WO2020216230A1 - 同种异体car-t细胞、其制备及应用 - Google Patents

同种异体car-t细胞、其制备及应用 Download PDF

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WO2020216230A1
WO2020216230A1 PCT/CN2020/086032 CN2020086032W WO2020216230A1 WO 2020216230 A1 WO2020216230 A1 WO 2020216230A1 CN 2020086032 W CN2020086032 W CN 2020086032W WO 2020216230 A1 WO2020216230 A1 WO 2020216230A1
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hla
protein
expression
cells
cell
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PCT/CN2020/086032
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French (fr)
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李俊
张鹏潮
徐昭
何玲
刘伟康
江雨辰
秦含笑
夏宇
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苏州方德门达新药开发有限公司
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Priority to US17/605,988 priority Critical patent/US20220340639A1/en
Priority to JP2021563394A priority patent/JP2022530139A/ja
Priority to AU2020261799A priority patent/AU2020261799A1/en
Priority to CA3137788A priority patent/CA3137788A1/en
Priority to EP20795581.6A priority patent/EP3960849A4/en
Priority to KR1020217037999A priority patent/KR20220018479A/ko
Publication of WO2020216230A1 publication Critical patent/WO2020216230A1/zh

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Definitions

  • the invention relates to a selective allogeneic CAR-T cell, its preparation and application.
  • CAR-T Chimeric Antigen Receptor-T
  • the CAR expressed by CAR-T cells generally contains an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • CAR-T cells can effectively recognize tumor antigens and cause specific anti-tumor immune responses without being restricted by major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • the US FDA has approved two autologous CAR-T cell products, namely Novartis’ Kymriah and Kate’s YesCAR-Ta, for the treatment of refractory relapsed non-Hodgkin’s lymphoma and acute B lymphocytic leukemia .
  • a large number of clinical trials have proved that CAR-T has great anti-tumor potential as a personalized living cell drug (Maude et al. 2018; Park et al. 2018; Schuster et al. 2017;).
  • Both of these CAR-T cell products on the market collect the patient’s own peripheral blood for T cell isolation, use lentivirus or retroviral vectors to transfect the CAR gene expression cassette into the T cells, expand the culture, and then infuse them. Into the patient.
  • the CAR-T of each patient needs to be prepared separately, the production cycle is long, and a variety of uncertain factors may cause the failure or failure of CART therapy, such as insufficient number of isolated cells, abnormal T cell quality or function of the patient, CAR-T Failure of cell preparation, rapid progress of the patient’s disease during cell preparation, etc.
  • TCR T cell receptor
  • HLA human leukocyte antigen
  • the first aspect of the present invention provides an engineered T cell, characterized in that the engineered T cell expresses a functional protein that can regulate the expression of HLA-I molecules on the cell surface, wherein the engineered T cell has The cell surface HLA-A and HLA-B expression and HLA-E expression are differentially regulated, or the cell surface HLA-A, HLA-B and HLA-E expression of the engineered T cell is down-regulated.
  • the differential regulation is that the expression of HLA-A and HLA-B on the cell surface of the engineered T cell is down-regulated but the expression of HLA-E is not down-regulated.
  • the expression level of HLA class I molecules on the cell surface of the engineered T cells is 50% or less of that of T cells that do not express the functional protein, preferably 10-50%.
  • the expression level of HLA-A and/or HLA-B molecules on the surface of the engineered T cell is 5-50% of that of the T cell that does not express the functional protein, 10-50 %, 10-45%, 10-40%, 15-50%, 20-50%, or 20-45%.
  • the expression level of HLA-E molecules on the surface of the engineered T cells is 65-100%, 70-100%, 75-100% of T cells that do not express the functional protein. , 80-100%, 85-100%, 90-100%, 95-100%.
  • the engineered T cell further expresses a chimeric antigen receptor, so that the engineered T cell is a CAR-T cell, wherein the CAR-T cell contains the chimeric antigen
  • the coding sequence of the receptor and the coding sequence of the functional protein are included in the engineered T cell.
  • the CAR-T cell contains the expression cassette of the chimeric antigen receptor and the expression cassette of the functional protein, or the coding sequence of the chimeric antigen receptor and the function
  • the coding sequence of the protein is in the same expression frame.
  • the chimeric antigen receptor specifically binds to one or more of the following tumor antigens: EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O -Glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT -2, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, folate receptor ⁇ , ERBB, Her2/neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl Base-GD2, folate receptor ⁇ , TEM1/CD248, TEM7R, FAP, legume protein, HPV E6 or E7, ML-
  • the functional protein capable of regulating the expression of HLA-I molecules on the cell surface is selected from: HSV, BHV-1, EHV-1/4, PRV, HSV-1/2, VZV, EBV , HCMV, mCMV, RhCMV, HHV-6/7, KSHV, MHV-68, vaccinia virus and adenovirus can directly target the functional proteins of HLA-I, and can down-regulate the expression of HLA-I molecules through TAP protein Functional proteins and functional proteins that can down-regulate the expression of HLA-I molecules via lysosomes.
  • the functional protein is selected from: proteins US11 and US6 from HCMV, protein UL49.5 from BHV-1, protein UL49.5 from EHV-1 and protein k5 from KSHV.
  • the functional protein is selected from the protein US11 from HCMV and the protein k5 from KSHV, and the cell surface HLA-A and HLA-B expression and HLA-E expression of the engineered T cells Differentially regulated.
  • the cell surface HLA-A and HLA-B expression of the engineered T cell is down-regulated but the HLA-E expression is not down-regulated.
  • the functional protein is selected from the group consisting of protein US6 from HCMV, protein UL49.5 from BHV-1 and protein UL49.5 from EHV-1, and HLA of the engineered T cell
  • the genotype of -E is 01:03/01:03, and the cell surface HLA-A and HLA-B expression and HLA-E expression of the engineered T cells are differentially regulated.
  • the cell surface HLA-A and HLA-B expression of the engineered T cell is down-regulated but the HLA-E expression is not down-regulated.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the functional protein is selected from the group consisting of protein US6 from HCMV, protein UL49.5 from BHV-1 and protein UL49.5 from EHV-1, and HLA of the engineered T cell
  • the genotype of -E is 01:01/01:01, and the cell surface expression of HLA-A, HLA-B and HLA-E of the engineered T cell is down-regulated.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the first aspect of the present invention also provides a nucleic acid molecule, characterized in that the nucleic acid molecule is selected from: (1) a coding sequence containing a chimeric antigen receptor and a functional protein capable of regulating the expression of HLA-I molecules on the cell surface A nucleic acid molecule encoding a sequence; and (2) the complementary sequence of the nucleic acid molecule described in (1).
  • the chimeric antigen receptor and the functional protein are as described in any one of the embodiments of the first aspect of the present invention.
  • the first aspect of the present invention also provides a nucleic acid construct, characterized in that the nucleic acid construct contains the nucleic acid molecule according to any one of the embodiments of the first aspect of the present invention.
  • the nucleic acid construct contains the expression cassette of the chimeric antigen receptor and the expression cassette of the functional protein; or the nucleic acid construct is an expression cassette, wherein the chimeric antigen receptor
  • the coding sequence of the antigen receptor and the coding sequence of the functional protein are in the expression frame.
  • the nucleic acid construct is a cloning vector or expression vector.
  • the first aspect of the present invention also provides a lentivirus, which contains the nucleic acid construct according to any one of the embodiments of the first aspect of the present invention.
  • the first aspect of the present invention also provides a host cell, which contains the nucleic acid molecule according to any one of the embodiments of the first aspect of the present invention or the nucleic acid construct of any one of the first aspects of the present invention or is entitled The lentivirus of one embodiment.
  • the first aspect of the present invention also provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains the engineered T cell according to any one of the embodiments of the first aspect of the present invention.
  • the first aspect of the present invention also provides the use of the functional protein or its coding sequence according to any one of the embodiments of the first aspect of the present invention in the preparation of engineered T cells that regulate the expression of HLA-I molecules on the cell surface, or Application in the preparation of engineered T cells for cancer treatment, wherein the cell surface HLA-A and HLA-B expression and HLA-E expression of the engineered T cells are differentially regulated, or the engineered The expression of HLA-A, HLA-B and HLA-E on the cell surface of T cells is down-regulated.
  • the differential regulation is that the expression of HLA-A and HLA-B on the cell surface of the engineered T cell is down-regulated but the expression of HLA-E is not down-regulated.
  • the expression level of HLA-A and/or HLA-B molecules on the surface of the engineered T cell is 5-50% of that of the T cell that does not express the functional protein, 10-50 %, 10-45%, 10-40%, 15-50%, 20-50%, or 20-45%.
  • the expression level of HLA-E molecules on the surface of the engineered T cells is 65-100%, 70-100%, 75-100% of T cells that do not express the functional protein. , 80-100%, 85-100%, 90-100%, 95-100%.
  • the functional protein is selected from the protein US11 from HCMV and the protein k5 from KSHV, and the cell surface HLA-A and HLA-B expression and HLA-E expression of the engineered T cells Differentially regulated.
  • the cell surface HLA-A and HLA-B expression of the engineered T cell is down-regulated but the HLA-E expression is not down-regulated.
  • the functional protein is selected from the group consisting of protein US6 from HCMV, protein UL49.5 from BHV-1 and protein UL49.5 from EHV-1, and HLA of the engineered T cell
  • the genotype of -E is 01:03/01:03, and the cell surface HLA-A and HLA-B expression and HLA-E expression of the engineered T cells are differentially regulated.
  • the cell surface HLA-A and HLA-B expression of the engineered T cell is down-regulated but the HLA-E expression is not down-regulated.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the functional protein is selected from the group consisting of protein US6 from HCMV, protein UL49.5 from BHV-1 and protein UL49.5 from EHV-1, and HLA of the engineered T cell
  • the genotype of -E is 01:01/01:01, and the cell surface expression of HLA-A, HLA-B and HLA-E of the engineered T cell is down-regulated.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the present invention also provides a method for inhibiting the expression of HLA-I molecules on the surface of T cells.
  • the method includes the step of expressing the functional protein according to any one of the embodiments herein in the T cell, wherein the T cell expressing the functional protein
  • the cell surface HLA-A and HLA-B expression and HLA-E expression are differentially regulated, or the cell surface HLA-A, HLA-B and HLA-E expressions of T cells expressing functional proteins are down-regulated.
  • the differential regulation is that the expression of HLA-A and HLA-B on the cell surface of the engineered T cell is down-regulated but the expression of HLA-E is not down-regulated.
  • the expression level of HLA-A and/or HLA-B molecules on the surface of the engineered T cell is 5-50% of that of the T cell that does not express the functional protein, 10-50 %, 10-45%, 10-40%, 15-50%, 20-50%, or 20-45%.
  • the expression level of HLA-E molecules on the surface of the engineered T cells is 65-100%, 70-100%, 75-100% of T cells that do not express the functional protein. , 80-100%, 85-100%, 90-100%, 95-100%.
  • the second aspect of the present invention provides a CAR-T cell, which contains a chimeric antigen receptor that specifically targets a tumor antigen and a functional protein capable of regulating the expression of HLA-I molecules on the cell surface.
  • the expression level of HLA class I molecules on the CAR-T cell surface is less than 50% of that of control CAR-T cells that express the same chimeric antigen receptor but do not express the functional protein.
  • the CAR-T cell contains the coding sequence of the chimeric antigen receptor and the coding sequence of the functional protein; preferably, the CAR-T cell contains the chimeric antigen
  • the expression cassette of the receptor and the expression cassette of the functional protein, or the coding sequence of the chimeric antigen receptor and the coding sequence of the functional protein are in the same expression cassette.
  • the chimeric antigen receptor specifically binds to one or more of the following tumor antigens: EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O -Glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT -2, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, folate receptor ⁇ , ERBB, Her2/neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl Base-GD2, folate receptor ⁇ , TEM1/CD248, TEM7R, FAP, legume protein, HPV E6 or E7, ML-
  • the functional protein capable of regulating the expression of HLA-I molecules on the cell surface is selected from: HSV, BHV-1, EHV-1/4, PRV, HSV-1/2, VZV, EBV , HCMV, mCMV, RhCMV, HHV-6/7, KSHV, MHV-68, vaccinia virus and adenovirus can directly target the functional proteins of HLA-I, and can down-regulate the expression of HLA-I molecules through TAP protein Functional proteins and functional proteins that can down-regulate the expression of HLA-I molecules via lysosomes.
  • the functional protein is selected from: proteins US11 and US6 from HCMV, protein UL49.5 from BHV-1, protein UL49.5 from EHV-1 and protein k5 from KSHV.
  • regulating the expression of HLA class I molecules on the cell surface includes: down-regulating the expression of HLA-A, HLA-B and HLA-E on the cell surface, or the expression of HLA-A and HLA-B on the cell surface and HLA- E expression is differentially regulated.
  • the differential regulation is that the expression of HLA-A and HLA-B on the cell surface of CAR-T cells is down-regulated but the expression of HLA-E is not down-regulated.
  • the cell surface HLA-A and HLA-B expression and HLA-E expression of CAR-T cells are differentially regulated, and the functional protein is selected from: the protein US11 from HCMV and the protein from KSHV ⁇ protein k5.
  • the expression of HLA-A and HLA-B on the cell surface of the CAR-T cell is down-regulated but the expression of HLA-E is not down-regulated.
  • the functional protein is selected from the group consisting of protein US6 from HCMV, protein UL49.5 from BHV-1 and protein UL49.5 from EHV-1, HLA-E from CAR-T cells
  • the genotype is 01:03/01:03
  • the cell surface HLA-A and HLA-B expression and HLA-E expression of the CAR-T cell are differentially regulated.
  • the cell surface HLA-A and HLA-B expression of the engineered T cell is down-regulated but the HLA-E expression is not down-regulated.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the functional protein is selected from the group consisting of protein US6 from HCMV, protein UL49.5 from BHV-1 and protein UL49.5 from EHV-1, and HLA of the engineered T cell
  • the genotype of -E is 01:01/01:01, and the cell surface expression of HLA-A, HLA-B and HLA-E of the engineered T cell is down-regulated.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the expression level of HLA-A and/or HLA-B molecules on the cell surface of CAR-T cells is 5-50%, 10-50% of T cells that do not express the functional protein , 10-45%, 10-40%, 15-50%, 20-50%, or 20-45%.
  • the expression level of HLA-E molecules on the cell surface of CAR-T cells is 65-100%, 70-100%, 75-100% of T cells that do not express the functional protein, 80-100%, 85-100%, 90-100%, 95-100%.
  • the present invention provides a CAR-T cell containing a chimeric antigen receptor that specifically targets tumor antigens and a functional protein capable of differentially regulating the expression of HLA-A and HLA-B and HLA-E on the cell surface, preferably
  • the functional protein is selected from: the protein US11 from HCMV and the protein k5 from KSHV.
  • the expression of HLA-A and HLA-B on the cell surface of the CAR-T cell is down-regulated but the expression of HLA-E is not down-regulated.
  • the present invention also provides a nucleic acid molecule selected from:
  • the chimeric antigen receptor and the functional protein are as described in any one of the embodiments of the second aspect herein.
  • the present invention also provides a nucleic acid construct, which contains the nucleic acid molecule described in the second aspect herein.
  • the nucleic acid construct contains the expression cassette of the chimeric antigen receptor and the expression cassette of the functional protein; or the nucleic acid construct is an expression cassette, wherein the chimeric antigen receptor
  • the coding sequence of the antigen receptor and the coding sequence of the functional protein are in the expression frame.
  • the nucleic acid construct is a cloning vector or expression vector.
  • the present invention also provides a lentivirus, which contains the nucleic acid construct described in the second aspect herein.
  • the present invention also provides a host cell, which contains the nucleic acid molecule, nucleic acid construct or lentivirus described in the second aspect herein.
  • the present invention also provides a pharmaceutical composition containing the CAR-T cell according to any one of the embodiments of the second aspect herein.
  • the present invention also provides the use of the functional protein or its coding sequence according to any one of the embodiments of the second aspect herein in the preparation of CAR-T cells whose cell surface expression of HLA-I molecules is regulated, or in the preparation of cancer therapy Used in CAR-T cells.
  • regulating the expression of HLA class I molecules on the cell surface includes: down-regulating the expression of HLA-A, HLA-B and HLA-E on the cell surface, or the expression of HLA-A and HLA-B on the cell surface and HLA- E expression is differentially regulated.
  • the differential regulation is that the expression of HLA-A and HLA-B on the cell surface of CAR-T cells is down-regulated but the expression of HLA-E is not down-regulated.
  • the functional protein is selected from the protein US11 from HCMV and protein k5 from KSHV, and the cell surface HLA-A and HLA-B expression and HLA-E expression of the CAR-T cell are controlled by Differentiated regulation.
  • the expression of HLA-A and HLA-B on the cell surface of CAR-T cells is down-regulated but the expression of HLA-E is not down-regulated.
  • the functional protein is selected from the protein US6 from HCMV, the protein UL49.5 from BHV-1 and the protein UL49.5 from EHV-1, the HLA- The genotype of E is 01:03/01:03, and the cell surface HLA-A and HLA-B expression and HLA-E expression of the CAR-T cell are differentially regulated.
  • the expression of HLA-A and HLA-B on the cell surface of CAR-T cells is down-regulated but the expression of HLA-E is not down-regulated.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the functional protein is selected from the protein US6 from HCMV, the protein UL49.5 from BHV-1 and the protein UL49.5 from EHV-1, the HLA- The genotype of E is 01:01/01:01, and the expression of HLA-A, HLA-B and HLA-E on the cell surface of the CAR-T cell is down-regulated.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the expression level of HLA-A and/or HLA-B molecules on the surface of the engineered T cell is 5-50% of that of the T cell that does not express the functional protein, 10-50 %, 10-45%, 10-40%, 15-50%, 20-50%, or 20-45%.
  • the expression level of HLA-E molecules on the surface of the engineered T cells is 65-100%, 70-100%, 75-100% of T cells that do not express the functional protein. , 80-100%, 85-100%, 90-100%, 95-100%.
  • the present invention also provides the application of the functional protein or its coding sequence in the preparation of CAR-T cells whose cell surface HLA-A and HLA-B expression and HLA-E expression are differentially regulated, or in the preparation of CAR-T cells for cancer treatment -Application in T cells, the functional protein is selected from: the protein US11 from HCMV and the protein k5 from KSHV.
  • the differential regulation is that the expression of HLA-A and HLA-B on the cell surface of CAR-T cells is down-regulated but the expression of HLA-E is not down-regulated.
  • the present invention also provides the application of the functional protein or its coding sequence in the preparation of CAR-T cells whose cell surface HLA-A and HLA-B expression and HLA-E expression are differentially regulated, or in the preparation of CAR-T cells for cancer treatment -T cell application, the HLA-E genotype of the CAR-T cell is 01:03/01:03, and the functional protein is selected from: the protein US6 from HCMV, the protein UL49 from BHV-1. 5 and the protein UL49.5 from EHV-1.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the differential regulation is that the expression of HLA-A and HLA-B on the cell surface of CAR-T cells is down-regulated but the expression of HLA-E is not down-regulated.
  • the present invention also provides the application of the functional protein or its coding sequence in the preparation of CAR-T cells with down-regulated expression of HLA-A, HLA-B and HLA-E on the cell surface, or in the preparation of CAR-T cells for cancer treatment
  • the HLA-E genotype of the CAR-T cell is 01:01/01:01
  • the functional protein is selected from the group consisting of: protein US6 from HCMV, protein UL49.5 from BHV-1, and protein from EHV -1 protein UL49.5.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the present invention also provides a method for inhibiting the expression of HLA-I molecules on the surface of CAR-T cells, the method comprising the step of simultaneously expressing the CAR and functional protein described in any of the embodiments herein in T cells.
  • the cell surface HLA-A and HLA-B expression and HLA-E expression of CAR-T cells are differentially regulated, and the functional protein is selected from: the protein US11 from HCMV and the protein from KSHV Protein k5.
  • the expression of HLA-A and HLA-B on the cell surface of CAR-T cells is down-regulated but the expression of HLA-E is not down-regulated.
  • the genotype of HLA-E of the CAR-T cell is 01:03/01:03
  • the cell surface HLA-A and HLA-B expression and HLA-E expression of the T cell Being differentially regulated, the functional protein is selected from the group consisting of: protein US6 from HCMV, protein UL49.5 from BHV-1 and protein UL49.5 from EHV-1.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the expression of HLA-A and HLA-B on the cell surface of CAR-T cells is down-regulated but the expression of HLA-E is not down-regulated.
  • the genotype of HLA-E of the CAR-T cell is 01:01/01:01, and the cell surface expression of HLA-A, HLA-B and HLA-E of the T cell is down-regulated
  • the functional protein is selected from: protein US6 from HCMV, protein UL49.5 from BHV-1 and protein UL49.5 from EHV-1.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the present invention also provides a method for differentially regulating the expression of HLA-A and HLA-B and HLA-E on the surface of CAR-T cells, the method comprising simultaneously expressing the CAR described in any of the embodiments herein in T cells And the step of a functional protein selected from: HCMV-derived protein US11 and KSHV-derived protein k5.
  • the differential regulation is that the expression of HLA-A and HLA-B on the cell surface of CAR-T cells is down-regulated but the expression of HLA-E is not down-regulated.
  • the present invention also provides a method for differentially regulating the expression of HLA-A and HLA-B and HLA-E on the surface of CAR-T cells, the method comprising simultaneously expressing the CAR described in any of the embodiments herein in T cells And a step of a functional protein selected from the group consisting of: protein US6 from HCMV, protein UL49.5 from BHV-1, and protein UL49.5 from EHV-1, the HLA-E genotype of the T cell is 01: 03/01:03.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the differential regulation is that the expression of HLA-A and HLA-B on the cell surface of CAR-T cells is down-regulated but the expression of HLA-E is not down-regulated.
  • the present invention also provides a method for inhibiting the expression of HLA-A, HLA-B and HLA-E on the surface of CAR-T cells, the method comprising simultaneously expressing in T cells the CAR described in any of the embodiments herein and selected from the following The steps of functional protein: protein US6 from HCMV, protein UL49.5 from BHV-1 and protein UL49.5 from EHV-1, the HLA-E genotype of the T cell is 01:01/01: 01.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • FIG. 1 Each group of lentiviral vectors transfected activated T cells. T cells were cultured in vitro to the 8th day, and the average fluorescence intensity of HLA-I molecules on the surface of CAR19 + T cells was measured by flow cytometry.
  • Figure 2 On the 9th day of in vitro culture of T cells in each group, the target cells K562-CD19 were used to stimulate the T cells in each group with an effective target ratio of 10:1. The stimulation was repeated for 2 days. Flow cytometry was used to detect the HLA- CAR19+ cell population of T cells in each group. The average fluorescence intensity of class I molecules, of which five carrier molecules PCTL200, PCTL201, PCTL205, PCTL206, and PCTL213 have the ability to prepare CAR-T cells with down-regulated expression of HLA-I molecules.
  • Figure 3 Flow cytometric analysis of the proportion of whole blood lymphocytes. 100ml peripheral blood Ficoll density gradient centrifugation separated 1.24 ⁇ 10 8 PBMC, flow cytometric detection CD3+T accounted for 62.4% of the albuginea cells, the ratio of CD4+T/CD8+T was 1.2, and the 24h activation efficiency was 68.9%.
  • Figure 4 The multiplication factor of each group of cells expanded in vitro for 8 days. The cells of each group were expanded in vitro for 8 days, and the expansion multiple was significantly higher than that of PCTL135 group.
  • Figure 5 Average fluorescence intensity of HLA-I molecules of T cells in each group. Through flow cytometry, the average fluorescence intensity of CAR-positive cell populations and HLA-I molecules in each group was significantly down-regulated, and the PCTL206 group was down-regulated the most.
  • FIG. 6 T cell killing efficiency of each group.
  • the cells of each group were used in the in vitro cytotoxicity experiment, and the killing efficiency reached more than 90% when the effective target ratio was 5:1, which was not significantly different from the control group.
  • Figure 7 Differentiation phenotype analysis of T cells in each group. The cell differentiation phenotype of each group of cells was detected, and more than 65% of T cells showed Group, there is no significant difference from the control group 135.
  • Figure 8 The average fluorescence intensity of HLA-I molecules in each group of T cells stimulated by target cell K562-CD19. After culturing to the 9th day in vitro, the target cells K526-CD19 were used to stimulate T cells in each group. After stimulation, the HLA-I molecules in the CAR-positive cell population in each group were detected by flow cytometry, and it was found that the average fluorescence intensity was significantly lower than that of the control group. .
  • Figure 9 Flow cytometric detection of the average fluorescence intensity of various HLA-I molecules in the CAR19+ T cell population with the HLA-E genotype heterozygous 01:01/01:03.
  • Figure 10 Flow cytometric detection of the average fluorescence intensity of various HLA-I molecules in CAR19+ T cell populations homozygous for the HLA-E genotype of 01:01/01:01.
  • Figure 11 Flow cytometric detection of the average fluorescence intensity of various HLA class I molecules in CAR19+ T cell populations homozygous for the HLA-E genotype of 01:03/01:03.
  • the basic principle of gene editing to prepare allogeneic T cells is to generate a site-specific double strand break (DSB) at a specific location in the genome (such as TCR gene, HLA-I molecular chaperone protein b2m gene), and then Repair by means of non-homologous end joining (NHEJ) or homologous directed recombination repair (HDR) to achieve complete deletion of targeted genes (such as TCR and b2m genes), Try to fundamentally avoid the rejection of allogeneic CAR-T cells mediated by GvHD and host immune cells.
  • DSB site-specific double strand break
  • NHEJ non-homologous end joining
  • HDR homologous directed recombination repair
  • Cellectis and Allogene Therapeutics use TALEN gene editing technology to knock out the TCR and CD52 genes of donor T cells (that is, without changing the expression of HLA-I molecules on the surface of CAR-T cells), and then combine with anti-CD52 monoclonal antibody drugs to use their cleansing Activity in order to achieve simultaneous suppression of the rejection of patient T cells and NK cells to allogeneic CAR-T cells.
  • Cellectis and Allogene Therapeutics on the premise of not changing the expression of HLA-I molecules on the surface of CAR-T cells, by deleting the CD52 gene of CAR-T cells and then using anti-CD52 antibodies to simultaneously relieve the immune system of patients’ T cells and NK cells from CAR. -T cell attack, this is an attempt to get out of the predicament and further proves this logical conclusion.
  • the immune system T cells and NK cells is at risk of attacking allogeneic T cells.
  • HLA-I molecules are composed of classic HLA-I molecules (HLA-A, HLA-B, etc.) and non-classical HLA-I molecules (HLA-E, etc.). HLA-E molecules can inhibit the biological activity of NK cells by binding to CD94/NKG2 receptors on the surface of NK cells.
  • the HLA-E gene in humans contains two alleles 01:01 and 01:03.
  • the functional protein when the functional protein is selected from the protein US11 from HCMV and the protein k5 from KSHV, the functional protein differentially regulates the expression of HLA-A and HLA-B and the expression of HLA-E. Therefore, retaining the expression of HLA-E molecules on the cell surface in the T cells of the present invention (especially CAR-T cells) can effectively avoid being cleared by the host NK cells, thereby improving the survival and survival of T cells (especially CAR-T cells). active.
  • the functional protein is selected from the protein US6 from HCMV, the protein UL49.5 from BHV-1 and the protein UL49.5 from EHV-1, and the T cell (especially CAR-T cell)
  • the functional protein differentially regulates the expression of HLA-A and HLA-B and HLA-E on the cell surface of T cells (especially CAR-T cells);
  • the protein is selected from the protein US6 from HCMV, the protein UL49.5 from BHV-1 and the protein UL49.5 from EHV-1, and the HLA-E genotype of T cells (especially CAR-T cells) is 01: At 01/01:01, the functional protein down-regulates the expression of HLA-A, HLA-B and HLA-E on the cell surface of T cells (especially CAR-T cells).
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the expression level of HLA-A and HLA-B molecules on the surface of allogeneic T cells is 5-50% of that of T cells that do not express the functional protein, 10- 50%, 10-45%, 10-40%, 15-50%, or 20-50%, or 20-45%; the expression level of HLA-E molecules is 65- of T cells that do not express the functional protein 100%, 70-100%, 75-100%, 80-100%, 85-100%, 90-100%, 95-100%.
  • allogeneic T cells express functional proteins that can differentially regulate the expression levels of HLA-A, HLA-B and HLA-E molecules, including but not limited to:
  • UL49.5 comes from BHV-1 (SEQ ID NO: 8) or EHV-1/4 (SEQ ID NO: 9), which can induce conformational arrest of TAP , Or induce the conformation of TAP to prevent and degrade or block the combination of ATP and TAP, thereby inhibiting the expression of MHC-I molecules on the cell surface.
  • the results have been verified on bovine kidney cells and horse epidermal cells (Koppers-Lalic et al. al.2008);
  • US6/gp21 (SEQ ID NO: 10), or a variant of US6/gp21, and SEQ ID NO: 10 are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology
  • US11/gp33 (SEQ ID NO: 11), or a variant of US11/gp33, and SEQ ID NO : 11 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% Homology
  • US6/gp21 and US11/gp33 are derived from hCMV, which can induce TAP conformational changes to prevent ATP binding, and target MHC-I to cause endoplasmic reticulum-mediated protein degradation, thereby inhibiting cell surface MHC-I
  • the expression of class molecules was verified on Chinese hamster
  • kK5/MIR2 (SEQ ID NO: 12), or a variant of kK5/MIR2, is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% of SEQ ID NO: 12 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology; kK5/MIR2 comes from KSHV, which makes MHC-I ubiquitinated to be internalized by lysosome Degradation, thereby inhibiting the expression of MHC-I molecules on the cell surface. This result has been verified on human cervical cancer cells (Coscoy et al. 2005).
  • HLA-I down-regulated protein functions are in specific cell types of specific species, or partially inhibited the expression of HLA-I molecules in naturally susceptible cells, in target cell types (such as humans) of target species (such as T Whether or not the expression of HLA-I molecules is down-regulated in cells) is unknown, especially when T cells express chimeric antigen receptors (CAR) at the same time.
  • CAR chimeric antigen receptors
  • mK3 of MHV-68, US3/gp23 of hCMV, CPXV012 of vaccinia virus, rh178/VIHCE of RhCMV, and E3-19K of adenovirus failed to show down-regulation of HLA- The ability of class I molecules.
  • the present inventors found that multiple viruses across species exhibited unexpected effects of down-regulating HLA-I molecules in human T cells, such as: EHV-1 (equine rhino pneumonia) Virus) UL49.5 and so on.
  • the present invention regulates cell surface HLA-I molecules by expressing related functional proteins, and prepares engineered T cells that can inhibit the expression of HLA-I molecules on the cell surface.
  • An example of the engineered T cells is allogeneic CAR -T cells.
  • the expression of HLA-I molecules is not completely inhibited.
  • These T cells still express a certain amount of HLA-I molecules on the cell surface, so they are well avoided.
  • the attack of the receptor's NK cells also solved the receptor's rejection.
  • the expression level of HLA-I molecules in the engineered T cells of the present invention is less than 80%, preferably less than 60% of the control engineered T cells that do not express the functional protein, It is more preferably 50% or less, more preferably 30% or less, and still more preferably 25% or less.
  • the expression level of HLA class I molecules in the engineered T cells of the present invention is 10-50% of that of the control engineered T cells that do not express the functional protein, such as 15-50%.
  • the expression level of HLA-I molecules in the engineered T cells of the present invention is still 10-50% of that of the control engineered T cells that do not express the functional protein. , Such as 15-50%.
  • the engineered T cells can be CAR-T cells. Therefore, the present invention provides a type of CAR-T cell, which contains a nucleic acid molecule encoding a chimeric antigen receptor (CAR) targeting a tumor antigen of interest and Nucleic acid molecules that can regulate the expression of functional proteins of HLA-I molecules on the cell surface.
  • suitable T cells can be various T cells well known in the art, especially various T cells conventionally used in cellular immunotherapy, including but not limited to peripheral blood T lymphocytes, cytotoxic killer T cells, and helper T cells. Cells, suppressor/regulatory T cells, ⁇ T cells, cytokine-induced killer cells, tumor infiltrating lymphocytes, etc., and any one or more mixtures of the above cells.
  • CAR-T cells refer to T cells that at least express chimeric antigen receptors.
  • the chimeric antigen receptor has a well-known meaning in the art. It is an artificially modified receptor that can anchor specific molecules (such as antibodies) that recognize tumor cell surface antigens on immune cells (such as T cells). , So that immune cells recognize tumor antigens and kill tumor cells.
  • Chimeric antigen receptors suitable for use herein may be various CARs known in the art.
  • CARs include polypeptides that bind tumor antigens, hinge regions, transmembrane regions, and intracellular signaling regions in sequence.
  • the polypeptide that binds to the tumor antigen may be a natural polypeptide or an artificially synthesized polypeptide; preferably, the artificially synthesized polypeptide is a single-chain antibody or a Fab fragment.
  • tumor antigens of interest include, but are not limited to, solid tumor antigens, myeloid tumor antigens, and antigens of hematological tumors of non-B cell lineage.
  • Suitable solid tumor antigens include but are not limited to EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT , IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, folate receptor ⁇ , ERBB( For example, ERBB2), Her2/neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acet
  • Suitable B cell antigens include, but are not limited to, CD5, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD37, CD38, CD40, CD53, CD69, CD72, CD73, CD74, CD75 , CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269, Flt3, ROR1, BCMA, FcRn5, FcRn2, CS-1, CXCR4, CXCR5, CXCR7 , IL-7/3R, IL7/4/3R and IL4R.
  • the tumor antigen-binding polypeptide of the present invention is a single-chain antibody that specifically binds to any of the above-mentioned tumor antigens.
  • a single-chain antibody refers to an antibody fragment that is formed by connecting the amino acid sequence of an antibody light chain variable region (VL region) and a heavy chain variable region (VH region) via a hinge.
  • the single chain antibody of interest can be derived from the antibody of interest.
  • the antibody of interest can be a human antibody, including a human-mouse chimeric antibody and a humanized antibody.
  • the antibody may be secreted or membrane-anchored; preferably it is membrane-anchored.
  • an antibody that specifically binds to a certain antigen means that the antibody has a concentration of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, The affinity (KD) of 10 -8 M, 10 -9 M, or 10 -10 M or less binds the antigen.
  • a single chain antibody may contain the variable region of the heavy chain and the variable region of the light chain of the antibody of interest, or be composed of the variable region of the heavy chain and the variable region of the light chain and an optional linker.
  • the variable region of the heavy chain and the variable region of the light chain can be connected by a well-known linker.
  • a linker or hinge is a polypeptide fragment that connects different proteins or polypeptides, and its purpose is to keep the connected proteins or polypeptides in their respective spatial conformations to maintain the function or activity of the protein or polypeptide.
  • Exemplary linkers include linkers containing G and/or S, and Furin 2A peptide (F2A).
  • the length of the linker can be 3-25 amino acid residues, such as 3-15, 5-15, 10-20 amino acid residues.
  • the linker sequence is a polyglycine linker sequence.
  • the number of glycines in the linker sequence is not particularly limited, and is usually 2-20, such as 2-15, 2-10, 2-8.
  • the linker can also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine Acid (F), Arginine (R), Glutamine (Q), etc.
  • the linker is usually 15-20 amino acids in length.
  • the linker is (GGGS)n, where n is an integer of 1-5.
  • the tumor antigen of interest is CD19
  • the single chain antibody of interest is a single chain antibody that specifically binds to CD19.
  • the amino acid sequence of an exemplary single-chain antibody that specifically binds to CD19 is shown in amino acid residues 23-267 of SEQ ID NO: 2, wherein the heavy chain variable region and the light chain variable region pass through the G and S Linker sequence connection.
  • CAR other parts contained in the CAR, such as the hinge region, the transmembrane region, and the intracellular signal region, can be the hinge region, transmembrane region, and intracellular signal region that are conventionally used to construct various CARs.
  • the hinge region refers to the region between the CH1 and CH2 functional regions of the immunoglobulin heavy chain. This region is rich in proline and does not form an alpha helix. It is prone to stretching and a certain degree of distortion, which is beneficial to the antigen binding site of the antibody and Complementary binding between epitopes.
  • Hinge regions suitable for use herein can be selected from CD8 extracellular hinge region, IgG1 Fc CH2CH3 hinge region, IgD hinge region, CD28 extracellular hinge region, IgG4 Fc CH2CH3 hinge region and CD4 extracellular hinge region. In certain embodiments, the CD8 ⁇ hinge region is used herein.
  • the transmembrane region may be selected from one or more of CD28 transmembrane region, CD8 transmembrane region, CD3 ⁇ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region and DAP10 transmembrane region .
  • the transmembrane region of the chimeric antigen receptor used herein is the CD8 transmembrane region.
  • Exemplary amino acid sequences of the hinge region and the transmembrane region may be as shown in the amino acid residues 268-336 of SEQ ID NO: 2.
  • the intracellular signal region can be selected from any one or more of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, DAP10, CD3 ⁇ and Fc310/, preferably 4- 1BB intracellular signal area and CD3 ⁇ intracellular signal area.
  • the amino acid sequence of the exemplary intracellular signal region herein may be shown in the amino acid residues 337-490 of SEQ ID NO: 2.
  • the chimeric antigen receptor may also include a signal peptide.
  • Signal peptide is a short peptide chain (5-30 amino acids in length) that guides the transfer of newly synthesized proteins to the secretory pathway. It often refers to the N-terminal amino acid sequence in the newly synthesized polypeptide chain that is used to guide the transmembrane transfer (positioning) of proteins.
  • the signal peptide may be a membrane protein signal peptide, such as a CD8 signal peptide, a CD28 signal peptide, and a CD4 signal peptide.
  • An exemplary signal peptide amino acid sequence may be shown in the amino acid residues 1-22 of SEQ ID NO: 2.
  • amino acid sequence of the chimeric antigen receptor of the present invention is usually optional signal peptide, single-chain antibody targeting heavy chain antigen of interest, hinge region, transmembrane region and intracellular signal Area.
  • the amino acid sequence of an exemplary chimeric antigen receptor may be shown in the amino acid residues 23-490 of SEQ ID NO: 2, or shown in the amino acid residues 1-490 of SEQ ID NO: 2.
  • the above-mentioned parts forming the chimeric antigen receptor herein can be directly connected to each other.
  • the functional protein capable of regulating the expression of HLA-I molecules on the cell surface may be a viral protein, preferably a protein derived from a virus whose natural host cell is not a T cell.
  • HIV-1 is the natural host of T cells
  • HIV-1 nef virus protein can down-regulate the expression of HLA-I molecules on the surface of T cells
  • the introduction of HIV-1 nef protein into the lentivirus to prepare CAR-T will cause RCL (replication competent lentivirus)
  • the present invention preferably uses materials such as HSV, BHV-1, EHV-1/4, PRV, HSV-1/2, VZV, EBV, hCMV, mCMV, RhCMV, HHV-6/7
  • the viral proteins of KSHV, MHV-68, vaccinia virus and adenovirus including but not limited to: UL41/vhs protein from HSV, UL49.5 from BHV-1, EHV-1/4 or PRV, and HSV-
  • the functional protein that regulates the cell surface HLA-I molecules is selected from the protein US11 from HCMV and the protein k5 from KSHV, so that the expression of HLA-A and HLA-B on the CAR-T cell surface is the same as that of HLA- E expression is differentially regulated.
  • the functional protein that regulates HLA class I molecules on the cell surface is selected from the group consisting of: protein US6 from HCMV, protein UL49.5 from BHV-1, and protein UL49.5 from EHV-1.
  • the expression of HLA-A and HLA-B and the expression of HLA-E on the cell surface are differentially regulated.
  • the expression of HLA-A, HLA-B and HLA-E on the cell surface was all down-regulated.
  • the functional protein is preferably the protein UL49.5 derived from EHV-1.
  • the preferred functional protein is a functional protein that can directly target and degrade MHC I, or can down-regulate the expression of HLA-I molecules through TAP protein (such as inhibiting TAP, including preventing TAP protein from binding ATP and/or inducing degradation of TAP protein)
  • Functional protein or a functional protein that can down-regulate the expression of HLA class I molecules via lysosomes.
  • Exemplary preferred proteins include, but are not limited to, viral proteins from HCMV, such as US11 and US6, viral proteins from BHV-1, such as UL49.5, viral proteins from EHV-1, such as UL49.5, and those from KSHV Viral protein, such as k5.
  • the present invention uses UL49.5 derived from EHV-1, and its amino acid sequence can be as shown in the amino acid residues 516-615 of SEQ ID NO: 2.
  • the viral protein can be connected to the CAR of the present invention through a linker commonly used in the art.
  • the linker is a conventional F2A sequence.
  • the amino acid sequence of an exemplary F2A may be shown in amino acid residues 494-515 of SEQ ID NO: 2.
  • F2A can also be connected to CAR through conventional G and S-containing linkers.
  • the nucleic acid molecule of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the nucleic acid molecule of the present invention can be a CAR coding sequence and a functional protein coding sequence capable of regulating the expression of HLA-I molecules on the cell surface, or a CAR expression cassette and an expression cassette of the functional protein.
  • the coding sequence refers to the part of the nucleic acid sequence that directly determines the amino acid sequence of its protein product (for example, CAR, single-chain antibody, hinge region, transmembrane region, intracellular signal region, viral protein or its fusion protein, etc.).
  • Coding sequences can include, but are not limited to DNA, cDNA, and recombinant nucleic acid sequences.
  • the expression cassette refers to the complete elements required to express the gene of interest, including promoter, gene coding sequence and PolyA tailing signal sequence.
  • the nucleic acid molecule described herein may be two independent nucleic acid molecules, each containing a CAR coding sequence and a functional protein coding sequence, such as a CAR expression cassette and a functional protein expression cassette; or, the containing The coding sequence of CAR and the coding sequence of the functional protein can be linked into one nucleic acid molecule via a linker.
  • the coding sequence of CAR and the coding sequence of the functional protein are in the same expression frame, or the two expression frames are linked via a suitable linker.
  • the nucleic acid molecule of the present invention is a nucleic acid molecule in which the coding sequence of the CAR and the coding sequence of the functional protein are in the same expression cassette, which contains a promoter, a nucleic acid encoding the chimeric antigen receptor and the functional protein. Sequence and PolyA tailed signal.
  • the coding sequence or expression cassette is integrated into the genome of the CAR-T cell. Therefore, in these embodiments, the genome of the CAR-T cell described herein stably integrates an expression cassette containing the CAR and functional protein described herein.
  • the nucleic acid molecule is a nucleic acid construct, which contains the CAR and/or functional protein coding sequences described herein, and one or more regulatory sequences operably linked to these sequences.
  • the control sequence can be a suitable promoter sequence.
  • the promoter sequence is usually operably linked to the coding sequence of the protein to be expressed.
  • the promoter can be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutant, truncated and hybrid promoters, and can be derived from extracellular sequences that encode homology or heterologous to the host cell. Or the intracellular polypeptide gene is obtained.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3'end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used herein.
  • the nucleic acid construct is a vector.
  • the vector can be a cloning vector, an expression vector, or a homologous recombination vector.
  • the CAR and/or functional protein coding sequences herein can be cloned into many types of vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids.
  • the cloning vector can be used to provide the coding sequence of the CAR and the functional protein of the present invention, such as a nucleic acid molecule containing the coding sequence of the CAR and the coding sequence of the functional protein.
  • the expression vector can be provided to the cell in the form of a viral vector.
  • Viruses that can be used as vectors include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus.
  • Homologous recombination vectors are used to integrate the expression cassettes described herein into the host genome.
  • a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.
  • the retroviral vector when a retroviral vector is used, the retroviral vector usually contains an origin of replication, 3'LTR, 5'LTR, the coding sequence of the fusion protein described herein, and an optional selectable marker.
  • Suitable promoters include but are not limited to the immediate early cytomegalovirus (CMV) promoter sequence, elongation growth factor-1 extension (EF-1 factor), simian virus 40 (SV40) early promoter, mouse breast cancer virus (MMTV) , Human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as But not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter.
  • the selectable marker includes either or both of a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from the cell population infected by the viral vector.
  • Useful selectable marker genes include, for example, antibiotic resistance genes such as neo.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein.
  • the nucleic acid molecules described herein can usually be obtained by PCR amplification.
  • primers can be designed according to the nucleotide sequences disclosed in this article, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as templates to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the nucleic acid molecules described herein can also be synthesized directly.
  • nucleotide sequence of the exemplary nucleic acid molecule containing the coding sequence of CAR and functional protein herein can be shown in SEQ ID NO:1.
  • Conventional methods can be used to introduce the nucleic acid molecules (especially vectors) herein into host cells. These methods include microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, and calcium phosphate precipitation. Wait.
  • the host cell contains the nucleic acid molecules described herein.
  • Host cells include not only T cells that are ultimately used for disease treatment purposes, but also various cells used in the process of producing CAR-T cells, such as E. coli cells, for example, to provide the coding sequence of the protein of the present invention or as described herein The carrier described.
  • a CAR-T cell stably expressing the functional protein described herein.
  • this document also includes the nucleic acid molecules described herein.
  • the nucleic acid molecules described herein can be prepared by conventional methods in the art.
  • this document also includes a lentivirus, which includes the expression cassette described herein and can integrate the expression cassette described herein into the genome of the host cell.
  • the lentivirus described herein can be prepared using methods well known in the art. For example, firstly, a lentiviral vector containing the expression cassette described herein is prepared, and then the virus is packaged in a suitable host cell, and the desired lentivirus is isolated and purified.
  • the reagents used for lentivirus packaging are well known in the art.
  • the conventional lentiviral vector system includes pRsv-REV, pMDlg-pRRE, pMD2G and the target interference plasmid.
  • This document also includes a CAR-T cell culture, which contains the CAR-T cell described herein and a suitable medium.
  • the medium may be a medium conventionally used in the art for culturing CAR-T cells.
  • compositions which contains the CAR-T cell described herein and pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients refer to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active ingredient, including but not limited to: pH regulators, surfactants, Adjuvant, ionic strength enhancer. More specifically, suitable pharmaceutically acceptable excipients may be those commonly used in the art for the administration of CAR-T cells.
  • the pharmaceutical composition contains a therapeutically effective amount of CAR-T cells.
  • a therapeutically effective amount refers to a dose that can treat, prevent, alleviate, and/or alleviate a disease or condition in a subject.
  • the therapeutically effective dose can be determined according to factors such as the patient's age, gender, the disease and its severity, and other physical conditions of the patient.
  • the subject or patient generally refers to a mammal, especially a human.
  • diseases suitable for the treatment of nucleic acid molecules, CAR-T cells and pharmaceutical compositions described herein are related to the nucleic acid molecules and single-chain antibodies in the chimeric antigen receptor expressed by the CAR-T cells. Therefore, the diseases described herein include various cancers related to the aforementioned tumor antigens, including solid tumors and hematological tumors, such as adenocarcinoma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, and gastric cancer.
  • kits containing the vector described herein may also contain various reagents suitable for transfecting the vector into cells, and optionally instructions for instructing those skilled in the art to transfect the recombinant expression vector into cells.
  • the present invention also provides a disease treatment method, which comprises administering to a subject in need a therapeutically effective amount of the engineered T cell according to any embodiment herein, preferably the engineered CAR according to any embodiment herein -T cells.
  • the drug can be administered by a suitable method (such as intravenous infusion).
  • the disease is the disease described in any of the embodiments herein.
  • the genotype of HLA-E of the patient's T cells is 01:03/01:03, and the patient is given a protein selected from the group consisting of HCMV-derived US11, KSHV-derived protein k5, and HCMV-derived protein.
  • the protein UL49.5 from BHV-1 and the protein UL49.5 from EHV-1 and/or T cells containing the coding sequence of one or more of these proteins preferably CAR -T cells.
  • the HLA-E genotype of the patient's T cells is or 01:01/01:01, and the patient is administered the protein US11 from HCMV and/or the protein k5 from KSHV and/or T cells with coding sequences for one or two of these proteins are preferably CAR-T cells.
  • a CAR-T cell characterized in that the CAR-T cell expresses a chimeric antigen receptor and a functional protein that can down-regulate the expression of HLA-I molecules on the cell surface; preferably, express the chimeric antigen
  • the expression level of the CAR-T cell surface HLA-I molecules of the receptor and the functional protein is less than 50% of the control CAR-T cell expressing the same chimeric antigen receptor but not expressing the functional protein.
  • Item 2 The CAR-T cell according to item 1, wherein the CAR-T cell contains the coding sequence of the chimeric antigen receptor and the coding sequence of the functional protein; preferably, the CAR The T cell contains the expression cassette of the chimeric antigen receptor and the expression cassette of the functional protein, or the coding sequence of the chimeric antigen receptor and the coding sequence of the functional protein are in the same expression frame.
  • the chimeric antigen receptor specifically binds to one or more of the following tumor antigens: EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-sugar Peptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, folate receptor ⁇ , ERBB, Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor ⁇ , TEM1/CD248, TEM7R, FAP, legume protein, HPV E6 or E7, ML-IAP, CLDN6,
  • the functional protein that can down-regulate the expression of HLA-I molecules on the cell surface is selected from: HSV, BHV-1, EHV-1/4, PRV, HSV-1/2, VZV, EBV, hCMV, mCMV, RhCMV, HHV- 6/7, KSHV, MHV-68, vaccinia virus and adenovirus can directly target the functional protein of HLA-I, the functional protein that can down-regulate the expression of HLA-I molecules through the TAP protein, and the functional protein that can be derived from the lysosome
  • a functional protein that down-regulates the expression of HLA-I molecules preferably, the functional protein is selected from: proteins US11 and US6 from HCMV, protein UL49.5 from BHV-1, protein UL49.5 from EHV-1 and KSHV protein k5.
  • nucleic acid molecule characterized in that the nucleic acid molecule is selected from:
  • the chimeric antigen receptor and the functional protein are as described in item 3.
  • nucleic acid construct wherein the nucleic acid construct contains the nucleic acid molecule described in item 4.
  • the nucleic acid construct contains the expression cassette of the chimeric antigen receptor and the expression cassette of the functional protein; or the nucleic acid construct is an expression cassette, wherein the coding sequence of the chimeric antigen receptor and the The coding sequence of the functional protein is in the expression frame; or
  • the nucleic acid construct is a cloning vector or expression vector.
  • Item 7 A lentivirus containing the nucleic acid construct according to item 5 or 6.
  • Item 8 A host cell containing the nucleic acid molecule of item 4 or the nucleic acid construct of item 5 or 6 or the lentivirus of item 7.
  • Item 9 A pharmaceutical composition, characterized in that the pharmaceutical composition contains the CAR-T cell according to any one of items 1-3.
  • Item 10 Application of the functional protein or its coding sequence of item 3 in the preparation of CAR-T cells with down-regulated expression of HLA-I molecules on the cell surface, or in the preparation of CAR-T cells for cancer treatment .
  • Anti-human Anti-HLA-I antibody (APC) (Biolegend, 311410), anti-human HLA-A antibody (Bio-Techne, NBP2-45320), anti-human HLA-B antibody (Bio-Techne, NBP2-45000) , Anti-human HLA-E antibody (Biolegend, 342612), Anti-CD3 antibody (BV421) (Biolegend, 300434), Anti-TCR antibody (PE-Cy7) (Biolegend, 306720), FBS (Lonsera, S711-001S), X-vivo15 (Lonza, 04-418Q), Dynabeads CD3/CD28 (Lifetechnology, 40203D), Ficoll (Dayou, DKW-LSH-0250), Tscm (Novoprotein, GMP-1647), Novonectin (Novoprotein, GMP-CH38) , Anti-human CCR7 (BV421) (BD, 562555), anti-human CD45RA (PE-Cy7) (BD, 560675
  • the human CD8 ⁇ hinge region, human CD8 transmembrane region, 41BB intracellular region, human CD3, and EHV1 UL49.5 gene sequence information were searched from the NCBI website database.
  • the anti-CD19 single chain antibody clone number is FMC63. These sequences are on the website https: Codon optimization is carried out on //www.thermofisher.com/order/geneartgenes to ensure that it is more suitable for human cell expression without changing the coding amino acid sequence.
  • the nucleotide sequence of the CAR molecule was seamlessly cloned into the Bamh1-Ecor1 site of the lentiviral plasmid pWPXL (Addgene), and transformed into competent E. coli (DH5 ⁇ , Beijing Bomad Biotechnology Co., Ltd.).
  • the recombinant plasmid was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing, and the sequencing result was compared with the proposed CD19-CAR-F2A-EHV1 UL49.5 sequence to verify whether the sequence was correct.
  • the sequencing primer is TCAAGCCTCAGACAGTGGTTC (SEQ ID NO: 3).
  • lentivirus PCTL135 (CAR19-F2A-GFP, control), PCTL199 (CAR19-F2A-HCMV US2), PCTL200 (CAR19-F2A-HCMV) were prepared.
  • PCTL201 CAR19-F2A-HCMV US6), PCTL202 (CAR19-F2A-HSV-1 ICP47), PCTL203 (CAR19-F2A-5 E3-19K), PCTL204 (CAR19-F2A-RhCMV Rh178), PCTL205 (CAR19 -F2A-BHV-1 UL49.5), PCTL207 (CAR19-F2A-EBV BNLF2a), PCTL208 (CAR19-F2A-CPXV012), PCTL209 (CAR19-F2A-HCMV US3), PCTL210 (CAR19-F2A-MHV68 mK3), PCTL211 (CAR19-F2A-CPXV203), PCTL212 (CAR19-F2A-KSHV k3), PCTL213 (CAR19-F2A-KSHV k5), including viral proteins HCMV US2, HSV-1 ICP47, human adenovirus 5E3-19K
  • HCMV US11 is shown in SEQ ID NO: 4, bases 1546-2193; the coding sequence of HCMV US6 is shown in SEQ ID NO: 5, bases 1546-2097; BHV-1 The coding sequence of UL49.5 is shown in SEQ ID NO: 6 bases 1546-2097; the coding sequence of KSHV k5 is shown in SEQ ID NO: 7 bases 1546-2316.
  • lentiviral transduction is performed.
  • the 24-well plate was coated with Novonectin and incubated at 37°C for 2 hours.
  • the guide system was placed in a coated 24-well plate, the cell density was adjusted to 1.0E+06/ml, and centrifuged at 500g for 30min. After centrifugation, it was allowed to stand in a CO 2 incubator at 37°C for 48h.
  • culture in Xvivo15 medium containing 5% FBS, supplement with Tscm (final concentration 2U/ml) every other day count cells, adjust cell density to 0.5E+06/ml, culture until 8-10 days to harvest cells.
  • Target cells stimulate each group of CAR-T cells
  • CAR-T cells in each group were cultured in vitro for 8-10 days, counted cells to take 1.0E+07 cells, adjusted the cell density to 1.0E+06/ml, cultured with Xvivo15 (excluding Tscm), according to the effective target ratio of 10:1 Target cell K562-CD19 was added to stimulate CAR-T cells, and the stimulation was repeated twice in two consecutive days. Flow cytometry was used to detect the CAR-positive rate of each group of cells and the average fluorescence intensity of CAR-positive cells HLA-I.
  • Count the CAR-T cells in each group take 5.0E+05 cells in different 1.5ml EP tubes, collect the cells by centrifugation at 2000rpm, 5min, discard the culture medium, resuspend and wash the cells with sterile 4% BSA twice, and then use 100ul Resuspend the cells in 4% BSA, add 8ul of anti-human Anti-HLA-I antibody (APC) (Biolegend, Cat#: 311410) antibody to each tube of cells, vortex to mix, and incubate at 4°C for 30 minutes; after staining, repeat washing the cells.
  • APC anti-human Anti-HLA-I antibody
  • CAR19 specific antibody diluted 1:500 resuspend the cells with the diluted antibody solution, 200ul per tube, vortex to mix, incubate at 4°C for 30min, after staining, repeat washing the cells, resuspend the cells in 500ul 4% BSA, add to each tube 4ul of 7AAD antibody, vortex to mix, and incubate for 10 minutes at room temperature in the dark. After the incubation is completed, transfer to a flow tube for testing.
  • Count the CAR-T cells in each group respectively take 1.0E+06 cells in different 1.5ml EP tubes, collect the cells by centrifugation at 2000rpm, 5min, discard the culture medium, resuspend the cells in sterile 4% BSA and wash the cells twice, then use 200ul Resuspend the cells in 4% BSA, add 5ul each of anti-human CD45RA (PE-Cy7) and anti-human CCR7 (BV421) antibodies to each tube of the sample tube, mix with a vortex mixer, and incubate at 4°C for 30 minutes. After staining, repeat washing the cells. Resuspend the cells with 500ul 4% BSA, add 4ul 7AAD antibody to each tube, vortex to mix, and incubate at room temperature for 10 minutes in the dark. After the incubation is completed, transfer to a flow tube for testing.
  • PE-Cy7 anti-human CD45RA
  • BV421 anti-human CCR7
  • NC-T T cells not transfected with lentivirus
  • CAR-T cells of each group observe whether the cell growth status is normal under a microscope, pipetting and mixing, collect NC-T and CAR-T cells of each group in In the centrifuge tube, count the cells, collect the cells by centrifugation, resuspend the cell pellet collected by the centrifugation with T cell culture medium X-VIVO15 (without Tscm), and adjust the cell density to 5.0E+07 cells/mL; remove the target cells, Observe whether the cell status is normal under a microscope, collect the target cells in a 15mL or 50mL centrifuge tube, and count the cells, resuspend the cell pellet collected by centrifugation with RPMI 1640 (without FBS), and adjust the cell density to 5.0E +06 cells/mL; in a 1.5mL centrifuge tube, the above-mentioned adjusted density effector cells NC-T and CAR-T are respectively compared with the target cells according to different
  • Table 1 The average fluorescence intensity of HLA-I of T cells in each group
  • the target cell K562-CD19 was used to stimulate the T cells of each group with an effect-to-target ratio of 10:1. The stimulation was repeated for 2 days.
  • the average fluorescence intensity of HLA-I of each group of T cells CAR19+ cell population was measured by flow cytometry.
  • the results are as follows As shown in Figure 2. The results show that the five lentiviruses PCTL200, PCTL201, PCTL205, PCTL206, and PCTL213 have a strong ability to produce CAR-T cells with down-regulated expression of HLA-I molecules.
  • CD3 + T accounts for 62.4% of buffy coat cells (monocytes and lymphocytes)
  • CD4 + T/CD8 + T is 1.2
  • DynaBeads CD3/CD28 sorts and activates CD3 + T, the activation efficiency is 68.9% after 24h ( Figure 3).
  • T cells were transfected with lentivirus in each group. T cells were counted every other day. T cells were cultured in vitro to the 8th day. The transfection efficiency of CAR19 was measured by flow cytometry and the cell proliferation was calculated (number of cells on day 8/cells used for transfection) number). The results are shown in Table 2 and Figure 4.
  • the positive rate of CAR19 in PCTL135 group was 65.6%
  • the positive rate of CAR19 in PCTL200 group was 37.4%
  • the positive rate of CAR19 in PCTL201 group was 46%
  • the positive rate of CAR19 in PCTL205 group was 55.8%.
  • the positive rate of CAR19 in the PCTL206 group was 54.6%
  • the positive rate of CAR19 in the PCTL213 group was 55.7%.
  • Table 3 Average fluorescence intensity of HLA-I of T cells in each group
  • the T cells of each group were cultured in vitro to the 8th day, and the target cell line k562-CD19-luc was cultured for 24 hours at the target ratio of 20:1, 10:1, 5:1, 2.5:1, and 1:1.
  • the cytotoxicity of each group of T cells was detected by the prime enzyme reporter gene detection system, and the results are shown in Table 4 and Figure 6.
  • T cells in each group were cultured in vitro to the 8th day.
  • the expression of CCR7 and CD45RA of T cells in each group was detected by flow cytometry, and the phenotype of T cells was analyzed. There was no significant difference in cell phenotype of each group. About 60% of cells were in the T-naive differentiation stage ( Figure 7).
  • the target cells K562-CD19 were used to stimulate the T cells of each group with an effective target ratio of 10:1, and the stimulation was repeated for 2 days.
  • the average fluorescence intensity of HLA-I of each group of T cells CAR19 + cell population was measured by flow cytometry. As shown in Table 5 and Figure 8. The results showed that the average fluorescence intensity of HLA-I in each group was significantly lower than that of the control group, and the PCTL206 group was the most significant.
  • Table 5 The average fluorescence intensity of HLA-I in each group of T cells stimulated by target cells
  • Figure 9 and Tables 6-7 show the average fluorescence intensity of each group in the CAR19+ T cell population for different HLA-I molecules (HLA-A, HLA-B, and HLA-E) and the ratio to the control.
  • the results of the PCTL135 group (control), PCTL200 group and PCTL213 group showed that compared with the control, the PCTL200 group and PCTL213 group significantly down-regulated the HLA-A and HLA-B molecules on the cell surface, but the HLA-E molecules were compared with the control There is no significant difference.
  • Table 7 The ratio of HLA average fluorescence intensity of each group of T cells to the control
  • Figure 9-11 and Table 8-11 show the average fluorescence intensity of various HLA-I molecules (HLA-A, HLA-B, and HLA-E) in CAR19+ T cell populations of different HLA-E genotypes and their comparison with the control ratio.
  • the PCTL201, PCTL205 and PCTL206 groups are selective for T cells of different HLA-E genotypes: for the CAR19+ T cell population homozygous for HLA-E genotype 01:03/01:03, PCTL206 group Down-regulate the HLA-A and HLA-B molecules on the cell surface, but the HLA-E molecules have no significant difference compared with the control (Figure 11); CAR19 that is homozygous for the HLA-E genotype 01:01/01:01 +T cell population, PCTL206 group down-regulated HLA-A, HLA-B and HLA-E molecules on the cell surface ( Figure 10).
  • Table 8 The average fluorescence intensity of HLA of T cells in each group ( Figure 10)
  • Table 9 The ratio of HLA average fluorescence intensity of each group of T cells to the control ( Figure 10)
  • Table 10 The average fluorescence intensity of HLA of T cells in each group ( Figure 11)
  • Table 11 The ratio of the average fluorescence intensity of HLA of each group of T cells to the control ( Figure 11)

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Abstract

提供了在T细胞中表达相关功能蛋白，调控细胞表面HLA-I类分子，从而制备得到同种异体CAR-T细胞。还提供了所述同种异体T细胞的应用。

Description

同种异体CAR-T细胞、其制备及应用 技术领域
本发明涉及选择性同种异体CAR-T细胞、其制备及应用。
背景技术
随着肿瘤治疗的发展，嵌合抗原受体T细胞(Chimeric antigen receptor-T，CAR-T)免疫疗法逐渐成为备受关注的治疗手段。CAR-T细胞所表达的CAR一般包含胞外抗原结合域、跨膜域和胞内信号传导域。CAR-T细胞可以有效的识别肿瘤抗原，引起特异性的抗肿瘤免疫应答，而不受主要组织相容性复合体(major histocompatibility complex，MHC)的限制。目前，美国FDA已经批准了两款自体CAR-T细胞产品上市，分别是诺华的Kymriah和凯特的YesCAR-Ta，用于难治性复发性非霍奇金淋巴瘤和急性B淋巴细胞白血病的治疗。大量临床试验证明，CAR-T作为个性化的活细胞药极具抗肿瘤潜力(Maude et al.2018；Park et al.2018；Schuster et al.2017；)。
这两款上市的CAR-T细胞产品均是采集患者自身的外周血，进行T细胞分离，利用慢病毒或逆转录病毒载体将CAR基因表达框转染到T细胞内，扩大培养后再回输到患者体内。每位患者的CAR-T需要单独制备，生产周期较长，而且多种不确定因素都可能导致CART治疗夭折或失败，例如分离得到的细胞数量不足、患者T细胞质量或功能失常、CAR-T细胞制备失败、细胞制备过程中患者疾病快速进展等。
利用健康供者的T细胞批量制备成即用型的同种异体CAR-T细胞，可有效解决上述自体CART细胞制备和应用中存在的问题。一个成功的同种异体CAR-T细胞技术，需要解决两个关键问题：①因为健康供者T细胞表面的TCR(T cell receptor，TCR)会识别患者的异体抗原，从而引发危险甚至致命的移植物抗宿主反应(graft-versus-host disease，GVHD)；②宿主的免疫系统会识 别健康供者T细胞表面的HLA(human leukocyte antigen)I类分子，导致回输的异体T细胞被快速清除，影响同种异体CAR-T的抗肿瘤疗效。
目前业内人士的共识是清除了TCR和HLA-I类分子的异体T细胞能够有效用于同种异体CAR-T细胞治疗。由于基因编辑技术能在基因组水平高效而彻底地清除一个或多个靶向基因的表达，它迅速成为制备同种异体CAR-T的主流技术手段，用以彻底敲除同种异体CAR-T细胞的TCR以及HLA-I类分子的表达，希望在临床上有效防止GVHD和宿主排斥。目前，多种基因编辑技术(zinc-finger nucleases，ZFN；transcription activator-like effector nucleases，TALENs；clustered regularly interspaced short palindromic repeats，CRISPR；ARCUS Genome Editing)被积极地应用于同种异体CAR-T细胞的临床前开发和临床试验(Provasi et al.2012；Berdien et al.2014；Ren et al.2017；McCreedy et al.2018)。
发明内容
本发明第一方面提供一种工程改造的T细胞，其特征在于，所述工程改造的T细胞表达能调控细胞表面HLA-I类分子表达的功能蛋白，其中，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控，或所述工程改造的T细胞的细胞表面HLA-A、HLA-B和HLA-E表达下调。
在一个或多个实施方案中，差异化调控是所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，所述工程改造的T细胞的细胞表面HLA-I类分子的表达水平为未表达所述功能蛋白的T细胞的50％或以下，优选10-50％。
在一个或多个实施方案中，所述工程改造的T细胞表面的HLA-A和/或HLA-B分子的表达水平为未表达所述功能蛋白的T细胞的5-50％，10-50％，10-45％，10-40％，15-50％，20-50％，或20-45％。
在一个或多个实施方案中，所述工程改造的T细胞表面的HLA-E分子的表达水平为未表达所述功能蛋白的T细胞的65-100％，70-100％，75-100％，80-100％，85-100％，90-100％，95-100％。
在一个或多个实施方案中，所述工程改造的T细胞进一步表达嵌合抗原受体，从而工程改造的T细胞是CAR-T细胞，其中，所述CAR-T细胞含有所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列。
在一个或多个实施方案中，所述CAR-T细胞含有所述嵌合抗原受体的表达框和所述功能蛋白的表达框，或所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列处于同一表达框内。
在一个或多个实施方案中，所述嵌合抗原受体特异性结合选自以下的肿瘤抗原中的一种或多种：EGFRvIII、间皮素、GD2、Tn抗原、sTn抗原、Tn-O-糖肽、sTn-O-糖肽、PSMA、CD97、TAG72、CD44v6、CEA、EPCAM、KIT、IL-13Ra2、leguman、GD3、CD171、IL-11Ra、PSCA、MAD-CT-1、MAD-CT-2、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、叶酸受体α、ERBB、Her2/neu、MUC1、EGFR、NCAM、肝配蛋白B2、CAIX、LMP2、sLe、HMWMAA、o-乙酰基-GD2、叶酸受体β、TEM1/CD248、TEM7R、FAP、豆荚蛋白、HPV E6或E7、ML-IAP、CLDN6、TSHR、GPRC5D、ALK、聚唾液酸、Fos-相关抗原、中性粒细胞弹性蛋白酶、TRP-2、CYP1B1、精子蛋白17、β人绒毛膜促性腺激素、AFP、甲状腺球蛋白、PLAC1、globoH、RAGE1、MN-CA IX、人端粒酶逆转录酶、肠羧基酯酶、mut hsp 70-2、NA-17、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、NY-ESO-1、GPR20、Ly6k、OR51E2、TARP、GFRα4和呈递在MHC上的这些抗原中任一者的多肽片段，以及CD5、CD10、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD27、CD30、CD34、CD37、CD38、CD40、CD53、CD69、CD72、CD73、CD74、CD75、CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85、CD86、CD123、CD135、CD138、CD179、CD269、Flt3、ROR1、BCMA、FcRn5、FcRn2、CS-1、CXCR4、CXCR5、CXCR7、IL-7/3R、IL7/4/3R和IL4R。
在一个或多个实施方案中，所述能调控细胞表面HLA-I类分子表达的功能蛋白选自：HSV、BHV-1、EHV-1/4、PRV、HSV-1/2、VZV、EBV、hCMV、mCMV、RhCMV、HHV-6/7、KSHV、MHV-68、牛痘病毒和腺病毒中能直接靶向降解HLA-I的功能蛋白、能经由TAP蛋白来下调HLA-I类分子表达的功 能蛋白和能经由溶酶体来下调HLA-I类分子表达的功能蛋白。
在一个或多个实施方案中，所述功能蛋白选自：来自HCMV的蛋白US11和US6、来自BHV-1的蛋白UL49.5、来自EHV-1的蛋白UL49.5和来自KSHV的蛋白k5。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US11和来自KSHV的蛋白k5，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。优选地，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述工程改造的T细胞的HLA-E的基因型为01:03/01:03，且所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。优选地，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述工程改造的T细胞的HLA-E的基因型为01:01/01:01，且所述工程改造的T细胞的细胞表面HLA-A、HLA-B和HLA-E表达下调。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
本发明第一方面还提供一种核酸分子，其特征在于，所述核酸分子选自：(1)含嵌合抗原受体的编码序列和能调控细胞表面HLA-I类分子表达的功能蛋白的编码序列的核酸分子；和(2)(1)所述核酸分子的互补序列。
在一个或多个实施方案中，所述嵌合抗原受体和所述功能蛋白如本发明第一方面任一实施方案所述。
本发明第一方面还提供一种核酸构建物，其特征在于，所述核酸构建物含有本发明第一方面任一实施方案所述的核酸分子。
在一个或多个实施方案中，所述核酸构建物含有所述嵌合抗原受体的表达框和所述功能蛋白的表达框；或所述核酸构建物为一表达框，其中所述嵌合抗 原受体的编码序列和所述功能蛋白的编码序列处于该表达框内。
在一个或多个实施方案中，所述核酸构建物是克隆载体或表达载体。
本发明第一方面还提供一种慢病毒，其含有本发明第一方面任一实施方案所述的核酸构建物。
本发明第一方面还提供一种宿主细胞，其含有本发明第一方面任一实施方案所述的核酸分子或本发明第一方面任一实施方案的核酸构建物或权本发明第一方面任一实施方案所述的慢病毒。
本发明第一方面还提供一种药物组合物，其特征在于，所述药物组合物含有本发明第一方面任一实施方案所述的工程改造的T细胞。
本发明第一方面还提供本发明第一方面任一实施方案所述的功能蛋白或其编码序列在制备细胞表面的HLA-I类分子的表达调控的工程改造的T细胞中的应用，或在制备癌症治疗用的工程改造的T细胞中的应用，其中，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控，或所述工程改造的T细胞的细胞表面HLA-A、HLA-B和HLA-E表达下调。
在一个或多个实施方案中，差异化调控是所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，所述工程改造的T细胞表面的HLA-A和/或HLA-B分子的表达水平为未表达所述功能蛋白的T细胞的5-50％，10-50％，10-45％，10-40％，15-50％，20-50％，或20-45％。
在一个或多个实施方案中，所述工程改造的T细胞表面的HLA-E分子的表达水平为未表达所述功能蛋白的T细胞的65-100％，70-100％，75-100％，80-100％，85-100％，90-100％，95-100％。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US11和来自KSHV的蛋白k5，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。优选地，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述工程改造的T 细胞的HLA-E的基因型为01:03/01:03，且所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。优选地，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述工程改造的T细胞的HLA-E的基因型为01:01/01:01，且所述工程改造的T细胞的细胞表面HLA-A、HLA-B和HLA-E表达下调。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
本发明还提供一种抑制T细胞表面表达HLA-I类分子的方法，所述方法包括在T细胞内表达本文任一实施方案所述的功能蛋白的步骤，其中，表达功能蛋白的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控，或表达功能蛋白的T细胞的细胞表面HLA-A、HLA-B和HLA-E表达下调。
在一个或多个实施方案中，差异化调控是所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，所述工程改造的T细胞表面的HLA-A和/或HLA-B分子的表达水平为未表达所述功能蛋白的T细胞的5-50％，10-50％，10-45％，10-40％，15-50％，20-50％，或20-45％。
在一个或多个实施方案中，所述工程改造的T细胞表面的HLA-E分子的表达水平为未表达所述功能蛋白的T细胞的65-100％，70-100％，75-100％，80-100％，85-100％，90-100％，95-100％。
本发明第二方面提供一种CAR-T细胞，其含有特异性靶向肿瘤抗原的嵌合抗原受体和能调控细胞表面HLA-I类分子表达的功能蛋白。
在一个或多个实施方案中，该CAR-T细胞细胞表面HLA-I类分子的表达水平为表达相同嵌合抗原受体但未表达所述功能蛋白的对照CAR-T细胞的50％以下。
在一个或多个实施方案中，所述CAR-T细胞含有所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列；优选地，所述CAR-T细胞含有所述嵌合 抗原受体的表达框和所述功能蛋白的表达框，或所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列处于同一表达框内。
在一个或多个实施方案中，所述嵌合抗原受体特异性结合选自以下的肿瘤抗原中的一种或多种：EGFRvIII、间皮素、GD2、Tn抗原、sTn抗原、Tn-O-糖肽、sTn-O-糖肽、PSMA、CD97、TAG72、CD44v6、CEA、EPCAM、KIT、IL-13Ra2、leguman、GD3、CD171、IL-11Ra、PSCA、MAD-CT-1、MAD-CT-2、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、叶酸受体α、ERBB、Her2/neu、MUC1、EGFR、NCAM、肝配蛋白B2、CAIX、LMP2、sLe、HMWMAA、o-乙酰基-GD2、叶酸受体β、TEM1/CD248、TEM7R、FAP、豆荚蛋白、HPV E6或E7、ML-IAP、CLDN6、TSHR、GPRC5D、ALK、聚唾液酸、Fos-相关抗原、中性粒细胞弹性蛋白酶、TRP-2、CYP1B1、精子蛋白17、β人绒毛膜促性腺激素、AFP、甲状腺球蛋白、PLAC1、globoH、RAGE1、MN-CA IX、人端粒酶逆转录酶、肠羧基酯酶、mut hsp 70-2、NA-17、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、NY-ESO-1、GPR20、Ly6k、OR51E2、TARP、GFRα4和呈递在MHC上的这些抗原中任一者的多肽片段，以及CD5、CD10、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD27、CD30、CD34、CD37、CD38、CD40、CD53、CD69、CD72、CD73、CD74、CD75、CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85、CD86、CD123、CD135、CD138、CD179、CD269、Flt3、ROR1、BCMA、FcRn5、FcRn2、CS-1、CXCR4、CXCR5、CXCR7、IL-7/3R、IL7/4/3R和IL4R。
在一个或多个实施方案中，所述能调控细胞表面HLA-I类分子表达的功能蛋白选自：HSV、BHV-1、EHV-1/4、PRV、HSV-1/2、VZV、EBV、hCMV、mCMV、RhCMV、HHV-6/7、KSHV、MHV-68、牛痘病毒和腺病毒中能直接靶向降解HLA-I的功能蛋白、能经由TAP蛋白来下调HLA-I类分子表达的功能蛋白和能经由溶酶体来下调HLA-I类分子表达的功能蛋白。
在一个或多个实施方案中，所述功能蛋白选自：来自HCMV的蛋白US11和US6、来自BHV-1的蛋白UL49.5、来自EHV-1的蛋白UL49.5和来自KSHV的蛋白k5。
在一个或多个实施方案中，调控细胞表面HLA-I类分子表达包括：下调细胞表面HLA-A、HLA-B和HLA-E表达，或细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。
在一个或多个实施方案中，差异化调控是CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，CAR-T细胞的细胞表面HLA-A和HLA-B表达与但HLA-E表达被差异化调控，所述功能蛋白选自：来自HCMV的蛋白US11和来自KSHV的蛋白k5。优选地，所述CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，CAR-T细胞的HLA-E的基因型为01:03/01:03，且所述CAR-T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。优选地，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述工程改造的T细胞的HLA-E的基因型为01:01/01:01，且所述工程改造的T细胞的细胞表面HLA-A、HLA-B和HLA-E表达下调。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
在一个或多个实施方案中，CAR-T细胞的细胞表面的HLA-A和/或HLA-B分子的表达水平为未表达所述功能蛋白的T细胞的5-50％，10-50％，10-45％，10-40％，15-50％，20-50％，或20-45％。
在一个或多个实施方案中，CAR-T细胞的细胞表面的HLA-E分子的表达水平为未表达所述功能蛋白的T细胞的65-100％，70-100％，75-100％，80-100％，85-100％，90-100％，95-100％。
本发明提供一种CAR-T细胞，其含有特异性靶向肿瘤抗原的嵌合抗原受体和能差异化调控细胞表面HLA-A和HLA-B表达与HLA-E表达的功能蛋白， 优选地，所述功能蛋白选自：来自HCMV的蛋白US11和来自KSHV的蛋白k5。优选地，所述CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
本发明还提供一种核酸分子，所述核酸分子选自：
(1)含嵌合抗原受体的编码序列和能调控细胞表面HLA-I类分子表达的功能蛋白的编码序列的核酸分子；和
(2)(1)所述核酸分子的互补序列；
在一个或多个实施方案中，所述嵌合抗原受体和所述功能蛋白如本文第二方面任一实施方案所述。
本发明还提供一种核酸构建物，所述核酸构建物含有本文第二方面所述的核酸分子。
在一个或多个实施方案中，所述核酸构建物含有所述嵌合抗原受体的表达框和所述功能蛋白的表达框；或所述核酸构建物为一表达框，其中所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列处于该表达框内。
在一个或多个实施方案中，所述核酸构建物是克隆载体或表达载体。
本发明还提供一种慢病毒，其含有本文第二方面所述的核酸构建物。
本发明还提供一种宿主细胞，其含有本文第二方面所述的核酸分子、核酸构建物或慢病毒。
本发明还提供一种药物组合物，所述药物组合物含有本文第二方面任一实施方案所述的CAR-T细胞。
本发明还提供本文第二方面任一实施方案所述的功能蛋白或其编码序列在制备其细胞表面的HLA-I类分子的表达被调控的CAR-T细胞中的应用，或在制备癌症治疗用的CAR-T细胞中的应用。
在一个或多个实施方案中，调控细胞表面HLA-I类分子表达包括：下调细胞表面HLA-A、HLA-B和HLA-E表达，或细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。
在一个或多个实施方案中，差异化调控是CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US11和来自KSHV的蛋白k5，所述CAR-T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。优选地，CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述CAR-T细胞的HLA-E的基因型为01:03/01:03，且所述CAR-T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。优选地，CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
在一个或多个实施方案中，所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述CAR-T细胞的HLA-E的基因型为01:01/01:01，且所述CAR-T细胞的细胞表面HLA-A、HLA-B和HLA-E表达下调。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
在一个或多个实施方案中，所述工程改造的T细胞表面的HLA-A和/或HLA-B分子的表达水平为未表达所述功能蛋白的T细胞的5-50％，10-50％，10-45％，10-40％，15-50％，20-50％，或20-45％。
在一个或多个实施方案中，所述工程改造的T细胞表面的HLA-E分子的表达水平为未表达所述功能蛋白的T细胞的65-100％，70-100％，75-100％，80-100％，85-100％，90-100％，95-100％。
本发明还提供功能蛋白或其编码序列在制备其细胞表面的HLA-A和HLA-B表达与HLA-E表达被差异化调控的CAR-T细胞中的应用，或在制备癌症治疗用的CAR-T细胞中的应用，所述功能蛋白选自：来自HCMV的蛋白US11和来自KSHV的蛋白k5。在一个或多个实施方案中，差异化调控是CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
本发明还提供功能蛋白或其编码序列在制备其细胞表面的HLA-A和HLA-B表达与HLA-E表达被差异化调控的CAR-T细胞中的应用，或在制备癌症治疗用的CAR-T细胞中的应用，所述CAR-T细胞的HLA-E的基因型为 01:03/01:03，所述功能蛋白选自：来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5。所述功能蛋白优选来自EHV-1的蛋白UL49.5。在一个或多个实施方案中，差异化调控是CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
本发明还提供功能蛋白或其编码序列在制备其细胞表面的HLA-A、HLA-B和HLA-E表达下调的CAR-T细胞中的应用，或在制备癌症治疗用的CAR-T细胞中的应用，所述CAR-T细胞的HLA-E的基因型为01:01/01:01，所述功能蛋白选自：来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
本发明还提供一种抑制CAR-T细胞表面表达HLA-I类分子的方法，所述方法包括在T细胞内同时表达本文任一实施方案所述的CAR和功能蛋白的步骤。
在一个或多个实施方案中，CAR-T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控，所述功能蛋白选自：来自HCMV的蛋白US11和来自KSHV的蛋白k5。优选地，CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，CAR-T细胞的HLA-E的基因型为01:03/01:03，且所述T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控，所述功能蛋白选自：来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5。所述功能蛋白优选来自EHV-1的蛋白UL49.5。优选地，CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
在一个或多个实施方案中，CAR-T细胞的HLA-E的基因型为01:01/01:01，且所述T细胞的细胞表面HLA-A、HLA-B和HLA-E表达下调，所述功能蛋白选自：来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
本发明还提供一种差异化调控CAR-T细胞表面表达HLA-A和HLA-B表达与HLA-E表达的方法，所述方法包括在T细胞内同时表达本文任一实施方 案所述的CAR和选自以下的功能蛋白的步骤：来自HCMV的蛋白US11和来自KSHV的蛋白k5。在一个或多个实施方案中，差异化调控是CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
本发明还提供一种差异化调控CAR-T细胞表面表达HLA-A和HLA-B表达与HLA-E表达的方法，所述方法包括在T细胞内同时表达本文任一实施方案所述的CAR和选自以下的功能蛋白的步骤：来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述T细胞的HLA-E的基因型为01:03/01:03。所述功能蛋白优选来自EHV-1的蛋白UL49.5。在一个或多个实施方案中，差异化调控是CAR-T细胞的细胞表面HLA-A和HLA-B表达下调但HLA-E表达不下调。
本发明还提供一种抑制CAR-T细胞表面表达HLA-A、HLA-B和HLA-E的方法，所述方法包括在T细胞内同时表达本文任一实施方案所述的CAR和选自以下的功能蛋白的步骤：来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述T细胞的HLA-E的基因型为01:01/01:01。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
附图说明
图1：各组慢病毒载体转染活化的T细胞，体外培养T细胞至第8天，流式检测CAR19 +T细胞表面HLA-I类分子的平均荧光强度。
图2：各组T细胞体外培养第9天，用靶细胞K562-CD19刺激各组T细胞，效靶比10：1，连续反复刺激2天，流式检测各组T细胞CAR19+细胞群体HLA-I类分子的平均荧光强度，其中PCTL200、PCTL201、PCTL205、PCTL206、PCTL213这5个载体分子具有制备HLA-I类分子表达下调的CAR-T细胞的能力。
图3：全血淋巴细胞比例流式分析。100ml外周血Ficoll密度梯度离心分离1.24x10 8PBMC，流式检测CD3+T占白膜细胞比例62.4％，CD4+T/CD8+T比例为1.2，24h活化效率68.9％。
图4：各组细胞体外扩增8天扩增倍数。各组细胞体外扩增8天，扩增倍 数显著高于PCTL135组。
图5：各组T细胞HLA-I类分子的平均荧光强度。经流式检测，各组细胞CAR阳性细胞群体，HLA-I类分子的平均荧光强度均显著下调，其中PCTL206组下调最显著。
图6：各组T细胞杀伤效率。各组细胞用于体外细胞毒性实验，杀伤效率在效靶比5：1时均达大于90％，与对照组无显著差异。
图7：各组T细胞的分化表型分析。各组细胞检测细胞分化表型，大于65％的T细胞显示为
Figure PCTCN2020086032-appb-000001
群，与对照组135无显著差异。
图8：经靶细胞K562-CD19刺激各组T细胞HLA-I类分子的平均荧光强度。体外培养至第9天，用靶细胞K526-CD19刺激各组T细胞，刺激后流式检测各组细胞中CAR阳性细胞群体的HLA-I类分子，发现其平均荧光强度均显著低于对照组。
图9：流式检测HLA-E基因型为杂合01:01/01:03的CAR19+T细胞群体的各种HLA-I类分子的平均荧光强度。
图10：流式检测HLA-E基因型为纯合01:01/01:01的CAR19+T细胞群体的各种HLA-I类分子的平均荧光强度。
图11：流式检测HLA-E基因型为纯合01:03/01:03的CAR19+T细胞群体的各种HLA-I类分子的平均荧光强度。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
由于基因编辑技术能在基因组水平高效而彻底地清除一个或多个靶向基因的表达，它们迅速成为制备同种异体CAR-T的主流技术手段，用以彻底敲除同种异体CAR-T细胞的TCR以及HLA-I类分子的表达，希望在临床上有效防止GVHD和宿主排斥。
基因编辑制备同种异体T细胞的基本原理是，在基因组的特定位置(如TCR基因、HLA-I类分子伴侣蛋白b2m基因)产生位点特异性双链断裂(Double  strand break，DSB)，然后通过非同源末端连接(Non-homologous end joining，NHEJ)或同源定向重组修复(Homology directed recombination repair，HDR)的方式进行修复，从而实现靶向基因的彻底缺失(如TCR及b2m基因)，试图从根本上避免GvHD和宿主免疫细胞介导的对异体CAR-T细胞的排斥反应。
但是迄今，基于基因编辑技术的同种异体CAR-T细胞在临床试验中均未展示出与自体CAR-T细胞同等的疗效优势。深入研究发现基因编辑产生的同种异体CAR-T细胞在患者体内的持久性差，原因之一是b2m基因敲除导致所有HLA-I类分子特别是HLA-A,HLA-B和HLA-E在同种异体CAR-T细胞上的表达缺失，易受到患者自身NK细胞的攻击，从而严重影响了同种异体CAR-T在患者体内的体内持久性和疗效(Torikai et al.2013)。
为了延长回输的同种异体CAR-T细胞的体内持久性，Precision BioSciences利用Arcus基因组编辑技术，仅敲除了TCR基因，但没有编辑b2m基因(即不改变CAR-T细胞表面HLA-I类分子的表达)，所以其同种异体CAR-T细胞表达正常的HLA-I类分子。在开展了第一和第二剂量爬坡试验后，其于2020年初向FDA提交同种异体CAR-T临床修正案，申请增强回输前的化疗预处理，并申请大幅提高同种异体CAR-T细胞的回输剂量以希望达到预期的治疗效果。
Cellectis和Allogene Therapeutics运用TALEN基因编辑技术敲除了供者T细胞的TCR和CD52基因(即不改变CAR-T细胞表面HLA-I类分子的表达)，然后联合抗CD52单抗药物，利用其清淋活性以求达到同时抑制患者T细胞和NK细胞对异体CAR-T细胞的排斥反应。
显然地，本领域技术人员面对的是一个无法逾越的困境；一方面，不改变HLA-I类分子的表达水平，患者的T细胞免疫系统对CAR-T细胞的攻击的危险依旧；另一方面，基因编辑导致的HLA-I类分子的表达完全缺失，导致患者NK细胞对CAR-T细胞的攻击；通过这些研究，本领域技术人员能够得出的一个令人沮丧的、逻辑的结论是调控HLA-I类分子的表达不能够消除患者免疫系统对CAR-T细胞的攻击的危险的。Cellectis和Allogene Therapeutics，在不改变CAR-T细胞表面HLA-I类分子的表达的前提下，通过CAR-T细胞CD52基因缺失然后用抗CD52的抗体同时解除患者T细胞和NK细胞免疫系统对 CAR-T细胞的攻击，这是一个走出困境的尝试，也进一步证明了这个逻辑结论。
可能有鉴于这些临床观察和发现，GraCell在CN109694854A专利申请中，公开了在基因敲除TCR和所有HLA-I分子的基础上，通过过表达HLA-E的单链三聚体，恢复HLA-E分子的表面表达，来同时避免宿主T细胞和NK细胞对同种异体CAR-T细胞的排斥反应。
本发明的发明人及其研究团队，经过多年的深层的研究，意外地发现，通过在同种异体T细胞(尤其是CAR-T细胞)中表达功能蛋白，能够差异化调控细胞表面的HLA-I类分子的表达水平，而同时能够保持或有限抑制细胞表面的HLA-E分子的表达水平；这样的同种异体的T细胞(尤其是CAR-T细胞)既能够避免GVHD，又能够消除患者免疫系统(T细胞和NK细胞)对同种异体T细胞的攻击的危险。
HLA-I类分子由经典HLA-I类分子(HLA-A、HLA-B等)和非经典HLA-I类分子(HLA-E等)组成。HLA-E分子通过与NK细胞表面的CD94/NKG2类受体结合，可抑制NK细胞的生物学活性。人体中HLA-E的基因包含2个等位基因01:01和01:03。
发明人发现，当功能蛋白选自来自HCMV的蛋白US11和来自KSHV的蛋白k5时，功能蛋白差异化调控HLA-A和HLA-B表达与HLA-E表达。因此，在本发明T细胞(尤其是CAR-T细胞)中保留细胞表面HLA-E分子的表达，能有效避免被宿主NK细胞清除，从而提高T细胞(尤其是CAR-T细胞)的存活和活性。
此外，发明人还发现，当功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，并且T细胞(尤其是CAR-T细胞)的HLA-E的基因型为01:03/01:03时，功能蛋白差异化调控T细胞(尤其是CAR-T细胞)的细胞表面HLA-A和HLA-B表达与HLA-E表达；当功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，并且T细胞(尤其是CAR-T细胞)的HLA-E的基因型为01:01/01:01时，功能蛋白下调T细胞(尤其是CAR-T细胞)的细胞表面HLA-A、HLA-B和HLA-E表达。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
在一些实施例中，同种异体T细胞(尤其是CAR-T细胞)表面的HLA-A和HLA-B分子的表达水平为未表达所述功能蛋白的T细胞的5-50％，10-50％，10-45％，10-40％，15-50％，或20-50％，或20-45％；HLA-E分子的表达水平为未表达所述功能蛋白的T细胞的65-100％，70-100％，75-100％，80-100％，85-100％，90-100％，95-100％。
在一些实施例中，同种异体T细胞(尤其是CAR-T细胞)表达能差异调节HLA-A，HLA-B和HLA-E分子表达水平的功能蛋白，包括但不限于：
UL49.5，或与UL49.5有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％的同源性；UL49.5来自BHV-1(SEQ ID NO:8)或EHV-1/4(SEQ ID NO:9)，其能诱导TAP的构象阻止(conformational arrest)、或诱导TAP的构象阻止和降解或阻断ATP与TAP的结合，从而抑制细胞表面MHC-I类分子的表达，该结果分别在牛肾细胞、马表皮细胞上得到了验证(Koppers-Lalic et al.2008)；
US6/gp21(SEQ ID NO:10)，或US6/gp21的变体，与SEQ ID NO:10有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％的同源性；US11/gp33(SEQ ID NO:11)，或US11/gp33的变体，与SEQ ID NO:11有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％的同源性；US6/gp21和US11/gp33来自hCMV，其分别能诱导TAP构象变化从而阻止ATP结合、以及靶向MHC-I以引起内质网介导的蛋白降解，从而抑制细胞表面MHC-I类分子的表达，该结果分别在中国仓鼠卵巢细胞、人类髓性白血病细胞、人肺肿瘤细胞、人星形细胞瘤细胞上得到了验证(Hewitt et al.2001，Wiertz et al.1996)；
kK5/MIR2(SEQ ID NO:12)，或kK5/MIR2的变体，与SEQ ID NO:12有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％的同源性；kK5/MIR2来自KSHV，其使MHC-I泛素化以被内化后被溶酶体降解，从而抑制细胞表面MHC-I类分子的表达，该结果在人宫颈癌细胞上得到了验证(Coscoy et al.2005)。
虽然上述HLA-I下调蛋白功能均在特定种属的特定细胞类型，或天然易感 细胞中部分抑制HLA-I类分子的表达，但在目标种属(例如人)的目标细胞类型(例如T细胞)中是否下调HLA-I类分子的表达是不得而知的，在T细胞同时表达嵌合抗原受体(CAR)的情况下就更是不得而知了。在一个或多个实施方案中，MHV-68的mK3、hCMV的US3/gp23、牛痘病毒的CPXV012、RhCMV的rh178/VIHCE以及腺病毒的E3-19K均未能在T细胞中表现出下调HLA-I类分子的能力。在一个或多个实施方案中，本发明人发现多个跨种属的病毒在人T细胞中展现出了出乎意料的下调HLA-I类分子的效果，如：EHV-1(马鼻肺炎病毒)UL49.5等。
本发明通过表达相关功能蛋白调控细胞表面HLA-I类分子，制备能抑制细胞表面HLA-I类分子的表达的工程改造的T细胞，所述工程改造的T细胞的一个示例是同种异体CAR-T细胞。采用本发明方法制备得到的工程改造的T细胞中，HLA-I类分子的表达并未完全被抑制，这些T细胞仍然在细胞表面表达一定量的HLA-I类分子，因此很好地避免了受体NK细胞的攻击，同时也解决了受体的排斥反应。优选地，以流式细胞术检测，本发明工程改造的T细胞中HLA-I类分子的表达水平为未表达所述功能蛋白的对照工程改造的T细胞的80％以下，优选60％以下，更优选50％以下，更优选30％以下，更以下25％以下。在一些实施方案中，以流式细胞术检测，本发明工程改造的T细胞中HLA-I类分子的表达水平为未表达所述功能蛋白的对照工程改造的T细胞的10-50％，如15-50％。在一些实施方案中，即便经过相同的靶细胞刺激，本发明工程改造的T细胞中HLA-I类分子的表达水平仍为未表达所述功能蛋白的对照工程改造的T细胞的10-50％，如15-50％。
工程改造的T细胞的可以是CAR-T细胞，因此，本发明提供一类CAR-T细胞，该类细胞含有编码靶向感兴趣肿瘤抗原的嵌合抗原受体(CAR)的核酸分子与编码能调控细胞表面HLA-I类分子表达的功能蛋白的核酸分子。本文中，合适的T细胞可以是本领域周知的各种T细胞，尤其是细胞免疫疗法中常规使用的各种T细胞，包括但不限于外周血T淋巴细胞、细胞毒杀伤T细胞、辅助T细胞、抑制/调节性T细胞、γδT细胞、细胞因子诱导的杀伤细胞和肿瘤浸润淋巴细胞等，以及上述细胞的任意一种或多种的混合物。本文中，CAR-T 细胞指至少表达嵌合抗原受体的T细胞。
本文中，嵌合抗原受体具有本领域周知的含义，它是一种人工改造受体，能够将识别肿瘤细胞表面抗原的特异性分子(如抗体)锚定在免疫细胞(如T细胞)上，使免疫细胞识别肿瘤抗原并杀死肿瘤细胞。
适用于本文的嵌合抗原受体可以是本领域周知的各种CAR。通常，CAR依次包含结合肿瘤抗原的多肽、铰链区、跨膜区和胞内信号区。结合肿瘤抗原的多肽可以是天然多肽或人工合成多肽；优选地，人工合成多肽为单链抗体或Fab片段。
本文中，感兴趣的肿瘤抗原包括但不限于实体瘤抗原、髓系肿瘤抗原以及非B细胞谱系血液肿瘤的抗原。合适的实体瘤抗原包括但不限于EGFRvIII、间皮素、GD2、Tn抗原、sTn抗原、Tn-O-糖肽、sTn-O-糖肽、PSMA、CD97、TAG72、CD44v6、CEA、EPCAM、KIT、IL-13Ra2、leguman、GD3、CD171、IL-11Ra、PSCA、MAD-CT-1、MAD-CT-2、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、叶酸受体α、ERBB(例如，ERBB2)、Her2/neu、MUC1、EGFR、NCAM、肝配蛋白B2、CAIX、LMP2、sLe、HMWMAA、o-乙酰基-GD2、叶酸受体β、TEM1/CD248、TEM7R、FAP、豆荚蛋白(Legumain)、HPV E6或E7、ML-IAP、CLDN6、TSHR、GPRC5D、ALK、聚唾液酸、Fos-相关抗原、中性粒细胞弹性蛋白酶、TRP-2、CYP1B1、精子蛋白17、β人绒毛膜促性腺激素、AFP、甲状腺球蛋白、PLAC1、globoH、RAGE1、MN-CA IX、人端粒酶逆转录酶、肠羧基酯酶、mut hsp 70-2、NA-17、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、NY-ESO-1、GPR20、Ly6k、OR51E2、TARP、GFRα4和呈递在MHC上的这些抗原中任一者的多肽片段。合适的B细胞抗原包括但不限于CD5、CD10、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD27、CD30、CD34、CD37、CD38、CD40、CD53、CD69、CD72、CD73、CD74、CD75、CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85、CD86、CD123、CD135、CD138、CD179、CD269、Flt3、ROR1、BCMA、FcRn5、FcRn2、CS-1、CXCR4、CXCR5、CXCR7、IL-7/3R、IL7/4/3R和IL4R。
在优选的实施方案中，本发明结合肿瘤抗原的多肽是特异性结合上述任一 种肿瘤抗原的单链抗体。本文中，单链抗体(scFv)指由抗体轻链可变区(VL区)氨基酸序列和重链可变区(VH区)氨基酸序列经铰链连接而成的具有结合抗原能力的抗体片段。感兴趣的单链抗体可来自感兴趣的抗体。感兴趣的抗体可以是人抗体，包括人鼠嵌合抗体和人源化抗体。抗体可以是分泌型或膜锚定型；优选地为膜锚定型。本文中，特异性结合是指抗体或其抗原结合片段与其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合该抗原。
单链抗体可含有感兴趣抗体的重链可变区和轻链可变区，或由重链可变区和轻链可变区以及任选的接头组成。重链可变区和轻链可变区之间可通过熟知的接头连接。本文中，接头或铰链是连接不同蛋白或多肽之间的多肽片段，其目的是使所连接的蛋白或多肽保持各自的空间构象，以维持蛋白或多肽的功能或活性。示例性的接头包括含有G和/或S的接头，以及Furin 2A肽(F2A)。接头的长度可以是3-25个氨基酸残基，例如3-15、5-15、10-20个氨基酸残基。在某些实施方案中，接头序列是多甘氨酸接头序列。接头序列中甘氨酸的数量无特别限制，通常为2-20个，例如2-15、2-10、2-8个。除甘氨酸和丝氨酸来，接头中还可含有其它已知的氨基酸残基，例如丙氨酸(A)、亮氨酸(L)、苏氨酸(T)、谷氨酸(E)、苯丙氨酸(F)、精氨酸(R)、谷氨酰胺(Q)等。接头长度通常为15-20个氨基酸。在某些实施方案中，接头为(GGGS)n，n为1-5的整数。
在某些实施方案中，感兴趣的肿瘤抗原是CD19，感兴趣的单链抗体是特异性结合CD19的单链抗体。示例性的特异性结合CD19的单链抗体的氨基酸序列如SEQ ID NO:2第23-267位氨基酸残基所示，其中，重链可变区和轻链可变区通过含G和S的接头序列连接。
CAR中所含的其它部分，如铰链区、跨膜区和胞内信号区可以是常规用于构建各类CAR的铰链区、跨膜区和胞内信号区。
本文中，铰链区指免疫球蛋白重链CH1和CH2功能区之间的区域，该区富含脯氨酸，不形成α螺旋，易发生伸展及一定程度扭曲，有利于抗体的抗原 结合部位与抗原表位间的互补性结合。适用于本文的铰链区可选自CD8胞外铰链区、IgG1 Fc CH2CH3铰链区、IgD铰链区、CD28胞外铰链区、IgG4 Fc CH2CH3铰链区和CD4胞外铰链区。在某些实施方案中，本文使用CD8α铰链区。
本文中，跨膜区可选自CD28跨膜区、CD8跨膜区、CD3ζ跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区和DAP10跨膜区中的一种或多种。优选地，用于本文的嵌合抗原受体的跨膜区为CD8跨膜区。示例性的铰链区和跨膜区的氨基酸序列可如SEQ ID NO:2第268-336位氨基酸残基所示。
本文中，胞内信号区可选自CD28、CD134/OX40、CD137/4-1BB、LCK、ICOS、DAP10、CD3ζ和Fc310/中的任意一种或多种的胞内信号区，优选为4-1BB胞内信号区和CD3ζ胞内信号区。本文示例性的胞内信号区的氨基酸序列可如SEQ ID NO:2第337-490位氨基酸残基所示。
嵌合抗原受体还可包括信号肽。信号肽是引导新合成的蛋白质向分泌通路转移的短肽链(长度5-30个氨基酸)，常指新合成多肽链中用于指导蛋白质的跨膜转移(定位)的N-末端的氨基酸序列。信号肽可以是膜蛋白信号肽，如CD8信号肽、CD28信号肽和CD4信号肽。示例性的信号肽氨基酸序列可如SEQ ID NO:2第1-22位氨基酸残基所示。
因此，本发明嵌合抗原受体的氨基酸序列，从N端到C端，通常为任选的信号肽、靶向感兴趣重链抗原的单链抗体、铰链区、跨膜区和胞内信号区。示例性的嵌合抗原受体的氨基酸序列可如SEQ ID NO:2第23-490位氨基酸残基所示，或者如SEQ ID NO:2第1-490位氨基酸残基所示。
形成本文嵌合抗原受体的上述各部分，如信号肽、单链抗体的轻链可变区和重链可变区、铰链区、跨膜区和胞内信号区等，相互之间可直接连接，或者可通过本领域周知的接头序列连接，例如前文所述的含G和S的接头序列。
本文中，能调控细胞表面HLA-I类分子表达的功能蛋白可以是病毒蛋白，优选是来自其天然宿主细胞不是T细胞的病毒的蛋白。虽然HIV-1是T细胞的天然宿主，且HIV-1nef病毒蛋白能下调T细胞表面的HLA-I类分子的表达，但在慢病毒中引入HIV-1nef蛋白来制备CAR-T时会引起RCL(replication  competent lentivirus)，因此，本发明优选使用来自诸如HSV、BHV-1、EHV-1/4、PRV、HSV-1/2、VZV、EBV、hCMV、mCMV、RhCMV、HHV-6/7、KSHV、MHV-68、牛痘病毒和腺病毒的病毒蛋白，包括但不限于：来自HSV的UL41/vhs蛋白，来自BHV-1、EHV-1/4或PRV的UL49.5，来自HSV-1/2的ICP47，来自VZV的ORF66，来自EBV的EBNA1、BNLF2a、BGLF5和BILF1，来自hCMV的US2/gp24、US3/gp23、US6/gp21、US10和US11/gp33，来自mCMV的m4/gp34、m6/gp48、m27和m152/gp40，来自RhCMV的rh178/VIHCE，来自HHV-6/7的U21和LANA1，来自KSHV的ORF37/SOX、kK3/MIR1和kK5/MIR2，来自MHV-68的mK3，来自牛痘病毒的CPXV012和CPXV203，以及来自腺病毒的E3-19K。
在一些实施方案中，调控细胞表面HLA-I类分子的功能蛋白选自来自HCMV的蛋白US11和来自KSHV的蛋白k5，从而获得的CAR-T细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。
在一些实施方案中，调控细胞表面HLA-I类分子的功能蛋白选自：来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5。对于表达了这些功能蛋白的HLA-E基因型为01:03/01:03的CAR-T细胞，细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控。对于表达了这些功能蛋白的HLA-E基因型为01:01/01:01的CAR-T细胞，细胞表面HLA-A、HLA-B和HLA-E表达均下调。所述功能蛋白优选来自EHV-1的蛋白UL49.5。
优选的功能蛋白是能直接靶向降解MHC I的功能蛋白，或者是能经由TAP蛋白(如抑制TAP，包括阻止TAP蛋白结合ATP和/或诱导TAP蛋白降解)来下调HLA-I类分子表达的功能蛋白，或者是能经由溶酶体来下调HLA-I类分子表达的功能蛋白。示例性的优选蛋白包括但不限于来自HCMV的病毒蛋白，如US11和US6，来自BHV-1的病毒蛋白，如UL49.5，来自EHV-1的病毒蛋白，如UL49.5，以及来自KSHV的病毒蛋白，如k5。在某些实施方案中，本发明使用来自EHV-1的UL49.5，其氨基酸序列可如SEQ ID NO:2第516-615位氨基酸残基所示。
病毒蛋白可通过本领域常用的接头与本发明的CAR相连。例如，在某些 实施方案中，该接头是常规的F2A序列。示例性的F2A的氨基酸序列可如SEQ ID NO:2第494-515位氨基酸残基所示。F2A也可通过常规的含G和S的接头与CAR相连。
本发明的核酸分子可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。本发明的核酸分子可以是CAR的编码序列和能调控细胞表面HLA-I类分子表达的功能蛋白的编码序列，或者是CAR的表达框和该功能蛋白的表达框。本文中，编码序列指核酸序列中直接确定其蛋白产物(例如CAR、单链抗体、铰链区、跨膜区、胞内信号区、病毒蛋白或其融合蛋白等)的氨基酸序列的部分。编码序列的边界通常是由紧邻mRNA 5’端开放读码框上游的核糖体结合位点(对于原核细胞)和紧邻mRNA 3’端开放读码框下游的转录终止序列确定。编码序列可以包括，但不限于DNA、cDNA和重组核酸序列。本文中，表达框指表达感兴趣基因所需的完整元件，包括启动子、基因编码序列和PolyA加尾信号序列。本文所述的核酸分子可以是独立的两个核酸分子，分别含CAR的编码序列和含所述功能蛋白的编码序列，如分别是CAR的表达框和功能蛋白的表达框；或者，所述含CAR的编码序列和所述功能蛋白的编码序列可经由接头连接为一个核酸分子，如CAR的编码序列和功能蛋白的编码序列在同一表达框内，或者是两个表达框经由合适的接头连接为同一核酸分子。在某些实施方案中，本发明的核酸分子为CAR的编码序列和功能蛋白的编码序列同处一表达框的核酸分子，其含有启动子、编码所述嵌合抗原受体和功能蛋白的核酸序列以及PolyA加尾信号。
在某些实施方案中，所述编码序列或表达框整合到CAR-T细胞的基因组中。因此，在这些实施方案中，本文所述的CAR-T细胞的基因组中稳定整合了包含编码本文所述CAR和功能蛋白的表达框。
在某些实施方案中，所述核酸分子是核酸构建物，其含有本文所述CAR和/或功能蛋白的编码序列，以及与这些序列操作性连接的一个或多个调控序列。调控序列可以是合适的启动子序列。启动子序列通常与待表达蛋白的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸 序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。调控序列也可以是合适的转录终止子序列，由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止子都可用于本文。
在某些实施方案中，所述核酸构建物是载体。载体可以是克隆载体，也可以是表达载体，或者是同源重组载体。具体而言，可将本文CAR和/或功能蛋白的编码序列克隆入许多类型的载体，包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。克隆载体可用于提供本发明CAR与功能蛋白的编码序列，如含CAR的编码序列与功能蛋白的编码序列的一个核酸分子。表达载体可以以病毒载体形式提供给细胞。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒等。同源重组载体用于将本文所述的表达框整合到宿主基因组中。
通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。例如，当使用逆转录病毒载体时，逆转录病毒载体通常含有复制起始位点、3’LTR、5’LTR、本文所述融合蛋白的编码序列以及任选的可选择的标记。
合适的启动子包括但不限于即时早期巨细胞病毒(CMV)启动子序列、延伸生长因子-1伸(EF-1因)、类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、EB病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子等。
可选择的标记包括可选择的标记基因或报道基因中的任一个或两者，以便于从被病毒载体感染的细胞群中鉴定和选择表达细胞。有用的可选择标记基因包括例如抗生素抗性基因，如neo。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色荧光蛋白的基因。
本文所述的核酸分子通常可以用PCR扩增法获得。具体而言，可根据本 文所公开的核苷酸序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增得到有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。或者，也可直接合成本文所述的核酸分子。
本文示例性的含CAR与功能蛋白的编码序列的核酸分子的核苷酸序列可如SEQ ID NO:1所示。可采用常规的方法将本文的核酸分子(尤其是载体)导入宿主细胞中，这些方法包括显微注射法、基因枪法、电穿孔法、病毒介导的转化法、电子轰击法、磷酸钙沉淀法等。
本文所述，宿主细胞含有本文所述的核酸分子。宿主细胞既包括最终用于疾病治疗目的的T细胞，也包括生产CAR-T细胞过程中使用到的各种细胞，如大肠杆菌细胞，以用于如提供本发明蛋白的编码序列或提供本文所述的载体。在某些实施方案中，本文提供一种稳定表达本文所述功能蛋白的CAR-T细胞。
本文也包括本文所述的核酸分子。如前文所述，可采用本领域常规的方法制备得到本文所述的核酸分子。在某些实施方案中，本文还包括慢病毒，其包括本文所述的表达框，并能将本文所述的表达框整合到宿主细胞的基因组中。可采用本领域周知的方法制备本文所述的慢病毒。例如，首先制备得到含有本文所述表达框的慢病毒载体，然后在合适的宿主细胞中进行病毒包装，并分离纯化得到所需的慢病毒。用于慢病毒包装的试剂为本领域所周知，如常规的慢病毒载体系统(Tronolab)包括pRsv-REV、pMDlg-pRRE、pMD2G和目的干扰质粒。
本文还包括一种CAR-T细胞培养物，该培养物含有本文所述的CAR-T细胞以及合适的培养基。培养基可以是本领域常规用于培养CAR-T细胞的培养基。
本文还提供一种药物组合物，该药物组合物中含有本文所述的CAR-T细胞以及药学上可接受的辅料。本文中，药学上可接受的辅料是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。更具体而言，合适的药学上可接受的辅料可以是本领域常用于CAR-T细胞给药的辅料。
通常，药物组合物中含有治疗有效量的CAR-T细胞。治疗有效量是指可在受试者中实现治疗、预防、减轻和/或缓解疾病或病症的剂量。可根据患者年龄、性别、所患病症及其严重程度、患者的其它身体状况等因素确定治疗有效量。本文中，受试者或患者通常指哺乳动物，尤其指人。
本文中，适合使用本文所述的核酸分子、CAR-T细胞以及药物组合物治疗的疾病与所述核酸分子以及CAR-T细胞所表达的嵌合抗原受体中的单链抗体有关。因此，本文所述的疾病包括与前文所述的肿瘤抗原相关的各类癌症，包括实体瘤和血液肿瘤，如腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌和前列腺癌等实体瘤，以及白血病和淋巴瘤，如B细胞淋巴瘤、套细胞淋巴瘤、急性淋巴细胞白血病、慢性淋巴细胞白血病、多毛细胞白血病和急性髓性白血病等。
在某些实施方案中，本文还提供了一种试剂盒，所述试剂盒含有本文所述的载体。试剂盒还可含有适用于将所述载体转染入细胞中的各种试剂，以及任选的指导本领域技术人员将所述重组表达载体转染入细胞的说明书。
本发明还提供一种疾病治疗方法，所述方法包括给予需要的对象治疗有效量的本文任一实施方案所述的工程改造的T细胞，优选是本文任一实施方案所述的工程改造的CAR-T细胞。可按照现有的细胞免疫疗法，根据患者的病症、严重程度、年龄、性别等因素，通过合适的方式(如静脉输注)给药。优选地，所述疾病为本文任一实施方案所述的疾病。在一些实施方案中，所述患者T细胞的HLA-E的基因型为01:03/01:03，给予所述患者含有选自来自HCMV的蛋白US11、来自KSHV的蛋白k5、来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5中的一种或多种和/或含有这些蛋白中的一种或多种的编码序列的T细胞，优选CAR-T细胞。在一些实施方案中，所述患者T细胞的HLA-E的基因型为或01:01/01:01，给予所述患者含有来自HCMV的蛋白US11和/或来自KSHV的蛋白k5和/或含有这些蛋白中的一种或两种的编码序列的T细胞，优选CAR-T细胞。
本发明包括如下实施方案：
项目1、一种CAR-T细胞，其特征在于，所述CAR-T细胞表达嵌合抗原 受体和能下调细胞表面HLA-I类分子表达的功能蛋白；优选地，表达所述嵌合抗原受体和功能蛋白的所述CAR-T细胞细胞表面HLA-I类分子的表达水平为表达相同嵌合抗原受体但未表达所述功能蛋白的对照CAR-T细胞的50％以下。
项目2、如项目1所述的CAR-T细胞，其特征在于，所述CAR-T细胞含有所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列；优选地，所述CAR-T细胞含有所述嵌合抗原受体的表达框和所述功能蛋白的表达框，或所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列处于同一表达框内。
项目3、如项目1或2所述的CAR-T细胞，其特征在于，
所述嵌合抗原受体特异性结合选自以下的肿瘤抗原中的一种或多种：EGFRvIII、间皮素、GD2、Tn抗原、sTn抗原、Tn-O-糖肽、sTn-O-糖肽、PSMA、CD97、TAG72、CD44v6、CEA、EPCAM、KIT、IL-13Ra2、leguman、GD3、CD171、IL-11Ra、PSCA、MAD-CT-1、MAD-CT-2、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、叶酸受体α、ERBB、Her2/neu、MUC1、EGFR、NCAM、肝配蛋白B2、CAIX、LMP2、sLe、HMWMAA、o-乙酰基-GD2、叶酸受体β、TEM1/CD248、TEM7R、FAP、豆荚蛋白、HPV E6或E7、ML-IAP、CLDN6、TSHR、GPRC5D、ALK、聚唾液酸、Fos-相关抗原、中性粒细胞弹性蛋白酶、TRP-2、CYP1B1、精子蛋白17、β人绒毛膜促性腺激素、AFP、甲状腺球蛋白、PLAC1、globoH、RAGE1、MN-CA IX、人端粒酶逆转录酶、肠羧基酯酶、mut hsp 70-2、NA-17、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、NY-ESO-1、GPR20、Ly6k、OR51E2、TARP、GFRα4和呈递在MHC上的这些抗原中任一者的多肽片段，以及CD5、CD10、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD27、CD30、CD34、CD37、CD38、CD40、CD53、CD69、CD72、CD73、CD74、CD75、CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85、CD86、CD123、CD135、CD138、CD179、CD269、Flt3、ROR1、BCMA、FcRn5、FcRn2、CS-1、CXCR4、CXCR5、CXCR7、IL-7/3R、IL7/4/3R和IL4R；或
所述能下调细胞表面HLA-I类分子表达的功能蛋白选自：HSV、BHV-1、EHV-1/4、PRV、HSV-1/2、VZV、EBV、hCMV、mCMV、RhCMV、HHV-6/7、 KSHV、MHV-68、牛痘病毒和腺病毒中能直接靶向降解HLA-I的功能蛋白、能经由TAP蛋白来下调HLA-I类分子表达的功能蛋白和能经由溶酶体来下调HLA-I类分子表达的功能蛋白；优选地，所述功能蛋白选自：来自HCMV的蛋白US11和US6、来自BHV-1的蛋白UL49.5、来自EHV-1的蛋白UL49.5和来自KSHV的蛋白k5。
项目4、一种核酸分子，其特征在于，所述核酸分子选自：
(1)含嵌合抗原受体的编码序列和能下调细胞表面HLA-I类分子表达的功能蛋白的编码序列的核酸分子；和
(2)(1)所述核酸分子的互补序列；
优选地，所述嵌合抗原受体和所述功能蛋白如项目3所述。
项目5、一种核酸构建物，其特征在于，所述核酸构建物含有项目4所述的核酸分子。
项目6、如项目5所述的核酸构建物，其特征在于，
所述核酸构建物含有所述嵌合抗原受体的表达框和所述功能蛋白的表达框；或所述核酸构建物为一表达框，其中所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列处于该表达框内；或
所述核酸构建物是克隆载体或表达载体。
项目7、一种慢病毒，其含有项目5或6所述的核酸构建物。
项目8、一种宿主细胞，其含有项目4所述的核酸分子或项目5或6所述的核酸构建物或项目7所述的慢病毒。
项目9、一种药物组合物，其特征在于，所述药物组合物含有项目1-3中任一项所述的CAR-T细胞。
项目10、项目3所述的功能蛋白或其编码序列在制备细胞表面的HLA-I类分子的表达下调的CAR-T细胞中的应用，或在制备癌症治疗用的CAR-T细胞中的应用。
下面将结合实施案例对本发明所涉及的实施方案进行详细描述。本领域技术人员将会理解，下面的实施案例仅用于说明本发明，而不应视为限定本发明的范围。实施案例中未注明具体技术或条件者，按照本领域内的文献所描述的 技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
一.实验材料和结果
(1)仪器和材料：
仪器：生物安全柜(海尔、HR40-IIA2)，CO 2培养箱(Thermo、3111)，流式细胞仪(BD、FACSCantoll)，酶标仪(Molecular Derices、SpectraMax M4)
试剂：抗人Anti-HLA-I抗体(APC)(Biolegend、311410)，抗人HLA-A抗体(Bio-Techne、NBP2-45320)，抗人HLA-B抗体(Bio-Techne、NBP2-45000)，抗人HLA-E抗体(Biolegend、342612)，Anti-CD3抗体(BV421)(Biolegend、300434)，Anti-TCR抗体(PE-Cy7)(Biolegend、306720)，FBS(Lonsera、S711-001S)，X-vivo15(Lonza、04-418Q)，Dynabeads CD3/CD28(Lifetechnology、40203D)，Ficoll(达优、DKW-LSH-0250)，Tscm(Novoprotein，GMP-1647)，Novonectin(Novoprotein、GMP-CH38)，抗人CCR7(BV421)(BD、562555)，抗人CD45RA(PE-Cy7)(BD、560675)，Luciferase检测(Promega、E6120)，抗人CD3(FITC)(BD、562555)，抗人CD4(BV510)(BD、563094)，抗人CD8(APC-Cy7)(BD、557834)，抗人CD25(PE)(BD、555432)、抗人CD69(APC)(BD、553237)、CAR19特异性单抗(自标)，IFN-gamma ELISA检测试剂盒(R&D、DIF50)。
(2)测试方法
实验方法
1)载体构建
从NCBI网站数据库搜索到人CD8α铰链区、人CD8跨膜区、41BB胞内区、人CD3、和EHV1 UL49.5基因序列信息，抗CD19单链抗体克隆号为FMC63，这些序列在网站https://www.thermofisher.com/order/geneartgenes上进行密码子优化，保证在编码氨基酸序列不变的情况下更适合人类细胞表达。
采用重叠PCR将上述序列依次按抗CD19-scFv基因、人CD8铰链区基因、 人CD8跨膜区基因、41BB胞内区基因、人CD3胞内区、F2A和EHV1 UL49.5基因序列进行连接，形成完整的CD19-CAR-F2A-EHV1 UL49.5基因序列信息(其包含信号肽编码序列的序列如SEQ ID NO:1所示，氨基酸序列如SEQ ID NO:2所示)。
该CAR分子的核苷酸序列经无缝克隆到慢病毒质粒pWPXL(Addgene)的Bamh1-Ecor1位点，转化到感受态大肠杆菌(DH5α，北京博迈德生物技术有限公司)。
将重组质粒送苏州金唯智生物科技有限公司进行测序，将测序结果与拟合成的CD19-CAR-F2A-EHV1 UL49.5序列比对来验证序列是否正确。测序引物为TCAAGCCTCAGACAGTGGTTC(SEQ ID NO:3)。
经测序正确后，使用Qiagen公司的质粒纯化试剂盒提取并纯化质粒，采用磷酸钙法将纯化的质粒转染293T细胞，进行慢病毒包装实验(Molecular Therapy-Methods&Clinical Development,2016,3:16017)，由此制备得到的慢病毒命名为PCTL206。
采用相同的方法，但用不同的病毒蛋白替换PCTL206中的病毒蛋白，制备得到慢病毒PCTL135(CAR19-F2A-GFP，对照)、PCTL199(CAR19-F2A-HCMV US2)、PCTL200(CAR19-F2A-HCMV US11)、PCTL201(CAR19-F2A-HCMV US6)、PCTL202(CAR19-F2A-HSV-1 ICP47)、PCTL203(CAR19-F2A-5 E3-19K)、PCTL204(CAR19-F2A-RhCMV Rh178)、PCTL205(CAR19-F2A-BHV-1 UL49.5)、PCTL207(CAR19-F2A-EBV BNLF2a)、PCTL208(CAR19-F2A-CPXV012)、PCTL209(CAR19-F2A-HCMV US3)、PCTL210(CAR19-F2A-MHV68 mK3)、PCTL211(CAR19-F2A-CPXV203)、PCTL212(CAR19-F2A-KSHV k3)、PCTL213(CAR19-F2A-KSHV k5)，其中病毒蛋白HCMV US2、HSV-1 ICP47、人腺病毒5E3-19K、RhCMV Rh178、EBV BNLF2a、牛痘病毒CPXV012、HCMV US3、MHV68 mK3、牛痘病毒CPXV203、KSHV k3、HCMV US11、HCMV US6、BHV-1 UL49.5和KSHV k5的氨基酸序列分别如以下UniprotKB登陆号所示的氨基酸序列所示：C8CFI0、P03170、Q8BEL5、Q7TFG4、P0C739、U5TIW7、Q910T7、O41933、G0XWR7、 A0A386AVI8、P09727、P14334、Q77CE4和F5H9K4。更具体而言，HCMV US11的编码序列如SEQ ID NO:4第1546-2193位碱基所示；HCMV US6的编码序列如SEQ ID NO:5第1546-2097位碱基所示；BHV-1 UL49.5的编码序列如SEQ ID NO:6第1546-2097位碱基所示；KSHV k5的编码序列如SEQ ID NO:7第1546-2316位碱基所示。
2)外周血PBMC的分离、T细胞分离活化、慢病毒转导、体外培养
选择HBV、HCV和HIV检测阴性的健康供者，肘正中静脉抽血100ml，Ficoll密度梯度离心分离PBMC白膜层，根据全血流式检测CD3+T细胞百分比，计算CD3+T细胞数，按DynaBeads CD3/CD28与CD3+T细胞比例3：1，吸取使用量磁珠，与白膜层细胞孵育30min，分离CD3+T细胞，CD3+T细胞经Dynabeads CD3/CD28(Lifetechnology、40203D)活化24小时后流式检测CD25+CD69+T细胞比例。CD3+T活化后，进行慢病毒转导。用Novonectin包被24孔板37℃孵育2小时，将细胞悬液与分别与前述制备得到的各种慢病毒(MOI＝8)、F108(10ug/ml)、Tscm(2U/ml)配置成转导体系置于包被的24孔板中，细胞密度调整至1.0E+06/ml，500g离心30min，离心后37℃CO 2培养箱静置培养48h。转染后以含5％FBS Xvivo15培养液培养，隔日补充Tscm(终浓度2U/ml)，计数细胞，调整细胞密度至0.5E+06/ml，培养至第8-10天收获细胞。
3)靶细胞刺激各组CAR-T细胞
各组CAR-T细胞体外培养至8-10天，计数细胞取1.0E+07细胞，调整细胞密度至1.0E+06/ml，以Xvivo15培养(不含Tscm)，按效靶比10：1添加靶细胞K562-CD19刺激CAR-T细胞，连续两天反复刺激两次，流式检测各组细胞CAR阳性率以及CAR阳性细胞HLA-I平均荧光强度。
4)流式检测各组CAR-T细胞CAR阳性率以及CAR +T细胞HLA-I平均荧光强度
计数各组CAR-T细胞，分别取5.0E+05细胞于不同1.5ml EP管，2000rpm，5min离心收集细胞，弃去培养液，用无菌4％BSA重悬洗涤细胞2次，后用100ul 4％BSA重悬细胞，每管细胞加入抗人Anti-HLA-I抗体(APC)(Biolegend，Cat#：311410)抗体8ul，旋涡混匀，4℃孵育30min；染色完毕，重复洗涤细胞，将CAR19特异性抗体1：500稀释，用稀释后的抗体溶液重悬细胞，每管200ul，旋涡混匀，4℃孵育30min，染色完毕，重复洗涤细胞，500ul 4％BSA重悬细胞，每管加入7AAD抗体4ul，旋涡混匀，常温避光孵育10min，孵育完成后，转移至流式管，上机检测。
5)细胞表型分析
计数各组CAR-T细胞，分别取1.0E+06细胞于不同1.5ml EP管，2000rpm，5min离心收集细胞，弃去培养液，用无菌4％BSA重悬洗涤细胞2次，后用200ul 4％BSA重悬细胞，样本管每管加入抗人CD45RA(PE-Cy7)、抗人CCR7(BV421)抗体各5ul，旋涡混匀仪混匀，4℃孵育30min，染色完毕，重复洗涤细胞，用500ul 4％BSA重悬细胞，每管加入7AAD抗体4ul，旋涡混匀，常温避光孵育10min，孵育完成后，转移至流式管，上机检测。
6)细胞杀伤
取出NC-T(未进行慢病毒转染的T细胞)、各组CAR-T细胞，于显微镜下观察细胞生长状态是否正常，吹打混匀，将NC-T、各组CAR-T细胞收集于离心管中，计数细胞，离心收集细胞，用T细胞培养液X-VIVO15(不含Tscm)重悬离心收集的细胞沉淀，并将细胞密度调整至5.0E+07细胞/mL；取出靶细胞，于显微镜下观察细胞状态是否正常，将靶细胞分别收集于15mL或50mL离心管中，并计数细胞，用RPMI 1640(不含FBS)重悬离心收集的细胞沉淀，并将细胞密度调整至5.0E+06细胞/mL；于1.5mL离心管中将上述调整好密度的效应细胞NC-T和CAR-T分别与靶细胞按不同效靶比(1:1、2.5:1、5:1、10:1、20:1)混合，用X-VIVO15将总体积补足200积补(靶细胞10ul，效应细胞量根据效靶比确定)，将上述配制的200配制杀伤体系分别移入96孔V型板中 共孵育24小时后，轻轻吹打混匀96孔板V型板中各孔细胞，并分别转移100μ0细胞悬液入白壁底不透96孔板中，加入80μL ONE-Glo TM Luciferase Assay Substrate，吹吸混匀，室温避光孵育10分钟后上Luminoskan Ascent化学发光分析仪检测荧光强度。
二.实验结果
1、筛选具有下调CAR-T细胞表面HLA-I类分子表达的功能蛋白
选取14种具有下调细胞表面功能的病毒蛋白合成构建到CAR19的慢病毒中，各组慢病毒转染T细胞，体外培养T细胞至第8天，流式检测CAR19转染效率以及CAR19 +T细胞群体HLA-I平均荧光强度(图1，表1)。
表1：各组T细胞HLA-I平均荧光强度
Figure PCTCN2020086032-appb-000002
体外培养第9天，用靶细胞K562-CD19刺激各组T细胞，效靶比10：1，连续反复刺激2天，流式检测各组T细胞CAR19+细胞群体HLA-I平均荧光强度，结果如图2所示。结果显示，PCTL200、PCTL201、PCTL205、PCTL206、 PCTL213这5种慢病毒具有较强的制备HLA-I类分子表达下调的CAR-T细胞的能力。
2、PCTL200、PCTL201、PCTL205、PCTL206、PCTL213分子的功能确定
1)外周血分离PBMC及T细胞活化
100ml外周血分离1.24E+08 PBMC，流式检测外周血CD3 +T、CD4 +T、CD8 +T比例，其中CD3 +T占白膜层细胞(单核细胞和淋巴细胞)62.4％，CD4 +T/CD8 +T为1.2，DynaBeads CD3/CD28分选并活化CD3 +T，24h后活化效率为68.9％(图3)。
2)T细胞转染效率、HLA-I平均荧光强度、体外扩增倍数、细胞毒性实验、T细胞表型分析
各组慢病毒转染T细胞，隔日计数T细胞，体外培养T细胞至第8天，流式检测CAR19转染效率，同时计算细胞增殖倍数(第8天的细胞数/用于转染的细胞数)。结果如下表2和图4所示，其中PCTL135组的CAR19阳性率为65.6％，PCTL200组的CAR19阳性率为37.4％，PCTL201组的CAR19阳性率为46％，PCTL205组的CAR19阳性率为55.8％，PCTL206组的CAR19阳性率为54.6％，PCTL213组的CAR19阳性率为55.7％。
表2：各组T细胞转染效率及扩增倍数
组别 病毒载体 滴度 转染效率(第8天) 扩增倍数
1 PCTL135 1.36E+07 65.6％ 165.60
2 PCTL200 7.20E+07 37.4％ 284.40
3 PCTL201 1.40E+08 46％ 206.15
4 PCTL205 1.32E+08 55.8％ 477.00
5 PCTL206 1.22E+08 54.6％ 274.00
6 PCTL213 1.04E+08 55.7％ 306.25
流式分析CAR19 +T细胞群体HLA-I平均荧光强度。结果如表3和图5所示，各实验组T细胞HLA-I平均荧光强度均较对照组PCTL135显著下调，表明测试的5种病毒蛋白能下调T细胞表面的HLA-I类分子的表达，可以通过表达相关病毒蛋白靶向抑制HLA-I类分子的技术制备同种异体CAR-T。
表3：各组T细胞HLA-I平均荧光强度
Figure PCTCN2020086032-appb-000003
各组T细胞体外培养至第8天，与靶细胞系k562-CD19-luc分别按效靶比20：1、10：1、5：1、2.5：1、1：1共培养24h，经荧光素酶报告基因检测系统检测各组T细胞细胞毒性，结果如表4和图6所示。
表4：各组T细胞体外杀伤效率
效靶比 PCTL135 PCTL200 PCTL201 PCTL205 PCTL206 PCTL213
1:1 -247％ 46％ 68％ 45％ -6％ 3％
2.5:1 -42％ 93％ 81％ 94％ 90％ 83％
5:1 91％ 99％ 95％ 99％ 97％ 97％
10:1 95％ 100％ 99％ 99％ 95％ 99％
20:1 95％ 100％ 99％ 100％ 100％ 100％
各组T细胞体外培养至第8天，经流式检测各组T细胞CCR7和CD45RA表达，分析T细胞表型，各组细胞表型无明显差异，约60％细胞在T-naive分化阶段(图7)。
3)经靶细胞刺激，各组T细胞HLA-I平均荧光强度
体外培养第9天，用靶细胞K562-CD19刺激各组T细胞，效靶比10：1，连续反复刺激2天，流式检测各组T细胞CAR19 +细胞群体HLA-I平均荧光强度，结果如表5和图8所示。结果显示，各组HLA-I平均荧光强度显著低于对照组，其中PCTL206组最显著。
表5：经靶细胞刺激各组T细胞HLA-I平均荧光强度
Figure PCTCN2020086032-appb-000004
3、PCTL200、PCTL213对HLA-I不同亚型的表达调节
图9和表6-7显示CAR19+T细胞群体中各组对不同HLA-I分子(HLA-A、HLA-B和HLA-E)的平均荧光强度以及与对照的比值。PCTL135组(对照)、PCTL200组和PCTL213组的结果显示，与对照相比，PCTL200组、PCTL213组显著下调细胞表面的HLA-A和HLA-B类分子，但HLA-E类分子与对照相比无显著差异。
表6：各组T细胞HLA平均荧光强度
Figure PCTCN2020086032-appb-000005
表7：各组T细胞HLA平均荧光强度与对照的比值
Figure PCTCN2020086032-appb-000006
4、在不同HLA-E基因型的T细胞中不同HLA-I亚型的表达调节
图9-11和表8-11显示不同HLA-E基因型的CAR19+T细胞群体中各种HLA-I分子(HLA-A、HLA-B和HLA-E)的平均荧光强度以及与对照的比值。可以看出，PCTL201、PCTL205和PCTL206组对不同的HLA-E基因型的T细胞具有选择性：对于HLA-E基因型为纯合01:03/01:03的CAR19+T细胞群体，PCTL206组下调细胞表面的HLA-A和HLA-B类分子，但HLA-E类分子与对 照相比无显著差异(图11)；对于HLA-E基因型为纯合01:01/01:01的CAR19+T细胞群体，PCTL206组下调细胞表面的HLA-A、HLA-B和HLA-E类分子(图10)。
表8：各组T细胞HLA平均荧光强度(图10)
Figure PCTCN2020086032-appb-000007
表9：各组T细胞HLA平均荧光强度与对照的比值(图10)
Figure PCTCN2020086032-appb-000008
表10：各组T细胞HLA平均荧光强度(图11)
Figure PCTCN2020086032-appb-000009
表11：各组T细胞HLA平均荧光强度与对照的比值(图11)
Figure PCTCN2020086032-appb-000010

Claims (13)

  1. 一种工程改造的T细胞，其特征在于，所述工程改造的T细胞表达能差异化调控细胞表面HLA-I类分子表达的功能蛋白，其中，所述工程改造的T细胞的细胞表面HLA-A和HLA-B的表达水平为未表达所述功能蛋白的T细胞的50％或以下，优选5-50％；其中，所述工程改造的T细胞的细胞表面HLA-E的表达水平为未表达所述功能蛋白的T细胞的50％以上，优选60-100％。
  2. 如权利要求1所述的工程改造的T细胞，其特征在于，所述工程改造的T细胞进一步表达嵌合抗原受体，从而工程改造的T细胞是CAR-T细胞，其中，所述CAR-T细胞含有所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列；优选地，所述CAR-T细胞含有所述嵌合抗原受体的表达框和所述功能蛋白的表达框，或所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列处于同一表达框内。
  3. 如权利要求2所述的工程改造的T细胞，其特征在于，
    所述嵌合抗原受体特异性结合选自以下的肿瘤抗原中的一种或多种：EGFRvIII、间皮素、GD2、Tn抗原、sTn抗原、Tn-O-糖肽、sTn-O-糖肽、PSMA、CD97、TAG72、CD44v6、CEA、EPCAM、KIT、IL-13Ra2、leguman、GD3、CD171、IL-11Ra、PSCA、MAD-CT-1、MAD-CT-2、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、叶酸受体α、ERBB、Her2/neu、MUC1、EGFR、NCAM、肝配蛋白B2、CAIX、LMP2、sLe、HMWMAA、o-乙酰基-GD2、叶酸受体β、TEM1/CD248、TEM7R、FAP、豆荚蛋白、HPV E6或E7、ML-IAP、CLDN6、TSHR、GPRC5D、ALK、聚唾液酸、Fos-相关抗原、中性粒细胞弹性蛋白酶、TRP-2、CYP1B1、精子蛋白17、β人绒毛膜促性腺激素、AFP、甲状腺球蛋白、PLAC1、globoH、RAGE1、MN-CA IX、人端粒酶逆转录酶、肠羧基酯酶、mut hsp 70-2、NA-17、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、NY-ESO-1、GPR20、Ly6k、OR51E2、TARP、GFRα4和呈递在MHC上的这些抗原中任一 者的多肽片段，以及CD5、CD10、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD27、CD30、CD34、CD37、CD38、CD40、CD53、CD69、CD72、CD73、CD74、CD75、CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85、CD86、CD123、CD135、CD138、CD179、CD269、Flt3、ROR1、BCMA、FcRn5、FcRn2、CS-1、CXCR4、CXCR5、CXCR7、IL-7/3R、IL7/4/3R和IL4R。
  4. 如权利要求1-3中任一项所述的工程改造的T细胞，其特征在于，所述调控细胞表面HLA-I类分子表达的功能蛋白选自：HSV、BHV-1、EHV-1/4、PRV、HSV-1/2、VZV、EBV、hCMV、mCMV、RhCMV、HHV-6/7、KSHV、MHV-68、牛痘病毒和腺病毒中能直接靶向降解HLA-I的功能蛋白、能经由TAP蛋白来下调HLA-I类分子表达的功能蛋白和能经由溶酶体来下调HLA-I类分子表达的功能蛋白；优选地，所述功能蛋白选自：来自HCMV的蛋白US11和US6、来自BHV-1的蛋白UL49.5、来自EHV-1的蛋白UL49.5和来自KSHV的蛋白k5。
  5. 如权利要求1-3中任一项所述的工程改造的T细胞，其特征在于，
    所述功能蛋白选自来自HCMV的蛋白US11和来自KSHV的蛋白k5，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控，或
    所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述工程改造的T细胞的HLA-E的基因型为01:03/01:03，且所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控，或
    所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述工程改造的T细胞的HLA-E的基因型为01:01/01:01，且所述工程改造的T细胞的细胞表面HLA-A、HLA-B和HLA-E表达下调。
  6. 一种核酸分子，其特征在于，所述核酸分子选自：
    (1)含嵌合抗原受体的编码序列和能调控细胞表面HLA-I类分子表达的功能蛋白的编码序列的核酸分子；和
    (2)(1)所述核酸分子的互补序列；
    优选地，所述嵌合抗原受体和所述功能蛋白如权利要求3-5中所述。
  7. 一种核酸构建物，其特征在于，所述核酸构建物含有权利要求4所述的核酸分子。
  8. 如权利要求7所述的核酸构建物，其特征在于，
    所述核酸构建物含有所述嵌合抗原受体的表达框和所述功能蛋白的表达框；或所述核酸构建物为一表达框，其中所述嵌合抗原受体的编码序列和所述功能蛋白的编码序列处于该表达框内；或
    所述核酸构建物是克隆载体或表达载体。
  9. 一种慢病毒，其含有权利要求7或8所述的核酸构建物。
  10. 一种宿主细胞，其含有权利要求6所述的核酸分子或权利要求7或8所述的核酸构建物或权利要求9所述的慢病毒。
  11. 一种药物组合物，其特征在于，所述药物组合物含有权利要求1-5中任一项所述的工程改造的T细胞。
  12. 权利要求4-5中所述的功能蛋白或其编码序列在制备细胞表面的HLA-I类分子的表达调控的工程改造的T细胞中的应用，或在制备癌症治疗用的工程改造的T细胞中的应用，其中，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控，或所述工程改造的T细胞的细胞表面HLA-A、HLA-B和HLA-E表达下调。
  13. 如权利要求12所述的应用，其特征在于，
    所述功能蛋白选自来自HCMV的蛋白US11和来自KSHV的蛋白k5，所述工程改造的T细胞的细胞表面HLA-A和HLA-B表达与HLA-E表达被差异化调控，或
    所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述工程改造的T细胞的HLA-E的基因型为01:03/01:03，或
    所述功能蛋白选自来自HCMV的蛋白US6、来自BHV-1的蛋白UL49.5和来自EHV-1的蛋白UL49.5，所述工程改造的T细胞的HLA-E的基因型为01:01/01:01。
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