WO2021147891A1 - 一种经修饰的免疫效应细胞及其用途 - Google Patents

一种经修饰的免疫效应细胞及其用途 Download PDF

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WO2021147891A1
WO2021147891A1 PCT/CN2021/072844 CN2021072844W WO2021147891A1 WO 2021147891 A1 WO2021147891 A1 WO 2021147891A1 CN 2021072844 W CN2021072844 W CN 2021072844W WO 2021147891 A1 WO2021147891 A1 WO 2021147891A1
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constant region
activity
expression
cell
cell receptor
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PCT/CN2021/072844
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French (fr)
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王文博
郭佩佩
冯爱华
林彦妮
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苏州克睿基因生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application relates to the field of biomedicine, specifically to a modified immune effector cell and its use.
  • CAR-T Chimeric Antigen Receptor-T cells
  • CAR-T therapy is a kind of genetically modified T cell therapy, by transferring chimeric antigen receptor (CAR) genes into primary T cells, giving T cells the ability to recognize and kill target cells.
  • CAR Chimeric Antigen Receptor
  • CAR is an artificial receptor that mimics the function of TCR. It consists of a target antigen binding domain, a transmembrane domain, a T cell costimulatory domain, and an intracellular signal transduction domain.
  • the source of T cells in clinical practice is generally from the patients themselves, so they are all autologous CAR-T cells.
  • Some researchers have developed a universal CAR-T (WO2019/011118), which uses T cells from healthy heterologous donors to prepare a large number of CAR-T cells for different patients in advance.
  • autologous CAR-T cell therapy and universal CAR-T cell therapy have similar problems, for example, leading to the loss of normal B cells and immunoglobulins.
  • some tumor-associated antigens that are regarded as reliable targets will also be expressed at low levels in normal cells, leading to the possibility of off-target toxicity in CAR-T therapy.
  • CAR-T cells will release a large amount of cytokines while killing tumor cells quickly, making it difficult to control cell activity.
  • a large number of cytokines may cause severe cytokine release syndrome (CRS) and nervous system toxicity, leading to high fever, low pressure, shock and even death in patients, increasing clinical risks.
  • CRS severe cytokine release syndrome
  • Bispecific monoclonal antibody refers to a special antibody containing two specific antigen binding sites modified by genetic engineering technology, which can simultaneously bind to two different antigens or two different epitopes on the same antigen.
  • a relatively special bispecific antibody is also called T cell adapters (Bispecific T cell engagers, BiTEs).
  • T cell adaptor is a bispecific recombinant antibody, including two linked scFvs, one end targets CD3 on the surface of T cells, and the other end targets the target protein on the surface of tumor cells. By tightly binding T cells and target cells at the same time, BiTEs can mediate the specific killing of target cells by T cells.
  • BiTEs can build a bridge between target cells and functional molecules (cells), stimulate a directed immune response, and have broad application prospects in tumor immunotherapy.
  • double-antibody drugs use T cells in patients, and some patients' autologous T-cell function is inhibited or T-cell dysfunction, which greatly reduces the efficacy of the double-antibody.
  • current double antibodies only activate TCR signals and lack co-activation signals, resulting in weak cell expansion and cytokine secretion capabilities.
  • T cells can be divided into ⁇ T cells and ⁇ T cells. The difference between the two lies in the type of TCR expressed.
  • ⁇ T cells express ⁇ TCR.
  • ⁇ T cells There are many subtypes of ⁇ TCR, which are expressed on the surface of ⁇ T cells and are responsible for recognizing specific antigens in a major histocompatibility complex (MHC) dependent form.
  • MHC major histocompatibility complex
  • ⁇ T cell refers to the T cell composed of ⁇ chain and ⁇ chain in the TCR of T cell. Its immune function is between innate immunity and adaptive immunity. It is MHC non-restricted T cell and has certain non-specific killing of tumor cells. Function, and has a broad anti-tumor spectrum.
  • ⁇ T cells The majority of human peripheral blood is ⁇ T cells, and ⁇ T cells account for only 2% to 5% of peripheral blood. They are mainly distributed in mucosa-associated lymphoid tissues and are one of the subgroups of T cells. Although there are certain differences in structure and function, both ⁇ TCR and ⁇ TCR can form complexes with CD3 molecular complexes to function.
  • This application provides a modified immune effector cell, wherein the T cell receptor ⁇ constant region protein and the T cell receptor ⁇ constant region are compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the expression and/or activity of at least one of the proteins is down-regulated, and the expression and/or activity of the transmembrane region of the T cell receptor gamma constant region protein and the transmembrane region of the T cell receptor delta constant region protein are up-regulated.
  • the expression and/or activity of the T cell receptor gamma constant region protein is up-regulated in the immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the expression and/or activity of the T cell receptor delta constant region protein is up-regulated in the immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, the T cell receptor ⁇ constant region protein and the T cell receptor ⁇
  • the expression and/or activity of the constant region protein is down-regulated.
  • the expression and/or activity of the two proteins are down-regulated in the immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, wherein the two proteins are down-regulated.
  • This protein is composed of T cell receptor ⁇ constant region protein and T cell receptor ⁇ constant region protein.
  • the expression and/or activity of the MHC complex is down-regulated in the immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the expression and/or activity of the two proteins are up-regulated in the immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • This protein is composed of the transmembrane region of the T cell receptor gamma constant region protein and the transmembrane region of the T cell receptor delta constant region protein.
  • the expression and/or activity of the two proteins are up-regulated in the immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • This protein is composed of T cell receptor gamma constant region protein and T cell receptor delta constant region protein.
  • the expression and/or activity of at least one of the T cell receptor ⁇ constant region protein and the T cell receptor ⁇ constant region protein of the immune effector cell is down-regulated compared with the corresponding wild type.
  • the expression and/or activity of the transmembrane region of the T cell receptor gamma constant region protein and the transmembrane region of the T cell receptor delta constant region protein are up-regulated.
  • the expression and/or activity of the T cell receptor gamma constant region protein of the immune effector cell is up-regulated compared with the corresponding wild-type.
  • the expression and/or activity of the T cell receptor ⁇ constant region protein of the immune effector cell is up-regulated compared with the corresponding wild-type.
  • the expression and/or activity of the T cell receptor ⁇ constant region protein and the T cell receptor ⁇ constant region protein of the immune effector cell are down-regulated compared with the corresponding wild type .
  • the expression and/or activity of the two proteins are down-regulated in comparison with the corresponding wild-type immune effector cells, wherein the two proteins are composed of T cell receptor alpha constant region protein and T cell receptor alpha constant region protein.
  • Cell receptor ⁇ constant region protein composition is composed of T cell receptor alpha constant region protein and T cell receptor alpha constant region protein.
  • the expression and/or activity of the MHC complex of the immune effector cell is down-regulated compared with the corresponding wild-type.
  • the expression and/or activity of the two proteins are up-regulated in the immune effector cell compared with the corresponding wild-type, wherein the two proteins are spanned by the T cell receptor gamma constant region protein.
  • the membrane region and the transmembrane region of the T cell receptor delta constant region protein are spanned by the T cell receptor gamma constant region protein.
  • the expression and/or activity of the two proteins are up-regulated in the immune effector cell compared with the corresponding wild-type, wherein the two proteins are composed of T cell receptor gamma constant region protein and T cell receptor ⁇ constant region protein. Cell receptor delta constant region protein composition.
  • the transmembrane region of the T cell receptor gamma constant region protein comprises the amino acid sequence shown in any one of SEQ ID Nos. 4, 6, and 9.
  • the nucleic acid molecule encoding the transmembrane region of the T cell receptor gamma constant region protein comprises the nucleotide sequence shown in any one of SEQ ID No. 26-27.
  • the transmembrane region of the T cell receptor delta constant region protein comprises the amino acid sequence shown in SEQ ID No.2.
  • the nucleic acid molecule encoding the transmembrane region of the T cell receptor delta constant region protein comprises the nucleotide sequence shown in SEQ ID No. 25.
  • the T cell receptor gamma constant region protein comprises the amino acid sequence shown in any one of SEQ ID Nos. 3, 5, 7 and 8.
  • the nucleic acid molecule encoding the T cell receptor gamma constant region protein comprises the nucleotide sequence shown in any one of SEQ ID No. 29-30.
  • the T cell receptor delta constant region protein comprises the amino acid sequence shown in SEQ ID No. 1.
  • the nucleic acid molecule encoding the T cell receptor delta constant region protein comprises the nucleotide sequence shown in SEQ ID No. 28.
  • the immune effector cells include T cells, natural killer cells (NK cells), monocytes, macrophages, NKT cells, dendritic cells, granulocytes, B cells, lymphocytes, white blood cells And/or peripheral blood mononuclear cells.
  • the expression level and/or activity being upregulated includes the expression of the transmembrane region of the T cell receptor gamma constant region protein and the transmembrane region of the T cell receptor delta constant region protein and/ Or the activity is up-regulated; and/or the expression of the nucleic acid molecule encoding the transmembrane region of the T cell receptor gamma constant region protein and the nucleic acid molecule encoding the transmembrane region of the T cell receptor delta constant region protein and/or Activity is up-regulated.
  • the upregulation of the expression level and/or activity includes up-regulating the expression and/or activity of the T cell receptor gamma constant region protein and the T cell receptor delta constant region protein; and/or The expression and/or activity of the nucleic acid molecule encoding the T cell receptor gamma constant region protein and the nucleic acid molecule encoding the T cell receptor delta constant region protein are up-regulated.
  • the expression level and/or activity being down-regulated includes down-regulating the expression and/or activity of the T cell receptor alpha constant region protein; and/or making the T cell receptor beta constant region The expression and/or activity of the protein is down-regulated.
  • the expression level and/or activity being down-regulated includes down-regulating the expression and/or activity of the nucleic acid molecule encoding the T cell receptor alpha constant region protein; and/or down-regulating the T cell The expression and/or activity of the nucleic acid molecule of the receptor ⁇ constant region protein is down-regulated.
  • the immune effector cell does not express a TCR containing a T cell receptor alpha constant region protein, and/or the immune effector cell does not express a TCR containing a T cell receptor beta constant region protein.
  • the immune effector cell expresses a heterodimer
  • the heterodimer includes a T cell receptor ⁇ constant region protein and a T cell receptor ⁇ constant region protein.
  • the heterodimer comprises a costimulatory domain.
  • the costimulatory domain comprises a polypeptide from a protein selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D , NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS and CD83 body.
  • a protein selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D , NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1,
  • the number of copies of the costimulatory domain is 1 or 2.
  • the N-terminus of the costimulatory domain is connected to the C-terminus of the T cell receptor delta constant region protein; or, the N-terminus of the costimulatory domain is connected to the T cell receptor The C-terminus of the gamma constant region protein is connected.
  • the present application provides a method for preparing the modified immune effector cell, which includes the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the immune effector cells include T cells, natural killer cells (NK cells), monocytes, macrophages, NKT cells, dendritic cells, granulocytes, B cells, lymphocytes, white blood cells And/or peripheral blood mononuclear cells.
  • the method includes up-regulating the expression and/or activity of T cell receptor gamma constant region protein.
  • the method includes up-regulating the expression and/or activity of the T cell receptor delta constant region protein.
  • the method includes up-regulating the expression and/or activity of two proteins, wherein the two proteins are affected by the transmembrane region of the T cell receptor gamma constant region protein and the T cell.
  • the transmembrane domain of the body delta constant region protein is affected by the two proteins, wherein the two proteins are affected by the transmembrane region of the T cell receptor gamma constant region protein and the T cell.
  • the method includes up-regulating the expression and/or activity of two proteins, wherein the two proteins are composed of the T cell receptor gamma constant region protein and the T cell receptor delta constant region protein. Protein composition.
  • the up-regulation includes up-regulating the expression and/or activity of the protein; and/or, it includes up-regulating the expression and/or activity of a nucleic acid molecule encoding the protein.
  • the transmembrane region of the T cell receptor gamma constant region protein comprises the amino acid sequence shown in any one of SEQ ID Nos. 4, 6, and 9.
  • the nucleic acid molecule encoding the transmembrane region of the T cell receptor gamma constant region protein comprises the nucleotide sequence shown in any one of SEQ ID No. 26-27.
  • the transmembrane region of the T cell receptor delta constant region protein comprises the amino acid sequence shown in SEQ ID No.2.
  • the nucleic acid molecule encoding the transmembrane region of the T cell receptor delta constant region protein comprises the nucleotide sequence shown in SEQ ID No. 25.
  • the T cell receptor gamma constant region protein comprises the amino acid sequence shown in any one of SEQ ID Nos. 3, 5, 7 and 8.
  • the nucleic acid molecule encoding the T cell receptor gamma constant region protein comprises the nucleotide sequence shown in any one of SEQ ID No. 29-30.
  • the T cell receptor delta constant region protein comprises the amino acid sequence shown in SEQ ID No. 1.
  • the nucleic acid molecule encoding the T cell receptor delta constant region protein comprises the nucleotide sequence shown in SEQ ID No. 28.
  • the up-regulation comprises administering to the immune effector cells a nucleic acid molecule comprising a transmembrane region encoding the T cell receptor gamma constant region protein and a nucleic acid molecule encoding the T cell receptor delta constant region protein Nucleic acid molecules in the transmembrane region of nucleic acid molecules.
  • the up-regulation comprises administering to the immune effector cells a nucleic acid comprising a nucleic acid molecule encoding the T cell receptor gamma constant region protein and a nucleic acid molecule encoding the T cell receptor delta constant region protein molecular.
  • the nucleic acid molecule is located on a vector.
  • the nucleic acid molecule encoding the T cell receptor gamma constant region protein and the nucleic acid molecule encoding the T cell receptor delta constant region protein are located on the same vector.
  • the vector comprises a nucleic acid molecule encoding a costimulatory domain.
  • the costimulatory domain comprises a polypeptide from a protein selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D , NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS and CD83 body.
  • a protein selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D , NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1,
  • the vector comprises a nucleotide sequence as shown in any one of SEQ ID NO: 20-24.
  • the method includes down-regulating the expression and/or activity of the nucleic acid molecule encoding the T cell receptor alpha constant region protein and the nucleic acid molecule encoding the T cell receptor beta constant region protein.
  • the method includes down-regulating the expression and/or activity of a nucleic acid molecule encoding an MHC complex.
  • the method includes down-regulating the expression and/or activity of two proteins, wherein the two proteins are composed of the T cell receptor ⁇ constant region protein and the T cell receptor ⁇ constant region. Protein composition.
  • the down-regulation includes down-regulation of the expression and/or activity of the nucleic acid molecule; and/or, the down-regulation of the expression and/or activity of the protein.
  • the down-regulation includes knocking out, knocking down, mutating, and/or silencing the nucleic acid molecule.
  • the down-regulation includes administering to the immune effector cells one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, CRISPR/Cas system, RNA editing system such as ADAR, RNA Guided endonucleases, zinc finger proteases, Mega-TAL nucleases, TALENs and Meganucleases.
  • one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, CRISPR/Cas system, RNA editing system such as ADAR, RNA Guided endonucleases, zinc finger proteases, Mega-TAL nucleases, TALENs and Meganucleases.
  • the down-regulation includes administering the CRISPR/Cas system to the immune effector cells.
  • the down-regulation comprises administering the Cas9 enzyme to the immune effector cells.
  • the down-regulation includes administering to the immune effector cells an sgRNA that targets the exon portion of the nucleic acid molecule encoding the T cell receptor alpha constant region protein.
  • the present application provides a composition, which includes the immune effector cell and a pharmaceutically acceptable carrier.
  • the present application provides the application of the immune effector cells in the preparation of CAR-T or TCR-T cells, or immune effector cells expressing at least one synthetic receptor.
  • the drug is used for allogeneic therapy.
  • this application provides the application of the immune effector cells in the preparation of medicines for the treatment of tumors.
  • this application provides the application of the immune effector cell combined antibody in the preparation of drugs for the treatment of tumors.
  • the antibody includes a bispecific antibody.
  • the bispecific antibody is derived from the immune effector cell.
  • the antibody targets tumor-specific antigens and/or immune effector cell-specific antigens.
  • the antibody targets CD3.
  • the antibody targets a tumor-specific antigen selected from the group consisting of CD19, CD20, CD123, EpCAM, and BCMA.
  • the tumor includes solid tumors and non-solid tumors.
  • the tumor is selected from the group consisting of leukemia, lymphoma, and multiple myeloma.
  • FIG. 1 schematically shows a partial structure of the TCR chimeric receptor molecule described in the present application.
  • FIG. 2 schematically shows the structure of the T cells of the control group 1 and the universal TCR chimeric receptor T cells transfected with the TCR chimeric receptor of the present application.
  • Figure 3 shows the double positive expression of Flag and CD3 in different groups of T cells.
  • FIG. 4 shows the expression of TCR and CD3 in different groups of T cells.
  • Figure 5 shows the double positive expression of Flag and CD3 in T cells of different groups.
  • FIG. 6 shows the expression of TCR and CD3 in different groups of T cells.
  • Figure 7 shows how different groups of T cells combined with bispecific antibodies kill tumors.
  • Figure 8 shows how different groups of T cells combined with bispecific antibodies kill tumors.
  • the term "immune effector cell” generally refers to a cell that participates in an immune response, for example, promotes an immune effector response.
  • the immune effector cells may include T cells (for example, ⁇ / ⁇ T cells and ⁇ / ⁇ T cells), natural killer cells (NK cells), monocytes, macrophages, NKT cells , Dendritic cells, granulocytes, B cells, lymphocytes, white blood cells and/or peripheral blood mononuclear cells.
  • the term "activity" generally includes various activities.
  • the activity may be the biological activity or biological function of a certain protein, and for example, the activity may be the binding specificity of a certain protein to tumor antigens. Affinity.
  • the term "corresponding cell without the modification” generally refers to a cell that has not undergone the modification described in this application, that is, the expression and/or activity of TRAC and TRBC are not down-regulated, and the TRGC transmembrane region The expression and/or activity of the TRDC transmembrane region was not up-regulated either.
  • the corresponding cells without the modification may include T cells, natural killer cells (NK cells), monocytes, macrophages, NKT cells, dendritic cells, granulocytes, B cells, Lymphocytes, white blood cells and/or peripheral blood mononuclear cells.
  • the corresponding cells that have not been modified may include corresponding cells that have undergone other modifications in addition to the modifications described in this application, for example, cells that have undergone individual amino acid substitutions, changes, and substitutions.
  • the term "modification” generally means that the state or structure of a molecule or cell is changed.
  • Molecules can be modified in many ways, including chemical, structural, and functional modifications.
  • Cells can be modified by the introduction of nucleic acids.
  • the modification may be gene editing, the modification causes the expression and/or activity of the protein in the cell to be changed, for example, the modification causes the expression and/or activity of at least one of TRAC and TRBC to be down-regulated, And the expression/or activity of TRGC transmembrane region and TRDC transmembrane region are up-regulated.
  • wild type is usually relative to the mutant type, and usually refers to cells or individuals obtained from nature, that is, cells or individuals that are not artificially mutagenized.
  • wild-type of modified immune effector cells generally refers to natural immune effector cells without any artificial modification.
  • the corresponding wild-type usually refers to immune effector cells that have not been modified, the expression and/or activity of TRAC and TRBC are not down-regulated, and the expression of TRGC transmembrane region and TRDC transmembrane region / Or the activity is not upregulated.
  • the term “leukocytes” generally refers to colorless, spherical, nucleated blood cells that have active mobility and can migrate from inside blood vessels to outside blood vessels, or from extravascular tissues to inside blood vessels.
  • the white blood cells may include granulocytes.
  • lymphocyte generally refers to any monocytes, non-phagocytic leukocytes, such as B lymphocytes and T lymphocytes, found in blood, lymph and lymphatic tissues.
  • peripheral blood mononuclear cells generally refers to cells with mononuclear nuclei in peripheral blood.
  • the peripheral blood mononuclear cells may include lymphocytes, monocytes and dendritic cells.
  • CAR-T or “CAR-T cell” generally refers to T cells capable of expressing CAR (also known as “chimeric antigen receptor”).
  • the CAR usually refers to a fusion protein comprising an extracellular domain capable of binding antigen and at least one intracellular domain.
  • CAR is the core component of chimeric antigen receptor T cells (CAR-T), which may include targeting parts (for example, parts that bind tumor-associated antigen (TAA)), hinge region, transmembrane region, and cell Inner domain.
  • TAA tumor-associated antigen
  • MHC complex is usually also called major histocompatibility complex (MHC), which is a collective term for a group of genes encoding animal major histocompatibility antigens.
  • MHC major histocompatibility complex
  • Human MHC is also called HLA (human leukocyte antigen, HLA) complex. Due to the multi-gene characteristics of MHC, based on the structure, tissue distribution and functional differences of its coding molecules, it can be divided into MHC class I, MHC class II, and MHC class III genes, which encode MHC class I molecules, MHC class II molecules, and MHC III respectively. Class molecule.
  • T cell generally refers to thymus-derived cells that participate in various cell-mediated immune responses.
  • TCR-T or "TCR-T cell” generally refers to a T cell capable of expressing TCR (also known as “T cell surface receptor”).
  • the TCR may include an ⁇ form or a ⁇ form of TCR.
  • T cell surface receptors TCR
  • T cells can be divided into ⁇ T cells and ⁇ T cells.
  • ⁇ T cells express ⁇ TCR.
  • MHC major histocompatibility complex
  • ⁇ T cell refers to the T cell composed of ⁇ chain and ⁇ chain in the TCR of T cell. Its immune function is between innate immunity and adaptive immunity. It is MHC non-restricted T cell and has certain non-specific killing of tumor cells. Function, and has a broad anti-tumor spectrum.
  • TRDC transmembrane region and “TRDC protein transmembrane region” are used interchangeably, and usually refer to the transmembrane of the T cell receptor delta constant region (TRDC) protein Area.
  • TRDC T cell receptor delta constant region
  • amino acid sequence of the TRDC transmembrane region may be as shown in SEQ ID NO.2.
  • TRGC transmembrane region and “TRGC protein transmembrane region” are used interchangeably, and generally refer to the transmembrane of the T cell receptor gamma constant region (TRGC) protein Area.
  • TRGC protein can be of two types, namely TRGC1 and TRGC2.
  • amino acid sequence of the TRGC transmembrane region may be as shown in any one of SEQ ID NOs. 4, 6, and 9.
  • sgRNA single guide RNA
  • synthetic guide RNA synthetic guide RNA
  • CRISPR generally refers to clusters of regularly spaced short palindrome repeats.
  • the CRISPR locus usually differs from other SSRs in the structure of repetitive sequences, which are called short regularly spaced repetitive sequences (SRSR).
  • SRSR short regularly spaced repetitive sequences
  • repetitive sequences are short elements, appearing in regularly spaced clusters, with unique insertion sequences of substantially constant length.
  • the repetitive sequences between strains are highly conserved, but the number of interspersed repetitive sequences and the sequence of spacers usually vary from strain to strain.
  • Cas9 enzyme or “Cas9 protein” generally refers to the RNA-guided DNA associated with the type II CRISPR (short palindromic repeats regularly spaced) adaptive immune system found in certain bacteria Endonucleases, such as bacteria such as Streptococcus pyogenes.
  • the Cas9 protein may not only include wild-type Cas9 found in Streptococcus pyogenes, but also various variants thereof, such as those described in WO2013/176772A1.
  • the Cas9 protein may include those derived from Streptococcus pyogenes, Neisseria meningitidis, Streptococcus thermophilus and The Cas9 sequence of dendritic nematode.
  • CRISPR/Cas system can also be referred to as “Cas-gRNA system”, and generally refers to tools for site-specific genome targeting in organisms. It is a prokaryotic adaptive immune response system that uses non-coding RNA to guide Cas nuclease to induce site-specific DNA cleavage. Through the cellular DNA repair mechanism, this DNA damage can be repaired through the non-homologous end joining DNA repair pathway (NHEJ) or the homology directed repair (HDR) pathway.
  • NHEJ non-homologous end joining DNA repair pathway
  • HDR homology directed repair
  • the CRISPR/Cas system can be used to create a simple RNA programmable method to mediate genome editing in mammalian cells, and can be used to generate gene knockouts (via insertion/deletion) or knock-ins (via HDR).
  • knock-in generally refers to a genetic engineering process involving the one-to-one replacement of DNA sequence information in a gene sequence or the insertion of sequence information not found in an endogenous locus. Knock-in may involve insertion of a gene at a specific locus, and therefore may be a "targeted” insertion.
  • synthetic receptor generally refers to an engineered cell surface protein or protein complex, which contains (1) a target binding domain that can specifically bind to a target molecule, and (2) a signal transduction pathway can be activated Functional domain.
  • the target binding domain includes the extracellular domain
  • the functional domain includes the intracellular domain.
  • Synthetic receptors also include transmembrane sequences.
  • Synthetic receptors can be protein complexes that include proteins expressed from exogenous nucleic acids.
  • Synthetic receptors can also be protein complexes that include at least one exogenously expressed protein and at least one endogenously expressed protein.
  • the engineered cells may be immune cells, such as T cells, natural killer (NK) cells, B cells, macrophages, etc., and the functional domain may directly or indirectly activate immune cells.
  • the synthetic receptor may be selected from: chimeric antigen receptor ("CAR"), T cell receptor ("TCR”), TCR receptor fusion construct ("TRuC"), T cell antigen Coupling agent (“TAC”), antibody TCR receptor ("AbTCR”) and chimeric CD3 epsilon receptor.
  • the synthetic receptor may be a CAR.
  • the synthetic receptor may be a TCR.
  • the synthetic receptor may be TRuC.
  • the synthetic receptor may be TAC.
  • the synthetic receptor may be AbTCR.
  • the synthetic receptor may be a chimeric CD3 epsilon receptor.
  • the present application provides a modified immune effector cell, wherein the expression and/or activity of at least one of TRAC and TRBC is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification. Down-regulation, and the expression and/or activity of TRGC transmembrane region and TRDC transmembrane region are up-regulated.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, the expression and/or activity of TRAC is down-regulated, and TRGC is transmembrane The expression and/or activity of the TRDC region and the TRDC transmembrane region are up-regulated.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, the expression and/or activity of TRBC is down-regulated, and TRGC is transmembrane The expression and/or activity of the TRDC region and the TRDC transmembrane region are up-regulated.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, the expression and/or activity of TRAC and TRBC are down-regulated, and TRGC The expression and/or activity of the transmembrane region and TRDC transmembrane region are up-regulated.
  • TRAC i.e. T cell receptor alpha constant
  • TRBC i.e. T cell receptor beta constant
  • TRGC i.e. T cell receptor gamma constant
  • TRDC ie T cell receptor delta constant
  • TRGC transmembrane region can be used interchangeably with “TRGC protein transmembrane region”
  • TRDC transmembrane region can be used interchangeably with “TRDC protein transmembrane region”.
  • the TRAC gene represents a nucleic acid molecule encoding the TRAC protein
  • the TRBC gene represents a nucleic acid molecule encoding the TRBC protein
  • the TRGC gene represents a nucleic acid molecule encoding the TRGC protein
  • the TRDC gene represents a nucleic acid molecule encoding the TRGC protein.
  • the nucleic acid molecule of the TRDC protein is the nucleic acid molecule of the TRDC protein.
  • the expression and/or activity of TRGC can be up-regulated in the modified immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the expression and/or activity of TRDC can be up-regulated in comparison with the expression and/or activity of the corresponding protein in the modified immune effector cell in the corresponding cell without the modification.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, the expression and/or activity of TRAC is down-regulated, and TRGC and TRDC The expression and/or activity of the transmembrane region is up-regulated.
  • the expression and/or activity of TRBC is down-regulated in the modified immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, and TRGC and TRDC
  • the expression and/or activity of the transmembrane region is up-regulated.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, the expression and/or activity of TRAC and TRBC are down-regulated, and TRGC The expression and/or activity of TRDC and TRDC transmembrane region are up-regulated.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, the expression and/or activity of TRAC is down-regulated, and TRGC spans The expression and/or activity of the membrane region and TRDC were up-regulated.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, the expression and/or activity of TRBC is down-regulated, and TRGC spans The expression and/or activity of the membrane region and TRDC were up-regulated.
  • the expression and/or activity of TRAC and TRBC are down-regulated in the modified immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, and The expression and/or activity of TRGC transmembrane region and TRDC are up-regulated.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, the expression and/or activity of TRAC is down-regulated, and TRGC and The expression and/or activity of TRDC is up-regulated.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, the expression and/or activity of TRBC is down-regulated, and TRGC and The expression and/or activity of TRDC is up-regulated.
  • the expression and/or activity of TRAC and TRBC are down-regulated in the modified immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, and the expression and/or activity of TRGC and TRDC are up-regulated.
  • the expression and/or activity of the MHC complex can be down-regulated in the modified immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • it may be that the expression and/or activity of ⁇ -2 microglobulin (ie, B2M) in the MHC complex is down-regulated.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, and the expression and/or activity of the two proteins can be down-regulated, wherein the The two proteins are composed of TRAC and TRBC.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, and the expression and/or activity of the two proteins can be up-regulated, wherein the The two proteins are composed of TRGC transmembrane protein and TRDC transmembrane protein.
  • the modified immune effector cell is compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification, and the expression and/or activity of the two proteins can be up-regulated, wherein the The two proteins are composed of TRGC and TRDC.
  • the expression and/or activity of the two proteins can be down-regulated in the modified immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the two proteins are composed of TRAC and TRBC, and the expression and/or activity of the two proteins can be up-regulated. These two proteins are composed of the TRGC transmembrane region protein and the TRDC transmembrane region protein.
  • the expression and/or activity of the two proteins can be down-regulated in the modified immune effector cell compared with the expression and/or activity of the corresponding protein in the corresponding cell without the modification,
  • These two proteins are composed of TRAC and TRBC, and the expression and/or activity of the two proteins can be up-regulated.
  • These two proteins are composed of TRGC and TRDC.
  • the “corresponding cell without the modification” generally refers to a cell that has not undergone the modification described in this application, that is, the expression and/or activity of TRAC and TRBC are not down-regulated, and TRGC The expression and/or activity of the transmembrane region and TRDC transmembrane region were also not up-regulated.
  • the corresponding cells that have not been modified may include corresponding cells that have undergone other modifications in addition to the modifications described in this application, for example, cells that have undergone individual amino acid substitutions, changes, and substitutions.
  • the "modification” generally means that the state or structure of a molecule or cell is changed. Molecules can be modified in many ways, including chemical, structural, and functional modifications.
  • Cells can be modified by the introduction of nucleic acids.
  • the modification may be gene editing, and the modification may cause the expression and/or activity of the protein in the cell to be changed, for example, the modification may cause the expression and/or activity of at least one of TRAC and TRBC to be down-regulated. , And make the expression/or activity of TRGC transmembrane region and TRDC transmembrane region up-regulated.
  • the “comparison” can be performed with respect to the "corresponding cell without the modification”, but also with respect to the "wild type”.
  • wild type wild type
  • the "wild type” is usually relative to the mutant type, and usually refers to cells or individuals obtained from nature, that is, cells or individuals that are not artificially mutagenized.
  • the wild-type of modified immune effector cells generally refers to natural immune effector cells without any artificial modification.
  • the expression and/or activity of at least one of TRAC and TRBC is down-regulated in the modified immune effector cell, and the expression and/or the expression of the TRGC transmembrane region and the TRDC transmembrane region are Or the activity is up-regulated.
  • the modified immune effector cell is compared with the corresponding wild-type, the expression and/or activity of TRAC is down-regulated, and the expression and/or activity of the TRGC transmembrane region and TRDC transmembrane region are reduced. Up.
  • the modified immune effector cell is compared with the corresponding wild-type, the expression and/or activity of TRBC is down-regulated, and the expression and/or activity of the TRGC transmembrane region and TRDC transmembrane region are reduced Up.
  • the expression and/or activity of TRAC and TRBC of the modified immune effector cell are down-regulated, and the expression and/or the expression of the TRGC transmembrane region and the TRDC transmembrane region are down-regulated.
  • Activity is up-regulated.
  • the expression and/or activity of TRGC can be up-regulated in the modified immune effector cell compared with the corresponding wild-type.
  • the expression and/or activity of TRDC can be up-regulated in the modified immune effector cell compared with the corresponding wild-type.
  • the expression and/or activity of TRAC of the modified immune effector cell is down-regulated, and the expression and/or activity of the TRGC and TRDC transmembrane regions are up-regulated.
  • the expression and/or activity of TRBC of the modified immune effector cells are down-regulated, and the expression and/or activity of the TRGC and TRDC transmembrane regions are up-regulated.
  • the expression and/or activity of TRAC and TRBC of the modified immune effector cell are down-regulated, and the expression and/or activity of the transmembrane region of TRGC and TRDC is up-regulated. .
  • the expression and/or activity of TRAC of the modified immune effector cell is down-regulated, and the expression and/or activity of the TRGC transmembrane region and TRDC are up-regulated.
  • the expression and/or activity of TRBC of the modified immune effector cell is down-regulated, and the expression and/or activity of the TRGC transmembrane region and TRDC are up-regulated.
  • the modified immune effector cell is compared with the corresponding wild-type, the expression and/or activity of TRAC and TRBC are down-regulated, and the expression and/or activity of the TRGC transmembrane region and TRDC are reduced Up.
  • the expression and/or activity of TRAC of the modified immune effector cell is down-regulated, and the expression and/or activity of TRGC and TRDC are up-regulated.
  • the expression and/or activity of TRBC is down-regulated, and the expression and/or activity of TRGC and TRDC are up-regulated in the modified immune effector cell compared with the corresponding wild-type.
  • the expression and/or activity of TRAC and TRBC are down-regulated, and the expression and/or activity of TRGC and TRDC are up-regulated in the modified immune effector cells.
  • the expression and/or activity of the MHC complex of the modified immune effector cell can be down-regulated compared with the corresponding wild-type. In some embodiments, it may be that the expression and/or activity of ⁇ -2 microglobulin (ie, B2M) in the MHC complex is down-regulated.
  • ⁇ -2 microglobulin ie, B2M
  • the expression and/or activity of the two proteins can be down-regulated in the modified immune effector cell compared with the corresponding wild-type, wherein the two proteins are composed of TRAC and TRBC.
  • the expression and/or activity of the two proteins can be up-regulated in the modified immune effector cell compared with the corresponding wild-type, wherein the two proteins are spanned by the TRGC transmembrane region protein and TRDC.
  • the protein composition of the membrane region is a region of the membrane region.
  • the expression and/or activity of two proteins can be up-regulated in the modified immune effector cell compared with the corresponding wild-type, wherein the two proteins are composed of TRGC and TRDC.
  • the modified immune effector cells can be down-regulated in expression and/or activity of two proteins compared with the corresponding wild-type.
  • These two proteins are composed of TRAC and TRBC, and the two proteins The expression and/or activity can be up-regulated.
  • These two proteins are composed of TRGC transmembrane proteins and TRDC transmembrane proteins.
  • the modified immune effector cells can be down-regulated in expression and/or activity of two proteins compared with the corresponding wild-type. These two proteins are composed of TRAC and TRBC, and the two proteins are composed of TRAC and TRBC. The expression and/or activity of proteins can be up-regulated, and these two proteins are composed of TRGC and TRDC.
  • the TRGC can be TRGC1 or TRGC2.
  • the nucleic acid molecule encoding the TRGC1 transmembrane region may include the nucleotide sequence shown in SEQ ID No. 27.
  • the nucleic acid molecule encoding the TRGC2 transmembrane region may include the nucleotide sequence shown in SEQ ID No. 26.
  • the nucleic acid molecule encoding the TRDC transmembrane region may include the nucleotide sequence shown in SEQ ID No. 25.
  • the nucleic acid molecule encoding the TRGC1 may include the nucleotide sequence shown in SEQ ID No. 30.
  • the nucleic acid molecule encoding the TRGC2 may include the nucleotide sequence shown in SEQ ID No. 29.
  • the nucleic acid molecule encoding the TRDC may include the nucleotide sequence shown in SEQ ID No. 28.
  • the modified immune effector cells may include T cells, natural killer cells (NK cells), monocytes, macrophages, NKT cells, dendritic cells, granulocytes, B cells, lymphocytes , White blood cells and/or peripheral blood mononuclear cells.
  • the white blood cells may include granulocytes.
  • the up-regulation of the expression level and/or activity may include up-regulating the expression and/or activity of the TRGC transmembrane region and the TRDC transmembrane region; and/or the nucleic acid encoding the TRGC transmembrane region
  • the expression and/or activity of the molecule and the nucleic acid molecule encoding the TRDC transmembrane region are up-regulated.
  • the expression level and/or activity being up-regulated may include up-regulating the expression and/or activity of the TRGC and TRDC; and/or the nucleic acid molecule encoding the TRGC and the nucleic acid molecule encoding the TRDC The expression and/or activity of is up-regulated.
  • the expression level and/or activity being down-regulated may include down-regulating the expression and/or activity of the TRAC; and/or down-regulating the expression and/or activity of the TRBC.
  • the expression level and/or activity being down-regulated may include down-regulating the expression and/or activity of the nucleic acid molecule encoding the TRAC; and/or reducing the expression and/or activity of the nucleic acid molecule encoding the TRBC Was downgraded.
  • the modified immune effector cell may not express TCR containing TRAC, and/or, the immune effector cell may not express TCR containing TRBC.
  • the modified immune effector cells may express heterodimers, and the heterodimers include TRDC and TRGC.
  • the modified immune effector cells may not express TCR containing TRAC, and may express heterodimers including TRDC and TRGC.
  • the modified immune effector cells may not express TCR containing TRBC, and may express heterodimers including TRDC and TRGC.
  • the modified immune effector cells may not express TCRs including TRAC and TRBC, and may express heterodimers including TRDC and TRGC.
  • the heterodimer may also include a costimulatory domain.
  • the costimulatory domain may comprise a polypeptide from a protein selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3 , 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS and CD83 ligands.
  • the number of copies of the costimulatory domain may be one or two.
  • the N-terminus of the costimulatory domain may be connected to the C-terminus of the TRDC; or, the N-terminus of the costimulatory domain may be connected to the C-terminus of the TRGC.
  • the number of copies of the costimulatory domain may be one, and its N-terminus is connected to the C-terminus of the TRDC (as shown by "2" in Figure 1).
  • the copy number of the costimulatory domain may be one, and its N-terminus is connected to the C-terminus of the TRGC (as shown by "3" in Figure 1).
  • the number of copies of the costimulatory domain may be two, the N terminal of one is connected to the C terminal of the TRDC, and the N terminal of the other is connected to the C terminal of the TRGC (as shown in Fig. "4" in 1).
  • the modified immune effector cells may express TCRs containing CG-pWW-001 protein, and the CG-pWW-001 protein may sequentially include CD8 ⁇ signal peptide, TRDC, and T2A from the N-terminus to the C-terminus. And TRGC1.
  • the amino acid sequence of the CG-pWW-001 protein may be as shown in SEQ ID NO.15.
  • the modified immune effector cells may express TCRs containing CG-pWW-002 protein, and the CG-pWW-002 protein may sequentially include CD8 ⁇ signal peptide, TRDC, and T2A from N-terminus to C-terminus. And TRGC2.
  • the amino acid sequence of the CG-pWW-002 protein may be as shown in SEQ ID NO.16.
  • the modified immune effector cells may express TCRs containing CG-pWW-003 protein, and the CG-pWW-003 protein may sequentially include CD8 ⁇ signal peptide, TRDC, CD28 from N-terminus to C-terminus. , T2A and TRGC2.
  • the amino acid sequence of the CG-pWW-003 protein may be as shown in SEQ ID NO.17.
  • the modified immune effector cell can express a TCR containing CG-pWW-004 protein
  • the CG-pWW-004 protein can include CD8 ⁇ signal peptide, TRDC, and linker in sequence from N-terminus to C-terminus.
  • the amino acid sequence of the CG-pWW-004 protein may be as shown in SEQ ID NO.18.
  • the modified immune effector cell can express a TCR containing the CG-pWW-005 protein, and the CG-pWW-005 protein can sequentially include CD8 ⁇ signal peptide, TRDC, and linker from N-terminus to C-terminus.
  • the amino acid sequence of the CG-pWW-005 protein may be as shown in SEQ ID NO.19.
  • the CD8 ⁇ signal peptide may include the amino acid sequence shown in SEQ ID NO.11.
  • the T2A may include the amino acid sequence shown in SEQ ID NO.12.
  • the CD28 may include the amino acid sequence shown in SEQ ID NO.10.
  • the connecting peptide 1 may include the amino acid sequence shown in SEQ ID NO.13.
  • the connecting peptide 2 may include the amino acid sequence shown in SEQ ID NO.14.
  • the TRDC transmembrane region may include the amino acid sequence shown in SEQ ID NO.2.
  • the TRDC may include the amino acid sequence shown in SEQ ID NO.1.
  • the TRGC1 transmembrane region may include the amino acid sequence shown in SEQ ID NO.4.
  • the TRGC1 may include an amino acid sequence as shown in any one of SEQ ID NOs. 3 and 7.
  • the TRGC2 transmembrane region may include an amino acid sequence as shown in any one of SEQ ID NOs. 6 and 9.
  • the TRGC2 may include the amino acid sequence shown in any one of SEQ ID NOs. 5 and 8.
  • the immune effector cell comprises an amino acid sequence as shown in any one of SEQ ID NOs: 15-19. In some embodiments, the immune effector cell comprises a nucleotide sequence as shown in any one of SEQ ID NO: 20-24.
  • the present application provides a method for preparing the modified immune effector cell, which comprises the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification The expression and/or activity of at least one of TRAC and TRBC in the immune effector cell; and the expression and/or activity of the TRGC transmembrane region and the TRDC transmembrane region are up-regulated.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the expression and/or activity of TRAC in the immune effector cells; and the expression and/or activity of TRGC transmembrane region and TRDC transmembrane region are up-regulated.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the expression and/or activity of TRBC in the immune effector cell may include the following steps: down-regulating the expression and/or activity of the TRGC transmembrane region and the TRDC transmembrane region.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the expression and/or activity of TRAC and TRBC in the immune effector cell; and the expression and/or activity of the TRGC transmembrane region and the TRDC transmembrane region are up-regulated.
  • the method includes up-regulating the expression and/or activity of TRGC.
  • the method includes up-regulating the expression and/or activity of TRDC.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the expression and/or activity of TRAC in the immune effector cells; and the expression and/or activity of TRGC and TRDC transmembrane regions are up-regulated.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the expression and/or activity of TRAC in the immune effector cell; and the expression and/or activity of TRGC transmembrane region and TRDC are up-regulated.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification.
  • the expression and/or activity of TRAC in the immune effector cell; and the expression and/or activity of TRGC and TRDC are up-regulated.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification The expression and/or activity of TRBC in the immune effector cell; and the expression and/or activity of TRGC and the transmembrane region of TRDC are up-regulated.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification The expression and/or activity of TRBC in the immune effector cell; and the expression and/or activity of TRGC transmembrane region and TRDC are up-regulated.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification The expression and/or activity of TRBC in the immune effector cell; and the expression and/or activity of TRGC and TRDC are up-regulated.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification The expression and/or activity of TRAC and TRBC in the immune effector cell; and the expression and/or activity of the transmembrane region of TRGC and TRDC are up-regulated.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification The expression and/or activity of TRAC and TRBC in the immune effector cell; and the expression and/or activity of TRGC transmembrane region and TRDC are up-regulated.
  • the method for preparing the modified immune effector cell may include the following steps: down-regulating the expression and/or activity of the corresponding protein in the corresponding cell without the modification The expression and/or activity of TRAC and TRBC in the immune effector cell; and the expression and/or activity of TRGC and TRDC are up-regulated.
  • the method may include up-regulating the expression and/or activity of two proteins, wherein the two proteins are composed of the TRGC transmembrane region and the TRDC transmembrane region.
  • the method may include up-regulating the expression and/or activity of two proteins, wherein the two proteins are composed of the TRGC and the TRDC.
  • the up-regulation may include up-regulating the expression and/or activity of the protein; and/or, including up-regulating the expression and/or activity of the nucleic acid molecule encoding the protein.
  • the up-regulation may include administering to the modified immune effector cells a nucleic acid molecule comprising a nucleic acid molecule encoding the TRGC transmembrane region and a nucleic acid molecule encoding the TRDC transmembrane region, so as to up-regulate the TRGC transmembrane region.
  • the expression and/or activity of nucleic acid molecules in the membrane region and the TRDC transmembrane region thereby up-regulating the expression and/or activity of the TRGC transmembrane region and the TRDC transmembrane region.
  • the up-regulation may include administering a nucleic acid molecule comprising a nucleic acid molecule encoding the TRGC and a nucleic acid molecule encoding the TRDC to the modified immune effector cell, so as to up-regulate the nucleic acid molecule encoding the TRGC and the TRDC The expression and/or activity of, thereby up-regulating the expression and/or activity of the TRGC and the TRDC.
  • the up-regulation can include knock-in.
  • knock-in generally refers to a genetic engineering process involving one-to-one replacement of DNA sequence information in a gene sequence or insertion of sequence information not found in an endogenous locus. Knock-in may involve insertion of a gene at a specific locus, and therefore may be a "targeted” insertion.
  • the method may include down-regulating the expression and/or activity of the nucleic acid molecule encoding the TRAC and the nucleic acid molecule encoding the TRBC.
  • the method may also include down-regulating the expression and/or activity of the nucleic acid molecule encoding the MHC complex.
  • it may be that the expression and/or activity of the nucleic acid molecule encoding ⁇ -2 microglobulin (ie, B2M) in the MHC complex is down-regulated.
  • the method may include down-regulating the expression and/or activity of two proteins, wherein the two proteins are composed of the TRAC and the TRBC.
  • the down-regulation may include down-regulating the expression and/or activity of the nucleic acid molecule; and/or, including down-regulating the expression and/or activity of the protein.
  • the method may include down-regulating the expression and/or activity of the nucleic acid molecule encoding the TRAC; and/or, including down-regulating the expression and/or activity of the TRAC.
  • the method may include down-regulating the expression and/or activity of the nucleic acid molecule encoding the TRBC; and/or, including down-regulating the expression and/or activity of the TRBC.
  • the method may include down-regulating the expression and/or activity of the nucleic acid molecule encoding the TRAC and the nucleic acid molecule encoding the TRBC; and/or, including down-regulating the expression of the TRAC and the TRBC And/or activity.
  • the down-regulation may include knocking out, knocking down, mutating, and/or silencing the nucleic acid molecule.
  • the down-regulation may include administering one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, CRISPR/Cas system, RNA editing system such as ADAR to the modified immune effector cell , RNA-guided endonuclease, zinc finger protease, Mega-TAL nuclease, TALENs and Meganucleases.
  • the down-regulation may include administering the CRISPR/Cas system to the modified immune effector cells.
  • the “CRISPR/Cas system” can also be referred to as the “Cas-gRNA system”, and generally refers to tools for site-specific genome targeting in organisms. It is a prokaryotic adaptive immune response system that uses non-coding RNA to guide Cas nuclease to induce site-specific DNA cleavage. Through the cellular DNA repair mechanism, this DNA damage can be repaired through the non-homologous end joining DNA repair pathway (NHEJ) or the homology directed repair (HDR) pathway.
  • NHEJ non-homologous end joining DNA repair pathway
  • HDR homology directed repair
  • the CRISPR/Cas system can be used to create a simple RNA programmable method to mediate genome editing in mammalian cells, and can be used to generate gene knockouts (via insertion/deletion) or knock-ins (via HDR).
  • the down-regulation may include administering the Cas9 enzyme to the modified immune effector cells.
  • the "Cas9 enzyme” or “Cas9 protein” generally refers to the RNA-guided DNA endonuclease associated with the type II CRISPR (short palindromic repeats regularly spaced) adaptive immune system found in certain bacteria , For example, bacteria such as Streptococcus pyogenes.
  • the Cas9 protein may not only include wild-type Cas9 found in Streptococcus pyogenes, but also various variants thereof, such as those described in WO2013/176772A1.
  • the Cas9 protein may include those derived from Streptococcus pyogenes, Neisseria meningitidis, Streptococcus thermophilus and The Cas9 sequence of dendritic nematode.
  • the down-regulation may include administering sgRNA targeting the exon part of the nucleic acid molecule encoding TRAC to the modified immune effector cell.
  • the "sgRNA”, “guide RNA”, “single guide RNA” and “synthetic guide RNA” are interchangeable and generally refer to a polynucleotide sequence containing a guide sequence.
  • the guide sequence is about 20 bp and is in the guide RNA at the designated target site.
  • the down-regulation method and the sgRNA please refer to the experimental procedures of the examples in the patent document WO2019/011118.
  • the TRGC can be TRGC1 or TRGC2.
  • the nucleic acid molecule encoding the TRGC1 transmembrane region may include the nucleotide sequence shown in SEQ ID No. 27.
  • the nucleic acid molecule encoding the TRGC2 transmembrane region may include the nucleotide sequence shown in SEQ ID No. 26.
  • the nucleic acid molecule encoding the TRDC transmembrane region may include the nucleotide sequence shown in SEQ ID No. 25.
  • the nucleic acid molecule encoding the TRGC1 may include the nucleotide sequence shown in SEQ ID No. 30.
  • the nucleic acid molecule encoding the TRGC2 may include the nucleotide sequence shown in SEQ ID No. 29.
  • the nucleic acid molecule encoding the TRDC may include the nucleotide sequence shown in SEQ ID No. 28.
  • the nucleic acid molecule may be located on a carrier.
  • nucleic acid molecule encoding TRGC and the nucleic acid molecule encoding TRDC may be located on the same vector.
  • the vector may include a nucleic acid molecule encoding a costimulatory domain.
  • the costimulatory domain may comprise a polypeptide selected from the following proteins: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, Ligands for NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS and CD83 .
  • proteins CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, Ligands for NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1
  • the vector comprises a nucleotide sequence as shown in any one of SEQ ID NO: 20-24.
  • the modified immune effector cells may include T cells, natural killer cells (NK cells), monocytes, macrophages, NKT cells, dendritic cells, granulocytes, B cells, lymphocytes , White blood cells and/or peripheral blood mononuclear cells.
  • the white blood cells may include granulocytes.
  • the present application provides a composition comprising the modified immune effector cell and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with drug administration, and are generally safe and non-toxic. , And neither biologically nor otherwise undesirable.
  • the composition may also include one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or Suitable formulations of preservatives.
  • the acceptable ingredients of the composition may be 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.
  • 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 composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the composition can 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.
  • the present application provides the application of the modified immune effector cells in the preparation of CAR-T or TCR-T cells, or immune effector cells expressing at least one synthetic receptor.
  • the "synthetic receptor” generally refers to an engineered cell surface protein or protein complex, which contains (1) a target binding domain that can specifically bind to a target molecule, and (2) can activate signal transduction The functional domain of the pathway.
  • the target binding domain includes the extracellular domain
  • the functional domain includes the intracellular domain.
  • Synthetic receptors also include transmembrane sequences.
  • Synthetic receptors can be protein complexes that include proteins expressed from exogenous nucleic acids.
  • Synthetic receptors can also be protein complexes that include at least one exogenously expressed protein and at least one endogenously expressed protein.
  • the engineered cells may be immune cells, such as T cells, national killer (NK) cells, B cells, macrophages, etc., and the functional domain may directly or indirectly activate immune cells.
  • the synthetic receptor may be selected from: chimeric antigen receptor ("CAR"), T cell receptor ("TCR”), TCR receptor fusion construct ("TRuC"), T cell antigen Coupling agent ("TAC", antibody TCR receptor (“AbTCR”)) and chimeric CD3 epsilon receptor.
  • the synthetic receptor may be a CAR.
  • the synthetic receptor may be a TCR.
  • the synthetic receptor may be TRuC.
  • the synthetic receptor may be TAC.
  • the synthetic receptor may be AbTCR.
  • the synthetic receptor may be Chimeric CD3 ⁇ receptor.
  • the present application provides the application of the modified immune effector cells in the preparation of medicines, which are used for allogeneic therapy.
  • the allograft treatment may include allograft.
  • this application provides the application of the modified immune effector cells in the preparation of medicines for the treatment of tumors.
  • the modified immune effector cells can be used for allogeneic therapy or for the treatment of tumors.
  • the present application provides a method for treating tumors, the method comprising administering the modified immune effector cells described in the present application to a subject in need.
  • this application provides the application of the modified immune effector cell combined with antibodies in the preparation of drugs for treating tumors.
  • the antibody can target tumor-specific antigens and/or immune effector cell-specific antigens.
  • the antibody can target CD3.
  • the antibody can target a tumor-specific antigen, and the tumor-specific antigen can be selected from the group consisting of CD19, CD20, CD123, EpCAM, and BCMA.
  • the antibody may include a bispecific antibody.
  • the bispecific antibody may be derived from the modified immune effector cells described in this application.
  • the tumor may include solid tumors and non-solid tumors.
  • the tumor may be selected from the group consisting of leukemia, lymphoma, and multiple myeloma.
  • Example 1 Construction of a lentiviral vector expressing the TCR chimeric receptor molecule of the present application
  • TCR chimeric receptor molecules search for genes required for designing TCR chimeric receptor molecules from the NCBI website database (https://www.ncbi.nlm.nih.gov/).
  • the gene sequence of the TCR chimeric receptor molecule includes two parts: the constant region of the TCR ⁇ chain and the constant region of the TCR ⁇ chain are connected in series, and the two genes are connected by the connecting molecule T2A gene. Because there are two constant regions of the TCR ⁇ chain, they are TRGC1 and TRGC2. Therefore, two molecules were designed, namely TRDC-T2A-TRGC1 and TRDC-T2A-TRGC2, named CG-pWW-001 and CG-pWW-002, respectively. At the same time, three other TCR chimeric receptor molecules were designed.
  • TRDC-CD28-T2A-TRGC2 TRDC-connecting peptide 1-CD28-T2A-TRGC2 and TRDC-connecting peptide 2-CD28-T2A-TRGC2, respectively named: CG-pWW-003, CG-pWW-004 and CG -pWW-005, wherein the amino acid sequences of connecting peptide 1 and connecting peptide 2 are shown in SEQ ID No. 13 and SEQ ID No. 14, respectively.
  • TCR chimeric receptor molecules The amino acid sequences of these five TCR chimeric receptor molecules are shown in Table 1, the DNA sequences are shown in Table 2, and the partial structures are shown in “1" and "2" in Figure 1.
  • the gene sequences of 5 TCR chimeric receptor molecules were synthesized by Nanjing GenScript and cloned into the pUC57 vector (Nanjing GenScript). When synthesizing genes, add specific restriction endonuclease sites: BamHI and SalI at both ends of the gene. Restriction enzymes BamHI (NEB; R3136S) and Sal I (NEB; R3138S) were used to digest the recombinant plasmid, the gene fragments were separated by DNA agarose gel electrophoresis, and the gel recovery kit (QIAGEN; 28706) was used for gel recovery. purification. And use Nanodrop (Thermo Fisher Scientific) to measure the concentration of recovered gene fragments.
  • T4 DNA ligase (NEB; M0202S) to ligate the purified gene sequence and the lentiviral vector (Addgene; article number: 12252) with T4 DNA ligase (NEB; M0202S) to obtain the lentiviral vector recombinant plasmid.
  • the sequencing verification of the recombinant plasmid of the lentiviral vector, the sequencing primers of the recombinant plasmid are: Lenti-For (as shown in SEQ ID No. 31) and Lenti-Rev (as shown in SEQ ID No. 32).
  • the constructed lentiviral vector recombinant plasmids are called: pLenti-CG-pWW-001, pLenti-CG-pWW-002, pLenti-CG-pWW-003, pLenti-CG-pWW-004 and pLenti-CG-pWW- 005.
  • lentiviral vector recombinant plasmids were transformed into Escherichia coli stbl3 (Beijing Crespo). Pick a single clone from the transformed plate into a shake tube.
  • the shake tube contains 3ml of liquid LB medium containing ampicillin, shake culture overnight at 220 rpm, 37°C on a shaker; press 1:500 in the activated bacterial solution
  • the amount of inoculation was inoculated into 250ml of liquid LB medium containing ampicillin, shaking culture at 220 rpm and 37°C for 12-16h.
  • the extracted plasmid was detected by DNA agarose gel electrophoresis for morphology and supercoiled plasmid content, and the plasmid concentration and purity were determined by Nanodrop (Thermo Fisher Scientific).
  • Resuscitation of 293T cells Take out the frozen 293T cells (ATCC) from liquid nitrogen, wipe the opening of the cryopreservation tube with sterile alcohol, and place it in a 37°C water bath with gentle shaking to thaw. Add 10ml of pre-warmed DMEM complete medium to a 15ml centrifuge tube, and blow gently; centrifuge at 1000 rpm at room temperature for 3 minutes, discard the supernatant; add 1ml DMEM complete medium to resuspend the cells, gently blow evenly and then inoculate to the existing 9ml DMEM complete medium in a 10cm culture dish, shake well and place it in a 37°C, 5% CO2 cell incubator;
  • the cultured 293T cells were digested with trypsin and mixed thoroughly, diluted and counted with a cell counter (NC200, chemometec), and then inoculated according to about (15 ⁇ 18) ⁇ 10 6 cells/T175 bottle (35-40ml culture medium) 293T cells, shake well and place them in a 37°C, 5% CO2 cell incubator overnight.
  • a cell counter NC200, chemometec
  • the medium before transfection needs to be changed to 30ml of 10% FBS medium without double antibody.
  • Prepare plasmid complex prepare 15ml centrifuge tube and add 1.5ml Opti-MEM (Thermo Fisher Scientific; 31985-070), and then add in sequence, virus vector plasmid: 18 ⁇ g, psPAX2 plasmid (Addgene; catalog number: 12260): 9 ⁇ g, pMD2 .G Plasmid (Addgene; Product Number: 12259): 18 ⁇ g, mix well and let stand for 5 min.
  • transfection reagent complex prepares the transfection reagent complex: add 67.5 ⁇ l (2mg/ml) PEI (polyscience:24765) to 1.5ml Opti-MEM, mix well after adding, and let stand at room temperature for 5 minutes; then add the transfection reagent complex to In the plasmid complex, mix the two and let it stand for 25 minutes; finally add the transfection complex to the cell culture medium and shake it gently.
  • PEI polyscience:24765
  • lentiviruses containing CG-pWW-001, CG-pWW-002, CG-pWW-003, CG-pWW-004 and CG-pWW-005 were prepared respectively.
  • PBMCs from healthy donor peripheral blood using a single harvester (purchased from Miaotong Biotechnology Co., Ltd.). Take out a tube of frozen PBMCs from liquid nitrogen, resuscitate them and dilute them to 2 ⁇ 10 6 /ml. According to the ratio of cells to magnetic beads 1:3, magnetic beads (Thermo Fisher Scientific) coupled with CD3/CD28 antibody were added to activate T cells, and 300 IU of IL-2 (PeproTech; 200-02) was added at the same time. On the second day after activation, the above-mentioned concentrated lentivirus was added to the T cell culture flask to transfect T cells.
  • a single harvester purchased from Miaotong Biotechnology Co., Ltd.
  • T cells prepared in Example 3 of the present application were collected separately, and the expression of the TCR chimeric receptor molecule in the transfected and untransfected T cells was detected by fluorescent antibody staining and flow cytometry. Adjust the concentration of T cells to 1 ⁇ 10 6 /ml, take 100 ⁇ l of cells, add fluorescent antibody to 4°C and incubate for 30min in the dark, centrifuge after incubation, wash with PBS and resuspend after centrifugation, and finally use flow cytometer (purchased from Thermo Fisher Scientific, model: Attune NxT) detects the positive rate of cell staining.
  • TCR chimeric receptor molecules in T cells ie, control group 2.
  • T cells in the control group 1 and the control group 2 have not been transfected with the TCR chimeric receptor molecule constructed in Example 1 of the application, and the T cells in the control group 1 are conventional ordinary T cells instead of the original T cells.
  • the universal TCR chimeric receptor T cells prepared in Example 3 of the application, and the T cells of the control group 2 are the universal TCR chimeric receptor T cells prepared in Example 3 of the application.
  • FIG. 2 schematically shows the structure of the T cells of the control group 1 and the universal TCR chimeric receptor T cells transfected with the TCR chimeric receptor of the present application.
  • the CD3 positive rate was 93.5% (control group 1). After TCR knockout, the CD3 positive rate was 4.77% (control group 2), indicating a higher knockout efficiency (see Figure 3).
  • T cells In virus-transfected T cells (that is, T cells (T cells in the control group 1 of the present application or T cells prepared in Example 3 of the present application) are transfected with the TCR chimeric receptor molecule constructed in Example 1 of the present application CG-pWW-001, CG-pWW-002, CG-pWW-003, CG-pWW-004 or CG-pWW-005), TCR chimeric receptors without costimulatory domain (ie CG-pWW-001 and CG The expression efficiency of -pWW-002) were 24.4% and 37.7% (see Figure 3).
  • the CG-pWW-001 and CG-pWW-002TCR chimeric receptors are Flag-positive and CD3-positive, respectively.
  • the efficiency is 57.7% and 71.7% (see Figure 3). It can be seen that when TCR is knocked out , T cells expressing the TCR chimeric receptor described in this application (ie, the modified immune effector cells described in this application) can still be recognized by the CD3 antibody.
  • TCR antibody BioLegend; 306718
  • CD3 antibody clone UCHT1 BD Biosciences; 555335
  • TCR knockout group was transfected with CG-pWW-001, CG -pWW-002, CG-pWW-003, CG-pWW-004 and CG-pWW-005 T cells can be recognized by CD3 antibody.
  • TCR-negative T cells that is, the TCR in the T cell is knocked out
  • 65.2% and 82.8% of the cells in the CG-pWW-001 and CG-pWW-002 groups were recognized by the CD3 antibody clone UCHT1 (see Figure 4).
  • the TCR chimeric receptors CG-pWW-003, CG-pWW-004 and CG-pWW-005 added with CD28 costimulatory domain have groups respectively.
  • 62.2%, 71.9% and 67.0% of the cells were recognized by the CD3 antibody (see Figure 6). Therefore, the TCR chimeric receptor described in the present application can restore the expression of the TCR-CD3 complex in ⁇ TCR knockout T cells, and this expression has nothing to do with ⁇ TCR expression.
  • Example 5 Universal TCR chimeric receptor T cells can recognize target cells and release cytokines through anti-CD3 and anti-CD19 bispecific antibodies
  • the anti-CD3 anti-CD19 bispecific antibody was purchased from Invivogene (Cat. No.: bimab-hcd19cd3).
  • the CD3 antibody clone number used in this double antibody is L2K-07.
  • the double antibody can simultaneously bind to CD3 and CD19, and mediate T cells to kill target cells expressing CD19.
  • the T cells of different groups were seeded into 96-well plates at 100 ⁇ l/well and a cell concentration of 1 ⁇ 10 6 /ml. Then adjust the concentration of Raji cells expressing CD19 (Cell Bank of Chinese Academy of Sciences) to 1 ⁇ 10 6 /ml at the same cell concentration, and add 100 ⁇ l per well to a 96-well plate containing T cells to make T cells and tumor cells (I.e. Raji cells) The ratio is 1:1.
  • the anti-CD3 and anti-CD19 bispecific antibodies to the wells where the two kinds of cells (ie T cells and tumor cells) are co-cultured, so that the final concentration of the double antibodies is 50ng/ml (the CD28 costimulatory domain group is not added) or 10ng/ ml (add CD28 costimulatory domain group). After mixing thoroughly, centrifuge at 500 rpm at room temperature for 3 minutes. The cells were placed in a 37°C, 5% CO 2 incubator for 24 hours.
  • the supernatant after 24 hours of co-cultivation of the above cells was transferred to a new 96-well plate, and an ELISA kit (Thermo Fisher Scientific; article number 88-7316) was used to detect the secretion of IFN- ⁇ cytokines from T cells. Plate preparation and supernatant cytokine detection were carried out in accordance with the procedures provided by the kit.
  • control group 1 refers to conventional ordinary T cells that have not been transfected with the TCR chimeric receptor molecule constructed in Example 1 and have not knocked out TCR
  • control group 2 Refers to T cells that have not been transfected with the TCR chimeric receptor molecule constructed in Example 1 and knocked out the TCR (that is, the universal TCR chimeric receptor T cells prepared in Example 3 of the present application)
  • CG-pWW- "001TCR knockout” refers to transfecting the TCR chimeric receptor molecule CG-pWW-001 constructed in Example 1 into the universal TCR chimeric receptor T cells prepared in Example 3 of the present application
  • CG-pWW-002TCR Knockout
  • the double antibody combined with tumor cells stimulated the general antibody receptor T cells expressing CG-pWW-003, CG-pWW-004 and CG-pWW-005 to secrete 668.6pg/ml, 806.1pg/ ml and 806.8pg/ml IFN- ⁇ (see Table 4). It can be seen that even in the case of TCR knockout, the universal TCR chimeric receptor T cells expressing the TCR chimeric receptor described in the present application can still respond to the stimulation of tumor cells and double antibodies to secrete cytokines.
  • Double antibodies mediate tumor cell stimulation of universal TCR chimeric receptors without costimulatory domains, T cells secrete cytokine IFN- ⁇ (pg/ml)
  • Example 6 Universal TCR chimeric receptor T cells combined with anti-CD3 and anti-CD19 bispecific antibodies efficiently kill target cells
  • Cell plating Use lentivirus with luciferase (GenBank: AAR29591.1) to transfect Raji cells (Cell Bank of Chinese Academy of Sciences) to prepare a cell line labeled with luciferase. See Example 2 for the lentivirus preparation process.
  • the Raji cells labeled with luciferase were plated into a 96-well flat-bottom opaque white plate at a cell concentration of 1 ⁇ 10 5 /ml, 50 ⁇ l/well.
  • Set the effective target ratio ie effector cell: target cell.
  • T cell Raji cell
  • the anti-CD3 anti-CD19 bispecific antibody (purchased from Invivogene, catalog number: bimab-hcd19cd3) was added to the co-cultured wells of two kinds of cells (ie T cells and target cells), so that the final concentration of the double antibody was 100ng/ml ( No costimulatory domain group added) or 10ng/ml (costimulatory domain group added). After mixing thoroughly, centrifuge at 500 rpm at room temperature for 3 minutes. The cells were placed in a 37°C, 5% CO 2 incubator for 24 hours.
  • luciferase activity relative light unit, RLU
  • the specific steps are: the co-cultured cells are centrifuged at 800 rpm for 5 min, the supernatant is aspirated, and 100ul of D-luciferin substrate (Thermo Fisher Scientific: 88293) is added and mixed for 5 min.
  • Scientific company, model: Varioskan LUX uses chemiluminescence mode to detect fluorescence intensity. Since luciferase is only expressed in target cells, the remaining luciferase activity in the well is directly related to the number of live target cells in the well. In the absence of effector cells, the maximum luciferase activity was obtained by adding medium to the target cells as a control.
  • control group refers to those that have not been transfected with the TCR chimeric receptor molecule constructed in Example 1 and have not been knocked out.
  • Conventional ordinary T cells control group TCR knockout” refers to T cells that have not been transfected with the TCR chimeric receptor molecule constructed in Example 1 and knocked out TCR (ie, the universal TCR chimeric prepared in Example 3 of this application) Recipient T cell);
  • CG-pWW-001TCR knockout refers to the universal TCR chimeric receptor T cell prepared in Example 3 of this application transfected with the TCR chimeric receptor molecule CG constructed in Example 1 -pWW-001;
  • CG-pWW-002TCR knockout refers to transfecting the TCR chimeric receptor molecule CG- constructed in Example 1 into the universal TCR chimeric receptor T cells prepared in Example 3 of the present application pWW-002;
  • the TCR knockout control cells may have a certain killing ability due to the remaining cells that have not been knocked out, but the killing effect is poor. It can be seen that even in the case of TCR knockout, T cells expressing the TCR chimeric receptor described in the present application can still effectively kill target cells under the mediation of double antibodies.

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蛋白质的表达和/或活性相比，T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白中至少一个的表达和/或活性被下调，且T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区的表达和/或活性被上调。还提供了所述经修饰的免疫效应细胞的制备方法和用途。

Description

一种经修饰的免疫效应细胞及其用途 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用途。
背景技术
近年来，肿瘤免疫疗法进入快速发展阶段，嵌合抗原受体T细胞(Chimeric Antigen Receptor-T cells，CAR-T)免疫疗法目前是最有前途的肿瘤细胞免疫疗法之一。CAR-T疗法是一种转基因T细胞疗法，通过将嵌合抗原受体(CAR)基因转入到原代T细胞内，赋予T细胞识别和杀伤靶细胞的能力。嵌合抗原受体(CAR)是模拟TCR功能的人工受体，由一个靶抗原结合结构域、跨膜结构域、T细胞共刺激结构域以及胞内信号传导结构域组成。
目前临床上的T细胞来源一般都为病人本身，因此都是自体CAR-T细胞。一些研究者开发了通用型CAR-T(WO2019/011118)，就是用健康异源供者的T细胞提前制备出大量供不同患者使用的CAR-T细胞。然而，自体CAR-T细胞疗法和通用型CAR-T细胞疗法都有类似的问题，例如，导致正常B细胞和免疫球蛋白的缺失。此外，一些被视为可靠靶点的一些肿瘤相关抗原，也会在正常细胞低水平表达，导致CAR-T治疗可能会产生脱靶毒性。另外，CAR-T细胞在快速杀死肿瘤细胞的同时会释放大量的细胞因子，细胞活性难以控制。大量的细胞因子可能导致严重的细胞因子释放综合征(CRS)和神经系统毒性，导致病人高烧、低压、休克甚至死亡，增加临床隐患。
双特异性抗体(bispecific monoclonal antibody)是指使用基因工程技术改造的含有两种特异性抗原结合位点的特殊抗体，其可以同时结合2个不同抗原或同一抗原上2个不同的表位。一种比较特殊的双特异性抗体又称为T细胞衔接器(Bispecific T cell engagers，BiTEs)。T细胞衔接器是一种双特异性的重组抗体，包括两个连接的scFv，一端靶向T细胞表面的CD3，另一端靶向肿瘤细胞表面的靶蛋白。通过同时紧密结合T细胞和靶细胞，BiTEs可以介导T细胞对靶细胞的特异性杀伤。BiTEs能在靶细胞和功能分子(细胞)之间架起桥梁，激发具有导向性的免疫反应，在肿瘤的免疫治疗中具有广阔的应用前景。然而，双抗药物利用病人体内的T细胞，一些病人自体T细胞的功能受到抑制或者T细胞功能不全，导致双抗的疗效大大降低。此外，目前的双抗只激活TCR信号，缺乏共激活信号，导致细胞扩增和细胞因子分泌能力较弱。
根据T细胞表面受体(TCR)类型的不同，可将T细胞分为αβT细胞和γδT细胞。两者的区别在于表达的TCR类型不同。αβT细胞表达αβTCR。αβTCR有很多亚型，其表达于αβT细胞表面，负责以主要组织相容性复合体(MHC)依赖的形式识别特异性抗原。γδT细胞是指T细胞的TCR由γ链和δ链构成的T细胞，其免疫作用介于固有免疫和适应性免疫之间，为MHC非限制型T细胞，具有一定的非特异性杀伤肿瘤细胞的作用，并且具有广泛的抗瘤谱。人类外周血中大多数为αβT细胞，γδT细胞仅占外周血的2％-5％，主要分布在粘膜相关淋巴组织，是T细胞的亚群之一。虽然结构和功能有一定的差异，但是αβTCR和γδTCR都可以和CD3分子复合物形成复合体从而发挥功能。
发明内容
本申请提供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蛋白质的表达和/或活性相比，T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白中至少一个的表达和/或活性被下调，且T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区的表达和/或活性被上调。
在某些实施方式中，所述的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，T细胞受体γ恒定区蛋白的表达和/或活性被上调。
在某些实施方式中，所述的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，T细胞受体δ恒定区蛋白的表达和/或活性被上调。
在某些实施方式中，所述的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，所述T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白的表达和/或活性被下调。
在某些实施方式中，所述的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，两种蛋白质的表达和/或活性被下调，其中所述两种蛋白质由T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白组成。
在某些实施方式中，所述的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，MHC复合物的表达和/或活性被下调。
在某些实施方式中，所述的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，两种蛋白质的表达和/或活性被上调，其中所述两种蛋白质由T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区的蛋白质组成。
在某些实施方式中，所述的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表 达和/或活性相比，两种蛋白质的表达和/或活性被上调，其中所述两种蛋白质由T细胞受体γ恒定区蛋白和T细胞受体δ恒定区蛋白组成。
在某些实施方式中，所述的免疫效应细胞与相应的野生型相比，T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白中的至少一个的表达和/或活性被下调，且所述T细胞受体γ恒定区蛋白的跨膜区和所述T细胞受体δ恒定区蛋白的跨膜区的表达和/或活性被上调。
在某些实施方式中，所述的免疫效应细胞与相应的野生型相比，所述T细胞受体γ恒定区蛋白的表达和/或活性被上调。
在某些实施方式中，所述的免疫效应细胞与相应的野生型相比，所述T细胞受体δ恒定区蛋白的表达和/或活性被上调。
在某些实施方式中，所述的免疫效应细胞与相应的野生型相比，所述T细胞受体α恒定区蛋白和所述T细胞受体β恒定区蛋白的表达和/或活性被下调。
在某些实施方式中，所述的免疫效应细胞与相应的野生型相比，两种蛋白质的表达和/或活性被下调，其中所述两种蛋白质由T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白组成。
在某些实施方式中，所述的免疫效应细胞与相应的野生型相比，MHC复合物的表达和/或活性被下调。
在某些实施方式中，所述的免疫效应细胞与相应的野生型相比，两种蛋白质的表达和/或活性被上调，其中所述两种蛋白质由T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区组成。
在某些实施方式中，所述的免疫效应细胞与相应的野生型相比，两种蛋白质的表达和/或活性被上调，其中所述两种蛋白质由T细胞受体γ恒定区蛋白和T细胞受体δ恒定区蛋白组成。
在某些实施方式中，所述T细胞受体γ恒定区蛋白的跨膜区包含SEQ ID No.4、6和9中任一项所示的氨基酸序列。
在某些实施方式中，编码所述T细胞受体γ恒定区蛋白的跨膜区的核酸分子包含SEQ ID No.26-27中任一项所示的核苷酸序列。
在某些实施方式中，所述T细胞受体δ恒定区蛋白的跨膜区包含SEQ ID No.2所示的氨基酸序列。
在某些实施方式中，编码所述T细胞受体δ恒定区蛋白的跨膜区的核酸分子包含SEQ ID No.25所示的核苷酸序列。
在某些实施方式中，所述T细胞受体γ恒定区蛋白包含SEQ ID No.3、5、7和8中任一项所示的氨基酸序列。
在某些实施方式中，编码所述T细胞受体γ恒定区蛋白的核酸分子包含SEQ ID No.29-30中任一项所示的核苷酸序列。
在某些实施方式中，所述T细胞受体δ恒定区蛋白包含SEQ ID No.1所示的氨基酸序列。
在某些实施方式中，编码所述T细胞受体δ恒定区蛋白的核酸分子包含SEQ ID No.28所示的核苷酸序列。
在某些实施方式中，所述免疫效应细胞包括T细胞、天然杀伤细胞(NK细胞)、单核细胞、巨噬细胞、NKT细胞、树突状细胞、粒细胞、B细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在某些实施方式中，所述表达水平和/或活性被上调包括使所述T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区的表达和/或活性上调；和/或使编码所述T细胞受体γ恒定区蛋白的跨膜区的核酸分子和编码所述T细胞受体δ恒定区蛋白的跨膜区的核酸分子的表达和/或活性被上调。
在某些实施方式中，所述表达水平和/或活性被上调包括使所述T细胞受体γ恒定区蛋白和T细胞受体δ恒定区蛋白的表达和/或活性上调；和/或使编码所述T细胞受体γ恒定区蛋白的核酸分子和编码所述T细胞受体δ恒定区蛋白的核酸分子的表达和/或活性被上调。
在某些实施方式中，所述表达水平和/或活性被下调包括使所述T细胞受体α恒定区蛋白的表达和/或活性下调；和/或使所述T细胞受体β恒定区蛋白的表达和/或活性被下调。
在某些实施方式中，所述表达水平和/或活性被下调包括使编码所述T细胞受体α恒定区蛋白的核酸分子的表达和/或活性下调；和/或使编码所述T细胞受体β恒定区蛋白的核酸分子的表达和/或活性被下调。
在某些实施方式中，所述免疫效应细胞不表达包含T细胞受体α恒定区蛋白的TCR，和/或，所述免疫效应细胞不表达包含T细胞受体β恒定区蛋白的TCR。
在某些实施方式中，所述免疫效应细胞表达异二聚体，所述异二聚体包括T细胞受体δ恒定区蛋白和T细胞受体γ恒定区蛋白。
在某些实施方式中，所述异二聚体包含共刺激结构域。
在某些实施方式中，所述共刺激结构域包含来自选自下述蛋白的多肽：CD28、CD137、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、 TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS和CD83的配体。
在某些实施方式中，所述共刺激结构域的拷贝数为1个或2个。
在某些实施方式中，所述共刺激结构域的N端与所述T细胞受体δ恒定区蛋白的C端连接；或者，所述共刺激结构域的N端与所述T细胞受体γ恒定区蛋白的C端连接。
另一方面，本申请提供制备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蛋白质的表达和/或活性相比，下调所述免疫效应细胞中T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白中的至少一个的表达和/或活性；且上调T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区的表达和/或活性。
在某些实施方式中，所述免疫效应细胞包括T细胞、天然杀伤细胞(NK细胞)、单核细胞、巨噬细胞、NKT细胞、树突状细胞、粒细胞、B细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在某些实施方式中，所述的方法包括上调T细胞受体γ恒定区蛋白的表达和/或活性。
在某些实施方式中，所述的方法包括上调T细胞受体δ恒定区蛋白的表达和/或活性。
在某些实施方式中，所述的方法包括上调两种蛋白质的表达和/或活性，其中所述两种蛋白质由所述T细胞受体γ恒定区蛋白的跨膜区和所述T细胞受体δ恒定区蛋白的跨膜区组成。
在某些实施方式中，所述的方法包括上调两种蛋白质的表达和/或活性，其中所述两种蛋白质由所述T细胞受体γ恒定区蛋白和所述T细胞受体δ恒定区蛋白组成。
在某些实施方式中，所述上调包括上调所述蛋白质的表达和/或活性；和/或，包括上调编码所述蛋白质的核酸分子的表达和/或活性。
在某些实施方式中，所述T细胞受体γ恒定区蛋白的跨膜区包含SEQ ID No.4、6和9中任一项所示的氨基酸序列。
在某些实施方式中，所述编码T细胞受体γ恒定区蛋白的跨膜区的核酸分子包含SEQ ID No.26-27中任一项所示的核苷酸序列。
在某些实施方式中，所述T细胞受体δ恒定区蛋白的跨膜区包含SEQ ID No.2所示的氨基酸序列。
在某些实施方式中，所述编码T细胞受体δ恒定区蛋白的跨膜区的核酸分子包含SEQ ID No.25所示的核苷酸序列。
在某些实施方式中，所述T细胞受体γ恒定区蛋白包含SEQ ID No.3、5、7和8中任一项所示的氨基酸序列。
在某些实施方式中，所述编码T细胞受体γ恒定区蛋白的核酸分子包含SEQ ID No.29-30 中任一项所示的核苷酸序列。
在某些实施方式中，所述T细胞受体δ恒定区蛋白包含SEQ ID No.1所示的氨基酸序列。
在某些实施方式中，所述编码T细胞受体δ恒定区蛋白的核酸分子包含SEQ ID No.28所示的核苷酸序列。
在某些实施方式中，所述上调包括向所述免疫效应细胞施用包含编码所述T细胞受体γ恒定区蛋白的跨膜区的核酸分子和编码所述T细胞受体δ恒定区蛋白的跨膜区的核酸分子的核酸分子。
在某些实施方式中，所述上调包括向所述免疫效应细胞施用包含编码所述T细胞受体γ恒定区蛋白的核酸分子和编码所述T细胞受体δ恒定区蛋白的核酸分子的核酸分子。
在某些实施方式中，所述核酸分子位于载体上。
在某些实施方式中，所述编码T细胞受体γ恒定区蛋白的核酸分子和所述编码T细胞受体δ恒定区蛋白的核酸分子位于同一载体上。
在某些实施方式中，所述载体包含编码共刺激结构域的核酸分子。
在某些实施方式中，所述共刺激结构域包含来自选自下述蛋白的多肽：CD28、CD137、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS和CD83的配体。
在某些实施方式中，所述载体包含如SEQ ID NO:20-24中任一项所示的核苷酸序列。
在某些实施方式中，所述的方法包括下调编码所述T细胞受体α恒定区蛋白的核酸分子和编码所述T细胞受体β恒定区蛋白的核酸分子的表达和/或活性。
在某些实施方式中，所述的方法包括下调编码MHC复合物的核酸分子的表达和/或活性。
在某些实施方式中，所述的方法包括下调两种蛋白质的表达和/或活性，其中所述两种蛋白质由所述T细胞受体α恒定区蛋白和所述T细胞受体β恒定区蛋白组成。
在某些实施方式中，所述下调包括下调所述核酸分子的表达和/或活性；和/或，包括下调所述蛋白质的表达和/或活性。
在某些实施方式中，所述下调包括敲除、敲低、突变、和/或沉默所述核酸分子。
在某些实施方式中，所述下调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、CRISPR/Cas系统、RNA编辑系统如ADAR、RNA指导的核酸内切酶、锌指蛋白酶、Mega-TAL核酸酶、TALENs和Meganucleases。
在某些实施方式中，所述下调包括向所述免疫效应细胞施用CRISPR/Cas系统。
在某些实施方式中，所述下调包括向所述免疫效应细胞施用Cas9酶。
在某些实施方式中，所述下调包括向所述免疫效应细胞施用靶向所述编码T细胞受体α恒定区蛋白的核酸分子外显子部分的sgRNA。
另一方面，本申请提供一种组合物，其包括所述的免疫效应细胞和药学上可接受的载体。
另一方面，本申请提供所述的免疫效应细胞在制备CAR-T或TCR-T细胞中，或表达至少一种合成受体的免疫效应细胞的应用。
在某些实施方式中，所述药物用于异体治疗。
另一方面，本申请提供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。
另一方面，本申请提供所述的免疫效应细胞联合抗体在制备治疗肿瘤的药物中的应用。
在某些实施方式中，所述抗体包括双特异性抗体。
在某些实施方式中，所述双特异性抗体源自所述的免疫效应细胞。
在某些实施方式中，所述抗体靶向肿瘤特异性抗原和/或免疫效应细胞特异性抗原。
在某些实施方式中，所述抗体靶向CD3。
在某些实施方式中，所述抗体靶向肿瘤特异性抗原，所述肿瘤特异性抗原选自以下组：CD19、CD20、CD123、EpCAM和BCMA。
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：白血病、淋巴瘤和多发性骨髓瘤。
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示意性地显示本申请所述的TCR嵌合受体分子的部分结构。
图2示意性地显示对照组1的T细胞和转染了本申请TCR嵌合受体的通用型TCR嵌合 受体T细胞的结构。
图3显示不同组的T细胞中Flag和CD3双阳性表达情况。
图4显示不同组的T细胞中TCR和CD3的表达情况。
图5显示不同组的T细胞中Flag和CD3双阳性表达情况。
图6显示不同组的T细胞中TCR和CD3的表达情况。
图7显示不同组的T细胞联合双特异性抗体杀伤肿瘤的情况。
图8显示不同组的T细胞联合双特异性抗体杀伤肿瘤的情况。
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。
术语定义
在本申请中，术语“免疫效应细胞”通常是指参与免疫应答，例如，促进免疫效应子应答的细胞。在本申请中，所述免疫效应细胞可以包括T细胞(例如，α/β的T细胞和γ/δ的T细胞)、天然杀伤细胞(NK细胞)、单核细胞、巨噬细胞、NKT细胞、树突状细胞、粒细胞、B细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在本申请中，术语“活性”通常包括各种活性，例如所述活性可以是某种蛋白质的生物活性或生物学功能，又例如所述活性可以是某种蛋白质对肿瘤抗原的结合特异性/亲和性。
在本申请中，术语“未经所述修饰的相应细胞”通常是指未经过本申请所述的修饰的细胞，即其TRAC和TRBC的表达和/或活性没有被下调，并且TRGC跨膜区和TRDC跨膜区的表达/或活性也没有被上调。在本申请中，所述未经所述修饰的相应细胞可以包括T细胞、天然杀伤细胞(NK细胞)、单核细胞、巨噬细胞、NKT细胞、树突状细胞、粒细胞、B细胞、淋巴细胞、白细胞和/或外周血单个核细胞。在本申请中，所述未经所述修饰的相应细胞可以包括经过除了本申请所述的修饰以外的其他修饰的相应细胞，例如进行个别氨基酸的取代、更改和替换后的细胞。
在本申请中，术语“修饰”通常是指分子或细胞的状态或结构被改变。分子可以以多种方式被修饰，包括化学、结构和功能修饰。细胞可以通过核酸引入而被修饰。在本申请中，所述修饰可以是基因编辑，所述修饰使得细胞中蛋白质的表达和/或活性被改变，例如，所述修 饰使得TRAC和TRBC中至少一个的表达和/或活性被下调，并且使得TRGC跨膜区和TRDC跨膜区的表达/或活性被上调。
在本申请中，术语“野生型”(wild type)通常是相对突变型而言的，通常是指从大自然中获得的细胞或个体，也就是非人工诱变的细胞或个体。例如，在本申请中，经修饰的免疫效应细胞的野生型，通常是指天然的、未经任何人工修饰的免疫效应细胞。在本申请中，所述相应的野生型通常是指未经过所述修饰的免疫效应细胞，其TRAC和TRBC的表达和/或活性没有被下调，且TRGC跨膜区和TRDC跨膜区的表达/或活性没有被上调。
在本申请中，术语“白细胞”通常是指无色、球形、有核的血细胞，具有活跃的移动能力，可以从血管内迁移到血管外，或从血管外组织迁移到血管内。在本申请中，所述白细胞可以包括粒细胞。
在本申请中，术语“淋巴细胞”通常是指在血液、淋巴和淋巴组织中发现的任何单核细胞、非吞噬白细胞，例如，B淋巴细胞和T淋巴细胞。
在本申请中，术语“外周血单个核细胞”(或PBMC)通常是指外周血中具有单个核的细胞。例如，在本申请中，所述外周血单个核细胞可以包括淋巴细胞、单核细胞和树突状细胞。
在本申请中，术语“CAR-T”或“CAR-T细胞”通常是指能够表达CAR(又称“嵌合抗原受体”)的T细胞。所述CAR通常是指包含能够结合抗原的胞外结构域和至少一个胞内结构域的融合蛋白。CAR是嵌合抗原受体T细胞(CAR-T)的核心部件，其可包括靶向部分(例如，结合肿瘤相关抗原(tumor-associatedantigen,TAA)的部分)、铰链区、跨膜区和细胞内结构域。
在本申请中，术语“MHC复合物”通常又称为主要组织相容性复合体(major histocompatibility complex，MHC)，是一组编码动物主要组织相容性抗原的基因群的统称。人类的MHC也叫做HLA(human leukocyte antigen，HLA)复合体。由于MHC的多基因特性，依据其编码分子的结构、组织分布与功能差异，可分为MHC I类、MHC II类、MHC III类基因，分别编码MHC I类分子、MHC II类分子、MHC III类分子。
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“TCR-T”或“TCR-T细胞”通常是指能够表达TCR(又称“T细胞表面受体”)的T细胞。所述TCR可以包括αβ形式或γδ形式的TCR。根据T细胞表面受体(TCR)类型的不同，可将T细胞分为αβT细胞和γδT细胞。αβT细胞表达αβTCR。αβTCR有很多亚型，其表达于αβT细胞表面，负责以主要组织相容性复合体(MHC)依赖的形式识别特异性抗原。γδT细胞是指T细胞的TCR由γ链和δ链构成的T细胞，其免疫作用介于固有 免疫和适应性免疫之间，为MHC非限制型T细胞，具有一定的非特异性杀伤肿瘤细胞的作用，并且具有广泛的抗瘤谱。
在本申请中，术语“TRDC跨膜区”和“TRDC蛋白的跨膜区”可互换地使用，通常是指T细胞受体δ恒定区(T cell receptor delta constant，TRDC)蛋白的跨膜区。在本申请中，TRDC跨膜区的氨基酸序列可以如SEQ ID NO.2所示。
在本申请中，术语“TRGC跨膜区”和“TRGC蛋白的跨膜区”可互换地使用，通常是指T细胞受体γ恒定区(T cell receptor gamma constant，TRGC)蛋白的跨膜区。所述TRGC蛋白可以有两种，分别为TRGC1和TRGC2。在本申请中，TRGC跨膜区的氨基酸序列可以如SEQ ID NO.4、6和9中任一项所示。
在本申请中，术语“sgRNA”，“指导RNA”，“单指导RNA”和“合成指导RNA”是可互换的，并且通常是指包含指导序列的多核苷酸序列。指导序列约20bp，并且在指定靶位点的指导RNA内。
在本申请中，术语“CRISPR”通常是指成簇的规则间隔的短回文重复序列。CRISPR基因座通常与其他SSR的不同之处在于重复序列的结构，这些重复序列被称为短规则间隔重复序列(SRSR)。通常，重复序列是短的元件，以规则间隔的簇出现，具有基本恒定长度的独特插入序列。菌株之间的重复序列高度保守，但是散布的重复序列的数目和间隔区的序列通常因菌株而异。
在本申请中，术语“Cas9酶”或“Cas9蛋白”通常是指与某些细菌中发现的II型CRISPR(有规律地间隔的短回文重复序列)自适应免疫系统相关的RNA引导的DNA核酸内切酶，比如，化脓性链球菌等细菌。例如，Cas9蛋白不仅可以包含在化脓链球菌中发现的野生型Cas9，而且可以包括其各种变体，例如WO2013/176772A1中描述的那些。在一些实施方案中，如Esvelt等人，Nature Methods，10(11)：1116-1121，2013中所述，Cas9蛋白可包含来自化脓性链球菌，脑膜炎奈瑟氏球菌，嗜热链球菌和树突线虫的Cas9序列。
在本申请中，术语“CRISPR/Cas系统”也可称为“Cas-gRNA系统”，通常是指用于生物体中位点特异性基因组靶向的工具。它是一种原核适应性免疫反应系统，该系统使用非编码RNA指导Cas核酸酶诱导位点特异性DNA切割。通过细胞DNA修复机制，可以通过非同源末端连接DNA修复途径(NHEJ)或同源性定向修复(HDR)途径修复这种DNA损伤。可以利用CRISPR/Cas系统创建一种简单的RNA可编程方法来介导哺乳动物细胞中的基因组编辑，并可以用于生成基因敲除(通过插入/缺失)或敲入(通过HDR)。
在本申请中，术语“敲入”通常是指涉及基因序列中DNA序列信息的一对一替换或内源基 因座中未发现的序列信息的插入的基因工程过程。敲入可能涉及插入特定基因座的基因，因此可以是“靶向”插入。
在本申请中，术语“合成受体”通常是指工程化的细胞表面蛋白或蛋白复合物，其包含(1)可以特异性结合靶分子的靶结合域，和(2)可以激活信号传导途径的功能域。在工程单元中，靶结合域包含细胞外结构域，功能结构域包含细胞内结构域。合成受体还包括跨膜序列。合成受体可以是蛋白质复合物，其包含从外源核酸表达的蛋白质。合成受体也可以是蛋白质复合物，其包含至少一种外源表达的蛋白质和至少一种内源表达的蛋白质。在一些实施方案中，工程细胞可以是免疫细胞，例如T细胞，自然杀伤(NK)细胞，B细胞，巨噬细胞等，并且功能域可以直接或间接激活免疫细胞。在某些实施方案中，合成受体可以选自：嵌合抗原受体(“CAR”)，T细胞受体(“TCR”)，TCR受体融合构建体(“TRuC”)，T细胞抗原偶联剂(“TAC”)，抗体TCR受体(“AbTCR”)和嵌合CD3ε受体。在一些实施方案中，合成受体可以是CAR。在一些实施方案中，合成受体可以是TCR。在一些实施方案中，合成受体可以是TRuC。在一些实施方案中，合成受体可以是TAC。在一些实施方案中，合成受体可以是AbTCR。在一些实施方案中，合成受体可以是嵌合CD3ε受体。
发明详述
经修饰的免疫效应细胞
一方面，本申请提供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蛋白质的表达和/或活性相比，TRAC和TRBC中至少一个的表达和/或活性被下调，且TRGC跨膜区和TRDC跨膜区的表达和/或活性被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRAC的表达和/或活性被下调，且TRGC跨膜区和TRDC跨膜区的表达和/或活性被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRBC的表达和/或活性被下调，且TRGC跨膜区和TRDC跨膜区的表达和/或活性被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRAC和TRBC的表达和/或活性被下调，且TRGC跨膜区和TRDC跨膜区的表达和/或活性被上调。
需要说明的是，在本申请中，TRAC(即T cell receptor alpha constant)指的是T细胞受体α恒定区，TRBC(即T cell receptor beta constant)指的是T细胞受体β恒定区，TRGC(即 T cell receptor gamma constant)指的是T细胞受体γ恒定区，TRDC(即T cell receptor delta constant)指的是T细胞受体δ恒定区。所述“TRGC跨膜区”可以与“TRGC蛋白的跨膜区”互换地使用，所述“TRDC跨膜区”可以与“TRDC蛋白的跨膜区”互换地使用。此外，所述TRAC基因表示编码所述TRAC蛋白的核酸分子，所述TRBC基因表示编码所述TRBC蛋白的核酸分子，所述TRGC基因表示编码所述TRGC蛋白的核酸分子，所述TRDC基因表示编码所述TRDC蛋白的核酸分子。
在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRGC的表达和/或活性可以被上调。
在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRDC的表达和/或活性可以被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRAC的表达和/或活性被下调，且TRGC和TRDC跨膜区的表达和/或活性被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRBC的表达和/或活性被下调，且TRGC和TRDC跨膜区的表达和/或活性被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRAC和TRBC的表达和/或活性被下调，且TRGC和TRDC跨膜区的表达和/或活性被上调。
又例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRAC的表达和/或活性被下调，且TRGC跨膜区和TRDC的表达和/或活性被上调。
又例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRBC的表达和/或活性被下调，且TRGC跨膜区和TRDC的表达和/或活性被上调。
又例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRAC和TRBC的表达和/或活性被下调，且TRGC跨膜区和TRDC的表达和/或活性被上调。
再例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRAC的表达和/或活性被下调，且TRGC和TRDC的表达和/或 活性被上调。
再例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRBC的表达和/或活性被下调，且TRGC和TRDC的表达和/或活性被上调。
再例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，TRAC和TRBC的表达和/或活性被下调，且TRGC和TRDC的表达和/或活性被上调。
在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，MHC复合物的表达和/或活性可以被下调。在某些实施方式中，可以是MHC复合物中的β-2微球蛋白(即B2M)的表达和/或活性被下调。
在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，两种蛋白质的表达和/或活性可以被下调，其中所述两种蛋白质由TRAC和TRBC组成。
在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，两种蛋白质的表达和/或活性可以被上调，其中所述两种蛋白质由TRGC跨膜区的蛋白质和TRDC跨膜区的蛋白质组成。
在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，两种蛋白质的表达和/或活性可以被上调，其中所述两种蛋白质由TRGC和TRDC组成。
例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，两种蛋白质的表达和/或活性可以被下调，这两种蛋白质由TRAC和TRBC组成，并且两种蛋白质的表达和/或活性可以被上调，这两种蛋白质由TRGC跨膜区的蛋白质和TRDC跨膜区的蛋白质组成。
又例如，在本申请中，所述经修饰的免疫效应细胞与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，两种蛋白质的表达和/或活性可以被下调，这两种蛋白质由TRAC和TRBC组成，并且两种蛋白质的表达和/或活性可以被上调，这两种蛋白质由TRGC和TRDC组成。
此外，需要说明的是，所述“未经所述修饰的相应细胞”通常是指未经过本申请所述的修饰的细胞，即其TRAC和TRBC的表达和/或活性没有被下调，并且TRGC跨膜区和TRDC跨膜区的表达/或活性也没有被上调。在某些实施方式中，所述未经所述修饰的相应细胞可以 包括经过除了本申请所述的修饰以外的其他修饰的相应细胞，例如进行个别氨基酸的取代、更改和替换后的细胞。所述“修饰”通常是指分子或细胞的状态或结构被改变。分子可以以多种方式被修饰，包括化学、结构和功能修饰。细胞可以通过核酸引入而被修饰。在本申请中，所述修饰可以是基因编辑，所述修饰可以使得细胞中蛋白质的表达和/或活性被改变，例如，所述修饰使得TRAC和TRBC中至少一个的表达和/或活性被下调，并且使得TRGC跨膜区和TRDC跨膜区的表达/或活性被上调。
在本申请中，所述“比较”除了可以相对于所述“未经所述修饰的相应细胞”进行，也可以相对于“野生型”进行。所述“野生型”(wild type)通常是相对突变型而言的，通常是指从大自然中获得的细胞或个体，也就是非人工诱变的细胞或个体。例如，在本申请中，经修饰的免疫效应细胞的野生型，通常是指天然的、未经任何人工修饰的免疫效应细胞。
在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRAC和TRBC中至少一个的表达和/或活性被下调，且TRGC跨膜区和TRDC跨膜区的表达和/或活性被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRAC的表达和/或活性被下调，且TRGC跨膜区和TRDC跨膜区的表达和/或活性被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRBC的表达和/或活性被下调，且TRGC跨膜区和TRDC跨膜区的表达和/或活性被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRAC和TRBC的表达和/或活性被下调，且TRGC跨膜区和TRDC跨膜区的表达和/或活性被上调。
在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRGC的表达和/或活性可以被上调。
在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRDC的表达和/或活性可以被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRAC的表达和/或活性被下调，且TRGC和TRDC跨膜区的表达和/或活性被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRBC的表达和/或活性被下调，且TRGC和TRDC跨膜区的表达和/或活性被上调。
例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRAC和TRBC的表达和/或活性被下调，且TRGC和TRDC跨膜区的表达和/或活性被上调。
又例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRAC的表达和/或活性被下调，且TRGC跨膜区和TRDC的表达和/或活性被上调。
又例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRBC的表达和/或活性被下调，且TRGC跨膜区和TRDC的表达和/或活性被上调。
又例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRAC和TRBC的表达和/或活性被下调，且TRGC跨膜区和TRDC的表达和/或活性被上调。
再例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRAC的表达和/或活性被下调，且TRGC和TRDC的表达和/或活性被上调。
再例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRBC的表达和/或活性被下调，且TRGC和TRDC的表达和/或活性被上调。
再例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，TRAC和TRBC的表达和/或活性被下调，且TRGC和TRDC的表达和/或活性被上调。
在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，MHC复合物的表达和/或活性可以被下调。在某些实施方式中，可以是MHC复合物中的β-2微球蛋白(即B2M)的表达和/或活性被下调。
在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，两种蛋白质的表达和/或活性可以被下调，其中所述两种蛋白质由TRAC和TRBC组成。
在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，两种蛋白质的表达和/或活性可以被上调，其中所述两种蛋白质由TRGC跨膜区的蛋白质和TRDC跨膜区的蛋白质组成。
在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，两种蛋白质的表达和/或活性可以被上调，其中所述两种蛋白质由TRGC和TRDC组成。
例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，两种蛋白质的表达和/或活性可以被下调，这两种蛋白质由TRAC和TRBC组成，并且两种蛋白质的表达和/或活性可以被上调，这两种蛋白质由TRGC跨膜区的蛋白质和TRDC跨膜区的蛋白质组成。
又例如，在本申请中，所述经修饰的免疫效应细胞与相应的野生型相比，两种蛋白质的表达和/或活性可以被下调，这两种蛋白质由TRAC和TRBC组成，并且两种蛋白质的表达和/或活性可以被上调，这两种蛋白质由TRGC和TRDC组成。
在本申请中，所述TRGC可以为TRGC1或TRGC2。
在本申请中，编码所述TRGC1跨膜区的核酸分子可以包含SEQ ID No.27所示的核苷酸序列。
在本申请中，编码所述TRGC2跨膜区的核酸分子可以包含SEQ ID No.26所示的核苷酸 序列。
在本申请中，编码所述TRDC跨膜区的核酸分子可以包含SEQ ID No.25所示的核苷酸序列。
在本申请中，编码所述TRGC1的核酸分子可以包含SEQ ID No.30所示的核苷酸序列。
在本申请中，编码所述TRGC2的核酸分子可以包含SEQ ID No.29所示的核苷酸序列。
在本申请中，编码所述TRDC的核酸分子可以包含SEQ ID No.28所示的核苷酸序列。
在本申请中，所述经修饰的免疫效应细胞可以包括T细胞、天然杀伤细胞(NK细胞)、单核细胞、巨噬细胞、NKT细胞、树突状细胞、粒细胞、B细胞、淋巴细胞、白细胞和/或外周血单个核细胞。在某些实施方式中，所述白细胞可以包括粒细胞。
在本申请中，所述表达水平和/或活性被上调可以包括使所述TRGC跨膜区和TRDC跨膜区的表达和/或活性上调；和/或使编码所述TRGC跨膜区的核酸分子和编码所述TRDC跨膜区的核酸分子的表达和/或活性被上调。
在本申请中，所述表达水平和/或活性被上调可以包括使所述TRGC和TRDC的表达和/或活性上调；和/或使编码所述TRGC的核酸分子和编码所述TRDC的核酸分子的表达和/或活性被上调。
在本申请中，所述表达水平和/或活性被下调可以包括使所述TRAC的表达和/或活性下调；和/或使所述TRBC的表达和/或活性被下调。
在本申请中，所述表达水平和/或活性被下调可以包括使编码所述TRAC的核酸分子的表达和/或活性下调；和/或使编码所述TRBC的核酸分子的表达和/或活性被下调。
在本申请中，所述经修饰的免疫效应细胞可以不表达包含TRAC的TCR，和/或，所述免疫效应细胞可以不表达包含TRBC的TCR。
在本申请中，所述经修饰的免疫效应细胞可以表达异二聚体，所述异二聚体包括TRDC和TRGC。
例如，在本申请中，所述经修饰的免疫效应细胞可以不表达包含TRAC的TCR，并且可以表达异二聚体，所述异二聚体包括TRDC和TRGC。
例如，在本申请中，所述经修饰的免疫效应细胞可以不表达包含TRBC的TCR，并且可以表达异二聚体，所述异二聚体包括TRDC和TRGC。
例如，在本申请中，所述经修饰的免疫效应细胞可以不表达包含TRAC和TRBC的TCR，并且可以表达异二聚体，所述异二聚体包括TRDC和TRGC。
在本申请中，所述异二聚体还可以包含共刺激结构域。所述共刺激结构域可以包含来自 选自下述蛋白的多肽：CD28、CD137、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS和CD83的配体。所述共刺激结构域的拷贝数可以为1个或2个。
在本申请中，所述共刺激结构域的N端可以与所述TRDC的C端连接；或者，所述共刺激结构域的N端可以与所述TRGC的C端连接。
例如，在本申请中，所述共刺激结构域的拷贝数可以为1个，其N端与所述TRDC的C端连接(如图1中的“2”所示)。
例如，在本申请中，所述共刺激结构域的拷贝数可以为1个，其N端与所述TRGC的C端连接(如图1中的“3”所示)。
例如，在本申请中，所述共刺激结构域的拷贝数可以为2个，一个的N端与所述TRDC的C端连接，另一个的N端与所述TRGC的C端连接(如图1中的“4”所示)。
在本申请中，所述经修饰的免疫效应细胞可以表达包含CG-pWW-001蛋白的TCR，所述CG-pWW-001蛋白从N端至C端可以依次包含：CD8α信号肽、TRDC、T2A和TRGC1。所述CG-pWW-001蛋白的氨基酸序列可以如SEQ ID NO.15所示。
在本申请中，所述经修饰的免疫效应细胞可以表达包含CG-pWW-002蛋白的TCR，所述CG-pWW-002蛋白从N端至C端可以依次包含：CD8α信号肽、TRDC、T2A和TRGC2。所述CG-pWW-002蛋白的氨基酸序列可以如SEQ ID NO.16所示。
在本申请中，所述经修饰的免疫效应细胞可以表达包含CG-pWW-003蛋白的TCR，所述CG-pWW-003蛋白从N端至C端可以依次包含：CD8α信号肽、TRDC、CD28、T2A和TRGC2。所述CG-pWW-003蛋白的氨基酸序列可以如SEQ ID NO.17所示。
在本申请中，所述经修饰的免疫效应细胞可以表达包含CG-pWW-004蛋白的TCR，所述CG-pWW-004蛋白从N端至C端可以依次包含：CD8α信号肽、TRDC、连接肽1、CD28、T2A和TRGC2。所述CG-pWW-004蛋白的氨基酸序列可以如SEQ ID NO.18所示。
在本申请中，所述经修饰的免疫效应细胞可以表达包含CG-pWW-005蛋白的TCR，所述CG-pWW-005蛋白从N端至C端可以依次包含：CD8α信号肽、TRDC、连接肽2、CD28、T2A和TRGC2。所述CG-pWW-005蛋白的氨基酸序列可以如SEQ ID NO.19所示。
在本申请中，所述CD8α信号肽可以包含如SEQ ID NO.11所示的氨基酸序列。所述T2A可以包含如SEQ ID NO.12所示的氨基酸序列。所述CD28可以包含如SEQ ID NO.10所示的氨基酸序列。所述连接肽1可以包含如SEQ ID NO.13所示的氨基酸序列。所述连接肽2 可以包含如SEQ ID NO.14所示的氨基酸序列。
在本申请中，所述TRDC跨膜区可以包含如SEQ ID NO.2所示的氨基酸序列。
在本申请中，所述TRDC可以包含如SEQ ID NO.1所示的氨基酸序列。
在本申请中，所述TRGC1跨膜区可以包含如SEQ ID NO.4所示的氨基酸序列。
在本申请中，所述TRGC1可以包含如SEQ ID NO.3和7中任一项所示的氨基酸序列。
在本申请中，所述TRGC2跨膜区可以包含如SEQ ID NO.6和9中任一项所示的氨基酸序列。
在本申请中，所述TRGC2可以包含如SEQ ID NO.5和8中任一项所示的氨基酸序列。
在某些实施方式中，所述免疫效应细胞包含如SEQ ID NO:15-19中任一项所示的氨基酸序列。在某些实施方式中，所述免疫效应细胞包含如SEQ ID NO:20-24中任一项所示的核苷酸序列。
制备方法
另一方面，本申请提供一种制备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蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRAC和TRBC中的至少一个的表达和/或活性；且上调TRGC跨膜区和TRDC跨膜区的表达和/或活性。
例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRAC的表达和/或活性；且上调TRGC跨膜区和TRDC跨膜区的表达和/或活性。
例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRBC的表达和/或活性；且上调TRGC跨膜区和TRDC跨膜区的表达和/或活性。
例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRAC和TRBC的表达和/或活性；且上调TRGC跨膜区和TRDC跨膜区的表达和/或活性。
在本申请中，所述方法包括上调TRGC的表达和/或活性。
在本申请中，所述方法包括上调TRDC的表达和/或活性。
例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中 TRAC的表达和/或活性；且上调TRGC和TRDC跨膜区的表达和/或活性。
例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRAC的表达和/或活性；且上调TRGC跨膜区和TRDC的表达和/或活性。
例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRAC的表达和/或活性；且上调TRGC和TRDC的表达和/或活性。
又例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRBC的表达和/或活性；且上调TRGC和TRDC跨膜区的表达和/或活性。
又例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRBC的表达和/或活性；且上调TRGC跨膜区和TRDC的表达和/或活性。
又例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRBC的表达和/或活性；且上调TRGC和TRDC的表达和/或活性。
再例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRAC和TRBC的表达和/或活性；且上调TRGC和TRDC跨膜区的表达和/或活性。
再例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRAC和TRBC的表达和/或活性；且上调TRGC跨膜区和TRDC的表达和/或活性。
再例如，在本申请中，制备所述的经修饰的免疫效应细胞的方法，其可以包括以下的步骤：与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，下调所述免疫效应细胞中TRAC和TRBC的表达和/或活性；且上调TRGC和TRDC的表达和/或活性。
在本申请中，所述方法可以包括上调两种蛋白质的表达和/或活性，其中所述两种蛋白质由所述TRGC跨膜区和所述TRDC跨膜区组成。
在本申请中，所述方法可以包括上调两种蛋白质的表达和/或活性，其中所述两种蛋白质由所述TRGC和所述TRDC组成。
在本申请中，所述上调可以包括上调所述蛋白质的表达和/或活性；和/或，包括上调编码 所述蛋白质的核酸分子的表达和/或活性。
例如，所述上调可以包括向所述经修饰的免疫效应细胞施用包含编码所述TRGC跨膜区的核酸分子和编码所述TRDC跨膜区的核酸分子的核酸分子，以便上调编码所述TRGC跨膜区和所述TRDC跨膜区的核酸分子的表达和/或活性，从而上调所述TRGC跨膜区和所述TRDC跨膜区的表达和/或活性。
例如，所述上调可以包括向所述经修饰的免疫效应细胞施用包含编码所述TRGC的核酸分子和编码所述TRDC的核酸分子的核酸分子，以便上调编码所述TRGC和所述TRDC的核酸分子的表达和/或活性，从而上调所述TRGC和所述TRDC的表达和/或活性。
在某些实施方式中，所述上调可以包括敲入。所述“敲入”通常是指涉及基因序列中DNA序列信息的一对一替换或内源基因座中未发现的序列信息的插入的基因工程过程。敲入可能涉及插入特定基因座的基因，因此可以是“靶向”插入。
在本申请中，所述方法可以包括下调编码所述TRAC的核酸分子和编码所述TRBC的核酸分子的表达和/或活性。
在本申请中，所述方法还可以包括下调编码MHC复合物的核酸分子的表达和/或活性。在某些实施方式中，可以是编码MHC复合物中的β-2微球蛋白(即B2M)的核酸分子的表达和/或活性被下调。
在本申请中，所述方法可以包括下调两种蛋白质的表达和/或活性，其中所述两种蛋白质由所述TRAC和所述TRBC组成。
在本申请中，所述下调可以包括下调所述核酸分子的表达和/或活性；和/或，包括下调所述蛋白质的表达和/或活性。
例如，在本申请中，所述方法可以包括下调编码所述TRAC的核酸分子的表达和/或活性；和/或，包括下调所述TRAC的表达和/或活性。
例如，在本申请中，所述方法可以包括下调编码所述TRBC的核酸分子的表达和/或活性；和/或，包括下调所述TRBC的表达和/或活性。
例如，在本申请中，所述方法可以包括下调编码所述TRAC的核酸分子和编码所述TRBC的核酸分子的表达和/或活性；和/或，包括下调所述TRAC和所述TRBC的表达和/或活性。
在本申请中，所述下调可以包括敲除、敲低、突变、和/或沉默所述核酸分子。
在本申请中，所述下调可以包括向所述经修饰的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、CRISPR/Cas系统、RNA编辑系统如ADAR、RNA指导的核酸内切酶、锌指蛋白酶、Mega-TAL核酸酶、TALENs和Meganucleases。
在本申请中，所述下调可以包括向所述经修饰的免疫效应细胞施用CRISPR/Cas系统。所述“CRISPR/Cas系统”也可称为“Cas-gRNA系统”，通常是指用于生物体中位点特异性基因组靶向的工具。它是一种原核适应性免疫反应系统，该系统使用非编码RNA指导Cas核酸酶诱导位点特异性DNA切割。通过细胞DNA修复机制，可以通过非同源末端连接DNA修复途径(NHEJ)或同源性定向修复(HDR)途径修复这种DNA损伤。可以利用CRISPR/Cas系统创建一种简单的RNA可编程方法来介导哺乳动物细胞中的基因组编辑，并可以用于生成基因敲除(通过插入/缺失)或敲入(通过HDR)。
在本申请中，所述下调可以包括向所述经修饰的免疫效应细胞施用Cas9酶。所述“Cas9酶”或“Cas9蛋白”通常是指与某些细菌中发现的II型CRISPR(有规律地间隔的短回文重复序列)自适应免疫系统相关的RNA引导的DNA核酸内切酶，比如，化脓性链球菌等细菌。例如，Cas9蛋白不仅可以包含在化脓链球菌中发现的野生型Cas9，而且可以包括其各种变体，例如WO2013/176772A1中描述的那些。在一些实施方案中，如Esvelt等人，Nature Methods，10(11)：1116-1121，2013中所述，Cas9蛋白可包含来自化脓性链球菌，脑膜炎奈瑟氏球菌，嗜热链球菌和树突线虫的Cas9序列。
在本申请中，所述下调可以包括向所述经修饰的免疫效应细胞施用靶向所述编码TRAC的核酸分子外显子部分的sgRNA。所述“sgRNA”，“指导RNA”，“单指导RNA”和“合成指导RNA”是可互换的，并且通常是指包含指导序列的多核苷酸序列。指导序列约20bp，并且在指定靶位点的指导RNA内。所述下调方法和所述sgRNA可以参考专利文件WO2019/011118中实施例的实验步骤。
在本申请中，所述TRGC可以为TRGC1或TRGC2。
在本申请中，编码所述TRGC1跨膜区的核酸分子可以包含SEQ ID No.27所示的核苷酸序列。
在本申请中，编码所述TRGC2跨膜区的核酸分子可以包含SEQ ID No.26所示的核苷酸序列。
在本申请中，编码所述TRDC跨膜区的核酸分子可以包含SEQ ID No.25所示的核苷酸序列。
在本申请中，编码所述TRGC1的核酸分子可以包含SEQ ID No.30所示的核苷酸序列。
在本申请中，编码所述TRGC2的核酸分子可以包含SEQ ID No.29所示的核苷酸序列。
在本申请中，编码所述TRDC的核酸分子可以包含SEQ ID No.28所示的核苷酸序列。
在本申请中，所述核酸分子可以位于载体上。
在本申请中，所述编码TRGC的核酸分子和所述编码TRDC的核酸分子可以位于同一载体上。
在本申请中，所述载体可以包含编码共刺激结构域的核酸分子。
在本申请中，所述共刺激结构域可以包含来自选自下述蛋白的多肽：CD28、CD137、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS和CD83的配体。
在某些实施方式中，所述载体包含如SEQ ID NO:20-24中任一项所示的核苷酸序列。
在本申请中，所述经修饰的免疫效应细胞可以包括T细胞、天然杀伤细胞(NK细胞)、单核细胞、巨噬细胞、NKT细胞、树突状细胞、粒细胞、B细胞、淋巴细胞、白细胞和/或外周血单个核细胞。在某些实施方式中，所述白细胞可以包括粒细胞。
组合物和用途
另一方面，本申请提供一种组合物，其包括所述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，所述药学上可接受的载体可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂等，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。
在某些实施方式中，所述组合物还可以包含一种或多种(药学上有效的)佐剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下可以对接受者无毒。本申请的组合物包括但不限于液体、冷冻和冻干组合物。
在某些实施方式中，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。在某些实施方案中，所述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。在某些实施方案中，所述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。
另一方面，本申请提供所述经修饰的免疫效应细胞在制备CAR-T或TCR-T细胞中，或表达至少一种合成受体的免疫效应细胞的应用。
在本申请中，所述“合成受体”通常是指工程化的细胞表面蛋白或蛋白复合物，其包含(1)可以特异性结合靶分子的靶结合域，和(2)可以激活信号传导途径的功能域。在工程单元中，靶结合域包含细胞外域，功能结构域包含细胞内结构域。合成受体还包括跨膜序列。合成受体可以是蛋白质复合物，其包含从外源核酸表达的蛋白质。合成受体也可以是蛋白质复合物，其包含至少一种外源表达的蛋白质和至少一种内源表达的蛋白质。在一些实施方案中，工程细胞可以是免疫细胞，例如T细胞，国家杀伤(NK)细胞，B细胞，巨噬细胞等，并且功能域可以直接或间接激活免疫细胞。在某些实施方案中，合成受体可以选自：嵌合抗原受体(“CAR”)，T细胞受体(“TCR”)，TCR受体融合构建体(“TRuC”)，T细胞抗原偶联剂(“TAC”，抗体TCR受体(“AbTCR”)和嵌合CD3ε受体。在一些实施方案中，合成受体可以是CAR。在一些实施方案中，合成受体可以是TCR。在一些实施方案中，合成受体可以是TRuC。在一些实施方案中，合成受体可以是TAC。在一些实施方案中，合成受体可以是AbTCR。在一些实施方案中，合成受体可以是嵌合CD3ε受体。
另一方面，本申请提供所述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。在某些实施方式中，所述异体治疗可以包括异体移植。
另一方面，本申请提供所述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。
在本申请中，所述经修饰的免疫效应细胞可以用于异体治疗或用于治疗肿瘤。
另一方面，本申请提供一种治疗肿瘤的方法，所述方法包括向有需要的受试者施用本申请所述经修饰的免疫效应细胞。
另一方面，本申请提供所述经修饰的免疫效应细胞联合抗体在制备治疗肿瘤的药物中的应用。
在本申请中，所述抗体可以靶向肿瘤特异性抗原和/或免疫效应细胞特异性抗原。在某些实施方式中，所述抗体可以靶向CD3。在某些实施方式中，所述抗体可以靶向肿瘤特异性抗原，所述肿瘤特异性抗原可以选自以下组：CD19、CD20、CD123、EpCAM和BCMA。
在本申请中，所述抗体可以包括双特异性抗体。在某些实施方式中，所述双特异性抗体可以源自本申请所述经修饰的免疫效应细胞。
在本申请中，所述肿瘤可以包括实体瘤和非实体瘤。在某些实施方式中，所述肿瘤可以选自以下组：白血病、淋巴瘤和多发性骨髓瘤。
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
实施例1：构建表达本申请的TCR嵌合受体分子的慢病毒载体
1.从NCBI网站数据库(https://www.ncbi.nlm.nih.gov/)搜索设计TCR嵌合受体分子所需要的基因。该TCR嵌合受体分子基因序列包括两部分，分别为TCRγ链的恒定区和TCRδ链的恒定区的基因串联，两个基因之间通过连接分子T2A基因连接。因TCRδ链的恒定区有两种，分别为TRGC1和TRGC2。因此设计了两种分子，即TRDC-T2A-TRGC1和TRDC-T2A-TRGC2，分别命名为：CG-pWW-001和CG-pWW-002。同时又设计了另外3种TCR嵌合受体分子，与CG-pWW-001和CG-pWW-002的区别在于，在TRDC部分又添加CD28共刺激区域以及不同长度的连接肽GGGGS。即TRDC-CD28-T2A-TRGC2、TRDC-连接肽1-CD28-T2A-TRGC2和TRDC-连接肽2-CD28-T2A-TRGC2，分别命名为：CG-pWW-003，CG-pWW-004和CG-pWW-005，其中连接肽1和连接肽2的氨基酸序列分别如SEQ ID No.13和SEQ ID No.14所示。为方便检测，在基因序列TRGC部分加入了Flag标签。这5种TCR嵌合受体分子的氨基酸序列见表1，DNA序列见表2，部分结构如图1中的“1”和“2”所示。
表1. 5种TCR嵌合受体分子的氨基酸序列
名称 SEQ ID No.
CG-pWW-001 15
CG-pWW-002 16
CG-pWW-003 17
CG-pWW-004 18
CG-pWW-005 19
表2. 5种TCR嵌合受体分子的DNA序列
名称 SEQ ID No.
CG-pWW-001 20
CG-pWW-002 21
CG-pWW-003 22
CG-pWW-004 23
CG-pWW-005 24
2. 5种TCR嵌合受体分子基因序列由南京金斯瑞公司合成后克隆至pUC57载体(南京金斯瑞)内。在合成基因时，在基因两端加入特异性限制性内切酶酶切位点：BamHⅠ和SalⅠ。使用限制性内切酶BamHⅠ(NEB；R3136S)和SalⅠ(NEB；R3138S)双酶切重组质粒，使用DNA琼脂糖凝胶电泳分离基因片段，利用胶回收试剂盒(QIAGEN；28706)分别进行胶回收纯化。并用Nanodrop(Thermo Fisher Scientific)测定回收基因片段的浓度。使用T4 DNA连接酶(NEB；M0202S)将纯化的基因序列和慢病毒载体(Addgene；货号：12252)通过T4 DNA连接酶(NEB；M0202S)进行连接得到慢病毒载体重组质粒。对慢病毒载体重组质粒进行测序验证，重组质粒测序引物为：Lenti-For(如SEQ ID No.31所示)和Lenti-Rev(如SEQ ID No.32所示)。构建成的慢病毒载体重组质粒分别称为：pLenti-CG-pWW-001、pLenti-CG-pWW-002、pLenti-CG-pWW-003、pLenti-CG-pWW-004和pLenti-CG-pWW-005。
实施例2：慢病毒制备
1.提取质粒
将测序验证正确的5种慢病毒载体重组质粒转化大肠杆菌stbl3(北京科瑞思博)。从转化好的平板上挑取单克隆到摇菌管中，摇菌管含有3ml氨卞青霉素的液体LB培养基，220rpm，37℃摇床振荡培养过夜；活化好的菌液中按1:500接种量接种到250ml含有氨卞青霉素的液体LB培养基中，220rpm，37℃摇床振荡培养12-16h。按照试剂盒提供的实验流程进行质粒提取，所使用试剂盒：Qiagen HiSpeed Plasmid Maxi Kit：12662。提取的质粒使用DNA琼脂糖凝胶电泳检测形态和超螺旋质粒含量并通过Nanodrop(Thermo Fisher Scientific)测定质粒浓度和纯度。
2. 293T细胞的复苏和传代
1)293T细胞的复苏：从液氮中取出冻存的293T细胞(ATCC)，消毒酒精擦拭冻存管口，放在37℃水浴锅内轻轻摇晃解冻后。加入已有10ml预热的DMEM完全培养基的15ml离心管中，轻轻吹匀；室温1000rpm离心3min，弃上清；加入1ml DMEM完全培养基重悬细胞，轻轻吹匀后接种到已有9ml DMEM完全培养基的10cm培养皿中，摇匀后置于37℃，5％CO2的细胞培养箱中培养；
2)293T细胞的传代：细胞培养24h，当密度达到80％-90％时，用移液管吸掉培养基，加入10ml PBS清洗1次；加入3ml含0.25％EDTA的胰蛋白酶，放入培养箱1-2min(期间需要拿出在显微镜下观察细胞是否变圆)；细胞变圆后加入1ml DMEM完全培养基终止胰酶消 化，将细胞悬液转移到15ml离心管中，1000rpm离心3min，吸尽上清接种到新的10cm皿中。根据实验需要，按照1:3或1:5的比例传代或者进行冻存。
3. 293T细胞种板
培养的293T细胞经胰酶消化充分混匀后，稀释后用细胞计数仪(NC200，chemometec)计数，然后按照约(15～18)×10 6个/T175瓶(35-40ml培养基培养)接种293T细胞，摇匀后置于37℃，5％CO2的细胞培养箱中培养过夜。
4.转染：PEI法转染细胞
第二天，转染前培养基需换成30ml的10％FBS但无双抗的培养基。准备质粒复合物：准备15ml离心管中加入1.5ml Opti-MEM(Thermo Fisher Scientific；31985-070)，然后再依次加入，病毒载体质粒：18μg，psPAX2质粒(Addgene；货号：12260)：9μg，pMD2.G质粒(Addgene；货号：12259)：18μg，充分混匀后静置5min。再准备转染试剂复合物：将67.5μl(2mg/ml)PEI(polyscience:24765)加入到1.5ml Opti-MEM内，加入后混匀，室温静置5min；再将转染试剂复合物加入到质粒复合物中，两者混匀后静置25min；最后将转染复合物加入到细胞培养基中，轻轻摇匀。
5.慢病毒的收集和浓缩
转染48h后收取细胞上清到50ml离心管内，2000rpm，离心10min；使用Millipore的0.45μm滤膜过滤上清，滤液转移到专用离心管中，配平后使用超速离心机25000rpm超速离心2h；倒掉上清后，使用1ml无血清培养基重悬慢病毒，分装慢病毒后保存在-80℃超低温冰箱冻存。按照该流程分别制备含有CG-pWW-001、CG-pWW-002、CG-pWW-003、CG-pWW-004和CG-pWW-005的慢病毒。
实施例3：通用型TCR嵌合受体T细胞制备
使用单采机分离健康供体外周血的PBMCs(购自妙通生物科技有限公司)。从液氮中取出一管冻存的PBMCs，对其复苏计数后稀释到2×10 6/ml。按照细胞与磁珠1:3的比例加入偶联有CD3/CD28抗体的磁珠(Thermo Fisher Scientific)来激活T细胞，同时添加300IU的IL-2(PeproTech；200-02)。在激活后第2天，将上述浓缩的慢病毒加入到T细胞培养瓶内，转染T细胞。在T细胞激活后的第5天，使用CRISPR/Cas9敲除T细胞中的TCR(主要是敲除TCR中α链的恒定区的TRAC基因)，构建通用型T细胞，具体操作流程参考专利WO2019/011118中的实施例3中有关敲除TCR的实验步骤进行，其中用到的sgRNA如专利WO2019/011118中的SEQ ID NO.14所示。
实施例4：TCR嵌合受体分子在细胞内表达情况的检测
分别收集本申请实施例3中制备的T细胞，通过荧光抗体染色和流式细胞术检测TCR嵌合受体分子在转染和未转染T细胞内的表达情况。调整T细胞浓度至1×10 6/ml，取100μl细胞，加入荧光抗体4℃避光孵育30min，孵育完成后离心，离心后用PBS洗涤后重悬，最后使用流式细胞仪(购于Thermo Fisher Scientific公司，型号：Attune NxT)检测细胞染色阳性率。
首先使用Flag抗体(BioLegend；637309)和CD3抗体克隆UCHT1(BD Biosciences；555335)对细胞进行染色，并用流式细胞术分析检测未敲除TCR的T细胞(即对照组1)和敲除TCR的T细胞(即对照组2)中TCR嵌合受体分子的表达情况。需要说明的是，对照组1和对照组2的T细胞均未转染本申请实施例1构建的TCR嵌合受体分子，且对照组1的T细胞为常规普通的T细胞，而非本申请实施例3制备的通用型TCR嵌合受体T细胞，对照组2的T细胞即为本申请的实施例3制备的通用型TCR嵌合受体T细胞。
图2示意性地显示了对照组1的T细胞和转染了本申请TCR嵌合受体的通用型TCR嵌合受体T细胞的结构。
在对照组的T细胞中，CD3阳性率为93.5％(对照组1)。TCR敲除后，CD3阳性率为4.77％(对照组2)，说明敲除效率较高(见图3)。
在病毒转染的T细胞中(即在T细胞(本申请对照组1的T细胞或本申请实施例3制备的T细胞)上转染了本申请实施例1构建的TCR嵌合受体分子CG-pWW-001、CG-pWW-002、CG-pWW-003、CG-pWW-004或CG-pWW-005)，无共刺激域的TCR嵌合受体(即CG-pWW-001和CG-pWW-002)的表达效率分别为：24.4％和37.7％(见图3)。当TCR被敲除后，CG-pWW-001和CG-pWW-002TCR嵌合受体的Flag阳性同时CD3阳性的效率分别为：57.7％和71.7％(见图3)，可见当TCR敲除后，表达本申请所述TCR嵌合受体的T细胞(即本申请所述经修饰的免疫效应细胞)仍然可以被CD3抗体识别。添加CD28共刺激域的TCR嵌合受体(即CG-pWW-003、CG-pWW-004和CG-pWW-005)，当TCR敲除后，Flag阳性同时CD3阳性的效率分别为：58.3％，71.9％和68.6％(见图5)。
使用TCR抗体(BioLegend；306718)和CD3抗体克隆UCHT1(BD Biosciences；555335)对表达TCR嵌合受体的T细胞染色并分析后发现，在TCR敲除组，转染CG-pWW-001、CG-pWW-002、CG-pWW-003、CG-pWW-004和CG-pWW-005的T细胞都可以被CD3抗体识别。在TCR阴性的T细胞(即T细胞中的TCR被敲除)中，CG-pWW-001、CG-pWW-002组分别有65.2％和82.8％细胞被CD3抗体克隆UCHT1所识别(见图4)。在TCR阴性的T 细胞(即T细胞中的TCR被敲除)中，添加CD28共刺激域的TCR嵌合受体CG-pWW-003、CG-pWW-004和CG-pWW-005组分别有62.2％，71.9％和67.0％的细胞被CD3抗体所识别(见图6)。因此，本申请所述TCR嵌合受体可以恢复αβTCR敲除T细胞中TCR-CD3复合体的表达，这种表达与αβTCR表达无关。
实施例5：通用型TCR嵌合受体T细胞可以通过抗CD3抗CD19双特异性抗体识别靶细胞并释放细胞因子
抗CD3抗CD19双特异性抗体购自Invivogene(货号：bimab-hcd19cd3)。该双抗使用的CD3抗体克隆号为L2K-07。该双抗可以同时结合CD3和CD19，介导T细胞杀伤表达CD19的靶细胞。
1.细胞共培养
分别将不同组别的T细胞按照100μl/孔，细胞浓度1×10 6/ml，接种到96孔板中。再按相同的细胞浓度将表达CD19的Raji细胞(中国科学院细胞库)浓度调整至1×10 6/ml，100μl每孔分别加入到含有T细胞的96孔板中，使其T细胞和肿瘤细胞(即Raji细胞)比例为1:1。将抗CD3抗CD19双特异性抗体加入到两种细胞(即T细胞和肿瘤细胞)共培养的孔中，使双抗的终浓度为50ng/ml(未添加CD28共刺激域组)或者10ng/ml(添加CD28共刺激域组)。充分混匀后室温500rpm，离心3分钟。将细胞置于37℃，5％CO 2培养箱中共培养24小时。
2.细胞因子分泌检测
将上述细胞的共培养24小时后的上清转移到新的96孔板，使用ELISA试剂盒(Thermo Fisher Scientific；货号88-7316)检测T细胞IFN-γ细胞因子的分泌。平板制备和上清细胞因子的检测按照试剂盒提供的流程进行。
结果显示，抗CD3抗CD19双特异性抗体可以介导通用型TCR嵌合受体T细胞分泌细胞因子IFN-γ(见表3和表4)。需要说明的是，在表3和表4中，“对照组1”指的是未转染实施例1构建的TCR嵌合受体分子且未敲除TCR的常规普通T细胞；“对照组2”指的是未转染实施例1构建的TCR嵌合受体分子且敲除TCR的T细胞(即本申请实施例3制备的通用型TCR嵌合受体T细胞)；“CG-pWW-001TCR敲除”指的是在本申请实施例3制备的通用型TCR嵌合受体T细胞中转染实施例1构建的TCR嵌合受体分子CG-pWW-001；“CG-pWW-002TCR敲除”指的是在本申请实施例3制备的通用型TCR嵌合受体T细胞中转染实施例1构建的TCR嵌合受体分子CG-pWW-002；“CG-pWW-003TCR敲除”指的是在本申请实施例3制备的通用型TCR嵌合受体T细胞中转染实施例1构建的TCR嵌合受体分子CG-pWW-003；“CG-pWW-004TCR敲除”指的是在本申请实施例3制备的通用型TCR嵌合受体T细胞中转染实施例1构建的TCR嵌合受 体分子CG-pWW-004；“CG-pWW-005TCR敲除”指的是在本申请实施例3制备的通用型TCR嵌合受体T细胞中转染实施例1构建的TCR嵌合受体分子CG-pWW-005。
从表3和表4可以看出，在TCR敲除的情况下，双抗联合肿瘤细胞分别刺激表达CG-pWW-001和CG-pWW-002的通用型抗体受体T细胞分泌2087.05pg/ml和3903.41pg/ml的IFN-γ(见表3)。较低浓度的抗CD3抗CD19双特异性抗体可以介导添加共刺激域的通用型TCR嵌合受体T细胞分泌细胞因子IFN-γ。在TCR敲除的情况下，双抗联合肿瘤细胞分别刺激表达CG-pWW-003、CG-pWW-004和CG-pWW-005的通用型抗体受体T细胞分泌668.6pg/ml，806.1pg/ml和806.8pg/ml的IFN-γ(见表4)。由此可见，即使在TCR敲除的情况下，表达本申请所述的TCR嵌合受体的通用型TCR嵌合受体T细胞仍然可以响应肿瘤细胞和双抗的刺激，从而分泌细胞因子。
表3.双抗(50ng/ml)介导肿瘤细胞刺激无共刺激域的通用型TCR嵌合受体T细胞分泌细胞因子IFN-γ(pg/ml)
Figure PCTCN2021072844-appb-000001
表4.双抗(10ng/ml)介导肿瘤细胞刺激添加共刺激域的通用型TCR嵌合受体T细胞分泌细胞因子IFN-γ(pg/ml)
Figure PCTCN2021072844-appb-000002
实施例6：通用型TCR嵌合受体T细胞联合抗CD3抗CD19双特异性抗体高效杀伤靶细胞
1.细胞铺板：使用带有荧光素酶(GenBank:AAR29591.1)的慢病毒转染Raji细胞(中国科学院细胞库)制备得到标记有荧光素酶的细胞系。慢病毒制备流程参见实施例2。将标记有荧光素酶的Raji细胞按照细胞浓度1×10 5/ml，50μl/孔铺至96孔平底不透明白板中。设置效靶比(即效应细胞：靶细胞。例如，T细胞：Raji细胞)10:1、5:1和1:1共3个梯度将 不同组别的T细胞加入到靶细胞(即Raji细胞)中。将抗CD3抗CD19双特异性抗体(购自Invivogene，货号：bimab-hcd19cd3)加入到两种细胞(即T细胞和靶细胞)共培养的孔中，使双抗的终浓度为100ng/ml(未添加共刺激域组)或者10ng/ml(添加共刺激域组)。充分混匀后室温500rpm，离心3分钟。将细胞置于37℃，5％CO 2培养箱中共培养24小时。
2.共培养24h后测定靶细胞剩余的荧光素酶活性(相对光单位，RLU)，来检测不同组别T对靶细胞的杀伤能力。具体步骤为：共培养后的细胞800rpm离心5min，吸掉上清后加入100ul的D-luciferin底物(Thermo Fisher Scientific：88293)混匀避光显色5min，在酶标仪(购于Thermo Fisher Scientific公司，型号：Varioskan LUX)用化学发光模式检测荧光强度。由于荧光素酶仅在靶细胞中表达，孔中的剩余荧光素酶活性与孔中活靶细胞的数量直接相关。在不存在效应细胞的情况下，通过将培养基加入靶细胞来获得最大荧光素酶活性作为对照。
结果如图7和图8所示，需要说明的是，在图7和图8中，“对照组”指的是未转染实施例1构建的TCR嵌合受体分子且未敲除TCR的常规普通T细胞；“对照组TCR敲除”指的是未转染实施例1构建的TCR嵌合受体分子且敲除TCR的T细胞(即本申请实施例3制备的通用型TCR嵌合受体T细胞)；“CG-pWW-001TCR敲除”指的是在本申请实施例3制备的通用型TCR嵌合受体T细胞中转染实施例1构建的TCR嵌合受体分子CG-pWW-001；“CG-pWW-002TCR敲除”指的是在本申请实施例3制备的通用型TCR嵌合受体T细胞中转染实施例1构建的TCR嵌合受体分子CG-pWW-002；“CG-pWW-003TCR敲除”指的是在本申请实施例3制备的通用型TCR嵌合受体T细胞中转染实施例1构建的TCR嵌合受体分子CG-pWW-003；“CG-pWW-004TCR敲除”指的是在本申请实施例3制备的通用型TCR嵌合受体T细胞中转染实施例1构建的TCR嵌合受体分子CG-pWW-004；“CG-pWW-005TCR敲除”指的是在本申请实施例3制备的通用型TCR嵌合受体T细胞中转染实施例1构建的TCR嵌合受体分子CG-pWW-005。
从图7和图8可以看出，在不同的效靶比，没有敲除TCR的对照组细胞和TCR敲除的转染本申请所述TCR嵌合受体的T细胞都可以很好的杀伤靶细胞。TCR敲除的CG-pWW-001和CG-pWW-002在效靶比是1:1时杀伤效率分别达到：52.3％和39.3％(见图7)。TCR敲除的添加共激活域的TCR嵌合受体CG-pWW-003，CG-pWW-004和CG-pWW-005在效靶比是1:1时杀伤效率分别达到：45.5％，56.3％和33.1％(见图8)。而TCR敲除的对照细胞可能由于残存的未被敲除的细胞还有一定的杀伤能力，但是杀伤效果较差。由此可见，即使在TCR敲除的情况下，表达本申请所述TCR嵌合受体的T细胞仍然可以在双抗介导下，高效地杀伤靶细胞。

Claims (78)

  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蛋白质的表达和/或活性相比，T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白中至少一个的表达和/或活性被下调，且T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区的表达和/或活性被上调。
  2. 根据权利要求2所述的免疫效应细胞，其中与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，T细胞受体γ恒定区蛋白的表达和/或活性被上调。
  3. 根据权利要求1-2中任一项所述的免疫效应细胞，其中与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，T细胞受体δ恒定区蛋白的表达和/或活性被上调。
  4. 根据权利要求1-3中任一项所述的免疫效应细胞，其中与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，所述T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白的表达和/或活性被下调。
  5. 根据权利要求1-4中任一项所述的免疫效应细胞，其中与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，两种蛋白质的表达和/或活性被下调，其中所述两种蛋白质由T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白组成。
  6. 根据权利要求1-5中任一项所述的免疫效应细胞，其中与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，MHC复合物的表达和/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，其中与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，两种蛋白质的表达和/或活性被上调，其中所述两种蛋白质由T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区的蛋白质组成。
  8. 根据权利要求1-7中任一项所述的免疫效应细胞，其中与未经所述修饰的相应细胞中相应蛋白质的表达和/或活性相比，两种蛋白质的表达和/或活性被上调，其中所述两种蛋白质由T细胞受体γ恒定区蛋白和T细胞受体δ恒定区蛋白组成。
  9. 根据权利要求1-8中任一项所述的免疫效应细胞，其中与相应的野生型相比，T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白中的至少一个的表达和/或活性被下调，且所述T细胞受体γ恒定区蛋白的跨膜区和所述T细胞受体δ恒定区蛋白的跨膜区的表达和/或活性被上调。
  10. 根据权利要求1-9中任一项所述的免疫效应细胞，其中与相应的野生型相比，所述T细胞受体γ恒定区蛋白的表达和/或活性被上调。
  11. 根据权利要求1-10中任一项所述的免疫效应细胞，其中与相应的野生型相比，所述T细胞受体δ恒定区蛋白的表达和/或活性被上调。
  12. 根据权利要求1-11中任一项所述的免疫效应细胞，其中与相应的野生型相比，所述T细胞受体α恒定区蛋白和所述T细胞受体β恒定区蛋白的表达和/或活性被下调。
  13. 根据权利要求1-12中任一项所述的免疫效应细胞，其中与相应的野生型相比，两种蛋白质的表达和/或活性被下调，其中所述两种蛋白质由T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白组成。
  14. 根据权利要求1-13中任一项所述的免疫效应细胞，其中与相应的野生型相比，MHC复合物的表达和/或活性被下调。
  15. 根据权利要求1-14中任一项所述的免疫效应细胞，其中与相应的野生型相比，两种蛋白质的表达和/或活性被上调，其中所述两种蛋白质由T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区组成。
  16. 根据权利要求1-15中任一项所述的免疫效应细胞，其中与相应的野生型相比，两种蛋白质的表达和/或活性被上调，其中所述两种蛋白质由T细胞受体γ恒定区蛋白和T细胞受体δ恒定区蛋白组成。
  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T细胞受体γ恒定区蛋白的跨膜区包含SEQ ID No.4、6和9中任一项所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的免疫效应细胞，其中编码所述T细胞受体γ恒定区蛋白的跨膜区的核酸分子包含SEQ ID No.26-27中任一项所示的核苷酸序列。
  19. 根据权利要求1-18中任一项所述的免疫效应细胞，其中所述T细胞受体δ恒定区蛋白的跨膜区包含SEQ ID No.2所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的免疫效应细胞，其中编码所述T细胞受体δ恒定区蛋白的跨膜区的核酸分子包含SEQ ID No.25所示的核苷酸序列。
  21. 根据权利要求1-20中任一项所述的免疫效应细胞，其中所述T细胞受体γ恒定区蛋白包含SEQ ID No.3、5、7和8中任一项所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的免疫效应细胞，其中编码所述T细胞受体γ恒定区蛋白的核酸分子包含SEQ ID No.29-30中任一项所示的核苷酸序列。
  23. 根据权利要求1-22中任一项所述的免疫效应细胞，其中所述T细胞受体δ恒定区蛋白包含SEQ ID No.1所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的免疫效应细胞，其中编码所述T细胞受体δ恒定区蛋白的核酸分子包含SEQ ID No.28所示的核苷酸序列。
  25. 根据权利要求1-24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、天然杀伤细胞(NK细胞)、单核细胞、巨噬细胞、NKT细胞、树突状细胞、粒细胞、B细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
  26. 根据权利要求1-25中任一项所述的免疫效应细胞，其中所述表达水平和/或活性被上调包括使所述T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区的表达和/或活性上调；和/或使编码所述T细胞受体γ恒定区蛋白的跨膜区的核酸分子和编码所述T细胞受体δ恒定区蛋白的跨膜区的核酸分子的表达和/或活性被上调。
  27. 根据权利要求1-26中任一项所述的免疫效应细胞，其中所述表达水平和/或活性被上调包括使所述T细胞受体γ恒定区蛋白和T细胞受体δ恒定区蛋白的表达和/或活性上调；和/或使编码所述T细胞受体γ恒定区蛋白的核酸分子和编码所述T细胞受体δ恒定区蛋白的核酸分子的表达和/或活性被上调。
  28. 根据权利要求1-27中任一项所述的免疫效应细胞，其中所述表达水平和/或活性被下调包括使所述T细胞受体α恒定区蛋白的表达和/或活性下调；和/或使所述T细胞受体β恒定区蛋白的表达和/或活性被下调。
  29. 根据权利要求1-28中任一项所述的免疫效应细胞，其中所述表达水平和/或活性被下调包括使编码所述T细胞受体α恒定区蛋白的核酸分子的表达和/或活性下调；和/或使编码所述T细胞受体β恒定区蛋白的核酸分子的表达和/或活性被下调。
  30. 根据权利要求1-29中任一项所述的免疫效应细胞，其中所述免疫效应细胞不表达包含T细胞受体α恒定区蛋白的TCR，和/或，所述免疫效应细胞不表达包含T细胞受体β恒定区蛋白的TCR。
  31. 根据权利要求1-30中任一项所述的免疫效应细胞，其中所述免疫效应细胞表达异二聚体，所述异二聚体包括T细胞受体δ恒定区蛋白和T细胞受体γ恒定区蛋白。
  32. 根据权利要求31所述的免疫效应细胞，其中所述异二聚体包含共刺激结构域。
  33. 根据权利要求32所述的免疫效应细胞，其中所述共刺激结构域包含源自选自下组中的一种或多种蛋白的共刺激结构域：CD28、CD137、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  34. 根据权利要求32-33中任一项所述的免疫效应细胞，其中所述共刺激结构域的拷贝数为1个或2个。
  35. 根据权利要求32-34中任一项所述的免疫效应细胞，其中所述共刺激结构域的N端与 所述T细胞受体δ恒定区蛋白的C端连接；或者，所述共刺激结构域的N端与所述T细胞受体γ恒定区蛋白的C端连接。
  36. 制备权利要求1-35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蛋白质的表达和/或活性相比，下调所述免疫效应细胞中T细胞受体α恒定区蛋白和T细胞受体β恒定区蛋白中的至少一个的表达和/或活性；且上调T细胞受体γ恒定区蛋白的跨膜区和T细胞受体δ恒定区蛋白的跨膜区的表达和/或活性。
  37. 根据权利要求36所述的方法，其中所述免疫效应细胞包括T细胞、天然杀伤细胞(NK细胞)、单核细胞、巨噬细胞、NKT细胞、树突状细胞、粒细胞、B细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
  38. 根据权利要求36-37中任一项所述的方法，其包括上调T细胞受体γ恒定区蛋白的表达和/或活性。
  39. 根据权利要求36-38中任一项所述的方法，其包括上调T细胞受体δ恒定区蛋白的表达和/或活性。
  40. 根据权利要求36-39中任一项所述的方法，其包括上调两种蛋白质的表达和/或活性，其中所述两种蛋白质由所述T细胞受体γ恒定区蛋白的跨膜区和所述T细胞受体δ恒定区蛋白的跨膜区组成。
  41. 根据权利要求36-40中任一项所述的方法，其包括上调两种蛋白质的表达和/或活性，其中所述两种蛋白质由所述T细胞受体γ恒定区蛋白和所述T细胞受体δ恒定区蛋白组成。
  42. 根据权利要求36-41中任一项所述的方法，其中所述上调包括上调所述蛋白质的表达和/或活性；和/或，包括上调编码所述蛋白质的核酸分子的表达和/或活性。
  43. 根据权利要求36-42中任一项所述的方法，其中所述T细胞受体γ恒定区蛋白的跨膜区包含SEQ ID No.4、6和9中任一项所示的氨基酸序列。
  44. 根据权利要求36-43中任一项所述的方法，其中所述编码T细胞受体γ恒定区蛋白的跨膜区的核酸分子包含SEQ ID No.26-27中任一项所示的核苷酸序列。
  45. 根据权利要求36-44中任一项所述的方法，其中所述T细胞受体δ恒定区蛋白的跨膜区包含SEQ ID No.2所示的氨基酸序列。
  46. 根据权利要求36-45中任一项所述的方法，其中所述编码T细胞受体δ恒定区蛋白的跨膜区的核酸分子包含SEQ ID No.25所示的核苷酸序列。
  47. 根据权利要求36-46中任一项所述的方法，其中所述T细胞受体γ恒定区蛋白包含SEQ ID No.3、5、7和8中任一项所示的氨基酸序列。
  48. 根据权利要求36-47中任一项所述的方法，其中所述编码T细胞受体γ恒定区蛋白的核酸分子包含SEQ ID No.29-30中任一项所示的核苷酸序列。
  49. 根据权利要求36-48中任一项所述的方法，其中所述T细胞受体δ恒定区蛋白包含SEQ ID No.1所示的氨基酸序列。
  50. 根据权利要求36-49中任一项所述的方法，其中所述编码T细胞受体δ恒定区蛋白的核酸分子包含SEQ ID No.28所示的核苷酸序列。
  51. 根据权利要求36-50中任一项所述的方法，其中所述上调包括向所述免疫效应细胞施用包含编码所述T细胞受体γ恒定区蛋白的跨膜区的核酸分子和编码所述T细胞受体δ恒定区蛋白的跨膜区的核酸分子的核酸分子。
  52. 根据权利要求36-51中任一项所述的方法，其中所述上调包括向所述免疫效应细胞施用包含编码所述T细胞受体γ恒定区蛋白的核酸分子和编码所述T细胞受体δ恒定区蛋白的核酸分子的核酸分子。
  53. 根据权利要求51-52中任一项所述的方法，其中所述核酸分子位于载体上。
  54. 根据权利要求51-53中任一项所述的方法，其中所述编码T细胞受体γ恒定区蛋白的核酸分子和所述编码T细胞受体δ恒定区蛋白的核酸分子位于同一载体上。
  55. 根据权利要求53-54中任一项所述的方法，其中所述载体包含编码共刺激结构域的核酸分子。
  56. 根据权利要求55所述的方法，其中所述共刺激结构域包含源自选自下组中的一种或多种蛋白的共刺激结构域：CD28、CD137、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  57. 根据权利要求53-56中任一项所述的方法，其中所述载体包含如SEQ ID NO:20-24中任一项所示的核苷酸序列。
  58. 根据权利要求36-57中任一项所述的方法，其包括下调编码所述T细胞受体α恒定区蛋白的核酸分子和编码所述T细胞受体β恒定区蛋白的核酸分子的表达和/或活性。
  59. 根据权利要求36-58中任一项所述的方法，其包括下调编码MHC复合物的核酸分子的表达和/或活性。
  60. 根据权利要求36-59中任一项所述的方法，其包括下调两种蛋白质的表达和/或活性，其中所述两种蛋白质由所述T细胞受体α恒定区蛋白和所述T细胞受体β恒定区蛋白组成。
  61. 根据权利要求36-60中任一项所述的方法，其中所述下调包括下调所述核酸分子的表达和/或活性；和/或，包括下调所述蛋白质的表达和/或活性。
  62. 根据权利要求36-61中任一项所述的方法，其中所述下调包括敲除、敲低、突变、和/或沉默所述核酸分子。
  63. 根据权利要求36-62中任一项所述的方法，其中所述下调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、CRISPR/Cas系统、RNA编辑系统如ADAR、RNA指导的核酸内切酶、锌指蛋白酶、Mega-TAL核酸酶、TALENs和Meganucleases。
  64. 根据权利要求36-63中任一项所述的方法，其中所述下调包括向所述免疫效应细胞施用CRISPR/Cas系统。
  65. 根据权利要求36-64中任一项所述的方法，其中所述下调包括向所述免疫效应细胞施用Cas9酶。
  66. 根据权利要求36-65中任一项所述的方法，其中所述下调包括向所述免疫效应细胞施用靶向所述编码T细胞受体α恒定区蛋白的核酸分子外显子部分的sgRNA。
  67. 组合物，其包括权利要求1-35中任一项所述的免疫效应细胞和药学上可接受的载体。
  68. 权利要求1-35中任一项所述的免疫效应细胞在制备CAR-T或TCR-T细胞中，或表达至少一种合成受体的免疫效应细胞的应用。
  69. 权利要求1-35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。
  70. 权利要求1-35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。
  71. 权利要求1-35中任一项所述的免疫效应细胞联合抗体在制备治疗肿瘤的药物中的应用。
  72. 根据权利要求71所述的应用，其中所述抗体包括双特异性抗体。
  73. 根据权利要求72所述的应用，其中所述双特异性抗体源自权利要求1-35中任一项所述的免疫效应细胞。
  74. 根据权利要求71-73中所述的应用，其中所述抗体靶向肿瘤特异性抗原和/或免疫效应细胞特异性抗原。
  75. 根据权利要求71-74中所述的应用，其中所述抗体靶向CD3。
  76. 根据权利要求71-75中所述的应用，其中所述抗体靶向肿瘤特异性抗原，所述肿瘤特 异性抗原选自以下组：CD19、CD20、CD123、EpCAM和BCMA。
  77. 根据权利要求71-76中所述的应用，其中所述肿瘤包括实体瘤和非实体瘤。
  78. 根据权利要求71-77中任一项所述的应用，其中所述肿瘤选自以下组：白血病、淋巴瘤和多发性骨髓瘤。
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