WO2020156335A1 - 一种嵌合抗原受体及其应用 - Google Patents

一种嵌合抗原受体及其应用 Download PDF

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WO2020156335A1
WO2020156335A1 PCT/CN2020/073304 CN2020073304W WO2020156335A1 WO 2020156335 A1 WO2020156335 A1 WO 2020156335A1 CN 2020073304 W CN2020073304 W CN 2020073304W WO 2020156335 A1 WO2020156335 A1 WO 2020156335A1
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seq
car
amino acid
acid sequence
antibody
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PCT/CN2020/073304
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French (fr)
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杨选明
傅阳心
李范林
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上海交通大学
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Priority to EP20748622.6A priority Critical patent/EP3919515A4/en
Priority to KR1020217027070A priority patent/KR20210121136A/ko
Priority to US17/426,548 priority patent/US20220144948A1/en
Priority to JP2021544328A priority patent/JP2022523502A/ja
Priority to CN202080011407.8A priority patent/CN113508137A/zh
Publication of WO2020156335A1 publication Critical patent/WO2020156335A1/zh

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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/565—Complementarity determining region [CDR]
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present application relates to the field of biomedicine, in particular to a chimeric antigen receptor that targets a part that specifically binds to the variable regions of T cell receptor (TCR) V ⁇ , V ⁇ and/or their fragments.
  • TCR T cell receptor
  • T cell tumors are common hematological malignancies. Compared with B-cell lymphoma or leukemia, T-cell lymphoma or leukemia has a poor prognosis. Currently, the main treatment for T-cell lymphoma or leukemia is chemotherapy, and there is no specific targeted immunotherapy.
  • Chimeric antigen receptor T cells are artificially modified tumor killer cells that combine the targeted recognition function of antibodies and the tumor killer function of T cells. It is a breakthrough in the field of tumor immunotherapy . However, CAR-T has the side effect of killing normal T cells, and there is a certain risk to the safety of tumor patients.
  • T-cell tumors Most mature T-cell tumors express TCR or complexes, and most of them are TCR.
  • the whole group of T cells is very heterogeneous, with various V ⁇ subtypes whose proportion ranges from 0.85% to 8.3%.
  • Therapies generally used to kill tumor cells will also affect normal T cells to a certain extent, which will adversely affect the patient’s immune system and hinder the treatment of tumors. Therefore, there is an urgent need to develop new and effective T cells.
  • the cell group affects less CAR.
  • This application provides a chimeric antigen receptor (CAR) and its application.
  • the targeting portion of the CAR specifically binds to the variable regions V ⁇ , V ⁇ of the T cell receptor (TCR) and/or their fragments.
  • the CAR provided in this application has one or more of the following properties: 1) It can specifically bind to the variable regions V ⁇ and V ⁇ of T cell receptor (TCR) and/or their fragments; 2) It can selectively kill T cells. Cell-derived tumor cells; 3) have little effect on normal T cell populations; 4) can be used to treat T cell lymphoma.
  • the present application provides a chimeric antigen receptor (CAR), wherein the CAR comprises a targeting moiety, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the targeting moiety It specifically binds to the variable regions V ⁇ , V ⁇ and/or their fragments of the T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • the targeting moiety specifically binds to the variable region V ⁇ of the TCR or a fragment thereof.
  • the V ⁇ or fragments thereof include V ⁇ 24 or fragments thereof.
  • the Va or fragments thereof also include J ⁇ or fragments thereof.
  • the J ⁇ or a fragment thereof includes J ⁇ 18 or a fragment thereof.
  • the targeting moiety specifically binds to the variable region V ⁇ of the TCR or a fragment thereof.
  • the V ⁇ or fragments thereof include V ⁇ 8 or fragments thereof.
  • the fragment of the TCR variable region comprises CDR3 of the TCR variable region.
  • variable regions V ⁇ and V ⁇ of the TCR and/or their fragments comprise the amino acid sequence shown in any one of SEQ ID NO: 58-61.
  • the targeting moiety includes an antibody or fragment thereof.
  • the antibody competes with a reference antibody for binding to the variable regions V ⁇ , V ⁇ and/or fragments of the TCR, wherein the reference antibody comprises a light chain variable region and a heavy chain variable region.
  • the light chain variable region of the reference antibody includes LCDR1, LCDR2, and LCDR3, and the LCDR1 includes the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 64, and SEQ ID NO: 21
  • the LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 65 and SEQ ID NO: 22;
  • the LCDR3 includes SEQ ID NO: 5, SEQ ID NO: 66 and SEQ ID NO: the amino acid sequence shown in any one of 23;
  • the heavy chain variable region of the reference antibody includes HCDR1, HCDR2 and HCDR3, and the HCDR1 includes SEQ ID NO: 11, SEQ ID NO: 67 and SEQ ID NO :
  • the light chain variable region of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 70, and SEQ ID NO: 27; and the reference antibody
  • the heavy chain variable region of the specific antibody includes the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 71, and SEQ ID NO: 35.
  • the reference antibody comprises a V ⁇ 24J ⁇ 18 antibody.
  • the antibody comprises a heavy chain complementarity determining region 1 (HCDR1), and the HCDR1 comprises an amino acid shown in any one of SEQ ID NO: 11, SEQ ID NO: 67, and SEQ ID NO: 29 sequence.
  • the antibody further includes a heavy chain complementarity determining region 2 (HCDR2), and the HCDR2 includes any one of SEQ ID NO: 12, SEQ ID NO: 68, and SEQ ID NO: 30. Amino acid sequence.
  • the antibody further includes a heavy chain complementarity determining region 3 (HCDR3), and the HCDR3 includes any one of SEQ ID NO: 13, SEQ ID NO: 69, and SEQ ID NO: 31. Amino acid sequence.
  • the antibody comprises a light chain complementarity determining region 1 (LCDR1), and the LCDR1 comprises an amino acid shown in any one of SEQ ID NO: 3, SEQ ID NO: 64 and SEQ ID NO: 21 sequence.
  • the antibody further includes a light chain complementarity determining region 2 (LCDR2), and the LCDR2 includes any one of SEQ ID NO: 4, SEQ ID NO: 65, and SEQ ID NO: 22. Amino acid sequence.
  • the antibody further includes a light chain complementarity determining region 3 (LCDR3), and the LCDR3 includes any one of SEQ ID NO: 5, SEQ ID NO: 66, and SEQ ID NO: 23. Amino acid sequence.
  • the antibody includes a heavy chain variable region, and the heavy chain variable region includes an amino acid shown in any one of SEQ ID NO: 17, SEQ ID NO: 71, and SEQ ID NO: 35 sequence. In some embodiments, the antibody includes a light chain variable region, and the light chain variable region includes an amino acid shown in any one of SEQ ID NO: 9, SEQ ID NO: 70, and SEQ ID NO: 27 sequence.
  • the antibody is a single chain antibody.
  • the antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 62 and SEQ ID NO: 19.
  • the transmembrane domain comprises a polypeptide derived from a protein selected from the group consisting of CD8, CD28, CD137 and/or CD3. In some embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:40.
  • the costimulatory domain comprises a polypeptide derived from a protein selected from: ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 and/or CD226.
  • the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:42.
  • the intracellular signaling domain comprises a polypeptide from CD3 ⁇ . In some embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:44.
  • the CAR further comprises a hinge region that connects the antibody and the transmembrane domain.
  • the hinge region comprises a polypeptide selected from the following proteins: CD8a and/or IgG.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO: 38.
  • the CAR includes the amino acid sequence shown in any one of SEQ ID NO: 46, SEQ ID NO: 72, and SEQ ID NO: 50.
  • this application provides an isolated nucleic acid molecule encoding the CAR described in this application.
  • the present application provides an isolated nucleic acid molecule encoding CAR, which comprises the nucleic acid sequence shown in SEQ ID NO: 47, SEQ ID NO: 73, or SEQ ID NO: 51.
  • the present application provides a vector, which contains the nucleic acid molecule described in the present application.
  • the vectors described in this application include mRNA vectors, plasmid vectors, retroviral vectors, and/or lentiviral vectors.
  • this application provides an immune effector cell, which contains and/or can express the CAR described in this application, the nucleic acid molecule described in this application, or the vector described in this application.
  • the immune effector cells are selected from T lymphocytes and natural killer (NK) cells.
  • the T lymphocytes and/or natural killer cells are of natural origin or induced by stem cells.
  • this application provides a method for preparing immune effector cells, which includes introducing the vector described in this application into the immune effector cells.
  • this application provides a composition comprising the immune effector cell described in this application.
  • this application provides the CAR described in this application, the nucleic acid molecule described in this application, the vector described in this application, or the use of the immune effector cells described in this application for the preparation of drugs, wherein the drug It is used to treat T-cell lymphoma or leukemia.
  • the T-cell lymphoma or leukemia comprises one or more selected from the group consisting of peripheral T-cell lymphoma, unspecified peripheral T-cell lymphoma, angioimmunoblastic lymphoma, Extranodal NKT cell lymphoma, anaplastic T cell lymphoma, ALK positive anaplastic cell lymphoma, ALK negative anaplastic cell lymphoma, T lymphoblastic lymphoma, cytotoxic T cell lymphoma, skin T cell lymphoma, Adult T cell leukemia/lymphoma (ATLL), T young lymphocytic leukemia (T-PLL), T-large granular lymphocyte (T-LGL) leukemia, hepatosplenic T cell lymphoma (HSTL), Sezari syndrome (SS), subcutaneous tonsilloid T-cell lymphoma and untyped T-cell lymphoma.
  • peripheral T-cell lymphoma unspecified peripheral T-cell lymphoma
  • FIG. 1 shows a schematic diagram of the CAR structure targeting TCR V ⁇ or V ⁇ subtypes described in this application.
  • Figure 2 shows the titer determination result of the T120BBZ lentivirus described in this application.
  • FIG. 3 shows the results of CAR expression analysis and detection of T120BBZ CAR-T described in this application.
  • Figure 4 shows the detection results of the killing of V ⁇ 24J ⁇ 18 + tumor cells by the T120BBZ CAR-T described in this application.
  • FIG. 5 shows the detection result that the V ⁇ 24J ⁇ 18 + tumor cells described in the present application can specifically induce T120BBZ CAR-T to secrete TNF.
  • FIGS 6A-6B show that the F231BBZ CAR-T described in this application can specifically kill V ⁇ 8 + tumor cells and release IFN.
  • Figures 7A-7C show the in vivo anti-tumor activity analysis of the T120BBZ CAR-T described in this application.
  • FIGS 8A-8C show the TCR expression analysis of the T120BBZ CAR-T described in this application.
  • Figure 9 shows the results of titer analysis of the 1168BBZ lentivirus described in this application.
  • Figure 10 shows the results of the CAR expression analysis and detection of 1168BBZ CAR-T described in this application.
  • Figure 11 shows the detection result of 1168BBZ CAR-T killing V ⁇ 8 + tumor cells according to the present application.
  • This application provides a CAR that can specifically bind to V ⁇ , V ⁇ and/or their fragments in the variable regions of T cell receptors, which can selectively kill T cell-derived tumor cells and has little impact on normal T cell populations .
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor T cells
  • TAA tumor-associated antigen
  • transmembrane domain the intracellular domain and the transmembrane domain may be derived from different proteins.
  • the targeting moiety generally refers to any oligopeptide or polypeptide in the chimeric antigen receptor that can specifically bind to the target protein or fragment thereof.
  • the targeting moiety may be an antigen binding region, for example, it may be an antigen binding portion of an antibody.
  • the targeting moiety may include a single-chain antibody (scFv) or a portion thereof that specifically recognizes a protein of interest (eg, an antigen).
  • the targeting moiety may be a tumor-associated antigen binding region.
  • the tumor-associated antigen may refer to a biomolecule that has antigenicity and whose expression is tumor-related.
  • the tumor-associated antigen may include a tumor-specific antigen that is only present in tumor cells but not in other normal cells; it may also include both tumor cells and other organs and tissues or abnormalities. Antigens in normal cells that are plasmonic and allogeneic, or antigens expressed during development and differentiation.
  • the transmembrane domain (Transmembrane Domain) generally refers to a domain that spans the cell membrane.
  • the transmembrane domain may be a domain of the chimeric antigen receptor that spans the cell membrane.
  • the transmembrane domain can be derived from a natural polypeptide, or can be obtained by artificial design.
  • the transmembrane domain may be a transmembrane domain derived from a protein selected from the group consisting of: T cell receptor alpha or beta chain, 3 ⁇ chain, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154 or GITR.
  • the "costimulatory domain” generally refers to an intracellular domain that can provide immunostimulatory molecules.
  • the costimulatory molecule may be a molecule required for lymphocytes to respond effectively to the antigen.
  • the costimulatory domain may include the costimulatory domain of CD28.
  • the costimulatory domain may include a costimulatory domain of the TNF receptor family, such as the costimulatory domain of OX40 and 4-1BB.
  • the intracellular signal transduction domain generally refers to a domain located inside a cell capable of transducing signals.
  • the intracellular signal transduction signal domain can transmit signals into the cell.
  • the intracellular signaling domain is the intracellular signaling domain of the chimeric antigen receptor.
  • the intracellular signal transduction domain may be derived from an intracellular domain selected from CD3 ⁇ , CD28, 4-1BB or OX40.
  • the intracellular signal transduction domain may be an intracellular domain derived from GITR.
  • the "T cell receptor (TCR)” generally refers to a specific receptor on the surface of T cells.
  • the T cell receptor is a heterodimer and can be composed of two different subunits. Most of the T cell receptors (for example, 95% and above, 96% and above, 97% and above, etc.) are composed of ⁇ subunits and ⁇ subunits. Each peptide chain can be divided into variable regions (V regions). ), constant region (C region), transmembrane region and cytoplasmic region.
  • the T cell receptor can be composed of gamma subunit and delta subunit, and the ratio of these two subunits can vary with individual development or disease.
  • the "TCR variable region” generally refers to the V regions of the two peptide chains ⁇ and ⁇ of TCR, namely V ⁇ and V ⁇ .
  • the V ⁇ and V ⁇ may have antigen specificity.
  • the V ⁇ and V ⁇ may each have three hypervariable regions CDR1, CDR2 and CDR3. Among them, the CDR3 of the TCR variable region has the greatest degree of change, and the CDR3 of the TCR variable region can determine the antigen binding specificity of the TCR.
  • V ⁇ generally refers to the variable region of the ⁇ chain of the TCR.
  • the term may also include the full-length sequence of V ⁇ and/or functional fragments of V ⁇ as long as it can be recognized by the antibody targeting it.
  • the V ⁇ mentioned in this application may be the V ⁇ with the Gene ID 6955 (human origin) and the V ⁇ with the Gene ID 21473 (mouse source) in GenBank.
  • the "V ⁇ 24” generally refers to a variant of the V ⁇ fragment.
  • the Va24 can be derived from human, rhesus monkey, mouse or rat and other different strains.
  • the V ⁇ 24 can be of rhesus monkey origin, and its accession number in GenBank is ABG76794.1; it can also be of human origin, and its PDB number is 4EN3_A.
  • J ⁇ generally refers to the Ja fragment of the variable region of the ⁇ chain of the TCR, which may have various variants.
  • the J ⁇ may be J ⁇ 18.
  • J ⁇ 18 generally refers to a variant of the J ⁇ fragment.
  • the J ⁇ 18 can be derived from human, macaque, mouse or rat and other different strains.
  • the J ⁇ 18 described in this application may be of human origin, and its sequence may be as shown in SEQ ID NO:58.
  • V ⁇ 24J ⁇ 18 generally refers to a variant obtained by recombining the variant V ⁇ 24 of V ⁇ and the variant J ⁇ 18 of the J ⁇ fragment.
  • sequence of V ⁇ 24J ⁇ 18 described in this application may be as shown in SEQ ID NO:59.
  • V ⁇ generally refers to the variable region of the ⁇ chain of the TCR.
  • the term may also include the full-length sequence of V ⁇ and/or functional fragments of V ⁇ as long as it can be recognized by the antibody targeting it.
  • the V ⁇ mentioned in this application may be V ⁇ with the registration numbers of Gene ID 6957 (human origin) and Gene ID 21577 (mouse origin) in GenBank.
  • V ⁇ 8 generally refers to a variant of the variable region of the ⁇ chain of the TCR.
  • the V ⁇ 8 described in the present application can be derived from human, macaque, mouse or rat and other different strains.
  • the V ⁇ 8 described in this application may be of human origin, and its sequence is shown in SEQ ID NO: 61; it may also be of mouse origin, and its sequence is shown in SEQ ID NO: 60.
  • the "variant" generally refers to substitution, deletion or addition in the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to the TCR variable region) A protein or polypeptide of one or more amino acids.
  • the functional variant may comprise at least one, such as 1-30, 1-20, or 1-10, and another example, 1, 2, 3, 4, or 5 amino acid substitutions.
  • the functional variant may substantially maintain the biological properties of the protein or polypeptide before the change (for example, substitution, deletion, or addition).
  • the functional variant can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide before the change.
  • the substitution may be a conservative substitution.
  • the "homolog” generally refers to the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to the TCR variable region) having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more ) Proteins or polypeptides with sequence homology.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • the “percentage of sequence homology” can be calculated in the following way: the two sequences to be aligned are compared in the comparison window, and it is determined that the same nucleic acid base (for example, A, T, C, G) or Positions of the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) To get the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to produce the sequence homology percentage.
  • the same nucleic acid base for example, A, T, C, G
  • Positions of the same amino acid residues e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu
  • the alignment to determine the percentage of sequence homology can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithms required to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.
  • the homology can also be determined by the following methods: FASTA and BLAST.
  • FASTA for a description of the FASTA algorithm, please refer to WRPearson and DJ Lipman's "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad.
  • the “CDR3 of the TCR variable region” generally refers to the complementarity determining region 3 of the TCR ⁇ chain.
  • the CDR3 of the TCR variable region may be a site that directly binds to the antigen peptide.
  • the CDR3 gene of the TCR variable region may be composed of a V terminal, a D segment, a J front end, and a rearranged insert sequence between V to D and D to J (VnDnJ).
  • V ⁇ 24J ⁇ 18 antibody generally refers to an antibody that can specifically bind to V ⁇ 24J ⁇ 18.
  • the V ⁇ 24J ⁇ 18 antibody may be 6B11 (Invitrogen Product#25-5806-41).
  • the V ⁇ 24J ⁇ 18 antibody may also be scFv T120 (its sequence may be as shown in SEQ ID NO:1).
  • V ⁇ 8 antibody generally refers to an antibody that can specifically bind to V ⁇ 8.
  • the V ⁇ antibody may be scFv F231 (its sequence may be as shown in SEQ ID NO: 19).
  • the V ⁇ antibody may be scFv 1168 (its amino acid sequence may be shown in SEQ ID NO: 62, and its nucleic acid sequence may be shown in SEQ ID NO: 63).
  • an antibody generally refers to a polypeptide molecule that can specifically recognize and/or neutralize a specific antigen.
  • an antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds, and includes any molecule comprising its antigen binding portion.
  • 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.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the 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.
  • the constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (for example, effector cells) and the first component (Clq) of the classical complement system.
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogs of any length isolated from their natural environment or artificially synthesized.
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction digestion and gel electrophoresis, or (iv) synthesis, for example, by chemical synthesis.
  • PCR polymerase chain reaction
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the nucleic acid encoding the antibody or its antigen-binding fragment can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the use of restriction fragment operations or the use of synthetic oligonucleotides. Overlap extension PCR. For specific operations, please refer to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing, and Wiley-Interscience New York NY, 1993.
  • 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 may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected 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-transcriptional 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.
  • the "mRNA vector” generally refers to an mRNA fragment that can express foreign genes or self-genes in host cells.
  • the mRNA vector may include F231mBBZ mRNA and 20mBBZ mRNA.
  • the "plasmid vector” generally refers to a plasmid constructed artificially on the basis of a natural plasmid in order to adapt to laboratory operations. Compared with natural plasmids, plasmid vectors usually carry one or more selectable marker genes (such as antibiotic resistance genes) and a synthetic multiple cloning site sequence containing multiple restriction endonuclease recognition sites. And removed most of the non-essential sequences to reduce the molecular weight as much as possible to facilitate genetic engineering operations.
  • selectable marker genes such as antibiotic resistance genes
  • the "retroviral vector” generally refers to a type of RNA virus whose genetic information is stored on ribonucleic acid. Most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, which contains genes for the proteins that make up the center and structure of the virus; pol, which contains genes for reverse transcriptase, and env, which contains genes that make up the virus coat. Through retroviral transfection, the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome. For example, the CAR molecule can be integrated into the host cell.
  • the "lentiviral vector” generally refers to a diploid RNA viral vector belonging to retrovirus.
  • the lentiviral vector is based on the genome of the lentivirus. Many of the sequence structures related to the viral activity are removed to make it biologically safe, and then the sequence of the target gene required for the experiment is introduced into the genome skeleton And expression structure prepared into a vector.
  • lentiviral vectors Compared with other retroviruses, lentiviral vectors have a wider host, and have the ability to infect both dividing and non-dividing cells. For some cells that are more difficult to transfect, such as primary cells, stem cells, and undifferentiated cells.
  • the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome.
  • the CAR molecule can be integrated into the host cell.
  • the "immune effector cells” generally refer to immune cells that participate in the elimination of foreign body antigens and exercise effector functions in an immune response.
  • immune cells For example, plasma cells, cytotoxic T cells, NK cells, APSC pluripotent cells, mast cells, etc.
  • pharmaceutically acceptable adjuvant generally refers to a pharmaceutically acceptable preparation carrier, solution or additive that enhances the characteristics of the preparation.
  • additives are well known to those skilled in the art.
  • T cell lymphoma generally refers to lymphoma that affects T cells.
  • the T cell lymphoma may include the following types: Angiocentric lymphoma, Cutaneous T cell lymphoma, Anaplastic large-cell lymphoma, and vascular lymphoma.
  • Immunoblastic T-cell lymphoma Angioimmunoblastic T-cell lymphoma
  • T-cell lymphoma can develop in lymphoid tissues such as lymph nodes and spleen or outside of lymphoid tissues (ie, gastrointestinal tract, liver, nasal cavity, skin, etc.).
  • leukemia is generally a broad definition of blood cell cancer.
  • the leukemia may be a disease caused by the proliferation and accumulation of leukemia cells.
  • the leukemia usually starts in the bone marrow.
  • the type of leukemia may depend on the type of blood cells that become cancer and/or the growth rate of the blood cells.
  • the leukemia may include subtypes such as adult T cell leukemia/lymphoma (ATLL), T young lymphocytic leukemia (T-PLL), and T large granular lymphocyte (T-LGL) leukemia.
  • ATLL adult T cell leukemia/lymphoma
  • T-PLL T young lymphocytic leukemia
  • T-LGL T large granular lymphocyte
  • the "natural source” generally refers to the way of obtaining wild type without artificial processing.
  • the natural source may be directly obtained from cells collected from a donor.
  • the donor may be a healthy or diseased mammal.
  • the disease may refer to cancer.
  • the disease may be T cell-derived leukemia or lymphoma (e.g., T cell lymphoma).
  • the "induction by stem cells” generally refers to the process of artificially inducing stem cells in vitro to differentiate.
  • the in vitro induction method is usually to add inducing factors to the adult stem cells cultured in vitro, and the inducing factors may include various substances that can affect cell differentiation, such as serum, sugar, vitamins, and various protein factors. Wait.
  • induced pluripotent stem cells iPS cells
  • the induced pluripotent stem cells can be obtained by transferring a combination of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) into differentiated somatic cells using viral vectors.
  • the iPS cell may be a cell type similar to embryonic stem cells and embryonic APSC pluripotent cells.
  • 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 chimeric antigen receptor may include a targeting moiety, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the targeting moiety can specifically bind to T cells
  • TCR variable regions of the receptor
  • the targeting moiety can specifically bind to the variable region V ⁇ of the TCR or a fragment thereof.
  • the V ⁇ or fragments thereof may include V ⁇ 24 or fragments thereof.
  • the V ⁇ or fragments thereof may also include J ⁇ or fragments thereof.
  • the J ⁇ or fragments thereof may include J ⁇ 18 or fragments thereof.
  • the targeting moiety may include a moiety that targets V ⁇ 24J ⁇ 18.
  • the part targeting V ⁇ 24J ⁇ 18 includes scFv T120, and its sequence is shown in SEQ ID NO:1.
  • the targeting moiety specifically binds to the variable region V ⁇ of the TCR or a fragment thereof.
  • the V ⁇ or fragments thereof may include V ⁇ 8 or fragments thereof.
  • the fragment of the TCR variable region may comprise CDR3 of the TCR variable region.
  • the targeting moiety may include a portion targeting V ⁇ 8.
  • the part that targets V ⁇ 8 may include scFv F231, the sequence of which is shown in SEQ ID NO: 19.
  • the part targeting V ⁇ 8 may also include scFv 1168, the sequence of which is shown in SEQ ID NO: 62.
  • variable region V ⁇ , V ⁇ or fragments of the TCR may include the amino acids shown in any one of SEQ ID NO: 58-61, Gene ID 21473, Gene ID 6955, Gene ID 21577, and Gene ID 6957 sequence.
  • the targeting moiety may include antibodies or fragments thereof.
  • the antibody can compete with the reference antibody for binding to the variable regions V ⁇ , V ⁇ and/or fragments of the TCR, wherein the reference antibody may comprise a light chain variable region and a heavy chain variable region.
  • the light chain variable region of the reference antibody may include LCDR1, LCDR2, and LCDR3, and the LCDR1 may include any of SEQ ID NO: 3, SEQ ID NO: 64, and SEQ ID NO: 21.
  • the LCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 65 and SEQ ID NO: 22; the LCDR3 may include SEQ ID NO: 5, SEQ ID NO: The amino acid sequence shown in any one of 66 and SEQ ID NO: 23; the heavy chain variable region of the reference antibody may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include SEQ ID NO: 11, SEQ ID NO : 67 and SEQ ID NO: 29; the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 68, and SEQ ID NO: 30; The HCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 13, SEQ ID NO: 69, and SEQ ID NO: 31.
  • the light chain variable region of the reference antibody may include the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 70, and SEQ ID NO: 27; and the reference The heavy chain variable region of the antibody may include the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 71, and SEQ ID NO: 35.
  • the reference antibody may comprise the V ⁇ 24J ⁇ 18 antibody.
  • the V ⁇ 24J ⁇ 18 antibody may be scFv T120, and its sequence is shown in SEQ ID NO:1.
  • the antibody may include the heavy chain complementarity determining region 1 (HCDR1), and the HCDR1 may include the amino acid shown in any one of SEQ ID NO: 11, SEQ ID NO: 67, and SEQ ID NO: 29 sequence.
  • the antibody may also include the heavy chain complementarity determining region 2 (HCDR2), and the HCDR2 may include any one of SEQ ID NO: 12, SEQ ID NO: 68, and SEQ ID NO: 30. Amino acid sequence.
  • the antibody may also include the heavy chain complementarity determining region 3 (HCDR3), and the HCDR3 includes the amino acids shown in any one of SEQ ID NO: 13, SEQ ID NO: 69 and SEQ ID NO: 31 sequence.
  • the antibody may include light chain complementarity determining region 1 (LCDR1), and the LCDR1 includes the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 64, and SEQ ID NO: 21 .
  • the antibody may also include the light chain complementarity determining region 2 (LCDR2), and the LCDR2 includes the amino acids shown in any one of SEQ ID NO: 4, SEQ ID NO: 65 and SEQ ID NO: 22 sequence.
  • the antibody may also include a light chain complementarity determining region 3 (LCDR3), and the LCDR3 includes the amino acids shown in any one of SEQ ID NO: 5, SEQ ID NO: 66, and SEQ ID NO: 23 sequence.
  • the antibody may include a heavy chain variable region, and the heavy chain variable region may include an amino acid shown in any one of SEQ ID NO: 17, SEQ ID NO: 71, and SEQ ID NO: 35 sequence.
  • the antibody includes a light chain variable region, and the light chain variable region includes an amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 70, and SEQ ID NO: 27.
  • the antibody may be a single chain antibody (scFv).
  • the single-chain antibody may comprise one or more scFv linked together.
  • the single chain antibody may comprise one scFv, and the single chain antibody may comprise more than one scFv (e.g., 2, 3, 4, 5, etc.).
  • the multiple scFvs can be directly linked.
  • the plurality of scFvs can be connected by connecting peptides.
  • the single-chain antibody may include scFv T120, whose sequence is shown in SEQ ID NO: 1; it may include the light chain variable region, whose sequence is shown in SEQ ID NO: 9; it may include LCDR1-3, Its sequence is shown in SEQ ID NO: 3-5; it may include heavy chain variable region, its sequence is shown in SEQ ID NO: 17; it may include HCDR1-3, its sequence is shown in SEQ ID NO: :Shown in 11-13; it can include the connecting peptide of VL and VH, and the connecting peptide sequence is shown in SEQ ID NO: 52.
  • the single-chain antibody may include scFv F231, whose sequence is shown in SEQ ID NO: 19; it may include the light chain variable region, whose sequence is shown in SEQ ID NO: 27; it may include LCDR1-3, Its sequence is shown in SEQ ID NO: 21-23 respectively; it may include the heavy chain variable region, its sequence is shown in SEQ ID NO: 35; it may include HCDR1-3, and its sequence is shown in SEQ ID NO, respectively : 29-31; it may include a connecting peptide between VL and VH, and its connecting peptide sequence is shown in SEQ ID NO: 52.
  • the single-chain antibody may include scFv 1168, its sequence is shown in SEQ ID NO: 62; it may include the light chain variable region, its sequence is shown in SEQ ID NO: 70; it may include LCDR1-3, Its sequence is shown in SEQ ID NO: 64-66 respectively; it may include the heavy chain variable region, and its sequence is shown in SEQ ID NO: 71; it may include HCDR1-3, and its sequence is shown in SEQ ID NO respectively. : Shown in 67-68; it can include the connecting peptide of VL and VH, and its connecting peptide sequence is shown in SEQ ID NO: 52.
  • the transmembrane domain may comprise a transmembrane domain from a protein selected from the group consisting of CD8, CD28, CD137 and/or CD3.
  • the transmembrane domain may include the amino acid sequence shown in SEQ ID NO:40.
  • the transmembrane domain may be the CD8 transmembrane region, and its sequence is shown in SEQ ID NO:40.
  • CAR T120BBZ and CAR 1168BBZ may include the transmembrane domain of CD8.
  • the costimulatory domain may include the costimulatory domains of the following proteins: ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 and/or CD226.
  • the costimulatory domain includes the amino acid sequence shown in SEQ ID NO:42.
  • the costimulatory domain may be 4-1BB, and its sequence is shown in SEQ ID NO:42.
  • CAR T120BBZ, CAR 1168BBZ and CAR F231mBBZ may include a costimulatory domain of 4-1BB.
  • the intracellular signaling domain may include a signaling domain from CD3 ⁇ .
  • the intracellular signaling domain may include the amino acid sequence shown in SEQ ID NO:44.
  • the signal transduction domain may be CD3 ⁇ , and its sequence is shown in SEQ ID NO:44.
  • CAR T120BBZ, CAR 1168BBZ and CAR F231mBBZ may include the CD3 ⁇ signal transduction domain.
  • the CAR may also include a hinge region, and the hinge region may connect the antibody and the transmembrane domain.
  • the hinge region may comprise a hinge region derived from a protein selected from the group consisting of CD8a and/or IgG.
  • the hinge region is CD8a, which may include the amino acid sequence shown in SEQ ID NO: 38.
  • the hinge region may be CD8a, and its sequence is shown in SEQ ID NO: 38.
  • CAR T120BBZ, CAR1168BBZ and CAR F231mBBZ may include the hinge region of CD8a.
  • the CAR described in this application may include the amino acid sequence shown in any one of SEQ ID NO: 46, SEQ ID NO: 64, and SEQ ID NO: 50.
  • the CAR may be selected from CAR T120BBZ, and its sequence is shown in SEQ ID NO: 46.
  • the CAR may be selected from CAR F231mBBZ, and its sequence is shown in SEQ ID NO:50.
  • the CAR may be selected from CAR 1168BBZ, and its sequence is shown in SEQ ID NO: 72.
  • the CAR described in this application may include a targeting moiety (the targeting moiety sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 19), a transmembrane domain ( The transmembrane domain sequence is shown in SEQ ID NO: 40), the costimulatory domain (the costimulatory domain sequence is shown in SEQ ID NO: 42) and the intracellular signaling domain (the intracellular The signal transduction domain sequence is shown in SEQ ID NO: 44).
  • a targeting moiety is shown in SEQ ID NO: 1 and SEQ ID NO: 19
  • the transmembrane domain sequence is shown in SEQ ID NO: 40
  • the costimulatory domain the costimulatory domain sequence is shown in SEQ ID NO: 42
  • the intracellular signaling domain the intracellular The signal transduction domain sequence is shown in SEQ ID NO: 44.
  • the CAR described in this application may be CAR T120BBZ, and its targeting part is scFv T120, and the amino acid sequence of scFv T120 is shown in SEQ ID NO: 46.
  • Its transmembrane domain is CD8a, and its amino acid sequence is shown in SEQ ID NO: 40.
  • the costimulatory domain can be 4-1BB, and its amino acid sequence is shown in SEQ ID NO:42.
  • the intracellular signal transduction domain can be CD3 ⁇ , and its amino acid sequence is shown in SEQ ID NO:44.
  • the amino acid sequence of CAR T120BBZ is shown in SEQ ID NO: 46.
  • the CAR described in this application may be CAR F231mBBZ, and its targeting part is scFv F231, and the amino acid sequence of scFv F231 is shown in SEQ ID NO: 19.
  • Its transmembrane domain is CD8a, and its amino acid sequence is shown in SEQ ID NO: 40.
  • the costimulatory domain can be 4-1BB, and its amino acid sequence is shown in SEQ ID NO:42.
  • the intracellular signaling domain can be CD3 ⁇ , and its amino acid sequence is shown in SEQ ID NO:44.
  • the amino acid sequence of CAR F231mBBZ is shown in SEQ ID NO: 50.
  • the CAR described in this application may be CAR 1168BBZ, and its targeting part is scFv 1168, and the amino acid sequence of scFv 1168 is shown in SEQ ID NO: 61.
  • Its transmembrane domain is CD8a, and its amino acid sequence is shown in SEQ ID NO: 40.
  • the costimulatory domain can be 4-1BB, and its amino acid sequence is shown in SEQ ID NO:42.
  • the intracellular signaling domain can be CD3 ⁇ , and its amino acid sequence is shown in SEQ ID NO:44.
  • the amino acid sequence of CAR 1168mBBZ is shown in SEQ ID NO: 72.
  • the protein, polypeptide, amino acid sequence and/or nucleotide sequence involved in this application should also be understood to include at least the following range: with the protein, polypeptide, or with the protein encoded by the nucleotide sequence, Polypeptides, variants or homologues with the same or similar functions.
  • this application provides an isolated nucleic acid molecule encoding the CAR described in this application.
  • the isolated nucleic acid molecule encoding CAR may include the nucleic acid sequence shown in SEQ ID NO: 47 or SEQ ID NO: 51.
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction digestion and gel electrophoresis, or (iv) synthesis, for example, by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the present application provides a vector, which may contain the nucleic acid molecule described in the present application.
  • the vector may include mRNA vector, plasmid vector, retroviral vector and/or lentiviral vector.
  • the vector described in this application may be a lentiviral vector, which may include the nucleic acid sequence shown in SEQ ID NO: 47 or SEQ ID NO: 51.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected 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. Such control elements are well known to those skilled in the art.
  • the expression control sequence 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-transcriptional 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.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage, an mRNA vector or other vectors commonly used in, for example, genetic engineering.
  • the vector may be a plasmid vector, for example, it may include a pCDH-MSCVEF vector.
  • the vector can be a lentiviral vector, for example, the lentiviral vector can be VSV-g, pMD Gag/Pol or RSV-REV, which can be co-transfected with a plasmid vector into host cells (for example, 293X Cells) then obtain the lentiviral vector loaded with plasmid.
  • the lentiviral vector loaded with the plasmid may include the T120BBZ virus, and may also include the 20BBZ virus.
  • the vector can be an mRNA vector, for example, the mRNA vector can be ARCA-capped RNA (AM1345, Thermo Fisher), which can be used to produce a plasmid-loaded mRNA vector.
  • the mRNA vector loaded with the plasmid may include F231mBBZ mRNA, and may also include 20BBZ mRNA.
  • the present application provides immune effector cells, which contain and/or can express the CAR described in this application, the nucleic acid molecule described in this application, or the vector described in this application.
  • the immune effector cells can be selected from T lymphocytes and natural killer (NK) cells.
  • T lymphocytes and/or natural killer cells may be of natural origin or induced by stem cells.
  • each or each cell may contain one or one of the vectors described in this application.
  • each or each cell may contain multiple (e.g., 2 or more) or multiple (e.g., 2 or more) vectors described in the present application.
  • the vectors described in this application can be introduced into the cells, such as eukaryotic cells (e.g., mammalian cells).
  • the mammalian cells may be 293T cells or 293X cells.
  • the vectors described in this application can be introduced into the cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the present application provides a composition, which may include the immune effector cells described in the present application. In some embodiments, it may comprise the immune effector cells and a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable adjuvant may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and/or non-ionic surfaces Active agent, etc.
  • the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration Administration or via subcutaneous depot.
  • the present application provides the use of the CAR, the nucleic acid molecule, the vector, or the immune effector cell for the preparation of a drug, wherein the drug is used for the treatment of T-cell lymphoma or leukemia.
  • this application provides the application of the CAR, the nucleic acid molecule, the vector, or the immune effector cell in the preparation of a medicine for the treatment of T cell lymphoma or leukemia.
  • the present application provides a method for treating T-cell lymphoma or leukemia, including administering the CAR, the nucleic acid molecule, the vector, or the immune effector cell to a patient.
  • the T-cell lymphoma or leukemia may comprise one or more selected from the group consisting of: peripheral T-cell lymphoma, non-specific peripheral T-cell lymphoma, angioimmunoblastic lymphoma, nodal External NKT cell lymphoma, anaplastic T cell lymphoma, ALK positive anaplastic cell lymphoma, ALK negative anaplastic cell lymphoma, T lymphoblastic lymphoma, cytotoxic T cell lymphoma, cutaneous T cell lymphoma, adult T cell leukemia/lymphoma (ATLL), T young lymphocytic leukemia (T-PLL), T large granular lymphocyte (T-LGL) leukemia, hepatosplenic T cell lymphoma (HSTL), Sezari syndrome (SS ), subcutaneous tonsilloid T-cell lymphoma and untyped T-cell lymphoma.
  • peripheral T-cell lymphoma non-specific peripheral T-cell lymphoma, an
  • the CAR, the nucleic acid molecule, the vector, or the immune effector cell can significantly improve the killing effect on tumor cells in vivo.
  • the tumor volume can be reduced by 50% or more (for example, 60% or more, 70% or more, 80% or more, 90% or more, etc.).
  • the CAR, the nucleic acid molecule, the vector, or the immune effector cell can significantly improve the killing effect on tumor cells in vitro.
  • the tumor volume can be reduced by 50% or more (for example, 60% or more, 70% or more, 80% or more, 90% or more, etc.).
  • the CAR, the nucleic acid molecule, the vector, or the immune effector cell can selectively kill tumor cells with specific antigens, but basically not kill normal T cells (For example, the normal T cells may be normal and healthy T cells that are not T cell lymphomas).
  • the killing effect on tumor cells with specific antigens can be increased by more than 50% (for example, 60% or more, 70% or more, 80% or more, 90% or more), and for normal T cells It is basically harmless (for example, the number of T cells is reduced by 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, etc.).
  • scFv T120 (SEQ ID NO: 1), hinge region (SEQ ID NO: 38), transmembrane region (SEQ ID NO: 40), 4-1BB costimulatory factor (SEQ ID NO: 42), CD3 ⁇ intracellular signaling domain (SEQ ID NO: 44).
  • the 4-1BB costimulatory factor and the CD3 ⁇ intracellular signaling domain can be connected end to end to obtain BBZ, the sequence of which is shown in SEQ ID NO: 54.
  • the scFv T120 (amino acid sequence SEQ ID NO: 1, nucleotide sequence SEQ ID NO: 2) and BBZ (SEQ ID NO: 54), which can specifically bind to TCR V ⁇ 24J ⁇ 18, were digested by overlapping PCR with EcoRI and BamHI at both ends Site cloning pCDH-MSCVEF vector. Carry out PCR amplification, and use extension PCR to sequentially bring EcoRI restriction site (including protective base), hinge region, transmembrane region, 4-1BB costimulatory factor, CD3 ⁇ intracellular signaling domain, BamHI at the 5'end. Restriction site, PCR amplification to obtain the CAR T120BBZ.
  • the clones with correct sequencing were used NucleoBond Xtra Midi Plus EF kit for endotoxin-free large-scale extraction, and co-transfected with lentivirus packaging plasmids (VSV-g, pMD Gag/Pol or RSV-REV) into 293X cells, 37°C, 5% CO 2 After 48 hours of incubation, the supernatant was collected. After 0.45 ⁇ M filtration, a Beckman ultracentrifuge and SW28 rotor were used to concentrate the virus at a speed of 25000RPM for 2 hours, which is pCDH-MSCVEF-T120BBZ virus (referred to as T120BBZ virus). Used for subsequent CAR-T cell production.
  • control pCDH-MSCVEF-20BBZ virus (20BBZ virus) was produced by the same method as the preparation of T120BBZ virus, and the obtained virus was infected with 293 cells, and the anti-mouse Fab antibody (JacksonImmunoResearch #115-605-006) was used for flow detection
  • the method for measuring virus titer is shown in Figure 2.
  • Figure 2 shows the flow detection results when 1 ⁇ L, 3 ⁇ L, and 9 ⁇ L of the T120BBZ virus were added, and no virus was added as a blank control.
  • the medium was RPMI complete medium containing 10% FBS, IL2( 50IU/ml), IL21 (4ng/ml), according to the use of artificial antigen presenting cells (Raji cells after X-ray 100Gray irradiation) or anti-hCD3 (0.1 ⁇ g/ml) or anti-hCD28 (0.25 ⁇ g/ ml) stimulation.
  • the cells obtained are T120BBZ CAR-T cells and 20BBZ CAR-T cells.
  • the T120BBZ CAR-T cells are stained with Alexa. 647AffiniPure F(ab') 2 Fragment Goat Anti-Human IgG, Fab fragment specific secondary antibody staining, the result is shown in Figure 3.
  • a CAR targeting mouse V ⁇ 8 was prepared, and the following sequences were artificially synthesized: scFv F231 (SEQ ID NO: 19), mBBZ (SEQ ID NO: 56).
  • scFv F231 SEQ ID NO: 19
  • mBBZ SEQ ID NO: 56
  • mBBZ is composed of mouse-derived 4-1BB costimulatory factor and CD3 ⁇ intracellular signal transduction domain.
  • the pCDH-MSCVEF vector was cloned by overlapping PCR with scFv F231 (SEQ ID 19) and mBBZ (SEQ ID NO: 56) that can specifically bind to TCR V ⁇ 8, and adding EcoRI and BamHI restriction sites at both ends. Carry out PCR amplification, and use extension PCR to sequentially bring EcoRI restriction site (including protective base), hinge region, transmembrane region, 4-1BB costimulatory factor, CD3 ⁇ intracellular signaling domain, BamHI at the 5'end.
  • Mouse spleen cells were purified by Mouse CD3T Cell Isolation Kit (purchased from biolegend#480031), inoculated into 96-well culture plates, activated by ConA (purchased from sigma#C2010) for 2 days, and then electrotransformed with F231mBBZ mRNA using ECM830 And 20BBZ mRNA, the resulting cells are F231mBBZ CAR-T cells and 20BBZ CAR-T cells.
  • a CAR targeting human V ⁇ 8 was first prepared, and the following sequences were artificially synthesized: scFv 1168 (SEQ ID NO: 62), hinge region (SEQ ID NO: 38), transmembrane region (SEQ ID NO: 40) ), 4-1BB costimulatory factor (SEQ ID NO: 42), CD3 ⁇ intracellular signaling domain (SEQ ID NO: 44).
  • the 4-1BB costimulatory factor and the CD3 ⁇ intracellular signaling domain can be connected end to end to obtain BBZ, the sequence of which is shown in SEQ ID NO: 54.
  • the pCDH-MSCVEF vector was cloned by overlapping PCR with scFv 1168 (SEQ ID NO: 62) and BBZ (SEQ ID NO: 54) that can specifically bind to human V ⁇ 8, and adding EcoRI and BamHI restriction sites at both ends. Carry out PCR amplification, and use extension PCR to sequentially bring EcoRI restriction site (including protective base), hinge region, transmembrane region, 4-1BB costimulatory factor, CD3 ⁇ intracellular signaling domain, BamHI at the 5'end. Restriction site, PCR amplification to obtain the CAR1168BBZ.
  • the clones with correct sequencing were used NucleoBond Xtra Midi Plus EF kit for endotoxin-free large-scale extraction, and co-transfected with lentivirus packaging plasmids (VSV-g, pMD Gag/Pol or RSV-REV) into 293X cells, 37°C, 5% CO 2 After 48 hours of incubation, the supernatant was collected. After 0.45 ⁇ M filtration, a Beckman ultracentrifuge and SW28 rotor were used to concentrate the virus at a speed of 25000RPM for 2 hours, which is pCDH-MSCVEF-1168BBZ virus (referred to as 1168BBZ virus). Used for subsequent CAR-T cell production.
  • control pCDH-MSCVEF-20BBZ virus (20BBZ virus for short) was produced by the same method as the 1168BBZ virus, and 293 cells were infected with the virus obtained, and the anti-mouse Fab antibody (JacksonImmunoResearch#115-605-006) was used for flow detection
  • the method for measuring virus titer is shown in Figure 9.
  • Figure 9 shows the results of flow cytometry when adding 1 ⁇ L, 3 ⁇ L, and 9 ⁇ L of the 1168BBZ virus, with no virus added as a blank control.
  • the medium is RPMI complete medium containing 10% FBS, IL2 (50IU/ml), IL21 (4ng/ml), using artificial antigen presenting cells (Raji cells after X-ray 100Gray irradiation) or anti-hCD3 (0.1 ⁇ g/ml) or anti-hCD28 (0.25 ⁇ g) every 6 days /ml) stimulation.
  • the cells obtained are 1168BBZ CAR-T cells and 20BBZ CAR-T cells, which are stained by flow cytometry with anti-mouse Fab antibody (JacksonImmunoResearch#115-605-006) Anti-staining, the results are shown in Figure 10.
  • T120BBZ CAR-T Take the T120BBZ CAR-T cells prepared in Example 1, and use 20BBZ CAR-T cells as a control, and co-culture with NALM6-V ⁇ 24J ⁇ 18 + cells at 37°C and 5% CO 2. After 24 hours, flow cytometry detects NALM6-V ⁇ 24J ⁇ 18 + Survival and TNF release, T120BBZ CAR-T can selectively kill V ⁇ 24J ⁇ 18 + tumor cells and release more TNF. The detection of tumor killing effect is shown in Figure 4, and the detection result of TNF release is shown in Figure 5.
  • T120BBZ CAR has specificity for V ⁇ 24J ⁇ 18.
  • Example 2.2 Obtained in Example 2.2 was prepared in step F231mBBZ CAR-T cells and 20BBZ CAR-T cells were seeded into 96-well plates 2, while non-infected T-cells as a negative control, and C1498V ⁇ 8 - CD20 - or C1498V ⁇ 8 + CD20 -
  • the tumor cells were co-cultured at 37°C with 5% CO 2 and 24 hours later, flow cytometry staining TCRVb8.1/8.2 (Biolegend#118406) to compare the survival ratio of tumor cells and the release of IFN ⁇ , the results are shown in Figure 6A-6B.
  • F231mBBZ CAR-T cells can selectively kill V ⁇ 8 + tumor cells and release more IFN ⁇ .
  • Example 6 1168BBZ CAR-T killing tumor cells in vitro and releasing effector molecules
  • Example 3 Take the 1168BBZ CAR-T cells prepared in Example 3, and use 20BBZ CAR-T cells as a control, and co-culture with V ⁇ 8 + Jurkat cells at 37°C and 5% CO 2. After 24 hours, flow cytometry is used to detect tumor cell survival , 1168BBZ CAR-T can selectively kill V ⁇ 8 + tumor cells. The detection of tumor killing effect is shown in Figure 11.
  • NALM6V ⁇ 24J ⁇ 18 + 10 6 tumor cells were inoculated by intravenous injection to mouse B-NDG (one hundred Orsay FIG.), Intravenous injection of 10 7 T120BBZ CAR-T cells after 6 days of treatment, while uninfected T cells as a negative control. On the 7th day, 5 drops of peripheral blood (100 ⁇ L) were taken to detect the contents of tumor cells and CAR-T cells in the peripheral blood by flow cytometry. The results are shown in Figures 7A-7C, respectively.
  • T120BBZ CAR-T cells reduced tumors in mice by 82.9% (3.798mm 3 to 0.646mm 3 ).
  • T120BBZ CAR-T cells significantly prolong the tumor burden of mice, and can expand and prolong the survival of mice in vivo.
  • T120BBZ CAR-T cells prepared in Example 1 and 20BBZ CAR-T cells were used as controls, using anti-TCR ⁇ (Biolegend#306708), anti-CD3 (Biolegend#317306) and anti-V ⁇ 24J ⁇ 18 + (Biolegend#342912) ) Staining, flow cytometry analysis of the changes in the composition of the specific target TCR (V ⁇ 24J ⁇ 18 + ), the results are shown in Figures 8A-8C.

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Abstract

本申请提供一种嵌合抗原受体(CAR)，其中所述CAR包含靶向部分、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述靶向部分特异性结合T细胞受体(TCR)的可变区Vα、Vβ和/或它们的片段。

Description

一种嵌合抗原受体及其应用 技术领域
本申请涉及生物医药领域，具体涉及一种靶向部分特异性结合T细胞受体(TCR)的可变区Vα、Vβ和/或它们的片段的嵌合抗原受体。
背景技术
T细胞肿瘤是常见的血液系统恶性疾病。与B细胞淋巴瘤或白血病相比，T细胞淋巴瘤或白血病的预后较差。目前T细胞淋巴瘤或白血病的主要治疗以化疗为主，尚无特异的靶向免疫治疗方案。
嵌和抗原受体T细胞(CAR-T细胞)是一种经人工修饰的肿瘤杀伤细胞，其结合了抗体的靶向识别功能和T细胞的肿瘤杀伤功能，是肿瘤免疫治疗领域的一大突破。然而，CAR-T存在杀死正常T细胞的副作用，对肿瘤患者的安全性存在一定的风险。
多数成熟T细胞肿瘤均表达TCR或复合物，其中多数为TCR。整个T细胞的类群是非常异质化的，具有各种Vα亚型其所占比例从0.85％-8.3％不等。一般用于杀伤肿瘤细胞(例如T细胞肿瘤细胞)的疗法也会一定程度影响正常T细胞从而对患者的免疫系统造成不利的影响，从而阻碍肿瘤的治疗，因此亟需开发新的、对于正常T细胞类群影响较小的CAR。
发明内容
本申请提供了一种嵌合抗原受体(CAR)及其应用，所述CAR的靶向部分特异性结合T细胞受体(TCR)的可变区Vα、Vβ和/或它们的片段。本申请提供的CAR具有下列性质中的一种或多种：1)能特异性结合T细胞受体(TCR)的可变区Vα、Vβ和/或它们的片段；2)能选择性杀伤T细胞来源的肿瘤细胞；3)对正常T细胞群体影响较小；4)可用于治疗T细胞淋巴瘤。
一方面，本申请提供了一种嵌合抗原受体(CAR)，其中所述CAR包含靶向部分、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述靶向部分特异性结合T细胞受体(TCR)的可变区Vα、Vβ和/或它们的片段。
在某些实施方式中，所述靶向部分特异性结合TCR的可变区Vα或其片段。在某些实施方式中，所述Vα或其片段包括Vα24或其片段。在某些实施方式中，所述Vα或其片段还包括Jα或其片段。在某些实施方式中，所述Jα或其片段包括Jα18或其片段。
在某些实施方式中，所述靶向部分特异性结合TCR的可变区Vβ或其片段。在某些实施 方式中，所述Vβ或其片段包括Vβ8或其片段。在某些实施方式中，所述TCR可变区的片段包含TCR可变区的CDR3。
在某些实施方式中，所述TCR的可变区Vα、Vβ和/或它们的片段包含SEQ ID NO：58-61中任一项所示的氨基酸序列。
在某些实施方式中，所述靶向部分包括抗体或其片段。
在某些实施方式中，所述抗体与参比抗体竞争结合所述TCR的可变区Vα、Vβ和/或它们的片段，其中所述参比抗体包含轻链可变区和重链可变区，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO：3、SEQ ID NO：64和SEQ ID NO：21中任一项所示的氨基酸序列；所述LCDR2包含SEQ ID NO：4、SEQ ID NO：65和SEQ ID NO：22中任一项所示的氨基酸序列；所述LCDR3包含SEQ ID NO：5、SEQ ID NO：66和SEQ ID NO：23中任一项所示的氨基酸序列；所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO：11、SEQ ID NO：67和SEQ ID NO：29中任一项所示的氨基酸序列；所述HCDR2包含SEQ ID NO：12、SEQ ID NO：68和SEQ ID NO：30中任一项所示的氨基酸序列；所述HCDR3包含SEQ ID NO：13、SEQ ID NO：69和SEQ ID NO：31中任一项所示的氨基酸序列。
在某些实施方式中，所述参比抗体的轻链可变区包含SEQ ID NO：9、SEQ ID NO：70和SEQ ID NO：27中任一项所示的氨基酸序列；且所述参比抗体的重链可变区包含SEQ ID NO：17、SEQ ID NO：71和SEQ ID NO：35中任一项所示的氨基酸序列。
在某些实施方式中，所述参比抗体包含Vα24Jα18抗体。
在某些实施方式中，所述抗体包含重链互补决定区1(HCDR1)，所述HCDR1包含SEQ ID NO：11、SEQ ID NO：67和SEQ ID NO：29中任一项所示的氨基酸序列。在某些实施方式中，所述抗体还包含重链互补决定区2(HCDR2)，所述HCDR2包含SEQ ID NO：12、SEQ ID NO：68和SEQ ID NO：30中任一项所示的氨基酸序列。在某些实施方式中，所述抗体还包含重链互补决定区3(HCDR3)，所述HCDR3包含SEQ ID NO：13、SEQ ID NO：69和SEQ ID NO：31中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体包含轻链互补决定区1(LCDR1)，所述LCDR1包含SEQ ID NO：3、SEQ ID NO：64和SEQ ID NO：21中任一项所示的氨基酸序列。在某些实施方式中，所述抗体还包含轻链互补决定区2(LCDR2)，所述LCDR2包含SEQ ID NO：4、SEQ ID NO：65和SEQ ID NO：22中任一项所示的氨基酸序列。在某些实施方式中，所述抗体还包含轻链互补决定区3(LCDR3)，所述LCDR3包含SEQ ID NO：5、SEQ ID NO：66和 SEQ ID NO：23中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体包含重链可变区，所述重链可变区包含SEQ ID NO：17、SEQ ID NO：71和SEQ ID NO：35中任一项所示的氨基酸序列。在某些实施方式中，所述抗体包含轻链可变区，所述轻链可变区包含SEQ ID NO：9、SEQ ID NO：70和SEQ ID NO：27中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体为单链抗体。
在某些实施方式中，所述抗体包含SEQ ID NO：1、SEQ ID NO：62和SEQ ID NO：19中任一项所示的氨基酸序列。
在某些实施方式中，所述跨膜结构域包含源自选自下述蛋白的多肽：CD8、CD28、CD137和/或CD3。在某些实施方式中，所述跨膜结构域包含SEQ ID NO：40所示的氨基酸序列。
在某些实施方式中，所述共刺激结构域包含源自选自下述蛋白的多肽：ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2和/或CD226。在某些实施方式中，所述共刺激结构域包含SEQ ID NO：42所示的氨基酸序列。
在某些实施方式中，所述胞内信号传导结构域包含来自CD3ζ的多肽。在某些实施方式中，所述胞内信号传导结构域包含SEQ ID NO：44所示的氨基酸序列。
在某些实施方式中，所述CAR还包含铰链区，所述铰链区连接所述抗体和所述跨膜结构域。在某些实施方式中，所述铰链区包含选自下述蛋白的多肽：CD8a和/或IgG。在某些实施方式中，所述铰链区包含SEQ ID NO：38所示的氨基酸序列。
在某些实施方式中，所述CAR包含SEQ ID NO：46、SEQ ID NO：72和SEQ ID NO：50中任一项所示的氨基酸序列。
另一方面，本申请提供了一种分离的核酸分子，其编码本申请所述的CAR。
另一方面，本申请提供了一种编码CAR的分离的核酸分子，其包含SEQ ID NO：47、SEQ ID NO：73或SEQ ID NO：51所示的核酸序列。
另一方面，本申请提供了一种载体，其包含本申请所述的核酸分子。在某些实施方式中，本申请所述的载体包括mRNA载体、质粒载体、逆转录病毒载体和/或慢病毒载体。
另一方面，本申请提供了一种免疫效应细胞，其包含和/或能够表达本申请所述的CAR，本申请所述的核酸分子，或本申请所述的载体。在某些实施方式中，所述免疫效应细胞选自T淋巴细胞和自然杀伤(NK)细胞。在某些实施方式中，所述T淋巴细胞和/或自然杀伤细胞是天然来源，或者经干细胞诱导。
另一方面，本申请提供了一种制备免疫效应细胞的方法，其包括向免疫效应细胞中引入本申请所述的载体。
另一方面，本申请提供了一种组合物，其包含本申请所述的免疫效应细胞。
另一方面，本申请提供了本申请所述的CAR，本申请所述的核酸分子，本申请所述的载体，或本申请所述的免疫效应细胞用于制备药物的用途，其中所述药物用于治疗T细胞淋巴瘤或白血病。
在某些实施方式中，所述T细胞淋巴瘤或白血病包含选自下组中的一种或多种：外周T细胞淋巴瘤、非特指型外周T细胞淋巴瘤、血管免疫母细胞淋巴瘤、结外NKT细胞淋巴瘤、间变性T细胞淋巴瘤、ALK阳性间变性细胞淋巴瘤、ALK阴性间变性细胞淋巴瘤、T淋巴母细胞淋巴瘤、细胞毒性T细胞淋巴瘤、皮肤T细胞淋巴瘤、成人T细胞白血病/淋巴瘤(ATLL)、T幼淋巴细胞白血病(T-PLL)、T大颗粒淋巴细胞(T-LGL)白血病、肝脾T细胞淋巴瘤(HSTL)、塞扎里综合征(SS)、皮下扁桃体样T细胞淋巴瘤以及未分型T细胞淋巴瘤。
本领域技术人员能够从下文的详细描述中容易地洞察到本公开的其它方面和优势。下文的详细描述中仅显示和描述了本公开的示例性实施方式。如本领域技术人员将认识到的，本公开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靶向TCR Vα或Vβ亚型的CAR结构示意图。
图2显示的是本申请所述T120BBZ慢病毒的滴度测定结果。
图3显示的是本申请所述T120BBZ CAR-T的CAR表达分析检测结果。
图4显示的是本申请所述T120BBZ CAR-T对Vα24Jα18 +肿瘤细胞的杀伤的检测结果。
图5显示的是本申请所述Vα24Jα18 +肿瘤细胞能特异诱导T120BBZ CAR-T分泌TNF的检测结果。
图6A-6B显示的是本申请所述F231BBZ CAR-T能特异杀伤Vβ8 +肿瘤细胞，并释放IFN。
图7A-7C显示的是本申请所述T120BBZ CAR-T的体内抗肿瘤活性分析。
图8A-8C显示的是本申请所述T120BBZ CAR-T的TCR表达分析。
图9显示的是本申请所述1168BBZ慢病毒的滴度分析检测结果。
图10显示的是本申请所述1168BBZ CAR-T的CAR表达分析检测结果。
图11显示的是本申请所述1168BBZ CAR-T对Vβ8 +肿瘤细胞的杀伤的检测结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。本申请提供了一种可以特异性结合T细胞受体可变区Vα、Vβ和/或它们的片段的CAR，其能够选择性杀伤T细胞来源的肿瘤细胞，并且对正常T细胞群体影响较小。
以下对本发明做进一步描述：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本申请中，所述“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，其可以包括肿瘤相关抗原(tumor-associated antigen，TAA)结合区、跨膜结构域、共刺激结构域和胞内信号结构域。在某些实施方式中，所述胞内结构域可以和所述跨膜结构域衍生自不同的蛋白质。
在本申请中，所述“靶向部分”通常是指嵌合抗原受体中可特异性结合目的蛋白或其片段的任何寡肽或多肽。在本申请中，所述靶向部分可为抗原结合区，例如，可以为抗体的抗原结合部分。在某些实施方式中，所述靶向部分可以包括特异性识别目的蛋白(例如，抗原)的单链抗体(scFv)或其部分。在本申请中，所述靶向部分可为肿瘤相关抗原结合区。在本申请中，所述肿瘤相关抗原可以指具有抗原性且其表达和肿瘤相关的生物分子。例如，所述肿瘤相关抗原可以包含仅在肿瘤细胞中存在、而在其它正常细胞中不存在的抗原的肿瘤特异性抗原；也可以包含既存在于肿瘤细胞中，也存在于其它器官和组织或异质和同种异体的正常细胞中的抗原，或在发育和分化过程中表达的抗原。
在本申请中，所述“跨膜结构域”(Transmembrane Domain)通常是指横跨细胞膜的结构域。例如，所述跨膜结构域可以为所述嵌合抗原受体的横跨细胞膜的结构域。在本申请中， 所述跨膜结构域可以衍生自天然多肽，或者可以经人工设计获得。例如，所述跨膜结构域可以为源自选自以下蛋白质的跨膜结构域：T细胞受体α或β链、3ζ链、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、ICOS、CD154或GITR。
在本申请中，所述“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。所述共刺激分子可以为淋巴细胞对抗原有效应答所需的分子。所述共刺激结构域可包括CD28的共刺激结构域。所述共刺激结构域可包括TNF受体家族的共刺激结构域，例如OX40和4-1BB的共刺激结构域。
在本申请中，所述“胞内信号传导结构域”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，所述胞内信号传导信号域可以将信号传导至细胞内。例如，所述胞内信号传导结构域是所述嵌合抗原受体的胞内信号传导结构域。例如，所述胞内信号转导结构域可以为源自选自CD3ζ、CD28、4-1BB或OX40的胞内域。又例如，所述胞内信号转导结构域可以为源自GITR的胞内结构域。
在本申请中，所述“T细胞受体(TCR)”通常是指T细胞表面的特异性受体。所述T细胞受体是异源二聚体，可以由两个不同的亚基所构成。大部分的T细胞受体(例如，95％及以上，96％及以上，97％及以上等)由α亚基和β亚基构成，每条肽链又可分为可变区(V区)、恒定区(C区)、跨膜区和胞质区等部分。在某些实施方式中，所述T细胞受体可由γ亚基和δ亚基构成，这两种亚基的比例可随个体发育或疾病而变化。
在本申请中，所述“TCR可变区”通常是指TCR的α、β两条肽链的V区，即Vα和Vβ。所述Vα和Vβ可具有抗原特异性。所述Vα和Vβ可各自有三个高变区CDR1、CDR2和CDR3。其中，TCR可变区的CDR3的变化程度最大，所述TCR可变区的CDR3可以决定TCR的抗原结合特异性。
在本申请中，所述“Vα”通常是指TCR的α链的可变区。该术语还可包括Vα全长序列和/或Vα功能性片段，只要能被靶向其的抗体识别即可。例如，本申请所述的Vα可以是在GenBank中的登记号为Gene ID 6955(人源)的Vα和Gene ID 21473(小鼠源)的Vα。在本申请中，所述“Vα24”通常是指Vα片段的一种变体。所述Vα24可来源于人、猕猴、小鼠或大鼠等不同种系。例如，所述Vα24可以是猕猴源的，其在GenBank中的登记号为ABG76794.1；也可以是人源的，其PDB号为4EN3_A。
在本申请中，所述“Jα”通常是指TCR的α链的可变区的Jα片段，其可以有多种变体。所述Jα可以为Jα18。在本申请中，所述“Jα18”通常是指Jα片段的一种变体。所述Jα18可来源于人、猕猴、小鼠或大鼠等不同种系。例如，本申请所述Jα18可以是人源的，其序列可 如SEQ ID NO：58所示。
在本申请中，所述“Vα24Jα18”通常是指由Vα的变体Vα24和Jα片段的变体Jα18重组后获得的一种变体。例如，本申请所述的Vα24Jα18的序列可以如SEQ ID NO：59所示。
在本申请中，所述“Vβ”通常是指TCR的β链的可变区。该术语还可包括Vβ全长序列和/或Vβ功能性片段，只要能被靶向其的抗体识别即可。例如，本申请所述的Vβ可以是在GenBank中的登记号为Gene ID 6957(人源)和Gene ID 21577(小鼠源)的Vβ。
在本申请中，所述“Vβ8”通常是指TCR的β链的可变区的一种变体。例如，本申请所述的Vβ8可来源于人源、猕猴、小鼠或大鼠等不同种系。例如，本申请所述Vβ8可以是人源的，其序列如SEQ ID NO：61所示；也可是小鼠源的，其序列如SEQ ID NO：60所示。
在本申请中，所述“变体”通常是指在所述蛋白质和/或所述多肽(例如，特异性结合TCR可变区的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述“同源物”通常是指与所述蛋白质和/或所述多肽(例如，特异性结合TCR可变区的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所 需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，所述“TCR可变区的CDR3”通常是指TCRβ链互补决定区3。所述TCR可变区的CDR3可以是直接与抗原肽结合的位点。所述TCR可变区的CDR3的基因可以由V末端、D片段、J前端以及重排V～D与D～J之间的插入序列(VnDnJ)组成。
在本申请中，所述“Vα24Jα18抗体”通常是指可以特异性结合Vα24Jα18的抗体。例如，在本申请中，所述Vα24Jα18抗体可以是6B11(Invitrogen Product#25-5806-41)。例如，在本申请中，所述Vα24Jα18抗体也可以是scFv T120(其序列可以如SEQ ID NO：1所示)。
在本申请中，所述“Vβ8抗体”通常是指可以特异性结合Vβ8的抗体。例如，在申请中，所述Vβ抗体可以是scFv F231(其序列可以如SEQ ID NO：19所示)。又例如，在本申请中，所述Vβ抗体可以是scFv 1168(其氨基酸序列可以如SEQ ID NO：62所示，其核酸序列可以如SEQ ID NO：63所示)。
在本申请中，所述“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(CH)构成。每条轻链可由轻链可变区(VL)和轻链恒定区(CL)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。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的多种细胞(例如，效应细胞)和经典补体系统的第一成分(Clq)。
在本申请中，所述“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。在本申请中，可以通过本领域已知的多种方法来制备编码所述抗体或其抗原结合片段的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR，具体操作可参见Sambrook等人，Molecular Cloning,A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor,N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology,Greene Publishing and Wiley-Interscience,New York N.Y.,1993。
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。在本申请中，所述载体中可包含一种或多种所述核酸分子。此外，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。
在本申请中，所述“mRNA载体”通常是指可将外源基因或自身基因在宿主细胞表达的mRNA片段。例如，在本申请中，所述mRNA载体可包括F231mBBZ mRNA和20mBBZ mRNA。
在本申请中，所述“质粒载体”通常是指在天然质粒的基础上为适应实验室操作而进行人工构建的质粒。与天然质粒相比，质粒载体通常带有一个或一个以上的选择性标记基因(如 抗生素抗性基因)和一个人工合成的含有多个限制性内切酶识别位点的多克隆位点序列，并去掉了大部分非必需序列，使分子量尽可能减少，以便于基因工程操作。
在本申请中，所述“逆转录病毒载体”通常是指RNA病毒的一种，其遗传信息存储在核糖核酸上，此类病毒多具有反转录酶。反转录病毒至少含有三种基因：gag，包含组成病毒中心和结构的蛋白质的基因；pol，包含反转录酶的基因和env，包含组成病毒外壳的基因。通过逆转录病毒转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，所述“慢病毒载体”通常是指属于逆转录病毒的一种二倍体RNA病毒载体。慢病毒载体是以慢病毒的基因组为基础，将其中多个和病毒活性相关的序列结构去除，使其具有生物学的安全性，然后再在这个基因组骨架中引入实验所需要的目标基因的序列和表达结构制备成的载体。与其他逆转录病毒相比，慢病毒载体有着更广泛的宿主，对于分裂和非分裂细胞均具有感染能力，对于一些较难转染的细胞，如原代细胞、干细胞、不分化的细胞等，能大大提高目的基因的转导效率(参见陈琛和万海粟，“慢病毒载体及其研究进展，Chinese Journal of Lung Cancer 17.12(2014):870–876.PMC”)。通过慢病毒载体转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，所述“免疫效应细胞”通常是指在免疫应答中参与清除异物抗原和行使效应功能的免疫细胞。例如浆细胞、细胞毒性T细胞、NK细胞、APSC多能细胞、肥大细胞等。
在本申请中，术语“药学上可接受的佐剂”通常是指药学可接受的制剂载体、溶液或加强制剂特性的添加剂。此类添加剂是所属领域技术人员熟知的。
在本申请中，“T细胞淋巴瘤”通常是指影响T细胞的淋巴瘤。所述T细胞淋巴瘤可包括以下的种类：血管中心性淋巴瘤(Angiocentric lymphoma)，皮肤T细胞淋巴瘤(Cutaneous T cell lymphoma)塞，间变性大细胞淋巴瘤(Anaplastic large-cell lymphoma)和血管免疫母细胞性T细胞淋巴瘤(Angioimmunoblastic T-cell lymphoma)。T细胞淋巴瘤可以在淋巴组织例如淋巴结和脾脏中或淋巴组织外部(即胃肠道、肝脏、鼻腔、皮肤等)中发展。
在本申请中，“白血病”通常是血细胞癌症的广义定义。例如，所述白血病可以为因白血病细胞大量增殖、累积所引起的疾病。所述白血病通常起始于骨髓中。在本申请中，所述白血病的类型可以取决于变成癌症的血细胞的类型和/或该血细胞的生长速度。例如，所述白血病可包括成人T细胞白血病/淋巴瘤(ATLL)、T幼淋巴细胞白血病(T-PLL)、T大颗粒 淋巴细胞(T-LGL)白血病等亚型。
在本申请中，所述“天然来源”通常是指未经人工处理的野生型的获取方式。在本申请中，所述天然来源可以是直接从供体采集的细胞获取方式。例如，可以是没有经过人工基因编辑或修饰的细胞获取方式。在本申请中，所述供体可是健康或患有疾病的哺乳动物。例如，人类，小鼠，猴等。在本申请中，所述疾病可指癌症。例如，所述疾病可以为T细胞来源的白血病或淋巴瘤(例如，T细胞淋巴瘤)。
在本申请中，所述“经干细胞诱导”通常是指在体外通过人工诱导使得干细胞分化的过程。在本申请中，体外诱导方法通常是在体外培养的成体干细胞中加人入诱导因子，所述诱导因子可包括各种能影响细胞分化的物质，例如血清、糖分、维生素、以及各种蛋白因子等。在本申请中，经干细胞诱导可为诱导的多能干细胞(induced pluripotent stem cells，iPS cells)。所述诱导的多能干细胞可用病毒载体将四个转录因子(Oct4、Sox2、Klf4和c-Myc)的组合转入分化的体细胞中而得到。在本申请中，所述iPS细胞可为类似胚胎干细胞和胚胎APSC多能细胞的一种细胞类型。
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“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。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
1.嵌合抗原受体
在本申请中，所述嵌合抗原受体(CAR)可包含靶向部分、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述靶向部分可特异性结合T细胞受体(TCR)的可变区Vα、Vβ和/或它们的片段。
a.靶向部分
在某些实施方式中，所述靶向部分可特异性结合TCR的可变区Vα或其片段。在某些实施方式中，所述Vα或其片段可包括Vα24或其片段。在某些实施方式中，所述Vα或其片段还可包括Jα或其片段。在某些实施方式中，所述Jα或其片段可包括Jα18或其片段。在某些实施方式中，所述靶向部分可包括靶向Vα24Jα18的部分。在本申请中，所述靶向Vα24Jα18的部分包括scFv T120，其序列如SEQ ID NO：1所示。
在某些实施方式中，所述靶向部分特异性结合TCR的可变区Vβ或其片段。在某些实施 方式中，所述Vβ或其片段可包括Vβ8或其片段。在某些实施方式中，所述TCR可变区的片段可包含TCR可变区的CDR3。在某些实施方式中，所述靶向部分可包括靶向Vβ8的部分。在本申请中，所述靶向Vβ8的部分可包括scFv F231，其序列如SEQ ID NO：19所示。又例如，在本申请中，所述靶向Vβ8的部分还可包括scFv 1168，其序列如SEQ ID NO：62所示。
在本申请中，所述TCR的可变区Vα、Vβ或其片段可包含SEQ ID NO：58-61、Gene ID21473、Gene ID 6955、Gene ID 21577和Gene ID 6957中任一项所示的氨基酸序列。
在本申请中，所述靶向部分可包括抗体或其片段。
在本申请中，所述抗体可与参比抗体竞争结合所述TCR的可变区Vα、Vβ和/或它们的片段，其中所述参比抗体可包含轻链可变区和重链可变区，所述参比抗体的轻链可变区可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO：3、SEQ ID NO：64和SEQ ID NO：21中任一项所示的氨基酸序列；所述LCDR2可包含SEQ ID NO：4、SEQ ID NO：65和SEQ ID NO：22中任一项所示的氨基酸序列；所述LCDR3可包含SEQ ID NO：5、SEQ ID NO：66和SEQ ID NO：23中任一项所示的氨基酸序列；所述参比抗体的重链可变区可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO：11、SEQ ID NO：67和SEQ ID NO：29中任一项所示的氨基酸序列；所述HCDR2可包含SEQ ID NO：12、SEQ ID NO：68和SEQ ID NO：30中任一项所示的氨基酸序列；所述HCDR3可包含SEQ ID NO：13、SEQ ID NO：69和SEQ ID NO：31中任一项所示的氨基酸序列。
在本申请中，所述参比抗体的轻链可变区可包含SEQ ID NO：9、SEQ ID NO：70和SEQ ID NO：27中任一项所示的氨基酸序列；且所述参比抗体的重链可变区可包含SEQ ID NO：17、SEQ ID NO：71和SEQ ID NO：35中任一项所示的氨基酸序列。
在本申请中，所述参比抗体可包含Vα24Jα18抗体。例如，所述Vα24Jα18抗体可为scFv T120，其序列如SEQ ID NO：1所示。
在本申请中，所述抗体可包含重链互补决定区1(HCDR1)，所述HCDR1可包含SEQ ID NO：11、SEQ ID NO：67和SEQ ID NO：29中任一项所示的氨基酸序列。在本申请中，所述抗体还可包含重链互补决定区2(HCDR2)，所述HCDR2可包含SEQ ID NO：12、SEQ ID NO：68和SEQ ID NO：30中任一项所示的氨基酸序列。在本申请中，所述抗体还可包含重链互补决定区3(HCDR3)，所述HCDR3包含SEQ ID NO：13、SEQ ID NO：69和SEQ ID NO：31中任一项所示的氨基酸序列。
在本申请中，所述抗体可包含轻链互补决定区1(LCDR1)，所述LCDR1包含SEQ ID NO：3、SEQ ID NO：64和SEQ ID NO：21中任一项所示的氨基酸序列。在本申请中，所述 抗体还可包含轻链互补决定区2(LCDR2)，所述LCDR2包含SEQ ID NO：4、SEQ ID NO：65和SEQ ID NO：22中任一项所示的氨基酸序列。在本申请中，所述抗体还可包含轻链互补决定区3(LCDR3)，所述LCDR3包含SEQ ID NO：5、SEQ ID NO：66和SEQ ID NO：23中任一项所示的氨基酸序列。
在本申请中，所述抗体可包含重链可变区，所述重链可变区可包含SEQ ID NO：17、SEQ ID NO：71和SEQ ID NO：35中任一项所示的氨基酸序列。在本申请中，所述抗体包含轻链可变区，所述轻链可变区包含SEQ ID NO：9、SEQ ID NO：70和SEQ ID NO：27中任一项所示的氨基酸序列。
在本申请中，所述抗体可为单链抗体(scFv)。在本申请中，所述单链抗体可包含一个或多个scFv连接而成。例如，所述单链抗体可包含一个scFv，所述单链抗体可包含一个以上scFv(例如，2个，3个，4个，5个等)。在某些实施方式中，所述多个scFv可直接相连。在某些实施方式中，所述多个scFv可通过连接肽连接而成。
例如，所述单链抗体可包括scFv T120，其序列如SEQ ID NO：1所示；其可包括轻链可变区，其序列如SEQ ID NO：9所示；其可包括LCDR1-3，其序列分别依次如SEQ ID NO：3-5所示；其可包括重链可变区，其序列如SEQ ID NO：17所示；其可包括HCDR1-3，其序列分别依次如SEQ ID NO：11-13所示；其可包含VL与VH的连接肽，其连接肽序列如SEQ ID NO：52所示。
例如，所述单链抗体可包括scFv F231，其序列如SEQ ID NO：19所示；其可包括轻链可变区，其序列如SEQ ID NO：27所示；其可包括LCDR1-3，其序列分别依次如SEQ ID NO：21-23所示；其可包括重链可变区，其序列如SEQ ID NO：35所示；其可包括HCDR1-3，其序列分别依次如SEQ ID NO：29-31所示；其可包含VL与VH的连接肽，其连接肽序列如SEQ ID NO：52所示。
例如，所述单链抗体可包括scFv 1168，其序列如SEQ ID NO：62所示；其可包括轻链可变区，其序列如SEQ ID NO：70所示；其可包括LCDR1-3，其序列分别依次如SEQ ID NO：64-66所示；其可包括重链可变区，其序列如SEQ ID NO：71所示；其可包括HCDR1-3，其序列分别依次如SEQ ID NO：67-68所示；其可包含VL与VH的连接肽，其连接肽序列如SEQ ID NO：52所示。
b.跨膜结构域
在本申请中，所述跨膜结构域可包含来自选自下述蛋白的跨膜结构域：CD8、CD28、CD137和/或CD3。在本申请中，所述跨膜结构域可以包含SEQ ID NO：40所示的氨基酸序 列。例如，在本申请中，所述跨膜结构域可为CD8跨膜区，其序列如SEQ ID NO：40所示。在本申请中，CAR T120BBZ和CAR 1168BBZ可包含CD8的跨膜结构域。
c.共刺激结构域
在本申请中，所述共刺激结构域可包含下述蛋白的共刺激结构域：ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2和/或CD226。在本申请中，所述共刺激结构域包含SEQ ID NO：42所示的氨基酸序列。例如，在本申请中，所述共刺激结构域可为4-1BB，其序列如SEQ ID NO：42所示。在本申请中，CAR T120BBZ、CAR 1168BBZ和CAR F231mBBZ可包含4-1BB的共刺激结构域。
d.信号传导结构域
在本申请中，所述胞内信号传导结构域可包含来自CD3ζ的信号传导结构域。在本申请中，所述胞内信号传导结构域可包含SEQ ID NO：44所示的氨基酸序列。例如，在本申请中，所述信号传导结构域可为CD3ζ，其序列如SEQ ID NO：44所示。在本申请中，CAR T120BBZ、CAR 1168BBZ和CAR F231mBBZ可包含CD3ζ的信号传导结构域。
e.铰链区
在本申请中，所述CAR还可包含铰链区，所述铰链区可连接所述抗体和所述跨膜结构域。在本申请中，所述铰链区可包含来自选自下述蛋白的铰链区：CD8a和/或IgG。在本申请中，所述铰链区为CD8a，其可包含SEQ ID NO：38所示的氨基酸序列。例如，在本申请中，所述铰链区可为CD8a，其序列如SEQ ID NO：38所示。在本申请中，CAR T120BBZ、CAR1168BBZ和CAR F231mBBZ可包含CD8a的铰链区。
f.CAR
本申请所述的CAR，其可包含SEQ ID NO：46、SEQ ID NO：64和SEQ ID NO：50中任一项所示的氨基酸序列。例如，所述CAR可选自CAR T120BBZ，其序列如SEQ ID NO：46所示。又例如，所述CAR可选自CAR F231mBBZ，其序列如SEQ ID NO：50所示。又例如，所述CAR可选自CAR 1168BBZ，其序列如SEQ ID NO：72所示。
在某些实施方式中，本申请所述CAR可自N端起依次包括靶向部分(所述靶向部分序列如SEQ ID NO：1和SEQ ID NO：19所示)、跨膜结构域(所述跨膜结构域序列如SEQ ID NO：40所示)、共刺激结构域(所述共刺激结构域序列如SEQ ID NO：42所示)和胞内信号传导结构域(所述胞内信号传导结构域序列如SEQ ID NO：44所示)。
例如，本申请所述的CAR可为CAR T120BBZ，其靶向部分为scFv T120，scFv T120的氨基酸序列如SEQ ID NO：46所示。其跨膜结构域为CD8a，其氨基酸序列如SEQ ID NO：40所示。共刺激结构域可为4-1BB，其氨基酸序列如SEQ ID NO：42所示。胞内信号传导结 构域可为CD3ζ，其氨基酸序列如SEQ ID NO：44所示。CAR T120BBZ的氨基酸序列如SEQ ID NO：46所示。
例如，本申请所述的CAR可为CAR F231mBBZ，其靶向部分为scFv F231，scFv F231的氨基酸序列如SEQ ID NO：19所示。其跨膜结构域为CD8a，其氨基酸序列如SEQ ID NO：40所示。共刺激结构域可为4-1BB，其氨基酸序列如SEQ ID NO：42所示。胞内信号传导结构域可为CD3ζ，其氨基酸序列如SEQ ID NO：44所示。CAR F231mBBZ的氨基酸序列如SEQ ID NO：50所示。
例如，本申请所述的CAR可为CAR 1168BBZ，其靶向部分为scFv 1168，scFv 1168的氨基酸序列如SEQ ID NO：61所示。其跨膜结构域为CD8a，其氨基酸序列如SEQ ID NO：40所示。共刺激结构域可为4-1BB，其氨基酸序列如SEQ ID NO：42所示。胞内信号传导结构域可为CD3ζ，其氨基酸序列如SEQ ID NO：44所示。CAR 1168mBBZ的氨基酸序列如SEQ ID NO：72所示。
在本申请中涉及的蛋白质、多肽、氨基酸序列和/或核苷酸序列，还应理解为至少包含以下的范围：与该所述蛋白质、多肽，或者与该核苷酸序列所编码的蛋白质、多肽，具备相同或类似功能的变体或同源物。
核酸、载体、细胞、组合物
另一方面，本申请提供了一种分离的核酸分子，其编码本申请所述的CAR。在本申请中，所述编码CAR的分离的核酸分子，其可包含SEQ ID NO：47或SEQ ID NO：51所示的核酸序列。本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
另一方面，本申请提供了一种载体，其可包含本申请所述的核酸分子。在本申请中，所述载体可包括mRNA载体、质粒载体、逆转录病毒载体和/或慢病毒载体。例如，本申请所述载体可为慢病毒载体，其可包含SEQ ID NO：47或SEQ ID NO：51所示的核酸序列。此外，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。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体、mRNA载体或者在例如遗传工程中通常使用的其他载体。
在某些实施方式中，所述载体可为质粒载体，例如，其可包括pCDH-MSCVEF载体。在某些实施方式中，所述载体可为慢病毒载体，例如，慢病毒载体可以是VSV-g，pMD Gag/Pol或RSV-REV，其可以与质粒载体共转染宿主细胞(例如，293X细胞)后获得装载有质粒的慢病毒载体。在本申请中，装载有质粒的慢病毒载体可包括T120BBZ病毒，还可包括20BBZ病毒。在某些实施方式中，所述载体可为mRNA载体，例如，所述mRNA载体可为ARCA-capped RNA(AM1345，Thermor Fisher)，其可用于生产装载有质粒的mRNA载体。在本申请中，装载有质粒的mRNA载体可包括F231mBBZ mRNA，还可包括20BBZ mRNA。
另一方面，本申请提供了免疫效应细胞，其包含和/或能够表达本申请所述的CAR，本申请所述的核酸分子，或本申请所述的载体。在本申请中，所述免疫效应细胞可选自T淋巴细胞和自然杀伤(NK)细胞。在本申请中，所述T淋巴细胞和/或自然杀伤细胞可是天然来源，或者经干细胞诱导。
另一方面，本申请提供了一种制备免疫效应细胞的方法，其包括向免疫效应细胞中引入本申请所述的载体。在某些实施方式中，每种或每个细胞可包含一个或一种本申请所述的载体。在某些实施方式中，每种或每个细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的载体。例如，可将本申请所述的载体引入所述细胞中，例如真核细胞(例如哺乳动物细胞)。例如，所述哺乳动物细胞可以是293T细胞，也可以是293X细胞。可通过本领域已知的方法将本申请所述的载体引入所述细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
另一方面，本申请提供了一种组合物，其可包含本申请所述的免疫效应细胞。在某些实施方式中，其可包含所述的免疫效应细胞和药学上可接受的佐剂。所述药学上可接受的佐剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。在本申请中，所述药物组合物可被配制用于口服给药，静脉内给药，肌肉内给药，在肿瘤部位的原位给药，吸入，直肠给药，阴道给药，经皮给药或通过皮下储存库给药。
制药用途
另一方面，本申请提供了所述的CAR，所述的核酸分子，所述的载体，或所述的免疫效应细胞用于制备药物的用途，其中所述药物用于治疗T细胞淋巴瘤或白血病。
另一方面，本申请提供了所述的CAR，所述的核酸分子，所述的载体，或所述的免疫效应细胞在制备治疗T细胞淋巴瘤或白血病的药物中的应用。
另一方面，本申请提供了一种治疗T细胞淋巴瘤或白血病的方法，包括向患者施用所述的CAR，所述的核酸分子，所述的载体，或所述的免疫效应细胞。
在本申请中，所述T细胞淋巴瘤或白血病可包含选自下组中的一种或多种：外周T细胞淋巴瘤、非特指型外周T细胞淋巴瘤、血管免疫母细胞淋巴瘤、结外NKT细胞淋巴瘤、间变性T细胞淋巴瘤、ALK阳性间变性细胞淋巴瘤、ALK阴性间变性细胞淋巴瘤、T淋巴母细胞淋巴瘤、细胞毒性T细胞淋巴瘤、皮肤T细胞淋巴瘤、成人T细胞白血病/淋巴瘤(ATLL)、T幼淋巴细胞白血病(T-PLL)、T大颗粒淋巴细胞(T-LGL)白血病、肝脾T细胞淋巴瘤(HSTL)、塞扎里综合征(SS)、皮下扁桃体样T细胞淋巴瘤以及未分型T细胞淋巴瘤。
在某些实施方式中，所述的CAR，所述的核酸分子，所述的载体，或所述的免疫效应细胞可显著提高对肿瘤细胞的体内杀伤效果。例如，可使肿瘤体积减少50％以上(例如，60％以上，70％以上，80％以上，90％以上等)。
在某些实施方式中，所述的CAR，所述的核酸分子，所述的载体，或所述的免疫效应细胞可显著提高对肿瘤细胞的体外杀伤效果。例如，可使肿瘤体积减少50％以上(例如，60％以上，70％以上，80％以上，90％以上等)。
在某些实施方式中，所述的CAR，所述的核酸分子，所述的载体，或所述的免疫效应细胞可选择性杀伤带有特定抗原的肿瘤细胞，而基本不杀伤正常的T细胞(例如，所述正常的T细胞可以为不属于T细胞淋巴瘤的正常、健康的T细胞)。例如，可以使对带有特定抗原(例如Vα24Jα18 +)肿瘤细胞的杀伤效果提高50％以上(例如，60％以上，70％以上，80％以上，90％以上等)，而对正常的T细胞的基本无伤害(例如，T细胞数减少5％以下，4％以下，3％以下，2％以下，1％以下等)。
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装置、方法和系统的工作方式，而不用于限制本申请发明的范围。
实施例
在本申请中，所述结合靶点、靶向部分(scFv)、CAR、载体、细胞名称的对应关系如下表1所示：
表1
Figure PCTCN2020073304-appb-000001
实施例1 抗Vα24Jα18CAR-T细胞(T120BBZ CAR-T细胞)的制备
1.1 慢病毒载体pCDH-MSCVEF-T120BBZ的构建及病毒生产
为构建本申请所述CAR(参见图1)。首先制备靶向Vα24Jα18的CAR，人工合成以下序列：scFv T120(SEQ ID NO：1)，铰链区(SEQ ID NO：38)，跨膜区(SEQ ID NO：40)，4-1BB共刺激因子(SEQ ID NO：42)，CD3ζ胞内信号传导结构域(SEQ ID NO：44)。其中，将4-1BB共刺激因子和CD3ζ胞内信号传导结构域首尾相连可获得BBZ，其序列如SEQ ID NO：54所示。
同时，构建scFv 20作为对照，其序列如SEQ ID NO：37所示。
将可特异性结合TCR Vα24Jα18的scFv T120(氨基酸序列SEQ ID NO：1，核苷酸序列SEQ ID NO：2)和BBZ(SEQ ID NO：54)通过overlap PCR，两端加入EcoRI和BamHI酶切位点克隆pCDH-MSCVEF载体。进行PCR扩增，并用延伸PCR在5’端依次带上EcoRI酶切位点(含保护碱基)、铰链区、跨膜区、4-1BB共刺激因子、CD3ζ胞内信号传导结构域、BamHI酶切位点，PCR扩增得到所述CAR T120BBZ。测序正确的克隆用NucleoBond Xtra Midi Plus EF试剂盒无内毒素大提，和慢病毒包装质粒(VSV-g，pMD Gag/Pol或RSV-REV)共同转染293X细胞，37℃、5％CO 2培养48小时后收取上清，0.45μM过滤后，使用贝克曼超速离心机和SW28转头，以25000RPM的速度离心2小时以浓缩病毒，即为pCDH-MSCVEF-T120BBZ病毒(简称为T120BBZ病毒)，用于后续CAR-T细胞生产。
同时用与制备T120BBZ病毒相同的方法生产对照pCDH-MSCVEF-20BBZ病毒(简称20BBZ病毒)，将所得病毒感染293细胞，使用anti-mouse Fab抗体(Jackson ImmunoResearch #115-605-006)用流式检测的方法测病毒滴度，如图2所示。图2中显示的是在添加1μL、3μL、9μL的所述T120BBZ病毒的时候的流式检测结果，以不添加病毒作为空白对照。
结果显示，随着所加病毒剂量的增加，所述CAR T120BBZ的表达量也随之增加。
1.2 T120BBZ CAR-T细胞和20BBZ CAR-T细胞的制备
将人PBMC经过Stemcell T细胞分离试剂盒(购自stem cell Catlog#19671纯化后，接种到anti-hCD3(购自Bioxcell#BE0001-2)和anti-hCD28(购自Bioxcell#BE0248)包被的96孔培养板，2天后，按照MOI＝10-20感染本实施例步骤1.1制备的T120BBZ病毒和20BBZ病毒，1天后换液继续细胞培养，培养基为含10％FBS的RPMI完全培养基，IL2(50IU/ml)，IL21(4ng/ml)，按照每6天使用人工抗原呈递细胞(X射线100Gray辐照后的Raji细胞)或anti-hCD3(0.1μg/ml)或anti-hCD28(0.25μg/ml)刺激，经过2轮刺激后，所得细胞即为T120BBZ CAR-T细胞和20BBZ CAR-T细胞，是通过流式染色，对T120BBZ CAR-T细胞用Alexa
Figure PCTCN2020073304-appb-000002
647AffiniPure F(ab') 2 Fragment Goat Anti-Human IgG,Fab fragment specific二抗染色，其结果如图3所示。
结果显示，所得细胞均为CAR阳性。
实施例2 抗Vβ8CAR-T(F231mBBZ)细胞的制备
2.1.抗Vβ8CAR-T(F231mBBZ)mRNA的生产
与实施例1类似，制备靶向鼠Vβ8的CAR，人工合成以下序列：scFv F231(SEQ ID NO：19)，mBBZ(SEQ ID NO：56)。其中mBBZ为鼠源的4-1BB共刺激因子和CD3ζ胞内信号传导结构域首尾连接而成。
将可特异性结合TCR Vβ8的scFv F231(SEQ ID 19)和mBBZ(SEQ ID NO：56)通过overlap PCR，两端加入EcoRI和BamHI酶切位点克隆pCDH-MSCVEF载体。进行PCR扩增，并用延伸PCR在5’端依次带上EcoRI酶切位点(含保护碱基)、铰链区、跨膜区、4-1BB共刺激因子、CD3ζ胞内信号传导结构域、BamHI酶切位点，PCR扩增得到所述CAR F231mBBZ(SEQ ID NO：51)，通过引物在5’端引入T7RNA聚合酶启动子序列，回收纯化PCR产物，使用mMESSAGE
Figure PCTCN2020073304-appb-000003
T7Ultra Kit生产F231mBBZ mRNA。同时用相同方法生产对照20BBZ mRNA。
2.2 F231mBBZ CAR-T细胞和20BBZ CAR-T细胞的制备
将小鼠脾脏细胞经Mouse CD3T Cell Isolation Kit细胞分离试剂盒(购自biolegend#480031)纯化后，接种到96孔培养板，经ConA(购自sigma#C2010)活化2天后，使用ECM830电 转F231mBBZ mRNA和20BBZ mRNA，所得细胞为F231mBBZ CAR-T细胞和20BBZ CAR-T细胞。
实施例3 抗1168CAR-T细胞(1168BBZ CAR-T细胞)的制备
3.1.慢病毒载体pCDH-MSCVEF-1168BBZ的构建及病毒生产
按照实施例1的方法，首先制备靶向人Vβ8的CAR，人工合成以下序列：scFv 1168(SEQ ID NO：62)，铰链区(SEQ ID NO：38)，跨膜区(SEQ ID NO：40)，4-1BB共刺激因子(SEQ ID NO：42)，CD3ζ胞内信号传导结构域(SEQ ID NO：44)。其中，将4-1BB共刺激因子和CD3ζ胞内信号传导结构域首尾相连可获得BBZ，其序列如SEQ ID NO：54所示。
同时，构建scFv 20作为对照，其序列如SEQ ID NO：37所示。
将可特异性结合人Vβ8的scFv 1168(SEQ ID NO：62)和BBZ(SEQ ID NO：54)通过overlap PCR，两端加入EcoRI和BamHI酶切位点克隆pCDH-MSCVEF载体。进行PCR扩增，并用延伸PCR在5’端依次带上EcoRI酶切位点(含保护碱基)、铰链区、跨膜区、4-1BB共刺激因子、CD3ζ胞内信号传导结构域、BamHI酶切位点，PCR扩增得到所述CAR1168BBZ。测序正确的克隆用NucleoBond Xtra Midi Plus EF试剂盒无内毒素大提，和慢病毒包装质粒(VSV-g，pMD Gag/Pol或RSV-REV)共同转染293X细胞，37℃、5％CO 2培养48小时后收取上清，0.45μM过滤后，使用贝克曼超速离心机和SW28转头，以25000RPM的速度离心2小时以浓缩病毒，即为pCDH-MSCVEF-1168BBZ病毒(简称为1168BBZ病毒)，用于后续CAR-T细胞生产。
同时用与制备1168BBZ病毒相同的方法生产对照pCDH-MSCVEF-20BBZ病毒(简称20BBZ病毒)，将所得病毒感染293细胞，使用anti-mouse Fab抗体(Jackson ImmunoResearch#115-605-006)用流式检测的方法测病毒滴度，如图9所示。图9中显示的是在添加1μL、3μL、9μL的所述1168BBZ病毒的时候的流式检测结果，以不添加病毒作为空白对照。
结果显示，随着所加病毒剂量的增加，所述CAR 1168BBZ的表达量也随之增加。
3.2. 1168BBZ CAR-T细胞和20BBZ CAR-T细胞的制备
将人PBMC经过Stemcell T细胞分离试剂盒(购自stem cell Catlog#19671)纯化后，接种到anti-hCD3(购自Bioxcell#BE0001-2)和anti-hCD28(购自Bioxcell#BE0248)包被的96孔培养板，2天后，按照MOI＝10-20感染本实施例步骤1.1制备的1168BBZ病毒和20BBZ病毒，1天后换液继续细胞培养，培养基为含10％FBS的RPMI完全培养基，IL2(50IU/ml),IL21(4ng/ml)，按照每6天使用人工抗原呈递细胞(X射线100Gray辐照后的Raji细胞)或anti-hCD3(0.1μg/ml)或anti-hCD28(0.25μg/ml)刺激，经过2轮刺激后，所得细胞即为1168BBZ  CAR-T细胞和20BBZ CAR-T细胞，是通过流式染色，用anti-mouse Fab抗体(Jackson ImmunoResearch#115-605-006)二抗染色，其结果如图10所示。
结果显示，所得细胞均为CAR阳性。
实施例4 T120BBZ CAR-T对肿瘤细胞的体外杀伤和效应分子释放
取实施例1制备所得的T120BBZ CAR-T细胞，并以20BBZ CAR-T细胞作为对照，和NALM6-Vα24Jα18 +细胞37℃，5％CO 2共培养，24小时后流式细胞仪检测NALM6-Vα24Jα18 +的存活和TNF的释放，T120BBZ CAR-T能选择性杀伤Vα24Jα18 +的肿瘤细胞，并释放更多的TNF。对肿瘤的杀伤效果检测如图4所示，TNF释放的检测结果如图5所示。
结果显示，与对照CAR-T细胞相比，T120BBZ CAR-T细胞对Vα24Jα18 +的肿瘤杀伤效果比对Vα24Jα18 -的肿瘤细胞杀伤效果更强。与对照CAR-T细胞相比，施用T120BBZ CAR-T细胞后，Vα24Jα18 -肿瘤细胞减少17.4％，Vα24Jα18 +肿瘤细胞减少72.2％，证明T120BBZ CAR具有对Vα24Jα18的特异性。并且，与对照CAR-T细胞相比，施用T120BBZ CAR-T细胞后，Vα24Jα18 -肿瘤细胞的TNF释放量略有增加(增加了23.7％)，Vα24Jα18 +肿瘤细胞的TNF释放量有显著增加(增加了188.6％)，其也可证明T120BBZ CAR具有对Vα24Jα18的特异性。
实施例5 F231mBBZ CAR-T细胞对肿瘤细胞的体外杀伤
将实施例2中步骤2.2制备所得的F231mBBZ CAR-T细胞和20BBZ CAR-T细胞接种到96孔板，同时以未被病毒感染的T细胞作为阴性对照，和C1498Vβ8 -CD20 -或C1498Vβ8 +CD20 -肿瘤细胞37℃，5％CO 2共培养，24小时后流式细胞仪染色TCRVb8.1/8.2(Biolegend#118406)比较肿瘤细胞的存活比例以及IFNγ的释放，结果如图6A-6B所示。
结果显示，F231mBBZ CAR-T细胞能选择性的杀伤Vβ8 +的肿瘤细胞，并释放更多IFNγ。
实施例6 1168BBZ CAR-T对肿瘤细胞的体外杀伤和效应分子释放
取实施例3制备所得的1168BBZ CAR-T细胞，并以20BBZ CAR-T细胞作为对照，和Vβ8 +Jurkat细胞37℃，5％CO 2共培养，24小时后流式细胞仪检测肿瘤细胞的存活，1168BBZ CAR-T能选择性杀伤Vβ8 +的肿瘤细胞。对肿瘤的杀伤效果检测如图11所示。
结果显示，与对照CAR-T细胞相比，1168BBZ CAR-T细胞对Vβ8 +的肿瘤杀伤效果比 20BBZ对Vβ8 +的肿瘤细胞杀伤效果更强。与对照CAR-T细胞相比，施用1168BBZ CAR-T细胞后，肿瘤细胞存活数量减少，证明1168BBZ CAR具有对Vβ8的特异性。
结果显示，1168BBZ CAR-T细胞能选择性的杀伤人Vβ8 +的肿瘤细胞。
实施例7 T120BBZ CAR-T细胞对肿瘤细胞的体内杀伤能力分析
将10 6个NALM6Vα24Jα18 +肿瘤细胞通过静脉注射接种到B-NDG小鼠(百奥赛图)，6天后静脉注射10 7个T120BBZ CAR-T细胞治疗，同时以未感染病毒的T细胞作为阴性对照。于第7天，取5滴外周血(100μL)流式检测其外周血中的肿瘤细胞和CAR-T细胞的含量，其结果分别如图7A-7C所示。
结果显示，与对照相比，T120BBZ CAR-T细胞使得小鼠体内肿瘤减少了82.9％(3.798mm 3到0.646mm 3)。T120BBZ CAR-T细胞显著延长了小鼠的肿瘤负荷，并在体内能扩增和延长小鼠的存活。
实施例8 T120BBZ CAR-T细胞的TCR组成分析
将实施例1制备所得的T120BBZ CAR-T细胞，并以20BBZ CAR-T细胞作为对照，使用anti-TCRαβ(Biolegend#306708)，anti-CD3(Biolegend#317306)和anti-Vα24Jα18 +(Biolegend#342912)染色，流式分析其特定靶向TCR(Vα24Jα18 +)组成的变化，结果如图8A-8C所示。结果显示，与对照CAR-T细胞相比，T120BBZ CAR-T中Vα24Jα18 +肿瘤细胞显著减少，证明T120BBZ CAR-T能选择性特异杀伤Vα24Jα18 +的T细胞，TCRαβ细胞、CD3 +细胞没有显著区别(NS，not siginificant)，证明保留了其他的TCRαβ亚型的T细胞和TCR复合物中的CD3组成部分，预示了其对绝大多数T细胞亚类的保留，对T细胞免疫的功能影响较小。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本文所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (44)

  1. 嵌合抗原受体(CAR)，其中所述CAR包含靶向部分、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述靶向部分特异性结合T细胞受体(TCR)的可变区Vα、Vβ和/或它们的片段。
  2. 根据权利要求1所述的CAR，其中所述靶向部分特异性结合TCR的可变区Vα或其片段。
  3. 根据权利要求2所述的CAR，其中所述Vα或其片段包括Vα24或其片段。
  4. 根据权利要求2-3中任一项所述的CAR，其中所述Vα或其片段还包括Jα或其片段。
  5. 根据权利要求4所述的CAR，其中所述Jα或其片段包括Jα18或其片段。
  6. 根据权利要求1所述的CAR，其中所述靶向部分特异性结合TCR的可变区Vβ或其片段。
  7. 根据权利要求6所述的CAR，其中所述Vβ或其片段包括Vβ8或其片段。
  8. 根据权利要求1-7中任一项所述的CAR，其中所述TCR可变区的片段包含TCR可变区的CDR3。
  9. 根据权利要求1-8中任一项所述的CAR，其中所述TCR的可变区Vα、Vβ和/或它们的片段包含SEQ ID NO：58-61中任一项所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的CAR，其中所述靶向部分包括抗体或其片段。
  11. 根据权利要求10所述的CAR，其中所述抗体与参比抗体竞争结合所述TCR的可变区Vα、Vβ和/或它们的片段，其中所述参比抗体包含轻链可变区和重链可变区，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO：3、SEQ ID NO：64和SEQ ID NO：21中任一项所示的氨基酸序列；所述LCDR2包含SEQ ID NO：4、SEQ ID NO：65和SEQ ID NO：22中任一项所示的氨基酸序列；所述LCDR3包含SEQ ID NO：5、SEQ ID NO：66和SEQ ID NO：23中任一项所示的氨基酸序列；且，
    所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO：11、SEQ ID NO：67和SEQ ID NO：29中任一项所示的氨基酸序列；所述HCDR2包含SEQ ID NO：12、SEQ ID NO：68和SEQ ID NO：30中任一项所示的氨基酸序列；所述HCDR3包含SEQ ID NO：13、SEQ ID NO：69和SEQ ID NO：31中任一项所示的氨基酸序列。
  12. 根据权利要求11所述的CAR，其中所述参比抗体的轻链可变区包含SEQ ID NO：9、SEQ ID NO：70和SEQ ID NO：27中任一项所示的氨基酸序列；且所述参比抗体的重链可变区包含SEQ ID NO：17、SEQ ID NO：71和SEQ ID NO：35中任一项所示的氨基酸序 列。
  13. 根据权利要求11-12中任一项所述的CAR，其中所述参比抗体包含Vα24Jα18抗体。
  14. 根据权利要求10-13中任一项所述的CAR，其中所述抗体包含重链互补决定区1(HCDR1)，所述HCDR1包含SEQ ID NO：11、SEQ ID NO：67和SEQ ID NO：29中任一项所示的氨基酸序列。
  15. 根据权利要求10-14中任一项所述的CAR，其中所述抗体还包含重链互补决定区2(HCDR2)，所述HCDR2包含SEQ ID NO：12、SEQ ID NO：68和SEQ ID NO：30中任一项所示的氨基酸序列。
  16. 根据权利要求10-15中任一项所述的CAR，其中所述抗体还包含重链互补决定区3(HCDR3)，所述HCDR3包含SEQ ID NO：13、SEQ ID NO：69和SEQ ID NO：31中任一项所示的氨基酸序列。
  17. 根据权利要求10-16中任一项所述的CAR，其中所述抗体包含轻链互补决定区1(LCDR1)，所述LCDR1包含SEQ ID NO：3、SEQ ID NO：64和SEQ ID NO：21中任一项所示的氨基酸序列。
  18. 根据权利要求10-17中任一项所述的CAR，其中所述抗体还包含轻链互补决定区2(LCDR2)，所述LCDR2包含SEQ ID NO：4、SEQ ID NO：65和SEQ ID NO：22中任一项所示的氨基酸序列。
  19. 根据权利要求10-18中任一项所述的CAR，其中所述抗体还包含轻链互补决定区3(LCDR3)，所述LCDR3包含SEQ ID NO：5、SEQ ID NO：66和SEQ ID NO：23中任一项所示的氨基酸序列。
  20. 根据权利要求10-19中任一项所述的CAR，其中所述抗体包含重链可变区，所述重链可变区包含SEQ ID NO：17、SEQ ID NO：71和SEQ ID NO：35中任一项所示的氨基酸序列。
  21. 根据权利要求10-20中任一项所述的CAR，其中所述抗体包含轻链可变区，所述轻链可变区包含SEQ ID NO：9、SEQ ID NO：70和SEQ ID NO：27中任一项所示的氨基酸序列。
  22. 根据权利要求10-21中任一项所述的CAR，其中所述抗体为单链抗体。
  23. 根据权利要求10-22中任一项所述的CAR，其中所述抗体包含SEQ ID NO：1、SEQ ID NO：62和SEQ ID NO：19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CAR，其中所述跨膜结构域包含源自选自下述蛋 白的多肽：CD8、CD28、CD137和/或CD3。
  25. 根据权利要求1-24中任一项所述的CAR，其中所述跨膜结构域包含SEQ ID NO：40所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的CAR，其中所述共刺激结构域包含源自选自下述蛋白的多肽：ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2和/或CD226。
  27. 根据权利要求1-26中任一项所述的CAR，其中所述共刺激结构域包含SEQ ID NO：42所示的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的CAR，其中所述胞内信号传导结构域包含来自CD3ζ的信号传导结构域。
  29. 根据权利要求1-28中任一项所述的CAR，其中所述胞内信号传导结构域包含SEQ ID NO：44所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的CAR，其中所述CAR还包含铰链区，所述铰链区连接所述抗体和所述跨膜结构域。
  31. 根据权利要求30所述的CAR，其中所述铰链区包含源自选自下述蛋白的多肽：CD8a和/或IgG。
  32. 根据权利要求30-31中任一项所述的CAR，其中所述铰链区包含SEQ ID NO：38所示的氨基酸序列。
  33. 根据权利要求1-32中任一项所述的CAR，其包含SEQ ID NO：46、SEQ ID NO：72和SEQ ID NO：50中任一项所示的氨基酸序列。
  34. 分离的核酸分子，其编码权利要求1-33中任一项所述的CAR。
  35. 编码CAR的分离的核酸分子，其包含SEQ ID NO：47、SEQ ID NO：73或SEQ ID NO：51所示的核酸序列。
  36. 载体，其包含权利要求34-35中任一项所述的核酸分子。
  37. 根据权利要求36所述的载体，其包括mRNA载体、质粒载体、逆转录病毒载体和/或慢病毒载体。
  38. 免疫效应细胞，其包含和/或能够表达权利要求1-33中任一项所述的CAR，权利要求34-35中任一项所述的核酸分子，或权利要求36-37中任一项所述的载体。
  39. 根据权利要求38所述的细胞，其中所述免疫效应细胞选自T淋巴细胞和自然杀伤(NK)细胞。
  40. 根据权利要求39所述的细胞，其中所述T淋巴细胞和/或自然杀伤细胞是天然来源，或者经干细胞诱导。
  41. 制备免疫效应细胞的方法，其包括向免疫效应细胞中引入权利要求36-37中任一项所述的载体。
  42. 组合物，其包含权利要求38-40中任一项所述的免疫效应细胞。
  43. 权利要求1-33中任一项所述的CAR，权利要求34-35中任一项所述的核酸分子，权利要求36-37中任一项所述的载体，或权利要求38-40中任一项所述的免疫效应细胞用于制备药物的用途，其中所述药物用于治疗T细胞淋巴瘤或白血病。
  44. 根据权利要求43所述的用途，其中所述T细胞淋巴瘤或白血病包含选自下组中的一种或多种：外周T细胞淋巴瘤、非特指型外周T细胞淋巴瘤、血管免疫母细胞淋巴瘤、结外NKT细胞淋巴瘤、间变性T细胞淋巴瘤、ALK阳性间变性细胞淋巴瘤、ALK阴性间变性细胞淋巴瘤、T淋巴母细胞淋巴瘤、细胞毒性T细胞淋巴瘤、皮肤T细胞淋巴瘤、成人T细胞白血病/淋巴瘤(ATLL)、T幼淋巴细胞白血病(T-PLL)、T大颗粒淋巴细胞(T-LGL)白血病、肝脾T细胞淋巴瘤(HSTL)、塞扎里综合征(SS)、皮下扁桃体样T细胞淋巴瘤和未分型T细胞淋巴瘤。
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