WO2019238015A1 - 一种基因工程化的免疫淋巴细胞及其制备方法 - Google Patents

一种基因工程化的免疫淋巴细胞及其制备方法 Download PDF

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WO2019238015A1
WO2019238015A1 PCT/CN2019/090604 CN2019090604W WO2019238015A1 WO 2019238015 A1 WO2019238015 A1 WO 2019238015A1 CN 2019090604 W CN2019090604 W CN 2019090604W WO 2019238015 A1 WO2019238015 A1 WO 2019238015A1
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cells
seq
engineered
hla
cell
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黄倬
林彦妮
李军
王文博
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苏州克睿基因生物科技有限公司
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the invention relates to a genetically engineered lymphocyte, in particular to a lymphocyte that is inactivated by the expression of histocompatibility antigen (MHC), and also relates to a preparation method of the lymphocyte and its use as an immunotherapy drug.
  • MHC histocompatibility antigen
  • Adoptive cellular immunotherapy is to isolate primary T cells from the human body, stimulate, expand and culture them in vitro under the conditions of ex vivo, and finally return these cells to the patient to achieve the purpose of treating diseases.
  • Chimeric antigen receptor T cell therapy (CAR-T) is one of the representatives of successful adoptive cellular immunotherapy.
  • CAR-T therapy refers to the isolation and activation of donor T cells in vitro, and the transfer of CAR genes including the antigen recognition region, co-activation region and activation region into T cells, and further large-scale expansion in vitro and back to patients. Curing disease.
  • Existing CAR-T technologies are individualized, and all use patient's own T cells to produce CAR-T cells.
  • the T cells of the universal CAR-T are derived from healthy donors and can be prepared in advance for use by any patient. There are two issues to consider in the preparation of a universal CAR-T. On the one hand, removing the expression of MHC class I molecules from donor cells can eliminate donor cell antigen presentation, escape recognition and killing of host T cells, and CAR-T cells can survive. On the other hand, removing the expression of the TCR complex of the donor cell can prevent the donor T cell from recognizing and killing the host cell.
  • Class I MHC molecules include two subunits, called heavy and light chains. Because of the different heavy chains, Class I MHC is divided into several different types: A / B / C / E / F / D.
  • the light chain is a universal subunit (B2M), and can form a type I MHC complex with any heavy chain.
  • the main role of the type I MHC complex in the cell is to present the polypeptide fragments of intracellular protein degradation. It combines with the polypeptide fragments in the cell to form a polypeptide-I type MHC complex and transports it to the cell surface.
  • the peptide-I MHC complex can be recognized by the TCR of specific T cell clones, resulting in T cell killing.
  • TCR consists of two subunits, the alpha ( ⁇ ) subunit and the beta ( ⁇ ) subunit. Both subunits have many subtypes, and different subtypes combine to form a large number of different TCR clones.
  • TCR is specifically expressed on the surface of T cells, and combines with a variety of different CD3 molecules to form a TCR complex.
  • the complex can recognize a peptide-I type MHC complex, which can cause the activation of T cells and the killing of the recognized cells.
  • Gene knockdown and gene editing technology caused by RNA silencing can simultaneously remove class I MHC complexes and TCR complexes in donor T cells.
  • Gene editing technology uses specific rare-cutting endonucleases to cut the intracellular genome. After the cleavage is completed, the non-homologous recombination repair mechanism inherent in the cell occurs. The repair is non-specific repair, which is prone to generate frameshift gene mutations. Causes gene knockout.
  • NK cells are a natural immune surveillance cell. There are many receptors on the surface of NK cells, including activating receptors and inhibitory receptors. Activating receptors are mainly NKG2D, and NKG2D ligands include MICA, MICB, ULBP1-6 and so on.
  • the inhibitory receptor is mainly KIR, and its ligand is a class I MHC molecule.
  • the inhibitory receptor also includes NKG2A, whose ligand is a class I MHC molecule family analog HLA-E. (Morvan and Lanier, Nat Rev Cancer 2016.16, 7-19).
  • NK cells are activated depends on the balance of activating and inhibitory signals.
  • Normal cells express both ligands that can activate and inhibit NK cell activation.
  • Class I MHC complexes expressed by normal cells bind to NK cell inhibitory receptors, thereby preventing activation and killing of NK cells.
  • Cells lacking class I MHC complex cannot bind inhibitory receptors, which leads to the inactivation of inhibitory receptors.
  • the balance tends to activate NK cells. Therefore, cells lacking class I MHC complex can be recognized and killed by NK cells. Many mechanisms can prevent the killing of NK cells.
  • Virus-infected cells express analogs of virus-derived class I MHC family proteins, which can bind to the inhibitory receptor NKG2A and inhibit NK cell activation.
  • the HLA-E molecule a member of the MHC family I, was introduced into cells, which can bind to the inhibitory receptor NKG2A and inhibit the activation of NK cells (CN 106103475 A).
  • NK cells CN 106103475 A
  • HLA class I null 721.221 cells the expression of class I MHC molecules is lost, and such cells will be recognized and killed by NK cells.
  • the artificial introduction of HLA-E or HLA-G can inhibit the activation of NK cells, thereby avoiding being killed by NK cells.
  • the introduction of immunosuppressive polypeptides such as viral class I MHC homologues or ligands of NKG2D can also resist the killing of NK cells.
  • HLA-E does not localize to the cell membrane after the universal CAR-T inhibits or knocks out B2M proteins.
  • membrane localization of HLA-E also depends on the presented polypeptide.
  • HLA-E specifically presents HLA-A, -B, -C and-G signal peptides and peptides of certain viruses.
  • Artificially constructed HLA-E chimeric single-chain molecules can increase the expression of HLA-E / B2M complexes on cells. Studies have shown that the expression of HLA-E chimeric single-chain molecules presenting HLA-G signal peptides in tumor cell lines or pluripotent stem cells can effectively resist the killing of allogeneic NK cells (WO2004 / 103149A2).
  • HLA-E chimeric single-stranded molecules expressing class I MHC molecules can effectively inhibit killing of NK cells, most of the data focus on model cell lines, virus-infected cells, pluripotent stem cells, or tumor cells.
  • the expression of this HLA-E chimeric single chain molecule in immune lymphocytes with class I MHC molecules has not been reported.
  • the expression pattern (expression, amount, and expression) and function of the same protein in different types of tissue cells can be completely different.
  • the different subtypes of the HLA-E heavy chain and the different signal peptides presented by the HLA-E / B2M complex are important for the expression, expression level, and immune function of HLA-E chimeric single-chain molecules on the cell membrane. .
  • the optimal combination was selected from different HLA-E heavy chain subtypes and many presenting peptides, and expressed in engineered immune lymphocytes, which is of great significance for allogeneic universal immune cell therapy.
  • the invention relates to a chimeric single-chain molecule containing a presented peptide, B2M, and HLA-E, which can realize the expression of HLA-E cell membrane in B2M-deleted lymphocytes, thereby solving the above problems.
  • the chimeric single-chain molecule containing the presenting peptide, B2M, and HLA-E can be further applied to the universal CAR-T lacking TCR and B2M to avoid killing of NK cells.
  • a modified lymphocyte having a class I MHC molecule missing from the cell surface and expressing a chimeric single-chain molecule on the cell surface; said chimeric single-chain molecule comprising: (a) a presenting peptide; (b) a B2M protein; c) an HLA-E heavy chain; and (d) a linker sequence for linking the aforementioned (a) and (b), and (b) and (c) fragments.
  • N-terminus to C-terminus of the chimeric single-chain molecule comprises: a presenting peptide segment-linking sequence-B2M protein-linking sequence-HLA-E heavy chain.
  • the presenting peptide is a signal peptide of a class I MHC molecule, preferably the presenting peptide comprises or is selected from one of the following sequences: SEQ ID NO : 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
  • the rare-cutting endonuclease is an RNA-directed endonuclease, TAL nuclease, homing endonuclease, zinc finger nuclease, or Mega -TAL nuclease.
  • RNA-directed endonuclease is a CRISPR-Cas system.
  • the engineered lymphocyte according to claim 14, wherein the domain that recognizes a specific DNA sequence is an RNA-guided dCas9 protein, or a Cas9 single-strand nickase.
  • deaminase is a cytosine deaminase, or an adenine deaminase.
  • linker sequence is selected from one or more of the following sequences: SEQ ID NO: 8 and SEQ ID NO: 9.
  • HLA-E heavy chain sequence is SEQ ID NO: 12 or SEQ ID NO: 13.
  • modified lymphocyte of claim 20 wherein the nucleic acid sequence encoding the chimeric multimer single-stranded molecule is shown in the sequence SEQ ID NO: 15 or SEQ ID NO: 19.
  • TCR T cell receptor
  • the protein sequence of the engineered lymphocyte according to claim 23, which simultaneously expresses the CAR molecule and the chimeric multimer single-chain molecule is shown in the sequence SEQ ID NO: 20.
  • the chimeric multimer single-stranded molecule comprises: (a) a presenting peptide, (b) a B2M protein, and (c) an HLA-E protein Strands, and (d) a linker sequence linking the aforementioned (a), (b), and (c) into a single strand;
  • step (i) is to inactivate at least one gene encoding a class I MHC molecular component of a lymphocyte by DNA cleavage or base editing.
  • lymphocytes according to any one of technical solutions 1-25 in inhibiting killing of NK cells.
  • a method for inhibiting NK cells comprising providing NK cells or lymphocytes according to any one of claims 1-15 in a patient.
  • lymphocytes are preferably T cells, and more preferably CAR-T cells.
  • a pharmaceutical composition for inhibiting NK cell killing comprising the lymphocytes according to any one of claims 1-25.
  • the genetically engineered lymphocytes of the present invention are characterized by the inactivation of the expression of class I major histocompatibility antigen (MHC), which avoids immune rejection of allogenes.
  • MHC major histocompatibility antigen
  • chimeric single-chain molecules containing presenting peptides, B2M, and HLA-E can be expressed in these modified T cells to avoid hosts caused by class I MHC molecule deletion Killer of NK cells.
  • This genetically modified lymphocyte is suitable for allogeneic use and reduces the host's response to rejection of the graft.
  • the genetically engineered T cells described in the present invention can further inactivate the expression of the T cell receptor (TCR) and reduce the graft-versus-host response, so as a universal T cell, it is more suitable for allogeneic use and further applicable for chimeric antigen receptor T cells (CAR-T) or T cell receptor T cells (TCR-T).
  • TCR T cell receptor
  • CAR-T chimeric antigen receptor T cells
  • TCR-T T cell receptor T cells
  • a type I MHC molecule also known as a type I major histocompatibility complex, is a heterodimeric glycoprotein composed of two peptide chains connected by non-covalent bonds; one of them is called a heavy chain and has a polymorphic structure The other is the light chain or ⁇ 2 microglobulin (B2M).
  • B2M microglobulin
  • MHC class I molecules present peptides that are not degraded intracellularly, thereby activating the immune system.
  • Human MHC molecules are called HLA molecules.
  • Human class I MHC molecules are divided into classic HLA molecules (HLA-A, HLA-B, HLA-C) and non-classical HLA molecules (HLA-E, HLA-G, HLA). -F).
  • the deletion of MHC class I molecules on the cell surface refers to human lymphocytes lacking HLA-A / B / C / E / F / G, which can be inactivated by editing B2M or corresponding heavy chain genes. achieve.
  • ⁇ -2 microglobulin also known as B2M protein
  • B2M protein is the light chain of class I MHC molecules and an integral part of class I MHC molecules.
  • the human B2M protein consists of 119 amino acids (SEQ ID NO: 16) and has a molecular weight of 11.800 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.
  • B2M molecules prevents cells from expressing class I MHC molecules, also called HLA-I molecules.
  • N-terminus to C-terminus of the chimeric single-chain molecule of the present invention are: presenting peptide segment-linking sequence-B2M protein-linking sequence-HLA-E heavy chain molecule.
  • the structure is shown in Figure 2.
  • the chimeric single-chain molecule is: a membrane-localized signal peptide-presenting peptide- (G 4 S) 3 -B2M mature protein- (G 4 S) 4 -HLA-E heavy chain molecule, wherein The membrane localization signal peptide is removed before the chimeric molecule is localized to the cell membrane.
  • the membrane localization signal peptide includes signal peptides of membrane localization proteins such as B2M and CD8.
  • Signal peptide about 20-80 amino acids in length, located at the N-terminus of the mature protein, guides the protein through the membrane, and is subsequently cut away.
  • Signal peptides of membrane-localized proteins such as B2M and CD8.
  • HLA-E heavy chain molecules include HLA-E * 01: 01 and HLA-E * 01: 03 subtypes; or variants of HLA-E * 01: 01 and HLA-E * 01: 03 subtypes .
  • the variant refers to a molecule that retains the function of the original protein after insertion, substitution, and / or deletion of one or more amino acids on the original protein sequence; for example, in HLA-E * 01: 01 or HLA-E * 01: A molecule that retains the function of the HLA-E heavy chain through the insertion, substitution, and / or deletion of one or more amino acids on the 03 sequence.
  • presenting peptide A peptide presented by the HLA-E complex, which can stabilize the stability and structural specificity of the HLA-E complex.
  • the polypeptide is derived from the signal peptides of class I MHC molecules, such as HLA-A2, HLA-B7 , HLA-B15, HLA-Cw3, HLA-Cw7, HLA-G, HLA-F, etc .; viral protein polypeptides, such as CMV (UL40), EBV, HIV, etc.
  • the linking sequence of the present invention is a linking amino acid sequence with flexible characteristics, which is used to link the presenting peptide segment in the chimeric single-chain molecule of the present invention with the B2M protein, and link the B2M protein with the HLA-E heavy chain.
  • the linking sequence is 3-4 repeats of 4 glycines and one serine, for example, the (G 4 S) 3 sequence is (SEQ ID NO: 2), and the (G 4 S) 4 sequence is (SEQ ID NO: 3)
  • Lymphocytes are various types of cells that are produced and mature in the lymphatic system and have an immune response function.
  • This patent includes T cells, B cells, natural killer cells (NK) cells, macrophages, and the like.
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • U uracil
  • Zinc-finger nucleases are composed of a DNA recognition domain and a non-specific endonuclease.
  • the DNA recognition domain consists of a series of Cys2-His2 zinc finger proteins in series. Each zinc finger unit includes approximately 30 amino acids for specific binding to DNA.
  • Non-specific endonucleases are FokI endonucleases, which form dimers to cleave DNA.
  • TALEN TranscriptionActivator-LikeEffectorNucleases
  • TALE protein is the core component of the DNA binding domain. It is generally composed of multiple basic repeating units connected in series. By designing and combining the tandem units, the DNA sequence can be specifically identified. After coupling with FokI endonuclease, specific DNA sequences can be achieved. Cutting.
  • CAR Chimeric Antigen Receptor
  • scFv Variable region
  • scFv transmembrane structure
  • intracellular signaling domain connecting extracellular and intracellular structures mainly contains T cell activation signals and costimulatory signals, where costimulatory molecules can include one or two, common costimulatory molecules
  • Stimulating molecules include CD28, CD137 (4-1BB), CD27, OX40, CD30, CD40 and so on.
  • the extracellular antigen receptor is 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.
  • the CRISPR / Cas system is a nuclease system consisting of a cluster of regularly spaced short palindromic repeats (CRISPR) and a CRISPR binding protein (i.e., Cas protein) .It can detect almost all motifs adjacent to the prototype spacer in eukaryotic cells ( protospacer-adjacent motif (PAM) adjacent genomic sequences are cut (Cong et al. Science 2013.339: 819-823).
  • CRISPR regularly spaced short palindromic repeats
  • Cas protein CRISPR binding protein
  • CRISPR / Cas system is used to collectively refer to transcripts involving CRISPR-related ("Cas") genes and other elements involved in their expression or directing their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (such as tracrRNA or active partial tracrRNA), tracr paired sequences (covering "direct repeats” and processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences, or other sequences and transcripts from CRISPR loci .
  • the CRISPR system is characterized by elements that facilitate the formation of a CRISPR complex at the site of a target sequence (also known as a prototype spacer in an endogenous CRISPR system). .
  • a CRISPR complex including a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins results in or near that target sequence (e.g., at 1, 2, 3, 4, 5, 6, (7, 8, 9, 10, 20, 50, or more base pairs).
  • the tracr sequence (which may comprise or consist of all or part of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, Or more nucleotides)) may also form part of a CRISPR complex, such as by hybridizing along at least a portion of the tracr sequence to all or part of a tracr pairing sequence operably linked to the guide sequence.
  • the tracr sequence is sufficiently complementary to a tracr pairing sequence to hybridize and participate in the formation of a CRISPR complex.
  • the tracr sequence when performing an optimal alignment, has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence along the length of the tracr paired sequence Complementarity.
  • one or more vectors that drive the expression of one or more elements of the CRISPR system are introduced into a host cell such that expression of these elements of the CRISPR system directs CRISPR complexes at one or more target sites ⁇ ⁇ ⁇ Formation.
  • a tracr pairing sequence includes a pair of tracr sequences that is sufficiently complementary to promote formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises a tracr pairing sequence that hybridizes to the tracr sequence.
  • the degree of complementarity is in terms of the optimal alignment of the length of the tracr paired sequence and the shorter of the two sequences along the tracr sequence.
  • the optimal alignment can be determined by any suitable alignment algorithm, and the effects caused by the secondary structure can be further taken into account, such as self-complementarity within the tracr sequence or tracr pairing sequence.
  • the degree of complementarity between the tracr sequence and the tracr paired sequence along the shorter of the two is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the tracr sequence is about or more than about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,40, 50 or more nucleotides.
  • Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, Csax15, Csax15 , Csf2, Csf3, Csf4, homologues thereof, or modified forms thereof.
  • the Cas protein is a Cas9 protein.
  • Cas9 also known as Csn1
  • Csn1 is a giant protein that participates in both crRNA biosynthesis and destroys invading DNA.
  • S. thermophiles Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and Streptococcus pyogenes Cas9 is described in (S. Pyogenes) (Deltcheva, Chylinski et al. 2011).
  • the giant Cas9 protein (> 1200 amino acids) contains two predicted nuclease domains, the HNH (McrA-like) nuclease domain and the split RuvC-like nuclease domain (RNAase H-fold) (Makarova, Grishin et al. (2006)).
  • the Cas9 variant may be a Cas9 endonuclease that does not occur naturally in nature and is obtained by protein engineering or by random mutagenesis.
  • the Cas9 variant according to the present invention can be obtained by mutation, that is, deletion or insertion or substitution of at least one residue in the amino acid sequence of Streptococcus pyogenes Cas9 endonuclease (COG3513).
  • the Cas9 protein is Streptococcus pneumoniae, Streptococcus pyogenes or Streptococcus thermophilus Cas9, and may include mutant Cas9 derived from these organisms, or variants with other amino acid sequences linked to Cas9, For example, FokI enzyme is linked to Cas9.
  • Cas9s are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found under the SwissProt database accession number Q99ZW2, and the amino acid sequence of the Neisseria meningitides Cas9 protein can be found in the UniProt database number A1IQ68, the amino acid sequence of Streptococcus thermomophilus Cas9 protein can be found in UniProt database number Q03LF7, and the amino acid sequence of Staphylococcus aureus Cas9 protein can be found in UniProt database number J7RUA5.
  • the method for preparing engineered lymphocytes includes: (i) preparing lymphocytes with surface-deleted MHC molecules of type I; (ii) expressing a chimeric single-chain molecule on the surface of the lymphocytes; the chimeric single-chain molecular body comprises: (a) Presenting peptides; (b) B2M protein; c) HLA-E heavy chain; and (d) linker sequence for linking the aforementioned (a) and (b) and (b) and (c) fragments.
  • the order of the above steps (i) and (ii) can be interchanged.
  • Lentivirus preparation method a three-plasmid system, a lentiviral target expression plasmid (Addgene ID: # 12252), a packaging helper plasmid psPAX2 (Addgene ID: # 12260), and pMD2.G (Addgene ID: # 12259).
  • Viral packaging was performed in HEK293T cells (sourced from Shanghai Institute of Cell Research, Chinese Academy of Sciences). The preparation process is as follows: the HEK293T cells in the frozen working cells are resuscitated, and the DMEM medium (+ 10% FBS + 1% P / S) (Cellgro 10-013-CMR) is cultured in a 10cm petri dish, and the second recovery is performed Change the fluid after the day.
  • the mixture of plasmid and PEI was added to Opti-MEM medium (Gibco, cat # 31985-070), and this mixed solution was added to HEK293T cells passaged to passage 4.
  • the medium was exchanged with 2% FBS fresh medium, and the culture was continued to 72 hours, and the supernatant of HEK293T cells was collected.
  • the collected virus supernatant was concentrated by ultra-ionization (82200g, centrifugation at 4-8 ° C for 2 hours), and the concentrated virus was filtered and sterilized with a 0.22um filter and resuspended for use.
  • the target gene (such as the HLA-E chimeric single-stranded molecule DNA sequence) is constructed into a lentiviral vector.
  • the lentiviral vector contains a long terminal repeat 5'LTR and a truncated 3'LTR, RRE, a rev response element (cPPT), a central termination sequence (CTS), and a post-translational regulatory element (WPRE).
  • the HLA-E chimeric single-chain molecule was constitutively expressed by the EF-1a (elongation factor-1a) promoter, and was digested with BamHI and SalI to construct a lentiviral vector.
  • PBMC peripheral blood
  • the medium used was a complete medium, ImmunoCult TM -XF T Cell Expansion Medium (Stem Cell Technology, cat # 10981) + 300IU / ml IL2 (Cayan, cat # HEILP-0201c). T-cells were activated using Dynabeads (Thermo, cat # 11141D), Dynabeads: cells
  • Gene editing method 4 days after T cell activation or 2 days after virus transfection, collect cells and wash 3 times with electrotransfection buffer, buffer T, T4, Opti-MEM (Gibco, cat # 31985-070), etc. Resuspend the cells in electrotransfection buffer and adjust the cell density to 1 ⁇ 10 8 / ml. Simultaneously mix the required sgRNA (a total of 300-400ng) with 1ug of Cas9 protein in vitro and incubate at room temperature for 10min, then add the mixture to the resuspended cells for electric shock. The total electric shock volume is 10ul, and electrophoresis with Neon The instrument performs electrical transfer. Shock voltage conditions are as follows: 1200-1600v, 10ms (3 times). The sgRNA candidate sequence is designed based on the prediction analysis of related websites; Cas9 protein is derived from Alt-R spCas9 Nuclease 3NLS protein from IDT DNA technology.
  • T cell activation Preparation of engineered CAR-T cells expressing chimeric single-chain molecules: T cell activation, virus transfection, and gene editing methods are as above. The procedure is: primary PBMC activation on day 0; virus transfection on day 2; gene editing (knock-out of TCR and B2M) on day 4; and protein expression on flow cytometry on days 6-7.
  • anti-CD3-APC (BD, 555335), anti-CD3-FITC (BD, 555916), anti-B2M-PE (BD, 551337), anti-MHC-I-APC (R & D , FAB7098A), anti-HLA-E-PE (Biolegend, 342604), anti-HLA-E-APC (Biolegend, 342606), anti-Fab-Biotin (Jackson ImmunoResearch, 115-065-072), Streptavidin-PE ( BD, 554061), Dye eFluor TM 670 (eBioscience, 65-0840-90)
  • FIG. 1 Editing of B2M genes by sgRNA and Cas9 protein targeting B2M using electroporation transfection method in primary T cells.
  • the B.B2M gene was edited; the cells lacked both MHC-I and B2M, showing a double negative population.
  • Figure 2 Schematic diagram of HLA-E chimeric single-chain molecular structure.
  • Figure 3 Flow cytometric detection of primary T cell chimeric single-chain molecule expression.
  • T cells expressing chimeric single-chain molecules are effective against in vitro killing of NK cells.
  • T cells lacking class I MHC molecules express chimeric single-chain molecules. (Left) Control group, (Right) After co-culture with NK-92 for 24 hours, the proportion of the cell population increased.
  • Figure 5 Schematic diagram of CAR and chimeric single-stranded molecules constructed on the same vector.
  • CAR / chimeric single chain molecules are efficiently co-expressed on T cells.
  • CAR-T cell CD3 / B2M dual gene editing efficiency is greater than 75%.
  • the efficiency of CD3 / B2M dual gene editing of CAR-T cells expressing chimeric single-chain molecules is greater than 75%.
  • Engineered CAR-T cells expressing chimeric single-chain molecules can effectively resist the killing of NK cells.
  • Example 1 Production of engineered T cells with surface-deleted class I MHC molecules
  • the Cas9 protein used in this example is Alt-Rs.p. Cas9 Nuclease 3NLS protein from IDT DNA Technology.
  • T cells were derived from peripheral blood (PBMC) from healthy human volunteers.
  • the medium used is complete medium, ImmunoCult TM -XF T Cell Expansion Medium (Stem Cell Technology, cat # 10981) +300
  • the efficiency of primary T cell editing was examined 3 days after the shock was completed. Remove approximately 1 ⁇ 10 6 cells, add 1 ml of PBS (Gibco, cat # C10010500BT) to wash the cells once, resuspend with 100 ⁇ l of PBS, add 20 ⁇ l of B2M-PE antibody to the cells, mix well, and incubate at 4 ° C for 30 min. After washing, use a flow cytometer to test on the machine. As shown in Figure 1, B2M negative cells are gene-edited cells. These cells also lack the expression of class I MHC molecules, indicating that the lack of B2M will affect the expression of class I MHC molecules.
  • Example 2 Production of engineered T cells expressing chimeric single-chain molecules
  • a three-plasmid system was used: a lentiviral expression plasmid (Addgene ID: # 12252), a packaging helper plasmid psPAX2 (Addgene ID: # 12260) and pMD2.G (Addgene ID: # 12259).
  • Viral 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 frozen working cells are resuscitated, and the DMEM medium (+ 10% FBS + 1% P / S) (Cellgro 10-013-CMR) is cultured in a 10cm petri dish, and the second recovery is performed Change the fluid after the day.
  • plasmid (mass ratio) 2: 1.
  • the mixture of plasmid and PEI was added to Opti-MEM medium (Gibco, cat # 31985-070), and this mixed solution was added to HEK293T cells passaged to passage 4. After 6 hours of transfection, the medium was exchanged with 2% FBS fresh medium, and the culture was continued to 72 hours, and the supernatant of HEK293T cells was collected.
  • the collected virus supernatant was concentrated by ultra-ionization (82200g, centrifugation at 4-8 ° C for 2 hours).
  • the concentrated virus was filtered and sterilized with a 0.22 ⁇ m filter membrane and resuspended for use.
  • T cell activation (method as in Example 1), 48 hours later, the lentivirus prepared in the above steps is transfected into activated T cells, and gene editing (knock-out B2M) is performed after 48 hours, and gene editing is performed 72 hours later. Cytometry was used to detect positive expression after infection. As shown in Figure 3, in the primary T cells, the chimeric single-chain molecule can be well expressed. In this embodiment, the expression efficiency is about 25%. At the same time, after B2M is edited, cells that lack B2M (or class I MHC molecules) are deleted. HLA-E chimeric single-chain molecules can still be expressed well (indicated by arrows). This shows that our designed B2M-editing sgRNA can specifically recognize and edit B2M in the genome, but does not recognize the B2M subunits that do not edit HLA-E chimeric single-stranded molecules.
  • Example 3 Engineered T-cells expressing class I MHC molecules with chimeric single-chain molecules can effectively inhibit the killing of NK cells
  • NK-92 cell killing flow cytometry detection method The culture medium ImmunoCult TM was derived from STEMCELL (same as Example 1). NK-92 cells were purchased from Shanghai Institute of Cell Research, Chinese Academy of Sciences. The target cells are: primary T cells derived from the peripheral blood of healthy human volunteers, T cells of class I MHC molecules deleted in Example 1 and class I MHC molecules of chimeric single chain molecules prepared in Example 2 deleted T cells. The final target cell concentration was 2 ⁇ 10 4 / ml.
  • CD56 protein was used to distinguish NK cells from T cells
  • MHC-I protein was used to identify edited T cells.
  • HLA-E chimeric single chain molecules can effectively inhibit the killing of NK-92 cells.
  • NK-92 cells after being killed by NK-92 cells, the proportion of cells expressing HLA-E chimeric single-chain molecules is significantly larger than the initial proportion (from 39.85% to 65.64% as shown by the arrow), while HLA-E is not expressed
  • the proportion of chimeric single-stranded molecules decreased significantly. It shows that NK-92 cells are more likely to kill cells with class I MHC molecule deletion, and expressing HLA-E chimeric single chain molecules will effectively inhibit this NK cell-mediated killing. According to statistical analysis, the proportion of cells with type I MHC molecules deleted was about 70%, and the proportion of lysed cells after the expression of HLA-E chimeric single-stranded molecules under the same conditions was about 7.5%.
  • Example 4 Various chimeric single-chain molecules can effectively resist the killing of NK cells
  • chimeric single-chain molecules were tested against the killing effect of NK cells.
  • the method is as in Example 3.
  • the target cells were Jurkat cell lines (T cell-derived), and the class I MHC molecules were deleted by gene editing (the method of Example 2).
  • the function of the chimeric multi-single-chain molecule was more fully confirmed K562 cell line is also used for target cells (K562 is a natural class I MHC molecule-deleted cell).
  • K562 is a natural class I MHC molecule-deleted cell.
  • Table 1 a variety of chimeric multiple single-stranded molecules can effectively resist NK cell killing to varying degrees in cells with class I MHC molecule deletions.
  • the chimeric single-chain molecule A sequence in Table 1 is SEQ ID NO: 22; the chimeric single-chain molecule B sequence is SEQ ID NO: 23; the chimeric single-chain molecule C sequence is SEQ ID NO: 24; chimera The single-chain molecule D sequence is SEQ ID NO: 25; the chimeric single-chain molecule E sequence is SEQ ID NO: 18.
  • Example 5 Generation of engineered CAR-T cells with surface-deleted class I MHC molecules and CD3 / TCR complexes
  • the methods for preparing lentivirus and gene editing are the same as those in Examples 1 and 2.
  • the TCR / CD3 complex is deleted using the gene editing TRAC gene.
  • the CAR gene and the chimeric single-stranded molecule gene are connected by 2A (self-shearing polypeptide 2A), as shown in FIG. 5, and the sequence is SEQ ID NO: 20.
  • CAR and chimeric single-chain molecules can be efficiently co-expressed on the surface of T cells by flow cytometry. As shown in Figure 6, the expression efficiency is more than 20%.
  • CAR expression detection uses antibodies against mouse Fab; CD3 / B2M dual genes Editing efficiency is over 75% (editing efficiency: the efficiency of gene editing to knock out corresponding genes, which is reflected in the proportion of cells with negative gene expression in total cells).
  • gene-edited CAR-T cells that is, CAR-edi (CAR-editing, CAR-T cells lacking class I MHC molecules and TCR / CD3 complexes); gene-edited expression chimeric single strands
  • CAR-edi-CM CAR-editing-Chimeric Molecule, CAR-T cells in which a class I MHC molecule and a TCR / CD3 complex are deleted while expressing a chimeric single-chain molecule.
  • Example 6 An engineered CAR-T cell expressing a chimeric single-chain molecule can effectively resist the killing of primary NK cells.
  • the in vitro NK killing experiment is the same as in Example 3. Specifically, the target cells are:
  • Example 5 The gene-edited CAR-T cells prepared in Example 5, that is, CAR-edi and the gene-edited CAR-T cells that express a chimeric single-chain molecule, that is, CAR-edi-CM;
  • Primary NK cells are derived from healthy human PBMC. Flow cytometry was used to detect the decrease in the number of CAR T-cells lacking class I MHC molecules and the decrease in the number of CAR-T cells expressing the chimeric single-strand class I MHC molecules to perform kill comparison.
  • Figure 6 shows that, regardless of NK92 or primary NK cells, the killing of CAR-edi-CM is significantly weaker, indicating that CAR-T cells expressing chimeric single-chain can effectively resist the killing of various NK cells.
  • Example 7 An engineered CAR-T cell expressing a chimeric single-chain molecule has high cell expansion activity while resisting NK cell killing.
  • This test simulates an in vivo environment, that is, tumor cells, CAR-T cells, and NK cells coexist, and detects whether edited CAR-T cells are still capable of tumor cell-induced proliferation while being killed by NK. ⁇ ⁇ same as in Example 6.
  • the cell co-culture, edited CAR-T cells: Raji: NK92 1: 5: 5, in which the number of NK-92 cells is 1 ⁇ 10 5 / ml, other cells are analogized by analogy, 24-well culture plates Incubate at 37 ° C and record the number of edited CAR-T cells at different time points (Figure 8).
  • FIG. 7 shows that edited CAR-Ti cells (CAR-edi-CM) expressing a chimeric single-stranded molecule exhibited efficient expansion capacity, suggesting that this group of cells can resist the killing of NK cells; instead, under the same conditions Even with the activation of Raji tumor cells, CAR-edi still cannot expand normally because NK cells kill them.
  • CAR-edi-CM edited CAR-Ti cells
  • Example 8 An engineered CAR-T cell expressing a chimeric single-chain molecule has a highly efficient cell expansion activity in the environment of an allogeneic PBMC.
  • This test further scientifically simulates the in vivo environment. Because the immune cells in the body are complex and contain not only NK cells and target cells, the establishment of a true allogeneic PBMC environment is closer to the in vivo environment.
  • the edited CAR-T cells were tested for their ability to expand in an allogeneic PBMC environment, where both allogeneic rejection (not only sourced from NK cells) and target cells (CD19-positive B cells) were present.
  • Allogeneic PBMCs were stained with Dye eFluor TM 670 before co-culture to distinguish between CAR-T cells and allogeneic PBMC cells: Adjust the cell density to 1 ⁇ 10 7 / ml, add e670 dye with a final concentration of 10 ⁇ M, and incubate at room temperature for 5 min in the dark After washing the medium three times, it was used for experiments.
  • Figure 8 shows that edited CAR-Ti cells (CAR-edi-CM) expressing a chimeric single-stranded molecule showed a highly efficient expansion capacity, suggesting that this group of cells can resist the killing of allogeneic immune cells; instead, under the same conditions However, CAR-edi failed to expand normally, suggesting that allogeneic PBMC cells killed or inhibited it.
  • the data show that the more allogeneic PBMCs, the more significant inhibition of CAR-edi, however, the CAR-edi-CM group is not affected, and can respond to B cells in allogeneic PBMCs for efficient expansion.

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Abstract

提供了一种改造的淋巴细胞，细胞表面缺失I类MHC分子并表达包含信号肽、呈递肽段、B2M蛋白、HLA-E重链和连接序列的嵌合多聚体单链分子。还提供了改造的淋巴细胞的制备方法和在抑制NK细胞杀伤中的用途。

Description

一种基因工程化的免疫淋巴细胞及其制备方法 技术领域
本发明涉及一种基因工程化的淋巴细胞，尤其涉及组织相容性抗原(MHC)的表达失活的淋巴细胞，还涉及该淋巴细胞的制备方法和作为免疫治疗药物的用途。
背景技术
过继性细胞免疫治疗是从人体内将原代的T细胞分离出来，在离体条件下体外刺激、扩增和培养，最终将这些细胞回输给病人，以达到治疗疾病的目的。嵌合抗原受体T细胞疗法(CAR-T)是目前过继性细胞免疫治疗成功的代表之一。CAR-T疗法是指体外分离激活供体的T细胞，并将包括抗原识别区、共激活区和激活区的CAR基因转入T细胞内，进一步体外大规模扩增后回输给病人，以治疗疾病。现有的CAR-T技术都是个体化的，都使用病人自体的T细胞来生产CAR-T细胞。自体T细胞制备CAR-T细胞有很多缺点，比如生产周期长、制备成本高、很多情况下病人T细胞状态不好等问题。因此CAR-T技术需要突破个体化限制，也就是使用来自于健康供体的异体T细胞作为细胞来源来生产CAR-T细胞，这种细胞没有受体限制，因此被称为通用型CAR-T。
通用型的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。通用型CAR-T的制备要考虑两个问题。一方面，去除供体细胞的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，CAR-T细胞得以存活。另外一方面，去除供体细胞TCR复合体的表达，可以防止供体T细胞识别杀伤宿主细胞。I类MHC分子包括两种亚基，称为重链和轻链。因为重链的不同，I类MHC分为A/B/C/E/F/D几种不同种类。轻链是通用性亚基(B2M)，可以和任意一种重链组成I类MHC复合物。I类MHC复合物在细胞内的主要作用是呈递细胞内蛋白降解后的多肽片段，它在细胞内和多肽片段结合后形成多肽-I类MHC复合物，并转运到细胞表面。多肽-I类MHC复合物可以被特异的T细胞克隆的TCR识别，从而导致T细胞的杀伤。TCR由两个亚基构成，alpha(α)亚基和beta(β)亚基。两种亚基都有很多亚型，不同的亚型组合，形成大量不同的TCR克隆。TCR特异表达于T细胞表面，和多种不同的CD3 分子结合形成TCR复合物，该复合物可以识别多肽-I类MHC复合物，识别后可以引起T细胞的激活和被识别细胞的杀伤。
去除细胞内蛋白表达的方法主要是两种：RNA沉默导致的基因敲低和核酸酶介导的基因编辑技术。RNA沉默导致的基因敲低和基因编辑技术可以在供体T细胞内同时实现I类MHC复合物和TCR复合物的去除。基因编辑技术是使用特异性的稀切核酸内切酶切割细胞内基因组，切割完成后，细胞内固有的非同源重组修复机制发生，该修复为非特异性修复，易产生移码基因突变，从而导致基因敲除。
虽然基因敲除可以实现I类MHC复合物表达的去除，避免受体T细胞的攻击，但是同时I类MHC的缺失会引起受体NK细胞的激活杀伤。NK细胞是一种天然免疫监视细胞。NK细胞表面有很多受体，其中包括激活性受体和抑制性受体。激活性受体主要有NKG2D等，NKG2D的配体包括MICA，MICB，ULBP1-6等。抑制性受体主要有KIR，其配体为I类MHC分子。另外，抑制性受体还包括NKG2A，该分子的配体是I类MHC分子家族类似物HLA-E。(Morvan and Lanier,Nat Rev Cancer 2016.16,7-19)。
NK细胞是否被激活，取决于激活性信号和抑制性信号的平衡。正常细胞同时表达可以激活和可以抑制NK细胞激活的配体。正常细胞表达的I类MHC复合物与NK细胞抑制性受体结合，从而避免NK细胞的激活和杀伤。缺乏I类MHC复合物的细胞不能结合抑制性受体，导致抑制性受体不能被激活，平衡倾向于激活NK细胞，因此，缺乏I类MHC复合物的细胞可以被NK细胞识别杀伤。很多机制可以避免NK细胞的杀伤。被病毒感染的细胞会表达病毒来源的I类MHC家族蛋白的类似物，该类似物分子可以和抑制性受体NKG2A结合，抑制NK细胞的激活。引入I类MHC家族成员HLA-E分子到细胞内，该分子可以和抑制性受体NKG2A结合，抑制NK细胞的激活(CN 106103475 A)。另外，在HLA class I null721.221细胞内，I类MHC分子表达缺失，这种细胞会被NK细胞识别和杀伤。人为导入HLA-E或者HLA-G可以抑制NK细胞的激活，从而避免被NK细胞杀伤。引入免疫抑制性多肽如病毒I类MHC同源物或者NKG2D的配体同样可以抵抗NK细胞的杀伤。
由于HLA-E的细胞膜表达依赖于B2M形成复合体，因此在通用型CAR-T抑制或敲除B2M蛋白后，HLA-E不会定位于细胞膜。另外，HLA-E的膜定位还依赖于呈递多肽。一般HLA-E特异呈递HLA-A,-B,-C and-G的信号肽和某些病毒的多肽。人为构建HLA-E嵌合单链分子，可以增加HLA-E/B2M复合体在细胞上的表达。有研究表明，在 肿瘤细胞系或多能干细胞中表达递呈HLA-G信号肽的HLA-E嵌合单链分子可以有效地抵抗同种异体NK细胞的杀伤(WO 2004/103149 A2)。
虽然I类MHC分子缺失的细胞表达HLA-E嵌合单链分子可以有效抑制NK细胞的杀伤，但多数的数据集中在模式细胞系、病毒感染的细胞、多能干细胞或是肿瘤细胞。这种HLA-E嵌合单链分子在I类MHC分子缺失的免疫淋巴细胞中的表达尚未有人报道。同一种蛋白在不同类别的组织细胞中的表达模式(是否表达、表达量)和功能可以完全不同，HLA-E嵌合单链分子在免疫淋巴细胞中是否可以高效表达、发挥对NK细胞的抑制功能，尚不清楚。此外，HLA-E重链的不同亚型、HLA-E/B2M复合体所递呈的不同信号肽对HLA-E嵌合单链分子在细胞膜上是否表达、表达量高低和免疫功能至关重要。在不同HLA-E重链亚型和众多呈递肽中筛选出最优的组合，并表达于工程化免疫淋巴细胞，对同种异体的通用型免疫细胞治疗意义重大。
本发明简述
本发明涉及一种包含呈递肽段、B2M、HLA-E的嵌合单链分子，可以在B2M缺失的淋巴细胞内实现HLA-E的细胞膜表达，从而解决上述问题。包含呈递肽段、B2M、HLA-E的嵌合单链分子可以进一步应用于缺失TCR和B2M的通用型CAR-T，避免NK细胞的杀伤。
本发明提供了如下技术方案：
1.一种改造的淋巴细胞，细胞表面缺失I类MHC分子，并且细胞表面表达嵌合单链分子；所述嵌合单链分子包含：(a)呈递肽段；(b)B2M蛋白；(c)HLA-E重链；和(d)连接序列，该连接序列用于连接前述(a)和(b)，以及(b)和(c)片段。
2.技术方案1所述的改造的淋巴细胞，其中，所述嵌合单链分子N端到C端依次包含：呈递肽段-连接序列-B2M蛋白-连接序列-HLA-E重链。
3.技术方案2所述的改造的淋巴细胞，其中，所述呈递肽段是I类MHC分子的信号肽,优选所述呈递肽段包含或者是选自下述序列中的一个：SEQ ID NO:1，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:6和SEQ ID NO:7。
4.技术方案3所述的改造的淋巴细胞，其中，所述呈递肽段是SEQ ID NO:1，SEQ ID NO:5所示。
5.技术方案1-4任一项所述的改造的淋巴细胞，其中，所述淋巴细胞是T细胞，NK细胞，B细胞或巨噬细胞。
6.技术方案5所述的改造的淋巴细胞，其中，所述细胞表面缺失I类MHC分子是通过DNA切割或碱基编辑导致至少一个编码I类MHC分子组件的基因失活。
7.技术方案6所述的改造的淋巴细胞，其中，所述编码I类MHC分子的组件的基因是B2M基因。
8.技术方案6所述的改造的淋巴细胞，其中，所述编码I类MHC分子的组件的基因是HLA-A,HLA-B,或HLA-C基因。
9.技术方案6或7或8所述的改造的淋巴细胞，其中，所述DNA切割由稀切核酸内切酶导致。
10.技术方案9所述的改造的淋巴细胞，其中，所述稀切核酸内切酶是RNA指导的核酸内切酶，TAL核酸酶，归巢核酸内切酶，锌指核酸酶，或Mega-TAL核酸酶。
11.技术方案10所述的改造的淋巴细胞，其中，所述稀切核酸内切酶是RNA指导的核酸内切酶。
12.技术方案11所述的改造的淋巴细胞，其中，所述RNA指导的核酸内切酶是CRISPR-Cas系统。
13.技术方案12所述的改造的淋巴细胞，其中，所述CRISPR-Cas系统中的Cas蛋白是Cas9蛋白，优选是来源于化脓链球菌的Cas9蛋白。
14.技术方案6所述的改造的淋巴细胞，其中，所述碱基编辑由识别特定DNA序列的结构域与脱氨酶组成的复合体导致。
15.技术方案14所述的改造的淋巴细胞，其中，所述识别特定DNA序列的结构域是RNA指导的dCas9蛋白，或Cas9单链切口酶。
16.技术方案14所述的改造的淋巴细胞，其中，所述脱氨酶是胞嘧啶脱氨酶，或腺嘌呤脱氨酶。
17.前述任一项技术方案所述的改造的淋巴细胞，其中，所述连接序列选自下述序列中的一个或多个：SEQ ID NO:8和SEQ ID NO:9。
18.前述任一项技术方案所述的改造的淋巴细胞，其中，所述HLA-E重链序列是SEQ ID NO:12或SEQ ID NO:13。
19.技术方案18所述的改造的淋巴细胞，其中，所述嵌合单链分子为SEQ ID NO：14或SEQ ID NO：18所示。
20.前述任一项技术方案所述的改造的淋巴细胞，其中，所述T细胞包含具有编码所述嵌合多聚体单链分子的核酸序列。
21.技术方案20所述的改造的淋巴细胞，其中，所述编码所述嵌合多聚体单链分子的核酸序列如序列SEQ ID NO：15或SEQ ID NO：19所示。
22.技术方案5至21中任一项所述的改造的淋巴细胞，进一步特征在于至少一个编码T细胞受体(TCR)的组件的基因失活。
23.技术方案5至22中任一项所述的改造的淋巴细胞，其中，所述T细胞是一种CAR-T细胞。
24.技术方案23所述的改造的淋巴细胞，其中，所述的CAR针对B淋巴细胞抗原CD19。
25.技术方案23所述的改造的淋巴细胞，同时表达CAR分子与嵌合多聚体单链分子的蛋白序列如序列SEQ ID NO：20所示。
26.一种制备技术方案1-25任一项所述的工程化淋巴细胞的方法，包括以下步骤：
(i)制备表面缺失I类MHC分子的淋巴细胞；
(ii)在淋巴细胞表面表达嵌合多聚体单链分子；所述嵌合多聚体单链分子体包含：(a)呈递肽段，(b)B2M蛋白，(c)HLA-E重链，和(d)将前述(a)、(b)和(c)连接成单链的连接序列；
其中上述步骤(i)和(ii)的顺序可以互换。
27.技术方案26所述的方法，其中步骤(i)是通过DNA切割或碱基编辑导致淋巴细胞至少一个编码I类MHC分子组件的基因失活。
28.技术方案1-25任一项所述的淋巴细胞在抑制NK细胞杀伤中的应用。
29.技术方案1-25任一项所述的淋巴细胞在制备抑制NK细胞杀伤的药物中的应用。
30.一种抑制NK细胞的方法，包括对NK细胞或在患者体内提供技术方案1-15任一项所述的淋巴细胞。
31.技术方案30所述的方法，其中该淋巴细胞优选为T细胞，更优选为CAR-T细胞。
32.一种抑制NK细胞杀伤的药物组合物，其包含任一项技术方案1-25所述的淋巴细胞。
本发明的基因工程化淋巴细胞特征在于I类主要组织相容性抗原(MHC)的表达失活，避免了同种异体的免疫排斥。为了进一步增加基因工程化淋巴细胞在宿主内的存活，可以在这些修饰的T细胞内表达包含呈递肽段、B2M、HLA-E的嵌合单链分子，避免因I类MHC分子缺失导致的宿主NK细胞的杀伤。这种基因改造的淋巴细胞适合同种异体使用，降低了宿主排斥移植物的反应。本发明描述的基因工程化的T细胞可以进一步使T细胞受体(TCR)的表达失活，降低移植物抗宿主反应，从而作为通用型T细胞，更适用于同种异体使用，进一步可应用于嵌合抗原受体T细胞(CAR-T)或者T细胞受体T细胞(TCR-T)。
本发明的详述
定义：
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分子称为HLA分子，人类的I类MHC分子分为经典的HLA分子(HLA-A、HLA-B、HLA-C)和非经典的HLA分子(HLA-E、HLA-G、HLA-F)。
在具体的实施方式中，细胞表面缺失I类MHC分子是指缺失HLA-A/B/C/E/F/G的人淋巴细胞，可以通过编辑B2M或相应的重链基因使之失活来实现。
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部分。人类B2M蛋白由119个氨基酸(SEQ ID NO：16)组成，并具有11.800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。
B2M分子的缺失会使得细胞无法在细胞膜上表达I类MHC分子，也称HLA-I分子。
HLA-E嵌合单链分子
本发明的嵌合的单链分子，N端到C端依次为：呈递肽段-连接序列-B2M蛋白-连接序列-HLA-E重链分子。结构如图2所示。
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-(G 4S) 3-B2M成熟蛋白-(G 4S) 4-HLA-E重链分子，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。
其中，膜定位信号肽包括，如B2M、CD8等膜定位蛋白的信号肽。
信号肽：长度约20-80个氨基酸，位于成熟蛋白的N端，引导蛋白穿膜，并且在后来被剪切掉。如B2M、CD8等膜定位蛋白的信号肽。
其中，HLA-E重链分子包括，HLA-E*01:01和HLA-E*01:03亚型；或者是HLA-E*01:01和HLA-E*01:03亚型的变体。
所述的变体是指在原蛋白序列上经过一个或多个氨基酸的插入、取代和/或缺失，而保留原蛋白功能的分子；例如在HLA-E*01:01或HLA-E*01:03序列上经过一个或多个氨基酸的插入、取代和/或缺失，而保留HLA-E重链功能的分子。
呈递肽段：HLA-E复合体递呈的一段多肽，可以稳定HLA-E复合体的稳定性和结构特异性，该多肽来源于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；病毒蛋白多肽，如CMV(UL40)，EBV，HIV等。
连接序列
本发明的连接序列是连接一段具备柔性特征的氨基酸序列，用于连接本发明嵌合的单链分子中的呈递肽段与B2M蛋白，以及连接B2M蛋白与HLA-E重链。连接序列可以是(EAAAK)n，(GGGGS)n等，n＝3或4或5等。
在具体实施方式中，连接序列是4个甘氨酸和一个丝氨酸的3-4次重复，例如(G 4S) 3序列为(SEQ ID NO：2)，(G 4S) 4序列为(SEQ ID NO：3)
淋巴细胞
淋巴细胞是产生和成熟于淋巴系统，具有免疫应答功能的多种类细胞。本专利包括T细胞、B细胞、天然杀伤细胞(NK)细胞、巨噬细胞等。
碱基编辑(base editing)
对核酸(DNA或RNA)中腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)和胸腺嘧啶(T)或尿嘧啶(U)进行精准定点的改变，使其之间进行转换。
ZFN
Zinc-finger nucleases锌指核糖核酸，由DNA识别域与非特异性核酸内切酶构成。DNA识别域由一系列Cys2-His2锌指蛋白串联组成，每个锌指单元包括大约30个氨基酸，用于特异性结合DNA。非特异性核酸内切酶是FokI内切酶，其形成二聚体对DNA进行切割。
TALEN
TALEN(Transcription Activator-Like Effector Nucleases)是转录激活因子效应物核酸酶。TALE蛋白是DNA结合结构域的核心组份，一般由多个基本重复单元串联组成，通过设计和组合的串联单元可以特异性识别DNA序列，偶联FokI核酸内切酶后，可以实现特异DNA序列的切割。
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)，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，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。
CRISPR/Cas体系
CRISPR/Cas体系是由成簇规律间隔短回文重复序列(CRISPR)和CRISPR结合蛋白(即Cas蛋白)组成了的核酸酶系统，能够对真核细胞中几乎所有与原型间隔子邻近基序(protospacer-adjacent motif,PAM)相邻的基因组序列进行切割(Cong et al.Science2013.339:819-823)。“CRISPR/Cas体系”用来统称涉及CRISPR相关("Cas")基因的转 录物、以及涉及其表达或指导其活性的其他元件，包括编码Cas基因的序列、tracr(反式激活CRISPR)序列(例如tracrRNA或有活性的部分tracrRNA)、tracr配对序列(在内源CRISPR系统背景下，涵盖"同向重复"和加工的部分同向重复)、指导序列、或来自CRISPR座位的其他序列和转录物。一般而言，CRISPR体系的特征为促进在靶序列(在内源CRISPR系统中又称为原型间隔子)的位点处的CRISPR复合物的形成的元件。。
CRISPR复合物(包含杂交到靶序列上并且与一种或多种Cas蛋白复合的指导序列)的形成导致在该靶序列中或其附近(例如在1、2、3、4、5、6、7、8、9、10、20、50、或更多个碱基对之内)的一条链或两条链的切割。该tracr序列(其可以包含或其组成为野生型tracr序列的全部或部分(例如野生型tracr序列的约或多于约20、26、32、45、48、54、63、67、85个、或更多个核苷酸))也可以形成CRISPR复合物的一部分，如通过沿着该tracr序列的至少一部分杂交到与该指导序列可操作地连接的tracr配对序列的全部或部分上。在一些实施方式中，该tracr序列与一个tracr配对序列具有足够的互补性以进行杂交，并参与一种CRISPR复合物的形成。在一些实施方式中，当进行最佳比对时，沿着该tracr配对序列的长度，该tracr序列具有至少50％、60％、70％、80％、90％、95％、或99％序列互补性。在一些实施方式中，将驱动CRISPR系统的一个或多个元件的表达的一个或多个载体引入到宿主细胞中，使得该CRISPR系统的这些元件的表达在一个或多个靶位点指导CRISPR复合物的形成。
一般而言，tracr配对序列包括与tracr序列具有足够互补性以促进CRISPR复合物在靶序列处的形成，其中该CRISPR复合物包含杂交到tracr序列上的tracr配对序列。通常，互补程度是就tracr配对序列与tracr序列沿着这两个序列的较短者的长度的最佳比对而言。可以通过任何适合的比对算法来确定最佳比对，并且可以进一步将二级结构造成的影响考虑进来，比如在该tracr序列或tracr配对序列之内的自我互补性。在进行最佳比对时，在该tracr序列与tracr配对序列之间沿着这两者的较短者的长度的互补程度是约或多于约25％、30％、40％、50％、60％、70％、80％、90％、95％、97.5％、99％、或更高。该tracr序列在长度上为约或多于约5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、40、50个、或更多个核苷酸。
Cas蛋白的非限制性实例包括：Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8，Cas9(也称为Csn1和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、 Csf1、Csf2、Csf3、Csf4、其同系物、或其修饰形式。在一些实施例中，该Cas蛋白是Cas9蛋白。
Cas9，也称为Csn1，是既参与crRNA生物合成又参与摧毁入侵DNA的巨型蛋白质。已经在不同的细菌物种如嗜热链球菌(S.thermophiles)、无害利斯特氏菌(Listeria innocua)(Gasiunas，Barrangou et al.2012；Jinek，Chylinski et al.2012)和化脓性链球菌(S.Pyogenes)(Deltcheva，Chylinski et al.2011)中描述了Cas9。巨型Cas9蛋白(>1200个氨基酸)含有两个预测的核酸酶结构域，即位于蛋白质中部的HNH(McrA样)核酸酶结构域和分裂的RuvC样核酸酶结构域(RNAase H折叠)(Makarova，Grishin et al.(2006))。Cas9变体可以是不天然存在于自然界中并且是由蛋白质工程或通过随机诱变获得的Cas9核酸内切酶。例如，可以通过突变，即化脓性链球菌Cas9核酸内切酶(COG3513)的氨基酸序列中至少一个残基的缺失或插入或取代获得根据本发明的Cas9变体。在一些实施例中，该Cas9蛋白是肺炎链球菌、化脓链球菌或嗜热链球菌Cas9，并且可包括源自于这些生物体的突变的Cas9，或在Cas9上连接其他氨基酸序列的变体，如在Cas9上连接FokI酶。这些Cas9是已知的；例如，化脓链球菌(Streptococcus pyogenes)Cas9蛋白的氨基酸序列可见于SwissProt数据库登录号Q99ZW2下，脑膜炎奈瑟氏菌(Neisseria meningitides)Cas9蛋白的氨基酸序列可见于UniProt数据库编号A1IQ68，嗜热链球菌(Streptococcus thermophilus)Cas9蛋白的氨基酸序列可见于UniProt数据库编号Q03LF7，金黃色葡萄球菌(Staphylococcus aureus)Cas9蛋白的氨基酸序列可见于UniProt数据库编号J7RUA5。
制备工程化淋巴细胞的方法
制备工程化淋巴细胞的方法包括：(i)制备表面缺失I类MHC分子的淋巴细胞；(ii)在淋巴细胞表面表达嵌合单链分子；所述嵌合单链分子体包含：(a)呈递肽段；(b)B2M蛋白；c)HLA-E重链；和(d)连接序列，该连接序列用于连接前述(a)和(b)以及(b)和(c)片段。其中，上述步骤(i)和(ii)的顺序可以互换。
慢病毒制备方法：采用三质粒系统，慢病毒目的表达质粒(Addgene ID：#12252)，包装辅助质粒psPAX2(Addgene ID：#12260)和pMD2.G(Addgene ID：#12259)。在HEK293T细胞(来源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。将质粒与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0.22um滤膜过滤除菌后重悬待用。
载体构建：目标基因(如HLA-E嵌合单链分子DNA序列)被构建到慢病毒载体中。慢病毒载体包含长末端重复序列5’LTR和截短的3’LTR，RRE，rev应答元件(cPPT)，中央终止序列(CTS)和翻译后调控元件(WPRE)。HLA-E嵌合单链分子由EF-1a(延伸因子-1a)启动子，组成型表达，通过BamHI和SalI酶切构建到慢病毒载体上。
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。
T细胞慢病毒转染方法：原代T细胞激活后，24小时-72小时细胞明显聚团、变大进入分裂期可以进行慢病毒转染。病毒用量：细胞数目＝10：1，即MOI＝10转染细胞。转染后48小时可以进行蛋白表达检测。
基因编辑方法：T细胞激活后4天，或病毒转染后2天，收集细胞，用电转缓冲液，缓冲液T，T4，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。在体外同时将所需的sgRNA(总量300-400ng)与1ug 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10ul，用Neon电转仪进行电转。电击电压条件如下：1200-1600v，10ms(3次)。sgRNA候选序列根据相关网站的预测分析设计；Cas9蛋白来源于IDT DNA technology公司的Alt-R s.p.Cas9 Nuclease 3NLS蛋白。
表达嵌合单链分子的工程化CAR-T细胞制备：T细胞激活、病毒转染、基因编辑方法如上。流程为：第0天，原代PBMC激活；第2天病毒转染，第4天基因编辑(敲除TCR和B2M),第6-7天流式细胞术检测各蛋白表达。
所需抗体和细胞染料信息：anti-CD3-APC(BD,555335),anti-CD3-FITC(BD,555916),anti-B2M-PE(BD,551337),anti-MHC-I-APC(R&D,FAB7098A),anti-HLA-E-PE(Biolegend,342604),anti-HLA-E-APC(Biolegend,342606),anti-Fab-Biotin(Jackson ImmunoResearch,115-065-072),Streptavidin-PE(BD,554061),Dye eFluor TM 670(eBioscience,65-0840-90)
附图说明
图1.在原代T细胞中，利用电穿孔转染方法，靶向B2M的sgRNA和Cas9蛋白对B2M基因的进行编辑。
A.对照组，未经基因编辑；细胞表现出MHC-I和B2M双阳性分群。
B.B2M基因被编辑组；细胞同时缺失MHC-I和B2M，呈双阴性群。
图2.HLA-E嵌合单链分子结构示意图。
图3.原代T细胞嵌合单链分子表达的流式细胞术检测。
A.嵌合单链分子在T细胞中的表达。
B.嵌合单链分子在I类MHC分子缺失的T细胞中的表达。箭头指示B2M被编辑的细胞能够有效表达嵌合单链分子。
图4.表达嵌合单链分子的T细胞有效抵抗NK细胞的体外杀伤。
A.缺失I类MHC分子的T细胞表达嵌合单链分子，(左)对照组，(右)与NK-92共培养24小时后，细胞群的比例升高。
B.NK细胞杀伤裂解率比较。
图5.CAR与嵌合单链分子构建于同一载体的示意图。
图6.免疫表型检测。
A.CAR/嵌合单链分子高效地共表达于T细胞。
B.CAR-T细胞CD3/B2M双基因编辑效率大于75％。
C.表达嵌合单链分子的CAR-T细胞CD3/B2M双基因编辑效率大于75％。
图7.表达嵌合单链分子的工程化CAR-T细胞可以有效抵抗NK细胞的杀伤。
A.NK92细胞杀伤试验。
B.1#供者的原代NK细胞杀伤试验。
C.2#供者的原代NK细胞杀伤试验。
图8.CAR-T细胞在抵抗NK细胞杀伤过程中的增殖实验；CAR-edi-CM由于可以有效抵抗NK细胞的杀伤，展现出更强的增殖活性。
图9.CAR-T细胞在异体PBMC环境中的增殖实验；CAR-edi-CM由于可以有效抵抗PBMC中NK细胞的杀伤，展现出更强的增殖活性，即使在40倍异体PBMC环境中依然保持高效的增殖活性。
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。
实施例1：产生表面缺失I类MHC分子的工程化T细胞
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：17，SEQ ID NO:21。
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。
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)+300
IU/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。
电击转化方法
T细胞激活后4天，收集细胞，用电转缓冲液，缓冲液T，T4，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。在体外同时将上述两组sgRNA(各150ng)与1ug 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10ul，用Neon电转仪进行电转。电击电压条件如下：1200v，10ms(3次)。
基因编辑检测
完成电击3天后检测原代T细胞编辑的效率。取出约1x10 6细胞，加入1ml PBS(Gibco，cat#C10010500BT)清洗细胞1遍，用100ul PBS重悬，再加入20ul B2M-PE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。如图1，B2M阴性的细胞为被基因编辑的细胞，这些细胞同时缺少I类MHC分子的表达，表明B2M的缺少会影响到I类MHC分子的表达。
实施例2：产生表达嵌合单链分子的工程化T细胞
慢病毒制备方法
采用三质粒系统：，慢病毒目的表达质粒(Addgene ID：#12252)，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。将质粒与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。
T细胞表达嵌合单链分子的检测
T细胞激活(方法如实施例1)，48小时后，将上述步骤制备好的慢病毒转染激活的T细胞，再48小时后进行基因编辑(敲除B2M)，基因编辑72小时后用流式细胞术的方法检测感染后阳性的表达。如图3，在原代T细胞中，嵌合单链分子能够很好地表达，本实施例中表达效率约25％左右，同时，B2M被编辑后，缺失B2M(或I类MHC分子)的细胞仍旧可以很好地表达HLA-E嵌合单链分子(箭头指示)。说明我们设计的编辑B2M的sgRNA可以特异性识别并编辑基因组中的B2M，而不识别不编辑HLA-E嵌合单链分子中的B2M亚基。
实施例3：表达嵌合单链分子的I类MHC分子缺失的工程化T细胞可以有效抑制NK细胞的杀伤
NK-92细胞杀伤流式细胞仪检测方法。培养基ImmunoCult TM来源STEMCELL公司(同实施例1)。NK-92细胞购自中科院上海细胞研究所。靶细胞分别为：来源于健康人志愿者外周血的原代T细胞、实施例1制备的I类MHC分子缺失的T细胞、实施例2制备的表达嵌合单链分子的I类MHC分子缺失的T细胞。靶细胞终浓度为2×10 4/ml。
效应细胞NK-92细胞：工程化T细胞＝1：1混合，96孔培养板，每孔100ul，37℃培养箱共培养24小时。流式细胞仪检测，CD56蛋白用于区分NK细胞和T细胞，MHC-I蛋白用于鉴定被编辑的T细胞。
表达HLA-E嵌合单链分子可有效抑制NK-92细胞的杀伤。如图4,被NK-92细胞杀伤后，表达HLA-E嵌合单链分子的细胞，比例明显大于起始的比例(如箭头所示从39.85％到65.64％)，而未表达HLA-E嵌合单链分子的细胞比例明显下降。说明，NK-92细胞更易杀伤I类MHC分子缺失的细胞，而表达HLA-E嵌合单链分子后会有效抑制这种NK细胞介导的杀伤。通过统计分析，I类MHC分子缺失的细胞被裂解的比例达70％左右，而同样条件下表达HLA-E嵌合单链体分子后，被裂解的比例约7.5％左右。
实施例4：多种嵌合单链分子均可以有效抵抗NK细胞的杀伤
多种嵌合单链分子被检测抵抗NK细胞杀伤的效果。方法如实施例3，靶细胞分别采用Jurkat细胞株(T细胞来源)，经基因编辑使其缺失I类MHC分子(实施例2的方法)；同时为更全面证实嵌合多单链分子的功能，K562细胞株也被用于靶细胞(K562为天然的I类MHC分子缺失的细胞)。如表1所示，多种嵌合多单链分子在I类MHC分子缺失的细胞中均可以不同程度地高效抵抗NK细胞的杀伤。
表1：NK细胞对靶细胞的杀伤率
Figure PCTCN2019090604-appb-000001
Figure PCTCN2019090604-appb-000002
其中，表1中的嵌合单链分子A序列为SEQ ID NO：22；嵌合单链分子B序列为SEQ ID NO：23；嵌合单链分子C序列为SEQ ID NO：24；嵌合单链分子D序列为SEQ ID NO：25；嵌合单链分子E序列为SEQ ID NO：18。
实施例5：产生表面缺失I类MHC分子和CD3/TCR复合体的工程化CAR-T细胞
慢病毒制备、基因编辑等方法同实施例1和2。TCR/CD3复合体缺失采用基因编辑TRAC基因，根据相关网站的预测分析，我们筛选并涉及了编辑TRAC基因的sgRNA，序列为SEQ ID NO:26。CAR基因与嵌合单链分子基因由2A(自剪切多肽2A)连接，如图5所示，序列为SEQ ID NO:20。流式细胞术检测CAR与嵌合单链分子可以高效地共表达于T细胞表面，图6所示，表达效率20％以上，CAR表达检测，利用抗鼠源Fab的抗体；CD3/B2M双基因编辑效率75％以上(编辑效率：即基因编辑敲除相应基因的效率，体现于该基因表达为阴性的细胞占总细胞的比例)。
其中，对于命名：基因编辑过的CAR-T细胞，即CAR-edi(CAR-editing，I类MHC分子和TCR/CD3复合体缺失的CAR-T细胞)；基因编辑过的表达嵌合单链分子的CAR-T细胞，即CAR-edi-CM(CAR-editing-Chimeric Molecule，I类MHC分子和TCR/CD3复合体缺失，同时表达嵌合单链分子的CAR-T细胞)。
实施例6：表达嵌合单链分子的工程化CAR-T细胞可以有效抵抗原代NK细胞的杀伤。
体外NK杀伤实验同实施例3。具体的，靶细胞分别为：
实施例5制备的基因编辑过的CAR-T细胞，即CAR-edi和基因编辑过的表达嵌合单链分子的CAR-T细胞，即CAR-edi-CM；
原代NK细胞来源于健康人PBMC。流式细胞术分别检测I类MHC分子缺失的CAR-T细胞减少数目和表达嵌合单链的I类MHC分子缺失的CAR-T细胞减少数目，进行杀伤对比。
图6显示，无论是NK92或是原代NK细胞，对CAR-edi-CM的杀伤均明显较弱，表明表达嵌合单链的CAR-T细胞可以有效抵抗各种NK细胞的杀伤。
实施例7：表达嵌合单链分子的工程化CAR-T细胞在抵抗NK细胞杀伤的同时具有高效的细胞扩增活性。
被编辑的CAR-T细胞、肿瘤Raji细胞、NK-92细胞共培养试验的建立。
该试验模拟一种体内环境，即肿瘤细胞、CAR-T细胞、NK细胞同时存在，检测被编辑的CAR-T细胞在被NK杀伤的同时是否仍然具备肿瘤细胞诱导的增殖能力。杀伤实验同实施例6。具体的，细胞共培养，被编辑的CAR-T细胞：Raji：NK92＝1:5:5,其中NK-92细胞细胞数为1×10 5/ml，其它细胞按比例类推，24孔培养板，37℃培养，在不同时间点记录被编辑CAR-T细胞的数目(图8)。
图7显示表达嵌合单链分子的被编辑的CAR-T细胞(CAR-edi-CM)表现出高效的扩增能力，暗示该组细胞可以抵抗NK细胞的杀伤；相反，在同样的条件下，即使存在Raji肿瘤细胞的激活作用，CAR-edi依旧无法正常扩增，因为NK细胞对其进行了杀伤。
实施例8：表达嵌合单链分子的工程化CAR-T细胞在异体PBMC的环境下具有高效的细胞扩增活性。
被编辑的CAR-T细胞与异体PBMC细胞共培养试验的建立。
该试验科学上进一步模拟体内环境，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(CD19阳性的B细胞)。
细胞共培养，被编辑的CAR-T细胞：异体PBMC＝1:10或1：40,其中异体PBMC是与制备CAR-T细胞不同来源志愿者的PBMC。被编辑CAR-T细胞,细胞数为5×10 4/ml，其它细胞按比例类推，24孔培养板，37℃培养，在不同时间点记录被编辑CAR-T细胞的数目(图8)。
异体PBMC共培养前用Dye eFluor TM 670染色，用于区分CAR-T细胞和异体PBMC细胞：将细胞密度调至1×10 7/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。
图8显示表达嵌合单链分子的被编辑的CAR-T细胞(CAR-edi-CM)表现出高效的扩增能力，暗示该组细胞可以抵抗异体免疫细胞的杀伤；相反，在同样的条件下，CAR-edi无法正常扩增，暗示异体PBMC细胞对其进行了杀伤或是某种抑制。特别的，数据显示异体PBMC越多，对CAR-edi的抑制约明显，然而，CAR-edi-CM组不受影响，能够响应异体PBMC中的B细胞，进行高效的扩增。

Claims (30)

  1. 一种改造的淋巴细胞，细胞表面缺失I类MHC分子，并且细胞表面表达嵌合单链分子；所述嵌合单链分子包含：(a)呈递肽段；(b)B2M蛋白；(c)HLA-E重链；和(d)连接序列，该连接序列用于连接前述(a)和(b)，以及(b)和(c)片段。
  2. 权利要求1所述的改造的淋巴细胞，其中，所述嵌合单链分子N端到C端依次包含：呈递肽段-连接序列-B2M蛋白-连接序列-HLA-E重链。
  3. 权利要求2所述的改造的淋巴细胞，其中，所述呈递肽段是I类MHC分子的信号肽,优选所述I类MHC分子的信号肽包含或者是选自下述序列中的一个：SEQ ID NO:1，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:6和SEQ ID NO:7。
  4. 权利要求3所述的改造的淋巴细胞，其中，所述呈递肽段是SEQ ID NO:1，SEQ ID NO:5所示。
  5. 权利要求1-4任一项所述的改造的淋巴细胞，其中，所述淋巴细胞是T细胞，NK细胞，B细胞或巨噬细胞。
  6. 权利要求5所述的改造的淋巴细胞，其中，所述细胞表面缺失I类MHC分子是通过DNA切割或碱基编辑导致至少一个编码I类MHC分子组件的基因失活。
  7. 权利要求6所述的改造的淋巴细胞，其中，所述编码I类MHC分子的组件的基因是B2M基因。
  8. 权利要求6所述的改造的淋巴细胞，其中，所述编码I类MHC分子的组件的基因是HLA-A,HLA-B,或HLA-C基因。
  9. 权利要求6或7或8所述的改造的淋巴细胞，其中，所述DNA切割由稀切核酸内切酶导致。
  10. 权利要求9所述的改造的淋巴细胞，其中，所述稀切核酸内切酶是RNA指导的核酸内切酶，TAL核酸酶，归巢核酸内切酶，锌指核酸酶，或Mega-TAL核酸酶。
  11. 权利要求10所述的改造的淋巴细胞，其中，所述稀切核酸内切酶是RNA指导的核酸内切酶。
  12. 权利要求11所述的改造的淋巴细胞，其中，所述RNA指导的核酸内切酶是CRISPR-Cas系统。
  13. 权利要求12所述的改造的淋巴细胞，其中，所述CRISPR-Cas系统中的Cas蛋白是Cas9蛋白，优选是来源于化脓链球菌的Cas9蛋白。
  14. 权利要求6所述的改造的淋巴细胞，其中，所述碱基编辑由识别特定DNA序列的结构域与脱氨酶组成的复合体导致。
  15. 权利要求14所述的改造的淋巴细胞，其中，所述识别特定DNA序列的结构域是RNA指导的dCas9蛋白，或Cas9单链切口酶。
  16. 权利要求14所述的改造的淋巴细胞，其中，所述脱氨酶是胞嘧啶脱氨酶，或腺嘌呤脱氨酶。
  17. 前述任一项所述的改造的淋巴细胞，其中，所述连接序列选自下述序列中的一个或多个：SEQ ID NO:8和SEQ ID NO:9。
  18. 前述任一项所述的改造的淋巴细胞，其中，所述HLA-E重链序列是SEQ ID NO:12或SEQ ID NO:13。
  19. 权利要求18所述的改造的淋巴细胞，其中，所述嵌合单链分子为SEQ ID NO：14或SEQ ID NO：18所示。
  20. 前述任一项所述的改造的淋巴细胞，其中，所述T细胞包含具有编码所述嵌合多聚体单链分子的核酸序列。
  21. 权利要求20所述的改造的淋巴细胞，其中，所述编码所述嵌合多聚体单链分子的核酸序列如序列SEQ ID NO：15或SEQ ID NO：19所示。
  22. 权利要求5至21中任一项所述的改造的淋巴细胞，进一步特征在于至少一个编码T细胞受体(TCR)的组件的基因失活。
  23. 权利要求5至22中任一项所述的改造的淋巴细胞，其中，所述T细胞是一种CAR-T细胞。
  24. 权利要求23所述的改造的淋巴细胞，其中，所述的CAR针对B淋巴细胞抗原CD19。
  25. 权利要求23所述的改造的淋巴细胞，同时表达CAR分子与嵌合多聚体单链分子的蛋白序列如序列SEQ ID NO：20所示。
  26. 一种制备工程化淋巴细胞的方法，包括以下步骤；
    (i)制备表面缺失I类MHC分子的淋巴细胞；
    (ii)在淋巴细胞表面表达任一项权利要求1-25所述的嵌合多聚体单链分子；所述嵌合多聚体单链分子体包含：(a)呈递肽段，(b)B2M蛋白，(c)HLA-E重链，和(d)将前述(a)，(b)和(c)连接成单链的连接序列；
    其中上述步骤(i)和(ii)的顺序可以互换。
  27. 权利要求26所述的方法，其中步骤(i)是通过DNA切割或碱基编辑导致淋巴细胞至少一个编码I类MHC分子组件的基因失活。
  28. 权利要求1-25任一项所述的淋巴细胞在抑制NK细胞杀伤中的应用。
  29. 一种抑制NK细胞的方法，包括对NK细胞或在患者体内提供权利要求1-15任一项所述的淋巴细胞。
  30. 权利要求29所述的方法，其中该淋巴细胞优选为T细胞，更优选为CAR-T细胞。
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