WO2023138661A9 - Nkg2a抗体及其应用 - Google Patents

Nkg2a抗体及其应用 Download PDF

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WO2023138661A9
WO2023138661A9 PCT/CN2023/073176 CN2023073176W WO2023138661A9 WO 2023138661 A9 WO2023138661 A9 WO 2023138661A9 CN 2023073176 W CN2023073176 W CN 2023073176W WO 2023138661 A9 WO2023138661 A9 WO 2023138661A9
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antibody
seq
cells
nkg2a
amino acid
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PCT/CN2023/073176
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French (fr)
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WO2023138661A1 (zh
WO2023138661A8 (zh
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周亮
王鹏
李宗海
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恺兴生命科技(上海)有限公司
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Priority to CN202380017128.6A priority Critical patent/CN118574853A/zh
Publication of WO2023138661A1 publication Critical patent/WO2023138661A1/zh
Publication of WO2023138661A9 publication Critical patent/WO2023138661A9/zh
Publication of WO2023138661A8 publication Critical patent/WO2023138661A8/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of tumor immunotherapy or diagnosis, and more specifically, to antibodies that recognize NKG2A and their applications.
  • NK cells Natural killer cells
  • NK cells are a type of lymphocyte that can non-specifically kill tumor cells and virus-infected cells without prior sensitization. They are one of the important components of the body's immune cells. There are many receptors on the surface of NK cells, which are divided into two categories: activating and inhibitory. NKG2A is one of the inhibitory receptors.
  • NKG2A protein also called C-type lectin, is expressed not only in NK cells, but also in NKT cells and CD8+ ⁇ T cells. NKG2A can form dimers with CD94 on the cell surface (Jiacheng Bi and Zhigang Tian.NK Cell Dysfunction and Checkpoint Immunotherapy. Front. Immunol, 2019.).
  • the non-classical MHC class I molecule HLA-E is the main ligand of NKG2A-CD94.
  • HLA-E Under normal circumstances, the expression of HLA-E is very low, but on the surface of most tumor cells, the expression of HLA-E increases, and NKG2A and The interaction of HLA-E inhibits the activation of NK cells, allowing tumor cells to avoid being killed by NK cells (Linda Borst, et al. The NKG2A-HLA-E axis as a novel checkpoint in the tumor microenvironment. American Association for Cancer, 2020 .). Therefore, the development of antibodies targeting NKG2A can block the binding of NKG2A to HLA-E, thereby activating NK cells and T cells.
  • NKG2A antibodies have great potential, their development is extremely challenging.
  • the NKG2 receptor family has multiple receptors, including NKG2A, NKG2C, NKG2D, and NKG2E, etc. Some of these receptors have inhibitory effects on immune cells, and some have activating effects.
  • the amino acid sequences of these receptors have a high degree of homology, with 90% homology between human NKG2A and NKG2C and 71% homology with NKG2E.
  • NKG2C and NKG2E are very similar to NKG2A in their extracellular sequences, they are completely opposite in function and require high antibody specificity.
  • NKG2A target monoclonal antibody drugs mainly including Monalizumab (US20170298131A1) from Innate/AstraZeneca and Mpb416 (CN111153995A) from Huaiyue Biotechnology.
  • Monalizumab US20170298131A1
  • Mpb416 CN111153995A
  • Huaiyue Biotechnology Huaiyue Biotechnology.
  • Preliminary clinical results show that the combination of NKG2A antibodies has a certain anti-tumor effect.
  • single drugs are less effective and have certain side effects.
  • the purpose of the present invention is to provide fully human antibodies that recognize NKG2A and their applications.
  • the present invention provides a fully human antibody that recognizes NKG2A, characterized in that the antibody includes a light chain variable region, and the light chain variable region includes RASQSISSWLA (SEQ ID NO: 4). and/or LCDR2 shown in DASSLES (SEQ ID NO:5); and/or LCDR3 shown in QQYDSYX 1 is R or S.
  • the antibody comprises a light chain variable region
  • the light chain variable region comprises LCDR1 shown in RASQSISSWLA (SEQ ID NO:4); and/or DASSLES (SEQ ID NO:5) LCDR2; and/or LCDR3 shown in QQYDSYIRT (SEQ ID NO:6).
  • the antibody comprises a light chain variable region
  • the light chain variable region comprises LCDR1 shown in RASQSISSWLA (SEQ ID NO:4); and/or DASSLES (SEQ ID NO:5) LCDR2; and/or LCDR3 shown in QQYDSYVST (SEQ ID NO:10).
  • the invention also provides a fully human antibody that recognizes NKG2A, characterized in that the antibody includes a heavy chain variable region, and the heavy chain variable region is selected from:
  • HCDR1 shown in SYAIS SEQ ID NO: 1
  • HCDR2 shown in GIIPIFGTAX 1 YAQKFQG SEQ ID NO: 130
  • X 1 is N or H
  • GFDGMDY SEQ ID HCDR3 shown in NO:3
  • ( 2) Contains HCDR1 represented by X 1 X 2 X 3 X 4 S (SEQ ID NO: 131), where X 1 is S, R or N, Or H, X 4 is M or V; and/ or HCDR2 shown in AIX 1 , X 3 is S, W, G or P, X 4 is G or V; and/or HCDR3 represented by GYDGFDY (SEQ ID NO: 9).
  • the antibody includes a heavy chain variable region comprising HCDR1 represented by SYAIS (SEQ ID NO: 1); and/or GIIPIFGTANYAQKFQG (SEQ ID NO: 2) HCDR2; and/or HCDR3 shown in GFDGMDY (SEQ ID NO:3).
  • the antibody includes a heavy chain variable region comprising HCDR1 shown in SYAIS (SEQ ID NO: 1); and/or GIIPIFGTAHYAQKFQG (SEQ ID NO: 11) HCDR2; and/or HCDR3 shown in GFDGMDY (SEQ ID NO:3).
  • the antibody includes a heavy chain variable region comprising HCDR1 represented by SYAMS (SEQ ID NO:7); and/or AISGSGGSTYYADSVKG (SEQ ID NO:8) HCDR2; and/or HCDR3 shown in GYDGFDY (SEQ ID NO:9).
  • the antibody includes a heavy chain variable region comprising HCDR1 shown in RFYMS (SEQ ID NO: 12); and/or AITGWGGSTYYADSVKG (SEQ ID NO: 13) HCDR2; and/or HCDR3 shown in GYDGFDY (SEQ ID NO:9).
  • the antibody includes a heavy chain variable region comprising HCDR1 shown in RVHMS (SEQ ID NO: 14); and/or AISAGGGSTYYADSVKG (SEQ ID NO: 15) HCDR2; and/or HCDR3 shown in GYDGFDY (SEQ ID NO:9).
  • the antibody includes a heavy chain variable region comprising HCDR1 shown in NFHVS (SEQ ID NO:16); and/or AINGPVGSTYYADSVKG (SEQ ID NO:17) HCDR2; and/or HCDR3 shown in GYDGFDY (SEQ ID NO:9).
  • the antibody is selected from any of the following:
  • An antibody comprising a heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 1, 7, 12, 14 or 16, and/or comprising SEQ ID NO: 2, 8 , HCDR2 shown in 11, 13, 15 or 17, and/or containing HCDR3 shown in any of SEQ ID NO: 3 or 9;
  • An antibody comprising a light chain variable region comprising LCDR1 shown in SEQ ID NO:4, and/or LCDR2 shown in SEQ ID NO:5, and/or comprising SEQ LCDR3 shown in either ID NO: 6 or 10;
  • An antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody;
  • the antibody is selected from any of the following:
  • Antibody which contains HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4, SEQ ID NO LCDR2 shown in :5 and LCDR3 shown in SEQ ID NO:6; or
  • Antibody which contains HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, SEQ ID NO LCDR2 shown in :5 and LCDR3 shown in SEQ ID NO:10; or
  • Antibody which contains HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:11 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4, SEQ ID NO LCDR2 shown in :5 and LCDR3 shown in SEQ ID NO:6; or
  • Antibody which contains HCDR1 shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO:13 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, SEQ ID NO LCDR2 shown in :5 and LCDR3 shown in SEQ ID NO:10; or
  • Antibody which contains HCDR1 shown in SEQ ID NO:14, HCDR2 shown in SEQ ID NO:15 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, SEQ ID NO LCDR2 shown in :5 and LCDR3 shown in SEQ ID NO:10; or
  • Antibody which contains HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, SEQ ID NO LCDR2 shown in :5 and LCDR3 shown in SEQ ID NO:10;
  • An antibody a variant of the antibody according to any one of (1) to (6), and having the same or similar activity as the antibody according to any one of (1) to (6).
  • the antibody is selected from any of the following:
  • An antibody comprising a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 28, 30 or 32, or a variant of the above sequence;
  • Antibody comprising a light chain variable region, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 or 24, or a variant of the above sequence;
  • An antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody.
  • the antibody is selected from any of the following:
  • an antibody the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:18, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:20;
  • An antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 30, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 24;
  • An antibody the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 32, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 24.
  • An antibody a variant of the antibody according to any one of (1) to (6), and having the same or similar activity as the antibody according to any one of (1) to (6).
  • the antibody described in the first aspect is a full antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') 2 fragment, Fd fragment, dAb fragment, multifunctional antibody or IgG4 antibodies.
  • the antibody of the first aspect does not significantly bind NKG2C, NKG2E, or a combination thereof.
  • the antibody of the first aspect binds to NKG2A/CD94 and does not significantly bind to NKG2C/CD94, NKG2E/CD94, or combinations thereof.
  • the antibody of the first aspect binds to cells expressing NKG2A/CD94 and does not significantly bind to cells expressing NKG2C/CD94, NKG2E/CD94 or combinations thereof.
  • the antibody of the first aspect is more effective in reducing CD94/NKG2A-mediated inhibition of cytotoxic activity of CD94/NKG2A-expressing cytotoxic lymphocytes.
  • the CD94/NKG2A-expressing cytotoxic lymphocytes are NK cells, NKT cells, alpha/beta T cells or gamma/delta T cells.
  • the CD94/NKG2A-expressing cytotoxic lymphocytes are NK cells.
  • the present invention provides an immunoconjugate, which includes the antibody described in the first aspect and a functional molecule connected thereto.
  • the present invention provides a chimeric receptor whose extracellular domain includes the antibody described in the first aspect, and the chimeric receptor includes: a chimeric antigen receptor (CAR), Chimeric T cell receptors, T cell antigen couplers (TACs), or combinations thereof.
  • CAR chimeric antigen receptor
  • TACs T cell antigen couplers
  • the chimeric receptor is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the CAR comprises sequentially connected: the antibody described in the first aspect, a transmembrane region and an intracellular signal region.
  • the intracellular signal region is selected from: intracellular signal region sequences of CD3 ⁇ , Fc ⁇ RI ⁇ , CD27, CD28, CD137, CD134, MyD88, CD40 or combinations thereof; and/or the transmembrane region Contains the transmembrane region of CD8 or CD28.
  • the CAR includes: the antibody described in the first aspect, the transmembrane region of CD8/CD28, and CD3 ⁇ ; or the antibody described in the first aspect, the transmembrane region of CD8/CD28, and the cytoplasmic region of CD137 Internal signal region and CD3 ⁇ ; or the antibody described in the first aspect, the transmembrane region of CD8/CD28, the intracellular signal region of CD28 and CD3 ⁇ ; or the antibody described in the first aspect, the transmembrane region of CD8/CD28, CD28 The intracellular signaling region, CD137 and CD3 ⁇ .
  • amino acid sequence of the chimeric receptor is shown in SEQ ID NO: 115 or 116.
  • the present invention provides nucleic acids encoding the antibody of the first aspect, the immunoconjugate of the second aspect, and the chimeric receptor of the third aspect.
  • the present invention provides an expression vector comprising the nucleic acid described in the fourth aspect.
  • the present invention provides a virus comprising the expression vector described in the fifth aspect or the nucleic acid described in the fourth aspect.
  • the nucleic acids, expression vectors and viruses involved in the fourth, fifth and sixth aspects are all biological materials of the present invention.
  • the biological material of the present invention is any one of the following:
  • the present invention provides a host cell expressing the chimeric receptor of the third aspect.
  • the host cell binds cells expressing NKG2A/CD94 and does not significantly bind NKG2C/CD94, NKG2E/CD94, or combinations thereof.
  • the host cell is resistant to NK cell attack or killing of NK cells.
  • the host cell also expresses chimeric receptors that recognize tumor antigens and/or pathogen antigens.
  • the host cell is used in combination with a second host cell that targets tumors and/or pathogens.
  • the host cell and/or second host cell does not express B2M, TCR/B2M, TCR/B2M/CIITA, TCR/B2M/NKG2A, and/or TCR/B2M/CIIA/NKG2A.
  • the host cell and/or the second host cell is a T cell, a natural killer cell, a cytotoxic T lymphocyte, a natural killer T cell, a DNT cell, a regulatory T cell, an NK92 cell, a stem cell derived Immune effector cells or combinations thereof.
  • the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells.
  • the T cells are autologous/allogeneic T cells.
  • the T cells are primary T cells.
  • the T cells are derived from human T cells.
  • the T cells include memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells (Tem), ⁇ T cells, or combinations thereof.
  • the present invention provides a combination drug, including the antibody described in the first aspect, the immunoconjugate described in the second aspect, the chimeric receptor described in the third aspect, and the host described in the seventh aspect.
  • the cells are administered in combination with an agent that enhances their function, preferably in combination with a chemotherapeutic agent; and/or in combination with an agent that ameliorates one or more side effects associated with them; and/or with expression of a chimeric gene other than targeting NKG2A.
  • Host cells containing antigen receptors are administered in combination.
  • the present invention provides a method for preparing the antibody described in the first aspect, the immunoconjugate described in the second aspect, and the chimeric receptor described in the third aspect, the method being comprised in a method suitable for Culturing the host cell of the seventh aspect under conditions for expressing the antibody, immunoconjugate, and chimeric receptor, and isolating the antibody, immunoconjugate, composition, and /or chimeric receptors.
  • the present invention provides a pharmaceutical composition, which includes: the antibody described in the first aspect or the nucleic acid encoding the antibody; or the immunoconjugate described in the second aspect or the nucleic acid encoding the conjugate. ; Or the chimeric receptor described in the third aspect or the nucleic acid encoding the chimeric receptor; or the host cell described in the seventh aspect; and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a kit comprising:
  • Container and the pharmaceutical composition of the tenth aspect located in the container;
  • Container and the antibody described in the first aspect or the nucleic acid encoding the antibody located in the container; or the immunoconjugate described in the second aspect or the nucleic acid encoding the conjugate; or the chimera described in the third aspect
  • Figure 1 shows a schematic diagram of using eukaryotic expression plasmid V152S to construct vectors expressing NKG2A/CD94 and NKG2C/CD94 heterodimers respectively;
  • Figure 2 shows the ELISA detection of the binding of antibodies A1 and A2 (Fab format) to NKG2A/CD94 and NKG2C/CD94;
  • Figure 3 shows the EC50 of ELISA detection of antibodies A1 and A2 (IgG4 form) binding to NKG2A/CD94;
  • FIG 4 shows the affinity of Biacore detection antibodies A1 and A2 (IgG4 form);
  • Figure 5 shows the vector map of the target genes CD94-Flag, NKG2A-CD94-Flag, NKG2C-CD94-Flag and NKG2E-CD94-Flag with Flag;
  • FIG. 6 shows the binding of FACs detection antibodies A1 and A2 (IgG4 form) to CHOK1-NKG2A-CD94, CHOK1-NKG2C-CD94, CHOK1-NKG2E-CD94 and CHOK1-CD94 cells;
  • Figure 7 shows the amino acid sequence comparison of the heavy chain variable regions of antibodies A1, A2, A3, A4, A5, and A6;
  • FIG. 8 shows the binding of FACs detection antibodies A1, A2, A3, A4, A5, and A6 (Fab format) to CHOK1-NKG2A-CD94, CHOK1-NKG2C-CD94 and CHOK1-NKG2E-CD94 cells;
  • Figure 9 shows the EC50 of ELISA detection of binding of antibodies A1, A2, A3, A4, A5, and A6 (Fab format) to NKG2A/CD94 heterodimer;
  • Figure 10 shows the EC50 of FACs detection antibodies A1, A2, A3, A4, A5, and A6 (Fab format) binding to CHO-K1 cells overexpressing NKG2A/CD94 heterodimer;
  • Figure 11 shows the EC50 of ELISA detection of binding of antibodies A1, A2, A3, A4, A5, and A6 (IgG4 form) to NKG2A/CD94 heterodimer;
  • Figure 12 shows the EC50 of FACs detection antibodies A1, A2, A3, A4, A5, A6 (IgG4 form) and CHOK1 cells overexpressing NKG2A/CD94;
  • FIG 13 shows the affinity of Biacore detection antibodies A3, A4, A5, and A6 (IgG4 form);
  • Figure 14 shows the vector maps of pET22b-HLA-E and pET22b- ⁇ 2m
  • Figure 15 shows the EC50 of HLA-E tetramer binding to NK cells expressing NKG2A/CD94;
  • Figure 16 shows the IC50 of FACs detection antibodies A1, A2, A3, A4, A5, and A6 (IgG4 form) blocking the binding of NKG2A to its ligand HLA-E;
  • Figure 17 shows HLA-E expression levels on tumor cells K562-HLA-E, K562 and FaDu cells
  • Figure 18 shows the FACs detection of CD107a expression after NK cells were co-incubated with K562, K562-HLA-E, and FaDu cells and added with antibodies A1, A2, A3, A4, A5, and A6 (IgG4 form);
  • Figure 19 shows the effect of FACs detection of anti-NKG2A antibodies on the killing effect of NK cells on K562, K562-HLA-E, and FaDu cells;
  • Figure 20 shows the changes in the proportion of NK cells after co-incubation of A4-BBZ CAR T cells, A5-BBZ CAR T cells and NK cells.
  • Figure 21 shows the change in the proportion of UCAR-T cells after co-incubation of A4 and A5 UCAR-T with NK cells.
  • Figure 22 shows the combined effect of A4, A5-UCAR-T and BCMA UCAR-T on RPMI-8226 cell subcutaneous transplanted tumors in the presence of NK cells.
  • the antibodies of the present invention can be used to prepare targeted anti-tumor drugs and drugs for diagnosing tumors.
  • any concentration range, percentage range, ratio range or integer range stated herein is to be understood to include any integer within the stated range and, where appropriate, fractions thereof (e.g., one-tenth of an integer and one-tenth of an integer). One percent) value.
  • NKG2A Natural killer group 2A
  • KLRC1 killer cell lectin like receptor C1
  • CD159a NK cell receptor A is one of the transmembrane proteins preferentially expressed on the surface of NK cells.
  • NKG2A is mainly expressed on the surface of NK cells and some T cells (CD8+ T cells, Th2 cells, ⁇ T cells and NKT cells).
  • NKG2A and CD94 molecules form a heterodimer NKG2A-CD94 connected by a disulfide bond, which is bound by the non-classical histocompatibility complex I (NKG2A-CD94) on the target cells.
  • major histocompatibility complex class I, MHC I) class molecule HLA-E recognition This molecule is low-expressed under normal circumstances, but on the surface of most tumor cells, the expression of HLA-E increases, thereby inducing a cascade of inhibitory signals. Inhibits NK cytotoxic activity and cytokine secretion. Certain viral infections, tumors, and immune diseases use this pathway to evade immune detection.
  • NKG2A and HLA-E can inhibit the activation of NK cells and T cells.
  • NK cells highly express NKG2A after being activated by IL15.
  • the NKG2 family also includes NKG2C, NKG2D and NKG2E.
  • NKG2A includes any native NKG2A from any vertebrate source, including mammals such as primates (eg, humans and monkeys) and rodents (eg, mice and rats).
  • the term includes "full-length” unprocessed NKG2A as well as any form of NKG2A derived from processing in the cell.
  • the term also includes naturally occurring variants of NKG2A, such as splice variants or allelic variants.
  • the anti-NKG2A antibodies described herein inhibit the binding of NKG2A protein to HLA-E and thus function as checkpoint inhibitors.
  • the full-length amino acid sequence of human NKG2A is shown in SEQ ID NO:68
  • the amino acid sequence of the extracellular segment of NKG2A is shown in SEQ ID NO:70
  • the full-length amino acid sequence of human NKG2C is shown in SEQ ID NO:123.
  • the amino acid sequence of the extracellular segment of NKG2C is shown in SEQ ID NO:72
  • the full-length amino acid sequence of human NKG2E is shown in SEQ ID NO:125
  • the amino acid sequence of the extracellular segment of NKG2E is shown in SEQ ID NO:74.
  • HLA Human leukocyte antigen
  • HLA Human leukocyte antigen
  • HLA includes class I, class II and class III gene parts.
  • the antigens expressed by HLA class I and class II genes are located on the cell membrane and are MHC-I (encoded by HLA-A, HLA-B, HLA-C sites) and MHC-II (encoded by HLA-D region), HLA I Class II is distributed on the surface of almost all cells in the body. It is a heterodimer composed of heavy chain ( ⁇ chain) and ⁇ 2 microglobulin (B2M). Class II is mainly a glycoprotein located on the surface of macrophages and B lymphocytes. .
  • HLA-E (OMIM 143010, gene number NM_005516.6) is a non-classical MHC molecule that is expressed on the cell surface and is regulated by the binding of peptides such as fragments of signal sequences derived from other MHC class I molecules .
  • HLA-E binds specifically to CD94/NKG2A, CD94/NKG2B, and CD94/NKG2C (see, e.g., Braud et al., (1998) Nature 391:795-799, the entire disclosure of which is incorporated herein by reference) and combines natural killer (NK) cells, natural killer T cells (NKT), and subsets of T cells (alpha/beta and gamma/delta).
  • HLA-E refers to any variant, derivative or isoform of the HLA-E gene or encoded protein.
  • SEQ ID NO:78 The amino acid sequence of the extracellular region of human HLA-E is shown in SEQ ID NO:78. HLA-E is widely distributed in cells throughout the body at low levels.
  • High levels of HLA-E are found in several tumors, including gynecological tumors (up to 90% of tumor samples), and up to 50% of breast cancer, non-small cell lung cancer (NSCLC), liver, pancreas, kidney, melanoma, prostate , head and neck, stomach, rectum and colorectal cancer.
  • NSCLC non-small cell lung cancer
  • Tumors with high HLA-E expression escape immune cell killing by binding to NKG2A of immune cells (such as NK cells and T cells).
  • the NKG2A antibodies disclosed in the present invention can inhibit immune escape of tumors with high HLA-E expression, thereby killing the tumor cells.
  • lysosome-associated membrane protein 1 (CD107a) is the main component of vesicle membrane proteins and mainly constitutes cytotoxic granules in the form of vesicles in the cell cytoplasm.
  • NK cells kill target cells, the released cytotoxic particles will reach the target cell membrane and fuse with the target cell membrane, causing the release of the contents of the particles, ultimately leading to the death of the target cell.
  • the spontaneous expression rate of CD107a on the surface of NK cell membrane is very low.
  • an increase in CD107a expression can be detected on the surface. Therefore, the increase in CD107a molecules after NK cell stimulation can reflect the cytotoxicity of NK cells. Level of cell killing activity.
  • polypeptide peptide
  • protein protein
  • protein protein
  • polymer may be linear, cyclic or branched, it may contain modified amino acids, especially conservatively modified amino acids, and it may be interrupted by non-amino acids.
  • the term also includes modified amino acid polymers such as those that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, isoprenylation, external digestion Amino acid polymers modified by rotation, selenoylation, transfer-RNA-mediated amino addition such as arginylation, ubiquitination, or any other operation such as conjugation with a labeling component.
  • amino acid refers to natural and/or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptide mimetics.
  • a polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide.
  • the term also includes polypeptides expressed from the specified nucleic acid sequence.
  • antibody is used herein in the broadest sense and includes a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the required The required antigen-binding activity is sufficient.
  • Antibody fragment refers to a molecule other than an intact antibody that contains the portion of the intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to (i) Fab fragments consisting of VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of VH and CH1 domains, (iii ) Fv fragment consisting of the VL and VH domains of a single antibody; (iv) dAb fragment consisting of a single variable region (Ward et al., 1989, Nature 341:544-546); (v) F(ab')2 Fragment, a bivalent fragment containing 2 linked Fab fragments; (vi) single-chain Fv molecule antigen-binding site; (vii) bispecific single-chain Fv dimer (PCT/US92/09965); (viii) " "Dibody” or "tribody", multivalent or multispecific
  • the "class" of an antibody refers to the type of constant domain or constant region its heavy chain has.
  • the heavy chain constant domains corresponding to different types of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • variable region or variable domain refers to the domain of an antibody heavy or light chain that is involved in antibody antigen binding.
  • the heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved FRs and three CDRs (see, e.g., Kindt et al., KubyImmunology, 6th ed., W.H. Freeman & Co., p. 91 (2007)).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • antibodies that bind a specific antigen can be isolated using a library of complementary VL or VH domains, respectively, from the VH or VL domains of the antibody that binds to the antigen. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • hypervariable region or “complementarity determining region” or “CDR” refers to sequences in an antibody variable domain that are hypervariable and/or form structurally defined loops ("hypervariable loops") and/or contain regions that contact the antigen. Regions of residues ("antigenic contacts").
  • antibodies typically contain six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3).
  • Fc region or “Fc” is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • FR Framework (FR) refers to the variable domain residues that are distinct from the hypervariable region (CDR) residues.
  • the FR of the variable domain usually consists of four FR domains: FR1, FR2, FR3 and FR4. Therefore, in VH (or VL) CDR and FR sequences usually appear in the following order: FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.
  • CDR residues and other residues in variable domains are numbered according to Kabat et al., above.
  • natural antibody refers to naturally occurring immunoglobulin molecules of various structures.
  • natural IgG antibodies are heterotetrameric proteins of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), which is also called a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3 ). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a light chain constant (CL) domain .
  • VH variable heavy chain domain
  • VL variable region
  • CL light chain constant domain
  • the light chains of antibodies can be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of their constant domains.
  • whole antibody full length antibody
  • intact antibody refers to having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein or having an antigen-binding region of the complete, full-length antibody.
  • Single domain antibody refers to a type of antibody that lacks the light chain of an antibody and only has the variable region of the heavy chain. Because of its small molecular weight, it is also called a Nanobody.
  • single domain antibody refers to an antibody that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain.
  • the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, U.S. Patent No. 6,248,516).
  • monoclonal antibody or “monoclonal antibody” refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising said population are identical and/or bind the same epitope, except where possible Variants other than antibodies that, for example, contain naturally occurring mutations or are generated during the preparation of monoclonal antibody preparations, are usually present in small amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.
  • the designation "monoclonal” indicates the nature of an antibody that is obtained from a substantially homogeneous population of antibodies and is not deemed to require that the antibody be prepared by any particular method.
  • they can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain of the antibody is derived from a specific source or species, while the remaining portion of the heavy and/or light chain is derived from a different source or species.
  • a chimeric antibody comprises a non-human variable region (eg, a variable region derived from a mouse, rat, hamster, rabbit, or a non-human primate such as a monkey) and a human constant region.
  • the chimeric antibodies are "class switched" antibodies in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • chimeric antibodies are "humanized antibodies.”
  • humanized is used for non-human antibodies, such as rodents or primates, etc., that are hybrid immunoglobulins, immunoglobulin chains or fragments thereof containing minimal sequences derived from non-human immunoglobulins.
  • Humanized antibody refers to a chimeric antibody that contains amino acid residues from a non-human CDR and amino acid residues from a human FR.
  • a humanized antibody will comprise substantially all of at least one (generally two) variable domains, wherein all or substantially all of the CDRs correspond to CDRs of a non-human antibody, and all or substantially All FRs above correspond to human antibody FRs.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized antibody” may include mutations, such as those introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo.
  • fully human antibody is an antibody that has an amino acid sequence corresponding to that of an antibody produced by humans or human cells or derived from a non-human source utilizing human antibody libraries or other human antibody coding sequences.
  • the definition of fully human specifically excludes humanized antibodies containing non-human antigen-binding residues.
  • the antibodies provided herein are "fully human antibodies” generated by phage display technology.
  • Antibodies of the invention can be isolated by screening combinatorial libraries of antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening the libraries for antibodies with desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., Human Press, Totowa, NJ, 2001) and are further described, for example, in McCafferty et al., Nature 348:552-554 ; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks, Meth. Mol.
  • VH and VL gene libraries are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage, as in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994).
  • Phages typically display antibody fragments as single chain Fv (scFv) fragments or Fab fragments. Libraries from the immunized source provide high affinity antibodies to the immunogen without the need to construct hybridomas.
  • antibodies or antibody fragments isolated from a fully human antibody library are considered to be fully human antibodies or fully human antibody fragments.
  • parent antibody refers to the antibody provided in this application or an antibody obtained by mutation, affinity maturation, etc. based on the antibody provided in this application.
  • the parent antibody may be a naturally occurring antibody, or a variant or engineered version of a naturally occurring antibody.
  • a parent antibody may refer to the antibody itself, a composition comprising the parent antibody, or its encoded amino acid sequence.
  • affinity matured antibody refers to an antibody that has one or more changes in one or more hypervariable regions (HVRs) compared to the parent antibody, such changes resulting in an increased affinity of the antibody for the antigen.
  • HVRs hypervariable regions
  • variant refers to a polypeptide that has substantially the same amino acid sequence or one or more activities encoded by substantially the same nucleotide sequence as the sequence of an antibody provided herein.
  • the variants have the same or similar activity as the antibodies provided in the examples of this application.
  • variable antibody or “antibody variant” includes antibody sequences that differ from the parent antibody sequence due to at least one amino acid modification compared to the parent.
  • Variant antibody sequences herein preferably are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%%, 96%, 97%, 98%, 99% amino acid sequence identity.
  • Antibody variants may refer to the antibodies themselves or to compositions containing the antibody variants.
  • Amino acid sequence variants of antibodies can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis.
  • amino acid modification includes amino acid substitution, addition and/or deletion.
  • amino acid substitution or “amino acid substitution” means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid.
  • amino acid insertion means replacing the amino acid at a specific position in the parent polypeptide sequence with another amino acid.
  • amino acid deletion means the removal of an amino acid at a specific position in the parent polypeptide sequence. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as binding to antigen.
  • Modification refers to a change in the state or structure of a protein or polypeptide of the invention. Modifications can be chemical, structural, or functional.
  • conservative modification or “conservative sequence modification” means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing the recited amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis. Families of amino acid residues with similar side chains have been defined in the art, as shown in Table 1.
  • amino acid residues in the CDR regions or in the framework regions of the antibodies of the invention can be replaced with other amino acid residues of the same side chain family, and the altered antibodies (variant antibodies) can be tested for retained functionality.
  • Nonconservative substitutions require exchanging a member of one of these groups for a member of the other group.
  • a substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody).
  • a parent antibody eg., a humanized or human antibody.
  • the resulting variants selected for further study will have altered (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will substantially Maintain certain biological properties of the parent antibody.
  • exemplary substitution variants are affinity matured antibodies, which can be routinely prepared, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and variant antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity).
  • Alterations can be made in the CDR regions, for example, to increase antibody affinity. Such changes can occur in HVR "hot spots," i.e., codon-encoded residues that are mutated at high frequencies during somatic cell maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008), and/or liberate the residues of the antigen, and test the resulting variant VH or VL for binding affinity.
  • Affinity maturation by construction of secondary libraries and reselection therefrom has been described, for example, by Hoogenboom et al.
  • affinity maturation by any of a variety of methods (e.g., , error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis) to introduce diversity into the variable gene species selected for maturation. Secondary libraries are then generated. The libraries are then screened to identify any antibodies with the desired affinity Variants. Another way to introduce diversity includes CDR-directed methods, in which several CDR residues (eg, 4-6 residues simultaneously) are randomized.
  • substitutions, insertions, or deletions may occur within one or more CDRs, so long as such changes do not significantly reduce the ability of the antibody to bind the antigen.
  • conservative changes that do not significantly reduce binding affinity eg, conservative modifications described herein
  • Such changes may be, for example, outside the residues in the CDRs that contact the antigen.
  • each CDR is unchanged or contains no more than one, two or three amino acid substitutions.
  • anti-NKG2A antibody refers to an antibody that is capable of binding NKG2A with sufficient affinity for use as a diagnostic agent for targeting NKG2A and /or therapeutic agents.
  • the anti-NKG2A antibody binds to an unrelated, non-NKG2A protein to a degree that is less than about 10% of that of the antibody to NKG2A, as determined by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • anti-NKG2A antibodies bind to an epitope of NKG2A that is conserved between NKG2A derived from different species.
  • antigen-binding proteins with Fab antigen-binding fragment-based antigen-binding regions
  • Fab antigen-binding fragment
  • antibody IgG4 antibody-binding fragment
  • NKG2A/CD94 heterodimer is used to select Fab from a fully human natural Fab phage library. These molecules exhibitaki specificity. For example, this antibody only recognizes NKG2A, but not NKG2C or NKG2E.
  • NKG2A refers to human NKG2A.
  • the invention includes antibodies having Fab, IgG4 sequences fused to one or more heavy chain constant regions to form antibodies with human immunoglobulin Fc regions to produce bivalent proteins, thereby increasing The overall affinity and stability of the antibody.
  • the Fc portion allows the direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to the antibody for use in, for example, antigen quantification studies, to immobilize the antibody for affinity measurements, for targeted delivery of therapeutics drugs, testing Fc-mediated cytotoxicity using immune effector cells, and many other applications.
  • the antibodies or antibody fragments of the present invention are based on the use of phage display to identify and select antigen-binding fragments (Fabs) whose amino acid sequences confer specificity to the antibody or antibody fragment for NKG2A and form the basis for all antigen-binding proteins of the present disclosure.
  • Fab antigen-binding fragments
  • the Fab can be used to design a range of different "antibodies or antibody fragments", including, for example, full-length antibodies, fragments thereof such as F(ab')2, fusion proteins, IgG4, multivalent antibodies, i.e., antibodies with the ability to target the same antigen. or more than one specific antibody for different antigens, e.g., bispecific T cell engaging antibodies (BiTE), tribodies, etc. (see Cuesta et al., Multivalent antibodies: when design surpasses evolution, Trends in Biotechnology 28:355- 362, 2010).
  • the invention provides full-length antibodies, the heavy and light chains of which may be full-length (e.g., the antibody may include at least one, preferably two complete heavy chains, and at least one, preferably two complete light chains) Alternatively, an antigen binding moiety (Fab, F(ab')2, Fv or scFv) may be included.
  • the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. The choice of antibody type will depend on the immune effector function that the antibody is designed to elicit. In constructing recombinant immunoglobulins, appropriate amino acid sequences for the constant regions of various immunoglobulin isotypes and methods for generating a wide variety of antibodies are known to those skilled in the art.
  • the invention provides fully human antibodies that recognize NKG2A, the antibodies comprising a light chain variable region comprising LCDR1 shown in RASQSISSWLA (SEQ ID NO: 4); and/or DASSLES (SEQ ID NO: 5) LCDR2 shown; and/or QQYDSYX 1 X 2 T shown in the LCDR3, wherein X 1 is selected from I, V, G, A, L, F or P, preferably, V; X 2 is selected from R, S, K, H, W, Y, C, M, N, Q or T. Preferably, X 2 is selected from R or S.
  • the invention provides fully human antibodies that recognize NKG2A.
  • the antibodies include a heavy chain variable region selected from:
  • HCDR1 represented by SYAIS (SEQ ID NO: 1); and/or HCDR2 represented by GIIPIFGTAX 1 YAQKFQG (SEQ ID NO: 130), wherein X 1 is selected from N, H, K, R, W, Y, S, C, M, Q or T, preferably, X 1 is selected from N or H; and/or HCDR3 shown in GFDGMDY (SEQ ID NO: 3); or
  • HCDR1 represented by X 1 X 2 X 3 X 4 S (SEQ ID NO: 131 ) ; and/or HCDR2 represented by AIX 1 / or HCDR3 represented by GYDGFDY (SEQ ID NO:9).
  • the amino acids at the X 1 X 2 X 3 X 4 positions in HCDR1 and HCDR2 are selected from Table 2 below.
  • the antibody further includes a framework region, wherein the 30th amino acid of the first framework region is selected from the group consisting of S, R, N, W, Y, C, M, Q, T, H, K, G, A, V, L, I, P or F, preferably, selected from S, R, N or G.
  • the invention provides an antibody that recognizes NKG2A, said antibody comprising a heavy chain variable region, said heavy chain variable region comprising any one of SEQ ID NO: 1, 7, 12, 14 or 16
  • the invention provides an antibody that recognizes NKG2A including: a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, and/or a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, And/or the light chain CDR3 comprising any of the amino acid sequences shown in SEQ ID NO: 6 or 10.
  • the invention provides an antibody that recognizes NKG2A including: a heavy chain CDR1 comprising any of the amino acid sequences shown in SEQ ID NO: 1, 7, 12, 14 or 16, and/or comprising SEQ ID NO:
  • CDR3 a heavy chain CDR1 comprising any of the amino acid sequences shown in SEQ ID NO: 1, 7, 12, 14 or 16, and/or comprising SEQ ID NO:
  • the antibody that recognizes NKG2A includes: a heavy chain CDR1 comprising any of the amino acid sequences shown in SEQ ID NO: 1, 7, 12, 14 or 16, and a heavy chain CDR1 comprising SEQ ID NO: 2, 8, 11, 13,
  • the antibody that recognizes NKG2A includes: a heavy chain CDR1 comprising any one of the amino acid sequences shown in SEQ ID NO: 1, 7, 12, 14 or 16, and a heavy chain CDR1 comprising SEQ ID NO: 2, 8, 11, 13 , the heavy chain CDR2 of any amino acid sequence shown in 15 or 17, and the heavy chain CDR3 including any amino acid sequence shown in SEQ ID NO:3 or 9, and the light chain CDR1 including the amino acid sequence shown in SEQ ID NO:4 , and a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO: 6 or 10.
  • the antibody that recognizes NKG2A includes: HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3; SEQ ID NO:4 LCDR1, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6; or containing HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8 and SEQ ID NO:9 HCDR3 shown; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:10; or containing HCDR1 shown in SEQ ID NO:1, SEQ ID NO :HCDR2 shown in SEQ ID NO:3 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6; or containing SEQ HCDR1 shown in SEQ ID NO
  • the invention provides an antibody that recognizes NKG2A, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 28, 30 or 32, or variants of the above sequences.
  • the invention provides an antibody that recognizes NKG2A, comprising a light chain variable region, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 or 24, or a variant of the above sequence.
  • heavy and light chain variable region sequences can bind NKG2A
  • heavy and light chain variable region sequences can be "mixed and matched" to generate anti-NKG2A binding molecules of the invention.
  • the invention provides variants of antibodies or fragments thereof that bind NKG2A.
  • the invention thus provides antibodies, or fragments thereof, having a heavy chain and/or light chain variable region that is at least 80% identical to the variable region sequence of the heavy chain or light chain.
  • the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, even more particularly 98% , most particularly 99%, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
  • Variants can be obtained by using the antibody described in this application as the parent antibody through yeast library screening, phage library screening, point mutation and other methods.
  • yeast library screening phage library screening
  • point mutation a method used in Example 2 of the present application
  • antibodies A1 and A2 are used as parent antibodies, and the phage library screening method is used for mutation transformation.
  • the present invention provides antibodies that recognize the same epitope as the aforementioned anti-NKG2A antibodies.
  • the invention provides an antibody that competitively binds to NKG2A with the aforementioned anti-NKG2A antibody.
  • the invention provides an antibody that specifically binds to NKG2A, said antibody being a whole anti-, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') 2 fragment, Fd fragment, dAb fragments, multifunctional antibodies or IgG4 antibodies.
  • the above-described antibodies do not significantly bind to NKG2C, NKG2E or combinations thereof.
  • the antibody binds to NKG2A/CD94 and does not significantly bind to NKG2C/CD94, NKG2E/CD94 or combinations thereof; and/or,
  • the antibody binds to cells expressing NKG2A/CD94 and does not bind significantly to cells expressing NKG2C/CD94, NKG2E/CD94, or combinations thereof.
  • anti-NKG2A antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. Includes, for example, ELISA, biacore, Western blotting and flow cytometry analysis. Suitable assays are described in detail in the Examples.
  • affinity refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • binding affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between the members of a binding pair (eg, antibody and antigen).
  • the affinity of a molecule X for its ligand Y can often be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including determining the affinity of an antibody using Biacore as described herein.
  • the "affinity" of the antibody for NKG2A/CD94 herein is expressed as the KD of the antibody.
  • the KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The greater the KD value of an antibody for binding to its antigen, the weaker its binding affinity for that particular antigen.
  • EC50 concentration for 50% of maximal effect (EC50) refers to the concentration that causes 50% of the maximum effect.
  • antigen refers to a substance recognized by and specifically bound by an antigen-binding unit.
  • Antigens may include peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens.
  • Antigen may also refer to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immunologically-competent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
  • epitope refers to an antigen or portion of an antigen recognized by an antibody, B cell, T cell, or engineered cell.
  • an epitope may be a tumor epitope or a pathogen epitope recognized by an antibody; an antibody recognizes multiple epitopes within an antigen. Epitopes can also be mutated.
  • antigenic epitope also known as “antigenic epitope” or “epitope” or “antigenic determinant” includes any determinant or region capable of being bound by an antibody.
  • An antigenic epitope is a region of an antigen that is bound by an antibody targeting that antigen, including specific amino acids that are in direct contact with the antibody.
  • the antigenic epitope can be composed of a continuous sequence of the NKG2A protein sequence, or it can be composed of a discontinuous three-dimensional structure of the NKG2A protein sequence.
  • the antigens used herein are NAG2A/CD94 heterodimers, NAG2C/CD94 heterodimers, and NAG2E formed by the NAG2A extracellular region, the NAG2C extracellular region, and the NAG2E extracellular region, respectively, and the CD94 extracellular region. /CD94 heterodimer.
  • the invention also provides immunoconjugates, which include the antibodies described herein and functional molecules linked thereto.
  • the antibody and the functional molecule can form a conjugate through covalent connection, coupling, attachment, cross-linking, etc.
  • connection or “fusion” are used interchangeably herein. These terms refer to the joining together of two more chemical elements or components by any means including chemical conjugation or recombinant methods.
  • “In-frame fusion” refers to the joining of two or more ORFs to form a continuous longer ORF in a manner that maintains the correct reading frame of the original open reading frame (ORF).
  • the resulting recombinant fusion protein is therefore a single protein containing two or more fragments that correspond to the polypeptide encoded by the original ORF (these fragments are not typically so linked in nature).
  • the reading frame is therefore continuous throughout the fusion fragments, the fragments may be physically or spatially separated, for example, by in-frame linking sequences (eg, "flexon").
  • Another aspect of the invention provides a nucleic acid molecule encoding at least one antibody, functional variant or immunoconjugate thereof of the invention.
  • the invention also relates to vectors comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • chimeric receptor refers to a fusion molecule formed by connecting DNA fragments from different sources or cDNA corresponding to proteins using genetic recombination technology, including extracellular domain, transmembrane domain and intracellular domain.
  • Chimeric receptors include, but are not limited to: chimeric antigen receptors (CAR), chimeric T cell receptors, and T cell antigen couplers (TAC).
  • chimeric antigen receptor includes extracellular antigen-binding domains, transmembrane domains, and intracellular signaling domains.
  • the intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and/or a costimulatory molecule.
  • the stimulatory molecule is a delta chain that binds to the T cell receptor complex; in one aspect, the cytoplasmic signaling domain Transduction domains further include functional signaling domains of one or more costimulatory molecules, such as 4-1BB (i.e. CD137), CD27 and/or CD28.
  • chimeric T cell receptor includes recombinant polypeptides derived from various polypeptides that constitute the TCR, which are capable of binding to surface antigens on target cells and interacting with other polypeptides of the complete TCR complex, usually co-located on T cell surface.
  • the chimeric T cell receptor consists of a TCR subunit and an antigen-binding domain composed of a human or humanized antibody domain.
  • the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, the TCR cellular domain, and the TCR extracellular domain.
  • the stimulation domain of the intracellular signaling domain of the inner domain; the TCR subunit and the antibody domain are effectively connected, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3 ⁇ or CD3 ⁇ , and , the chimeric T cell receptor is integrated into the TCR expressed on T cells.
  • T cell antigen coupler includes three functional domains: 1. Antigen-binding domain, including single-chain antibodies and designed ankyrin repeat protein (DARPin) or other targeting groups; 2. Extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor and TCR receptor close to each other; 3. The transmembrane region and the intracellular region of the CD4 co-receptor, in which , the intracellular domain-linked protein kinase LCK, catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • signaling domain refers to a functional portion of a protein that functions by transmitting information within a cell, serves to regulate the cell via a defined signaling pathway, by producing a second messenger or acting as an effector by responding to such a messenger. activity.
  • the intracellular signaling domain may include the entire intracellular portion of the molecule, or the entire native intracellular signaling domain, or functional fragments or derivatives thereof.
  • the term "primary signaling domain” regulates the initial activation of the TCR complex in a stimulatory manner.
  • the primary signaling domain is initiated by, for example, the binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating T cell responses (including, but not limited to, proliferation, activation, differentiation, etc.).
  • the primary signaling domain that acts in a stimulatory manner may contain an immunoreceptor tyrosine activation motif or a signaling motif of an ITAM.
  • ITAM-containing primary signaling domains examples include, but are not limited to, those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS" ) and the sequence of CD66d.
  • the intracellular signaling domain in any one or more CARs of the present invention includes an intracellular signaling sequence, such as the primary signaling domain of CD3 ⁇ .
  • costimulatory signaling domain refers to a "costimulatory molecule”, which refers to a signal that binds to a cell stimulatory signaling molecule, such as TCR/CD3, and the combination results in T cell proliferation and/or up-regulation or down-regulation of key molecules. Is a cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are non-antigen receptor cell surface molecules or their ligands that are required for an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137 ).
  • a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain containing a protein derived from Functional signaling domains of stimulatory molecules.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain containing functionality derived from a costimulatory molecule sexual signaling domains and functional signaling domains derived from stimulatory molecules.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain derived from one or more costimulatory molecules at least two functional signaling domains and a functional signaling domain derived from a stimulatory molecule.
  • the CAR contains an optional leader sequence at the amino acid (ND end) of the CAR fusion protein.
  • the CAR also contains a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.
  • CD3 delta also known as CD3 Zeta
  • CD3 delta domain is defined as the amino acid residues from the cytoplasmic domain of the ⁇ chain that are sufficient to functionally transmit the initial signal required for T cell activation.
  • the cytoplasmic domain of ⁇ includes residues 52 to 164 of GenBan accession number BAG36664.1, its functional orthologs—from non-human species such as mice, rodents, monkeys, apes, etc. Valence residue.
  • the chimeric receptors of the invention are chimeric antigen receptors.
  • the invention provides a chimeric antigen receptor (Chimeric Antigen Receptor, CAR), which includes an extracellular binding domain, a transmembrane domain and an intracellular domain as described herein.
  • CAR Chimeric Antigen Receptor
  • the extracellular binding domain (or structural region) of a CAR is derived from mouse or humanized or human monoclonal antibodies.
  • Chimeric antigen receptors usually contain extracellular antigen-binding regions or antibodies.
  • the extracellular antigen binding region may be fully human. In other cases, the extracellular antigen binding region can be humanized. In other cases, the extracellular antigen-binding region may be of murine origin, or the chimera in the extracellular antigen-binding region may consist of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region may be non-human.
  • Chimeric antigen receptors can be designed with a variety of antigen-binding regions, including single-chain variable fragments (scFv) derived from antibodies, fragmented antigen-binding regions (Fab) selected from libraries, single-domain fragments, or conjugated to their homologous receptors. body’s natural ligand.
  • the extracellular antigen binding region may comprise scFv, Fab or natural ligands, as well as any derivatives thereof.
  • An extracellular antigen-binding region may refer to a molecule other than an intact antibody, which may comprise a portion of an intact antibody and which may bind the antigen to which the intact antibody binds.
  • antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies, linear antibodies; single chain antibody molecules (eg, scFv); and those formed from antibody fragments Multispecific antibodies.
  • Extracellular antigen-binding regions such as scFv, Fab or natural ligands, can be part of the CAR that determines antigen specificity.
  • the extracellular antigen-binding region can bind any complementary target.
  • the extracellular antigen-binding region can be derived from antibodies of known variable region sequences.
  • Extracellular antigen-binding regions can be derived from antibody sequences obtained from available mouse hybridomas. Alternatively, extracellular antigen-binding regions can be obtained from total exo-cleavage sequencing of tumor cells or primary cells such as tumor-infiltrating lymphocytes (TILs).
  • TILs tumor-infiltrating lymphocytes
  • the binding specificity of the extracellular antigen-binding region of the CAR can be determined by complementarity determining regions or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, binding specificity can be determined by light chain CDRs and heavy chain CDRs.
  • the extracellular antigen-binding region of the CAR includes a hinge or spacer region.
  • the terms hinge and spacer are used interchangeably.
  • the hinge can be thought of as the part of the CAR that provides flexibility to the extracellular antigen-binding region.
  • the hinge can be used to detect CAR on the cell surface of cells, particularly when antibodies that detect extracellular antigen-binding regions are ineffective or available.
  • the length of a hinge derived from an immunoglobulin may need to be optimized depending on the location of the epitope on the target targeted by the extracellular antigen-binding region.
  • the hinge may not belong to an immunoglobulin, but to another molecule, such as the native hinge of the CD8 ⁇ molecule.
  • the CD8 alpha hinge may contain cysteine and proline residues known to play a role in the interaction of CD8 coreceptors and MHC molecules.
  • the cysteine and proline residues can affect the performance of the CAR.
  • the hinge can be adjusted depending on the extracellular antigen binding region used. Hinges can be of any length.
  • the transmembrane domain (or structural region) of CAR can anchor the CAR to the plasma membrane of the cell.
  • the native transmembrane portion of CD28 can be used in CARs. In other cases, the native transmembrane portion of CD8 ⁇ can also be used in CARs.
  • CD8 may be a protein that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to NCBI reference number: NP_001759, or a fragment thereof having stimulatory activity.
  • a "CD8 nucleic acid molecule” may be a polynucleotide encoding a CD8 polypeptide. In some cases, the transmembrane region may be the native transmembrane portion of CD28.
  • CD28 may refer to the same protein as NCBI reference number: NP_006130 or its stimulatory activity. Fragments are proteins with at least 85, 90, 95, 96, 97, 98, 99, or 100% identity.
  • a "CD28 nucleic acid molecule” may be a polynucleotide encoding a CD28 polypeptide.
  • the transmembrane portion may comprise a CD8 alpha region.
  • the intracellular signaling domain of a CAR may be responsible for activating at least one of the effector functions of immune response cells containing the CAR.
  • CAR can induce the effector function of T cells.
  • the effector function is cytolytic activity or auxiliary activity, including the secretion of cytokines, such as IL-2, TNF- ⁇ , ⁇ -IFN, etc.
  • the term intracellular signaling domain refers to the portion of a protein that transduces effector functional signals and directs the cell to perform specific functions. Although typically the entire intracellular signaling region can be used, in many cases it is not necessary to use the entire chain of signaling domains. In some embodiments, truncated portions of intracellular signaling regions are used. In some embodiments, the term intracellular signaling region is therefore intended to include any truncated portion of an intracellular signaling region sufficient to transduce an effector function signal.
  • Preferred examples of signaling domains (or regions) used in CAR may include the cytoplasmic sequence of a T cell receptor (TCR) and a coreceptor that cooperates to initiate signal transduction after target-receptor binding, and any derivative or variant sequences thereof and any synthetic sequences of these sequences having the same functionality.
  • TCR T cell receptor
  • the intracellular signaling region of the CAR may contain a signaling motif known as an immunoreceptor tyrosine activation motif (ITAM).
  • ITAMs containing cytoplasmic signaling sequences include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling domain is derived from the CD3 delta chain.
  • CD3 delta domain also known as the T cell receptor CD3 delta chain or CD247.
  • This domain is part of the T cell receptor-CD3 complex and plays an important role in coupling antigen recognition by several intracellular signaling pathways with primary effector activation of T cells.
  • CD3 delta refers primarily to human CD3 delta and its isoforms, as known from Swissprot entry P20963, including proteins with essentially identical sequences.
  • the full T cell receptor CD3 delta chain is not required to be part of a chimeric antigen receptor, and any derivative thereof containing the signaling domain of the T cell receptor CD3 delta chain is suitable, including any functional equivalents thereof.
  • the intracellular signaling domain can be selected from any costimulatory domain in Table 3.
  • a domain may be modified such that the identity to a reference domain may be from about 50% to about 100%.
  • Any of the domains of Table 3 can be modified such that the modified form can comprise about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% of identity.
  • the intracellular signaling domain of the CAR may further comprise one or more costimulatory domains.
  • the intracellular signaling region can comprise a single costimulatory domain, such as the delta chain (first generation CAR) or it together with CD28 or 4-1BB (second generation CAR).
  • the intracellular signaling domain may contain two costimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).
  • costimulatory domains can produce downstream activation of kinase pathways, thereby supporting gene transcription and functional cellular responses.
  • the costimulatory domain of CAR can activate pathways associated with CD28 (phosphatidylinositol-4,5-bisphosphate 3-kinase) or 4-1BB/OX40 (TNF-receptor-associated factor adapter protein) pathways as well as MAPK and Akt activation. Proximal signaling protein.
  • signals generated through CARs may be combined with auxiliary or costimulatory signals.
  • costimulatory signaling domains chimeric antigen receptor-like complexes can be designed to contain several possible costimulatory signaling domains. It is well known in the art that in naive T cells, T cell receptor binding alone is insufficient to induce full activation of the T cell into cytotoxic T cells. Complete productive T cell activation requires a second costimulatory signal.
  • Several receptors have been reported to provide costimulation for T cell activation, including but not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4- 1BB.
  • the signaling pathways used by these costimulatory molecules all work synergistically with primary T cell receptor activation signals.
  • the signals provided by these costimulatory signaling domains can synergize with primary effector activation signals derived from one or more ITAM motifs (e.g., CD3zeta signaling domain) and can fulfill the requirements for T cell activation.
  • adding costimulatory domains to chimeric antigen receptor-like complexes can enhance the efficacy and durability of engineered cells.
  • the T cell signaling domain and the costimulatory domain are fused to each other to form the signaling domain.
  • the present invention also provides a variety of chimeric antigen receptors (CARs), which contain the antibodies of the present invention or fragments thereof, and the CAR-T cells exhibit anti-tumor properties.
  • CARs chimeric antigen receptors
  • cells eg, T cells
  • the viral vector is a lentiviral vector.
  • cells can stably express a CAR.
  • the NKG2A binding part of the CAR is a scFv, which maintains equivalent affinity binding compared with the Fab antibody from which it is derived, for example, it binds the same antigen with comparable efficacy.
  • the antibody fragment is functional, whereby it provides a biochemical response, such as activating an immune response, inhibiting the initiation of signaling from its target antigen, inhibiting kinase activity, etc.
  • the anti-NKG2A antigen-binding domain of CAR is a fully human antibody fragment.
  • the CAR of the present invention combines the antigen-binding domain of a specific antibody and an intracellular signaling molecule.
  • intracellular signaling molecules include, but are not limited to, CD3 ⁇ , 4-1BB, and CD28 signaling modules, and combinations thereof.
  • the NKG2A-CAR includes at least one intracellular signaling domain selected from a CD137(4-1BB) signaling domain, a CD28 signaling domain, a CD3 ⁇ signaling domain, and any combination thereof. In one aspect, the NKG2A-CAR contains at least one intracellular signaling domain derived from one or more costimulatory molecules other than CD137 (4-1BB) or CD28.
  • sequence of NKG2A-CAR can be:
  • Chimeric antigen receptor one having an extracellular domain shown in SEQ ID NO: 64, a hinge domain shown in SEQ ID NO: 95, a transmembrane domain shown in SEQ ID NO: 97, and a covalent domain shown in SEQ ID NO: 101
  • Chimeric antigen receptor II has an extracellular domain shown in SEQ ID NO: 66, a hinge domain shown in SEQ ID NO: 95, a transmembrane domain shown in SEQ ID NO: 97, and a covalent domain shown in SEQ ID NO: 101.
  • amino acid sequence of the chimeric receptor is shown in SEQ ID NO: 115 or 116.
  • nucleic acid molecule encoding refers to the sequence or sequence of deoxyribonucleotides along a deoxyribonucleic acid strand. The order of these deoxyribonucleotides determines the order of the amino acids along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.
  • sequence when used to refer to a nucleotide sequence, the term "sequence" as used herein includes DNA or RNA, and may be single-stranded or double-stranded.
  • target sequence refers to a sequence that is complementary to a guide sequence, and the complementary pairing between the target sequence and the guide sequence promotes the formation of the CRISPR complex.
  • a target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide.
  • the target sequence is located in the nucleus or cytoplasm of the cell.
  • sequence identity determines percent identity by comparing two best-matched sequences over a comparison window (eg, at least 20 positions), where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions. (ie, a gap), such as a gap of 20% or less (eg, 5 to 15%, or 10 to 12%) for the two sequences that best match compared to the reference sequence (which contains no additions or deletions).
  • the percentage is usually calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to yield the number of positions that are correctly matched, dividing the number of positions that are correctly matched by the total number of positions in the reference sequence ( i.e. window size) and multiply the result by 100 to yield a percentage of sequence identity.
  • transfection refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • expression vector refers to a vector comprising a recombinant polynucleotide that contains expression control sequences operably linked to the nucleotide sequence to be expressed.
  • the expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in vitro expression system.
  • Expression vectors include all those known in the art, such as plasmids, viruses (eg, lentiviruses, retroviruses, adenoviruses and adeno-associated viruses).
  • vector is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell.
  • vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • Non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells may also be included, such as polylysine compounds, liposomes, and the like.
  • lentivirus refers to the genus of the family Retroviridae. Retroviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, and therefore they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide the means to achieve significant levels of gene transfer in vivo.
  • endogenous refers to a nucleic acid molecule or polypeptide etc. that comes from the organism itself.
  • exogenous refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or the expression level is insufficient to achieve the function when overexpressed.
  • exogenous protein used herein may be an exogenously introduced protein into a cell that recognizes a target antigen, such as an exogenous receptor (i.e., the "chimeric receptor” mentioned herein).
  • host refers to a recipient of a graft, which in some embodiments may be an individual, such as a human, to whom exogenous cells are implanted.
  • isolated means separated from cellular components or other components with which the polynucleotide, peptide, polypeptide, protein, antibody or fragments thereof are normally associated in their natural state.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof does not need to be “isolated” to distinguish it from its naturally occurring counterpart.
  • a “concentrated,” “isolated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof may be distinguished from its naturally occurring counterpart because the concentration or number of molecules per volume is greater than (“concentrated” ”) or less than (“dilute”) the concentration of its naturally occurring counterpart.
  • the degree of enrichment may be measured on an absolute basis, such as weight per solution volume, or may be measured relative to another potential interfering substance present in the source mixture.
  • the preferred enrichment degree of the technical solutions of the present invention is higher. Therefore, for example, 2-fold enrichment is preferred, 10-fold enrichment is more preferred, 100-fold enrichment is more preferred, and 1000-fold enrichment is more preferred.
  • "Isolated" substances may also be provided by artificial assembly methods, such as chemical synthesis or recombinant expression.
  • the present invention provides isolated nucleic acids encoding antibodies or fragments thereof that recognize NKG2A, vectors, and host cells containing the nucleic acids or vectors.
  • the nucleic acid may be in intact cells, in cell lysates, or in partially purified or substantially purified form.
  • the nucleic acids of the invention can be obtained using standard molecular biology techniques, for example, cDNA encoding the light and heavy chains of the antibody or the VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques.
  • cDNA encoding the light and heavy chains of the antibody or the VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques.
  • antibodies obtained from an immunoglobulin gene library eg, using phage display technology
  • one or more nucleic acids encoding the antibody can be recovered from the library.
  • Methods for introducing exogenous nucleic acids into host cells are generally known in the art and may vary depending on the host cell used.
  • the nucleic acid molecule of the present invention is selected from SEQ ID NO: 19, 23, 27, 29, 31 or 33 encoding the heavy chain variable region, and/or selected from SEQ ID NO: 21 encoding the light chain variable region. or 25. More preferably, it is a nucleic acid molecule comprising the heavy chain variable region sequence of SEQ ID NO: 19, and the light chain variable region sequence comprising SEQ ID NO: 21; or comprising SEQ ID NO: 23 A heavy chain variable region sequence, and a light chain variable region sequence comprising SEQ ID NO: 25; or a heavy chain variable region sequence comprising SEQ ID NO: 27, and a light chain variable region sequence comprising SEQ ID NO: 21 region sequence; or a heavy chain variable region sequence comprising SEQ ID NO: 29, and a light chain variable region sequence comprising SEQ ID NO: 25; or a heavy chain variable region sequence comprising SEQ ID NO: 31, and comprising The light chain variable region sequence of SEQ ID NO:25; or the heavy chain variable region sequence comprising SEQ ID NO:
  • one or more vectors comprising the nucleic acids described above are provided.
  • cell refers to cells of human or non-human animal origin.
  • host cell refers to a cell into which exogenous nucleic acid is introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include transformed primary cells and progeny derived therefrom (regardless of passage number).
  • the nucleic acid content of the progeny may not be identical to that of the parent cell and may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the original transformed cell are included herein.
  • NKG2A or NKG2A/CD94-positive host cells refers to host cells that express NKG2A/CD94 on their cell surface, which cells can be detected, for example, by flow cytometry using antibodies that specifically recognize CD94 and NKG2A. Combination epitopes or epitopes on NKG2A alone.
  • the host cells are immune effector cells.
  • immune effector cells refers to cells that participate in immune responses and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, and macrophages. , mast cells, etc.
  • the immune effector cells are T cells, NK cells, or NKT cells.
  • the T cells can be autologous T cells, xenogeneic T cells, or allogeneic T cells.
  • the NK cells may be allogeneic NK cells.
  • Immunune effector function or immune effector response refers to an immune effector cell, such as a function or response that enhances or promotes an immune attack on a target cell.
  • immune effector functions or responses refer to properties of T cells or NK cells that promote killing of target cells or inhibit growth or proliferation.
  • artificially modified cells with immune effector cell functions refers to cells or cell lines that do not have immune effectors that have acquired immune effector cell functions after being artificially modified or stimulated by stimulants.
  • 293T cells have been artificially modified to function as immune effector cells; such as stem cells, which have been induced in vitro to differentiate into immune effector cells.
  • T cells can be pluripotent stem cells derived from bone marrow that differentiate and mature into immune-competent mature T cells within the thymus.
  • T cells can be a population of cells with specific phenotypic characteristics, or a mixed population of cells with different phenotypic characteristics, such as “T cells” can be cells that contain at least one T cell subpopulation: memory Stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and/or effector memory T cells ( Temp).
  • a "T cell” may be a specific subtype of T cell, such as a gamma delta T cell.
  • T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells may be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as FicollTM isolation.
  • cells from the circulating blood of an individual are obtained by apheresis. Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or culture medium for subsequent processing steps.
  • T cells can be obtained from healthy donors, or from cells derived from patients diagnosed with tumors.
  • peripheral blood mononuclear cell refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, etc.
  • activation and “activation” are used interchangeably and can refer to the process by which cells transition from a quiescent to an active state. The process may include responses to phenotypic or genetic changes in antigen, migration and/or functional activity states. For example, the term “activation” can refer to the process of gradual activation of NK cells and T cells.
  • T cell activation or “T cell activation” refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • a host cell comprising the nucleic acid described above.
  • the host cell comprises (e.g., is transduced with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH, or (2) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL a first vector comprising a nucleic acid sequence of an amino acid sequence, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH.
  • the host cell is eukaryotic, eg, Chinese hamster ovary (CHO) cells or lymphocytes (eg, YO, NSO, Sp20 cells).
  • the host cell expresses a chimeric receptor of the invention.
  • the host cells include T cells, natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, regulatory T cells, NK92 cells, and/or stem cell-derived immune effector cells .
  • the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells; preferably, the T cells are autologous/allogeneic T cells; preferably, The T cells are primary T cells; preferably, the T cells are derived from human autologous T cells.
  • the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells cells (Tem), ⁇ T cells or combinations thereof.
  • the host cell binds to cells expressing NKG2A/CD94 without significantly binding to NKG2C/CD94, NKG2E/CD94, or combinations thereof.
  • the host cell further carries a coding sequence for an exogenous cytokine.
  • the host cell may also express another chimeric antigen receptor in addition to the above-described antigen-binding receptor.
  • the host cell may also express a chemokine receptor.
  • the host cell may also express a safety switch.
  • the host cell is capable of killing activated NK cells.
  • a method of preparing an anti-NKG2A antibody includes culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody as described above, and optionally from the host cell (or host cell culture medium) to recover the antibody.
  • the nucleic acid encoding the antibody of the invention can be integrated into an expression vector.
  • expression vectors are available for protein expression.
  • Expression vectors may include self-replicating extrachromosomal vectors, or vectors that integrate into the host genome.
  • Expression vectors useful in the present invention include, but are not limited to, those enabling protein expression in mammalian cells, bacteria, insect cells, yeast and in vitro systems. As is known in the art, a variety of expression vectors are commercially or otherwise available. Can be used in the present invention to express antibodies.
  • the host cell is administered in combination with an agent that enhances its function, preferably in combination with a chemotherapy drug; and/or the host cell is administered in combination with an agent that improves one or more side effects associated with it. ; and/or the host cell is administered in combination with a host cell expressing a chimeric antigen receptor targeting other than NKG2A.
  • a CRISPR system containing the gRNA provided by the invention is used to knock out endogenous TCR, B2M, NKG2A and/or CIITA in cells.
  • Genetic modification of cells can be accomplished by transducing a substantially homogeneous population of cells with recombinant nucleic acid molecules.
  • TCR, B2M, NKG2A and/or HLA-II are low-expressed or not expressed in the cells.
  • Low or no expression of TCR, B2M, NKG2A and/or HLA-II means that the expression of TCR, B2M, NKG2A and/or HLA-II in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, At least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.
  • low expression or no expression of TCR, B2M, NKG2A and/or HLA-II means that the content of TCR, B2M, NKG2A and/or HLA-II in cells is reduced by at least 1%, at least 5%, and at least 10%, respectively. %, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.
  • Specific antibodies for TCR, B2M, NKG2A and/or HLA-II can be used to determine the content of cells in cells by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry. protein expression or content.
  • the donor due to the immunogenetic differences between the donor and the recipient (or host), when performing an exogenous donor transplant, the donor as an exogenous graft will be recognized and recognized by immune cells (such as NK cells) in the host. Attack, thereby inhibiting or eliminating the donor, resulting in host versus graft response (HVGR).
  • HVGR host versus graft response
  • the deletion of HLA class I molecules in allogeneic cells can reduce the host's CD8+-mediated cellular immune rejection.
  • the invention provides immune cells with low or no expression of endogenous HLA-II/B2M.
  • GVHD graft-versus-host disease
  • the donor T lymphocytes will recognize the antigens on the host's normal tissues and expand upon expansion. It increases and releases a series of cytokines, which greatly enhances the immune response of the graft to host antigens and attacks host cells.
  • the invention provides immune cells with low or no expression of endogenous HLA-II/TCR.
  • the present invention uses the CRISPR system to knock out the gene TRAC of the ⁇ chain of the endogenous TCR to prepare cells with low or no expression of the endogenous TCR.
  • the expression of endogenous NKG2A in the donor immune cells of the exogenous transplant is up-regulated and will be killed by the immune cells that recognize NKG2A in the composition of the present invention.
  • low or no expression of NKG2A may relieve the inhibitory effect of immune cells themselves, thereby exerting stronger anti-tumor ability.
  • the invention provides immune cells with low or no expression of endogenous HLA-II/NKG2A.
  • the above immune cells do not significantly activate allogeneic immune cells.
  • the above immune cells can reduce allogeneic immune rejection.
  • the above-mentioned immune cells that recognize tumor antigens and/or the immune cells that recognize NKG2A polypeptide and tumor antigens can significantly kill tumor cells without significantly activating allogeneic immune cells.
  • the above-mentioned immune cells that recognize tumor antigens and/or the immune cells that recognize NKG2A polypeptides and tumor antigens can significantly kill tumor cells, and have low allogeneic immune rejection.
  • the antibodies of the present invention, immunoconjugates containing the antibodies, chimeric receptors, and host cells can be used to prepare pharmaceutical compositions or diagnostic reagents.
  • the composition may also include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • the composition includes another therapeutic agent.
  • the other therapeutic agent is a chemotherapeutic agent, such as those described in US20140271820 and/or a pharmaceutically acceptable salt or analog thereof.
  • the therapeutic agents include, but are not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and novibine(TM) (vinorelbine, 5' -dehydrogen sulfide); topoisomerase I inhibitors, such as camptothecin compounds, including CamptosarTM (irinotecan HCL), HycamtinTM (topotecan HCL) and others derived from camptothecin and its analogs Compounds; podophyllotoxin derivatives such as etoposide, teniposide, and midozozide; alkylating agents cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphamide, carmustine ,
  • the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin, and 5-fluorouracil.
  • the additional therapeutic agents include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides), as well as other vascular Inhibitors such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, retinoic acid and tissue inhibitors of metalloproteinases-1 and -2, etc.
  • anti-angiogenic agents including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides), as well as other vascular Inhibitors such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, retinoic acid and tissue inhibitors of
  • sugars such as lactose, glucose and sucrose
  • starches such as corn starch and potato starch
  • cellulose and its derivatives such as carboxymethyl cellulose Sodium vegetarian, ethylcellulose and methylcellulose
  • tragacanth powder malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter
  • polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol
  • wetting agents such as sodium lauryl sulfate
  • Colorants flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions
  • compositions described herein may contain one or more pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not produce any adverse toxicological effects (see, e.g., Berge, S.M. et al., 1977, J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include salts derived from nontoxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, as well as salts derived from nontoxic organic acids such as aliphatic monocarboxylic acids and Salts of dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
  • nontoxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like
  • nontoxic organic acids such as aliphatic monocarboxylic acids and Salts of dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids
  • Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., as well as salts derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucose Glucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.
  • compositions described herein may also include antioxidants.
  • antioxidants include, but are not limited to: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, Phosphoric acid etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
  • composition of the present invention can be made into various dosage forms as needed, and can be administered by a physician to determine a dose that is beneficial to the patient based on the patient's type, age, weight, general disease status, administration method and other factors.
  • the administration method may, for example, adopt parenteral administration (such as injection) or other treatment methods.
  • parenteral administration of immunogenic compositions includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection or infusion techniques.
  • the compositions may be isotonic, that is, they may have the same osmotic pressure as blood and tears.
  • the desired isotonicity of the compositions of the invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • the viscosity of the composition can be maintained at a selected level using pharmaceutically acceptable thickening agents.
  • Suitable thickeners include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like.
  • the preferred concentration of thickening agent will depend on the agent chosen. Obviously, the selection of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., a liquid dosage form.
  • kits comprising the antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells described herein.
  • a kit may include a therapeutic or prophylactic composition containing an effective amount of an antibody, chimeric receptor, nucleic acid, or host cell described herein in one or more unit dosage forms.
  • the kit includes a sterile container that may contain the therapeutic or prophylactic composition; such container may be a box, ampoule, bottle, vial, tube, bag, blister pack, or other suitable container known in the art. container form.
  • Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding the medication.
  • the kit includes an antibody, immunoconjugate, chimeric receptor, nucleic acid, or host cell described herein, and an antibody, immunoconjugate, chimeric receptor, Instructions for administering nucleic acid or host cells to an individual. Instructions generally include methods of treating or preventing cancer or tumors using the antibodies, immunoconjugates, chimeric receptors, nucleic acids, or host cells described herein.
  • a kit includes a host cell described herein, and may include about 1 ⁇ 10 4 cells to about 1 ⁇ 10 6 cells.
  • the kit can include at least about 1 ⁇ 10 cells, at least about 1 ⁇ 10 cells, at least about 1 ⁇ 10 cells , at least about 4 ⁇ 10 cells, at least about 5 ⁇ 10 cells, at least about 6 ⁇ 10 cells, at least about 6 ⁇ 10 cells, 8 ⁇ 10 cells, at least about 9 ⁇ 10 cells, at least about 1 ⁇ 10 cells, at least about 2 ⁇ 10 cells, at least about 3 ⁇ 10 cells, at least about 4 ⁇ 10 cells, at least about 5 ⁇ 10 cells, at least about 6 ⁇ 10 cells, at least about 6 ⁇ 10 cells , at least about 8 ⁇ 10 8 cells, at least about 9 ⁇ 10 8 cells, at least about 1 ⁇ 10 9 cells, at least about 2 ⁇ 10 9 cells, at least about 3 ⁇ 10 9 cells, at least about 4 ⁇ 10 9 cells 9 cells, at least about 5 ⁇ 10 9 cells, at least about 6 ⁇ 10 9 cells, at least about 8 ⁇ 10 9 cells, at least about 9 ⁇ 10 9 cells, at least about 1 ⁇ 10 9 cells, at least about 2 ⁇ 10
  • the kit may include allogeneic cells.
  • a kit can include cells that can contain genomic modifications.
  • the kit may contain "ready-made" cells.
  • a kit can include cells that can be expanded for clinical use. In some cases, kits may contain contents for research purposes.
  • the instructions include at least one of the following: a description of the therapeutic agent; dosage regimens and administration for treating or preventing tumors or symptoms thereof; precautions, warnings, contraindications, overdose information, adverse reactions, animals Pharmacology, clinical studies, and/or cited literature. Instructions may be printed directly on the container (if available), as a label on the container, or as a separate sheet, booklet, card or folder on or provided with the container. In some embodiments, the instructions provide methods of administering the antibodies of the invention for treating or preventing tumors. In some cases, the instructions provide for administering an antibody of the invention before, after, or concurrently with administration of a chemotherapeutic agent.
  • modulation refers to positive or negative change. Adjustment examples include 1%, 2%, 10%, 25%, 50%, 75%, or 100% changes. In a specific embodiment, it refers to a negative change.
  • treatment refers to interventions that attempt to modify the course of a disease, either preventatively or in the clinical pathological process.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing down the progression of the disease, improving or alleviating the condition, alleviating or improving the prognosis, etc.
  • prevention refers to interventions that attempt to prevent disease (such as rejection of cell transplantation) before it occurs.
  • tumor antigen refers to antigens that are newly emerged or overexpressed during the development and progression of hyperproliferative diseases.
  • a hyperproliferative disorder of the invention refers to a tumor.
  • the tumor antigen of the present invention may be a solid tumor antigen or a hematological tumor antigen.
  • Tumor antigens of the present invention include, but are not limited to: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13R ⁇ ); interleukin 11 receptor ⁇ (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; casein Amidase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor
  • the pathogen antigen is selected from: viral, bacterial, fungal, protozoan, or parasite antigens; the viral antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.
  • Subjects of the present invention include, but are not limited to, humans, non-human primates (eg, rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
  • non-human primates eg, rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
  • an effective amount refers to an amount that provides a therapeutic or prophylactic benefit.
  • anti-NKG2A antibodies Any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions, or kits provided herein can be used in methods of treatment.
  • any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit for use as a medicament is provided.
  • any anti-NKG2A antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition, or kit for treating a disease is provided.
  • any anti-NKG2A antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition, or kit for use in a method of treatment is provided.
  • the invention provides any anti-NKG2A antibody, immunoconjugate, chimeric antigen receptor modified host cell, pharmaceutical composition, or kit for use in a method of treating an individual suffering from a disease
  • the methods include administering to the individual an effective amount of any anti-NKG2A antibody, immunoconjugate, chimeric antigen receptor modified immune host cell, pharmaceutical composition, or kit.
  • the method further includes administering to the individual an effective amount of at least one additional therapeutic agent.
  • the "individual" is preferably a human.
  • the invention provides the use of any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit in the preparation or formulation of a medicament.
  • the medicament is used to treat a disease.
  • the medicament is used in a method of treating a disease, the method comprising administering an effective amount of the medicament to an individual suffering from the disease.
  • the method further includes administering to the individual an effective amount of at least one additional therapeutic agent.
  • the "individual" is preferably a human.
  • the invention provides methods for treating disease.
  • the method includes administering an effective amount of any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition, or kit to an individual suffering from a disease that expresses HLA-E.
  • the method further includes administering to the individual an effective amount of at least one additional therapeutic agent.
  • the "individual" is preferably a human.
  • the invention provides pharmaceutical formulations comprising any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit provided herein, eg for use in any of the above-described methods of treatment.
  • the pharmaceutical formulation comprises any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit provided herein and a pharmaceutical carrier.
  • the pharmaceutical formulation comprises any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit provided herein and at least one additional therapeutic agent.
  • the pharmaceutical formulation is used to treat disease.
  • the pharmaceutical formulation is administered to a diseased individual.
  • An "individual" according to any of the above embodiments is preferably a human.
  • the invention provides methods for the preparation of a medicament or pharmaceutical formulation, comprising combining any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits provided herein with a pharmaceutically acceptable
  • the carrier is admixed, for example, for use in any of the above treatment methods.
  • a method for preparing a medicament or pharmaceutical formulation further includes adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.
  • any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions, or kits of the invention can be used in therapy alone or in combination with other agents.
  • any anti-NKG2A antibody, immunoconjugate, chimeric antigen receptor modified host cell, pharmaceutical composition, or kit of the invention can be co-administered with at least one additional therapeutic agent.
  • Such combination treatments as described above include combined administration (in which two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case any of the anti-NKG2A antibodies, immunoconjugates, Administration of the host cell, pharmaceutical composition, or kit can occur before, simultaneously with, and/or after the administration of the additional therapeutic agent or agent.
  • the administration of any anti-NKG2A antibody, immunoconjugate, chimeric antigen receptor modified host cell, pharmaceutical composition, or kit of the invention and the administration of the additional therapeutic agent occur about one day apart from each other. Within a month, or within about a week, two weeks, or three weeks, or within about one, two, three, four, five, or six days.
  • Any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition, or kit of the invention may be administered by any suitable means, including parenteral, intrapulmonary, or intranasal administration administration, and, if therapeutically necessary, intralesional administration.
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenously or subcutaneously, depending in part on whether the administration is transient or long-term.
  • a variety of dosing regimens are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • a formulation comprising a population of immunoreactive cells administered to an individual contains a plurality of immunoreactive cells effective in treating and/or preventing a particular indication or disease.
  • a therapeutically effective population of immunoreactive cells can be administered to an individual.
  • a preparation containing about 1 ⁇ 10 4 to about 1 ⁇ 10 10 immunoreactive cells is administered.
  • the preparation will contain about 1 ⁇ 10 5 to about 1 ⁇ 10 9 immunoreactive cells, about 5 ⁇ 10 5 to about 5 ⁇ 10 8 immunoreactive cells, or about 1 ⁇ 10 6 to Approximately 1 ⁇ 10 7 immunoreactive cells.
  • the number of CAR immunoreactive cells administered to an individual will vary within a wide range depending on the location, origin, identity, extent and severity of the tumor, the age and physical condition of the individual to be treated, etc. Your doctor will ultimately determine the appropriate dosage to use.
  • chimeric receptors are used to stimulate host cell-mediated immune responses.
  • a T cell-mediated immune response is an immune response involving activation of T cells.
  • Activated antigen-specific cytotoxic T cells are capable of inducing apoptosis in target cells displaying foreign antigen epitopes on their surface, such as cancer cells displaying tumor antigens.
  • chimeric antigen receptors are used to provide anti-tumor immunity in mammals. The subject will develop anti-tumor immunity as a result of the T cell-mediated immune response.
  • methods of treating a subject with a tumor may involve administering one or more host cells of the invention to the subject in need of treatment.
  • the host cells can bind tumor target molecules and induce cancer cell death.
  • the invention also provides methods of treating a pathogen infection in an individual, comprising administering to said individual a therapeutically effective amount of a host cell of the invention.
  • the frequency of administration of the host cells of the invention will depend on factors including the disease being treated, the components of the particular host cell, and the mode of administration. For example, it can be administered 4 times, 3 times, 2 times a day, or once a day, every other day, every three days, every four days, every five days, every six days, once a week, once every eight days, or every day. Dosing is given once every nine days, every ten days, once a week, or twice a month. As described herein, due to the improved viability of the host cells of the present application, the host cells of the present application can be administered not only in lower therapeutically effective amounts than similar host cells that do not express exogenous type I interferon, but also in more therapeutically effective amounts. Less frequent dosing to achieve at least similar, and preferably more significant, therapeutic effects.
  • the present invention provides antibodies that specifically bind to NKG2A, which are fully human antibodies with low immunogenicity and few possible clinical side effects;
  • the antibody of the present invention can effectively block the combination of HLA-E of tumor cells and NKG2A/CD94 of NK cells, reduce the inhibitory effect of tumor cells expressing HLA-E on NK cells through the NKG2A/CD94 pathway, and enhance the effect of NK cells on NK cells. It has a killing effect on tumor cells and shows good anti-tumor effect.
  • T cells expressing NKG2A-CAR prepared by the antibody of the present invention can kill NK cells; T cells using tandem CARs prepared by the antibody of the present invention that target both tumors and NK cells can kill NK cells and can also enhance Its anti-tumor effect; UCAR-T cells expressing NKG2A-CAR prepared by the antibody of the present invention can resist killing by NK cells, enhance their survival ability, and can cooperate with the anti-tumor effect of tumor-targeting T cells or CAR-T cells .
  • the eukaryotic expression plasmid V152S was used to construct vectors expressing NKG2A/CD94 and NKG2C/CD94 heterodimers respectively. It will include mFc (sequence shown in SEQ ID NO: 84), NKG2A extracellular segment (sequence shown in SEQ ID NO: 71), (G 4 S) 3 (sequence shown in SEQ ID NO: 110), CD94
  • the fragment mFc-NKG2A-CD94 of the extracellular segment (sequence shown in SEQ ID NO: 77) was inserted into the eukaryotic expression plasmid V152S to construct the vector V152S-mFc-NKG2A-CD94; the mFc (sequence shown in SEQ ID NO: 84 shown), NKG2C extracellular segment (sequence shown in SEQ ID NO: 73), (G 4 S) 3 (sequence shown in SEQ ID NO: 110), CD94 extracellular segment (se
  • V152S-mFc-NKG2A-CD94 and V152S-mFc-NKG2C-CD94 were transfected into HEK293 cells (China Type Culture Collection Center (CCTCC)) respectively and then cultured for 7 days.
  • the culture medium was centrifuged to take the supernatant, and Mabselect Sure columns were used to and purification to obtain antigens NKG2A/CD94 and NKG2C/CD94 heterodimers respectively.
  • the phage display library used in the present invention is a phage library constructed by our company, with a library capacity of 1E+11. Fab fragments highly specific for the NKG2A/CD94 heterodimer were obtained using screening methods known to those skilled in the art.
  • mFc-NKG2A-CD94 and mFc-NKG2C-CD94 prepared above were coated on immune tubes respectively.
  • MPBS phosphate buffered saline
  • the phage library was added to the immune tube coated with mFc-NKG2C-CD94 and combined for 1 hour. The supernatant was added to an immune tube coated with mFc-NKG2A-CD94 and bound for 1.5 hours.
  • a total of 1504 clones were screened, of which 50 clones only bound mFc-NKG2A-CD94 and did not bind mFc-NKG2C-CD94. After sequencing, 2 clones were obtained. These two clones were expressed and purified to obtain antibodies A1 and A2 in Fab form.
  • the amino acid sequence of HCDR1 of A1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3, and the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:6.
  • the amino acid sequence of the heavy chain variable region of A1 is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:20.
  • the amino acid sequence of the heavy chain of A1 is shown in SEQ ID NO: 34, and the amino acid sequence of the light chain is shown in SEQ ID NO: 42.
  • the amino acid sequence of HCDR1 of A2 is shown in SEQ ID NO:7
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:8
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:9
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:5
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:10.
  • the amino acid sequence of the heavy chain variable region of A2 is shown in SEQ ID NO:22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:24.
  • the amino acid sequence of the A2 heavy chain is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain is shown in SEQ ID NO: 46.
  • antibodies in the form of IgG4 were constructed and purified by eukaryotic expression, and two antibodies specifically binding to the NKG2A/CD94 heterodimer were obtained, named A1-IgG4 and A2-IgG4.
  • the heavy chain amino acid sequence of A1-IgG4 is shown in SEQ ID NO:40, and the light chain amino acid sequence is shown in SEQ ID NO:42.
  • the heavy chain amino acid sequence of A2-IgG4 is shown in SEQ ID NO:44, and the light chain amino acid sequence is shown in SEQ ID NO:46.
  • the antigens mFc-NKG2A-CD94 and mFc-NKG2C-CD94 were diluted with PBS to a concentration of 5 ⁇ g/ml and coated overnight at 4°C.
  • NA is the blank control.
  • Both antibodies A1 and A2 specifically bind to the NKG2A/CD94 heterodimer but do not bind to the NKG2C/CD94 heterodimer.
  • the mFc-NKG2A-CD94 prepared above was diluted with PBS to a concentration of 2.5 ⁇ g/ml and coated overnight at 4°C. Block with 2% MPBS (skimmed milk powder/PBS) at room temperature for 1 hour; wash 3 times with PBS, add primary antibodies (A1-IgG4, A2-IgG4: 5-fold dilution starting from 10 ⁇ g/ml and 8 gradients), and incubate at room temperature for 1 hour; wash with PBST for 5 After the first pass, the secondary antibody (anti-Fc-HRP, 1:10000, Sigma) was added and continued to incubate at room temperature for 1 hour; after washing with PBST 5 times, TMB developed color and the OD450 value was read with a microplate reader.
  • CD94 full length (including CD94 full length, Flag tag), NKG2A-CD94 (including NKG2A full length, F2A, CD94 full length, Flag tag), NKG2C -CD94 (contains NKG2C full length, F2A, CD94 full length, Flag tag), NKG2E-CD94 (contains NKG2E full length, F2A, CD94 full length, Flag tag), respectively transferred into CHO-K1 cells (also known as CHOK1) A mixed clone cell line was constructed to overexpress the target gene. Anti-Flag antibody was used to confirm that the mixed clone strain was successfully constructed, and then the limiting dilution method was used for monoclonal screening.
  • the CHO-K1 stably transduced cell line CHOK1-CD94 expressing CD94 was successfully obtained.
  • -CD94 see Figure 5 for the vector map
  • the positive rates were all greater than 90%.
  • the full-length base sequence of CD94 is SEQ ID No. 128, the full-length base sequence of NKG2A is SEQ ID No. 69, the full-length base sequence of NKG2C is SEQ ID No. 124, and the full-length base sequence of NKG2E is SEQ ID No. .126, the Flag tag base sequence is SEQ ID No.112, and the F2A base sequence is SEQ ID No.108.
  • Antibodies A1-IgG4 and A2-IgG4 respectively bind to CHO-K1 cells overexpressing NKG2A/CD94 heterodimer, but do not bind to CHO-K1 cells overexpressing NKG2C/CD94, NKG2E/CD94 and CD94.
  • A1 and A2 as parent antibodies respectively, two phage libraries were constructed using conventional biological techniques, one randomized the CDR1 and CDR2 of the light chain, and the other randomized the CDR1 and CDR2 of the heavy chain. Then the antigen is screened, and high-affinity antibodies, namely A1 or A2 variants, are screened out through ELISA technology.
  • a template plasmid was constructed based on antibody A1 (Fab).
  • Fab antibody A1
  • PCR amplified fragment 1 using primers LMF (nucleic acid sequence as shown in SEQ ID NO: 85) and IL1R (nucleic acid sequence as shown in SEQ ID NO: 91); using primers IL2F (the nucleic acid sequence is shown in SEQ ID NO: 92) and FdR (the nucleic acid sequence is shown in SEQ ID NO: 86), PCR amplified fragment 2; then fragment 1 and fragment 2 were connected by bridge PCR to obtain the fragment containing the randomized sequence.
  • LMF nucleic acid sequence as shown in SEQ ID NO: 85
  • IL1R nucleic acid sequence as shown in SEQ ID NO: 91
  • primers IL2F the nucleic acid sequence is shown in SEQ ID NO: 92
  • FdR the nucleic acid sequence is shown in SEQ ID NO: 86
  • the full-length Fab was then digested with NcoI and NotI, ligated into the template plasmid digested by the same enzyme through T4 ligase, and electrotransformed into TG1 competent cells with a library capacity of 2.6x10 9 .
  • the primers LMF the nucleic acid sequence is shown in SEQ ID NO: 85
  • F10H1R the nucleic acid sequence is shown in SEQ ID NO: 87
  • PCR amplify fragment 3 use the primers F10H2F (nucleic acid sequence as shown in SEQ ID NO: 88) and FdR (nucleic acid sequence as shown in SEQ ID NO: 86), PCR amplified fragment 4; and then connected fragment 3 and fragment 4 by bridge PCR to obtain the fragment containing the randomized sequence
  • the full-length Fab was then digested with NcoI and NotI, ligated into the template plasmid digested by the same enzyme through T4 ligase, and electrotransformed into TG1 competent cells, with a library capacity of 3.2x10 9 .
  • antibody A2 affinity maturation library is similar to A1, and the template plasmid is constructed based on antibody A2 (Fab).
  • the CDR1 and CDR2 of the light chain were randomized using the same primers as A1, and the resulting phage library size was 1.89x10 9 .
  • the primers LMF the nucleic acid sequence is shown in SEQ ID NO: 85
  • BH1R the nucleic acid sequence is shown in SEQ ID NO: 89
  • PCR amplify fragment 5 use the primers BH2F (the nucleic acid sequence is shown in SEQ ID NO: 90) and FdR (the nucleic acid sequence is shown in SEQ ID NO: 86), PCR amplified fragment 6; then fragment 5 and fragment 6 were connected by bridge PCR to obtain the fragment containing the randomized sequence.
  • the full-length Fab was then digested with NcoI and NotI, ligated into the template plasmid digested by the same enzyme through T4 ligase, and electrotransformed into TG1 competent cells with a library capacity of 1.22x10 9 .
  • Example 1 two rounds of screening were performed.
  • the first round of screening was coated with the antigen mFc-NKG2A-CD94 at a concentration of 5 ⁇ g/ml and cleared with the antigen mFc-NKG2C-CD94 at a concentration of 5 ⁇ g/ml; the second round of screening used the antigen mFc-NKG2A-CD94 at a concentration of 1 ⁇ g/ml.
  • positive clones were determined by ELISA.
  • the amino acid sequences of the heavy chain variable regions of six antibodies A1, A2, A3, A4, A5 and A6 were compared.
  • sequence similarity between the heavy chain variable regions of A3 and the parent A1 was 98.3%.
  • the heavy chain variable region sequence similarity between A4 and the parent A2 is 94.8%.
  • the heavy chain variable region sequence similarity between A5 and the parent A2 is 94.8%.
  • the heavy chain variable region sequence similarity between A6 and the parent A2 is 93.1%.
  • the antigen mFc-NKG2A-CD94 was diluted with PBS to a concentration of 2 ⁇ g/ml and coated overnight at 4°C.
  • MFI mean fluorescence intensity
  • GraphPad Prism5 software uses GraphPad Prism5 software to calibrate the mean fluorescence intensity with the primary antibody concentration as the abscissa.
  • MFI mean fluorescence intensity
  • the results are shown in Figure 10.
  • the EC50 value of the antibody (Fab form) binding to CHO-K1 cells overexpressing NKG2A/CD94 heterodimer is shown in Table 9. The binding ability of the antibody cells after affinity maturation is significantly better than that of the corresponding mother cells. This antibody.
  • the antigen mFc-NKG2A-CD94 was diluted with PBS to a concentration of 2 ⁇ g/ml and coated overnight at 4°C. Block with 2% MPBS (skimmed milk powder/PBS) at room temperature for 1 hour; wash with PBS 3 times and then add primary antibodies (A1-IgG4, A2-IgG4, A3-IgG4, A4-IgG4, A5-IgG4, A6-IgG4: starting from 5 ⁇ g/mL (Start with 5-fold gradient dilution (eight gradients)) and incubate at room temperature for 1 hour; after washing with PBST 5 times, add secondary antibody (anti-Fc-HRP, 1:10000, Sigma) and continue incubating at room temperature for 1 hour; after washing with PBST 5 times, TMB develops the color and uses enzyme The standard instrument reads the OD450 value.
  • the GraphPad Prism5 software uses the GraphPad Prism5 software to perform four-parameter fitting with the primary antibody concentration as the abscissa and the OD450 value as the ordinate, and calculate the EC50 value.
  • the results are shown in Figure 11.
  • the antibody (IgG4 form) binds to the NKG2A/CD94 heterodimer in a concentration gradient dependence.
  • the EC50 value is shown in Table 10.
  • primary antibodies A1-IgG4, A2-IgG4, A3-IgG4, A4-IgG4, A5-IgG4, A6 -IgG4: 25 ⁇ g/mL starting with 5-fold gradient dilution (8 gradients) and incubation with secondary antibody (Anti-Fab-FITC: 1:200, Jackson ImmunoResearch), followed by fluorescence intensity detection using a flow cytometer.
  • MFI mean fluorescence intensity
  • GraphPad Prism5 software uses GraphPad Prism5 software to calibrate the mean fluorescence intensity with the primary antibody concentration as the abscissa.
  • MFI mean fluorescence intensity
  • the results are shown in Figure 12.
  • the antibodies (IgG4 form) all bind to CHO-K1 cells overexpressing NKG2A/CD94 heterodimers in a concentration gradient dependence.
  • the EC50 values are shown in Table 11, which shows that the antibodies after affinity maturation The binding activity was improved.
  • the fragments HLA-E-avi- including the human HLA-E extracellular segment (sequence shown in SEQ ID NO:79) and avi tag (sequence shown in SEQ
  • the plasmids pET22b-HLA-E and pET22b- ⁇ 2m (sequence shown in SEQ ID NO: 81) and fragment ⁇ 2m (sequence shown in SEQ ID NO: 81) were transformed into the BL21 strain respectively (see Figure 14 for the vector).
  • the BL21 strain was used for prokaryotic induction and expression, and the bacterial cells were collected and disrupted by sonication to collect inclusion body precipitates. The inclusion body precipitate was washed and then dissolved in 8M urea. After further purification with an anion exchange column, the purity was determined by electrophoresis to be over 90%. Add 5 mg of VMA nonapeptide (the amino acid sequence is shown in SEQ ID NO: 82, synthesized by Gill Biochemical) into 100 ml of refolding solution, and then add purified HLA-E and ⁇ 2m at a molar ratio of 1:2 to obtain a refolding complex. Then dialyze against PBS 5% glycerol.
  • VMA nonapeptide the amino acid sequence is shown in SEQ ID NO: 82, synthesized by Gill Biochemical
  • the product is purified by molecular sieves.
  • the target peak is collected, concentrated and replaced with 10mM Tris pH 8.0, and stored in aliquots at -80°C.
  • the complex was labeled with biotin using birA enzyme, and the labeled product and SA-PE (PE-labeled SA antibody (BD Horizon TM )) were gently mixed in a ratio of 1:4 to obtain the final HLA-E tetramer.
  • NK cells were purified from peripheral blood PBMCs by NK Cell Isolation Kit (Miltenyi Biotec) and treated with NK cells containing 500IU/ml IL-2 and 150IU/ml IL-15.
  • NKG2A-positive NK cells (NKG2A + NK) were collected after 8 days of culture in Medium (Miltenyi Biotec), and the positivity rate was 86%.
  • the HLA-E tetramer was used at a concentration starting from 5 ⁇ g/ml, with a 5-fold gradient dilution, and the blank well concentration was 0.
  • HLA-E tetramer significantly binds to NK cells expressing NKG2A/CD94 in a concentration gradient-dependent manner, with an EC50 of 0.1029 ⁇ g/ml.
  • Example 4 Take the NKG2A-positive NK cells (NKG2A + NK) prepared in Example 4, add HLA-E-PE and anti-NKG2A antibody at the same time, incubate them at 4°C for 45 minutes, and detect the PE fluorescence signal intensity.
  • the HLA-E-PE concentration uses the EC50 value, which is 0.103 ⁇ g/ml.
  • the antibody concentration starts from 10 ⁇ g/ml and is diluted 5 times to 0.000128 ⁇ g/ml.
  • the blank group only contains HLA-E-PE. After the co-incubation, the cells were washed 3 times with PBS containing 1% FBS, and then the PE fluorescence signal was detected by a flow cytometer to obtain the MFI value.
  • the inhibition rate calculation formula is: (MFI (blank group) - MFI (experimental group) ))/MFI(blank group) ⁇ 100%.
  • the results are shown in Figure 16.
  • the obtained affinity matured antibody can completely inhibit the binding of HLA-E tetramer to NK cells at concentrations above 1 ⁇ g/ml.
  • the IC50 value of the half inhibitory effective concentration is shown in Table 13. Experiments show that the ability of affinity matured antibodies to competitively inhibit the binding of HLA-E tetramers to NK cells is significantly improved compared to the parent antibody.
  • NKG2A antibody can reduce the inhibition of NK cell activity by target cells expressing HLA-E
  • the NKG2A + NK cells prepared in Example 4 were used as effector cells.
  • Target cells were selected from K562 cells that do not express HLA-E (human myeloid leukemia cells, cell bank of the Chinese Academy of Sciences), K562 cells that overexpress HLA-E (called K562-HLA-E cells), and endogenously express HLA-E. FaDu cells (human pharyngeal squamous cells, ATCC), HLA-E expression levels are shown in Figure 17.
  • K562-HLA-E cells were obtained by transferring the human HLA-E extracellular region (sequence shown in SEQ ID NO: 78) into K562 cells through lentivirus-mediated method.
  • NK cells 1 ⁇ 10 5 NK cells were co-incubated with three target cells at an effect-to-target ratio of 1:1, and CD107a-APC antibody (purchased from BD Biosciences, 5 ⁇ l/test) and NKG2A antibody (IgG4 form, 10 ⁇ g/ml) were added. , the blank group (no NKG2A antibody added) was incubated for 1 hour, and the protein transport inhibitor (Brefeldin A/Monensin Mix) was added to continue incubating for 3 hours.
  • CD107a-APC antibody purchased from BD Biosciences, 5 ⁇ l/test
  • NKG2A antibody IgG4 form, 10 ⁇ g/ml
  • CD56 antibody with PE-Cy7 fluorescence CD56-PE-Cy7, purchased from eBioscience, 5 ⁇ l/test, used to detect NK cells
  • anti-Fc antibody with FITC fluorescence anti-Fc-FITC Antibodies, purchased from Jackson Immunoresearch, 1:200, used to detect NKG2A-positive NK cells
  • CD56-PE-Cy7 and anti-NKG2A-PE antibodies purchased from Miltenyi Biotec, 2 ⁇ l/test
  • NKG2A + NK cells were co-incubated with K562-HLA-E cells or FaDu cells, the expression level of CD107a was low, indicating that the combination of HLA-E on tumor cells and NKG2A on NK cells can significantly inhibit NK cell activity; and Adding NKG2A antibodies competitively binds to NKG2A on NK cells, blocking the binding of NKG2A on NK cells to HLA-E on tumor cells, thereby reducing the inhibition of NK cell activity by tumor cells with high HLA-E expression. Therefore, compared with no Adding antibody group, adding NKG2A antibody, the expression level of CD107a in each group increased significantly. Due to the low endogenous expression of HLA-E in FaDu cells, the increase in CD107a expression level was lower than that in the HLA-E high expression group K562-HLA-E.
  • NKG2A antibody enhances the killing effect of primary NK cells on target cells expressing HLA-E
  • NKG2A + NK cells were incubated with target cells (K562, K562-HLA-E and FaDu cells) respectively, with an effect-to-target ratio of 3:1, and 10 ⁇ g/ml NKG2A antibody ( IgG4 form, no antibody was added to the blank group), the culture supernatant was collected after 4 hours of incubation, and the killing effect was calculated by LDH detection method.
  • target cells K562, K562-HLA-E and FaDu cells
  • Cytotoxicity % [LDH release amount of experimental group (Avg.) – Spontaneous LDH release amount of effector cells (Avg.) – Spontaneous LDH release amount of target cells (Avg.)]/[Maximum LDH release amount of target cells (Avg.) – Spontaneous LDH release from target cells (Avg.) – Volume calibration (Avg.)] ⁇ 100%
  • NKG2A + NK cells have a killing rate of approximately 55% on K562 cells, and the killing rates on K562-HLA-E cells and FaDu cells that highly express HLA-E are both less than 20%. This shows that HLA-E expressed on target cells binds to NKG2A expressed on NK cells, inhibiting NK cell activity, thereby reducing the killing of target cells by NK cells. Adding NKG2A antibodies can block the binding of HLA-E of target cells to NKG2A of NK cells, reduce the inhibition of NK cell activity by target cells, thereby increasing NK cell activity and increasing NK cell killing of tumor cells that highly express HLA-E.
  • Example 8 Killing effect of anti-NKG2A-specific CAR-T cells on NK cells
  • PRRLSIN-cPPT.EF-1 ⁇ purchased from Addgene
  • PRRLSIN-cPPT.EF-1 ⁇ purchased from Addgene
  • lentiviral plasmids expressing second-generation chimeric antigen receptors of antibodies A4 and A5 were constructed, namely PRRLSIN-A4-BBZ and PRRLSIN-A5-BBZ.
  • the A4-BBZ (SEQ ID NO: 115) sequence consists of CD8 ⁇ signal peptide, A4scFv, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signaling domain and CD3 ⁇ sequence connection.
  • the A5-BBZ (SEQ ID NO: 116) sequence consists of CD8 ⁇ signal peptide, A5scFv, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signaling domain and CD3 ⁇ sequence connection.
  • CD8 ⁇ signal peptide SEQ ID NO: 93
  • A4scFv SEQ ID NO: 64
  • A5scFv SEQ ID NO: 66
  • CD8 hinge region SEQ ID NO: 95
  • CD8 transmembrane region SEQ ID NO: 97
  • CD137 intracellular signaling domain SEQ ID NO: 101
  • CD3 ⁇ SEQ ID NO: 105
  • the lentivirus was packaged using the calcium phosphate method, and the viral supernatant was purified with PEG8000/NaCl. After purification, the virus was infected with CD3/CD28 magnetic beads at an MOI value of 10 and activated for 48 hours on T cells expressing A4-BBZ and A5 respectively. -BBZ's CAR-T cells, T cells not transfected with virus are considered UTD. On the 6th day after infection, the FACS method was used to detect the CAR positivity rate. The detection antigen was Bio-NKG2A-CD94, and the secondary antibody was BV421-labeled SA antibody (BD Horizon TM ). It was diluted at 1:200. The results showed that the CAR of A4-BBZ CAR T The positive rate was 62.8%, and the CAR positive rate of A5-BBZ CAR T was 59%.
  • NK cells were purified from peripheral blood PBMCs of two donors (#1 and #2) by NK Cell Isolation Kit (Miltenyi Biotec) and treated with NK containing 500IU/ml IL-2 and 150IU/ml IL-15. Medium (Miltenyi Biotec) was cultured until harvested on the 14th day.
  • the above-mentioned NK cells were incubated with APC-labeled NKG2A antibody (Invitrogen) (diluted at 1:200) for 5 min at 4°C, and then the expression level of NKG2A in the NK cells was detected by FACS method. The results showed that the positive rate of NKG2A in NK cells from donor #1 was 80.4%, and the positive rate of NKG2A in NK cells from donor #2 was 61.5%.
  • Target cells Take 5 ⁇ 10 4 of the above NKG2A-positive NK cells as target cells and inoculate them into a 96-well plate.
  • Effector cells Inoculate UTD cells, A4-BBZ CAR-T cells, and A5-BBZ CAR-T cells into the corresponding 96-well plates according to the effect-to-target ratio of 1:1 and 2:1 respectively.
  • Flow cytometry staining was used to conduct in vitro cell killing experiments. Flow cytometry staining was performed at 0hr, 4hr, and 24hr to detect the proportion of NK cells in the co-culture system. The results are shown in Figure 20. As the co-culture time increases, the proportion of NK cells in the UTD cell group does not change significantly, while the proportion of NK cells in the A4-BBZ CAR-T and A5-BBZ CAR-T groups decreases significantly. This shows that both A4-BBZ CAR-T cells and A5-BBZ CAR-T cells can effectively kill NK cells expressing NKG2A.
  • Example 9 Anti-NKG2A UCAR-T cells can effectively resist killing by NK cells
  • UCAR-TKO NKG2A knockout NKG2A-UCAR-T cells
  • gRNA targeting the TCR/B2M/NKG2A gene was synthesized in vitro, and the sequences are shown in SEQ ID NO: 117, 118, and 119 respectively.
  • CRISPR/Cas9 technology Use conventional CRISPR/Cas9 technology to knock out endogenous TCR/B2M or TCR/B2M/NKG2A in T cells.
  • CRISPR/Cas 9 enzyme Kaika Biotechnology
  • gRNA were mixed at a molar ratio of 1:4 to form an RNP complex (the final concentration of Cas 9 enzyme was 1uM). After incubation at room temperature for 10 minutes, the RNP complex was complexed using a MaxCyte electroporation instrument. introduced into T cells.
  • the A4-CAR-T and A5-CAR-T cells in Example 8 were respectively subjected to TCR/B2M double knockout to obtain A4-UCAR-T and A5-UCAR-T cells; A4-CAR-T and A5- CAR-T cells were triple-knocked out of TCR/B2M/NKG2A to obtain A4-UCAR-T-TKO and A5-UCAR-T-TKO cells.
  • the proportion of UTD UCAR-T and UTD UCAR-T-TKO cells gradually decreased over time, indicating that NK cells inhibited the growth of UTD UCAR-T and UTD UCAR-T-TKO cells; while expressing NKG2A-CAR
  • the proportion of UCAR-T or UCAR-T-TKO cells increased significantly 48h after co-incubation. This shows that anti-NKG2A UCAR-T or UCAR-T-TKO cells can effectively resist killing by NK cells.
  • BCMA-targeting CAR-T cells Conventional molecular biology methods in this field are used to construct chimeric antigen receptors targeting BCMA, package lentivirus and transfect T cells to prepare BCMA-targeting CAR-T cells.
  • the amino acid sequence of BCMA-scFv is shown in SEQ ID NO: 120
  • the amino acid sequence of BCMA-CAR is shown in SEQ ID NO: 121.
  • the method of Example 9 was used to knock out the B2M/TCR/NKG2A gene of BCMA CAR-T cells to obtain BCMA UCAR-T cells (named BCMA UCAR-T-TKO).
  • the BCMA-expressing multiple myeloma cell line RPMI-8226 (Cell Bank of the Chinese Academy of Sciences) was cultured in vitro, and 5 ⁇ 10 6 cells/mouse were subcutaneously inoculated into NPG immunodeficient mice (recorded as D0). The average tumor volume 10 days after inoculation is about 200 mm 3 , and the mice are divided into 4 groups. On D10, D14, D17, D21, and D24, 1 ⁇ 10 6 NK cells were injected into the tail veins of groups 2, 3, and 4, respectively, for a total of 5 injections. On D11, T cells were injected into the tail vein of each group. The details of each group are as follows:
  • FIG. 22 The results are shown in Figure 22.
  • anti-NKG2A UCAR-T cells exerted a synergistic anti-tumor effect with BCMA UCAR-T cells: on D32, tumors in mice in groups 3 and 4 were almost completely eliminated.
  • CAR-T cells targeting both NKG2A and BCMA were constructed to observe their anti-tumor activity and resistance to NK cell killing.
  • BCMA-NKG2A CAR-T cells expressing tandem CAR SEQ ID NO: 122
  • the B2M/TCR/NKG2A gene of BCMA-NKG2A CAR-T cells was knocked out to obtain BCMA-NKG2A UCAR-T-TKO cells.
  • dual-target tandem UCAR-T cells targeting NKG2A and tumor antigens can effectively resist NK cell killing and inhibit tumor growth.
  • This application relates to the following sequence:
  • N represents any base in the base sequence.

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Abstract

本发明涉及靶向NKG2A的抗体及其应用。本发明公开了新的特异性识别NKG2A的全人源抗体。本发明的抗体能够有效阻断肿瘤细胞的HLA-E与NK细胞的NKG2A/CD94结合，降低了表达HLA-E的肿瘤细胞通过NKG2A/CD94通路对NK细胞的抑制作用，增强NK细胞对肿瘤细胞的杀伤作用。

Description

NKG2A抗体及其应用
相关申请
本专利申请要求于2022年1月24日递交的申请号为202210080479.9的中国专利申请的优先权。
同时提交的序列表文件
下列XML文件的全部内容通过整体引用并入本文：计算机可读格式(CRF)的序列表(名称：FF00718PCT-sequence listing.xml，日期：20230119，大小：148KB)。
技术领域
本发明涉及肿瘤免疫治疗或诊断领域，更具体地，涉及识别NKG2A的抗体及其应用。
 背景技术
自然杀伤细胞(natural killer cell，NK细胞)是一类无需预先致敏就能非特异性杀伤肿瘤细胞和病毒感染细胞的淋巴细胞，是机体免疫细胞的重要组成之一。NK细胞表面有众多受体，分为激活性和抑制性两类，NKG2A便是其中一类抑制性受体。
NKG2A蛋白又叫C型凝集素，不仅表达在NK细胞中，在NKT细胞和CD8+αβT细胞中也有表达，NKG2A可与细胞表面的CD94形成二聚体(Jiacheng Bi and Zhigang Tian.NK Cell Dysfunction and Checkpoint Immunotherapy.Front.Immunol,2019.)。非经典MHC I类分子HLA-E是NKG2A-CD94的主要配体，在正常情况下HLA-E的表达量很低，但是在大部分的肿瘤细胞表面，HLA-E的表达量上升，NKG2A与HLA-E的相互作用抑制了NK细胞的激活，使得肿瘤细胞避免被NK细胞杀伤(Linda Borst,et al.The NKG2A-HLA-E axis as a novel checkpoint in the tumor microenvironment.American Association for Cancer,2020.)。因此开发靶向NKG2A的抗体可阻断NKG2A与HLA-E的结合，进而激活NK细胞和T细胞。
虽然NKG2A抗体极具潜力，但是其开发极具挑战。NKG2受体家族具有多种受体，包括NKG2A，NKG2C，NKG2D和NKG2E等。这些受体对于免疫细胞有些具有抑制作用，有些具有激活作用。这些受体的氨基酸序列具有高度的同源性，其中人的NKG2A和NKG2C同源性为90％，和NKG2E的同源为71％。虽然NKG2C和NKG2E与NKG2A在胞外段序列非常类似，但是在功能上完全相反，对抗体的特异性要求很高。
目前已有相关NKG2A靶点单克隆抗体药物报道，主要包括Innate/阿斯利康的Monalizumab(US20170298131A1)和怀越生物的Mpb416(CN111153995A)，初步临床结果显示NKG2A抗体联合用药具有一定的抗肿瘤效果，但也存在单药效果较差和具有一定副作用的问题。
发明内容
本发明的目的在于提供识别NKG2A的全人源抗体及其应用。
在第一方面，本发明提供了一种识别NKG2A的全人源抗体，其特征在于，所述抗体包含轻链可变区，所述轻链可变区包含RASQSISSWLA(SEQ ID NO:4)所示的LCDR1；和/或DASSLES(SEQ ID NO:5)所示的LCDR2；和/或QQYDSYX1X2T(SEQ ID NO:129)所示的LCDR3，其中X1是I或V，X2是R或S。
在具体实施方式中，所述抗体包含轻链可变区，所述轻链可变区包含RASQSISSWLA(SEQ ID NO:4)所示的LCDR1；和/或DASSLES(SEQ ID NO:5)所示的LCDR2；和/或QQYDSYIRT(SEQ ID NO:6)所示的LCDR3。
在具体实施方式中，所述抗体包含轻链可变区，所述轻链可变区包含RASQSISSWLA(SEQ ID NO:4)所示的LCDR1；和/或DASSLES(SEQ ID NO:5)所示的LCDR2；和/或QQYDSYVST(SEQ ID NO:10)所示的LCDR3。
本发明还提供了一种识别NKG2A的全人源抗体，其特征在于，所述抗体包括重链可变区，所述重链可变区选自：
(1)包含SYAIS(SEQ ID NO:1)所示的HCDR1；和/或GIIPIFGTAX1YAQKFQG(SEQ ID NO:130)所示的HCDR2，其中X1是N或H；和/或GFDGMDY(SEQ ID NO:3)所示的HCDR3；或
(2)包含X1X2X3X4S(SEQ ID NO:131)所示的HCDR1，其中X1是S、R或N，X2是Y、F或V，X3是A、Y或H，X4是M或V；和/或AIX1X2X3X4GSTYYADSVKG(SEQ ID NO:132)所示的HCDR2，其中X1是S、T或N，X2是G或A，X3是S、W、G或P，X4是G或V；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含SYAIS(SEQ ID NO:1)所示的HCDR1；和/或GIIPIFGTANYAQKFQG(SEQ ID NO:2)所示的HCDR2；和/或GFDGMDY(SEQ ID NO:3)所示的HCDR3。
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含SYAIS(SEQ ID NO:1)所示的HCDR1；和/或GIIPIFGTAHYAQKFQG(SEQ ID NO:11)所示的HCDR2；和/或GFDGMDY(SEQ ID NO:3)所示的HCDR3。
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含SYAMS(SEQ ID NO:7)所示的HCDR1；和/或AISGSGGSTYYADSVKG(SEQ ID NO:8)所示的HCDR2；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含RFYMS(SEQ ID NO:12)所示的HCDR1；和/或AITGWGGSTYYADSVKG(SEQ ID NO:13)所示的HCDR2；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含RVHMS(SEQ ID NO:14)所示的HCDR1；和/或AISAGGGSTYYADSVKG(SEQ ID NO:15)所示的HCDR2；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含NFHVS(SEQ ID NO:16)所示的HCDR1；和/或AINGPVGSTYYADSVKG(SEQ ID NO:17)所示的HCDR2；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。
在具体实施方式中，所述抗体选自以下的任一种：
(1)抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO:1、7、12、14或16所示的HCDR1，和/或包含SEQ ID NO:2、8、11、13、15或17所示的HCDR2，和/或包含SEQ ID NO:3或9任一所示的HCDR3；
(2)抗体，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:4所示的LCDR1，和/或包含SEQ ID NO:5所示的LCDR2，和/或包含SEQ ID NO:6或10任一所示的LCDR3；
(3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；
(4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
在具体实施方式中，所述抗体选自以下的任一种：
(1)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或
(2)抗体，其包含SEQ ID NO:7所示的HCDR1，SEQ ID NO:8所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或
(3)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:11所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或
(4)抗体，其包含SEQ ID NO:12所示的HCDR1，SEQ ID NO:13所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或
(5)抗体，其包含SEQ ID NO:14所示的HCDR1，SEQ ID NO:15所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或
(6)抗体，其包含SEQ ID NO:16所示的HCDR1，SEQ ID NO:17所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；
(7)抗体，(1)～(6)中任一项所述的抗体的变体，且具备与(1)～(6)中任一项所述的抗体相同或相似的活性。
在具体实施方式中，所述抗体选自以下的任一种：
(1)抗体，包含重链可变区，所述重链可变区包含SEQ ID NO:18、22、26、28、30或32所示的氨基酸序列、或上述序列的变体；
(2)抗体，包含轻链可变区，该轻链可变区包含SEQ ID NO:20或24所示的氨基酸序列、或上述序列的变体；
(3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区。
在具体实施方式中，所述抗体选自以下的任一种：
(1)抗体，所述抗体的重链可变区具有SEQ ID NO:18所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:20所示的氨基酸序列；
(2)抗体，所述抗体的重链可变区具有SEQ ID NO:22所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列；
(3)抗体，所述抗体的重链可变区具有SEQ ID NO:26所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:20所示的氨基酸序列；
(4)抗体，所述抗体的重链可变区具有SEQ ID NO:28所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列；
(5)抗体，所述抗体的重链可变区具有SEQ ID NO:30所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列；
(6)抗体，所述抗体的重链可变区具有SEQ ID NO:32所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列。
(7)抗体，(1)～(6)中任一项所述的抗体的变体，且具备与(1)～(6)中任一项所述的抗体相同或相似的活性。
在具体实施方式中，第一方面所述的抗体是全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体或IgG4抗体。
在具体实施方式中，第一方面所述的抗体不显著结合NKG2C、NKG2E或其组合。
在具体实施方式中，第一方面所述的抗体结合NKG2A/CD94，不显著结合NKG2C/CD94、NKG2E/CD94或其组合。
在具体实施方式中，第一方面所述的抗体结合表达NKG2A/CD94的细胞，不显著结合表达NKG2C/CD94、NKG2E/CD94或其组合的细胞。
在具体实施方式中，第一方面所述的抗体在降低CD94/NKG2A介导的抑制表达CD94/NKG2A的细胞毒性淋巴细胞的细胞毒性活性中更有效。
在具体实施方式中，所述表达CD94/NKG2A的细胞毒性淋巴细胞是NK细胞、NKT细胞、α/βT细胞或γ/δT细胞。
在具体实施方式中，所述表达CD94/NKG2A的细胞毒性淋巴细胞是NK细胞。
在第二方面，本发明提供一种免疫缀合物，所述的免疫辍合物包括第一、方面所述的抗体，以及与之连接的功能性分子。
在第三方面，本发明提供一种嵌合受体，所述嵌合受体的胞外域包含第一方面所述的抗体，所述嵌合受体包括：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)或其组合。
在具体实施方式中，所述嵌合受体是嵌合抗原受体(CAR)。
在具体实施方式中，所述CAR包含顺序连接的：第一方面所述的抗体、跨膜区和胞内信号区。
在具体实施方式中，所述的胞内信号区选自：CD3δ、FcεRIγ、CD27、CD28、CD137、CD134、MyD88、CD40的胞内信号区序列或其组合；和/或所述的跨膜区包含CD8或CD28的跨膜区。
在具体实施方式中，所述的CAR包括：第一方面所述的抗体、CD8/CD28的跨膜区和CD3δ；或第一方面所述的抗体、CD8/CD28的跨膜区、CD137的胞内信号区和CD3δ；或第一方面所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区和CD3δ；或第一方面所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区、CD137和CD3δ。
在具体实施方式中，所述的嵌合受体的氨基酸序列如SEQ ID NO：115或116所示。
在第四方面，本发明提供编码第一方面所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体的核酸。
在第五方面，本发明提供一种表达载体，其包含第四方面所述的核酸。
在第六方面，本发明提供一种病毒，其包含第五方面所述的表达载体或第四方面所述的核酸。
在第四方面、第五方面和第六方面涉及的核酸、表达载体和病毒均为本发明的生物材料。本发明的生物材料，其为如下中的任意一种：
1)编码第一方面的抗体、第二方面的免疫缀合物、第三方面所述的嵌合受体的核酸；
2)包含1)所述的表达载体；或者
3)包含1)或2)所述的病毒。
在第七方面，本发明提供一种宿主细胞，所述宿主细胞表达第三方面所述的嵌合受体。
在具体实施方式中，所述宿主细胞结合表达NKG2A/CD94的细胞，不显著结合NKG2C/CD94、NKG2E/CD94或其组合。
在具体实施方式中，所述宿主细胞能抵抗NK细胞攻击或杀伤NK细胞。
在具体实施方式中，所述宿主细胞还表达识别肿瘤抗原和/或病原体抗原的嵌合受体。
在具体实施方式中，所述宿主细胞与靶向肿瘤和/或病原体的第二宿主细胞联合应用。
在具体实施方式中，所述宿主细胞和/或第二宿主细胞不表达B2M、TCR/B2M、TCR/B2M/CIITA、TCR/B2M/NKG2A、和/或TCR/B2M/CIIA/NKG2A。
在具体实施方式中，所述宿主细胞和/或第二宿主细胞是T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、调节性T细胞、NK92细胞、干细胞衍生的免疫效应细胞或其组合。
在具体实施方式中，所述T细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞。
在具体实施方式中，所述T细胞为自体/同种异体T细胞。
在具体实施方式中，所述T细胞为原代T细胞。
在具体实施方式中，所述T细胞来源于人的T细胞。
在具体实施方式中，所述T细胞包含记忆性干细胞样T细胞(Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef)、调节性T细胞(Tregs)，效应记忆T细胞(Tem)、γδT细胞或其组合。
在第八方面，本发明提供一种联合用药，第一方面所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体、第七方面所述的宿主细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；和/或与改善其相关的一种或多种副作用的药剂联合施用；和/或与表达靶向NKG2A之外的嵌合抗原受体的宿主细胞联合施用。
在第九方面，本发明提供一种制备第一方面所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体的方法，所述方法包含在适于表达所述抗体、免疫缀合物、嵌合受体的条件下培养第七方面所述的宿主细胞，以及分离出由所述宿主细胞表达的所述抗体、免疫缀合物、组合物、和/或嵌合受体。
在第十方面，本发明提供一种药物组合物，其包括：第一方面所述的抗体或编码该抗体的核酸；或第二方面所述的免疫缀合物或编码该缀合物的核酸；或第三方面所述的嵌合受体或编码该嵌合受体的核酸；或第七方面所述的宿主细胞；以及药学上可接受的载体或赋形剂。
在第十一方面，本发明提供一种试剂盒，其包括：
容器，以及位于容器中的第十方面所述的药物组合物；或
容器，以及位于容器中的第一方面所述的抗体或编码该抗体的核酸；或第二方面所述的免疫缀合物或编码该缀合物的核酸；或第三方面所述的嵌合受体或编码该嵌合受体的核酸；或第七方面所述的宿主细胞。
应理解，在本发明范围内中，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，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
 附图说明
图1显示了利用真核表达质粒V152S构建分别表达NKG2A/CD94、NKG2C/CD94异源二聚体的载体示意图；
图2显示了ELISA检测抗体A1、A2(Fab形式)与NKG2A/CD94、NKG2C/CD94的结合；
图3显示了ELISA检测抗体A1、A2(IgG4形式)结合NKG2A/CD94的EC50；
图4显示了Biacore检测抗体A1、A2(IgG4形式)的亲和力；
图5显示了带Flag的目的基因CD94-Flag、NKG2A-CD94-Flag、NKG2C-CD94-Flag和NKG2E-CD94-Flag的载体图；
图6显示了FACs检测抗体A1、A2(IgG4形式)与CHOK1-NKG2A-CD94、CHOK1-NKG2C-CD94、CHOK1-NKG2E-CD94和CHOK1-CD94细胞的结合；
图7显示了抗体A1、A2、A3、A4、A5、A6的重链可变区的氨基酸序列对比；
图8显示了FACs检测抗体A1、A2、A3、A4、A5、A6(Fab形式)与CHOK1-NKG2A-CD94、CHOK1-NKG2C-CD94和CHOK1-NKG2E-CD94细胞的结合；
图9显示了ELISA检测抗体A1、A2、A3、A4、A5、A6(Fab形式)与NKG2A/CD94异源二聚体结合的EC50；
图10显示了FACs检测抗体A1、A2、A3、A4、A5、A6(Fab形式)与过表达NKG2A/CD94异源二聚体的CHO-K1细胞结合的EC50；
图11显示了ELISA检测抗体A1、A2、A3、A4、A5、A6(IgG4形式)与NKG2A/CD94异源二聚体结合的EC50；
图12显示了FACs检测抗体A1、A2、A3、A4、A5、A6(IgG4形式)与过表达NKG2A/CD94的CHOK1细胞的EC50；
图13显示了Biacore检测抗体A3、A4、A5、A6(IgG4形式)的亲和力；
图14显示了pET22b-HLA-E和pET22b-β2m的载体图；
图15显示了HLA-E四聚体与表达NKG2A/CD94的NK细胞结合的EC50；
图16显示了FACs检测抗体A1、A2、A3、A4、A5、A6(IgG4形式)阻断NKG2A与其配体HLA-E结合的IC50；
图17显示了肿瘤细胞K562-HLA-E、K562和FaDu细胞上的HLA-E表达水平；
图18显示了FACs检测NK细胞与K562、K562-HLA-E、FaDu细胞共孵育及加入抗体A1、A2、A3、A4、A5、A6(IgG4形式)后CD107a的表达情况；
图19显示了FACs检测抗NKG2A抗体对NK细胞在K562、K562-HLA-E、FaDu细胞上发挥杀伤作用的影响；
图20显示了A4-BBZ CAR T细胞、A5-BBZ CAR T细胞与NK细胞共孵育后NK细胞比例变化。
图21显示了A4、A5 UCAR-T与NK细胞共孵育后UCAR-T细胞比例变化。
图22显示了A4、A5-UCAR-T与BCMA UCAR-T在NK细胞存在下联合作用于RPMI-8226细胞皮下移植瘤的情况。
 具体实施方式
本发明人经过深入的研究筛选，获得了特异性识别NKG2A的全人源抗体，包括Fab形式的抗体、IgG4形式的抗体。本发明的抗体可以被应用于制备靶向抗肿瘤药物以及诊断肿瘤的药物。
术语
除非专门定义，本文所用的所有技术和科学术语具有在基因治疗、生物化学、遗传学和分子生物学领域内的技术人员通常理解的相同含义。类似或等效于本文中描述的所有方法和材料都可以在本发明的实践或测试中使用，其中，本文描述的是合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书，包括定义为准。此外，除非另有规定，材料、方法和实施例仅是说明性的，而并非旨在进行限制。
除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。
公开内容中，请求保护的主题的各个方面均以范围形式呈现。应当理解，范围形式的描述仅仅是为了方便和简洁，并且不应被解释为对所要求保护的主题的范围的硬性限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，在提供值的范围的情况下，应当理解，在该范围的上限和下限之间的每个中间值以及在所述范围内的任何其他所述的或中间的值均被包括在要求保护的主题内，所述范围的上下限也属于请求保护的主题的范围。所述较小范围内可独立地包含这些较小范围的上下限，它们也属于请求保护的主题的范围，除非明确地排除所述范围的上下限。设定范围包含一个或两个限值时，请求保护的主题也包括排除所述限值之一个或两个的范围。这适用而无关范围的宽度。
术语“约”是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中述及“约”值或参数，包括(并描述)指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”的描述。例如，“约”或“包含”可意指按照在该领域中的实际的标准偏差在1以内或多于1。或者“约”或“包含”可意指至多10％(即±10％)的范围。例如，约5uM可包括在4.5uM与5.5uM之间的任何数目。当在申请案与申请专利范围中提供特定值或组成时，除非另外指出，否则“约”或“包含”应假定为在该特定值或组成的可接受误差范围内。
除非另外指出，本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值。
为了更易于理解本发明，首先定义一些术语。
术语“NKG2A”(Natural killer group 2A)是NKG2凝集素受体家族中抑制性的受体，也被称为自然杀伤细胞凝集素样受体C1(killer cell lectin like receptor C1,KLRC1),CD159a、NK细胞受体A，是NK细胞表面优先表达的跨膜蛋白之一。NKG2A主要表达在NK细胞表面和部分T细胞(CD8+T细胞、Th2细胞、γδT细胞以及NKT细胞)。在人的免疫细胞表面,NKG2A与CD94分子(NK细胞表面膜蛋白)以二硫键连接形式形成的异源二聚体NKG2A-CD94,被靶细胞上非经典的组织相容性复合体I(major histocompatibility complex class I,MHC I)类分子HLA-E识别,该分子在正常情况下低表达，但在大部分的肿瘤细胞表面，HLA-E的表达量上升，从而诱导级联的抑制信号,抑制NK的细胞毒活性和细胞因子的分泌。某些病毒感染，肿瘤和免疫性疾病通过该途径逃避免疫检查。NKG2A与HLA-E的相互作用能抑制NK细胞和T细胞的激活。NK细胞经IL15活化后高表达NKG2A。NKG2家族还包括NKG2C、NKG2D和NKG2E。
“NKG2A”包括来自任何脊椎动物来源，包括哺乳动物如灵长类(例如，人和猴)和啮齿类(例如，小鼠和大鼠)的任何天然NKG2A。该术语包括“全长”未加工的NKG2A以及来源于细胞中的加工的任何形式的NKG2A。该术语还包括天然存在的NKG2A的变体，例如剪接变体或等位变体。在一实施例中，本文描述的抗NKG2A抗体抑制NKG2A蛋白与HLA-E的结合，因此起到检查点抑制剂的作用。示例性，人NKG2A全长的氨基酸序列如SEQ ID NO:68所示，NKG2A胞外段的氨基酸序列如SEQ ID NO:70所示，人NKG2C全长的氨基酸序列如SEQ ID NO:123所示,NKG2C胞外段的氨基酸序列如SEQ ID NO:72所示，人NKG2E全长的氨基酸序列如SEQ ID NO:125所示,NKG2E胞外段的氨基酸序列如SEQ ID NO:74所示。
术语“人类白细胞抗原”(Human leukocyte antigen，HLA)是人类的主要组织相容性复合体MHC的编码基因，与人类的免疫系统功能密切相关。HLA包括有I类、II类和III类基因部分。HLA的I类和II类基因所表达的抗原位于细胞膜上，为MHC-I(HLA-A、HLA-B、HLA-C位点编码)和MHC-II(HLA-D区编码)，HLA I类几乎分布于身体全部细胞表面，是一个异二聚体，由重链(α链)与β2微球蛋白组成(B2M)，II类主要是定位于巨噬细胞和B淋巴细胞表面的糖蛋白。
术语“HLA-E”(OMIM 143010、基因编号NM_005516.6)是非经典MHC分子，其在细胞表面表达，并且通过肽(例如源于其他MHC I类分子的信号序列的片段)的结合而被调节。HLA-E通过特异性结合CD94/NKG2A、CD94/NKG2B和CD94/NKG2C(参见，例如，布劳德(Braud)等人，(1998)自然(Nature)391：795-799，其全部公开内容通过引用并入本文)而结合自然杀伤(NK)细胞、自然杀伤T细胞(NKT)和T细胞(α/β和γ/δ)的亚群。细胞表面表达HLA-E的靶细胞免受CD94/NKG2A阳性的NK、T、或NKT细胞克隆的裂解。如本文所使用的，“HLA-E”是指HLA-E基因或编码的蛋白的任何变体、衍生物或同种型。人HLA-E胞外区氨基酸序列如SEQ ID NO:78所示。HLA-E广泛低水平分布于全身细胞。在几种肿瘤发现高水平的HLA-E，包括妇科肿瘤(高达90％的肿瘤样本)，以及高达50％的乳腺癌、非小细胞肺癌(NSCLC)、肝脏、胰腺、肾脏、黑色素瘤、前列腺、头颈部、胃、直肠和结肠直肠癌。(《The NKG2A-HLA-E axis as a novel checkpoint in the tumor microenvironment》Clin Cancer Res.2020 Nov 1；26(21):5549-5556.)。HLA-E高表达肿瘤通过与免疫细胞(如NK细胞、T细胞)的NKG2A结合来逃逸免疫细胞杀伤，本发明公开的NKG2A抗体能够抑制HLA-E高表达肿瘤免疫逃逸从而杀伤该肿瘤细胞。
术语“溶酶体相关膜蛋白1(CD107a)”，是囊泡膜蛋白的主要成分，在细胞胞浆中主要构成以囊泡形式存在的细胞毒性颗粒。NK细胞杀伤靶细胞时，释放的细胞毒性颗粒将到达靶细胞膜并与靶细胞膜融合，引起颗粒内容物释放，最终导致靶细胞的死亡。静息状态下，NK细胞膜表面CD107a自发表达率很低，经靶细胞刺激后可以在其表面检测到CD107a表达的增加，因此，NK细胞受刺激后CD107a分子增加的幅度可反映NK细胞的细胞毒性细胞杀伤活性水平。
术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换使用，指任何长度的氨基酸的聚合物。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，特别是保守修饰的氨基酸，并且它可以被非氨基酸中断。该术语还包括改性的氨基酸聚合物例如已经通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解加工、异戊二烯化、外消旋化、硒酰化、转移-RNA介导的氨基加成如精氨酸化、泛在化、或任何其他操作如与标记组分缀合等改性的氨基酸聚合物。如本文所用，术语“氨基酸”是指天然和/或非天然或合成氨基酸，包括甘氨酸以及D或L光学异构体，以及氨基酸类似物和肽模拟物。“衍生自”指定的蛋白质的多肽或氨基酸序列是指多肽的来源。该术语还包括由指定的核酸序列表达的多肽。
术语“抗体”在本文中以最广义使用并且包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要其显示所需的抗原结合活性即可。
“抗体片段”是指不同于完整抗体的分子，其包含完整抗体结合完整抗体所结合的抗原的部分。抗体片段的实例包括但不限于(i)由VL、VH、CL和CH1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)VH和CH1结构域组成的Fd片段，(iii)由单个抗体的VL和VH结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段(Ward等，1989，Nature 341:544-546)；(v)F(ab’)2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点；(vii)双特异性单链Fv二聚体(PCT/US92/09965)；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段；和(ix)与相同或不同抗体遗传融合的scFv。
抗体的“分类”是指其重链所具有的恒定结构域或恒定区的类型。主要有五类抗体：IgA、IgD、IgE、IgG和IgM，并且这些中的一些可以被进一步划分成亚类(同种异型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同的类型的免疫球蛋白的重链恒定结构域被分别称为α，δ，ε，γ，和μ。
术语“可变区或可变结构域”是指参与抗体抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链可变结构域(分别为VH和VL)通常具有相似的结构，其中各结构域包含四个保守的FR和三个CDR(参见，例如，Kindt等，KubyImmunology,6th ed.,W.H.Freeman&Co.,第91页(2007))。单个VH或VL结构域可足以给予抗原结合特异性。此外，结合特定抗原的抗体可以分别使用来自与所述抗原结合的抗体的VH或V L结构域筛选互补VL或VH结构域的文库来分离。参见，例如，Portolano等,J.Immunol.150:880-887(1993)；Clarkson等,Nature352:624-628(1991)。
术语“高变区”或“互补决定区”或“CDR”是指抗体可变结构域中序列高变和/或形成结构确定的环(“高变环”)和/或含有与抗原接触的残基(“抗原触点”)的各区域。通常，抗体包含六个CDR：VH中的三个(HCDR1，HCDR2，HCDR3)和VL中的三个(LCDR1，LCDR2，LCDR3)。
术语“Fc区”或“Fc”被用于限定含有恒定区的至少一部分的免疫球蛋白重链的C-端区域。该术语包括天然序列Fc区和变体Fc区。
“框架(FR)”是指不同于高变区(CDR)残基的可变结构域残基。可变结构域的FR通常由四个FR结构域组成：FR1，FR2，FR3和FR4。因此，在VH(或VL)中CDR和FR序列通常按以下顺序出现：FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。
除非另外指出，在本文中，CDR残基和可变结构域中的其他残基(例如，FR残基)根据以上Kabat等编号。
术语“天然抗体”是指天然存在的具有多种结构的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异源四聚糖蛋白，由通过二硫键键合的两个相同的轻链和两个相同的重链组成。从N端到C端，各重链具有可变区(VH)，其也被称为可变重链结构域或重链可变结构域，之后是三个恒定结构域(CH1，CH2和CH3)。类似地，从N端到C端，各轻链具有可变区(VL)，其也被称为可变轻链结构域或轻链可变结构域，之后是轻链恒定(CL)结构域。抗体的轻链基于其恒定结构域的氨基酸序列可以被分配至两种类型之一，被称为κ(κ)和λ(λ)。
术语“全抗”、“全长抗体”、“完整抗体”可交换使用，是指具有与天然抗体结构基本类似的结构或具有含有如本文中所限定的Fc区的重链或包括具有抗原结合区域的完整的全长抗体。
术语“单域抗体(Single domain antibody,sdAb)”是指缺失抗体轻链而只有重链可变区的一类抗体，因其分子量小，也被称为纳米抗体(Nanobody)。
术语“单结构域抗体”是指包含抗体的全部或部分的重链可变结构域或全部或部分的轻链可变结构域。在某些实施方案中，单结构域抗体是人单结构域抗体(Domantis,Inc.,Waltham,MA；参加，例如，美国专利号6248516)。
术语“单克隆抗体”、“单抗”是指获自基本上同源的抗体的群体的抗体，即，包括所述群体的个体抗体是相同的和/或结合相同的表位，除可能的变体抗体以外，例如，含天然存在的突变或在单克隆抗体制剂的制备过程中产生，此种变体通常少量存在。对比于多克隆抗体制剂(通常包括针对不同的决定簇(表位)的不同抗体)，单克隆抗体制剂中的各单克隆抗体是针对抗原上的单个决定簇。因此，定于“单克隆”指示抗体的性质为获得自基本上同源的抗体群体，并且不视为要求通过任何特定的方法制备所述抗体。例如，可以通过多种技术制备，包括但不限于杂交瘤方法、重组DNA法、噬菌体展示法，以及利用含有所有或部分的人免疫球蛋白基因座的转基因动物的方法。
术语“嵌合抗体”是指抗体重链和/或轻链的一部分来源于特定来源或物种，而重链和/或轻链的剩余部分来源于不同的来源或物种的抗体。在某些实施方案中，嵌合抗体包含非人可变区(例如，来源于小鼠、大鼠、仓鼠、兔或非人灵长类动物如猴的可变区)和人恒定区。在另外的实施方案中，嵌合抗体是“类型转换”抗体，其中类型或亚类已经由亲本抗体的类型或亚类改变。嵌合抗体包括其抗原结合片段。在某些实施方案中，嵌合抗体是“人源化抗体”。
术语“人源化”用于非人抗体，例如啮齿动物或灵长类动物等，是含有来源于非人免疫球蛋白的最小序列的杂合免疫球蛋白，免疫球蛋白链或其片段。“人源化抗体”是指一种嵌合抗体，其包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基。在某些实施例中，人源化抗体将包含基本上全部的至少一个(一般为两个)可变结构域，其中所有或基本上所有的CDR对应于非人抗体的CDR，并且所有或基本上所有的FR对应于人抗体的FR。人源化抗体任选地可以包含来源于人抗体的抗体恒定区的至少一部分。
在一些实施方案中，“人源化抗体”可包括突变，例如通过体外随机或定点诱变或通过体内体细胞突变引入的突变。
术语“全人源抗体”是一种抗体，其具有的氨基酸序列对应于由人或人细胞产生的抗体或来源于利用人抗体库或其他人抗体编码序列的非人来源的抗体的氨基酸序列。全人源体的定义明确排除了包含非人抗原结合残基的人源化抗体。在某些实施方案中，本文中提供的抗体是“全人源抗体”，是由噬菌体展示技术生成。
本发明的抗体可以通过筛选具有所需一种或多种活性的抗体的组合文库来分离。例如，本领域中已知多种方法用于生成噬菌体展示文库并且针对具有所需结合性质的抗体筛选所述文库。此种方法被综述于例如Hoogenboom等，Methods in Molecular Biology 178:1-37(0’Brien等，Human Press,Totowa,NJ,2001)中并被进一步描述于例如McCafferty等，Nature 348：552-554；Clackson等，Nature 352：624-628(1991)；Marks等，J.Mol.Biol.222:581-597(1992)；Marks,Meth.Mol.Biol.,248:161-175(Lo,ed.,Human Press,Totowa,NJ,2003)；Sidhu等,J.Mol.Biol.338(2):299-310(2004)；Lee等，J.Mol.Biol.340(5):1073-1093(2004)；Fellouse,Proc.Natl.Acad.Sci.USA101(34):12467-12472(2004)；Lee等，J.Immunol.Methods 284(1-2):119-132(2004)中。
[根据细则91更正 11.07.2023]
在某些噬菌体展示方法中，VH和VL基因库通过聚合酶链式反应(PCR)分别克隆并且在噬菌体文库中随机重组，然后可以针对结合抗原的噬菌体筛选所述文库，如Winter等，Ann.Rev.Immunol.12:433-455(1994)中所述。噬菌体典型地将抗体片段展示为单链Fv(scFv)片段或Fab片段。来自被免疫的来源的文库向免疫原提供高亲和力抗体而不需要构建杂交瘤。备选地，可以克隆天然库(例如，由人)从而向多种非自身抗原以及自身抗原提供单一来源的抗体而不需要进行任何免疫，如Griffiths等，EMBO J,12:725-734(1993)所述。最后，也可以通过以下方式合成制备天然文库：自干细胞克隆未重排的V-基因区段，并且使用含随机序列的PCR引物以编码高变CDR3区并且在体外实现重排，如Hoogenboom，J.Mol.Biol.227:381-388(1992)所述。
在本文中，分离自全人源抗体文库的抗体或抗体片段被认为是全人源抗体或全人源抗体片段。
在某些实施方案中，本文中提供抗体的氨基酸序列变体。术语“亲本抗体”指本申请所提供的抗体或依据本申请所提供的抗体进行突变、或亲和力成熟等处理后所得的抗体。所述亲本抗体可以是天然存在的抗体，或者天然存在的抗体的变体或改造版本。亲本抗体可以指抗体本身，包含所述亲本抗体的组合物，或其编码氨基酸序列。
术语“亲和力成熟”抗体是指相比于亲本抗体在一个或多个高变区(HVR)中具有一个或多个改变的抗体，此种改变导致抗体对抗原的亲和力提高。
术语“变体”指与本申请所提供的抗体的序列具有基本上相同氨基酸序列或由基本上相同的核苷酸序列编码的一种或多种活性的多肽。所述变体与本申请实施例中所提供的抗体具有相同或相似的活性。
术语“变体抗体”或“抗体变体”包括由于相比亲本的至少一个氨基酸修饰，而不同于亲本抗体序列的抗体序列。本文中的变体抗体序列优选的具有与亲本抗体序列至少约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％％、91％、92％、93％、94％、95％％、96％、97％、98％、99％的氨基酸序列同一性。抗体变体可以指抗体本身，也可以指包含所述抗体变体的组合物。抗体的氨基酸序列变体可以通过向编码所述抗体的核苷酸序列引入合适的修饰或通过肽合成来制备。术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”、“氨基酸置换”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸，“氨基酸插入”意指在亲本多肽序列中的特定位置添加氨基酸，“氨基酸缺失”意指去除亲本多肽序列中特定位置上的氨基酸。可以进行缺失、插入和取代的任意组合以获得最终的构建体，前提是最终的构建体具有所需的特征，例如：结合抗原。
术语“修饰”一词是指本发明的蛋白或多肽的状态或结构的改变。修饰的方式可以是化学的、结构的和功能上的。
术语“保守修饰”或“保守序列修饰”意指不显著影响或改变含有所述氨基酸序列的抗体的结合特征的氨基酸修饰。此类保守修饰包括氨基酸取代、插入和缺失。可通过本领域已知的标准技术将修饰导入本发明的抗体中，例如定点诱变、PCR介导的诱变。本领域已经定义了具有相似侧链的氨基酸残基家族，如表1所示。
表1：具有相似侧链的氨基酸残基家族
因而，可以用其他相同侧链家族的氨基酸残基替换本发明抗体的CDR区中或框架区中的一个或多个氨基酸残基，并可以测试所改变的抗体(变体抗体)保留的功能。
非保守置换需要将这些组之一的成员换成另一组的成员。
一种置换变体包括置换亲本抗体(例如，人源化或人抗体)的一个或多个高变区残基。通常，被选择用于进一步研究的所得的变体相对于亲本抗体将具有某些生物学性质(例如，增加的亲和力、减小的免疫原性)的改变(例如，提高)和/或将基本上保持亲本抗体的某些生物学性质。示例性替换变体是亲和力成熟抗体，其可以例如，使用基于噬菌体展示的亲和力成熟技术(如本文中描述的那些)常规制备。简言之，将一个或多个CDR残基突变并将变体抗体展示在噬菌体上并且筛选特定的生物学活性(例如，结合亲和力)。
改变(例如，置换)可以在CDR区进行，例如，以提高抗体亲和力。此种改变可以在HVR“热点”中进行，所述“热点”即在体细胞成熟过程期间以高频率进行突变的密码子编码的残基(参见，例如，Chowdhury,Methods Mol.Biol.207:179-196(2008)，和/或解除抗原的残基，测试所得的变体VH或VL的结合亲和力。通过构建二级文库并自其进行再选择的亲和力成熟已被描述于例如Hoogenboom等，Methods in Molecular Biology 178:1-37(0’Brien等，ed.,Human press,Totowa,NJ,(2001))中。在亲和力成熟的一些实施方案中，通过多种方法中任一种(例如，易错PCR、链改组或寡核苷酸定向诱变)将多样性引入选择用于成熟的可变基因种。然后产生二级文库。然后筛选所述文库以鉴定任何具有所需亲和力的抗体变体。引入多样性的另一种方法包括CDR定向方法，其中若干CDR残基(例如，同时4-6个残基)被随机化。
在某些实施方案种，置换、插入或缺失可以发生在一个或多个CDR内，只要这样的改变不显著减小抗体结合抗原的能力。例如，不显著减小结合亲和力的保守改变(例如，本文中所述的保守修饰)可以在CDR中进行。此种改变可以例如在CDR中接触抗原的残基的外部。在上述提供的变体VH和VL序列的某些实施方案中，各CDR是未改变的，或含不超过一个、两个或三个氨基酸置换。
术语“抗NKG2A抗体”、“结合NKG2A的抗体”、“NKG2A抗体”、“识别NKG2A的抗体”是指能够以足够的亲和力结合NKG2A的抗体，所述抗体可用作用于靶向NKG2A的诊断剂和/或治疗剂。在一个实施方案中，抗NKG2A抗体与不相关的、非NKG2A蛋白的结合程度小于所述抗体与NKG2A的约10％，如通过酶联免疫吸附实验(ELISA)测定。在某些实施方案中，抗NKG2A抗体结合NKG2A的表位，所述表位在来源于不同物种的NKG2A之间是保守的。
在本文中，描述了具有基于Fab(抗原结合片段)的抗原结合区域的抗原结合蛋白，包括抗体Fab和抗体IgG4。其中使用NKG2A/CD94异源二聚体，从全人的天然的Fab噬菌体文库选择Fab。这些分子展示了精细的特异性。例如，该抗体仅识别NKG2A,不识别NKG2C、NKG2E。该抗体仅识别NKG2A/CD94异源二聚体，不识别NKG2C/CD94异源二聚体；该抗体仅识别过表达NKG2A/CD94异源二聚体的CHO-K1细胞，不识别过表达NKG2C/CD94、NKG2E/CD94以及CD94的CHO-K1细胞。本发明中如果没有特别说明，NKG2A指人的NKG2A。
在一些实施方案中，本发明包括具有Fab、IgG4序列的抗体，所述Fab序列与一个或多个重链恒定区域融合以形成具有人免疫球蛋白Fc区的抗体以产生双价蛋白，从而增加抗体的总体亲和力和稳定性。此外，Fc部分允许将其他分子(包括但不限于荧光染料、细胞毒素、放射性同位素等)与例如用于抗原定量研究中的抗体直接缀合，以便固定抗体用于亲和力测量、用于定向递送治疗药、使用免疫效应细胞测试Fc介导的细胞毒性和许多其它应用。
本文提供的结果突出本发明抗体在靶向NKG2A时的特异性、灵敏性和效用。
本发明的抗体或抗体片段基于使用噬菌体展示鉴定和选择抗原结合片段(Fab)，所述抗原结合片段的氨基酸序列赋予抗体或抗体片段针对NKG2A的特异性并且形成本公开的全部抗原结合蛋白的基础。因此，所述Fab可以用来设计一系列不同“抗体或抗体片段”，包括例如全长抗体、其片段如F(ab’)2、融合蛋白、IgG4、多价抗体，即，具有针对相同抗原或不同抗原的多于一种特异性的抗体，例如，双特异性T细胞结合抗体(BiTE)、三抗体等(见Cuesta等人，Multivalent antibodies:when design surpasses evolution,Trends in Biotechnology 28:355-362,2010)。
在具体实施例中，本发明提供全长抗体，其重链和轻链可以是全长(例如，抗体可以包括至少一条，优选两条完整重链，和至少一条，优选两条完整轻链)或可以包括抗原结合部分(Fab、F(ab’)2、Fv或scFv)。在其他实施方案中，抗体重链恒定区选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE。抗体类型的选择将取决于所设计的抗体欲引发的免疫效应子功能。在构建重组免疫球蛋白时，各种免疫球蛋白同种型的恒定区的适宜氨基酸序列和用于产生广泛种类抗体的方法是本领域技术人员已知的。
本发明提供了识别NKG2A的全人源抗体，所述抗体包含轻链可变区，所述轻链可变区包含RASQSISSWLA(SEQ ID NO:4)所示的LCDR1；和/或DASSLES(SEQ ID NO:5)所示的LCDR2；和/或QQYDSYX1X2T所示的LCDR3，其中X1选自I、V、G、A、L、F或P，优选地，X1选自I或V；X2选自R、S、K、H、W、Y、C、M、N、Q或T，优选地，X2选自R或S。
本发明提供了识别NKG2A的全人源抗体，所述抗体包括重链可变区，所述重链可变区选自：
(1)包含SYAIS(SEQ ID NO:1)所示的HCDR1；和/或GIIPIFGTAX1YAQKFQG(SEQ ID NO:130)所示的HCDR2，其中X1选自N、H、K、R、W、Y、S、C、M、Q或T，优选地，X1选自N或H；和/或GFDGMDY(SEQ ID NO:3)所示的HCDR3；或
(2)包含X1X2X3X4S(SEQ ID NO:131)所示的HCDR1；和/或AIX1X2X3X4GSTYYADSVKG(SEQ ID NO:132)所示的HCDR2；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。其中HCDR1和HCDR2中X1X2X3X4位点的氨基酸选自下表2。
在一优选方案中，所述抗体还包括框架区，其中第1框架区的第30位氨基酸选自S、R、N、W、Y、C、M、Q、T、H、K、G、A、V、L、I、P、或F，优选地，选自S、R、N或G。
表2.抗体HCDR1和HCDR2中X1X2X3X4位点的氨基酸
在一优选方案中，本发明提供了识别NKG2A的抗体，所述抗体包含重链可变区，所述重链可变区包含SEQ ID NO:1、7、12、14或16所示任一氨基酸序列的重链CDR1，和/或包含SEQ ID NO:2、8、11、13、15或17所示任一氨基酸序列的重链CDR2，和/或包含SEQ ID NO:3或9所示任一氨基酸序列的重链CDR3。在另一优选方案中，本发明提供了识别NKG2A的抗体包括：包含SEQ ID NO:4所示氨基酸序列的轻链CDR1，和/或包含SEQ ID NO:5所示氨基酸序列的轻链CDR2，和/或包含SEQ ID NO:6或10所示任一氨基酸序列的轻链CDR3。在另一优选方案中，本发明提供了识别NKG2A的抗体包括：包含SEQ ID NO:1、7、12、14或16所示任一氨基酸序列的重链CDR1，和/或包含SEQ ID NO:2、8、11、13、15或17所示任一氨基酸序列的重链CDR2，和/或包含SEQ ID NO:3或9所示任一氨基酸序列的重链CDR3，和/或包含SEQ ID NO:4所示氨基酸序列的轻链CDR1，和/或包含SEQ ID NO:5所示氨基酸序列的轻链CDR2，和/或包含SEQ ID NO:6或10所示任一氨基酸序列的轻链CDR3。优选地，所述识别NKG2A的抗体包括：包含SEQ ID NO:1、7、12、14或16所示任一氨基酸序列的重链CDR1，和包含SEQ ID NO:2、8、11、13、15或17所示任一氨基酸序列的重链CDR2，和包含SEQ ID NO:3或9所示任一氨基酸序列的重链CDR3，和/或包含SEQ ID NO:4所示氨基酸序列的轻链CDR1，和包含SEQ ID NO:5所示氨基酸序列的轻链CDR2，和包含SEQ ID NO:6或10所示任一氨基酸序列的轻链CDR3。更优选地，所述识别NKG2A的抗体包括：包含SEQ ID NO:1、7、12、14或16所示任一氨基酸序列的重链CDR1，和包含SEQ ID NO:2、8、11、13、15或17所示任一氨基酸序列的重链CDR2，和包含SEQ ID NO:3或9所示任一氨基酸序列的重链CDR3，和包含SEQ ID NO:4所示氨基酸序列的轻链CDR1，和包含SEQ ID NO:5所示氨基酸序列的轻链CDR2，和包含SEQ ID NO:6或10所示任一氨基酸序列的轻链CDR3。更优选地，所述识别NKG2A的抗体包括：包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或包含SEQ ID NO:7所示的HCDR1，SEQ ID NO:8所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:11所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或包含SEQ ID NO:12所示的HCDR1，SEQ ID NO:13所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或包含SEQ ID NO:14所示的HCDR1，SEQ ID NO:15所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或包含SEQ ID NO:16所示的HCDR1，SEQ ID NO:17所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3。
在另一方面，本发明提供了识别NKG2A的抗体，包含重链可变区，所述重链可变区包含SEQ ID NO:18、22、26、28、30或32所示的氨基酸序列、或上述序列的变体。
在另一方面，本发明提供了识别NKG2A的抗体，包含轻链可变区，该轻链可变区包含SEQ ID NO:20或24所示的氨基酸序列、或上述序列的变体。
考虑到这些重链可变区和轻链可变区序列各自可以结合NKG2A，可以“混合和匹配”重链和轻链可变区序列来产生本发明的抗NKG2A的结合分子。
在另一个方面，本发明提供了结合NKG2A的抗体的变体或其片段的变体。因而本发明提供了抗体或其片段，具有与重链或轻链的可变区序列至少80％相同的重链和/或轻链可变区。优选的，重链和/或轻链可变区的氨基酸序列同一性是至少85％，更优选至少90％，最优选至少95％，特别是96％，更特别97％，甚至更特别98％，最特别99％，包括例如80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％和100％。变体可以以本申请所述的抗体为母本抗体，通过酵母库筛选、噬菌体库筛选、点突变等方法得到。如本申请实施例2采用的方法，以抗体A1和A2为母本抗体，采用噬菌体库筛选的方法进行突变改造。
在另一个方面，本发明提供了与前述的抗NKG2A的抗体识别相同的抗原决定部位的抗体。
在另一个方面，本发明提供了与前述的抗NKG2A的抗体竞争性结合NKG2A的抗体。
在另一个方面，本发明提供了特异性结合NKG2A的抗体，所述抗体是全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体或IgG4抗体。
在一优选方案中，以上所述的抗体不显著结合NKG2C、NKG2E或其组合。
在一优选方案中，所述抗体结合NKG2A/CD94，不显著结合NKG2C/CD94、NKG2E/CD94或其组合；和/或，
所述抗体结合表达NKG2A/CD94的细胞，不显著结合表达NKG2C/CD94、NKG2E/CD94或其组合的细胞。
抗体测定
通过本领域已知的多种测定可以鉴定、筛选本文中提供的抗NKG2A抗体或表征其物理/化学性质和/或生物学活性。包括例如ELISA、biacore、Western印迹和流式细胞仪分析。合适的测定详细描述在实施例中。
术语“亲和力”是指分子(例如：抗体)的单个结合位点及其结合配体(例如：抗原)之间非共价相互作用的力量总和。除非另外指出，如本文中使用的“结合亲和力”是指固有结合亲和力，其反应结合对的成员(例如：抗体和抗原)之间1：1相互作用。分子X对其配体Y的亲和力通常可以由解离常数(Kd)代表。亲和力可以通过本领域中已知的常规方法测量，所述方法包括本文中所述的利用Biacore测定抗体的亲和力。本文中的抗体对NKG2A/CD94的“亲和力”以抗体的KD表示。抗体的KD是指抗体——抗原相互作用的平衡解离常数。抗体结合其抗原的KD值越大，其对所述具体抗原的结合亲和力越弱。
术语“EC50”，半最大效应浓度(concentration for 50％of maximal effect,EC50)是指能引起50％最大效应的浓度。
抗原
术语“抗原”是指被抗原结合单元识别并特异性结合的物质。抗原可以包括肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合。非限制性示例性抗原包括肿瘤抗原或病原体抗原。“抗原”也可以指引发免疫反应的分子。这种免疫反应可能涉及抗体产生或特定免疫活性细胞(immunologically-competent cells)的活化，或两者兼有。本领域技术人员将理解，任何大分子，包括实际上所有的蛋白质或肽，都可以作为抗原。
术语“表位”指可被抗体、B细胞、T细胞或工程化细胞识别的抗原或部分抗原。例如，表位可以是被抗体识别的肿瘤表位或病原体表位；抗体识别抗原内的多个表位。表位也可以突变。
术语“抗原决定部位”又称“抗原表位”或“表位”或“抗原决定簇”，包括任何能够被抗体结合的决定簇或区域。抗原表位是抗原中被靶向所述抗原的抗体结合的区域，包括与抗体直接接触的特定氨基酸。示例性，抗原表位可以由NKG2A蛋白序列的连续序列组成，也可以由NKG2A蛋白序列不连续的三维结构组成。示例性，本文中使用的抗原是NAG2A胞外区、NAG2C胞外区、NAG2E胞外区分别与CD94胞外区形成的NAG2A/CD94异源二聚体、NAG2C/CD94异源二聚体、NAG2E/CD94异源二聚体。
免疫缀合物
本发明还提供了免疫缀合物，所述的免疫辍合物包括本文所述的抗体，以及与之连接的功能性分子。所述抗体与所述功能性分子可以通过共价连接、偶联、附着、交联等方式构成缀合物。
术语“连接”或“融合”在本文中可互换使用。这些术语是指通过包括化学缀合或重组方法的任何手段将两个以上化学元件或组件连接在一起。“框内融合”是指以维持原始开放阅读框(ORF)的正确阅读框的方式连接两个或更多个ORF以形式连续的较长的ORF。因此，所得到的重组融合蛋白是含有两个或更多个片段的单一蛋白质，这些片段对应于由原始ORF编码的多肽(这些片段在自然状态通常不是如此连接)。尽管阅读框因此在整个融合片段中是连续的，但这些片段可以在物理上或空间上通过例如框内连接序列(例如“flexon”)分开。
本发明另一方面提供了编码本发明的至少一种抗体、其功能变体或免疫缀合物的核酸分子。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。
嵌合受体
术语“嵌合受体”，即用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子，包括胞外域、跨膜域和胞内域。嵌合受体包括但不限于：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)。
术语“嵌合抗原受体”(CAR)包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域。胞内信号传导结构域包括刺激性分子和/或共刺激性分子的功能信号传导结构域，在一个方面，刺激性分子为与T细胞受体复合体结合的δ链；在一个方面，细胞质信号传导结构域进一步包括一种或多种共刺激性分子的功能性信号传导结构域，例如4-1BB(即CD137)、CD27和/或CD28。
术语“嵌合T细胞受体”，包括构成TCR的各种多肽衍生的重组多肽，其能够结合到靶细胞上的表面抗原，和与完整的TCR复合物的其他多肽相互作用，通常同定位在T细胞表面。嵌合T细胞受体由一个TCR亚基与人或人源化抗体结构域组成的一个抗原结合结构域组成，其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内结构域的胞内信号结构域的刺激结构域；该TCR亚基和该抗体结构域有效连接，其中，TCR亚基的胞外、跨膜、胞内信号结构域来源于CD3ε或CD3γ，并且，该嵌合T细胞受体整合进T细胞上表达的TCR。
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：1、抗原结合结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2、胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3、跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。
术语“信号传导结构域”是指通过在细胞内传递信息而起作用的蛋白质的功能性部分，用来通过产生第二信使或通过响应这样的信使起效应物作用经由确定的信号传导途径调节细胞的活性。胞内信号传导结构域可以包括分子的全部细胞内部分、或全部天然胞内信号传导结构域、或其功能片段或衍生物。
术语“初级信号域”以刺激性方式调节TCR复合物的初始活化。一方面，初级信号域由例如TCR/CD3复合物与加载了肽的MHC分子的结合而引发，由此介导T细胞反应(包括但不限于，增殖、活化、分化等)。以刺激性方式起作用的初级信号域可以包含免疫受体酪氨酸激活基序或ITAM的信号传导基序。在本发明中尤其有用的包含ITAM的初级信号域的例子包括但不限于，源自TCRξ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε，CD5，CD22，CD79a，CD79b，CD278(也称作“ICOS”)和CD66d的序列，在特例的本发明CAR中，在任何一个或多个本发明CAR中胞内信号传导结构域包含胞内信号传导序列，例如CD3δ的初级信号域。
术语“共刺激信号域”指“共刺激分子”，指与细胞刺激信号分子，例如TCR/CD3结合，组合导致T细胞增殖和/或关键分子的上调或下调的信号。为T细胞上的关连结合性配偶体，其特异性结合共刺激配体，由此介导T细胞的共刺激反应，例如，但不限于，增殖。共刺激分子是有效免疫反应所需的、非抗原受体的细胞表面分子或其配体。共刺激分子包括但不限于，MHC I类分子、BTLA和Toll配体受体、以及OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)和4-1BB(CD137)。
在本发明中，一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、和胞内信号传导结构域，所述胞内信号传导结构域含有源自刺激分子的功能信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、和胞内信号传导结构域，所述胞内信号传导结构域含有源自共刺激分子的功能性信号传导结构域和源自刺激分子的功能性信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域和胞内传导结构域，所述胞内信号传导结构域包含源自一个或多个共刺激分子的至少两个功能性信号传导结构域和源自刺激分子的功能性信号传导结构域。一方面，CAR在CAR融合蛋白的氨基酸(ND端)包含可选的前导序列。一方面，CAR在胞外抗原识别结构域的N端还包含前导序列，其中前导序列任选地在CAR的细胞加工和定位至细胞膜的过程中从抗原识别结构域(例如scFv)切下。
术语“CD3δ(也称为CD3 Zeta)”定义为GenBan登录号BAG36664.1提供的蛋白质、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD3δ结构域”定义为来自ξ链的胞质结构域的氨基酸残基，其足以功能性地传递T细胞活化所需的初始信号。一方面，ξ的胞质结构域包含GenBan登录号BAG36664.1的残基52至164、其功能性直向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。
在一些实施方案中，本发明嵌合受体是嵌合抗原受体。
本发明提供了嵌合抗原受体(Chimeric Antigen Receptor，CAR)，其包含本文所述的胞外结合结构域、跨膜域和胞内域。常见地，CAR的胞外结合结构域(或称为结构区)来源于小鼠或人源化或人的单克隆抗体。
嵌合抗原受体通常包含胞外抗原结合区或者抗体。在一些实施例中，胞外抗原结合区可以是完全人的。在其他情况下，胞外抗原结合区可以被人源化。在其他情况下，胞外抗原结合区可以是鼠源的，或者所述胞外抗原结合区中的嵌合体由来自至少两种不同动物的氨基酸序列组成。在一些实施例中，所述胞外抗原结合区可以是非人的。
嵌合抗原受体可以设计多种抗原结合区，包括衍生自抗体的单链可变片段(scFv)、选自文库的片段抗原结合区(Fab)、单结构域片段或与接合其同源受体的自然配体。在一些实施例中，胞外抗原结合区可以包含scFv、Fab或天然配体，以及它们的任何衍生物。胞外抗原结合区可以指除完整抗体之外的分子，其可以包含完整抗体的一部分并且可以与完整抗体所结合的抗原结合。抗体片段的实例可以包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2；双功能抗体、线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。胞外抗原结合区，例如scFv、Fab或天然配体，可以是确定抗原特异性的CAR的一部分。胞外抗原结合区可以结合任何互补靶。胞外抗原结合区可以衍生自已知可变区序列的抗体。胞外抗原结合区可以从获自可获得的小鼠杂交瘤的抗体序列中得到。或者，可以从肿瘤细胞或原代细胞例如肿瘤浸润淋巴细胞(TIL)的全外切割测序获得胞外抗原结合区。
在一些实施例中，CAR的胞外抗原结合区的结合特异性可以通过互补决定区或CDR，如轻链CDR或重链CDR来确定。在许多情况下，结合特异性可以通过轻链CDR和重链CDR来确定。
在一些实施例中，CAR的胞外抗原结合区包括铰链或间隔区。术语铰链和间隔区可以互换使用。铰链可以被认为是用于向胞外抗原结合区提供柔性的CAR的一部分。在一些实施例中，铰链可用于检测细胞的细胞表面上的CAR，特别是当检测胞外抗原结合区的抗体不起作用或可用时。例如，衍生自免疫球蛋白的铰链的长度可能需要优化，这取决于胞外抗原结合区靶向靶上的表位的位置。在一些实施例中，铰链可能不属于免疫球蛋白，而是属于另一种分子，如CD8α分子的天然铰链。CD8α铰链可以含有已知在CD8辅助受体和MHC分子的相互作用中起作用的半胱氨酸和脯氨酸残基。所述半胱氨酸和脯氨酸残基可影响所述CAR的性能。可以根据所使用的胞外抗原结合区来调节铰链。铰链可以是任何长度的。
CAR的跨膜结构域(或称为结构区)可以将CAR锚定在细胞的质膜上。CD28的天然跨膜部分可用于CAR。在其他情况下，也可以在CAR中使用CD8α的天然跨膜部分。“CD8”可以是与NCBI参考号：NP_001759或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD8核酸分子”可以是编码CD8多肽的多核苷酸，在某些情况下，跨膜区可以是CD28的天然跨膜部分，“CD28”可以指与NCBI参考号：NP_006130或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD28核酸分子”可以是编码CD28多肽的多核苷酸。在一些实施例中，跨膜部分可以包含CD8α区。
CAR的(细)胞内信号传导区可以负责活化包含所述CAR的免疫应答细胞的效应子功能中的至少一种。CAR可以诱导T细胞的效应子功能，例如，所述效应子功能是细胞溶解活性或辅助活性，包括细胞因子的分泌，如IL-2，TNF-α,γ-IFN等。因此，术语细胞内信号传导区是指转导效应子功能信号并引导细胞进行特异功能的蛋白质部分。虽然通常可以使用整个细胞内信号传导区，但是在许多情况下，不必使用信号结构域的整个链。在一些实施例中，使用细胞内信号传导区的截短部分。在一些实施例中，术语细胞内信号传导区因此意在包括足以转导效应子功能信号的细胞内信号传导区的任何截短部分。
在CAR中使用的信号结构域(或称为结构区)的优选实例可以包括T细胞受体(TCR)的细胞质序列和协同作用以在靶-受体结合之后启动信号转导的共同受体，以及它们的任何衍生物或变体序列和这些序列的具有相同功能性的任何合成序列。
在一些实施例中，所述CAR的细胞内信号传导区可以含有已知的免疫受体酪氨酸激活基序(ITAM)的信号基序。含有细胞质信号传导序列的ITAM的实例包括衍生自TCRδ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。然而，在优选的实施例中，细胞内信号结构域衍生自CD3δ链。
含有一个或多个ITAM基序的T细胞信号结构域的实例是CD3δ结构域，也称为T细胞受体CD3δ链或CD247。该结构域是T细胞受体-CD3复合物的一部分，并且在将几种细胞内信号转导途径的抗原识别与T细胞的主效应激活相结合方面起重要作用。如本文所用，CD3δ主要是指人类CD3δ及其同种型，如从Swissprot条目P20963所知的，包括具有基本相同序列的蛋白质。作为嵌合抗原受体的一部分，不需要全T细胞受体CD3δ链，并且其包含T细胞受体CD3δ链的信号结构域的任何衍生物都是合适的，包括其任何功能等同物。
[根据细则91更正 11.07.2023]
细胞内信号传导结构域(或称为结构区)可以选自表3的任何一个共刺激结构域。在一些实施例中，可以修饰结构域，使得与参考结构域的同一性可以为约50％至约100％。可以修饰表3的任何一个结构域，使得修饰形式可以包含约50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或至多约100％的同一性。
CAR的细胞内信号传导区可以进一步包含一个或多个共刺激结构域。细胞内信号传导区可以包含单个共刺激结构域，例如δ链(第一代CAR)或其与CD28或4-1BB(第二代CAR)。在其他实例中，细胞内信号传导区可以包含两个共刺激结构域，例如CD28/OX40或CD28/4-1BB(第三代)。
与细胞内信号结构域如CD8一起，这些共刺激结构域可以产生激酶途径的下游激活，从而支持基因转录和功能性细胞反应。CAR的共刺激结构域可以激活与CD28(磷脂酰肌醇-4,5-二磷酸3-激酶)或4-1BB/OX40(TNF-受体相关因子衔接蛋白)途径以及MAPK和Akt激活相关的近端信号蛋白。
在某些情况下，通过CAR产生的信号可能与辅助或共刺激信号相结合。对于共刺激信号结构域，嵌合抗原受体样复合物可被设计成包含若干可能的共刺激信号结构域。在本领域众所周知的，在幼稚T细胞中，T细胞受体的单独结合不足以诱导T细胞的完全活化为细胞毒性T细胞。完整的生产性T细胞激活需要第二共刺激信号。已经报道对T细胞活化提供共刺激的几种受体，包括但不限于CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB。这些共刺激分子使用的信号传导途径均能与主T细胞受体激活信号协同作用。这些共刺激信号传导区提供的信号可以与源自一个或多个ITAM基序(例如CD3zeta信号转导域)的主效应激活信号协同作用，并且可以完成T细胞激活的要求。
在一些实施方案中，向嵌合抗原受体样复合物添加共刺激结构域可以增强工程细胞的功效和耐久性。在另一些实施方案中，T细胞信号结构域和共刺激结构域彼此融合从而构成信号传导区。
表3.共刺激结构域
含有抗NKG2A抗体的嵌合抗原受体
本发明还提供多种嵌合抗原受体(CAR)，其中包含本发明的抗体或其片段，该CAR-T细胞表现出抗肿瘤性质。一些实施案例中，用编码CAR的病毒载体转导细胞(例如T细胞)。在一些实施案例中，病毒载体是慢病毒载体。一些实施方案中，细胞可以稳定地表达CAR。
在一优选例中，CAR的NKG2A结合部分是scFv，与其所来自的Fab抗体相比，保持等价的亲和结合力，例如其以相当的功效结合相同抗原。该抗体片段是功能性的，由此其提供生物化学反应，例如激活免疫反应、抑制从其靶抗原的信号传导起始、抑制激酶活性等。
一方面，CAR的抗NKG2A抗原结合结构域是全人源抗体片段。
一方面，本发明CAR将特定抗体的抗原结合结构域和胞内信号传导分子组合在一起。例如，一些方面，胞内信号传导分子包括但不限于，CD3δ、4-1BB和CD28信号传导模块及其组合。
一方面，NKG2A-CAR包含至少一个胞内信号传导结构域，其选择CD137(4-1BB)信号传导结构域、CD28信号传导结构域、CD3δ信号结构域，及其任何组合。一方面，NKG2A-CAR包含至少一个胞内信号传导结构域，其来自一个或多个非CD137(4-1BB)或CD28的共刺激分子。
作为示例性的，NKG2A-CAR的序列可以是：
嵌合抗原受体一，具有SEQ ID NO:64所示的胞外域、SEQ ID NO：95所示的铰链域、SEQID NO：97所示的跨膜域、SEQ ID NO：101所示的共刺激信号域、以及SEQ ID NO：105所示的初级信号域(A4-BBZ)；或
嵌合抗原受体二，具有SEQ ID NO:66所示的胞外域、SEQ ID NO：95所示的铰链域、SEQID NO：97所示的跨膜域、SEQ ID NO：101所示的共刺激信号域、以及SEQ ID NO：105所示的初级信号域(A5-BBZ)。
示例性的，嵌合受体的氨基酸序列如SEQ ID NO：115或116所示。
上述嵌合抗原受体的跨膜域和胞内域，本领域技术人员可以选择常规的跨膜域和胞内域进行替换，且均落入本申请的保护范围。
核酸、载体、病毒、宿主细胞
术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。
当用于指核苷酸序列时，本文所用的术语“序列”包括DNA或RNA，并且可以是单链或双链。
术语“靶序列”是指与指导序列具有互补性的序列，靶序列与指导序列之间互补配对促进CRISPR复合物的形成。一个靶序列可以包含任何多核苷酸，如DNA或RNA多核苷酸。在一些实施例中，靶序列位于细胞的细胞核或细胞质中。
术语序列“同一性”通过在比较窗口(例如至少20个位置)上比较两个经最佳匹配的序列来确定同一性百分比，其中比较窗口中多核苷酸或多肽序列的部分可以包含添加或缺失(即间隙)，例如对于最佳匹配的两个序列而言与参考序列(其不包含添加或缺失)相比20％或更少的间隙(例如5至15％、或10至12％)。通常通过确定在两个序列中发生相同的核酸碱基或氨基酸残基的位置的数目来计算百分比，以产生正确匹配的位置的数目，将正确匹配位置的数目除以参考序列中的位置总数(即窗口大小)，并将结果乘以100，以产生序列同一性的百分比。
术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
本文所用的术语“表达载体”是指包含重组多核苷酸的载体，其包含与待表达的核苷酸序列有效连接的表达调控序列。表达载体包含用于表达的足够的顺式作用元件(cis-acting elements)；用于表达的其它元件可以由宿主细胞或体外表达系统提供。表达载体包括本领域所有已知的那些，如质粒、病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。还可以包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。
本文使用的术语“慢病毒”是指逆转录病毒科的属。逆转录病毒在能够感染非分裂细胞方面是逆转录病毒中独特的；它们可以将大量的遗传信息递送到宿主细胞的DNA中，因此它们是基因递送载体最有效的方法之一。HIV、SIV和FIV都是慢病毒的实例。源自慢病毒的载体提供了在体内实现显著水平的基因转移的手段。
术语“内源”是指一个核酸分子或多肽等来自生物体自身。
本文所用的术语“外源”指的是一个核酸分子或多肽、细胞、组织等没有在生物体自身内源性表达，或表达水平不足以实现过表达时具有的功能。
本文所用的术语“外源蛋白”可以是识别靶抗原的外源转入细胞的蛋白，如外源受体(即本文中前述“嵌合受体”)。
本文中使用的术语“宿主”是指接受移植物移植的受体，在一些实施方式中，可以是接受外源细胞植入的个体，如人。
术语“分离的”是指与细胞成分或其他成分相分离，在这些成分中，多核苷酸、肽、多肽、蛋白质、抗体或其片段在自然状态通常是相关联的。如本领域技术人员将理解，非天然存在的多核苷酸、肽、多肽、蛋白质、抗体或其片段不需要“分离”以将其与天然存在的对应物区分开。此外，“浓缩”、“分离”或“稀释”的多核苷酸、肽、多肽、蛋白质、抗体或其片段可与其天然存在的对应物区分开，因为每体积分子的浓度或数量大于(“浓缩”)或小于(“稀释”)其天然存在的配对物的浓度。富集程度可以以绝对基础测量，例如每溶液体积的重量，或者可以相对于存在于源混合物中的另一潜在的干扰物测量。在一些实施方案中，本发明的技术方案优选的富集程度更高。因此，例如，优选2倍富集、更优选10倍富集、更优选100倍富集、更优选1000倍富集。也可以通过人工组装的方法，例如通过化学合成或重组表达，从而提供“分离的”物质。
本发明提供了编码识别NKG2A的抗体或其片段分离的核酸、载体以及包含所述核酸或载体的宿主细胞。核酸可位于完整细胞中、细胞裂解液中或者以部分纯化的或基本纯化的形式。
可以使用标准的分子生物学技术获得本发明的核酸，例如可以通过标准的PCR扩增或cDNA克隆技术，获得编码抗体的轻链和重链或者编码VH和VL区段的cDNA。对于从免疫球蛋白基因文库获得的抗体(例如，使用噬菌体展示技术)，可以从文库回收编码抗体的一种或多种核酸。向宿主细胞中导入外源核酸的方法是本领域普遍已知的，并可随所使用的宿主细胞而变化。
优选的，本发明核酸分子是选自编码重链可变区的SEQ ID NO:19、23、27、29、31或33，和/或选自编码轻链可变区的SEQ ID NO:21或25。更优选的，是这样的核酸分子，所述核酸分子包含SEQ ID NO:19的重链可变区序列，和包含SEQ ID NO:21的轻链可变区序列；或者包含SEQ ID NO:23的重链可变区序列，和包含SEQ ID NO:25的轻链可变区序列；或者包含SEQ ID NO:27的重链可变区序列，和包含SEQ ID NO:21的轻链可变区序列；或者包含SEQ ID NO:29的重链可变区序列，和包含SEQ ID NO:25的轻链可变区序列；或者包含SEQ ID NO:31的重链可变区序列，和包含SEQ ID NO:25的轻链可变区序列；或者包含SEQ ID NO:33的重链可变区序列，和包含SEQ ID NO:25的轻链可变区序列。
在一个实施方案中，提供一种或多种包含上述核酸的载体(例如，表达载体)。
术语“细胞”指人或非人动物来源的细胞。
术语“宿主细胞”指被引入外源核酸的细胞，包括此种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括转化的原代细胞及来源于其的后代(不考虑传代次数)。后代的核酸内容可以与亲本细胞不完全相同，并且可以含有突变。本文中包括具有与对于原始转化的细胞中筛选或选择的相同的功能或生物学活性的突变体后代。
术语“NKG2A或NKG2A/CD94阳性宿主细胞”是指在细胞表面上表达NKG2A/CD94的宿主细胞，这些细胞可以通过例如使用抗体的流式细胞术来检测，这些抗体特异性识别CD94和NKG2A上的组合表位或单独NKG2A上的表位。
在一些实施方案中，所述宿主细胞是免疫效应细胞。
术语“免疫效应细胞”是指参与免疫应答，产生免疫效应的细胞，如T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、树突细胞、CIK细胞、巨噬细胞、肥大细胞等。在一些实施方案中，所述的免疫效应细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞可以是同种异体NK细胞。“免疫效应功能或免疫效应应答”是指免疫效应细胞，例如增强或促进靶细胞的免疫攻击的功能或反应。例如，免疫效应功能或应答是指促进靶细胞的杀伤或者抑制生长或增殖的T细胞或NK细胞的属性。
术语“经人工改造的具有免疫效应细胞功能的细胞”是指不具有免疫效应的细胞或细胞系经人工改造或接受刺激物刺激后，该细胞获得了免疫效应细胞功能。如293T细胞，经人工改造，使其具有免疫效应细胞的功能；如干细胞，经体外诱导，使其分化成免疫效应细胞。
在一些情况下，“T细胞”可以是来自骨髓的多能干细胞，在胸腺内分化成熟成为具有免疫活性的成熟的T细胞。在一些情况下，“T细胞”可以是具有特定表型特征的细胞群，或不同表型特征的混合细胞群体，如“T细胞”可以是包含至少一种T细胞亚群的细胞：记忆性干细胞样T细胞(stem cell-like memory T cells，Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef、Teff)、调节性T细胞(tregs)和/或效应记忆T细胞(Tem)。在一些情况下，“T细胞”可以是某种特定亚型的T细胞，如γδT细胞。
T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从个体收集的血液获得T细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆分子并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。在一个实施方案中，可以从健康供体，或来自诊断患有肿瘤的患者衍生细胞获得T细胞。
术语“外周血单个核细胞”(peripheral blood mononuclear cell,PBMC)是指外周血中具有单个核的细胞，包含淋巴细胞、单核细胞等。
术语“激活”和“活化”可互换使用，可以指细胞从静止状态转变为活性状态的过程。该过程可以包括对抗原、迁移和/或功能活性状态的表型或遗传变化的响应。例如，术语“激活”可以指NK细胞、T细胞逐步活化的过程。
术语“T细胞活化”或“T细胞激活”指被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。
在另一实施方案中，提供包含上述核酸的宿主细胞。宿主细胞包含(例如，转导有)：(1)载体，所述载体包含核酸，所述核酸编码包含抗体VL的氨基酸序列和包含抗体VH的氨基酸序列，或(2)包含编码包含抗体VL的氨基酸序列的核酸的第一载体，和包含编码包含抗体VH的氨基酸序列的核酸的第二载体。在一个实施方案中，宿主细胞是真核的，例如，中国仓鼠卵巢(CHO)细胞或淋巴细胞(例如，YO、NSO、Sp20细胞)。
在另一实施方案中，所述宿主细胞表达本发明所述的嵌合受体。
在另一实施方案中，所述宿主细胞包括T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、调节性T细胞、NK92细胞、和/或干细胞衍生的免疫效应细胞。
在另一实施方案中，所述T细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞；优选地，所述T细胞为自体/同种异体T细胞；优选地，所述T细胞为原代T细胞；优选地，所述T细胞来源于人的自体T细胞。
在另一实施方案中，所述T细胞包含记忆性干细胞样T细胞(Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef)、调节性T细胞(Tregs)，效应记忆T细胞(Tem)、γδT细胞或其组合。
在另一实施方案中，所述宿主细胞结合表达NKG2A/CD94的细胞，不显著结合NKG2C/CD94、NKG2E/CD94或其组合。
在另一实施方案中，所述宿主细胞还携带外源的细胞因子的编码序列。
在另一实施方案中，所述宿主细胞还可以表达除了上述结合抗原的受体以外的另一种嵌合抗原受体。
在另一实施方案中，所述宿主细胞还可以表达趋化因子受体。
在另一实施方案中，所述宿主细胞还可以表达安全开关。
在另一实施方案中，所述宿主细胞能杀伤活化的NK细胞。
在一个实施例中，提供制备抗NKG2A抗体的方法，其中所述方法包括在适合于表达如上所述的抗体的条件下培养包含编码所述抗体的核酸的宿主细胞，和任选地从宿主细胞(或宿主细胞培养基)回收抗体。
为了表达蛋白质，可以将编码本发明抗体的核酸整合到表达载体中。多种表达载体可用于蛋白质表达。表达载体可包括自我复制的染色体外载体，或整合到宿主基因组中的载体。用于本发明的表达载体包括但不限于使蛋白质能够在哺乳动物细胞、细菌、昆虫细胞、酵母和体外系统中表达的那些。如本领域已知的，多种表达载体是可商业或其他方式获得的。可用于本发明中来表达抗体。
在一优选例中，所述宿主细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；和/或所述宿主细胞与改善其相关的一种或多种副作用的药剂联合施用；和/或所述宿主细胞与表达靶向NKG2A之外的嵌合抗原受体的宿主细胞联合施用。
基因编辑的细胞
在一实例中，采用包含本发明提供的gRNA的CRISPR系统敲除细胞内源性TCR、B2M、NKG2A和/或CIITA。对细胞(例如，T细胞或NKT细胞)的遗传修饰可以通过用重组核酸分子转导基本上均质的细胞群来完成。
[根据细则91更正 11.07.2023]
在一实例中，细胞内源性TCR、B2M、NKG2A和/或CIITA基因敲除后，细胞中TCR、B2M、NKG2A和/或HLA-II低表达或不表达。TCR、B2M、NKG2A和/或HLA-II低表达或不表达是指细胞中TCR、B2M、NKG2A和/或HLA-II的表达减少至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。更具体而言，TCR、B2M、NKG2A和/或HLA-II低表达或不表达分别是指细胞中TCR、B2M、NKG2A和/或HLA-II的含量降低至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。可以通过本领域内已知的任何合适的方法，如ELISA、免疫组织化学、免疫印迹(Western Blotting)或流式细胞术使用TCR、B2M、NKG2A和/或HLA-II的特异性抗体测定细胞中蛋白的表达或含量。
由于供体和受体(或称为宿主)之间的免疫遗传学差异，在进行外源供体移植时，作为外源移植物的供体会受到宿主体内的免疫细胞(例如NK细胞)识别和攻击，进而抑制或者清除供体，产生宿主抗移植物反应(HVGR)。如在异体细胞移植中，当异体细胞的HLA-I类分子的缺失，可以降低宿主CD8+介导的细胞免疫排斥作用。在一实例中，本发明提供内源性HLA-II/B2M低表达或不表达的免疫细胞。
移植物抗宿主病(GVHD)是由于外源移植供体T淋巴细胞的TCR的多样性，以及与宿主HLA分子的不兼容性，供体T淋巴细胞会识别宿主正常组织上的抗原，经扩增并释放一系列细胞因子，大大增强了移植物对宿主抗原的免疫反应，攻击宿主细胞。在一实例中，本发明提供内源性HLA-II/TCR低表达或不表达的免疫细胞。在一实例中，本发明采用CRISPR系统敲除内源性TCR的α链的基因TRAC制备得到内源性TCR低表达或不表达的细胞。
在靶细胞(例如表达靶抗原的肿瘤细胞)反复刺激下，外源移植物的供体免疫细胞中内源性NKG2A表达上调，会被本发明组合物中识别NKG2A的免疫细胞杀伤。此外，NKG2A低表达或不表达可能解除免疫细胞本身的抑制作用，从而发挥更强的抗肿瘤能力。在一实例中，本发明提供内源性HLA-II/NKG2A低表达或不表达的免疫细胞。
上述免疫细胞不显著活化异体免疫细胞。上述免疫细胞能降低异体免疫排斥反应。
上述识别肿瘤抗原的免疫细胞和/或识别NKG2A多肽和肿瘤抗原的免疫细胞，能显著杀伤肿瘤细胞，且不显著活化异体免疫细胞。上述识别肿瘤抗原的免疫细胞和/或识别NKG2A多肽和肿瘤抗原的免疫细胞，能显著杀伤肿瘤细胞，且异体免疫排斥反应低。
药物组合物
本发明的抗体、包含该抗体的免疫辍合物、嵌合受体、宿主细胞可以应用于制备药物组合物或诊断试剂。所述的组合物除了包括有效量的所述抗体、免疫辍合物、嵌合受体、核酸或宿主细胞，还可包含药学上可接受的载体。
术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
在一些实施方案中，所述组合物包含另一治疗剂。在一些实施方案中，所述另一治疗剂是化疗剂，如US20140271820中记载的那些和/或其药学上可接受的盐或类似物。在一些实施方案中，所述治疗剂包括但不限于有丝分裂抑制剂(长春花生物碱)，包括长春新碱、长春花碱、长春地辛和诺维宾(TM)(长春瑞滨，5'-去氢硫化氢)；拓扑异构酶I抑制剂，例如喜树碱化合物，包括CamptosarTM(伊立替康HCL)、HycamtinTM(托泊替康HCL)和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂顺铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、5-氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、霉素霉素、丝裂霉素、肉瘤霉素C和道诺霉素；以及其它化疗药物，包括但不限于抗肿瘤抗体、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。在一些实施方案中，所述另外的治疗剂选自表柔比星、奥沙利铂和5-氟尿嘧啶中的一种或多种。在一些实施方案中，所述另外的治疗剂包括但不限于抗血管生成剂，包括抗VEGF抗体(包括人源化和嵌合抗体、抗VEGF适体和反义寡核苷酸)以及其他血管发生抑制剂，例如血管抑素、内皮抑制素、干扰素、白细胞介素1(包括α和β)白介素12、视黄酸和金属蛋白酶-1和-2的组织抑制剂等。
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。
本文所述的药物组合物可包含一种或多种药学可接受的盐。“药学可接受的盐”指这样一种盐，其保留亲本化合物的期望生物学活性且不产生任何不利的毒物学效果(参见例如，Berge,S.M等人.,1977,J.Pharm.Sci.66:1-19)。此类盐的例子包括酸加成盐和碱加成盐。
酸加成盐包括衍生自无毒无机酸，诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸、亚磷酸等的盐，以及衍生自无毒有机酸，诸如脂肪族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、芳香族酸、脂肪族和芳香族磺酸等的盐。碱加成盐包括衍生自碱土金属(诸如钠、钾、镁、钙等)的盐，以及衍生自无毒有机胺的盐，诸如N,N'-二苄乙二胺、N-甲基葡糖胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、普鲁卡因等。
本文所述的药物组合物还可包含抗氧化剂。抗氧化剂的实例包括但不限于:水溶性抗氧化剂，诸如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠等；油溶性抗氧化剂，诸如抗坏血酸棕榈酸酯、丁基化羟基茴香醚(BHA)、丁基化羟基甲苯(BHT)，卵磷脂、没食子酸丙酯、α-生育酚等；和金属螯合剂，诸如柠檬酸、乙二胺四乙酸(EDTA)、山梨醇、酒石酸、磷酸等。
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用肠胃外给药(如注射)或其它治疗方式。
免疫原性组合物的“肠胃外”施用包括例如皮下(s.c.)、静脉内(i.v.)、肌内(i.m.)或胸骨内注射或输注技术。
在一些实施方案中，组合物可以是等渗的，即它们可以具有与血液和泪液相同的渗透压。本发明组合物的期望等渗性可以使用氯化钠或其它药学上可接受的试剂如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。如果需要，组合物的粘度可以使用药学上可接受的增稠剂维持在选定的水平。合适的增稠剂包括，例如，甲基纤维素、黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的优选浓度将取决于所选择的试剂。显然，合适的载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型。
试剂盒
本发明还提供了包含本文所述的抗体、免疫缀合物、嵌合受体、核酸或宿主细胞的试剂盒。在一些实施方案中，试剂盒可以包括含有有效量的包含一种或多种单位剂型的本文所述的抗体、嵌合受体、核酸或宿主细胞的治疗或预防组合物。在一些实施方案中，试剂盒包含可含有治疗或预防性组合物的无菌容器；这样的容器可以是盒、安瓿、瓶、小瓶、管、袋、泡罩包装或本领域已知的其它合适的容器形式。这种容器可以由塑料、玻璃、层压纸、金属箔或其他适合于保持药物的材料制成。在一些实施方案中，所述试剂盒包含本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞，以及将本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞给予个体的说明书。说明书中通常包含使用本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞来治疗或预防癌症或肿瘤的方法。在一些实施方案中，试剂盒包含本文所述的宿主细胞，并且可以包括约1×104个细胞至约1×106个细胞。在一些实施方案中，试剂盒可以包括至少约1×105个细胞，至少约1×106个细胞，至少约1×107个细胞，至少约4×107个细胞，至少约5×107个细胞，至少约6×107个细胞，至少约6×107个细胞，8×107个细胞，至少约9×107个细胞，至少约1×108个细胞，至少约2×108个细胞，至少约3×108个细胞，至少约4×108个细胞，至少约5×108个细胞，至少约6×108个细胞，至少约6×108细胞，至少约8×108个细胞，至少约9×108细胞，至少约1×109个细胞，至少约2×109个细胞，至少约3×109个细胞，至少约4×109个细胞，至少约5×109个细胞，至少约6×109个细胞，至少约8×109个细胞，至少约9×109个细胞，至少约1×1010个细胞，至少约2×1010个细胞，至少约3×1010个细胞，至少约4×1010个细胞，至少约5×1010个细胞，至少约6×1010个细胞，至少约7×1010个细胞、至少约8×1010个细胞、至少约9×1010个细胞，至少约1×1011个细胞，至少约2×1011个细胞，至少约3×1011个细胞，至少约4×1011个细胞，至少约5×1011个细胞，至少约8×1011个细胞，至少约9×1011个细胞，或至少约1×1012个细胞。例如，可以在试剂盒中包括大约5×1010个细胞。在另一个实例中，试剂盒可以包括3×106个细胞；细胞可以扩增至约5×1010个细胞并施用于受试者。
在一些实施方案中，试剂盒可以包括同种异体细胞。在一些实施方案中，试剂盒可以包括可以包含基因组修饰的细胞。在一些实施方案中，试剂盒可以包含“现成的”细胞。在一些实施方案中，试剂盒可以包括可以扩展用于临床使用的细胞。在某些情况下，试剂盒可能包含用于研究目的的内容物。
在一些实施方案中，说明书包括以下中的至少一个：治疗剂的描述；用于治疗或预防肿瘤或其症状的剂量方案和给药；预防措施、警示、禁忌症、过量信息、不良反应、动物药理学、临床研究、和/或引用文献。说明书可以直接打印在容器上(如果有的话)，或作为容器上的标签，或作为容器内或容器中提供的单独的纸张、小册子、卡片或文件夹打印。在一些实施方案中，说明书提供施用本发明所述的抗体用于治疗或预防肿瘤的方法。在某些情况下，说明书提供了施用化学治疗剂之前、之后或同时给与本发明的抗体的方法。
用于诊断/检测/治疗的方法
术语“调控”是指正向或负向改变。调节范例包括1％、2％、10％、25％、50％、75％、或100％变化。在一具体实施方式中，是指负向改变。
术语“治疗”是指在试图改变疾病过程的干预措施，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
术语“预防”是指在试图在疾病(如细胞移植产生的排斥反应)产生前进行的干预措施。
术语“肿瘤抗原”指的是过度增生性疾病发生、发展过程中新出现的或过度表达的抗原。在某些方面，本发明的过度增生性病症是指肿瘤。
本发明所述的肿瘤抗原可以是实体瘤抗原，也可以是血液瘤抗原。
本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P4501B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。优选的，所述肿瘤抗原为CS1、Claudin18.2、GPC3、BCMA或者CD19。
病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原,或流感病毒抗原。
本文中使用的术语“个体”是指任何动物，例如哺乳动物或有袋动物。本发明的个体包括但不限于人类、非人类灵长类动物(例如恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
本文所用的术语“有效量”是指提供治疗或预防益处的量。
本文中提供的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒都可以用于治疗方法中。
在一个方面，提供用作药物的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒。在另一方面，提供用于治疗疾病的任一抗NKG2A抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在某些实施方案中，提供用于治疗方法的任一抗NKG2A抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在某些实施方案中，本发明提供任一抗NKG2A抗体、免疫缀合物、嵌合抗原受体修饰的宿主细胞、药物组合物或试剂盒，其用于治疗患有疾病的个体的方法，所述方法包括向个体施用有效量的任一抗NKG2A抗体、免疫缀合物、嵌合抗原受体修饰的免疫宿主细胞、药物组合物或试剂盒。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。
在另一个方面，本发明提供任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒在制备或配制药物中的用途。在一个实施方案中，所述药物用于治疗疾病。在另一个实施方案中，所述药物用于治疗疾病的方法，所述方法包括向患病个体施用有效量的药物。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。
在另一个方面，本发明提供用于治疗疾病的方法。在一个实施方案中，所述方法包括向患有表达HLA-E的疾病的个体施用有效量的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。
在另一个方面，本发明提供例如用于任一上述治疗方法的包含本文中提供的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒的药物制剂。在一个实施方案中，所述药物制剂包含本文中提供的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒和药用载体。在另一个实施方案中，所述药物制剂包含本文中提供的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒和至少一种另外的治疗剂。
在另一个方面，所述药物制剂用于治疗疾病。在一个实施方案中，向患病个体施用所述药物制剂。根据任一以上实施方案的“个体”优选是人。
在另一个方面，本发明提供用于制备药物或药物制剂的方法，所述方法包括将本文中提供的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒与药用载体混合，例如，以用于任一上述治疗方法。在一个实施方案中，用于制备药物或药物制剂的方法还包括添加至少一种另外的治疗剂至药物或药物制剂。
本发明的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒可以单独地用于治疗或与其他试剂组合地用于治疗。例如，本发明的任一抗NKG2A抗体、免疫缀合物、嵌合抗原受体修饰的宿主细胞、药物组合物或试剂盒可以与至少一种另外的治疗剂共同施用。
上述的此种组合治疗包括组合施用(其中两种以上治疗剂被包含在同一或分开的制剂中)和分开施用，在此种情况中，本发明的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒的施用可以发生在另外的治疗剂或试剂的施用之前、同时、和/或之后。在一个实施方案中，本发明的任一抗NKG2A抗体、免疫缀合物、嵌合抗原受体修饰的宿主细胞、药物组合物或试剂盒的施用和另外的治疗剂的施用彼此发生在约一个月以内、或在约一周、两周或三周以内，或在约一天、两天、三天、四天、五天或六天以内。
本发明的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒(以及任意另外的治疗剂)可以通过任何合适的手段施用，包括肠胃外施用、肺内施用或鼻内施用，以及，如果具备治疗需要，病变内施用。肠胃外输注包括肌肉内施用、静脉内施用、动脉内施用、腹膜内施用或皮下施用。用药可以是通过任何合适的途径，例如，通过注射，如静脉内或皮下注射，这部分取决于施用是短暂的还是长期的。本文中考虑多种用药方案，包括但不限于单次施用或在多个时间点的多次施用、推注施用，和脉冲注入。
给予个体的包含免疫反应性细胞群体的制剂包含有效治疗和/或预防特定适应症或疾病的多个免疫反应性细胞。因此，可以向个体施用免疫反应性细胞的治疗有效群体。通常，施用包含约1×104至约1×1010个免疫反应性细胞的制剂。在大多数情况下，制剂将包含约1×105至约1×109个免疫反应性细胞、约5×105至约5×108个免疫反应性细胞、或约1×106至约1×107个免疫反应性细胞。然而，根据肿瘤的位置、来源、身份、程度和严重程度、待治疗的个体的年龄和身体状况等，对个体施用的CAR免疫反应性细胞的数量将在宽的范围之间变化。医生将最终确定要使用的适当剂量。
在一些实施方案中，使用嵌合受体来刺激宿主细胞介导的免疫应答。例如，T细胞介导的免疫应答是涉及T细胞活化的免疫应答。活化的抗原特异性细胞毒性T细胞能够在表面上显示外源抗原表位的靶细胞中诱导细胞凋亡，例如显示肿瘤抗原的癌细胞。在另一些实施方案中，使用嵌合抗原受体在哺乳动物中提供抗肿瘤免疫。由于T细胞介导的免疫应答，受试者将产生抗肿瘤免疫。
在某些情况下，治疗患有肿瘤的受试者的方法可以涉及向需要治疗的受试者施用一种或多种本发明所述的宿主细胞。所述宿主细胞可结合肿瘤靶分子并诱导癌细胞死亡。如前文所述，本发明还提供治疗个体中的病原体感染的方法，包括向所述个体施用治疗有效量的本发明的宿主细胞。
本发明的宿主细胞的给药频率将根据包括所治疗疾病的因素、特定宿主细胞的元件和给药方式。例如可以每日给药4次、3次、2次或每日一次、每隔一天、每三天、每四天、每五天、每六天一次、每周一次、每八天一次、每九天一次、每十天、每周一次、或者每月两次给药。如本文所述，由于本申请的宿主细胞具有改善的活力，从而可以不仅以与类似的但不表达外源性I型干扰素的宿主细胞更低的治疗有效的量给药，并且可以以更低的频率给药，以获得至少类似、并且优选更加显著的疗效。
本发明的优点：
1.本发明提供了特异性结合NKG2A的抗体，是全人源抗体，免疫原性低，可能的临床副反应少；
2.本发明的抗体能够有效阻断肿瘤细胞的HLA-E与NK细胞的NKG2A/CD94结合，降低了表达HLA-E的肿瘤细胞通过NKG2A/CD94通路对NK细胞的抑制作用，增强NK细胞对肿瘤细胞的杀伤作用，表现出良好的抗肿瘤效果。
3.表达本发明的抗体制备的NKG2A-CAR的T细胞能杀伤NK细胞；利用本发明的抗体制备的既靶向肿瘤又靶向NK细胞的串联CAR的T细胞能杀伤NK细胞，也能增强其抗肿瘤作用；表达本发明的抗体制备的NKG2A-CAR的UCAR-T细胞能抵抗NK细胞的杀伤，增强其存活能力，并且能协同靶向肿瘤的T细胞或CAR-T细胞的抗肿瘤作用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1.NKG2A抗体的筛选和鉴定
1、使用全人噬菌体展示文库筛选针对NKG2A特异的抗体
(1)NKG2A/CD94、NKG2C/CD94异源二聚体的制备
利用真核表达质粒V152S构建分别表达NKG2A/CD94、NKG2C/CD94异源二聚体的载体。将包括mFc(序列如SEQ ID NO：84所示)、NKG2A胞外段(序列如SEQ ID NO：71所示)、(G4S)3(序列如SEQ ID NO：110所示)、CD94胞外段(序列如SEQ ID NO：77所示)的片段mFc-NKG2A-CD94插入真核表达质粒V152S，构建成载体V152S-mFc-NKG2A-CD94；将包括mFc(序列如SEQ ID NO：84所示)、NKG2C胞外段(序列如73所示)、(G4S)3(序列如SEQ ID NO：110所示)、CD94胞外段(序列如SEQ ID NO：77所示)的片段mFc-NKG2C-CD94插入真核表达质粒V152S，构建成载体V152S-mFc-NKG2C-CD94(见图1)。
将载体V152S-mFc-NKG2A-CD94、V152S-mFc-NKG2C-CD94分别转染HEK293细胞(中国典型培养物保藏中心(CCTCC))后培养7天，培养液离心取上清，使用Mabselect Sure柱亲和纯化分别得到抗原NKG2A/CD94、NKG2C/CD94异源二聚体。
(2)NKG2A抗体的筛选
本发明使用的噬菌体展示文库为本公司构建的噬菌体文库，库容为1E+11。利用本领域技术人员已知的筛选方法得到针对NKG2A/CD94异源二聚体高度特异的Fab片段。
简言之，分别包被上述制备的异源二聚体10μg/ml抗原mFc-NKG2A-CD94、mFc-NKG2C-CD94于免疫管。用含有2％脱脂奶粉的磷酸缓冲液(MPBS)室温静置封闭2小时。为了筛选到特异性结合NKG2A的抗体，将噬菌体文库加入包被mFc-NKG2C-CD94的免疫管中结合1小时。取上清液加入包被mFc-NKG2A-CD94的免疫管中结合1.5小时，随后将非特异性的噬菌体洗掉，将结合的噬菌体洗脱下来并感染对数生长期的大肠杆菌TG1。扩大培养洗脱下来的噬菌体，并使用PEG/NaCl沉淀纯化扩大后的噬菌体文库用于下一轮的筛选。筛选一共进行三轮，富集与NKG2A/CD94异源二聚体特异性结合的Fab噬菌体克隆。通过针对NKG2A的标准ELISA方法确定阳性克隆。ELISA使用mFc-NKG2C-CD94作为无关抗原来验证抗体的特异性。一共筛选了1504个克隆，其中50个克隆只结合mFc-NKG2A-CD94，不结合mFc-NKG2C-CD94。经过测序，得到了2个克隆。表达纯化这2个克隆，得到Fab形式的抗体A1和A2。
A1的HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:3所示、LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR2的氨基酸序列如SEQ ID NO:5所示、LCDR3的氨基酸序列如SEQ ID NO:6所示。A1的重链可变区的氨基酸序列如SEQ ID NO:18所示、轻链可变区的氨基酸序列如SEQ ID NO:20所示。A1的重链的氨基酸序列如SEQ ID NO：34所示，轻链的氨基酸序列如SEQ ID NO：42所示。A2的HCDR1的氨基酸序列如SEQ ID NO:7所示、HCDR2的氨基酸序列如SEQ ID NO:8所示、HCDR3的氨基酸序列如SEQ ID NO:9所示、LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR2的氨基酸序列如SEQ ID NO:5所示、LCDR3的氨基酸序列如SEQ ID NO:10所示。A2的重链可变区的氨基酸序列如SEQ ID NO:22所示、轻链可变区的氨基酸序列如SEQ ID NO:24所示。A2重链的氨基酸序列如SEQ ID NO：35所示，轻链的氨基酸序列如SEQ ID NO：46所示。
同时构建和真核表达纯化出IgG4形式的抗体，得到两个特异性结合NKG2A/CD94异源二聚体的抗体，命名为A1-IgG4和A2-IgG4。A1-IgG4的重链氨基酸序列如SEQ ID NO:40所示，轻链氨基酸序列如SEQ ID NO:42所示。A2-IgG4的重链氨基酸序列如SEQ ID NO:44所示，轻链氨基酸序列如SEQ ID NO:46所示。
2、通过标准ELISA检测抗体A1和A2的特异性
抗原mFc-NKG2A-CD94和mFc-NKG2C-CD94分别用PBS稀释至浓度为5μg/ml，4℃包被过夜。2％MPBS(脱脂奶粉/PBS)室温封闭1h；PBS清洗3遍后分别加入一抗A1(10μg/ml)或A2(10μg/ml)，室温孵育1h；PBST清洗5遍后加入二抗(anti-Flag-HRP，1:4000，Sigma)继续室温孵育1h；PBST清洗5遍后TMB显色并用酶标仪读数OD450值。NA为空白对照。结果见图2，抗体A1、A2均特异性结合NKG2A/CD94异源二聚体，不结合NKG2C/CD94异源二聚体。
3、利用ELISA分别测定抗体A1-IgG4、A2-IgG4与NKG2A/CD94异源二聚体的EC50
上述制备的mFc-NKG2A-CD94用PBS稀释至浓度为2.5μg/ml，4℃包被过夜。2％MPBS(脱脂奶粉/PBS)室温封闭1h；PBS清洗3遍后分别加入一抗(A1-IgG4、A2-IgG4:10μg/ml开始5倍稀释8个梯度)，室温孵育1h；PBST清洗5遍后加入二抗(anti-Fc-HRP，1:10000，Sigma)继续室温孵育1h；PBST清洗5遍后TMB显色并用酶标仪读数OD450值。随后通过GraphPad Prism5软件以一抗浓度为横坐标，OD450值为纵坐标进行四参数拟合，计算EC50值。结果见图3，抗体A1-IgG4和A2-IgG4均与NKG2A/CD94异源二聚体结合，且呈现浓度梯度依赖性，EC50值分别为0.012μg/ml，0.011μg/ml。
4、利用Biacore测定抗体A1-IgG4和A2-IgG4的亲和力
用Anti-huFc antibody包被芯片CM5(ID:180925-0245:1738291)，将A1-IgG4和A2-IgG4抗体分别作为配体，mFc-NKG2A-CD94作为流动相(图4中的曲线从上到下分别代表浓度为150nM、50nM、16.67nM、5.56nM和1.85nM)，再生试剂为3M MgCl2，25℃，实验数据用Biacore T200Evaluation Software2.0软件处理。选择“surface bound”，Rmax选择“local”，使用1:1的langmuir模型进行拟合。结果见图4，A1-IgG4和A2-IgG4结合NKG2A/CD94异源二聚体的亲和力KD值分别为4.41nM和4.30nM。
5、利用FACs测定抗体A1-IgG4、A2-IgG4与靶细胞结合的特异性
利用常规分子生物学技术，通过pWPT慢病毒载体介导的方式将CD94全长(包含CD94全长，Flag标签)、NKG2A-CD94(包含NKG2A全长，F2A，CD94全长，Flag标签)、NKG2C-CD94(包含NKG2C全长，F2A，CD94全长，Flag标签)、NKG2E-CD94(包含NKG2E全长，F2A，CD94全长，Flag标签)，分别转入CHO-K1细胞(也称为CHOK1)构建成过表达目的基因的混合克隆细胞株，使用抗Flag抗体确认混合克隆株构建成功后用有限稀释法进行单克隆筛选，最后成功得到表达CD94的CHO-K1稳转细胞株CHOK1-CD94、表达NKG2A-CD94的CHO-K1稳转细胞株CHOK1-NKG2A-CD94、表达NKG2C-CD94的CHO-K1稳转细胞株CHOK1-NKG2C-CD94及表达NKG2E-CD94的CHO-K1稳转细胞株CHOK1-NKG2E-CD94(载体图见图5)，阳性率均大于90％。
其中CD94全长碱基序列为SEQ ID No.128，NKG2A全长碱基序列为SEQ ID No.69，NKG2C全长碱基序列为SEQ ID No.124，NKG2E全长碱基序列为SEQ ID No.126，Flag标签碱基序列为SEQ ID No.112，F2A碱基序列为SEQ ID No.108。
分别取CHOK1-NKG2A-CD94，CHOK1-NKG2C-CD94，CHOK1-NKG2E-CD94和CHOK1-CD94细胞计数铺U型底板，每孔约2×105个细胞，然后分别进行一抗(A1-IgG4、A2-IgG4:5μg/mL)和二抗(Anti-Fc-FITC:1:200,Jackson ImmunoResearch)孵育，随后用流式分析仪进行荧光强度检测。结果见图6，抗体A1-IgG4、A2-IgG4分别结合过表达NKG2A/CD94异源二聚体的CHO-K1细胞，不结合过表达NKG2C/CD94、NKG2E/CD94以及CD94的CHO-K1细胞。
实施例2.抗体的亲和力成熟
1、利用噬菌体展示技术进行亲和力成熟
分别以A1和A2为亲本抗体，采用常规生物学技术分别构建两个噬菌体文库，一个随机化轻链的CDR1以及CDR2，另一个随机化重链的CDR1以及CDR2。然后针对抗原进行筛选，通过ELISA技术等筛选出高亲和力的抗体，即A1或A2的变体。
首先基于抗体A1(Fab)构建模板质粒。对于轻链CDR1和CDR2随机化的噬菌体文库，使用引物LMF(核酸序列如SEQ ID NO：85所示)和IL1R(核酸序列如SEQ ID NO：91所示)，PCR扩增片段1；使用引物IL2F(核酸序列如SEQ ID NO：92所示)和FdR(核酸序列如SEQ ID NO：86所示)，PCR扩增片段2；然后通过搭桥PCR连接片段1和片段2得到含有随机化序列的Fab全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中，并电转化至TG1感受态细胞中，库容为2.6ⅹ109。对于重链CDR1和CDR2随机化的噬菌体文库，使用引物LMF(核酸序列如SEQ ID NO：85所示)和F10H1R(核酸序列如SEQ ID NO：87所示)，PCR扩增片段3；使用引物F10H2F(核酸序列如SEQ ID NO：88所示)和FdR(核酸序列如SEQ ID NO：86所示)，PCR扩增片段4；然后通过搭桥PCR连接片段3和片段4得到含有随机化序列的Fab全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中，并电转化至TG1感受态细胞中，库容为3.2ⅹ109。
抗体A2亲和力成熟文库的构建与A1类似，基于抗体A2(Fab)构建模板质粒。使用与A1相同的引物随机化轻链的CDR1和CDR2，得到的噬菌体文库库容为1.89ⅹ109。对于重链CDR1和CDR2随机化的噬菌体文库，使用引物LMF(核酸序列如SEQ ID NO：85所示)和BH1R(核酸序列如SEQ ID NO：89所示)，PCR扩增片段5；使用引物BH2F(核酸序列如SEQ ID NO：90所示)和FdR(核酸序列如SEQ ID NO：86所示)，PCR扩增片段6；然后通过搭桥PCR连接片段5和片段6得到含有随机化序列的Fab全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中，并电转化至TG1感受态细胞中，库容为1.22ⅹ109。
2、噬菌体文库的筛选
参照实施例1方法，进行两轮筛选。第一轮筛选用浓度为5μg/ml的抗原mFc-NKG2A-CD94包被，并使用浓度为5μg/ml的抗原mFc-NKG2C-CD94清除；第二轮筛选用浓度为1μg/ml的抗原mFc-NKG2A-CD94包被，同时使用浓度为2μg/ml的抗原mFc-NKG2E-CD94清除。两轮筛选后通过ELISA确定阳性克隆，共挑取79个阳性克隆进行测序，并选取22个克隆原核表达纯化得到Fab抗体。最终得到4个克隆A3、A4、A5、A6。经测序，抗体的序列如表4、5所示.
表4抗体VH和VL序列
表5抗体CDR序列
表6抗体重链和轻链序列
将这4个克隆构建成IgG4形式，进行表达纯化。经测序，4个抗体的序列如表7所示。
表7抗体(huIgG4)序列
如图7所示，比较了6个抗体A1、A2、A3、A4、A5和A6的重链可变区的氨基酸序列。经测序，A3与母本A1的重链可变区序列相似性为98.3％。A4与母本A2的重链可变区序列相似性为94.8％。A5与母本A2的重链可变区序列相似性为94.8％。A6与母本A2的重链可变区序列相似性为93.1％。
3、利用FACs测定抗体(Fab形式)与靶细胞结合的特异性
分别取CHOK1-NKG2A-CD94，CHOK1-NKG2C-CD94和CHOK1-NKG2E-CD94细胞计数铺U型底板，每孔约2×105个细胞，然后分别进行一抗(A1、A2、A3、A4、A5、A6:10μg/mL)和二抗(Anti-Fab-FITC:1:200,Jackson ImmunoResearch)孵育，随后用流式分析仪进行荧光强度检测。结果见图8，6个抗体(Fab形式)都特异性结合过表达NKG2A/CD94异源二聚体的CHO-K1细胞，不结合过表达NKG2C/CD94以及NKG2E/CD94的CHO-K1细胞。
4、利用ELISA测定抗体(Fab形式)与NKG2A/CD94异源二聚体的EC50
抗原mFc-NKG2A-CD94用PBS稀释至浓度为2μg/ml，4℃包被过夜。2％MPBS(脱脂奶粉/PBS)室温封闭1h；PBS清洗3遍后分别加入一抗(A3、A4、A5、A6:25μg/mL起始5倍梯度稀释8个梯度)，室温孵育1h；PBST清洗5遍后加入二抗(anti-Flag-HRP，1：4000，Sigma)继续室温孵育1h；PBST清洗5遍后TMB显色并用酶标仪读数OD450值。随后通过GraphPad Prism5软件以一抗浓度为横坐标，OD450值为纵坐标进行四参数拟合，计算EC50值。结果见图9，4个抗体(Fab形式)均与NKG2A/CD94异源二聚体结合，且呈现浓度梯度依赖性，亲和力成熟后的抗体的结合能力明显优于对应的母本抗体。EC50值如表8所示。
表8抗体(Fab形式)与NKG2A/CD94异源二聚体结合的EC50值
5、利用FACs测定抗体(Fab形式)与靶细胞结合的EC50
取CHOK1-NKG2A-CD94细胞计数铺U型底板，每孔约2×105个细胞，然后分别进行一抗(A1、A2、A3、A4、A5、A6:25μg/mL起始5倍梯度稀释8个梯度)和二抗(Anti-Fab-FITC:1:200,Jackson ImmunoResearch)孵育，随后用流式分析仪进行荧光强度检测。实验数据用FlowJo分析软件计算平均荧光强度(MFI)再根据“校准MFI＝实测MFI-阴性对照MFI”计算各浓度点校准MFI，随后通过GraphPad Prism5软件以一抗浓度为横坐标，校准平均荧光强度(MFI)为纵坐标进行四参数拟合，计算EC50值。结果见图10，抗体(Fab形式)结合过表达NKG2A/CD94异源二聚体的CHO-K1细胞的EC50值如表9所示，亲和力成熟后的抗体胞的结合能力明显优于对应的母本抗体。
表9抗体(Fab形式)与过表达NKG2A/CD94异源二聚体的CHO-K1细胞结合的EC50
实施例3.抗体(IgG4形式)与抗原的结合活性检测
1、利用ELISA检测抗体(IgG4形式)与NKG2A/CD94异源二聚体的结合活性
抗原mFc-NKG2A-CD94用PBS稀释至浓度为2μg/ml，4℃包被过夜。2％MPBS(脱脂奶粉/PBS)室温封闭1h；PBS清洗3遍后加入一抗(A1-IgG4、A2-IgG4、A3-IgG4、A4-IgG4、A5-IgG4、A6-IgG4:5μg/mL起始5倍梯度稀释8个梯度)，室温孵育1h；PBST清洗5遍后加入二抗(anti-Fc-HRP，1：10000，Sigma)继续室温孵育1h；PBST清洗5遍后TMB显色并用酶标仪读数OD450值。通过GraphPad Prism5软件以一抗浓度为横坐标，OD450值为纵坐标进行四参数拟合，计算EC50值。结果见图11，抗体(IgG4形式)与NKG2A/CD94异源二聚体结合，且呈现浓度梯度依赖性，EC50值如表10所示。
表10抗体(IgG4形式)与NKG2A/CD94异源二聚体结合的EC50值
2、利用FACS检测抗体(IgG4形式)与靶细胞的结合
取CHOK1-NKG2A-CD94细胞计数铺U型底板，每孔约2×105个细胞，然后分别进行一抗(A1-IgG4、A2-IgG4、A3-IgG4、A4-IgG4、A5-IgG4、A6-IgG4:25μg/mL起始5倍梯度稀释8个梯度)和二抗(Anti-Fab-FITC:1:200,Jackson ImmunoResearch)孵育，随后用流式分析仪进行荧光强度检测。实验数据用FlowJo分析软件计算平均荧光强度(MFI)再根据“校准MFI＝实测MFI-阴性对照MFI”计算各浓度点校准MFI，随后通过GraphPad Prism5软件以一抗浓度为横坐标，校准平均荧光强度(MFI)为纵坐标进行四参数拟合，计算EC50值。结果见图12，抗体(IgG4形式)均结合过表达NKG2A/CD94异源二聚体的CHO-K1细胞，且呈现浓度梯度依赖性，EC50值如表11所示，这表明亲和力成熟后的抗体的结合活性均有提高。
表11抗体(IgG4形式)与过表达NKG2A/CD94异源二聚体的CHO-K1细胞结合的EC50
3、利用Biacore检测抗体(IgG4形式)与NKG2A/CD94异源二聚体的亲和力
用Anti-huFc antibody包被芯片CM5(ID:180925-0245:1738291)，将A3-IgG4、A4-IgG4、A5-IgG41、A6-IgG4抗体作为配体，mFc-NKG2A-CD94作为流动相(图13中的曲线从上到下分别代表浓度为100nM、33.33nM、11.11nM、3.70nM和1.23nM)，再生试剂为3M MgCl2，25℃。实验数据用Biacore T200 Evaluation Software2.0软件处理。选择“surface bound”，Rmax选择“local”，使用1:1的langmuir模型进行拟合。结果见图13，所得抗体(IgG4形式)与NKG2A/CD94异源二聚体的结合亲和力KD值如表12所示，经过亲和力成熟后的抗体的亲和力与母本抗体相比有约12～18倍的提高。
表12抗体(IgG4形式)与NKG2A/CD94异源二聚体结合的亲和力
实施例4.NKG2A的天然配体HLA-E四聚体的制备及活性检测
1、NKG2A的天然配体HLA-E四聚体的制备
利用常规分子生物学技术，通过质粒转导的方式将分别包含片段HLA-E-avi-(包含人HLA-E胞外段(序列如SEQ ID NO:79所示)、avi标签(序列如SEQ ID NO:114所示)、片段β2m(序列如SEQ ID NO:81所示)的质粒pET22b-HLA-E、pET22b-β2m，分别转入BL21菌株(载体见图14)。
使用BL21菌株原核诱导表达，收集菌体再超声破碎后收集包涵体沉淀。包涵体沉淀经过洗涤后再用8M尿素溶解，经过阴离子交换柱的进一步纯化后电泳检测，纯度90％以上。100ml复性液中加入5mg VMA九肽(氨基酸序列如SEQ ID NO:82所示，吉尔生化合成)，再以1:2的摩尔比加入纯化的HLA-E和β2m，得到复性复合物，再用PBS 5％甘油透析。透析后产物经过分子筛纯化，收集目的峰后浓缩并置换到10mMTris pH8.0，分装-80℃保存。使用birA酶对复合物进行生物素标记，标记产物和SA-PE(PE标记的SA抗体(BD HorizonTM))1:4的比例轻柔混合，得到最终的HLA-E四聚体。
2、将纯化后的HLA-E四聚体与内源性表达NKG2A/CD94的原代NK细胞进行结合活性检测
NK细胞通过NK Cell Isolation Kit(Miltenyi Biotec)从外周血PBMCs中纯化得到，并用含500IU/ml IL-2和150IU/ml IL-15的NKMedium(Miltenyi Biotec)培养8天后收集得到NKG2A阳性NK细胞(NKG2A+NK)，阳性率为86％。HLA-E四聚体使用浓度从5μg/ml开始，5倍梯度稀释，空白孔浓度为0，与NK细胞4℃共孵育45min后，用含1％FBS的PBS洗3遍，随后用流式细胞仪检测PE荧光信号。结果见图15，HLA-E四聚体与表达NKG2A/CD94的NK细胞有明显结合，且呈现浓度梯度依赖性，EC50为0.1029μg/ml。
实施例5.检测NKG2A抗体(IgG4形式)阻断NKG2A与其配体HLA-E的结合
取实施例4中制备的NKG2A阳性的NK细胞(NKG2A+NK)，同时加入HLA-E-PE和抗NKG2A抗体在4℃共孵育45min，检测PE荧光信号强度。根据实施例4，HLA-E-PE浓度选用EC50值，即0.103μg/ml，抗体浓度从10μg/ml开始，5倍梯度稀释至0.000128μg/ml，空白组只含HLA-E-PE。共孵育结束后，用含1％FBS的PBS洗3遍，随后通过流式细胞分析仪进行PE荧光信号检测，得到MFI值，抑制率计算公式为：(MFI(空白组)-MFI(实验组))/MFI(空白组)×100％。结果见图16，所得亲和力成熟抗体在1μg/ml以上浓度时均可完全抑制HLA-E四聚体与NK细胞的结合，其中半数抑制有效浓度IC50值如表13所示。实验表明亲和力成熟抗体竞争抑制HLA-E四聚体与NK细胞结合的能力相比较母本有明显提高。
表13抗体(IgG4形式)阻断HLA-E与NK细胞结合的IC50值
实施例6.NKG2A抗体能够降低表达HLA-E的靶细胞对NK细胞活性的抑制
取实施例4中制备的NKG2A+NK细胞为效应细胞。靶细胞选用不表达HLA-E的K562细胞(人髓性白血病细胞，中科院细胞库)、过表达HLA-E的K562细胞(称为K562-HLA-E细胞)以及内源性表达HLA-E的FaDu细胞(人咽鳞状细胞，ATCC)，HLA-E表达水平见图17。通过慢病毒介导的方式将人HLA-E胞外区(序列如SEQ ID NO:78所示)转入K562细胞得到K562-HLA-E细胞。
将1×105个NK细胞与三种靶细胞分别按照效靶比1:1共孵育，同时加入CD107a-APC抗体(购自BD Biosciences，5μl/test)及NKG2A抗体(IgG4形式，10μg/ml，空白组不添加NKG2A抗体)共孵育1h，添加蛋白转运抑制剂(Brefeldin A/Monensin Mix)继续孵育3h。收集细胞，在实验组添加带PE-Cy7荧光的CD56抗体(CD56-PE-Cy7，购自eBioscience，5μl/test，用于检测NK细胞)和带FITC荧光的抗Fc抗体(anti-Fc-FITC抗体，购自Jackson Immunoresearch，1:200，用于检测NKG2A阳性NK细胞)共孵育后进行FACs检测，空白组添加CD56-PE-Cy7和anti-NKG2A-PE抗体(购自Miltenyi Biotec,2μl/test)共孵育后进行FACs检测，分析各组NKG2A+NK细胞中CD107a的表达情况。结果见图18，NKG2A+NK细胞与K562细胞共孵育后，CD107a表达水平较高，说明肿瘤细胞K562没有结合NK细胞上的NKG2A而抑制NK细胞活性，因此添加NKG2A抗体对CD107a表达水平无显著影响。而NKG2A+NK细胞与K562-HLA-E细胞、或与FaDu细胞共孵育后，CD107a表达水平较低，说明肿瘤细胞上的HLA-E与NK细胞上的NKG2A结合能够明显抑制NK细胞活性；而添加NKG2A抗体竞争性地与NK细胞上的NKG2A结合，阻断NK细胞上的NKG2A与肿瘤细胞上的HLA-E结合，从而降低HLA-E高表达肿瘤细胞对NK细胞活性抑制，因此相对于没有添加抗体组，添加NKG2A抗体各组的CD107a表达水平明显提高。由于FaDu细胞内源性表达HLA-E的量较低，导致CD107a表达水平提高程度低于HLA-E高表达组K562-HLA-E。
实施例7.NKG2A抗体增强原代NK细胞对表达HLA-E的靶细胞的杀伤作用
同实施例6，将6×104个NKG2A+NK细胞分别与靶细胞(K562，K562-HLA-E和FaDu细胞)共孵育，效靶比为3:1，同时加入10μg/ml NKG2A抗体(IgG4形式,空白组不添加抗体)，共孵育4h后收集培养基上清，通过LDH检测法计算杀伤作用。
按照下面公式计算杀伤率：
细胞毒性％＝[实验组LDH释放量(Avg.)–效应细胞自发LDH释放量(Avg.)–靶细胞自发LDH释放量(Avg.)]/[靶细胞最大LDH释放量(Avg.)-靶细胞自发LDH释放量(Avg.)–体积校准(Avg.)]×100％
结果见图19，NKG2A+NK细胞对K562细胞有约55％杀伤率，对高表达HLA-E的K562-HLA-E细胞和FaDu细胞的杀伤率均低于20％。说明了靶细胞上表达的HLA-E与NK细胞上表达的NKG2A结合，抑制NK细胞活性，从而降低NK细胞对靶细胞的杀伤。添加NKG2A抗体后可以阻断靶细胞的HLA-E与NK细胞的NKG2A结合，降低靶细胞对NK细胞活性抑制，从而提高NK细胞活性，提高NK细胞杀伤高表达HLA-E的肿瘤细胞。
实施例8.抗NKG2A特异性CAR-T细胞对NK细胞的杀伤作用
1、抗NKG2A抗体A4、A5嵌合抗原受体质粒的构建
以PRRLSIN-cPPT.EF-1α(购自Addgene公司)为载体，构建了表达抗体A4、A5的二代嵌合抗原受体的慢病毒质粒，即PRRLSIN-A4-BBZ和PRRLSIN-A5-BBZ。
A4-BBZ(SEQ ID NO：115)序列由CD8α信号肽、A4scFv、CD8铰链区、CD8跨膜区、CD137胞内信号传导结构域以及CD3δ顺序连接组成。A5-BBZ(SEQ ID NO：116)序列由CD8α信号肽、A5scFv、CD8铰链区、CD8跨膜区、CD137胞内信号传导结构域以及CD3δ顺序连接组成。其中，CD8α信号肽(SEQ ID NO：93)、A4scFv(SEQ ID NO：64)、A5scFv(SEQ ID NO：66)、CD8铰链区(SEQ ID NO：95)、CD8跨膜区(SEQ ID NO：97)、CD137胞内信号传导结构域(SEQ ID NO：101)、CD3δ(SEQ ID NO：105)。
2、NKG2A-CAR T细胞的制备
用磷酸钙法进行慢病毒的包装，病毒上清用PEG8000/NaCl进行纯化，纯化后病毒按MOI值为10感染CD3/CD28磁珠活化48小时后的T细胞，分别得到表达A4-BBZ、A5-BBZ的CAR-T细胞，未转染病毒的T细胞视为UTD。感染后第6天用FACS法检测CAR阳性率，检测抗原为Bio-NKG2A-CD94，二抗为BV421标记SA抗体(BD HorizonTM)，1:200稀释使用，结果显示A4-BBZ CAR T的CAR阳性率为62.8％、A5-BBZ CAR T的CAR阳性率为59％。
3、NKG2A-CAR T细胞靶向NKG2A阳性NK细胞的体外杀伤毒性检测
NK细胞通过NK Cell Isolation Kit(Miltenyi Biotec)从两位供者(#1和#2)外周血PBMCs中纯化得到，并用含500IU/ml IL-2和150IU/ml IL-15的NKMedium(Miltenyi Biotec)培养至第14天收集。取上述NK细胞分别与APC标记的NKG2A抗体(Invitrogen)(1:200稀释使用)4℃共孵育5min后，用FACS法检测NK细胞中NKG2A的表达水平。结果显示，#1供者NK细胞中NKG2A的阳性率为80.4％，#2供者NK细胞中NKG2A的阳性率为61.5％。
靶细胞：取5×104个上述NKG2A阳性的NK细胞作为靶细胞接种到96孔板。
效应细胞：按效靶比1：1及2：1分别接种UTD细胞、A4-BBZ CAR-T细胞、A5-BBZ CAR-T细胞细胞到相应的96孔板。
采用流式染色法进行体外细胞杀伤实验，分别在0hr、4hr、24hr进行流式染色，检测共培养体系中NK细胞比例。结果见图20，随着共培养时间延长，UTD细胞组中NK细胞比例无明显变化，而A4-BBZ CAR-T、A5-BBZ CAR-T组中NK细胞比例显著下降。这表明A4-BBZ CAR-T细胞和A5-BBZ CAR-T细胞均能有效杀伤表达NKG2A的NK细胞。
实施例9抗NKG2A的UCAR-T细胞能够有效抵抗NK细胞的杀伤
B2M缺失的T细胞会引起NK细胞的排斥作用，通过构建TCR/B2M缺失的NKG2A-UCAR-T细胞来验证UCAR-T细胞对NK细胞的抵抗。为避免NKG2A表达造成的自我攻击作用，同时制备了NKG2A敲除的NKG2A-UCAR-T细胞(UCAR-TKO)。
体外合成靶向TCR/B2M/NKG2A基因的gRNA，序列分别如SEQ ID NO：117、118、119所示。采用常规CRISPR/Cas9技术敲除T细胞内源性TCR/B2M、或TCR/B2M/NKG2A。CRISPR/Cas 9酶(恺佧生物)和gRNA按摩尔比1：4比例进行混合，形成RNP复合物(Cas 9酶的终浓度为1uM)，室温孵育10分钟后，利用MaxCyte电转仪将RNP复合物导入到T细胞中。将实施例8中的A4-CAR-T、A5-CAR-T细胞分别进行TCR/B2M双敲除，得到A4-UCAR-T、A5-UCAR-T细胞；将A4-CAR-T、A5-CAR-T细胞分别进行TCR/B2M/NKG2A三敲除，得到A4-UCAR-T-TKO、A5-UCAR-T-TKO细胞。以同样方法敲除TCR/B2M、TCR/B2M/NKG2A基因但未转染CAR的UTD细胞(命名为UTD UCAR-T、UTD UCAR-T-TKO)进行对照，调整细胞浓度至5×105/mL，接种至96孔板，按原代扩增的#2供者NK细胞与T细胞比例1：1或1：2接种细胞，于培养箱中分别共孵育0hr、24hr、48hr。利用流式细胞术标记HLA-ABC阳性的NK细胞，检测共孵育不同时间点UCAR-T细胞比例。结果见图21，UTD UCAR-T、UTD UCAR-T-TKO细胞的比例随着时间延长而逐渐下降，表明NK细胞抑制UTD UCAR-T、UTD UCAR-T-TKO细胞生长；而表达NKG2A-CAR的UCAR-T或UCAR-T-TKO细胞在共孵育后48h比例均显著上升。这表明，抗NKG2A的UCAR-T或UCAR-T-TKO细胞能够有效抵抗NK细胞的杀伤。
实施例10抗NKG2A的UCAR-T细胞协同靶向肿瘤抗原的CAR-T细胞的抗肿瘤作用
采用本领域常规分子生物学方法，构建靶向BCMA的嵌合抗原受体，包装慢病毒并转染T细胞，制备成靶向BCMA的CAR-T细胞。示例性，BCMA-scFv的氨基酸序列如SEQ ID NO:120所示，BCMA-CAR的氨基酸序列如SEQ ID NO:121所示。采用实施例9的方法对BCMA CAR-T细胞的B2M/TCR/NKG2A基因进行敲除得到BCMA UCAR-T细胞(命名为BCMA UCAR-T-TKO)。
体外培养表达BCMA的多发性骨髓瘤细胞系RPMI-8226(中国科学院细胞库)，皮下接种5×106细胞/只于NPG免疫缺陷的小鼠中(记为D0)，接种10天后瘤体积平均为200mm3左右，将小鼠分4组。D10，D14,D17,D21,D24第2、3、4组尾静脉分别注射1×106NK细胞，一共注射5次。D11，按分组尾静脉分别注射T细胞。各组具体情况如下：
第1组：0.6×106UTD UCAR-T
第2组：0.6×106UTD UCAR-T+NK
第3组：0.6×106BCMA UCAR-T-TKO+1×106A4UCAR-T+NK
第4组：0.6×106BCMA UCAR-T-TKO+1×106A5 UCAR-T+NK
T细胞注射后，每周2次测量体重(包括分组给药及安乐死当天)，并用游标卡尺测量并记录肿瘤长径、短径，计算肿瘤体积，根据肿瘤体积绘制肿瘤生长曲线，并比较各组间肿瘤生长曲线的差异(肿瘤体积：V＝1/2×长径×短径2)。结果见图22，在NK细胞存在下，抗NKG2A的UCAR-T细胞发挥协同BCMA UCAR-T细胞抗肿瘤作用：在D32,第3、4组小鼠体内肿瘤几乎被完全清除。
实施例11靶向NKG2A及肿瘤抗原的双靶点UCAR-T细胞的抗肿瘤能力及抵抗NK活性
以靶向肿瘤抗原BCMA为例，构建了同时靶向NKG2A和BCMA的CAR-T细胞，观察其抗肿瘤活性以及抵抗NK细胞杀伤的效果。使用载体PRRLsin，构建表达串联CAR(SEQ ID NO：122)的BCMA-NKG2A CAR-T细胞。采用实施例9的方法，对BCMA-NKG2A CAR-T细胞的B2M/TCR/NKG2A基因进行敲除得到BCMA-NKG2A UCAR-T-TKO细胞。体内外实验结果显示，靶向NKG2A及肿瘤抗原的双靶点的串联UCAR-T细胞能够有效抵抗NK细胞的杀伤，抑制肿瘤生长。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
本申请涉及如下序列：
上述序列表中当N在碱基序列中表示任意碱基。

Claims (30)

  1. 识别NKG2A的全人源抗体，其特征在于，所述抗体包含轻链可变区，所述轻链可变区包含RASQSISSWLA(SEQ ID NO:4)所示的LCDR1；和/或
    DASSLES(SEQ ID NO:5)所示的LCDR2；和/或
    QQYDSYX1X2T(SEQ ID NO:129)所示的LCDR3，其中X1是I或V，X2是R或S。
  2. 识别NKG2A的全人源抗体，其特征在于，所述抗体包括重链可变区，所述重链可变区选自：
    (1)包含SYAIS(SEQ ID NO:1)所示的HCDR1；和/或
    GIIPIFGTAX1YAQKFQG(SEQ ID NO:130)所示的HCDR2，其中X1是N或H；和/或
    GFDGMDY(SEQ ID NO:3)所示的HCDR3；或
    (2)包含X1X2X3X4S(SEQ ID NO:131)所示的HCDR1，其中X1是S、R或N，X2是Y、F或V，X3是A、Y或H，X4是M或V；和/或
    AIX1X2X3X4GSTYYADSVKG(SEQ ID NO:132)所示的HCDR2，其中X1是S、T或N，X2是G或A，X3是S、W、G或P，X4是G或V；和/或
    GYDGFDY(SEQ ID NO:9)所示的HCDR3。
  3. 如权利要求1或2所述的抗体，其特征在于，所述抗体选自：
    (1)抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO:1、7、12、14或16所示的HCDR1，和/或包含SEQ ID NO:2、8、11、13、15或17所示的HCDR2，和/或包含SEQ ID NO:3或9任一所示的HCDR3；
    (2)抗体，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:4所示的LCDR1，和/或包含SEQ ID NO:5所示的LCDR2，和/或包含SEQ ID NO:6或10任一所示的LCDR3；
    (3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；
    (4)抗体，(1)～(3)中任一项所述抗体的变体，且具备与(1)～(3)中任一项所述抗体相同或相似的活性。
  4. 如权利要求3所述的抗体，其特征在于，所述抗体选自：
    (1)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或
    (2)抗体，其包含SEQ ID NO:7所示的HCDR1，SEQ ID NO:8所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或
    (3)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:11所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或
    (4)抗体，其包含SEQ ID NO:12所示的HCDR1，SEQ ID NO:13所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或
    (5)抗体，其包含SEQ ID NO:14所示的HCDR1，SEQ ID NO:15所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或
    (6)抗体，其包含SEQ ID NO:16所示的HCDR1，SEQ ID NO:17所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；
    (7)抗体，(1)～(6)中任一项所述抗体的变体，且具备与(1)～(6)中任一项所述抗体相同或相似的活性。
  5. 如权利要求1-4中任一项所述的抗体，其特征在于，所述抗体选自：
    (1)抗体，包含重链可变区，所述重链可变区包含SEQ ID NO:18、22、26、28、30或32所示的氨基酸序列、或上述序列的变体或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；
    (2)抗体，包含轻链可变区，该轻链可变区包含SEQ ID NO:20或24所示的氨基酸序列、或上述序列的变体或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；
    (3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区。
  6. 如权利要求5所述的抗体，其特征在于，所述抗体选自：
    (1)抗体，所述抗体的重链可变区具有SEQ ID NO:18所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:20所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；
    (2)抗体，所述抗体的重链可变区具有SEQ ID NO:22所示的氨基酸序列或与上述序列具有95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；
    (3)抗体，所述抗体的重链可变区具有SEQ ID NO:26所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:20所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；
    (4)抗体，所述抗体的重链可变区具有SEQ ID NO:28所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；
    (5)抗体，所述抗体的重链可变区具有SEQ ID NO:30所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；
    (6)抗体，所述抗体的重链可变区具有SEQ ID NO:32所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列。
    (7)抗体，(1)～(6)中任一项所述抗体的变体，且具备与(1)～(6)中任一项所述抗体相同或相似的活性。
  7. 如权利要求1-6任一项所述的抗体，其特征在于，所述抗体是全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体或IgG4抗体。
  8. 如权利要求1-7任一所述的抗体，其特征在于，所述抗体不显著结合NKG2C、NKG2E或其组合。
  9. 如权利要求1-8任一所述的抗体，其特征在于，所述抗体结合NKG2A/CD94，不显著结合NKG2C/CD94、NKG2E/CD94或其组合；和/或，
    所述抗体结合表达NKG2A/CD94的细胞，不显著结合表达NKG2C/CD94、NKG2E/CD94或其组合的细胞。
  10. 如权利要求1-9任一所述的抗体，其特征在于，所述抗体在降低CD94/NKG2A介导的抑制表达CD94/NKG2A的细胞毒性淋巴细胞的细胞毒性中更有效。
  11. 如权利要求10所述的抗体，其特征在于，所述表达CD94/NKG2A的细胞毒性淋巴细胞是NK细胞、NKT细胞、α/βT细胞或γ/δT细胞。
  12. 如权利要求10所述的抗体，其特征在于，所述表达CD94/NKG2A的细胞毒性淋巴细胞是NK细胞。
  13. 一种免疫辍合物，其特征在于，所述免疫辍合物包括：权利要求1-12任一所述的抗体，以及与之连接的功能性分子。
  14. 嵌合受体，其特征在于，所述嵌合受体的胞外域包含权利要求1-12任一所述的抗体，所述嵌合受体选自：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)或其组合。
  15. 如权利要求14所述嵌合受体，其特征在于，所述嵌合受体包含顺序连接的如下结构区域：权利要求1-12任一所述的抗体、跨膜区和胞内信号区。
  16. 如权利要求15所述的嵌合受体，所述的胞内信号区选自：CD3δ、FcεRIγ、CD27、CD28、CD137、CD134、MyD88、CD40的胞内信号区序列或其组合；和/或
    所述的跨膜区包含CD8或CD28的跨膜区。
  17. 如权利要求16所述的嵌合受体，其特征在于，所述嵌合受体选自如下中的任意：
    权利要求1-12任一所述的抗体、CD8/CD28的跨膜区和CD3δ；或
    权利要求1-12任一所述的抗体、CD8/CD28的跨膜区、CD137的胞内信号区和CD3δ；或
    权利要求1-12任一所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区和CD3δ；或
    权利要求1-12任一所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区、CD137和CD3δ。
  18. 如权利要求14所述的嵌合受体，其特征在于，所述嵌合受体的氨基酸序列如SEQ ID NO：115或116所示。
  19. 一种生物材料，其为如下中的任意一种：
    1)编码权利要求1-12任一所述的抗体、权利要求13所述的免疫缀合物、权利要求14-18任一所述的嵌合受体的核酸；
    2)包含1)所述的表达载体；或者
    3)包含1)或2)所述的病毒。
  20. 一种宿主细胞，其包含权利要求14-18任一所述的嵌合受体。
  21. 如权利要求20所述的宿主细胞，其特征在于，所述宿主细胞结合表达NKG2A/CD94的细胞，不显著结合NKG2C/CD94、NKG2E/CD94或其组合。
  22. 如权利要求20或21所述的宿主细胞，其特征在于，所述宿主细胞能抵抗NK细胞攻击或杀伤NK细胞。
  23. 如权利要求20-22任一所述的宿主细胞，其特征在于，所述宿主细胞还表达识别肿瘤抗原和/或病原体抗原的嵌合受体。
  24. 如权利要求20-22任一所述的宿主细胞，其特征在于，所述宿主细胞与靶向肿瘤和/或病原体的第二宿主细胞联合应用。
  25. 如权利要求20-24任一所述的宿主细胞，其特征在于，所述宿主细胞和/或第二宿主细胞不表达B2M、TCR/B2M、TCR/B2M/CIITA、TCR/B2M/NKG2A、和/或TCR/B2M/CIITA/NKG2A。
  26. 如权利要求20-25任一所述的宿主细胞，其特征在于，所述宿主细胞和/或第二宿主细胞是T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、调节性T细胞、NK92细胞、干细胞衍生的免疫效应细胞或其组合。
  27. 如权利要求26所述的宿主细胞，其特征在于，所述T细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞；
    优选地，所述T细胞为自体/同种异体T细胞；
    优选地，所述T细胞为原代T细胞；
    优选地，所述T细胞来源于人的自体T细胞。
  28. 联合用药，其特征在于，权利要求1-12任一所述的抗体、权利要求13所述的免疫缀合物、权利要求14-18任一所述的嵌合受体、权利要求20-27任一所述的宿主细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；
    和/或与改善其相关的一种或多种副作用的药剂联合施用；
    和/或与表达靶向NKG2A之外的嵌合受体的宿主细胞联合施用。
  29. 一种药物组合物，其特征在于，其包括：
    权利要求1-12任一所述的抗体或编码该抗体的核酸；或
    权利要求13所述的免疫缀合物或编码该免疫缀合物的核酸；或
    权利要求14-18任一所述的嵌合受体或编码该嵌合受体的核酸；或
    权利要求20-27任一所述的宿主细胞；
    以及药学上可接受的载体或赋形剂。
  30. 一种试剂盒，其特征在于，其包括：
    容器，以及位于容器中的权利要求30所述的药物组合物；或
    容器，以及位于容器中的权利要求1-12任一所述的抗体或编码该抗体的核酸；或权利要求13所述的免疫缀合物或编码该免疫缀合物的核酸；或权利要求14-18任一所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或权利要求20-27任一所述的宿主细胞。
PCT/CN2023/073176 2022-01-24 2023-01-19 Nkg2a抗体及其应用 WO2023138661A1 (zh)

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