WO2022095802A1 - 靶向cd7的嵌合抗原受体及其用途 - Google Patents

靶向cd7的嵌合抗原受体及其用途 Download PDF

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WO2022095802A1
WO2022095802A1 PCT/CN2021/127567 CN2021127567W WO2022095802A1 WO 2022095802 A1 WO2022095802 A1 WO 2022095802A1 CN 2021127567 W CN2021127567 W CN 2021127567W WO 2022095802 A1 WO2022095802 A1 WO 2022095802A1
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cells
engineered immune
immune cell
seq
hla
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PCT/CN2021/127567
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English (en)
French (fr)
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周亚丽
姜小燕
陈功
任江涛
贺小宏
王延宾
韩露
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南京北恒生物科技有限公司
江苏浦珠生物医药科技有限公司
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Priority to JP2023526562A priority Critical patent/JP2023548844A/ja
Priority to EP21888499.7A priority patent/EP4219693A4/en
Priority to US18/034,177 priority patent/US20230390336A1/en
Priority to KR1020237014559A priority patent/KR20230095083A/ko
Publication of WO2022095802A1 publication Critical patent/WO2022095802A1/zh

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Definitions

  • the present invention belongs to the field of immunotherapy. More specifically, the present invention relates to chimeric antigen receptors targeting CD7 and their use in the treatment of diseases.
  • CAR-T chimeric antigen receptor T cell
  • CAR-T cells According to the source of T cells used to construct CAR-T cells, they can be divided into two categories: autologous CAR-T cells and allogeneic CAR-T cells (also known as universal CAR-T).
  • autologous CAR-T cells are that there is no risk of immune rejection, because the initial T cells come from the patients themselves who receive CAR-T therapy; but their disadvantages are also very obvious: high cost and long preparation cycle. Therefore, more and more researches focus on the development of general-purpose CAR-T cells.
  • Universal CAR-T can be prepared from T cells isolated from the peripheral blood of healthy donors, which greatly shortens the waiting time of patients for treatment. In addition, the viability and function of T cells obtained from healthy donors was also superior to that of patient-derived T cells, which could increase the infection rate of CAR cells and improve the therapeutic effect.
  • CD7 is a cell surface glycoprotein with a molecular weight of about 40 kDa, which belongs to the immunoglobulin superfamily. CD7 is expressed in most T cells, NK cells, myeloid cells, T cell acute lymphoblastic leukemia/lymphoma, acute myeloid leukemia and chronic myeloid leukemia. It has been reported that the CD7 molecule acts as a costimulatory signal during T cell activation through binding to its ligand K12/SECTM1. Furthermore, disruption of the CD7 molecule in mouse T progenitor cells still resulted in normal T cell development and homeostasis, suggesting that CD7 does not appear to have a critical impact on T cell development and function, making it a promising candidate for the treatment of T cell acuteness. Very suitable therapeutic target for lymphocytic leukemia (T-ALL). Indeed, CD7 has been extensively studied as a target for cytotoxic molecules for the treatment of leukemias and lymphomas.
  • T-ALL lymphocytic leukemia
  • the present invention aims to provide an efficient universal CAR-T cell targeting CD7 and its use in the treatment of diseases.
  • the present invention provides an engineered immune cell, characterized by: (1) expressing a chimeric antigen receptor comprising an antigen-binding region, the antigen-binding region comprising an anti-CD7 antibody; (2) endogenous The expression of sex CD7, at least one TCR/CD3 gene, and at least one MHC class II-related gene is suppressed or silenced.
  • the chimeric antigen receptor comprises an anti-CD7 antibody, a transmembrane domain and an intracellular signaling domain.
  • the anti-CD7 antibody comprises CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 1, 2 and 3, and CDR-L3 as set forth in SEQ ID NOs: 4, 5 and 6, respectively CDR-H1, CDR-H2 and CDR-H3 shown.
  • the anti-CD7 antibody comprises a light chain variable region having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 10, 13, 16 and 19 and an Heavy chain variable regions having at least 90% identity from the amino acid sequences set forth in SEQ ID NOs: 8, 11, 14, 17 and 20.
  • the amino acid sequence of the anti-CD7 antibody is selected from the group consisting of SEQ ID NOs: 9, 12, 15, 18 and 21.
  • the antigen binding region of the chimeric antigen receptor further comprises an antibody targeting a second antigen selected from the group consisting of: TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171 , CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, CD20, AFP, Folate receptor ⁇ , ERBB2(Her2/neu), MUC1, EGFR, CS1, CD138 , NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2,
  • the antibody targeting the second antigen is an antibody targeting CD19.
  • the anti-CD19 antibody comprises a light chain variable region having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 52 and 55 and a variable region to a light chain selected from the group consisting of SEQ ID NO: 53 A heavy chain variable region having at least 90% identity to the amino acid sequence shown in 56.
  • the amino acid sequence of the anti-CD19 antibody is selected from the group consisting of SEQ ID NOs: 54 and 57.
  • the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain is selected from the transmembrane domains of CD8 ⁇ , CD4 and CD28.
  • the intracellular signaling domain is selected from the intracellular regions of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling domain comprises the CD3 ⁇ intracellular domain.
  • the chimeric antigen receptor further comprises one or more costimulatory domains selected from the intracellular regions of the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18(LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD270(HVEM) , CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof.
  • the costimulatory domain is the intracellular region of CD27, CD28, CD134, CD137 or CD278 or a combination thereof.
  • the TCR/CD3 gene is selected from the group consisting of TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and combinations thereof.
  • the MHC-II-related genes are selected from the group consisting of: HLA-DPA, HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK, CIITA and combinations thereof, preferably RFX5, RFXAP, RFXANK, CIITA and their combinations.
  • the engineered immune cells of the present invention comprise endogenous CD7, at least one TCR/CD3 gene selected from TRAC and TRBC, and at least one selected from RFX5, RFXAP, RFXANK and CIITA
  • the expression of MHC class II genes is suppressed or silenced.
  • the engineered immune cells of the present invention comprise endogenous CD7, at least one TCR/CD3 gene selected from TRAC and TRBC, and the expression of RFX5 is inhibited or silenced.
  • the engineered immune cells further express a NK inhibitory molecule comprising one or more NK inhibitory ligands, a transmembrane domain, and a costimulatory domain.
  • the NK inhibitory ligand is an antibody targeting a NK inhibitory receptor selected from the group consisting of NKG2/CD94 components (eg, NKG2A, NKG2B, CD94); killer cell Ig like receptor (KIR) family members (e.g. KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3); leukocyte Ig-like receptor (LIR) family members (e.g.
  • KLRG1 killer lectin-like receptor G1
  • the NK inhibitory receptor is preferably selected from NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM1, LAIR1, NKR- P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4 and KLRG1.
  • the NK inhibitory receptor is selected from the group consisting of NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, LAIR1 and KLRG1. Still more preferably, the NK inhibitory receptor is selected from the group consisting of NKG2A, NKG2B, LIR1, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, LAIR1 and KLRG1.
  • the NK inhibitory ligand is a natural ligand for an NK inhibitory receptor or an NK inhibitory receptor binding region it comprises.
  • the NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, immune checkpoint ligands (eg PD-L1/PD-L2 , CTLA4, CD155, CD112, CD113, Gal-9, FGL1, etc.), and the NK inhibitory receptor binding domains they contain.
  • the NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PD-L1/PD-L2, CTLA-4, CD155 , CD112, CD113, Gal-9, FGL1, or the NK inhibitory receptor binding region they comprise; more preferably selected from HLA-E, HLA-F, HLA-G, cadherin, PD-L1, PD-L2 , or the NK inhibitory receptor binding domains they contain.
  • the NK inhibitory ligand is selected from the group consisting of HLA-E extracellular domain, HLA-G extracellular domain, E-cadherin extracellular domain, PD-L1 extracellular domain and PD-L2 extracellular region.
  • the NK inhibitory ligand is the E-cadherin extracellular domain comprising EC1 and EC2, more preferably EC1, EC2, EC3, EC4 and EC5.
  • the NK inhibitory ligand is the extracellular domain of PD-L1 or PD-L2.
  • the NK inhibitory ligand is the extracellular domain of HLA-E.
  • the NK inhibitory ligand is the HLA-G extracellular domain.
  • the NK inhibitory molecule further comprises the CD3 ⁇ intracellular domain as an intracellular signaling domain.
  • the engineered immune cells are T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells.
  • the engineered immune cells are T cells, such as CD4+/CD8+ T cells, CD4+ helper T cells (eg Th1 and Th2 cells), CD8+ T cells (eg, cytotoxic T cells), tumor infiltrating cells, Memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the engineered immune cells of the present invention as an active agent, and a plurality of pharmaceutically acceptable excipients.
  • the present invention also provides a method of treating a subject with a disease associated with CD7 expression, comprising administering to the subject an effective amount of an engineered immune cell or a pharmaceutical composition according to the present invention. Accordingly, the present invention also encompasses the use of engineered immune cells in the manufacture of a medicament for the treatment of diseases associated with CD7 expression.
  • the present invention provides an engineered immune cell, characterized by: (1) expressing a chimeric antigen receptor comprising an antigen-binding region, the antigen-binding region comprising an anti-CD7 antibody; (2) endogenous The expression of sex CD7, at least one TCR/CD3 gene, and at least one MHC class II-related gene is suppressed or silenced.
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide that generally includes an antigen-binding region (eg, an antibody or antigen-binding portion thereof), a transmembrane domain, any Selected co-stimulatory domains and intracellular signaling domains, each of which is connected by a linker.
  • CARs can exploit the antigen-binding properties of antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets in a non-MHC-restricted manner.
  • Non-MHC-restricted antigen recognition confers CAR-expressing immune cells the ability to recognize antigen independent of antigen processing, thus bypassing the primary mechanism of tumor escape.
  • the CAR expressed by the engineered immune cells provided by the present invention comprises an antibody targeting CD7 or an antigen-binding fragment thereof, a transmembrane domain and an intracellular signaling domain.
  • antibody has the broadest meaning as understood by those skilled in the art, and includes monoclonal antibodies (including whole antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (eg, bispecific antibodies) ), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences capable of exhibiting the desired biological activity.
  • the antibodies of the invention can be of any class (eg, IgG, IgE, IgM, IgD, IgA, etc.) or subclass (eg, IgGl, IgG2, IgG2a, IgG3, IgG4, IgAl, IgA2, etc.).
  • Antibodies of the present invention also include recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, chimeric antibodies, and antigen-binding portions thereof.
  • antibody fragment or "antigen-binding portion” refers to a portion of an intact antibody, typically comprising the antigen-binding site of the intact antibody and thus retaining the ability to bind antigen.
  • antibody fragments in the present invention include, but are not limited to: Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, scFv, disulfide-linked Fv (sdFv), heavy Chain variable region (VH) or light chain variable region (VL), linear antibodies, "diabodies” with two antigen binding sites, single domain antibodies, nanobodies, natural ligands for said antigen or functional fragments, etc. Accordingly, an “antibody” of the present invention encompasses an antibody fragment or antigen-binding portion of an antibody as defined above.
  • the anti-CD7 antibody of the invention is an anti-CD7 scFv.
  • Single-chain antibody and “scFv” are used interchangeably herein and refer to an antibody comprising an antibody heavy chain variable region (VH) and light chain variable region (VL) linked by a linker.
  • the optimal length and/or amino acid composition of the linker can be determined as desired.
  • the length of the linker significantly affects the variable region folding and interaction of scFv. In fact, intrachain folding can be prevented if shorter linkers are used (eg between 5-10 amino acids).
  • linker size and composition see, eg, Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A.
  • the scFv can comprise VH and VL linked in any order, eg, VH-linker-VL or VL-linker-VH.
  • the CAR of the invention comprises an antibody targeting CD7 comprising CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 1, 2, and 3, respectively, and as set forth in SEQ ID NO: 1, 2, and 3, respectively CDR-H1, CDR-H2 and CDR-H3 shown in NO: 4, 5 and 6.
  • the CD7-targeting antibody of the present invention comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95% amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 10, 13, 16 and 19.
  • the chimeric antigen receptor of the present invention comprises an anti-CD7 antibody whose amino acid sequence is shown in SEQ ID NO: 9, 12, 15, 18 or 21.
  • the antigen binding region included in the chimeric antigen receptor of the present invention also includes an antibody targeting a second antigen selected from the group consisting of: TSHR, CD19, CD123 , CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, CD20, AFP, Folate receptor ⁇ , ERBB2 (Her2/ neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin
  • the tumor antigen is selected from CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUCl, AFP, Folate receptor alpha, CEA, PSCA, PSMA, Her2, EGFR, IL13Ra2, GD2, NKG2D, EGFRvIII, CS1, BCMA, mesothelin and any combination thereof.
  • Antibodies known in the art targeting the above tumor antigens can be used in the present invention.
  • the antibody targeting the second antigen is an antibody targeting CD19 comprising at least 70%, preferably at least 80%, more preferably the amino acid sequence shown in SEQ ID NO: 52 or 55
  • a light chain variable region sequence of at least 90%, 95%, 97%, 99% or 100% sequence identity Preferably a light chain variable region sequence of at least 90%, 95%, 97%, 99% or 100% sequence identity and at least 70%, preferably at least 80%, with the amino acid sequence shown in SEQ ID NO: 53 or 56, More preferred are heavy chain variable region sequences of at least 90%, 95%, 97%, 99% or 100% sequence identity.
  • the chimeric antigen receptor of the present invention comprises an antibody targeting CD7 and an antibody targeting CD19, and the amino acid sequence of the antibody targeting CD19 is shown in SEQ ID NO: 54 or 57.
  • the term "functional variant” or “functional fragment” refers to a variant that substantially comprises the amino acid sequence of a parent but contains at least one amino acid modification (ie, substitution, deletion or insertion) compared to the parent amino acid sequence, provided that all The variants retain the biological activity of the parent amino acid sequence.
  • the amino acid modification is preferably a conservative modification.
  • conservative modification refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced by amino acid residues having similar side chains.
  • Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) ), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine) acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • threonine valine, isoleucine
  • aromatic side chains eg tyrosine, phenylalanine, tryptophan, histidine.
  • Conservative modifications can be selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • a “functional variant” or “functional fragment” is at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% of the parent amino acid sequence %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, And retain the biological activity of the parent amino acid, such as binding activity.
  • sequence identity refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies with the exact same sequence are 100% identical.
  • sequence identity can be determined using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197) and ClustalW.
  • transmembrane domain refers to a polypeptide that enables expression of a chimeric antigen receptor on the surface of immune cells (eg, lymphocytes, NK cells, or NKT cells) and directs the cellular response of the immune cells against target cells structure.
  • immune cells eg, lymphocytes, NK cells, or NKT cells
  • the transmembrane domain can be natural or synthetic, and can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain is capable of signaling when the chimeric antigen receptor binds to the target antigen.
  • Transmembrane domains particularly useful in the present invention may be derived from, for example, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and their functional fragments.
  • the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from the CD8 alpha chain or CD28, which is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% of the amino acid sequence shown in SEQ ID NO: 22 or 24 % or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptors of the present invention may further comprise a hinge region between the antigen binding region and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the antigen binding region.
  • the hinge region serves to provide greater flexibility and accessibility to the antigen binding region.
  • the hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the hinge region can be derived in whole or in part from a native molecule, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.
  • the hinge region comprises a hinge region portion of a CD8 ⁇ chain, CD28, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably a hinge from CD8 ⁇ , CD28 or IgG4, which is the same as SEQ ID NO: 38, 40 or
  • the amino acid sequence shown in 42 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence and SEQ ID NO: 39, 41
  • the nucleotide sequence shown in or 43 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs cells to perform specified functions.
  • the intracellular signaling domain is responsible for primary signaling in cells following antigen binding at the antigen binding region, resulting in the activation of immune cells and immune responses.
  • the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed.
  • the effector function of T cells can be cytolytic activity or helper activity, including secretion of cytokines.
  • the chimeric antigen receptors of the invention comprise intracellular signaling domains that may be cytoplasmic sequences of T cell receptors and co-receptors that act together upon antigen receptor binding to initiate primary signaling conduction, as well as any derivatives or variants of these sequences and any synthetic sequences that have the same or similar function.
  • the intracellular signaling domain can contain a number of immunoreceptor tyrosine-based activation motifs (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motifs
  • intracellular signaling domains of the invention include, but are not limited to, the intracellular regions of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the signaling domain of the CAR of the present invention may comprise a CD3 ⁇ intracellular region which is at least 70%, preferably at least 80%, more preferably at least 90% identical to the amino acid sequence shown in SEQ ID NO: 30 or 32 %, 95%, 97% or 99% or 100% sequence identity, or its coding sequence is at least 70%, preferably at least 80%, more preferably at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 31 or 33 90%, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptors of the invention further comprise one or more costimulatory domains.
  • a costimulatory domain may be an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof.
  • a "costimulatory molecule” refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (eg, proliferation) of the T cell.
  • Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA and Toll ligand receptors.
  • Non-limiting examples of costimulatory domains of the invention include, but are not limited to, the intracellular regions of the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18(LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM1), CD83, CD134(OX40), CD137(4-1BB), CD270(HVEM), CD272(BTLA), CD276(B7- H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, CD94, LTB and ZAP70 and combinations thereof.
  • the costimulatory domain comprises one or more intracellular domains selected from the group consisting of DAP10, DAP12, CD27, CD28, CD134, 4-1BB or CD278.
  • the costimulatory domain comprises the intracellular region of 4-1BB.
  • the costimulatory domain comprises the intracellular region of CD28.
  • the costimulatory domain comprises the intracellular domain of 4-1BB and the intracellular domain of CD28.
  • the intracellular region of 4-1BB is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence set forth in SEQ ID NO:28 % sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% with the nucleotide sequence shown in SEQ ID NO: 29 sequence identity.
  • the intracellular region of CD28 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence set forth in SEQ ID NO:26
  • the sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence with the nucleotide sequence shown in SEQ ID NO: 27 identity.
  • the CAR of the present invention may further comprise a signal peptide such that when it is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface.
  • the core of the signal peptide may contain a long segment of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • signal peptidases At the end of the signal peptide, there is usually a segment of amino acids that is recognized and cleaved by signal peptidases.
  • the signal peptidase can cleave during or after translocation to generate the free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases.
  • Signal peptides useful in the present invention are well known to those skilled in the art, eg, signal peptides derived from CD8 ⁇ , IgG1, GM-CSFR ⁇ , B2M, and the like.
  • the signal peptide useful in the present invention is from B2M or CD8 ⁇ , which is at least 70%, preferably at least 80%, more preferably at least 90%, 95% of the amino acid sequence shown in SEQ ID NO: 34 or 36 , 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95% with the nucleotide sequence shown in SEQ ID NO: 35 or 37 %, 97% or 99% or 100% sequence identity.
  • the CAR of the present invention may further comprise a switch structure to regulate the expression time of the CAR.
  • the switch structure can be in the form of a dimerization domain that, upon binding to its corresponding ligand, induces a conformational change, exposing the extracellular antigen-binding region for binding to the targeted antigen, thereby activating a signaling pathway.
  • switch structures can be used to connect the antigen-binding and signaling domains, respectively, and the antigen-binding and signaling domains can be dimerized together only when the switch structures bind to each other (eg, in the presence of an inducing compound). together, thereby activating the signaling pathway.
  • the switch structure can also be in the form of a masked peptide.
  • the masking peptide can mask the extracellular antigen-binding region and prevent it from binding to the targeted antigen.
  • the masking peptide is cleaved by, for example, protease, the extracellular antigen-binding region can be exposed, making it a "common" CAR structure .
  • Various switch structures known to those skilled in the art can be used in the present invention.
  • the CAR of the present invention may further comprise a suicide gene, i.e., causing it to express a cell death signal that can be induced by a foreign substance, to clear the CAR cells when needed (eg, when severe toxic side effects occur).
  • suicide genes can be in the form of inserted epitopes, such as CD20 epitopes, RQR8, etc., and when desired, CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes.
  • the suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which causes cell death induced by ganciclovir treatment.
  • HSV-TK herpes simplex virus thymidine kinase
  • the suicide gene can also be iCaspase-9, which can be induced to dimerize by chemical inducing drugs such as AP1903, AP20187, etc., thereby activating the downstream Caspase3 molecule, leading to apoptosis.
  • chemical inducing drugs such as AP1903, AP20187, etc.
  • Various suicide genes known to those skilled in the art can be used in the present invention.
  • the expression of endogenous CD7, at least one TCR/CD3 gene and at least one MHC class II-related gene of the engineered immune cells provided by the present invention is inhibited or silenced.
  • CD7 is not only expressed in tumor cells such as T cell acute lymphoblastic leukemia/lymphoma, acute myeloid leukemia and chronic myeloid leukemia, but also in most normal T cells and NK cells. Therefore, in order to avoid mutual recognition and killing between CD7-targeting CAR cells, it is necessary to suppress or silence the endogenous CD7 gene expression of CAR cells.
  • T cell receptor is a characteristic marker on the surface of all T cells. It binds to CD3 in a non-covalent bond to form a TCR/CD3 complex, and presents a specific MHC-antigen on the cell surface through a non-covalent bond.
  • the peptide complexes bind to generate specific antigen-stimulating signals, activate T cells, and play a killing role. Therefore, inhibiting or silencing the expression of endogenous TCR/CD3 genes in CAR-T cells can avoid their attack on normal cells or tissues in patients, thereby avoiding or reducing the risk of graft-versus-host disease (GvHD).
  • TCR is a heterodimer composed of two different peptide chains, usually divided into two categories: ⁇ / ⁇ type and ⁇ / ⁇ type, among which more than 95% of peripheral T lymphocytes express TCR ⁇ / ⁇ .
  • the TCR ⁇ chain is encoded by the TRAC gene and the ⁇ chain is encoded by the TRBC gene.
  • Each peptide chain of TCR includes a variable region (V region), a constant region (C region), a transmembrane region and a cytoplasmic region, wherein the cytoplasmic region is very short and does not have the ability to transmit antigen-stimulating signals.
  • the TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region has three hypervariable regions, CDR1, CDR2, and CDR3. Among them, CDR3 has the greatest variation, which directly determines the antigen-binding specificity of TCR.
  • CDR1 and CDR2 recognize and bind to MHC molecules, while CDR3 directly binds to the antigen peptide.
  • CD3 includes four subunits: ⁇ , ⁇ , ⁇ , and ⁇ , which usually exist in the form of dimers ⁇ , ⁇ , and ⁇ .
  • TCR/CD3 gene is selected from the group consisting of: TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ .
  • CD4+CD7-T cells By detecting the composition of peripheral blood lymphocytes in patients, the inventors found that CD4+CD7-T cells accounted for about 20% of the total CD3+T cells (Fig. 1). Since CAR cells targeting CD7 cannot recognize the CD4+CD7-T population, they cannot effectively kill this population. Instead, CD4+CD7-T cells will kill exogenous CAR cells by recognizing MHC-II molecules. Therefore, in order to avoid the killing of exogenous CAR-T cells by CD4+CD7-T cells in patients, the inventors considered inhibiting or silencing the expression of endogenous MHC-II-related genes in CAR-T cells.
  • MHC-II related genes include MHC-II genes themselves, as well as genes that interact with MHC-II genes or regulate their expression.
  • MHC The major histocompatibility complex
  • H-2 The major histocompatibility complex
  • HLA The major histocompatibility complex
  • Class I MHC proteins are heterodimers of two proteins: a transmembrane protein alpha chain encoded by the MHC I gene, and a beta2 microprotein of an extracellular protein encoded by genes not located within the MHC gene cluster globulin chain.
  • the alpha chain consists of three domains, and the foreign peptide binds to two domains, alpha1, alpha2, which are also the most variable at the N-terminus.
  • Class II MHC proteins are also heterodimers, comprising two transmembrane proteins encoded by genes within the MHC complex.
  • Class I MHC/antigen complexes interact with cytotoxic T cells (eg, CD8+ T cells), while class II MHCs present antigens to helper T cells (eg, CD4+ T cells).
  • class I MHC proteins tend to be expressed in nearly all nucleated cells and platelets (and red blood cells in mice), while class II MHC proteins are expressed more selectively.
  • MHC class II proteins are expressed on B cells, some macrophages and monocytes, activated T cells, Langerhans cells, and dendritic cells.
  • the human class II HLA cluster contains three major loci, DP, DQ and DR.
  • Class II molecules are heterodimers consisting of alpha and beta chains, both anchored in the membrane, where the alpha chain is approximately 33-35 kDa and contains 5 exons.
  • Exon 1 encodes the leader peptide
  • exons 2 and 3 encode the two extracellular domains
  • exon 4 encodes the transmembrane domain
  • exon 5 encodes the cytoplasmic tail.
  • the MHC class II associated genes are selected from the group consisting of: HLA-DPA, HLA-DQ and HLA-DRA.
  • MHC-II genes also depends on a variety of important positive regulatory proteins, such as RFX complex, CIITA and so on.
  • the RFX complex consists of three subunits: RFXANK (also known as RFXB), RFX5 and RFX accessory protein (also known as RFXAP).
  • RFXANK also known as RFXB
  • RFX5 also known as RFX5
  • RFXAP RFX accessory protein
  • the RFX complex promotes the expression of MHC class II molecules by facilitating the binding of other transcription factors to the promoters of MHC class II molecules and enhancing the specificity of promoter binding.
  • CIITA is the master controller of MHC class II expression.
  • CIITA includes an N-terminal rich in acidic amino acids, a PST region rich in Pro, Ser, and Thr, a GTP-binding region in the middle, and a C-terminal rich in Leu repeats (LRR), where the N-terminal acidic region and the PST region are transcriptional activators area.
  • the MHC class II associated genes are selected from the group consisting of: RFX5, RFXAP, RFXANK and CIITA.
  • the MHC class II associated genes are selected from the group consisting of: HLA-DPA, HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK and CIITA, preferably selected from RFX5, RFXAP, RFXANK and CIITA, more preferably RFX5.
  • the endogenous MHC class I genes eg, HLA-A, HLA-B, HLA-C, B2M, etc.
  • the expression of endogenous MHC class I genes in the CAR-T cells is also inhibited or silenced.
  • the engineered immune cells of the present invention expressing a CD7-targeting chimeric antigen receptor include endogenous CD7, at least one TCR/CD3 gene, and at least one MHC-class II-related gene. Expression is suppressed or silenced, wherein the at least one TCR/CD3 gene is selected from TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and combinations thereof; the at least one MHC-II-related gene is selected from HLA-DPA , HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK and CIITA.
  • the engineered immune cells expressing a chimeric antigen receptor targeting CD7 comprise endogenous CD7, at least one TCR/CD3 gene selected from TRAC and TRBC, and a gene selected from The expression of at least one MHC class II gene of RFX5, RFXAP, RFXANK and CIITA is inhibited or silenced. More preferably, the engineered immune cells of the present invention expressing a chimeric antigen receptor targeting CD7 include endogenous CD7, at least one TCR/CD3 gene selected from TRAC and TRBC, and the expression of RFX5 is inhibited or silence. In one embodiment, the expression of MHC class I genes, eg, HLA-A, HLA-B, HLA-C, and B2M, in the engineered immune cells is functional.
  • MHC class I genes eg, HLA-A, HLA-B, HLA-C, and B2M
  • the engineered immune cells of the present invention may comprise at least one gene selected from the group consisting of: CD52, GR, dCK and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2,
  • Methods of inhibiting gene expression or silencing genes include, but are not limited to, DNA fragmentation mediated by, for example, meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system, or by Antisense oligonucleotides, RNAi, shRNA and other technologies inactivate genes.
  • the inventors also found that there are a large proportion (about 20%) of CD7-NK cells in patients (Fig. 1), which cannot be targeted and killed by CD7CAR. Moreover, under the condition that MHC-II gene expression is inhibited or silenced, these cells may kill CAR cells.
  • the engineered immune cells in order to inhibit the killing of CAR-T cells by NK cells in the patient, the engineered immune cells further express NK inhibitory molecules comprising one or more NK inhibitory ligands. body, transmembrane domain, and costimulatory domain.
  • the NK inhibitory molecule comprises one or two NK inhibitory ligands, a transmembrane domain and a costimulatory domain.
  • transmembrane domain and the costimulatory domain included in the NK inhibitory molecule are the same as the definitions of the transmembrane domain and the costimulatory domain included in the above-mentioned chimeric antigen receptor.
  • the NK inhibitory ligand is an antibody targeting an NK inhibitory receptor selected from the group consisting of NKG2/CD94 components (eg, NKG2A, NKG2B, CD94); killer cell Ig like receptor (KIR) family members (e.g. KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3); leukocyte Ig-like receptor (LIR) family members (e.g.
  • KLRG1 killer lectin-like receptor G1
  • the NK inhibitory receptor is preferably selected from NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM1, LAIR1, NKR- P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4 and KLRG1.
  • the NK inhibitory receptor is selected from the group consisting of NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, LAIR1 and KLRG1. Still more preferably, the NK inhibitory receptor is selected from the group consisting of NKG2A, NKG2B, LIR1, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, LAIR1 and KLRG1.
  • the NK inhibitory ligand is an antibody targeting an NK inhibitory receptor, the antibody is an intact antibody, Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, linear antibody , sdAb or Nanobody.
  • the NK inhibitory ligand is an antibody targeting NKG2A.
  • the antibody targeting NKG2A comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% of the amino acid sequence shown in SEQ ID NO: 59, 62 or 77 % or 100% sequence identity to the light chain variable region sequence and the amino acid sequence shown in SEQ ID NO: 58, 61 or 76 having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% %, 99% or 100% sequence identity of heavy chain variable region sequences. More preferably, the amino acid sequence of the antibody targeting NKG2A is shown in SEQ ID NO: 60, 63 or 78.
  • NKG2A-targeting antibodies known in the art can also be used in the present invention, such as Z270 (available from Immunotech, France), Z199 (available from Beckman Coulter, USA), 20D5 (available from BD Biosciences Pharmingen, USA) , P25 (available from Morettaetal, Univ. Genova, Italy) and the like.
  • the NK inhibitory ligand is an antibody targeting KIRs, eg, KIR2DL1, KIR2DL2/3 and KIR3DL1. More preferably, the antibody targeting KIR comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or A light chain variable region sequence with 100% sequence identity and at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% with the amino acid sequence shown in SEQ ID NO: 66 or 80 or heavy chain variable region sequences with 100% sequence identity. More preferably, the amino acid sequence of the KIR-targeting antibody is shown in SEQ ID NO: 67 or 81.
  • KIR-targeting antibodies known in the art can also be used in the present invention, such as GL183 (targeting KIR2DL2/L3, available from Immunotech, France and Beckton Dickinson, USA), EB6 (targeting KIR2DL1, available from Immunotech, France and Beckton Dickinson, USA), AZ138 (targets KIR3DL1, available from Morettaetal, Univ. Genova, Italy), Q66 (targets KIR3DL2, available from Immunotech, France), Z27 (targets KIR3DL1, available from Immunotech) , France and Beckton Dickinson, USA) et al.
  • GL183 targeting KIR2DL2/L3, available from Immunotech, France and Beckton Dickinson, USA
  • EB6 targeting KIR2DL1, available from Immunotech, France and Beckton Dickinson, USA
  • AZ138 targets KIR3DL1, available from Morettaetal, Univ. Genova, Italy
  • Q66 targets KIR3DL2, available from Immuno
  • the NK inhibitory ligand is an antibody targeting LIR1. More preferably, the antibody targeting LIR1 comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity of light chain variable region sequence and amino acid sequence shown in SEQ ID NO: 69 or 72 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or heavy chain variable region sequences with 100% sequence identity. More preferably, the amino acid sequence of the antibody targeting LIR1 is shown in SEQ ID NO: 70 or 73.
  • the NK inhibitory ligand is a natural ligand for an NK inhibitory receptor or an NK inhibitory receptor binding region it comprises.
  • the NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, Cadherin, collagen, OCIL, sialic acid, immune checkpoint ligands (eg PD-L1/ PD-L2, CD155, CD112, CD113, Gal-9, FGL1, etc.), and the NK inhibitory receptor binding domains they contain.
  • the NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PD-L1/PD-L2, CTLA-4, CD155 , CD112, CD113, Gal-9, FGL1, or the NK inhibitory receptor binding regions they contain; more preferably selected from HLA-E, HLA-F, HLA-G, cadherin, or the NK inhibitory regions they contain receptor binding region.
  • the NK inhibitory ligand is selected from the group consisting of HLA-E extracellular domain, HLA-G extracellular domain, E-Cadherin (E-Cadherin) extracellular domain, PD-L1 extracellular domain and PD-L1 extracellular domain. L2 extracellular region.
  • the NK inhibitory ligand is the E-cadherin extracellular domain comprising domains EC1 and EC2, more preferably domains EC1, EC2, EC3, EC4 and EC5.
  • the E-cadherin extracellular region is the same as the one set forth in SEQ ID NO: 44 (comprising domains EC1 and EC2) or SEQ ID NO: 48 (comprising domains EC1, EC2, EC3, EC4 and EC5)
  • the amino acid sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity.
  • the NK inhibitory ligand is the extracellular domain of PD-L1 or PD-L2.
  • the PD-L1 extracellular region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence with the amino acid sequence shown in SEQ ID NO: 45 Identity
  • the PD-L2 extracellular region and the amino acid sequence shown in SEQ ID NO: 46 have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity.
  • the NK inhibitory ligand is the HLA-E extracellular domain or the HLA-G extracellular domain.
  • the HLA-E extracellular region is at least 70%, preferably at least 80% identical to the amino acid sequence shown in SEQ ID NO: 50 (wild type) or SEQ ID NO: 51 (mutant comprising the Y84C mutation), More preferably at least 90%, 95%, 97%, 99% or 100% sequence identity
  • the HLA-G extracellular region has at least 70%, preferably at least 80%, with the amino acid sequence shown in SEQ ID NO: 49, More preferably at least 90%, 95%, 97%, 99% or 100% sequence identity.
  • non-classical HLA-class I molecules eg HLA-E, HLA-G
  • B2M need to be knocked out
  • non-canonical HLA-I molecules to normally exert their inhibitory function
  • B2M with DNA sequence synonymous mutation it is necessary to introduce B2M with DNA sequence synonymous mutation to avoid being knocked out by gene editing tools.
  • the NK inhibitory ligand is a fusion molecule of B2M with either the HLA-E extracellular domain or the HLA-G extracellular domain.
  • the NK inhibitory molecule comprises at least two NK inhibitory ligands selected from the group consisting of anti-NKG2A scFv, anti-KIR scFv, anti-LIR1 scFv, HLA-E extracellular domain , HLA-G extracellular domain, E-cadherin extracellular domain, PD-L1 extracellular domain and PD-L2 extracellular domain, more preferably PD-L1 extracellular domain and HLA-E extracellular domain.
  • the NK inhibitory molecule further comprises an intracellular signaling domain.
  • the intracellular signaling domain is selected from the intracellular regions of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling domain comprises the intracellular region of CD3 ⁇ .
  • the NK inhibitory molecule may further comprise a signal peptide, eg, a signal peptide from PDL1 or B2M.
  • the NK inhibitory molecule comprises a PD-L1 signal peptide having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity.
  • Those skilled in the art can also select other suitable signal peptides as needed.
  • the present invention also provides an engineered immune cell, characterized by: (1) expressing a chimeric antigen receptor comprising an antigen binding region comprising an anti-CD7 antibody and an optional second antigen region; (2) ) expression of endogenous CD7, at least one TCR/CD3 gene, and at least one MHC class II-related gene is inhibited or silenced.
  • the engineered immune cells of the invention further express a NK inhibitory molecule comprising one or more NK inhibitory ligands, a transmembrane domain, and a costimulatory domain.
  • the term "immune cell” refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • the immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, or derived from stem cells such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, Immune cells such as bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells.
  • the immune cells are T cells.
  • the T cells can be any T cells, such as T cells cultured in vitro, eg, primary T cells, or T cells from T cell lines cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified.
  • T cells can be at any stage of development, including, but not limited to, CD4+/CD8+ T cells, CD4+ helper T cells (eg, Th1 and Th2 cells), CD8+ T cells (eg, cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • the immune cells are human T cells.
  • T cells can be obtained by separating the blood of a subject using a variety of techniques known to those of skill in the art, such as Ficoll.
  • immune cells are engineered to express chimeric antigen receptors and to inhibit or silence the expression of endogenous CD7, at least one TCR/CD3 gene, and at least one MHC class II-related gene.
  • Nucleic acid sequences encoding chimeric antigen receptor polypeptides and, optionally, NK inhibitory molecules can be introduced into immune cells using conventional methods known in the art (eg, by transduction, transfection, transformation, etc.).
  • Transfection is the process of introducing a nucleic acid molecule or polynucleotide, including a vector, into a target cell.
  • An example is RNA transfection, the process of introducing RNA (eg, in vitro transcribed RNA, ivtRNA) into a host cell.
  • the term is mainly used for non-viral methods in eukaryotic cells.
  • transduction is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides.
  • Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of material.
  • Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to create liposomes that fuse with cell membranes and deposit their cargo inside.
  • Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation.
  • transformation is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells).
  • transformation is the genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by the direct uptake of the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Conversion can be achieved by manual means.
  • the cells or bacteria must be in a competent state.
  • techniques can include heat shock-mediated uptake, fusion of bacterial protoplasts with intact cells, microinjection, and electroporation.
  • nucleic acid or vector After the nucleic acid or vector is introduced into immune cells, those skilled in the art can expand and activate the resulting immune cells by conventional techniques.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the engineered immune cells of the present invention as an active agent, and a variety of pharmaceutically acceptable excipients.
  • the term "pharmaceutically acceptable excipient” means pharmacologically and/or physiologically compatible with the subject and the active ingredient (ie, capable of eliciting the desired therapeutic effect without causing any inconvenience desired local or systemic effect) carriers and/or excipients, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, antiadherents, glidants, antioxidants, flavoring agents, colorants, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity modifiers . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention.
  • excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose and water.
  • suitable excipients depends, among other things, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
  • compositions according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally.
  • Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
  • compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular ointments, creams, transdermal patches, gels, powders, tablets, solutions, gaseous In the form of aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts, or in a form particularly suitable for the desired method of administration.
  • Processes known in the present invention for the manufacture of pharmaceuticals may include, for example, conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Pharmaceutical compositions comprising immune cells such as those described herein are typically provided in solution and preferably comprise a pharmaceutically acceptable buffer.
  • compositions according to the present invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated.
  • agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetate glucuronate, auristatin E, vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, act
  • the present invention also provides a method of treating a subject suffering from a disease associated with CD7 expression, comprising administering to the subject an effective amount of an immune cell or a pharmaceutical composition according to the present invention. Therefore, the present invention also covers the use of the engineered immune cells and the pharmaceutical composition in the manufacture of a medicament for treating diseases associated with CD7 expression.
  • an effective amount of an immune cell and/or pharmaceutical composition of the invention is administered directly to a subject.
  • the method of treatment of the present invention is an ex vivo treatment.
  • the method comprises the steps of: (a) providing a sample comprising immune cells; (b) introducing the chimeric antigen receptor of the present invention and an exogenous gene (eg, NK inhibitory molecule) into the body in vitro said immune cells, and inhibit or silence the expression of specific genes in said immune cells (such as endogenous CD7, TCR/CD3 genes and MHC-II related genes) to obtain modified immune cells, (c) to have this The modified immune cells are administered to a subject in need thereof.
  • an exogenous gene eg, NK inhibitory molecule
  • the immune cells provided in step (a) are selected from macrophages, dendritic cells, monocytes, T cells, NK cells and/or NKT cells; Conventional methods are obtained from a subject's sample, particularly a blood sample. However, other immune cells capable of expressing the chimeric antigen receptors of the invention and performing the desired biological effector functions as described herein may also be used. Furthermore, the immune cells are typically selected to be compatible with the subject's immune system, ie preferably the immune cells do not elicit an immunogenic response. For example, "universal recipient cells" can be used, ie, universally compatible lymphocytes that can be grown and expanded in vitro that perform the desired biological effector function.
  • step (c) can be carried out by introducing the nucleic acids or vectors described herein into immune cells via electroporation or by infecting immune cells with a viral vector, such as a lentiviral vector, adenoviral Viral vector, adeno-associated viral vector or retroviral vector.
  • a viral vector such as a lentiviral vector, adenoviral Viral vector, adeno-associated viral vector or retroviral vector.
  • transfection reagents such as liposomes
  • transient RNA transfection transient RNA transfection.
  • the immune cells are autologous or allogeneic cells, preferably T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, more preferably T cells, NK cells cells or NKT cells.
  • autologous refers to any material derived from an individual to be later reintroduced into that same individual.
  • allogeneic refers to any material derived from a different animal or different patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are different. In some cases, allogeneic material from individuals of the same species may be genetically different enough for antigenic interactions to occur.
  • the term "subject" is a mammal.
  • the mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples.
  • Mammals other than humans can advantageously be used as subjects representing animal models of cancer.
  • the subject is a human.
  • diseases associated with CD7 expression include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) and solid tumors.
  • Hematological neoplasms are cancers of the blood or bone marrow, including but not limited to acute leukemias such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia and myeloblasts, promyelocytic, myelocytic -monocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myeloid (myeloid) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodge Gold lymphoma, non-Hodgkin lymphoma (painless and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic syndrome,
  • a solid tumor is an abnormal mass of tissue that does not usually contain cysts or areas of fluid, which can be benign or malignant.
  • Different types of solid tumors are named after the cell type that forms them (such as sarcomas, carcinomas, and lymphomas).
  • Examples of solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, pancreatic cancer, ovarian cancer, peritoneal, omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, Small bowel cancer, melanoma, kidney cancer, laryngeal cancer, soft tissue cancer, stomach cancer, testicular cancer, colon cancer, esophagus cancer, cervical cancer, acinar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal cancer , cancer of the eye, intrahepatic bile duct, joint, neck
  • the disease associated with CD7 expression is preferably selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), T lymphoblastic lymphoma (T-LBL), early pre-T lymphoblastoma Leukemia (ETP-ALL) and extranodal NK/T cell lymphoma.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • T-LBL T lymphoblastic lymphoma
  • EDP-ALL early pre-T lymphoblastoma Leukemia
  • extranodal NK/T cell lymphoma extranodal NK/T cell lymphoma.
  • Figure 1 Shows the ratio of CD7+ and CD7- cells in lymphocytes in peripheral blood of patients.
  • Figure 2 Shows the expression level of scFv on CAR7-dKO T cells and CAR7-tKO T cells.
  • Figure 3 Shows the killing ability of CAR7-dKO T cells and CAR7-tKO T cells against target cells.
  • Figure 4 Shows the cytokine release levels after co-culture of CAR7-dKO T cells and CAR7-tKO T cells with target cells.
  • Figure 5 Shows the inhibitory effect of NK inhibitory molecules on NK cell killing.
  • Figure 6 Killing of NK cells by CD4+ T cells (A) and CD8+ T cells (B) expressing NK inhibitory molecules comprising CD3 ⁇ .
  • Figure 7 Shows the expression level of scFv on CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells.
  • Figure 8 Shows the expression levels of NK inhibitory molecules in CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells.
  • Figure 9 Shows the killing ability of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells to target cells.
  • Figure 10 Shows cytokine release levels after co-culture of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells with target cells.
  • Figure 11 Shows scFv expression levels on CAR7-E T cells, CAR7-PDL1 T cells and CAR7-EPDL1 T cells.
  • Figure 12 Shows the expression level of HLA-E in CAR7-E T cells and the expression level of PDL1 in CAR7-PDL1 T cells.
  • Figure 13 Shows the expression levels of HLA-E and PDL1 in CAR7-EPDL1 T cells.
  • Figure 14 Shows the killing ability of CAR7-E T cells, CAR7-PDL1 T cells and CAR7-EPDL1 T cells to target cells.
  • Figure 15 Shows the cytokine release levels after co-culture of CAR7-E T cells, CAR7-PDL1 T cells and CAR7-EPDL1 T cells with target cells.
  • Figure 16 Shows the expression levels of CD7 scFv and CD19 scFv in CAR7-19 T cells.
  • Figure 17 Shows the killing ability of CAR7-19 T cells against two target cells.
  • Figure 18 Shows cytokine release levels after co-culture of CAR7-19 T cells with two target cells.
  • Opti-MEM 3ml Opti-MEM (Gibco, Item No. 31985-070) to a sterile tube to dilute the above plasmid, and then add the packaging vector psPAX2 (Addgene, Cat. No. 12260) and the enveloped vector pMD2.G (Addgene, Cat. No. 12259). Then, add 120ul of X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001), mix immediately, incubate at room temperature for 15 min, and then add the plasmid/vector/transfection reagent mixture dropwise to the culture flask of 293T cells . Viruses were collected at 24 hours and 48 hours, pooled, and ultracentrifuged (25000 g, 4°C, 2.5 hours) to obtain concentrated lentiviruses.
  • Wild-type T cells were activated with DynaBeads CD3/CD28CTS TM (Gibco, Cat. No. 40203D) and cultured at 37°C and 5% CO2 for an additional day. Then use the CRISPR system to knock out TCR/CD3 components (specifically TRAC gene), CD7 gene and optional MHC-II related genes (specifically RFX5) in wild-type T cells to obtain TCR/CD7 double knockout dKO -T cells and TCR/CD7/RFX5 triple knockout tKO-T cells. Wild-type T cells without gene knockout (ie, NT cells) served as controls.
  • TRAC gene specifically CD7 gene
  • MHC-II related genes specifically RFX5
  • the concentrated lentivirus was added to dKO-T cells and tKO-T cells to obtain CAR7-dKO T cells and CAR7-tKO T cells.
  • Biotin-SP long spacer AffiniPure Goat Anti-human IgG, F(ab') 2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 109-065-097) was used as primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) were used as secondary antibodies to detect the expression levels of scFv on CAR7-dKO T cells and CAR7-tKO T cells by flow cytometry. The results are shown in Figure 2. Show.
  • Example 2 Killing effect and cytokine release of CAR T cells on target cells
  • both CAR7-dKO T cells and CAR7-tKO T cells have specific killing ability to target cells, and the killing ability of CAR7-tKO T cells is higher than that of CAR7-dKO T cells.
  • ELISA enzyme-linked immunosorbent assay
  • Jurkat target cells were plated in 96 - well plates at 1x105/well, then CAR T and NT cells (negative control) were co-cultured with target cells at a ratio of 0.125:1, and the cell co-culture supernatant was collected 18-24 hours later liquid.
  • the 96-well plate was coated with the capture antibody Purified anti-human IFN- ⁇ Antibody (Biolegend, Cat. No. 506502) and incubated at 4°C overnight, then the antibody solution was removed, and 250 ⁇ L of PBST containing 2% BSA (sigma, Cat. No. V900933-1kg) was added. 0.1% Tween in 1X PBS) for 2 hours at 37°C. Plates were then washed 3 times with 250 [mu]L of PBST (1XPBS with 0.1% Tween). 50 ⁇ L of cell co-culture supernatant or standards were added to each well and incubated at 37° C.
  • the coding sequences of the following proteins were synthesized and cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): B2m signal peptide (SEQ ID NO: 34), NK inhibitory ligand, CD28 hinge region (SEQ ID NO:40), CD28 transmembrane region (SEQ ID NO:24), wherein the NK inhibitory ligand is the extracellular region of E-cadherin (SEQ ID NO:47, corresponding to ECad0 plasmid),
  • the fusion molecule of B2M and HLA-E extracellular domain comprising presentation peptide SEQ ID NO: 75, B2M SEQ ID NO: 74 and HLA-E extracellular domain mutant SEQ ID NO: 51, wherein the coding sequence of B2M is SEQ ID NO: : 82 contains synonymous mutation, corresponding to EO plasmid) or fusion molecule of B2M and HLA-G extracellular
  • the CD28 co-stimulatory domain (SEQ ID NO: 26) was further included in the ECad0, EO and G0 plasmids to obtain the ECad28, E28 and G28 plasmids, respectively. Confirm the correct insertion of the target sequence in the plasmid by sequencing.
  • the coding sequences of the following proteins were synthesized and cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): B2m signal peptide (SEQ ID NO: 34), NK inhibitory ligand, IgG4 hinge region (SEQ ID NO:42), CD8 ⁇ transmembrane region (SEQ ID NO:22), CD28 costimulatory domain (SEQ ID NO:26), wherein the NK inhibitory ligand is an anti-NKG2A scFv (SEQ ID NO:26) 63, corresponding to A28 plasmid), anti-KIR scFv (SEQ ID NO: 67, corresponding to KIRG4 plasmid) or anti-LIR1 scFv (SEQ ID NO: 70, corresponding to LIR1-1 plasmid). Confirm the correct insertion of the target sequence in the plasmid by sequencing.
  • PPL Public Protein/Pla
  • the coding sequences of the following proteins were synthesized and cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): CD8 ⁇ signal peptide (SEQ ID NO: 36), anti-LIR1 scFv (SEQ ID NO: 36) : 73), CD28 hinge region (SEQ ID NO: 40), CD28 transmembrane region (SEQ ID NO: 24), 4-1BB costimulatory domain (SEQ ID NO: 28) to obtain LIR1-2 plasmid. Confirm the correct insertion of the target sequence in the plasmid by sequencing.
  • PPL Public Protein/Plasmid Library
  • PPL00157-4a CD8 ⁇ signal peptide
  • SEQ ID NO: 40 anti-LIR1 scFv
  • SEQ ID NO: 40 CD28 hinge region
  • SEQ ID NO: 24 CD28 transmembrane region
  • 4-1BB costimulatory domain SEQ ID NO: 28
  • Opti-MEM 3ml Opti-MEM (Gibco, Item No. 31985-070) to a sterile tube to dilute the above plasmid, and then add the packaging vector psPAX2 (Addgene, Cat. No. 12260) and the envelope vector pMD2.G (Addgene, Cat. No. 12259). Then, add 120ul of X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001), mix immediately, incubate at room temperature for 15 min, and then add the plasmid/vector/transfection reagent mixture dropwise to the culture flask of 293T cells . Viruses were collected at 24 hours and 48 hours, pooled, and ultracentrifuged (25000 g, 4°C, 2.5 hours) to obtain concentrated lentiviruses.
  • T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, Cat. No. 40203D) and cultured for 1 day at 37°C and 5% CO2. Then, the concentrated lentivirus was added and cultured for 3 days to obtain T cells expressing NK inhibitory molecules, namely UNKi-T cells.
  • the CRISPR system was then used to knock out TCR/CD3 components (specifically TRAC genes) and MHC-related genes (specifically B2M and RFX5) in wild-type T cells (Mock T cells, used as controls) and the UNKi-T cells. , and confirmed by flow cytometry that each gene was effectively knocked out.
  • TRAC genes specifically TRAC genes
  • MHC-related genes specifically B2M and RFX5
  • the inhibitory effect of the UNKi-T cells prepared by the present invention on NK cell killing was detected according to the following method: the UNKi-T cells and Mock-T cells prepared by the present invention were labeled with Far-Red (invitrogen, product number C34564). Labeled UNKi-T cells and Mock T cells were then plated into 96-well plates at a concentration of 1x104 cells/well, and NK92 cells (for the expression of HLA-E extracellular domain) were added at a 2:1 effector-target ratio.
  • HLA-G ectodomain, anti-NKG2A scFv, anti-KIR scFv or anti-LIR1 scFv for UNKi-T cells and Mock T cells) or NK92-KLRG1 cells (for UNKi-T cells expressing E-cadherin ectodomain cells, prepared by introducing the KLRG1 gene into NK92 cells) were co-cultured. After 16-18 hours, the proportion of T cells in the culture was detected by flow cytometry, and then the killing effect of NK cells on T cells was calculated. The results are shown in FIG. 5 .
  • inhibitory ligands such as anti-KIR scFv, anti-LIR1 scFv, anti-NKG2A scFv, HLA-G extracellular domain, HLA-E cells UNKi-T cells in the extracellular domain and E-cadherin extracellular domain can significantly reduce the killing effect of NK cells on T cells.
  • the addition of costimulatory domains can further significantly enhance T cell response to T cells (G0, EO, Ecad0) compared to T cells (G0, EO, Ecad0) expressing only the inhibitory ligand and transmembrane domain (ie, not containing the costimulatory domain). Inhibition of NK cell killing (G28, E28, ECad28). Therefore, the NK inhibitory molecule comprising an inhibitory ligand, a transmembrane domain and a costimulatory domain according to the present invention can significantly reduce the killing effect of NK cells on UNKi-T cells, thereby effectively reducing the risk of HvGD.
  • the inventors further included the CD3 ⁇ intracellular signaling domain (SEQ ID NO: 30) on the basis of the E28 plasmid and A28 plasmid cells, and packaged it into a lentivirus according to the above method, and infected the TCR/CD3 group.
  • T cells specifically, TRAC gene
  • MHC-related genes specifically, B2M and RFX5
  • NK92 cells killing of NK cells by UNKi -T cells was detected by plating target cells (NK92 cells) in 96-well plates at a concentration of 1x10 cells/well, and then adding Mock T cells at a ratio of 1:1 to each well , E28z-UNKi-T cells and A28z-UNKi-T cells, and 10 ⁇ l PE-anti-human CD107a (BD Pharmingen, Cat. No. 555801) was added at the same time, and co-cultured at 37°C under 5% CO2 culture conditions. After 1 h, Goigstop (BD Pharmingen, Cat. No. 51-2092KZ) was added and incubated for 2.5 hours.
  • Goigstop BD Pharmingen, Cat. No. 51-2092KZ
  • CD8 ⁇ signal peptide SEQ ID NO:36
  • anti-CD7 scFv SEQ ID NO:21
  • CD8 ⁇ hinge region SEQ ID NO:38
  • CD8 ⁇ span Membrane region SEQ ID NO:22
  • 4-1BB intracellular region SEQ ID NO:28
  • CD3 ⁇ intracellular signaling domain SEQ ID NO:30
  • F2A E-cadherin extracellular region
  • SEQ ID NO: 48 E-cadherin extracellular region
  • CD28 hinge region SEQ ID NO: 40
  • CD28 transmembrane region SEQ ID NO: 24
  • CD28 intracellular region SEQ ID NO: 26
  • TCR/CD7 double knockout dKO T cells and TCR/CD7/RFX5 triple knockout tKO T cells were prepared with reference to the knockout method in Example 1. Wild-type T cells without gene knockout (ie, NT cells) served as controls.
  • the 24-well plate was coated with Retronectin and incubated at 4°C overnight, then the solution was removed, and 300 ⁇ L of PBS solution containing 5% FBS (Gibco, Cat. No.) was added, and left at room temperature for 30 min. The supernatant was then removed and the plate was washed 2 times with 1 mL of PBS. Add 2 mL of retroviral supernatant and 0.5M dKO-T cells or tKO-T cells to each well, centrifuge at 2000g for 2 hours at 32°C, and place them in a carbon dioxide incubator to culture to obtain CAR7-NKi-dKO T cells and CAR7-NKi -tKO T cells.
  • Biotin-SP long spacer AffiniPure Goat Anti-Mouse IgG, F(ab') 2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) was used as Primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) as secondary antibody, was used to detect CD7 scFv in CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells by flow cytometry The expression levels are shown in Figure 7.
  • E-Cadherin in CAR T cells with E-cadherin monoclonal antibody (Invitrogen Cat. No. 13-5700) and Goat anti-Mouse IgG(H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, Cat. No. A-11001) expression, and the results are shown in Figure 8.
  • the cytokine release levels of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells after co-culture with Jurkat target cells were detected according to the method described in 2.2 in Example 2, and the results are shown in Figure 10. It can be seen that the cytokine release water of the CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells of the present invention is significantly higher than that of the control NT cells, and the release level of the CAR7-NKi-tKO T cell group is significantly higher in the CAR7-NKi-dKO T cell group.
  • CD8 ⁇ signal peptide SEQ ID NO:36
  • anti-CD7 scFv SEQ ID NO:21
  • CD28 hinge region SEQ ID NO:40
  • CD8 ⁇ span Membrane region SEQ ID NO:22
  • CD28 intracellular region SEQ ID NO:26
  • CD3 ⁇ intracellular signaling domain SEQ ID NO:32
  • F2A F2A
  • PD-L1 signal peptide SEQ ID NO:44
  • PD-L1 extracellular region SEQ ID NO: 45
  • CD28 transmembrane region SEQ ID NO: 24
  • 4-1BB intracellular region SEQ ID NO: 28
  • the above plasmids were packaged into retroviruses according to the method described in Example 3, and tKO-T cells were infected to obtain CAR7-E T cells, CAR7-PDL1 T cells and CAR7-EPDL1 T cells, respectively.
  • Biotin-SP long spacer AffiniPure Goat Anti-human IgG, F(ab') 2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 109-065-097) was used as The primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) was used as the secondary antibody, and the expression levels of scFv in the three cells were detected by flow cytometry. The results are shown in Figure 11.
  • HLA-E and PDL1 in CAR T cells were detected using PE mouse anti-human HLA-E (biolegend, Cat. No. 342604) and PE anti-human PDL1 (biolegend, Cat. No. 329706), respectively. The results are shown in Figure 12 and Figure 13. Show.
  • the anti-CD7 scFv and NK inhibitory molecules (HLA-E and PD-L1) in the CAR T cells prepared by the present invention can be effectively expressed.
  • the cytokine release levels of the three CAR-T cells after co-culture with Jurkat target cells were detected, and the results are shown in Figure 15. Compared with the NT group, the cytokine release levels of all three CAR-T cells were significantly increased.
  • the above plasmid vector was packaged into lentivirus according to the method of Example 1, and infected tkO-T cells to obtain CAR7-19 T cells. Unmodified wild-type T cells were used as negative controls (NT).
  • Biotin-SP long spacer AffiniPure Goat Anti-Mouse IgG, F(ab') 2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) or Biotin-SP ( long spacer) AffiniPure Goat Anti-human IgG, F(ab') 2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 109-065-097) as primary antibody, FITC Streptavidin (BD Pharmingen, Cat. No. 554060) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) were used as secondary antibodies to detect the expression level of scFv in CAR7-19 T cells by flow cytometry. The results are shown in Figure 16.
  • CD7 scFv and CD19 scFv in the CAR T cells prepared by the present invention can be effectively expressed.
  • Example 2 According to the method in Example 2, the killing function and cytokine release level of CAR7-19 T cells were detected, and the results were shown in Figure 17 and Figure 18, respectively. It can be seen that CAR7-19 T cells have specific killing and significantly increased cytokine release against both Nalm6 and Jurkat target cells.

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Abstract

提供了一种工程化免疫细胞，该细胞表达包含抗原结合区的嵌合抗原受体，所述抗原结合区包含抗CD7抗体，以及内源性CD7、至少一种TCR/CD3基因和至少一种MHC-II类相关基因的表达被抑制或沉默。还提供了所述工程化免疫细胞在治疗与CD7表达相关的疾病中的用途。

Description

靶向CD7的嵌合抗原受体及其用途 技术领域
本发明属于免疫治疗领域。更具体地，本发明涉及靶向CD7的嵌合抗原受体，及其在治疗疾病中的用途。
背景技术
近几年，癌症免疫治疗技术发展迅速，尤其是嵌合抗原受体T细胞(CAR-T)相关的免疫疗法，作为一种新型的过继性免疫治疗技术，已经在多种实体肿瘤和血液肿瘤的治疗中显示出非常显著的临床疗效。
根据构建CAR-T细胞所用的T细胞的来源不同，可以分成自体CAR-T细胞和同种异体CAR-T细胞(也称为通用型CAR-T)两类。自体CAR-T细胞的优势在于不会产生免疫排斥风险，因为起始T细胞来自于接受CAR-T疗法的患者本身；但其缺点也非常明显：造价高昂、制备周期长。因此，越来越多的研究聚焦于通用型CAR-T细胞的开发。通用型CAR-T可以用健康供体外周血分离的T细胞进行制备，大大缩短患者等待治疗的时间。此外，从健康供体获得的T细胞的活力和功能也优于患者来源的T细胞，这可以增加CAR细胞的感染率，提高治疗效果。
CD7是一种细胞表面糖蛋白，分子量为约40kDa，属于免疫球蛋白超家族的一员。CD7在大多数的T细胞、NK细胞、髓细胞、T细胞急性淋巴细胞白血病/淋巴瘤、急性粒细胞性白血病和慢性粒细胞白血病中均有表达。据报道，CD7分子通过与其配体K12/SECTM1的结合在T细胞活化期间起到共刺激信号的作用。并且，破坏小鼠T祖细胞中的CD7分子依然会产生正常的T细胞发育和体内平衡，表明CD7似乎对T细胞的发育和功能不会产生关键性的影响，这使其 成为治疗T细胞急性淋巴细胞白血病(T-ALL)的非常合适的治疗靶点。实际上，CD7作为治疗白血病和淋巴瘤的细胞毒性分子的靶标已被广泛研究。
目前针对CD7的CAR-T疗法，尤其是通用型CAR-T疗法仍然非常有限。因此，本发明旨在提供一种高效的靶向CD7的通用型CAR-T细胞，及其在治疗疾病中的用途。
发明内容
在第一个方面，本发明提供一种工程化免疫细胞，其特征在于：(1)表达包含抗原结合区的嵌合抗原受体，所述抗原结合区包含抗CD7抗体；(2)内源性CD7、至少一种TCR/CD3基因和至少一种MHC-II类相关基因的表达被抑制或沉默。
在一个实施方案中，所述嵌合抗原受体包含抗CD7抗体、跨膜结构域和胞内信号传导结构域。
在一个实施方案中，所述抗CD7抗体包含分别如SEQ ID NO：1、2和3所示的CDR-L1、CDR-L2和CDR-L3，和如SEQ ID NO：4、5和6所示的CDR-H1、CDR-H2和CDR-H3。在一个优选的实施方案中，所述抗CD7抗体包含与选自SEQ ID NO：7、10、13、16和19所示的氨基酸序列具有至少90％同一性的轻链可变区和与选自SEQ ID NO：8、11、14、17和20所示的氨基酸序列具有至少90％同一性的重链可变区。在一个优选的实施方案中，所述抗CD7抗体的氨基酸序列选自SEQ ID NO：9、12、15、18和21。
在一个实施方案中，所述嵌合抗原受体的抗原结合区还包含靶向第二抗原的抗体，所述第二抗原选自：TSHR、CD19、CD123、CD22、BAFF-R、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、GPRC5D、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-l lRa、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、AFP、Folate受体α、ERBB2(Her2/neu)、MUC1、 EGFR、CS1、CD138、NCAM、Claudin18.2、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gploo、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD 179a、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、豆荚蛋白、HPV E6、E7、MAGE Al、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋白、存活蛋白和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D和它们的任意组合。更优选地，所述靶向第二抗原的抗体是靶向CD19的抗体。在一个优选的实施方案中，所述抗CD19抗体包含与选自SEQ ID NO：52和55所示的氨基酸序列具有至少90％同一性的轻链可变区和与选自SEQ ID NO：53和56所示的氨基酸序列具有至少90％同一性的重链可变区。在一个优选的实施方案中，所述抗CD19抗体的氨基酸序列选自SEQ ID NO：54和57。
在一个实施方案中，所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154。优选地，跨膜结构域选自CD8α、CD4和CD28的跨膜结构域。
在一个实施方案中，所述胞内信号传导结构域选自以下蛋白的胞内区：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。优选地，所述胞内信号传导结构域包含CD3ζ胞内区。
在一个实施方案中，所述嵌合抗原受体还包含一个或多个共刺激结构域，其选自以下蛋白质的胞内区：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18(LFA-1)、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM、ZAP70以及它们的组合。优选地，所述共刺激结构域是CD27、CD28、CD134、CD137或CD278的胞内区或它们的组合。
在一个实施方案中，所述TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ和它们的组合。
在一个实施方案中，所述MHC-II类相关基因选自：HLA-DPA、HLA-DQ、HLA-DRA、RFX5、RFXAP、RFXANK、CIITA和它们的组合，优选选自RFX5、RFXAP、RFXANK、CIITA和它们的组合。
在一个优选的实施方案中，本发明所述的工程化免疫细胞包括内源性CD7、选自TRAC和TRBC的至少一种TCR/CD3基因和选自RFX5、RFXAP、RFXANK和CIITA的至少一种MHC-II类基因的表达被抑制或沉默。更优选地，本发明所述的工程化免疫细胞包括内源性CD7、选自TRAC和TRBC的至少一种TCR/CD3基因和RFX5的表达被抑制或沉默。
在一个实施方案中，所述工程化免疫细胞进一步表达NK抑制性分子，所述NK抑制性分子包含一个或多个NK抑制性配体、跨膜结构域和共刺激结构域。
在一个实施方案中，所述NK抑制性配体是靶向NK抑制性受体的抗体，所述NK抑制性受体选自NKG2/CD94组分(例如NKG2A、 NKG2B、CD94)；杀伤细胞Ig样受体(KIR)家族成员(例如KIR2DL1、KIR2DL2/3、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2和KIR3DL3)；白细胞Ig样受体(LIR)家族成员(例如LIR1、LIR2、LIR3、LIR5和LIR8)；NK细胞受体蛋白1(NKR-P1)家族成员(例如NKR-P1B和NKR-P1D)；免疫检查点受体(如PD-1、TIGIT和CD96、TIM3、LAG3)；癌胚抗原相关的细胞黏附分子1(CEACAM1)；唾液酸结合性免疫球蛋白样凝集素(SIGLEC)家族成员(例如SIGLEC7和SIGLEC9)；白细胞相关的免疫球蛋白样受体1(LAIR1)；Ly49家族成员(例如Ly49A、Ly49C、Ly49F、Ly49G1和Ly49G4)和杀伤细胞凝集素样受体G1(KLRG1)。优选地，所述NK抑制性受体优选自NKG2A、NKG2B、CD94、LIR1、LIR2、LIR3、LIR5、LIR8、KIR2DL1、KIR2DL2/3、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、CEACAM1、LAIR1、NKR-P1B、NKR-P1D、PD-1、TIGIT、CD96、TIM3、LAG3、SIGLEC7、SIGLEC9、Ly49A、Ly49C、Ly49F、Ly49G1、Ly49G4和KLRG1。更优选地，所述NK抑制性受体选自NKG2A、NKG2B、CD94、LIR1、LIR2、LIR3、KIR2DL1、KIR2DL2/3、KIR3DL1、CEACAM1、LAIR1和KLRG1。还更优选地，所述NK抑制性受体选自NKG2A、NKG2B、LIR1、KIR2DL1、KIR2DL2/3、KIR3DL1、CEACAM1、LAIR1和KLRG1。
在一个实施方案中，所述NK抑制性配体是NK抑制性受体的天然配体或其包含的NK抑制性受体结合区。优选的，所述NK抑制性配体选自HLA-E、HLA-F、HLA-G、钙黏素、胶原蛋白、OCIL、唾液酸、免疫检查点配体(例如PD-L1/PD-L2、CTLA4、CD155、CD112、CD113、Gal-9、FGL1等)，和它们包含的NK抑制性受体结合区。更优选的，所述NK抑制性配体选自HLA-E、HLA-F、HLA-G、钙黏素、胶原蛋白、OCIL、唾液酸、PD-L1/PD-L2、CTLA-4、CD155、CD112、CD113、Gal-9、FGL1，或它们包含的NK抑制性受体结合区；更优选选自HLA-E、HLA-F、HLA-G、钙黏素、PD-L1、PD-L2，或它们包含的NK抑制性受体结合区。在一个优选的实施方案中， 所述NK抑制性配体选自HLA-E胞外区、HLA-G胞外区、E-钙黏素胞外区、PD-L1胞外区和PD-L2胞外区。在一个优选的实施方案中，所述NK抑制性配体是E-钙黏素胞外区，其包含EC1和EC2，更优选包含EC1、EC2、EC3、EC4和EC5。在一个优选的实施方案中，所述NK抑制性配体是PD-L1或PD-L2的胞外区。在一个优选的实施方案中，所述NK抑制性配体是HLA-E胞外区。在一个优选的实施方案中，所述NK抑制性配体是HLA-G胞外区。
在一个实施方案中，所述NK抑制性分子进一步包含CD3ζ胞内区作为胞内信号传导结构域。
在一个实施方案中，所述工程化免疫细胞是T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞。优选的，所述工程化免疫细胞是T细胞，例如CD4+/CD8+T细胞、CD4+辅助T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如，细胞毒性T细胞)、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞。
在一个方面，本发明还提供一种药物组合物，其包含本发明所述的工程化免疫细胞作为活性剂，和一种多种药学上可接受的赋型剂。
在一个方面，本发明还提供一种治疗与CD7表达相关的疾病的受试者的方法，包括向所述受试者施用有效量的根据本发明所述的工程化免疫细胞或药物组合物。因此，本发明还涵盖工程化免疫细胞在制备用于治疗与CD7表达相关的疾病的药物中的用途。
发明详述
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本发明所属领域的普通技术人员通常所了解的相同。
嵌合抗原受体
在第一个方面，本发明提供一种工程化免疫细胞，其特征在于：(1)表达包含抗原结合区的嵌合抗原受体，所述抗原结合区包含抗 CD7抗体；(2)内源性CD7、至少一种TCR/CD3基因和至少一种MHC-II类相关基因的表达被抑制或沉默。
如本文所用，术语“嵌合抗原受体”或“CAR”是指人工构建的杂合多肽，该杂合多肽一般包括抗原结合区(例如抗体或其抗原结合部分)、跨膜结构域、任选的共刺激结构域和胞内信号传导结构域，各个结构域之间通过接头连接。CAR能够利用抗体的抗原结合特性以非MHC限制性的方式将T细胞和其它免疫细胞的特异性和反应性重定向至所选择的靶标。非MHC限制性的抗原识别给予表达CAR的免疫细胞与抗原处理无关的识别抗原的能力，因此绕过了肿瘤逃逸的主要机制。
在一个实施方案中，本发明提供的工程化免疫细胞表达的CAR包含靶向CD7的抗体或其抗原结合片段、跨膜结构域和胞内信号传导结构域。
如本文所用，术语“抗体”具有本领域技术人员所理解的最广泛的含义，并且包括单克隆抗体(包含完整抗体)、多克隆抗体、多价抗体、多特异性抗体(例如双特异性抗体)、和能够表现期望的生物活性的携带一个或多个CDR序列的抗体片段或合成多肽。本发明所述抗体可为任何种类(例如IgG、IgE、IgM、IgD、IgA等)或亚类(例如IgG1、IgG2、IgG2a、IgG3、IgG4、IgA1、IgA2等)。本发明的抗体也包含重组抗体、人抗体、人源化抗体、鼠源抗体、嵌合抗体及其抗原结合部分。
如本文所用，“抗体片段”或“抗原结合部分”是指完整抗体的一部分，一般包含完整抗体的抗原结合位点并因此保留结合抗原的能力。本发明中的抗体片段的实例包括但不限于：Fab、Fab'、F(ab')2、Fd片段、Fd′、Fv片段、scFv、二硫键-连接的Fv(sdFv)、抗体的重链可变区(VH)或轻链可变区(VL)、线性抗体、具有两个抗原结合位点的“双体”、单结构域抗体、纳米抗体、所述抗原的天然配体或其功能性片段等。因此，本发明的“抗体”涵盖如上定义的抗体的抗体片段或抗原结合部分。
在一个实施方案中，本发明的抗CD7抗体是抗CD7 scFv。“单链抗体”和“scFv”在本文中可互换使用，是指由抗体重链可变区(VH)和轻链可变区(VL)通过接头连接而成的抗体。接头的最佳长度和/或氨基酸组成可以根据需要确定。接头的长度会明显影响scFv的可变区折叠和相互作用情况。事实上，如果使用较短的接头(例如在5-10个氨基酸之间)，则可以防止链内折叠。关于接头的大小和组成的选择，参见例如，Hollinger等人,1993Proc Natl Acad.Sci.U.S.A.90:6444-6448；美国专利申请公布号2005/0100543、2005/0175606、2007/0014794；以及PCT公布号WO2006/020258和WO2007/024715，其全文通过引用并入本文。scFv可以包含以任何顺序连接的VH和VL，例如VH-接头-VL或VL-接头-VH。
在一个实施方案中，本发明的CAR包含靶向CD7的抗体，其包含分别如SEQ ID NO：1、2、和3所示的CDR-L1、CDR-L2和CDR-L3，和如SEQ ID NO：4、5和6所示的CDR-H1、CDR-H2和CDR-H3。优选地，本发明的靶向CD7的抗体包含与选自SEQ ID NO：7、10、13、16和19所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性的轻链可变区和与选自SEQ ID NO：8、11、14、17和20所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性的重链可变区。更优选地，本发明的嵌合抗原受体包含抗CD7抗体，其氨基酸序列如SEQ ID NO：9、12、15、18或21所示。
在一个实施方案中，除了靶向CD7的抗体外，本发明的嵌合抗原受体包含的抗原结合区还包含靶向第二抗原的抗体，所述第二抗原选自：TSHR、CD19、CD123、CD22、BAFF-R、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、GPRC5D、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-l lRa、PSCA、 PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、AFP、Folate受体α、ERBB2(Her2/neu)、MUC1、EGFR、CS1、CD138、NCAM、Claudin18.2、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gploo、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD 179a、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、豆荚蛋白、HPV E6、E7、MAGE Al、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋白、存活蛋白和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D和它们的任意组合。优选地，所述肿瘤抗原选自CD19、CD20、CD22、CD30、CD33、CD38、CD123、CD138、CD171、MUC1、AFP、Folate受体α、CEA、PSCA、PSMA、Her2、EGFR、IL13Ra2、GD2、NKG2D、EGFRvIII、CS1、BCMA、间皮素和它们的任意组合。本领域已知的靶向上述肿瘤抗原的抗体均可用于本发明。
在一个优选的实施方案中，所述靶向第二抗原的抗体是靶向CD19的抗体，其包含与SEQ ID NO：52或55所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性的轻链可变区序列和与SEQ ID NO：53或 56所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性的重链可变区序列。更优选地，本发明的嵌合抗原受体包含靶向CD7的抗体和靶向CD19的抗体，所述靶向CD19的抗体的氨基酸序列如SEQ ID NO：54或57所示。
术语“功能性变体”或“功能性片段”是指基本上包含亲本的氨基酸序列但与该亲本氨基酸序列相比含有至少一个氨基酸修饰(即取代、缺失或插入)的变体，条件是所述变体保留亲本氨基酸序列的生物活性。在一个实施方案中，所述氨基酸修饰优选是保守型修饰。
如本文所用，术语“保守性修饰”是指不会明显影响或改变含有该氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰。这些保守修饰包括氨基酸取代、添加及缺失。修饰可以通过本领域中已知的标准技术，如定点诱变和PCR介导的诱变而引入本发明的嵌合抗原受体中。保守氨基酸取代是氨基酸残基被具有类似侧链的氨基酸残基置换的取代。具有类似侧链的氨基酸残基家族已在本领域中有定义，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。保守性修饰可以例如基于极性、电荷、溶解度、疏水性、亲水性和/或所涉及残基的两亲性质的相似性来进行选择。
因此，“功能性变体”或“功能性片段”与亲本氨基酸序列具有至少75％，优选至少76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性，并且保留亲本氨基酸的生物活性，例如结合活性。
如本文所用，术语“序列同一性”表示两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同一性。本领域技术人员将认识到，一些算法可以用于使用标准参数来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25：3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215：403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147：195-197)和ClustalW。
如本文所用，术语“跨膜结构域”是指能够使嵌合抗原受体在免疫细胞(例如淋巴细胞、NK细胞或NKT细胞)表面上表达，并且引导免疫细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合抗原受体与靶抗原结合时，跨膜结构域能够进行信号传导。特别适用于本发明中的跨膜结构域可以源自例如TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154及其功能性片段。或者，跨膜结构域可以是合成的并且可以主要地包含疏水性残基如亮氨酸和缬氨酸。优选地，所述跨膜结构域源自CD8α链或CD28，其与SEQ ID NO:22或24所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:23或25所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的嵌合抗原受体还可以包含位于抗原结合区和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至抗原结合区的任何寡肽或多肽。具体地，铰链区用来为抗原结合区提供更大的灵活性和可及性。铰 链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。在优选的实施方式中，所述铰链区包含CD8α链、CD28、FcγRIIIα受体、IgG4或IgG1的铰链区部分，更优选来自CD8α、CD28或IgG4的铰链，其与SEQ ID NO:38、40或42所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或者其编码序列与SEQ ID NO:39、41或43所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
如本文所用，术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分。胞内信号传导结构域负责在抗原结合区结合抗原以后的细胞内初级信号传递，从而导致免疫细胞和免疫反应的活化。换言之，胞内信号传导结构域负责活化其中表达CAR的免疫细胞的正常的效应子功能的至少一种。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。
在一个实施方案中，本发明的嵌合抗原受体包含的胞内信号传导结构域可以是T细胞受体和共受体的细胞质序列，其在抗原受体结合以后一同起作用以引发初级信号传导，以及这些序列的任何衍生物或变体和具有相同或相似功能的任何合成序列。胞内信号传导结构域可以包含许多免疫受体酪氨酸激活基序(Immunoreceptor Tyrosine-based Activation Motifs,ITAM)。本发明的胞内信号传导结构域的非限制性施例包括但不限于FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的胞内区。在优选的实施方式中，本发明CAR的信号传导结构域可以包含CD3ζ胞内区，其与SEQ ID NO:30或32所示的氨基酸序列具有至少70％， 优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:31或33所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的嵌合抗原受体还包含一个或多个共刺激结构域。共刺激结构域可以是来自共刺激分子的细胞内功能性信号传导结构域，其包含所述共刺激分子的整个细胞内部分，或其功能片段。“共刺激分子”是指在T细胞上与共刺激配体特异性结合，由此介导T细胞的共刺激反应(例如增殖)的同源结合配偶体。共刺激分子包括但不限于1类MHC分子、BTLA和Toll配体受体。本发明的共刺激结构域的非限制性施例包括但不限于以下蛋白质的胞内区：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18(LFA-1)、CD27、CD28、CD30、CD40、CD54(ICAM1)、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM、CD94、LTB以及ZAP70以及它们的组合。
在一个优选的实施方案中，所述共刺激结构域包含一个或多个选自以下蛋白质的胞内区：DAP10、DAP12、CD27、CD28、CD134、4-1BB或CD278。例如，在一个实施方案中，所述共刺激结构域包含4-1BB的胞内区。在一个实施方案中，所述共刺激结构域包含CD28的胞内区。在一个实施方案中，所述共刺激结构域包含4-1BB的胞内区和CD28的胞内区。
在一个实施方案中，4-1BB的胞内区与SEQ ID NO:28所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:29所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个实施方案中， CD28的胞内区与SEQ ID NO:26所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:27所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的CAR还可以包含信号肽，使得当其在细胞例如T细胞中表达时，新生蛋白质被引导至内质网并随后引导至细胞表面。信号肽的核心可以含有长的疏水性氨基酸区段，其具有形成单个α-螺旋的倾向。在信号肽的末端，通常有被信号肽酶识别和切割的氨基酸区段。信号肽酶可以在移位期间或完成后切割，以产生游离信号肽和成熟蛋白。然后，游离信号肽被特定蛋白酶消化。可用于本发明的信号肽是本领域技术人员熟知的，例如衍生自CD8α、IgG1、GM-CSFRα、B2M等的信号肽。在一个实施方案中，可用于本发明的信号肽来自B2M或CD8α，其与SEQ ID NO:34或36所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:35或37所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的CAR还可以包含开关结构，以调控CAR的表达时间。例如，开关结构可以是二聚化结构域的形式，通过与其相应配体的结合引起构象变化，暴露胞外的抗原结合区，使其与被靶向抗原结合，从而激活信号传导通路。或者，也可以使用开关结构分别连接抗原结合区和信号传导结构域，仅当开关结构互相结合(例如在诱导化合物的存在下)时，抗原结合区和信号传导结构域才能通过二聚体连接在一起，从而激活信号通路。开关结构还可以是掩蔽肽的形式。掩蔽肽可以遮蔽胞外的抗原结合区，阻止其与被靶向抗原的结合，当通过例如蛋白酶切割掩蔽肽后，就可以暴露胞外的抗原结合区，使其成为一个“普通”的CAR结构。本领域技术人员知晓的各种开关结构均可用于本发明。
在一个实施方案中，本发明的CAR还可以包含自杀基因，即，使其表达一个可通过外源物质诱导的细胞死亡信号，以在需要时(例如产生严重的毒副作用时)清除CAR细胞。例如，自杀基因可以是插入的表位的形式，例如CD20表位、RQR8等，当需要时，可以通过加入靶向这些表位的抗体或试剂来消除CAR细胞。自杀基因也可以是单纯疱疹病毒胸苷激酶(HSV-TK)，该基因可使细胞在接受更昔洛韦治疗诱导下死亡。自杀基因还可以是iCaspase-9，可以通过化学诱导药物如AP1903、AP20187等诱导iCaspase-9发生二聚化，从而激活下游的Caspase3分子，导致细胞凋亡。本领域技术人员知晓的各种自杀基因均可用于本发明。
内源性基因表达的抑制或沉默
在一个实施方案中，本发明的提供的工程化免疫细胞的内源性CD7、至少一种TCR/CD3基因和至少一种MHC-II类相关基因的表达被抑制或沉默。
CD7不仅在肿瘤细胞，例如T细胞急性淋巴细胞白血病/淋巴瘤、急性粒细胞性白血病和慢性粒细胞白血病中表达，在大多数正常T细胞和NK细胞中也有表达。因此，为了避免靶向CD7的CAR细胞之间的互相识别和杀伤，有必要抑制或沉默CAR细胞的内源性CD7基因的表达。
T细胞表面受体(T cell receptor,TCR)是所有T细胞表面的特征性标志，以非共价键与CD3结合形成TCR/CD3复合物，并通过与抗原呈递细胞表面的特异性MHC-抗原肽复合物结合，产生特异性抗原刺激信号，激活T细胞，发挥杀伤作用。因此，抑制或沉默CAR-T细胞中内源性TCR/CD3基因的表达能够避免其对患者体内正常细胞或组织的攻击，进而避免或降低发生移植物抗宿主病(GvHD)的风险。TCR是由两条不同肽链构成的异二聚体，通常分为两类：α/β型和γ/δ型，其中95％以上的外周T淋巴细胞都表达TCRα/β。TCRα链由TRAC基因编码，β链由TRBC基因编码。TCR的每条肽链包括可变区(V区)、恒定区(C区)、跨膜区和胞质区，其中胞质 区很短，不具备传递抗原刺激信号的能力。TCR分子属于免疫球蛋白超家族，其抗原特异性存在于V区；V区又各有三个高变区CDR1、CDR2、CDR3，其中以CDR3变异最大，直接决定了TCR的抗原结合特异性。在TCR识别MHC-抗原肽复合物时，CDR1、CDR2识别并结合MHC分子，而CDR3直接与抗原肽相结合。CD3包括四种亚基：γ、δ、ε、ζ，通常以二聚体εγ、εδ、ζζ的形式存在。这四种亚基均包含保守的免疫受体酪氨酸激活基序(Immunoreceptor tyrosine-based activation motif,ITAM),其中的2个酪氨酸残基被酪氨酸蛋白激酶磷酸化后，向T细胞传递活化信号。因此，在一个实施方案中，至少一种TCR/CD3基因选自：TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ。
通过检测患者体内外周血淋巴细胞的组成，发明人发现，CD4+CD7-T细胞占总CD3+T细胞的20％左右(图1)。由于靶向CD7的CAR细胞无法识别CD4+CD7-T这一群体，因此不能对该群体进行有效杀伤。反而，CD4+CD7-T细胞会通过识别MHC-II类分子进而对外源性的CAR细胞进行杀伤。因此，为了避免患者体内CD4+CD7-T细胞对外源性CAR-T细胞的杀伤，发明人考虑抑制或沉默CAR-T细胞中内源性MHC-II类相关基因的表达。
在本发明中，MHC-II类相关基因包括MHC-II类基因本身，以及与MHC-II类基因相互作用或调控其表达的基因。
主要组织相容性复合物(major histocompatibility complex，MHC)最初被表征为在移植反应中起主要作用的蛋白，其在所有高等脊椎动物的表面上上表达，并且在小鼠中称为H-2，在人细胞中称为HLA。MHC主要有两类：I类和II类。I类MHC蛋白是两种蛋白质的异二聚体：一种是由MHC I基因编码的跨膜蛋白α链，另一种是由不位于MHC基因簇内的基因编码的细胞外蛋白质的β2微球蛋白链。α链包括三个结构域，外来肽与两个在N末端也最可变的结构域α1、α2结合。II类MHC蛋白也是异二聚体，包含两个由MHC复合物内的基因编码的跨膜蛋白。I类MHC/抗原复合物与细胞毒性T细胞 (例如CD8+T细胞)相互作用，而II类MHC向辅助T细胞(例如CD4+T细胞)呈递抗原。此外，I类MHC蛋白倾向于在几乎所有有核细胞和血小板(以及小鼠中的红血细胞)中表达，而II类MHC蛋白更具选择性地表达。通常，II类MHC蛋白在B细胞、一些巨噬细胞和单核细胞、激活的T细胞、郎格罕氏细胞(Langerhans cell)和树突细胞上表达。
人的II类HLA簇包含三个主要基因座DP、DQ和DR。II类分子是由均锚定在膜中的α链和β链组成的异二聚体，其中α链为约33-35kDa，并且含有5个外显子。外显子1编码前导肽，外显子2和3编码两个细胞外结构域，外显子4编码跨膜结构域，外显子5编码细胞质尾部。因此，在一个实施方案中，MHC-II类相关基因选自：HLA-DPA、HLA-DQ和HLA-DRA。
MHC-II类基因的表达还取决于多种重要的正向调控蛋白，例如RFX复合物、CIITA等。RFX复合物由三个亚基组成：RFXANK(也称为RFXB)、RFX5和RFX辅助蛋白(也称为RFXAP)。RFX复合物通过促进其他转录因子与II类MHC分子的启动子结合并增强启动子结合的特异性来促进II类MHC分子的表达。CIITA是II类MHC表达的主控制因子。CIITA包括富含酸性氨基酸的N端，富含Pro、Ser、Thr的PST区域，中间的GTP结合区域以及富含Leu重复序列(LRR)的C端，其中N端酸性区域和PST区域是转录激活区域。因此，在一个实施方案中，MHC-II类相关基因选自：RFX5、RFXAP、RFXANK和CIITA。
因此，在一个实施方案中，MHC-II类相关基因选自：HLA-DPA、HLA-DQ、HLA-DRA、RFX5、RFXAP、RFXANK和CIITA，优选选自RFX5、RFXAP、RFXANK和CIITA，更优选RFX5。
在一个实施方案中，CAR-T细胞中的内源性MHC-I类基因(例如HLA-A、HLA-B、HLA-C、B2M等)是功能性的。在另一个实施方案中，CAR-T细胞中的内源性MHC-I类基因的表达也被抑制或沉默。
在一个实施方案中，本发明所述的表达靶向CD7的嵌合抗原受体的工程化免疫细胞包括内源性CD7、至少一种TCR/CD3基因和至少一种MHC-II类相关基因的表达被抑制或沉默，其中所述至少一种TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ和它们的组合；所述至少一种MHC-II类相关基因选自HLA-DPA、HLA-DQ、HLA-DRA、RFX5、RFXAP、RFXANK和CIITA。在一个优选的实施方案中，本发明所述的表达靶向CD7的嵌合抗原受体的工程化免疫细胞包括内源性CD7、选自TRAC和TRBC的至少一种TCR/CD3基因和选自RFX5、RFXAP、RFXANK和CIITA的至少一种MHC-II类基因的表达被抑制或沉默。更优选地，本发明所述的表达靶向CD7的嵌合抗原受体的工程化免疫细胞包括内源性CD7、选自TRAC和TRBC的至少一种TCR/CD3基因和RFX5的表达被抑制或沉默。在一个实施方案中，所述工程化免疫细胞中的MHC-I类基因，例如HLA-A、HLA-B、HLA-C和B2M的表达是功能性的。
在一个实施方案中，除了CD7、MHC-II类相关基因和TCR/CD3基因，本发明的工程化免疫细胞还可以包含至少一种选自以下的基因的表达被抑制或沉默：CD52、GR、dCK和免疫检查点基因，如PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。
抑制基因表达或使基因沉默的方法是本领域技术人员熟知的，包括但不限于例如通过大范围核酸酶、锌指核酸酶、TALE核酸酶或CRISPR系统中的Cas酶介导DNA断裂、或通过反义寡核苷酸、RNAi、shRNA等技术使基因失活。
NK抑制性分子
发明人还发现，患者体内存在较大比例(约20％左右)的CD7-NK细胞(图1)，其不能被CD7CAR靶向杀伤。并且，在MHC-II基因表达被抑制或沉默的情况下，这部分细胞可能对CAR细胞产生杀伤。
因此，在一个实施方案中，为了抑制患者体内NK细胞对CAR-T细胞的杀伤，所述工程化免疫细胞进一步表达NK抑制性分子，所述NK抑制性分子包含一个或多个NK抑制性配体、跨膜结构域和共刺激结构域。例如，所述NK抑制性分子包含一个或两个NK抑制性配体、跨膜结构域和共刺激结构域。
NK抑制性分子中包含的跨膜结构域和共刺激结构域的定义与上述嵌合抗原受体中包含的跨膜结构域和共刺激结构域的定义相同。
在一个实施方案中，所述NK抑制性配体是靶向NK抑制性受体的抗体，所述NK抑制性受体选自NKG2/CD94组分(例如NKG2A、NKG2B、CD94)；杀伤细胞Ig样受体(KIR)家族成员(例如KIR2DL1、KIR2DL2/3、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2和KIR3DL3)；白细胞Ig样受体(LIR)家族成员(例如LIR1、LIR2、LIR3、LIR5和LIR8)；NK细胞受体蛋白1(NKR-P1)家族成员(例如NKR-P1B和NKR-P1D)；免疫检查点受体(如PD-1、TIGIT和CD96、TIM3、LAG3)；癌胚抗原相关的细胞黏附分子1(CEACAM1)；唾液酸结合性免疫球蛋白样凝集素(SIGLEC)家族成员(例如SIGLEC7和SIGLEC9)；白细胞相关的免疫球蛋白样受体1(LAIR1)；Ly49家族成员(例如Ly49A、Ly49C、Ly49F、Ly49G1和Ly49G4)和杀伤细胞凝集素样受体G1(KLRG1)。优选地，所述NK抑制性受体优选自NKG2A、NKG2B、CD94、LIR1、LIR2、LIR3、LIR5、LIR8、KIR2DL1、KIR2DL2/3、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、CEACAM1、LAIR1、NKR-P1B、NKR-P1D、PD-1、TIGIT、CD96、TIM3、LAG3、SIGLEC7、SIGLEC9、Ly49A、Ly49C、Ly49F、 Ly49G1、Ly49G4和KLRG1。更优选地，所述NK抑制性受体选自NKG2A、NKG2B、CD94、LIR1、LIR2、LIR3、KIR2DL1、KIR2DL2/3、KIR3DL1、CEACAM1、LAIR1和KLRG1。还更优选地，所述NK抑制性受体选自NKG2A、NKG2B、LIR1、KIR2DL1、KIR2DL2/3、KIR3DL1、CEACAM1、LAIR1和KLRG1。
在一个实施方案中，NK抑制性配体是靶向NK抑制性受体的抗体，所述抗体是完整抗体、Fab、Fab’、F(ab’)2、Fv片段、scFv抗体片段、线性抗体、sdAb或纳米抗体。
在一个优选的实施方案中，NK抑制性配体是靶向NKG2A的抗体。更优选地，所述靶向NKG2A的抗体包含与SEQ ID NO：59、62或77所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性的轻链可变区序列和与SEQ ID NO：58、61或76所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性的重链可变区序列。更优选地，所述靶向NKG2A的抗体的氨基酸序列如SEQ ID NO：60、63或78所示。本领域已知的其他靶向NKG2A的抗体也可用于本发明，例如Z270(可获自Immunotech，France)、Z199(可获得自Beckman Coulter，USA)、20D5(可获得自BD Biosciences Pharmingen，USA)、P25(可获自Morettaetal，Univ.Genova，Italy)等。
在一个优选的实施方案中，NK抑制性配体是靶向KIR，例如KIR2DL1、KIR2DL2/3和KIR3DL1的抗体。更优选地，所述靶向KIR的抗体包含与SEQ ID NO：65或79所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性的轻链可变区序列和与SEQ ID NO：66或80所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性的重链可变区序列。更优选地，所述靶向KIR的抗体的氨基酸序列如SEQ ID NO：67或81所示。本领域已知的其他靶向KIR的抗体也可用于本发明，例如GL183(靶向 KIR2DL2/L3，可获自Immunotech，France和Beckton Dickinson，USA)、EB6(靶向KIR2DL1，可获自Immunotech，France和Beckton Dickinson，USA)、AZ138(靶向KIR3DL1，可获自Morettaetal，Univ.Genova，Italy)、Q66(靶向KIR3DL2，可获自Immunotech，France)、Z27(靶向KIR3DL1，可获自Immunotech，France和Beckton Dickinson，USA)等。
在一个优选的实施方案中，NK抑制性配体是靶向LIR1的抗体。更优选地，所述靶向LIR1的抗体包含与SEQ ID NO：68或71所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性的轻链可变区序列和与SEQ ID NO：69或72所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性的重链可变区序列。更优选地，所述靶向LIR1的抗体的氨基酸序列如SEQ ID NO：70或73所示。
在一个实施方案中，所述NK抑制性配体是NK抑制性受体的天然配体或其包含的NK抑制性受体结合区。优选的，所述NK抑制性配体选自HLA-E、HLA-F、HLA-G、钙黏素(Cadherin)、胶原蛋白、OCIL、唾液酸、免疫检查点配体(例如PD-L1/PD-L2、CD155、CD112、CD113、Gal-9、FGL1等)，和它们包含的NK抑制性受体结合区。更优选的，所述NK抑制性配体选自HLA-E、HLA-F、HLA-G、钙黏素、胶原蛋白、OCIL、唾液酸、PD-L1/PD-L2、CTLA-4、CD155、CD112、CD113、Gal-9、FGL1，或它们包含的NK抑制性受体结合区；更优选选自HLA-E、HLA-F、HLA-G、钙黏素，或它们包含的NK抑制性受体结合区。更优选的，所述NK抑制性配体选自HLA-E胞外区、HLA-G胞外区、E-钙黏素(E-Cadherin)胞外区、PD-L1胞外区和PD-L2胞外区。
在一个优选的实施方案中，所述NK抑制性配体是E-钙黏素胞外区，其包含结构域EC1和EC2，更优选包含结构域EC1、EC2、EC3、EC4和EC5。优选地，所述E-钙黏素胞外区与SEQ ID NO： 44(包含结构域EC1和EC2)或SEQ ID NO：48(包含结构域EC1、EC2、EC3、EC4和EC5)所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性。
在一个实施方案中，NK抑制性配体是PD-L1或PD-L2的胞外区。优选地，所述PD-L1胞外区与SEQ ID NO：45所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性，所述PD-L2胞外区与SEQ ID NO：46所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性。
在一个实施方案中，NK抑制性配体是HLA-E胞外区或HLA-G胞外区。优选地，所述HLA-E胞外区与SEQ ID NO：50(野生型)或SEQ ID NO：51(包含Y84C突变的突变体)所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性，所述HLA-G胞外区与SEQ ID NO：49所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性。本领域知晓，非经典HLA-I类分子(例如HLA-E、HLA-G)需要与B2M形成复合物才能发挥其功能。因此，当B2M需要被敲除时，为了使非经典HLA-I类分子正常发挥其抑制功能，需要引入DNA序列同义突变的B2M以避免被基因编辑工具敲除。换言之，在B2M被敲除的细胞中，NK抑制性配体是B2M与HLA-E胞外区或HLA-G胞外区的融合分子。
还在一个实施方案中，所述NK抑制性分子包含至少两个NK抑制性配体，所述NK抑制性配体选自抗NKG2A scFv、抗KIR scFv、抗LIR1 scFv、HLA-E胞外区、HLA-G胞外区、E-钙黏素胞外区、PD-L1胞外区和PD-L2胞外区，更优选是PD-L1胞外区和HLA-E胞外区。
在一个实施方案中，所述NK抑制性分子进一步包含胞内信号传导结构域。在一个实施方案中，所述胞内信号传导结构域选自以下蛋白的胞内区：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、 CD79a、CD79b和CD66d。优选地，所述胞内信号传导结构域包含CD3ζ的胞内区。
在一个实施方案中，所述NK抑制性分子还可以包含信号肽，例如来自PDL1或B2M的信号肽。在一个实施方案中，所述NK抑制性分子包含PD-L1信号肽，其与SEQ ID NO：44所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％序列同一性。本领域技术人员也可以根据需要选择其他合适的信号肽。
工程化免疫细胞
本发明还提供一种工程化免疫细胞，其特征在于：(1)表达包含抗原结合区的嵌合抗原受体，所述抗原结合区包含抗CD7抗体和任选的第二抗原区；(2)内源性CD7、至少一种TCR/CD3基因和至少一种MHC-II类相关基因的表达被抑制或沉默。在一个实施方案中，本发明的工程化免疫细胞还进一步表达NK抑制性分子，所述NK抑制性分子包含一个或多个NK抑制性配体、跨膜结构域和共刺激结构域。
如本文所用，术语“免疫细胞”是指免疫系统的具有一种或多种效应子功能(例如，细胞毒性细胞杀伤活性、分泌细胞因子、诱导ADCC和/或CDC)的任何细胞。例如，免疫细胞可以是T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞，或者是衍生自干细胞，例如成体干细胞、胚胎干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞等的免疫细胞。优选地，免疫细胞是T细胞。T细胞可以是任何T细胞，如体外培养的T细胞，例如原代T细胞，或者来自体外培养的T细胞系例如Jurkat、SupT1等的T细胞，或获得自受试者的T细胞。受试者的实例包括人、狗、猫、小鼠、大鼠及其转基因物种。T细胞可以从多种来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐血、胸腺组织、来自感染部位的组织、腹水、胸膜积液、脾组织及肿瘤。T细胞也可以被浓缩或纯化。T细胞可以处于任何 发育阶段，包括但不限于，CD4+/CD8+T细胞、CD4+辅助T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如，细胞毒性T细胞)、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞等。在一个优选的实施方案中，免疫细胞是人T细胞。可以使用本领域技术人员已知的多种技术，如Ficoll分离受试者的血液获得T细胞。在本发明中，免疫细胞被工程化以表达嵌合抗原受体并抑制或沉默内源性CD7、至少一种TCR/CD3基因和至少一种MHC-II类相关基因的表达。
采用本领域已知的常规方法(如通过转导、转染、转化等)可以将编码嵌合抗原受体多肽的核酸序列以及任选的NK抑制性分子的编码核酸序列引入免疫细胞。“转染”是将核酸分子或多核苷酸(包括载体)引入靶细胞的过程。一个例子是RNA转染，即将RNA(比如体外转录的RNA，ivtRNA)引入宿主细胞的过程。该术语主要用于真核细胞中的非病毒方法。术语“转导”通常用于描述病毒介导的核酸分子或多核苷酸的转移。动物细胞的转染通常涉及在细胞膜中打开瞬时的孔或“洞”，以允许摄取材料。可以使用磷酸钙、通过电穿孔、通过细胞挤压或通过将阳离子脂质与材料混合以产生与细胞膜融合并将它们的运载物沉积入内部的脂质体，进行转染。用于转染真核宿主细胞的示例性技术包括脂质囊泡介导的摄取、热休克介导的摄取、磷酸钙介导的转染(磷酸钙/DNA共沉淀)、显微注射和电穿孔。术语“转化”用于描述核酸分子或多核苷酸(包括载体)向细菌中、也向非动物真核细胞(包括植物细胞)中的非病毒转移。因此，转化是细菌或非动物真核细胞的基因改变，其通过细胞膜从其周围直接摄取并随后并入外源遗传材料(核酸分子)而产生。转化可以通过人工手段实现。为了发生转化，细胞或细菌必须处于感受态的状态。对于原核转化，技术可包括热休克介导的摄取、与完整细胞的细菌原生质体融合、显微注射和电穿孔。
将核酸或载体引入免疫细胞后，本领域技术人员可以通过常规技术对所得免疫细胞进行扩增和活化。
药物组合物
本发明还提供一种药物组合物，其包含本发明所述的工程化免疫细胞作为活性剂，和一种多种药学上可接受的赋型剂。
如本文所用，术语“药学上可接受的赋型剂”是指在药理学和/或生理学上与受试者和活性成分相容(即，能够引发所需的治疗效果而不会引起任何不希望的局部或全身作用)的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)。药学上可接受的赋型剂的实例包括但不限于填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、表面活性剂、稀释剂、润湿剂、防腐剂、乳化剂、包覆剂、等渗剂、吸收延迟剂、稳定剂和张力调节剂。本领域技术人员已知选择合适的赋型剂以制备本发明期望的药物组合物。用于本发明的药物组合物中的示例性赋型剂包括盐水、缓冲盐水、葡萄糖和水。通常，合适的赋形剂的选择尤其取决于所使用的活性剂、待治疗的疾病和药物组合物的期望剂型。
根据本发明的药物组合物可适用于多种途径施用。通常，通过胃肠外完成施用。胃肠外递送方法包括局部、动脉内、肌内、皮下、髓内、鞘内、心室内、静脉内、腹膜内、子宫内、阴道内、舌下或鼻内施用。
根据本发明的药物组合物也可以制备成各种形式，如固态、液态、气态或冻干形式，特别可以是软膏、乳膏、透皮贴剂、凝胶、粉末、片剂、溶液、气雾剂、颗粒、丸剂、混悬剂、乳剂、胶囊、糖浆、酏剂、浸膏剂、酊剂或流浸膏提取物的形式，或者是特别适用于所需施用方法的形式。本发明已知的用于生产药物的过程可包括例如常规混合、溶解、制粒、制糖衣、研磨、乳化、包封、包埋或冻干过程。包含例如本文所述的免疫细胞的药物组合物通常以溶液形式提供，并且优选包含药学上可接受的缓冲剂。
根据本发明的药物组合物还可以与一种或多种适用于治疗和/或预防待治疗疾病的其它药剂组合施用。适用于组合的药剂的优选实例包括已知的抗癌药物，比如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimer sodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreate glucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；肽细胞毒素，比如蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，比如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，比如抗体定向的酶前药；免疫刺激剂，比如血小板因子4、黑色素瘤生长刺激蛋白等；抗体或其片段，比如抗CD3抗体或其片段，补体活化剂，异种蛋白结构域，同种蛋白结构域，病毒/细菌蛋白结构域和病毒/细菌肽。此外，本发明的药物组合物也可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。
治疗应用
本发明还提供一种治疗患有与CD7表达相关的疾病的受试者的方法，包括向所述受试者施用有效量的根据本发明所述的免疫细胞或药物组合物。因此，本发明还涵盖所述工程化免疫细胞以及药物组合物在制备治疗与CD7表达相关的疾病的药物中的用途。
在一个实施方案中，直接向受试者施用有效量的本发明的免疫细胞和/或药物组合物。
在另一个实施方案中，本发明的治疗方法是离体治疗。具体地，该方法包括以下步骤：(a)提供样品，所述样品包含免疫细胞；(b)在体外将本发明的嵌合抗原受体以及外源性基因(例如NK抑制性分子)引入所述免疫细胞，并抑制或沉默所述免疫细胞中特定基因的表达(例如内源性CD7、TCR/CD3基因和MHC-II类相关基因)，获得经修饰的免疫细胞，(c)向有此需要的受试者施用所述经修饰的免疫细胞。优选地，步骤(a)中提供的免疫细胞选自巨噬细胞、树突 状细胞、单核细胞、T细胞、NK细胞和/或NKT细胞；并且所述免疫细胞可以通过本领域已知的常规方法从受试者的样品(特别是血液样品)中获得。然而，也可以使用能够表达本发明的嵌合抗原受体并发挥如本文所述的所需生物效应功能的其它免疫细胞。此外，通常选择的免疫细胞与受试者的免疫系统相容，即优选所述免疫细胞不引发免疫原性响应。例如，可以使用“通用接受体细胞”，即发挥所需生物效应功能的普遍相容的可在体外生长和扩增的淋巴细胞。使用此类细胞将不需要获得和/或提供受试者自身淋巴细胞。步骤(c)的离体引入可以通过经由电穿孔将本文所述的核酸或载体引入免疫细胞或通过用病毒载体感染免疫细胞来实施，所述病毒载体为如前所述的慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体。其它可想到的方法包括使用转染试剂(比如脂质体)或瞬时RNA转染。
在一个实施方案中，所述免疫细胞是自体或同种异体的细胞，优选T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞，更优选T细胞、NK细胞或NKT细胞。
如本文所用，术语“自体”是指来源于个体的任何材料稍后将被再引入该相同个体中。
如本文所用，术语“同种异体”是指任何材料来源于与引入该材料的个体相同物种的不同动物或不同患者。当在一个或多个基因座处的基因不同时，认为两个或更多个体彼此为同种异体的。在一些情况下，来自同一物种的各个体的同种异体材料在基因上的不同可能足以发生抗原相互作用。
如本文所用，术语“受试者”是哺乳动物。哺乳动物可以是人、非人灵长类动物、小鼠、大鼠、狗、猫、马或牛，但不限于这些实例。除人以外的哺乳动物可以有利地用作代表癌症动物模型的受试者。优选地，所述受试者是人。
在一个实施方案中，与CD7表达相关的疾病包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)和实体瘤。血液学肿瘤是血液或 骨髓的癌症，包括但不限于急性白血病(诸如急性淋巴细胞白血病(ALL)、急性髓细胞白血病(AML)、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、骨髓增生异常综合征、多毛细胞白血病、伯基特淋巴瘤、弥漫性大细胞淋巴瘤、套细胞淋巴瘤、T淋巴母细胞性淋巴瘤(T-LBL)、早期前T淋巴母细胞白血病(ETP-ALL)、结外NK/T细胞淋巴瘤、小淋巴细胞淋巴瘤(SLL)和脊髓发育不良。实体瘤是通常不包含囊肿或液体区的组织的异常肿块，其可以是良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤的实例包括但不限于纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、胰腺癌、卵巢癌、腹膜、大网膜和肠系膜癌、咽癌、前列腺癌、直肠癌、肾癌、皮肤癌、小肠癌、黑素瘤、肾癌，喉癌、软组织癌、胃癌、睾丸癌、结肠癌，食道癌，宫颈癌、腺泡型横纹肌肉瘤、膀胱癌、骨癌、脑癌、乳腺癌、肛门癌、眼癌、肝内胆管癌、关节癌、颈癌、胆囊癌、胸膜癌、鼻癌、中耳癌、口腔癌、外阴癌、甲状腺癌和输尿管癌。
在一个实施方案中，与CD7表达相关的疾病优选选自急性淋巴细胞白血病(ALL)、急性髓细胞白血病(AML)、T淋巴母细胞性淋巴瘤(T-LBL)、早期前T淋巴母细胞白血病(ETP-ALL)和结外NK/T细胞淋巴瘤。
下面将参考附图并结合实例来详细说明本发明。需要说明的是，本领域的技术人员应该理解本发明的附图及其实施例仅仅是为了例举的目的，并不能对本发明构成任何限制。在不矛盾的情况下，本申请中的实施例及实施例中的特征可以相互组合。
附图说明
图1：示出了患者外周血淋巴细胞中CD7+细胞和CD7-细胞的比例。
图2：示出了CAR7-dKO T细胞和CAR7-tKO T细胞上scFv的表达水平。
图3：示出了CAR7-dKO T细胞和CAR7-tKO T细胞对靶细胞的杀伤能力。
图4：示出了CAR7-dKO T细胞和CAR7-tKO T细胞与靶细胞共培养后的细胞因子释放水平。
图5：示出了NK抑制性分子对NK细胞杀伤作用的抑制效果。A：NK抑制性分子包含抗KIR scFv；B:NK抑制性分子包含抗LIR scFv；C：NK抑制性分子包含抗NKG2A scFv、HLA-G胞外区、HLA-E胞外区或E-Cad胞外区。
图6：表达包含CD3ζ的NK抑制性分子的CD4+T细胞(A)和CD8+T细胞(B)对NK细胞的的杀伤作用。
图7：示出了CAR7-NKi-dKO T细胞和CAR7-NKi-tKO T细胞上scFv的表达水平。
图8：示出了CAR7-NKi-dKO T细胞和CAR7-NKi-tKO T细胞的NK抑制性分子的表达水平。
图9：示出了CAR7-NKi-dKO T细胞和CAR7-NKi-tKO T细胞对靶细胞的杀伤能力。
图10：示出了CAR7-NKi-dKO T细胞和CAR7-NKi-tKO T细胞与靶细胞共培养后的细胞因子释放水平。
图11：示出了CAR7-E T细胞、CAR7-PDL1 T细胞和CAR7-EPDL1 T细胞上的scFv表达水平。
图12：示出了CAR7-E T细胞中的HLA-E的表达水平以及CAR7-PDL1 T细胞中的PDL1表达水平。
图13：示出了CAR7-EPDL1 T细胞中的HLA-E和PDL1的表达水平。
图14：示出了CAR7-E T细胞、CAR7-PDL1 T细胞和 CAR7-EPDL1 T细胞对靶细胞的杀伤能力。
图15：示出了CAR7-E T细胞、CAR7-PDL1 T细胞和CAR7-EPDL1 T细胞与靶细胞共培养后的细胞因子释放水平。
图16：示出了CAR7-19 T细胞中CD7 scFv和CD19 scFv的表达水平。
图17：示出了CAR7-19 T细胞对两种靶细胞的杀伤能力。
图18：示出了了CAR7-19 T细胞与两种靶细胞共培养后的细胞因子释放水平。
具体实施方式
实施例1.制备抗CD7的通用型CAR T细胞
合成编码以下蛋白的序列，并将其依次克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号:PPL00157-4a)：CD8α信号肽(SEQ ID NO：36)、抗CD7 scFv(SEQ ID NO：21)、CD8α铰链区(SEQ ID NO：38)、CD8α跨膜区(SEQ ID NO：24)、4-1BB胞内区(SEQ ID NO：28)、CD3ζ胞内信号传导结构域(SEQ ID NO：30)，并通过测序确认目标序列的正确插入。
在无菌管中加入3ml Opti-MEM(Gibco，货号31985-070)稀释上述质粒后，再根据质粒:病毒包装载体：病毒包膜载体＝4:2:1的比例加入包装载体psPAX2(Addgene，货号12260)和包膜载体pMD2.G(Addgene，货号12259)。然后，加入120ul X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，于室温下孵育15min，然后将质粒/载体/转染试剂混合物逐滴加入到293T细胞的培养瓶中。在24小时和48小时收集病毒，将其合并后，超速离心(25000g，4℃，2.5小时)获得浓缩的慢病毒。
用DynaBeads CD3/CD28CTS TM(Gibco,货号40203D)激活野生型T细胞，并在37℃和5％CO2下继续培养1天。然后采用CRISPR系统敲除野生型T细胞中的TCR/CD3组分(具体为TRAC基因)、CD7基因和任选的MHC-II类相关基因(具体RFX5)，获得TCR/CD7 双敲除的dKO-T细胞和TCR/CD7/RFX5三敲除的tKO-T细胞。未敲除基因的野生型T细胞(即，NT细胞)作为对照。
使用FITC Mouse Anti-Human CD3(BD Pharmingen，货号555916)抗体、PE mouse anti-human CD7(biolegend货号395604)和APC anti-human DR,DP,DQ(biolegend，货号361714)抗体，通过流式细胞仪检测T细胞中的TCR/CD7/RFX5的基因编辑效率，结果如表1所示。
表1.T细胞中的基因表达效率
名称 TCR/CD3 CD7 RFX5/MHC-II
dKO-T 2.2％ 5.3％ 92％
tKO-T 3％ 8.9％ 11.2％
NT 98％ 94％ 92％
从表1可以看出，本发明制备的dKO-T细胞和tKO-T细胞中相关基因的表达被有效抑制或沉默。
将浓缩后的慢病毒加入dKO-T细胞和tKO-T细胞，获得CAR7-dKO T细胞和CAR7-tKO T细胞。使用Biotin-SP(long spacer)AffiniPure Goat Anti-human IgG,F(ab') 2Fragment Specific(min X Hu,Bov,Hrs Sr Prot)(jackson immunoresearch，货号109-065-097)作为一抗，APC Streptavidin(BD Pharmingen，货号554067)或PE Streptavidin(BD Pharmingen，货号554061)作为二抗，通过流式细胞仪检测CAR7-dKO T细胞和CAR7-tKO T细胞上scFv的表达水平，结果如图2所示。
可以看出，本发明制备的CAR T细胞中的scFv均可以有效表达。
实施例2：CAR T细胞对靶细胞的杀伤效果和细胞因子释放
2.1 CAR-T细胞对靶细胞的杀伤效果
为了检测CAR-T细胞对靶细胞的杀伤能力，首先以1x10 4/孔将携带荧光素基因的Jurkat靶细胞铺入96孔板中，然后以0.5:1、0.25:1和0.125:1的效靶比(即效应T细胞与靶细胞之比)将CAR T细胞和NT细胞铺入到96孔板进行共培养，16-18小时后利用酶标仪测定荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶 细胞荧光均值×100％，计算得到杀伤效率，结果如图3所示。
可以看出，与NT相比，CAR7-dKO T细胞和CAR7-tKO T细胞对靶细胞均有特异性杀伤，并且CAR7-tKO T细胞的杀伤能力高于CAR7-dKO T细胞。
2.2 CAR-T细胞的细胞因子释放
T细胞杀伤靶细胞时，靶细胞数量减少的同时也会释放细胞因子。根据以下步骤，使用酶联免疫吸附法(ELISA)来测定本发明的CAR T细胞杀伤靶细胞时细胞因子IFNγ的释放水平。
(1)收集细胞共培养上清液
以1x10 5/孔将Jurkat靶细胞铺于96孔板中，然后以0.125:1的比例将CAR T和NT细胞(阴性对照)与靶细胞共培养，18-24小时后收集细胞共培养上清液。
(2)ELISA检测上清中IFNγ分泌量
使用捕获抗体Purified anti-human IFN-γAntibody(Biolegend，货号506502)包被96孔板4℃孵育过夜，然后移除抗体溶液，加入250μL含有2％BSA(sigma，货号V900933-1kg)的PBST(含0.1％吐温的1XPBS)溶液，37℃孵育2小时。然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。每孔加入50μL细胞共培养上清液或标准品，并在37℃孵育1小时，然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。然后向各孔分别加入50μL检测抗体Anti-Interferon gamma抗体[MD-1](Biotin)(abcam，货号ab25017)，在37℃孵育1小时后，用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。再加入HRP Streptavidin(Biolegend，货号405210)，在37℃孵育30分钟后，弃上清液，加入250μL PBST(含0.1％吐温的1XPBS)，清洗5次。向各孔加入50μL TMB底物溶液。使反应在室温下于暗处发生30分钟，之后向各孔中加入50μL 1mol/L H 2SO 4以停止反应。在停止反应的30分钟内，使用酶标仪检测450nm处吸光度，并根据标准曲线(根据标准品的读值和浓度绘制)计算细胞因子的含量，结果如图4所示。
可以看出，CAR7-dKO T细胞和CAR7-tKO T细胞对靶细胞的因子释放均显著高于对照NT组，且CAR7-tKO T细胞的释放水平高于CAR7-dKO T细胞。
从以上结果可以看出，CAR7-tKO T细胞对靶细胞的杀伤能力和细胞因子的释放水平均高于CAR7-dKO T细胞，表明MHC-II类基因的敲除增强了CAR-T细胞的杀伤活性。这是出乎意料的，因为现有报道一般认为MHC-II类基因的表达与免疫排斥相关，尚未见任何有关于MHC-II类基因与CAR-T细胞本身活性之间关系的报道。
实施例3.NK抑制性分子对NK细胞杀伤作用的抑制效果
合成以下蛋白的编码序列，并将其依次克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号:PPL00157-4a)：B2m信号肽(SEQ ID NO：34)、NK抑制性配体、CD28铰链区(SEQ ID NO：40)、CD28跨膜区(SEQ ID NO：24)，其中NK抑制性配体是E-钙黏素的胞外区(SEQ ID NO：47，对应ECad0质粒)、B2M和HLA-E胞外区的融合分子(包含提呈肽SEQ ID NO：75、B2MSEQ ID NO：74和HLA-E胞外区突变体SEQ ID NO：51，其中B2M的编码序列SEQ ID NO：82包含同义突变，对应E0质粒)或B2M和HLA-G胞外区的融合分子(包含B2M SEQ ID NO：74和HLA-G胞外区SEQ ID NO：49，其中B2M的编码序列SEQ ID NO：82包含同义突变，对应G0质粒)。在ECad0、E0和G0质粒中进一步包括CD28共刺激结构域(SEQ ID NO：26)，分别获得ECad28、E28和G28质粒。通过测序确认目标序列在质粒中的正确插入。
合成以下蛋白的编码序列，并将其依次克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号:PPL00157-4a)：B2m信号肽(SEQ ID NO：34)、NK抑制性配体、IgG4铰链区(SEQ ID NO：42)、CD8α跨膜区(SEQ ID NO：22)、CD28共刺激结构域(SEQ ID NO：26)，其中NK抑制性配体是抗NKG2A scFv(SEQ ID NO: 63，对应A28质粒)、抗KIR scFv(SEQ IDNO：67，对应KIRG4质粒)或抗LIR1 scFv(SEQ ID NO：70，对应LIR1-1质粒)。通过测序确认目标序列在质粒中的正确插入。
合成以下蛋白的编码序列，并将其依次克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号:PPL00157-4a)：CD8α信号肽(SEQ ID NO：36)、抗LIR1 scFv(SEQ ID NO：73)、CD28铰链区(SEQ ID NO：40)、CD28跨膜区(SEQ ID NO：24)、4-1BB共刺激结构域(SEQ ID NO：28)，获得LIR1-2质粒。通过测序确认目标序列在质粒中的正确插入。
在无菌管中加入3ml Opti-MEM(Gibco，货号31985-070)稀释上述质粒后，再根据质粒:病毒包装载体：病毒包膜载体＝4:2:1的比例加入包装载体psPAX2(Addgene，货号12260)和包膜载体pMD2.G(Addgene，货号12259)。然后，加入120ul X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，于室温下孵育15min，然后将质粒/载体/转染试剂混合物逐滴加入到293T细胞的培养瓶中。在24小时和48小时收集病毒，将其合并后，超速离心(25000g，4℃，2.5小时)获得浓缩的慢病毒。
用DynaBeads CD3/CD28 CTSTM(Gibco,货号40203D)激活T细胞，并在37℃和5％CO2下培养1天。然后，加入浓缩的慢病毒，持续培养3天后，获得表达NK抑制性分子的T细胞，即UNKi-T细胞。
然后采用CRISPR系统敲除野生型T细胞(Mock T细胞，用作对照)和所述UNKi-T细胞中的TCR/CD3组分(具体为TRAC基因)和MHC相关基因(具体为B2M和RFX5)，并用流式细胞仪确认各基因被有效敲除。
然后根据以下方法检测本发明制备的UNKi-T细胞对NK细胞杀伤作用的抑制效果：用Far-Red(invitrogen，货号C34564)标记本发明制备的UNKi-T细胞和Mock-T细胞。然后按照1x10 4个细胞/孔的浓度将标记好的UNKi-T细胞和Mock T细胞铺入96孔板，并 以2:1的效靶比加入NK92细胞(用于表达HLA-E胞外区、HLA-G胞外区、抗NKG2A scFv、抗KIR scFv或抗LIR1 scFv的UNKi-T细胞和Mock T细胞)或NK92-KLRG1细胞(用于表达E-钙黏素胞外区的UNKi-T细胞，通过将KLRG1基因引入NK92细胞来制备)进行共培养。16-18小时后，用流式细胞仪检测培养物中T细胞的比例，进而计算NK细胞对T细胞的杀伤效果，结果如图5所示。
从图5可以看出，与不表达NK抑制性分子的Mock T细胞相比，表达抑制性配体例如抗KIR scFv、抗LIR1 scFv、抗NKG2A scFv、HLA-G胞外区、HLA-E胞外区、E-钙黏素胞外区的UNKi-T细胞均能显著降低NK细胞对T细胞的杀伤作用。并且，与仅表达抑制性配体和跨膜结构域(即，不包含共刺激结构域)的T细胞(G0、E0、Ecad0)相比，共刺激结构域的加入可以进一步显著增强T细胞对NK细胞杀伤的抑制(G28、E28、ECad28)。因此，本发明所述的包含抑制性配体、跨膜结构域和共刺激结构域的NK抑制性分子可以显著降低NK细胞对UNKi-T细胞的杀伤作用，从而能够有效降低HvGD风险。
此外，在某些情况下，不仅需要抑制NK细胞对CAR-T细胞的杀伤，甚至进一步需要T细胞对NK细胞进行杀伤。因此，发明人在E28质粒和A28质粒细胞的基础上进一步包含了CD3ζ胞内信号传导结构域(SEQ ID NO：30)，并根据上述方法将其包装为慢病毒，并感染其中TCR/CD3组分(具体为TRAC基因)和MHC相关基因(具体为B2M和RFX5)被有效敲除的T细胞，获得E28z-UNKi-T细胞核A28z-UNKi-T细胞。
通过以下方法检测UNKi-T细胞对NK细胞的杀伤：以1x10 5个细胞/孔的浓度将靶细胞(NK92细胞)铺于96孔板中，然后每孔以1:1的比例加入Mock T细胞、E28z-UNKi-T细胞和A28z-UNKi-T细胞，同时加入10μl PE-anti-human CD107a(BD Pharmingen，货号555801)，于37℃，5％CO2培养条件下共培养。1h后，加入Goigstop(BD Pharmingen，货号51-2092KZ)继续孵育2.5小时。然后向每 孔加入5μl APC-anti human CD8(BD Pharmingen，货号：555369)和5μl FITC-anti human CD4(BD Pharmingen，货号：561005)，于37℃孵育30分钟后，用流式细胞仪检测CD107a的表达情况，结果如图6A(CD4+T细胞毒性)和图6B(CD8+T细胞毒性)所示。
可以看出，不表达NK抑制性分子的Mock T细胞对靶细胞几乎没有杀伤。与此相反，本发明制备的E28z-UNKi-T细胞和A28z-UNKi-T细胞与靶细胞共培养后，CD107a的表达率显著提高，表明本发明的UNKi-T细胞可显著杀伤NK细胞。
实施例4.制备表达NK抑制性分子的通用型CAR T细胞
合成编码以下蛋白的序列，并将其克隆至MSCV载体：CD8α信号肽(SEQ ID NO：36)、抗CD7 scFv(SEQ ID NO：21)、CD8α铰链区(SEQ ID NO：38)、CD8α跨膜区(SEQ ID NO：22)、4-1BB胞内区(SEQ ID NO：28)、CD3ζ胞内信号传导结构域(SEQ ID NO：30)、F2A、E-钙黏素胞外区(SEQ ID NO：48)、CD28铰链区(SEQ ID NO：40)、CD28跨膜区(SEQ ID NO：24)和CD28胞内区(SEQ ID NO：26)，并通过测序确认目标序列的正确插入。
在无菌管中加入3ml Opti-MEM(Gibco，货号31985-070)稀释上述质粒后，再根据质粒:病毒包装载体＝3:1的比例加入包装载体体pCL-Eco(上海禾午生物科技有限公司，货号P3029)。然后，加入120ul X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，于室温下孵育15min，然后将质粒/载体/转染试剂混合物逐滴加入到293GP细胞的培养瓶中。在72小时和96小时收集病毒，将其合并后，离心(2000rpm，4℃，10min)去碎片后获得逆转录病毒上清液。
参照实施例1中的敲除方式制备TCR/CD7双敲除的dKO T细胞和TCR/CD7/RFX5三敲除的tKO T细胞。未敲除基因的野生型T细胞(即，NT细胞)作为对照。
采用Retronectin包被24孔板4℃孵育过夜，然后移除溶液，加 入300μL含有5％FBS(Gibco，货号)的PBS溶液，室温放置30min。然后移除上清，用1mL PBS清洗板2次。每孔加入2mL逆转录病毒上清液和0.5M dKO-T细胞或tKO-T细胞，于2000g，32℃离心2h后放置二氧化碳培养箱中培养，获得CAR7-NKi-dKO T细胞和CAR7-NKi-tKO T细胞。
培养7天后，使用Biotin-SP(long spacer)AffiniPure Goat Anti-Mouse IgG,F(ab') 2Fragment Specific(min X Hu,Bov,Hrs Sr Prot)(jackson immunoresearch，货号115-065-072)作为一抗，APC Streptavidin(BD Pharmingen，货号554067)或PE Streptavidin(BD Pharmingen，货号554061)作为二抗，通过流式细胞仪检测CAR7-NKi-dKO T细胞和CAR7-NKi-tKO T细胞的CD7 scFv的表达水平，结果如图7所示。用E-cadherin monoclonal antibody(invitrogen货号13-5700)和Goat anti-Mouse IgG(H+L)Cross-Adsorbed Secondary Antibody,Alexa Fluor 488(Invitrogen，货号A-11001)检测CAR T细胞中的E-Cadherin表达,结果如图8所示。
可以看出，本发明制备的CAR T细胞中的抗CD7 scFv和NKi抑制性分子均可有效表达。
根据实施例2中2.1所述的方法检测CAR7-NKi-dKO T细胞和CAR7-NKi-tKO T细胞对Jurkat靶细胞的杀伤效果，结果如图9所示。可以看出，两种CAR-T细胞在各种效靶比下均能够显著杀伤靶细胞，并且在0.125：1的效靶比下，CAR7-NKi-tKO T细胞的杀伤效果优于CAR7-NKi-dKO T细胞。
根据实施例2中2.2所述的方法检测CAR7-NKi-dKO T细胞和CAR7-NKi-tKO T细胞分别与Jurkat靶细胞共培养后的细胞因子释放水平，结果如图10所示。可以看出，本发明的CAR7-NKi-dKO T细胞和CAR7-NKi-tKO T细胞的细胞因子释放水均显著高于对照的NT细胞，且CAR7-NKi-tKO T细胞组的释放水平显著高于CAR7-NKi-dKO T细胞组。
实施例5.制备表达NK抑制性分子的通用型CAR T细胞并验证其功能
合成编码以下蛋白的序列，并将其克隆至MSCV载体：CD8α信号肽(SEQ ID NO：36)、抗CD7 scFv(SEQ ID NO：21)、CD28铰链区(SEQ ID NO：40)、CD8α跨膜区(SEQ ID NO：22)、CD28胞内区(SEQ ID NO：26)、CD3ζ胞内信号传导结构域(SEQ ID NO：32)、F2A、PD-L1信号肽(SEQ ID NO：44)、PD-L1胞外区(SEQ ID NO：45)、CD28跨膜区(SEQ ID NO：24)、4-1BB胞内区(SEQ ID NO：28)，并通过测序确认目标序列的正确插入(质粒命名：CAR7-PDL1)。
合成编码以下蛋白的序列，并将其克隆至MSCV载体：CD8α信号肽(SEQ ID NO：36)、抗CD7 scFv(SEQ ID NO：21)、CD8α铰链区(SEQ ID NO：38)、CD28跨膜区(SEQ ID NO：24)、4-1BB胞内区(SEQ ID NO：28)、CD3ζ胞内信号传导结构域(SEQ ID NO：32)、F2A、B2M信号肽(SEQ ID NO：34)、HLA-E胞外区(SEQ ID NO：50)、CD28跨膜区(SEQ ID NO：24)、CD28胞内区(SEQ ID NO：26)，并通过测序确认目标序列的正确插入(质粒命名：CAR7-E)。
合成编码以下蛋白的序列，并将其克隆至MSCV载体：CD8α信号肽(SEQ ID NO：36)、抗CD7 scFv(SEQ ID NO：21)、CD8α铰链区(SEQ ID NO：38)、CD8α跨膜区(SEQ ID NO：22)、4-1BB胞内区(SEQ ID NO：28)、CD3ζ胞内信号传导结构域(SEQ ID NO：32)、F2A、B2M信号肽(SEQ ID NO：34)、HLA-E胞外区(SEQ ID NO：50)、连接肽(SEQ ID NO：64)、PD-L1胞外区(SEQ ID NO：45)、CD28跨膜区(SEQ ID NO：24)、CD28胞内区(SEQ ID NO：26)，并通过测序确认目标序列的正确插入(质粒命名：CAR7-EPDL1)。
根据实施例3所述的方法将上述质粒包装为逆转录病毒，并感染tKO-T细胞，分别获得CAR7-E T细胞、CAR7-PDL1 T细胞和 CAR7-EPDL1 T细胞。
培养7天后，使用Biotin-SP(long spacer)AffiniPure Goat Anti-human IgG,F(ab') 2Fragment Specific(min X Hu,Bov,Hrs Sr Prot)(jackson immunoresearch，货号109-065-097)作为一抗，APC Streptavidin(BD Pharmingen，货号554067)或PE Streptavidin(BD Pharmingen，货号554061)作为二抗，通过流式细胞仪检测三种细胞的scFv的表达水平，结果如图11所示。使用PE mouse anti-human HLA-E(biolegend，货号342604)和PE anti-human PDL1(biolegend，货号329706)分别检测CAR T细胞中的HLA-E和PDL1的表达,结果如图12和图13所示。
可以看出，本发明制备的CAR T细胞中的抗CD7 scFv和NK抑制性分子(HLA-E和PD-L1)均可有效表达。
根据实施例2中2.1所述的方法检测三种CAR-T细胞对Jurkat靶细胞的杀伤效果，结果如图14所示。可以看出，三种CAR-T细胞均能够显著杀伤靶细胞。
根据实施例2中2.2所述的方法检测三种CAR-T细胞分别与Jurkat靶细胞共培养后细胞因子释放水平，结果如图15所示。与NT组相比，三种CAR-T细胞细胞因子释放水平均显著升高。
实施例6.制备双靶点CAR-T细胞并验证其功能
合成编码以下蛋白的序列，并将其依次克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号:PPL00157-4a)：CD8α信号肽(SEQ ID NO：36)、抗CD7 scFv(SEQ ID NO：20)、连接肽(SEQ ID NO：64)、抗CD19 scFv(SEQ ID NO：54)、CD8α铰链区(SEQ ID NO：38)、CD8α跨膜区(SEQ ID NO：22)、4-1BB胞内区(SEQ ID NO：28)、CD3ζ胞内信号传导结构域(SEQ ID NO：32)并通过测序确认目标序列的正确插入。
按照实施例1的方法将上述质粒载体包装为慢病毒，并感染tkO-T细胞，获得CAR7-19 T细胞。未经修饰的野生型T细胞用作 阴性对照(NT)。
使用Biotin-SP(long spacer)AffiniPure Goat Anti-Mouse IgG,F(ab') 2Fragment Specific(min X Hu,Bov,Hrs Sr Prot)(jackson immunoresearch，货号115-065-072)或Biotin-SP(long spacer)AffiniPure Goat Anti-human IgG,F(ab') 2Fragment Specific(min X Hu,Bov,Hrs Sr Prot)(jackson immunoresearch，货号109-065-097)作为一抗，FITC Streptavidin(BD Pharmingen，货号554060)或PE Streptavidin(BD Pharmingen，货号554061)作为二抗，通过流式细胞仪检测CAR7-19 T细胞scFv的表达水平，结果如图16所示。
可以看出，本发明制备的CAR T细胞中的CD7 scFv和CD19 scFv均可以有效表达。
根据实施例2中的方法分别检测CAR7-19 T细胞的杀伤功能和细胞因子释放水平，结果分别如图17和图18所示。可以看出，CAR7-19 T细胞对Nalm6和Jurkat两种靶细胞均具有特异性杀伤和显著升高的细胞因子释放。
需要说明的是，以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。本领域技术人员理解的是，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (18)

  1. 一种工程化免疫细胞，其特征在于：(1)表达包含抗原结合区的嵌合抗原受体，所述抗原结合区包含抗CD7抗体；(2)内源性CD7、至少一种TCR/CD3基因和至少一种MHC-II类相关基因的表达被抑制或沉默。
  2. 权利要求1所述的工程化免疫细胞，其中所述嵌合抗原受体包含抗CD7抗体、跨膜结构域和胞内信号传导结构域。
  3. 权利要求1或2所述的工程化免疫细胞，其中所述抗CD7抗体包含分别如SEQ ID NO：1、2和3所示的CDR-L1、CDR-L2和CDR-L3，和如SEQ ID NO：4、5和6所示的CDR-H1、CDR-H2和CDR-H3。
  4. 权利要求1-3任一项所述的工程化免疫细胞，其中所述嵌合抗原受体的抗原结合区还包含靶向第二抗原的抗体或其功能性片段，所述第二抗原选自：TSHR、CD19、CD123、CD22、BAFF-R、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、GPRC5D、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-l lRa、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、AFP、Folate受体α、ERBB2(Her2/neu)、MUC1、EGFR、CS1、CD138、NCAM、Claudin18.2、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gploo、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD 179a、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、豆荚蛋白、HPV E6、E7、MAGE Al、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原 1、p53、p53突变体、前列腺特异性蛋白、存活蛋白和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2 ETS融合基因)、NA17、PAX3、雄激素受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D和它们的任意组合。
  5. 权利要求1-4任一项所述的工程化免疫细胞，其中所述嵌合抗原受体包含抗CD7抗体和抗CD19抗体。
  6. 权利要求1-5任一项所述的工程化免疫细胞，其中所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
  7. 权利要求1-6任一项所述的工程化免疫细胞，其中所述胞内信号传导结构域选自以下蛋白的胞内区：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。
  8. 权利要求1-7任一项所述的工程化免疫细胞，其中所述嵌合抗原受体还包含一个或多个共刺激结构域，其选自以下蛋白质的胞内区：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18(LFA-1)、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、 LIGHT、TRIM、ZAP70以及它们的组合。
  9. 权利要求1所述的工程化免疫细胞，其中所述TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ和它们的组合。
  10. 权利要求1所述的工程化免疫细胞，其中所述MHC-II类相关基因选自：HLA-DPA、HLA-DQ、HLA-DRA、RFX5、RFXAP、RFXANK、CIITA和它们的组合。
  11. 权利要求1-10任一项所述的工程化免疫细胞，其中所述工程化免疫细胞的内源性CD7、选自TRAC和TRBC的至少一种TCR/CD3基因和选自RFX5、RFXAP、RFXANK和CIITA的至少一种MHC-II类基因的表达被抑制或沉默。
  12. 权利要求1-11任一项所述的工程化免疫细胞，其中所述工程化免疫细胞进一步表达NK抑制性分子，所述NK抑制性分子包含一个或多个NK抑制性配体、跨膜结构域和共刺激结构域。
  13. 权利要求12所述的工程化免疫细胞，其中所述NK抑制性配体是靶向NK抑制性受体的抗体或其功能性片段，所述NK抑制性受体优选自NKG2A、NKG2B、CD94、LIR1、LIR2、LIR3、LIR5、LIR8、KIR2DL1、KIR2DL2/3、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、CEACAM1、LAIR1、NKR-P1B、NKR-P1D、PD-1、TIGIT、CD96、TIM3、LAG3、SIGLEC7、SIGLEC9、Ly49A、Ly49C、Ly49F、Ly49G1、Ly49G4和KLRG1。
  14. 权利要求12所述的工程化免疫细胞，其中所述NK抑制性配体是HLA-E、HLA-F、HLA-G、钙黏素、胶原蛋白、OCIL、唾液酸、PD-L1/PD-L2、CTLA-4、CD155、CD112、CD113、Gal-9、FGL1，或它们包含的NK抑制性受体结合区。
  15. 权利要求12所述的工程化免疫细胞，其中所述NK抑制性分子进一步包含CD3ζ胞内区作为胞内信号传导结构域。
  16. 权利要求1-15任一项所述的工程化免疫细胞，其中所述工程化免疫细胞是T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞。
  17. 一种药物组合物，其包含权利要求1-16任一项所述的工程化免疫细胞，和一种多种药学上可接受的赋型剂。
  18. 权利要求1-16任一项所述的工程化免疫细胞或权利要求17所述的药物组合物在制备用于治疗与CD7表达相关的疾病的药物中的用途。
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