WO2021249462A1 - 表达nk抑制性分子的工程化免疫细胞及其用途 - Google Patents

表达nk抑制性分子的工程化免疫细胞及其用途 Download PDF

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WO2021249462A1
WO2021249462A1 PCT/CN2021/099314 CN2021099314W WO2021249462A1 WO 2021249462 A1 WO2021249462 A1 WO 2021249462A1 CN 2021099314 W CN2021099314 W CN 2021099314W WO 2021249462 A1 WO2021249462 A1 WO 2021249462A1
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seq
cdr
cells
inhibitory
hla
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PCT/CN2021/099314
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English (en)
French (fr)
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周亚丽
陈功
姜小燕
任江涛
贺小宏
王延宾
韩露
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南京北恒生物科技有限公司
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Priority to US17/910,007 priority Critical patent/US20230242661A1/en
Priority to KR1020227039055A priority patent/KR20220166837A/ko
Priority to JP2022549899A priority patent/JP2023514386A/ja
Priority to AU2021286676A priority patent/AU2021286676A1/en
Priority to CA3171344A priority patent/CA3171344A1/en
Priority to EP21821147.2A priority patent/EP4112721A4/en
Publication of WO2021249462A1 publication Critical patent/WO2021249462A1/zh

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Definitions

  • the invention belongs to the field of immunotherapy. More specifically, the present invention relates to a NK inhibitory molecule comprising one or more NK inhibitory ligands, transmembrane domains and costimulatory domains, wherein the NK inhibitory ligands specifically bind to NK inhibitory
  • the receptor can inhibit the killing of NK cells to engineered immune cells expressing the NK inhibitory molecule.
  • CAR-T cell immunotherapy is to genetically modify T cells in vitro so that they can recognize tumor antigens, and after being amplified to a certain number, they are returned to the patient's body to kill cancer cells, thereby achieving the purpose of treating tumors.
  • the universal CAR-T can be prepared with T cells isolated from the peripheral blood of healthy donors, thereby realizing allogeneic reinfusion and greatly shortening the waiting time of patients for treatment.
  • the vitality and function of T cells obtained from healthy donors are also better than those of patient-derived T cells, which can increase the rate of CAR infection and improve the therapeutic effect.
  • knocking out CD52 can make universal CAR-T cells resistant to alemtuzumab (CD52 antibody), thereby avoiding killing the introduced CAR-T cells when alemtuzumab is used to eliminate T cells in patients.
  • alemtuzumab will increase the production and treatment costs of universal CAR-T products.
  • knocking out HLA molecules can ensure that CAR-T cells are prevented from being eliminated by patient T cells without the use of antibodies or other treatments, cells that knock out HLA molecules will be recognized by the patient’s NK cells and will be rejected. .
  • the present invention provides an NK inhibitory molecule comprising one or more NK inhibitory ligands, transmembrane domains and costimulatory domains, wherein the NK inhibitory ligands specifically bind to NK
  • An inhibitory receptor (NK inhibitory receptor, NKIR) is used to inhibit NK cells from killing the engineered immune cells expressing the NK inhibitory molecule.
  • the NK inhibitory ligand is an antibody or a functional fragment thereof that targets NKIR, or a natural ligand of NKIR or a NKIR binding fragment contained therein.
  • the NKIR is selected from NKG2/CD94 components (e.g., NKG2A, NKG2B, CD94); killer cell Ig-like receptor (KIR) family members (e.g., KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3); Leukocyte Ig-like receptor (LIR) family members (such as LIR1, LIR2, LIR3, LIR5, and LIR8); NK cell receptor protein 1 (NKR-P1) family members (such as NKR-P1B and NKR-P1D) ); immune checkpoint receptors (such as PD-1, TIGIT and CD96, TIM3, LAG3); carcinoembryonic antigen-related cell adhe
  • KIR killer
  • the NKIR is selected from PD1, NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, LAIR1, SIGLEC7, SIGLEC9 and KLRG1. More preferably, the NKIR is selected from PD1, NKG2A, CD94, KIR2DL1, KIR2DL2/3, KIR3DL1, LIR1, CEACAM1, LAIR1, SIGLEC7, SIGLEC9 and KLRG1.
  • the NK inhibitory ligand is an antibody that targets NKIR, which is a complete antibody, Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, linear antibody, sdAb or nano antibody.
  • the NK inhibitory ligand is an antibody or a functional fragment thereof that targets PD1, NKG2A, LIR1, KIR, SIGLEC7, SIGLEC9 and/or KLRG1.
  • the NK inhibitory ligand is a natural ligand of NKIR or a NKIR binding fragment contained therein.
  • the NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, immune checkpoint ligands (such as PD-L1/PD-L2 , CD155, CD112, CD113, Gal-9, FGL1, etc.), and the NKIR binding region they contain. More preferably, the NK inhibitory ligand is sialic acid, HLA-E, HLA-F, HLA-G, cadherin, PD-L1, PD-L2, or the NKIR binding region they contain.
  • the NK inhibitory ligand is selected from the group consisting of sialic acid, HLA-E extracellular domain, HLA-G extracellular domain, E-cadherin extracellular domain, PD-L1 extracellular domain and PD-L2 cell Outer zone. More preferably, the NK inhibitory ligand is the extracellular domain of E-cadherin, which comprises EC1 and EC2, more preferably EC1, EC2, EC3, EC4 and EC5.
  • the transmembrane domain contained in the NK inhibitory molecule is selected from the transmembrane domains of the following proteins: TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and the transmembrane domain of NKIR natural ligands, such as HLA- E, HLA-F, HLA-G, cadherin, collagen, OCIL transmembrane domain.
  • the transmembrane domain is selected from the transmembrane domains of CD8 ⁇ , CD4, CD28 and CD278.
  • the transmembrane domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% with the amino acid sequence shown in SEQ ID NO: 9 or 11. 100% sequence identity.
  • the costimulatory domain contained in the NK inhibitory molecule is selected from the costimulatory signaling domains of the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7- H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70 and combinations thereof.
  • the costimulatory domain of the present invention is derived from 4-1BB, CD28, CD27, OX40, CD278 or a combination thereof.
  • the costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or the amino acid sequence shown in SEQ ID NO: 13 or 15 100% sequence identity.
  • the NK inhibitory molecule does not contain an intracellular signaling domain. In another embodiment, the NK inhibitory molecule further comprises an intracellular signaling domain.
  • the intracellular signaling domain is selected from the signaling domains of the following proteins: FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling domain comprises the signaling domain of CD3 ⁇ .
  • the intracellular signaling domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the amino acid sequence shown in SEQ ID NO: 17 or 19. % Or 100% sequence identity.
  • the present invention also provides a nucleic acid encoding the aforementioned NK inhibitory molecule, and a vector containing the nucleic acid.
  • the present invention provides an engineered immune cell characterized by: (1) expressing the NK inhibitory molecule of the present invention, and (2) the expression of at least one MHC-related gene is suppressed or silenced.
  • the engineered immune cell of the present invention further expresses a chimeric antigen receptor, the chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling structure area.
  • the present invention provides an engineered immune cell characterized by: (1) expressing a fusion protein of the NK inhibitory molecule of the present invention and a chimeric antigen receptor, the fusion protein comprising an NK inhibitory ligand, The ligand binding domain, transmembrane domain, costimulatory domain, and intracellular signaling domain, and (2) the expression of at least one MHC-related gene is suppressed or silenced.
  • the MHC-related genes are selected from HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK , CIITA and their combinations, preferably HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA and their combinations.
  • the engineered immune cell further comprises suppressed or silenced expression of at least one TCR/CD3 gene selected from the group consisting of TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and their combination.
  • the expression of at least one TCR/CD3 gene and at least one MHC-related gene of the engineered immune cell is suppressed or silenced, wherein the at least one TCR/CD3 gene is selected from TRAC, TRBC and combinations thereof, the at least one MHC-related gene is B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.
  • the expression of TRAC or TRBC, and B2M of the engineered immune cells is suppressed or silenced.
  • the expression of TRAC or TRBC, and CIITA of the engineered immune cells is suppressed or silenced.
  • the expression of TRAC or TRBC, B2M and CIITA of the engineered immune cells is suppressed or silenced.
  • the expression of TRAC or TRBC, B2M and RFX5 of the engineered immune cells is suppressed or silenced.
  • the engineered immune cell of the present invention is further characterized in that the expression of one or more genes selected from the group consisting of CD52, GR, dCK and immune checkpoint genes, such as PD1, LAG3, is suppressed or silenced.
  • CD52, dCK, PD1, LAG3, TIM3, CTLA4, TIGIT or a combination of the engineered immune cells are inhibited or silenced.
  • the ligand binding domain binds to a target selected from: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25 , CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, CD179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BC
  • the engineered immune cells are B cells, T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells.
  • the engineered immune cells are T cells, such as CD4+/CD8+ T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), tumor infiltrating cells, Memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the NK inhibitory molecule, nucleic acid molecule, carrier or engineered immune cell of the present invention as an active agent, and one or more pharmaceutically acceptable Excipients.
  • the present invention also provides a method for treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of the NK inhibitory molecule according to the present invention, Nucleic acid molecules, vectors, engineered immune cells or pharmaceutical compositions. Therefore, the present invention also covers the use of NK inhibitory molecules, nucleic acid molecules, vectors, and engineered immune cells in the preparation of drugs for the treatment of cancer, infection or autoimmune diseases.
  • the cancer is a solid tumor or hematological tumor. More specifically, the cancer is selected from: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer , Choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma (GBM), liver cancer, hepatocytoma, Intraepithelial tumor, kidney cancer, laryngeal cancer, liver tumor, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectum Cancer, cancer of the respiratory system, saliva
  • the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.
  • the autoimmune diseases include but are not limited to type I diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Eddy Illness, Sjogren’s syndrome, Hashimoto’s thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus, etc.
  • the advantage of the present invention is that compared with expressing only NK inhibitory ligand, the NK inhibitory molecule of the present invention also contains a costimulatory domain, which can further reduce/inhibit the influence of NK cells on engineered immune cells in the subject. Even when the NK inhibitory molecule contains an intracellular signaling domain, it can enhance the killing of NK cells in the subject by engineered immune cells, thereby better reducing the risk of HvGD and realizing a true allogeneic return. lose.
  • cells in which the expression of one or more HLA-class I molecules are reduced or eliminated can be recognized as non-self by NK cells and thus targeted for killing. Therefore, the expression of one or more NK inhibitory molecules on the cell can protect it from NK cell killing.
  • the present invention provides an NK inhibitory molecule comprising one or more NK inhibitory ligands, transmembrane domains and costimulatory domains, wherein the NK inhibitory ligands are specific Combining with NK inhibitory receptor (NKIR) to inhibit NK cells from killing the engineered immune cells expressing the NK inhibitory molecule.
  • NKIR NK inhibitory receptor
  • NK inhibitory ligand refers to a molecule capable of binding to NKIR and inhibiting NK cell function (e.g., killing function).
  • the NK inhibitory ligand is an antibody or a functional fragment thereof that targets NKIR, or a natural ligand of NKIR or a NKIR binding fragment contained therein.
  • Non-limiting examples of NKIR include immunoreceptor tyrosine-based inhibitory motifs (Immunoreceptor tyrosine-based inhibitory motif, ITIM) or NK cell surface receptors bound thereto.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • NK cell surface receptors bound thereto include but are not limited to NKG2/CD94 components (e.g.
  • KIR killer cell Ig-like receptor
  • KIR killer cell Ig-like receptor family members
  • Leukocyte Ig-like receptor (LIR) family members such as LIR1, LIR2, LIR3, LIR5, and LIR8
  • NK cell receptor protein 1 (NKR-P1) family members such as NKR-P1B and NKR-P1D
  • immunity Checkpoint receptors such as PD-1, TIGIT and CD96, TIM3, LAG3
  • CEACAM1 carcinoembryonic antigen-associated cell adhesion molecule 1
  • CEACAM1 carcinoembryonic antigen-associated cell adhesion molecule 1
  • SIGLEC sialic acid-binding immunoglobulin-like lectin
  • SIGLEC9 sialic acid-binding immunoglobulin-like lectin
  • LAIR1 leukocyte-associated immunoglobulin-like receptor 1
  • Ly49 family members such as Ly49 family members
  • the NK inhibitory ligand is an antibody or a functional fragment thereof that targets NKIR, such as monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, murine antibody, chimeric antibody And its functional fragments.
  • antibodies or functional fragments thereof include, but are not limited to, intact antibodies, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, linear antibodies, sdAb (VH or VL), Nanobodies (Nanobody, Nb ) Etc., preferably selected from Fab, scFv, sdAb and Nanobody.
  • the NK inhibitory ligand is an antibody or a functional fragment thereof that targets the NKG2A/CD94 component.
  • the NK inhibitory ligand is an antibody targeting NKG2A, NKG2B or CD94.
  • NKG2/CD94 is a heterodimer composed of CD94 combined with another NKG2 subunit through a disulfide bond.
  • the cytoplasmic region of CD94 has only 7 amino acid residues and does not have a signal-transmitting structure.
  • the NKG2 family includes members such as NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, and NKG2F.
  • NKG2A and NKG2B are different splices of the same gene and have a high degree of homology.
  • the tail of the cytoplasmic region of NKG2A/2B contains two ITIMs, which can deliver inhibitory signals by recruiting SHP1 or SHP-2.
  • the natural ligand of NKG2A/2B is HLA-E. Since NKG2A/2B binds to a ligand with a higher affinity than the activating receptor NKG2C, when both the inhibitory receptor and the activated receptor of NK cells can bind to target cells expressing HLA-E, the inhibitory NKG2A/CD94 Will dominate and ultimately inhibit NK cell activity.
  • NKG2A antibody well known to those skilled in the art can be used in the present invention, such as Z270 (available from Immunotech, France), Z199 (available from Beckman Coulter, USA), 20D5 (available from BD Biosciences Pharmingen, USA), P25 (available from BD Biosciences Pharmingen, USA), Obtained from Morettaetal, Univ. Genova, Italy) and so on.
  • the NK inhibitory ligand is an antibody that targets NKG2A, which comprises (1) CDR-L1 shown in SEQ ID NO: 72, CDR-L1 shown in SEQ ID NO: 73, such as CDR-L3 shown in SEQ ID NO: 74, CDR-H1 shown in SEQ ID NO: 75, CDR-H2 shown in SEQ ID NO: 76, and CDR-H3 shown in SEQ ID NO: 77 , Or (2) CDR-L1 as shown in SEQ ID NO: 78, CDR-L2 as shown in SEQ ID NO: 79, CDR-L3 as shown in SEQ ID NO: 80, as shown in SEQ ID NO: 81 CDR-H1 as shown in SEQ ID NO: 82 and CDR-H2 as shown in SEQ ID NO: 83.
  • the NK inhibitory ligand is an antibody targeting NKG2A, which comprises a light chain variable region and a heavy chain variable region, and the light chain variable region is identical to SEQ ID NO: 3, 7, or 68.
  • the amino acid sequence shown has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, and the heavy chain variable region is identical to SEQ ID NO:1. 5.
  • the amino acid sequence shown in 67 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the NK inhibitory ligand is an anti-NKG2A antibody comprising SEQ ID NO: 1 and 3, an anti-NKG2A antibody comprising SEQ ID NO: 5 and 7, or an anti-NKG2A antibody comprising SEQ ID NO: 67 and 68 The anti-NKG2A antibody.
  • the NK inhibitory ligand is an antibody or functional fragment thereof that targets KIR.
  • the KIR molecule is a type I transmembrane protein, belonging to the immunoglobulin superfamily, and its structure includes the extramembrane domain, the transmembrane domain, and the cytoplasmic domain. According to the number of Ig-like domains contained in the extramembrane region, KIR can be divided into KIR2D and KIR3D subfamily. According to the length of the cytoplasmic region, KIR can also be divided into long type (L) and short type (S), such as KIR2DL, KIR2DS, KIR3DL, and KIR3DS.
  • L long type
  • S short type
  • the cytoplasmic region of KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, and KIR3DL3 contains two immunoreceptor tyrosine-based inhibitory motifs (ITIM), and the cytoplasmic region of KIR2DL5 contains one ITIM is an inhibitory KIR receptor.
  • ITIM immunoreceptor tyrosine-based inhibitory motifs
  • phosphorylation of ITIM contained in the cytoplasmic region of inhibitory KIR receptors can recruit phosphatase SHP1 and SHP2, leading to dephosphorylation of cell substrates, and ultimately inhibiting or terminating the effector functions of NK cells, such as cytotoxicity and cellular Secretion of factors.
  • KIR is expressed on most NK cells, although the level of expression varies among individuals.
  • KIR3DL2 recognizes HLA-A alleles -A3 and -A11
  • KIR3DL1 recognizes HLA-Bw-4
  • KIR2DL1 recognizes HLA-Cw2, HLA-Cw4, and HLA-Cw6 isotypes.
  • the mouse homologue of KIR is gp49B1, which is 335 amino acids in length and contains 2 ITIM structures in the cytoplasm.
  • the KIR-targeting antibody is an antibody that targets one or more targets selected from the group consisting of KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DL5, and gp49B1.
  • KIR antibodies well known to those skilled in the art can be used in the present invention, such as GL183 (targeting KIR2DL2/L3, available from Immunotech, France and Beckton Dickinson, USA), EB6 (targeting KIR2DL1, available from Immunotech, France and Beckton Dickinson , USA), AZ138 (targeting KIR3DL1, available from Morettaetal, Univ. Genova, Italy), Q66 (targeting KIR3DL2, available from Immunotech, France), Z27 (targeting KIR3DL1, available from Immunotech, France and Beckton Dickinson, USA) and so on.
  • GL183 targeting KIR2DL2/L3, available from Immunotech, France and Beckton Dickinson, USA
  • the NK inhibitory ligand is an antibody targeting KIR, which comprises CDR-L1 as shown in SEQ ID NO: 84, CDR-L2 as shown in SEQ ID NO: 85, and CDR-L2 as shown in SEQ ID NO: CDR-L3 shown in: 86, CDR-H1 shown in SEQ ID NO: 87, CDR-H2 shown in SEQ ID NO: 88, and CDR-H3 shown in SEQ ID NO: 89.
  • the NK inhibitory ligand is an antibody targeting KIR, which comprises a light chain variable region and a heavy chain variable region, and the light chain variable region corresponds to the amino acid sequence shown in SEQ ID NO: 58 Having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO: 59 Have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the NK inhibitory ligand is an anti-KIR antibody comprising SEQ ID NO: 58 and SEQ ID NO: 59, and its amino acid sequence is as shown in SEQ ID NO: 57 or 60, for example.
  • the NK inhibitory ligand is an LIR-targeting antibody or functional fragment thereof.
  • LIR is also called immunoglobulin-like transcripts (ILT) or monocyte-macrophage inhibitory receptor (MIR).
  • the LIR family contains 8 members, among which the cytoplasmic regions of LIR1 (also called ILT2), LIR2 (also called ILT4), LIR3 (also called ILT5), LIR5 (also called ILT3) and LIR8 contain 2-4
  • the ITIM structure at least one of which is a VXYXXL/V motif, belongs to the inhibitory LIR receptor.
  • LIR-1 can inhibit the killing of target cells expressing HLA-I molecules by NK cell line NKL and CD16-mediated activation of NKL.
  • the homologue of LIR in mice is PIR (Paired Ig-like receptor), including PIR-A and PIR-B. Among them, PIR-A transmits activation signals with the help of FcR ⁇ homodimer, while PIR-B is The inhibitory signal is transmitted through the four ITIM structures contained in its cytoplasmic region.
  • the NK inhibitory ligand is an antibody that targets LIR1, LIR2, LIR3, LIR5, LIR8, or PIR-B.
  • the NK inhibitory ligand is an antibody targeting LIR1, which comprises: (1) CDR-L1 as shown in SEQ ID NO: 90, CDR-L2 as shown in SEQ ID NO: 91 CDR-L3 shown in SEQ ID NO: 92, CDR-H1 shown in SEQ ID NO: 93, CDR-H2 shown in SEQ ID NO: 94, and CDR-H2 shown in SEQ ID NO: 95 H3, or (2) CDR-L1 as shown in SEQ ID NO: 96, CDR-L2 as shown in SEQ ID NO: 97, CDR-L3 as shown in SEQ ID NO: 98, as shown in SEQ ID NO: CDR-H1 shown in 99, CDR-H2 shown in SEQ ID NO: 100, and CDR-H3 shown in SEQ ID NO: 101.
  • the NK inhibitory ligand is an antibody targeting LIR1, which comprises a light chain variable region and a heavy chain variable region, and the light chain variable region is identical to that shown in SEQ ID NO: 61 or 65
  • the amino acid sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity
  • the heavy chain variable region is identical to SEQ ID NO: 62 or 64.
  • the amino acid sequence shown has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the NK inhibitory ligand is an anti-LIR1 antibody, the amino acid sequence of which is shown in SEQ ID NO: 63 or 66.
  • the NK inhibitory ligand is an antibody or a functional fragment thereof that targets immune checkpoint receptors (eg, PD-1, TIGIT, CD96, TIM3, LAG3).
  • the inhibitory ligand of NK is an antibody or a functional fragment thereof that targets PD-1.
  • PD-1 is mainly expressed in activated NK cells and belongs to the CD28 family. It is a type I transmembrane glycoprotein composed of 268 amino acids. Its structure mainly includes extracellular immunoglobulin variable domain (IgV)-like structure, hydrophobic transmembrane domain and intracellular domain.
  • IgV immunoglobulin variable domain
  • tyrosine residues at the end of the intracellular domain There are two independent tyrosine residues at the end of the intracellular domain.
  • the tyrosine residue at the nitrogen terminal is involved in the formation of an ITIM
  • the tyrosine residue at the carbon terminal is involved in the formation of an immunoreceptor tyrosine conversion motif (immunoreceptor tyrosine). based switch motif, ITSM).
  • PD-1 After PD-1 binds to its ligands (such as PD-L1 and PD-L2), it promotes the phosphorylation of tyrosine in the ITSM domain of PD-1, which in turn causes the dephosphorylation of downstream protein kinases Syk and PI3K, and inhibits The activation of downstream AKT, ERK and other pathways ultimately inhibits the activity of NK.
  • ligands such as PD-L1 and PD-L2
  • the NK inhibitory ligand is an antibody or functional fragment that targets TIGIT.
  • TIGIT is a member of the immunoglobulin superfamily. It consists of an extracellular immunoglobulin variable region (IgV) domain, a type 1 transmembrane domain, and an intracellular with ITIM and immunoglobulin tyrosine tail (ITT) motifs. Structure domain composition. When it binds to ligands (such as CD155, CD112, CD113), it can induce intracellular inhibition of signal transmission, thereby inhibiting the activity of NK cells.
  • ligands such as CD155, CD112, CD113
  • the NK inhibitory ligand is an antibody or functional fragment targeting LAG3.
  • LAG3 is a member of the protein Ig superfamily. It is a type 1 transmembrane protein expressed on activated T cells, NK cells, B cells and plasmacytoid dendritic cells. The four IgG domains of LAG3 have high structural homology with the CD4 molecule, but their amino acid homology is less than 20%. Studies have shown that LAG3 has a negative regulatory effect on the proliferation and persistent memory of T cells and NK cells. Once activated by its ligand (such as FGL1), it can promote tumor cells and other "bad cells" to escape the immune system.
  • FGL1 ligand
  • the NK inhibitory ligand is an antibody or functional fragment that targets TIM3.
  • TIM3 is a receptor protein of the TIM family, which is expressed on the surface of T cells, Treg cells, and innate immune cells (dendritic cells, natural killer cells, monocytes).
  • the TIM family members are encoded by three genes, specifically HAVCR1 encodes TIM1, HAVCR2 encodes TIM3, and TIMD4 encodes TIM4.
  • TIM3 has a variety of ligands, such as phosphatidylserine (phosphatidylserine), galectin-9 (galectin-9 or Gal-9), HMGB1 and CEACAM-1.
  • TIM3 When expressed in NK cells, TIM3 is considered to be a marker of dysfunctional NK cells, and TIM3 blockade has been shown to reverse the dysfunction of NK cells.
  • the NK inhibitory ligand is an antibody or functional fragment thereof that targets NKR-P1.
  • NKR-P1 belongs to type II transmembrane glycoprotein and is expressed in human, mouse and rat NK cells.
  • Six NKR-P1 members have been found in mice, namely NKR-P1A, NKR-P1B, NKR-P1C, NKR-P1D, NKR-P1E, NKR-P1F, and only NKR-P1A ( Also known as CD161).
  • NKR-P1 The extracellular domain of NKR-P1 molecule belongs to the NK receptor domain (NKD) in the C-type lectin-like superfamily, which is similar in structure to Ly49, Cd69, and CD94/NKG2 molecules.
  • NKR-P1 mainly exists as a homodimer
  • human NKR-P1A may exist in a monomeric form.
  • the cytoplasmic regions of NKR-P1 family molecules differ in structure in different species.
  • the cytoplasmic regions of NKR-P1B and NKR-P1D contain ITIM motifs, which recruit SHP-1 to deliver inhibition after tyrosine phosphorylation.
  • the NK inhibitory ligand is an antibody that targets NKR-P1B or NKR-P1D.
  • the NK inhibitory ligand is an antibody or functional fragment thereof that targets CEACAM1.
  • CEACAM1 also known as CD66a, bile glycoprotein (BGP) or C-CAM1
  • BGP bile glycoprotein
  • C-CAM1 is a member of the carcinoembryonic antigen (CEA) gene family and belongs to the immunoglobulin (Ig) superfamily.
  • Ig immunoglobulin
  • CEACAM1 interacts with other known CEACAM proteins including CD66a (CEACAM1), CD66c (CEACAM6) and CD66e (CEACAM5, CEA) proteins.
  • CEACAM1 CD66a
  • CEACAM6 CD66c
  • CEACAM5 CD66e
  • CEACAM1 The naming of the CEACAM1 isotype and the number of extracellular immunoglobulin-like domains (for example, CEACAM1 with 4 extracellular immunoglobulin-like domains is called CEACAM1-4), and the length of the cytoplasmic tail ( For example, CEACAM1-4 with a long cytoplasmic tail is called CEACAM1-4L, and CEACAM1-4 with a short cytoplasmic tail is called CEACAM1-4S).
  • the N-terminal domain of CEACAM1 starts immediately after the signal peptide, and its structure is considered to be IgV-type.
  • the NK inhibitory ligand is an antibody or functional fragment thereof that targets SIGLEC.
  • SIGLEC proteins 16 SIGLEC proteins have been identified in humans and 9 SIGLEC proteins have been identified in mice.
  • the SIGLEC protein consists of 2-17 extracellular Ig domains including an amino-terminal V-type domain.
  • the -type domain contains a sialic acid binding site.
  • Siglec is usually divided into two groups: the first subset consisting of Siglec1, Siglec2, Siglec4, and Siglec15, and the CD33-related ones including Siglec3, Siglec5, Siglec6, Siglec7, Siglec8, Siglec9, Siglec10, Siglec11, Siglec12, Siglec14, and Siglec16 The second subset.
  • Siglec7 also known as p75, CD328 or AIRM, contains an extracellular N-terminal Ig-like V-type domain, two Ig-like C2-type domains, and an intracytoplasm containing an ITIM motif and an ITIM-like motif area.
  • Siglec7 is constitutively expressed on NK cells, dendritic cells, monocytes and neutrophils. It has been observed that Siglec7 has an inhibitory effect on NK cell-mediated tumor clearance.
  • the structure of Siglec9 is very similar to Siglec7, and their N-terminal V-group Ig domain has about 77% total amino acid sequence identity and shows different sialic acid binding specificities.
  • NK cells In view of the functional studies of NK cells, it has been proved that tumor cells expressing Siglec9 binding sialic acid ligand inhibit NK cell activation and tumor cell killing. Many people's tumors robustly upregulate the sialic acid ligand that binds to Siglec9, which allows the tumor to evade immunity and cause cancer progression.
  • the NK inhibitory ligand is an antibody that targets Siglec7 or Siglec9, such as those known in the art.
  • the anti-Siglec7 antibody can be derived from human Siglec7/CD328 antibody (AF1138, R&D Systems), clone #194212 (MAB1138, R&D Systems), clone #194211 (MAB11381, R&D Systems), clone Z176 (A22330, Beckman Coulter) , 6-434 (339202, Biolegend), REA214 (Miltenyl Biotec), S7.7 (MCA5782GA, BioRad), 10B2201 (MBS604764, MyBioSource), 8D8 (MBS690562, MyBioSource), 10B2202 (MBS608694, MyBioSource), 5-386 ( MBS214370, MyBioSource).
  • Anti-Siglec9 antibodies can be derived from MAB1139 (clone #191240, R&D Systems), AF1139 (R&D Systems), D18 (SC-34936, Santa Cruz Biotechnology), Y-12 SC34938 (SC3-4938, Santa Cruz Biotechnology), AB 197981 (Abeam ), AB96545 (Abeam), AB89484 (clone #MM0552-6K12, Abeam), AB 130493 (Abeam), AB117859 (clone #3G8, Abeam), E10-286 (Becton Dickinson).
  • antibodies targeting both can also act as the NK inhibitory ligand of the present invention.
  • the NK inhibitory ligand is an antibody that targets SIGLEC7, SIGLEC9, or both, and it comprises (1) CDR-L1 as shown in SEQ ID NO: 102, as shown in SEQ ID NO: 103 CDR-L2, CDR-L3 shown in SEQ ID NO: 104, CDR-H1 shown in SEQ ID NO: 105, CDR-H2 shown in SEQ ID NO: 106, and CDR-H2 shown in SEQ ID NO: 107 CDR-H3 shown in SEQ ID NO: 122, (2) CDR-L1 shown in SEQ ID NO: 122, CDR-L2 shown in SEQ ID NO: 123, CDR-L3 shown in SEQ ID NO: 124, as shown in SEQ CDR-H1 shown in ID NO: 125, CDR-H2 shown in SEQ ID NO: 126 and CDR-H3 shown in SEQ ID NO: 1277, (3) CDR shown in SEQ ID NO: 131 -L1, CDR-L2
  • the NK inhibitory ligand is an antibody that targets SIGLEC7, SIGLEC9, or both, which comprises a light chain variable region and a heavy chain variable region, and the light chain variable region is identical to SEQ ID NO: 108
  • the amino acid sequence shown in, 128, 137, 146, 182, 185 or 194 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, so
  • the variable region of the heavy chain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% of the amino acid sequence shown in SEQ ID NO: 109, 129, 138, 147, 183, 186 or 195 Or 99% or 100% sequence identity.
  • the NK inhibitory ligand is an antibody against SIGLEC7, SIGLEC9 or both, and its amino acid sequence is shown in SEQ ID NO: 110, 130, 139, 148, 184, 187 or 196.
  • the NK inhibitory ligand is an antibody or functional fragment thereof that targets LAIR1.
  • LAIR1 contains 10 exons and encodes a type I transmembrane glycoprotein consisting of 287 amino acids, which contains a single extracellular C2 type Ig-like domain, followed by a stem region connected to a single transmembrane domain and 2 transmissions ITIM motifs that inhibit signaling.
  • LAIR1 has a certain degree of homology with LIR and KIR family members in structure, indicating that these molecules may be derived from the same ancestral gene. LAIR1 is expressed in T cells, B cells, natural killer (NK) cells, macrophages and dendritic cells, and hematopoietic progenitor cells including human CD34+ cells. Due to the existence of the ITIM motif, studies so far in humans and mice have found that LAIR1 exerts an immunosuppressive effect. Further studies have shown that LAIR1 can not only inhibit resting NK cells, but also inhibit the killing of target cells by activated NK cells.
  • the NK inhibitory ligand is an antibody or functional fragment thereof that targets Ly49.
  • Ly49 is a type II transmembrane glycoprotein, which can be connected by disulfide bonds to form a homodimer, which has a function similar to that of human KIR, that is, it transmits signals through the interaction with MHC-I molecular ligands, thereby adjusting NK Cell activity. So far, it has been found that the mouse Ly49 family includes 11 members, namely Ly49A, Ly49B, Ly49C, Ly49D, Ly49E, Ly49F, Ly49G, Ly49H, Ly49I, Ly49P, Ly49Q.
  • Ly49A, Ly49C, Ly49F, Ly49G, and Ly49Q all contain ITIM motifs in the cytoplasm, which can bind to and activate tyrosine kinase SHP-1, and inhibit NK cell activation by interfering with the production of phosphorylated tyrosine . Therefore, in a preferred embodiment, the NK inhibitory ligand is an antibody that targets Ly49A, Ly49C, Ly49F, Ly49G or Ly49Q.
  • the NK inhibitory ligand is an antibody or functional fragment thereof that targets KLRG1.
  • KLRG1 is a type II transmembrane protein surface co-inhibitory receptor that regulates the activities of T cells and NK cells. Its extracellular part contains a C-type lectin domain, a known ligand is cadherin, and its intracellular part contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) domain.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • the NK inhibitory ligand is an antibody targeting KLRG1, which comprises: (1) CDR-L1 shown in SEQ ID NO: 111, CDR-L2 shown in SEQ ID NO: 112 CDR-L3 shown in SEQ ID NO: 113, CDR-H1 shown in SEQ ID NO: 114, CDR-H2 shown in SEQ ID NO: 115, and CDR-H2 shown in SEQ ID NO: 116 H3, (2) CDR-L1 as shown in SEQ ID NO: 149, CDR-L2 as shown in SEQ ID NO: 150, CDR-L3 as shown in SEQ ID NO: 151, as shown in SEQ ID NO: 152 CDR-H1 shown in SEQ ID NO: 153 and CDR-H3 shown in SEQ ID NO: 154, (3) CDR-L1 shown in SEQ ID NO: 158, as shown in CDR-L2 shown in SEQ ID NO: 159, CDR-L3 shown in SEQ ID NO: 160, CDR-H
  • the NK inhibitory ligand is an antibody targeting KLRG1, which comprises a light chain variable region and a heavy chain variable region, and the light chain variable region is identical to SEQ ID NO: 117, 155, 164 or
  • the amino acid sequence shown in 173 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity
  • the heavy chain variable region is identical to SEQ ID NO:
  • the amino acid sequence represented by 118, 156, 165 or 174 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the NK inhibitory ligand is an anti-KLRG1 antibody, the amino acid sequence of which is shown in SEQ ID NO: 119, 157, 166 or 175.
  • the NK inhibitory ligand is the NKIR natural ligand or the NKIR binding region (such as the extracellular region) contained therein.
  • natural ligands include, but are not limited to, non-classical HLA-I molecules (such as HLA- E, HLA-F and HLA-G), Cadherin, collagen, OCIL, sialic acid, immune checkpoint ligands (e.g. PD-L1/PD-L2, CD155, CD112, CD113, Gal-9 , FGL1), etc.
  • the NK inhibitory ligand is a non-classical HLA-class I molecule or its extracellular region, more preferably the ⁇ 1 and ⁇ 2 domains of a non-classical HLA-class I molecule.
  • Non-classical HLA-I molecules are located in the same chromosome region 6p21.3, the short arm of chromosome 6, which consists of a heavy chain ( ⁇ chain) and a light chain ( ⁇ chain, encoded by the B2M gene) joined by non-covalent bonds .
  • the ⁇ chain includes the extracellular domain (including the three domains of ⁇ 1, ⁇ 2, and ⁇ 3), the transmembrane domain and the cytoplasmic region.
  • Non-classical HLA-I molecules include three members: HLA-E, HLA-F and HLA-G.
  • HLA-E regulates the activity of NK cells by binding to the CD94/NKG2 receptor on the surface of NK cells.
  • HLA-E The function of HLA-E is to bind peptides derived from the leader sequence of class I HLA molecules (HLA-A, -B, -C, and -G) and present them through the interaction with the inhibitory receptor CD94/NKG2A To NK cells, thereby inhibiting the lysis of cells expressing normal levels of class I HLA molecules by NK cells. Because the affinity of HLA-E and the inhibitory receptor CD94/NKG2A is significantly higher than that of the activating receptor CD94/NKG2C under physiological conditions, the up-regulation of the expression level of HLA-E can protect target cells from NK cells. Killing effect.
  • HLA-F can bind to NK inhibitory receptors ILT2 and ILT4, and this binding can be effectively inhibited by ILT2 and ILT4 antibodies.
  • HLA-G can recognize a variety of NK inhibitory receptors, such as CD94/NKG2A, LIR-1, LIR-2, KIR2DL1, etc.
  • NK inhibitory receptors such as CD94/NKG2A, LIR-1, LIR-2, KIR2DL1, etc.
  • Studies have found that HLA-G molecules on the surface of fetal cells may bind to the KIR on the surface of maternal NK cells to inhibit the killing activity of NK cells, leading to the mother’s immune tolerance to HLA semi-heterogeneous fetuses.
  • the high expression of HLA-G molecules on the surface of solid tumors such as melanoma, sarcoma, and lymphoma, can also enable tumor cells to escape the killing and lysis effects of NK cells.
  • the NK inhibitory ligand is the extracellular region of HLA-E, which has at least 70% of the amino acid sequence shown in SEQ ID NO: 31, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% with the nucleotide sequence shown in SEQ ID NO: 32 % Or 99% or 100% sequence identity.
  • the NK inhibitory ligand is a mutant of the extracellular region of HLA-E (including the Y84C mutation), which has at least 70%, preferably at least 80%, of the amino acid sequence shown in SEQ ID NO: 33, More preferably, the sequence identity is at least 90%, 95%, 97%, 99%, or 100%, or its coding sequence has at least 70%, preferably at least 80%, and the nucleotide sequence shown in SEQ ID NO: 34, and more Preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the NK inhibitory ligand is the extracellular region of HLA-G, which has at least 70% of the amino acid sequence shown in SEQ ID NO: 35, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% with the nucleotide sequence shown in SEQ ID NO: 36 % Or 99% or 100% sequence identity.
  • the endogenous B2M of the engineered immune cells needs to be knocked out and non-classical HLA-I molecules are expressed as NK inhibitory ligands
  • it is necessary to introduce synonymous mutations ie, only the nucleotide sequence The B2M gene is changed without changing the amino acid sequence
  • the B2M gene with synonymous mutations can also avoid being targeted to endogenous B2M gene editing tools knockout.
  • the NK inhibitory ligand comprises a fusion molecule of B2M and the extracellular region of a non-classical HLA-class I molecule.
  • the NK inhibitory ligand includes a fusion molecule of B2M and the extracellular domain of HLA-E or the extracellular domain of HLA-G.
  • the NK inhibitory ligand comprises a fusion molecule of B2M and the extracellular domain of HLA-E.
  • the HLA-E extracellular domain comprises a Y84C mutation (SEQ ID NO: 33).
  • the NK inhibitory ligand comprises a presentation peptide and a fusion molecule of the extracellular region of B2M and HLA-E, and the presentation peptide is selected from SEQ ID NO: 46-53.
  • the method of synonymously mutating the B2M gene is well known to those skilled in the art.
  • B2M has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 37 Sex; the nucleotide sequence of the synonymously mutated B2M gene is shown in SEQ ID NO: 38, for example.
  • the NK inhibitory ligand is osteoclast inhibitory lectin (OCIL) or its NKIR binding domain.
  • Mouse OCIL includes three members: OCIL (also known as Clr-b), OCILrP1 (also known as Clr-d), and mOCILrP2 (also known as Clr-g).
  • OCIL is widely expressed in various tissues, and its expression pattern is similar to that of MHC-I molecules.
  • OCIL is the ligand of NKR-P1B/D. Studies have shown that expressing OCL on tumor cells can inhibit the killing effect of NK cells on tumor cells, and OCL-specific antibodies can reverse this inhibition.
  • the NK inhibitory ligand is cadherin or its extracellular domain, such as E-cadherin (E-cad), N-cadherin (N-cad) or R-cadherin ( R-cad), preferably the extracellular region of E-cadherin.
  • Cadherin is a calcium-dependent transmembrane protein that mainly mediates homogenous adhesion between cells, which binds to the NK inhibitory receptor KLRG1.
  • the cadherin molecule is a type I membrane protein, composed of about 723-748 amino acids.
  • the structure includes the extracellular domain responsible for binding to the ligand, the transmembrane domain, and the highly conserved cytoplasmic domain.
  • the extracellular region has several cadherin repeat domains (EC), and contains a repetitive sequence composed of 4 to 5 amino acid residues, which is responsible for binding to the ligand.
  • Human E-cadherin is encoded by the CDH 1 gene and is currently the most studied member of the cadherin family. Therefore, in a preferred embodiment, the NK inhibitory ligand is the extracellular domain of E-cadherin, which contains EC1 and EC2. More preferably, the NK inhibitory ligand is the extracellular domain of E-cadherin, which includes EC1, EC2, EC3, EC4, and EC5.
  • the NK inhibitory ligand is E-Cad, which has at least 70% of the amino acid sequence shown in SEQ ID NO: 39 or 41, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% with the nucleotide sequence shown in SEQ ID NO: 40 or 42 Or 99% or 100% sequence identity.
  • the NK inhibitory ligand is collagen or its NKIR binding domain, which binds LAIR1.
  • Collagen molecule is a trimer composed of 3 alpha chains, each alpha chain contains (glycine-proline-hydroxyproline) n repeat sequence.
  • LAIR1 recognizes and interacts with Gly-Pro-Hyp repeats. Due to the widespread existence of this repetitive sequence, it has been proven that LAIR1 can bind to a wide range of collagen molecules, including but not limited to transmembrane collagen, such as collagen XVII, XIII, XXIII; and non-transmembrane collagen, such as collagen I, II, III, etc. .
  • Tumor cells or tumor stromal cells often highly express a variety of collagen molecules, which may be combined with the inhibitory receptor LAIR1 on the surface of immune cells to transmit inhibitory signals to immune cells, so as to achieve the purpose of immune escape.
  • the NK inhibitory ligand is sialic acid or its NKIR binding domain, which binds to a member of the SIGLEC family (eg, SIGLEC7 and/or SIGLEC9).
  • SIGLEC family eg, SIGLEC7 and/or SIGLEC9.
  • Sialic acid is an important part of the innate immune system of vertebrates, and the killing activity of NK cells is related to the sialylation on the surface of tumor cells.
  • the sialylation of tumor cells can not only hinder the physical interaction between tumor cells and NK cells, but also mask the activating ligands that can bind to the surface of tumor cells.
  • sialylation on the surface of tumor cells will cause the formation of immune synapses between tumor cells and NK cells to be blocked, thereby weakening the killing toxicity of NK cells to tumors.
  • Siglec7 is expressed on the surface of most NK cells, and after the sialic acid linked by ⁇ -2,8 glycosidic bonds on the surface of tumor cells binds to Siglec7 on the surface of NK cells, it inhibits the activation of NK cells, thereby allowing tumor cells to escape the NK cell mediation. Guided killing function.
  • the NK inhibitory ligand is PD-L1/PD-L2 or its extracellular region, which binds PD1.
  • PD-L1 is constitutively under-expressed in antigen presenting cells (APC), and non-hematopoietic cells such as vascular endothelial cells, pancreatic islet cells, and immune-privileged sites (such as placenta, testes, and eyes).
  • APC antigen presenting cells
  • non-hematopoietic cells such as vascular endothelial cells, pancreatic islet cells, and immune-privileged sites (such as placenta, testes, and eyes).
  • Inflammatory cytokines such as type I and type II interferons, TNF- ⁇ and VEGF can induce the expression of PD-L1.
  • PD-L2 is only expressed in activated macrophages and dendritic cells.
  • Tumor cells themselves can up-regulate the expression of PD-L1, and the inflammatory factors in the tumor microenvironment can also induce the expression of PD-L1 and PDL2.
  • the up-regulated expression of PD-L1 and PD-L2 on the surface of tumor cells can trigger the transmission of immunosuppressive signals mediated by PD-1 to inhibit the killing activity of NK cells.
  • the NK inhibitory ligand is the extracellular region of PD-L1 or the extracellular region of PD-L2, which has at least 70% of the amino acid sequence shown in SEQ ID NO: 70 or 71, preferably at least 80%. %, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the NK inhibitory ligand is CD155, CD112, or CD113 or its NKIR binding region, all of which bind TIGIT.
  • CD155 is a high-affinity ligand of TIGIT. Once CD155, which is highly expressed on the tumor surface, binds to the TIGIT on the NK surface, the killing effect of NK cells on tumor cells will be inhibited.
  • CD112 and CD113 also bind to TIGIT, although the affinity is relatively weak.
  • the NK inhibitory ligand is galectin 9 (also known as Gal-9) or its NKIR binding domain, which binds TIM3.
  • Gal-9 is a C-type lectin widely expressed and secreted by many hematopoietic cells, which can bind to carbohydrates on cell surface proteins.
  • Gal-9 binds to the carbohydrate motif on its IgV domain and can induce calcium influx and cell death in TIM3-positive NK cells.
  • a large number of evidences have shown that the TIM3/Gal-9 interaction plays a role in suppressing the immune response.
  • the NK inhibitory ligand is FGL1 or its NKIR binding region, which binds LAG3.
  • FGL1 belongs to the fibrinogen family and is a newly discovered ligand of LAG3, but it does not have characteristic domains such as platelet binding sites and thrombin sensitive sites.
  • FGL1 protein is mainly distributed in tumor cells, and the expression of tumor stroma is low.
  • the FGL1/LAG3 interaction is another tumor immune escape pathway independent of the B7-H1/PD-1 pathway. Blocking this pathway can have a synergistic effect with blocking the PD-1 pathway.
  • transmembrane domain refers to a polypeptide structure that enables chimeric antigen receptors to be expressed on the surface of immune cells (for example, lymphocytes or NKT cells) and guides immune cells to respond to target cells.
  • the transmembrane domain can be natural or synthetic, and can also be derived from any membrane-bound protein or transmembrane protein. When the chimeric antigen receptor binds to the target antigen, the transmembrane domain is capable of signal transduction.
  • Transmembrane domains particularly suitable for use in the present invention can be derived from, for example, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and transmembrane domains of natural ligands of NKIR, such as non-classical HLA-I molecules (such as HLA-E, HLA-F and HLA-G), Cadherin (Cadherin), collagen, OCIL and other transmembrane domains.
  • NKIR transmembrane domains of natural ligands of NKIR, such as non-classical HLA-I molecules (such as HLA-E, HLA-F and HLA-G), Cadherin (Cadherin), collagen,
  • the transmembrane domain is selected from the transmembrane domains of CD8 ⁇ , CD4, CD28 and CD278.
  • the transmembrane domain may be synthetic and may contain mainly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from CD8 ⁇ or CD28, and more preferably has at least 70% of the amino acid sequence shown in SEQ ID NO: 9 or 11, preferably at least 80%, more preferably at least 90%, 95%, 97 % Or 99% or 100% sequence identity, or the coding sequence of the transmembrane domain has at least 70%, preferably at least 80%, more preferably at least the nucleotide sequence shown in SEQ ID NO: 10 or 12 90%, 95%, 97% or 99% or 100% sequence identity.
  • the NK inhibitory molecule of the present invention may further comprise a hinge region located between the NK inhibitory ligand and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the ligand binding domain. Specifically, the hinge region is used to provide greater flexibility and accessibility to the ligand binding domain.
  • the hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the hinge region may be derived in whole or in part from natural molecules, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from the constant region of an antibody.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.
  • the hinge region comprises the hinge region part of CD8 ⁇ , CD28, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably CD8 ⁇ , CD28 or IgG4 hinge, which is shown in SEQ ID NO: 25, 27 or 29
  • the amino acid sequence of has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or the coding sequence of the CD28 hinge is the same as SEQ ID NO: 26, 28 or
  • the nucleotide sequence shown by 30 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the NK inhibitory molecule of the present invention includes a costimulatory domain. Therefore, when the NK inhibitory ligand binds to NKIR, on the one hand, it can transmit inhibitory signals to NK cells through the signal transduction area of NKIR, reducing its killing of target cells (for example, the engineered immune cells of the present invention); on the other hand;
  • the costimulatory domain contained in the NK inhibitory molecule can transmit stimulating signals to the target cells (for example, the engineered immune cells of the present invention) to stimulate their proliferation and survival, so as to better resist the killing of NK cells.
  • NK inhibitory molecules targeting NK inhibitory receptors can inhibit NK cells.
  • the present invention found for the first time that the expression of NK inhibitory molecules targeting NK inhibitory receptors can inhibit NK cells.
  • the present invention also found that, compared with the NK inhibitory molecule without the costimulatory domain, the NK inhibitory molecule containing the costimulatory domain has a better inhibitory effect on the killing effect of NK cells.
  • the costimulatory domain may be an intracellular functional signaling domain derived from a costimulatory molecule, which includes the entire intracellular part of the costimulatory molecule, or a functional fragment thereof.
  • a "costimulatory molecule” refers to a homologous binding partner that specifically binds to a costimulatory ligand on T cells, thereby mediating a costimulatory response (for example, proliferation) of T cells.
  • Co-stimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors.
  • Non-limiting examples of costimulatory domains of the present invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 , TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7 -H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70 and combinations thereof.
  • costimulatory signaling domains derived from the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 , T
  • the costimulatory domain of the present invention is derived from 4-1BB, CD28, CD27, OX40, CD278 or a combination thereof, more preferably 4-1BB, CD28 or a combination thereof.
  • the costimulatory domain of the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the amino acid sequence shown in SEQ ID NO: 13 or 15 Or 100% sequence identity, or the coding sequence of the costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95% with the nucleotide sequence shown in SEQ ID NO: 14 or 16 , 97% or 99% or 100% sequence identity.
  • the NK inhibitory molecule does not comprise an intracellular signaling domain.
  • the NK inhibitory molecule further comprises an intracellular signaling domain. That is, the NK inhibitory molecule includes an NK inhibitory ligand, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.
  • the binding of NK inhibitory ligand to NKIR will transmit activation signals to target cells (for example, the engineered immune cells of the present invention) through the costimulatory domain and intracellular signal transduction domain, so that the target cells will respond to NK. Cell killing, thereby further enhancing the inhibitory effect on NK cell killing.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specified function.
  • the intracellular signal transduction domain is responsible for the primary signal transmission in the cell after the ligand binding domain binds to the antigen, which leads to the activation of immune cells and immune response.
  • the intracellular signaling domain is responsible for activating at least one of the normal effector functions of immune cells in which NK inhibitory molecules are expressed.
  • the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
  • the intracellular signaling domain of the present invention may be the cytoplasmic sequences of T cell receptors and co-receptors, which act together to initiate primary signaling after antigen receptor binding, and any of these sequences Derivatives or variants and any synthetic sequence with the same or similar functions.
  • the intracellular signal transduction domain can contain many immunoreceptor tyrosine activation motifs (Immunoreceptor Tyrosine-based Activation Motifs, ITAM).
  • intracellular signaling domains of the present invention include, but are not limited to, those derived from FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the signaling domain of the CAR of the present invention may include a CD3 ⁇ signaling domain, which has at least 70%, preferably at least 80%, of the amino acid sequence shown in SEQ ID NO: 17 or 19 , More preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70% of the nucleotide sequence shown in SEQ ID NO: 18 or 20, preferably at least 80% %, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the NK inhibitory molecule of the present invention may also contain a signal peptide so that when it is expressed in a cell, such as a T cell, the nascent protein is directed to the endoplasmic reticulum and then to the cell surface.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids, which has a tendency to form a single ⁇ -helix.
  • signal peptidase At the end of the signal peptide, there is usually an amino acid segment that is recognized and cleaved by signal peptidase.
  • Signal peptidase can cut during or after translocation to produce free signal peptide and mature protein. Then, the free signal peptide is digested by a specific protease.
  • the signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8 ⁇ , IgG1, GM-CSFR ⁇ , and the like.
  • the signal peptide that can be used in the present invention has at least 70% of the amino acid sequence shown in SEQ ID NO: 21 or 23, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% Or 100% sequence identity, or the coding sequence of the signal peptide has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or the amino acid sequence shown in SEQ ID NO: 22 or 24. 99% or 100% sequence identity.
  • the engineered immune cell expressing the NK inhibitory molecule further includes that the expression of at least one MHC-related gene is inhibited or silenced, for example, the expression of at least one MHC gene is inhibited or silenced, or the expression of at least one MHC gene is inhibited or silenced, or at least The expression of a gene that interacts with an MHC gene or regulates its expression is suppressed or silenced.
  • MHC The major histocompatibility complex
  • H-2 The major histocompatibility complex
  • MHC protein is a heterodimer of two proteins: one is a transmembrane protein ⁇ chain encoded by the MHC I gene, and the other is ⁇ 2 microspheres of extracellular proteins encoded by genes not located in the MHC gene cluster Protein chain.
  • the ⁇ chain includes three domains, and the foreign peptide binds to the two most variable domains ⁇ 1 and ⁇ 2 at the N-terminus.
  • Class II MHC proteins are also heterodimers, containing two transmembrane proteins encoded by genes in the MHC complex. MHC class I/antigen complexes interact with cytotoxic T cells, while MHC class II present antigens to helper T cells. In addition, MHC class I proteins tend to be expressed in almost all nucleated cells and platelets (and red blood cells in mice), while MHC class II proteins are more selectively expressed. Generally, MHC class II proteins are expressed on B cells, some macrophages and monocytes, Langerhans cells and dendritic cells.
  • the human class I HLA gene cluster contains three main loci B, C, and A.
  • HLA-A, HLA-B and HLA-C are class I HLA heavy chain paralogs.
  • Class I molecules are heterodimers composed of MHC ⁇ heavy chain (encoded by HLA-A, HLA-B or HLA-C) and light chain ( ⁇ -2 microglobulin, encoded by B2M).
  • the heavy chain is anchored in the membrane, is about 45 kDa, and contains 8 exons.
  • suppressing or silencing the expression of MHC-related genes refers to suppressing or silencing the expression of one or more genes selected from: HLA-A, HLA-B, HLA-C, and B2M.
  • the human class II HLA cluster also contains three main loci DP, DQ, and DR, and both the class I gene cluster and the class II gene cluster are polymorphic.
  • HLA-DPA1, HLA-DQA1 and HLA-DRA belong to class II HLA ⁇ chain paralogs.
  • Class II molecules play a major role in the immune system by presenting exogenous peptides, and are mainly expressed in antigen-presenting cells (such as B lymphocytes, dendritic cells, and macrophages).
  • Class II molecules are heterodimers composed of alpha and beta chains that are both anchored in the membrane, where the alpha chain is about 33-35 kDa and contains 5 exons.
  • suppressing or silencing the expression of MHC-related genes refers to suppressing or silencing the expression of one or more genes selected from the group consisting of HLA-DPA, HLA-DQ, and HLA-DRA.
  • MHC class I and class II also depends on a variety of accessory proteins.
  • the Tap1 and Tap2 subunits are part of the TAP transporter complex necessary for loading peptide antigens on the class I HLA complex.
  • the LMP2 and LMP7 proteosome subunits play a role in the proteolytic degradation of antigens into peptides for display on HLA. It has been shown that reducing LMP7 reduces the expression of MHC class I on the cell surface.
  • the expression of MHC class II is induced and expressed by some positive regulatory factors, such as RFX complex, CIITA and so on.
  • the RFX complex is composed of three subunits: RFXANK (also called RFXB), RFX5, and RFX accessory protein (also called RFXAP).
  • the RFX complex promotes the expression of MHC class II molecules by promoting the binding of other transcription factors to the promoters of MHC class II molecules and enhancing the specificity of the promoter binding.
  • CIITA is the main control factor for the expression of MHC class II.
  • CIITA includes an N-terminal rich in acidic amino acids, a PST region rich in Pro, Ser, and Thr, a GTP binding region in the middle, and a C-terminal rich in Leu repeats (LRR).
  • LRR Leu repeats
  • suppressing or silencing the expression of MHC-related genes refers to suppressing or silencing the expression of one or more genes selected from: TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, and CIITA .
  • suppressing or silencing the expression of MHC-related genes refers to suppressing or silencing the expression of one or more genes selected from: HLA-A, HLA-B, HLA-C, B2M, HLA -DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA and combinations thereof, preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA and their combination.
  • the engineered immune cell expressing the NK inhibitory molecule further includes that the expression of at least one TCR/CD3 gene is suppressed or silenced.
  • T cell receptor is a characteristic mark on the surface of all T cells. It binds to CD3 by a non-covalent bond to form a TCR/CD3 complex, and passes through the specific MHC-antigen on the surface of the antigen presenting cell
  • the peptide complexes combine to generate specific antigen stimulation signals, activate T cells, and play a killing effect.
  • TCR is a heterodimer composed of two different peptide chains, usually divided into two types: ⁇ / ⁇ type and ⁇ / ⁇ type, in which more than 95% of peripheral T lymphocytes express TCR ⁇ / ⁇ .
  • the TCR ⁇ chain is encoded by the TRAC gene, and the ⁇ chain is encoded by the TRBC gene.
  • Each peptide chain of TCR includes a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region.
  • the cytoplasmic region is very short and does not have the ability to transmit antigen stimulation signals.
  • TCR molecules belong to the immunoglobulin superfamily, and their antigen specificity exists in the V region; each V region has three hypervariable regions, CDR1, CDR2, and CDR3, of which CDR3 has the largest variation, which directly determines the antigen binding specificity of TCR.
  • CDR1 and CDR2 recognize and bind to MHC molecules, and CDR3 directly binds to the antigen peptide.
  • CD3 includes four subunits: ⁇ , ⁇ , ⁇ , and ⁇ , which usually exist in the form of dimers ⁇ , ⁇ , and ⁇ . These four subunits all contain a conservative immunoreceptor tyrosine-based activation motif (ITAM), and two of the tyrosine residues are phosphorylated by tyrosine protein kinases, and they turn to T Cells transmit activation signals. Therefore, in one embodiment, suppressing or silencing the expression of at least one TCR/CD3 gene refers to suppressing or silencing the expression of one or more genes selected from: TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ .
  • TRAC immunoreceptor tyrosine-based activation motif
  • the engineered immune cells expressing NK inhibitory molecules include at least one TCR/CD3 gene and at least one MHC-related gene whose expression is suppressed or silenced, wherein the at least one TCR/CD3 gene is suppressed or silenced.
  • the CD3 gene is selected from TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and combinations thereof; the at least one MHC-related gene is selected from HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA- DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA and combinations thereof, preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA And their combination.
  • the at least one TCR/CD3 gene is selected from TRAC, TRBC, and combinations thereof, and the at least one MHC-related gene is selected from B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof .
  • the expression of TRAC or TRBC, and B2M of the engineered immune cells is suppressed or silenced.
  • the expression of TRAC or TRBC, and CIITA of the engineered immune cells is suppressed or silenced.
  • the expression of TRAC or TRBC, B2M and CIITA of the engineered immune cells is suppressed or silenced.
  • the expression of TRAC or TRBC, B2M and RFX5 of the engineered immune cells is suppressed or silenced.
  • the engineered immune cells of the present invention may also contain suppressed or silenced expression of at least one gene selected from: CD52, GR, dCK and Immune checkpoint genes, such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2,
  • Methods of inhibiting gene expression or silencing genes are well known to those skilled in the art, including but not limited to, for example, DNA fragmentation mediated by meganucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in the CRISPR system, or by Antisense oligonucleotides, RNAi, shRNA and other technologies can inactivate genes.
  • the engineered immune cells expressing NK inhibitory molecules of the present invention can also express chimeric antigen receptors. That is, in this embodiment, the engineered immune cell expresses the NK inhibitory molecule and the chimeric antigen receptor, and preferably, the expression of at least one MHC-related gene of the engineered immune cell is suppressed or silenced. In a preferred embodiment, the engineered immune cell further includes suppressed or silenced expression of at least one TCR/CD3 gene.
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide that generally includes one or more ligand binding domains (such as the antigen binding portion of an antibody), The membrane domain, costimulatory domain and intracellular signal transduction domain are connected by a joint.
  • CAR can use the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets in a non-MHC-restricted manner.
  • Non-MHC-restricted antigen recognition gives CAR cells the ability to recognize antigens unrelated to antigen processing, thus bypassing the main mechanism of tumor escape.
  • the CAR advantageously does not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).
  • ligand binding domain refers to any structure or functional variant thereof that can bind to a ligand (eg, an antigen).
  • the ligand binding domain can be an antibody structure, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, chimeric antibodies and functional fragments thereof.
  • the ligand binding domain includes, but is not limited to, intact antibodies, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, linear antibodies, sdAb (VH or VL), Nanobody (Nanobody, Nb) , Recombinant fibronectin domain, anticalin and DARPIN, etc., preferably selected from Fab, scFv, sdAb and Nanobody.
  • the ligand binding domain can be monovalent or bivalent, and can be a monospecific, bispecific or multispecific antibody.
  • Fab refers to any of the two identical fragments produced by papain cleavage of immunoglobulin molecules, consisting of a complete light chain and a heavy chain N-terminal part connected by a disulfide bond, wherein the heavy chain N-terminal part includes Heavy chain variable region and CH1. Compared with intact IgG, Fab has no Fc fragment, has higher fluidity and tissue penetration, and can bind to antigen monovalently without mediating antibody effects.
  • a “single chain antibody” or “scFv” is an antibody in which the variable region of the heavy chain (VH) of the antibody and the variable region of the light chain (VL) are connected by a linker.
  • the optimal length and/or amino acid composition of the linker can be selected.
  • the length of the linker will significantly affect the folding and interaction of the variable region of scFv. In fact, if a shorter linker (for example, between 5-10 amino acids) is used, intra-chain folding can be prevented.
  • a shorter linker for example, between 5-10 amino acids
  • the scFv may comprise VH and VL connected in any order, such as VH-linker-VL or VL-linker-VH.
  • Single domain antibody or “sdAb” refers to an antibody that naturally lacks light chain.
  • the antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as “heavy chain” antibody”.
  • VHH heavy chain variable region
  • CH2 and CH3 regions also known as “heavy chain” antibody”.
  • Nemobody or “Nb” refers to a separately cloned and expressed VHH structure, which has structural stability and antigen binding activity equivalent to that of the original heavy chain antibody, and is the smallest unit currently known to bind the target antigen .
  • the term "functional variant” or “functional fragment” refers to a variant that essentially contains the amino acid sequence of the parent but contains at least one amino acid modification (ie substitution, deletion or insertion) compared to the parent amino acid sequence, provided that all The variant retains the biological activity of the parent amino acid sequence.
  • its functional fragment is its antigen-binding portion.
  • the amino acid modification is preferably a conservative modification.
  • conservative modification refers to an amino acid modification that does not significantly affect or change the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the chimeric antigen receptor of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain.
  • Amino acid residue families with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid) ), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine Acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chains (e.g.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, t
  • amino acids involved threonine, valine, isoleucine
  • aromatic side chains such as tyrosine, phenylalanine, tryptophan, histidine.
  • Conservative modifications can be selected, for example, based on polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or similarity in the amphipathic properties of the residues involved.
  • the "functional variant” or “functional fragment” has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% of the parent amino acid sequence. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, And retain the biological activity of the parent amino acid, such as binding activity.
  • sequence identity refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in the alignment, and is usually expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Therefore, two copies with exactly the same sequence have 100% identity.
  • Those skilled in the art will recognize that some algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197) and ClustalW.
  • the ligand binding domain of the present invention binds to one or more targets selected from: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-1 1Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, CD20, Folate receptor ⁇ , ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor
  • the target is selected from: CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2 (Her2/neu), MUC1, EGFR, CAIX, WT1, NY- ESO-1, CD79a, CD79b, GPC3, Claudin 18.2, NKG2D and any combination thereof.
  • the CAR of the present invention can be designed to include a ligand binding domain specific for the antigen.
  • a CD19 antibody can be used as the ligand binding domain of the present invention.
  • transmembrane domain for the definitions of the transmembrane domain, costimulatory domain, intracellular signal transduction domain, optional hinge region, signal peptide and other structures contained in the CAR that can be used in the present invention, please refer to the above.
  • the CAR of the present invention may also include a switch structure to regulate the expression time of the CAR.
  • the switch structure may be in the form of a dimerization domain, which causes a conformational change by binding to its corresponding ligand, exposing the extracellular binding domain to bind to the targeted antigen, thereby activating the signal transduction pathway.
  • a switch domain can be used to connect the binding domain and the signal transduction domain respectively. Only when the switch domains bind to each other (for example, in the presence of an inducing compound), the binding domain and the signal transduction domain can pass through the dimer. Connected together to activate the signal path.
  • the switch structure can also be in the form of a masking peptide.
  • the masking peptide can mask the extracellular binding domain and prevent it from binding to the targeted antigen.
  • the masking peptide is cleaved by, for example, a protease, the extracellular binding domain can be exposed, making it a "normal" CAR structure.
  • Various switch structures known to those skilled in the art can be used in the present invention.
  • the CAR of the present invention may also contain a suicide gene, that is, it can express a cell death signal that can be induced by exogenous substances, so as to eliminate CAR cells when needed (for example, when serious toxic side effects are caused).
  • the suicide gene can be in the form of inserted epitopes, such as CD20 epitopes, RQR8, etc.
  • CAR cells can be eliminated by adding antibodies or reagents that target these epitopes.
  • the suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which can cause cells to die under induction of ganciclovir treatment.
  • HSV-TK herpes simplex virus thymidine kinase
  • the suicide gene can also be iCaspase-9, which can induce the dimerization of iCaspase-9 through chemically inducing drugs such as AP1903, AP20187, etc., thereby activating downstream Caspase3 molecules and leading to cell apoptosis.
  • iCaspase-9 which can induce the dimerization of iCaspase-9 through chemically inducing drugs such as AP1903, AP20187, etc., thereby activating downstream Caspase3 molecules and leading to cell apoptosis.
  • Various suicide genes known to those skilled in the art can be used in the present invention.
  • the engineered immune cell of the present invention express the fusion protein of the NK inhibitory molecule of the present invention and the chimeric antigen receptor, the fusion protein comprising NK inhibitory ligand, ligand The binding domain, transmembrane domain, costimulatory domain, and intracellular signaling domain, and (2) the expression of at least one MHC-related gene is suppressed or silenced.
  • the present invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding the NK inhibitory molecule of the present invention.
  • the nucleic acid molecule may also include a nucleic acid sequence encoding a chimeric antigen receptor.
  • nucleic acid molecule includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each of which is linear or circular in single-stranded and/or double-stranded form Shapes, or their mixtures (including hybrid molecules). Therefore, the nucleic acid according to the present invention includes DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.
  • Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)).
  • the nucleic acid of the present invention may also contain one or more modified bases, such as, for example, tritylated bases and unusual bases (such as inosine). Other modifications are also conceivable, including chemical, enzymatic or metabolic modifications, as long as the multi-chain CAR of the present invention can be expressed from polynucleotides.
  • the nucleic acid can be provided in an isolated form.
  • the nucleic acid may also include regulatory sequences, such as transcription control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.
  • the nucleic acid sequence of the present invention can be codon-optimized for optimal expression in desired host cells (eg, immune cells); or for expression in bacteria, yeast, or insect cells.
  • Codon optimization refers to the replacement of codons that are generally rare in highly expressed genes of a given species in the target sequence with codons that are generally common in highly expressed genes of such species, and the codons before and after the replacement Code the same amino acid. Therefore, the choice of the best codon depends on the codon usage preference of the host genome.
  • the present invention also provides a vector, which contains the nucleic acid molecule of the present invention.
  • the nucleic acid sequence encoding the NK inhibitory molecule and the nucleic acid sequence encoding the chimeric antigen receptor may be located in the same vector or in different vectors.
  • vector is a nucleic acid molecule used as a vehicle for transferring (exogenous) genetic material into a host cell, where the nucleic acid molecule can be replicated and/or expressed, for example.
  • Targeting vector is a medium that delivers an isolated nucleic acid to the inside of a cell by, for example, homologous recombination or a hybrid recombinase using a specific targeting site sequence.
  • An “expression vector” is a vector used for the transcription of heterologous nucleic acid sequences (such as those encoding the chimeric antigen receptor polypeptide of the present invention) in a suitable host cell and the translation of their mRNA. Suitable vectors that can be used in the present invention are known in the art, and many are commercially available.
  • the vectors of the present invention include, but are not limited to, plasmids, viruses (e.g.
  • the vector itself is usually a nucleotide sequence, usually a DNA sequence containing an insert (transgene) and a larger sequence as the "backbone" of the vector
  • the engineered vector usually also contains a starting point for autonomous replication in the host cell (if stable expression of the polynucleotide is required), a selection marker and a restriction enzyme cleavage site (such as multiple cloning site, MCS).
  • the vector may additionally contain a promoter. , Polyadenylic acid tail (polyA), 3'UTR, enhancer, terminator, insulator, operon, selectable marker, reporter gene, targeting sequence and/or protein purification tag and other elements.
  • polyA Polyadenylic acid tail
  • 3'UTR Polyadenylic acid tail
  • enhancer polyadenylic acid tail
  • terminator e.g., 3'UTR
  • insulator e.g., enhancer, terminator, insulator, operon, selectable marker, reporter gene, targeting sequence and/or protein purification tag and other elements.
  • the vector is an in vitro transcribed vector.
  • the present invention also provides an engineered immune cell that expresses the NK inhibitory molecule of the present invention, and wherein the expression of at least one MHC-related gene is suppressed or silenced.
  • the engineered immune cell of the present invention further expresses a chimeric antigen receptor comprising one or more ligand binding domains, transmembrane domains, costimulatory domains and cellular Internal signaling domain.
  • the engineered immune cell of the present invention further includes that the expression of at least one TCR/CD3 gene is suppressed or silenced.
  • the engineered immune cells of the present invention may also contain suppressed or silenced expression of at least one gene selected from: CD52, GR, dCK and Immune checkpoint genes, such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2,
  • the engineered immune cell of the present invention when the NK inhibitory molecule contains an intracellular signal transduction domain and the cell expresses CAR, the NK inhibitory molecule of the present invention and the CAR may share other structures other than the binding region, such as costimulatory domains and intracellular signals Conduction domain. Therefore, in this embodiment, the engineered immune cell of the present invention: (1) express the fusion protein of the NK inhibitory molecule of the present invention and the chimeric antigen receptor, the fusion protein comprising NK inhibitory ligand, ligand The binding domain, transmembrane domain, costimulatory domain, and intracellular signaling domain, and (2) the expression of at least one MHC-related gene is suppressed or silenced. In a preferred embodiment, the engineered immune cell further includes suppressed or silenced expression of at least one TCR/CD3 gene.
  • the term "immune cell” refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • the immune cells may be B cells, T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells, or immune cells derived from stem cells such as cord blood.
  • the immune cells are T cells.
  • the T cell may be any T cell, such as a T cell cultured in vitro, such as a primary T cell, or a T cell derived from a T cell line cultured in vitro, such as Jurkat, SupT1, etc., or a T cell obtained from a subject.
  • a T cell cultured in vitro such as a primary T cell
  • a T cell derived from a T cell line cultured in vitro such as Jurkat, SupT1, etc.
  • T cell obtained from a subject examples include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified.
  • T cells can be at any stage of development, including, but not limited to, CD4+/CD8+ T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • the immune cells are human T cells.
  • Various techniques known to those skilled in the art, such as Ficoll isolation, can be used to obtain T cells from the blood of the subject.
  • the NK inhibitory molecule and optional chimeric antigen receptor can be introduced into immune cells using conventional methods known in the art (such as by transduction, transfection, transformation, etc.).
  • Transfection is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells.
  • RNA transfection the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells.
  • the term is mainly used for non-viral methods in eukaryotic cells.
  • transduction is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells usually involves opening transient holes or "holes" in the cell membrane to allow uptake of material.
  • Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to produce liposomes that fuse with cell membranes and deposit their cargoes inside.
  • Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation.
  • transformation is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and non-animal eukaryotic cells (including plant cells).
  • transformation is a genetic modification of bacteria or non-animal eukaryotic cells, which is produced by direct ingestion from its surroundings through the cell membrane and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Conversion can be achieved by manual means. In order for transformation to occur, the cell or bacteria must be in a competent state. For prokaryotic transformation, techniques can include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation.
  • the present invention provides a kit comprising the NK inhibitory molecule, nucleic acid molecule, vector or engineered immune cell of the present invention.
  • kit of the present invention also contains instructions.
  • the present invention also provides a pharmaceutical composition comprising the NK inhibitory molecule, engineered immune cell, nucleic acid molecule or carrier of the present invention as an active agent, and a variety of pharmaceutically acceptable excipients. Therefore, the present invention also covers the use of the NK inhibitory molecules, nucleic acid molecules, vectors or engineered immune cells in the preparation of pharmaceutical compositions or medicines.
  • the term "pharmaceutically acceptable excipient” refers to pharmacologically and/or physiologically compatible with the subject and the active ingredient (that is, capable of eliciting the desired therapeutic effect without causing any undesirable
  • the carriers and/or excipients for the desired local or systemic effects are well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adherents, glidants, antioxidants, flavoring agents, coloring agents, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity regulators . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical composition of the present invention.
  • Exemplary excipients used in the pharmaceutical composition of the present invention include saline, buffered saline, dextrose, and water.
  • suitable excipients depends inter alia on the active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.
  • composition according to the present invention can be applied to various routes of administration. Usually, administration is accomplished parenterally.
  • Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.
  • the pharmaceutical composition according to the present invention can also be prepared in various forms, such as solid, liquid, gaseous or freeze-dried forms, especially ointments, creams, transdermal patches, gels, powders, tablets, solutions, gas
  • the processes known in the present invention for producing drugs may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or freeze-drying processes.
  • Pharmaceutical compositions containing immune cells such as those described herein are usually provided in the form of a solution, and preferably contain a pharmaceutically acceptable buffer.
  • the pharmaceutical composition according to the present invention can also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated.
  • agents suitable for the combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , Gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, Austria Auristatin E (auristatin E), vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth
  • the present invention also provides a method for treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of the NK inhibitory molecule, nucleic acid molecule, and vector according to the present invention , Engineered immune cells or pharmaceutical compositions. Therefore, the present invention also covers the use of the NK inhibitory molecules, nucleic acid molecules, vectors or engineered immune cells in the preparation of drugs for the treatment of cancer, infection or autoimmune diseases.
  • an effective amount of the immune cells and/or pharmaceutical composition of the present invention is directly administered to the subject.
  • the treatment method of the present invention is ex vivo treatment.
  • the method includes the following steps: (a) providing a sample, the sample comprising immune cells; (b) inhibiting the expression of at least one TCR/CD3 gene and at least one MHC-related gene of the immune cell in vitro Or silence, and introduce the NK inhibitory molecule of the present invention and the optional chimeric antigen receptor into the immune cell to obtain a modified immune cell, (c) administer the modified immune cell to a subject in need Of immune cells.
  • the immune cells provided in step (a) are selected from B cells, macrophages, dendritic cells, monocytes, T cells, NK cells or NKT cells; and the immune cells may be known in the art
  • the conventional method is obtained from a sample of a subject (especially a blood sample).
  • other immune cells capable of expressing the chimeric antigen receptor and NK inhibitory molecule of the present invention and exerting the desired biological effect function as described herein can also be used.
  • selected immune cells are compatible with the subject's immune system, that is, it is preferred that the immune cells do not elicit an immunogenic response.
  • step (c) can be carried out by introducing the nucleic acid or vector described herein into immune cells via electroporation or by infecting immune cells with a viral vector, the viral vector being the aforementioned lentiviral vector, adenoma Viral vector, adeno-associated virus vector or retroviral vector.
  • a viral vector being the aforementioned lentiviral vector, adenoma Viral vector, adeno-associated virus vector or retroviral vector.
  • transfection reagents such as liposomes
  • transient RNA transfection transient RNA transfection.
  • the immune cells are autologous or allogeneic cells, preferably B cells, T cells, macrophages, dendritic cells, monocytes or NK cells, NKT cells, more preferably T cells, NK cells or NKT cells.
  • autologous refers to any material derived from an individual that will later be reintroduced into that same individual.
  • allogeneic refers to any material derived from a different animal or a different patient of the same species as the individual into which the material is introduced. When the genes at one or more loci are different, two or more individuals are considered to be allogeneic to each other. In some cases, the genetic differences of allogeneic materials from individual individuals of the same species may be sufficient for antigenic interaction to occur.
  • the term "subject" is a mammal.
  • the mammal may be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples.
  • Mammals other than humans can be advantageously used as subjects representing animal models of cancer.
  • the subject is a human.
  • the cancer is a cancer associated with the expression of a target bound by a ligand binding domain.
  • the cancer includes, but is not limited to: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer , Choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), Liver cancer, hepatocellular tumor, intraepithelial tumor, kidney cancer, laryngeal cancer, liver tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer, glandular lung cancer and squamous lung cancer), lymphoma (including Hodgkin's lymphoma and Non-Hodgkin’s lympho
  • the disease that can be treated with the engineered immune cell or the pharmaceutical composition of the present invention is selected from the group consisting of leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer and the like.
  • the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.
  • the autoimmune diseases include but are not limited to type I diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Eddy Illness, Sjogren’s syndrome, Hashimoto’s thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus, etc.
  • the method further comprises administering one or more additional chemotherapeutic agents, biological agents, drugs, or treatments to the subject.
  • the chemotherapeutic agent, biological agent, drug or treatment is selected from radiotherapy, surgery, antibody agents and/or small molecules and any combination thereof.
  • Figure 1 Shows the expression level of HLA-E in E0-UNKi-T and E28-UNKi-T cells.
  • Figure 2 Shows the expression level of HLA-G in G0-UNKi-T and G28-UNKi-T cells.
  • Figure 3 Shows the expression level of E-cadherin in ECad0-UNKi-T and ECad28-UNKi-T cells.
  • Figure 4 shows the expression level of NKG2A scFv in A28-UNKi-T cells.
  • Figure 5 shows the expression level of KLRG1 in NK92-KLRG1 cells.
  • Figure 6 shows the inhibitory effect of the UNKi-T cells of the present invention on the killing effect of NK cells.
  • Two-way ANOVA was used for analysis, and T test was used for statistical analysis. * Indicates that the P value is less than 0.05, ** indicates that the P value is less than 0.01, and *** indicates that the P value is less than 0.001, reaching a significant level.
  • Figure 7 shows the expression level of NKG2A ScFv in A28z-UNKi-T cells.
  • Figure 8 Shows the expression level of HLA-E in E28z-UNKi-T cells.
  • Figure 9 shows the killing effect of UNKi-T cells of the present invention on NK cells.
  • Two-way ANOVA was used for analysis, and T test was used for statistical analysis. * Indicates that the P value is less than 0.05, ** indicates that the P value is less than 0.01, and *** indicates that the P value is less than 0.001, reaching a significant level.
  • Figure 10 shows the release level of IFN- ⁇ after the UNKi-T cells of the present invention are co-cultured with NK cells. Two-way ANOVA was used for analysis, and T test was used for statistical analysis. *** means that the P value is less than 0.001, reaching a significant level.
  • Figure 11 shows the expression level of KIR scFv in KIRG4-UNKi-T cells of the present invention.
  • Figure 12 shows the expression level of LIR1 scFv in LIRG4-UNKi-1-T and LIRG4-UNKi-2-T cells of the present invention.
  • Figure 13 shows the inhibitory effect of the UNKi-T cells of the present invention on the killing effect of NK cells.
  • Figure 14 shows the expression levels of scFv in SC7G4-T cells, SC7/SC9G4-T cells and K1G4-T cells.
  • Figure 15 shows the inhibitory effects of SC7G4-T cells, SC7/SC9G4-T cells and K1G4-T cells on the killing effect of NK cells.
  • Figure 16 shows the expression level of PDL1 in PDL1-T cells.
  • Figure 17 Shows the inhibitory effect of PDL1-T cells on the proliferation of NK cells.
  • Figure 18 shows the expression levels of KIR scFv in NKi-B cells and NKi-Huh7 cells.
  • Figure 19 shows the inhibitory effects of NKi-B cells and NKi-Huh7 cells on the killing effect of NK cells.
  • the T cells used in all the examples of the present invention are primary human CD4+CD8+T cells isolated from healthy donors by Ficoll-PaqueTM PREMIUM (GE Healthcare, article number 17-5442-02) using leukocyte separation.
  • Example 1 Construction of UNKi-T immune cells expressing NK inhibitory molecules and knocking out TCR/HLA-I/HLA-II
  • B2m signal peptide SEQ ID NO: 21
  • NK inhibitory ligand CD28 hinge region
  • CD28 transmembrane region SEQ ID NO: 11
  • the NK inhibitory ligand is the extracellular region of E-cadherin (SEQ ID NO: 41, corresponding to the ECad0 plasmid)
  • B2M and HLA-E extracellular region The fusion molecule (including the presenting peptide SEQ ID NO: 46, B2M SEQ ID NO: 37 and the HLA-E extracellular region mutant SEQ ID NO: 33, wherein the B2M nucleic acid sequence is a synonymous mutation of SEQ ID NO: 38 , Corresponding to E0 plasmid), or a fusion molecule of B2M and HLA-G extracellular region (including B2M SEQ ID NO:
  • the ECad0, E0, and G0 plasmids further include the CD28 costimulatory domain (SEQ ID NO: 13), and the ECad28, E28, and G28 plasmids were obtained, respectively. Confirm the correct insertion of the target sequence in the plasmid by sequencing.
  • B2m signal peptide SEQ ID NO: 21
  • anti-NKG2A-scFv including SEQ ID NO: 5 and 7
  • IgG4 Hinge region SEQ ID NO: 29
  • CD28 transmembrane region SEQ ID NO: 11
  • CD28 costimulatory domain SEQ ID NO: 13
  • Opti-MEM After adding 3ml Opti-MEM (Gibco, article number 31985-070) to the sterile tube to dilute the above plasmid, add the packaging vector psPAX2 (Addgene, Item No. 12260) and the envelope vector pMD2.G (Addgene, Item No. 12259). Then, add 120ul X-treme GENE HP DNA transfection reagent (Roche, catalog number 0636236601), mix immediately, incubate at room temperature for 15 minutes, and then add the plasmid/vector/transfection reagent mixture dropwise to the 293T cell culture flask . The virus was collected at 24 hours and 48 hours, and after combining them, ultracentrifugation (25000g, 4°C, 2.5 hours) was used to obtain concentrated lentivirus.
  • T cells were activated with DynaBeads CD3/CD28CTSTM (Gibco, catalog number 40203D), and cultured at 37°C and 5% CO2 for 1 day. Then, the concentrated lentivirus was added and the culture was continued for 3 days to obtain T cells expressing NK inhibitory molecules.
  • the CRISPR system is used to knock out the TCR/CD3 component (specifically TRAC gene) and MHC-related genes (specifically B2M and RFX5) in the T cells expressing the NK inhibitory molecule.
  • TRAC gene specifically TRAC gene
  • MHC-related genes specifically B2M and RFX5 genes
  • B2M and RFX5sgRNA was electrotransfected into activated NKi-T cells.
  • TCR/B2M/RFX5 Three knockout UNKi-T cells.
  • the wild-type T cells ie, Mock T cells
  • TCR/B2M/RFX5 with the CRISPR system and the wild-type T cells (ie, NT cells) that did not knock out the gene were used as controls.
  • the structure of the NK inhibitory molecule contained in the UNKi-T cells prepared in this example is shown in Table 1 below.
  • PE mouse anti-human HLA-E (biolegend catalog number 342604) was used to detect the HLA-E expression in UNKi-T cells and Mock T cells ( Figure 1), and PE mouse anti-human HLA-E G (biolegend catalog number 335906) detects HLA-G expression in UNKi-T cells and Mock T cells ( Figure 2), using E-cadherin monoclonal antibody (invitrogen catalog number 13-5700) and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, catalog number A-11001) to detect the expression of E-cadherin in UNKi-T cells and Mock T cells ( Figure 3), use Biotin-SP (long spacer) AffiniPure Goat Anti -Human IgG, F(ab') fragment specific antibody (Jackson ImmunoResearch, catalog number 109-065-097) and APC Streptavidin
  • Example 2 The inhibitory effect of UNKi-T cells on the killing effect of NK cells
  • the effector cells used in this example are NK92 cells. Since the NK92 cell line does not express E-cadherin receptor KLRG1, NK92 cells overexpressing KLRG1 were first prepared.
  • the nucleic acid sequence encoding KLRG1 (SEQ ID NO: 54) was synthesized and cloned into the pGEM-T Easy vector (Promega, catalog number A1360) in sequence, and the correct insertion of the target sequence was confirmed by sequencing.
  • the vector was digested with SpeI enzyme, and the linearized vector was obtained after purification and recovery. Then, according to the manufacturer’s recommendation, use the linearized vector as a template and use mMESSAGE T7 Ultra Kit (Invitrogen, article number AM1345) was used to prepare mRNA, and the Fastpure cell/Tissue total RNA isolation kit (Vazyme, article number RC101-01) was used for purification to obtain purified mRNA.
  • NK92 cells not transfected with KLRG1 served as control.
  • NK92-KLRG1 cells can effectively express KLRG1.
  • the inhibitory effect of the UNKi-T cells prepared in the present invention on the killing effect of NK cells was tested according to the following method: the UNKi-T cells and Mock-T cells prepared in the present invention were labeled with Far-Red (invitrogen, catalog number C34564).
  • NK92 cells used to express HLA-E, HLA-E, HLA- G or NKG2A scFv UNKi-T cells and Mock T cells
  • NK92-KLRG1 cells NK92-KLRG1 cells
  • NK inhibitory molecules containing inhibitory ligands such as NKG2A scFv, HLA-G, HLA-E, and E-cadherin UNKi-T cells can significantly reduce the killing effect of NK cells on T cells.
  • the addition of costimulatory domains can further significantly enhance the inhibition of NK cell killing by T cells (see G0 vs G28; E0 vs E28; ECad0 vs. ECad28).
  • the NK inhibitory molecule containing inhibitory ligand, transmembrane domain and costimulatory domain prepared by the present invention can significantly reduce the killing effect of NK cells on UNKi-T cells, thereby effectively reducing the risk of HvGD.
  • E28z-UNKi-T and A28z-UNKi-T cells according to the method described in Example 1, which are different from E28-UNKi-T and A28-UNKi-T cells only in that they further contain CD3 ⁇ intracellular signaling domain (SEQ ID NO: 17).
  • NK inhibitory molecules in E28z-UNKi-T cells and A28z-UNKi-T cells prepared by the present invention can be effectively expressed.
  • Cytotoxic T lymphocytes contain high concentrations of cytotoxic particles in the form of vesicles in the cytoplasm.
  • Lysosome-associated membrane protein I (CD107a) is the main component of vesicle membrane proteins.
  • CD107a Lysosome-associated membrane protein I
  • the CD107a molecule is a sensitive marker of CTL cell degranulation, which can reflect the cell killing activity.
  • NK92 cells target cells
  • Mock T cells E28z-UNKi-T cells
  • A28z-UNKi-T cells at a ratio of 1:1 to each well.
  • Co-cultivation was conducted at °C, 5% CO2, 18-24 hours later, the cell co-cultivation supernatant was collected.
  • cytokine IFN- ⁇ release level of E28z-UNKi-T cells and A28z-UNKi-T cells of the present invention is much higher than that of Mock T cells, which also indicates that the killing of NK92 target cells is significantly increased.
  • the following coding sequences were synthesized and cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), catalog number: PPL00157-4a): B2m signal peptide (SEQ ID NO: 21), anti-KIR-scFv (including SEQ ID NO) : 55 and 56) or anti-LIR1 scFv (including SEQ ID NO: 57 and 58, or SEQ ID NO: 59 and 60), IgG4 hinge region (SEQ ID NO: 29), CD8 ⁇ transmembrane region (SEQ ID NO: 9) CD28 costimulatory domain (SEQ ID NO: 13), obtain KIRG4, LIRG4-1 and LIRG4-2 plasmids, and confirm the correct insertion of the target sequence in the plasmid by sequencing.
  • PPL Public Protein/Plasmid Library
  • the following coding sequences were synthesized and cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), catalog number: PPL00157-4a): B2m signal peptide (SEQ ID NO: 21), anti-SIGLEC7-scFv (SEQ ID NO: 130), anti-SIGLEC7/SIGLEC9-scFv (SEQ ID NO: 184) or anti-KLRG1-scFv (SEQ ID NO: 119), IgG4 hinge region (SEQ ID NO: 29), CD8 ⁇ transmembrane region (SEQ ID NO: 9) ), CD28 costimulatory domain (SEQ ID NO: 13) to obtain SC7G4, SC7/SC9G4 and K1G4 plasmids, and confirm the correct insertion of the target sequence in the plasmid by sequencing.
  • PPL Public Protein/Plasmid Library
  • the above plasmid was transferred into T cells according to the method in Example 1, and the B2M gene was knocked out to obtain NKi-T cells expressing NK inhibitory molecules and B2M knockout (ie, SC7G4-T cells, SC7/SC9G4- T cells and K1G4-T cells). Only B2M knockout T cells were used as a negative control (NT).
  • the above-mentioned NKi-T cells were co-cultured with NK92-KLRG1 cells to detect their inhibitory effect on NK cell killing.
  • the results are shown in FIG. 15. It can be seen that, compared with NT, the T cells targeting SIGLEC7, SIGLEC9 or KLRG1 prepared in this example can significantly reduce the killing effect of NK cells on T cells.
  • PPL Public Protein/Plasmid Library
  • PPL00157-4a PDL1 signal peptide
  • SEQ ID NO: 70 PDL1 extracellular region
  • SEQ ID NO: 120 PDL1 transmembrane region
  • SEQ ID NO: 13 CD28 costimulatory domain
  • NKi-T cells expressing NK inhibitory molecules namely PDL1-T cells
  • Anti-PD-L1 Antibody manufactured by Soleibao, article number: 10084-R312-A
  • Figure 16 It can be seen that PDL1 is effectively expressed.
  • Untreated T cells were used as a negative control (NT).
  • NKi-T cells were cultured and treated with mitomycin C.
  • the number of NK cells was calculated by the ratio of total cells * NK cell population, and the results are shown in Figure 17. It can be seen that NKi-T cells targeting PD-1 can significantly inhibit the proliferation of NK cells.
  • Example 7 The inhibitory effect of KIR-targeted B cells and Huh7 cells on the killing effect of NK cells
  • the KIRG4 plasmid prepared in Example 4 was transferred to B cells and Huh7 cells (liver cancer cells) according to the method in Example 1, and the B2M gene was knocked out to obtain NKi-B expressing NK inhibitory molecules and knocking out B2M Cells and NKi-Huh7 cells.
  • B cells and Huh7 cells (NC cells) with only the B2M gene knocked out were used as negative controls.
  • the cells prepared above and NK cells were co-cultured according to the method in Example 2 to detect the inhibitory effect of the cells on the killing effect of NK cells.
  • the results are shown in FIG. 19. It can be seen that, compared with NC cells, the NKi-B cells and NKi-Huh7 cells prepared in this example can significantly reduce the killing effect of NK cells on them.

Abstract

提供一种NK抑制性分子，其包含一个或多个NK抑制性配体、跨膜结构域和共刺激结构域，其中所述NK抑制性配体特异性结合NK抑制性受体以抑制NK细胞对表达所述NK抑制性分子的工程化免疫细胞的杀伤。一种工程化免疫细胞，其表达本发明的NK抑制性分子，且其中至少一种MHC相关基因的表达被抑制或沉默。提供了该工程化免疫细胞在治疗癌症、感染或自身免疫性疾病中的用途。与传统的工程化免疫细胞相比，该工程化免疫细胞能够显著抑制受试者体内的NK细胞的杀伤作用，从而降低HvGD风险。

Description

表达NK抑制性分子的工程化免疫细胞及其用途 技术领域
本发明属于免疫治疗领域。更具体地，本发明涉及一种NK抑制性分子，其包含一个或多个NK抑制性配体、跨膜结构域和共刺激结构域，其中所述NK抑制性配体特异性结合NK抑制性受体以抑制NK细胞对表达所述NK抑制性分子的工程化免疫细胞的杀伤。
背景技术
近几年，癌症免疫治疗技术发展迅速，尤其是嵌合抗原受体T细胞(CAR-T)相关的免疫疗法在血液瘤的治疗上获得了优异的临床效果。CAR-T细胞免疫疗法是将T细胞在体外进行基因改造，使其能够识别肿瘤抗原，在扩增到一定数量后回输至病人体内，进行癌细胞杀伤，从而达到治疗肿瘤的目的。
2017年，两款自体型CAR-T疗法获FDA批准在美国上市，一款针对B细胞急性白血病，另一款针对弥漫性B细胞非霍奇金氏淋巴瘤。虽然这两款CAR-T细胞在临床上治疗效果优异，但其定价十分高昂，制备周期较长，使得大规模推广变得非常困难。因此，有必要发展通用型CAR-T产品以解决上述问题。通用型CAR-T可以用健康供体外周血分离的T细胞进行制备，从而实现同种异体回输，大大缩短患者等待治疗的时间。此外，从健康供体获得的T细胞的活力和功能也优于患者来源的T细胞，这可以增加CAR感染率，提高治疗效果。
然而，通用型CAR-T细胞的开发仍然面临如下两个问题：(1)工程改造的CAR-T细胞进入患者体内并增殖到一定程度后，可能攻击患者的正常细胞或组织，从而产生移植物抗宿主病(GvHD)；(2)患者体内的正常免疫系统可能会排斥异体来源的CAR-T细胞，从而产生宿主抗移植物病(HvGD)。目前，对于HvGD，主要通过敲除CD52或HLA的方式来降低或避免。具体地，敲除CD52可以使通用型CAR-T细胞对阿仑单抗(CD52抗体)产生抗性，从而避免在用阿伦单抗清除患者体内T细胞时对引入的CAR-T细胞产生杀伤。然而，使用阿仑单抗会增加通用型CAR-T产品的生产和治疗成本。另一方面，敲除HLA分子虽然可以保证在不使用抗体或者其他处理的情况下避免 CAR-T细胞被患者T细胞清除，但敲除HLA分子的细胞会被病人的NK细胞识别而发生排斥反应。
因此，仍然需要对现有的通用型CAR细胞疗法进行改进，尤其是降低NK细胞对CAR细胞的杀伤作用，从而进一步降低或避免HvGD风险。
发明简述
在第一个方面，本发明提供一种NK抑制性分子，其包含一个或多个NK抑制性配体、跨膜结构域和共刺激结构域，其中所述NK抑制性配体特异性结合NK抑制性受体(NK inhibitory receptor，NKIR)以抑制NK细胞对表达所述NK抑制性分子的工程化免疫细胞的杀伤。
在一个实施方案中，NK抑制性配体是靶向NKIR的抗体或其功能性片段，或NKIR的天然配体或其包含的NKIR结合片段。在一个实施方案中，所述NKIR选自NKG2/CD94组分(例如NKG2A、NKG2B、CD94)；杀伤细胞Ig样受体(KIR)家族成员(例如KIR2DL1、KIR2DL2/3、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2和KIR3DL3)；白细胞Ig样受体(LIR)家族成员(例如LIR1、LIR2、LIR3、LIR5和LIR8)；NK细胞受体蛋白1(NKR-P1)家族成员(例如NKR-P1B和NKR-P1D)；免疫检查点受体(如PD-1、TIGIT和CD96、TIM3、LAG3)；癌胚抗原相关的细胞黏附分子1(CEACAM1)；唾液酸结合性免疫球蛋白样凝集素(SIGLEC)家族成员(例如SIGLEC7和SIGLEC9)；白细胞相关的免疫球蛋白样受体1(LAIR1)；Ly49家族成员(例如Ly49A、Ly49C、Ly49F、Ly49G1和Ly49G4)和杀伤细胞凝集素样受体G1(KLRG1)。优选地，所述NKIR选自PD1、NKG2A、NKG2B、CD94、LIR1、LIR2、LIR3、KIR2DL1、KIR2DL2/3、KIR3DL1、CEACAM1、LAIR1、SIGLEC7、SIGLEC9和KLRG1。更优选地，所述NKIR选自PD1、NKG2A、CD94、KIR2DL1、KIR2DL2/3、KIR3DL1、LIR1、CEACAM1、LAIR1、SIGLEC7、SIGLEC9和KLRG1。
在一个实施方案中，NK抑制性配体是靶向NKIR的抗体，所述抗体是完整抗体、Fab、Fab’、F(ab’)2、Fv片段、scFv抗体片段、线性抗体、sdAb或纳米抗体。在一个优选的实施方案中，NK抑制性配体是靶向PD1、NKG2A、LIR1、KIR、SIGLEC7、SIGLEC9和/或KLRG1的抗体或其功能性片段。
在一个实施方案中，NK抑制性配体是NKIR的天然配体或其包含的NKIR结合片段。优选的，所述NK抑制性配体选自HLA-E、HLA-F、HLA-G、钙黏素、胶原蛋白、 OCIL、唾液酸、免疫检查点配体(例如PD-L1/PD-L2、CD155、CD112、CD113、Gal-9、FGL1等)，和它们包含的NKIR结合区。更优选的，所述NK抑制性配体是唾液酸、HLA-E、HLA-F、HLA-G、钙黏素、PD-L1、PD-L2，或它们包含的NKIR结合区。更优选的，所述NK抑制性配体选自唾液酸、HLA-E胞外区、HLA-G胞外区、E-钙黏素胞外区、PD-L1胞外区和PD-L2胞外区。更优选的，所述NK抑制性配体是E-钙黏素胞外区，其包含EC1和EC2，更优选包含EC1、EC2、EC3、EC4和EC5。
在一个实施方案中，NK抑制性分子包含的跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154，和NKIR天然配体的跨膜结构域，例如HLA-E、HLA-F、HLA-G、钙黏素、胶原蛋白、OCIL的跨膜结构域。在一个优选的实施方案中，跨膜结构域选自CD8α、CD4、CD28和CD278的跨膜结构域。
在一个实施方案中，所述跨膜结构域与SEQ ID NO：9或11所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，NK抑制性分子包含的共刺激结构域选自以下蛋白质的共刺激信号传导结构域：LTB、CD94、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM、ZAP70以及它们的组合。优选地，本发明的共刺激结构域来自4-1BB、CD28、CD27、OX40、CD278或其组合。
在一个实施方案中，所述共刺激结构域与SEQ ID NO：13或15所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，NK抑制性分子进不包含胞内信号传导结构域。在另一个实施方案中，NK抑制性分子进一步包含胞内信号传导结构域。
在一个实施方案中，所述胞内信号传导结构域选自以下蛋白的信号传导结构域：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。优选地，所述胞内信号传导结构域包含CD3ζ的信号传导结构域。
在一个实施方案中，所述胞内信号传导结构域与SEQ ID NO：17或19所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
本发明还提供编码上述NK抑制性分子的核酸，和包含所述核酸的载体。
在第二个方面，本发明提供一种工程化免疫细胞，其特征在于：(1)表达本发明的NK抑制性分子，和(2)至少一种MHC相关基因的表达被抑制或沉默。在一个实施方案中，本发明的工程化免疫细胞进一步表达嵌合抗原受体，所述嵌合抗原受体包含配体结合结构域、跨膜结构域、共刺激结构域和细胞内信号传导结构域。
在一个方面，本发明提供一种工程化免疫细胞，其特征在于：(1)表达本发明的NK抑制性分子和嵌合抗原受体的融合蛋白，所述融合蛋白包含NK抑制性配体、配体结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，和(2)至少一种MHC相关基因的表达被抑制或沉默。
在一个实施方案中，MHC相关基因选自HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA和它们的组合，优选HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA和它们的组合。
在一个实施方案中，所述工程化免疫细胞还包含至少一种TCR/CD3基因的表达被抑制或沉默，所述TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ和它们的组合。
在一个优选的实施方案中，所述工程化免疫细胞的至少一种TCR/CD3基因和至少一种MHC相关基因的表达被抑制或沉默，其中所述至少一种TCR/CD3基因选自TRAC、TRBC和它们的组合，所述至少一种MHC相关基因是B2M、RFX5、RFXAP、RFXANK、CIITA和它们的组合。在一个实施方案中，所述工程化免疫细胞的TRAC或TRBC，和B2M的表达被抑制或沉默。在一个实施方案中，所述工程化免疫细胞的TRAC或TRBC，和CIITA的表达被抑制或沉默。在一个优选的实施方案中，所述工程化免疫细胞的TRAC或TRBC、B2M和CIITA的表达被抑制或沉默。在一个优选的实施方案中，所述工程化免疫细胞的TRAC或TRBC、B2M和RFX5的表达被抑制或沉默。
在一个实施方案中，本发明的工程化免疫细胞的特征还在于，其中选自以下的一个或多个基因的表达被抑制或沉默：CD52、GR、dCK和免疫检查点基因，如PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、 CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。优选地，所述工程化免疫细胞的CD52、dCK、PD1、LAG3、TIM3、CTLA4、TIGIT或其组合被抑制或沉默。
在一个实施方案中，所述配体结合结构域与选自以下的靶标结合：TSHR、CD2、CD3、CD4、CD5、CD7、CD8、CD14、CD15、CD19、CD20、CD21、CD23、CD24、CD25、CD37、CD38、CD40、CD40L、CD44、CD46、CD47、CD52、CD54、CD56、CD70、CD73、CD80、CD97、CD123、CD22、CD126、CD138、CD 179a、DR4、DR5、TAC、TEM1/CD248、VEGF、GUCY2C、EGP40、EGP-2、EGP-4、CD133、IFNAR1、DLL3、kappa轻链、TIM3、tEGFR、IL-22Ra、IL-2、ErbB3、ErbB4、MUC16、MAGE-A3、MAGE-A6、NKG2DL、BAFF-R、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、GPRC5D、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-1 1Ra、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、AFP、Folate受体α、ERBB2(Her2/neu)、MUC1、EGFR、CS1、CD138、NCAM、Claudin18.2、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gploo、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD 179a、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、豆荚蛋白、HPV E6、E7、MAGE-A4、MART-1、WT-1、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋白、存活蛋白和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2 ETS融合基因)、NA17、PAX3、雄激素受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D和它们的任意组合。
在一个实施方案中，所述工程化免疫细胞是B细胞、T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞。优选的，所述工程化免疫细胞是T细胞，例如CD4+/CD8+T细胞、CD4+辅助T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如，细胞毒性T细胞)、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞。
在一个方面，本发明还提供一种药物组合物，其包含本发明所述的NK抑制性分子、核酸分子、载体或工程化免疫细胞作为活性剂，和一种或多种药学上可接受的赋型剂。
在一个方面，本发明还提供一种治疗患有癌症、感染或自身免疫性疾病的受试者的方法，包括向所述受试者施用有效量的根据本发明所述的NK抑制性分子、核酸分子、载体、工程化免疫细胞或药物组合物。因此，本发明还涵盖NK抑制性分子、核酸分子、载体、工程化免疫细胞在制备治疗癌症、感染或自身免疫性疾病的药物中的用途。
在一个实施方案中，所述癌症是实体瘤或血液肿瘤。更具体地，所述癌症选自：脑神经胶质瘤、胚细胞瘤、肉瘤、白血病、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、肝肿瘤、肺癌、淋巴瘤、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌、卵巢癌、胰腺癌、前列腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌以及其它癌和肉瘤、以及B细胞淋巴瘤、T细胞淋巴瘤、套细胞淋巴瘤、AIDS相关淋巴瘤、以及Waldenstrom巨球蛋白血症、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、B细胞急性淋巴细胞白血病(B-ALL)、T细胞急性淋巴细胞白血病(T-ALL)、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特氏淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、慢性骨髓性白血病(CML)、恶性淋巴组织增生疾病、MALT淋巴瘤、毛细胞白血病、边缘区淋巴瘤、多发性骨髓瘤、骨髓发育不良、浆母细胞性淋巴瘤、白血病前期、浆细胞样树突状细胞瘤、以及移植后淋巴细胞增生性紊乱(PTLD)。
在一个实施方案中，所述感染包括但不限于由病毒、细菌、真菌和寄生虫引起的感染。
在一个实施方案中，所述自身免疫性疾病包括但不限于I型糖尿病、腹腔疾病、格雷夫斯病、炎症性肠病、多发性硬化症、银屑病、类风湿性关节炎、艾迪生病、干燥综 合征、桥本甲状腺炎、重症肌无力、血管炎、恶性贫血与系统性红斑狼疮等。
本发明的优势之处在于，与仅表达NK抑制性配体相比，本发明的NK抑制性分子还包含共刺激结构域，这能够进一步降低/抑制受试者体内NK细胞对工程化免疫细胞的杀伤，甚至当NK抑制性分子包含胞内信号传导结构域时，其能够增强工程化免疫细胞对受试者体内NK细胞的杀伤，从而更好地降低HvGD风险，实现真正的同种异体回输。
发明详述
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本发明所属领域的普通技术人员通常所了解的相同。
NK抑制性分子
据报道，其中一种或多种HLA-I类分子表达被减少或消除的细胞可以被NK细胞识别为非自我，从而被靶向杀伤。因此，一种或多种NK抑制性分子在所述细胞上的表达可以保护它免受NK细胞杀伤。
因此，在第一个方面，本发明提供一种NK抑制性分子，其包含一个或多个NK抑制性配体、跨膜结构域和共刺激结构域，其中所述NK抑制性配体特异性结合NK抑制性受体(NK inhibitory receptor，NKIR)以抑制NK细胞对表达所述NK抑制性分子的工程化免疫细胞的杀伤。
如本文所用，术语“NK抑制性配体”是指能够与NKIR结合并抑制NK细胞功能(例如杀伤功能)的分子。在一个实施方案中，NK抑制性配体是靶向NKIR的抗体或其功能性片段，或NKIR的天然配体或其包含的NKIR结合片段。NKIR的非限制性实例包括具有基于免疫受体酪氨酸的抑制性基序(Immunoreceptor tyrosine-based inhibitory motif，ITIM)或与之结合的NK细胞表面受体。这类受体包括但不限于NKG2/CD94组分(例如NKG2A、NKG2B、CD94)；杀伤细胞Ig样受体(KIR)家族成员(例如KIR2DL1、KIR2DL2/3、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2和KIR3DL3)；白细胞Ig样受体(LIR)家族成员(例如LIR1、LIR2、LIR3、LIR5和LIR8)；NK细胞受体蛋白1(NKR-P1)家族成员(例如NKR-P1B和NKR-P1D)；免疫检查点受体(例如PD-1、TIGIT和CD96、TIM3、LAG3)；癌胚抗原相关的细胞黏附分子1(CEACAM1)；唾液酸结合性免疫球蛋白样凝集素(SIGLEC)家族成员(例如SIGLEC7和SIGLEC9)；白细胞相关的免疫球蛋白样受体1(LAIR1)；Ly49家族成员(例如Ly49A、Ly49C、Ly49F、Ly49G1和Ly49G4)和杀伤细胞凝集素样受体G1(KLRG1)。
在一个实施方案中，NK抑制性配体是靶向NKIR的抗体或其功能性片段，例如单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、鼠源抗体、嵌合抗体及其功能性片段。抗体或其功能性片段的实例包括但不限于完整抗体、Fab、Fab’、F(ab’)2、Fv片段、scFv抗体片段、线性抗体、sdAb(VH或VL)、纳米抗体(Nanobody，Nb)等，优选选自Fab、scFv、sdAb和纳米抗体。
在一个实施方案中，NK抑制性配体是靶向NKG2A/CD94组分的抗体或其功能性片段。在一个优选的实施方案中，NK抑制性配体是靶向NKG2A、NKG2B或CD94的抗体。NKG2/CD94是由CD94通过二硫键与另一个NKG2亚单位结合组成的异二聚体。CD94胞质区只有7个氨基酸残基，不具备传递信号的结构。NKG2家族包括NKG2A、NKG2B、NKG2C、NKG2D、NKG2E、NKG2F等成员，其中NKG2A和NKG2B是同一基因的不同剪接物，具有高度同源性。NKG2A/2B的胞质区尾部包含2个ITIM，通过募集SHP1或SHP-2来传递抑制信号。NKG2A/2B的天然配体是HLA-E。由于NKG2A/2B与配体结合的亲和力高于活化性受体NKG2C，因此当NK细胞的抑制性受体和活化受体都可与表达HLA-E的靶细胞结合时，抑制性的NKG2A/CD94将占据主导，最终抑制NK细胞活性。本领域技术人员熟知的NKG2A抗体可用于本发明，例如Z270(可获自Immunotech，France)、Z199(可获得自Beckman Coulter，USA)、20D5(可获得自BD Biosciences Pharmingen，USA)、P25(可获自Morettaetal，Univ.Genova，Italy)等。
在一个实施方案中，NK抑制性配体是靶向NKG2A的抗体，其包含(1)如SEQ ID NO：72所示的CDR-L1、如SEQ ID NO：73所示的CDR-L2、如SEQ ID NO：74所示的CDR-L3、如SEQ ID NO：75所示的CDR-H1、如SEQ ID NO：76所示的CDR-H2和如SEQ ID NO：77所示的CDR-H3，或(2)如SEQ ID NO：78所示的CDR-L1、如SEQ ID NO：79所示的CDR-L2、如SEQ ID NO：80所示的CDR-L3、如SEQ ID NO：81所示的CDR-H1、如SEQ ID NO：82所示的CDR-H2和如SEQ ID NO：83所示的CDR-H3。在一个实施方案中，NK抑制性配体是靶向NKG2A的抗体，其包含轻链可变区和重链可变区，所述轻链可变区与SEQ ID NO：3、7或68所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，所述重链可变区与SEQ ID NO：1、5、67所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个优选的实施方案中，NK抑制性配体是包含SEQ ID NO：1和3的抗NKG2A抗体、包含SEQ ID NO：5和7的抗NKG2A抗体，或是包含SEQ ID NO：67和68的抗NKG2A抗体。
在一个实施方案中，NK抑制性配体是靶向KIR的抗体或其功能性片段。KIR分子是I型跨膜蛋白，属于免疫球蛋白超家族，其结构包括膜外区、跨膜区和胞质区。根据膜外区包含的Ig样结构域的数量，KIR可以分为KIR2D和KIR3D亚家族。根据胞质区的长短，KIR还可以分为长型(L)和短型(S)，例如KIR2DL、KIR2DS、KIR3DL、KIR3DS。其中，KIR2DL1、KIR2DL2、KIR2DL3、KIR3DL1、KIR3DL2、KIR3DL3的胞质区包含2个基于免疫受体酪氨酸的抑制基序(Immunoreceptor tyrosine-based inhibitory motif，ITIM)，KIR2DL5的胞质区包含1个ITIM，属于抑制性KIR受体。具体地，抑制性KIR受体胞质区包含的ITIM发生磷酸化时可以募集磷酸酶SHP1和SHP2，导致细胞底物去磷酸化，最终抑制或终止NK细胞的效应功能，例如细胞毒性作用和细胞因子的分泌。KIR在绝大部分NK细胞上表达，尽管在不同个体上的表达水平有差异。甚至在同一个体中，不同的NK细胞表达的KIR种类也不尽相同，且同一个NK细胞可表达几种不同的KIR分子。KIR的识别配体是经典HLA-I类分子，包括HLA-A、HLA-B和HLA-C的某些多态性表位。例如，KIR3DL2识别HLA-A等位基因-A3和-A11，KIR3DL1识别HLA-Bw-4，KIR2DL1识别HLA-Cw2、HLA-Cw4和HLA-Cw6同种型。KIR的小鼠同源物是gp49B1，其长度为335个氨基酸，并且在胞质区含有2个ITIM结构。在一个优选的实施方案中，靶向KIR的抗体是靶向选自以下的一个或多个靶标的抗体：KIR2DL1、KIR2DL2、KIR2DL3、KIR3DL1、KIR3DL2、KIR3DL3、KIR2DL5和gp49B1。本领域技术人员熟知的KIR抗体可用于本发明，例如GL183(靶向KIR2DL2/L3，可获自Immunotech，France和Beckton Dickinson，USA)、EB6(靶向KIR2DL1，可获自Immunotech，France和Beckton Dickinson，USA)、AZ138(靶向KIR3DL1，可获自Morettaetal，Univ.Genova，Italy)、Q66(靶向KIR3DL2，可获自Immunotech，France)、Z27(靶向KIR3DL1，可获自Immunotech，France和Beckton Dickinson，USA)等。
在一个实施方案中，NK抑制性配体是靶向KIR的抗体，其包含如SEQ ID NO：84所示的CDR-L1、如SEQ ID NO：85所示的CDR-L2、如SEQ ID NO：86所示的CDR-L3、如SEQ ID NO：87所示的CDR-H1、如SEQ ID NO：88所示的CDR-H2和如SEQ ID NO：89所示的CDR-H3。在一个实施方案中，NK抑制性配体是靶向KIR的抗体，其包含轻链可变区和重链可变区，所述轻链可变区与SEQ ID NO：58所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，所述重链可变区与SEQ ID NO：59所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个优选的实施方案中， NK抑制性配体是包含SEQ ID NO：58和SEQ ID NO：59的抗KIR抗体，其氨基酸序列例如如SEQ ID NO：57或60所示。
在一个实施方案中，NK抑制性配体是靶向LIR的抗体或其功能性片段。LIR也称为免疫球蛋白样转录物(Immunoglobulin-like transcripts，ILT)或单核细胞-巨噬细胞抑制性受体(Monocyte-macrophage inhibitory receptor，MIR)。LIR家族包含8个成员，其中LIR1(也称为ILT2)、LIR2(也称为ILT4)、LIR3(也称为ILT5)、LIR5(也称为ILT3)和LIR8的胞质区含有2-4个ITIM结构，其中至少一个为VXYXXL/V基序，属于抑制性LIR受体。已有报道证明LIR-1能够抑制NK细胞系NKL对表达HLA-I类分子的靶细胞的杀伤以及CD16介导的NKL的活化。小鼠中LIR的同源物是PIR(Paired Ig-like receptor)，包括PIR-A和PIR-B，其中PIR-A在FcRγ同源二聚体的协助下传递活化信号，而PIR-B则通过其胞质区包含的4个ITIM结构传递抑制信号。在一个优选的实施方案中，NK抑制性配体是靶向LIR1、LIR2、LIR3、LIR5、LIR8或PIR-B的抗体。
在一个实施方案中，NK抑制性配体是靶向LIR1的抗体，其包含：(1)如SEQ ID NO：90所示的CDR-L1、如SEQ ID NO：91所示的CDR-L2、如SEQ ID NO：92所示的CDR-L3、如SEQ ID NO：93所示的CDR-H1、如SEQ ID NO：94所示的CDR-H2和如SEQ ID NO：95所示的CDR-H3，或(2)如SEQ ID NO：96所示的CDR-L1、如SEQ ID NO：97所示的CDR-L2、如SEQ ID NO：98所示的CDR-L3、如SEQ ID NO：99所示的CDR-H1、如SEQ ID NO：100所示的CDR-H2和如SEQ ID NO：101所示的CDR-H3。在一个实施方案中，NK抑制性配体是靶向LIR1的抗体，其包含轻链可变区和重链可变区，所述轻链可变区与SEQ ID NO：61或65所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，所述重链可变区与SEQ ID NO：62或64所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个优选的实施方案中，NK抑制性配体是抗LIR1抗体，其氨基酸序列如SEQ ID NO：63或66所示。本领域已知的其他靶向LIR家族成员的抗体也可用于本发明。在一个实施方案中，NK抑制性配体是靶向免疫检查点受体(例如PD-1、TIGIT、CD96、TIM3、LAG3)的抗体或其功能性片段。在一个优选的实施方案中，NK的抑制性配体是靶向PD-1的抗体或其功能性片段。PD-1主要在激活的NK细胞中表达，属于CD28家族成员，是由268个氨基酸组成的I型跨膜糖蛋白。它的结构主要包括胞外免疫球蛋白可变区(IgV)样结构、疏水的跨膜区以 及胞内区。胞内区尾部有2个独立的酪氨酸残基，氮端的酪氨酸残基参与构成一个ITIM，碳端酪氨酸残基则参与构成一个免疫受体酪氨酸转换基序(immunoreceptor tyrosine based switch motif，ITSM)。PD-1与其配体(例如PD-L1和PD-L2)结合后，促使PD-1的ITSM结构域中的酪氨酸发生磷酸化，进而引起下游蛋白激酶Syk和PI3K的去磷酸化，抑制下游AKT、ERK等通路的活化，最终抑制NK的活性。
在一个优选的实施方案实施方案中，NK抑制性配体是靶向TIGIT的抗体或功能性片段。TIGIT属于免疫球蛋白超家族成员，它由细胞外免疫球蛋白可变区(IgV)结构域、1型跨膜结构域和具有ITIM和免疫球蛋白酪氨酸尾(ITT)基序的细胞内结构域组成。当其与配体(例如CD155、CD112、CD113)结合后，可诱发胞内抑制信号的传递，从而抑制NK细胞的活性。
在一个优选的实施方案中，NK抑制性配体是靶向LAG3的抗体或功能性片段。LAG3是蛋白质Ig超家族的成员。它是1型跨膜蛋白，表达于活化的T细胞、NK细胞、B细胞和浆细胞样树突状细胞上。LAG3的四个IgG域与CD4分子具有高的结构同源性，但其氨基酸的同源性低于20％。研究表明，LAG3对T细胞和NK细胞的增殖及持久记忆具有负调控作用。一旦被其配体(例如FGL1)激活以后，可以促进肿瘤细胞等“坏细胞”逃脱免疫系统的追杀。
在一个优选的实施方案中，NK抑制性配体是靶向TIM3的抗体或功能性片段。TIM3是TIM家族的一个受体蛋白，在T细胞、Treg细胞、先天免疫细胞(树突细胞、自然杀伤细胞、单核细胞)表面表达。TIM家族成员由3个基因编码，具体为HAVCR1编码TIM1、HAVCR2编码TIM3以及TIMD4编码TIM4。TIM3有多种配体，如磷脂酰丝氨酸(phosphatidylserine)、半乳凝素9(galectin-9或Gal-9)、HMGB1和CEACAM-1等。在NK细胞中表达时，TIM3被认为可能是功能障碍的NK细胞的标志物，TIM3阻断已被证明可逆转NK细胞的功能障碍。
在一个实施方案中，NK抑制性配体是靶向NKR-P1的抗体或其功能性片段。NKR-P1属于II型跨膜糖蛋白，在人、小鼠和大鼠NK细胞中均有表达。目前已发现小鼠的6个NKR-P1成员，分别为NKR-P1A、NKR-P1B、NKR-P1C、NKR-P1D、NKR-P1E、NKR-P1F，而在人体中只发现了NKR-P1A(又称为CD161)。NKR-P1分子胞外区属于C型凝集素样超家族中NK受体结构域(NKD)，在结构上与Ly49、Cd69、CD94/NKG2分子相似。虽然NKR-P1主要以同源二聚体形式存在，但人NKR-P1A可能存在单体形式。NKR-P1家族分子的胞质区在不同物种中结构有所差异，例如NKR-P1B和NKR-P1D的 胞质区含有ITIM基序，其在酪氨酸磷酸化后募集SHP-1来传递抑制性信号，而NKR-P1C则通过跨膜区的带正电氨基酸与Fc受体结合，进而募集Syk传递活化信号。因此，在一个优选的实施方案中，NK抑制性配体是靶向NKR-P1B或NKR-P1D的抗体。
在一个实施方案中，NK抑制性配体是靶向CEACAM1的抗体或其功能性片段。CEACAM1也称为CD66a、胆汁糖蛋白(BGP)或C-CAM1，是癌胚抗原(CEA)基因家族的成员并属于免疫球蛋白(Ig)超家族。在活化的NK细胞中，CEACAM1表达被上调，且其嗜同性相互作用(homophilic interaction)导致淋巴细胞细胞毒性效应的抑制。CEACAM1与其他已知CEACAM蛋白包括CD66a(CEACAM1)、CD66c(CEACAM6)和CD66e(CEACAM5、CEA)蛋白相互作用。在人中，迄今为止已检测11个不同的CEACAM1剪接变体。CEACAM1同种型的命名与胞外免疫球蛋白-样结构域的数目(例如，具有4个胞外免疫球蛋白样结构域的CEACAM1被称为CEACAM1-4)、以及胞质尾区的长度(例如，具有长胞质尾区的CEACAM1-4被称为CEACAM1-4L，具有短胞质尾区的CEACAM1-4被称为CEACAM1-4S)有关。CEACAM1的N-末端结构域紧接在信号肽之后开始，并且其结构被认为是IgV-型。
在一个实施方案中，NK抑制性配体是靶向SIGLEC的抗体或其功能性片段。已经在人类中鉴定了16种SIGLEC蛋白并且在小鼠中鉴定了9种SIGLEC蛋白，所述SIGLEC蛋白由包括氨基末端V-型结构域的2-17个细胞外Ig结构域组成，所述V-型结构域含有唾液酸结合位点。Siglec通常被分为两组：由Siglec1、Siglec2、Siglec4和Siglec15组成的第一子集，以及包括Siglec3、Siglec5、Siglec6、Siglec7、Siglec8、Siglec9、Siglec10、Siglec11、Siglec12、Siglec14和Siglec16的CD33相关的第二子集。Siglec7，也称为p75、CD328或AIRM，含有细胞外N-末端Ig样V-型结构域、两个Ig样C2-型结构域以及含有一个ITIM基序和一个ITIM样基序的胞质内区域。Siglec7在NK细胞、树突细胞、单核细胞和嗜中性粒细胞上组成型表达。已经观察到Siglec7对NK细胞介导的肿瘤清除具有抑制作用。Siglec9的结构与Siglec7非常相似，它们的N-末端V-组Ig结构域具有约77％的总氨基酸序列同一性，并且显示不同的唾液酸结合特异性。鉴于对NK细胞的功能研究已经证明，表达Siglec9结合唾液酸配体的肿瘤细胞抑制NK细胞活化和肿瘤细胞杀伤。许多人肿瘤稳健地上调结合Siglec9的唾液酸配体，这使得肿瘤能够逃避免疫，发生癌症进展。
在一个优选的实施方案中，NK抑制性配体是靶向Siglec7或Siglec9的抗体，例如本领域已知的那些。举例而言，抗Siglec7抗体可以源自人Siglec7/CD328抗体(AF1138， R&D Systems)、克隆#194212(MAB1138，R&D Systems)、克隆#194211(MAB11381，R&D Systems)、克隆Z176(A22330，Beckman Coulter)、6-434(339202，Biolegend)、REA214(Miltenyl Biotec)、S7.7(MCA5782GA，BioRad)、10B2201(MBS604764，MyBioSource)、8D8(MBS690562，MyBioSource)、10B2202(MBS608694，MyBioSource)、5-386(MBS214370，MyBioSource)。抗Siglec9抗体可以源自MAB1139(克隆#191240，R&D Systems)、AF1139(R&D Systems)、D18(SC-34936，Santa Cruz Biotechnology)、Y-12SC34938(SC3-4938，Santa Cruz Biotechnology)、AB 197981(Abeam)、AB96545(Abeam)、AB89484(克隆#MM0552-6K12，Abeam)、AB 130493(Abeam)、AB117859(克隆#3G8，Abeam)、E10-286(Becton Dickinson)。鉴于Siglec7和Siglec9在细胞外结构的相似性，同时靶向这两者的抗体也可作用本发明的NK抑制性配体。
在一个实施方案中，NK抑制性配体是靶向SIGLEC7、SIGLEC9或两者的抗体，其包含(1)如SEQ ID NO：102所示的CDR-L1、如SEQ ID NO：103所示的CDR-L2、如SEQ ID NO：104所示的CDR-L3、如SEQ ID NO：105所示的CDR-H1、如SEQ ID NO：106所示的CDR-H2和如SEQ ID NO：107所示的CDR-H3，(2)如SEQ ID NO：122所示的CDR-L1、如SEQ ID NO：123所示的CDR-L2、如SEQ ID NO：124所示的CDR-L3、如SEQ ID NO：125所示的CDR-H1、如SEQ ID NO：126所示的CDR-H2和如SEQ ID NO：1277所示的CDR-H3，(3)如SEQ ID NO：131所示的CDR-L1、如SEQ ID NO：132所示的CDR-L2、如SEQ ID NO：133所示的CDR-L3、如SEQ ID NO：134所示的CDR-H1、如SEQ ID NO：135所示的CDR-H2和如SEQ ID NO：136所示的CDR-H3，(4)如SEQ ID NO：140所示的CDR-L1、如SEQ ID NO：141所示的CDR-L2、如SEQ ID NO：142所示的CDR-L3、如SEQ ID NO：143所示的CDR-H1、如SEQ ID NO：144所示的CDR-H2和如SEQ ID NO：155所示的CDR-H3，(5)如SEQ ID NO：176所示的CDR-L1、如SEQ ID NO：177所示的CDR-L2、如SEQ ID NO：178所示的CDR-L3、如SEQ ID NO：179所示的CDR-H1、如SEQ ID NO：180所示的CDR-H2和如SEQ ID NO：181所示的CDR-H3，或(6)如SEQ ID NO：188所示的CDR-L1、如SEQ ID NO：189所示的CDR-L2、如SEQ ID NO：190所示的CDR-L3、如SEQ ID NO：191所示的CDR-H1、如SEQ ID NO：192所示的CDR-H2和如SEQ ID NO：193所示的CDR-H3。上述抗体(1)-(4)靶向SIGLEC7，抗体(5)-(6)同时靶向SIGLEC7和SIGLEC9两者。在一个实施方案中，NK抑制性配体是靶向SIGLEC7、SIGLEC9或两者的抗体，其包含轻链可变区和重链可变区，所述轻链可变区与SEQ ID NO：108、128、137、146、 182、185或194所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，所述重链可变区与SEQ ID NO：109、129、138、147、183、186或195所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个优选的实施方案中，NK抑制性配体是抗SIGLEC7、SIGLEC9或两者的抗体，其氨基酸序列如SEQ ID NO：110、130、139、148、184、187或196所示。在一个实施方案中，NK抑制性配体是靶向LAIR1的抗体或其功能性片段。LAIR1含有10个外显子，编码由287个氨基酸组成的I型跨膜糖蛋白，其包含单个细胞外C2型Ig样结构域，随后是与单个跨膜结构域连接的茎区域和2个传递抑制信号的ITIM基序。LAIR1在结构上与LIR和KIR家族成员有一定同源性，表明这些分子可能来源于同一祖先基因。LAIR1在T细胞、B细胞、自然杀伤(NK)细胞、巨噬细胞和树突状细胞以及包括人CD34+细胞的造血祖细胞中表达。由于ITIM基序的存在，目前为止在人和小鼠体内的研究均发现LAIR1发挥免疫抑制的作用。进一步的研究表明，LAIR1不仅可抑制静止NK细胞，还可抑制活化的NK细胞对靶细胞的杀伤。
在一个实施方案中，NK抑制性配体是靶向Ly49的抗体或其功能性片段。Ly49是II型跨膜糖蛋白，可以通过二硫键连接形成同源二聚体，发挥与人KIR相似的功能，即通过与MHC-I类分子配体的相互作用来传递信号，进而调整NK细胞的活性。至今发现小鼠Ly49家族包括11个成员，分别是Ly49A、Ly49B、Ly49C、Ly49D、Ly49E、Ly49F、Ly49G、Ly49H、Ly49I、Ly49P、Ly49Q。其中，Ly49A、Ly49C、Ly49F、Ly49G和Ly49Q均在胞质区含有ITIM基序，能与酪氨酸激酶SHP-1结合并使之活化，通过干扰磷酸化酪氨酸的生成来抑制NK细胞活化。因此，在一个优选的实施方案中，NK抑制性配体是靶向Ly49A、Ly49C、Ly49F、Ly49G或Ly49Q的抗体。
在一个实施方案中，NK抑制性配体是靶向KLRG1的抗体或其功能性片段。KLRG1是调节T细胞和NK细胞的活性的II型跨膜蛋白表面共抑制受体。其细胞外部分包含C型凝集素结构域，已知的配体是钙粘素，并且其细胞内部分包含基于免疫受体酪氨酸的抑制基序(ITIM)结构域。有文献报道在丙肝病人外周血NK细胞上的KLRG1受体表达会促进NK细胞数量减少和功能受损，其机制主要是抑制NK细胞的增殖、促进NK细胞的凋亡和减少NK细胞炎症细胞因子的释放。
在一个实施方案中，NK抑制性配体是靶向KLRG1的抗体，其包含：(1)如SEQ ID NO：111所示的CDR-L1、如SEQ ID NO：112所示的CDR-L2、如SEQ ID NO：113所示的CDR-L3、如SEQ ID NO：114所示的CDR-H1、如SEQ ID NO：115所示的CDR-H2 和如SEQ ID NO：116所示的CDR-H3，(2)如SEQ ID NO：149所示的CDR-L1、如SEQ ID NO：150所示的CDR-L2、如SEQ ID NO：151所示的CDR-L3、如SEQ ID NO：152所示的CDR-H1、如SEQ ID NO：153所示的CDR-H2和如SEQ ID NO：154所示的CDR-H3，(3)如SEQ ID NO：158所示的CDR-L1、如SEQ ID NO：159所示的CDR-L2、如SEQ ID NO：160所示的CDR-L3、如SEQ ID NO：161所示的CDR-H1、如SEQ ID NO：162所示的CDR-H2和如SEQ ID NO：163所示的CDR-H3，或(4)如SEQ ID NO：167所示的CDR-L1、如SEQ ID NO：168所示的CDR-L2、如SEQ ID NO：169所示的CDR-L3、如SEQ ID NO：170所示的CDR-H1、如SEQ ID NO：171所示的CDR-H2和如SEQ ID NO：172所示的CDR-H3。在一个实施方案中，NK抑制性配体是靶向KLRG1的抗体，其包含轻链可变区和重链可变区，所述轻链可变区与SEQ ID NO：117、155、164或173所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，所述重链可变区与SEQ ID NO：118、156、165或174所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个优选的实施方案中，NK抑制性配体是抗KLRG1抗体，其氨基酸序列如SEQ ID NO：119、157、166或175所示。
在一个实施方案中，NK抑制性配体是NKIR天然配体或其包含的NKIR结合区(例如胞外区)，此类天然配体包括但不限于非经典HLA-I类分子(例如HLA-E、HLA-F和HLA-G)、钙黏素(Cadherin)、胶原蛋白、OCIL、唾液酸、免疫检查点配体(例如PD-L1/PD-L2、CD155、CD112、CD113、Gal-9、FGL1)等。
在一个实施方案中，NK抑制性配体是非经典HLA-I类分子或其胞外区，更优选非经典HLA-I类分子的α1和α2结构域。非经典HLA-I类分子位于相同的染色体区域6p21.3，第6号染色体短臂，由通过非共价键结合的重链(α链)和轻链(β链，由B2M基因编码)组成。α链包括胞外区(包括α1、α2、α3三个结构域)、跨膜结构域和胞质区三部分，其中α1和α2形成抗原结合槽，负责与进入槽内的抗原肽结合，α3与免疫球蛋白的恒定区结构域同源，与T细胞表面分子CD8结合。非经典HLA-I类分子包括三个成员：HLA-E、HLA-F和HLA-G。HLA-E通过与NK细胞表面的CD94/NKG2受体结合，调节NK细胞活性。HLA-E的功能是结合源自于I类HLA分子(HLA-A、-B、-C和-G)的前导序列的肽，并通过与抑制性受体CD94/NKG2A的相互作用将它们呈递给NK细胞，从而抑制NK细胞对表达正常水平的I类HLA分子的细胞的裂解。由于生理情况下HLA-E与抑制性受体CD94/NKG2A的亲和力明显高于其与活化性受体 CD94/NKG2C之前的亲和力，因此HLA-E的表达水平上调可以保护靶细胞免受NK细胞的杀伤作用。HLA-F能够与NK抑制性受体ILT2和ILT4结合，并且这种结合能被ILT2和ILT4抗体有效抑制。目前对HLA-F功能仍在探索，但推测其与ILT2和ILT4的结合可能具有免疫抑制作用。HLA-G可识别多种NK抑制性受体，例如CD94/NKG2A、LIR-1、LIR-2、KIR2DL1等。研究发现，胎儿细胞表面的HLA-G分子可能通过与母体NK细胞表面的KIR结合，抑制NK细胞杀伤活性，从而导致母体对HLA半异源性胎儿产生免疫耐受。此外，在实体肿瘤，例如黑素瘤、肉瘤、淋巴瘤等细胞表面高表达的HLA-G分子还能使肿瘤细胞逃避NK细胞的杀伤、溶解作用。
在一个实施方案中，NK抑制性配体是HLA-E胞外区，其与SEQ ID NO：31所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO：32所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在另一个实施方案中，NK抑制性配体是HLA-E胞外区的突变体(包含Y84C突变)，其与SEQ ID NO：33所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO：34所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，NK抑制性配体是HLA-G胞外区，其与SEQ ID NO：35所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO：36所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，当需要将工程化免疫细胞的内源性B2M敲除并且表达非经典HLA-I类分子作为NK抑制性配体时，需要引入同义突变(即，仅核苷酸序列改变而氨基酸序列不改变)的B2M基因以使其能与非经典HLA-I分子形成复合物从而发挥抑制功能，同时同义突变的B2M基因也能避免被靶向内源性B2M的基因编辑工具敲除。在该实施方案中，NK抑制性配体包含B2M和非经典HLA-I类分子的胞外区的融合分子。例如，NK抑制性配体包含B2M和HLA-E胞外区或HLA-G胞外区的融合分子。在一个具体的实施方案中，NK抑制性配体包含B2M和HLA-E胞外区的融合分子，优选的，所述HLA-E胞外区包含Y84C突变(SEQ ID NO：33)。在一个优选的实施方案中，NK抑制性配体包含提呈肽、以及B2M和HLA-E胞外区的融合分子，所述提呈肽选自SEQ  ID NO：46-53。将B2M基因进行同义突变的方法是本领域技术人员熟知的。在一个优选的实施方案中，B2M与SEQ ID NO：37所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；同义突变的B2M基因的核苷酸序列例如如SEQ ID NO：38所示。
在一个实施方案中，NK抑制性配体是破骨细胞抑制凝集素(Osteoclast inhibitory lectin，OCIL)或其NKIR结合区。小鼠OCIL包括三个成员：OCIL(也称为Clr-b)、OCILrP1(也称为Clr-d)和mOCILrP2(也称为Clr-g)。OCIL广泛表达在各组织中，其表达模式与MHC-I类分子相似。OCIL是NKR-P1B/D的配体。研究表明，在肿瘤细胞上表达OCL可以抑制NK细胞对肿瘤细胞的杀伤作用，而OCL特异性抗体则可以逆转这种抑制作用。
在一个实施方案中，NK抑制性配体是钙黏素或其胞外区，例如E-钙粘素(E-cad)、N-钙粘素(N-cad)或R-钙粘素(R-cad)，优选E-钙粘素的胞外区。钙黏素是一类主要介导细胞间同质黏附的钙依赖性跨膜蛋白，其结合NK抑制性受体KLRG1。钙黏素分子是I型膜蛋白，由约723-748个氨基酸组成，结构上包括负责与配体结合的胞外区、跨膜区和高度保守的胞质区。其中，胞外区有数个钙黏蛋白重复结构域(EC)，并含有由4～5个氨基酸残基组成的重复序列，负责与配体结合。人E-钙黏素由CDH 1基因编码，是目前研究最多的钙黏素家族成员。因此，在一个优选的实施方案中，NK抑制性配体是E-钙粘素的胞外区，其包含EC1和EC2。更优选的，NK抑制性配体是E-钙粘素的胞外区，其包含EC1、EC2、EC3、EC4和EC5。更优选地，NK抑制性配体是E-Cad，其与SEQ ID NO：39或41所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO：40或42所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，NK抑制性配体是胶原蛋白或其NKIR结合区，其结合LAIR 1。胶原分子是由3条α链组成的三聚体，每条α链都含有(甘氨酸-脯氨酸-羟脯氨酸)n重复序列。LAIR1识别Gly-Pro-Hyp重复序列并与其相互作用。由于该重复序列的广泛存在，已经证明LAIR1可与多种胶原分子广泛结合，包括但不限于跨膜胶原，例如胶原XVII、XIII、XXIII；和非跨膜胶原，例如胶原I、II、III等。肿瘤细胞或肿瘤间质细胞常常高表达多种胶原分子，这可能通过与免疫细胞表面的抑制性受体LAIR1结合来向免疫细胞内传递抑制性信号，从而达到免疫逃逸的目的。
在一个实施方案中，NK抑制性配体是唾液酸或其NKIR结合区，其结合SIGLEC家族成员(例如SIGLEC7和/或SIGLEC9)。唾液酸是脊椎动物先天性免疫系统的重要组成部分，NK细胞杀伤活性与肿瘤细胞表面的唾液酸化相关。肿瘤细胞的唾液酸化不仅可以阻碍肿瘤细胞与NK细胞之间的物理作用，而且可以遮蔽肿瘤细胞表面能够与之结合的活化性配体。此外，肿瘤细胞表面的唾液酸化会导致肿瘤细胞和NK细胞之间的免疫突触形成受阻，从而削弱NK细胞对肿瘤的杀伤毒性。研究发现，肿瘤细胞表面的唾液酸化还可以通过触发由Siglec介导的免疫抑制信号来抑制NK细胞的杀伤活性。Siglec7表达于大部分的NK细胞表面，而肿瘤细胞表面以α-2，8糖苷键连接的唾液酸与NK细胞表面的Siglec7结合后，会抑制NK细胞的活化，从而使肿瘤细胞逃逸NK细胞介导的杀伤功能。
在一个实施方案中，NK抑制性配体为PD-L1/PD-L2或其胞外区，其结合PD1。PD-L1组成性地低表达于抗原递呈细胞(APC)、以及非造血细胞如血管内皮细胞、胰岛细胞以及免疫豁免部位(如胎盘、睾丸和眼睛)。炎性细胞因子如I型和II型干扰素、TNF-α和VEGF等均可以诱导PD-L1的表达。PD-L2只在被激活的巨噬细胞和树突细胞中有表达。肿瘤细胞本身可上调PD-L1的表达，同时肿瘤微环境中的炎症因子同样可以诱导PD-L1和PDL2的表达。肿瘤细胞表面PD-L1和PD-L2的上调表达可触发由PD-1介导的免疫抑制信号的传递进而抑制NK细胞的杀伤活性。在一个优选的实施方案中，NK抑制性配体是PD-L1胞外区或PD-L2胞外区，其与SEQ ID NO：70或71所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，NK抑制性配体为CD155、CD112或CD113或其NKIR结合区，其均结合TIGIT。CD155是TIGIT的高亲和力配体。肿瘤表面高表达的CD155一旦与NK表面的TIGIT结合，NK细胞对肿瘤细胞的杀伤作用就会被抑制。CD112和CD113也与TIGIT结合，尽管亲和力相对较弱。
在一个实施方案中，NK抑制性配体是半乳凝素9(也称为Gal-9)或其NKIR结合区，其结合TIM3。Gal-9是一种由许多造血细胞广泛表达和分泌的C型凝集素，可与细胞表面蛋白上的碳水化合物部分结合。在TIM3上，Gal-9结合其IgV域上的碳水化合物基序，可诱导TIM3阳性NK细胞钙内流和细胞死亡。已有大量证明表明，TIM3/Gal-9相互作用在抑制免疫反应中发挥了作用。
在一个实施方案中，NK抑制性配体是FGL1或其NKIR结合区，其结合LAG3。FGL1 属于纤维蛋白原家族，是新进发现的LAG3的配体，但它并没有特征性的血小板结合位点、凝血酶敏感位点等结构域。FGL1蛋白主要分布于肿瘤细胞中，肿瘤间质表达较低。FGL1/LAG3相互作用是独立于B7-H1/PD-1通路的另一条肿瘤免疫逃逸通路，阻断这条通路能和阻断PD-1通路起到协同作用。
如本文所用，术语“跨膜结构域”是指能够使嵌合抗原受体在免疫细胞(例如淋巴细胞或NKT细胞)表面上表达，并且引导免疫细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合抗原受体与靶抗原结合时，跨膜结构域能够进行信号传导。特别适用于本发明中的跨膜结构域可以源自例如TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154，和NKIR天然配体的跨膜结构域，例如非经典HLA-I类分子(例如HLA-E、HLA-F和HLA-G)、钙黏素(Cadherin)、胶原蛋白、OCIL等的跨膜结构域。优选地，跨膜结构域选自CD8α、CD4、CD28和CD278的跨膜结构域。或者，跨膜结构域可以是合成的并且可以主要地包含疏水性残基如亮氨酸和缬氨酸。优选地，所述跨膜结构域源自CD8α或CD28，更优选与SEQ ID NO：9或11所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或所述跨膜结构域的编码序列与SEQ ID NO：10或12所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的NK抑制性分子还可以包含位于NK抑制性配体和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至配体结合结构域的任何寡肽或多肽。具体地，铰链区用来为配体结合结构域提供更大的灵活性和可及性。铰链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。在优选的实施方式中，所述铰链区包含CD8α、CD28、FcγRIIIα受体、IgG4或IgG1的铰链区部分，更优选CD8α、CD28或IgG4铰链，其与SEQ ID NO：25、27或29所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或者CD28铰链的编码序列与SEQ ID NO：26、28或30所示的核苷酸序列具有至 少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
本发明的NK抑制性分子包括共刺激结构域。因此，当NK抑制性配体与NKIR结合时，一方面可以通过NKIR的信号传导区向NK细胞传递抑制信号，降低其对靶细胞(例如本发明的工程化免疫细胞)的杀伤；另一方面可以通过NK抑制性分子包含的共刺激结构域向靶细胞(例如本发明的工程化免疫细胞)内部传递刺激信号，刺激其增殖和存活，从而更好地抵抗NK细胞的杀伤。之前有研究发现，使用抗体靶向NK细胞NKG2A、KIR、ILT2等抑制性受体，可以竞争结合HLA-E、HLA-G等抑制分子的结合位点或中和其抑制效果，从而起到激活NK细胞的作用。与此相反，本发明首次发现，表达靶向NK抑制性受体的NK抑制性分子可以抑制NK细胞。本发明还发现，与不含共刺激结构域的NK抑制性分子相比，包含共刺激结构域的NK抑制性分子对NK细胞的杀伤作用抑制效果更好。
共刺激结构域可以是来自共刺激分子的细胞内功能性信号传导结构域，其包含所述共刺激分子的整个细胞内部分，或其功能片段。“共刺激分子”是指在T细胞上与共刺激配体特异性结合，由此介导T细胞的共刺激反应(例如增殖)的同源结合配偶体。共刺激分子包括但不限于I类MHC分子、BTLA和Toll配体受体。本发明的共刺激结构域的非限制性施例包括但不限于源自以下蛋白质的共刺激信号传导结构域：LTB、CD94、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM、ZAP70以及它们的组合。优选地，本发明的共刺激结构域来自4-1BB、CD28、CD27、OX40、CD278或其组合，更优选4-1BB、CD28或其组合。在一个实施方案中，本发明的共刺激结构域与SEQ ID NO：13或15所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或该共刺激结构域的编码序列与SEQ ID NO：14或16所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，NK抑制性分子不包含胞内信号传导结构域。在另一个实施方案中，NK抑制性分子进一步包含胞内信号传导结构域。即，NK抑制性分子包含NK抑制性配体、跨膜结构域、共刺激结构域和胞内信号传导结构域。在该实施方案中，NK抑制性配体与NKIR的结合会通过共刺激结构域和胞内信号传导结构域向靶细胞(例如 本发明的工程化免疫细胞)传递活化信号，促使靶细胞对NK细胞的杀伤，从而进一步增强对NK细胞杀伤作用的抑制效果。
如本文所用，术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分。胞内信号传导结构域负责在配体结合结构域结合抗原以后的细胞内初级信号传递，从而导致免疫细胞和免疫反应的活化。换言之，胞内信号传导结构域负责活化其中表达NK抑制性分子的免疫细胞的正常的效应子功能的至少一种。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。
在一个实施方案中，本发明的胞内信号传导结构域可以是T细胞受体和共受体的细胞质序列，其在抗原受体结合以后一同起作用以引发初级信号传导，以及这些序列的任何衍生物或变体和具有相同或相似功能的任何合成序列。胞内信号传导结构域可以包含许多免疫受体酪氨酸激活基序(Immunoreceptor Tyrosine-based Activation Motifs，ITAM)。本发明的胞内信号传导结构域的非限制性施例包括但不限于源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。在优选的实施方式中，本发明CAR的信号传导结构域可以包含CD3ζ信号传导结构域，该信号传导结构域与SEQ ID NO：17或19所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO：18或20所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的NK抑制性分子还可以包含信号肽，使得当其在细胞例如T细胞中表达时，新生蛋白质被引导至内质网并随后引导至细胞表面。信号肽的核心可以含有长的疏水性氨基酸区段，其具有形成单个α-螺旋的倾向。在信号肽的末端，通常有被信号肽酶识别和切割的氨基酸区段。信号肽酶可以在移位期间或完成后切割，以产生游离信号肽和成熟蛋白。然后，游离信号肽被特定蛋白酶消化。可用于本发明的信号肽是本领域技术人员熟知的，例如衍生自B2M、CD8α、IgG1、GM-CSFRα等的信号肽。在一个实施方案中，可用于本发明的信号肽与SEQ ID NO：21或23所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或该信号肽的编码序列与SEQ ID NO：22或24所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
至少一种MHC相关基因的抑制或沉默
在一个实施方案中，所述表达NK抑制性分子的工程化免疫细胞进一步包括至少一 种MHC相关基因表达被抑制或沉默，例如使至少一种MHC基因的表达被抑制或沉默，或使与至少一种MHC基因相互作用或调控其表达的基因的表达被抑制或沉默。
主要组织相容性复合物(major histocompatibility complex，MHC)最初被表征为在移植反应中起主要作用的蛋白，其在所有高等脊椎动物的表面上上表达，并且在小鼠中称为H-2，在人细胞中称为HLA。MHC主要有两类：I类和II类。I类MHC蛋白是两种蛋白质的异二聚体：一种是由MHCI基因编码的跨膜蛋白α链，另一种是由不位于MHC基因簇内的基因编码的细胞外蛋白质的β2微球蛋白链。α链包括三个结构域，外来肽与两个在N末端，也是最可变的结构域α1、α2结合。II类MHC蛋白也是异二聚体，包含两个由MHC复合物内的基因编码的跨膜蛋白质。I类MHC/抗原复合物与细胞毒性T细胞相互作用，而II类MHC向辅助T细胞呈递抗原。此外，I类MHC蛋白倾向于在几乎所有有核细胞和血小板(以及小鼠中的红血细胞)中表达，而II类MHC蛋白更具选择性地表达。通常，II类MHC蛋白在B细胞、一些巨噬细胞和单核细胞、郎格罕氏细胞(Langerhans cell)和树突细胞上表达。
人的I类HLA基因簇包含三个主要基因座B、C和A。HLA-A、HLA-B和HLA-C是I类HLA重链旁系同源物。I类分子是由MHCα重链(由HLA-A、HLA-B或HLA-C编码)和轻链(β-2微球蛋白，由B2M编码)组成的异二聚体。重链锚定在膜中，为约45kDa，并且含有8个外显子。外显子1编码前导肽，外显子2和3编码α1和α2结构域，其两者均结合肽，外显子4编码α3结构域，外显子5编码跨膜区域，外显子6和7编码细胞质尾部。外显子2和外显子3内的多态性导致每一类分子的肽结合特异性。因此，在一个实施方案中，使MHC相关基因表达被抑制或沉默是指使选自以下的一个或多个基因的表达被抑制或沉默：HLA-A、HLA-B、HLA-C和B2M。
人的II类HLA簇也包含三个主要基因座DP、DQ和DR，并且I类基因簇与II类基因簇两者均是多态性的。HLA-DPA1、HLA-DQA1和HLA-DRA属于II类HLAα链旁系同源物。II类分子通过呈递外源性肽而在免疫系统中起主要作用，主要在抗原呈递细胞(例如B淋巴细胞、树突细胞、巨噬细胞)中表达。II类分子是由均锚定在膜中的α链和β链组成的异二聚体，其中α链为约33-35kDa，并且含有5个外显子。外显子1编码前导肽，外显子2和3编码两个细胞外结构域，外显子4编码跨膜结构域，外显子5编码细胞质尾部。因此，在一个实施方案中，使MHC相关基因表达被抑制或沉默是指使选自以下的一个或多个基因的表达被抑制或沉默：HLA-DPA、HLA-DQ和HLA-DRA。
I类和II类MHC的表达还取决于多种辅助蛋白质。例如，Tap1和Tap2亚单位是将 肽抗原装载于I类HLA复合物上所必需的TAP转运体复合物的部分。LMP2和LMP7蛋白体亚单位在使抗原蛋白水解降解成肽以在HLA上展示中起作用。已显示，降低LMP7会降低细胞表面处I类MHC的表达量。II类MHC表达则受到一些正调控因子的诱导和表达，例如RFX复合物、CIITA等。RFX复合物由三个亚基组成：RFXANK(也称为RFXB)、RFX5和RFX辅助蛋白(也称为RFXAP)。RFX复合物通过促进其他转录因子与II类MHC分子的启动子结合并增强启动子结合的特异性促进II类MHC分子的表达。CIITA是II类MHC表达的主控制因子。CIITA包括富含酸性氨基酸的N端，富含Pro、Ser、Thr的PST区域，中间的GTP结合区域以及富含Leu重复序列(LRR)的C端，其中N端酸性区域和PST区域是转录激活区域。因此，在一个实施方案中，使MHC相关基因表达被抑制或沉默是指使选自以下的一个或多个基因的表达被抑制或沉默：TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK和CIITA。
因此，在一个实施方案中，使MHC相关基因表达被抑制或沉默是指使选自以下的一个或多个基因的表达被抑制或沉默：HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA和它们的组合，优选选自HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA和它们的组合。
在一个实施方案中，所述表达NK抑制性分子的工程化免疫细胞进一步包括至少一种TCR/CD3基因的表达被抑制或沉默。
T细胞表面受体(T cell receptor，TCR)是所有T细胞表面的特征性标志，以非共价键与CD3结合形成TCR/CD3复合物，并通过与抗原呈递细胞表面的特异性MHC-抗原肽复合物结合，产生特异性抗原刺激信号，激活T细胞，发挥杀伤作用。TCR是由两条不同肽链构成的异二聚体，通常分为两类：α/β型和γ/δ型，其中95％以上的外周T淋巴细胞都表达TCRα/β。TCRα链由TRAC基因编码，β链由TRBC基因编码。TCR的每条肽链包括可变区(V区)、恒定区(C区)、跨膜区和胞质区，其中胞质区很短，不具备传递抗原刺激信号的能力。TCR分子属于免疫球蛋白超家族，其抗原特异性存在于V区；V区又各有三个高变区CDR1、CDR2、CDR3，其中以CDR3变异最大，直接决定了TCR的抗原结合特异性。在TCR识别MHC-抗原肽复合物时，CDR1、CDR2识别并结合MHC分子，而CDR3直接与抗原肽相结合。CD3包括四种亚基：γ、δ、ε、ζ，通常以二聚体εγ、εδ、ζζ的形式存在。这四种亚基均包含保守的免疫受体酪氨酸激活基序(Immunoreceptor tyrosine-based activation motif，ITAM)，其中的2个酪氨酸残 基被酪氨酸蛋白激酶磷酸化后，向T细胞传递活化信号。因此，在一个实施方案中，使至少一种TCR/CD3基因表达被抑制或沉默是指使选自以下的一个或多个基因的表达被抑制或沉默：TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ。
在一个优选的实施方案中，所述表达NK抑制性分子的工程化免疫细胞包括至少一种TCR/CD3基因和至少一种MHC相关基因的表达被抑制或沉默，其中所述至少一种TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ和它们的组合；所述至少一种MHC相关基因选自HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA和它们的组合，优选选自HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA和它们的组合。
在一个优选的实施方案中，所述至少一种TCR/CD3基因选自TRAC、TRBC和它们的组合，所述至少一种MHC相关基因选自B2M、RFX5、RFXAP、RFXANK、CIITA和它们的组合。在一个实施方案中，所述工程化免疫细胞的TRAC或TRBC，和B2M的表达被抑制或沉默。在一个实施方案中，所述工程化免疫细胞的TRAC或TRBC，和CIITA的表达被抑制或沉默。在一个优选的实施方案中，所述工程化免疫细胞的TRAC或TRBC、B2M和CIITA的表达被抑制或沉默。在一个优选的实施方案中，所述工程化免疫细胞的TRAC或TRBC、B2M和RFX5的表达被抑制或沉默。
在一个实施方案中，除了MHC相关基因和任选的TCR/CD3基因，本发明的工程化免疫细胞还可以包含至少一种选自以下的基因的表达被抑制或沉默：CD52、GR、dCK和免疫检查点基因，如PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。
抑制基因表达或使基因沉默的方法是本领域技术人员熟知的，包括但不限于例如通过大范围核酸酶、锌指核酸酶、TALE核酸酶或CRISPR系统中的Cas酶介导DNA断裂、或通过反义寡核苷酸、RNAi、shRNA等技术使基因失活。
嵌合抗原受体
在另一个方面，本发明的表达NK抑制性分子的工程化免疫细胞还可以表达嵌合抗 原受体。即，在该实施方案中，工程化免疫细胞表达NK抑制性分子和嵌合抗原受体，优选地，所述工程化免疫细胞的至少一种MHC相关基因的表达被抑制或沉默。在一个优选的实施方案中，所述工程化免疫细胞还包括至少一种TCR/CD3基因的表达被抑制或沉默。
如本文所用，术语“嵌合抗原受体”或“CAR”是指人工构建的杂合多肽，该杂合多肽一般包括一个或多个配体结合结构域(例如抗体的抗原结合部分)、跨膜结构域、共刺激结构域和细胞内信号传导结构域，各个结构域之间通过接头连接。CAR能够利用单克隆抗体的抗原结合特性以非MHC限制性的方式将T细胞和其它免疫细胞的特异性和反应性重定向至所选择的靶标。非MHC限制性的抗原识别给予CAR细胞与抗原处理无关的识别抗原的能力，因此绕过了肿瘤逃逸的主要机制。此外，当在T细胞内表达时，CAR有利地不与内源性T细胞受体(TCR)的α链和β链二聚化。
如本文所用，“配体结合结构域”是指可以与配体(例如抗原)结合的任何结构或其功能性变体。配体结合结构域可以是抗体结构，包括但不限于单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、鼠源抗体、嵌合抗体及其功能性片段。例如，配体结合结构域包括但不限于完整抗体、Fab、Fab’、F(ab’)2、Fv片段、scFv抗体片段、线性抗体、sdAb(VH或VL)、纳米抗体(Nanobody，Nb)、重组纤连蛋白结构域、anticalin和DARPIN等，优选选自Fab、scFv、sdAb和纳米抗体。在本发明中，配体结合结构域可以是单价或二价，且可以是单特异性、双特异性或多特异性的抗体。
“Fab”是指免疫球蛋白分子被木瓜蛋白酶裂解后产生的两个相同片段中的任一个，由通过二硫键连接的完整轻链和重链N端部分组成，其中重链N端部分包括重链可变区和CH1。与完整的IgG相比，Fab没有Fc片段，流动性和组织穿透能力较高，并且无需介导抗体效应即可单价结合抗原。
“单链抗体”或“scFv”是由抗体重链可变区(VH)和轻链可变区(VL)通过接头连接而成的抗体。可以选择接头的最佳长度和/或氨基酸组成。接头的长度会明显影响scFv的可变区折叠和相互作用情况。事实上，如果使用较短的接头(例如在5-10个氨基酸之间)，则可以防止链内折叠。关于接头的大小和组成的选择，参见例如，Hollinger等人，1993Proc Natl Acad.Sci.U.S.A.90：6444-6448；美国专利申请公布号2005/0100543、2005/0175606、2007/0014794；以及PCT公布号WO2006/020258和WO2007/024715，其全文通过引用并入本文。scFv可以包含以任何顺序连接的VH和VL，例如VH-接头-VL或VL-接头-VH。
“单结构域抗体”或“sdAb”是指一种天然缺失轻链的抗体，该抗体只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，也称为“重链抗体”。
“纳米抗体”或“Nb”是指单独克隆并表达出来的VHH结构，其具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是目前已知的可结合目标抗原的最小单位。
术语“功能性变体”或“功能性片段”是指基本上包含亲本的氨基酸序列但与该亲本氨基酸序列相比含有至少一个氨基酸修饰(即取代、缺失或插入)的变体，条件是所述变体保留亲本氨基酸序列的生物活性。例如，对于抗体，其功能性片段是其抗原结合部分。在一个实施方案中，所述氨基酸修饰优选是保守型修饰。
如本文所用，术语“保守性修饰”是指不会明显影响或改变含有该氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰。这些保守修饰包括氨基酸取代、添加及缺失。修饰可以通过本领域中已知的标准技术，如定点诱变和PCR介导的诱变而引入本发明的嵌合抗原受体中。保守氨基酸取代是氨基酸残基被具有类似侧链的氨基酸残基置换的取代。具有类似侧链的氨基酸残基家族已在本领域中有定义，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。保守性修饰可以例如基于极性、电荷、溶解度、疏水性、亲水性和/或所涉及残基的两亲性质的相似性来进行选择。
因此，“功能性变体”或“功能性片段”与亲本氨基酸序列具有至少75％，优选至少76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性，并且保留亲本氨基酸的生物活性，例如结合活性。
如本文所用，术语“序列同一性”表示两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同一性。本领域技术人员将认识到，一些算法可以用于使用标准参数来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25：3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215：403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147：195-197) 和ClustalW。
配体结合结构域的选择取决于待识别的与具体疾病状态相关的靶细胞上的细胞表面标记，例如肿瘤特异性抗原或肿瘤相关抗原。因此，在一个实施方案中，本发明的配体结合结构域与选自以下的一个或多个靶标结合：TSHR、CD19、CD123、CD22、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-1 1Ra、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、Folate受体α、ERBB2(Her2/neu)、MUC1、EGFR、NCAM、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gplOO、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD 179a、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、豆荚蛋白、HPV E6、E7、MAGE Al、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋白、存活蛋白和端粒酶、PCTA-1/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、Claudin18.2、NKG2D和它们的任意组合。优选地，所述靶标选自：CD19、CD20、CD22、BAFF-R、CD33、EGFRvIII、BCMA、GPRC5D、PSMA、ROR1、FAP、ERBB2(Her2/neu)、MUC1、EGFR、CAIX、WT1、NY-ESO-1、CD79a、CD79b、GPC3、Claudin18.2、NKG2D和它们的任意组合。根据待靶向的抗原，本发明的CAR可以被设计为包括对该抗原具有特异性的配体结合结构域。例如，如果CD19是待靶向的抗原，则CD19抗体可用作本发明的配体结合结构域。
可用于本发明的CAR包含的跨膜结构域、共刺激结构域、胞内信号传导结构域以及任选的铰链区、信号肽等结构的定义参见上文。
在一个实施方案中，本发明的CAR还可以包含开关结构，以调控CAR的表达时间。例如，开关结构可以是二聚化结构域的形式，通过与其相应配体的结合引起构象变化， 暴露胞外结合结构域，使其与被靶向抗原结合，从而激活信号传导通路。或者，也可以使用开关结构域分别连接结合结构域和信号传导结构域，仅当开关结构域互相结合(例如在诱导化合物的存在下)时，结合结构域和信号传导结构域才能通过二聚体连接在一起，从而激活信号通路。开关结构还可以是掩蔽肽的形式。掩蔽肽可以遮蔽胞外结合结构域，阻止其与被靶向抗原的结合，当通过例如蛋白酶切割掩蔽肽后，就可以暴露胞外结合结构域，使其成为一个“普通”的CAR结构。本领域技术人员知晓的各种开关结构均可用于本发明。
在一个实施方案中，本发明的CAR还可以包含自杀基因，即，使其表达一个可通过外源物质诱导的细胞死亡信号，以在需要时(例如产生严重的毒副作用时)清除CAR细胞。例如，自杀基因可以是插入的表位的形式，例如CD20表位、RQR8等，当需要时，可以通过加入靶向这些表位的抗体或试剂来消除CAR细胞。自杀基因也可以是单纯疱疹病毒胸苷激酶(HSV-TK)，该基因可使细胞在接受更昔洛韦治疗诱导下死亡。自杀基因还可以是iCaspase-9，可以通过化学诱导药物如AP1903、AP20187等诱导iCaspase-9发生二聚化，从而激活下游的Caspase3分子，导致细胞凋亡。本领域技术人员知晓的各种自杀基因均可用于本发明。
根据需要，当NK抑制性分子包含胞内信号传导结构域且细胞表达CAR时，本发明的NK抑制性分子和CAR可以共享除结合区之外的其他结构，例如共刺激结构域和胞内信号传导结构域。因此，在该实施方案中，本发明的工程化免疫细胞：(1)表达本发明的NK抑制性分子和嵌合抗原受体的融合蛋白，所述融合蛋白包含NK抑制性配体、配体结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，和(2)至少一种MHC相关基因的表达被抑制或沉默。
核酸和载体
本发明还提供一种核酸分子，其包含编码本发明的NK抑制性分子的核酸序列。任选地，所述核酸分子还可以包含编码嵌合抗原受体的核酸序列。
如本文所用，术语“核酸分子”包括核糖核苷酸和脱氧核糖核苷酸的序列，如经修饰的或未经修饰的RNA或DNA，各自为单链和/或双链形式的线性或环状，或它们的混合物(包括杂合分子)。因此，根据本发明的核酸包括DNA(比如dsDNA、ssDNA、cDNA)、RNA(比如dsRNA、ssRNA、mRNA、ivtRNA)，它们的组合或衍生物(比如PNA)。优选地，所述核酸是DNA或RNA，更优选mRNA。
核酸可以包含常规的磷酸二酯键或非常规的键(如酰胺键，比如在肽核酸(PNA)中发 现的)。本发明的核酸还可含有一种或多种经修饰的碱基，比如，例如三苯甲基化的碱基和不常见的碱基(比如肌苷)。也可以想到其它修饰，包括化学、酶促或代谢修饰，只要本发明的多链CAR可以从多核苷酸表达即可。核酸可以以分离的形式提供。在一个实施方案中，核酸也可以包括调节序列，比如转录控制元件(包括启动子、增强子、操纵子、抑制子和转录终止信号)、核糖体结合位点、内含子等。
可以对本发明的核酸序列进行密码子优化以在所需的宿主细胞(如，免疫细胞)中进行最佳表达；或者用于在细菌、酵母菌或昆虫细胞中表达。密码子优化是指将目标序列中存在的在给定物种的高度表达的基因中一般罕见的密码子替换为在这类物种的高度表达的基因中一般常见的密码子，而替换前后的密码子编码相同的氨基酸。因此，最佳密码子的选择取决于宿主基因组的密码子使用偏好。
本发明还提供一种载体，其包含本发明所述的核酸分子。任选地，编码NK抑制性分子的核酸序列和编码嵌合抗原受体的核酸序列可以位于相同载体或不同载体。
如本文所用，术语“载体”是用作将(外源)遗传材料转移到宿主细胞中的媒介核酸分子，在该宿主细胞中所述核酸分子可以例如复制和/或表达。
载体一般包括靶向载体和表达载体。“靶向载体”是通过例如同源重组或使用特异性靶向位点处序列的杂合重组酶将分离的核酸递送至细胞内部的介质。“表达载体”是用于异源核酸序列(例如编码本发明的嵌合抗原受体多肽的那些序列)在合适的宿主细胞中的转录以及它们的mRNA的翻译的载体。可用于本发明的合适载体是本领域已知的，并且许多可商购获得。在一个实施方案中，本发明的载体包括但不限于质粒、病毒(例如逆转录病毒、慢病毒、腺病毒、牛痘病毒、劳氏肉瘤病毒(RSV、多瘤病毒和腺相关病毒(AAV)等)、噬菌体、噬菌粒、粘粒和人工染色体(包括BAC和YAC)。载体本身通常是核苷酸序列，通常是包含插入物(转基因)的DNA序列和作为载体“骨架”的较大序列。工程化载体通常还包含在宿主细胞中自主复制的起点(如果需要多核苷酸的稳定表达)、选择标记和限制酶切割位点(如多克隆位点，MCS)。载体可另外包含启动子、多聚腺苷酸尾(polyA)、3’UTR、增强子、终止子、绝缘子、操纵子、选择标记、报告基因、靶向序列和/或蛋白质纯化标签等元件。在一个具体的实施方案中，所述载体是体外转录的载体。
工程化免疫细胞
本发明还提供一种工程化免疫细胞，其表达本发明的NK抑制性分子，且其中至少一种MHC相关基因的表达被抑制或沉默。在一个实施方案中，本发明的工程化免疫细 胞进一步表达嵌合抗原受体，所述嵌合抗原受体包含一个或多个配体结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域。在一个优选的实施方案中，本发明的工程化免疫细胞还包括至少一种TCR/CD3基因的表达被抑制或沉默。
在一个实施方案中，除了MHC相关基因和任选的TCR/CD3基因，本发明的工程化免疫细胞还可以包含至少一种选自以下的基因的表达被抑制或沉默：CD52、GR、dCK和免疫检查点基因，如PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。
根据需要，当NK抑制性分子包含胞内信号传导结构域且细胞表达CAR时，本发明的NK抑制性分子和CAR可以共享除结合区之外的其他结构，例如共刺激结构域和胞内信号传导结构域。因此，在该实施方案中，本发明的工程化免疫细胞：(1)表达本发明的NK抑制性分子和嵌合抗原受体的融合蛋白，所述融合蛋白包含NK抑制性配体、配体结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，和(2)至少一种MHC相关基因的表达被抑制或沉默。在一个优选的实施方案中，所述工程化免疫细胞还包括至少一种TCR/CD3基因的表达被抑制或沉默。
如本文所用，术语“免疫细胞”是指免疫系统的具有一种或多种效应子功能(例如，细胞毒性细胞杀伤活性、分泌细胞因子、诱导ADCC和/或CDC)的任何细胞。例如，免疫细胞可以是B细胞、T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞，或者是从细胞脐带血等干细胞来源获得的免疫细胞。优选地，免疫细胞是T细胞。T细胞可以是任何T细胞，如体外培养的T细胞，例如原代T细胞，或者来自体外培养的T细胞系例如Jurkat、SupT1等的T细胞，或获得自受试者的T细胞。受试者的实例包括人、狗、猫、小鼠、大鼠及其转基因物种。T细胞可以从多种来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐血、胸腺组织、来自感染部位的组织、腹水、胸膜积液、脾组织及肿瘤。T细胞也可以被浓缩或纯化。T细胞可以处于任何发育阶段，包括但不限于，CD4+/CD8+T细胞、CD4+辅助T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如，细胞毒性T细胞)、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞等。在一个优选的实施方案中，免疫细胞是人T细胞。可以使用本领域技术 人员已知的多种技术，如Ficoll分离从受试者的血液获得T细胞。
采用本领域已知的常规方法(如通过转导、转染、转化等)可以NK抑制性分子和任选的嵌合抗原受体引入免疫细胞。“转染”是将核酸分子或多核苷酸(包括载体)引入靶细胞的过程。一个例子是RNA转染，即将RNA(比如体外转录的RNA，ivtRNA)引入宿主细胞的过程。该术语主要用于真核细胞中的非病毒方法。术语“转导”通常用于描述病毒介导的核酸分子或多核苷酸的转移。动物细胞的转染通常涉及在细胞膜中打开瞬时的孔或“洞”，以允许摄取材料。可以使用磷酸钙、通过电穿孔、通过细胞挤压或通过将阳离子脂质与材料混合以产生与细胞膜融合并将它们的运载物沉积入内部的脂质体，进行转染。用于转染真核宿主细胞的示例性技术包括脂质囊泡介导的摄取、热休克介导的摄取、磷酸钙介导的转染(磷酸钙/DNA共沉淀)、显微注射和电穿孔。术语“转化”用于描述核酸分子或多核苷酸(包括载体)向细菌中、也向非动物真核细胞(包括植物细胞)中的非病毒转移。因此，转化是细菌或非动物真核细胞的基因改变，其通过细胞膜从其周围直接摄取并随后并入外源遗传材料(核酸分子)而产生。转化可以通过人工手段实现。为了发生转化，细胞或细菌必须处于感受态的状态。对于原核转化，技术可包括热休克介导的摄取、与完整细胞的细菌原生质体融合、显微注射和电穿孔。
试剂盒和药物组合物
本发明该提供一种试剂盒，其包含本发明的NK抑制性分子、核酸分子、载体或工程化免疫细胞。
在一个优选的实施方案中，本发明的试剂盒还包含说明书。
本发明还提供一种药物组合物，其包含本发明所述的NK抑制性分子、工程化免疫细胞、核酸分子或载体作为活性剂，和一种多种药学上可接受的赋型剂。因此，本发明还涵盖所述NK抑制性分子、核酸分子、载体或工程化免疫细胞在制备药物组合物或药物中的用途。
如本文所用，术语“药学上可接受的赋型剂”是指在药理学和/或生理学上与受试者和活性成分相容(即，能够引发所需的治疗效果而不会引起任何不希望的局部或全身作用)的载体和/或赋形剂，其是本领域公知的(参见例如Remington′s Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania：Mack Publishing Company，1995)。药学上可接受的赋型剂的实例包括但不限于填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、表面活性剂、稀释剂、润湿剂、防腐剂、乳化剂、包覆剂、等渗剂、吸收延迟剂、 稳定剂和张力调节剂。本领域技术人员已知选择合适的赋型剂以制备本发明期望的药物组合物。用于本发明的药物组合物中的示例性赋型剂包括盐水、缓冲盐水、葡萄糖和水。通常，合适的赋形剂的选择尤其取决于所使用的活性剂、待治疗的疾病和药物组合物的期望剂型。
根据本发明的药物组合物可适用于多种途径施用。通常，通过胃肠外完成施用。胃肠外递送方法包括局部、动脉内、肌内、皮下、髓内、鞘内、心室内、静脉内、腹膜内、子宫内、阴道内、舌下或鼻内施用。
根据本发明的药物组合物也可以制备成各种形式，如固态、液态、气态或冻干形式，特别可以是软膏、乳膏、透皮贴剂、凝胶、粉末、片剂、溶液、气雾剂、颗粒、丸剂、混悬剂、乳剂、胶囊、糖浆、酏剂、浸膏剂、酊剂或流浸膏提取物的形式，或者是特别适用于所需施用方法的形式。本发明已知的用于生产药物的过程可包括例如常规混合、溶解、制粒、制糖衣、研磨、乳化、包封、包埋或冻干过程。包含例如本文所述的免疫细胞的药物组合物通常以溶液形式提供，并且优选包含药学上可接受的缓冲剂。
根据本发明的药物组合物还可以与一种或多种适用于治疗和/或预防待治疗疾病的其它药剂组合施用。适用于组合的药剂的优选实例包括已知的抗癌药物，比如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimer sodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreate glucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；肽细胞毒素，比如蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，比如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，比如抗体定向的酶前药；免疫刺激剂，比如血小板因子4、黑色素瘤生长刺激蛋白等；抗体或其片段，比如抗CD3抗体或其片段，补体活化剂，异种蛋白结构域，同种蛋白结构域，病毒/细菌蛋白结构域和病毒/细菌肽。此外，本发明的药物组合物也可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。
治疗应用
本发明还提供一种治疗患有癌症、感染或自身免疫性疾病的受试者的方法，包括向所述受试者施用有效量的根据本发明所述的NK抑制性分子、核酸分子、载体、工程化免疫细胞或药物组合物。因此，本发明还涵盖所述NK抑制性分子、核酸分子、载体或工程化免疫细胞在制备治疗癌症、感染或自身免疫性疾病的药物中的用途。
在一个实施方案中，直接向受试者施用有效量的本发明的免疫细胞和/或药物组合物。
在另一个实施方案中，本发明的治疗方法是离体治疗。具体地，该方法包括以下步骤：(a)提供样品，所述样品包含免疫细胞；(b)在体外将所述免疫细胞的至少一种TCR/CD3基因和至少一种MHC相关基因的表达抑制或沉默，并将本发明的NK抑制性分子以及任选的嵌合抗原受体引入所述免疫细胞，获得经修饰的免疫细胞，(c)向有此需要的受试者施用所述经修饰的免疫细胞。优选地，步骤(a)中提供的免疫细胞选自B细胞、巨噬细胞、树突状细胞、单核细胞、T细胞、NK细胞或NKT细胞；并且所述免疫细胞可以通过本领域已知的常规方法从受试者的样品(特别是血液样品)中获得。然而，也可以使用能够表达本发明的嵌合抗原受体和NK抑制性分子并发挥如本文所述的所需生物效应功能的其它免疫细胞。此外，通常选择的免疫细胞与受试者的免疫系统相容，即优选所述免疫细胞不引发免疫原性响应。例如，可以使用“通用接受体细胞”，即发挥所需生物效应功能的普遍相容的可在体外生长和扩增的淋巴细胞。使用此类细胞将不需要获得和/或提供受试者自身淋巴细胞。步骤(c)的离体引入可以通过经由电穿孔将本文所述的核酸或载体引入免疫细胞或通过用病毒载体感染免疫细胞来实施，所述病毒载体为如前所述的慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体。其它可想到的方法包括使用转染试剂(比如脂质体)或瞬时RNA转染。
在一个实施方案中，所述免疫细胞是自体或同种异体的细胞，优选B细胞、T细胞、巨噬细胞、树突状细胞、单核细胞或、NK细胞NKT细胞，更优选T细胞、NK细胞或NKT细胞。
如本文所用，术语“自体”是指来源于个体的任何材料稍后将被再引入该相同个体中。
如本文所用，术语“同种异体”是指任何材料来源于与引入该材料的个体相同物种的不同动物或不同患者。当在一个或多个基因座处的基因不同时，认为两个或更多个体彼此为同种异体的。在一些情况下，来自同一物种的各个体的同种异体材料在基因上的不同可能足以发生抗原相互作用。
如本文所用，术语“受试者”是哺乳动物。哺乳动物可以是人、非人灵长类动物、小鼠、大鼠、狗、猫、马或牛，但不限于这些实例。除人以外的哺乳动物可以有利地用作代表癌症动物模型的受试者。优选地，所述受试者是人。
在一个实施方案中，所述癌症是与配体结合结构域结合的靶标表达有关的癌症。例 如，所述癌症包括但不限于：脑神经胶质瘤、胚细胞瘤、肉瘤、白血病、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌(包括胃肠癌)、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、肝肿瘤、肺癌(例如小细胞肺癌、非小细胞肺癌、腺状肺癌和鳞状肺癌)、淋巴瘤(包括霍奇金淋巴瘤和非霍奇金淋巴瘤)、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌(例如唇、舌、口和咽)、卵巢癌、胰腺癌、前列腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌以及其它癌和肉瘤、以及B细胞淋巴瘤(包括低级/滤泡性非霍奇金淋巴瘤(NHL)、小淋巴细胞性(SL)NHL、中间级/滤泡性NHL、中间级扩散性NHL、高级成免疫细胞性NHL、高级成淋巴细胞性NHL、高级小型非裂化细胞性NHL、大肿块病NHL)、套细胞淋巴瘤、AIDS相关淋巴瘤、以及Waldenstrom巨球蛋白血症、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、B细胞急性淋巴细胞白血病(B-ALL)、T细胞急性淋巴细胞白血病(T-ALL)、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特氏淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、慢性骨髓性白血病(CML)、恶性淋巴组织增生疾病、MALT淋巴瘤、毛细胞白血病、边缘区淋巴瘤、多发性骨髓瘤、骨髓发育不良、浆母细胞性淋巴瘤、白血病前期、浆细胞样树突状细胞瘤、以及移植后淋巴细胞增生性紊乱(PTLD)；以及其他与靶标表达有关的疾病。优选地，可以用本发明的工程化免疫细胞或药物组合物治疗的疾病选自：白血病、淋巴瘤、多发性骨髓瘤、脑神经胶质瘤、胰腺癌、胃癌等。
在一个实施方案中，所述感染包括但不限于由病毒、细菌、真菌和寄生虫引起的感染。
在一个实施方案中，所述自身免疫性疾病包括但不限于I型糖尿病、腹腔疾病、格雷夫斯病、炎症性肠病、多发性硬化症、银屑病、类风湿性关节炎、艾迪生病、干燥综合征、桥本甲状腺炎、重症肌无力、血管炎、恶性贫血与系统性红斑狼疮等。
在一个实施方案中，所述方法还进一步包括向所述受试者施用一种或多种额外的化疗剂、生物制剂、药物或治疗。在该实施方案中，化疗剂、生物制剂、药物或治疗选自放射疗法、手术、抗体试剂和/或小分子和它们的任意组合。
下面将参考附图并结合实例来详细说明本发明。需要说明的是，本领域的技术人员应该理解本发明的附图及其实施例仅仅是为了例举的目的，并不能对本发明构成任何限 制。在不矛盾的情况下，本申请中的实施例及实施例中的特征可以相互组合。
附图说明
图1：示出了E0-UNKi-T和E28-UNKi-T细胞中HLA-E的表达水平。
图2：示出了G0-UNKi-T和G28-UNKi-T细胞中HLA-G的表达水平。
图3：示出了ECad0-UNKi-T和ECad28-UNKi-T细胞中E-钙黏素的表达水平。
图4：示出了A28-UNKi-T细胞中NKG2A scFv的表达水平。
图5：示出了NK92-KLRG1细胞中KLRG1的表达水平。
图6：示出了本发明的UNKi-T细胞对NK细胞杀伤作用的抑制效果。用Two-way ANOVA分析，并用T test进行统计学分析。*表示P值小于0.05，**表示P值小于0.01，***表示P值小于0.001，达到显著水平。
图7：示出了A28z-UNKi-T细胞中NKG2A ScFv的表达水平。
图8：示出了E28z-UNKi-T细胞中HLA-E的表达水平。
图9：示出了本发明的UNKi-T细胞对NK细胞的杀伤效果。用Two-way ANOVA分析，并用T test进行统计学分析。*表示P值小于0.05，**表示P值小于0.01，***表示P值小于0.001，达到显著水平。
图10：示出了本发明的UNKi-T细胞与NK细胞共培养后的IFN-γ释放水平。用Two-way ANOVA分析，并用T test进行统计学分析。***表示P值小于0.001，达到显著水平。
图11：示出了本发明KIRG4-UNKi-T细胞中KIR scFv的表达水平。
图12：示出了本发明LIRG4-UNKi-1-T和LIRG4-UNKi-2-T细胞中LIR1 scFv的表达水平。
图13：示出了本发明的UNKi-T细胞对NK细胞杀伤作用的抑制效果。
图14：示出了SC7G4-T细胞、SC7/SC9G4-T细胞和K1G4-T细胞中scFv的表达水平。
图15：示出了SC7G4-T细胞、SC7/SC9G4-T细胞和K1G4-T细胞对NK细胞杀伤作用的抑制效果。
图16：示出了PDL1-T细胞中PDL1的表达水平。
图17：示出了PDL1-T细胞对NK细胞增殖的抑制效果。
图18：示出了NKi-B细胞和NKi-Huh7细胞中KIR scFv的表达水平。
图19：示出了NKi-B细胞和NKi-Huh7细胞对NK细胞杀伤作用的抑制效果。
具体实施方式
本发明所有实施例中使用的T细胞是通过Ficoll-PaqueTM PREMIUM(GE Healthcare，货号17-5442-02)采用白细胞分离术从健康供体分离的原代人CD4+CD8+T细胞。
实施例1：构建表达NK抑制性分子且敲除TCR/HLA-I/HLA-II的UNKi-T免疫细胞
合成以下的编码序列，并将其依次克隆至pGEM-T Easy载体(Promega，货号A1360)：B2m信号肽(SEQ ID NO：21)、NK抑制性配体、CD28铰链区(SEQ ID NO：27)、CD28跨膜区(SEQ ID NO：11)，其中NK抑制性配体是E-钙黏素的胞外区(SEQ ID NO：41，对应ECad0质粒)、B2M和HLA-E胞外区的融合分子(包含提呈肽SEQ ID NO：46、B2M SEQ ID NO：37和HLA-E胞外区突变体SEQ ID NO：33，其中B2M的核酸序列是同义突变的SEQ ID NO：38，对应E0质粒)，或B2M和HLA-G胞外区的融合分子(包含B2M SEQ ID NO：37和HLA-G胞外区SEQ ID NO：35，其中B2M的核酸序列是同义突变的SEQ ID NO：38，对应G0质粒)。在ECad0、E0和G0质粒中进一步包括CD28共刺激结构域(SEQ ID NO：13)，分别获得ECad28、E28和G28质粒。通过测序确认目标序列在质粒中的正确插入。
合成以下编码序列，并将其依次克隆至pGEM-T Easy载体(Promega，货号A1360)：B2m信号肽(SEQ ID NO：21)、抗NKG2A-scFv(包含SEQ ID NO：5和7)、IgG4铰链区(SEQ ID NO：29)、CD28跨膜区(SEQ ID NO：11)、CD28共刺激结构域(SEQ ID NO：13)，获得A28质粒，并通过测序确认目标序列在质粒中的正确插入。
在无菌管中加入3ml Opti-MEM(Gibco，货号31985-070)稀释上述质粒后，再根据质粒∶病毒包装载体∶病毒包膜载体＝4∶2∶1的比例加入包装载体psPAX2(Addgene，货号12260)和包膜载体pMD2.G(Addgene，货号12259)。然后，加入120ul X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，于室温下孵育15min，然后将质粒/载体/转染试剂混合物逐滴加入到293T细胞的培养瓶中。在24小时和48小时收集病毒，将其合并后，超速离心(25000g，4℃，2.5小时)获得浓缩的慢病毒。
用DynaBeads CD3/CD28CTSTM(Gibco，货号40203D)激活T细胞，并在37℃和5％CO2下培养1天。然后，加入浓缩的慢病毒，持续培养3天后，获得表达NK抑制性 分子的T细胞。
然后采用CRISPR系统敲除所述表达NK抑制性分子的T细胞中的TCR/CD3组分(具体为TRAC基因)和MHC相关基因(具体为B2M和RFX5)。具体地，使用BTX Agile Pulse Max电穿孔仪(Harvard Apparatus BTX)，以400V、0.7ms将10ug Cas9蛋白和10ug sgRNA(3.3ug TRAC sgRNA(SEQ ID NO：43)+3.3ug B2m sgRNA(SEQ ID NO：44)+3.3ug RFX5sgRNA(SEQ ID NO：45))电转染进激活的NKi-T细胞。电转染之后，立即将NKi-T细胞放入1ml预热的培养基中，并在IL-2(300IU/m1)存在下，在37℃和5％CO2下培养，获得TCR/B2M/RFX5三敲除的UNKi-T细胞。用CRISPR系统敲除TCR/B2M/RFX5的野生型T细胞(即，Mock T细胞)和未敲除基因的野生型T细胞(即，NT细胞)作为对照。
本实施例中制备的UNKi-T细胞包含的NK抑制性分子的结构如下表1所示。
表1.包含NK抑制性分子的UNKi-T细胞
Figure PCTCN2021099314-appb-000001
11天之后，使用FITC Mouse Anti-Human CD3(BD Pharmingen，货号555916)抗体、PE mouse anti-human HLA-I(R&D货号FAB7098P)和APC anti-human DR，DP，DQ(biolegend，货号361714)抗体，通过流式细胞仪检测UNKi-T细胞、Mock T细胞和NT细胞中的TCR/HLA-I/HLA-II的表达效率，结果如下表2所示。
表2.UNKi-T细胞中的基因表达效率
细胞名称 TCR/CD3 B2M/HLA-I RFX5/HLA-II
Ecad0-UNKi-T 3.6％ 19％ 11％
Ecad28-UNKi-T 2.9％ 18.3％ 10.4％
E0-UNKi-T 4.8％ 18.9％ 11.3％
E28-UNKi-T 3.4％ 20％ 12％
G0-UNKi-T 4％ 19％ 9.8％
G28-UNKi-T 2.9％ 19.7％ 10％
A28-UNKi-T 3.8％ 21.1％ 12.1％
Mock T 2.7％ 17.8％ 9.8％
NT 97％ 98％ 60％
从表2可以看出，本发明制备的UNKi-T细胞以及Mock T细胞中的TCR/B2M/RFX5的表达被有效抑制或沉默。
此外，使用流式细胞仪，用PE mouse anti-human HLA-E(biolegend货号342604)检测UNKi-T细胞和Mock T细胞中的HLA-E表达(图1)，用PE mouse anti-human HLA-G(biolegend货号335906)检测UNKi-T细胞和Mock T细胞中的HLA-G表达(图2)，用E-cadherin monoclonal antibody(invitrogen货号13-5700)和Goat anti-Mouse IgG(H+L)Cross-Adsorbed Secondary Antibody，Alexa Fluor 488(Invitrogen，货号A-11001)检测UNKi-T细胞和Mock T细胞中的E-钙黏素表达(图3)，用Biotin-SP(long spacer)AffiniPure Goat Anti-Human IgG，F(ab′)fragment specific antibody(Jackson ImmunoResearch，货号109-065-097)和APC Streptavidin(BD，货号554067)检测UNKi-T细胞和Mock T细胞中的anti-NKG2A scFv表达(图4)。
从图1-4可以看出，本发明制备的UNKi-T细胞中的NK抑制性分子均有效表达。
实施例2.UNKi-T细胞对NK细胞杀伤作用的抑制效果
本实施例中使用的效应细胞是NK92细胞。由于NK92细胞系不表达E-钙黏素的受体KLRG1，因此首先制备过表达KLRG1的NK92细胞。
合成编码KLRG1(SEQ ID NO：54)的核酸序列，并将其依次克隆至pGEM-T Easy载体(Promega，货号A1360)，并通过测序确认目标序列的正确插入。用SpeI酶对该载体进行酶切，纯化回收后获得线性化载体。然后，根据制造商的建议，以线性化载体为模板，用mMESSAGE
Figure PCTCN2021099314-appb-000002
T7 Ultra Kit试剂盒(Invitrogen，货号AM1345)制备mRNA，并用Fastpure cell/Tissue total RNA isolation kit试剂盒(Vazyme，货号RC101-01)进行纯化，获得纯化的mRNA。然后，使用BTX Agile Pulse Max电穿孔仪(Harvard Apparatus BTX)，以200V、2ms将上述制备的20ug纯化mRNA电转染进NK92细胞内，获得NK92-KLRG1细胞。16h后，检测KLRG1的表达，结果如图5所示。未转染KLRG1的NK92细胞作为对照。
从图5可以看出，NK92-KLRG1细胞可有效表达KLRG1。
然后根据以下方法检测本发明制备的UNKi-T细胞对NK细胞杀伤作用的抑制效果：用Far-Red(invitrogen，货号C34564)标记本发明制备的UNKi-T细胞和Mock-T细胞。 然后按照1x10 4个细胞/孔的浓度将标记好的UNKi-T细胞和Mock T细胞铺入96孔板，并以2∶1的效靶比加入NK92细胞(用于表达HLA-E、HLA-G或NKG2A scFv的UNKi-T细胞和Mock T细胞)或NK92-KLRG1细胞(用于表达E-钙黏素的UNKi-T细胞)进行共培养。16-18小时后，用流式细胞仪检测培养物中T细胞的比例，进而计算NK细胞对T细胞的杀伤率，结果如图6所示。
从图6可以看出，与不表达NK抑制性分子的Mock T细胞相比，表达包含抑制性配体例如NKG2A scFv、HLA-G、HLA-E、E-钙黏素的NK抑制性分子的UNKi-T细胞均能显著降低NK细胞对T细胞的杀伤作用。并且，与仅表达抑制性配体和跨膜结构域的T细胞相比，共刺激结构域的加入可以进一步显著增强T细胞对NK细胞杀伤的抑制(参见G0 vs G28；E0 vs E28；ECad0 vs ECad28)。因此，本发明制备的包含抑制性配体、跨膜结构域和共刺激结构域的NK抑制性分子可以显著降低NK细胞对UNKi-T细胞的杀伤作用，从而能够有效降低HvGD风险。
实施例3.UNKi-T细胞对NK细胞的杀伤效果
根据实施例1所述的方法制备E28z-UNKi-T和A28z-UNKi-T细胞，其与E28-UNKi-T和A28-UNKi-T细胞的区别仅在于其进一步包含CD3ζ胞内信号传导结构域(SEQ ID NO：17)。
(1)检测NK抑制性分子的表达
用Biotin-SP(long spacer)AffiniPure Goat Anti-Human IgG，F(ab′)fragment specific antibody(Jackson ImmunoResearch，货号109-065-097)和APC Streptavidin(BD，货号554067)检测A28z-UNKi-T细胞和Mock T细胞中的anti-NKG2A scFv表达(图7)。使用流式细胞仪，用PE mouse anti-human HLA-E(biolegend货号342604)检测E28z-UNKi-T细胞和Mock T细胞中的HLA-E表达(图8)。
可以看出，本发明制备的E28z-UNKi-T细胞和A28z-UNKi-T细胞中的NK抑制性分子能够有效表达。
(2)检测CD107a的表达
细胞毒性T淋巴细胞(CTL细胞)胞浆内含有高浓度以囊泡形式存在的细胞毒性颗粒，溶酶体相关膜蛋白I(CD107a)是囊泡膜蛋白的主要成分。CTL细胞杀伤靶细胞时，毒性颗粒将到达细胞膜并与细胞膜融合(此时CD107a分子被转运到细胞膜表面)，引起颗粒内容物释放，最终导致靶细胞的死亡。因此，CD107a分子是CTL细胞脱颗粒的一种敏感标志，可反应细胞杀伤活性。
以1x10 5个细胞/孔的浓度将靶细胞(NK92细胞)铺于96孔板中，然后每孔以1∶1的比例加入Mock T细胞、E28z-UNKi-T细胞和A28z-UNKi-T细胞，同时加入10μl PE-anti-human CD107a(BD Pharmingen，货号555801)，于37℃，5％CO 2培养条件下共培养。1h后，加入Goigstop(BD Pharmingen，货号51-2092KZ)继续孵育2.5小时。然后向每孔加入5μl APC-anti human CD8(BD Pharmingen，货号：555369)和5μl FITC-anti human CD4(BD Pharmingen，货号：561005)，于37℃孵育30分钟后，用流式细胞仪检测CD107a的表达情况，结果如图9A(CD4+T细胞毒性)和图9B(CD8+T细胞毒性)所示。
可以看出，不表达NK抑制性分子的Mock T细胞对靶细胞几乎没有杀伤。与此相反，本发明制备的E28z-UNKi-T细胞和A28z-UNKi-T细胞与靶细胞共培养后，CD107a的表达率显著提高，表明本发明的UNKi-T细胞可显著杀伤NK细胞。
(3)检测IFN-γ的分泌
以1x10 5个细胞/孔将靶细胞(NK92细胞)铺于96孔板中，每孔以1∶1的比例加入Mock T细胞、E28z-UNKi-T细胞和A28z-UNKi-T细胞，于37℃，5％CO2培养条件下共培养，18-24小时后收集细胞共培养上清液。
使用捕获抗体Purified anti-human IFN-γ Antibody(Biolegend，货号506502)包被96孔板4℃孵育过夜，然后移除抗体溶液。加入250μL含有2％BSA(sigma，货号V900933-1kg)的PBST(含0.1％吐温的1XPBS)溶液，37℃孵育2小时。然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。每孔加入50μL细胞共培养上清液或标准品，并在37℃孵育1小时，然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。然后向各孔分别加入50μL检测抗体Anti-Interferon gamma抗体[MD-1](Biotin)(abcam，货号ab25017)，在37℃孵育1小时后，用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。再加入HRP Streptavidin(Biolegend，货号405210)，在37℃孵育30分钟后，弃上清液，加入250μL PBST(含0.1％吐温的1XPBS)，清洗5次。向各孔加入50μL TMB底物溶液。使反应在室温下于暗处发生30分钟，之后向各孔中加入50μL 1mol/L H 2SO 4以停止反应。在停止反应的30分钟内，使用酶标仪检测450nm处吸光度，并根据标准曲线(根据标准品的读值和浓度绘制)计算细胞因子的含量，结果如图10所示。
可以看出，本发明的E28z-UNKi-T细胞和A28z-UNKi-T细胞的细胞因子IFN-γ释放水平远远高于Mock T细胞，这也表明其对NK92靶细胞的杀伤显著增加。
实施例4.靶向KIR或LIR1的UNKi-T细胞及其对NK细胞杀伤作用的抑制效果
合成以下编码序列，并将其依次克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号：PPL00157-4a)：B2m信号肽(SEQ ID NO：21)、抗KIR-scFv(包含SEQ ID NO：55和56)或抗LIR1 scFv(包含SEQ ID NO：57和58，或包含SEQ ID NO：59和60)、IgG4铰链区(SEQ ID NO：29)、CD8α跨膜区(SEQ ID NO：9)、CD28共刺激结构域(SEQ ID NO：13)，获得KIRG4、LIRG4-1和LIRG4-2质粒，并通过测序确认目标序列在质粒中的正确插入。
根据实施例1中的敲除和感染方式制备UNKi-T细胞，并使用FITC Mouse Anti-Human CD3(BD Pharmingen，货号555916)抗体、PE mouse anti-human HLA-I(R&D货号FAB7098P)和APC anti-human DR，DP，DQ(biolegend，货号361714)抗体，通过流式细胞仪检测UNKi-T细胞、Mock T细胞和NT细胞中的CD3/HLA-I/HLA-II的表达效率，结果如下表3所示。
表3.UNKi-T细胞中的基因表达效率
细胞名称 TCR/CD3 B2M/HLA-I RFX5/HLA-II
KIRG4-UNKi-T 4.5％ 16％ 10.8％
LIRG4-UNKi-1-T 3.5％ 17.6％ 10.9％
LIRG4-UNKi-2-T 4.3％ 16.9％ 10.3％
Mock T 2.6％ 15.8％ 9.3％
NT 98％ 98％ 83％
从表3可以看出，本发明制备的UNKi-T细胞以及Mock T细胞中的CD3/HLA-I/HLA-II的表达被有效抑制或沉默。
用Biotin-SP(long spacer)AffiniPure Goat Anti-Human IgG，F(ab′)fragment specific antibody(Jackson ImmunoResearch，货号109-065-097)和APC Streptavidin(BD，货号554067)检测KIRG4-UNKi-T细胞中的scFv表达，结果如图11所示。用recombinant human LILRB1protein(sino biological，货号16014-H02H)作为一抗，APC anti-human IgG Fc(biolegend，货号409306)作为二抗检测LIRG4-UNKi-1T细胞和LIRG4-UNKi-2T细胞中scFv的表达，结果如图12所示。从图11和图12中可以看出，本发明制备的UNKi-T细胞的scFv均有效表达。
根据实施例2中的方法将UNKi-T细胞和NK92细胞共培养，以检测UNKi-T细胞对NK细胞杀伤作用的抑制效果，结果如图13所示。可以看出，与Mock T相比，本实施例制备的靶向KIR(图13A)或LIR1(图13B)的UNKi-T细胞均能显著降低NK细胞对T细胞的杀伤作用。
实施例5.靶向SIGLC7、SIGLEC9或KLRG1的T细胞及其对NK细胞杀伤作用 的抑制效果
合成以下编码序列，并将其依次克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号：PPL00157-4a)：B2m信号肽(SEQ ID NO：21)、抗SIGLEC7-scFv(SEQ ID NO：130)、抗SIGLEC7/SIGLEC9-scFv(SEQ ID NO：184)或抗KLRG1-scFv(SEQ ID NO：119)、IgG4铰链区(SEQ ID NO：29)、CD8α跨膜区(SEQ ID NO：9)、CD28共刺激结构域(SEQ ID NO：13)，获得SC7G4、SC7/SC9G4和K1G4质粒，并通过测序确认目标序列在质粒中的正确插入。
将上述质粒根据实施例1中的方法转入T细胞，并敲除其中的B2M基因，获得表达NK抑制性分子且B2M敲除的NKi-T细胞(即，SC7G4-T细胞、SC7/SC9G4-T细胞和K1G4-T细胞)。仅敲除B2M的T细胞用作阴性对照(NT)。
用Biotin-SP(long spacer)AffiniPure Goat Anti-mouse IgG，F(ab′)fragment specific antibody(Jackson ImmunoResearch，货号115-066-072)和APC Streptavidin(BD，货号554067)检测SC7G4、SC7/SC9G4和K1G4T细胞中的scFv表达，结果如图14所示。可以看出，本发明制备的NKi-T细胞的scFv均有效表达。
根据实施例2所述的方法，将上述NKi-T细胞与NK92-KLRG1细胞共培养，以检测其对NK细胞杀伤作用的抑制效果，结果如图15所示。可以看出，与NT相比，本实施例制备的靶向SIGLEC7、SIGLEC9或KLRG1的T细胞均能显著降低NK细胞对T细胞的杀伤作用。
实施例6.靶向PD-1的T细胞及其对NK细胞增殖的抑制效果
合成以下编码序列，并将其依次克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号：PPL00157-4a)：PDL1信号肽(SEQ ID NO：121)、PDL1胞外区(SEQ ID NO：70)、PDL1跨膜区(SEQ ID NO：120)、CD28共刺激结构域(SEQ ID NO：13)，获得PDL1质粒，并通过测序确认目标序列在质粒中的正确插入。
将上述质粒根据实施例1中的方法转入T细胞，获得表达NK抑制性分子的NKi-T细胞，即PDL1-T细胞。采用Anti-PD-L1Antibody(厂家：索莱宝，货号：10084-R312-A)检测其表达，结果如图16所示。可以看出，PDL1被有效表达。未经处理的T细胞用作阴性对照(NT)。
将上述NKi-T细胞进行培养并经细胞丝裂霉素C处理，同时将两个异体来源的PBMC(donor1和donor2)采用Far-Red进行标记后，按照T细胞∶PBMC＝1∶2的比例进行共培养。每2-3天进行半换液处理。8d后细胞计数并采用PE anti-human CD3(厂 家biolegend，货号：317308)和FITC anti-human CD56(厂家：biolegend，货号：362546)染色，然后通过流式细胞术检测NK细胞群比例。通过细胞总量*NK细胞群比例来计算NK细胞数量，结果如图17所示。可以看出，靶向PD-1的NKi-T细胞可显著抑制NK细胞的增殖。
实施例7.靶向KIR的B细胞和Huh7细胞对NK细胞杀伤作用的抑制效果
将实施例4中制备的KIRG4质粒根据实施例1中的方法转入B细胞和Huh7细胞(肝癌细胞)，并敲除其中的B2M基因，获得表达NK抑制性分子且B2M敲除的NKi-B细胞和NKi-Huh7细胞。仅敲除B2M基因的B细胞和Huh7细胞(NC细胞)用作阴性对照。
用Biotin-SP(long spacer)AffiniPure Goat Anti-Human IgG，F(ab′)fragment specific antibody(Jackson ImmunoResearch，货号109-065-097)和APC Streptavidin(BD，货号554067)检测NKi-B细胞和NKi-Huh7细胞中的scFv表达，结果如图18所示。可以看出，NK抑制性分子在B细胞和Huh7细胞中均有效表达。
根据实施例2中的方法将上述制备的细胞和NK细胞共培养，以检测上述细胞对NK细胞杀伤作用的抑制效果，结果如图19所示。可以看出，与NC细胞相比，本实施例制备的NKi-B细胞和NKi-Huh7细胞均能显著降低NK细胞对其的杀伤作用。
要说明的是，以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。本领域技术人员理解的是，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (35)

  1. 一种NK抑制性分子，其包含一个或多个NK抑制性配体、跨膜结构域和共刺激结构域，其中所述NK抑制性配体特异性结合NK抑制性受体(NK inhibitory receptor，NKIR)以抑制NK细胞对表达所述NK抑制性分子的工程化免疫细胞的杀伤。
  2. 权利要求1所述的NK抑制性分子，其中所述NK抑制性配体是靶向NKIR的抗体，或NKIR的天然配体或其包含的NKIR结合区。
  3. 权利要求1或2所述的NK抑制性分子，其中所述NKIR选自：NKG2/CD94组分；杀伤细胞Ig样受体(KIR)家族成员；白细胞Ig样受体(LIR)家族成员；NK细胞受体蛋白1(NKR-P1)家族成员；免疫检查点受体；癌胚抗原相关的细胞黏附分子1(CEACAM1)；唾液酸结合性免疫球蛋白样凝集素(SIGLEC)家族成员；白细胞相关的免疫球蛋白样受体1(LAIR1)；Ly49家族成员和杀伤细胞凝集素样受体G1(KLRG1)。
  4. 权利要求3所述的NK抑制性分子，其中所述NKG2/CD94组分选自NKG2A、NKG2B和CD94；所述KIR家族成员选自KIR2DL1、KIR2DL2/3、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2和KIR3DL3；所述LIR家族成员选自LIR1、LIR2、LIR3、LIR5和LIR8；所述NKR-P1家族成员选自NKR-P1B和NKR-P1D；所述免疫检查点受体选自PD-1、TIGIT、CD96、TIM3和LAG3；所述SIGLEC家族成员选自SIGLEC7和SIGLEC9；所述Ly49家族成员选自Ly49A、Ly49C、Ly49F、Ly49G1和Ly49G4。
  5. 权利要求3或4所述的NK抑制性分子，其中所述NKIR选自：NKG2A、NKG2B、CD94、LIR1、LIR2、LIR3、KIR2DL1、KIR2DL2/3、KIR3DL1、CEACAM1、PD1、LAIR1、SIGLEC7、SIGLEC9和KLRG1。
  6. 权利要求1-5任一项所述的NK抑制性分子，其中所述NK抑制性配体是靶向NKIR的抗体或其功能性片段，所述抗体或其功能性片段选自完整抗体、Fab、Fab’、F(ab’)2、Fv片段、scFv抗体片段、线性抗体、sdAb或纳米抗体。
  7. 权利要求1-5任一项所述的NK抑制性分子，其中所述NK抑制性配体是靶向PD1、NKG2A、LIR1、KIR、SIGLEC7、SIGLEC9和/或KLRG1的抗体。
  8. 权利要求7所述的NK抑制性分子，其中：
    (i)所述靶向NKG2A的抗体包含(1)如SEQ ID NO：72所示的CDR-L1、如SEQ ID NO：73所示的CDR-L2、如SEQ ID NO：74所示的CDR-L3、如SEQ ID NO：75所示的CDR-H1、如SEQ ID NO：76所示的CDR-H2和如SEQ ID NO：77所示的CDR-H3，或(2)如SEQ ID NO：78所示的CDR-L1、如SEQ ID NO：79所示的CDR-L2、如SEQ ID NO：80所示的CDR-L3、如SEQ ID NO：81所示的CDR-H1、如SEQ ID NO：82所示的CDR-H2和如SEQ ID NO：83所示的CDR-H3；
    (ii)所述靶向LIR1的抗体包含(1)如SEQ ID NO：90所示的CDR-L1、如SEQ ID NO：91所示的CDR-L2、如SEQ ID NO：92所示的CDR-L3、如SEQ ID NO：93所示的CDR-H1、如SEQ ID NO：94所示的CDR-H2和如SEQ ID NO：95所示的CDR-H3，或(2)如SEQ ID NO：96所示的CDR-L1、如SEQ ID NO：97所示的CDR-L2、如SEQ ID NO：98所示的CDR-L3、如SEQ ID NO：99所示的CDR-H1、如SEQ ID NO：100所示的CDR-H2和如SEQ ID NO：101所示的CDR-H3；
    (iii)所述靶向KIR的抗体包含如SEQ ID NO：84所示的CDR-L1、如SEQ ID NO：85所示的CDR-L2、如SEQ ID NO：86所示的CDR-L3、如SEQ ID NO：87所示的CDR-H1、如SEQ ID NO：88所示的CDR-H2和如SEQ ID NO：89所示的CDR-H3；
    (iv)所述靶向SIGLEC7、SIGLEC9或两者的抗体包含(1)如SEQ ID NO：102所示的CDR-L1、如SEQ ID NO：103所示的CDR-L2、如SEQ ID NO：104所示的CDR-L3、如SEQ ID NO：105所示的CDR-H1、如SEQ ID NO：106所示的CDR-H2和如SEQ ID NO：107所示的CDR-H3，(2)如SEQ ID NO：122所示的CDR-L1、如SEQ ID NO：123所示的CDR-L2、如SEQ ID NO：124所示的CDR-L3、如SEQ ID NO：125所示的CDR-H1、如SEQ ID NO：126所示的CDR-H2和如SEQ ID NO：1277所示的CDR-H3，(3)如SEQ ID NO：131所示的CDR-L1、如SEQ ID NO：132所示的CDR-L2、如SEQ ID NO：133所示的CDR-L3、如SEQ ID NO：134所示的CDR-H1、如SEQ ID NO：135所示的CDR-H2和如SEQ ID NO：136所示的CDR-H3，(4)如SEQ ID NO：140所示的CDR-L1、如SEQ ID NO：141所示的CDR-L2、如SEQ ID NO：142所示的CDR-L3、如SEQ ID NO：143所示的CDR-H1、如SEQ ID NO：144所示的CDR-H2和如SEQ ID NO：155所示的 CDR-H3，(5)如SEQ ID NO：176所示的CDR-L1、如SEQ ID NO：177所示的CDR-L2、如SEQ ID NO：178所示的CDR-L3、如SEQ ID NO：179所示的CDR-H1、如SEQ ID NO：180所示的CDR-H2和如SEQ ID NO：181所示的CDR-H3，或(6)如SEQ ID NO：188所示的CDR-L1、如SEQ ID NO：189所示的CDR-L2、如SEQ ID NO：190所示的CDR-L3、如SEQ ID NO：191所示的CDR-H1、如SEQ ID NO：192所示的CDR-H2和如SEQ ID NO：193所示的CDR-H3；和/或
    (v)所述靶向KLRG1的抗体包含(1)如SEQ ID NO：111所示的CDR-L1、如SEQ ID NO：112所示的CDR-L2、如SEQ ID NO：113所示的CDR-L3、如SEQ ID NO：114所示的CDR-H1、如SEQ ID NO：115所示的CDR-H2和如SEQ ID NO：116所示的CDR-H3，(2)如SEQ ID NO：149所示的CDR-L1、如SEQ ID NO：150所示的CDR-L2、如SEQ ID NO：151所示的CDR-L3、如SEQ ID NO：152所示的CDR-H1、如SEQ ID NO：153所示的CDR-H2和如SEQ ID NO：154所示的CDR-H3，(3)如SEQ ID NO：158所示的CDR-L1、如SEQ ID NO：159所示的CDR-L2、如SEQ ID NO：160所示的CDR-L3、如SEQ ID NO：161所示的CDR-H1、如SEQ ID NO：162所示的CDR-H2和如SEQ ID NO：163所示的CDR-H3，或(4)如SEQ ID NO：167所示的CDR-L1、如SEQ ID NO：168所示的CDR-L2、如SEQ ID NO：169所示的CDR-L3、如SEQ ID NO：170所示的CDR-H1、如SEQ ID NO：171所示的CDR-H2和如SEQ ID NO：172所示的CDR-H3。
  9. 权利要求1-5任一项所述的NK抑制性分子，其中所述NK抑制性配体选自HLA-E、HLA-F、HLA-G、钙黏素、胶原蛋白、OCIL、唾液酸、PD-L1/PD-L2、CD155、CD112、CD113、Gal-9、FGL1，和它们包含的NKIR结合区。
  10. 权利要求9所述的NK抑制性分子，其中所述NK抑制性配体选自唾液酸、HLA-E胞外区、HLA-F胞外区、HLA-G胞外区、E-钙黏素胞外区、PD-L1胞外区和PD-L2胞外区。
  11. 权利要求10所述的NK抑制性分子，其中：
    (i)所述HLA-E的胞外区与SEQ ID NO：31或33所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；
    (ii)所述HLA-G的胞外区与SEQ ID NO：35所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；
    (iii)E-钙黏素胞外区与SEQ ID NO：39或41所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；
    (iv)PD-L1胞外区与SEQ ID NO：70所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；
    (v)PD-L2胞外区与SEQ ID NO：71所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
  12. 权利要求1所述的NK抑制性分子，其中所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154、HLA-E、HLA-F、HLA-G、钙黏素、胶原蛋白、OCIL。
  13. 权利要求1所述的NK抑制性分子，其中所述共刺激结构域选自以下蛋白质的共刺激信号传导结构域：LTB、CD94、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM、ZAP70以及它们的组合。
  14. 权利要求1所述的NK抑制性分子，其中所述NK抑制性分子不包含胞内信号传导结构域。
  15. 权利要求1所述的NK抑制性分子，其中所述NK抑制性分子进一步包含胞内信号传导结构域。
  16. 权利要求14或15所述的NK抑制性分子，其中所述胞内信号传导结构域选自以下蛋白的信号传导结构域：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。
  17. 权利要求14或15所述的NK抑制性分子，其中所述胞内信号传导结构域包含CD3ζ信号传导结构域。
  18. 一种核酸分子，其编码权利要求1-17任一项所述的NK抑制性分子。
  19. 一种载体，其包含权利要求18所述的核酸分子。
  20. 一种工程化免疫细胞，其特征在于：(1)表达权利要求1-17任一项所述的NK抑制性分子，和(2)至少一种MHC相关基因的表达被抑制或沉默。
  21. 权利要求20所述的工程化免疫细胞，其特征还在于，所述工程化免疫细胞还表达嵌合抗原受体，所述嵌合抗原受体包含配体结合结构域、跨膜结构域、共刺激结构域和细胞内信号传导结构域。
  22. 一种工程化免疫细胞，其特征在于：(1)表达NK抑制性分子和嵌合抗原受体的融合蛋白，所述融合蛋白包含NK抑制性配体、配体结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，和(2)至少一种MHC相关基因的表达被抑制或沉默。
  23. 权利要求20-22任一项所述的工程化免疫细胞，其中所述至少一种MHC相关基因选自HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA和它们的组合。
  24. 权利要求23所述的工程化免疫细胞，其中所述至少一种MHC相关基因选自B2M、RFX5、RFXAP、RFXANK、CIITA和它们的组合。
  25. 权利要求24所述的工程化免疫细胞，其中所述MHC相关基因包含B2M，其中所述NK抑制性配体是B2M和非经典HLA-I类分子的胞外区的融合分子。
  26. 权利要求25所述的工程化免疫细胞，其中所述非经典HLA-I类分子是HLA-E或HLA-G。
  27. 权利要求25所述的工程化免疫细胞，其中所述NK抑制性分子还包含提呈肽，其选自SEQ ID NO：46-53。
  28. 权利要求20-27任一项所述的工程化免疫细胞，其中所述工程化免疫细胞的至少一种TCR/CD3基因的表达被抑制或沉默，所述TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ和它们的组合。
  29. 权利要求20-28任一项所述的工程化免疫细胞，其特征还在于，其中选自以下的一个或多个基因的表达被抑制或沉默：CD52、GR、dCK、PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、 CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。
  30. 权利要求20-29任一项所述的工程化免疫细胞，其中所述配体结合结构域与选自以下的靶标结合：TSHR、CD2、CD3、CD4、CD5、CD7、CD8、CD14、CD15、CD19、CD20、CD21、CD23、CD24、CD25、CD37、CD38、CD40、CD40L、CD44、CD46、CD47、CD52、CD54、CD56、CD70、CD73、CD80、CD97、CD123、CD22、CD126、CD138、CD 179a、DR4、DR5、TAC、TEM1/CD248、VEGF、GUCY2C、EGP40、EGP-2、EGP-4、CD133、IFNAR1、DLL3、kappa轻链、TIM3、BAFF-R、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、tEGFR、GD2、GD3、BCMA、GPRC5D、Tn抗原、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、IL-22Ra、IL-2、间皮素、IL-llRa、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、AFP、Folate受体α、ERBB2(Her2/neu)、ErbB3、ErbB4、MUC1、MUC16、EGFR、CS1、CD138、NCAM、Claudin18.2、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gploo、bcr-abl、酪氨酸酶、EphA2、Fucosyl GM1、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD 179a、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、MAGE-A3、MAGE-A6、豆荚蛋白、HPV E6、E7、MAGE-A4、MART-1、WT-1、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋白、存活蛋白和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、 CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D、NKG2DL和它们的任意组合。
  31. 权利要求30所述的工程化免疫细胞，其中所述靶标选自CD7、CD19、CD20、CD22、CD30、CD33、CD38、CD123、CD138、CD171、MUC1、AFP、Folate受体α、CEA、PSCA、PSMA、Her2、EGFR、IL13Ra2、GD2、NKG2D、EGFRvIII、CS1、BCMA、间皮素、Cluadin18.2、ROR1、NY-ESO-1、MAGE-A4和它们的任意组合。
  32. 权利要求20-31任一项所述的工程化免疫细胞，其中所述工程化免疫细胞是B细胞、T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞。
  33. 权利要求32所述的工程化免疫细胞，其中所述工程化免疫细胞是CD4+/CD8+T细胞、CD4+辅助T细胞、CD8+T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞或αβ-T细胞。
  34. 一种药物组合物，其包含权利要求1-17任一项所述的NK抑制性分子、权利要求18所述的核酸分子、权利要求19所述的载体或权利要求20-33任一项所述的工程化免疫细胞，和一种多种药学上可接受的赋型剂。
  35. 权利要求1-17任一项所述的NK抑制性分子、权利要求18所述的核酸分子、权利要求19所述的载体或权利要求20-33任一项所述的工程化免疫细胞在制备治疗癌症、感染或自身免疫性疾病的药物中的用途。
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