WO2021073624A1 - 用于免疫治疗的嵌合抗原受体、制备方法及其应用 - Google Patents

用于免疫治疗的嵌合抗原受体、制备方法及其应用 Download PDF

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WO2021073624A1
WO2021073624A1 PCT/CN2020/121672 CN2020121672W WO2021073624A1 WO 2021073624 A1 WO2021073624 A1 WO 2021073624A1 CN 2020121672 W CN2020121672 W CN 2020121672W WO 2021073624 A1 WO2021073624 A1 WO 2021073624A1
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amino acid
acid sequence
tcr
seq
cell
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French (fr)
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芦志华
杜宝华
刘永峰
王方圆
戴卫国
朱滨
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北京门罗生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the invention relates to the field of immunotherapy, and relates to a chimeric antigen receptor for immunotherapy, a preparation method and application thereof.
  • T cells expressing chimeric antigen receptors (hereinafter referred to as “CAR") (hereinafter referred to as “CAR-T cells”) refer to recombinant T cells in which the code recognition is specific on the surface of cancer cells The gene of the receptor of the surface antigen of the cancer cell that is sexually expressed is introduced into the T cell to kill the cancer cell.
  • CAR chimeric antigen receptors
  • Dr. Zelig Fshhar and others have successfully prepared T cells with chimeric antigen receptors by obtaining such a theory: that is, when artificially manufactured When T cells have receptors that bind to antigens specifically expressed in cancer cells, an immune response to cancer cells only occurs, thereby killing cancer cells. This fact was subsequently reported on PNAS in 1989.
  • CAR-T cells produced in the early stage that is, the first generation CAR-T cells only use CD3 ⁇ as the signal transduction domain. In addition to their insignificant therapeutic effect, they also have the shortcoming of short duration. Therefore, efforts have been made to improve the responsiveness of CAR-T cells, and as a result, second-generation CAR-T cells were produced.
  • the costimulatory domain CD28 or CD137/4 -1BB
  • CD3 ⁇ are combined, and the number of CAR-T cells present in the body is significantly increased compared with the number of first-generation CAR-T cells.
  • second-generation CAR-T cells use one type of costimulatory domain
  • CAR-T cells that use two types of costimulatory domains are called third-generation CAR-T.
  • Most recent studies have focused on second-generation CAR-T cells and third-generation CAR-T cells.
  • CAR-T cells to treat cancer, there are reports that when cytotoxic T cells transformed to recognize CD19 are injected into 3 patients with end-stage chronic lymphoid leukemia (CCL), two of them The patient’s leukemia was thoroughly treated, and the condition lasted for about 10 months (N. Engl J Med 2011; 365: 725-733, August 25, 2011, Sic. Transl. Med 2011 Aug 10; 3(95): 95ra73).
  • the CAR-T used herein corresponds to the second generation, using 4-1BB as the costimulatory domain and CD3 ⁇ as the signal transduction domain.
  • the antigen binding domain of CAR-T cells recognizes CD19 as an antigen found on the surface of leukemia cancer cells.
  • the present invention provides an anti-TCR chimeric antigen receptor, which comprises a target antigen binding domain, a transmembrane domain, and a T cell activation signal transmission region.
  • the target antigen is TCR.
  • chimeric antigen receptor refers to a chimeric protein containing a target antigen binding domain, a transmembrane domain, and a T cell activation signal transmission region. It should be noted that the chimeric protein refers to a protein containing sequences derived from two or more different proteins. CAR is not limited to only including the above three areas, and may also include other areas.
  • the CAR of this embodiment includes a target antigen binding domain whose target antigen is TCR.
  • the "target antigen binding domain” refers to the extracellular region that binds to the target antigen outside the cell when the T cell expresses the CAR.
  • the CAR expressed in CAR-T cells is transferred to the cell membrane, and the target antigen-binding domain outside the cell and the T cell activation signal transmission domain inside the cell are connected via a transmembrane domain penetrating the cell membrane.
  • the target antigen binding domain binds to the target antigen, and thus the T cell activation signal is transmitted from the T cell activation signal transmission area to the T cell, and the T cell is activation.
  • the target antigen-binding domain in the specific embodiment of the present invention is not particularly limited as long as it can specifically bind to TCR. It is preferably an antigen containing a monoclonal antibody capable of specifically binding to TCR (hereinafter, also referred to as "anti-TCR antibody”) Combine the area.
  • the "antigen binding region" of an antibody refers to a region related to antigen binding in an antibody, and specifically refers to a region including a complementarity determining region (CDR).
  • CDR complementarity determining region
  • the antigen binding region of an antibody contains at least one CDR of the antibody. In a preferred mode, the antigen binding region of the antibody contains all 6 CDRs of the antibody.
  • CDR can be determined by an arbitrary definition known as the definition of CDR, and for example, the definitions of Kabat, Chothia, AbM, cotact, etc. can be used. Preferably, the CDR defined by Kabat can be cited.
  • the anti-TCR antibody that can be used in the target antigen-binding region is not particularly limited, and it may be a known antibody or a newly prepared antibody.
  • an anti-TCR antibody is newly prepared, the preparation of the anti-TCR antibody may be performed by a known method. For example, a method of immunizing an animal with TCR to obtain a hybridoma, a phage display method, and the like can be used.
  • anti-TCR antibodies include those having the amino acid sequence described in SEQ ID NO: 7 as the variable heavy chain (VH) region, and those having the amino acid sequence described in SEQ ID NO: 8 as the variable light chain (VL) region Antibody.
  • the amino acid sequences of CDR1 to 3 defined by Kabat in the VH region composed of the amino acid sequence described in SEQ ID NO: 7 are shown in SEQ ID NO: 1-3, respectively.
  • the amino acid sequences of CDRs 1 to 3 defined by Kabat in the VL region composed of the amino acid sequence described in SEQ ID NO: 8 are shown in SEQ ID NOs: 4-6, respectively.
  • the target antigen binding region may include the VH region and the VL region of the anti-TCR antibody.
  • a single-chain antibody (scFv) polypeptide comprising the VH region and the VL region of an anti-TCR antibody is a preferred example of the target antigen-binding region.
  • the scFv is a polypeptide formed by linking the VH region and the VL region of an antibody with a peptide linker, and is generally used as the target antigen binding region of the CAR.
  • the peptide linker connecting the VH region and the VL region is not particularly limited, and peptides commonly used in scFv can be used.
  • Examples of peptide linkers include linkers of the amino acid sequence shown in SEQ ID NO: 9, but are not limited to these.
  • VH region and VL region used in scFv the VH region and VL region of an anti-TCR antibody can be used.
  • Preferred examples of anti-TCR antibodies are as described above.
  • part of the sequence may be changed.
  • scFv the following sequence can be preferably used:
  • a scFv comprising a VH region including CDR1-3 of the VH region composed of the amino acid sequence described in SEQ ID NO: 7; and CDR1 including a VL region composed of the amino acid sequence described in SEQ ID NO: 8 -3 VL region has the ability to bind to TCR.
  • a scFv which includes a VH region composed of the amino acid sequence described in SEQ ID NO: 7 and a VL region composed of the amino acid sequence described in SEQ ID NO: 8, and has the ability to bind to TCR.
  • a scFv comprising a VH region composed of one or more amino acid sequences in the amino acid sequence described in SEQ ID NO: 7 mutated, and a VH region in the amino acid sequence described in SEQ ID NO: 8
  • the VL region composed of an amino acid sequence formed by one or more amino acid mutations has the ability to bind to TCR.
  • a scFv comprising a VH region composed of an amino acid sequence having 95% or more sequence identity (identity) with the amino acid sequence described in SEQ ID NO: 7, and having the amino acid sequence described in SEQ ID NO: 8
  • the VL region composed of amino acid sequences with 95% or more sequence identity (identity) has the ability to bind to TCR.
  • human antibody framework sequences for sequences other than CDRs (framework sequences).
  • the framework sequence of the amino acid sequence of the human antibody registered in the well-known sequence database such as GenBank, and the common sequence derived from each subgroup of the human antibody (Human Most Homologous Consensus Sequence; Kabat, EA, etc. Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991) selected amino acid sequence and so on.
  • a plurality may be, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5.
  • “variation” may be any of deletion, substitution, addition, and insertion, or a combination thereof.
  • the position of mutation is preferably a region other than CDR1 to 3 (that is, a framework region).
  • sequence identity is 95% or more, it is not particularly limited. It is preferably 96% or more, more preferably 97% or more, still more preferably 98% or more, and still more preferably 99% or more. Preferably, it is 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
  • sequence identity (identity) between the amino acid sequences means that the two amino acid sequences are arranged side by side by adding gaps in the part corresponding to the insertion and deletion in such a way that the corresponding amino acids are the same.
  • the ratio of amino acids whose entire amino acid sequence other than the gaps in the alignment coincides with each other is determined.
  • the sequence identity between amino acid sequences can be determined using various identity search software known in the technical field. For example, the value of the sequence identity of the amino acid sequence can be calculated based on the alignment obtained by the well-known identity search software BLASTP.
  • scFv a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 9; comprising an amino acid sequence formed by mutation of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 9 and having a TCR A polypeptide having binding ability; or a polypeptide comprising an amino acid sequence having 95% or more sequence identity (identity) with the amino acid sequence shown in SEQ ID NO: 9 and having binding ability to TCR, etc.
  • the "plurality” and “variation” are the same as described above.
  • sequence identity is also the same as described above.
  • the transmembrane domain refers to a region that penetrates the cell membrane and connects the extracellular region and the intracellular region when T cells express CAR.
  • the transmembrane domain is not particularly limited as long as it is a polypeptide having a function of penetrating the cell membrane.
  • the transmembrane domain can be derived from natural proteins or artificially designed.
  • the transmembrane domain derived from a natural protein can be obtained from any membrane-bound protein or membrane penetrating protein.
  • the transmembrane domain can transmit an activation signal to the T cell activation signal transmission region corresponding to the binding of the target antigen to the target antigen binding domain.
  • transmembrane domains include the ⁇ chain and ⁇ chain of T cell receptors, CD3 ⁇ , CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137, ICOS, CD154, GITR and other transmembrane domains.
  • the organism from which these proteins are derived is not particularly limited, but humans are preferred.
  • the amino acid sequences of these proteins can be obtained from well-known sequence databases such as GenBank.
  • the transmembrane domain is often connected to the extracellular hinge region.
  • the "extracellular hinge region” refers to the area that connects the target antigen binding region outside the cell with the transmembrane domain.
  • the CAR of this embodiment includes an extracellular hinge region.
  • the extracellular hinge region is not particularly limited as long as it can connect the target antigen-binding region and the transmembrane domain. It can be derived from natural protein, or it can be artificially designed.
  • the extracellular hinge region may be composed of, for example, about 1 to 100 amino acids, preferably about 10 to 70 amino acids. Preferably, the extracellular hinge region does not hinder the TCR binding ability of the target antigen binding region, and does not hinder the signal transmission of the T cell activation signal transmission region.
  • extracellular hinge region examples include extracellular hinge regions such as CD8, CD28, and CD4.
  • extracellular hinge regions such as CD8, CD28, and CD4.
  • the hinge region of immunoglobulins for example, IgG4, etc.
  • the organism from which the above-mentioned protein is derived is not particularly limited, but is preferably human.
  • amino acid sequences of these proteins can be obtained from well-known sequence databases such as GenBank.
  • extracellular hinge region and transmembrane domain may be variants of the extracellular hinge region and transmembrane domain derived from the natural protein described above.
  • the following examples can be given.
  • a polypeptide that is composed of one or more amino acid mutations in the amino acid sequence of the extracellular hinge region and transmembrane domain of natural protein (such as SEQ ID NO: 11), and has the ability of membrane penetration .
  • sequence identity is 95% or more, it is not particularly limited. It is preferably 96% or more, more preferably 97% or more, still more preferably 98% or more, and even more preferably 99% or more. Preferably, it is 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
  • a plurality may be, for example, 2 to 10, preferably 2 to 5, more preferably 2 to 4, and still more preferably 2 or 3.
  • “variation” may be any of deletion, substitution, addition, and insertion, or a combination thereof.
  • T cell activation signal transmission area refers to the area that is located in the cell and transmits T cell activation signal to the T cell when the T cell expresses the CAR.
  • T cell when the MHC-peptide complex and the T cell receptor (T cell Receptor : TCR) binding, the T cell activation signal is transmitted to the cell through the TCR ⁇ CD3 complex, and causes various phosphorylation signals (primary signal transmission).
  • costimulatory molecules expressed on the cell surface of T cells By binding with ligands specific to each costimulatory molecule expressed on the cell surface of antigen-presenting cells, costimulatory signals are delivered into the cells, and the activation of T cells is assisted (secondary signal transmission).
  • T cell activation signaling includes both the aforementioned primary signaling and secondary signaling.
  • the “T cell activation signal transmission region” refers to the intracellular region of the protein involved in the above-mentioned primary signal transmission and the secondary signal transmission, which participates in the signal transmission.
  • the T cell activation signal transmission region is not particularly limited as long as it is a T cell activation signal transmission region of a protein involved in T cell activation signal transmission.
  • ITAM immunoreceptor tyrosine-based activation motif
  • a T cell activation signal transmission region of a protein having ITAM can be cited.
  • proteins having ITAM include CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, and the like.
  • the T cell activation signaling region of ITAM containing these proteins is a preferred example of the T cell activation signaling region for CAR.
  • a T cell activation signal transmission region such as CD3 ⁇ can be cited.
  • costimulatory molecules participate in secondary signal transmission. Therefore, as an example of the T cell activation signal transmission area, the signal transmission area of a costimulatory molecule can also be cited.
  • costimulatory molecules include CD2, CD4, CD5, CD8, CD27, CD28, OXO40 (CD134), 4-1BB (CD137), ICOS, CD154, HVEM, GITR, Fc Receptor-associated gamma chain, and the like.
  • the T cell activation signal transmission region of these proteins is also a preferred example of the T cell activation signal transmission region for CAR.
  • T cell activation signal transmission regions such as CD28 and 4-1BB can be cited.
  • the organism from which the above-mentioned protein is derived is not particularly limited, but is preferably human.
  • the amino acid sequences of these proteins can be obtained from well-known sequence databases such as GenBank.
  • the T cell activation signal transmission region may be a variant of the above-mentioned natural protein-derived T cell activation signal transmission region.
  • variants derived from the activation signal transmission region of a natural protein include the following variants.
  • sequence identity is 95% or more, it is not particularly limited. It is preferably 96% or more, more preferably 97% or more, still more preferably 98% or more, and even more preferably 99% or more. Preferably, it is 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
  • a plurality of for example, in the case of using a protein involved in one signal transmission, may be 2 to 30, preferably 2 to 20, more preferably 2 to 10, and still more preferably 2 to 5 pieces.
  • the number of "plurality” may be 2 to 15, preferably 2 to 10, more preferably 2 to 5, and still more preferably 2 or 3.
  • “variation” may be any of deletion, substitution, addition, and insertion, or a combination thereof.
  • the number of T cell activation signal transmission regions included in the CAR of the present invention is not limited to one, and may include multiple T cell activation signal transmission regions. Multiple T cell activation signal transmission regions can be the same or different.
  • the CAR contains more than two T cell activation signal transmission regions.
  • the T cell activation signal transmission region contained in the CAR is preferably a combination of the T cell activation signal transmission area involved in the primary signal transmission and the T cell activation signal transmission area involved in the secondary signal transmission.
  • T cell activation signal transmission area When only one T cell activation signal transmission area is used, it is preferable to use a T cell activation signal transmission area that participates in one signal transmission, and it is more preferable to use a T cell activation signal transmission area of CD3 ⁇ .
  • the CAR of the present invention may include a signal peptide and the like in addition to the above-mentioned regions.
  • a “signal peptide” is a peptide that indicates the localization of membrane proteins or secreted proteins.
  • the signal peptide is usually a peptide composed of about 5 to 60 amino acids present at the N-terminus of the membrane protein, and is removed in the mature protein that has been localized.
  • the signal peptide used in the CAR of the present invention is preferably a signal peptide indicating localization to the cell membrane, preferably a signal peptide of a membrane protein.
  • signal peptides include the ⁇ chain and ⁇ chain of T cell receptors, CD3 ⁇ , CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 , ICOS, CD154, GITR, immunoglobulin heavy chain, immunoglobulin light chain and other signal peptides.
  • the amino acid sequence of the signal peptide the amino acid sequence described in SEQ ID NO: 10 can be cited.
  • the signal peptide is arranged at the N-terminus of the CAR.
  • the above-mentioned regions of the CAR of the present invention can be arranged in the order of the target antigen binding region, the transmembrane domain, and the T cell activation signal transmission region starting from the N-terminal. These respective regions may be directly connected to each other, or may be connected via other regions or interval sequences.
  • the CAR is composed of a signal peptide, a target antigen binding region, an extracellular hinge region, a transmembrane domain, a T cell activation signal transmission region for secondary signal transmission, and a primary signal transmission from the N-terminus.
  • T cell activation signal transmission region is a sequence of polypeptides configured.
  • the anti-TCR CAR of the present invention has an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 14.
  • the present invention also provides cells expressing the above CAR.
  • the cells are preferably mammalian cells, for example, human cells, or non-human mammalian cells such as mice, rats, cows, sheep, horses, dogs, pigs, and monkeys, and more preferably human cells.
  • the type of cells is not particularly limited, and examples include cells collected from blood, bone marrow fluid, spleen, thymus, lymph nodes, etc.; immune cells infiltrated in cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites.
  • Preferred examples include immune cells, and peripheral blood mononuclear cells isolated from peripheral blood can be preferably used.
  • effector cells are preferred, and particularly preferred cells include T cells and their precursor cells.
  • T cells are not particularly limited, and include ⁇ T cells, ⁇ T cells, CD8-positive T cells, cytotoxic T cells, CD4-positive T cells, helper T cells, memory T cells, naive T cells, tumor-infiltrating T cells, Any T cell such as natural killer T cell.
  • CD8-positive T cells or cytotoxic T cells are more preferred.
  • the cell of the present invention can be obtained by introducing a polynucleotide or vector containing the base sequence encoding the CAR of the present invention described later into the cell.
  • the present invention provides a polynucleotide comprising a base sequence encoding the CAR of the present invention.
  • the polynucleotide of the present invention is not particularly limited as long as it contains the base sequence encoding the CAR of the present invention.
  • the polynucleotide of the present invention preferably includes a base sequence encoding the aforementioned CAR amino acid sequence.
  • the base sequence encoding the amino acid sequence described in SEQ ID NO: 1-6 may be included. It may include the base sequence encoding the amino acid sequence described in SEQ ID NO: 7 and 8. It may include a base sequence encoding the amino acid sequence described in SEQ ID NO: 9.
  • the base sequence encoding the extracellular hinge region and the transmembrane domain may include a base sequence encoding the amino acid sequence described in SEQ ID NO: 11.
  • the base sequence encoding the T cell activation signal transmission region the base sequence encoding the amino acid sequence described in SEQ ID NO: 12 or 13 may be included.
  • a base sequence encoding the amino acid sequence described in SEQ ID NO: 10 may be included.
  • the base sequence encoding CAR the base sequence encoding the amino acid sequence described in SEQ ID NO: 14 may be included.
  • the base sequence encoding each of the above-mentioned regions is not limited to known ones, and any sequence may be used as long as it is a base sequence encoding each of the above-mentioned regions. Due to the polycondensation of gene encoding, there are multiple codons corresponding to one amino acid. Therefore, there are multiple base sequences encoding the same amino acid sequence.
  • the base sequence encoding each of the above-mentioned regions may be any one of a plurality of base sequences generated by condensation polymerization of the gene code as long as it encodes these regions.
  • the base sequence encoding each of the above-mentioned regions it is preferable to perform codon optimization according to the biological species of the introduced cell, and in the case of introducing into a human cell, it is preferable to perform human codon optimization.
  • the base sequence encoding each region described above may be a base sequence encoding a variant derived from each region of a natural protein.
  • the polynucleotide of the present invention can be obtained by linking a polynucleotide consisting of the base sequence encoding each region of the CAR of the present invention directly or via a spacer sequence.
  • the polynucleotide encoding each region of the CAR of the present invention can also be obtained by chemical synthesis using a known method based on the base sequence of each region.
  • cDNA obtained by reverse transcription of DNA extracted from T cells, etc., or RNA extracted from T cells, etc. can be used as a template to amplify the polynucleotides encoding each region by PCR, isothermal amplification, etc. Increase to obtain.
  • the polynucleotide encoding each region obtained in this way may be changed by substitution, deletion, addition, insertion, etc., within a range that does not lose the function of each region after translation.
  • the polynucleotide of the present invention may also contain control sequences such as promoters, enhancers, poly A additional signals, terminators, and base sequences encoding other proteins.
  • the present invention provides a vector comprising a polynucleotide encoding the CAR of the present invention.
  • the polynucleotide described above may also be in the form of a vector.
  • the type of vector is not particularly limited, and commonly used expression vectors and the like can be used.
  • the vector can be linear or circular, and can be a non-viral vector such as a plasmid, a viral vector, or a transposon-based vector. Examples of vectors include viral vectors, plasmid vectors, episomal vectors, and artificial chromosomal vectors.
  • viral vectors include Sendai virus vectors, retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, polio virus vectors, and Greek viruses. Erbis virus vector, rhabdovirus vector, paramyxovirus vector, orthomyxovirus vector, etc.
  • plasmid vectors examples include plasmid vectors for animal cell expression such as pA1-11, pXT1, pRc/CMV, pRc/RSV, and pcDNAI/Neo.
  • Episomal vectors are vectors that can replicate autonomously outside the chromosome.
  • a vector containing a sequence required for autonomous replication from EBV, SV40, etc., as a vector element can be cited.
  • a vector element required for autonomous replication specifically, a gene encoding an origin of replication and a protein that binds to the origin of replication to control replication can be cited.
  • EBV may include the origin of replication oriP and the EBNA-1 gene
  • SV40 may include the origin of replication ori and the SV40LT gene.
  • artificial chromosome vectors examples include YAC (Yeast artificial chromosome) vectors, BAC (Bacterial artificial chromosome) vectors, and PAC (P1-derived artificial chromosome) vectors.
  • a viral vector can be cited, and as a more preferred example, a retroviral vector can be cited.
  • a retroviral vector As the retroviral vector, pMSGV1 vector (Tamada k et al., Clin Cancer Res18: 6436-6445 (2012)) or pMSCV vector (manufactured by Takara Bio) can be exemplified.
  • the genes in the vector are combined into the genome of the host cell and can be stably expressed in the host cell for a long time.
  • the vector of the present invention may further include a base sequence encoding a replication origin, a protein that binds to the replication origin and controls replication, a base sequence encoding a marker gene such as a drug resistance gene, a reporter gene, and the like.
  • the present invention provides a pharmaceutical composition comprising the aforementioned polynucleotide, vector or CAR expressing cell.
  • the pharmaceutical composition of the present invention may also contain other ingredients such as a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for example, in addition to pharmaceutically acceptable carriers, T cell activating factors such as cytokines, immune activating agents, immune checkpoint inhibitors, other CAR-expressing cells, anti-inflammatory agents, etc., but not It is not limited to these.
  • pharmaceutically acceptable carriers include cell culture media, physiological saline, phosphate buffer, and citrate buffer.
  • the pharmaceutical composition of the present invention can be administered by a known method, and preferably can be administered to a patient by injection or infusion.
  • administration route intravenous administration is preferred, but it is not limited to this, and administration may be by injection into a tumor or the like.
  • the pharmaceutical composition of the present invention can contain a therapeutically effective amount of CAR expressing cells.
  • the "therapeutically effective dose” refers to the dose of the drug effective for the treatment or prevention of diseases.
  • the therapeutically effective amount can vary according to the state of the disease, age, sex, and weight of the subject of administration.
  • the therapeutically effective amount of the CAR-expressing cells may be, for example, an amount that the CAR-expressing cells can inhibit the proliferation of tumors.
  • the dosage and interval of administration of the pharmaceutical composition of the present invention can be appropriately selected according to the age, sex, weight, etc. of the subject of administration, the type of disease, the degree of progression, symptoms, etc., and the administration method.
  • a therapeutically effective amount can be administered.
  • the number of administered cells includes 1 ⁇ 10 4 to 1 ⁇ 10 10 , preferably 1 ⁇ 10 5 to 1 ⁇ 10 9 and more preferably 5 ⁇ 10 6 to 5 ⁇ 10 8 A.
  • the administration interval of the pharmaceutical composition of the present embodiment can be, for example, every 1 week, every 10 to 30 days, every 1 month, every 3 to 6 months, every 1 year, or the like.
  • CAR-expressing cells can proliferate autonomously in the body of the administration target, they can also be administered all at once.
  • the number of CAR-expressing cells in the body can also be monitored after administration, and the period of administration can be determined based on the results.
  • the pharmaceutical composition of the present invention may be used in combination with other anticancer agents.
  • other anticancer agents include alkylating agents such as cyclophosphamide, metabolic antagonists such as pentostatin, molecular targeted drugs such as rituximab, kinase inhibitors such as imatinib, and bortezomib Proteasome inhibitors, calcineurin inhibitors such as cyclosporine, anti-cancer antibiotics such as idarubicin, plant alkaloids such as irinotecan, platinum preparations such as cisplatin, and hormones such as tamoxifen Therapeutic drugs, immunological control drugs such as Odivo, pembrolizumab, etc. are not limited to these.
  • the present invention provides a kit for manufacturing CAR expressing cells, which includes the aforementioned vector.
  • the kit is not particularly limited as long as it contains the aforementioned vector, and may contain instructions for producing CAR-expressing cells, reagents for introducing the vector into cells, and the like.
  • the present invention provides a method of immunotherapy or anti-transplant rejection, the method comprising administering the aforementioned polynucleotide, vector, cell or pharmaceutical composition to a patient.
  • the patients include patients with immune diseases and cancer patients.
  • the cancer patients include acute myeloid leukemia patients, chronic myeloid leukemia patients, acute lymphocytic leukemia patients, Hodgkin lymphoma patients, neuroblastoma patients, Ewing sarcoma patients, multiple myeloma patients, and myelodysplastic syndromes Sign patients, BPDCN patients, glioma patients, or other solid tumor patients: including pancreatic cancer patients, lung cancer patients, colorectal cancer patients, breast cancer patients, and bladder cancer patients.
  • the cancer patient is a T-cell lymphoma patient (such as, for example, anaplastic large cell lymphoma (ALCL), peripheral T-cell lymphoma-non-specific (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL) and other T-cell lymphomas).
  • a T-cell lymphoma patient such as, for example, anaplastic large cell lymphoma (ALCL), peripheral T-cell lymphoma-non-specific (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL) and other T-cell lymphomas.
  • the cancer is characterized by the expression or overexpression of TCR.
  • the transplant rejection reaction includes graft versus host reaction and host versus graft reaction.
  • the present invention provides an anti-cancer gene therapy.
  • the method comprises administering the aforementioned polynucleotide, vector, or pharmaceutical composition to a patient.
  • the cancer patients include acute myeloid leukemia patients, chronic myeloid leukemia patients, acute lymphocytic leukemia patients, Hodgkin lymphoma patients, neuroblastoma patients, Ewing sarcoma patients, multiple myeloma patients, and myelodysplastic syndromes Sign patients, BPDCN patients, glioma patients, or other solid tumor patients: including pancreatic cancer patients, lung cancer patients, colorectal cancer patients, breast cancer patients, and bladder cancer patients. Cancer is characterized by the expression or overexpression of TCR.
  • the cancer is T cell lymphoma (e.g., for example, anaplastic large cell lymphoma (ALCL), peripheral T cell lymphoma-non-specific (PTCL-NOS), angioimmunoblastic T cell Lymphoma (AITL) and other T-cell lymphomas).
  • T cell lymphoma e.g., for example, anaplastic large cell lymphoma (ALCL), peripheral T cell lymphoma-non-specific (PTCL-NOS), angioimmunoblastic T cell Lymphoma (AITL) and other T-cell lymphomas.
  • ACL anaplastic large cell lymphoma
  • PTCL-NOS peripheral T cell lymphoma-non-specific
  • AITL angioimmunoblastic T cell Lymphoma
  • the cancer is characterized by the expression or overexpression of TCR.
  • the present invention provides the application of the aforementioned polynucleotide and carrier in the preparation of the aforementioned cell or pharmaceutical composition.
  • the present invention provides the use of the aforementioned cells in the preparation of the aforementioned pharmaceutical composition.
  • the present invention provides the application of the aforementioned polynucleotide, carrier, cell, or pharmaceutical composition in the preparation of drugs for immunotherapy.
  • the present invention provides the application of the aforementioned polynucleotide, carrier, cell, or pharmaceutical composition in the preparation of anti-cancer drugs.
  • the cancer limit is the same as before.
  • the present invention provides the application of the aforementioned polynucleotide, carrier, cell, or pharmaceutical composition in the preparation of a medicine for resisting transplantation rejection.
  • the invention provides the application of TCR in the preparation of anti-TCR chimeric antigen receptors.
  • the present invention provides the application of an antibody against TCR or its antigen binding region in preparing chimeric antigen receptors against TCR.
  • the chimeric antigen receptor for anti-TCR is the same as the chimeric antigen receptor described above.
  • the present invention also provides a method for destroying TCR positive cells.
  • the method includes the following steps:
  • anti-TCR chimeric antigen receptor is the same as above.
  • the present invention also provides a method for producing engineered cells, the method comprising the following steps:
  • step 3 Introduce the polynucleotide encoding the recombinant anti-TCR chimeric antigen receptor into the cell obtained by the treatment in step 3;
  • the method also includes the following steps: 4) at least one polynucleotide encoding a recombinant chimeric antigen receptor (not an anti-TCR chimeric antigen receptor) is introduced into the cells obtained by the treatment in step 2;
  • Step 3 and step 4 can be performed at the same time, or step 3 and then step 4 can be performed, or step 4 and step 3 can be performed first.
  • the donor is a healthy person and not a patient.
  • the anti-TCR chimeric antigen receptor is specifically directed to the TCR epitope, specifically to the epitope of the TCR-related protein, or specifically to the epitope of the TCR subunit.
  • polynucleotide encoding the recombinant anti-TCR chimeric antigen receptor includes a base sequence encoding the amino acid sequence described in SEQ ID NO: 14.
  • the technique for inhibiting the expression of endogenous TCR in the immune cells obtained in step 1 includes introducing mRNA encoding a rare-cutting endonuclease against the genome sequence.
  • the rare-cutting endonuclease includes TAL effector, CRISPR CAS9, and ZFN.
  • RNA interference technology can also be used to inhibit the expression of endogenous TCR in immune cells.
  • the method of introducing the polynucleotide encoding the recombinant chimeric antigen receptor into the cells obtained through the step 2 treatment is not particularly limited, and a known method can be used.
  • a virus infection method a liposome transfection method, a microinjection method, a calcium phosphate method, a DEAE-dextran method, an electroporation method, a method using a transposon, a particle gun method, etc. can be mentioned.
  • the present invention can also be produced by using a known gene editing technique or the like to assemble a polynucleotide containing a CAR-encoding base sequence into the genome of a cell so that it can be expressed under the control of an appropriate promoter.
  • gene editing techniques include the use of zinc finger nuclease, TALEN (transcription activator-like effector nuclease), CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system, PPR (pentatricopeptide repeat) and other endonucleases.
  • immune cells obtained from a donor include T cells.
  • T cells include regulatory T cells, cytotoxic T cells, helper T cells, and memory T cells.
  • the T cells include CD4+ T cells and CD8+ T cells.
  • the chimeric antigen receptor in step 4 above specifically targets cell surface antigens.
  • Cell surface antigens that can be used in the present invention include CAIX, ROR1, CD20, CD44v7/8, CEA, EGP-2, EGP-40, erb-B2/3/4, FBP, fetal acetylcholine receptor, EGFRvIII, BCMA, CD33, GD3, CD19, CD38, HSP70, CD30, FAP, HER2, CD79a, CD79b, CD123, CD22, CLL-1, MUC-1, GD2, O acetyl GD2, CS1, KDR, LEY, MAGE-A1, Mesothelin, PSCA, PSMA, TAG-72, VEGF-R2.
  • the chimeric antigen receptor in step 4 above is single-chain or multi-chain.
  • Figure 1 shows a schematic diagram of the LV-TCRCAR plasmid constructed in the present invention
  • Figure 2 shows the result of detecting the transduction rate of lentivirus by flow cytometry
  • Figure 3 shows the result of using flow cytometry to detect the effect of TCR knockout
  • Figure 4 shows the results of using flow cytometry to detect the killing effect of CAR-T cells on Jurkat-GFP cells
  • Fig. 5 shows a result diagram of using an animal model to study the effect of CAR-T cells constructed in the present invention on tumors
  • Figure 6 shows a statistical graph of fluorescence intensity in mice
  • Figure 7 shows a statistical chart of the survival time of mice
  • Figure 8 shows a statistical diagram of the clearance effect of LV-TCRCAR-T on TCR-positive cells.
  • each partial sequence is sequentially connected to form an anti-TCR expression CAR nucleic acid molecule, sequence code TCRCAR (SEQ ID NO: 15).
  • Insert TCRCAR into the expression vector pLVX-Puro according to restriction digestion and connection (the linear sequence of the vector is shown in SEQ ID NO: 16) to construct the LV-TCRCAR expression plasmid.
  • the schematic diagram of the LV-TCRCAR plasmid is shown in Figure 1 (intracellular The costimulatory domain is 4-1BB, and EGFR D III-D VI can be used as CAR expression detection markers and the suicide gene of CAR-T cells, increasing the safety of the product).
  • Restriction site XbaI, EcoRI. Transform, plate, and sequence to confirm that the plasmid is constructed successfully. Large-scale extraction of plasmids to obtain endotoxin-free expression plasmids for packaging lentivirus.
  • PEI transfection method for T75 culture flask.
  • Virus packaging will be performed in the afternoon of day6. Observe the cell status before transfection, and proceed to transfection when the confluence is about 90%. The culture medium in the bottle was discarded, replaced with 15ml fresh DMEM medium (without antibiotics), and cultured for 30 minutes.
  • Solution A Take 17.7 ⁇ g of LV-TCRCAR expression plasmid, helper plasmid pRSV-REV 8.8 ⁇ g, helper plasmid pMDLg/pRRE 8.8 ⁇ g and helper plasmid pMD2.G 4.4 ⁇ g, the transfection ratio is 4:2:2:1, total The amount is 40 ⁇ g, after mixing, dilute to 0.75ml with serum-free DMEM, and let stand at room temperature for 5min after mixing.
  • Solution B preparation Take 630 ⁇ l DMEM, and then add 120 ⁇ l PEI working solution (1mg/ml, stored at 4°C), mix well, and let stand for 5min at room temperature.
  • T cell complete medium preparation OpTmizer TM CTS TM T-cell Expansion SFM + 5% CTS Immune cell SR + 1% L-glutamine + 10ng/ml IL-7/15.
  • the starting cell number is 3M+Human T-Activator TCR/CD28 Dynabeads 75 ⁇ l.
  • the initial cell concentration is 1M/ml. Cultivate in a 37°C incubator. Activate for 48 hours.
  • the CRISPR/cas9 system was used to design sgRNA and knock out TCR by electrotransformation.
  • Cas9 protein and sgRNA were purchased from ThermFisher Company.
  • TCR sgRNA sequence is as follows:
  • TCR knockout effect After 48 hours of electroporation, flow cytometry was used to detect the TCR knockout effect. The results are shown in Figure 2.
  • the TCR knockout rate reached 80-90%, and LV-TCRCAR-T (knock out LV-TCRCAR-transfected T cells, CAR-T cells) cells had a small amount of TCR/ ⁇ TCR/ ⁇ , TCR positive
  • PanT represents untreated T cells
  • PanT TCRKO represents TCR knock-out T cells
  • LV-TCRCAR-T represents CAR-T cells.
  • the results are shown in Figure 3.
  • the CAR expression rate is not less than 50% after 3 days of lentiviral transduction.
  • PanT represents untreated T cells
  • PanT TCRKO represents TCR knocked out T cells
  • LV-TCRCAR-T represents CAR-T cells.
  • the Jurkat-GFP cell line is co-cultured with the CAR-T cells prepared in Example 1, and the E/T (Jurkat-GFP: CAR-T) ratio is 8:1, 4:1, 2:1, and 1:1 respectively. , 0.5:1, 0:1.
  • Jurkat-GFP group 0.5M per well, three multiple wells;
  • PanT TCRKO (T cell knockout TCR) group 0.5M per well, three duplicate wells;
  • PanT TCRKO T cell knockout TCR + Jurkat-GFP group: 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1;
  • LV-TCRCAR(CAR-T)+Jurkat-GFP group 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1;
  • NPG mice aged 5-8 weeks, all female, were injected with 1 ⁇ 10 6 Jurkat-Fluc cells through the tail vein. One week later, the biofluorescence test confirmed that the NPG mouse tumor model was successfully constructed.
  • mice were divided into tumor model group (negative control group), LV-CD3CAR-T group (positive control group), LV-TCRCAR-T group, a total of three groups, each with three mice.
  • the results of in vivo effectiveness are shown in Figure 5.
  • the CAR-T group significantly inhibited tumor growth, and the biofluorescence intensity was significantly lower than that of the tumor group.
  • the survival time of mice in the CAR-T group was significantly prolonged.
  • the mice in the experimental group were still alive during the observation period.
  • the LV-TCRCAR-T group has a better tumor suppression effect.
  • the anti-TCR CAR-T constructed in the present invention can effectively kill TCR positive cells, and can be used to treat T cell-derived lymphocytic leukemia and T cell-derived lymphoma.
  • the results are shown in Figure 8.
  • the CAR-T cells constructed in the present invention can effectively eliminate TCR-positive cells in the allogeneic body. Therefore, the TCRCAR-T of the present invention can be used to inhibit the occurrence of transplant rejection.
  • UCAR-T stands for LV-TCRCAR-T.

Abstract

用于免疫治疗的嵌合抗原受体、制备方法及其应用。编码核酸、表达载体、宿主细胞及药物组合物以及使用其来治疗各种疾病例如癌症的方法。

Description

用于免疫治疗的嵌合抗原受体、制备方法及其应用 技术领域
本发明涉及免疫治疗领域，涉及用于免疫治疗的嵌合抗原受体、制备方法及其应用。
背景技术
表达嵌合抗原受体(在下文被称为“CAR”，)的T细胞(在下文被称为“CAR-T细胞”)是指这样的重组T细胞：其中编码识别在癌细胞表面上特异性表达的癌细胞表面抗原的受体的基因被引入到该T细胞中以杀伤癌细胞。作为以色列魏兹曼科学院(Weizmann Institute of Science in Israel)的化学家和免疫学家的Zelig Fshhar博士等人通过获得这样的理论成功地制备了具有嵌合抗原受体的T细胞：即当人工制造具有与在癌细胞中特异性表达的抗原结合的受体的T细胞时，发生仅针对癌细胞免疫应答，从而杀伤癌细胞，这一事实随后于1989年报道在PNAS上。
然而，早期生产的CAR-T细胞，即第一代CAR-T细胞仅使用CD3ζ作为信号转导结构域，除了其治疗效果不明显外，同样还存在持续时间短的缺点。因此，已经进行努力以改善CAR-T细胞的反应性，并且结果是产生了第二代CAR-T细胞，在第二代CAR-T细胞中，产生的共刺激结构域(CD28或CD137/4-1BB)和CD3ζ是结合的，其中存在于体内的CAR-T细胞的数量与第一代CAR-T细胞的数量相比显著增加。同时，第二代CAR-T细胞使用一种类型的共刺激结构域，而使用两种类型共刺激结构域的CAR-T细胞被称为第三代CAR-T。最近的研究大部分集中在第二代CAR-T细胞和第三代CAR-T细胞。同时，关于使用CAR-T细胞治疗癌症的方法，有报道称当转化以识别CD19的细胞毒性T细胞被注射到3例终末期慢性淋巴细胞白血病(chronic lymphoid leukemia，CCL)患者中时，其中两例患者的白血病得到彻底治疗，并且病情持续约10个月(N.Engl J Med 2011；365:725-733,2011年8月25日,Sic.Transl.Med 2011 Aug 10；3(95):95ra73)。 本文使用的CAR-T对应于第二代，使用4-1BB作为共刺激结构域并且使用CD3ζ作为信号转导结构域。CAR-T细胞的抗原结合结构域识别在白血病癌细胞表面发现的CD19作为抗原。
此外，有报道称，当通过给予CTL019治疗患有急性白血病患者时，30例患者中有27例完全缓解，所有患者中有67％的患者完全缓解2年，并且78％的患者存活2年。鉴于受试者患者是复发性或难治性患者，这个结果是非常令人惊讶的(N Engl j Med 2014；371:1507-1517,2014年10月16日)。
目前，对于使用各种CAR-T细胞的治疗方法，已经进行了各种血液癌症如淋巴瘤(lymphoma)、骨髓瘤(myeloma)等的临床试验，并且预计CAR-T将成为市场上可获得的可用药物。由于使用CAR-T细胞的癌症治疗是自源方法，因此该产品不能大规模生产；然而，这是患者特异性治疗，因此其治疗效果之高是现有的抗癌药物无法相比的。
发明内容
在一个实施方式中，本发明提供了一种抗TCR的嵌合抗原受体，其包含目标抗原结合结构域、跨膜结构域、T细胞活化信号传递区域。所述目标抗原是TCR。
在本说明书中，“嵌合抗原受体(CAR)”是指包含目标抗原结合结构域、跨膜结构域和T细胞活化信号传递区域的嵌合蛋白质。需要说明的是，嵌合蛋白质是指包含源自两种以上异种蛋白质的序列的蛋白质。CAR不限于仅包含上述3个区域，也可包含其他区域。
本实施方式的CAR包含目标抗原为TCR的目标抗原结合结构域。
“目标抗原结合结构域”是指在T细胞表达CAR时，在细胞外与目标抗原结合的细胞外区域。在CAR-T细胞中表达的CAR转移至细胞膜，位于细胞外的目标抗原结合结构域和位于细胞内的T细胞活化信号传递区域经由贯通细胞膜的跨膜结构域而成为连结的状态。当CAR-T细胞与具有目标抗原作为膜抗原的细胞接触时，目标抗原结合结构域与该目标抗原结合，由此T细胞活化信号从T细胞活化信号传递区域传递到T细胞内，T细胞被活化。
本发明具体实施方式中的目标抗原结合结构域只要能够与TCR特异性结合就没有特别限定，优选包含能够与TCR特异性结合的单克隆抗体(以下，也称为 “抗TCR抗体”)的抗原结合区域。抗体的“抗原结合区域”是指在抗体中与抗原结合有关的区域，具体而言，是指包含互补性决定区域(Complementarity Determining Region：CDR)的区域。抗体的抗原结合区域包含该抗体的至少一个CDR。在优选的方式中，抗体的抗原结合区域包含该抗体的全部6个CDR。另外，CDR能够通过作为CDR的定义已知的任意的定义来决定，例如能够使用Kabat、Chothia、AbM、cotact等的定义。优选的是，可举出由Kabat定义的CDR。
能够在目标抗原结合区域中使用的抗TCR抗体没有特别限定，可以是公知的抗体，也可以是新制作的抗体。新制作抗TCR抗体的情况下，抗TCR抗体的制作用公知的方法进行即可。例如，可以使用TCR免疫动物而得到杂交瘤的方法、噬菌体显示法等。
作为抗TCR抗体的例子，可举出具有SEQ ID NO：7记载的氨基酸序列作为重链可变(VH)区域、具有SEQ ID NO：8记载的氨基酸序列作为轻链可变(VL)区域的抗体。SEQ ID NO：7记载的氨基酸序列构成的VH区域的Kabat定义的CDR1～3的氨基酸序列分别示于SEQ ID NO：1-3。另外，由SEQ ID NO：8记载的氨基酸序列构成的VL区域的Kabat定义的CDR1～3的氨基酸序列分别示于SEQ ID NO：4-6。
在优选的方式中，目标抗原结合区域可以包含抗TCR抗体的VH区域和VL区域。例如，包含抗TCR抗体的VH区域和VL区域的单链抗体(scFv)的多肽是目标抗原结合区域的优选例。scFv是用肽连接子将抗体的VH区域和VL区域连结而成的多肽，一般用作CAR的目标抗原结合区域。
在使用scFv的情况下，连结VH区域和VL区域的肽连接子没有特别限定，可以使用scFv中通常使用的肽。作为肽连接子的例子，例如可举出SEQ ID NO：9所示的氨基酸序列的连接子，但并不限定于这些。
scFv中使用的VH区域和VL区域可以使用抗TCR抗体的VH区域和VL区域。抗TCR抗体的优选的例子如上所述。另外，scFv所使用的VH区域和VL区域只要维持对TCR的结合能力，也可以改变一部分的序列。例如，作为scFv，可以优选使用以下所述的序列：
(1)一种scFv，其包括包含由SEQ ID NO：7记载的氨基酸序列构成的VH区 域的CDR1-3的VH区域；和包含由SEQ ID NO：8记载的氨基酸序列构成的VL区域的CDR1-3的VL区域，具有对TCR的结合能力。
(2)一种scFv，其包括由SEQ ID NO：7记载的氨基酸序列构成的VH区域、和由SEQ ID NO：8记载的氨基酸序列构成的VL区域，具有对TCR的结合能力。
(3)一种scFv，其包含由SEQ ID NO：7记载的氨基酸序列中的一个或多个氨基酸变异而成的氨基酸序列构成的VH区域、和由SEQ ID NO：8记载的氨基酸序列中的一个或多个氨基酸变异而成的氨基酸序列构成的VL区域，具有对TCR的结合能力。
(4)一种scFv，其包含与SEQ ID NO：7记载的氨基酸序列具有95％以上的序列同一性(相同性)的氨基酸序列构成的VH区域、与SEQ ID NO：8记载的氨基酸序列具有95％以上的序列同一性(相同性)的氨基酸序列构成的VL区域，具有对TCR的结合能力。
在上述(1)中，CDR以外的序列(框架序列)优选使用已知的人抗体的框架序列。例如，可以由从GenBank等公知的序列数据库中登录的人抗体的氨基酸序列的框架序列、来源于人抗体的各亚组的共同序列(Human Most Homologous Consensus Sequence；Kabat，E.A.等Sequences of Proteins of Immunological Interest，US Dept.Health and Human Services,1991)中选择的氨基酸序列等进行选择。
在上述(3)中，“多个”例如可以为2～30个，优选为2～20个，更优选为2～10个，进一步优选为2～5个。另外，“变异”可以是缺失、替换、附加以及插入中的任一种，也可以是它们的组合。另外，变异的位置优选为CDR1～3以外的区域(即，框架区域)。
在上述(4)中，只要序列同一性为95％以上，就没有特别限定，优选为96％以上，更优选为97％以上，进一步优选为98％以上，进一步更优选为99％以上，特别优选为99.1％以上、99.2％以上、99.3％以上、99.4％以上、99.5％以上、99.6％以上、99.7％以上、99.8％以上、99.9％以上。需要说明的是，氨基酸序列彼此的序列同一性(相同性)是将2个氨基酸序列以对应的氨基酸最多一致的方式，在与 插入和缺失相当的部分加入间隙并排设置，相对于除去所得到的比对中的间隙以外的氨基酸序列整体一致的氨基酸的比例而求出。氨基酸序列之间的序列同一性可以使用该技术领域中公知的各种相同性检索软件求出。例如，氨基酸序列的序列同一性的值可以通过以公知的相同性检索软件BLASTP得到的比对为基础计算而得到。
作为scFv的具体例，包含SEQ ID NO：9所示的氨基酸序列的多肽；包含SEQ ID NO：9所示的氨基酸序列中的一个或多个氨基酸变异而成的氨基酸序列构成且具有对TCR的结合能力的多肽；或包含与SEQ ID NO：9所示的氨基酸序列具有95％以上的序列同一性(相同性)的氨基酸序列构成且具有对TCR的结合能力的多肽等。另外，关于“多个”及“变异”，与上述相同。另外，关于“序列同一性”，也与上述相同。
“跨膜结构域”是指在T细胞表达CAR时，贯通细胞膜而存在、将细胞外区域与细胞内区域连结的区域。跨膜结构域只要是具有贯通细胞膜的功能的多肽就没有特别限定。跨膜结构域可以来自天然蛋白质，也可以是人为设计的。来自天然蛋白质的跨膜结构域可以从任意的膜结合蛋白质或膜贯通蛋白质取得。在优选的方式中，跨膜结构域能够与目标抗原相对于目标抗原结合结构域的结合相对应地向T细胞活化信号传递区域传递活化信号。
作为跨膜结构域，例如可举出T细胞受体的α链及β链、CD3ζ、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、ICOS、CD154、GITR等跨膜结构域。这些蛋白质来自的生物没有特别限定，优选为人。这些蛋白质的氨基酸序列可从GenBank等公知的序列数据库获得。
跨膜结构域常与细胞外铰链区域连接，“细胞外铰链区域”是指将细胞外的目标抗原结合区域与跨膜结构域连结的区域。
在优选的方式中，本实施方式的CAR包含细胞外铰链区域。
细胞外铰链区域只要是能够将目标抗原结合区域与跨膜结构域连结的区域就没有特别限定。可以来自天然蛋白质，也可以是人为设计的。细胞外铰链区域例如可以由1～100个左右的氨基酸、优选10～70个左右的氨基酸构成。优选细 胞外铰链区域不妨碍目标抗原结合区域的TCR结合能力，且不妨碍T细胞活化信号传递区域的信号传递。
作为细胞外铰链区域，例如可举出CD8、CD28、CD4等细胞外铰链区域。另外，也可以使用免疫球蛋白(例如IgG4等)的铰链区域。上述蛋白质所来源的生物没有特别限定，优选为人。另外，这些蛋白质的氨基酸序列可从GenBank等公知的序列数据库获得。
另外，细胞外铰链区域和跨膜结构域也可以是来自上述天然蛋白质的细胞外铰链区域和跨膜结构域的变异体，例如可以举出以下的例子。
(1)由与来自天然蛋白质的细胞外铰链区域和跨膜结构域的氨基酸序列(例如SEQ ID NO：11具有95％以上的序列同一性(相同性)的氨基酸序列构成，且具有膜贯通能力的多肽。
(2)由源自天然蛋白质的细胞外铰链区域和跨膜结构域的氨基酸序列(例如SEQ ID NO：11)中一个或多个氨基酸变异而成的氨基酸序列构成，并且具有膜贯通能力的多肽。
在上述(1)中，只要序列同一性为95％以上，就没有特别限定，优选为96％以上，更优选为97％以上，进一步优选为98％以上，进一步更优选为99％以上，特别优选为99.1％以上、99.2％以上、99.3％以上、99.4％以上、99.5％以上、99.6％以上、99.7％以上、99.8％以上、99.9％以上。
在上述(2)中，“多个”例如可以为2～10个，优选为2～5个，更优选为2～4个，进一步优选为2或3个。另外，“变异”可以是缺失、替换、附加以及插入中的任一种，也可以是它们的组合。
T细胞活化信号传递区域”是指在T细胞表达CAR时位于细胞内、向T细胞内传递T细胞活化信号的区域。T细胞中，当MHC-肽复合体与T细胞受体(T cell Receptor：TCR)结合时，T细胞活化信号通过TCR·CD3复合体传递到细胞中，并且引起各种磷酸化信号(一次信号传递)。另外，已知在T细胞的细胞表面上表达的共刺激分子通过与表达在抗原呈递细胞的细胞表面上的各共刺激分子特异性的配体结合而向细胞内传递共刺激信号，辅助T细胞的活化(二次信号传递)。
在本说明书中，“T细胞活化信号传递”包括前述一次信号传递和二次信号传递两者。“T细胞活化信号传递区域”是指参与上述一次信号传递以及二次信号传递的蛋白质的、参与该信号传递的细胞内区域。
T细胞活化信号传递区域只要是参与T细胞活化信号传递的蛋白质的T细胞活化信号传递区域，就没有特别限定。例如，已知免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motif：ITAM)参与一次信号传递。因此，作为T细胞活化信号传递区域的例子，可以举出具有ITAM的蛋白质的T细胞活化信号传递区域。作为具有ITAM的蛋白质的例子，例如可列举出：CD3ζ、FcRγ、FcRβ、CD3γ，CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d等。包含这些蛋白质的ITAM的T细胞活化信号传递区域是用于CAR的T细胞活化信号传递区域的优选例。作为更优选的例子，可列举出CD3ζ等T细胞活化信号传递区域。
此外，如上所述，共刺激分子参与二次信号传递。因此，作为T细胞活化信号传递区域的例子，也可以举出共刺激分子的信号传递区域。作为共刺激分子的例子，例如有CD2、CD4、CD5、CD8、CD27、CD28，OXO40(CD134)、4-1BB(CD137)、ICOS，CD154、HVEM、GITR、Fc Receptor-associatedγchain等。这些蛋白质的T细胞活化信号传递区域也是用于CAR的T细胞活化信号传递区域的优选例。作为更优选的例子，可以举出CD28、4-1BB等T细胞活化信号传递区域。
上述蛋白质所来源的生物没有特别限定，优选为人。另外，这些蛋白质的氨基酸序列可从GenBank等公知的序列数据库获得。
另外，T细胞活化信号传递区域也可以是上述那样的来自天然蛋白质的T细胞活化信号传递区域的变异体。作为来自天然蛋白质的活化信号传递区域的变异体的例子，例如可举出以下的变异体。
(1)由与来自天然蛋白质的T细胞活化信号传递区域的氨基酸序列(例如SEQ ID NO：12或13)具有95％以上的序列同一性(相同性)的氨基酸序列构成、且具有T细胞活化信号传递能力的多肽。
(2)来自天然蛋白质的T细胞活化信号传递区域的氨基酸序列(例如SEQ ID  NO：12或13)中一个或多个氨基酸变异而成的氨基酸序列构成、并且具有T细胞活化信号传递能力的多肽。
在上述(1)中，只要序列同一性为95％以上，就没有特别限定，优选为96％以上，更优选为97％以上，进一步优选为98％以上，进一步更优选为99％以上，特别优选为99.1％以上、99.2％以上、99.3％以上、99.4％以上、99.5％以上、99.6％以上、99.7％以上、99.8％以上、99.9％以上。
在上述(2)中，“多个”例如在使用与一次信号传递有关的蛋白质的情况下，可以为2～30个，优选为2～20个，更优选为2～10个，进一步优选为2～5个。另外，例如，在使用共刺激分子的情况下，“多个”可以为2～15个，优选为2～10个，更优选为2～5个，进一步优选为2或3个。另外，“变异”可以是缺失、替换、附加以及插入中的任一种，也可以是它们的组合。
本发明的CAR所包含的T细胞活化信号传递区域的数量不限定于1个，也可以包含多个T细胞活化信号传递区域。多个T细胞活化信号传递区域既可以是相同的，也可以是不同的。在优选的方式中，CAR包含2个以上T细胞活化信号传递区域。在此情况下，CAR包含的T细胞活化信号传递区域优选为参与一次信号传递的T细胞活化信号传递区域与参与二次信号传递的T细胞活化信号传递区域的组合。作为具体例，可列举出CD3ζ与CD28的T细胞活化信号传递区域的组合、CD3ζ与4-1BB的T细胞活化信号传递区域的组合、CD3ζ与CD28与4-1BB的T细胞活化信号传递区域的组合等。
在仅使用一个T细胞活化信号传递区域的情况下，优选使用参与一次信号传递的T细胞活化信号传递区域，更优选使用CD3ζ的T细胞活化信号传递区域。
本发明的CAR除了上述的区域以外，还可以包含信号肽等。
“信号肽”是指示膜蛋白质或分泌蛋白质的局部化的肽。信号肽通常是由存在于膜蛋白质的N末端的5～60个左右的氨基酸构成的肽，在局部化完成的成熟蛋白质中被除去。
本发明的CAR中使用的信号肽优选为指示向细胞膜局部化的信号肽，优选为膜蛋白质的信号肽。信号肽的例子可列举T细胞受体的α链及β链、CD3ζ、 CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、ICOS、CD154、GITR、免疫球蛋白重链、免疫球蛋白轻链等信号肽。作为信号肽的氨基酸序列的具体例，可以举出SEQ ID NO：10记载的氨基酸序列。
在本发明的具体实施方式中，信号肽配置于CAR的N末端。
本发明的CAR的上述各区域可以从N末端开始按照目标抗原结合区域、跨膜结构域、T细胞活化信号传递区域的顺序配置。这些各区域既可以相互直接连结，也可以经由其他区域或间隔序列等连结。
作为本发明的CAR的具体例，CAR是由从N末端按照信号肽、目标抗原结合区域、细胞外铰链区域、跨膜结构域、二次信号传递的T细胞活化信号传递区域和一次信号传递的T细胞活化信号传递区域的顺序配置的多肽。
本发明的抗TCR的CAR具有与SEQ ID NO：14所示的氨基酸序列具有至少95％以上的序列同一性的氨基酸序列。
本发明还提供表达上述CAR的细胞。
细胞优选为哺乳动物细胞，例如，人细胞，或小鼠、大鼠、牛、羊、马、狗、猪、猴等非人哺乳动物细胞，更优选为人细胞。细胞的种类没有特别限定，可以举出从血液、骨髓液、脾脏、胸腺、淋巴结等采集的细胞；原发性肿瘤、转移性肿瘤、癌性腹水等癌组织中浸润的免疫细胞等。作为优选的例子，可以举出免疫细胞，可以优选使用从末梢血分离的末梢血单核细胞等。在末梢血单核细胞包含的细胞中，优选效应细胞，作为特别优选的细胞，可以举出T细胞及其前体细胞。T细胞的种类没有特别限定，可举出αβT细胞、γδT细胞、CD8阳性T细胞、细胞毒性T细胞，CD4阳性T细胞、辅助性T细胞、记忆T细胞、初始T细胞、肿瘤浸润T细胞、自然杀伤T细胞等的任意T细胞。其中，更优选为CD8阳性T细胞或细胞毒性T细胞。
本发明的细胞可以通过将包含后面所述的编码本发明的CAR的碱基序列的多核苷酸或载体导入细胞而得到。
本发明提供包含编码本发明的CAR的碱基序列的多核苷酸。
本发明的多核苷酸只要包含编码本发明的CAR的碱基序列，就没有特别限 定。本发明的多核苷酸优选包含编码前面所述的CAR氨基酸序列的碱基序列。
在本发明的具体实施方案中，作为编码目标抗原结合区域的碱基序列，可包含编码SEQ ID NO：1-6记载的氨基酸序列的碱基序列。可包含编码SEQ ID NO：7和8记载的氨基酸序列的碱基序列。可包含编码SEQ ID NO：9记载的氨基酸序列的碱基序列。
作为编码细胞外铰链区和跨膜结构域的碱基序列，可包含编码SEQ ID NO：11记载的氨基酸序列的碱基序列。
作为编码T细胞活化信号传递区域的碱基序列，可包含编码SEQ ID NO：12或13记载的氨基酸序列的碱基序列。
作为编码信号肽的碱基序列，可包含编码SEQ ID NO：10记载的氨基酸序列的碱基序列。
作为编码CAR的碱基序列，可包含编码SEQ ID NO：14记载的氨基酸序列的碱基序列。
编码上述各区域的碱基序列不限于公知的，只要是编码上述各区域的碱基序列，则可以是任意的序列。由于基因编码的缩聚，与1个氨基酸对应的密码子存在多个。因此，编码同一氨基酸序列的碱基序列存在多个。编码上述各区域的碱基序列只要编码这些区域，则可以是因基因编码的缩聚而产生的多个碱基序列中的任一种。作为编码上述各区域的碱基序列，优选根据导入的细胞的生物种，进行密码子优化，在导入人细胞的情况下，优选进行人密码子优化。另外，编码上述各区域的碱基序列也可以是编码来自天然蛋白质的各区域的变异体的碱基序列。
本发明的多核苷酸可通过直接或经由间隔序列连结由编码本发明的CAR的各区域的碱基序列构成的多核苷酸而得到。编码本发明的CAR的各区域的多核苷酸也可以通过基于各区域的碱基序列，利用公知的方法化学合成来取得。另外，也可以将对从T细胞等提取的DNA、从T细胞等提取的RNA进行逆转录而得到的cDNA作为模板，通过PCR法、等温扩增法等对编码各区域的多核苷酸进行扩增来取得。对这样得到的各区域进行编码的多核苷酸也可以在不丧失翻译后的各区域的功能的范围内进行替换、缺失、附加、插入等的改变。
本发明的多核苷酸除了编码本发明的CAR的碱基序列之外，还可以包含启动子、增强子、多聚A附加信号、终止子等控制序列、编码其他蛋白质的碱基序列等。
本发明提供包含编码本发明的CAR的多核苷酸的载体。
上面所述的多核苷酸也可以是载体的形态。载体的种类没有特别限定，可以使用通常使用的表达载体等。载体可以是直链状也可以是环状，可以是质粒等非病毒载体，也可以是病毒载体，还可以是基于转座子的载体。作为载体，例如可以举出病毒载体、质粒载体、附加型载体和人工染色体载体等。
作为病毒载体，例如可以举出仙台病毒载体、逆转录病毒(包括慢病毒)载体，腺病毒载体、腺伴随病毒载体、疱疹病毒载体、痘苗病毒载体、痘病毒载体，脊髓灰质炎病毒载体、希尔比斯病毒载体、弹状病毒载体，副粘液病毒载体、正粘液病毒载体等。
作为质粒载体，例如可举出pA1-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo等动物细胞表达用质粒载体。
附加型载体是能够在染色体外进行自主复制的载体。作为附加型载体，例如可以举出包含来自EBV、SV40等的自主复制所需的序列作为载体要素的载体。作为自主复制所需的载体要素，具体而言，可以举出编码复制起始点和与复制起始点结合来控制复制的蛋白质的基因。例如，EBV可以举出复制起始点oriP与EBNA-1基因，SV40可以举出复制起始点ori和SV40LT基因。
作为人工染色体载体，例如可举出YAC(Yeast artificial chromosome)载体、BAC(Bacterial artificial chromosome)载体、PAC(P1–derived artificial chromosome)载体等。
作为本发明的载体的优选例，可以举出病毒载体，作为更优选的例子，可以举出逆转录病毒载体。作为逆转录病毒载体，可以例示pMSGV1载体(Tamada k et al.，Clin Cancer Res18：6436-6445(2012))或pMSCV载体(Takara Bio公司制)。通过使用逆转录病毒载体，载体中的基因被组合到宿主细胞的基因组中，能够在宿主细胞中长时间稳定地表达。
本发明的载体还可以包含编码复制起始点、与复制起始点结合并控制复制的 蛋白质的碱基序列、编码抗药性基因、报告基因等标记基因的碱基序列等。
本发明提供包含前面所述的多核苷酸、载体或CAR表达细胞的药物组合物。
本发明的药物组合物还可以含有药学上可接受的载体等其他成分。作为其他成分，例如，除了药学上可接受的载体以外，还可以举出细胞因子等T细胞活化因子、免疫活化剂、免疫检验点抑制剂、其他表达CAR的细胞、抗炎症剂等，但并不限定于这些。作为药学上可接受的载体，可以举出细胞培养培养基、生理盐水、磷酸缓冲液、柠檬酸缓冲液等。
本发明的药物组合物可以通过公知的方法投与，优选可以通过注射或输液投与给患者。作为投与途径，优选静脉内投与，但并不限定于此，也可以通过向肿瘤内注入等投与。
本发明的药物组合物能够包含治疗有效量的CAR表达细胞。“治疗有效量”是指对于疾病的治疗或预防有效的药剂量。治疗有效量可根据投与对象的疾病的状态、年龄、性别及体重等而变动。在本发明的药物组合物中，上述CAR表达细胞的治疗有效量例如可以是CAR表达细胞能够抑制肿瘤的增殖的量。
本发明的药物组合物的投与量及投与间隔可以根据投与对象的年龄、性别及体重等、疾病的种类、发展度及症状等、以及投与方法等适当选择。作为投与量，可以投与治疗有效量，例如，在1次投与中，作为投与细胞的个数，可以举出1X10 4～1X10 10个、优选1X10 5～1X10 9个、更优选5X10 6～5X10 8个。
作为本实施方式的药物组合物的投与间隔，例如可以设为每1周、每10～30天、每1个月、每3～6月、每1年等。另外，CAR表达细胞能够在投与对象的体内自主地增殖，因此也可以一次性投与。另外，也可以在投与后监测体内的CAR表达细胞的数量，根据其结果决定投与时期。
另外，本发明的药物组合物可以与其他抗癌剂并用。作为其他抗癌剂，可列举环磷酰胺等烷基化剂、喷司他丁等代谢拮抗剂、利妥昔单抗等分子靶向药、伊马替尼等的激酶抑制剂、硼替佐米等蛋白酶体抑制剂、环孢霉素等钙调磷酸化酶抑制剂、伊达比星等抗癌性抗生物质、伊立替康等植物生物碱，顺铂等铂制剂、他莫昔芬等激素疗法药、欧狄沃、派姆单抗等免疫控制药等，但并不限定于这些。
本发明提供一种用于制造CAR表达细胞的试剂盒，其包括前面所述的载体。 该试剂盒只要包含前面所述的载体就没有特别限制，可以含有用于制造CAR表达细胞的说明书、用于将载体导入细胞的试剂等。
本发明提供一种免疫治疗或抗移植排异反应的方法，所述方法包括给患者施用前面所述的多核苷酸、载体、细胞或药物组合物。
所述患者包括身免疫性疾病患者、癌症患者。
所述癌症患者包括急性髓系白血病患者、慢性髓系白血病患者、急性淋巴细胞白血病患者、霍奇金淋巴瘤患者、神经母细胞瘤患者、尤文肉瘤患者、多发性骨髓瘤患者、骨髓增生异常综合征患者、BPDCN患者、胶质瘤患者，或其他实体瘤患者：包括胰腺癌患者、肺癌患者、结直肠癌患者、乳腺癌患者、膀胱癌患者。
在特别优选的实施方案中，癌症患者为T细胞淋巴瘤患者(如例如，间变性大细胞淋巴瘤(ALCL)、外周T细胞淋巴瘤-非特指型(PTCL-NOS)、血管免疫母细胞性T细胞淋巴瘤(AITL)和其它T细胞淋巴瘤)。优选地，癌症的特征在于TCR的表达或过表达。
所述移植排异反应包括移植物抗宿主反应、宿主抗移植物反应。
本发明提供一种抗癌基因疗法，所述方法包括给患者施用前面所述的多核苷酸、载体、或药物组合物。
所述癌症患者包括急性髓系白血病患者、慢性髓系白血病患者、急性淋巴细胞白血病患者、霍奇金淋巴瘤患者、神经母细胞瘤患者、尤文肉瘤患者、多发性骨髓瘤患者、骨髓增生异常综合征患者、BPDCN患者、胶质瘤患者，或其他实体瘤患者：包括胰腺癌患者、肺癌患者、结直肠癌患者、乳腺癌患者、膀胱癌患者。癌症的特征在于TCR的表达或过表达。
在特别优选的实施方案中，癌症为T细胞淋巴瘤(如例如，间变性大细胞淋巴瘤(ALCL)、外周T细胞淋巴瘤-非特指型(PTCL-NOS)、血管免疫母细胞性T细胞淋巴瘤(AITL)和其它T细胞淋巴瘤)。优选地，癌症的特征在于TCR的表达或过表达。
本发明提供了前面所述的多核苷酸、载体在制备前面所述的细胞或药物组合物中的应用。
本发明提供了前面所述的细胞在制备前面所述的药物组合物中的应用。
本发明提供了前面所述的多核苷酸、载体、细胞、或药物组合物在制备用于免疫治疗的药物中的应用。
本发明提供了前面所述的多核苷酸、载体、细胞、或药物组合物在制备抗癌症的药物中的应用。
癌症限定同前。
本发明提供了前面所述的多核苷酸、载体、细胞、或药物组合物在制备抗移植排异反应的药物中的应用。
本发明提供了TCR在制备抗TCR的嵌合抗原受体中的应用。
本发明提供了针对TCR的抗体或其抗原结合区域在制备抗TCR的嵌合抗原受体中的应用。
抗TCR的嵌合抗原受体正如前面所述的嵌合抗原受体一样。
本发明还提供了一种破坏TCR阳性细胞的方法，所述方法包括如下步骤：
1)获取TCR阳性细胞作为靶细胞；
2)将细胞膜表面表达抗TCR的嵌合抗原受体的细胞作为第二细胞；
3)将步骤2获得的第二细胞与步骤1的靶细胞接触。
进一步，所述抗TCR的嵌合抗原受体的限定同前。
本发明还提供了一种生产工程化细胞的方法，所述方法包括以下步骤：
1)从供体处获得免疫细胞；
2)抑制步骤1获得的免疫细胞中内源性TCR表达；
3)将编码重组抗TCR的嵌合抗原受体的多核苷酸导入经步骤3处理获得的细胞中；
优选地，
所述方法还包括如下步骤：4)将至少一种编码重组嵌合抗原受体(并非抗TCR的嵌合抗原受体)的多核苷酸导入经过步骤2处理获得的细胞中；
步骤3和步骤4可同时进行，或先进行步骤3再进行步骤4，或先进行步骤4再进行步骤3。
进一步，所述供体是健康人而不是患者。
进一步，所述抗TCR的嵌合抗原受体特异性针对TCR表位，特异性针对TCR相关蛋白的表位，或特异性针对TCR亚基的表位。
进一步，所述编码重组抗TCR的嵌合抗原受体的多核苷酸包括编码SEQ ID NO：14记载的氨基酸序列的碱基序列。
抑制步骤1获得的免疫细胞中内源性TCR表达的技术包括引入编码针对基因组序列的稀有切割核酸内切酶的mRNA。
所述稀有切割核酸内切酶包括TAL效应因子、CRISPR CAS9、ZFN。
也可使用常用的RNA干扰技术抑制免疫细胞中内源性TCR表达。
将编码重组嵌合抗原受体的多核苷酸导入经过步骤2处理获得的细胞中的方法没有特别限定，可以使用公知的方法。例如，可以举出病毒感染法、脂质体转染法、微注入法、磷酸钙法、DEAE-葡聚糖法、电穿孔法、使用转座子的方法、粒子枪法等。
另外，本发明的也可以通过使用公知的基因编辑技术等，将包含编码CAR的碱基序列的多核苷酸以能够在适当的启动子的控制下表达的方式组装到细胞的基因组中而制造。作为基因编辑技术，例如，可举出使用锌指核酸酶、TALEN(转录活化因子样效应核酸酶)、CRISPR(Clustered Regularly Interspaced Short Palindromic Repeat)-Cas系统、PPR(pentatricopeptide repeat)等内切核酸酶的技术。
进一步，从供体处获得的免疫细胞包括T细胞。T细胞包括调节性T细胞、细胞毒性T细胞、辅助性T细胞、记忆T细胞。
进一步，所述T细胞包括CD4+T细胞、CD8+T细胞。
上述步骤4中的嵌合抗原受体特异性针对细胞表面抗原。
可用于本发明的细胞表面抗原包括CAIX、ROR1、CD20、CD44v7/8、CEA、EGP-2、EGP-40、erb-B2/3/4、FBP、胎儿乙酰胆碱受体、EGFRvIII、BCMA、CD33、GD3、CD19、CD38、HSP70、CD30、FAP、HER2、CD79a、CD79b、CD123、 CD22、CLL-1、MUC-1、GD2、O acetyl GD2、CS1、KDR、LEY、MAGE-A1、间皮素、PSCA、PSMA、TAG-72、VEGF-R2。
上述步骤4中的嵌合抗原受体是单链的或多链的。
附图说明
图1显示本发明构建的LV-TCRCAR质粒示意图；
图2显示利用流式细胞仪检测慢病毒转导率的结果图；
图3显示利用流式细胞仪检测TCR敲除效果的结果图；
图4显示利用流式细胞仪检测CAR-T细胞对Jurkat-GFP细胞的杀伤作用结果图；
图5显示利用动物模型研究本发明构建的CAR-T细胞对肿瘤影响的结果图；
图6显示小鼠体内荧光强度统计图；
图7显示小鼠生存时间统计图；
图8显示LV-TCRCAR-T对TCR阳性细胞的清除效果统计图。
具体实施方式
以下实施例进一步说明本发明。下述实施例只是为了说明本发明，而不应被解释为是限制性的。
实施例1 抗TCR的CAR表达
1、合成抗TCR的CAR的核酸分子
按照信号肽-ScFv-铰链区及跨膜结构域-二次信号传递的T细胞活化信号传递区域-一次信号传递的T细胞活化信号传递区域的顺序将各部分序列依次连接形成可表达抗TCR的CAR的核酸分子，序列代号TCRCAR(SEQ ID NO：15)。
3、构建LV-TCRCAR表达质粒
将TCRCAR按照酶切连接的方式插入表达载体pLVX-Puro中(载体的线性序列如SEQ ID NO：16所示)，构建LV-TCRCAR表达质粒，LV-TCRCAR质粒示意图如图1所示(胞内共刺激域为4-1BB，EGFR D III-D VI可作为CAR表达检测标志物以及CAR-T细胞的自杀基因，增加该产品的安全性)。酶切位点： XbaI，EcoRI。转化，涂板，小提测序，确认质粒构建成功。质粒大提获得无内毒素的表达质粒，以备包装慢病毒。
4、LV-TCRCAR慢病毒包装
PEI转染法(针对T75培养瓶)步骤如下：
(1)day1复苏293T/17细胞至1*T75内，培养基体积15ml；
(2)day3将293T/17细胞传代至1*T225内，培养基体积45ml；
(3)day5将293T/17细胞传代至3*T225内，接种密度大概为6*10 7个细胞/T225瓶；
(4)day6下午进行病毒包装。转染前观察细胞状态，汇聚度约90％时进行转染。弃去瓶内培养基，更换为15ml新鲜的DMEM培养基(无抗生素)，培养30min。
溶液A配制：取LV-TCRCAR表达质粒17.7μg，辅助质粒pRSV-REV 8.8μg、辅助质粒pMDLg/pRRE 8.8μg及辅助质粒pMD2.G 4.4μg，转染比例为4:2:2:1，总量为40μg，混匀后用无血清DMEM稀释定容至0.75ml，混匀后室温静置5min。
溶液B配制：取630μl DMEM，再加入120μl PEI工作液(1mg/ml、4℃保存)，充分混匀，室温静置5min。
将B液逐滴加入到A液中，并轻柔混匀，室温孵育20min。将混合液逐滴加入到细胞中，轻柔混匀，置于5％二氧化碳培养17h。
(5)day7上午弃去原培养基，加入15ml的不含血清及抗生素的DMEM培养基，培养31h后收获病毒，之后再加入培养基培养24h，再收获一次病毒。收取细胞上清液，2000rpm离心5min。之后将上清液转移至高速离心管内，配平，然后30000g，4℃离心4h，吸净上清，加入500μl无菌PBS缓冲液重悬病毒颗粒，混匀200μl/支分装并于-80℃冰箱保存。
5、T细胞分离
从中心血站或医院获得健康捐献者的血液样品。经过如下疾病的检测(不仅局限于这些检测)合格的患者。包括：甲肝，乙肝，丙肝，艾滋病，梅毒抗体，结核，遗传性疾病等。采用美天旎公司的Pan T Isolation Kit human(Order no.130- 096-535)，按提供的protocol分离T细胞。
6、T细胞激活
T细胞完全培养基配制：OpTmizer TMCTS TMT-cell Expansion SFM+5％CTS Immune cell SR+1％L-glutamine+10ng/ml IL-7/15。
起始细胞数为3M+Human T-Activator TCR/CD28 Dynabeads 75μl。起始细胞浓度为1M/ml。37℃培养箱培养。激活48小时。
7、T细胞基因编辑
采用CRISPR/cas9系统，设计sgRNA，以电转方式敲除TCR。Cas9蛋白及sgRNA购买自ThermFisher公司。
电转体系：
Figure PCTCN2020121672-appb-000001
电转条件：1600V，10ms，3pulses
其中，TCR sgRNA序列如下：
cagggttctggatatctgt(SEQ ID NO：17)
8、LV-TCRCAR慢病毒转导
T细胞基因编辑12小时以后，LV-TCRCAR慢病毒转导，病毒MOI：3-20，聚凝胺1.5μl(5-10μg/ml)。6-12小时后去除含有慢病毒的培养基，更换新鲜培养基，进行CAR-T细胞扩增。
9、CAR-T细胞扩增
更换新鲜培养基后，在IL-7/15持续存在下，以1M/ml为起始细胞密度，进行细胞传代，每2天检测细胞密度及活率，补充新鲜培养基及细胞因子。保持细胞密度在1M/ml。
10、CAR-T细胞TCR敲除效率检测
电转48小时后，采用流式细胞仪检测TCR敲除效果。结果如图2所示，TCR敲除率达到80-90％，LV-TCRCAR-T(敲除转染LV-TCRCAR的T细胞，CAR-T细胞)细胞残留少量TCR/αβTCR/γδ，TCR阳性的细胞＜1％，图中PanT代表未经处理的T细胞，PanT TCRKO代表TCR敲除的T细胞，LV-TCRCAR-T代表CAR-T细胞。
11、LV-TCRCAR慢病毒转导率检测
慢病毒转导之后2-7天，采用流式细胞仪检测转导率。结果如图3所示，慢病毒转导3天之后CAR表达率不低于50％，图中PanT代表未经处理的T细胞，PanT TCRKO代表TCR敲除的T细胞，LV-TCRCAR-T代表CAR-T细胞。
实施例2 CAR-T细胞体外杀伤能力检测
1、Jurkat-GFP细胞系与实施例1制备的CAR-T细胞共培养，E/T(Jurkat-GFP：CAR-T)比例分别为8:1，4:1，2:1，1:1，0.5:1，0:1。
2、分组如下：
Jurkat-GFP组：0.5M每孔，三个复孔；
PanT TCRKO(敲除TCR的T细胞)组：0.5M每孔，三个复孔；
PanT TCRKO(敲除TCR的T细胞)+Jurkat-GFP组：8:1，4:1，2:1，1:1，0.5:1，0:1；
LV-TCRCAR(CAR-T)+Jurkat-GFP组：8:1，4:1，2:1，1:1，0.5:1，0:1；
每个比例设三个复孔。
3、24小时后流式细胞仪检测Jurkat-GFP细胞的GFP荧光。
4、结果
结果如图4所示，CAR-T(LV-TCRCAR-T)细胞在E:T＝2:1情况下，1天即可100％杀伤TCR/TCR阳性Jurkat-GFP细胞。
实施例3 CAR-T细胞体内杀伤功能检测
一、步骤
1、Jurkat-Fluc细胞系构建NPG鼠肿瘤模型
NPG小鼠5-8周龄，均为雌性，尾静脉注射1×10 6个Jurkat-Fluc细胞。一周后生物荧光检测，确认NPG鼠肿瘤模型构建成功。
2、、一周后将NPG小鼠分为肿瘤模型组(阴性对照组)，LV-CD3CAR-T组(阳性对照组)，LV-TCRCAR-T组，共三组，每组三只鼠。
3、经NPG小鼠尾静脉回输CAR阳性细胞1×10 7个。观察期限8周。
4、每周观察各组NPG鼠生物荧光强度，体重，状态，以及生存时间。
二、结果
体内有效性结果如图5所示，CAR-T组明显抑制肿瘤生长，生物荧光强度显著低于肿瘤组。CAR-T组小鼠生存时间显著延长。在观察期限内实验组小鼠仍存活。与阳性对照组相比，LV-TCRCAR-T组具有更好的肿瘤抑制效果。
体内有效性结果如图6所示：CAR-T组小鼠体内荧光强度显著低于肿瘤模型组，反映出CAR-T组的肿瘤负荷显著低于肿瘤模型组，证明CAR-T在小鼠体内可以有效杀伤肿瘤细胞。
体内有效性结果如图7所示：CAR-T组小鼠的生存时间明显优于肿瘤模型组，可以说明CAR-T组抑制肿瘤的生长，减缓病情的发展，可显著延长小鼠的生存期。
体外和体内实验证明本发明构建的抗TCR的CAR-T可有效杀伤TCR阳性细胞，可以用于治疗T细胞来源的淋巴细胞白血病以及T细胞来源的淋巴瘤。
实施例4 CAR-T细胞抑制移植排异反应的功能检测
一、步骤
1、获取异体健康献血者的PBMC，提取Pan T细胞(未处理的T细胞)，计数；
2、细胞计数确定LV-TCRCAR-T的数量；
3、流式检测确定LV-TCRCAR-T转导率；
4、将LV-TCRCAR-T与Pan T细胞数量分别按0.5：1，1：1和2：1的比例混合；
5、第0h、24h、48h流式检测TCR阳性细胞数量。
二、结果
结果如图8所示，本发明构建的CAR-T细胞可有效清除异体体内的TCR阳性细胞。因此本发明的TCRCAR-T可用于抑制移植排异反应的发生。图8中：UCAR-T代表LV-TCRCAR-T。
在此将本文引用的所有参考文献，包括出版物、专利申请和专利通过引用并入，其程度如同将各参考文献单独且明确指明通过引用并入，并且在本文整体示出一样。
术语“一个/一种(a)”和“一个/一种(an)”和“所述(the)”以及类似的指代物在描述本发明的上下文中(特别是在下述权利要求的上下文中)的使用被解释为既涵盖单数又涵盖复数，除非本文另外指明或者上下文明显矛盾。术语“包含(comprising)”、“具有(having)”、“包括(including)”以及“含有(containing)”被解释为开放式术语(即，意为“包括但不限于”)除非另外标注。本文数值范围的叙述仅意图作为单独指落入范围内的各独立数值的速记法，除非本文另外指明，并且各独立的数值并入说明书如同本文单独对其进行叙述一样。可以以任何合适的顺序实施本文所述的所有方法，除非本文另外指明或者在其它方面与上下文明显矛盾。本文提供的任何或所有实施例或者示例性语言(例如“如”)的使用仅意图更好地阐明本发明，并且不对本发明的范围构成限制，除非另外声明。说明书中的语言均不应被解释为指示任何未要求保护的元素对于本发明的实践是必要的。
本文描述了本发明的优选实施方案，包括发明人已知的实施本发明的最佳方式。经阅读前述描述，那些优选实施方案的改变对于本领域普通技术人员而言可以变得显而易见。发明人期望本领域技术人员视情况应用此类改变，并且发明人意图以与本文具体所述的不同的方式实践本发明。因此，如适用的法律所允许的，本发明包括在此所附的权利要求中所述的主题的所有修饰和等同物。此外，本发明涵盖以上所述元素的所有可能的改变的任何组合，除非本文另外指明或者在其 它方面与上下文明显矛盾。

Claims (67)

  1. 一种破坏TCR阳性细胞的方法，其特征在于，所述方法包括如下步骤：
    1)获取TCR阳性细胞作为靶细胞；
    2)将细胞膜表面表达抗TCR的嵌合抗原受体的细胞作为第二细胞；
    3)将步骤2获得的第二细胞与步骤1的靶细胞接触。
  2. 根据权利要求1所述的方法，其特征在于，所述抗TCR的嵌合抗原受体包含目标抗原结合结构域、跨膜结构域、T细胞活化信号传递区域；目标抗原结合结构域包含抗TCR抗体的抗原结合区域。
  3. 根据权利要求1所述的方法，其特征在于，所述抗TCR抗体的抗原结合区域是包含抗TCR抗体的重链可变区域和轻链可变区域的单链抗体的多肽。
  4. 根据权利要求3所述的方法，其特征在于，所述单链抗体的序列选自以下任一个：
    其包括包含由SEQ ID NO：7记载的氨基酸序列构成的VH区域的CDR1-3的VH区域；和包含由SEQ ID NO：8记载的氨基酸序列构成的VL区域的CDR1-3的VL区域，具有对TCR的结合能力；
    其包括由SEQ ID NO：7记载的氨基酸序列构成的VH区域，和由SEQ ID NO：8记载的氨基酸序列构成的VL区域，具有对TCR的结合能力；
    其包含由SEQ ID NO：7记载的氨基酸序列中的一个或多个氨基酸变异而成的氨基酸序列构成的VH区域，和由SEQ ID NO：8记载的氨基酸序列中的一个或多个氨基酸变异而成的氨基酸序列构成的VL区域，具有对TCR的结合能力；
    或其包含与SEQ ID NO：7记载的氨基酸序列具有95％以上的序列同一性的氨基酸序列构成的VH区域、与SEQ ID NO：8记载的氨基酸序列具有95％以上的序列同一性的氨基酸序列构成的VL区域，具有对TCR的结合能力。
  5. 根据权利要求4所述的方法，其特征在于，所述单链抗体包含以下任一项：
    包含SEQ ID NO：9所示的氨基酸序列的多肽；
    包含SEQ ID NO：9所示的氨基酸序列中的一个或多个氨基酸变异而成的氨基酸序列构成且具有对TCR的结合能力的多肽；
    或包含与SEQ ID NO：9所示的氨基酸序列具有95％以上的序列同一性的氨基酸序列构成且具有对TCR的结合能力的多肽。
  6. 根据权利要求2所述的方法，其特征在于，跨膜结构域包含以下任一种或多种分子的跨膜结构域：T细胞受体的α链及β链、CD3ζ、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、ICOS、CD154、GITR。
  7. 根据权利要求1-6中任一项所述的方法，其特征在于，所述抗TCR的嵌合抗原受体还包括细胞外铰链区域，所述跨膜结构域与细胞外铰链区域连接。
  8. 根据权利要求7所述的方法，其特征在于，所述细胞外铰链区域和跨膜结构域的氨基酸序列包括：
    与SEQ ID NO：11所示的氨基酸序列具有95％以上的序列同一性的氨基酸序列构成，且具有膜贯通能力的多肽；
    或由SEQ ID NO：11所示的氨基酸序列中一个或多个氨基酸变异而成的氨基酸序列构成，并且具有膜贯通能力的多肽。
  9. 根据权利要求2所述的方法，其特征在于，所述T细胞活化信号传递区域包含具有ITAM的蛋白质的T细胞活化信号传递区域，具有ITAM的蛋白质包括CD3ζ、FcRγ、FcRβ、CD3γ，CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d；和/或
    包含共刺激分子的T细胞活化信号传递区域，共刺激分子包括CD2、CD4、CD5、CD8、CD27、CD28，OXO40、4-1BB、ICOS，CD154、HVEM、GITR、Fc受体相关γ链。
  10. 根据权利要求9所述的方法，其特征在于，所述T细胞活化信号传递区域包含：
    (1)由SEQ ID NO：12或13的氨基酸序列具有95％以上的序列同一性的氨 基酸序列构成、且具有T细胞活化信号传递能力的多肽；
    (2)由SEQ ID NO：12或13的氨基酸序列中一个或多个氨基酸变异而成的氨基酸序列构成、并且具有T细胞活化信号传递能力的多肽。
  11. 根据权利要求2所述的方法，其特征在于，所述抗TCR的嵌合抗原受体还包括信号肽，所述信号肽来源包括以下分子：T细胞受体的α链及β链、CD3ζ、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、ICOS、CD154、GITR、免疫球蛋白重链、免疫球蛋白轻链。
  12. 根据权利要求11所述的方法，其特征在于，所述信号肽包含SEQ ID NO：10所示的氨基酸序列。
  13. 根据权利要求1-12任一项所述的方法，其特征在于，所述抗TCR的嵌合抗原受体具有与SEQ ID NO：14所示的氨基酸序列具有至少95％以上的序列同一性的氨基酸序列。
  14. 一种生产工程化细胞的方法，其特征在于，所述方法包括以下步骤：
    1)从供体处获得免疫细胞；
    2)抑制步骤1获得的免疫细胞中内源性TCR表达；
    3)将编码重组抗TCR的嵌合抗原受体的多核苷酸导入经步骤3处理获得的细胞中；
    优选地，
    所述方法还包括如下步骤：4)将至少一种编码重组嵌合抗原受体的多核苷酸导入经过步骤2处理获得的细胞中；
    步骤3和步骤4可同时进行，或先进行步骤3再进行步骤4，或先进行步骤4再进行步骤3。
  15. 根据权利要求14所述的方法，其特征在于，所述供体是健康人而不是患者。
  16. 根据权利要求14所述的方法，其特征在于，所述抗TCR的嵌合抗原受体特异性针对TCR表位，特异性针对TCR相关蛋白的表位，或特异性针对TCR 亚基的表位。
  17. 根据权利要求14所述的方法，其特征在于，所述编码重组抗TCR的嵌合抗原受体的多核苷酸包括编码与SEQ ID NO：14记载的氨基酸序列具有至少95％以上的序列同一性的氨基酸序列的碱基序列。
  18. 根据权利要求14所述的方法，其特征在于，抑制步骤1获得的免疫细胞中内源性TCR表达的技术包括引入编码针对基因组序列的稀有切割核酸内切酶的mRNA，RNA干扰技术。
  19. 根据权利要求18所述的方法，其特征在于，所述稀有切割核酸内切酶包括TAL效应因子、CRISPR CAS9、ZFN。
  20. 根据权利要求14所述的方法，其特征在于，将编码重组嵌合抗原受体的多核苷酸导入经过步骤2处理获得的细胞中的方法包括使用病毒载体。
  21. 根据权利要求14所述的方法，其特征在于，从供体处获得的免疫细胞包括T细胞。
  22. 根据权利要求21所述的方法，其特征在于，所述T细胞包括调节性T细胞、细胞毒性T细胞、辅助性T细胞、记忆T细胞。
  23. 根据权利要求14所述的方法，其特征在于，步骤4中的嵌合抗原受体特异性针对细胞表面抗原。
  24. 根据权利要求23所述的方法，其特征在于，所述细胞表面抗原包括CAIX、ROR1、CD20、CD44v7/8、CEA、EGP-2、EGP-40、erb-B2/3/4、FBP、胎儿乙酰胆碱受体、EGFRvIII、BCMA、CD33、GD3、CD19、CD38、HSP70、CD30、FAP、HER2、CD79a、CD79b、CD123、CD22、CLL-1、MUC-1、GD2、O acetyl GD2、CS1、KDR、LEY、MAGE-A1、间皮素、PSCA、PSMA、TAG-72、VEGF-R2。
  25. 根据权利要求14所述的方法，其特征在于，步骤4中的嵌合抗原受体是单链的或多链的。
  26. 一种抗TCR的嵌合抗原受体，其特征在于，所述抗TCR的嵌合抗原受体包含目标抗原结合结构域、跨膜结构域、T细胞活化信号传递区域；所述目标抗原是TCR。
  27. 根据权利要求26所述的抗TCR的嵌合抗原受体，其特征在于，目标抗原结合结构域包含抗TCR抗体的抗原结合区域。
  28. 根据权利要求26所述的抗TCR的嵌合抗原受体，其特征在于，抗TCR抗体的抗原结合区域是包含抗TCR抗体的重链可变区域和轻链可变区域的单链抗体的多肽。
  29. 根据权利要求28所述的抗TCR的嵌合抗原受体，其特征在于，所述单链抗体的序列选自以下任一个：
    其包括包含由SEQ ID NO：7记载的氨基酸序列构成的VH区域的CDR1-3的VH区域；和包含由SEQ ID NO：8记载的氨基酸序列构成的VL区域的CDR1-3的VL区域，具有对TCR的结合能力；
    其包括由SEQ ID NO：7记载的氨基酸序列构成的VH区域，和由SEQ ID NO：8记载的氨基酸序列构成的VL区域，具有对TCR的结合能力；
    其包含由SEQ ID NO：7记载的氨基酸序列中的一个或多个氨基酸变异而成的氨基酸序列构成的VH区域，和由SEQ ID NO：8记载的氨基酸序列中的一个或多个氨基酸变异而成的氨基酸序列构成的VL区域，具有对TCR的结合能力；
    或其包含与SEQ ID NO：7记载的氨基酸序列具有95％以上的序列同一性的氨基酸序列构成的VH区域、与SEQ ID NO：8记载的氨基酸序列具有95％以上的序列同一性的氨基酸序列构成的VL区域，具有对TCR的结合能力。
  30. 根据权利要求29所述的抗TCR的嵌合抗原受体，其特征在于，所述单链抗体包含以下任一项：
    包含SEQ ID NO：9所示的氨基酸序列的多肽；
    包含SEQ ID NO：9所示的氨基酸序列中的一个或多个氨基酸变异而成的氨基酸序列构成且具有对TCR的结合能力的多肽；
    或包含与SEQ ID NO：9所示的氨基酸序列具有95％以上的序列同一性的氨基酸序列构成且具有对TCR的结合能力的多肽。
  31. 根据权利要求26所述的抗TCR的嵌合抗原受体，其特征在于，跨膜结构域包含以下任一种或多种分子的跨膜结构域：T细胞受体的α链及β链、CD3 ζ、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、ICOS、CD154、GITR。
  32. 根据权利要求26-31中任一项所述的抗TCR的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包括细胞外铰链区域，所述跨膜结构域与细胞外铰链区域连接。
  33. 根据权利要求32所述的抗TCR的嵌合抗原受体，其特征在于，所述细胞外铰链区域和跨膜结构域的氨基酸序列包括：
    与SEQ ID NO：11所示的氨基酸序列具有95％以上的序列同一性的氨基酸序列构成，且具有膜贯通能力的多肽；
    或由SEQ ID NO：11所示的氨基酸序列中一个或多个氨基酸变异而成的氨基酸序列构成，并且具有膜贯通能力的多肽。
  34. 根据权利要求26所述的抗TCR的嵌合抗原受体，其特征在于，所述T细胞活化信号传递区域包含具有ITAM的蛋白质的T细胞活化信号传递区域，具有ITAM的蛋白质包括CD3ζ、FcRγ、FcRβ、CD3γ，CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d；和/或
    包含共刺激分子的T细胞活化信号传递区域，共刺激分子包括CD2、CD4、CD5、CD8、CD27、CD28，OXO40(CD134)、4-1BB(CD137)、ICOS，CD154、HVEM、GITR、Fc受体相关γ链。
  35. 根据权利要求34所述的抗TCR的嵌合抗原受体，其特征在于，所述T细胞活化信号传递区域包含：
    (1)由SEQ ID NO：12或13的氨基酸序列具有95％以上的序列同一性的氨基酸序列构成、且具有T细胞活化信号传递能力的多肽；
    (2)由SEQ ID NO：12或13的氨基酸序列中一个或多个氨基酸变异而成的氨基酸序列构成、并且具有T细胞活化信号传递能力的多肽。
  36. 根据权利要求26所述的抗TCR的嵌合抗原受体，其特征在于，所述抗TCR的嵌合抗原受体还包括信号肽，所述信号肽来源包括以下分子：T细胞受体的α链及β链、CD3ζ、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、 CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、ICOS、CD154、GITR、免疫球蛋白重链、免疫球蛋白轻链。
  37. 根据权利要求36所述的抗TCR的嵌合抗原受体，其特征在于，所述信号肽包含SEQ ID NO：10所示的氨基酸序列。
  38. 根据权利要求26-37任一项所述的抗TCR的嵌合抗原受体，其特征在于，所述嵌合抗原受体具有与SEQ ID NO：14所示的氨基酸序列具有至少95％以上的序列同一性的氨基酸序列。
  39. 包含编码权利要求14-26中任一项所述的抗TCR的嵌合抗原受体的碱基序列的多核苷酸。
  40. 根据权利要求39所述的多核苷酸，其特征在于，作为编码目标抗原结合区域的碱基序列，包含编码SEQ ID NO：1-6记载的氨基酸序列的碱基序列；或包含编码SEQ ID NO：7和8记载的氨基酸序列的碱基序列；或包含编码SEQ ID NO：10记载的氨基酸序列的碱基序列。
  41. 根据权利要求39所述的多核苷酸，其特征在于，作为编码细胞外铰链区和跨膜结构域的碱基序列，包含编码SEQ ID NO：11记载的氨基酸序列的碱基序列。
  42. 根据权利要求39所述的多核苷酸，其特征在于，作为编码T细胞活化信号传递区域的碱基序列，可包含编码SEQ ID NO：12或13记载的氨基酸序列的碱基序列。
  43. 根据权利要求39所述的多核苷酸，其特征在于，作为编码信号肽的碱基序列，可包含编码SEQ ID NO：10记载的氨基酸序列的碱基序列。
  44. 根据权利要求39所述的多核苷酸，其特征在于，作为编码CAR的碱基序列，包含编码SEQ ID NO：14记载的氨基酸序列的碱基序列。
  45. 包含权利要求39-44任一项所述的多核苷酸的载体。
  46. 一种工程化细胞，其特征在于，所述细胞包含权利要求26-38中任一项所述的抗TCR的嵌合抗原受体、权利要求39-44中任一项所述的多核苷酸、或权利要求45所述的载体。
  47. 根据权利要求46所述的细胞，其特征在于，所述细胞的来源包括T细胞。
  48. 一种药物组合物，其特征在于，所述药物组合物包括权利要求39-44中任一项所述的多核苷酸、权利要求45所述的载体、权利要求46或47所述的细胞。
  49. 根据权利要求48所述的药物组合物，其特征在于，所述药物组合物还包括药学上可接受的载体、T细胞活化因子、免疫活化剂、免疫检验点抑制剂、其他表达CAR的细胞、或抗炎症剂。
  50. 一种制造表达抗TCR的嵌合抗原受体的细胞的试剂盒，其特征在于，所述试剂盒包括权利要求45所述的载体。
  51. 一种免疫治疗的方法，其特征在于，所述方法包括给患者施用权利要求46或47所述的细胞、或权利要求48或49所述的药物组合物。
  52. 根据权利要求51所述的方法，其特征在于，所述患者包括自身免疫性疾病患者、癌症患者。
  53. 根据权利要求52所述的方法，其特征在于，所述癌症患者包括急性髓系白血病患者、慢性髓系白血病患者、急性淋巴细胞白血病患者、霍奇金淋巴瘤患者、神经母细胞瘤患者、尤文肉瘤患者、多发性骨髓瘤患者、骨髓增生异常综合征患者、BPDCN患者、胶质瘤患者，或其他实体瘤患者：包括胰腺癌患者、肺癌患者、结直肠癌患者、乳腺癌患者、膀胱癌患者。
  54. 一种抗移植排异反应的方法，其特征在于，所述方法包括给患者施用权利要求46或47所述的细胞、或权利要求48或49所述的药物组合物。
  55. 根据权利要求54所述的方法，其特征在于，所述移植排异反应包括移植物抗宿主反应、宿主抗移植物反应。
  56. 一种抗癌基因疗法，其特征在于，所述方法包括给患者施用权利要求39-44中任一项所述的多核苷酸、权利要求45所述的载体或权利要求48或49所述的药物组合物。
  57. 根据权利要求56所述的方法，其特征在于，所述患者包括急性髓系白 血病患者、慢性髓系白血病患者、急性淋巴细胞白血病患者、霍奇金淋巴瘤患者、神经母细胞瘤患者、尤文肉瘤患者、多发性骨髓瘤患者、骨髓增生异常综合征患者、BPDCN患者、胶质瘤患者，或其他实体瘤患者：包括胰腺癌患者、肺癌患者、结直肠癌患者、乳腺癌患者、膀胱癌患者。
  58. 权利要求39-44中任一项所述的多核苷酸、或权利要求45所述的载体在制备权利要求46或47所述的细胞中的应用。
  59. 权利要求39-44中任一项所述的多核苷酸、或权利要求45所述的载体、或权利要求46或47所述的细胞在制备权利要求48或49所述的药物组合物中的应用。
  60. 权利要求39-44中任一项所述的多核苷酸、或权利要求45所述的载体在制备权利要求50所述的试剂盒中的应用。
  61. 权利要求39-44中任一项所述的多核苷酸、或权利要求45所述的载体、权利要求46或47所述的细胞，或权利要求48或49所述的药物组合物在制备治疗疾病的药物中的应用，其特征在于，所述疾病包括自身免疫性疾病、癌症。
  62. 根据权利要求61所述的应用，其特征在于，所述癌症包括急性髓系白血病、慢性髓系白血病、急性淋巴细胞白血病、霍奇金淋巴瘤、神经母细胞瘤、尤文肉瘤、多发性骨髓瘤、骨髓增生异常综合征、BPDCN、胶质瘤，或其他实体瘤：包括胰腺癌、肺癌、结直肠癌、乳腺癌、膀胱癌。
  63. 权利要求39-44中任一项所述的多核苷酸、或权利要求45所述的载体、权利要求46或47所述的细胞，或权利要求48或49所述的药物组合物在制备抗移植排异反应的药物中的应用。
  64. 权利要求39-44中任一项所述的多核苷酸、或权利要求45所述的载体、权利要求46或47所述的细胞，或权利要求48或49所述的药物组合物在制备用于免疫治疗的药物中的应用。
  65. TCR在制备抗TCR的嵌合抗原受体中的应用。
  66. 针对TCR的抗体或其抗原结合区域在制备抗TCR的嵌合抗原受体中的应用。
  67. 根据权利要求65或66所述的应用，其特征在于，抗TCR的嵌合抗原受体包括权利要求26-38中任一项所述的抗TCR的嵌合抗原受体。
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