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WO2021004400A1 - 一种表达cd3抗体受体复合物的免疫细胞及其用途 - Google Patents

一种表达cd3抗体受体复合物的免疫细胞及其用途 Download PDF

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WO2021004400A1
WO2021004400A1 PCT/CN2020/100231 CN2020100231W WO2021004400A1 WO 2021004400 A1 WO2021004400 A1 WO 2021004400A1 CN 2020100231 W CN2020100231 W CN 2020100231W WO 2021004400 A1 WO2021004400 A1 WO 2021004400A1
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immune cell
cells
cell according
modified immune
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PCT/CN2020/100231
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English (en)
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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Priority to CN202080003009.1A priority Critical patent/CN112204135A/zh
Publication of WO2021004400A1 publication Critical patent/WO2021004400A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This application relates to the field of biomedicine, and specifically relates to an immune cell expressing a CD3 antibody receptor complex, which may be independent of TCR expression.
  • CAR-T chimeric antigen receptor T cell
  • the present application provides a modified immune cell which contains a CD3 antibody receptor complex and does not express T cell receptor (TCR).
  • the CD3 antibody receptor complex described in this application can be expressed in a TCR-independent form. And can be recognized by common CD3 antibodies.
  • the modified immune cells described in this application can be activated and secrete cytokines.
  • the modified immune cells described in this application can also be combined with anti-CD3 and anti-CD19 bispecific antibodies to kill tumor cells.
  • the present application provides a modified immune cell comprising a CD3 antibody receptor complex, the CD3 antibody receptor complex comprising a first CD3 recombinant protein and a second CD3 recombinant protein, wherein the first CD3
  • the recombinant protein comprises: (1) a first extracellular domain, the first extracellular domain comprising an extracellular domain derived from the CD3 epsilon domain, (2) a first transmembrane domain, (3) a first intracellular domain;
  • the second CD3 recombinant protein includes: (1) a second extracellular domain, the second extracellular domain comprising an extracellular domain derived from any one of CD3 gamma domain and CD3 delta domain, (2) second transmembrane Domain, (3) the second intracellular domain, and it does not express T cell receptor (TCR).
  • TCR T cell receptor
  • the modified immune cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes , White blood cells and/or peripheral blood mononuclear cells.
  • NK cells natural killer cells
  • macrophages include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes , White blood cells and/or peripheral blood mononuclear cells.
  • the extracellular domain of the CD3 epsilon domain comprises the amino acid sequence shown in SEQ ID NO:1.
  • the second extracellular domain comprises an extracellular domain derived from the CD3 gamma domain.
  • the extracellular domain of the CD3 gamma domain comprises an amino acid sequence as shown in SEQ ID NO: 2.
  • the second extracellular domain comprises an extracellular domain derived from the CD3 delta domain.
  • the extracellular domain of the CD3 delta domain comprises the amino acid sequence shown in SEQ ID NO:4.
  • the first transmembrane domain and the second transmembrane domain are the same or different.
  • the transmembrane domain does not comprise a transmembrane domain derived from CD3
  • the transmembrane domain comprises a transmembrane domain derived from any one protein selected from the group consisting of CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • any one protein selected from the group consisting of CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30,
  • the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:7.
  • the first intracellular domain and the second intracellular domain are the same or different.
  • At least one of the first intracellular domain and the second intracellular domain comprises a costimulatory domain and/or a signal transduction domain.
  • the costimulatory domain contained in the first intracellular domain and the costimulatory domain contained in the second intracellular domain are the same or different.
  • the costimulatory domain comprises a costimulatory domain derived from any one or more proteins selected from the following group: 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, CD83 ligand, CD40 and MyD88.
  • any one or more proteins selected from the following group: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3 , SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10
  • the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 8.
  • the signal transduction domain contained in the first intracellular domain and the signal transduction domain contained in the second intracellular domain are the same or different.
  • the signal transduction domain comprises at least one immunoreceptor tyrosine activation motif (ITAM).
  • ITAM immunoreceptor tyrosine activation motif
  • the signal transduction domain comprises a signal transduction domain derived from any one or more proteins selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpes virus
  • the signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO: 9.
  • the extracellular domain and the transmembrane domain further comprise a hinge region.
  • the hinge region comprises a hinge region derived from any one or more proteins selected from the group consisting of CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, and PD-1.
  • the hinge region comprises the amino acid sequence shown in any one of SEQ ID NO: 6.
  • the first CD3 recombinant protein includes an extracellular domain derived from the epsilon domain of CD3, a transmembrane region derived from CD28, an intracellular domain derived from CD28, and an intracellular domain derived from CD3 zeta. In some embodiments, the first CD3 recombinant protein includes the amino acid sequence shown in SEQ ID NO: 10.
  • the second CD3 recombinant protein includes an extracellular domain derived from CD3 gamma domain, a transmembrane region derived from CD28, and an intracellular domain derived from CD3 gamma.
  • the second CD3 recombinant protein includes the amino acid sequence shown in SEQ ID NO: 11.
  • the second CD3 recombinant protein includes an extracellular domain derived from the CD3 gamma domain, a transmembrane region derived from CD28, and the peptide fragment.
  • the second CD3 recombinant protein includes the amino acid sequence shown in SEQ ID NO: 12.
  • the modified immune cell further comprises a third CD3 recombinant protein, the third CD3 recombinant protein comprising: (1) a third extracellular domain, the third extracellular domain comprising The extracellular domain of CD3 gamma domain or CD3 delta domain, (2) the third transmembrane domain, and (3) the third intracellular domain.
  • the third extracellular domain is the same or different from the second extracellular domain.
  • the third transmembrane domain is the same as or different from the first transmembrane domain and/or the second transmembrane domain.
  • the third intracellular domain is the same as or different from the first intracellular domain and/or the second intracellular domain.
  • the expression and/or activity of the major histocompatibility complex (MHC) of the modified immune cell is down-regulated.
  • the MHC complex includes B2M.
  • the modified immune cell comprises a chimeric antigen receptor (CAR) and/or a chimeric autoantibody receptor (CAAR).
  • CAR chimeric antigen receptor
  • CAAR chimeric autoantibody receptor
  • the present application provides a pharmaceutical composition, which comprises the modified immune cell and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition includes antibodies.
  • the antibody can recognize and/or bind to the CD3 antibody receptor complex.
  • the antibody includes a bispecific antibody.
  • the bispecific antibody is derived from the immune cell.
  • the bispecific antibody can recognize and/or bind to receptors on the surface of target cells.
  • the target cell is a tumor cell.
  • the receptor on the target cell surface is selected from one of the following group: CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, T
  • the present application provides a nucleic acid molecule that encodes the CD3 antibody receptor complex in the modified immune cell.
  • the present application provides a vector, which contains the nucleic acid molecule.
  • the vector is a viral vector.
  • the vector is a lentiviral vector.
  • the present application provides a cell, which contains the nucleic acid molecule and/or the vector.
  • the present application provides the use of the modified immune cells and/or the pharmaceutical composition in the preparation of medicines for the treatment of tumors.
  • the tumor includes solid tumors and non-solid tumors.
  • the tumor is selected from the group consisting of lymphoma, leukemia, and multiple myeloma.
  • the present application provides a method of treating tumors, the method comprising administering the modified immune cells and the pharmaceutical composition to a subject in need.
  • the tumor includes solid tumors and non-solid tumors.
  • the tumor is selected from the group consisting of lymphoma, leukemia, and multiple myeloma. .
  • Figure 1 shows a schematic diagram of the modified immune cells described in this application combined with bispecific antibodies to kill target cells.
  • FIG. 2 shows that the modified immune cells described in this application express CD3 antibody receptor complexes.
  • Figure 3 shows that the CD3 antibody receptor complex can be recognized by the CD3 antibody UCHT1 in TCR KO human primary T cells.
  • Figure 4 shows that the CD3 antibody receptor complex can be recognized by the CD3 antibody HIT3a in TCR KO human primary T cells.
  • Figure 5 shows that the CD3 antibody receptor complex cannot be recognized by the CD3 antibody SP34-2 in TCR KO human primary T cells
  • Figure 6 shows that the CD3 antibody receptor complex can be recognized by the CD3 antibody OKT3 in TCR KO human primary T cells.
  • Figure 7 shows that the modified immune cells described in this application are activated by the CD3 antibody to express the cell activation tag CD137.
  • Figure 8 shows that the modified immune cells described in this application are activated by tumor cells mediated by anti-CD3 and anti-CD19 bispecific antibodies.
  • Figure 9 shows that the modified immune cells described in this application kill tumor cells through anti-CD3 and anti-CD19 bispecific antibodies.
  • CD3 antibody usually refers to an antibody that can specifically recognize CD3 subunits (for example, CD3 epsilon, CD3 gamma, CD3 delta, or the above-mentioned complexes).
  • the antibody may be an antibody that can only recognize CD3.
  • CD3 antibody receptor complex generally refers to a receptor that can be recognized by a CD3 antibody, which may include at least two (e.g., three) CD3 subunits (e.g., CD3 epsilon and CD3 delta, Or CD3 epsilon and CD3 gamma).
  • the CD3 antibody receptor complex is composed of at least 2 (for example, 3, 4, 5, 6 or more) CD3 recombinant proteins, and the CD3 recombinant protein may include a CD3 subunit (for example, CD3 epsilon , CD3 gamma and/or CD3 delta) extracellular domain, transmembrane domain and intracellular domain.
  • CD3 epsilon and CD3 gamma or CD3 delta ectodomain is close to the natural conformation of CD3 epsilon heterodimer in TCR complex, including the epitope recognized by most CD3 antibodies such as UCHT1 and OKT3.
  • the conformation formed by the CD3 epsilon extracellular domain alone does not include the binding epitope of most CD3 antibody clones.
  • first CD3 recombinant protein generally refers to a recombinant protein containing an extracellular domain, a transmembrane domain, and an intracellular domain derived from CD3 (eg, CD3 epsilon).
  • the CD3 antibody receptor complex may include one or more (for example, 2, 3, 4 or more) of the first CD3 recombinant protein.
  • first extracellular domain generally refers to the extracellular domain part of the first CD3 recombinant protein, and the first extracellular domain may include the extracellular domain derived from the CD3 epsilon domain.
  • second CD3 recombinant protein generally refers to a recombinant protein containing an extracellular domain, a transmembrane domain, and an intracellular domain derived from CD3 (for example, CD3 delta and or CD3 gamma).
  • the CD3 antibody receptor complex may include one or more (for example, 2, 3, 4 or more) second CD3 recombinant protein.
  • second extracellular domain generally refers to the extracellular domain part of the second CD3 recombinant protein, and the second extracellular domain may include an extracellular domain derived from CD3 delta and or CD3 gamma domain.
  • third CD3 recombinant protein generally refers to a recombinant protein containing an extracellular domain, a transmembrane domain, and an intracellular domain derived from CD3 (for example, CD3 delta and or CD3 epsilon).
  • the CD3 antibody receptor complex may include one or more (for example, two, three, four or more) third CD3 recombinant proteins.
  • third extracellular domain generally refers to the extracellular domain part of the third CD3 recombinant protein, and the third extracellular domain may include an extracellular domain derived from the CD3 delta and or CD3 epsilon domains.
  • the third extracellular domain and the second extracellular domain may be derived from the same or different CD3 subunit (for example, CD3 delta and or CD3 gamma).
  • an antibody generally refers to a polypeptide molecule that can specifically recognize and/or neutralize a specific antigen.
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds, and includes any molecule comprising an antigen binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dAb), single chain antibodies (e.g., scFv), And antibody fragments that bind to the antigen (for example, Fab, Fab', and (Fab)2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described in this application.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • transmembrane domain generally refers to a sequence of cell surface proteins that spans the cell membrane, which may include a hydrophobic alpha helix.
  • the transmembrane domain can be connected to the intracellular signal transduction domain and play a role in transmitting signals.
  • the transmembrane domain can be derived from any type I, type II or type III transmembrane protein.
  • the transmembrane domain may not include a transmembrane protein derived from CD3 (for example, CD3 epsilon, CD3 gamma, and/or CD3 delta).
  • immunoreceptor tyrosine activation motif usually refers to a conserved sequence composed of more than ten amino acids, which often appears in the intracellular of transmembrane proteins of certain cells of the immune system Area. TAM is an important transduction signal of immune cells. Therefore, they often appear in the intracellular regions of important cell signaling molecules, such as the CD3 and ⁇ chains in the T cell receptor complex, the CD79 ⁇ and ⁇ chains in the B cell receptor complex, and certain Fc receptors.
  • the tyrosine residues on the intracellular ITAM will be phosphorylated, and the phosphorylated ITAM can bind to free proteins in the envelope with the SH2 domain to make immune cells
  • the signal is transmitted to the downstream signal molecule.
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor T cells
  • antigen for example, tumor-specific antigen and/or tumor-associated antigen binding domain
  • transmembrane domain for example, tumor-specific antigen and/or tumor-associated antigen binding domain
  • costimulatory domain for example, tumor-associated antigen binding domain
  • Intracellular signal domain for example, tumor-associated antigen binding domain
  • the CAR can be combined with the T cell receptor activation intracellular domain based on the specificity of the antigen (eg CD70) of the antibody.
  • Genetically modified T cells expressing CAR can specifically recognize and eliminate malignant cells expressing target antigens.
  • CAAR chimeric autoantibody receptor
  • CAAR can direct genetically modified immune cells expressing CAAR to attack B cells expressing antibodies capable of recognizing the antigen.
  • bispecific antibody generally refers to an antibody that has binding sites for two different antigens within a single antibody molecule.
  • one of the antigens can be CD3.
  • peptide generally refers to a polypeptide consisting of at least two (for example, four, five, six or more) amino acids. It can include all molecules with amino functionality and acid functionality and including naturally occurring amino acid polymers, including natural amino acids and artificial amino acids.
  • costimulatory domain generally refers to an intracellular domain that can provide immune costimulatory molecules, which are cell surface molecules required for effective response of lymphocytes to antigens.
  • hinge region generally refers to the junction region between the extracellular domain (for example, the CD3 extracellular domain) and the transmembrane region.
  • the term "signal transduction domain” generally refers to a domain located inside a cell capable of transducing signals.
  • the intracellular signal transduction domain can transmit signals into the cell.
  • a signal transduction domain is any continuous amino acid sequence used to direct protein targeting.
  • the signaling domain can be derived from CD3 ⁇ .
  • CD3 ⁇ can form a T cell receptor-CD3 complex with T cell receptor subunits and CD3-gamma, -delta, and -epsilon.
  • CD3 ⁇ contains three ITAM motifs, and the ITAM sequence mediates the activation of TCR intracellular signals.
  • the zeta chain is a receptor-activated protein tyrosine kinase substrate.
  • CD3 ⁇ plays a key role in antigen recognition and TCR signal transduction.
  • the term "pharmaceutically acceptable adjuvant” generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient.
  • Such formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans.
  • Other contemplated carriers, excipients, and/or additives that can be used in the formulations described herein include: for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, Protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), etc.
  • immune cell generally refers to a cell that participates in an immune response, such as promoting an immune effector response.
  • immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages.
  • NK natural killer
  • the term also includes engineered immune cells, such as immune cells that have been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.
  • the "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, and is used to transfer the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation.
  • the carrier also includes a carrier having multiple functions described above.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.
  • the vector can cover additional features besides the transgene insert sequence and main chain: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag.
  • Vectors called expression vectors are specifically used to express transgenes in target cells, and usually have control sequences.
  • the vector described in this application may be an expression vector, including viral vectors (lentiviral vectors and/or retroviral vectors), phage vectors, phagemids, cosmids, cosmid, artificial chromosomes such as yeast artificial chromosomes (YAC), Bacterial artificial chromosome (BAC) or P1 derived artificial chromosome (PAC) and/or plasmid.
  • viral vectors lentiviral vectors and/or retroviral vectors
  • phage vectors phagemids
  • cosmids cosmid
  • artificial chromosomes such as yeast artificial chromosomes (YAC), Bacterial artificial chromosome (BAC) or P1 derived artificial chromos
  • treatment generally refers to: (i) preventing the disease, disorder, or condition from appearing in patients who may be susceptible to the disease, disorder, and/or condition but have not yet been diagnosed with the disease; (ii) inhibiting the disease , Disease or condition, that is, curb its development; and (iii) alleviate the disease, disease, or condition, that is, make the disease, condition, and/or condition and/or symptoms associated with the disease, condition, and/or condition Subside.
  • MHC major histocompatibility complex
  • MHC mainly includes class I MHC molecules and class II MHC molecules.
  • Class I MHC molecules can span the membranes of almost all cells in the organism, while Class II molecules are usually found on immune cells.
  • class I MHC molecules are also called class I major histocompatibility complexes, which are heterodimeric glycoproteins composed of two peptide chains connected by non-covalent bonds; one of them is called a heavy chain and the structure is polymorphic , The other is the light chain or called ⁇ 2 microglobulin (B2M).
  • B2M microglobulin
  • class I MHC molecules present polypeptides that are not self-proteins degraded in the cell, thereby activating the immune system.
  • Human Class I MHC molecules are divided into classic HLA molecules (HLA-A, HLA-B, HLA-C) and non-classical HLA molecules (HLA-E, HLA-G, HLA-F).
  • the modified immune cell may not express active MHC, and the "not expressing active MHC" may include that the expressed MHC loses the activity of activating the immune system and/or the cell surface lacks class I MHC Molecule (e.g., missing HLA-A/B/C/E/F/G).
  • B2M or the corresponding heavy chain gene can be edited to delete MHC class I molecules on the cell surface.
  • B2M usually refers to ⁇ -2 microglobulin, usually refers to the light chain of MHC class I molecules, and therefore is an indispensable part of the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • B2M is encoded by the b2m gene located on chromosome 15, while other MHC genes exist as clusters on chromosome 6.
  • the human B2M protein has 119 amino acids (see UniProt database code P61769).
  • B2M is necessary for the presentation of class I MHC molecules on the cell surface and the stability of the polypeptide binding groove.
  • Class I MHC molecules exist on the surface of all nucleated cells in the human body. MHC mismatches can cause immune rejection and result in the destruction of the graft. Knockout of the B2M gene to remove the class I MHC molecules on the cell surface can prevent mismatches. .
  • CD3 generally refers to a CD3 protein multi-subunit complex, which is composed of 6 different polypeptide chains (subunits).
  • the polypeptide chain of CD3 can include one CD3 gamma ( ⁇ ) chain, one CD3 gamma ( ⁇ ) chain, two CD3 epsilon ( ⁇ ) chains, and two CD3 zeta ( ⁇ ) chains.
  • CD3 refers to any natural CD3 from any vertebrate source, including mammals, such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. small Rats and rats).
  • mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. small Rats and rats).
  • the term encompasses "full-length” and unprocessed CD3 proteins as well as any form of protein or one or more CD3 chains (polypeptides) (e.g. mature polypeptides) derived from processing in the cell.
  • the term also encompasses naturally occurring variants and isoforms of CD3, such as splice variants or allelic variants.
  • CD3 epsilon refers to any natural CD3 from any vertebrate source, including mammals, such as primates (such as humans). ), non-human primates (such as cynomolgus monkeys) and rodents (such as mice and rats).
  • CD3 epsilon covers “full length” and unprocessed CD3 epsilon, “CD3 gamma”, “CD3 gamma” and/or “CD3 zeta”, respectively "Protein and any form of CD3 chain (polypeptide) derived from processing in the cell (e.g. mature polypeptide).
  • the term also encompasses naturally occurring variants and isoforms of the CD3 chain, such as splice variants or allelic variants.
  • An exemplary CD3delta amino acid sequence can be found in UniProt database accession number P04234, an exemplary CD3 epsilon amino acid sequence can be seen in UniProt database accession number P07766, and an exemplary CD3 gamma amino acid sequence can be seen in UniProt database accession number P09693.
  • T cell receptor generally refers to a transmembrane protein complex that participates in the activation of T cells after recognizing an antigen.
  • TCR is a heterodimer formed by two different protein subunits. In humans, 95% of T cells express one alpha ( ⁇ ) chain and one beta ( ⁇ ) chain. The remaining 5% express gamma ( ⁇ ) and delta ( ⁇ ) chains. Each chain of a TCR molecule can contain two extramembrane domains: a variable region and a constant region.
  • variable region can bind to the polypeptide/major histocompatibility complex, and the variable regions of both the ⁇ chain and the ⁇ chain contain three complementarity determining regions (CDRs), which are responsible for the recognition of the antigen/MHC complex.
  • CDRs complementarity determining regions
  • the constant region is close to the cell membrane and connected to the transmembrane region.
  • the extracellular constant domains of the two heterologous subunits of TCR both contain short binding sequences with cysteine residues. A disulfide bond is formed between the cysteine residues to bind the two heterologous TCR subunits together.
  • the TCR transmembrane domain contains positively charged amino acids and is responsible for binding to CD3 molecules.
  • the intracellular region of TCR is very short and has no active domain.
  • TCR can recognize processed polypeptide fragments that bind to MHC molecules. Because recognition requires the presentation of MHC molecules, it is also called MHC restriction. When the donor and recipient have different MHC molecules, TCR can recognize the difference in MHC and cause the activation and expansion of T cells, which may cause graft-versus-host disease (GvHD). Knockout of the TRAC gene can remove the expression of the TCR ⁇ chain, thereby removing the TCR complex from the surface of the T cell, and thus can prevent the TCR from recognizing allogeneic antigens and causing graft-versus-host disease.
  • GvHD graft-versus-host disease
  • the CD3 multi-subunit complex and TCR can form a functional complex through non-covalent bonds, which is called the TCR-CD3 complex.
  • the CD3 epsilon molecule forms a heterodimer with CD3 gamma and CD3 delta
  • CD3 zeta forms a homodimer with itself.
  • a TCR-CD3 complex includes a CD epsilon:delta heterodimer, a CD epsilon:gamma heterodimer and a CD zeta:zeta homodimer and a TCR alpha:beta heterodimer .
  • CD3 gamma, CD3 delta, and CD3 epsilon molecules are very related immunoglobulin superfamily membrane proteins, all of which contain a single extracellular immunoglobulin domain, while the extracellular domain of CD3zeta is very short.
  • the intracellular regions of CD3 gamma, CD3 delta and CD3 epsilon molecules all contain a single conserved region immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • the CD3 zeta chain contains three ITAM motifs.
  • a single TCR-CD3 complex contains 10 ITAM motifs, and these ITAM motifs determine the degree of TCR activation.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • proteins are used interchangeably and generally refer to polymers of amino acids of any length.
  • the polymer can be linear or branched, it can contain modified amino acids, and can be interrupted by non-amino acids. These terms also cover amino acid polymers that have been modified. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeling component).
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.
  • polynucleotide used interchangeably, and generally refer to nucleosides of any length.
  • the polymerized form of acid such as deoxyribonucleotides or ribonucleotides, or their analogs.
  • the polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of genes or gene fragments, multiple loci (one loci) defined according to linkage analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence Of isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • Transfer RNA transfer RNA
  • ribosomal RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro-RNA
  • ribozyme ribozyme
  • cDNA recombinant polynucleotide
  • branched polynucleotide plasmid
  • vector any sequence
  • a polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.
  • the application may also include functional variants, derivatives, analogs, homologs and fragments thereof.
  • the term "functional variant” refers to a polypeptide that has substantially the same amino acid sequence as or is encoded by substantially the same nucleotide sequence and is capable of having one or more activities of the naturally occurring sequence.
  • a variant of any given sequence means that the specific sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide essentially retains at least one A sequence of endogenous functions.
  • the variant sequence can be obtained by the addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in the naturally-occurring protein and/or polynucleotide, as long as the The original functional activity is sufficient.
  • the term "derivative” generally refers to any substitution, variation, modification, substitution, deletion, and deletion of one (or more) amino acid residues of the self/pair sequence in the polypeptide or polynucleotide of the present application. /Or addition, as long as the resulting polypeptide or polynucleotide substantially retains at least one endogenous function.
  • analog generally refers to a polypeptide or polynucleotide, including any mimic of a polypeptide or polynucleotide, that is, possessing at least one endogenous function of the polypeptide or polynucleotide mimicked by the mimic Chemical compounds.
  • amino acid substitutions can be made, for example, at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made, as long as the modified sequence basically remains as required The activity or ability.
  • Amino acid substitutions may include the use of non-naturally occurring analogs.
  • the protein or polypeptide used in the present application may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Intentional amino acid substitutions can be made based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or similarity of the amphoteric properties of the residues, as long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with a similar hydrophilicity value without an electrical head group include day Paraffin, glutamine, serine, threonine and tyrosine.
  • homolog generally refers to an amino acid sequence or a nucleotide sequence that has certain homology with the wild-type amino acid sequence and the wild-type nucleotide sequence.
  • the term “homology” can be equated with sequence "identity”.
  • the homologous sequence may include an amino acid sequence that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • the homologue will contain the same active site as the subject amino acid sequence, etc.
  • homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), and homology can also be expressed in terms of sequence identity.
  • any one of the SEQ ID NO of the mentioned amino acid sequence or the nucleotide sequence has the percent identity sequence refers to the sequence having the percentage identity over the entire length of the mentioned SEQ ID NO the sequence of.
  • sequence alignment can be performed, which can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithms needed to achieve optimal alignment among the full-length sequences being compared.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the T cells used in this application are universal T cells that express a CD3 antibody receptor and undergo gene knockout.
  • the CD3 antibody receptor is composed of two recombinant CD3 protein molecules.
  • the T cell expresses a CD3 antibody receptor that can be recognized by bispecific antibodies.
  • the difference from similar chimeric antibody receptor therapy is that the CD3 antibody receptor of the present application can be recognized by bispecific antibodies, and The expression does not depend on the TCR complex and can be directly expressed on the surface of T cells independently.
  • a modified lymphocyte the lymphocyte lacks TCR molecules, the lymphocyte expresses the CD3 antibody receptor complex, and the CD3 antibody receptor complex contains at least two CD3 recombinant proteins; wherein the first CD3 recombinant protein contains :
  • the second CD3 recombinant protein contains:
  • the intracellular domain is composed of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain, or the intracellular domain is a peptide fragment of at least two amino acids;
  • the first CD3 recombinant protein and the second At least one intracellular domain in the CD3 recombinant protein is composed of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain.
  • lymphocyte of technical solution 1 wherein the lymphocyte is T cell, B cell, NK cell or macrophage.
  • lymphocyte of technical solution 2 wherein the lymphocyte is a T cell.
  • the extracellular domain comprises the extracellular domain of CD3 epsilon shown in SEQ ID No:1, or a variant thereof.
  • the lymphocyte of technical solution 4 wherein a.
  • the extracellular domain is the extracellular domain of CD3 epsilon shown in SEQ ID No:1.
  • the extracellular domain comprises the extracellular domain of the CD3 gamma domain.
  • the extracellular domain comprises the extracellular domain of CD3 gamma shown in SEQ ID No: 2, or a variant thereof.
  • the extracellular domain is the extracellular domain of CD3 gamma shown in SEQ ID No: 2.
  • the extracellular domain comprises the extracellular domain of the CD3 delta domain, or a variant thereof.
  • the extracellular domain comprises the extracellular domain of CD3 delta shown in SEQ ID No: 4, or a variant thereof.
  • transmembrane domains b and e comprise at least one of the following: CD8 ⁇ transmembrane domain, CD28 transmembrane domain, 4-1BB transmembrane domain, CD4 transmembrane domain Membrane domain, transmembrane domain of CD27, transmembrane domain of CD7, transmembrane domain of PD-1, transmembrane domain of TRAC, and transmembrane domain of TRBC.
  • transmembrane domain of CD28 comprises the transmembrane domain of CD28 shown in SEQ ID No: 7, or a variant thereof.
  • the intracellular costimulatory signal transduction domain comprises at least one of the following: CD28, 4-1BB, CD40L, TIM1, CD226, DR3, SLAM, ICOS, OX40, NKG2D , 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD27, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT and DAP12 in the costimulatory signal transduction regions and the combination of costimulatory molecules.
  • the lymphocyte of technical solution 14, wherein the intracellular costimulatory signal transduction domain comprises the costimulatory signal transduction region of CD28 shown in SEQ ID No: 8, or a variant thereof.
  • the lymphocyte of technical solution 14, wherein the intracellular costimulatory signal transduction domain comprises the costimulatory signal transduction region of 4-1BB, or a variant thereof.
  • the intracellular signal transduction domain comprises at least one of the following: CD3zeta activation region, CD3delta activation region, CD3gamma activation region, FceRI ⁇ activation region, FceRI activation region, immunoglobulin alpha (Iga) activation area, Igbeta activation area, bovine leukemia virus gp30 activation area, Ep-stein-Barr virus (EBV) LMP2A activation area, simian immunodeficiencyvirus PBj14 Nef activation area, HSKV activation area, DAP-12 activation area, at least including An ITAM (tyrosine activation motif) domain, and a domain formed by combining the above domains.
  • Iga immunoglobulin alpha
  • Igbeta activation area bovine leukemia virus gp30 activation area
  • Ep-stein-Barr virus (EBV) LMP2A activation area Ep-stein-Barr virus (EBV) LMP2A activation area
  • the lymphocyte of technical solution 17, wherein the CD3 zeta activation region comprises the sequence shown in SEQ ID No: 9, or a variant thereof.
  • CD3 recombinant protein further has a hinge region between the extracellular domain and the transmembrane region.
  • the lymphocyte of technical solution 19, wherein the hinge region comprises at least one of the following: CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7 and PD-1 hinge region.
  • lymphocyte according to any one of the preceding technical solutions, wherein the intracellular domain is a peptide fragment of at least two amino acids.
  • the first CD3 recombinant protein is selected from one of the following recombinant proteins: CD3 ⁇ extracellular domain-CD8 ⁇ hinge region-CD8 ⁇ transmembrane region-4-1BB costimulation Signaling area-CD3 ⁇ activation area, CD3 epsilon extracellular domain-CD8 ⁇ transmembrane area-4-1BB costimulatory signaling area-CD3 ⁇ activation area, CD3 epsilon extracellular domain-CD28 hinge area-CD28 transmembrane area-CD28 costimulatory signaling area- CD3 ⁇ activation region, and CD3 ⁇ extracellular domain-CD28 transmembrane region-CD28 co-stimulatory signal transduction region-CD3 ⁇ activation region.
  • the lymphocyte of technical solution 22, wherein the first CD3 recombinant protein has the sequence shown in SEQ ID No: 10, or a variant thereof.
  • the second CD3 recombinant protein is selected from one of the following recombinant proteins: CD3 ⁇ extracellular region-CD8 ⁇ hinge region-CD8 ⁇ transmembrane region-CD3 ⁇ costimulatory signal Conduction region, CD3 ⁇ extracellular region-CD8 ⁇ transmembrane region-CD3 ⁇ costimulatory signaling region, CD3 ⁇ extracellular region-CD28 hinge region-CD28 transmembrane region-CD3 ⁇ costimulatory signaling region, and CD3 ⁇ extracellular region-CD28 transmembrane Region-CD3 ⁇ costimulatory signal transduction region, CD3 ⁇ extracellular region-CD28 hinge region-CD28 transmembrane region-peptide, wherein the peptide can be a peptide of at least 2, 4 or at least 6 amino acids.
  • the lymphocyte of technical solution 24, wherein the second CD3 recombinant protein has the sequence shown in SEQ ID No: 11 and SEQ ID No: 12, or a variant thereof.
  • the second CD3 recombinant protein is selected from one of the following recombinant proteins: CD3 ⁇ extracellular region-CD8 ⁇ hinge region-CD8 ⁇ transmembrane region-CD3 ⁇ Co-stimulatory signal transduction zone, CD3 ⁇ extracellular zone-CD8 ⁇ transmembrane zone-CD3 ⁇ costimulatory signal transduction zone, CD3 ⁇ extracellular zone-CD28 hinge zone-CD28 transmembrane zone-CD3 ⁇ costimulatory signal transduction zone, and CD3 ⁇ extracellular zone- CD28 transmembrane zone-CD3 ⁇ costimulatory signal transduction zone.
  • the intracellular domain of the first CD3 recombinant protein is composed of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain.
  • lymphocyte according to any one of the preceding technical solutions, wherein the intracellular domain of the second CD3 recombinant protein is composed of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain.
  • the CD3 antibody receptor complex comprises three CD3 recombinant proteins
  • the first CD3 recombinant protein comprises:
  • the second CD3 recombinant protein contains:
  • the third type of CD3 recombinant protein includes:
  • the intracellular domain is composed of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain, or the intracellular domain is a peptide fragment of at least two amino acids; among them, at least one of the three CD3 recombinant proteins
  • the intracellular domain consists of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain.
  • a pharmaceutical composition comprising the lymphocyte of any one of technical solutions 1-30, and a bispecific antibody, which can bind to the CD3 antibody receptor complex on the surface of the lymphocyte and can simultaneously Binding to receptors on the surface of target cells.
  • the pharmaceutical composition according to technical solution 31 or 32, wherein the bispecific antibody can bind to the CD3 antibody receptor complex on the surface of lymphocytes and can simultaneously bind to the CD19 receptor on the surface of target cells.
  • lymphocytes are T cells, B cells, NK cells or macrophages.
  • the drug further includes a bispecific antibody that can bind to the CD3 antibody receptor complex on the surface of lymphocytes and can simultaneously bind to receptors on the surface of target cells.
  • a method of treating a disease comprising injecting into a subject:
  • An effective amount of bispecific antibody that can bind to the CD3 antibody receptor complex on the surface of lymphocytes and can simultaneously bind to receptors on the surface of target cells.
  • the receptor of the target cell is selected from one of the following: CD19, CD20, CD22, CD123, CD33, BCMA, IL13R alpha, PSMA, EGFR, HER2, Mesothelin, and Claudin18 .2.
  • the present application provides a modified immune cell, which may comprise a CD3 antibody receptor complex, the CD3 antibody receptor complex comprising a first CD3 recombinant protein and a second CD3 recombinant protein.
  • the first CD3 recombinant protein may comprise (1) a first extracellular domain, (2) a first transmembrane domain, and (3) a first intracellular domain;
  • the second CD3 recombinant protein may Contains (1) a second extracellular domain, (2) a second transmembrane domain, and (3) a second intracellular domain.
  • the first extracellular domain may include an extracellular domain derived from the CD3 epsilon domain, for example, the first extracellular domain may include an amino acid sequence as shown in SEQ ID NO:1.
  • the first extracellular domain may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the second extracellular domain may include an extracellular domain derived from the CD3 gamma domain, for example, the second extracellular domain may include an amino acid sequence as shown in SEQ ID NO: 2.
  • the second extracellular domain may comprise an amino acid sequence that is at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the second extracellular domain may include an extracellular domain derived from the CD3 delta domain, for example, the second extracellular domain may include an amino acid sequence as shown in SEQ ID NO:4.
  • the second extracellular domain may comprise an amino acid sequence that is at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the CD3 recombinant protein (for example, the first CD3 recombinant protein and/or the second CD3 recombinant protein) may comprise a transmembrane domain (for example, the first and/or second transmembrane domain ).
  • the transmembrane domain (for example, the first transmembrane domain and/or the second transmembrane domain) may be derived from any type I transmembrane protein as long as it is not the transmembrane domain of CD3.
  • the exemplary transmembrane domains may include, but are not limited to, transmembrane domains derived from lower histones: CD8, CD28 , 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, ⁇ chain of T cell receptor, CD3 ⁇ , CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM , DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, SLAM, and any other independent Transmembrane domain expressed in TCR, or a mutant of the above transmembrane domain.
  • transmembrane domains derived from lower histones: CD8, CD28 , 4-1BB, CD4, CD27, CD7, PD
  • the transmembrane domain (eg, the first transmembrane domain and/or the second transmembrane domain) may be a transmembrane domain derived from human CD28.
  • the first transmembrane domain may include the amino acid sequence shown in SEQ ID NO:7.
  • the first transmembrane domain may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in SEQ ID NO: 7. , 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the CD3 recombinant protein may comprise an intracellular domain (for example, a first intracellular domain and/or a second intracellular domain).
  • the intracellular domain e.g., the first intracellular domain and/or the second intracellular domain
  • the intracellular domain may include an intracellular costimulatory domain and/or an intracellular signaling domain.
  • the intracellular signal transduction domain may include a domain with at least one ITAM motif.
  • Exemplary signal transduction domains can be derived from signal transduction domains selected from the following group, including but not limited to CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30 activation region, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12, and variants of the above.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpes virus
  • the signal transduction domain may be a signal transduction domain derived from the intracellular domain of CD3zeta.
  • the signal transduction domain from the intracellular domain of CD3zeta may include the signal transduction domain shown in SEQ ID NO: 9.
  • the signal transduction domain from the intracellular domain of CD3zeta may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 93%, at least 85%, 90%, 91%, 92%, 93%) of the amino acid sequence shown in SEQ ID NO: 9 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the costimulatory domain may include, but is not limited to, the following groups: 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, CD83 ligand, CD40 and MyD88 co-stimulation Co-stimulatory molecules composed of signal transduction regions and their combinations.
  • the costimulatory domain may be a costimulatory domain derived from the intracellular domain of human CD28.
  • the costimulatory domain of the human CD28 intracellular domain may include the costimulatory domain shown in SEQ ID NO: 8.
  • the costimulatory domain of the human CD28 intracellular domain may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, at least 80%) of the amino acid sequence shown in SEQ ID NO: 8 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the intracellular domain may include the costimulatory domain from the above-mentioned human CD28 intracellular domain and the above-mentioned signal from the CD3zeta intracellular domain Conduction domain.
  • the intracellular domain may comprise an intracellular domain derived from CD3 gamma.
  • the intracellular domain of CD3 gamma may include an amino acid sequence as shown in SEQ ID NO: 3.
  • the intracellular domain of the CD3 gamma may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 94%, at least 85%, 90%, 91%, 92%, 94%, etc.) of the amino acid sequence shown in SEQ ID NO: 3. 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the intracellular domain (for example, the first intracellular domain and/or the second intracellular domain) may comprise at least two (for example, at least four, at least six or more ) A peptide of amino acids.
  • the intracellular domain may be a peptide fragment of at least two (eg, at least four, at least six or more) amino acids.
  • the peptide fragment may include the amino acid sequence shown in SEQ ID NO:19.
  • the first CD3 recombinant protein and the second CD3 recombinant protein except for the extracellular region, other parts (for example, transmembrane domain, intracellular domain) may be the same or different.
  • the first transmembrane domain of the first CD3 recombinant protein and the second transmembrane domain of the second CD3 recombinant protein may be the same or different.
  • the first intracellular domain of the first CD3 recombinant protein and the second CD3 recombinant protein The two intracellular domains can be the same or different.
  • the signal transduction domain of the first intracellular domain and the signal transduction domain of the second intracellular domain may be the same or different.
  • the costimulatory domain of the first intracellular domain and the costimulatory domain of the second intracellular domain may be the same or different.
  • at least one of the first intracellular domain and the second intracellular domain (for example, at least two, three, four or more) comprises a costimulatory domain and/or signal transduction The domain, or, as long as it can provide sufficient signal transduction for antibody stimulation to activate the engineered immune cells.
  • the intracellular domain may be an intracellular domain derived from CD3 gamma and/or CD3delta.
  • the intracellular domain may include any one of SEQ ID NO: 3 and 5.
  • the intracellular domain may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, at least 85%, 90%, 91%, 92%, 93%) of the amino acid sequence shown in any one of SEQ ID NO: 3 and 5. 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the first intracellular domain of the first CD3 recombinant protein may include a costimulatory domain and a signal transduction domain
  • An intracellular domain can be a peptide fragment of at least two (for example, at least four, at least six or more) amino acids.
  • the first intracellular domain of the second CD3 recombinant protein may include a costimulatory domain and a signal transduction domain.
  • the second CD3 recombinant protein is the first intracellular domain.
  • An intracellular domain can be a peptide fragment of at least two (for example, at least four, at least six or more) amino acids.
  • the CD3 recombinant protein may further include a hinge region.
  • the hinge region may be between the extracellular domain and the transmembrane domain.
  • the hinge region may comprise a hinge region derived from any one or more proteins selected from the group consisting of CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, and PD-1.
  • the hinge region may include an amino acid sequence as shown in any one of SEQ ID NO: 6.
  • the hinge region may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in any one of SEQ ID NO: 6 , 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the first CD3 recombinant protein may be derived from the extracellular domain of CD3 epsilon domain, the transmembrane region derived from CD28, the intracellular domain derived from CD28, and the intracellular domain derived from CD3 zeta.
  • the first CD3 recombinant protein may include the amino acid sequence shown in SEQ ID NO: 10.
  • the first CD3 recombinant protein may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in SEQ ID NO: 10 , 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the second CD3 recombinant protein may include an extracellular domain derived from CD3 gamma domain, a transmembrane region derived from CD28, and an intracellular domain derived from CD3 gamma.
  • the second CD3 recombinant protein may include the amino acid sequence shown in SEQ ID NO: 11.
  • the second CD3 recombinant protein may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in SEQ ID NO: 11. , 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the second CD3 recombinant protein may include an extracellular domain derived from the CD3 gamma domain, a transmembrane region derived from CD28, and the peptide fragment.
  • the second CD3 recombinant protein may include the amino acid sequence shown in SEQ ID NO: 12.
  • the second CD3 recombinant protein may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in SEQ ID NO: 12 , 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the CD3 antibody receptor complex may also include one or more (for example, two, three, four or more) first recombinant proteins.
  • the CD3 antibody receptor complex may also include one or more (for example, two, three, four or more) second recombinant proteins.
  • the CD3 antibody receptor complex may further include a third recombinant protein, and the third recombinant protein may include (1) a third extracellular domain, the third extracellular domain comprising a source An extracellular domain selected from any of CD3 gamma domain or CD3 delta domain, (2) the third transmembrane domain, and (3) the third intracellular domain.
  • the third transmembrane domain may be within the range of the transmembrane domain described above, and may be the same as or different from the first transmembrane domain and/or the second transmembrane domain.
  • the third intracellular domain may be within the scope of the intracellular domain described above, and may be the same as or different from the first intracellular domain and/or the second intracellular domain.
  • the intracellular domain of at least one (for example, at least two, three, four or more) CD3 recombinant protein in the CD3 antibody receptor complex contains a costimulatory domain and/or a signal transduction domain Or, as long as it can provide sufficient signal transduction for antibody stimulation to activate the modified immune cells.
  • the present application provides a modified immune cell.
  • the immune cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells .
  • the immune cells may include T lymphocytes.
  • the T lymphocytes may include thymocytes, natural T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes.
  • the T cell may be a helper T cell (Th), for example, a helper T cell 1 (Th1) or a helper T cell 2 (Th2) cell.
  • the T lymphocytes may be CD4 + helper T cells (HTL; CD4 + T cells), cytotoxic T cells (CTL; CD8 + T cells), tumor infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + / CD8 + T cells, CD4 - / CD8 - T cells or any other subtypes of T lymphocytes.
  • the modified T cells are human T cells.
  • T cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some cases, any number of T cell lines available and known to those skilled in the art can be used. In other cases, the cells may be derived from a healthy donor, from a patient diagnosed with cancer, or obtained from a patient diagnosed with an infection. In other cases, the cell is part of a mixed population of cells with different phenotypic characteristics.
  • the immune cells may include B cells.
  • the B cells may include effector B cells (plasma cells) and memory B cells.
  • the B cells may include B2 cells, B1 cells, marginal zone B cells, follicular B cells, and regulatory B cells.
  • the immune cells may include macrophages.
  • the B cells may include type I macrophages (M1) and type II macrophages (such as M2a, M2B, M2c).
  • the immune cells may include NK cells.
  • the NK cells may include CD56bright and CD56dim.
  • the NK cells may include NK1 and NK2.
  • the NK cells may include A-NK and NA-NK.
  • the immune cells may include white blood cells.
  • White blood cells usually refer to a kind of nucleated blood cells that have active mobility and can migrate from inside blood vessels to outside blood vessels, or from tissues outside blood vessels into blood vessels.
  • white blood cells can also be present in the lymphatic system, spleen, tonsils, and other tissues of the body.
  • the white blood cells may include granulocytes (such as neutrophils, eosinophils, basophils), agranulocytes (such as lymphocytes, monocytes, macrophages, phagocytes, Mast cells).
  • the immune cells may include lymphocytes, and the lymphocytes may include any monocytes, non-phagocytic leukocytes found in blood, lymph and lymphatic tissues, for example, B lymphocytes, T lymphocytes, Natural killer (NK) cells.
  • lymphocytes may include any monocytes, non-phagocytic leukocytes found in blood, lymph and lymphatic tissues, for example, B lymphocytes, T lymphocytes, Natural killer (NK) cells.
  • the immune cells may include peripheral blood mononuclear cells, which may include any cell having a mononucleus in peripheral blood.
  • the peripheral blood mononuclear cells may include T cells, B cells, NK cells, lymphocytes, monocytes, and dendritic cells.
  • the immune cells may include macrophages.
  • Macrophages are a kind of material that can swallow and digest cell debris, microorganisms, cancer cells, and all other substances that lack the surface markers expressed on the surface of normal cells. This process is called phagocytosis. Macrophages are found in almost all tissues, looking for possible pathogens through the movement of amoeba. In addition to playing an important role in the non-specific natural immune response, they can also help initiate adaptive immunity by recruiting other immune cell types, such as lymphocytes.
  • TCR T cell receptor
  • the non-expression of T cell receptor (TCR) may include down-regulation of the expression and/or activity of T cell receptor (TCR).
  • TCR T cell receptor
  • the down-regulation may include not expressing active TCR, not expressing endogenous TCR, not expressing exogenous TCR, not including TCR structure, including inactivated TCR, and/or missing TCR.
  • the expression and/or activity of the T cell receptor alpha constant region protein and/or the T cell receptor beta constant region protein in the immune cell can be down-regulated.
  • the down-regulation may include down-regulating the expression and/or activity of the nucleic acid molecule encoding the cell receptor alpha constant region protein and/or the T cell receptor beta constant region protein; and/or, including down-regulating the The expression and/or activity of cell receptor alpha constant region protein and/or T cell receptor beta constant region protein.
  • the expression of the CD3 antibody receptor complex may not depend on the expression of TCR.
  • the expression and/or activity of the MHC complex in the immune cell is down-regulated.
  • the down-regulation may include down-regulating the expression and/or activity of the nucleic acid molecule encoding the cellular MHC complex; and/or, including down-regulating the expression and/or activity of the cellular MHC complex protein.
  • the down-regulation can be achieved by knockout, knockdown, gene mutation, gene deletion, gene silencing, or any combination of the above to make the TCR of the immune cell and/ Or the expression and/or activity of the MHC complex is down-regulated.
  • one or more substances selected from the following group can be administered to the immune cells to achieve down-regulation: antisense RNA, siRNA, shRNA, CRISPR/Cas system, RNA editing system such as RNA adenosine deaminase (ADAR) , RNA-guided endonuclease, zinc finger nuclease (ZFN), Mega-TAL nuclease, transcription activator-like effector nuclease (TALEN), meganuclease (Meganuclease), base editing, CRISPR interference, And, zinc finger protein (Zinc finger) gene repressor and/or transcription activator-like effector (TALE) gene repressor mediated transcriptional inhibition.
  • RNA editing system such as RNA adenosine deaminase (ADAR) , RNA-guided endonuclease, zinc finger nuclease (ZFN), Mega-TAL nuclease, transcription activator-like effector nucle
  • the down-regulation may include administering to the immune effector cell a guide RNA targeting the exon portion of the nucleic acid molecule (e.g., a nucleic acid molecule encoding the MHC complex of the cell).
  • a guide RNA targeting the nucleic acid molecule encoding the B2M can use the guide RNA in the prior art, and the full text of WO2019/011118 is incorporated herein by reference.
  • the modified immune cells described in this application may also include chimeric antigen receptors (CAR) and/or chimeric autoantibody receptors (CAAR).
  • CAR chimeric antigen receptors
  • CAAR chimeric autoantibody receptors
  • This application provides a method for preparing modified lymphocytes, which may include the following steps: 1. Obtain peripheral blood T cells from healthy donors; 2. Use magnetic beads loaded with CD3 and CD28 antibodies to activate T cells; 3. T After cell activation, use lentivirus to transfer the chimeric antibody receptor gene into T cells; 4. Remove magnetic beads; 5. Use gene editing technology to knock out the important genes TRAC and B2M that produce immune rejection; 6. Continue to culture and Harvest the cells.
  • the TRAC and B2M genes in lymphocytes are inactivated, which can effectively reduce the immune rejection of lymphocytes in allogeneic cell therapy, and at the same time express two recombinant CD3 surface proteins in lymphocytes, which are co-expressed
  • the CD3 surface protein is the CD3 antibody receptor, which enables the engineered lymphocytes to bind bispecific antibodies.
  • the method for preparing modified lymphocytes includes: (i) preparing lymphocytes with surface deletion of class I MHC molecules and TCR molecules; (ii) expressing two recombinant CD3 surface proteins on the surface of lymphocytes.
  • the present application provides a vector that can be used to transfer the isolated nucleic acid molecule encoding the CD3 antibody receptor complex into cells.
  • the vector may be selected from one or more of plasmids, retroviral vectors and lentiviral vectors.
  • the vector may also contain other genes, such as a marker gene that allows selection of the vector in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • One or more nucleic acid molecules described in this application can be operably linked to the expression control element.
  • Nucleic acid non-viral delivery methods include lipofection, nuclear transfection, microinjection, gene gun, viral particles, liposomes, immunoliposomes, polycations or lipid nucleic acid conjugates, naked DNA, artificial virions And reagents to enhance DNA uptake.
  • RNA or DNA virus-based systems can be used to deliver nucleic acids.
  • the virus can be targeted to specific cells in the body to effectively load the virus payload into the nucleus.
  • the viral vector may be directly administered to the patient (in vivo) or may be in an indirect form, for example, the virus is used to treat cells in vitro, and then the treated cells are administered to the patient (ex vivo).
  • Retroviral vectors can include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors for gene transfer.
  • retroviruses, lentiviruses, and adeno-associated viruses can be used to transfer genes into the host genome to allow long-term expression of the inserted genes.
  • Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce higher viral titers.
  • the lentiviral vector may include a long terminal repeat sequence 5'LTR and a truncated 3'LTR, RRE, rev response element (cPPT), central termination sequence (CTS) and/or post-translational regulatory element (WPRE).
  • the molecule can be constructed on a lentiviral vector by digestion with BamHI and SalI.
  • the present application provides a pharmaceutical composition.
  • the pharmaceutical composition may include the modified immune cells described in this application, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable adjuvant generally refers to any and all solvents, dispersion media, coatings, antibacterial agents that are compatible with the administration of immune cells and/or cell populations of this application. Agents and antifungal agents, isotonic agents and absorption delay agents. Unless it is incompatible with the immune cells of the application and/or the cell population of the application, any conventional medium or reagent can be considered for use in the pharmaceutical composition of the application.
  • the pharmaceutical composition described in this application may include an antibody.
  • the antibody and the modified immune cell may be present in the same or different container.
  • the antibody can be administered before, during or after the administration of the modified immune cells.
  • the antibody may be a bispecific antibody.
  • the bispecific antibody can simultaneously bind to two targets, and the two targets can be on the same target protein or different target proteins.
  • T cell adapter is a relatively special bispecific antibody.
  • the T cell adaptor may include two linked scFvs, one end targeting CD3 on the surface of T cells, and the other end targeting receptors on the surface of target cells (eg, tumor cells), thereby mediating T cells to kill tumor cells.
  • the receptors on the target cell surface may include, but are not limited to, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56 , CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2 /HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125 , CTLA-4, GITR, BTLA, TGFBR1, TGFBR2,
  • the basic principle of this combination therapy is that bispecific antibodies are responsible for finding the target and guiding T cells to the target cells, while the modified immune cells are responsible for the powerful killing of target cells.
  • TCR In primary T cells, TCR is not knocked out, and the bispecific antibody can mediate the killing of target cells by control T cells through the naturally expressed TCR-CD3 complex.
  • the CD3 antibody receptor As shown in Figure 1, among the universal CD3 antibody receptor T cells, the CD3 antibody receptor is expressed on the surface of the modified immune cells.
  • Bispecific antibodies can simultaneously activate the co-activation signal and CD3 zeta activation signal through the CD3 antibody receptor to promote the killing of tumor cells and the proliferation of T cells.
  • the advantage of the combined use is that cell therapy can be combined with bispecific antibodies for multiple targets, and it is easy to control, making the therapy safer.
  • the bispecific antibody may be derived from the engineered immune cells described in this application.
  • the bispecific antibody may include a bispecific antibody secreted by the engineered immune cells described in this application
  • the recurrence after the use of CAR-T cells includes target protein negative recurrence and target protein positive recurrence.
  • the positive recurrence of the target protein is mainly caused by the failure of CAR-T cells after reinfusion.
  • This therapy can achieve continuous use of bispecific antibodies after cell regression, using chimeric antibody receptor T cells and the body's own T cells to suppress tumor recurrence. For patients with poor physical conditions in the late stage, they can also achieve good therapeutic effects through flexible adjustments on the basis of ensuring safety, and increase the scope of application for patients.
  • the application also provides the use of the modified immune cells and/or the pharmaceutical composition in the preparation of medicines for the treatment of tumors.
  • this application also provides a method for treating tumors, the method comprising administering the modified immune cells and the pharmaceutical composition to a subject in need.
  • the subject can first receive certain chemotherapy pretreatment, and then the transformed lymphocytes and the bispecific antibody are infused into the subject simultaneously or sequentially. Take cell transfusion as an example. After the cells are transfused, the bispecific antibodies are transfused in different doses. If the patient has side effects, adjust the input dose of the bispecific antibody.
  • the transformed lymphocytes and double antibodies can be re-infused until remission or serious side effects appear.
  • the modified lymphocytes of the present application can kill cancer cells, such as blood cancer and solid tumor cancer cells, after binding bispecific antibodies.
  • the tumor may include non-solid tumors, including but not limited to leukemia, lymphoma, and/or multiple myeloma, and solid tumors, including but not limited to lung cancer, gastric cancer, esophageal cancer, colon Cancer, breast cancer, ovarian cancer, bladder cancer, renal cell carcinoma, prostate cancer, melanoma, head and neck tumors, glioma and soft tissue sarcoma, etc.
  • non-solid tumors including but not limited to leukemia, lymphoma, and/or multiple myeloma
  • solid tumors including but not limited to lung cancer, gastric cancer, esophageal cancer, colon Cancer, breast cancer, ovarian cancer, bladder cancer, renal cell carcinoma, prostate cancer, melanoma, head and neck tumors, glioma and soft tissue sarcoma, etc.
  • the tumor may include lymphoma.
  • CG-UST-1 consists of two parts, which are the tandem of two CD3 recombinant protein genes.
  • the first CD3 recombinant protein contains CD3 epsilon extracellular domain, CD28 transmembrane region, CD28 intracellular domain and CD3 zeta intracellular domain (SEQ ID NO: 10).
  • the second CD3 recombinant protein includes CD3 gamma extracellular domain, CD28 transmembrane region, and CD3 gamma intracellular domain (SEQ ID NO: 11).
  • the two CD3 recombinant proteins are connected by the linker T2A gene (SEQ ID NO. 16).
  • the CG-UST-1 gene sequence was synthesized by Nanjing GenScript and cloned into pUC57 vector (Nanjing GenScript). When synthesizing genes, add specific restriction endonuclease sites: BamH1 and Sal1 at both ends of the gene.
  • the recombinant plasmid of gene synthesis was digested with restriction enzymes BamH1 (NEB; R3136S) and Sal1 (NEB; R3138S), and the gene fragments were separated by agarose gel electrophoresis and then purified by gel recovery (QIAGEN; 28706). Determine the concentration of recovered gene fragments.
  • T4 DNA ligase (NEB; M0202S) was used to ligate the synthesized gene sequence to the BamH1-Sal1 site of the lentiviral vector (Addgene; catalog number: 12252).
  • the cloned lentiviral vector is called: pL-CG-UST-1.
  • the lentiviral vector plasmid was sequenced and verified, and the recombinant plasmid sequencing primers were: Lenti-For (TCAAGCCTCAGACAGTGGTTC; SEQ ID NO: 17) and Lenti-Rev (CCTCATAAAGAGACAGCAACCAGG; SEQ ID NO: 18).
  • the plasmid construction of CG-UST-2 and CG-UST-3 uses the same construction process.
  • the constructed lentiviral vector plasmids are called pL-CG-UST-2 and pL-CG-UST-3 respectively.
  • the lentiviral vector plasmid constructed above was retransformed into E. coli.
  • Use Qiagen HiSpeed Plasmid Maxi Kit (Cat. No. 12662) to perform plasmid extraction according to the experimental procedure provided by the kit. After the plasmid was extracted, Nanodrop (Thermo Fisher Scientific) was used to detect the plasmid concentration and the DNA agarose gel was used to detect the supercoiled plasmid content.
  • inoculate 293T cells inoculate cells according to about 15-16 ⁇ 10 6 cells/T175 flask (cultured in 35-40 ml medium).
  • plasmid transfection Before transfection, the medium needs to be replaced with a medium with 10% FBS but no double antibody.
  • Viral vector plasmid 18 ⁇ g, psPAX2 plasmid (Addgene; article number: 12260): 9 ⁇ g, pMD2.G plasmid (Addgene; article number: 12259): 18 ⁇ g.
  • transfection reagent complex prepares 100 ⁇ l Lipofectamine 2000 (invitrogen; 11668-019) to 1.5ml Opti-MEM, mix well after adding, and let stand at room temperature for 5 minutes; then add the plasmid complex to the transfection reagent complex After mixing, let it stand for 25 minutes; finally add the transfection complex to the cell culture medium and shake gently.
  • PMBCs purchased from Miaotong Biotechnology
  • the concentrated lentivirus was added to the T cell culture flask to transfect the T cells.
  • CRISPR/Cas9 was used to knock out TCR and B2M in T cells to construct universal T cells.
  • the gRNA sequence and operation procedure used are carried out with reference to Example 3 of patent WO2019/011118.
  • Detect the expression of CD3 antibody receptor complex in cells by fluorescent antibody staining and flow cytometry The basic steps are as follows: centrifuge to collect a certain volume of modified T cells cultured, and use Flag antibody (BioLegend; 637309) and APC- TCR antibody (BioLegend; 306718) stained the cells, incubated in the dark for 30 minutes, washed once with PBS, resuspended in an appropriate volume of PBS, and finally used flow cytometry to detect the expression and expression of CD3 antibody receptor complex in T cells Expression in TCR-negative cells.
  • the expression efficiencies of the CD3 antibody receptor complex in TCR-negative T cells were: CG-UST-1: 30.2%, CG-UST-2: 25.0% and CG-UST-3: 65.6 %(figure 2).
  • the expression efficiency is similar to the corresponding non-knockout group, so the CD3 antibody receptor complex can be independently expressed on human T cells in a TCR-independent manner.
  • T cells expressing CG-UST-1 and CG-UST-2 can be recognized by the CD3 antibody HIT3a clone (see Figure 4), but not by the clone SP34-2 (see Figure 5) ). Further, the cells were stained with SP34-2 and OKT3 antibodies at the same time, and it was found that these cells can be recognized by the OKT3 antibody (see Figure 6). However, T cells expressing CG-UST-3 containing the extramembrane domain of CD3 epsilon alone cannot be recognized by common CD3 antibody clones (see Figure 3 to Figure 6).
  • the CD3 antibody receptor can be expressed in TCR knockout primary T cells in a TCR-independent form.
  • T cells co-expressing the CD3 antibody receptor complex of CD3 epsilon and CD3 gamma extracellular domain can be recognized by conventional CD3 antibodies, including CD3 antibodies UCHT1, HIT3a and OKT3.
  • Coated tablet Dilute OKT3 antibody with PBS to 0.25 ⁇ g/ml. The diluted antibody was added to a 96-well plate at 100 ⁇ l per well, and incubated at 37°C for 3 hours. After the incubation is complete, wash the plate with 1 ⁇ PBS to remove the PBS.
  • T cells (2) Activate T cells. Adjust the T cell density of different groups to 1 ⁇ 10 6 /ml. Then the cells were seeded into the plate according to 100 ⁇ l per well and incubated at 37°C for 24 hours.
  • cytokine secretion Take the supernatant of the above cells, transfer to a new 96-well plate, and use an ELISA kit (Thermo Fisher Scientific; Catalog No. 88-7316) to detect the secretion of IFN- ⁇ cytokines from T cells. Plate preparation and supernatant cytokine detection were performed in accordance with the procedures provided by the kit.
  • OKT3 antibody stimulated general antibody receptor T cells expressing CG-UST-1 and CG-UST-2 to secrete 341.28 and 248.76 pg/ml IFN- ⁇ , respectively. See Table 1. Therefore, the coated CD3 antibody can stimulate universal T cells expressing the CD3 antibody receptor complex CG-UST-1 and CG-UST-2 to secrete the cytokine IFN- ⁇ .
  • OKT3 antibody stimulates universal CD3 antibody receptor T cells to secrete cytokine IFN- ⁇ (pg/ml)
  • the anti-CD3 anti-CD19 bispecific antibody was purchased from Invivogen (Cat. No.: bimab-hcd19cd3).
  • the double antibody can simultaneously bind CD3 epsilon and CD19, and mediate T cells to kill target cells expressing CD19.
  • the CD3 antibody clone number used in this double antibody is L2K-07.
  • the cells are co-cultured first.
  • the steps are as follows: adjust the T cell concentration of different groups to 1 ⁇ 10 6 /ml, and then plant the T cells into a 96-well plate according to 100 ⁇ l per well. Place the seeded T cells temporarily at 37°C and incubate; adjust the concentration of Raji cells expressing CD19 (Cell Bank of the Chinese Academy of Sciences) to 1 ⁇ 10 6 /ml, and then seed the Raji cells to 96 cells containing T cells according to 100 ⁇ l per well Orifice plate species. The final ratio of T cells to tumor cells is 1:1.
  • the anti-CD3 anti-CD19 bispecific antibody was added to the cells.
  • the final concentration of the double antibody is 50ng/ml. After mixing thoroughly, centrifuge at 500 rpm for 3 minutes. The cells were incubated in a 37°C incubator for 24 hours.
  • CD19 negative cells are T cells.
  • CD137 fluorescent antibody eBioscience, 11-0199-42
  • TCR antibody TCR antibody
  • FIG. 8 By detecting the cell membrane activation label CD137, it is found that Raji cells with double antibodies can activate universal T cells expressing CD3 antibody receptors CG-UST-1 and CG-UST-2, as shown in Figure 8.
  • the positive rates of CD137 were 7.15% and 6.47%, respectively.
  • Example 5 After the incubation was completed, the supernatant was taken, as in Example 5, and the ELISA experiment was performed.
  • the results show that the anti-CD3 and anti-CD19 bispecific antibodies can mediate the secretion of the cytokine IFN- ⁇ from the universal CD3 antibody receptor T cells, as shown in Table 2.
  • TCR knockout the double antibody combined with tumor cells stimulated the general antibody receptor T cells expressing CG-UST-1 and CG-UST-2 to secrete 287.639 and 286.512 pg/ml IFN- ⁇ , respectively.
  • Double antibodies mediated tumor cells to stimulate universal CD3 chimeric antibody receptor T cells to secrete cytokine IFN- ⁇ (pg/ml)

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Abstract

一种改造的免疫细胞，其不表达T细胞受体(TCR)，并包含CD3抗体受体复合物。还涉及包含所述改造的免疫细胞和双特异性抗体的药物组合物，以及所述药物组合物在制备药物中的用途。

Description

一种表达CD3抗体受体复合物的免疫细胞及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种表达CD3抗体受体复合物的免疫细胞，所述所述CD3抗体受体复合物可以不依赖于TCR表达。
背景技术
近年来，因在癌症治疗领域明显的临床疗效，嵌合抗原受体T细胞(CAR-T)疗法的免疫治疗方法逐渐成为世界范围内的研究热点。目前临床上制备CAR-T细胞发热来源一般都为病人本身，因此都是自体CAR-T细胞。但是自体CAR-T在临床应用上有很多问题，例如制备周期长，制备成本高。病人免疫功能状态比较差的时候不能成功制备CAR-T细胞。
也有采用CRISPR/Cas9 nucleases等基因编辑工具特异性的敲除某些免疫相关基因制备通用型CAR-T的方法。但是长期存在的CAR-T细胞可能导致正常B细胞和免疫球蛋白的缺失，针对实体瘤的CAR-T出现在靶但脱肿瘤的细胞毒性等安全性问题。
因此，仍需要开发一种更具安全性的可调控的细胞疗法。
发明内容
本申请提供了一种改造的免疫细胞，其包含CD3抗体受体复合物，且，其不表达T细胞受体(TCR)。本申请所述CD3抗体受体复合物能够以不依赖TCR的形式表达。且能够被常见的CD3抗体识别。在CD3抗体刺激下，本申请所述的改造的免疫细胞能够被激活，并分泌细胞因子。本申请所述的改造的免疫细胞还能够联合抗CD3抗CD19双特异性抗体杀伤肿瘤细胞。
一方面，本申请提供了一种改造的免疫细胞，其包含CD3抗体受体复合物，所述CD3抗体受体复合物包含第一CD3重组蛋白和第二CD3重组蛋白，其中所述第一CD3重组蛋白包含：(1)第一胞外域，所述第一胞外域包含源自CD3 epsilon结构域的胞外域，(2)第一跨膜域，(3)第一胞内结构域；所述第二CD3重组蛋白包含：(1)第二胞外域，所述第二胞外域包含源自选自CD3 gamma结构域和CD3 delta结构域中任意一种的胞外域，(2)第二跨膜域，(3)第二胞内结构域，且，其不表达T细胞受体(TCR)。
在某些实施方式中，所述的改造的免疫细包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、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CD3 epsilon结构域的胞外域包含如SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述第二胞外域包含源自CD3 gamma结构域的胞外域。
在某些实施方式中，所述CD3 gamma结构域的胞外域包含如SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述第二胞外域包含源自CD3 delta结构域的胞外域。
在某些实施方式中，所述CD3 delta结构域的胞外域包含如SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述第一跨膜域和所述第二跨膜域相同或不同。
在某些实施方式中，所述跨膜域不包含源自CD3的跨膜域
在某些实施方式中，所述跨膜域包含源自选自下组任意一种蛋白的跨膜域：CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在某些实施方式中，所述跨膜域包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述第一胞内结构域和所述第二胞内结构域相同或不同。
在某些实施方式中，所述第一胞内结构域和所述第二胞内结构域中的至少一种包含共刺激结构域和/或信号转导结构域。
在某些实施方式中，所述第一胞内结构域中包含的共刺激结构域和所述第二胞内结构域中包含的共刺激结构域相同或不同。
在某些实施方式中，所述共刺激结构域包含源自选自下组任意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。
在某些实施方式中，所述共刺激结构域包含如SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述第一胞内结构域中包含的信号转导结构域和所述第二胞内结构域中包含的信号转导结构域相同或不同。
在某些实施方式中，所述信号转导结构域包含至少包含一个免疫受体酪氨酸活化基序(ITAM)。
在某些实施方式中，所述信号转导结构域包含源自选自下组任意一种或多种蛋白的信号转导结构域：CD3zeta、CD3delta、CD3gamma、CD3ε、CD79a、CD79b、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10和DAP-12。
在某些实施方式中，所述信号转导结构域包含如SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，所述胞外域和跨膜域之间还包含铰链区。
在某些实施方式中，所述铰链区包含源自选自下组任意一种或多种蛋白的铰链区：CD8α、CD28、4-1BB、CD4、CD27、CD7和PD-1。
在某些实施方式中，所述铰链区包含SEQ ID NO:6中任一项所示的氨基酸序列。
在某些实施方式中，所述第一胞内结构域和所述第二胞内结构域中的一种包含至少两个氨基酸的肽段。
在某些实施方式中，所述第一CD3重组蛋白包括源自CD3 epsilon结构域的胞外域、源自CD28的跨膜区、源自CD28的胞内域和源自CD3 zeta的胞内域。在某些实施方式中，所述第一CD3重组蛋白包括SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述第二CD3重组蛋白包括源自CD3 gamma结构域的胞外域、源自CD28的跨膜区和源自CD3 gamma的胞内域。
在某些实施方式中，所述第二CD3重组蛋白包括SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述第二CD3重组蛋白包括源自CD3 gamma结构域的胞外域、源自CD28的跨膜区和所述肽段。
在某些实施方式中，所述第二CD3重组蛋白包括SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述的改造的免疫细胞还包含第三CD3重组蛋白，所述第三CD3重组蛋白包含：(1)第三胞外域，所述第三胞外域包含源自选自CD3 gamma结构域或CD3 delta结构域中任意一种的胞外域，(2)第三跨膜域，和(3)第三胞内结构域。
在某些实施方式中，所述第三胞外域与所述第二胞外域相同或不同。
在某些实施方式中，所述第三跨膜域与所述第一跨膜域和/或所述第二跨膜域相同或不同。
在某些实施方式中，述第三胞内结构域与所述第一胞内结构域和/或第二胞内结构域相同或不同。
在某些实施方式中，所述的改造的免疫细胞的主要组织相容性复合体(MHC)的表达和 /或活性下调。
在某些实施方式中，所述MHC复合体包括B2M。
在某些实施方式中，所述的改造的免疫细胞包含嵌合抗原受体(CAR)和/或嵌合自身抗体受体(CAAR)。
另一方面，本申请提供了药物组合物，其包含所述的改造的免疫细胞和药学上可接受的佐剂。
在某些实施方式中，所述的药物组合物包括抗体。
在某些实施方式中，所述抗体能够识别和/或结合所述CD3抗体受体复合物。
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细胞。
在某些实施方式中，所述双特异性抗体能够识别和/或结合靶细胞表面的受体。
在某些实施方式中，所述靶细胞为肿瘤细胞。
在某些实施方式中，所述靶细胞表面的受体选自下组中的一种：CD19、CD2、CD3、CD4、CD5、CD7、CD8、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD38、CD40、CD44V6、CD47、CD52、CD56、CD57、CD58、CD79b、CD80、CD86、CD81、CD123、CD133、CD137、CD151、CD171、CD276、CLL1、B7H4、BCMA、VEGFR-2、EGFR、GPC3、PMSA、CEACAM6、c-Met、EGFRvIII、ErbB2/HER2、ErbB3、HER-2、HER3、ErbB4/HER-4、EphA2、IGF1R、GD2、O-乙酰基GD2、O-乙酰基GD3、GHRHR、GHR、Flt1、KDR、Flt4、Flt3、CEA、CA125、CTLA-4、GITR、BTLA、TGFBR1、TGFBR2、TGFBR1、IL6R、gp130、Lewis、TNFR1、TNFR2、PD1、PD-L1、PD-L2、PSCA、HVEM、MAGE-A、MSLN、NY-ESO-1、PSMA、RANK、RORl、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、MUC1、MUC16、TCRα、TCRβ、TLR7、TLR9、PTCH1、WT-1、Robol、卷曲受体(Frizzled)、OX40、Notch-1-4、APRIL、CS1、MAGE3、Claudin 18.2、叶酸受体α、叶酸受体β、GPC2、CD70、BAFF-R和TROP-2。
另一方面，本申请提供了核酸分子，其编码所述的改造的免疫细胞中的CD3抗体受体复合物。
另一方面，本申请提供了载体，其包含所述的核酸分子。
在某些实施方式中，所述载体为病毒载体。
在某些实施方式中，所述载体为慢病毒载体。
另一方面，本申请提供了细胞，其包含所述的核酸分子和/或所述的载体。
另一方面，本申请提供了所述的改造的免疫细胞和/或所述的药物组合物在制备药物中的 用途，所述药物用于治疗肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和非实体瘤。
在某些实施方式中，所述肿瘤选自以下组：淋巴瘤、白血病和多发性骨髓瘤。
另一方面，本申请提供了治疗肿瘤的方法，所述方法包括向有需要的受试者施用所述的改造的免疫细胞、所述的药物组合物。
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：淋巴瘤、白血病和多发性骨髓瘤。。
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显示的是本申请所述改造的免疫细胞联合双特异性抗体杀伤靶细胞示意图。
图2显示的是本申请所述改造的免疫细胞表达CD3抗体受体复合物。
图3显示的是CD3抗体受体复合物可以在TCR KO的人原代T细胞中被CD3抗体UCHT1识别。
图4显示的是CD3抗体受体复合物可以在TCR KO的人原代T细胞中被CD3抗体HIT3a识别。
图5显示的是CD3抗体受体复合物在TCR KO的人原代T细胞中不能被CD3抗体SP34-2识别
图6显示的是CD3抗体受体复合物可以在TCR KO的人原代T细胞中被CD3抗体OKT3识别。
图7显示的是本申请所述的改造的免疫细胞被CD3抗体激活表达细胞激活标签CD137。
图8显示的是本申请所述的改造的免疫细胞被抗CD3抗CD19双特异性抗体介导的肿瘤细胞激活。
图9显示的是本申请所述的改造的免疫细胞通过抗CD3抗CD19双特异性抗体介导杀伤 肿瘤细胞。
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“CD3抗体”通常是指能够特异性识别CD3亚基(例如，CD3 epsilon、CD3 gamma、CD3 delta，或上述的复合物)的抗体，该抗体可以为只能够识别CD3的单克隆抗体，或者是可以同时识别CD3和其他靶点的多靶点抗体。
在本申请中，术语“CD3抗体受体复合物”通常是指能够被CD3抗体识别的受体，其可以至少包含2种(例如，3种)CD3亚基(例如，CD3 epsilon和CD3 delta，或CD3 epsilon和CD3 gamma)。CD3抗体受体复合物由至少2条(例如，3条、4条、5条、6条或更多条)CD3重组蛋白组成，对于所述CD3重组蛋白可包含CD3亚基(例如，CD3 epsilon、CD3 gamma和/或CD3 delta)的胞外域、跨膜域和胞内结构域。CD3 epsilon和CD3 gamma或者CD3 delta胞外域结合后的构象接近于CD3 epsilon异二聚体在TCR复合物中的天然构象，包括大多数CD3抗体如UCHT1，OKT3等识别的表位。单独CD3 epsilon胞外域形成的构象不包括大多数CD3抗体克隆的结合表位。
在本申请中，术语“第一CD3重组蛋白”通常是指包含源自CD3(例如，CD3 epsilon)的胞外域、跨膜域和胞内结构域的重组蛋白。所述CD3抗体受体复合物可包含1条或多条(例如，2条、3条、4条或更多条)第一CD3重组蛋白。
在本申请中，术语“第一胞外域”通常是指第一CD3重组蛋白的胞外域部分，所述第一胞外域可包含源自CD3 epsilon结构域的胞外域。
在本申请中，术语“第二CD3重组蛋白”通常是指包含源自CD3(例如，CD3 delta和或CD3 gamma)的胞外域、跨膜域和胞内结构域的重组蛋白。所述CD3抗体受体复合物可包含1条或多条(例如，2条、3条、4条或更多条)第二CD3重组蛋白。
在本申请中，术语“第二胞外域”通常是指第二CD3重组蛋白的胞外域部分，所述第二胞外域可包含源自CD3 delta和或CD3gamma结构域的胞外域。
在本申请中，术语“第三CD3重组蛋白”通常是指包含源自CD3(例如，CD3 delta和或CD3 epsilon)的胞外域、跨膜域和胞内结构域的重组蛋白。所述CD3抗体受体复合物可包含1条或多条(例如，2条、3条、4条或更多条)第三CD3重组蛋白。
在本申请中，术语“第三胞外域”通常是指第三CD3重组蛋白的胞外域部分，所述第三胞外域可包含源自CD3 delta和或CD3 epsilon结构域的胞外域。所述第三胞外域可以与所述第二胞外域源自相同或不同的CD3亚基(例如，CD3 delta和或CD3 gamma)。
在本申请中，术语“抗体”通常是指一种能够特异性识别和/或中和特定抗原的多肽分子。例如，抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球蛋白，并且包括任何包含其抗原结合部分的分子。术语“抗体”包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于人抗体、人源化抗体、嵌合抗体、单域抗体(例如，dAb)，单链抗体(例如，scFv)，以及与抗原结合的抗体片段(例如，Fab、Fab’和(Fab)2片段)。术语“抗体”还包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及本申请所述的任何与抗原结合的抗体片段及其衍生物。每条重链可由重链可变区(VH)和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
在本申请中，术语“跨膜域”通常是指细胞表面蛋白中一段跨越细胞膜的序列，其可以包含疏水性alpha螺旋。跨膜域可以与细胞内信号转导结构域相连接，起着传递信号的作用在本申请中，跨膜域可以源自任意的I型、II型或III型跨膜蛋白。在本申请中，跨膜域可不包含源自CD3(例如，CD3 epsilon、CD3 gamma和/或CD3 delta)的跨膜蛋白。
在本申请中，术语“免疫受体酪氨酸活化基序(ITAM)”通常是指是一个由十多个氨基酸构成的保守序列，常出现在免疫系统某些细胞的跨膜蛋白的胞内区。TAM是免疫细胞的重要转导信号。因此，他们常出现在的重要的细胞信号分子的胞内区，如T细胞受体复合物的CD3和ζ链，B细胞受体复合物中CD79α和β链，与某些Fc受体。当这些受体分子与他们的配体发生相互作用时，胞内ITAM上的酪氨酸残基会磷酸化，磷酸化的ITAM可与带有SH2结构域的包内游离蛋白结合，使免疫细胞信号传送到下游的信号分子。
在本申请中，术语“嵌合抗原受体(Chimeric Antigen Receptor，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，其可包括抗原(例如，肿瘤特异性抗原和/或肿瘤相关抗原)结合结构域、跨膜结构域、共刺激结构域和胞内信号结构域。在本申请中，所述CAR可以基于抗体的抗原(例如CD70)特异性与T细胞受体活化胞内结构域组合在一起。经遗传修饰表达CAR 的T细胞可以特异地识别和消除表达靶抗原的恶性细胞。关于CAR和CAR-T细胞的描述，可参见例如Sadelain M,Brentjens R,Rivi`ere I.The basicprinciples of chimeric antigen receptor design.Cancer Discov.2013；3(4):388-398；Turtle CJ,Hudecek M,Jensen MC,Riddell SR.Engineered T cells for anti-cancer therapy.Curr Opin Immunol.2012；24(5):633-639；Dotti G,Gottschalk S,Savoldo B,Brenner MK.Design and development of therapies using chimeric antigen receptor-expressing T cells.Immunol Rev.2014；257(1):107-126；以及WO2013154760、WO2016014789。
在本申请中，术语“嵌合自身抗体受体(CAAR)”通常是指包含自身抗原、能够被自身抗体识别的蛋白，英文名称为chimeric autoantibody receptors。CAAR可以指导经遗传修饰表达CAAR的免疫细胞攻击表达能够识别该抗原的抗体的B细胞。
在本申请中，术语“双特异性抗体”通常是指在单个抗体分子内具有两种不同抗原的结合位点的抗体。例如，其中一种抗原可以是CD3。
在本申请中，术语“肽段”通常是指一段至少由两个(例如，四个、五个、六个或更多各)氨基酸构成的多肽。其可包括任何具有氨基功能性和酸功能性并且包含天然存在的氨基酸聚合物在内的全部分子，包括天然氨基酸和人工氨基酸。
在本申请中，术语“共刺激结构域”通常是指可以提供免疫共刺激分子的胞内结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。
在本申请中，术语“铰链区”通常是指胞外域(例如，CD3胞外域)和跨膜区之间的连接区。
在本申请中，术语“信号传导结构域”通常是指位于细胞内部能够转导信号的结构域。在本申请中，所述胞内信号传导结构域可以将信号传导至细胞内。通常，信号传导结构域为用于指导蛋白质寻靶的任何一段连续的氨基酸序列。在某些情形中，信号传导结构域可以源自CD3ζ。CD3ζ可以与T细胞受体亚基和CD3-gamma,-delta,和-epsilon形成T细胞受体-CD3复合体。CD3ζ含有三个ITAM基序，ITAM序列介导TCR的胞内信号激活。ζ链是一种受体激活的蛋白酪氨酸激酶底物，当TCR受体与多肽MHC复合物结合后，ζ链可以很快发生酪氨酸磷酸化，参与淋巴细胞活化信号的转导。因此CD3ζ在抗原识别和TCR信号转导中起着关键作用。
在本申请中，术语“药学上可接受的佐剂”通常是指不干扰活性成分的生物活性的有效性的一种或多种非毒性材料。这类制剂常规地可以含有盐、缓冲剂、防腐剂、相容的载体、以及任选地其他治疗剂。这类药学上可接受的制剂还可以含有适合于给予人的相容的固体或 液体填料、稀释剂或包封物质。可以用于在此所描述的配制品中的其他设想的载体、赋形剂、和/或添加剂包括：例如，调味剂、抗微生物剂、增甜剂、抗氧化剂、抗静电剂、脂质、蛋白质赋形剂(如血清白蛋白、明胶、酪蛋白)、成盐平衡离子(如钠)等等。
在本申请中，术语“免疫细胞”通常是指参与免疫应答，例如促进免疫效应应答的细胞。免疫细胞的示例包括但不限于T细胞、B细胞、天然杀伤(NK)细胞、肥大细胞、粒细胞、单核细胞、淋巴细胞以及巨噬细胞。该术语还包括工程化的免疫细胞，如通过将DNA或RNA形式的外源遗传物质加入细胞的总遗传物质而被基因修饰的免疫细胞。
在本申请中，所述“载体”通常是指能够在合适的宿主中自我复制的核酸分子，用以将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。载体可涵盖除转基因插入序列和主链以外的额外特征：启动子、遗传标记、抗生素抗性、报告基因、靶向序列、蛋白质纯化标签。称为表达载体(表达构建体)的载体具体地讲用于在靶细胞中表达转基因，且通常具有控制序列。本申请所述的载体可以是表达载体，可包括病毒载体(慢病毒载体和/或逆转录病毒载体)、噬菌体载体、噬菌粒、粘粒、cosmid、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)和/或质粒。
在本申请中，术语“治疗”通常是指：(i)预防可能易患疾病、病症和/或病状、但尚未诊断出患病的患者出现该疾病、病症或病状；(ii)抑制该疾病、病症或病状，亦即遏制其发展；以及(iii)缓解该疾病、病症或病状，亦即使得该疾病、病症和/或病状和/或与该疾病、病症和/或病状相关联的症状消退。
在本申请中，术语“主要组织相容性复合体(MHC)”通常是指位于细胞表面的帮助免疫系统识别异物的一系列的蛋白质，MHC主要包含I类MHC分子和II类MHC分子。I类MHC分子可以横跨生物体几乎所有细胞的膜，而II类分子则通常存在于免疫细胞上。其中I类MHC分子也称为I类主要组织相容复合体，由非共价键连接的两条肽链组成的异源二聚体糖蛋白；其中一条称为重链，结构呈多态性，另一条为轻链或称为β2微球蛋白(B2M)。功能上，I类MHC分子会呈递胞内所降解的非自身蛋白的多肽，从而激活免疫系统。人类的I类MHC分子分为经典的HLA分子(HLA-A、HLA-B、HLA-C)和非经典的HLA分子(HLA-E、HLA-G、HLA-F)。在本申请中，所述的改造的免疫细胞可以不表 达有活性的MHC，所述“不表达有活性的MHC”可包括表达的MHC失去激活免疫系统的活性和/或细胞表面缺失I类MHC分子(例如，缺失HLA-A/B/C/E/F/G)。在某些情形中，可以通过编辑B2M或相应的重链基因来使细胞表面缺失I类MHC分子。
在本申请中，术语“B2M”通常是指β-2微球蛋白，通常是指I类MHC分子的轻链，因此是主要组织相容性复合体(MHC)不可缺少的部分。在人类基因组中，B2M被位于15号染色体上的b2m基因编码，而其他MHC基因在6号染色体上以基因簇的形式存在。人类的B2M蛋白有119个氨基酸(见UniProt数据库编码P61769)。在缺乏β-2微球蛋白的小鼠模型中可以说明，B2M对I类MHC分子在细胞表面的呈递、多肽结合槽的稳定性是必须的。I类MHC分子存在于人体所有的有核细胞表面，MHC的错配会引起免疫排斥，导致移植物的破坏，而通过敲除Β2Μ基因来去除细胞表面的I类MHC分子可以防止错配的发生。
在本申请中，术语“CD3”通常是指CD3蛋白质多亚基复合体，CD3蛋白质多亚基复合体由6个不同的多肽链(亚基)构成。在哺乳动物中，CD3的多肽链可包含一条CD3 gamma(γ)链，一条CD3 gamma(δ)链，两条CD3 epsilon(ε)链和两条CD3 zeta(ζ)链。
在本申请中，术语“CD3”指来自任何脊椎动物来源的任何天然CD3，包括哺乳动物，诸如灵长动物(例如人)，非人灵长动物(例如食蟹猴)和啮齿动物(例如小鼠和大鼠)。该术语涵盖“全长”和未加工的CD3蛋白质以及源自细胞中加工的任何形式的蛋白质或一种或多种CD3链(多肽)(例如成熟多肽)。该术语还涵盖CD3的天然发生变体和同等型，例如剪接变体或等位变体。
同样的，本申请的术语“CD3 epsilon”、“CD3 gamma”、“CD3 gamma”和/或“CD3 zeta”指来自任何脊椎动物来源的任何天然CD3，包括哺乳动物，诸如灵长动物(例如人)，非人灵长动物(例如食蟹猴)和啮齿动物(例如小鼠和大鼠)。“CD3 epsilon”、“CD3 gamma”、“CD3 gamma”和/或“CD3 zeta”分别涵盖“全长”和未加工的CD3 epsilon”、“CD3 gamma”、“CD3 gamma”和/或“CD3 zeta”蛋白质以及源自细胞中加工的任何形式的CD3链(多肽)(例如成熟多肽)。该术语还涵盖CD3链的天然发生变体和同等型，例如剪接变体或等位变体。例如，示例性的CD3delta的氨基酸序列可参见UniProt数据库登录号P04234，示例性的CD3 epsilon的氨基酸序列可参见UniProt数据库登录号P07766，和示例性的CD3 gamma的氨基酸序列可参见UniProt数据库登录号P09693。
在本申请中，术语“T细胞受体(TCR)”通常是指参与T细胞识别抗原后激活的跨膜蛋白复合物。TCR是由两条不同的蛋白亚基形成的异源二聚体。在人类体内，有95％的T细胞 表达一条alpha(α)链和一条beta(β)链。剩余的5％表达gamma(γ)和delta(δ)链。TCR分子的每条链可包含两个膜外结构域：可变区和恒定区。可变区可结合多肽/主要组织相容性复合物，α链和β链的可变区都含有三个互补决定区(CDRs)，负责抗原/MHC复合物的识别。恒定区靠近细胞膜，与跨膜区连接。TCR两个异源亚基胞外的恒定结构域都包含短的结合序列，该序列内有半胱氨酸残基。半胱氨酸残基之间形成二硫键从而使两个异源TCR亚基结合在一起。TCR跨膜域含有带正电的氨基酸，负责与CD3分子结合。TCR的胞内区很短，没有活性结构域。
TCR能识别与MHC分子结合的经过处理的多肽片段，由于识别需要MHC分子的呈递，也称为MHC限制性。当供体和受体的MHC分子不同时，TCR能够识别MHC的不同，并导致T细胞的激活和扩增，可能引起移植物抗宿主疾病(GvHD)。敲除TRAC基因可以去除TCRα链的表达，从而可以将TCR复合物从T细胞表面除去，因此能够防止TCR识别同种异体抗原而引起的移植物抗宿主疾病。
CD3多亚基复合体和TCR可以过非共价键形成功能性的复合体，称为TCR-CD3复合体。在形成TCR-CD3复合体的过程中，CD3 epsilon分子分别和CD3 gamma和CD3 delta形成异源二聚体，CD3 zeta与自身形成同源二聚体。一个TCR-CD3复合体包括一个CD epsilon:delta异源二聚体,一个CD epsilon:gamma异源二聚体和一个CD zeta:zeta同源二聚体和一个TCR alpha:beta异源二聚体。因此，这四种二聚体在一个TCR-CD3复合体中呈1:1:1:1的关系。CD3分子的跨膜区因为含有天冬氨酸残基而带有负电，从而使CD3可以与跨膜区带正电的一对TCR亚基结合。CD3 gamma、CD3 delta和CD3 epsilon分子是非常相关的免疫球蛋白超家族细胞膜蛋白，都包含单个胞外免疫球蛋白结构域，而CD3 zeta的胞外区非常短。CD3 gamma、CD3 delta和CD3 epsilon分子的胞内区都含有单个的保守区免疫受体酪氨酸激活基序(immunoreceptor tyrosine-based activation motif，ITAM)。CD3 zeta链含有三个ITAM基序。单个TCR-CD3复合物共含有10个ITAM基序，这些ITAM基序决定了TCR激活的程度。
在本申请中，术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换地使用，通常是指具有任何长度的氨基酸的聚合物。该聚合物可以是直链或支链的，它可以包含修饰的氨基酸，并且可以被非氨基酸中断。这些术语还涵盖已经被修饰的氨基酸聚合物。这些修饰可以包含：二硫键形成、糖基化、脂化(lipidation)、乙酰化、磷酸化、或任何其他操纵(如与标记组分结合)。术语“氨基酸”包括天然的和/或非天然的或者合成的氨基酸，包括甘氨酸以及D和L旋光异构体、以及氨基酸类似物和肽模拟物。
在本申请中，术语“多核苷酸”、“核苷酸”、“核苷酸序列”、“核酸”和“寡核苷酸”可互 换地使用，通常是指具有任何长度的核苷酸的聚合形式，如脱氧核糖核苷酸或核糖核苷酸、或其类似物。多核苷酸可具有任何三维结构，并且可以执行已知或未知的任何功能。以下是多核苷酸的非限制性实例：基因或基因片段的编码区或非编码区、根据连接分析定义的多个座位(一个座位)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、短干扰RNA(siRNA)、短发夹RNA(shRNA)、micro-RNA(miRNA)、核酶、cDNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针、和引物。多核苷酸可以包含一个或多个经修饰的核苷酸，如甲基化的核苷酸和核苷酸类似物。如果存在，可以在聚合物组装之前或之后进行核苷酸结构的修饰。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以在聚合后，如通过与标记的组分缀合来进一步修饰。
除了本文提到的特定蛋白质和核苷酸之外，本申请还可包括其功能性变体、衍生物、类似物、同源物及其片段。
术语“功能性变体”指与天然存在序列具有基本上同一的氨基酸序列或由基本上同一的核苷酸序列编码并能够具有天然存在序列的一种或多种活性的多肽。在本申请的上下文中，任何给定序列的变体是指其中残基的特定序列(无论是氨基酸或核苷酸残基)已经经过修饰而使得所述多肽或多核苷酸基本上保留至少一种内源功能的序列。可以通过天然存在的蛋白质和/或多核苷酸中存在的至少一个氨基酸残基和/或核苷酸残基的添加、缺失、取代、修饰、替换和/或变异来获得变体序列，只要保持原来的功能活性即可。
在本申请中，术语“衍生物”通常是指本申请的多肽或多核苷酸而言包括自/对序列的一个(或多个)氨基酸残基的任何取代、变异、修饰、替换、缺失和/或添加，只要所得的多肽或多核苷酸基本上保留其至少一种内源功能。
在本申请中，术语“类似物”通常对多肽或多核苷酸而言，包括多肽或多核苷酸的任何模拟物，即拥有该模拟物模拟的多肽或多核苷酸的至少一种内源功能的化学化合物。
通常，可以进行氨基酸取代，例如至少1个(例如，1、2、3、4、5、6、7、8、9、10或20个以上)氨基酸取代，只要经修饰的序列基本上保持需要的活性或能力。氨基酸取代可包括使用非天然存在的类似物。
用于本申请的蛋白质或多肽也可以具有氨基酸残基的缺失、插入或取代，所述氨基酸残基产生沉默的变化并导致功能上等同的蛋白质。可以根据残基的极性、电荷、溶解性、疏水性、亲水性和/或两性性质的相似性进行有意的氨基酸取代，只要保留内源性功能即可。例如，带负电荷的氨基酸包括天冬氨酸和谷氨酸；带正电荷的氨基酸包括赖氨酸和精氨酸；并且含具有相似亲水性值的不带电极性头基的氨基酸包括天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸和 酪氨酸。
在本申请中，术语“同源物”通常是指与野生型氨基酸序列和野生型核苷酸序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。
为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
本申请开发了一种可调控的细胞疗法，该疗法充分利用了双特异性抗体疗法和细胞疗法的优势，为一种双特异性抗体疗法和细胞疗法的联合疗法。本申请使用的T细胞为表达一种CD3抗体受体并且经过基因敲除的通用型T细胞。CD3抗体受体由两种重组CD3蛋白分子构成。该T细胞表达一种可以被双特异性抗体识别的CD3抗体受体，相对于类似的嵌合抗体受体疗法的不同之处在于，本申请CD3抗体受体可以被双特异性抗体识别，并且表达不依赖于TCR复合物，可以独立直接表达于T细胞表面。
本申请提供了如下技术方案：
1.一种改造的淋巴细胞，该淋巴细胞缺失TCR分子，该淋巴细胞表达CD3抗体受体复合物，该CD3抗体受体复合物包含至少两种CD3重组蛋白；其中第一种CD3重组蛋白包含：
a.包含CD3 epsilon结构域的胞外域；
b.跨膜域；
c.胞内结构域；和
其中第二种CD3重组蛋白包含：
d.包含CD3 delta和/或CD3 gamma结构域的胞外域；
e.跨膜域；
f.胞内结构域；
其中，胞内结构域由胞内共刺激信号传导结构域和胞内信号转导结构域组成，或者胞内结构域是至少两个氨基酸的肽段；其中第一种CD3重组蛋白和第二种CD3重组蛋白中的至少一种胞内结构域，由胞内共刺激信号传导结构域和胞内信号转导结构域组成。
2.技术方案1所述的淋巴细胞，其中该淋巴细胞是T细胞、B细胞、NK细胞或巨噬细胞。
3.技术方案2所述的淋巴细胞，其中该淋巴细胞是T细胞。
4.前述任一项技术方案所述的淋巴细胞，其中a.胞外域包含SEQ ID No:1所示的CD3 epsilon的胞外域，或其变体。
5.技术方案4所述的淋巴细胞，其中a.胞外域为SEQ ID No:1所示的CD3 epsilon的胞外域。
6.前述任一项技术方案所述的淋巴细胞，其中d.胞外域包含CD3 gamma结构域的胞外域。
7.技术方案6所述的淋巴细胞，其中d.胞外域包含SEQ ID No:2所示的CD3 gamma的胞外域，或其变体。
8.技术方案7所述的淋巴细胞，其中d.胞外域为SEQ ID No:2所示的CD3 gamma的胞外域。
9.前述任一项技术方案所述的淋巴细胞，其中d.胞外域包含CD3 delta结构域的胞外域，或其变体。
10.技术方案9所述的淋巴细胞，其中d.胞外域包含SEQ ID No:4所示的CD3 delta的胞外域，或其变体。
11.技术方案10所述的淋巴细胞，其中d.胞外域为SEQ ID No:4所示的CD3 delta的胞外域。
12.前述任一项技术方案所述的淋巴细胞，其中跨膜域b和e包含以下至少一项：CD8α的跨膜域、CD28的跨膜域、4-1BB的跨膜域、CD4的跨膜域、CD27的跨膜域、CD7的跨膜 域、PD-1的跨膜域、TRAC的跨膜域和TRBC的跨膜域。
13.技术方案12所述的淋巴细胞，其中CD28的跨膜域包含SEQ ID No:7所示的CD28的跨膜域，或其变体。
14.前述任一项技术方案所述的淋巴细胞，其中胞内共刺激信号传导结构域包含以下至少一项：CD28、4-1BB、CD40L、TIM1、CD226、DR3、SLAM、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD27、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT和DAP12中的共刺激信号传导区及其组合组成的共刺激分子。
15.技术方案14所述的淋巴细胞，其中胞内共刺激信号传导结构域包含SEQ ID No:8所示的CD28的共刺激信号传导区，或其变体。
16.技术方案14所述的淋巴细胞，其中胞内共刺激信号传导结构域包含4-1BB的共刺激信号传导区，或其变体。
17.前述任一项技术方案所述的淋巴细胞，其中的胞内信号转导结构域包含以下至少一项：CD3zeta激活区、CD3delta激活区、CD3gamma激活区、FceRIγ激活区、FceRI激活区、immunoglobulin alpha(Iga)激活区、Igbeta激活区、bovine leukemia virus gp30激活区、Ep-stein-Barr virus(EBV)LMP2A激活区、simian immunodeficiencyvirus PBj14 Nef激活区、HSKV激活区、DAP-12激活区、至少包含一个ITAM(酪氨酸激活基序)的结构域，及以上结构域组合形成的结构域。
18.技术方案17所述的淋巴细胞，其中的CD3 zeta激活区包含SEQ ID No:9所示的序列，或其变体。
19.前述任一项技术方案所述的淋巴细胞，其中CD3重组蛋白的胞外域和跨膜区之间还具有铰链区。
20.技术方案19所述的淋巴细胞，其中的铰链区包含以下至少一项：CD8α、CD28、4-1BB、CD4、CD27、CD7和PD-1的铰链区。
21.前述任一项技术方案所述的淋巴细胞，其中胞内结构域是至少为两个氨基酸的肽段。
22.前述任一项技术方案所述的淋巴细胞，其中第一种CD3重组蛋白选自下述重组蛋白中的一种：CD3ε胞外域-CD8α铰链区-CD8α跨膜区-4-1BB共刺激信号传导区-CD3ζ激活区，CD3ε胞外域-CD8α跨膜区-4-1BB共刺激信号传导区-CD3ζ激活区，CD3ε胞外域-CD28铰链区-CD28跨膜区-CD28共刺激信号传导区-CD3ζ激活区，和CD3ε胞外域-CD28跨膜区-CD28共刺激信号传导区-CD3ζ激活区。
23.技术方案22所述的淋巴细胞，其中第一种CD3重组蛋白具有SEQ ID No:10所示的 序列，或其变体。
24.前述任一项技术方案所述的淋巴细胞，其中第二种CD3重组蛋白选自下述重组蛋白中的一种：CD3γ胞外区-CD8α铰链区-CD8α跨膜区-CD3γ共刺激信号传导区，CD3γ胞外区-CD8α跨膜区-CD3γ共刺激信号传导区，CD3γ胞外区-CD28铰链区-CD28跨膜区-CD3γ共刺激信号传导区，和CD3γ胞外区-CD28跨膜区-CD3γ共刺激信号传导区，CD3γ胞外区-CD28铰链区-CD28跨膜区-肽段，其中的肽段可以为至少2个、4个或者至少6个氨基酸的肽段。
25.技术方案24所述的淋巴细胞，其中第二种CD3重组蛋白具有SEQ ID No:11和SEQ ID No:12所示的序列，或其变体。
26.前述技术方案1-25任一项所述的淋巴细胞，其中第二种CD3重组蛋白选自下述重组蛋白中的一种：CD3δ胞外区-CD8α铰链区-CD8α跨膜区-CD3δ共刺激信号传导区，CD3δ胞外区-CD8α跨膜区-CD3δ共刺激信号传导区，CD3δ胞外区-CD28铰链区-CD28跨膜区-CD3δ共刺激信号传导区，和CD3δ胞外区-CD28跨膜区-CD3δ共刺激信号传导区。
27.前述技术方案任一项所述的淋巴细胞，该淋巴细胞缺失MHC分子。
28.前述任一项技术方案所述的淋巴细胞，其中第一种CD3重组蛋白的胞内结构域由胞内共刺激信号传导结构域和胞内信号转导结构域组成。
29.前述任一项技术方案所述的淋巴细胞，其中第二种CD3重组蛋白的胞内结构域由胞内共刺激信号传导结构域和胞内信号转导结构域组成。
30.前述技术方案任一项所述的淋巴细胞，该CD3抗体受体复合物包含三种CD3重组蛋白，第一种CD3重组蛋白包含：
a.包含CD3 epsilon结构域的胞外域；
b.跨膜域；
c.胞内结构域；和
其中第二种CD3重组蛋白包含：
d.包含CD3 delta结构域的胞外域；
e.跨膜域；
f.胞内结构域；和
其中第三种CD3重组蛋白包含：
g.包含CD3 gamma结构域的胞外域；
h.跨膜域；
i.胞内结构域；
其中，胞内结构域由胞内共刺激信号传导结构域和胞内信号转导结构域组成，或者胞内结构域是至少两个氨基酸的肽段；其中三种CD3重组蛋白中的至少一种胞内结构域，由胞内共刺激信号传导结构域和胞内信号转导结构域组成。
31.一种药物组合物，包含技术方案1-30任一项所述的淋巴细胞，以及一种双特异性抗体，该抗体能与淋巴细胞表面的CD3抗体受体复合物结合，并能同时与靶细胞表面的受体结合。
32.技术方案31所述的药物组合物，其中该靶细胞的受体选自下述中的一种：CD19、CD20、CD22、CD123、CD33、BCMA、IL13R alpha、PSMA、EGFR、HER2、Mesothelin和Claudin18.2。
33.技术方案31或32所述的药物组合物，其中所述的双特异性抗体能与淋巴细胞表面的CD3抗体受体复合物结合，并能同时与靶细胞表面的CD19受体结合。
34.技术方案31-33任一项所述的药物组合物，其中所述的靶细胞为肿瘤细胞。
35.技术方案31-34任一项所述的药物组合物，其中所述的淋巴细胞是T细胞、B细胞、NK细胞或巨噬细胞。
36.技术方案1-24任一项所述的淋巴细胞在制备用于治疗肿瘤的药物中的应用。
37.技术方案30所述的应用，其中的药物还包括一种双特异性抗体，该抗体能与淋巴细胞表面的CD3抗体受体复合物结合，并能同时与靶细胞表面的受体结合。
38.一种疾病的治疗方法，该方法包括给受试者体内注入：
c.有效量的技术方案1-30任一项所述的淋巴细胞，和
d.有效量的双特异性抗体，该抗体能与淋巴细胞表面的CD3抗体受体复合物结合，并能同时与靶细胞表面的受体结合。
39.技术方案38所述的方法，其中该靶细胞的受体选自下述中的一种：CD19、CD20、CD22、CD123、CD33、BCMA、IL13R alpha、PSMA、EGFR、HER2、Mesothelin和Claudin18.2。
40.技术方案39所述的方法，其中所述的靶细胞为肿瘤细胞。
CD3抗体受体复合物
本申请提供了一种改造的免疫细胞，其可包含CD3抗体受体复合物，所述CD3抗体受体复合物包含第一CD3重组蛋白和第二CD3重组蛋白。
在本申请中，所述第一CD3重组蛋白可包含(1)第一胞外域，(2)第一跨膜域，和(3)第一胞内结构域；所述第二CD3重组蛋白可包含(1)第二胞外域，(2)第二跨膜域，和(3)第二胞内结构域。
在某些情形中，所述第一胞外域可包含源自CD3 epsilon结构域的胞外域，例如，所述第一胞外域可包含如SEQ ID NO:1所示的氨基酸序列。例如，所述第一胞外域可包含与SEQ ID NO:1所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在某些情形中，所述第二胞外域可包含源自CD3 gamma结构域的胞外域，例如，所述第二胞外域可包含如SEQ ID NO:2所示的氨基酸序列。例如，所述第二胞外域可包含与SEQ ID NO:2所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在某些情形中，所述第二胞外域可包含源自CD3 delta结构域的胞外域，例如，所述第二胞外域可包含如SEQ ID NO:4所示的氨基酸序列。例如，所述第二胞外域可包含与SEQ ID NO:4所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在本申请中，所述CD3重组蛋白(例如，第一CD3重组蛋白和/或第二CD3重组蛋白)可包含跨膜域(例如，所述第一跨膜域和/或第二跨膜域)。所述跨膜域(例如，所述第一跨膜域和/或第二跨膜域)可以源自任意的I型跨膜蛋白，只要其不是CD3的跨膜域即可。在本申请中，示例性的所述跨膜域(例如，所述第一跨膜域和/或第二跨膜域)可包含但不限于源自下组蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、T细胞受体的ζ链、CD3ε、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154、SLAM、和其他任何不依赖于TCR表达的跨膜域，或者以上跨膜域的突变体。
例如，所述跨膜域(例如，所述第一跨膜域和/或第二跨膜域)可以是源自人CD28的跨膜域。例如，所述第一跨膜域可包含如SEQ ID NO:7所示的氨基酸序列。例如，所述第一跨膜域可包含与SEQ ID NO:7所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在本申请中，所述CD3重组蛋白可包含胞内结构域(例如，第一胞内结构域和/或第二胞内结构域)。在某些情形中，所述胞内结构域(例如，第一胞内结构域和/或第二胞内结构域)可包含胞内共刺激结构域和/或胞内信号传导结构域。
在本申请中，所述胞内信号转导结构域可以包含至少有一个ITAM基序的结构域。示例性的信号传导结构域可源自选自下组的信号转导结构域，包括但不限于CD3zeta、CD3delta、 CD3gamma、CD3ε、CD79a、CD79b、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒gp30激活区、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10和DAP-12，及上述的变体。
例如，所述信号转导结构域可以为来自CD3zeta胞内域的信号传导结构域。例如，所述来自CD3zeta胞内域的信号传导结构域可以包含SEQ ID NO:9所示的信号转导结构域。例如，所述来自CD3zeta胞内域的信号传导结构域可包含与SEQ ID NO:9所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
所述CD3抗体受体复合物受到刺激后，可以将激活信号传输到细胞内部。有时候，信号转导结构域的激活并不足以提供足够的活化信号，还需要共刺激结构域提供刺激信号。所述共刺激结构域可以包括但不限于下组：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中的共刺激信号传导区及其组合组成的共刺激分子。
例如，所述共刺激结构域可以是来自人CD28胞内域的共刺激结构域。例如，所述人CD28胞内域的共刺激结构域可以包含SEQ ID NO:8所示的共刺激结构域。例如，所述人CD28胞内域的共刺激结构域可包含与SEQ ID NO:8所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
例如，所述胞内结构域(例如，第一胞内结构域和/或第二胞内结构域)可包含来自上述人CD28胞内域的共刺激结构域和上述来自CD3zeta胞内域的信号传导结构域。
又例如，所述胞内结构域(例如，第一胞内结构域和/或第二胞内结构域)可包含来自CD3 gamma的胞内结构域。例如，所述CD3 gamma的胞内结构域可包含如SEQ ID NO:3所示的氨基酸序列。例如，所述CD3 gamma的胞内结构域可包含与SEQ ID NO:3所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在某些情形中，所述胞内结构域(例如，第一胞内结构域和/或第二胞内结构域)可以包含至少两个(例如，至少四个、至少六个或更多个)氨基酸的肽段。在某些情形中，所述胞内结构域可以为至少两个(例如，至少四个、至少六个或更多个)氨基酸的肽段。例如，所 述肽段可包含如SEQ ID NO:19所示的氨基酸序列。
在本申请的所述CD3抗体受体复合物中，对于第一CD3重组蛋白和第二CD3重组蛋白，除胞外区以外，其他部分(例如，跨膜域、胞内结构域)可以相同或不同。例如，第一CD3重组蛋白的第一跨膜域和第二CD3重组蛋白的第二跨膜域可以相同或不同，第一CD3重组蛋白的第一胞内结构域和第二CD3重组蛋白的第二胞内结构域可以相同或不同。例如，所述第一胞内结构域的信号转导结构域和第二胞内结构域的信号转导结构域可以相同或不同。例如，所述第一胞内结构域的共刺激结构域和第二胞内结构域的共刺激结构域可以相同或不同。只要所述第一胞内结构域和所述第二胞内结构域中的至少一个(例如，至少两个、三个、四个或更多个)包含共刺激结构域和/或信号转导结构域，或者，只要能够为抗体刺激提供足够的信号转导，从而激活改造的免疫细胞即可。
在某些情形中给，所述胞内结构域可以是来自CD3 gamma和/或CD3delta的胞内结构域，例如，所述胞内结构域可包含如SEQ ID NO:3和5中任一项所示的胞内结构域。或者，所述胞内结构域可包含与SEQ ID NO:3和5中任一项所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在本申请的所述CD3抗体受体复合物中，所述第一CD3重组蛋白的第一胞内结构域可以包含共刺激结构域和信号转导结构域，所述第二CD3重组蛋白的第一胞内结构域可以为至少两个(例如，至少四个、至少六个或更多个)氨基酸的肽段。
在本申请的所述CD3抗体受体复合物中，所述第二CD3重组蛋白的第一胞内结构域可以包含共刺激结构域和信号转导结构域，所述第以CD3重组蛋白的第一胞内结构域可以为至少两个(例如，至少四个、至少六个或更多个)氨基酸的肽段。
在本申请中，所述CD3重组蛋白(第一CD3重组蛋白和/或第二CD3重组蛋白)还可包含铰链区。所述铰链区可以在胞外域和跨膜域之间。例如，所述铰链区可以包含源自选自下组任意一种或多种蛋白的铰链区：CD8α、CD28、4-1BB、CD4、CD27、CD7和PD-1。
例如，所述铰链区可以包含如SEQ ID NO:6中任一项所示的氨基酸序列。或者所述铰链区可以包含与SEQ ID NO:6中任一项所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
例如，在本申请中，所述第一CD3重组蛋白可源自CD3 epsilon结构域的胞外域、源自CD28的跨膜区、源自CD28的胞内域和源自CD3 zeta的胞内域。例如，所述第一CD3重组 蛋白可包含如SEQ ID NO:10所示的氨基酸序列。或者，所述第一CD3重组蛋白可包含与SEQ ID NO:10所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
例如，所述第二CD3重组蛋白可包含源自CD3 gamma结构域的胞外域、源自CD28的跨膜区和源自CD3 gamma的胞内域。例如，所述第二CD3重组蛋白可包含如SEQ ID NO:11所示的氨基酸序列。或者，所述第二CD3重组蛋白可包含与SEQ ID NO:11所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
例如，所述第二CD3重组蛋白可包含源自CD3 gamma结构域的胞外域、源自CD28的跨膜区和所述肽段。例如，所述第二CD3重组蛋白可包含如SEQ ID NO:12所示的氨基酸序列。或者，所述第二CD3重组蛋白可包含与SEQ ID NO:12所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
在本申请中，所述CD3抗体受体复合物还可包含一个或多个(例如，两个、三个、四个或更多个)第一重组蛋白。在本申请中，所述CD3抗体受体复合物也可包含一个或多个(例如，两个、三个、四个或更多个)第二重组蛋白。在某些情形中，本申请中，所述CD3抗体受体复合物还可包含第三重组蛋白，所述第三重组蛋白可包含(1)第三胞外域，所述第三胞外域包含源自选自CD3 gamma结构域或CD3 delta结构域中任意一种的胞外域，(2)第三跨膜域，和(3)第三胞内结构域。所述第三跨膜域可以在上文所述的跨膜域的范围内，并且可以与第一跨膜域和/或第二跨膜域相同或不同。所述第三胞内结构域可以在上文所述的胞内结构域的范围内，并且可以与第一胞内结构域和/或第二胞内结构域相同或不同。只要所述CD3抗体受体复合物中的至少一个(例如，至少两个、三个、四个或更多个)CD3重组蛋白的胞内域包含共刺激结构域和/或信号转导结构域，或者，只要能够为抗体刺激提供足够的信号转导，从而激活改造的免疫细胞即可。
免疫细胞
另一方面，本申请提供了一种经修饰的免疫细胞。所述免疫细胞可包括T细胞、B细胞、天然杀伤(NK)细胞、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在某些情形中，所述免疫细胞可包括T淋巴细胞。所述T淋巴细胞可包括胸腺细胞、天然T淋巴细胞、未成熟T淋巴细胞、成熟T淋巴细胞、静息T淋巴细胞或活化的T淋巴细 胞。所述T细胞可以是辅助T细胞(Th)，例如辅助T细胞1(Th1)或辅助T细胞2(Th2)细胞。所述T淋巴细胞可以是CD4 +辅助T细胞(HTL；CD4 +T细胞)、细胞毒性T细胞(CTL；CD8 +T细胞)、肿瘤浸润细胞毒性T细胞(TIL；CD8 +T细胞)、CD4 +/CD8 +T细胞、CD4 -/CD8 -T细胞或任何其他T淋巴细胞亚型。在某些情形中，所述经修饰的T细胞是人类T细胞。在扩增和遗传修饰本申请的细胞之前，可以通过各种非限制性方法从受试者，例如患者，获得细胞来源。T细胞可以获自许多非限制性来源，包括外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、感染位点的组织、腹水、胸腔积液、脾脏组织和肿瘤。在某些情形中，可以使用本领域技术人员可利用的和已知的任何数量的T细胞系。在另一些情形中，所述细胞可以源自健康供体、源自确诊患有癌症的患者或获自确诊感染的患者。在另一些情形中，所述细胞是存在不同表型特性的细胞的混合群体的一部分。
在某些情形中，所述免疫细胞可包括B细胞。在某些情形中，所述B细胞可包括效应B细胞(浆细胞)、记忆B细胞。所述B细胞可包括B2细胞、B1细胞、边缘区B细胞、滤泡B细胞、调节性B细胞。在某些情形中，所述免疫细胞可包括巨噬细胞。所述B细胞可包括I型巨噬细胞(M1)、II型巨噬细胞(如M2a、M2B、M2c)。
在某些情形中，所述免疫细胞可包括NK细胞。在某些情形中，所述NK细胞可包括CD56bright和CD56dim。在某些情形中，所述NK细胞可包括NK1和NK2。在某些情形中，所述NK细胞可包括A-NK和NA-NK。
在某些情形中，所述免疫细胞可包括白细胞。白细胞通常是指一种有核的血细胞，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。除了在血液外，白细胞还可以存在于淋巴系统、脾，扁桃腺以及身体的其他组织。在本申请中，所述白细胞可以包括粒细胞(如中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞)、无粒白细胞(如淋巴细胞、单核细胞、巨噬细胞、吞噬细胞、肥大细胞)。
在某些情形中，所述免疫细胞可包括淋巴细胞，所述淋巴细胞可以包括在血液、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、T淋巴细胞、天然杀伤(NK)细胞。
在某些情形中，所述免疫细胞可包括外周血单个核细胞，其可包括在外周血中具有单个核的任何细胞。例如，在本申请中，所述外周血单个核细胞可以包括T细胞、B细胞、NK细胞、淋巴细胞、单核细胞和树突状细胞。
在某些情形中，所述免疫细胞可包括巨噬细胞。巨噬细胞是一种可以吞噬和消化细胞碎片、微生物、癌细胞和那些所有缺少正常细胞表面表达的表面标志的其他物质，这个过程叫 做吞噬作用。巨噬细胞几乎存在于所有组织中，通过阿米巴运动寻找可能的病原物。它们除了在非专一的天然免疫反应中起重要作用外，还可以通过招募其他免疫细胞类型，如淋巴细胞，帮助启动获得性免疫。
本申请所述的改造的免疫细胞，其不表达T细胞受体(TCR)。所述不表达T细胞受体(TCR)可以包括T细胞受体(TCR)的表达和/或活性下调。所述下调可以包含不表达有活性的TCR，不表达内源性TCR，不表达外源性TCR，不包含TCR结构，包含失活的TCR，和/或缺失TCR。
在某些情形中，所述免疫细胞中的T细胞受体α恒定区蛋白和/或T细胞受体β恒定区蛋白的表达和/或活性可以下调。在某些情形中，所述下调可包括下调编码所述细胞受体α恒定区蛋白和/或T细胞受体β恒定区蛋白的核酸分子的表达和/或活性；和/或，包括下调所述细胞受体α恒定区蛋白和/或T细胞受体β恒定区蛋白的表达和/或活性。在本申请所述改造的免疫细胞中，CD3抗体受体复合物的表达可以不依赖于TCR的表达。
所述免疫细胞中的MHC复合物的表达和/或活性下调。在某些情形中，所述下调可包括下调编码所述细胞MHC复合物的核酸分子的表达和/或活性；和/或，包括下调所述细胞MHC复合物蛋白的表达和/或活性。
在某些情形中，所述下调可通过基因敲除(knock out)、基因敲减(knock down)、基因突变、基因缺失、基因沉默或上述的任意组合来使所述免疫细胞的TCR和/或MHC复合物的表达和/或活性下调。
例如，可以向所述免疫细胞施用一种或多种选自下组的物质来实现下调：反义RNA、siRNA、shRNA、CRISPR/Cas系统、RNA编辑系统如RNA腺苷脱氨酶(ADAR)、RNA指导的核酸内切酶、锌指核酸酶(ZFN)、Mega-TAL核酸酶、转录激活子样效应物核酸酶(TALEN)、大范围核酸酶(Meganuclease)、碱基编辑、CRISPR干扰，和，锌指蛋白(Zinc finger)基因阻遏物和/或转录激活子样效应物(TALE)基因阻遏物介导的转录抑制。
在某些情形中，所述下调可包括向所述免疫效应细胞施用靶向所述核酸分子(例如，编码所述细胞MHC复合物的核酸分子)外显子部分的指导RNA。靶向编码所述B2M的核酸分子的指导RNA可使用现有技术中的指导RNA，WO2019/011118的全文通过引用的方式并入本文。
本申请所述的改造的免疫细胞还可包含嵌合抗原受体(CAR)和/或嵌合自身抗体受体(CAAR)。
制备方法
本申请提供了制备改造的淋巴细胞的方法，其可包括以下步骤：1.从健康供体中获取外周血T细胞；2.使用负载有CD3和CD28抗体的磁珠激活T细胞；3.T细胞激活后，使用慢病毒将嵌合抗体受体基因转入到T细胞内；4.去除磁珠；5.使用基因编辑技术敲除产生免疫排斥的重要基因TRAC和B2M；6.继续培养并收获细胞。
经过基因编辑而使淋巴细胞中的TRAC和B2M基因失活，能有效降低淋巴细胞在异体细胞治疗中的免疫排斥，并且同时在淋巴细胞中表达两种重组的CD3表面蛋白，这两种共表达的CD3表面蛋白即为CD3抗体受体，能使改造的淋巴细胞结合双特异性抗体。
制备改造的淋巴细胞的方法包括：(i)制备表面缺失I类MHC分子和TCR分子的淋巴细胞；(ii)在淋巴细胞表面表达两种重组的CD3表面蛋白。
另一方面，本申请提供了一种载体，其可用于将编码所述CD3抗体受体复合物的分离的核酸分子转入细胞。在本申请中，所述载体可选自质粒、逆转录病毒载体和慢病毒载体中的一种或多种。所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。
核酸的非病毒递送方法包括脂转染、核转染、显微注射、基因枪、病毒颗粒、脂质体、免疫脂质体、聚阳离子或脂质核酸共轭物、裸DNA、人工病毒体以及增强DNA摄取的试剂。可以使用基于RNA或DNA病毒的系统递送核酸，例如，利用病毒能够靶向体内的特定细胞的性质，将病毒有效加载(payload)运至细胞核中。可以将病毒载体直接给予至患者(体内)或可以通过间接的形式，例如，在体外使用病毒处理细胞，然后将处理过的细胞给予至患者(离体)。常规的基于病毒的系统可以包括用于基因转移的逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关病毒载体以及单纯疱疹病毒载体。在某些情形中，可以用逆转录病毒、慢病毒和腺相关病毒的方法将基因转移整合进宿主基因组中，使插入的基因长期表达。慢病毒载体是能够转导或感染非分裂细胞并典型地产生较高病毒效价的逆转录病毒载体。慢病毒载体可包含长末端重复序列5’LTR和截短的3’LTR、RRE、rev应答元件(cPPT)、中央终止序 列(CTS)和/或翻译后调控元件(WPRE)。所述分子可以通过BamHI和SalI酶切构建到慢病毒载体上。
另一方面，本申请提供了一种药物组合物。所述药物组合物可包含本申请所述改造的免疫细胞，以及药学上可接受的载体。在本申请中，术语“药学上可接受的佐剂”通常是指可与本申请的免疫细胞和/或本申请的细胞群施用相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。除非与本申请的免疫细胞和/或本申请的细胞群不相容，否则任何常规介质或试剂均可以考虑用于本申请的药物组合物中。
本申请所述药物组合物可以包括抗体。所述抗体可以和所述改造的免疫细胞存在于相同或不同的容器中。所述的抗体可以在所述改造的免疫细胞施用前、施用中或施用后施用。例如，所述抗体可以为双特异性抗体。所述双特异性抗体可以同时结合两个靶点，两个靶点可以在同一个靶蛋白上，也可以是不同的靶蛋白。T细胞衔接器是一种比较特殊的双特异性抗体。T细胞衔接器可以包括两个连接的scFv，一端靶向T细胞表面的CD3，另一端靶向靶细胞(例如，肿瘤细胞)表面的受体，从而介导T细胞杀伤肿瘤细胞。靶细胞表面的受体可以包括但不限于CD19、CD2、CD3、CD4、CD5、CD7、CD8、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD38、CD40、CD44V6、CD47、CD52、CD56、CD57、CD58、CD79b、CD80、CD86、CD81、CD123、CD133、CD137、CD151、CD171、CD276、CLL1、B7H4、BCMA、VEGFR-2、EGFR、GPC3、PMSA、CEACAM6、c-Met、EGFRvIII、ErbB2/HER2、ErbB3、HER-2、HER3、ErbB4/HER-4、EphA2、IGF1R、GD2、O-乙酰基GD2、O-乙酰基GD3、GHRHR、GHR、Flt1、KDR、Flt4、Flt3、CEA、CA125、CTLA-4、GITR、BTLA、TGFBR1、TGFBR2、TGFBR1、IL6R、gp130、Lewis、TNFR1、TNFR2、PD1、PD-L1、PD-L2、PSCA、HVEM、MAGE-A、MSLN、NY-ESO-1、PSMA、RANK、RORl、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、MUC1、MUC16、TCRα、TCRβ、TLR7、TLR9、PTCH1、WT-1、Robol、卷曲受体(Frizzled)、OX40、Notch-1-4、APRIL、CS1、MAGE3、Claudin 18.2、叶酸受体α、叶酸受体β、GPC2、CD70、BAFF-R和TROP-2。
这种联合疗法的基本原理是双特异性抗体负责寻找到靶点，将T细胞引导到靶细胞，而改造的免疫细胞则负责对靶细胞的强力杀伤。在原代T细胞中，TCR未被敲除，双特异性抗体可以通过自然表达的TCR-CD3复合体介导对照T细胞对靶细胞的杀伤。如图1所示，在通用型CD3抗体受体T细胞中，CD3抗体受体表达于改造的免疫细胞表面。双特异性抗体能够通过CD3抗体受体，同时激活共激活信号和CD3 zeta激活信号，促进肿瘤细胞的杀伤和T细胞的增殖。联合使用的优势是细胞疗法可以和双特异性抗体进行多靶点组合，同时易于 调控，使该疗法更加安全。在某些情形中，所述双特异性抗体可源自本申请所述的改造的免疫细胞。例如，所述双特异性抗体可以包括由本申请所述的改造的免疫细胞自身分泌的双特异性抗体。
本申请不仅可以应用于通用型T细胞，还可以应用于没有TCR结构的其他杀伤细胞，如NK细胞，或者吞噬细胞，如巨噬细胞等。目前CAR-T细胞使用后的复发包括靶蛋白阴性复发和靶蛋白阳性复发。靶蛋白阳性复发主要是CAR-T细胞在回输后的衰竭导致的。该疗法可以实现细胞消退后持续使用双特异性抗体，利用嵌合抗体受体T细胞和人体内自身的T细胞压制肿瘤的复发。针对晚期身体状态不好的病人也能通过灵活的调控，在保证安全性基础上实现好的治疗效果，增大了病人的适用范围。
方法和用途
另一方面，本申请还提供了所述的改造的免疫细胞和/或所述的药物组合物在制备药物中的用途，所述药物用于治疗肿瘤。
另一方面，本申请还提供了治疗肿瘤的方法，所述方法包括向有需要的受试者施用所述的改造的免疫细胞、所述的药物组合物。
所述受试者首先可以接受一定的化疗预处理，然后将改造的淋巴细胞与双特异性抗体同时或者先后通过静脉输给受试者。以先输细胞为例，细胞在输入后，再按照不同剂量输入双特异性抗体。如果病人出现副反应则调整双特异性抗体的输入剂量。可以重复输入改造的淋巴细胞和双抗，直到出现缓解或者严重的副反应。本申请的改造的淋巴细胞结合双特异性抗体后能够杀伤癌细胞，如血液癌与实体瘤癌细胞。
在本申请中，所述肿瘤可以包括非实体瘤，可以包括但不局限于白血病、淋巴瘤、和/或多发性骨髓瘤，以及实体肿瘤，包括但不局限于肺癌、胃癌、食管癌、结肠癌、乳腺癌、卵巢癌、膀胱癌、肾细胞癌、前列腺癌、黑色素瘤、头颈部肿瘤、胶质瘤及软组织肉瘤等。
例如，所述肿瘤可以包括淋巴瘤。
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融合蛋白、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 设计CD3抗体受体复合物分子和构建质粒
(1)设计分子
从NCBI网站数据库(https://www.ncbi.nlm.nih.gov/)搜索获取基因序列信息(表1)，设 计编码CD3抗体受体复合物的基因CG-UST-1(SEQ ID NO.13)，CG-UST-1包括两部分，为两个CD3重组蛋白基因的串联，第一CD3重组蛋白包含CD3 epsilon胞外域、CD28跨膜区、CD28胞内域和CD3 zeta胞内域(SEQ ID NO:10)。第二CD3重组蛋白包含CD3 gamma胞外域、CD28跨膜区和CD3 gamma胞内域(SEQ ID NO:11)。两个CD3重组蛋白之间通过连接分子T2A基因(SEQ ID NO.16)连接。
将CG-UST-1的编码第二CD3重组蛋白基因的CD3 gamma胞内域替换为编码短肽段的基因，得到CG-UST-2(SEQ ID NO.14)，编码第二CD3重组蛋白(SEQ ID NO:12)。同时设计了CG-UST-1的第一CD3重组蛋白的核酸分子CG-UST-3(SEQ ID NO.15)。为方便检测，在基因序列中加入了Flag标签。
(2)构建质粒
CG-UST-1基因序列由南京金斯瑞公司合成并克隆至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：BamH1和Sal1。将基因合成的重组质粒使用限制性内切酶BamH1(NEB；R3136S)和Sal1(NEB；R3138S)双酶切，使用琼脂糖凝胶电泳分离基因片段后分别进行胶回收纯化(QIAGEN；28706)。测定回收基因片段的浓度。使用T4DNA连接酶(NEB；M0202S)将合成的基因序列连接到慢病毒载体(Addgene；货号：12252)的BamH1-Sal1位点。克隆的慢病毒载体称为：pL-CG-UST-1。克隆后对慢病毒载体质粒进行测序验证，重组质粒测序引物为：Lenti-For(TCAAGCCTCAGACAGTGGTTC；SEQ ID NO:17)和Lenti-Rev(CCTCATAAAGAGACAGCAACCAGG；SEQ ID NO:18)。CG-UST-2和CG-UST-3的质粒构建采用相同的构建流程。构建成的慢病毒载体质粒分别称为：pL-CG-UST-2和pL-CG-UST-3。
实施例2 制备慢病毒
(1)提取质粒
将上述构建的慢病毒载体质粒重新转化大肠杆菌。从转化好的平板上挑取单克隆到3ml含有氨卞青霉素的液体LB培养基的摇菌管中，转速220rpm，摇床振荡培养8h；从活化好的菌液中吸取500μl接种到250ml含有氨卞青霉素的液体LB培养基中，220rpm，摇床振荡培养12-16h。使用Qiagen HiSpeed Plasmid Maxi Kit试剂盒(货号：12662)按照试剂盒提供的实验流程进行质粒提取。提取质粒后使用Nanodrop(Thermo Fisher Scientific)检测质粒浓度并通过DNA琼脂糖凝胶检测超螺旋质粒含量。
(2)培养293T细胞
将冻存的293T细胞(ATCC)从液氮中取出后，在37℃水浴锅内不断摇动促进其融化。 移入提前加入10ml预热的DMEM完全培养基的15ml离心管中，轻轻吹匀；1000rpm离心3min，吸弃上清；加入10ml DMEM完全培养基，轻轻吹匀后接种到10cm皿中，在37℃含5％CO2的细胞培养箱中培养；当细胞密度达到80％-90％时，弃培养基，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，转移到15ml离心管中，1000rpm离心3min，弃上清。根据实验需要，按照1:3或1:5的比例传代，接种到新的10cm皿中，或者进行冻存。
(3)转染293T细胞，收获慢病毒
1)第1天，接种293T细胞：按照约15-16×10 6个/T175瓶(35-40ml培养基培养)接种细胞。
2)第2天，质粒转染：转染前培养基需换成有10％FBS但无双抗的培养基。首先准备质粒复合物：将以下质粒加入到1.5ml Opti-MEM(Thermo Fisher Scientific；31985-070)内，加入后混匀。病毒载体质粒：18μg，psPAX2质粒(Addgene；货号：12260)：9μg，pMD2.G质粒(Addgene；货号：12259)：18μg。再准备转染试剂复合物：将100μl Lipofectamine 2000(invitrogen；11668-019)加入到1.5ml Opti-MEM内，加入后混匀，室温静置5min；再将质粒复合物加入到转染试剂复合物中，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。
3)第4天，收获病毒：收取细胞上清，2000rpm，离心10min；使用0.45um滤膜过滤上清，滤液转移到专用离心管中，配平；使用超速离心机20000rpm超速离心2-3h；倒掉上清后，使用无血清培养基重悬慢病毒，分装慢病毒后保存在-80℃。按照该流程分别制备含有CG-UST-1、CG-UST-2和CG-UST-3的慢病毒。
实施例3 制备通用型表达CD3抗体受体复合物的T细胞
使用单采机分离健康供体外周血的PMBCs(购自妙通生物)。将PBMCs稀释到2×106。按照细胞与磁珠1:3的比例使用CD3/CD28磁珠(Thermo Fisher Scientific)激活T细胞，同时添加IL-2(PeproTech；200-02)。在激活后第3天，将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和B2M，构建通用型T细胞。使用的gRNA序列和操作流程参考专利WO2019/011118实施例3进行。
实施例4 检测CD3抗体受体在细胞内的表达情况
通过荧光抗体染色和流式细胞术检测CD3抗体受体复合物在细胞内的表达情况，基本步骤如下：分别离心收集一定体积培养的改造的T细胞，使用Flag抗体(BioLegend；637309)和APC-TCR抗体(BioLegend；306718)对细胞进行染色，避光30min孵育，用PBS洗涤一次后使用适量体积PBS重悬，最后使用流式细胞仪检测CD3抗体受体复合物在T细胞内的表达情况和在TCR阴性细胞中的表达情况。
Flag抗体和APC-TCR抗体的染色结果如图2所示。在未转染的对照组，TCR未敲除时，TCR阳性率为95.09％；TCR敲除后，TCR阳性率为1.95％。说明敲除效率很高。在TCR未敲除的组别，CD3抗体受体复合物的表达效率分别为：CG-UST-1：27.21％、CG-UST-2：25.41％和CG-UST-3：68.88％。在TCR敲除组，TCR阴性的T细胞中，CD3抗体受体复合物的表达效率分别为：CG-UST-1：30.2％、CG-UST-2：25.0％和CG-UST-3：65.6％(图2)。在敲除组，表达效率与对应的未敲除组类似，因此CD3抗体受体复合物都可以以TCR不依赖的形式独立表达于人T细胞。
使用不同的CD3抗体UCHT1(BD Biosciences；555335)、HIT3a(BD Biosciences；561804)、SP34-2(BD Biosciences；552127)和OKT3(BD Biosciences；566686)对表达CD3抗体受体的T细胞染色，并用流式细胞仪进行分析。结果分别如图3-图6所示。结果显示，表达CG-UST-1和CG-UST-2的T细胞可以被常见CD3抗体克隆UCHT1识别。如图3所示，TCR敲除组，在TCR阴性的T细胞中，CG-UST-1和CG-UST-2组分别有22.0％和19.9％的细胞被UCHT1所识别。采用同样的染色和分析方法发现，表达CG-UST-1和CG-UST-2的T细胞可以被CD3抗体HIT3a克隆所识别(见图4)，但是不能被克隆SP34-2识别(见图5)。进一步同时对细胞进行SP34-2和OKT3抗体染色发现，这些细胞可以被OKT3抗体识别(见图6)。但是单独表达包含CD3 epsilon膜外域的CG-UST-3的T细胞不能被常见CD3抗体克隆识别(见图3-图6)。
因此，CD3抗体受体可以以不依赖于TCR的形式表达于TCR敲除的原代T细胞内。共表达CD3 epsilon和CD3 gamma胞外域的CD3抗体受体复合物的T细胞可以被常规CD3抗体识别，其中包括CD3抗体UCHT1、HIT3a和OKT3。
实施例5 CD3抗体OKT3激活通用型CD3抗体受体T细胞
(1)包被平板。使用PBS稀释OKT3抗体，稀释到0.25μg/ml。将稀释的抗体按照100μl每孔加入到96孔板中，37℃孵育3小时。孵育完成后，使用1×PBS清洗平板，去除PBS。
(2)激活T细胞。将不同组别的T细胞密度调整为1×10 6/ml。然后将细胞按照100μl每孔种到平板内，37℃孵育24小时。
(3)检测T细胞的激活。细胞孵育完成后，取一定体积的细胞，使用CD137荧光抗体(BD Biosciences；555956)和TCR抗体(Biolegend；306718)进行细胞染色，使用流式细胞术检测TCR敲除T细胞中激活标签CD137蛋白的表达情况。在表达CD3抗体受体CG-UST-1和CG-UST-2的T细胞中，TCR阴性切CD137阳性的细胞分别为7.01％和4.05％，见图7。说明包被的CD3抗体可以激活通用型CD3抗体受体T细胞。
(4)检测细胞因子分泌。取上述细胞的上清，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细胞因子的分泌情况。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。
结果发现，OKT3抗体分别刺激表达CG-UST-1和CG-UST-2的通用型抗体受体T细胞分泌341.28和248.76pg/ml的IFN-γ，分泌情况见表1。因此，包被的CD3抗体可以刺激表达CD3抗体受体复合物CG-UST-1和CG-UST-2的通用型T细胞分泌细胞因子IFN-γ。
表1.OKT3抗体刺激通用型CD3抗体受体T细胞分泌细胞因子IFN-γ(pg/ml)
  对照组 CG-UST-1 CG-UST-2 CG-UST-3
无抗体 -64.32 -60.24 -61.68 -53.52
加抗体 6.48 341.28 248.76 -6.6
实施例6 通用型CD3抗体受体复合物T细胞联合抗CD3抗CD19双特异性抗体杀伤肿瘤细胞
抗CD3抗CD19双特异性抗体购自Invivogen(货号：bimab-hcd19cd3)。该双抗可以同时结合CD3 epsilon和CD19，介导T细胞杀伤表达CD19的靶细胞。该双抗使用的CD3抗体克隆号为L2K-07。
(1)细胞共培养。
首先进行细胞共培养。步骤如下：将不同组别的T细胞浓度调整至1×10 6/ml，然后将T细胞按照100μl每孔种到96孔板中。将种好的T细胞暂时放置于37℃孵育；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，然后将Raji细胞按照100μl每孔种到含有T细胞的96孔板种。最终T细胞和肿瘤细胞比例为1:1。将抗CD3抗CD19双特异性抗体加入到细胞中。双抗的终浓度为50ng/ml。充分混匀后离心，500rpm，离心3分钟。将细胞置于37℃温箱孵育24小时。
(2)CD137表达检测。
取一定体积的细胞，使用CD19抗体(eBioscience，11-0199-42)、CD137荧光抗体和TCR抗体进行细胞染色，使用流式细胞术检测。CD19阴性的细胞即为T细胞。通过检测细胞膜激活标签CD137，发现添加双抗的Raji细胞可以激活表达CD3抗体受体CG-UST-1和CG-UST-2的通用型T细胞，见图8。在TCR阴性的T细胞中，两者的CD137阳性率分别为7.15％和6.47％。
(3)细胞因子分泌检测。
孵育完成后取上清，如实施例5，进行ELISA实验。结果显示抗CD3抗CD19双特异性抗体可以介导通用型CD3抗体受体T细胞分泌细胞因子IFN-γ，见表2。在TCR敲除的情况下，双抗联合肿瘤细胞分别刺激表达CG-UST-1和CG-UST-2的通用型抗体受体T细胞分泌287.639和286.512pg/ml的IFN-γ。
表2.双抗介导肿瘤细胞刺激通用型CD3嵌合抗体受体T细胞分泌细胞因子IFN-γ(pg/ml)
  对照组 CG-UST-1 CG-UST-2 CG-UST-3
无双抗 -32.478 -18.807 -28.117 -6.018
加双抗 50.773 287.639 286.512 195.127
(4)CD107a表达检测。
1)取96孔板，每孔加T细胞和靶细胞各2×10 5个，离心沉淀后使用100μlRPMI-1640完全培养基重悬，将抗CD3抗CD19双抗加入到细胞中。双抗的终浓度为50ng/ml。每孔按照1:50比例加入CD107a-PE抗体(BD Biosciences；555801)，孵育一小时。之后加入1:30,000比例稀释的BD GolgiStop(BD Biosciences；554724)，37℃孵育2.5小时；
2)将样品离心去除培养基，使用无血清培养基洗细胞一次，1600rpm离心6分钟。弃上清，重悬细胞，重悬体积100μl。每管加入适量CD3抗体UCHT1，4℃避光孵育30分钟；
3)每管用PBS清洗细胞1次，1600rpm离心5分钟。仔细吸去上清；
4)加入适量PBS重悬细胞，流式细胞仪检测CD107a表达量。
结果显示表达CG-UST-1和CG-UST-2的通用型抗体受体T细胞可以被UCHT1识别，同时抗CD3抗CD19双特异性抗体可以介导通用型抗体受体T细胞释放杀伤颗粒(见图9)。在经过双抗和肿瘤细胞刺激后，表达CG-UST-1和CG-UST-2的通用型CD3抗体受体T细胞分别有9.49％和10.75％的细胞表达CD107a。因此，通用型CD3抗体受体T细胞可以和双特异性抗体联合使用杀伤肿瘤细胞。

Claims (57)

  1. 一种改造的免疫细胞，其包含CD3抗体受体复合物，所述CD3抗体受体复合物包含第一CD3重组蛋白和第二CD3重组蛋白，其中所述第一CD3重组蛋白包含：
    (1)第一胞外域，所述第一胞外域包含源自CD3 epsilon结构域的胞外域，
    (2)第一跨膜域，
    (3)第一胞内结构域；
    所述第二CD3重组蛋白包含：
    (1)第二胞外域，所述第二胞外域包含源自选自CD3 gamma结构域和CD3 delta结构域中任意一种的胞外域，
    (2)第二跨膜域，
    (3)第二胞内结构域；且，
    其不表达T细胞受体(TCR)。
  2. 根据权利要求1所述的改造的免疫细胞，其包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
  3. 根据权利要求1-2中任一项所述的改造的免疫细胞，其中所述CD3 epsilon结构域的胞外域包含如SEQ ID NO:1所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的改造的免疫细胞，其中所述第二胞外域包含源自CD3gamma结构域的胞外域。
  5. 根据权利要求1-4中任一项所述的改造的免疫细胞，其中所述CD3 gamma结构域的胞外域包含如SEQ ID NO:2所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的改造的免疫细胞，其中所述第二胞外域包含源自CD3delta结构域的胞外域。
  7. 根据权利要求1-6中任一项所述的改造的免疫细胞，其中所述CD3 delta结构域的胞外域包含如SEQ ID NO:4所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的改造的免疫细胞，其中所述第一跨膜域和所述第二跨膜域相同或不同。
  9. 根据权利要求1-8中任一项所述的改造的免疫细胞，其中所述跨膜域不包含源自CD3的跨膜域。
  10. 根据权利要求1-9中任一项所述的改造的免疫细胞，其中所述跨膜域包含源自选自下组任意一种蛋白的跨膜域：CD8α、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、 GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
  11. 根据权利要求1-10中任一项所述的改造的免疫细胞，其中所述跨膜域包含SEQ ID NO:7所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的改造的免疫细胞，其中所述第一胞内结构域和所述第二胞内结构域相同或不同。
  13. 根据权利要求1-12中任一项所述的改造的免疫细胞，其中所述第一胞内结构域和所述第二胞内结构域中的至少一种包含共刺激结构域和/或信号转导结构域。
  14. 根据权利要求13中所述的改造的免疫细胞，其中所述第一胞内结构域中包含的共刺激结构域和所述第二胞内结构域中包含的共刺激结构域相同或不同。
  15. 根据权利要求13-14中任一项所述的改造的免疫细胞，其中所述共刺激结构域包含源自选自下组任意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。
  16. 根据权利要求13-15中任一项所述的改造的免疫细胞，其中所述共刺激结构域包含如SEQ ID NO:8所示的氨基酸序列。
  17. 根据权利要求13-16中任一项所述的改造的免疫细胞，其中所述第一胞内结构域中包含的信号转导结构域和所述第二胞内结构域中包含的信号转导结构域相同或不同。
  18. 根据权利要求13-17中任一项所述的改造的免疫细胞，其中所述信号转导结构域包含至少包含一个免疫受体酪氨酸活化基序(ITAM)。
  19. 根据权利要求13-18中任一项所述的改造的免疫细胞，其中所述信号转导结构域包含源自选自下组任意一种或多种蛋白的信号转导结构域：CD3zeta、CD3delta、CD3gamma、CD3ε、CD79a、CD79b、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10和DAP-12。
  20. 根据权利要求13-19中任一项所述的改造的免疫细胞，其中所述信号转导结构域包含如SEQ ID NO:9所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的改造的免疫细胞，其中所述胞外域和跨膜域之间还包 含铰链区。
  22. 根据权利要求21所述的改造的免疫细胞，其中所述铰链区包含源自选自下组任意一种或多种蛋白的铰链区：CD8α、CD28、4-1BB、CD4、CD27、CD7和PD-1。
  23. 根据权利要求21-22中任一项所述的改造的免疫细胞，其中所述铰链区包含SEQ ID NO:6中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的改造的免疫细胞，其中所述第一胞内结构域和所述第二胞内结构域中的一种包含至少两个氨基酸的肽段。
  25. 根据权利要求1-24中任一项所述的改造的免疫细胞，其中所述第一CD3重组蛋白包括源自CD3 epsilon结构域的胞外域、源自CD28的跨膜区、源自CD28的胞内域和源自CD3 zeta的胞内域。
  26. 根据权利要求25所述的改造的免疫细胞，其中所述第一CD3重组蛋白包括SEQ IDNO:10所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的改造的免疫细胞，其中所述第二CD3重组蛋白包括源自CD3 gamma结构域的胞外域、源自CD28的跨膜区和源自CD3 gamma的胞内域。
  28. 根据权利要求27所述的改造的免疫细胞，其中所述第二CD3重组蛋白包括SEQ ID NO:11所示的氨基酸序列。
  29. 根据权利要求1-26中任一项所述的改造的免疫细胞，其中所述第二CD3重组蛋白包括源自CD3 gamma结构域的胞外域、源自CD28的跨膜区和所述肽段。
  30. 根据权利要求29所述的改造的免疫细胞，其中所述第二CD3重组蛋白包括SEQ ID NO:12所示的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的改造的免疫细胞，其还包含第三CD3重组蛋白，所述第三CD3重组蛋白包含：
    (1)第三胞外域，所述第三胞外域的至少一部分包含源自选自CD3 gamma结构域或CD3 delta结构域中任意一种的胞外域，
    (2)第三跨膜域，和
    (3)第三胞内结构域。
  32. 根据权利要求31所述的改造的免疫细胞，其中所述第三胞外域与所述第二胞外域相同或不同。
  33. 根据权利要求31-32中任一项所述的改造的免疫细胞，其中所述第三跨膜域与所述第一跨膜域和/或所述第二跨膜域相同或不同。
  34. 根据权利要求31-33中任一项所述的改造的免疫细胞，其中所述第三胞内结构域与所述 第一胞内结构域和/或第二胞内结构域相同或不同。
  35. 根据权利要求1-34中任一项所述的改造的免疫细胞，其中主要组织相容性复合体(MHC)的表达和/或活性下调。
  36. 根据权利要求35所述的改造的免疫细胞，其中所述MHC复合体包括B2M。
  37. 根据权利要求1-36中任一项所述的改造的免疫细胞，其中所述的改造的免疫细胞包含嵌合抗原受体(CAR)和/或嵌合自身抗体受体(CAAR)。
  38. 药物组合物，其包含权利要求1-37中任一项所述的改造的免疫细胞和药学上可接受的佐剂。
  39. 根据权利要求38中所述的药物组合物，其包括抗体。
  40. 根据权利要求38-39中任一项所述的药物组合物，其中所述抗体能够识别和/或结合所述CD3抗体受体复合物。
  41. 根据权利要求38-40中任一项所述的药物组合物，其中所述抗体包括双特异性抗体。
  42. 根据权利要求38-41中任一项所述的药物组合物，其中所述双特异性抗体源自权利要求1-37中任一项所述的改造的免疫细胞。
  43. 根据权利要求38-42中任一项所述的药物组合物，其中所述双特异性抗体能够识别和/或结合CD3。
  44. 根据权利要求38-43中任一项所述的药物组合物，其中所述双特异性抗体能够识别和/或结合靶细胞表面的受体。
  45. 根据权利要求44所述的药物组合物，其中所述靶细胞为肿瘤细胞。
  46. 根据权利要求44-45中任一项所述的药物组合物，其中所述靶细胞表面的受体选自下组中的一种：CD19、CD2、CD3、CD4、CD5、CD7、CD8、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD38、CD40、CD44V6、CD47、CD52、CD56、CD57、CD58、CD79b、CD80、CD86、CD81、CD123、CD133、CD137、CD151、CD171、CD276、CLL1、B7H4、BCMA、VEGFR-2、EGFR、GPC3、PMSA、CEACAM6、c-Met、EGFRvIII、ErbB2/HER2、ErbB3、HER-2、HER3、ErbB4/HER-4、EphA2、IGF1R、GD2、O-乙酰基GD2、O-乙酰基GD3、GHRHR、GHR、Flt1、KDR、Flt4、Flt3、CEA、CA125、CTLA-4、GITR、BTLA、TGFBR1、TGFBR2、TGFBR1、IL6R、gp130、Lewis、TNFR1、TNFR2、PD1、PD-L1、PD-L2、PSCA、HVEM、MAGE-A、MSLN、NY-ESO-1、PSMA、RANK、RORl、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、MUC1、MUC16、TCRα、TCRβ、TLR7、TLR9、PTCH1、WT-1、Robol、卷曲受体(Frizzled)、OX40、Notch-1-4、APRIL、CS1、MAGE3、 Claudin 18.2、叶酸受体α、叶酸受体β、GPC2、CD70、BAFF-R和TROP-2。
  47. 核酸分子，其编码权利要求1-37中任一项所述的改造的免疫细胞中的CD3抗体受体复合物。
  48. 载体，其包含权利要求47所述的核酸分子。
  49. 根据权利要求48所述的载体，其为病毒载体。
  50. 根据权利要求48-49中任一项所述的载体，其为慢病毒载体。
  51. 细胞，其包含权利要求47所述的核酸分子和/或权利要求48-50中任一项所述的载体。
  52. 权利要求1-37中任一项所述的改造的免疫细胞和/或权利要求38-46中任一项所述的药物组合物在制备药物中的用途，所述药物用于治疗肿瘤。
  53. 根据权利要求52所述的用途，其中所述肿瘤包括实体瘤和非实体瘤。
  54. 根据权利要求52-53中任一项所述的用途，其中所述肿瘤选自以下组：淋巴瘤、白血病和多发性骨髓瘤。
  55. 治疗肿瘤的方法，所述方法包括向有需要的受试者施用权利要求1-37中任一项所述的改造的免疫细胞、权利要求38-46中任一项所述的药物组合物。
  56. 根据权利要求55所述的方法，其中所述肿瘤包括实体瘤和非实体瘤。
  57. 根据权利要求55-56中任一项所述的方法，其中所述肿瘤选自以下组：淋巴瘤、白血病和多发性骨髓瘤。
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