WO2022171179A1 - 一种扩增增强子及其应用 - Google Patents

一种扩增增强子及其应用 Download PDF

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WO2022171179A1
WO2022171179A1 PCT/CN2022/075873 CN2022075873W WO2022171179A1 WO 2022171179 A1 WO2022171179 A1 WO 2022171179A1 CN 2022075873 W CN2022075873 W CN 2022075873W WO 2022171179 A1 WO2022171179 A1 WO 2022171179A1
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cell
cells
amino acids
mhc
cancer
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WO2022171179A9 (zh
WO2022171179A8 (zh
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黄倬
林彦妮
赵珣
郑小翠
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苏州克睿基因生物科技有限公司
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Priority to EP22752339.6A priority Critical patent/EP4293041A1/en
Priority to US18/276,145 priority patent/US20240117007A1/en
Priority to CN202280001808.4A priority patent/CN115066433A/zh
Publication of WO2022171179A1 publication Critical patent/WO2022171179A1/zh
Publication of WO2022171179A9 publication Critical patent/WO2022171179A9/zh
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    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
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    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to an amplification enhancer, in particular to a protein molecule comprising an intracellular domain, a transmembrane domain and an extracellular domain capable of triggering cellular STAT5 and/or STAT3 signaling pathways, and a protein molecule comprising the amplification Enhancer lymphocytes and their use as immunotherapy drugs.
  • Immune cell therapy such as chimeric antigen receptor T-cell (CAR-T) therapy
  • CAR-T chimeric antigen receptor T-cell
  • UAR-T universal CAR-T
  • T cells are derived from healthy donors and pre-prepared for use by patients
  • UAR-T universal CAR-T
  • the timeliness and standardization of drugs, and the cost is low.
  • the ability of allogeneic immune cells to survive and proliferate in vivo is limited. On the one hand, there is rejection among different individuals, and on the other hand, the low cytokine environment in the body is not conducive to the survival and expansion of universal CAR-T. Earthly limits the effectiveness of such treatments.
  • CN109952309 discloses a constitutively active cytokine receptor for cell therapy, especially a constitutively active IL-7 cytokine formed from the transmembrane and intracellular domains of IL-7R ⁇ and the extracellular domain of CD34
  • the receptor C7R promotes the in vivo expansion of T cells for use in the treatment of medical conditions including cancer.
  • CD34 is very large, and its use in combination with CAR will affect the expression efficiency and stability of CAR molecules in T cells, and also affect the preparation and large-scale production of products.
  • T cells for general-purpose CAR-T needs to solve the problem of bidirectional rejection between allogenes.
  • the current preparation technology of CAR-T removes the expression of the TCR complex of the donor T cell to prevent the donor T cell from recognizing and killing the host cell; on the other hand, removes the expression of the class I MHC molecule of the donor T cell, Can eliminate donor cell antigen presentation, escape the recognition and killing of host T cells (CN 106103475A). But the absence of class I MHC molecules triggers receptor NK cell-mediated killing.
  • the amplification enhancer comprises the following components: a) one or more cytokine receptor intracellular domains capable of initiating signaling in the cell through the STAT5 and/or STAT3 pathway; b) a transmembrane structure domain; c) one or more extracellular domains, the extracellular domain is the extracellular domain of DAP12 or a variant thereof; wherein the transmembrane domain and the extracellular domain comprise a homologue that promotes the formation of the amplification enhancer The structure of the source dimerization.
  • the present invention further provides a genetically engineered cell, characterized in that the cell surface expresses the amplification enhancer of the present invention.
  • the present invention provides a CAR-T cell expressing the amplified enhancer of the present invention.
  • cytokines such as IL-2
  • the ability to expand the gene-edited CAR-T cells was improved.
  • the present invention further provides a CAR-T cell that simultaneously expresses the amplification enhancer of the present invention and a chimeric single-chain molecule;
  • the chimeric single-chain molecule comprises: (a) a presentation peptide, ( b) a B2M protein, and (c) a linker sequence for linking the aforementioned (a) and (b) fragments, wherein the chimeric single-chain molecule and the heavy chain molecule of the MHC or MHC analog are on the cell membrane A complex is formed, and the presented peptide segment is a polypeptide sequence of 5-30 amino acids.
  • An amplification enhancer comprising the following components: a) one or more intracellular domains of cytokine receptors capable of triggering signaling in cells through the STAT5 and/or STAT3 pathway; b ) a transmembrane domain; c) one or more extracellular domains, the extracellular domain being an extracellular domain of DAP12 or a variant thereof; wherein the transmembrane domain and the extracellular domain are comprised of facilitating said expansion Enhancers form homodimerized structures.
  • the amplification enhancer of claim 1, wherein the receptor intracellular domain is from IL-7 cytokine receptor alpha, IL-21 cytokine receptor alpha, IL-23 cytokine receptor alpha , IL-12 cytokine receptor alpha, CD122, or a combination thereof.
  • the amplification enhancer of claim 1, wherein the receptor intracellular domain is the amino acid at positions 47-241 of SEQ ID No: 3, and the amino acids at positions 47-245 of SEQ ID No: 4 Amino acids, amino acids 47-251 of SEQ ID No:5 or amino acids 47-266 of SEQ ID No:6.
  • transmembrane domain is the endogenous transmembrane domain of one or more cytokine receptor intracellular domains of component a) or Variants of the endogenous transmembrane domain.
  • transmembrane domain is an IL-7 cytokine receptor alpha transmembrane domain or a variant thereof, or the transmembrane domain is the transmembrane domain of DAP12 or a variant thereof.
  • transmembrane domain comprises at least one cysteine.
  • transmembrane domain is an amino acid sequence of 21-33 in length.
  • transmembrane domain is amino acids 19-46 of SEQ ID No: 3, or the transmembrane domain is SEQ ID NO: 11, Alternatively, the transmembrane domain is amino acids 20-40 of SEQ ID NO: 15.
  • the extracellular domain is the extracellular domain of DAP12 shown in amino acids 7-18 of SEQ ID No: 3, or is A polypeptide sequence comprising the extracellular domain of DAP12, or a variant of the extracellular domain of DAP12 derived by substitution, deletion or addition of one or several amino acids to the extracellular domain of DAP12.
  • a genetically engineered cell wherein the cell surface expresses the amplification enhancer of any one of claims 1-10.
  • lymphocytes are T cells, NKT cells, ⁇ T cells, mucosa-associated invariant T cells, NK cells, or B cells.
  • TCR T cell receptor
  • T cell is a CAR-T cell or a TCR-T cell.
  • the cell has a nucleic acid sequence expressing a chimeric single-chain molecule, the chimeric single-chain molecule comprising: (a) presenting a peptide segment, (b) B2M protein, and (c) a linker sequence for linking the aforementioned (a) and (b) fragments; wherein the chimeric single-chain molecule is complexed with a heavy chain molecule of MHC or an MHC analog on the cell membrane body; the presentation peptide segment is a polypeptide sequence of 5-30 amino acids.
  • N-terminal to C-terminal of the chimeric single-chain molecule sequentially comprises: a presenting peptide segment-linking sequence-B2M protein, or a B2M protein-linking sequence-presenting peptide segment.
  • the heavy chain molecule of MHC is endogenously expressed in the cell and is selected from the heavy chain molecule of classical class I MHC molecules, preferably HLA-A, HLA-B or A heavy chain molecule of HLA-C; or a heavy chain molecule of MHC that is endogenously expressed in cells and selected from the group of non-classical MHC class I molecules, preferably HLA-E, HLA-F or HLA-G
  • the heavy chain molecule; or the heavy chain molecule of MHC is endogenously expressed in the cell and is selected from heavy chain molecules of other non-classical MHC analogs, preferably heavy chain molecules of CD1 or MR1 or UL18.
  • a method of treating cancer comprising administering to an individual the genetically engineered cells of claims 12-23.
  • cancer small bowel cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer or thyroid cancer.
  • Cytokines refer to cell signaling molecules that regulate the immune system's response to inflammation and infection and aid in intercellular communication in the immune response.
  • Amplification enhancers contain one or more intracellular domains.
  • the intracellular domain can be derived from the same molecule as the transmembrane domain, but is otherwise not the case.
  • the intracellular domain comprises binding sites for STAT5 and STAT3, and thus can function in the signaling pathways of STAT5 and STAT3.
  • Immunostimulatory cytokine intracellular domains of receptors useful in the context of the present invention are derived from e.g. IL-7 cytokine receptor alpha, IL-21 cytokine receptor alpha, IL-23 cytokine receptor alpha, IL-12 cells Factor receptor alpha, CD122, or a combination thereof.
  • the intracellular domain comprises the intracellular domain from the alpha chain of the IL7 receptor; preferably amino acids 47-241 of SEQ ID NO:3, amino acids 47-245 of SEQ ID NO:4, Amino acids 47-251 of SEQ ID NO:5 and amino acids 47-266 of SEQ ID NO:6.
  • the intracellular domain is 70-250 amino acids, 70-200 amino acids, 80-100 amino acids, 90-250 amino acids, 90-100 amino acids, 100-250 amino acids, 100 amino acids in length -125 amino acids, 125-150 amino acids, 150-175 amino acids, 175-200 amino acids, 200-225 amino acids, etc.
  • these fragments of these lengths retain STAT5 and STAT3 signaling activity.
  • Amplification enhancers of the present invention comprise a transmembrane domain operably linked to the extracellular and intracellular domains.
  • the transmembrane domain may be derived from the same natural molecule as the intracellular domain to which it is operably linked, or it may be derived from a different natural molecule.
  • the transmembrane domain comprises one or more structures that cause or promote homodimerization, such as disulfide bonds.
  • the transmembrane domain utilizes mutations identified in tumor patients as gain-of-function mutations in the transmembrane domain.
  • mutant forms include cysteine insertions that induce disulfide bond formation in the transmembrane domain.
  • one or more of the mutations lack insertion of cysteine.
  • transmembrane domain derivatives can be used which do not have cysteine insertions (and thus no disulfide bonds) but still signal and are constitutively active, for example because mutations make the transmembrane domain different from the transmembrane structure The domain undergoes a conformational change from the native form, allowing for the induction of signaling.
  • the transmembrane domain is from the IL-7R ⁇ receptor and the mutation in the transmembrane domain is in the sequence PILLTISILSFSVALLVILACVLW (SEQ ID NO: 11).
  • the transmembrane domain is a transmembrane domain from DAP12 or a variant thereof, such as the transmembrane region in the amino acid sequence of DAP12 with Genbank accession NP_003323.1 (SEQ ID NO: 15 20-40 amino acids).
  • the mutation is or comprises insertion of one or more cysteines and/or one or more prolines into amino acids 20-40 of SEQ ID NO: 11, or SEQ ID NO: 15
  • the amino acid sequence of wherein the mutation enables or promotes homodimerization of the receptor.
  • the mutation involves insertion of a trimeric peptide of cysteine, proline, threonine (CPT) into the transmembrane domain.
  • This mutation confers disulfide bond formation between the -SH (thiol) groups of the cysteine residues of two molecules (as an example, the two IL7RP2 receptor alpha chains), allowing the formation of homology between them Dimers (in specific embodiments, the proline next to the cysteine helps to twist the homodimer into the correct orientation).
  • the threonine inserted by the CPT is not threonine but another amino acid, and in at least certain instances, the other amino acid may or may not be cysteine or proline.
  • the insertion may be at SEQ ID NO: 11 or SEQ ID NO: 15: Between any two amino acids of amino acids 20-40 of 15.
  • the insertion is located at the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, first in SEQ ID NO: 11 twelve, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third or after the twenty-fourth amino acid.
  • a known disclosed transmembrane domain can be used, such as the transmembrane domain disclosed in Chinese Patent CN201780065147.0 (referred to as TM sequence in the patent).
  • the extracellular domain of the amplification enhancer of the present invention is derived from the extracellular domain of DAP12, corresponding to amino acids 7-18 of SEQ ID NO: 3, and SEQ ID NOs: 3, 4, 5 and 6 have the same extracellular domain .
  • the DAP12 gene is a transmembrane receptor that is widely present on the surface of natural killer cells, granulocytes, and monocytes/macrophages and plays an important role in transmitting active signals.
  • the transmembrane protein DAP12 consists of a short extracellular domain, a single-transmembrane transmembrane domain, and an intracellular domain containing an immunotyrosine activation motif (ITAM). DAP12 can form a disulfide bond-containing homodimer.
  • ITAM immunotyrosine activation motif
  • the ectodomain of the invention is the ectodomain of DAP12 or a variant thereof.
  • a variant of the extracellular domain of DAP12 is a polypeptide sequence comprising amino acids 7-18 of SEQ ID NO: 3, eg, a fusion polypeptide in which the extracellular domain of DAP12 is combined with other functional fragments.
  • a variant of the DAP12 ectodomain may also be a DAP12 ectodomain derived from amino acids 7-18 of SEQ ID NO: 3 by substitution, deletion or addition of one or several amino acids.
  • the length of the extracellular domain is no more than 130 amino acids; preferably no more than 120 amino acids; preferably no more than 40 amino acids; preferably no more than 20 amino acids; preferably the length of the extracellular domain is 30-120 amino acids , 30-100 amino acids, 30-90 amino acids, 30-70 amino acids, 30-60 amino acids, 25-60 amino acids, 30-50 amino acids, 35-45 amino acids, 36-42 amino acids, 38 -40 amino acids, 39-40 amino acids, or 39 amino acids, 10-40 amino acids, 12-20 amino acids, or 12 amino acids.
  • a variant in the present invention refers to a sequence that is at least 80%, 83%, 85%, 86% identical to the sequence, such as the extracellular domain of DAP12, the IL-7 cytokine receptor alpha transmembrane domain, etc. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to sequences, and have the same or similar function; or a protein sequence with the same or similar function obtained by deletion, substitution or addition of one or several amino acids to the sequence; preferably the variant has 10 or 9 of the sequence , 8, 7, 6, 5, 4, 3, 2 or 1 amino acid deletion, substitution or addition.
  • the degree of identity between two amino acid sequences is determined using the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), It is preferably determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in version 3.0.0 or higher.
  • the optional parameters used were a gap penalty of 10, a gap extension penalty of 0.5 and the EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62).
  • Amplification enhancers of the present invention are constitutively active cytokine receptors that have no ligand requirement, and their transmembrane structure/or intracellular domain components are configured to The ectodomain can transmit an activation signal when it receives the corresponding signal.
  • the enhancers of the present invention exist in a homodimerized form such that the extracellular domain remains in a state that transmits activation signals to intracellular signaling pathways.
  • the amplification enhancer has the amino acid sequence of Ev1-5 set forth in SEQ ID NO: 3, 4, 5, 6 or 15.
  • Ev1-5 represents a sequence of amplifying enhancers Ev1, Ev2, Ev3, Ev4 and Ev5.
  • MHC class I molecules also known as class I major histocompatibility complexes, are heterodimeric glycoproteins composed of two non-covalently linked peptide chains; one of them, called the heavy chain, is structurally polymorphic , and the other is a light chain or called ⁇ 2 microglobulin (B2M). Functionally, class I MHC molecules present intracellularly degraded peptides that are not self-proteins, thereby activating the immune system.
  • Human MHC molecules are also known as HLA molecules.
  • Classical class I MHC molecules also called MHC-Ia
  • Non-classical MHC class I molecules also called MHC-Ib
  • HLA-E HLA-E
  • HLA-F HLA-G.
  • MHC analog molecules also referred to as MHC molecular analogs in this patent, refer to the complexes formed by non-covalent bonding of heavy chains and B2M proteins, which do not belong to class I MHC molecules, but are structurally similar to class I MHC molecules.
  • non-classical MHC-like molecules including but not limited to CD1, MR1.
  • Another example is the human cytomegalovirus HCMV-derived UL18 protein, which has homology with the heavy chain of class I MHC molecules, and binds to B2M to form a complex that is expressed on the cell membrane, allowing the cell to escape the killing of NK cells.
  • chimeric single-chain molecule expressed on the cell surface of the present invention, from the N-terminus to the C-terminus, is: presenting peptide segment-connecting sequence-B2M protein, or B2M protein-connecting sequence-presenting peptide segment .
  • Chimeric single-chain molecules have the effect of inhibiting rejection.
  • the T cells of the universal CAR-T come from healthy donors and can be prepared in advance for use by any patient, but the problem of two-way rejection between allogenes needs to be solved.
  • the current preparation technology of universal CAR-T removes the expression of the TCR complex of the donor T cells to prevent the donor T cells from recognizing and killing the host cells; on the other hand, removes the expression of the class I MHC molecules of the donor T cells , can eliminate donor cell antigen presentation and escape the recognition and killing of host T cells (CN 106103475 A). But the absence of class I MHC molecules triggers receptor NK cell-mediated killing.
  • the engineered universal immune cells of the present invention by expressing a specific type of presentation peptide (such as a presentation peptide that specifically binds to HLA-E/HLA-G/HLA-C) and a chimeric single-chain molecule coupled with B2M, it can be Avoid the killing of allogeneic (or autologous) NK cells and other immune cells, and improve the compatibility of allogeneic transplantation.
  • a specific type of presentation peptide such as a presentation peptide that specifically binds to HLA-E/HLA-G/HLA-C
  • B2M chimeric single-chain molecule coupled with B2M
  • the chimeric single-chain molecule is: a membrane-localized signal peptide-presenting peptide segment-(G4S)3-B2M mature protein, wherein the membrane-localized signal peptide is removed before the chimeric molecule is localized to the cell membrane.
  • presenting peptide is also referred to as “presenting peptide” and “presenting polypeptide” in the present invention.
  • Presenting peptides are a class of short amino acid peptides, which can be stably combined with the antigen-binding groove of MHC molecules to form a stable MHC complex.
  • Most of the presenting peptides are 7-30 amino acids in length, preferably 7-17 amino acids, more preferably 7-12 amino acids, most preferably 8-10 amino acids.
  • the "presenting peptide-coupled B2M protein" ie, a chimeric single-chain molecule
  • presenting peptide-coupled B2M protein ie, a chimeric single-chain molecule
  • a specific peptide segment that matches the target HLA heavy chain can be selected depending on the expressed target HLA. For example, selecting a presentation peptide segment matched to the HLA-C heavy chain can make the chimeric single-chain molecule heavy with HLA-C.
  • the complex molecule formed by the chain is stably presented on the cell membrane; for another example, selecting the presenting peptide segment matched with the HLA-E heavy chain can make the complex formed by the chimeric single-chain molecule and the HLA-E heavy chain stably presented on the cell membrane.
  • the presenting polypeptides are derived from the signal peptides of class I MHC molecules, such as HLA-A2, HLA-B7, HLA-B15, HLA-Cw3, Signal peptides of HLA-Cw7, HLA-G, HLA-F, etc.; proteins derived from viral protein polypeptides, such as CMV (UL40), EBV, HIV, etc.
  • most of the presented polypeptides derived from the signal peptides of class I MHC molecules have the following general structure: VM(A/P)PRT(L/V)(V/L/I/F)L or V (T/A)(A/P)PRT(L/V)(V/L/I/F)L, the general formula represents a peptide segment of 9 amino acids, wherein “()" represents the same amino acid position, The "/" symbol stands for "or”.
  • the signal peptide derived from a class I MHC molecule is one of the following sequences:
  • HLA-E complexes The presenting peptides that can be bound by HLA-E complexes are also listed in the following documents: Celik et al. Immunogenetics 2016. 68: 29-41; Hannoun et al. Immunology Letters 2018. 202: 65-72; et al. Cell Rep 2018.14(8):1967-1976; et al. Front Immunol 2018. 9:2410. For example a 10 amino acid peptide: YLLPRRGPRL.
  • Beta-2 microglobulin also known as B2M protein, is the light chain of MHC class I molecules and is an indispensable part of MHC class I molecules.
  • the human B2M protein consists of 119 amino acids (SEQ ID NO: 7) and has a molecular weight of 11800 Daltons. The absence of B2M molecules prevents cells from expressing class I MHC molecules normally on the cell membrane. Such cells are recognized and killed by NK cells.
  • CAR Chimeric Antigen Receptor
  • scFv single domain antibody/nanobody
  • transmembrane structure connecting extracellular and intracellular structures and intracellular signaling domain or domain binding to signaling domain/complex, mainly containing T cells
  • Activation signal and co-stimulatory signal which can contain one or two co-stimulatory molecules, common co-stimulatory molecules are CD28, CD137 (4-1BB), CD27, OX40, CD30, CD40 and so on.
  • Extracellular antigen receptors are mainly derived from related tumor antigens and can be selected from the following antigens: CD19, CD20, CD22, CD123, CD33/IL3Ra, CD138, CD33, BCMA, CS1, C-Met, EGFRvIII, CEA, Her2 , GD2, MAG3, GPC3, NY-ESO-1, etc.
  • Absence of a molecule on the cell surface means that the level of the molecule detected on the cell surface is reduced compared to the level in normal physiological conditions.
  • the expression of the molecule is detected on the cell surface by using the molecule-specific antibody to decrease or below the detection limit; or after the cell membrane is separated, the molecule cannot be detected by Western blot technology, or the expression of the molecule is detected to decrease.
  • Endogenously expressed molecules refer to molecules expressed by cells without modification or transformation. For example, in one embodiment, cells without virus transfection, gene editing, or RNA interference, etc. The molecules expressed by the unengineered cells are referred to as endogenously expressed molecules.
  • the deletion of at least one class I MHC molecule on the cell surface refers to the deletion of one or more of HLA-A/B/C/E/F/G and other molecules; the deletion of at least one MHC on the cell surface Molecular analogs refer to, including but not limited to, deletion of one or more of CD1, MR1, UL18 and other molecules. If cells lack B2M molecules, the level of MHC class I molecules or MHC molecule analogs on the cell surface is reduced. In one embodiment, deletion of at least one endogenously expressed MHC molecule or MHC molecule analog on the cell surface can be achieved by inactivating B2M or the corresponding heavy chain gene. Removing or partially removing the expression of MHC class I molecules in donor cells can reduce or eliminate donor cell antigen presentation and escape the recognition and killing of host T cells, which is especially beneficial for improving the compatibility of allografts.
  • Immune cells are a variety of cells that are produced and mature in the immune system and have immune response functions.
  • the immune cells of this patent may be a mixture of one or more of lymphocytes, macrophages, stem cells, progenitor cells or immune effector cells.
  • the lymphocytes can be one of T cells, NKT cells, ⁇ T cells, mucosa-associated invariant T cells (MAIT cells), NK cells, B cells, tumor-infiltrating lymphocytes (TILs) and innate lymphocytes or Various mixtures.
  • TILs tumor-infiltrating lymphocytes
  • the T cell can also be a T cell comprising a chimeric antigen receptor (CAR-T cell) or a TCR-T cell.
  • CAR-T cell chimeric antigen receptor
  • TCR-T cell chimeric antigen receptor
  • Universal immune cells refer to immune cells that can be used in allogeneic transplantation and that do not produce or produce a controlled GvHD response.
  • a method of treating cancer comprising administering to an individual suffering from said cancer an effective amount of a genetically engineered cell of the invention, particularly administering a CAR-T cell expressing a CAR-T cell specific for the cancer, wherein the CAR T cell otherwise expresses Amplification enhancers of the present invention.
  • the cancer is a solid tumor, including, for example, glioblastoma.
  • the CAR T cells target the antigen GD2.
  • the amplification enhancer comprises a transmembrane domain derived from IL-7 receptor alpha, wherein the transmembrane domain comprises one or more mutations that promote cytokine receptor homodimerization.
  • the present invention further provides the application of the genetically engineered cells in the preparation of medicines for treating cancer.
  • the cancer is glioblastoma.
  • the cancer is breast cancer, prostate cancer, lung cancer (eg, small cell lung cancer or non-small cell lung cancer), brain cancer, colon cancer, head and neck cancer, skin cancer (eg, melanoma cancer), ovarian, endometrial, cervix, kidney, stomach, small bowel, liver, pancreas, gallbladder, bile duct, esophagus, salivary gland or thyroid cancer.
  • FIG. 1 Schematic diagram of the co-expression of Ev1-5 protein and CM protein (Ev1-5 is localized to the cell membrane and self-assembles into a dimer by the extracellular domain and transmembrane domain, and CM and HLA-E heavy chain molecules assemble into an HLA-E protein complex )
  • Example 1 Generation of universal CAR-T cells co-expressing amplifying enhancers and chimeric molecules (CMs)
  • Step 1 Construction of DNA expressing the amplified enhancer
  • the DNA sequences of 5 kinds of following amplification enhancers were synthesized and expressed respectively, and the 5' end of the following sequences was added with a DNA sequence expressing a signal peptide that helps cell membrane localization, and the amino acid sequence of the signal peptide was SEQ ID NO: 14.
  • the amino acid sequence of amplified enhancer 1 is SEQ ID NO: 3, wherein the intracellular domain is the amino acids 47-241 of SEQ ID NO: 3, and the transmembrane domain is 19-46 of SEQ ID NO: 3 , the extracellular domain is amino acids 7-18 of SEQ ID NO: 3.
  • the amino acid sequence of amplified enhancer 2 is SEQ ID NO: 4, wherein the intracellular domain is amino acids 47-245 of SEQ ID NO: 4, and the transmembrane domain is 19-46 of SEQ ID NO: 4 , the extracellular domain is amino acids 7-18 of SEQ ID NO:4.
  • the amino acid sequence of amplified enhancer 3 is SEQ ID NO: 5, wherein the intracellular domain is amino acids 47-251 of SEQ ID NO: 5, and the transmembrane domain is 19-46 of SEQ ID NO: 5 , the extracellular domain is amino acids 7-18 of SEQ ID NO: 5.
  • the amino acid sequence of amplified enhancer 4 is SEQ ID NO:6, wherein the intracellular domain is amino acids 47-266 of SEQ ID NO:6, and the transmembrane domain is 19-46 of SEQ ID NO:6 , the extracellular domain is amino acids 7-18 of SEQ ID NO: 6.
  • the amino acid sequence of amplified enhancer 5 is SEQ ID NO: 15, wherein the intracellular domain is amino acids 44-242 of SEQ ID NO: 15, and the transmembrane domain is 19-43 of SEQ ID NO: 15 , the extracellular domain is amino acids 7-18 of SEQ ID NO: 15.
  • CJP molecule Construct an anti-CD19 CAR molecule (called CJP molecule), amplifying enhancer (denoted by Ev) and chimeric molecule (denoted by CM) co-expressed lentiviral vector, viral expression plasmid (Addgene ID: #12252)
  • Ev anti-CD19 CAR molecule
  • CM chimeric molecule
  • viral expression plasmid Adgene ID: #12252212
  • BamHI/SalI digestion as the backbone, the core gene structure is CJP-Ev-CM connected in sequence, with 2A self-cleaving peptides (P2A and T2A) in the middle.
  • the CJP sequence is SEQ ID NO: 1.
  • CJP is connected to one of the 5 variants of the amplified enhancer (represented by Ev1/2/3/4/5) by the GSG-P2A sequence, and then by the GSG-T2A sequence and the CM connect.
  • the CJP-P2A-(Ev1-5)-T2A-CM sequence or the CJP-P2A-C7R-T2A-CM whole gene was synthesized and linked into the digested viral expression backbone (Fig. 1).
  • Eight viral expression plasmids containing CJP, amplifying enhancers and chimeric molecules were constructed:
  • the target expression plasmid containing CJP-P2A-C7R-T2A-CM is a target expression plasmid containing CJP-P2A-C7R-T2A-CM
  • the target expression plasmid containing CJP The target expression plasmid containing CJP
  • the C7R sequence as a control is derived from patent CN109952309, amino acid sequence SEQ ID NO: 2.
  • the amino acid sequence of Ev1 is SEQ ID NO:3, the amino acid sequence of Ev2 is SEQ ID NO:4, the amino acid sequence of Ev3 is SEQ ID NO:5, and the amino acid sequence of Ev4 is SEQ ID NO:6.
  • the amino acid sequence of Ev5 is SEQ ID NO:15.
  • the amino acid sequence of CM is SEQ ID NO:7.
  • P2A and T2A are self-cleaving polypeptide linking sequences, which are commonly used linking sequences for the simultaneous expression of two independent proteins in one transcript in a vector. During translation, the proteins are split at the ends of the P2A and T2A sequences, so that the P2A and T2A sequences are separated from each other. The two proteins before and after T2A are connected are separated and function respectively.
  • the amino acid sequence of P2A is ATNFSLLKQAGDVEENPGP (SEQ ID NO: 12), and the amino acid sequence of T2A is EGRGSLLTCGDVEENPGP (SEQ ID NO: 13).
  • lentiviral target expression plasmid target expression plasmid constructed in step 2
  • packaging helper plasmids psPAX2 Additional helper plasmids
  • psPAX2 Additional helper plasmids
  • pMD2.G Additional helper plasmids
  • Virus packaging was performed in HEK293T cells (purchased from Shanghai Institute of Cell Research, Chinese Academy of Sciences). The preparation process is as follows: the HEK293T cells in the cryopreserved working cells are recovered, placed in a 10cm culture dish with DMEM medium (+10%FBS+1%P/S) (Cellgro 10-013-CMR), and the second Change the fluid after a few days.
  • DMEM medium +10%FBS+1%P/S
  • the cells After the cells become confluent, the cells can be passaged (usually, 1 petri dish can be transferred to 5 petri dishes), and the plasmids can be transfected after the cells are passaged for 4 generations.
  • PEI as the transfection reagent
  • Opti-MEM medium Gibco, cat#31985-070
  • this mixed solution was added to HEK293T cells passaged to passage 4.
  • the medium was replaced with fresh medium of 2% FBS, and then the culture was continued to 72 hours, and the supernatant of HEK293T cells was collected.
  • the collected virus supernatant was concentrated by ultracentrifugation (82200g, 4-8°C centrifugation for 2 hours), and the concentrated virus was filtered and sterilized with a 0.22 ⁇ m filter membrane, and then resuspended for use.
  • T cells were derived from the peripheral blood (PBMC) of healthy human volunteers.
  • the medium used was complete medium, ImmunoCult TM -XF T Cell Expansion Medium (Stem Cell Technology, cat #10981) + 300 IU/ml IL2 (Cayan, cat #HEILP-0201c).
  • Supplement medium DMEM medium (+10% FBS+1% P/S)
  • Step 6 sgRNA candidate sequences
  • the Cas9 protein selected in this example is the Alt-R s.p.Cas9 Nuclease 3NLS protein of IDT DNA technology company.
  • Step 9 Detection of gene transduction efficiency and gene editing efficiency
  • the efficiency of primary T cell editing was examined 3 days after completion of the shock. Take out a small amount of cells, add 1mL PBS (Gibco, cat#C10010500BT) to wash the cells once, resuspend with 100 ⁇ L PBS, add 3 ⁇ L anti-FMC63 primary antibody (to detect CAR19), incubate at 4°C for 30min, add 1mL PBS to wash the cells, 300g Centrifuge for 3 min and remove the supernatant.
  • PBS Gibco, cat#C10010500BT
  • Resuspend cells in 100 ⁇ L PBS add 0.5 ⁇ L secondary antibody to detect CAR19, 3 ⁇ L anti-HLA-ABC antibody, 3 ⁇ L antibody CD3 antibody (to detect gene editing efficiency) and 3 ⁇ L anti-HLA-E antibody to the cells, mix well, and incubate at 4°C 30min. After washing, use a flow cytometer to detect on the machine.
  • Example 2 Compared with the prior art C7R, Ev1-5 can effectively improve the lentiviral packaging and toxin production efficiency of CAR-X-CM (X refers to C7R or Ev1-5)
  • the length of the inserted gene will affect the packaging and toxin production efficiency of the virus.
  • the CAR gene is about 1.5kb. If a gene sequence larger than 1.5kb is added, it is close to the load of the lentiviral expression gene (the additional promoter is about 1.5-2kb, and the terminator WPRE is about 0.5kb). Although it can be packaged Toxicity is produced, but the toxin production efficiency will be significantly reduced, which is not conducive to large-scale lentivirus preparation, and affects the yield and subsequent transfection effect.
  • the Ev1-5 gene length (786bp-816bp) is much smaller than C7R (1539bp)
  • viruses prepared by the lentivirus preparation method in steps 1-3 of Example 1 including CJP-P2A-C7R-T2A-CM, CJP-P2A-Ev1-T2A-CM, CJP-P2A-Ev2-T2A-CM , CJP-P2A-Ev3-T2A-CM, CJP-P2A-Ev4-T2A-CM, CJP-P2A-Ev5-T2A-CM target expression plasmid lentivirus.
  • the comparison of toxin production efficiency does not require purification of virus, but directly performs titer detection on the supernatant of lentiviral packaging.
  • the biological titer of the lentiviral supernatant was calculated by detecting the positive ratio of CAR molecules.
  • Cultivate 293T cells digest the cultured 293T cells with TrypLE Express Enzyme (1x) and phenol red (gibco, 12605-028) (the medium is DMEM medium containing 10% FBS), adjust the cell density, and inoculate to 24
  • the inoculation amount of each well is 2.5E5 cells, and each well is 1.5ml of medium, and then placed in a 37°C 5% CO 2 incubator for 24 hours.
  • Washing Add 1ml PBS to each tube to resuspend the cells, centrifuge at 350g for 5min, and discard the supernatant.
  • washing After incubation, add 1ml PBS to each tube, mix well, centrifuge at 350g for 5min, discard the supernatant, repeat twice, and resuspend with 100 ⁇ l PBS.
  • the cells in the NC wells were divided into 3 groups: the first group was not stained. After the cells were digested and collected, they were washed once with PBS and then resuspended in 100 ⁇ l of PBS, marked as NC not stained. In the second group, no primary antibody was added, only secondary antibody was added to exclude specific staining, which was marked as NC PE-strep. The third group of normal staining, marked as NC staining
  • On-machine detection first collect NC unstained samples, perform voltage adjustment and gate setting, and then collect alternate samples in turn to obtain CAR expression rate results.
  • Example 3 Compared with the prior art C7R, Ev1-5 can effectively improve the transfection efficiency of universal CAR-T cells
  • the primary T cells were transfected at the same MOI after lentiviruses of each group were purified.
  • the preparation method of lentivirus is the same as that of step 3 of Example 1, and five kinds of viruses of Example 2 are prepared.
  • Example 4 Compared with the prior art C7R, Ev1-5 can effectively improve the stability of CAR expression in universal CAR-T cells
  • CAR is a protein expressed on the cell membrane. After the CAR gene is transcribed and translated, it is transported to the surface of the cell membrane through vesicles.
  • the expression of CAR is a dynamic process of cell membrane display-endocytosis.
  • the stability of CAR expression is crucial to the function of CAR-T cells. If the expression of CAR on the cell membrane is blocked due to factors such as expression efficiency and endocytosis speed, the activity of CAR-T cells will be seriously affected. Therefore, we further compared the effects of C7R and Ev1-5 on the stability of CAR expression.
  • the experimental method was the same as in Example 3. After the fifth day of the experiment, the dynamic expression changes of CAR during the preparation and expansion of universal CAR-T cells were continued to be tracked. As shown in Figures 3-1, 3-2 and 3-3, on the 5th, 8th and 11th days, the positive ratio of CAR in each group was detected, and the positive ratio of CAR in the CJP-P2A-C7R-T2A-CM group gradually decreased. Compared with the initial transfection efficiency, the other groups maintained a stable expression ratio.
  • Figure 4 is the statistical data of each group. The results show that the introduction of C7R makes the expression efficiency of CAR in primary T cells unstable, and the expression tends to decline with the culture time. The introduction of Ev1-5 can stabilize the stability of CAR molecules. Express.
  • Example 5 Ev1-5 can effectively activate STAT5 and/or STAT3 signaling
  • STAT5 and STAT3 are important signaling proteins in the IL-7 and IL-2 cytokine signaling pathways.
  • pSTAT5 and pSTAT3 will activate a series of downstream genes, which is beneficial to the survival and proliferation of T cells.
  • Ev1-5 can form a dimer and efficiently activate STAT5 and STAT3 signaling.
  • the expression of pSTAT5 and pSTAT3 were used as assays for each group of cells.
  • the positive control cells (T cells expressing CJP) were treated with 600IU of IL-2 for 30min in advance to activate the expression of pSTAT5 and pSTAT3. Cells of other groups were not treated.
  • Anti-pSTAT5 (pY694) (BD Pharmingen, 612567) or Anti-pSTAT3 (pY705) (BD Pharmingen, 612569) antibody and incubate at room temperature for 30-60 minutes. Avoid light.
  • the purpose of this example is to detect the expression of pSTAT5 and pSTAT3 in each group of cells.
  • each group of CAR-T cells was prepared by using Jurkat cell line.
  • Jurkat cell is a common model cell for studying T cell signal transduction.
  • pSTAT5 For pSTAT5, as shown in Figure 5-1, most cells in the positive control group expressed pSTAT5 after being activated by IL-2, and were not related to the expression of CAR ( Figure 5-1A); the prior art C7R group ( Figure 5-1D) ), the expression of pSTAT5 can be detected in some CAR-positive cells, and the expression efficiency is 21.9%, of which the expression of pSTAT5 in CAR-positive cells accounts for about 46.7%; Ev1-5 groups also have effective pSTAT5 expression, accounting for the total 33.8%, 43.8%, 36.0%, 10.0%, and 11.4% of the cells. Except for Ev4 and Ev5, the expression ratio of pSTAT5 in CAR-positive cells in other groups was similar to that of C7R, ranging from 45% to 55%.
  • Example 6 Ev1-5 can effectively improve the in vitro expansion ability of universal CAR-T cells
  • Example 1 was used to prepare universal CAR-T cells in each group (U denotes universal T cells), and cultured until the 14th day.
  • 1E5CAR-positive cells were selected from each group for in vitro tumor cell-stimulated proliferation assay, and CD19-positive Raji cells were selected for tumor cells, and Raji cells stably expressed GFP fluorescent protein to distinguish CAR-T cells from tumor cells.
  • Example 1 Sample 100ul every 2-3 days, and use the method of Example 1 for flow cytometry detection of CAR-positive cells, that is, add 1mL of PBS (Gibco, cat#C10010500BT) to 100uL of detection solution to wash the cells once, and resuspend with 100 ⁇ L of PBS , 3 ⁇ L of anti-FMC63 primary antibody (to detect CAR19) and 0.5 ⁇ L of secondary antibody were added to the cells in sequence, mixed well, and incubated at 4°C for 30 min. After washing, use a flow cytometer on the machine for detection. On the 7th day, repeated stimulation of tumor cells was carried out, 1E5 Raji cells were injected into each group, and the medium was supplemented to 1 mL, and the above test was repeated subsequently.
  • PBS Gibco, cat#C10010500BT
  • the expansion ability of CAR-positive cells in each group was not significantly different after the first tumor stimulation. After repeated stimulation on the 7th day, the expansion of the U-CJP group was limited and was in a state of slow proliferation.
  • U-CJP-P2A-CM cells with chimeric molecular CM expanded to a certain extent after repeated stimulation on the 7th day, about 40-50 times in 14 days. about 70 times; the introduction of C7R can significantly improve the cell expansion ability, which can reach 80-100 times after repeated stimulation on the 7th day; in contrast, Ev1-4 can significantly improve the cell expansion ability.
  • the 14th day ranged from 70-130 times.
  • U-CJP means universal T cells express CAR molecule CJP
  • U-CJP-P2A-CM means universal CAR-T cells express CAR molecule CJP and chimeric molecule CM
  • U-CJP-P2A-Ev1-T2A-CM means universal Type CAR-T cells express the CAR molecule CJP, the amplifying enhancer Ev1, and the chimeric molecule CM.
  • Example 7 Universal CAR-T cells expressing Ev molecules have stronger expansion ability than universal CAR-T cells expressing C7R molecules in the context of immune rejection
  • the experimental strategy with the same number of CAR positive cells in each group was adopted, so the CAR positive rate of 14.8% did not affect the subsequent functional evaluation.
  • HLA-E can effectively inhibit the toxic response of NK cells and T by interacting with NKG2A/CD94, and the expression of HLA-E by universal CAR-T cells can effectively alleviate the killing of NK cells and allogeneic PBMCs. Therefore, universal CAR-T cells expressing higher abundance of HLA-E molecules can enhance the resistance of universal CAR-T cells to immune rejection.
  • the detection method is as in Example 4, that is, the average fluorescence expression abundance of HLA-E is analyzed in HLA-ABC, CD3 double-negative, and CAR-positive cells.
  • the MFI expression of HLA-E in U-CJP cells is 523, which belongs to the negative background expression. 5 times.
  • the HLA-E expression of U-CJP-P2A-C7R-T2A-CM was 1891, which was 3.6 times the background expression.
  • the HLA-E expression of U-CJP-P2A-Ev1-T2A-CM and U-CJP-P2A-Ev2-T2A-CM groups were 4335 and 5478, respectively, which were 8.3 and 10.5 times higher than background.
  • the introduction of Ev1/2 significantly increased the expression abundance of HLA-E, suggesting that this group of general-purpose CAR-T cells may have a stronger ability to resist rejection and killing.
  • This test is a scientific simulation of the in vivo environment. Since the immune cells in the body are complex, not only including NK cells and target cells, the establishment of a real allogeneic PBMC environment can be closer to the in vivo environment. The ability of the edited CAR-T cells to expand in the context of allogeneic PBMCs was examined, in which there were both allogeneic immune rejection (not only source NK cells), but also target cells (tumor Raji cells).
  • Allogeneic PBMC and Raii cells were stained with Dye eFluor TM 670 before co-culture to differentiate universal CAR-T cells: adjust the cell density to 1E7/ml, add e670 dye at a final concentration of 10 ⁇ M, incubate at room temperature for 5 min in the dark, and incubate After washing three times, the base was used for the experiment.
  • the rejected PBMCs came from 3 different healthy donors, and the data of donors 1, 2, and 3 are shown in Figures 9-1, -2, and -3, respectively.
  • donor 1 and the PBMC of the prepared universal CAR-T cells belong to the same source, that is, belong to autologous, and the others are allogeneic.

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Abstract

涉及一种扩增增强子，该增强子包含一种能引发细胞STAT5和/或STAT3信号传导途径的胞内结构域、跨膜结构域和细胞外结构域的蛋白分子；还涉及表达该扩增增强子的淋巴细胞及其作为免疫治疗药物的用途。

Description

一种扩增增强子及其应用 技术领域
本发明涉及一种扩增增强子，尤其是一种包含能引发细胞STAT5和/或STAT3信号传导途径的胞内结构域、跨膜结构域和细胞外结构域的蛋白分子，以及包含该扩增增强子的淋巴细胞及其作为免疫治疗药物的用途。
背景技术
免疫细胞疗法，如嵌合抗原受体T细胞(CAR-T)疗法在癌症治疗中显示出了良好的治疗效果。现有的自体CAR-T技术需要个性化的细胞制备，受限于生产周期长、成本高以及在许多情况下缺乏足够的患者T细胞。通用免疫细胞疗法，如通用CAR-T(UCAR-T)疗法，其T细胞来源于健康供体，并预先准备好供患者使用的细胞，生产过程可以实现标准化和统一化，临床应用可实现给药的及时性和规范性，且成本低。然而，同种异体免疫细胞在体内的生存和增殖能力有限，一方面是不同个体间存在排异，二是体内低细胞因子的环境，不利于通用型CAR-T的存活和扩增，这极大地限制了此类治疗的有效性。
CN109952309公开了一种用于细胞治疗的组成型活性细胞因子受体，尤其是来自IL-7Rα的跨膜和胞内结构域与CD34的胞外结构域所形成的组成型活性IL-7细胞因子受体C7R能促进T细胞的体内扩增，用作医学病症包括癌症的治疗。但是该方案中，CD34非常大，与CAR组合使用会影响T细胞中CAR分子的表达效率和稳定性，也影响产品的制备和规模化生产。
另一方面，通用型CAR-T的T细胞使用需要解决异体间双向排异问题。目前型CAR-T的制备技术，一方面，去除供体T细胞TCR复合体的表达，以防止供体T细胞识别杀伤宿主细胞；另一方面去除供体T细胞的I类MHC分子的表达，可以消除供体细胞抗原呈递，逃离宿主T细胞的识别和杀伤(CN 106103475A)。但I类MHC分子的缺失会引发受体NK细胞介导的杀伤。
因此，基因工程的免疫细胞在克服同种异体移植排斥反应的同时，如何能够提高扩增和存续能力并增强持久的抗肿瘤活性，是细胞治疗领域长久以来未得到满足的需求。
发明内容
为了提高淋巴细胞的扩增能力，本发明提供了一种扩增增强子。该扩增增强子包含以下组分：a)一个或多个细胞因子受体胞内结构域，该胞内结构域能引发细胞中通过STAT5和/或STAT3途径的信号传导；b)跨膜结构域；c)一个或多个胞外结构域，该胞外结构域是DAP12的胞外结构域或其变体；其中跨膜结构域和胞外结构域包含促进所述扩增增强子形成同源二聚化的结构。
本发明进一步提供了一种基因工程改造的细胞，其特征在于，该细胞表面表达本发明所述的扩增增强子。
在一个具体实施方式中，本发明提供了一种表达本发明扩增增强子的CAR-T细胞。在无细胞因子(如IL-2)条件下，该基因编辑后的CAR-T细胞的扩增能力得到了提高。
在一个具体实施方式中，本发明进一步提供了同时表达本发明扩增增强子和一种嵌合单链分子的CAR-T细胞；该嵌合单链分子包含：(a)呈递肽段，(b)B2M蛋白，和(c)连接序列，该连接序列用于连接前述(a)和(b)片段，其中，所述嵌合单链分子与MHC或MHC类似物的重链分子在细胞膜上形成复合体，所述呈递肽段为5-30个氨基酸的多肽序列。
本发明公开了以下序号所示的技术方案：
1.一种扩增增强子，包含以下组分：a)一个或多个细胞因子受体胞内结构域，该胞内结构域能引发细胞中通过STAT5和/或STAT3途径的信号传导；b)跨膜结构域；c)一个或多个胞外结构域，该胞外结构域是DAP12的胞外结构域或其变体；其中跨膜结构域和胞外结构域包含促进所述扩增增强子形成同源二聚化的结构。
2.权利要求1所述的扩增增强子，其中所述的受体胞内结构域来自IL-7细胞因子受体α、IL-21细胞因子受体α、IL-23细胞因子受体α、IL-12细胞因子受体α、CD122或其组合。
3.权利要求1所述的扩增增强子，其中所述的受体胞内结构域是序列为SEQ ID No：3的第47-241位氨基酸，SEQ ID No：4的第47-245位氨基酸，SEQ ID No：5的第47-251位氨基酸或SEQ ID No：6的第47-266位氨基酸。
4.前述任一项权利要求所述的扩增增强子，其中所述的跨膜结构域是组分a)的一个或多个细胞因子受体胞内结构域的内源跨膜结构域或内源跨膜结构域的变体。
5.前述任一项权利要求所述的扩增增强子，其中所述的跨膜结构域是IL-7细胞因子受体α跨膜结构域或其变体，或者所述的跨膜结构域是DAP12的跨膜结构域或其变 体。
6.前述任一项权利要求所述的扩增增强子，其中所述的跨膜结构域包含至少一个半胱氨酸。
7.前述任一项权利要求所述的扩增增强子，其中所述的跨膜结构域的长度为21-33的氨基酸序列。
8.权利要求7所述的扩增增强子，其中所述的跨膜结构域是SEQ ID No：3的第19-46位氨基酸，或者所述的跨膜结构域是SEQ ID NO：11，或者所述的跨膜结构域是SEQID NO：15的第20-40位氨基酸。
9.前述任一项权利要求所述的扩增增强子，其中所述的胞外结构域是序列为SEQ IDNo：3的第7-18位氨基酸所示的DAP12的胞外结构域，或者是包含DAP12的胞外结构域的多肽序列，或者是对DAP12的胞外结构域经过一个或几个氨基酸的取代、缺失或添加而衍生的DAP12胞外结构域的变体。
10.权利要求9所述的扩增增强子，其中所述的胞外结构域的长度不超过130个氨基酸；优选不超过120个氨基酸；优选不超过40个氨基酸；优选不超过20个氨基酸；优选胞外结构域的长度为10-40个氨基酸、12-20个氨基酸。
11.一种表达前述任一项权利要求所述的扩增增强子的核酸序列。
12.一种基因工程改造的细胞，其特征在于，该细胞表面表达权利要求1-10任一项所述的扩增增强子。
13.权利要求12所述的细胞，其中，该细胞是免疫细胞，进一步该免疫细胞优选为淋巴细胞。
14.权利要求13所述的细胞，其中，所述淋巴细胞是T细胞、NKT细胞、γδT细胞、粘膜相关不变T细胞、NK细胞或B细胞。
15.权利要求12-14任一项所述的细胞，其中，所述的细胞表面缺失至少一种内源性表达的MHC分子或MHC分子类似物。
16.权利要求12-15任一项所述的细胞，其中，所述细胞中至少一个编码内源T细胞受体(TCR)的组件的基因失
17.权利要求12-16任一项所述的细胞，其中所述的T细胞是一种CAR-T细胞或TCR-T细胞。
18.权利要求12-17任一项所述的细胞，其中所述的细胞具有表达嵌合单链分子的核酸序列，所述嵌合单链分子包含：(a)呈递肽段，(b)B2M蛋白，和(c)连接序列，该连接序列用于连接前述(a)和(b)片段；其中，所述嵌合单链分子与MHC或MHC类似 物的重链分子在细胞膜上形成复合体；所述呈递肽段为5-30个氨基酸的多肽序列。
19.权利要求18所述的细胞，其中所述的嵌合单链分子N端到C端依次包含：呈递肽段-连接序列-B2M蛋白，或B2M蛋白-连接序列-呈递肽段。
20.权利要求18或19所述的细胞，其中，MHC的重链分子是细胞内源性表达的且选自经典的I类MHC分子的重链分子，优选为HLA-A，HLA-B或HLA-C的重链分子；或MHC的重链分子是细胞内源性表达的且选自非经典的I类MHC分子的重链分子，优选为HLA-E，HLA-F或HLA-G的重链分子；或MHC的重链分子是细胞内源性表达的且选自其他非经典MHC类似分子的重链分子，优选CD1或MR1或UL18的重链分子。
21.权利要求18-20任一项所述的细胞，其中，所述呈递肽段来源于I类MHC分子的信号肽。
22.权利要求17-21任一项所述的细胞，其中所述的细胞缺失内源性表达的MHC分子，并缺失内源性表达的T细胞受体。
23.权利要求22所述的细胞，其中所述的细胞表达的嵌合单链分子与MHC或MHC类似物的重链分子在细胞膜上形成复合体。
24.权利要求12-23任一项所述的细胞在制备治疗癌症的药物中的应用。
25.一种治疗癌症的方法，所述方法包括向个体施用权利要求12-23所述的基因工程改造的细胞。
26.权利要求24或25所述的方法和应用，其中的癌症是乳腺癌、前列腺癌、肺癌、脑
癌、结肠癌、头颈癌、皮肤癌、卵巢癌、子宫内膜癌、子宫颈癌、肾癌、肺癌、胃
癌、小肠癌、肝癌、胰腺癌、胆囊癌、胆管癌、食道癌、唾液腺癌或甲状腺癌。
本发明的详述
定义
细胞因子
细胞因子是指细胞信号传导分子，用于调节免疫系统对炎症和感染的应答并帮助免疫应答中的细胞间通信。
胞内结构域
扩增增强子包含一个或多个胞内结构域。胞内结构域可以来自与跨膜结构域相同的分子，但是在其他情况下并非如此。在具体的实施方案中，胞内结构域包含STAT5和 STAT3的结合位点，因此能STAT5和STAT3的信号传导途径中起作用。可用于本发明内容的受体的免疫刺激细胞因子胞内结构域来自例如IL-7细胞因子受体α、IL-21细胞因子受体α、IL-23细胞因子受体α、IL-12细胞因子受体α、CD122或其组合。
在特定实施方案中，胞内结构域包含来自IL7受体α链的胞内结构域；优选SEQ ID NO：3的第47-241位氨基酸，SEQ ID NO：4的第47-245位氨基酸，SEQ ID NO：5的第47-251位氨基酸和SEQ ID NO：6的第47-266位氨基酸。
在一些实施方案中，胞内结构域的长度为70-250个氨基酸、70-200个氨基酸、80-100个氨基酸、90-250个氨基酸、90-100个氨基酸、100-250个氨基酸、100-125个氨基酸、125-150个氨基酸、150-175个氨基酸、175-200个氨基酸、200-225个氨基酸等等。在具体实施方案中，这些长度的这些片段保留STAT5和STAT3信号传导活性。
跨膜结构域
本发明的扩增增强子包含与胞外结构域和胞内结构域可操作地连接的跨膜结构域。跨膜结构域可以来自与其可操作连接的胞内结构域相同的天然分子，也可以来自不同的天然分子。在具体实施方案中，跨膜结构域包含一个或多个引起或促进同源二聚化的结构，如二硫键。
在一些情况下，跨膜结构域利用在肿瘤患者中鉴定的突变作为跨膜结构域中的功能获得突变。在至少一些情况下，突变体形式包括诱导跨膜结构域中的二硫键形成的半胱氨酸插入。然而，在其他实施方案中，一个或多个突变缺乏半胱氨酸的插入。例如，可以使用跨膜结构域衍生物，其不具有半胱氨酸插入(并因此没有二硫键)但仍然发出信号并且是组成型活性的，例如因为突变使得跨膜结构域与跨膜结构域的天然形式相比发生了构象变化，从而允许诱导信号传导。
在具体的实施方案中，跨膜结构域来自IL-7Rα受体，跨膜结构域中的突变是在序列PILLTISILSFFSVALLVILACVLW(SEQ ID NO：11)中。在另一具体的实施方案中，跨膜结构域是来自DAP12的跨膜结构域或其变体，例如Genbank编号为NP_003323.1的DAP12氨基酸序列中的跨膜区(SEQ ID NO：15的第20-40位氨基酸)。在某些实施方案中，突变是或包括将一个或多个半胱氨酸和/或一个或多个脯氨酸插入SEQ ID NO：11，或SEQ ID NO：15的第20-40位氨基酸的氨基酸序列，其中所述突变使得受体能够同源二聚化或促进受体的同源二聚化。在某些情况下，突变包括将半胱氨酸、脯氨酸、苏氨酸(CPT)的三聚体肽插入跨膜结构域。这种突变赋予两个分子(作为实例，两个IL7RP2受体α链)的半胱氨酸残基的-SH(巯基)基团之间的二硫键形成，允许在它们之间 形成同源二聚体(在具体的实施方案中，紧接着半胱氨酸的脯氨酸有助于将同源二聚体扭转成正确的方向)。在具体的实施方案中，CPT插入的苏氨酸不是苏氨酸而是另一种氨基酸，并且在至少特定情况下，其他氨基酸是或不是半胱氨酸或脯氨酸。在其中将一个或多个氨基酸插SEQ ID NO：11或SEQ ID NO：15的第20-40位氨基酸以用于受体的实施方案中，插入可以在SEQ ID NO：11或SEQ ID NO：15的第20-40位氨基酸的任意两个氨基酸之间。在特定实施方案中，插入位于SEQ ID NO：11中的第一、第二、第三、第四、第五、第六、第七、第八、第九、第十、第十一、第十二、第十三、第十四、第十五、第十六、第十七、第十八、第十九、第二十、第二十一、第二十二、第二十三或第二十四个氨基酸之后。
在具体实施方式中，可以采用已知公开的跨膜结构域，例如中国专利CN201780065147.0中公开的跨膜结构域(专利中称为TM序列)。
胞外结构域
本发明的扩增增强子的胞外结构域来自DAP12的胞外域，对应SEQ ID NO：3的第7-18位氨基酸，SEQ ID NO：3，4，5和6具有相同的胞外结构域。
DAP12基因是一个跨膜受体，广泛存在于自然杀伤细胞、粒细胞、单核/巨噬细胞表面，在传递活性信号中发挥重要作用。跨膜蛋白DAP12由一个较短的胞外区、一个单跨膜的跨膜区以及含有免疫酪氨酸激活基元(ITAM)的胞内区这三部分组成，DAP12能形成含二硫键的同源二聚体。
在具体实施方式中，本发明的胞外结构域为DAP12的胞外域或其变体。DAP12胞外域的变体是包含SEQ ID NO：3的第7-18位氨基酸的多肽序列，例如DAP12的胞外域与其他功能片段结合的融合多肽。DAP12胞外域的变体也可以是对SEQ ID NO：3的第7-18位氨基酸经过一个或几个氨基酸的取代、缺失或添加而衍生的DAP12胞外结构域。
其中所述的胞外结构域的长度不超过130个氨基酸；优选不超过120个氨基酸；优选不超过40个氨基酸；优选不超过20个氨基酸；优选胞外结构域的长度为30-120个氨基酸，30-100个氨基酸，30-90个氨基酸，30-70个氨基酸，30-60个氨基酸，25-60个氨基酸、30-50个氨基酸、35-45个氨基酸，36-42个氨基酸、38-40个氨基酸、39-40个氨基酸或39个氨基酸，10-40个氨基酸、12-20个氨基酸，或12个氨基酸。
变体
本发明中的变体是指与所述序列，例如DAP12的胞外结构域、IL-7细胞因子受体α跨膜结构域等序列，具有至少80％，83％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性(identity)的序列，并且有相同或相似的功能；或者是与所述序列具有一个或几个氨基酸的缺失、取代或添加而得到的具有相同或相似功能的蛋白序列；优选变体与所述序列具有10个，9个，8个，7个，6个，5个，4个，3个，2个或1个的氨基酸的缺失、取代或添加。
由参数“同一性”描述两个氨基酸序列之间的相关性。
就本发明而言，两个氨基酸序列之间的同一性程度使用如EMBOSS软件包(EMBOSS：The European Molecular Biology Open Software Suite，Rice等，2000，Trends in Genetics 16：276-277)的Needle程序，优选3.0.0版或更高版本中执行的Needleman-Wunsch算法(Needleman和Wunsch，1970，J.Mol.Biol.48：443-453)来确定。使用的任选参数为缺口罚分(gap penalty)10，缺口延伸罚分(gap extension penalty)0.5和EBLOSUM62取代矩阵(BLOSUM62的EMBOSS版)。使用Needle标记为“最高同一性(longest identity)”(使用-nobrief选项获得)的输出结果作为百分比同一性，并计算如下：
(相同的残基×100)/(比对长度-比对中缺口的总数)。
扩增增强子
本发明的扩增增强子是一种组成型活性细胞因子受体，该细胞因子受体没有配体要求，它们的跨膜结构/或胞内结构域组分被配置成在没有从它们的胞外结构域接收相应信号的情况下就能传递活化信号。本发明的增强子是以同源二聚化的形式存在，从而使得胞外结构域保持在将活化信号传递到胞内信号传导途径的状态。
在具体实施方案中，扩增增强子具有SEQ ID NO：3，4，5，6或15所示的Ev1-5的氨基酸序列。Ev1-5表示扩增增强子Ev1、Ev2、Ev3、Ev4和Ev5中的一个序列。
I类MHC分子
I类MHC分子，也称为I类主要组织相容复合体，由非共价键连接的两条肽链组成的异源二聚体糖蛋白；其中一条称为重链，结构呈多态性，另一条为轻链或称为β2微球蛋白(B2M)。功能上，I类MHC分子会呈递胞内所降解的非自身蛋白的多肽，从而激活免疫系统。人类的MHC分子又称为HLA分子。经典的I类MHC分子(也叫做MHC-Ia)，包括HLA-A，HLA-B，HLA-C。非经典的I类MHC分子(也叫做MHC-Ib)，包括HLA-E，HLA-F，HLA-G。
MHC类似分子
MHC类似分子，本专利也称MHC分子类似物，指不属于I类MHC分子，但结构类似于I类MHC分子，也由重链和B2M蛋白通过非共价键结合形成的复合体。比如非经典MHC类似分子，包括但不限于CD1，MR1。又如人巨细胞病毒HCMV来源UL18蛋白，与I类MHC分子的重链具有同源性，与B2M结合形成复合体表达在细胞膜上，可以使该细胞逃避NK细胞的杀伤。
嵌合单链分子(Chimeric single chain molecule，CM)
本发明细胞表面表达的嵌合单链分子(又称为“嵌合分子”)，N端到C端依次为：呈递肽段-连接序列-B2M蛋白，或者B2M蛋白-连接序列-呈递肽段。嵌合单链分子(CM)具有抑制排异的作用。
通用型CAR-T的T细胞来自于健康供体，可以提前制备好供任意病人使用，但需要解决异体间双向排异问题。目前通用型CAR-T的制备技术，一方面，去除供体T细胞TCR复合体的表达，以防止供体T细胞识别杀伤宿主细胞；另一方面去除供体T细胞的I类MHC分子的表达，可以消除供体细胞抗原呈递，逃离宿主T细胞的识别和杀伤(CN 106103475 A)。但I类MHC分子的缺失会引发受体NK细胞介导的杀伤。本发明在工程化通用型免疫细胞中，通过表达特异类型的呈递肽(如与HLA-E/HLA-G/HLA-C特异结合的呈递肽)与B2M偶联的嵌合单链分子，能避免异体(或自体)NK细胞其他免疫细胞的杀伤，提异体移植的相容性。
在具体的实施方式中，该嵌合单链分子为：膜定位信号肽-呈递肽段-(G4S)3-B2M成熟蛋白，其中的膜定位信号肽在嵌合分子定位到细胞膜之前被去除。
呈递肽段
“呈递肽段”在本发明中也称为“呈递肽”、“呈递多肽”。呈递肽段是一类氨基酸短肽，可以与MHC分子的抗原结合槽稳定结合，从而形成稳定的MHC复合体，呈递肽段长度多数在7-30个氨基酸，优选7-17个氨基酸，更优选7-12个氨基酸，最优选8-10个氨基酸。呈递肽段只有与MHC分子的抗原结合槽匹配，才能使“呈递肽偶联的B2M蛋白”(即，嵌合单链分子)与MHC的重链分子形成复合体，稳定地在细胞上呈现，进而被免疫细胞识别。不同的MHC分子具备独特匹配的呈递肽段。
特别的，根据所表达的目的HLA不同，可选择与目的HLA重链匹配的特异性肽 段，如选择HLA-C重链所匹配的呈递肽段可以使嵌合单链分子与HLA-C重链形成的复合体分子在细胞膜上稳定呈现；又如选择HLA-E重链所匹配的呈递肽段，可以使嵌合单链分子与HLA-E重链形成复合体在细胞膜上稳定呈现。特别的，对于能与HLA-E重链分子形成复合体的嵌合分子，该呈递多肽来源于I类MHC分子的信号肽，如HLA-A2，HLA-B7，HLA-B15，HLA-Cw3，HLA-Cw7，HLA-G，HLA-F等的信号肽；来源于病毒蛋白多肽，如CMV(UL40)，EBV，HIV等的蛋白多
在具体的实施方式中，来源于I类MHC分子的信号肽的呈递多肽多数具有如下通式结构：VM(A/P)PRT(L/V)(V/L/I/F)L或V(T/A)(A/P)PRT(L/V)(V/L/I/F)L，该通式代表了9个氨基酸的肽段，其中“()”代表同一个氨基酸位置，“/”符号代表“或”。
优选，来源于I类MHC分子的信号肽是选自下述序列中的一个：
Val Met Ala Pro Arg Thr Leu Ile Leu，
Val Met Ala Pro Arg Thr Leu Val Leu，
Val Met Ala Pro Arg Thr Leu Phe Leu，
Val Met Ala Pro Arg Thr Leu Leu Leu，
Val Met Ala Pro Arg Thr Val Leu Leu，和
Val Thr Ala Pro Arg Thr Leu Val Leu。
在下述文献中，也列举了HLA-E复合物可结合的呈递肽段：Celik et al.Immunogenetics 2016.68：29-41；Hannoun et al.Immunology Letters 2018.202：65-72；
Figure PCTCN2022075873-appb-000001
et al.Cell Rep 2018.14(8)：1967-1976；
Figure PCTCN2022075873-appb-000002
et al.Front Immunol 2018.9：2410。例如10个氨基酸的肽段：YLLPRRGPRL。
B2M
β-2微球蛋白，也称为B2M蛋白，是I类MHC分子的轻链，是I类MHC分子不可缺少的一部分。人类B2M蛋白由119个氨基酸(SEQ ID NO：7)组成，并具有11800道尔顿的分子量。B2M分子的缺失使得细胞无法正常在细胞膜上表达I类MHC分子。这种细胞会被NK细胞识别并杀伤。
嵌合抗原受体(CAR)
CAR(Chimeric Antigen Receptor)是指一段特异性的抗原受体上同时表达一些胞内的激活信号，主要由3个部分组成：在胞外表达的抗原受体，主要是来自于单链抗体的可变区(scFv)或单域抗体/纳米抗体，连接胞外与胞内结构的跨膜结构及胞内信号传导 的结构域或与信号传导结构域/复合体结合的结构域，主要含有T细胞激活信号与共刺激信号，其中共刺激分子可以包含一个或两个，常见的共刺激分子有CD28，CD137(4-1BB)，CD27，OX40，CD30，CD40等。胞外的抗原受体主要是来自于相关肿瘤抗原的，可选自如下抗原：CD19，CD20，CD22，CD123，CD33/IL3Ra，CD138，CD33，BCMA，CS1，C-Met，EGFRvIII，CEA，Her2，GD2，MAG3，GPC3，NY-ESO-1等。
细胞表面缺失至少一种内源性表达的MHC分子或MHC分子类似物
细胞表面缺失某种分子是指在细胞表面检测到该分子的水平与正常生理状态下的水平相比降低。例如利用该分子特异性抗体在细胞表面检测到该分子的表达下降或低于检测限；或分离细胞膜后，利用Western blot技术无法检测到该分子，或检测该分子表达下降。内源性表达的分子，是指细胞在没有经过修饰、改造的情况下表达的分子，例如在一种实施方式中，细胞在没有经过病毒转染，基因编辑，或RNA干扰等改造处理时，该未经过改造的细胞表达的分子称为内源性表达的分子。
在具体的实施方式中，细胞表面缺失至少一种I类MHC分子是指缺失HLA-A/B/C/E/F/G等分子中的一种或多种；细胞表面缺失至少一种MHC分子类似物是指，包括但不限于缺失CD1、MR1、UL18等分子中的一种或多种。如果细胞缺失B2M分子，会使得在细胞表面的I类MHC分子或MHC分子类似物水平降低。在一种实施方式中，细胞表面缺失至少一种内源性表达的MHC分子或MHC分子类似物可以通过使B2M或相应的重链基因失活来实现。去除或部分去除供体细胞的I类MHC分子的表达，可以降低或消除供体细胞抗原呈递，逃离宿主T细胞的识别和杀伤，对于提高异体移植的相容性尤其有利。
免疫细胞
免疫细胞是产生和成熟于免疫系统，具有免疫应答功能的多种类细胞。本专利的免疫细胞可以是淋巴细胞、巨噬细胞、干细胞、祖细胞或免疫效应细胞中的一种或多种的混合物。
进一步，所述淋巴细胞可以是T细胞、NKT细胞、γδT细胞、粘膜相关不变T细胞(MAIT细胞)、NK细胞、B细胞、肿瘤浸润淋巴细胞(TIL)和先天淋巴细胞中的一种或多种的混合物。
在具体实施方案中，T细胞也可以是一种包含嵌和抗原受体的T细胞(CAR-T细胞)或TCR-T细胞。
通用型免疫细胞是指可以应用于异体移植的、不产生或产生可控的GvHD反应的免疫细胞。
药用用途
本文提供了治疗癌症的方法，其包括向患有所述癌症的个体施用有效量的本发明的基因工程改造的细胞，尤其是施用表达对癌症特异的CAR-T细胞，其中CAR T细胞另外表达本发明的扩增增强子。在更具体的实施方案中，癌症是实体瘤，包括例如胶质母细胞瘤。在另一个具体实施方案中，CAR T细胞靶向抗原GD2。在更具体的实施方案中，扩增增强子包含衍生自IL-7受体α的跨膜结构域，其中跨膜结构域包含一个或多个促进细胞因子受体同源二聚化的突变。
本发明进一步提供了基因工程改造的细胞在制备治疗癌症的药物中的应用。
在本文提供的任何治疗方法的具体实施方案中，癌症是成胶质细胞瘤。在本文提供的方法的某些具体实施方案中，癌症是乳腺癌、前列腺癌、肺癌(例如，小细胞肺癌或非小细胞肺癌)、脑癌、结肠癌、头颈癌、皮肤癌(例如，黑色素瘤)、卵巢癌、子宫内膜癌、子宫颈癌、肾癌、胃癌、小肠癌、肝癌、胰腺癌、胆囊癌、胆管癌、食道癌、癌症唾液腺或甲状腺癌。
附图说明
图1.表达载体示意图。
图2.Ev1-5蛋白和CM蛋白共表达示意图(Ev1-5定位于细胞膜依靠胞外域和跨膜域自组装成二聚体，CM与HLA-E重链分子组装成HLA-E蛋白复合体)
图3.流式细胞术检测原代T细胞CAR表达效率和表达稳定性分析。
图4.原代T细胞CAR表达效率动态分析(Day5，Day8，Day11)。
图5.流式细胞术检测pSTAT5和pSTAT3表达。
图6.通用型CAR-T细胞在无细胞因子环境中的扩增。
图7.流式细胞术检测通用型CAR-T细胞基因编辑效率和CAR表达效率。
图8.通用型CAR-T细胞HLA-E表达丰度检测。
图9.通用型CAR-T细胞在不同来源的具有免疫抑制环境的PBMC中增殖的检测。
具体实施方式
以下用实施例对本发明作进一步阐述。这些实施例仅仅用于举例说明，而不对本 发明的范围构成任何限制。
缩写词意义如下：“h”指小时，“min”指分钟，“s”指秒，“ms”指毫秒，“d”指天，“μL”指微升，“mL”指毫升，“L“指升，“bp”指碱基对，“mM”指毫摩尔，“μM”指微摩尔。
材料
Figure PCTCN2022075873-appb-000003
实施例1：产生共表达扩增增强子和嵌合分子(CM)的通用型CAR-T细胞
步骤1：表达扩增增强子的DNA的构建
分别合成表达5种如下扩增增强子的DNA序列，并在下述序列的5’端加上表达帮助细胞膜定位的信号肽的DNA序列，信号肽的氨基酸序列为SEQ ID NO：14。
扩增增强子1(Ev1)的氨基酸序列为SEQ ID NO：3，其中胞内结构域为SEQ ID NO：3的47-241的氨基酸，跨膜结构域为SEQ ID NO：3的19-46的氨基酸，胞外结构域为SEQ ID NO：3的7-18的氨基酸。
扩增增强子2(Ev2)的氨基酸序列为SEQ ID NO：4，其中胞内结构域为SEQ ID NO：4的47-245的氨基酸，跨膜结构域为SEQ ID NO：4的19-46的氨基酸，胞外结构域为SEQ ID NO：4的7-18的氨基酸。
扩增增强子3(Ev3)的氨基酸序列为SEQ ID NO：5，其中胞内结构域为SEQ ID NO：5的47-251的氨基酸，跨膜结构域为SEQ ID NO：5的19-46的氨基酸，胞外结构域为SEQ ID NO：5的7-18的氨基酸。
扩增增强子4(Ev4)的氨基酸序列是SEQ ID NO：6，其中胞内结构域为SEQ ID NO：6的47-266的氨基酸，跨膜结构域为SEQ ID NO：6的19-46的氨基酸，胞外结构域为SEQ ID NO：6的7-18的氨基酸。
扩增增强子5(Ev5)的氨基酸序列是SEQ ID NO：15，其中胞内结构域为SEQ ID NO：15的44-242的氨基酸，跨膜结构域为SEQ ID NO：15的19-43的氨基酸，胞外结构域为SEQ ID NO：15的7-18的氨基酸。
步骤2：质粒构建方法
构建一种抗CD19的CAR分子(称为CJP分子)、扩增增强子(用Ev表示)和嵌合分子(用CM表示)共同表达的慢病毒载体，病毒表达质粒(Addgene ID：#12252)用BamHI/SalI酶切作为骨架，核心基因结构是CJP-Ev-CM顺次相连，中间用2A自切割肽(P2A和T2A)连接。CJP序列是SEQ ID NO：1，CJP分别与扩增增强子的5种变(用Ev1/2/3/4/5表示)之一由GSG-P2A序列连接，再由GSG-T2A序列与CM连接。CJP-P2A-(Ev1-5)-T2A-CM序列或CJP-P2A-C7R-T2A-CM全基因合成后连入已酶切的病毒表达骨架中(图1)。构建得到8个包含CJP、扩增增强子和嵌合分子的病毒表达质粒：
包含CJP-P2A-Ev1-T2A-CM的目的表达质粒，
包含CJP-P2A-Ev2-T2A-CM的目的表达质粒，
包含CJP-P2A-Ev3-T2A-CM的目的表达质粒，
包含CJP-P2A-Ev4-T2A-CM的目的表达质粒，
包含CJP-P2A-Ev5-T2A-CM的目的表达质粒，
包含CJP-P2A-C7R-T2A-CM的目的表达质粒，
包含CJP的目的表达质粒，
包含CJP-CM的目的表达质粒。
作为对照的C7R序列来源于专利CN109952309，氨基酸序SEQ ID NO：2。Ev1的氨基酸序列是SEQ ID NO：3，Ev2的氨基酸序列是SEQ ID NO：4，Ev3的氨基酸序列是SEQ ID NO：5，Ev4的氨基酸序列是SEQ ID NO：6。Ev5的氨基酸序列是SEQ ID NO：15。CM的氨基酸序列是SEQ ID NO：7。
P2A和T2A是一种自切割多肽连接序列，是在一个载体中一个转录本同时表达两个独立蛋白常用的连接序列，蛋白在翻译过程中在P2A和T2A序列的末端断裂分开，使得由P2A和T2A所连接的前后两个蛋白分开，分别发挥功能，P2A的氨基酸序列为ATNFSLLKQAGDVEENPGP(SEQ ID NO：12)，T2A的氨基酸序列为EGRGSLLTCGDVEENPGP(SEQ ID NO：13)。
步骤3：慢病毒制备方法
采用三质粒系统：慢病毒目的表达质粒(步骤2构建的目的表达质粒)，包装辅助质粒psPAX2(Addgene ID：#12260)和pMD2.G(Addgene ID：#12259)。在HEK293T细胞(购自中科院上海细胞研究所)中进行病毒包装。制备流程如下：将冻存的工作细胞中的HEK293T细胞复苏，用DMEM培养基(+10％FBS+1％P/S)(Cellgro 10-013-CMR)置于10cm培养皿培养，复苏第2天后换液。待细胞长满后开始传代(通常1个培养皿长满之后可传至5个培养皿)，将细胞传代4代后可进行质粒转染。我们系统优选的转染采用PEI作为转染试剂，PEI：质粒(质量比)＝2：1的条件转染。将三质粒系统与PEI的混合物加入到Opti-MEM培养基(Gibco，cat#31985-070)中，再将此混合溶液加入到传代至第4代的HEK293T细胞中。转染6小时后用2％FBS的新鲜培养基换液，之后继续培养至72小时，收集HEK293T细胞上清。收集的病毒上清采用超离的方式(82200g，4-8℃离心2小时)进行浓缩，浓缩好的病毒用0.22μm滤膜过滤除菌后重悬待用。
步骤4.原代T细胞激活
原代T细胞来源于健康人志愿者的外周血(PBMC)。所用培养基为完全培养基，ImmunoCult TM-XF T Cell Expansion Medium(Stem Cell Technology，cat#10981)+300IU/ml IL2(Cayan，cat#HEILP-0201c)。利用Dynabeads(Thermo，cat#11141D)激活T细胞，Dynabeads：细胞＝3：1。T细胞被激活24小时后形态上明显聚团、变大。
步骤5.慢病毒基因转导方法
T细胞激活后48h，取3E5个细胞于24孔培养板，以细胞数的3倍量加入制备好的慢病毒，即MOI＝3(Multiplicity ofInfection，感染复数，病毒量与细胞数的比值)，补充培养基(DMEM培养基(+10％FBS+1％P/S))至500μL。24h后补充培养基至1mL，以利于细胞生长。
步骤6.sgRNA候选序列
根据相关网站的信息，我们筛选并设计了编辑B2M基因的sgRNA SEQ ID NO：8，SEQ ID NO：9；TRAC基因的sgRNA，序列为SEQ ID NO：10。
步骤7.Cas9蛋白的获取
本实施例选用Cas9蛋白是IDT DNA technology公司的Alt-R s.p.Cas9 Nuclease 3NLS蛋白。
步骤8.电击转化制备通用型-T细胞
T细胞慢病毒转染后48h(Jurkat模式细胞株不需激活)，收集细胞，用电转缓冲液，缓冲液T，T4(Invitrogen，Lot#1E14211)，Opti-MEM(Gibco，cat#31985-070)等清洗3遍，将细胞重悬于电转缓冲液中，细胞密度调整为1x10 8/mL。在体外同时将上述三组sgRNA(各150ng)与1μg Cas9蛋白混合均匀后在室温孵育10min，再将混合物加入至重悬好的细胞中进行电击，电击总体积为10μL，用Neon电转仪进行电转。电击电压条件如下：1200v，10ms(3次)。
将细胞以1x106/mL的密度，于完全培养基中培养至14天，即通用型CAR-T细胞，培养基同上，ImmunoCult TM-XF T Cell Expansion Medium(Stem Cell Technology，cat#10981)+300IU/ml IL2(Cayan，cat#HEILP-0201c)。Ev1-5蛋白和CM蛋白共表达示意图参见图2。
步骤9.基因转导效率和基因编辑效率检测
完成电击3天后检测原代T细胞编辑的效率。取出少量细胞，加入1mL PBS(Gibco，cat#C10010500BT)清洗细胞1遍，用100μL PBS重悬，加入3μL抗FMC63的抗体一抗(检测CAR19)，4℃孵育30min，加入1mL PBS清洗细胞，300g离心3min，去上清。用100μL PBS重悬细胞，加入0.5μL检测CAR19的二抗、3μL抗HLA-ABC抗体、3μL抗体CD3抗体(检测基因编辑效率)和3μL抗HLA-E抗体至细胞中，混合均匀，4℃孵育30min。清洗后再用流式细胞仪上机检测。
实施例2：与现有技术C7R相比，Ev1-5可以有效提高CAR-X-CM的慢病毒包装产毒效率(X指C7R或Ev1-5)
慢病毒的制备包装过程中，插入基因的长度会影响病毒的包装和产毒效率。通常情况下CAR基因约1.5kb，如果再加上大于1.5kb的基因序列，则已接近慢病毒表达基因的载量(额外启动子约1.5-2kb，终止子WPRE约0.5kb)，虽可以包装产毒，但产毒效率会明显下降，不利于大规模的慢病毒制备，影响产量和后续的转染效果。考虑到的Ev1-5基因长度(786bp-816bp)远小于C7R(1539bp)，我们进一步探索Ev1-5是 否可以提高CAR-X-CM的慢病毒包装和产毒效率。
采用实施例1步骤1-3慢病毒制备方法制备得到的6种病毒，即包含CJP-P2A-C7R-T2A-CM、CJP-P2A-Ev1-T2A-CM、CJP-P2A-Ev2-T2A-CM、CJP-P2A-Ev3-T2A-CM、CJP-P2A-Ev4-T2A-CM、CJP-P2A-Ev5-T2A-CM的目的表达质粒的慢病毒。产毒效率比较不需要纯化病毒，而直接对慢病毒包装的上清液进行滴度检测。
慢病毒上清液滴度检测方法
用293T细胞作为被转染的靶细胞，通过检测CAR分子阳性比例计算慢病毒上清的生物滴度。
1.培养293T细胞，用TrypLE Express Enzyme(1x)，phenol red(gibco，12605-028)消化培养的293T细胞(培养基为含10％FBS的DMEM培养基)，调整细胞密度后，接种至24孔细胞培养板中，使每孔细胞接种量为2.5E5个，每孔1.5ml培养基，然后置于37℃ 5％CO 2培养箱中培养24小时。
2.根据样品数量决定铺板孔数，同时另接种3个孔，标记为阴性对照NC。NC对照组不接种病毒。
3.接病毒前取少量3个NC孔中细胞进行计数，取均值，再乘以1.5ml即为病毒侵染时细胞数目。
4.取5种病毒样品分别按下表梯度稀释并加入稀释后样品，混匀后37℃ 5％CO 2培养箱中培养48小时。
编号 稀释倍数 加入体积(μl) 对应病毒原液的体积(μl)
1 1 20 20
2 2 20 10
3 20 20 1
4 200 20 0.1
5 2000 20 0.01
5.每孔加入100μl TrypLE Express Enzyme(1x)，phenol red消化细胞，静置2min后加入900μl含10％FBS的DMEM培养基中和，混匀吹打并收集细胞。取少量细胞进行计数，根据计数结果，取1E 6个细胞置于离心管中，350g离心5min后，去除上清。
6.洗涤：每管加入1ml PBS重悬细胞，350g离心5min，弃上清。
7.重悬：每管加入100μl PBS重悬细胞。
8.加一抗：每管分别加入1μl一抗(BiotinRabbitAnti-MouseFMC63scFv)，充分混匀，4℃避光孵育40min。
9.洗涤：孵育结束后，每管加入1ml PBS，充分混匀，350g离心5min后弃上清，重复一次。
10.加二抗：每管加入100μl PBS悬匀细胞，然后每管加入0.5μl二抗(streptavidinPE)，4℃避光孵育40min。
11.洗涤：孵育结束后，每管加入1ml PBS，充分混匀，350g离心5min后弃上清，重复2次后，用100μl PBS重悬。
12.NC孔细胞分为3组：第一组不染色，将细胞消化收集后用PBS洗涤一次后加100μl PBS重悬，标记为NC不染色。第二组不加一抗，只加二抗，用于排除特异性染色，标记为NC PE-strep。第三组正常染色，标记为NC染色
13.上机检测：先采集NC不染色样本，进行电压调节与设门，然后依次采集替他样本，获得CAR表达率结果。
14.慢病毒滴度(TU/ml)的计算公式如下：
TU/ml＝(C×N×0.01×1000×D)/V
C＝CAR表达率(％)；N＝侵染时细胞的数目(约为5E 5)；D＝病毒样品的稀释倍数；V＝加入的稀释病毒的体积(μl)。
实施例3：与现有技术C7R相比，Ev1-5可以有效提高通用型CAR-T细胞的转染效率
为平行比较原代T细胞中通用型CAR-T细胞的转染效率，各组别慢病毒经纯化后，按相同的MOI对原代T细胞进行转染。慢病毒制备方法同实施例1步骤3，制备得到实施例2的5种病毒。
相同的MOI转染可以确保转染结果的差异不是由上清产毒效率造成的。
在PBMC激活后48h，各组慢病毒以MOI＝3的条件转染原代T细胞，转染后第3天(即激活后的第5天，图3-1)检测CAR的表达效率。如图3-1，在相同转染条件下，只表达CJP的组别有81.5％的效率(图3-1B)，表达CJP-P2A-CM的组别有66.8％的效率(图3-1C)，而表达CJP-P2A-C7R-T2A-CM的组别效率显著下降，只有26.1％(图3-1D)，说明C7R的引入对CAR的转染效率和表达效率有不利影响。相比之下，把C7R替换成Ev1-5，各组别表达效率有明显提高，在50％-60％之间，近似于CJP-P2A-CM。
实施例4：与现有技术C7R相比，Ev1-5可以有效提高通用型CAR-T细胞中CAR表 达的稳定性
CAR是表达在细胞膜上的蛋白，CAR基因经过转录、翻译后，通过囊泡运输至细胞膜表面，CAR的表达是一个细胞膜展示-内吞的动态过程。CAR表达稳定性对于CAR-T细胞的功能至关重要，如果由于表达效率、内吞速度等因素造成CAR在细胞膜上的表达受阻，则会严重影响CAR-T细胞的活性。因此我们进一步比较C7R和Ev1-5对CAR表达稳定性的影响。
实验方法同实施例3，在试验的第5天后，继续跟踪通用型CAR-T细胞制备扩增过程中，CAR的动态表达变化。如图3-1，3-2和3-3，在第5、8、11天检测各组的CAR阳性比例，CJP-P2A-C7R-T2A-CM组别的CAR阳性比例出现了逐步下降，分别是26.1％、17.5％和6.7％，其它各组别较起始的转染效率相比，均保持稳定的表达比例。图4是各组的统计数据，结果表面，C7R的引入，使得原代T细胞中CAR的表达效率不稳定，随培养时间表达有下降趋势，而Ev1-5的引入，可以稳定CAR分子的稳定表达。
实施例5：Ev1-5可以有效激活STAT5和(或)STAT3信号
STAT5和STAT3是IL-7、IL-2细胞因子信号通路的重要信号蛋白，响应信号后，pSTAT5和pSTAT3会激活下游一系列基因，利于T细胞存活和增殖。通过对胞外域的筛选，虽然Ev1-5分子的大小远小于C7R，但Ev1-5可以形成二聚体，并高效激活STAT5和STAT3信号。pSTAT5和pSTAT3的表达被用作各组细胞的检测。
胞外CAR和胞内pSTAT5/3表达流式细胞术检测方法
1.阳性对照组细胞(表达CJP的T细胞)提前用600IU的IL-2处理30min，激活pSTAT5和pSTAT3的表达。其它组别细胞不做处理。
2.胞外CAR染色。各组别细胞以5E6/mL的密度，1mL重悬于在PBS中，参考实施例1加入检测CAR的抗体对胞外CAR进行染色。
3.细胞固定。将细胞重悬于250uL PBS中，加入等体积的IC固定液(BioLegend，420801)，涡旋混合固定细胞。
4.在室温下孵育样品30min，避光。
5.在室温下500g离心样品5min，弃上清。
6.将细胞沉淀重悬，加入1mL预冷的90-100％甲醇(BBI life science，MT1617)。涡旋混合并在2-8℃下孵育30min。
7.清洗甲醇。用过量体积的PBS洗涤细胞，本实施例采用10mL。
8.在室温下500g离心细胞5min。弃上清。
9.用PBS液以1E7/mL的密度重悬细胞，并将100μL细胞分于流式管中。
10.加入Anti-pSTAT5(pY694)(BD Pharmingen，612567)或Anti-pSTAT3(pY705)(BD Pharmingen，612569)抗体，并在室温下孵育30-60分钟。避光。
11.加入2mL流式细胞术染色液，在室温下500g离心5min。弃上清。
12.重复第11步，进行两次细胞洗涤。
13.将染色细胞重悬于200uL PBS中，流式细胞术分析。
表达CJP-P2A-C7R-T2A-CM和CJP-P2A-(Ev1-5)-T2A-CM的细胞制备
本实施例目的为检测各组细胞中pSTAT5和pSTAT3的表达，为降低检测背景干扰，采用Jurkat细胞株制备各组CAR-T细胞，Jurkat细胞是研究T细胞信号转导的常用模式细胞。取1E6 Jurkat细胞重悬至1mL铺于24孔培养板，MOI＝0.5转染各组别慢病毒，24h后换液。培养3天后，可检测CAR表达并用来检测pSTAT5和pSTAT3的表达。特别的由于CJP-P2A-C7R-T2A-CM的表达效率低，相同条件下该组采用MOI＝1的转染条件。
pSTAT5和pSTAT3的表达分析
对于pSTAT5，如图5-1，阳性对照组绝大部分细胞经过IL-2激活后，均表达pSTAT5，且与CAR的表达无关(图5-1A)；现有技术C7R组(图5-1D)，pSTAT5的表达在部分CAR阳性细胞中可以检测到，表达效率为21.9％，其中CAR阳性细胞中pSTAT5的表达约占46.7％；Ev1-5各组也具备有效的pSTAT5的表达，分别占总细胞的33.8％、43.8％、36.0％、10.0％、和11.4％，其中除Ev4和Ev5外，其它各组CAR阳性细胞中pSTAT5的表达比例与C7R类似，45％-55％之间。
对于pSTAT3，如图5-2，阳性对照组约一半的细胞经过IL-2激活后，表达pSTAT3，且与CAR的表达无关(图5-2A)；现有技术C7R组(图5-2D)，仅微弱检测到pSTAT3的表达约5.9％，说明C7R仅微弱激活STAT3通路；Ev1-5各组pSTAT3的表达效率具有不同程度的提高，说明Ev1-5可同时激活STAT5和STAT3通路，强度依分子不同而易。
实施例6：Ev1-5可以有效提高通用型CAR-T细胞的体外扩增能力
采用实施例1制备各组通用型CAR-T细胞(用U表示通用型T细胞)，培养至第 14天。各组细胞取1E5CAR阳性细胞用于体外肿瘤细胞刺激的增殖试验，肿瘤细胞选择CD19阳性的Raji细胞，Raji细胞稳定表达GFP荧光蛋白，用于区分CAR-T细胞和肿瘤细胞。培养基是X-VIVO TM 15 Medium(Lonza，04-418Q，无IL-2添加)，CAR阳性细胞∶Raji细胞＝1∶1，细胞投入24孔细胞培养板，培养基补充至1mL。每2-3天取样100ul，采用实施例1的方法进行CAR阳性细胞的流式细胞书检测，即100uL检测液中加入1mL PBS(Gibco，cat#C10010500BT)清洗细胞1遍，用100μL PBS重悬，顺次加入3μL抗FMC63的抗体一抗(检测CAR19)、0.5μL二抗至细胞中，混合均匀，4℃孵育30min。清洗后再用流式细胞仪上机检测。第7天，进行肿瘤细胞的重复刺激，各组投入1E5的Raji细胞，补充培养基至1mL，后续重复上述试验检测。
如图6，各组CAR阳性细胞在第一次肿瘤刺激后，扩增能力差异不大，在第7天重复刺激后，U-CJP组的扩增受限，处于缓慢增殖的状态，10-14天之间扩增至40-50倍左右；相比之下，嵌合分子CM的U-CJP-P2A-CM细胞，在第7天的重复刺激后，扩增有一定程度的提高，约70倍左右；C7R的引入可以明显提高细胞扩增能力，在第7天的重复刺激后，可达80-100倍；相比之下，Ev1-4均可以显著提高细胞的扩增能力，在第14天达70-130倍不等。特别的，U-CJP-P2A-Ev1-T2A-CM和U-CJP-P2A-Ev2-T2A-CM两组扩增最强，达到110倍以上，而且，在第10至第14天之间，依旧保持扩增能力。综上，实验数据显示Ev1-4可以有效提高通用型CAR-T细胞的体外扩增能力，Ev1和Ev2更优，分别选出继续后续抵抗排异的功能试验。
U-CJP表示通用型T细胞表达CAR分子CJP；U-CJP-P2A-CM表示通用型CAR-T细胞表达CAR分子CJP和嵌合分子CM；U-CJP-P2A-Ev1-T2A-CM表示通用型CAR-T细胞表达CAR分子CJP，扩增增强子Ev1，以及嵌合分子CM。
实施例7：在免疫排斥环境中，表达Ev分子的通用型CAR-T细胞比表达C7R分子的通用型CAR-T细胞具备更强扩增能力
通用型CAR-T细胞的制备和细胞表型检测
各组通用型CAR-T细胞的制备和流式细胞术检测同实施例1，考虑到C7R的转染效率在相同的条件下比较低，且不稳定，为完成功能试验，我们提高了MOI，本实施例采用MOI＝4，同时C7R组别再增加一组MOI＝8。结果如图7-1和7-2，各组别均实现高效的基因编辑效率，即CD3/HLA-ABC双阴性细胞比例大于80％；除C7R组别外，各组通用型CAR-T细胞的CAR阳性比例均在50％-70％，且CAR与HLA-E(HLA-E用来检测CM表达)具备高效的共表达；如图7-2，在同等病毒转染条件下(MOI＝4)， U-CJP-P2A-C7R-T2A-CM仅表达12.3％，即使MOI＝8时，也仅14.8％，与实施例3结果类似，说明C7R显著影响了CAR的表达。后续的功能评价，采用各组别CAR阳性细胞数相同的试验策略，因此14.8％的CAR阳性率，不影响后续功能评价。
通用型CAR-T细胞HLA-E表达丰度检测
HLA-E可以通过与NKG2A/CD94相互作用有效地抑制NK细胞和T的毒性反应，通用型CAR-T细胞表达HLA-E可以有效缓解NK细胞和异体PBMC的杀伤。因此通用型CAR-T细胞表达更高丰度的HLA-E分子可以增强通用型CAR-T细胞对免疫排斥的抵抗能力。
取出约1E5细胞，加入1mL PBS(Gibco，cat#C10010500BT)清洗细胞1遍，用100μL PBS重悬，加入3μL抗FMC63的抗体一抗(检测CAR19)，4℃孵育30min，加入1mL PBS清洗细胞，300g离心3min，去上清。用100μL PBS重悬细胞，加入0.5μL检测CAR19的二抗、3μL抗HLA-ABC抗体、3μL抗体CD3抗体(检测基因编辑效率)和3μL抗HLA-E抗体至细胞中，混合均匀，4℃孵育30min。清洗后再用流式细胞仪上机检测。流式细胞术分析，检测方法如实施例4，即在HLA-ABC、CD3双阴性，CAR阳性细胞中分析HLA-E的平均荧光表达丰度。
如图8，U-CJP细胞中HLA-E的MFI表达是523，属于阴性背景表达，表达异源嵌合分子CM的组别U-CJP-P2A-CM的表达是2612，表达丰度提高到了5倍。引入C7R后，U-CJP-P2A-C7R-T2A-CM的HLA-E表达是1891，是背景表达的3.6倍。U-CJP-P2A-Ev1-T2A-CM和U-CJP-P2A-Ev2-T2A-CM组的HLA-E表达分别是4335和5478，分别是背景的8.3和10.5倍。Ev1/2的引入显著提高了HLA-E的表达丰度，暗示可能该组通用型CAR-T细胞具备更强的抵抗排异杀伤的能力。
通用型CAR-T细胞与异体PBMC细胞共培养试验的建立。
该试验为科学上模拟体内环境，由于体内的免疫细胞复杂，不仅仅包含NK细胞和靶细胞，因此建立真实的异体PBMC环境更能接近体内环境。检测被编辑的CAR-T细胞在异体PBMC环境下的扩增能力，该环境中，即存在异体免疫排异(不仅仅来源NK细胞)，也存在靶细胞(肿瘤Raji细胞)。
细胞共培养，通用型CAR-T细胞∶肿瘤Raji细胞∶异体PBMC＝1∶5∶20。通用型CAR-T细胞的CAR阳性细胞数为5E4/ml，其它细胞按比例类推，24孔培养板，37℃培养，在不同时间点记录不同组别CAR阳性细胞的数目(图9)。
异体PBMC和Raii细胞共培养前用Dye eFluor TM 670染色，用于区分通用型CAR-T细胞：将细胞密度调至1E7/ml，加入终浓度为10μM的e670染料，室温避光孵育5min，培养基洗涤三遍后，用于实验。
本实施例中排异性PBMC来自3个不同的健康供者，供者1、2、3的数据分别如图9-1，-2，-3。其中供者1与制备的通用型CAR-T细胞的PBMC属相同来源，即属于自体，其它是异体。如图9所示，在3个免疫排异PBMC的环境中，U-CJP的扩增均是最弱的，暗示该组抵抗排斥能力最差；U-CJP-P2A-CM的扩增能力优于U-CJP，但C7R的引入U-CJP-P2A-C7R-T2A-CM较U-CJP-P2A-CM未能体现扩增优势，推测可能与CAR表达效率低、表达不稳定或是HLA-E丰度低有关。特别的，在三组排异试验中，U-CJP-P2A-Ev1-T2A-CM和U-CJP-P2A-Ev1-T2A-CM均体现出明显扩增优势，表明Ev和CM的组合表达可以有效提高通用型CAR-T细胞的扩增和抵抗排异杀伤能力。

Claims (26)

  1. 一种扩增增强子，包含以下组分：a)一个或多个细胞因子受体胞内结构域，该胞内结构域能引发细胞中通过STAT5和/或STAT3途径的信号传导；b)跨膜结构域；c)一个或多个胞外结构域，该胞外结构域是DAP12的胞外结构域或其变体；其中跨膜结构域和胞外结构域包含促进所述扩增增强子形成同源二聚化的结构。
  2. 权利要求1所述的扩增增强子，其中所述的受体胞内结构域来自IL-7细胞因子受体α、IL-21细胞因子受体α、IL-23细胞因子受体α、IL-12细胞因子受体α、CD122或其组合。
  3. 权利要求1所述的扩增增强子，其中所述的受体胞内结构域是序列为SEQ ID No：3的第47-241位氨基酸，SEQ ID No：4的第47-245位氨基酸，SEQ ID No：5的第47-251位氨基酸或SEQ ID No：6的第47-266位氨基酸。
  4. 前述任一项权利要求所述的扩增增强子，其中所述的跨膜结构域是组分a)的一个或多个细胞因子受体胞内结构域的内源跨膜结构域或内源跨膜结构域的变体。
  5. 前述任一项权利要求所述的扩增增强子，其中所述的跨膜结构域是IL-7细胞因子受体α跨膜结构域或其变体，或者所述的跨膜结构域是DAP12的跨膜结构域或其变体。
  6. 前述任一项权利要求所述的扩增增强子，其中所述的跨膜结构域包含至少一个半胱氨酸。
  7. 前述任一项权利要求所述的扩增增强子，其中所述的跨膜结构域的长度为21-33的氨基酸序列。
  8. 权利要求7所述的扩增增强子，其中所述的跨膜结构域是SEQ ID No：3的第19-46位氨基酸，或者所述的跨膜结构域是SEQ ID NO：11，或者所述的跨膜结构域是SEQ ID NO：15的第20-40位氨基酸。
  9. 前述任一项权利要求所述的扩增增强子，其中所述的胞外结构域是序列为SEQ ID No：3的第7-18位氨基酸所示的DAP12的胞外结构域，或者是包含DAP12的胞外结构域的多肽序列，或者是对DAP12的胞外结构域经过一个或几个氨基酸的取代、缺失或添加而衍生的DAP12胞外结构域的变体。
  10. 权利要求9所述的扩增增强子，其中所述的胞外结构域的长度不超过130个氨基酸；优选不超过120个氨基酸；优选不超过40个氨基酸；优选不超过20个氨基酸；优选胞外结构域的长度为10-40个氨基酸、12-20个氨基酸。
  11. 一种表达前述任一项权利要求所述的扩增增强子的核酸序列。
  12. 一种基因工程改造的细胞，其特征在于，该细胞表面表达权利要求1-10任一项所述的扩增增强子。
  13. 权利要求12所述的细胞，其中，该细胞是免疫细胞，进一步该免疫细胞优选为淋巴细胞或巨噬细胞。
  14. 权利要求13所述的细胞，其中，所述淋巴细胞是T细胞、NKT细胞、γδT细胞、粘膜相关不变T细胞、NK细胞或B细胞。
  15. 权利要求12-14任一项所述的细胞，其中，所述的细胞表面缺失至少一种内源性表达的MHC分子或MHC分子类似物。
  16. 权利要求12-15任一项所述的细胞，其中，所述细胞中至少一个编码内源T细胞受体(TCR)的组件的基因失活。
  17. 权利要求12-16任一项所述的细胞，其中所述的T细胞是一种CAR-T细胞或TCR-T细胞。
  18. 权利要求12-17任一项所述的细胞，其中所述的细胞具有表达嵌合单链分子的核酸序列，所述嵌合单链分子包含：(a)呈递肽段，(b)B2M蛋白，和(c)连接序列，该连接序列用于连接前述(a)和(b)片段；其中，所述嵌合单链分子与MHC或MHC类似物的重链分子在细胞膜上形成复合体；所述呈递肽段为5-30个氨基酸的多肽序列。
  19. 权利要求18所述的细胞，其中所述的嵌合单链分子N端到C端依次包含：呈递肽段-连接序列-B2M蛋白，或B2M蛋白-连接序列-呈递肽段。
  20. 权利要求18或19所述的细胞，其中，MHC的重链分子是细胞内源性表达的且选自经典的I类MHC分子的重链分子，优选为HLA-A，HLA-B或HLA-C的重链分子；或MHC的重链分子是细胞内源性表达的且选自非经典的I类MHC分子的重链分子，优选为HLA-E，HLA-F或HLA-G的重链分子；或MHC的重链分子是细胞内源性表达的且选自其他非经典MHC类似分子的重链分子，优选CD1或MR1或UL18的重链分子。
  21. 权利要求18-20任一项所述的细胞，其中，所述呈递肽段来源于I类MHC分子的信号肽。
  22. 权利要求17-21任一项所述的细胞，其中所述的细胞缺失内源性表达的MHC分子，并缺失内源性表达的T细胞受体。
  23. 权利要求22所述的细胞，其中所述的细胞表达的嵌合单链分子与MHC或MHC类似物的重链分子在细胞膜上形成复合体。
  24. 权利要求12-23任一项所述的细胞在制备治疗癌症的药物中的应用。
  25. 一种治疗癌症的方法，所述方法包括向个体施用权利要求12-23所述的基因工程改造的细胞。
  26. 权利要求24或25所述的方法和应用，其中的癌症是乳腺癌、前列腺癌、肺癌、脑癌、结肠癌、头颈癌、皮肤癌、卵巢癌、子宫内膜癌、子宫颈癌、肾癌、肺癌、胃癌、小肠癌、肝癌、胰腺癌、胆囊癌、胆管癌、食道癌、唾液腺癌或甲状腺癌。
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