAC+B2M+CIITA三敲指将NK细胞与B2M、CIITA和TRAC三基因敲除的T细胞共培养的情况。
实施例12 不同的T细胞异体免疫排斥反应的区别
供者1来源的外周血制备实施例2、5、7和9中的无基因敲除、双基因敲除、三种基因敲除和双基因敲低的T细胞。供者2来源的外周血制备CD3 +T细胞。将供者1外周血制备的各组细胞分别与供者2外周血按照实施例2制备的CD3 +T细胞等比例混合，24小时后检测细胞混合体系中的IFN-γ的表达水平。结果显示，双基因敲除的T细胞组IFN-γ的表达水平低于三基因敲除的T细胞组。
结果如图16所示，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基 因和HLA-A基因双基因敲除的T细胞的结果；TRAC+HLA-A+CIITA三敲指TRAC、HLA-A和CIITA三基因敲除的T细胞的结果；其中TRAC+B2M+CIITA三敲指B2M、CIITA和TRAC三基因敲除的T细胞的结果；TRAC+HLA-A敲低指实施例9制备的TRAC基因和HLA-A基因敲低的T细胞的结果。
实施例13.制备TRAC基因和HLA-A基因双基因敲除的CAR-T细胞，TRAC基因，HLA-A基因和CIITA基因三基因敲除的CAR-T细胞以及TRAC基因，B2M基因和CIITA基因敲除的CAR-T细胞
(1)按照实施例2的方式获得CD3 +T细胞(D0天)，并用CD3/CD28抗体磁珠激活，激活后于D1天进行慢病毒载体(包含CD19-CAR、CD20-CAR或BCMA-CAR等的慢病毒)转染，D2天洗去慢病毒载体，D3天对CAR阳性的T细胞进行分选并继续培养至D5天。
(2)取D5天的CAR-T细胞为初始细胞，分别按照实施例5和实施例7中的方式制备TRAC基因和HLA-A基因双基因基因敲除的细胞，TRAC基因、HLA-A基因和CIITA基因以及TRAC基因、B2M基因和CIITA基因三基因敲除的CAR-T细胞。
(3)通过流式细胞技术检测可得到上述双基因敲除和三基因敲除的CAR-T细胞，其中双基因敲除CAR-T细胞的得率高于三基因敲除CAR-T细胞。
结果如图17A-17D所示。其中，图17A-17C依次为TRAC、HLA-A和B2M蛋白质水平的敲除情况。图17D显示了CIITA蛋白质水平的敲除情况。其中，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的CAR-T细胞的结果；TRAC+HLA-A+CIITA三敲指TRAC、HLA-A和CIITA三基因敲除的CAR-T细胞的结果；其中TRAC+B2M+CIITA三敲指B2M、CIITA和TRAC三基因敲除的CAR-T细胞的结果。
其中，CD19CAR的转染效率如图18A-18B所示。其中，CAR30％+即代表CD19 CAR的转染效率。
图19显示了不同细胞的扩增倍数。其中，TRAC基因和HLA-A基因双基因基因敲除的CAR-T细胞扩增倍数最高。
实施例14.TRAC基因和HLA-A基因双基因敲除的CAR-T细胞抗肿瘤效果
制备实施例14中的TRAC基因和HLA-A基因双敲除的CAR-T细胞(靶向CD19、CD20或BCMA)，接种表达荧光素酶基因的靶细胞(靶基因阳性的白血病或淋巴瘤细胞系，如Raji、Jurkat、MM1S等)至孔板中，再分别以不同效靶比(1∶2.5，1∶1，5：1，10：1)加入双基 因敲除的CAR-T细胞、三基因敲除的CAR-T细胞或无基因敲除的T细胞，共培养24小时后将细胞转移至检测孔板中，加入荧光素酶底物，酶标仪检测荧光值。杀伤效率＝1-靶细胞T细胞共培养荧光值/单独培养的靶细胞荧光值。
结果显示TRAC基因和HLA-A基因双敲除的CAR-T细胞对肿瘤细胞有显著的杀伤效果。
图20显示了对CD19靶细胞Raji-Luciferase的杀伤效果，其中TRAC基因和HLA-A基因双敲除的CAR-T细胞的杀伤效果最为显著。其中每个E/T比下从左到右依次是A-D的图注所对应的结果。
实施例15.TRAC基因和HLA-A基因双基因敲除的CAR-T细胞抗肿瘤效果
NSG小鼠静脉注射肿瘤细胞，肿瘤成功建立后向小鼠体内回输TRAC基因和HLA-A基因双基因敲除的CAR-T细胞、三基因敲除的CAR-T细胞或无基因敲除的T细胞，监测小鼠肿瘤体积。
回输双基因敲除CAR-T细胞的小鼠，肿瘤生长速度明显减缓。
结果如图21-22所示。其中，图21显示了对小鼠的给药方式，i.v.表示静脉注射，CAR-T细胞代表表达CD19 CAR的双基因敲除的CAR-T细胞、三基因敲除的CAR-T细胞。图20显示了小鼠在施用CAR-T细胞后体内肿瘤的体积情况。其中，图20从左至右列依次显示了分别施用生理盐水、未改造的T细胞、TRAC基因和HLA-A基因双基因敲除的CD19 CAR-T细胞、TRAC、HLA-A和CIITA三基因敲除的CD19 CAR-T细胞、B2M、CIITA和TRAC三基因敲除的CD19 CAR-T细胞后，小鼠体内肿瘤的体积情况。结果发现回输TRAC基因和HLA-A基因双基因敲除的CAR-T细胞的小鼠，肿瘤生长的速度明显减缓。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (49)

  1. 经修饰的免疫效应细胞，其中与未经所述修饰的相应细胞中相应基因的表达和/或活性相比，TRAC基因和HLA-A基因的表达和/或活性被下调，B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调。
  2. 根据权利要求1所述的免疫效应细胞，其中所述修饰使得两种基因的表达和/或活性被下调，其中所述两种基因由TRAC基因和HLA-A基因组成。
  3. 根据权利要求1-2中任一项所述的免疫效应细胞，其中与相应的野生型细胞相比，TRAC基因和HLA-A基因的表达和/或活性被下调，B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调。
  4. 根据权利要求1-3中任一项所述的免疫效应细胞，其中与相应的野生型细胞相比，两种基因的表达和/或活性被下调，其中所述两种基因由TRAC基因和HLA-A基因组成。
  5. 根据权利要求1-4中任一项所述的免疫效应细胞，其中所述免疫效应细胞包括T细胞。
  6. 根据权利要求1-5中任一项所述的免疫效应细胞，其中所述基因的表达水平和/或活性被下调包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
  7. 根据权利要求1-6中任一项所述的免疫效应细胞，其中所述修饰包括：基因突变和/或基因沉默。
  8. 根据权利要求1-7中任一项所述的免疫效应细胞，其中所述修饰包括向所述免疫效应细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和CRISPR/Cas9系统。
  9. 根据权利要求1-8中任一项所述的免疫效应细胞，其中所述修饰包括向所述免疫效应细胞施用CRISPR/Cas9系统。
  10. 根据权利要求1-9中任一项所述的免疫效应细胞，其中所述修饰包括向所述免疫效应细胞施用靶向所述HLA-A基因外显子部分的sgRNA。
  11. 根据权利要求1-10中任一项所述的免疫效应细胞，其中所述修饰还包括向所述免疫效应细胞施用靶向所述TRAC基因外显子部分的sgRNA。
  12. 根据权利要求10所述的免疫效应细胞，其中所述靶向所述HLA-A基因外显子部分的sgRNA包含SEQ ID No.16-54、91-92中任一项所示的核苷酸序列。
  13. 根据权利要求12所述的免疫效应细胞，其中所述靶向所述TRAC基因外显子部分的sgRNA包含SEQ ID No.1-15中任一项所示的核苷酸序列。
  14. 根据权利要求8-13中任一项所述的免疫效应细胞，其中所述反义RNA包含SEQ ID No.93-96中任一项所示的核苷酸序列。
  15. 根据权利要求1-14中任一项所述的免疫效应细胞，其中所述修饰还包括向所述细胞施用 Cas酶。
  16. 根据权利要求15所述的免疫效应细胞，其中Cas酶包括Cas9蛋白。
  17. 根据权利要求1-16中任一项所述的免疫效应细胞，其中所述免疫效应细胞包含编码嵌合抗原受体(CAR)的核酸，所述CAR包括抗原结合结构域、铰链区、跨膜结构域、共刺激结构和初级信号传导结构域。
  18. 根据权利要求17所述的免疫效应细胞，其中所述抗原结合结构域特异性地结合肿瘤抗原。
  19. 根据权利要求17所述的免疫效应细胞，其中所述肿瘤抗原选自以下组：CD19、CD20和BCMA。
  20. 根据权利要求17-19中任一项所述的免疫效应细胞，其中所述抗原结合结构域选自以下组：单克隆抗体、多克隆抗体、人抗体、人源化抗体、单域抗体和其抗原结合片段。
  21. 根据权利要求17-20中任一项所述的免疫效应细胞，其中所述抗原结合结构域靶向实体瘤。
  22. 根据权利要求21所述的免疫效应细胞，其中所述实体瘤选自以下组：肝癌、胃癌、肺癌、乳腺癌和非小细胞肺癌。
  23. 根据权利要求17-20中任一项所述的免疫效应细胞，其中所述抗原结合结构域靶向非实体瘤。
  24. 根据权利要求23所述的免疫效应细胞，其中所述非实体瘤选自以下组：B淋巴细胞瘤、霍奇金淋巴瘤、慢性髓性白血病和急性髓样白血病。
  25. 根据权利要求17-24中任一项所述的免疫效应细胞，其中所述跨膜结构域包含源自选自下述蛋白的跨膜结构域：CD28、CD3e、CD27、CD3ε和CD45。
  26. 根据权利要求17-25中任一项所述的免疫效应细胞，其中所述共刺激结构域包含选自下述蛋白的共刺激结构域：CD137、CD28、CD27、OX40和CD30。
  27. 根据权利要求17-26中任一项所述的免疫效应细胞，其中所述铰链区连接所述抗原结合结构域和所述跨膜结构域，所述铰链区包含源自选自下述蛋白的铰链区：人免疫球蛋白铰链区、GS接头、KIR2DS2铰链区和CD8a铰链区。
  28. 制备权利要求1-27中任一项所述的经修饰的免疫效应细胞的方法，其包括以下的步骤：与未经所述修饰的相应细胞中相应基因的表达和/或活性相比，下调所述免疫效应细胞中TRAC基因和HLA-A基因的表达和/或活性；不下调B2M基因的表达和/或活性，且不下调CIITA基因的表达和/或活性。
  29. 根据权利要求28所述的方法，其中所述修饰使得两种基因的表达和/或活性被下调，其 中所述两种基因由TRAC基因和HLA-A基因组成。
  30. 根据权利要求28-29中任一项所述的方法，与相应的野生型细胞相比，TRAC基因和HLA-A基因的表达和/或活性被下调，B2M基因的表达和/或活性未被下调，且CIITA基因的表达和/或活性未被下调。
  31. 根据权利要求28-30中任一项所述的方法，与相应的野生型细胞相比，两种基因的表达和/或活性被下调，其中所述两种基因由TRAC基因和HLA-A基因组成。
  32. 根据权利要求28-31中任一项所述的方法，其中所述下调基因的表达水平和/或活性包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
  33. 根据权利要求28-32中任一项所述的方法，其中所述修饰包括：基因突变和/或基因沉默。
  34. 根据权利要求28-33中任一项所述的方法，其中所述修饰包括向所述免疫效应细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和CRISPR/Cas9系统。
  35. 根据权利要求28-34中任一项所述的方法，其中所述修饰包括向所述免疫效应细胞施用CRISPR/Cas9系统。
  36. 根据权利要求28-35中任一项所述的方法，其中所述修饰包括向所述免疫效应细胞施用靶向所述HLA-A基因外显子部分的sgRNA。
  37. 根据权利要求28-36中任一项所述的方法，其中所述修饰包括向所述免疫效应细胞施用靶向所述TRAC基因外显子部分的sgRNA。
  38. 根据权利要求36所述的方法，其中所述靶向所述HLA-A基因外显子部分的sgRNA包含SEQ ID No.16-54、91-92中任一项所示的核苷酸序列。
  39. 根据权利要求37所述的方法，其中所述靶向所述TRAC基因外显子部分的sgRNA包含SEQ ID No.1-15中任一项所示的核苷酸序列。
  40. 根据权利要求34-39中任一项所述的方法，其中所述反义RNA包含SEQ ID No.93-96中任一项所示的核苷酸序列。
  41. 根据权利要求34-40任一项所述的方法，其中所述修饰还包括向所述细胞施用Cas酶。
  42. 根据权利要求41所述的方法，其中Cas酶包括Cas9蛋白。
  43. 组合物，其包括权利要求1-27中任一项所述的免疫效应细胞和药学上可接受的载体。
  44. 根据权利要求43所述的组合物，其包含细胞群，其中所述细胞群包含权利要求1-27中任一项所述的免疫效应细胞。
  45. 权利要求1-27中任一项所述的免疫效应细胞在制备CAR-T细胞中的应用。
  46. 权利要求1-27中任一项所述的免疫效应细胞在制备药物中的应用，所述药物用于异体治疗。
  47. 权利要求1-27中任一项所述的免疫效应细胞在制备药物中的应用，所述药物用于治疗肿瘤。
  48. 根据权利要求47所述的应用，其中所述肿瘤包括实体瘤和非实体瘤。
  49. 根据权利要求47-48中任一项所述的应用，其中所述肿瘤选自以下组：肝癌、胃癌、肺癌、乳腺癌、非小细胞肺癌、B淋巴细胞瘤、霍奇金淋巴瘤、慢性髓性白血病和急性髓样白血病。
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