WO2024067821A1 - 一种修饰细胞及其用途 - Google Patents

一种修饰细胞及其用途 Download PDF

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WO2024067821A1
WO2024067821A1 PCT/CN2023/122707 CN2023122707W WO2024067821A1 WO 2024067821 A1 WO2024067821 A1 WO 2024067821A1 CN 2023122707 W CN2023122707 W CN 2023122707W WO 2024067821 A1 WO2024067821 A1 WO 2024067821A1
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cells
antibody
cell
dct
well
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PCT/CN2023/122707
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English (en)
French (fr)
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梁兆端
田野
李振娟
柳其
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瑅安生物医药（杭州）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present disclosure relates to the field of immunology, and in particular to a modified cell and use thereof in treating diseases.
  • T cells redirected by T cell receptors T cell receptor, TCR
  • chimeric antigen receptors Chimeric Antigen Receptor, CAR
  • T cell receptor T cell receptor
  • CAR Chimeric Antigen Receptor
  • the present disclosure provides a modified cell, wherein the modified cell is formed by a complex protein bound to a cell surface marker molecule of the modified cell, wherein the complex protein comprises a portion that specifically binds to the cell surface marker molecule of the modified cell and a portion that specifically binds to a target cell surface antigen.
  • the portion that specifically binds to a target cell surface antigen is a TCR molecule, a single-chain ⁇ TCR or a TCR ⁇ chain/TCR ⁇ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.
  • the portion that specifically binds to a cell surface marker molecule of the modified cell is an antibody or a functional fragment thereof that specifically binds to a cell surface marker molecule of the modified cell.
  • the cell surface marker molecule of the modified cell is selected from CD8, CD4, CD3, NKG2D, CD16, CD2, CD56, CD28 or CD26.
  • the modified cell is an immune cell, preferably a T cell, a ⁇ T cell, a CD4 and CD8 double negative T cell, a CD4 + T cell, a CD8 + T cell, a NK cell, a NKT cell or a monocyte.
  • an immune cell preferably a T cell, a ⁇ T cell, a CD4 and CD8 double negative T cell, a CD4 + T cell, a CD8 + T cell, a NK cell, a NKT cell or a monocyte.
  • the target cell surface antigen is selected from pMHC.
  • the structure of the complex protein from N-terminus to C-terminus is as shown in Formula Ia: ALB (Ia) BLA (Ib)
  • Element A comprises an antibody or a functional fragment thereof that specifically binds to a cell surface marker molecule of the modified cell
  • Element B comprises a portion that specifically binds to a target cell surface antigen
  • Element L is a flexible joint; the flexible joint is optional;
  • the element A in the aforementioned modified cells, is an immune effector molecule; in one embodiment, in the aforementioned modified cells, the element A may also be a non-immune effector molecule.
  • the antibody contained in the element A is a single-chain antibody scFv.
  • the antibody comprised by the element A is an anti-CD3 antibody.
  • the heavy chain variable region in the antibody comprises the sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the sequence shown in SEQ ID NO:3.
  • the single-chain antibody comprises OKT3, UCHT-1, TR66, BMA031 or 12F6.
  • the target cell surface antigen is selected from pMHC.
  • the portion that specifically binds to the target cell surface antigen is an antibody, a TCR-like antibody or a TCR mimicking antibody, a TCR molecule, a single-chain ⁇ TCR or a TCR ⁇ chain/TCR ⁇ chain heterodimer, a TCR-like antibody or a TCR mimicking antibody.
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:5, 25 or 40, and the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:7, 27, 42 or 44.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the sequence shown in SEQ ID NO:3
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:5
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:7.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the sequence shown in SEQ ID NO:3
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:25
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:27.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the sequence shown in SEQ ID NO:3
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:25
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:44.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the sequence shown in SEQ ID NO:3
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:40
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:42.
  • the structure of the complex protein from N-terminus to C-terminus is as shown in Formula Ic or Id: A2...A1-LB (Ic); BL-A1...A2 (Id);
  • Elements A1 and A2 each independently comprise a non-immune effector molecule; preferably, the non-immune effector molecule comprises an antibody heavy chain variable region or an antibody light chain variable region;
  • Element B is a portion that specifically binds to a target cell surface antigen
  • Element L is a flexible joint; the flexible joint is optional;
  • A1 comprises the heavy chain variable region of an antibody
  • A2 comprises the light chain variable region of an antibody
  • A1 comprises the light chain variable region of an antibody
  • A1 and A2 form a dimer through a disulfide bond, preferably, the dimer is a non-immune effector molecule.
  • the heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO:9.
  • the light chain variable region of the antibody comprises the sequence shown in SEQ ID NO:11.
  • the portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain ⁇ TCR or a TCR ⁇ chain/TCR ⁇ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:13, 17 or 21, and the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:15, 19 or 23.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:9
  • the light chain variable region comprises the sequence shown in SEQ ID NO:11
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:13
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:15.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:9
  • the light chain variable region comprises the sequence shown in SEQ ID NO:11
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:17
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:19.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:9
  • the light chain variable region comprises the sequence shown in SEQ ID NO:11
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:21
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:23.
  • the present disclosure provides a combination or kit, comprising a. a complex protein; b. a cell; wherein the cell expresses a cell surface marker molecule, and wherein the complex protein comprises a portion that binds to the cell surface marker molecule of the cell and a portion that specifically binds to a target cell surface antigen.
  • the specific binding target cell surface antibody is a TCR molecule, a single-chain ⁇ TCR or a TCR ⁇ chain/TCR ⁇ chain heterodimer, a TCR-like antibody or a TCR-mimicking antibody.
  • the portion that specifically binds to the cell surface marker molecule of the modified cell is an antibody or a functional fragment thereof that specifically binds to the cell surface marker molecule of the modified cell.
  • the cell surface marker molecule of the cell is selected from CD8, CD4, CD3, NKG2D, CD16, CD2, CD56, CD28 or CD26.
  • the cell is an immune cell, preferably a T cell, a ⁇ T cell, a CD4 and CD8 double negative T cell, a CD4 + T cell, a CD8 + T cell, a NK cell, a NKT cell or a monocyte.
  • an immune cell preferably a T cell, a ⁇ T cell, a CD4 and CD8 double negative T cell, a CD4 + T cell, a CD8 + T cell, a NK cell, a NKT cell or a monocyte.
  • the target cell surface antigen is selected from pMHC.
  • the structure of the complex protein from N-terminus to C-terminus is as shown in Formula Ia: ALB (Ia) BLA (Ib)
  • Element A comprises an antibody or a functional fragment thereof that specifically binds to a cell surface marker molecule of the modified cell
  • Element B comprises a portion that specifically binds to a target cell surface antigen
  • Element L is a flexible joint; the flexible joint is optional;
  • the element A in the aforementioned combination or kit, is an immune effector molecule; in one embodiment, in the aforementioned combination or kit, the element A is a non-immune effector molecule.
  • the antibody contained in the element A is a single-chain antibody scFv.
  • the antibody comprised by the element A is an anti-CD3 antibody.
  • the heavy chain variable region of the antibody comprises The light chain variable region comprises the sequence shown in SEQ ID NO:1, and the light chain variable region comprises the sequence shown in SEQ ID NO:3.
  • the single-chain antibody comprises OKT3, UCHT-1, TR66, BMA031 or 12F6.
  • the target cell surface antigen is selected from pMHC.
  • the portion that specifically binds to a target cell surface antigen is a TCR molecule, a single-chain ⁇ TCR or a TCR ⁇ chain/TCR ⁇ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:5, 25 or 40, and the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:7, 27, 42 or 44.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the sequence shown in SEQ ID NO:3
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:5
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:7.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the sequence shown in SEQ ID NO:3
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:25
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:27.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the sequence shown in SEQ ID NO:3
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:25
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:44.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the sequence shown in SEQ ID NO:3
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:40
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:42.
  • the structure of the complex protein from N-terminus to C-terminus is as shown in Formula Ic or Id: A2...A1-LB (Ic); BL-A1...A2 (Id);
  • Elements A1 and A2 each independently comprise a non-immune effector molecule; preferably, the non-immune effector molecule comprises an antibody heavy chain variable region or an antibody light chain variable region;
  • Element B is a portion that specifically binds to a target cell surface antigen
  • Element L is a flexible joint; the flexible joint is optional;
  • A1 comprises the heavy chain variable region of an antibody
  • A2 comprises the light chain variable region of an antibody
  • A1 comprises the light chain variable region of an antibody
  • A1 and A2 form a dimer through a disulfide bond, preferably, the dimer is a non-immune effector molecule.
  • the heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO:9.
  • the light chain variable region of the antibody comprises the sequence shown in SEQ ID NO:11.
  • the portion that specifically binds to a target cell surface antigen is a TCR molecule, a single-chain ⁇ TCR or a TCR ⁇ chain/TCR ⁇ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:13, 17 or 21, and the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:15, 19 or 23.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:9
  • the light chain variable region comprises the sequence shown in SEQ ID NO:11
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:13
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:15.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:9
  • the light chain variable region comprises the sequence shown in SEQ ID NO:11
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:17
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:19.
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO:9
  • the light chain variable region comprises the sequence shown in SEQ ID NO:11
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:21
  • the TCR ⁇ chain amino acid sequence comprises the sequence shown in SEQ ID NO:23.
  • the combination or kit further comprises an optional pharmaceutically acceptable carrier, preferably a buffer, more preferably, the buffer comprises albumin or plasma.
  • the present disclosure provides a method for treating a disease, comprising administering the modified cell as described above or the combination or kit as described above to a subject in need thereof.
  • the present disclosure provides use of a modified cell as described above or a combination or kit as described above in a method for treating a disease in a subject in need thereof.
  • the present disclosure provides use of the modified cell as described above or the combination or kit as described above in the preparation of a medicament for treating a disease in a subject in need thereof.
  • the disease comprises cancer, a microbial infectious disease, an autoimmune disease, or a disease of aging.
  • a method for preparing the modified cell as described above wherein the complex protein is incubated with immune cells to form the modified cell.
  • the incubation time is about 1 hour or more.
  • the incubation temperature is about 4°C, room temperature, or 37°C.
  • the buffer used to incubate the complex protein with immune cells contains albumin or plasma.
  • Figure 1 shows the structure and purity of the complex protein detected by SDS-PAGE.
  • e Anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) complex protein;
  • Lane 3 Multi-domain bifunctional fusion molecule under reducing state.
  • FIG 2 shows the levels of binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules to T cells at different concentrations.
  • Figure 3 shows the level of binding of the same concentration of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules to different numbers of T cells.
  • (a) The positive rate of the same concentration of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules binding to different numbers of T cells.
  • FIG 4 shows the levels of binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules to T cells at different incubation temperatures.
  • FIG 5 shows the levels of binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules to T cells at different incubation times.
  • Figure 6 shows the level of binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules to T cells under different incubation buffers.
  • the control group is an RPMI-1640 culture medium group without anti-CD3 antibody (UCHT1)-1G4 (HATima).
  • Figure 7 shows the effect of the combination of anti-CD3 antibody (UCHT1)-1G4 (HATima) on T cell activation within a certain concentration range.
  • Flow cytometry was used to detect the CD137 positive rate of the CD3 positive cell population in the co-culture system.
  • the target cells were NCI-H1299-A2 cells.
  • Figure 8 shows the effect of the number of molecules bound to the complex protein on the tumor recognition function of T cells.
  • DCT prepared with the same concentration of anti-CD3 antibody (UCHT1)-1G4 (HATima) and different numbers of T cells, flow cytometry detection of the positive rate of T cells bound to the complex protein.
  • HATima anti-CD3 antibody
  • Lactate dehydrogenase release experiment to detect the difference in tumor cell recognition function of DCT prepared by different methods.
  • Figure 9 shows the difference in tumor recognition function between DCT and TCR-T cells.
  • Flow cytometry was used to detect the positive rate of T cells and TCR-T cells that bind to the complex protein.
  • Lactate dehydrogenase release assay was used to detect the difference in tumor cell recognition function between DCT and TCR-T cells.
  • FIG 10 shows that anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects T cells to kill tumor cells.
  • Flow cytometry was used to detect the proportion and phenotype of T cells.
  • Flow cytometry was used to detect the positive rate of CD3 + T-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of CD3 + T-DCT cells on target cells.
  • Flow cytometry was used to detect the expression level of CD137 in CD3 + T-DCT cells under different conditions.
  • FIG 11 shows that anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects peripheral blood mononuclear cells (PBMC) to kill tumor cells.
  • PBMC peripheral blood mononuclear cells
  • Flow cytometry was used to detect the proportion of T cells in PBMC.
  • Flow cytometry was used to detect the positive rate of PBMC-DCT cells prepared with anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of PBMC-DCT cells on target cells.
  • Flow cytometry was used to detect the CD137 expression level of PBMC-DCT cells in different situations.
  • Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein of PBMC-DCT cells in different situations.
  • Figure 12 shows that anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects peripheral blood lymphocytes (PBL) to kill tumor cells.
  • Flow cytometry was used to detect the proportion of T cells in PBL.
  • Flow cytometry was used to detect the positive rate of PBL-DCT cells prepared with anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of PBL-DCT cells on target cells.
  • Flow cytometry was used to detect the CD137 expression level of PBL-DCT cells in different situations.
  • Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein of PBL-DCT cells in different situations.
  • Figure 13 shows that anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects ⁇ T cells to kill tumor cells.
  • Flow cytometry was used to detect the proportion of ⁇ T cells.
  • Flow cytometry was used to detect the positive rate of ⁇ T-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of tumor cells by ⁇ T-DCT cells and ⁇ T cells.
  • Flow cytometry was used to detect the CD137 expression level of ⁇ T-DCT cells and ⁇ T cells in different situations.
  • Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein in ⁇ T-DCT cells and ⁇ T cells in different situations.
  • Figure 14 shows that anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects T cells to kill tumor cells.
  • Flow cytometry was used to detect the positive rate of CD3 + T-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of CD3 + T-DCT cells on target cells.
  • Flow cytometry was used to detect the CD137 expression level of CD3 + T-DCT cells in different situations.
  • Flow cytometry was used to detect the anti-CD3 + T-DCT cells in different situations.
  • In vitro HepG2 cell tumor model was used to detect the killing effect of CD3 + T-DCT cells on tumors.
  • Figure 15 shows that anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects peripheral blood mononuclear cells (PBMC) to kill tumor cells.
  • PBMC peripheral blood mononuclear cells
  • Flow cytometry was used to detect the positive rate of PBMC-DCT cells prepared with anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of PBMC-DCT cells on target cells.
  • Flow cytometry was used to detect the CD137 expression level of PBMC-DCT cells in different situations.
  • Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein of PBMC-DCT cells in different situations.
  • Figure 16 shows that anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects peripheral blood lymphocytes (PBL) to kill tumor cells.
  • Flow cytometry was used to detect the positive rate of PBL-DCT cells prepared with anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of PBL-DCT cells on target cells.
  • Flow cytometry was used to detect the CD137 expression level of PBL-DCT cells in different situations.
  • Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein of PBL-DCT cells in different situations.
  • Figure 17 shows that anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects ⁇ T cells to kill tumor cells.
  • Flow cytometry was used to detect the positive rate of ⁇ T-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of tumor cells by ⁇ T-DCT cells and ⁇ T cells.
  • Flow cytometry was used to detect the CD137 expression level of ⁇ T-DCT cells and ⁇ T cells in different situations.
  • Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein by ⁇ T-DCT cells and ⁇ T cells in different situations.
  • Figure 18 shows that anti-CD3 antibody (UCHT1)-AFP (HATima) redirects T cells to kill tumor cells.
  • Flow cytometry was used to detect the positive rate of CD3 + T-DCT cells prepared with anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of target cells by CD3 + T-DCT cells.
  • Flow cytometry was used to detect the CD137 expression level of CD3 + T-DCT cells in different situations.
  • Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein of CD3 + T-DCT cells in different situations.
  • Figure 19 shows that anti-CD3 antibody (UCHT1)-AFP (HATima) redirects peripheral blood mononuclear cells (PBMC) to kill tumor cells.
  • PBMC peripheral blood mononuclear cells
  • (a) Flow cytometry detection of the positive rate of PBMC-DCT cells prepared by anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein.
  • (b) Lactate dehydrogenase release experiment to detect the killing level of PBMC-DCT cells on target cells.
  • c Flow cytometry detection The expression level of CD137 in PBMC-DCT cells under different conditions.
  • Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein in PBMC-DCT cells under different conditions.
  • Figure 20 shows that anti-CD3 antibody (UCHT1)-AFP (HATima) redirects peripheral blood lymphocytes (PBL) to kill tumor cells.
  • Flow cytometry was used to detect the positive rate of PBL-DCT cells prepared with anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of PBL-DCT cells on target cells.
  • Flow cytometry was used to detect the CD137 expression level of PBL-DCT cells in different situations.
  • Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein of PBL-DCT cells in different situations.
  • Figure 21 shows that anti-CD3 antibody (UCHT1)-AFP (HATima) redirects ⁇ T cells to kill tumor cells.
  • Flow cytometry was used to detect the positive rate of ⁇ T-DCT cells prepared with anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein.
  • Lactate dehydrogenase release experiment was used to detect the killing level of target cells by ⁇ T-DCT and ⁇ T cells.
  • Flow cytometry was used to detect the CD137 expression levels of ⁇ T-DCT and ⁇ T cells in different situations.
  • Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein in ⁇ T-DCT cells in different situations.
  • FIG. 22 shows that anti-CD3 antibody (UCHT1)-gp100 (HATima) redirects immune cells to kill tumor cells.
  • Flow cytometry was used to detect the positive rates of T-DCT, PBMC-DCT, PBL-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells prepared with anti-CD3 antibody (UCHT1)-gp100 (HATima) complex proteins.
  • Lactate dehydrogenase release experiment was used to detect the killing level of target cells by T-DCT, PBMC-DCT, PBL-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells.
  • Flow cytometry was used to detect the expression levels of CD137 in T-DCT, PBMC-DCT, PBL-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells under different situations.
  • Flow cytometry was used to detect the binding levels of anti-CD3 antibody (UCHT1)-gp100 (HATima) complex protein in T-DCT, PBMC-DCT, PBL-DCT and CD4 + T-DCT cells under different conditions.
  • UCHT1-gp100 HATima
  • Figure 23 shows that anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) redirects immune cells to kill tumor cells.
  • Flow cytometry was used to detect the positive rates of T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT, CD8 + T-DCT, PBMC-DCT and PBL-DCT cells prepared with anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) complex proteins.
  • Lactate dehydrogenase release assay was used to detect the effects of T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT, CD8 + T-DCT, PBMC-DCT and PBL-DCT cells on target cells.
  • Figure 24 shows that anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) redirects immune cells to kill tumor cells.
  • Flow cytometry was used to detect the positive rates of T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT, CD8 + T-DCT, PBMC-DCT and PBL-DCT cells prepared with anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) complex proteins.
  • Lactate dehydrogenase release experiment was used to detect the killing level of target cells by T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT, CD8 + T-DCT, PBMC-DCT and PBL-DCT cells.
  • Flow cytometry was used to detect the CD137 expression levels of T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT, CD8 + T-DCT, PBMC-DCT and PBL-DCT cells under different situations.
  • Figure 25 shows that anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) redirects immune cells to kill tumor cells.
  • Flow cytometry was used to detect the positive rates of T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells prepared with anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) complex proteins.
  • Lactate dehydrogenase release experiment was used to detect the killing level of target cells by T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells.
  • Flow cytometry was used to detect the expression levels of CD137 in T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells under different circumstances.
  • Flow cytometry was used to detect the binding levels of anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) complex protein in T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells under different conditions.
  • UCHT1-igp100 ImmTAC
  • Figure 26 shows that anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects immune cells to kill tumor cells.
  • (a) Flow cytometry was used to detect the positive rate of DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells prepared with anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex proteins.
  • (b) Lactate dehydrogenase release experiment was used to detect the killing level of DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells on target cells.
  • Flow cytometry was used to detect the CD137 expression levels of DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells in different situations.
  • (d) Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex proteins of CD4 + T-DCT and CD8 + T-DCT cells in different situations.
  • Figure 27 shows that anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects immune cells to kill tumor cells.
  • (a) Flow cytometry detection of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein The positive rates of the prepared DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells.
  • (b) Lactate dehydrogenase release assay to detect the killing level of DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells on target cells.
  • (d) Flow cytometry to detect the binding level of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein in CD4 + T-DCT and CD8 + T-DCT cells under different conditions.
  • Figure 28 shows that anti-CD3 antibody (UCHT1)-AFP (HATima) redirects immune cells to kill tumor cells.
  • (a) Flow cytometry was used to detect the positive rate of DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells prepared with anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein.
  • (b) Lactate dehydrogenase release experiment was used to detect the killing level of DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells on target cells.
  • Flow cytometry was used to detect the CD137 expression level of DNT-DCT, CD4 + T-DCT and CD8 + T-DCT cells in different situations.
  • (d) Flow cytometry was used to detect the binding level of anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein of CD8 + T-DCT cells in different situations.
  • Figure 29 shows the comparison of biological activity functions of anti-CD3 antibody (UCHT1) in the HATima molecule and ImmTAC molecule structure used to modify cells.
  • SDS-PAGE method was used to detect the structure and purity of VH-VL and scFv proteins. Lane 1: molecular weight marker; Lane 2: protein in non-reduced state; Lane 3: protein in reduced state.
  • Lactate dehydrogenase release experiment was used to detect the killing level of target cells by T cells redirected by VH-VL and scFv proteins.
  • Flow cytometry was used to detect the CD137 expression level of T cells redirected by VH-VL and scFv proteins at a concentration of 1E-9M in different situations.
  • complex protein refers to a structure comprising a portion that specifically binds to a cell surface marker molecule of the modified cell and a portion that specifically binds to a target cell surface antigen, which may be optionally connected by a flexible linker peptide, wherein the portion that specifically binds to a cell surface marker molecule of the modified cell may be a portion that specifically binds to a target cell surface antigen.
  • the antibody or fragment thereof of the cell surface marker molecule of the modified cell; the antibody can be a complete antibody, a single-chain antibody scFv, a heavy chain variable region or a light chain variable region, or a functional fragment of these antibodies.
  • the part that specifically binds to the target cell surface antigen can be a TCR molecule, a single-chain ⁇ TCR, or a TCR ⁇ chain/TCR ⁇ chain heterodimer.
  • binding means a binding or connection that can be covalent (e.g., by chemical coupling) or non-covalent (e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc.).
  • the binding or connection is a non-covalent interaction.
  • binding means the connection of an associated antigen binding moiety to its antigen.
  • Immune cell includes cells of hematopoietic origin and that play a role in immune response.
  • Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; bone marrow cells, such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes. It should be understood by those skilled in the art that these immune cells can be of any origin, including but not limited to primary cells, passaged cells, or cells obtained after induction of stem cells.
  • a “disease” is any condition that would benefit from medical treatment using the modified cells or combinations of the present disclosure.
  • disorders include cancer, microbial infectious diseases, autoimmune diseases, or diseases of aging.
  • autoimmune disease generally refers to a disease characterized by a self-recognized component.
  • autoimmune diseases include, but are not limited to, autoimmune hepatitis, multiple sclerosis, systemic lupus erythematosus, idiopathic thrombocytopenic purpura, myasthenia gravis, type I diabetes, rheumatoid arthritis, psoriasis, Hashimoto's thyroiditis, Graves' disease, ankylosing spondylitis, Sjogrens disease, CREST syndrome, scleroderma, IgA nephropathy, bullous pemphigoid, pemphigus vulgaris, ANCA-associated vasculitis, antiphospholipid syndrome and more.
  • autoimmune diseases are also chronic inflammatory diseases. It is defined as a disease process associated with the long-term (>6 months) activation of inflammatory cells (leukocytes). Chronic inflammation leads to damage to the patient's organs or tissues. Many diseases are chronic inflammatory conditions, but are not known to have an autoimmune basis. Examples include atherosclerosis, congestive heart failure, Crohn's disease, ulcerative colitis, polyarteritis nodosa, Whipple's disease, primary sclerosing cholangitis and more.
  • cancer refers to a physiological condition in mammals that is generally characterized by unregulated cell growth/proliferation.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, and leukemia. More specific examples of cancer include, but are not limited to, colorectal cancer, chronic lymphocytic leukemia (CLL), Cancers of particular interest for treatment using the methods of the present disclosure include gliomas, medulloblastomas, colon cancer, colorectal cancer, melanomas, breast cancer, lung cancer, liver cancer, and gastric cancer.
  • the term “subject” includes any human or non-human animal.
  • the term “non-human animal” includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, amphibians, reptiles, etc. Unless otherwise indicated, the terms “patient” or “subject” are used interchangeably. In the present disclosure, the preferred subject is a human.
  • treatment refers to administering to a subject an effective amount of cells having a polynucleotide sequence of a target gene altered in vitro according to the methods described herein, so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, a beneficial or desired clinical outcome.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or total relief), whether detectable or undetectable.
  • Treatment may refer to prolonging survival compared to the expected survival if not receiving treatment. Therefore, those skilled in the art recognize that treatment may improve the disease condition, but may not be a complete cure for the disease.
  • the term “treatment” includes prevention. Alternatively, treatment is “effective” in the case of a reduction or cessation of the progression of the disease. “Treatment” may also mean prolonging survival compared to the expected survival if not receiving treatment.
  • Patients in need of treatment include those who have been diagnosed with a condition associated with the expression of a polynucleotide sequence, and those who may develop such a condition due to genetic susceptibility or other factors.
  • an "immune effector molecule” immune effector polypeptide is a molecule that induces or stimulates an immune response by directly or indirectly activating the humoral or cellular components of the immune system, such as through T cell activation.
  • An immune effector molecule can be a scFv antibody, such as an anti-CD3 scFv.
  • Non-immune effector molecules do not activate the humoral or cellular components of the immune system, such as do not induce or stimulate an immune response.
  • the genes encoding the complex proteins were cloned into the expression plasmid of pET-28a.
  • the plasmids were transformed into E. coli strain BL21-DE3, and the kanamycin-resistant single clones were grown in LB medium (kanamycin 50 ⁇ g/mL) at 37°C until OD600 was about 1.0, and then protein expression was induced with 1 mM IPTG. Three hours after induction, the cells were collected by centrifugation at 4000g for 15 minutes in a Thermo Scientific HERAEUS X1R centrifuge.
  • TCR refolding buffer (5M urea, 100mM Tris pH 8.1, 0.4M L-arginine, 2mM EDTA, 6.5mM cysteamine and 1.87mM cystamine) and precool to 4°C in advance.
  • TCR ⁇ chain and 11.2mg of antibody heavy chain-TCR ⁇ chain, and 6mg of antibody light chain inclusion body from the cryopreservation solution Add them to 6mL 6M guanidine hydrochloride solution, add DTT with a final concentration of 15 ⁇ M, mix well, and place in a 37°C incubator for incubation for 40 minutes.
  • Soluble and correctly folded multi-domain fusion molecules are separated from misfolded, degraded products and impurities by the three-step purification method described below.
  • anion exchange purification is used.
  • the refolded and dialyzed sample is placed in a pre-cooled high-speed centrifuge, 8000 ⁇ g, 15 minutes, 4°C to remove the precipitate by centrifugation, and the supernatant is filtered again with a 0.45 ⁇ m filter membrane.
  • Use an anion exchange column Q HP GE Healthcare
  • PBMC peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • the cloud layer was aspirated and placed in a 50 mL centrifuge tube, and more than 5 times the volume of phosphate buffered saline (PBS) was added.
  • the cells were centrifuged (1800 rpm, 10 minutes, room temperature RT) to wash the cells. The supernatant was discarded, 8 mL of preheated red blood cell lysis solution was added, blown evenly, and incubated in a 37°C incubator for 10 minutes. 30 mL of phosphate buffered saline (PBS) was added to stop the process and centrifuged (1200 rpm, 10 minutes, RT). The supernatant was discarded, and the cells were resuspended in complete medium (RPMI-1640 + 10% FBS), and the cell number and viability were detected by Countstar cell sorter.
  • PBS phosphate buffered saline
  • PBL peripheral blood lymphocytes
  • PBMC Peripheral blood mononuclear cells
  • PBL peripheral blood lymphocytes
  • Isolate T cells from peripheral blood mononuclear cells using Stemcell's EasySep TM Human T Cell Isolation Kit (Cat. No. 17951). Collect PBMCs, count cells using Countstar, centrifuge (300g, 5 minutes, room temperature), and discard supernatant. Resuspend cells with EasySep TM Buffer and adjust the cell density to 5 ⁇ 10 7 cells/mL, with a cell suspension volume of 0.25mL-2mL. Transfer the cell suspension to a polystyrene round-bottom tube (5mL volume). Add Isolation Cocktail at a ratio of 50 ⁇ L/mL. Mix by "flicking" with your fingers and incubate at room temperature for 5 minutes.
  • the cells poured out are T cells. Count, centrifuge (300g, 5 minutes, room temperature RT), and discard the supernatant. Add complete RPMI-1640 medium containing IL-2 (RPMI-1640 + 10% FBS + 100U/mL IL-2, hereinafter referred to as "medium"), resuspend, and adjust the cell density to 1 ⁇ 10 6 cells/mL. Take a certain amount of sample for flow cytometry phenotyping and purity testing. Add at a ratio of 1:1. Human T-Activator CD3/CD28, mixed, inoculated in 48-well plates, 500 ⁇ L per well, placed in a 37°C 5% CO 2 incubator for culture.
  • medium RPMI-1640 + 10% FBS + 100U/mL IL-2
  • the antibodies used for flow cytometry are: FITC anti-human CD3 antibody (biolegend, catalog number: 317306); APC anti-human CD4 antibody (biolegend, catalog number: 300514); PE anti-human CD8 antibody (biolegend, catalog number: 344706).
  • CD4 MicroBeads human (Cat. No. 130-045-101) to positively select CD4 + T cells from T cells.
  • Prepare separation buffer which consists of phosphate buffer containing 0.5% bovine serum albumin and 2mM ethylenediaminetetraacetic acid (EDTA). Collect T cells, count cells using Countstar, centrifuge (300g, 5 minutes, room temperature), and discard the supernatant. Resuspend cells with separation buffer and adjust cell density 1 ⁇ 10 7 cells/80 ⁇ L. Add CD4 MicroBeads at a ratio of 20 ⁇ L CD4 MicroBeads per 1 ⁇ 10 7 cells, mix well, and place at 4°C for 15 minutes.
  • separation buffer which consists of phosphate buffer containing 0.5% bovine serum albumin and 2mM ethylenediaminetetraacetic acid (EDTA). Collect T cells, count cells using Countstar, centrifuge (300g, 5 minutes, room temperature), and discard the supernatant. Resuspend cells with separation buffer and adjust cell density 1 ⁇
  • CD8 + T cells were positively selected from T cells using Miltenyi's CD8 MicroBeads, human (Cat. No. 130-045-201). Separation buffer was prepared, consisting of phosphate buffer containing 0.5% bovine serum albumin and 2mM ethylenediaminetetraacetic acid (EDTA). T cells were collected, cell counted using Countstar, centrifuged (300g, 5 minutes, RT), and the supernatant was discarded. Cells were resuspended in separation buffer and the cell density was adjusted to 1 ⁇ 10 7 cells/80 ⁇ L. CD8 MicroBeads were added at a ratio of 20 ⁇ L CD8 MicroBeads per 1 ⁇ 10 7 cells, mixed, and placed at 4 degrees for 15 minutes.
  • Separation buffer was prepared, consisting of phosphate buffer containing 0.5% bovine serum albumin and 2mM ethylenediaminetetraacetic acid (EDTA). T cells were collected, cell counted using Countstar, centrifuged (300g,
  • 2mL of separation buffer was added for every 1 ⁇ 10 7 cells , centrifuged (300g, 5 minutes, RT), and the supernatant was discarded.
  • Add 500 ⁇ L of separation buffer resuspend the cells, and add them to the separation column that has been moistened and hung on the magnet.
  • After the cell suspension flows through the column add 1 mL of separation buffer, rinse the column, and repeat once.
  • Add 2 mL of separation buffer to the column move the column away from the magnet, and use the piston to push 2 mL of separation buffer into a 15 mL centrifuge tube.
  • the separation buffer contains CD8 + T cells, count them, centrifuge (300g, 5 minutes, RT room temperature), and discard the supernatant.
  • RPMI-1640 + 10% FBS + 100U/mL IL-2 hereinafter referred to as "medium”
  • medium RPMI-1640 + 10% FBS + 100U/mL IL-2
  • the antibodies used for flow cytometry are: FITC anti-human CD3 antibody (biolegend, catalog number: 317306); PE anti-human CD8 antibody (biolegend, catalog number: 344706).
  • CD8 MicroBeads human (Cat. No. 130-045-201) and CD4 MicroBeads, human (Cat. No. 130-045-101) were used to negatively select CD4 - CD8 - double-negative T cells from T cells, hereinafter referred to as double-negative T cells.
  • Separation buffer was prepared, the components of which were phosphate buffer containing 0.5% bovine serum albumin and 2mM ethylenediaminetetraacetic acid (EDTA). T cells were collected, counted by Countstar, centrifuged (300g, 5 minutes, RT), and the supernatant was discarded. Cells were resuspended in separation buffer and the cell density was adjusted to 1 ⁇ 107 /80 ⁇ L.
  • CD8 MicroBeads and CD4 MicroBeads were added at the same time, mixed, and placed at 4 degrees for 15 minutes. For every 1 ⁇ 10 7 cells, add 2 mL of separation buffer, centrifuge (300g, 5 minutes, RT), and discard the supernatant. Add 1 mL of separation buffer, resuspend the cells, add to the separation column that has been moistened and hung on the magnet, collect the liquid that flows through the column, which contains double-negative T cells, count, centrifuge (300g, 5 minutes, RT), and discard the supernatant.
  • RPMI-1640 + 10% FBS + 100U/mL IL-2 hereinafter referred to as "culture medium”
  • culture medium RPMI-1640 + 10% FBS + 100U/mL IL-2, hereinafter referred to as "culture medium”
  • culture medium RPMI-1640 + 10% FBS + 100U/mL IL-2, hereinafter referred to as "culture medium”
  • sample Take a certain amount of sample for flow cytometry phenotyping and purity testing.
  • Human T-Activator CD3/CD28 mixed, inoculated in 48-well plates, 500 ⁇ L per well, placed in a 37°C 5% CO 2 incubator for culture.
  • the antibodies used for flow cytometry are: FITC anti-human CD3 antibody (biolegend, catalog number: 317306); PE anti-human CD8 antibody (biolegend, catalog number: 344706); APC anti-human CD4 antibody (biolegend, catalog number: 300514).
  • peripheral blood mononuclear cells Collect peripheral blood mononuclear cells, count, centrifuge (300g, 5 minutes, room temperature), discard the supernatant. Resuspend the cells in complete culture medium (ImmunoCult TM -XF T cell expansion medium + 5% FBS + 100U/mL IL-2 + 10ng/ml IL-15), adjust the cell density to 2 ⁇ 10 6 cells/mL, add zoledronic acid (Zoledronate, InvivoChem, Catalog No.: V1560), the working concentration is 5 ⁇ M. The cells were inoculated in a 24-well plate and cultured in a 37°C 5% CO 2 incubator.
  • the medium was changed every 2-3 days, the complete medium was replaced and the culture was expanded, but zoledronic acid was not added; after 12-14 days, a certain amount of cells was taken for flow cytometry phenotype and purity detection; the cells were collected for freezing and functional experiments.
  • the antibodies used for flow cytometry detection were: FITC anti-human CD3 antibody (biolegend, Catalog No.: 317306); PE anti-human TCR V ⁇ 2 antibody (biolegend, Catalog No.: 331408).
  • the complex proteins were added to the above-mentioned immune cells for incubation according to different combinations of parameters such as the concentration of the complex protein, the number of immune cells, the concentration of serum/plasma protein/albumin in the buffer, the volume of the incubation system, the incubation temperature, and the incubation time.
  • the proportion and fluorescence intensity of the immune cells bound by the complex proteins were detected by flow cytometry, the concentration of the residual complex protein in the system was detected by ELISA, and its tumor recognition function was detected and compared.
  • the complex protein takes anti-CD3 antibody (UCHT1)-1G4 (HATima) as an example.
  • the complex protein was added to a cell suspension of 3 ⁇ 10 5 T cells/100 ⁇ L RPMI-1640 medium, with final concentrations of 3 ⁇ 10 -7 mol/L, 3 ⁇ 10 -8 mol/L, 3 ⁇ 10 -9 mol/L, 3 ⁇ 10 -10 mol/L and 0 mol/L, respectively, and incubated at 4 degrees for 30 minutes. Centrifuge (500g, 5 minutes, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells. Add 1 ⁇ L pHLA-tetramer-APC and incubate at 4 degrees for 1 hour.
  • the fluorescence signal of pHLA-tetramer-APC represents the proportion and intensity of T cells bound to the complex protein, which is positively correlated with the number of complex proteins bound to the surface of T cells.
  • the complex protein is an example of anti-CD3 antibody (UCHT1)-1G4 (HATima).
  • T cells were added to 100 ⁇ L RPMI-1640 medium containing 1 ⁇ 10 -9 mol/L complex protein, respectively 4 ⁇ 10 6 , 2 ⁇ 10 6 , 1 ⁇ 10 6 and 0.3 ⁇ 10 6 , incubate at 4 degrees for 1 hour. Centrifuge (500g, 5 minutes, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells. Add 1 ⁇ L pHLA-tetramer-APC and incubate at 4 degrees for 1 hour.
  • the fluorescence signal of pHLA-tetramer-APC represents the proportion and intensity of T cells bound to the complex protein, which is positively correlated with the number of complex proteins bound to the surface of T cells.
  • the complex protein takes the anti-CD3 antibody (UCHT1) 1G4 (HATima) as an example.
  • UCHT1 1G4
  • pHLA-tetramer-APC Add 1 ⁇ L pHLA-tetramer-APC and incubate at 4 degrees for 1 hour. Add 1mL RPMI-1640 medium, centrifuge (500g, 5 minutes, 4°C), discard the supernatant, and repeat once. 100 ⁇ L RPMI-1640 medium was added, and the fluorescence signal of pHLA-tetramer-APC was detected by flow cytometry.
  • the fluorescence signal of pHLA-tetramer-APC represents the proportion and intensity of T cells bound to the complex protein, which is positively correlated with the number of complex proteins bound to the surface of T cells.
  • the complex protein is an example of anti-CD3 antibody (UCHT1)-1G4 (HATima).
  • the complex protein is added to a cell suspension of 3 ⁇ 10 5 T cells/100 ⁇ L RPMI-1640 medium, and the final concentration is 3 ⁇ 10 -8 mol/L, 3 ⁇ 10 -9 mol/L, 2 ⁇ 10 -9 mol/L and 1 ⁇ 10 -9 mol/L, incubated at 37 degrees for 1 hour and 18 hours respectively. Centrifuge (500g, 5 minutes, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells. Add 1 ⁇ L pHLA-tetramer-APC and incubate at 4 degrees for 1 hour.
  • the fluorescence signal of pHLA-tetramer-APC represents the proportion and intensity of T cells bound to the complex protein, which is positively correlated with the number of complex proteins bound to the surface of T cells.
  • the complex protein takes anti-CD3 antibody (UCHT1)-1G4 (HATima) as an example.
  • Prepare different incubation buffers namely RPMI-1640 medium, phosphate buffer, phosphate buffer containing 30 ⁇ g/mL mouse albumin, and phosphate buffer containing 10% mouse plasma, and store at 4 degrees.
  • Dilute the complex protein with different incubation buffers to a working concentration of 2 ⁇ 10 -9 mol/L, place at 37 degrees for 1 hour, cool to 4 degrees for use; resuspend T cells to a cell density of 3 ⁇ 10 5 /50 ⁇ L.
  • Mix the same incubation buffer that is, mix 50 ⁇ L T cell suspension with 50 ⁇ L complex protein, and incubate at 4 degrees for 1 hour.
  • the complex protein takes anti-CD3 antibody (UCHT1)-1G4 (HATima) as an example.
  • T cell suspension In a 96-well U-bottom plate, add 100 ⁇ L of T cell suspension, then add the complex protein, mix well, and the final concentrations are 3 ⁇ 10 -8 mol/L, 3 ⁇ 10 -9 mol/L, 2 ⁇ 10 -9 mol/L and 1 ⁇ 10 -9 mol/L, respectively.
  • DCT prepared with 3 ⁇ 10-8 mol/L, 3 ⁇ 10-9 mol/L, 2 ⁇ 10-9 mol/L and 1 ⁇ 10-9 mol/L composite protein when not in contact with target cells, had T cell activation rates of 17.2%, 3.91%, 2.01% and 1.07% after 18 hours of culture; when DCT was co-cultured with NCI-H1299-A2 target cells for 18 hours, the T cell activation rates were 78.7%, 54.2%, 69.1% and 69.3% respectively.
  • the complex protein takes anti-CD3 antibody (UCHT1)-1G4 (HATima) as an example.
  • the method for preparing DCT is: in 300 ⁇ L HIPP-T009 medium containing 1 ⁇ 10-9 mol/L complex protein, 0.9 ⁇ 106 and 6 ⁇ 106 T cells are added respectively, and incubated at 4 degrees for 1 hour. Equivalent to: in 100 ⁇ L HIPP-T009 medium containing 1 ⁇ 10-9 mol/L complex protein, 0.3 ⁇ 106 and 2 ⁇ 106 T cells are added respectively, and incubated at 4 degrees for 1 hour.
  • the prepared DCT was used to prepare cell suspensions of different densities, namely 1.25 ⁇ 10 3 /75 ⁇ L, 2.5 ⁇ 10 3 /75 ⁇ L, 5 ⁇ 10 3 /75 ⁇ L and 1 ⁇ 10 4 /75 ⁇ L; HepG2-NYESO1 (NY-ESO-1 positive, A2 positive), HepG2 (NY-ESO-1 negative, A2 positive), NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1 ⁇ 10 4 /75 ⁇ L.
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and each had 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • the 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250g, 4 minutes). 50 ⁇ L of supernatant was taken out to a 96-well flat-bottom plate, and 50 ⁇ L of substrate solution was added. The reaction was kept at room temperature away from light for 30 minutes. After the reaction was completed, 50 ⁇ L of stop solution was added, and the absorbance at 490 nm was immediately measured with an ELISA reader. According to the product manual, the efficiency of specific killing was calculated as (experimental wells-spontaneous wells of effector cells-spontaneous wells of target cells)/(maximum lysis wells of target cells-spontaneous wells of target cells) ⁇ 100%.
  • Example 3 Modifying tumor recognition and killing functions of cells
  • Preparation method of TCR-T cells Use NheI and SalI restriction sites to insert TCR ⁇ and ⁇ chain sequences into the lentiviral expression vector pGZ178, mix with packaging plasmids (pMDLg/pRRE, pRSV-REV and pMD.2G), and transfect 293T cells under the action of transfection reagent PEI-MAX, and culture for 3 days. Collect the culture supernatant containing lentiviral particles, concentrate with a concentration tube, freeze at -80°C after aliquoting, and detect its titer. Stimulate T cells with magnetic beads coated with anti-CD3/CD28 antibodies and IL-2 (100IU/mL).
  • the protein uses anti-CD3 antibody (UCHT1)-1G4 (HATima) as an example.
  • the method for preparing DCT is as follows: add 5 ⁇ 10 6 T cells to 200 ⁇ L HIPP-T009 medium containing 1 ⁇ 10 -7 mol/L complex protein, and incubate at 4 degrees for 1 hour. Take 0.3 ⁇ 10 6 cells each, centrifuge (500g, 5 minutes, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells.
  • HIPP-T009 medium was used to prepare cell suspensions of different densities of 1.25 ⁇ 10 3 cells/75 ⁇ L, 2.5 ⁇ 10 3 cells/75 ⁇ L, 5 ⁇ 10 3 cells/75 ⁇ L and 1 ⁇ 10 4 cells/75 ⁇ L, respectively.
  • HepG2-NYESO1 (NY-ESO-1 positive, A2 positive), HepG2 (NY-ESO-1 negative, A2 positive), NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1 ⁇ 10 4 cells/75 ⁇ L. In a 96-well U-bottom plate, 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • the 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250g, 4 minutes).
  • the efficiency of specific killing is calculated as (experimental wells-spontaneous wells of effector cells-spontaneous wells of target cells)/(maximum lysis wells of target cells-spontaneous wells of target cells) ⁇ 100%.
  • Anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects T cells to kill tumor cells
  • CD3 + T-DCT was prepared by the scheme of "adding 5x10 6 T cells to 200 ⁇ L HIPP-T009 culture medium containing 1x10 -7 mol/L complex protein anti - CD3 antibody (UCHT1)-1G4 (HATima), and incubating at 4 degrees for 1 hour", and the positivity rate of DCT was detected by flow cytometry.
  • the results showed that more than 99% of T cells were coated with CD3 antibody (UCHT1)-1G4 (HATima) complex protein (Figure 10b).
  • the prepared CD3 + T-DCT was adjusted to different cell suspension densities using HIPP-T009 medium, namely 1x10 3 cells/75 ⁇ L, 1.25x10 3 cells/75 ⁇ L, 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L and 8x10 4 /75 ⁇ L; HepG2-NYESO1 (NY-ESO-1 positive, A2 positive), HepG2 (NY-ESO-1 negative, A2 positive), NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 /75 ⁇ L.
  • 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added to the 96-well U-bottom plate in sequence, and the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well and culture medium plus lysis solution spontaneous well were set at the same time.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells for each.
  • the 96-well U-bottom plate with cells was placed in a cell culture incubator at 37 degrees and 5% CO 2 for 18 hours. Add 15 ⁇ L of lysis solution to the maximum lysis well, mix well, and place in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes. Place the 96-well U-bottom cell culture plate in a centrifuge and centrifuge (250g, 4 minutes).
  • Cells were collected from HepG2-NYESO-1/DCT, HepG2/DCT, and DCT groups with a target-effector ratio of 1:2 in 96-well U-bottom plates; NCI-H1299-A2/DCT, NCI-H1299/DCT, and DCT groups with a target-effector ratio of 4:1. Cells. Centrifuge (500g, 5min, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells.
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • CD137 and pHLA-tetramer in the CD3 positive cell population were analyzed, which represented the activation degree of T cells and the ratio of surface-bound complex proteins, respectively.
  • the results showed that CD3 + T-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (HATima) induced T cells to upregulate CD137 expression only after encountering positive target cells, that is, T cells showed an activated state; for negative tumor cells and in the absence of tumor cells, DCT cells did not upregulate CD137 expression and showed a resting state (Figure 10d), although a high level of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein was bound to the surface of T cells ( Figure 10e).
  • UCHT1-1G4 HATima
  • B-NDG mice (purchased from Biocytogen) were used to establish a HepG2-NYESO-1 tumor model.
  • B-NDG mice were subcutaneously injected with 5x10 6 HepG2-NYESO-1 cells.
  • the tumors grew to 14 days, and the average tumor volume was 226mm 3.
  • the mice were randomly divided into a control group and a CD3 + T-DCT group, with 6 mice in each group. 2x10 7 T cells were intravenously infused.
  • 5x10 6 CD3 + T-DCT cells were infused into the DCT group.
  • the preparation of DCT was the same as that used in the cell experiment; the T cells in the control group were not loaded with complex proteins.
  • CD3 + T-DCT cells were infused once every other day, for a total of 7 infusions.
  • IL-2 (50000U) was injected intraperitoneally once a day for 14 consecutive days. Every 3-4 days, the length and width of the tumor were measured with a vernier caliper, and the volume of the tumor was calculated using the formula "(length x width x width) x 0.5".
  • the mice were euthanized by carbon dioxide and the experiment reached the end point.
  • T cells redirected by CD3 antibody (UCHT1)-1G4 (HATima) can effectively kill tumor cells both in vivo and in vitro, without causing "spontaneous" activation of T cells.
  • Anti-CD3 antibody (UCHT1)-1G4 HATima) redirects peripheral blood mononuclear cells (PBMCs) to kill tumor cells
  • PBMC Peripheral blood mononuclear cells
  • Flow cytometry showed that the CD3 positive cell population was 62.3 % ( Figure 11a).
  • 5x10 6 PBMCs were added to 200 ⁇ L HIPP-T009 culture medium containing the pHLA-tetramer anti-CD3 antibody (UCHT1)-1G4 (HATima) and incubated at 4 degrees for 1 hour to prepare PBMC-DCT, and the positive rate of DCT was detected by flow cytometry.
  • the results showed that the CD3 cell population and the pHLA-tetramer cell population were mutually positive, that is, the complex protein only bound to T cells, and the binding rate was 100% (Figure 11b).
  • the prepared PBMC-DCT were adjusted to different cell suspension densities using HIPP-T009 medium, namely 1.25x10 3 cells/75 ⁇ L, 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L and 8x10 4 cells/75 ⁇ L; HepG2-NYESO1 (NY-ESO-1 positive, A2 positive), HepG2 (NY-ESO-1 negative, A2 positive), NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 /75 ⁇ L.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • the fluorescence signal of CD137 and pHLA-tetramer in CD3-positive cell population was analyzed, which represented the activation degree of T cells and the ratio of surface-bound complex proteins, respectively.
  • the results showed that PBMC-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (HATima) induced T cells in PBMC to upregulate the expression of CD137 only after encountering positive target cells, that is, T cells showed an activated state; for negative tumor cells and in the absence of tumor cells, DCT cells did not upregulate the expression of CD137 and showed a resting state (Figure 11d), although a high level of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein was bound to the surface of T cells ( Figure 11e).
  • UCHT1-1G4 HATima
  • Anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects peripheral blood lymphocytes (PBL) to kill tumor cells
  • PBL-DCT peripheral blood mononuclear cells
  • the prepared PBL-DCT was adjusted to different cell suspension densities using HIPP-T009 medium, namely 1.25x10 3 cells/75 ⁇ L, 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L and 8x10 4 cells/75 ⁇ L; HepG2-NYESO1 (NY-ESO-1 positive, A2 positive), HepG2 (NY-ESO-1 negative, A2 positive), NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 /75 ⁇ L.
  • the 96-well U-bottom plate add 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells in sequence, and set up the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well.
  • the final volume of each well is 150 ⁇ L, and there are 2 duplicate wells.
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • the ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, which represented the activation degree of T cells and the ratio of surface-bound complex proteins, respectively.
  • the results showed that PBL-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (HATima) induced T cells in PBL to upregulate the expression of CD137 only after encountering positive target cells, that is, T cells showed an activated state; for negative tumor cells and in the absence of tumor cells, DCT cells did not upregulate the expression of CD137 and showed a resting state (Figure 12d), although high levels of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex proteins were bound to the surface of T cells ( Figure 12e).
  • UCHT1-1G4 HATima
  • Anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects ⁇ T cells to kill tumor cells
  • Zoledronic acid (5 ⁇ M) was used to expand the ⁇ T cells in peripheral blood mononuclear cells, and they were cultured in complete medium (ImmunoCult TM -XF T cell expansion medium + 5% FBS + 200U/mL IL-2 + 10ng/mL IL-15) for 12 days, and their phenotype and purity were detected by flow cytometry.
  • the results showed that 87.8% of the cells were TCR Vd2/CD3 double positive, that is, the proportion of ⁇ T cells in the expanded and cultured cells was 87.8% ( Figure 13a).
  • ⁇ T-DCT was prepared by the scheme of "adding 5x10 6 ⁇ T cells to 200 ⁇ L HIPP-T009 medium containing 1x10 -8 mol/L complex protein anti-CD3 antibody (UCHT1)-1G4 (HATima), and incubating at 4 degrees for 1 hour", and the positive rate of DCT was detected by flow cytometry.
  • the results showed that 97.8% of ⁇ T cells were coated with the anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein ( FIG. 13 b ).
  • the prepared ⁇ T-DCT and ⁇ T cells were adjusted to different cell suspension densities, namely 5x10 2 cells/75 ⁇ L, 1x10 3 cells/75 ⁇ L, 2x10 3 cells/75 ⁇ L and 4x10 3 cells /75 ⁇ L; 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L and 8x10 4 cells/75 ⁇ L; HepG2-NYESO1 (NY-ESO-1 positive, A2 positive), HepG2 (NY-ESO-1 negative, A2 positive), NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 cells/75 ⁇ L.
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • Centrifuge 500g, 5min, 4°C
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • ⁇ T-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (HATima) induced T cells to upregulate the expression of CD137 only after encountering positive target cells.
  • the cells showed an activated state; when negative tumor cells or no tumor cells were present, ⁇ T-DCT cells did not regulate the expression of CD137 and showed a quiescent state, which was consistent with the results of ⁇ T ( Figure 13d).
  • the ⁇ T-DCT prepared with the complex protein CD3 antibody (UCHT1)-1G4 (HATima) at a concentration of 1x10-8 mol/L could not detect the anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein bound to the cell surface by flow cytometry (Figure 13e).
  • Anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects T cells to kill tumor cells
  • CD3 + T-DCT was prepared by the scheme of "adding 5x10 6 T cells to 200 ⁇ L HIPP-T009 culture medium containing 1x10 -7 mol/L composite protein anti-CD3 antibody (UCHT1)-1G4 (ImmTAC), and incubating at 4 degrees for 1 hour", and the positivity rate of DCT was detected by flow cytometry.
  • the results showed that more than 99% of T cells were coated with the anti - CD3 antibody (UCHT1)-1G4 (ImmTAC) composite protein ( Figure 14a).
  • the prepared CD3 + T-DCT was adjusted to different cell suspension densities using HIPP-T009 medium, namely 1x10 3 cells/75 ⁇ L, 1.25x10 3 cells/75 ⁇ L, 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L and 8x10 4 /75 ⁇ L; HepG2-NYESO1 (NY-ESO-1 positive, A2 positive), HepG2 (NY-ESO-1 negative, A2 positive), NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 /75 ⁇ L.
  • 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added to the 96-well U-bottom plate in sequence, and the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well and culture medium plus lysis solution spontaneous well were set at the same time.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells for each.
  • the 96-well U-bottom plate with cells was placed in a cell culture incubator at 37 degrees and 5% CO 2 for 18 hours. Add 15 ⁇ L of lysis solution to the maximum lysis well, mix well, and place in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. Place the 96-well U-bottom cell culture plate in a centrifuge and centrifuge (250g, 4 minutes).
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • the ratio of CD137 and pHLA-tetramer in the CD3 positive cell population was analyzed, which represented the activation degree of T cells and the ratio of surface-bound complex proteins, respectively.
  • the results showed that CD3 + T-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) induced T cells to upregulate CD137 expression only after encountering positive target cells, that is, T cells showed an activated state; for negative tumor cells and when there were no tumor cells, DCT cells did not upregulate CD137 expression and showed a resting state (Figure 14c), although a high level of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein was bound to the surface of T cells ( Figure 14d).
  • UCHT1-1G4 ImmTAC
  • B-NDG mice (purchased from Biocytogen) were used to establish a HepG2-NYESO-1 tumor model.
  • B-NDG mice were subcutaneously injected with 5x10 6 HepG2-NYESO-1 cells.
  • the tumors grew to 14 days, and the average tumor volume was 226mm 3.
  • the mice were randomly divided into a control group and a CD3 + T-DCT group, with 6 mice in each group. 2x10 7 T cells were intravenously infused.
  • 5x10 6 CD3 + T-DCT cells were infused into the DCT group.
  • the preparation of DCT was the same as that used in the cell experiment; the T cells in the control group were not loaded with complex proteins.
  • CD3 + T-DCT cells were infused once every other day, for a total of 7 infusions.
  • IL-2 (50000U) was injected intraperitoneally once a day for 14 consecutive days. Every 3-4 days, the length and width of the tumor were measured with a vernier caliper, and the volume of the tumor was calculated using the formula "(length x width x width) x 0.5".
  • the mice were euthanized by carbon dioxide and the experiment reached the end point.
  • T cells redirected by anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) can effectively kill tumor cells both in vivo and in vitro. At the same time, it does not cause "spontaneous" activation of T cells.
  • Anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects peripheral blood mononuclear cells (PBMCs) to kill tumor cells
  • PBMC-DCT Peripheral blood mononuclear cells
  • the prepared PBMC-DCT were adjusted to different cell suspension densities using HIPP-T009 medium, namely 1.25x10 3 cells/75 ⁇ L, 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L and 8x10 4 cells/75 ⁇ L; HepG2-NYESO1 (NY-ESO-1 positive, A2 positive), HepG2 (NY-ESO-1 negative, A2 positive), NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 /75 ⁇ L.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • the ratio of CD137 and pHLA-tetramer in the CD3 positive cell population was analyzed, which represented the activation degree of T cells and the ratio of surface-bound complex proteins, respectively.
  • the results showed that PBMC-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) induced T cells in PBMC to upregulate the expression of CD137 only after encountering positive target cells, that is, T cells showed an activated state; for negative tumor cells and when there were no tumor cells, DCT cells did not upregulate the expression of CD137 and showed a resting state (Figure 15c), although a high level of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein was bound to the surface of T cells ( Figure 15d).
  • UCHT1-1G4 ImmTAC
  • Anti-CD3 antibody (UCHT1)-1G4 redirects peripheral blood lymphocytes (PBL) to kill tumor cells
  • PBL-DCT peripheral blood mononuclear cells
  • the prepared PBL-DCT was adjusted to different cell suspension densities using HIPP-T009 medium, namely 1.25x10 3 cells/75 ⁇ L, 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L and 8x10 4 cells/75 ⁇ L; HepG2-NYESO1 (NY-ESO-1 positive, A2 positive), HepG2 (NY-ESO-1 negative, A2 positive), NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 /75 ⁇ L.
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence, and the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium addition well were set.
  • the final volume of each well of the spontaneous pores of the lysis solution is 150 ⁇ L, and there are 2 duplicate wells.
  • the 96-well U-bottom plate with cells added is placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. Add 15 ⁇ L of lysis solution to the largest lysis well, mix well, and place in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • the ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, which represented the activation degree of T cells and the ratio of surface-bound complex proteins, respectively.
  • the results showed that PBL-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) induced T cells in PBL to upregulate the expression of CD137 only after encountering positive target cells, that is, T cells showed an activated state; for negative tumor cells and in the absence of tumor cells, DCT cells did not upregulate the expression of CD137 and showed a resting state (Figure 16c), although high levels of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex proteins were bound to the surface of T cells ( Figure 16d).
  • UCHT1-1G4 ImmTAC
  • Anti-CD3 antibody (UCHT1)-1G4 redirects ⁇ T cells to kill tumor cells
  • Zoledronic acid (5 ⁇ M) was used to expand the ⁇ T cells in peripheral blood mononuclear cells, and they were cultured in complete medium (ImmunoCult TM -XF T cell expansion medium + 5% FBS + 200U/mL IL-2 + 10ng/mL IL-15) for 12 days. The phenotype and purity were detected by flow cytometry. The results showed that 87.8% of the cells were TCR Vd2/CD3 double positive, that is, the proportion of ⁇ T cells in the expanded cultured cells is 87.8% ( Figure 13a).
  • ⁇ T-DCT was prepared by the scheme of "adding 5x10 6 ⁇ T cells to 200 ⁇ L HIPP-T009 culture medium containing 1x10 -8 mol/L composite protein anti-CD3 antibody (UCHT1)-1G4 (ImmTAC), and incubating at 4 degrees for 1 hour", and the positive rate of DCT was detected by flow cytometry.
  • the results showed that 98.8% of ⁇ T cells were coated with the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) composite protein (Figure 17a).
  • the prepared ⁇ T-DCT and ⁇ T cells were adjusted to different cell suspension densities using HIPP-T009 medium, namely 5x10 2 cells/75 ⁇ L, 1x10 3 cells/75 ⁇ L, 2x10 3 cells/75 ⁇ L, and 4x10 3 cells/75 ⁇ L; 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L, and 8x10 4 /75 ⁇ L; HepG2-NYESO1 (NY-ESO-1 positive, A2 positive), HepG2 (NY-ESO-1 negative, A2 positive), NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 /75 ⁇ L.
  • 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added to the 96-well U-bottom plate in sequence, and the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well and culture medium plus lysis solution spontaneous well were set at the same time.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells for each.
  • the 96-well U-bottom plate with cells was placed in a cell culture incubator at 37 degrees and 5% CO 2 for 18 hours. Add 15 ⁇ L of lysis solution to the maximum lysis well, mix well, and place in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes. Place the 96-well U-bottom cell culture plate in a centrifuge and centrifuge (250g, 4 minutes).
  • the ⁇ T-DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) induced T cells to upregulate the expression of CD137 only after encountering positive target cells, that is, the ⁇ T-DCT cells were in an activated state; when there were negative tumor cells or no tumor cells, the ⁇ T-DCT cells did not upregulate the expression of CD137 and were in a quiescent state, which was consistent with the results of ⁇ T ( Figure 17c).
  • the ⁇ T-DCT prepared by the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein bound to the cell surface could not be detected by flow cytometry (Figure 17d).
  • Anti-CD3 antibody (UCHT1)-AFP (HATima) redirects T cells to kill tumor cells
  • CD3 + T-DCT was prepared according to the scheme of "adding 5x10 6 T cells to 200 ⁇ L HIPP-T009 culture medium containing 1x10 -8 mol/L complex protein anti - CD3 antibody (UCHT1)-AFP (HATima), and incubating at 4 degrees for 1 hour", and the positivity rate of DCT was detected by flow cytometry. The results showed that more than 98% of T cells were coated with anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein (Figure 18a).
  • the prepared CD3 + T-DCT was adjusted to cell suspensions of different densities, namely 5x10 3 /75 ⁇ L, 1x10 4 /75 ⁇ L, 2x10 4 /75 ⁇ L and 4x10 4 /75 ⁇ L; HepG2 (AFP positive, A2 positive) and NCI-H1299-A2 (AFP negative, A2 positive) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 /75 ⁇ L.
  • 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added to the 96-well U-bottom plate in sequence, and the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, medium spontaneous well and medium plus lysis solution spontaneous well were set at the same time.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells for each.
  • CD3 + T-DCT cells prepared by anti-CD3 antibody (UCHT1)-AFP (HATima) induced T cells to upregulate the expression of CD137 only after encountering positive target cells, that is, T cells showed an activated state; for negative tumor cells and when there were no tumor cells, DCT cells did not upregulate the expression of CD137 and showed a resting state (Figure 18c).
  • CD3 + T-DCT prepared with 1x10 -8 mol/L concentration of the complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) could not be detected by flow cytometry on the cell surface after 18 hours of cell culture ( FIG. 18 d ).
  • Anti-CD3 antibody (UCHT1)-AFP (HATima) redirects peripheral blood mononuclear cells (PBMC) to kill tumor cells
  • PBMC-DCT Peripheral blood mononuclear cells
  • the prepared PBMC-DCT was adjusted to cell suspensions of different densities, namely 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L and 4x10 4 cells/75 ⁇ L; HepG2 (AFP positive, A2 positive) and NCI-H1299-A2 (AFP negative, A2 positive) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 cells/75 ⁇ L.
  • HepG2 AFP positive, A2 positive
  • NCI-H1299-A2 AFP negative, A2 positive
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • PBMC-DCT cells prepared by anti-CD3 antibody (UCHT1)-AFP (HATima) induced T cells in PBMC to upregulate the expression of CD137 only after encountering positive target cells, that is, T cells were in an activated state; for negative tumor cells and when there were no tumor cells, DCT cells did not upregulate the expression of CD137 and were in a resting state (Figure 19c).
  • PBMC-DCT prepared by the complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) at a concentration of 1x10-8 mol/L showed no significant difference in CD137 expression after 18 hours of cell culture. No cell surface-bound anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein was detected ( FIG. 19 d ).
  • Anti-CD3 antibody (UCHT1)-AFP (HATima) redirects peripheral blood lymphocytes (PBL) to kill tumor cells
  • PBL-DCT peripheral blood mononuclear cells
  • the prepared PBL-DCT was adjusted to cell suspensions of different densities, namely 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L and 4x10 4 cells/75 ⁇ L; HepG2 (AFP positive, A2 positive) and NCI-H1299-A2 (AFP negative, A2 positive) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 cells/75 ⁇ L.
  • HepG2 AFP positive, A2 positive
  • NCI-H1299-A2 AFP negative, A2 positive
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • PBL-DCT cells prepared by anti-CD3 antibody (UCHT1)-AFP (HATima) induced T cells in PBL to upregulate the expression of CD137 only after encountering positive target cells, that is, T cells were in an activated state; when facing negative tumor cells or when there were no tumor cells, DCT cells did not upregulate the expression of CD137 and were in a resting state (Figure 20c).
  • Anti-CD3 antibody (UCHT1)-AFP (HATima) redirects ⁇ T cells to kill tumor cells
  • Zoledronic acid (5 ⁇ M) was used to expand the ⁇ T cells in peripheral blood mononuclear cells, and they were cultured in complete medium (ImmunoCult TM -XF T cell expansion medium + 5% FBS + 200U/mL IL-2 + 10ng/mL IL-15) for 12 days, and their phenotype and purity were detected by flow cytometry.
  • the results showed that 87.8% of the cells were TCR Vd2/CD3 double positive, that is, the proportion of ⁇ T cells in the expanded cultured cells was 87.8% ( Figure 13a).
  • ⁇ T-DCT was prepared by the scheme of "adding 5x10 6 ⁇ T cells to 200 ⁇ L HIPP-T009 medium containing 1x10 -9 mol/L complex protein anti-CD3 antibody (UCHT1)-AFP (HATima), and incubating at 4 degrees for 1 hour", and the positive rate of DCT was detected by flow cytometry.
  • the results showed that 100% of the ⁇ T cells were coated with the anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein ( FIG. 21 a ).
  • the prepared ⁇ T-DCT and ⁇ T cells were adjusted to different cell suspension densities, namely 5x10 3 /75 ⁇ L, 1x10 4 /75 ⁇ L, 2x10 4 /75 ⁇ L and 4x10 4 /75 ⁇ L; HepG2 (AFP positive, A2 positive) and NCI-H1299-A2 (AFP negative, A2 positive) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 /75 ⁇ L.
  • 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added to the 96-well U-bottom plate in sequence, and the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, medium spontaneous well and medium plus lysis solution spontaneous well were set at the same time.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells for each.
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • the ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, which represented the activation degree of ⁇ T-DCT or ⁇ T cells and the ratio of surface-bound complex proteins, respectively.
  • the results showed that ⁇ T-DCT cells prepared by anti-CD3 antibody (UCHT1)-AFP (HATima) induced T cells to upregulate the expression of CD137 only after encountering positive target cells, that is, ⁇ T-DCT cells showed an activated state; when facing negative tumor cells or without tumor cells, ⁇ T-DCT cells did not upregulate the expression of CD137 and showed a resting state, which was consistent with the results of ⁇ T (Figure 21c).
  • ⁇ T-DCT prepared with the complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) at a concentration of 1x10 -9 mol/L
  • no anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein bound to the cell surface could be detected by flow cytometry after 18 hours of cell culture ( FIG. 21 d ).
  • Anti-CD3 antibody (UCHT1)-gp100 (HATima) redirects immune cells to kill tumor cells
  • immune cells were prepared, including T cells, ⁇ T cells, CD4 and CD8 double negative T cells (DNT), CD4 + T cells, CD8 + T cells, PBMC and PBL, and their phenotype and purity were detected by flow cytometry.
  • DCT preparation method in the presence of 1x10 -8 mol/L complex protein anti-CD3 antibody (UCHT1) -gp100 (HATima) HIPP-T009 medium (200 ⁇ L), 5x10 6 immune cells were added, incubated at 4 degrees for 1 hour, DCT was prepared, and the positive rate of DCT was detected by flow cytometry.
  • the prepared DCT cells or control cells were adjusted to cell suspensions with different densities, namely 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L, 8x10 4 cells/75 ⁇ L, 16x10 4 cells/75 ⁇ L, and 32x10 4 cells/75 ⁇ L; T2 cells were collected, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 cells /75 ⁇ L, and gp100 peptide (YLEPGPVTA, positive target) and NY-ESO-1 peptide (SLLMWITQC, negative control) with a final concentration of 1x10 -8 M were loaded respectively.
  • YLEPGPVTA positive target
  • SLLMWITQC NY-ESO-1 peptide
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • T-DCT, PBMC-DCT, PBL-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT redirected by anti-CD3 antibody (UCHT1)-gp100 (HATima) efficiently killed T2-positive target tumor cells loaded with gp100 peptide, and had a gradient dependence of effector-target ratio; it did not recognize T2-negative tumor cells loaded with NY-ESO-1 peptide, or was equivalent to the recognition level of control cells (DNT and ⁇ T cells) on tumor cells ( Figure 22b).
  • Collect cells from experimental wells with different effector-target ratios in a 96-well U-bottom plate Centrifuge (500g, 5min, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells.
  • DCT cells prepared by anti-CD3 antibody (UCHT1)-gp100 (HATima) induced T cells, PBMC, PBL, ⁇ T cells, DNT, CD4 + T cells and CD8 + T cells to upregulate the expression of CD137 only after encountering positive target cells (T2-gp100 peptide), that is, DCT cells showed an activated state; when facing negative tumor cells (T2-NYESO-1 peptide) and in the absence of tumor cells, DCT cells did not regulate the expression of CD137 and showed a resting state (Figure 22c).
  • UCHT1-gp100 HATima
  • UCHT1-gp100 HATima
  • Anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) redirects immune cells to kill tumor cells
  • immune cells including T cells, ⁇ T cells, CD4 and CD8 double negative T cells (DNT), CD4 + T cells, CD8 + T cells, PBMC and PBL were prepared, and their phenotype and purity were detected by flow cytometry.
  • the method for preparing DCT was as follows: 5x10 6 immune cells were added to HIPP-T009 culture medium (200 ⁇ L) containing 1x10 -8 mol/L composite protein anti-CD3 antibody (UCHT1)-gp100 (ImmTAC), incubated at 4 degrees for 1 hour, prepared DCT, and the positive rate of DCT was detected by flow cytometry.
  • the prepared DCT cells or control cells were adjusted to cell suspensions with different densities, namely 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L, 8x10 4 cells/75 ⁇ L, and 16x10 4 cells/75 ⁇ L; T2 cells were collected, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 cells/75 ⁇ L, and gp100 peptide (YLEPGPVTA, positive target) and AFP peptide (FMNKFIYEI, negative control) were loaded with a final concentration of 1x10 -8 M, respectively.
  • YLEPGPVTA positive target
  • FMNKFIYEI AFP peptide
  • a 96-well U-bottom plate add 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells in sequence, and set up the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well.
  • the final volume of each well is 150 ⁇ L, and there are 2 duplicate wells for each.
  • T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT, CD8 + T-DCT, PBMC-DCT and PBL-DCT redirected by anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) efficiently killed T2-positive target tumor cells loaded with gp100 peptide, and had a gradient dependence of effector-target ratio; it did not recognize T2-negative tumor cells loaded with AFP peptide, or its recognition level of tumor cells was equivalent to that of control cells (DNT and ⁇ T cells) ( Figure 23b).
  • Collect cells from experimental wells with different effector-target ratios in a 96-well U-bottom plate Centrifuge (500g, 5min, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells.
  • DCT cells prepared by anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) induced T cells, ⁇ T cells, DNT, CD4 + T cells, CD8 + T, PBMC and PBL cells to upregulate the expression of CD137 only after encountering positive target cells (T2-gp100 peptide), that is, DCT cells were in an activated state; for negative tumor cells (T2-AFP peptide) and in the absence of tumor cells, DCT cells did not regulate the expression of CD137 and were in a resting state (Figure 23c).
  • Anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) redirects immune cells to kill tumor cells
  • immune cells were prepared, including T cells, ⁇ T cells, CD4 and CD8 double negative T cells (DNT), CD4 + T cells, CD8 + T cells, PBMC and PBL, and their phenotype and purity were detected by flow cytometry.
  • the method for preparing DCT was as follows: 5x10 6 immune cells were added to the HIPP-T009 culture medium (200 ⁇ L) containing 1x10 -7 mol/L to 1x10 -10 mol/L of the complex protein anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC), incubated at 4 degrees for 1 hour, and DCT was prepared. The positive rate of DCT was detected by flow cytometry.
  • the prepared DCT cells or control cells were adjusted to cell suspensions with different densities, namely 0.625x10 3 cells/75 ⁇ L, 1.25x10 3 cells/75 ⁇ L, 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L, and 8x10 4 cells/75 ⁇ L; the positive (HepG2-NYESO-1(NYESO-1 + /HLA-A0201 + )) and negative (HepG2(NYESO-1 - /HLA-A0201 + )) target cells were digested with trypsin, washed once with phosphate buffer, and resuspended in HIPP-T009 medium to adjust the cell density to 2x10 4 cells/75 ⁇ L.
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • ImmTAC anti-CD3 antibody
  • Collect cells from the experimental wells with different effector-target ratios in the 96-well U-bottom plate Centrifuge (500g, 5min, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells. Add 1 ⁇ L FITC anti-human CD3 antibody (biolegend, catalog number: 317306), PEcy7 anti-human CD137 antibody (biolegend, catalog number: 309818) and pHLA-tetramer-APC, respectively, and set up an unstained group and an isotype control group at the same time, and incubate at 4 degrees for 30 minutes.
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • DCT prepared by anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) After encountering positive target cells (HepG2-NYESO-1), DCT cells induced T cells, ⁇ T cells, DNT, CD4 + T cells, CD8 + T, PBMC and PBL cells to upregulate the expression of CD137, that is, DCT cells showed an activated state; when facing negative tumor cells (HepG2) and when there were no tumor cells, DCT cells did not upregulate the expression of CD137 and showed a quiescent state (Figure 24c).
  • UCHT1-i1G4 ImmTAC
  • T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT, CD8 + T - DCT, PBMC-DCT and PBL-DCT prepared by the composite protein anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) at a concentration of 1x10 -7 mol/L, after 18 hours of cell culture, the surface-bound anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) composite protein was relatively low (Figure 24d).
  • Anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) redirects immune cells to kill tumor cells
  • immune cells including T cells, ⁇ T cells, CD4 and CD8 double negative T cells (DNT), CD4 + T cells and CD8 + T cells were prepared, and their phenotype and purity were detected by flow cytometry.
  • the method for preparing DCT was as follows: 5x10 6 immune cells were added to the HIPP-T009 culture medium (200 ⁇ L) containing 1x10 -8 mol/L composite protein anti-CD3 antibody (UCHT1)-igp100 (ImmTAC), incubated at 4 degrees for 1 hour, and DCT was prepared. The positive rate of DCT was detected by flow cytometry. The results showed that 100% of T cells, ⁇ T cells, DNT, CD4 + T cells and CD8 + T cells were coated with anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) composite protein (Figure 25a).
  • UCHT1-igp100 ImmTAC
  • the prepared DCT cells or control cells were adjusted to cell suspensions with different densities, namely 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L, 8x10 4 cells/75 ⁇ L, 16x10 4 cells/75 ⁇ L, and 32x10 4 cells/75 ⁇ L; T2 cells were collected, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 cells/75 ⁇ L, and gp100 peptide (YLEPGPVTA, positive target) and AFP peptide (FMNKFIYEI, negative control) were loaded with a final concentration of 1x10 -8 M, respectively.
  • YLEPGPVTA positive target
  • FMNKFIYEI AFP peptide
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT redirected by anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) efficiently killed T2-positive target tumor cells loaded with gp100 peptide, and had a gradient dependence of effect-target ratio; it did not recognize T2-negative tumor cells loaded with AFP peptide, or was equivalent to the recognition level of control cells (DNT and ⁇ T cells) on tumor cells ( Figure 25b).
  • Collect cells from the experimental wells with different effector-target ratios in the 96-well U-bottom plate Centrifuge (500g, 5min, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells. Add 1 ⁇ L FITC anti-human CD3 antibody (biolegend, catalog number: 317306), PEcy7 anti-human CD137 antibody (biolegend, catalog number: 309818) and pHLA-tetramer-APC, respectively, and set up an unstained group and an isotype control group at the same time, and incubate at 4 degrees for 30 minutes.
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • DCT cells prepared by anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) induced T cells, ⁇ T cells, DNTs, CD4 + T cells and CD8 + T cells to upregulate the expression of CD137 only after encountering positive target cells (T2-gp100 peptide), that is, DCT cells showed an activated state; when facing negative tumor cells (T2-AFP peptide) and when there were no tumor cells, DCT cells did not upregulate the expression of CD137 and showed a resting state (Figure 25c).
  • T-DCT, ⁇ T-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT prepared with 1x10-8 mol/L concentration of complex protein anti-CD3 antibody (UCHT1)-igp100 (ImmTAC), after 18 hours of cell culture, the anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) complex protein bound to the surface of DCT cells co-cultured with positive target cells (T2-gp100 peptide) decreased significantly (Figure 25d).
  • UCHT1-igp100 ImmTAC
  • Anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects immune cells to kill tumor cells
  • immune cells were prepared, including CD4 and CD8 double negative T cells (DNT), CD4 + T cells and CD8 + T cells, and their phenotype and purity were detected by flow cytometry.
  • Method for preparing DCT 5x10 6 immune cells were added to HIPP-T009 culture medium (200 ⁇ L) containing 1x10 -7 mol/L (for CD4 + T cells and CD8 + T cells) or 1x10 -8 mol/L (for DNT) complex protein anti-CD3 antibody (UCHT1) -1G4 (ImmTAC), incubated at 4 degrees for 1 hour to prepare DCT, and the positive rate of DCT was detected by flow cytometry. The results showed that 100% of DNT, CD4 + T cells and CD8 + T cells were coated with anti-CD3 antibody (UCHT1) -1G4(ImmTAC) complex protein (Figure 26a).
  • the prepared DCT cells or control cells were adjusted to cell suspensions with different densities, namely 1.25x10 3 cells/75 ⁇ L, 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L, 8x10 4 cells/75 ⁇ L, and 16x10 4 cells/75 ⁇ L; positive (HepG2-NYESO-1(NYESO-1 + /HLA-A0201 + )) and negative (HepG2(NYESO-1 - /HLA-A0201 + )) target cells were digested with trypsin, washed once with phosphate buffer, and resuspended in HIPP-T009 medium to adjust the cell density to 2x10 4 cells/75 ⁇ L.
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • the 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250g, 4 minutes). 50 ⁇ L of supernatant was taken out to a 96-well flat-bottom plate, 50 ⁇ L of substrate solution was added, and the reaction was carried out at room temperature in the dark for 30 minutes. After the reaction was completed, 50 ⁇ L of stop solution was added, and the absorbance at 490 nm was immediately measured with an ELISA reader. According to the product manual, the efficiency of specific killing was calculated as (experimental wells - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum lysis wells of target cells - spontaneous pores of target cells) x 100%.
  • Centrifuge 500g, 5min, 4°C
  • discard the supernatant add 100 ⁇ L RPMI-1640 medium, and resuspend the cells.
  • 1 ⁇ L of FITC anti-human CD3 antibody (biolegend, catalog number: 317306), PEcy7 anti-human CD137 antibody (biolegend, catalog number: 309818) and pHLA-tetramer-APC were added respectively, and the unstained group and isotype control group were set up at the same time, and incubated at 4 degrees for 30 minutes.
  • 100 ⁇ L of RPMI-1640 medium was added, centrifuged (500g, 5min, 4°C), the supernatant was discarded, and repeated once.
  • CD137 The cells up-regulated the expression of CD137, that is, the DCT cells were in an activated state; for negative tumor cells (HepG2) and when there were no tumor cells, the DCT cells did not up-regulate the expression of CD137 and were in a quiescent state (Figure 26c).
  • UCHT1-1G4 ImmTAC
  • Anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects immune cells to kill tumor cells
  • immune cells including CD4 and CD8 double negative T cells (DNT), CD4 + T cells and CD8 + T cells were prepared, and their phenotype and purity were detected by flow cytometry.
  • Method for preparing DCT 5x10 6 immune cells were added to HIPP-T009 culture medium (200 ⁇ L) containing 1x10 -7 mol/L (for CD4 + T cells and CD8 + T cells) or 1x10 -8 mol/L (for DNT) composite protein anti-CD3 antibody (UCHT1) -1G4 (HATima), incubated at 4 degrees for 1 hour, prepared DCT, and the positive rate of DCT was detected by flow cytometry. The results showed that 100% of DNT, CD4 + T cells and CD8 + T cells were coated with anti-CD3 antibody (UCHT1)-1G4 (HATima) composite protein (Figure 27a).
  • the prepared DCT cells or control cells were adjusted to cell suspensions with different densities, namely 1.25x10 3 cells/75 ⁇ L, 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L, 8x10 4 cells/75 ⁇ L, and 16x10 4 cells/75 ⁇ L; positive (HepG2-NYESO-1(NYESO-1 + /HLA-A0201 + )) and negative (HepG2(NYESO-1 - /HLA-A0201 + )) target cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 cells/75 ⁇ L.
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells for each.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • Collect cells from the experimental wells with different effector-target ratios in the 96-well U-bottom plate Centrifuge (500g, 5min, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells. Add 1 ⁇ L FITC anti-human CD3 antibody (biolegend, catalog number: 317306), PEcy7 anti-human CD137 antibody (biolegend, catalog number: 309818) and pHLA-tetramer-APC, respectively, and set up an unstained group and an isotype control group at the same time, and incubate at 4 degrees for 30 minutes.
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (HATima) induced DNT, CD4 + T cells and CD8 + T cells to upregulate the expression of CD137 only after encountering positive target cells (HepG2-NYESO-1), that is, DCT cells showed an activated state; when facing negative tumor cells (HepG2) or when there were no tumor cells, DCT cells did not upregulate the expression of CD137 and showed a resting state (Figure 27c).
  • UCHT1-1G4 HATima
  • Anti-CD3 antibody (UCHT1)-AFP (HATima) redirects immune cells to kill tumor cells
  • immune cells including CD4 and CD8 double negative T cells (DNT), CD4 + T cells and CD8 + T cells were prepared, and their phenotype and purity were detected by flow cytometry.
  • Method for preparing DCT 5x10 6 immune cells were added to HIPP-T009 culture medium (200 ⁇ L) containing 1x10 -8 mol/L composite protein anti-CD3 antibody (UCHT1)-AFP (HATima), incubated at 4 degrees for 1 hour, prepared DCT, and the positive rate of DCT was detected by flow cytometry. The results showed that 100% of DNT, CD4 + T cells and CD8 + T cells were coated with anti-CD3 antibody (UCHT1)-AFP (HATima) composite protein (Figure 28a).
  • UCHT1-AFP HATima
  • the prepared DCT cells or control cells were adjusted using HIPP-T009 culture medium.
  • the cell suspensions were prepared to different densities, namely 2.5x10 3 cells/75 ⁇ L, 5x10 3 cells/75 ⁇ L, 1x10 4 cells/75 ⁇ L, 2x10 4 cells/75 ⁇ L, 4x10 4 cells/75 ⁇ L, 8x10 4 cells/75 ⁇ L, 16x10 4 cells/75 ⁇ L, and 32x10 4 cells/75 ⁇ L; the positive (HepG2 (AFP + /HLA-A0201 + )) and negative (NCI-H1299-A2 (AFP - /HLA-A0201 + )) target cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 cells/75 ⁇ L.
  • a 96-well U-bottom plate 75 ⁇ L of effector cells and 75 ⁇ L of target tumor cells were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and there were 2 duplicate wells.
  • the 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO 2 and cultured for 18 hours. 15 ⁇ L of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • Collect cells from the experimental wells with different effector-target ratios in the 96-well U-bottom plate Centrifuge (500g, 5min, 4°C), discard the supernatant, add 100 ⁇ L RPMI-1640 medium, and resuspend the cells. Add 1 ⁇ L FITC anti-human CD3 antibody (biolegend, catalog number: 317306), PEcy7 anti-human CD137 antibody (biolegend, catalog number: 309818) and pHLA-tetramer-APC, respectively, and set up an unstained group and an isotype control group at the same time, and incubate at 4 degrees for 30 minutes.
  • FITC anti-human CD3 antibody biolegend, catalog number: 317306
  • PEcy7 anti-human CD137 antibody biolegend, catalog number: 309818
  • pHLA-tetramer-APC pHLA-tetramer-APC
  • DCT cells prepared by anti-CD3 antibody (UCHT1)-AFP (HATima) induced DNT, CD4 + T cells and CD8 + T cells to upregulate the expression of CD137 only after encountering positive target cells (HepG2), that is, DCT cells showed an activated state; when facing negative tumor cells (NCI-H1299-A2) and when there were no tumor cells, DCT cells did not regulate the expression of CD137, showing Resting state ( Figure 28c).
  • the anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein bound to the surface of CD8 + T-DCT prepared with 1x10 -8 mol/L concentration of the complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) decreased, and there was no difference between the groups (Figure 28d).
  • Example 4 Comparison of biological activity functions of anti-CD3 antibody (UCHT1) in the HATima molecule and ImmTAC molecule structure used to modify cells
  • the anti-CD3 antibody (UCHT1) in the HATima molecular structure is a heavy chain variable region (SEQ ID NO:9 and SEQ ID NO:10) and a light chain variable region (SEQ ID NO:11 and SEQ ID NO:12), connected by an interchain disulfide bond, namely VH-VL;
  • the anti-CD3 antibody (UCHT1) in the ImmTAC molecular structure is a heavy chain variable region and a light chain variable region, connected by a linker, namely scFv (SEQ ID NO:51 and SEQ ID NO:52).
  • Vector construction and inclusion body expression and purification are the same as 1.1.1.
  • Protein renaturation is the same as 1.1.2, and the dialysis buffer is 10mM PB pH 6.0; the amount of VH and VL inclusion bodies added is 6mg each, and the amount of scFv inclusion bodies added is 6mg. Protein purification is the same as 1.1.3.
  • the results in Figure 29a show that: in the non-reduced state, VH-VL is a single band with a molecular weight of 22kDa; in the reduced state, VH-VL is still a single band, but the molecular weight is reduced to 15kDa, indicating that VH-VL is decomposed into two chains, and the purity of the protein is greater than 90%.
  • scFv is a single band with a molecular weight of 27kDa; in the reduced state, scFv is still a single band with a molecular weight of 27kDa, indicating that scFv is a single chain as a whole, and the purity of the protein is greater than 90%.
  • T cells were adjusted to a cell suspension density of 1x10 4 cells/50 ⁇ L using HIPP-T009 medium.
  • NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were digested with trypsin, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 cells/75 ⁇ L.
  • the protein drugs namely anti-CD3 antibody (UCHT1)-1G4 (HATima), anti-CD3 antibody (UCHT1)-1G4 (ImmTAC), VH-VL and scFv, were diluted with HIPP-T009 medium to different molar concentrations, namely 3x10 -7 M/50 ⁇ L, 3x10 -8 M/50 ⁇ L, 3x10 -9 M/50 ⁇ L, 3x10 -10 M/50 ⁇ L, 3x10 -11 M/50 ⁇ L, 3x10 -12 M/50 ⁇ L, 3x10 -13 M/50 ⁇ L and 0 ⁇ L, respectively.
  • HIPP-T009 medium namely 3x10 -7 M/50 ⁇ L, 3x10 -8 M/50 ⁇ L, 3x10 -9 M/50 ⁇ L, 3x10 -10 M/50 ⁇ L, 3x10 -11 M/50 ⁇ L, 3x10 -12 M/50 ⁇ L, 3x10 -13 M/50 ⁇ L and 0 ⁇ L, respectively
  • a 96-well U-bottom plate 50 ⁇ L of effector cells, 50 ⁇ L of target tumor cells, and 50 ⁇ L of the corresponding concentration of the test protein were added in sequence.
  • the target cell maximum lysis well, target cell spontaneous well, effector cell spontaneous well, culture medium spontaneous well, and culture medium plus lysis solution spontaneous well were set.
  • the final volume of each well was 150 ⁇ L, and Two replicate wells each. Place the 96-well U-bottom plate with cells in a cell culture incubator at 37 degrees and 5% CO 2 and culture for 18 hours. Add 15 ⁇ L of lysis solution to the largest lysis well, mix well, and place in a cell culture incubator at 37 degrees and 5% CO 2 for 45 minutes.
  • VH-VL cannot induce T cells to upregulate CD137 expression.
  • the results in Figure 29d show that when the protein concentration increased to 1E-7M, the expression of NY-ESO-1 in NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) cell group, NCI-H1299 (NY-ESO-1 positive, A2 negative) group and tumor-free cell group increased significantly.
  • VH-VL still could not induce T cells to upregulate the expression of CD137; scFv showed target-independent T cell activation, and only in the NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) cell group, the activation of T cells induced by scFv was lower than that of CD3 antibody (UCHT1)-1G4 (HATima) and anti-CD3 antibody (UCHT1)-1G4 (ImmTAC). The results showed that scFv is an immune effector molecule, and VH-VL is a non-immune effector molecule.

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Abstract

提供了一种修饰细胞，其中所述修饰细胞是由一种复合蛋白结合于所述修饰细胞的细胞表面标记分子而成，其中所述复合蛋白包含特异性结合所述修饰细胞的细胞表面标记分子的部分，以及包含特异性结合靶细胞表面抗原的部分。并提供了一种制备该修饰细胞的方法和使用该修饰细胞治疗疾病的方法。

Description

一种修饰细胞及其用途
优先权
本申请要求2022年9月30日提交的PCT国际申请PCT/CN2022/123635的权益和优先权，其全部内容通过引用并入本文。
技术领域
本公开涉及免疫学领域，特别是涉及一种修饰细胞及其在治疗疾病中的用途。
背景技术
T细胞受体(T cell receptor，TCR)或者嵌合抗原受体(Chimeric Antigen Receptor，CAR)重定向的T细胞，目前使用最多的是病毒载体系统，利用了慢病毒等转基因技术将外源治疗基因插入T细胞的基因组中，以实现基因稳定性表达。这一复杂的过程造成了治疗细胞的基因改造，改变基因的方式可造成治疗后与疗效无关的细胞的滞留，同时基因改造的潜在风险仍然有待深入研究以确保安全，因此病毒载体的生物安全性有待进一步提高。由于导入T细胞的外源治疗基因的整合，可能无意中插入进驱动基因中，使得基因修饰的T细胞存在一定的致瘤风险。目前有研究企图避免细胞滞留的问题，比如引入自杀基因等，可是一旦在某些患者体内出现脱靶效应，即使有自杀基因的引入，也很难清除体内已回输的免疫细胞。因此有研究希望通过非病毒载体系统实现重定向T细胞的目的，认为这样较为安全，但是这种方法向细胞导入目的基因的能力较差，其重定向效率需进一步提升。
发明内容
修饰细胞
一方面，本公开提供了一种修饰细胞，其中所述修饰细胞是由一种复合蛋白结合于所述修饰细胞的细胞表面标记分子而成，其中所述复合蛋白包含特异性结合所述修饰细胞的细胞表面标记分子的部分和特异性结合靶细胞表面抗原的部分。
在一个实施方案中，所述特异性结合靶细胞表面抗原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
在一个实施方案中，所述特异性结合所述修饰细胞的细胞表面标记分子的部分为特异性结合所述修饰细胞的细胞表面标记分子的抗体或其功能片段。
在一个实施方案中，所述修饰细胞的细胞表面标记分子选自CD8、CD4、CD3、NKG2D、CD16、CD2、CD56、CD28或CD26。
在一个实施方案中，所述修饰细胞为免疫细胞，优选为T细胞、γδT细胞、CD4和CD8双阴性T细胞、CD4+T细胞、CD8+T细胞、NK细胞、NKT细胞或单核细胞。
在一个实施方案中，所述靶细胞表面抗原选自pMHC。
在一个实施方案中，在前述的修饰细胞中，所述复合蛋白的结构从N端到C端如式Ia所示：
A-L-B  (Ia)
B-L-A  (Ib)
其中，
元件A包含特异性结合所述修饰细胞的细胞表面标记分子的抗体或其功能片段；
元件B包含特异性结合靶细胞表面抗原的部分；和
元件L为柔性接头；所述柔性接头是可选择的；
“-”为肽键。
在一个实施方案中，在前述的修饰细胞中，所述元件A是一种免疫效应分子；在一个实施方案中，在前述的修饰细胞中，所述元件A也可以是一种非免疫效应分子。
在一个实施方案中，在前述的修饰细胞中，所述元件A包含的抗体为单链抗体scFv。
在一个实施方案中，在前述的修饰细胞中，所述元件A包含的抗体为抗CD3抗体。
在一个实施方案中，在前述的修饰细胞中，所述抗体中的重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列。
在一个实施方案中，在前述的修饰细胞中，所述单链抗体包含OKT3、UCHT-1、 TR66、BMA031或12F6。
在一个实施方案中，在前述的修饰细胞中，所述靶细胞表面抗原选自pMHC。
在一个实施方案中，在前述的修饰细胞中，所述特异性结合靶细胞表面抗原的部分为抗体、TCR样抗体或TCR模拟抗体、TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
在一个实施方案中，在前述的修饰细胞中，所述TCRβ链氨基酸序列包含如SEQ ID NO:5、25或40所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:7、27、42或44所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:5所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:7所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:25所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:27所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:25所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:44所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:40所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:42所示的序列。
在一个实施方案中，在前述的修饰细胞中，所述复合蛋白的结构从N端到C端如式Ic或Id所示：
A2…A1-L-B  (Ic)；
B-L-A1…A2  (Id)；
其中，
元件A1、A2各自独立地包含非免疫效应分子；优选地，所述非免疫效应分子包含抗体的重链可变区或抗体的轻链可变区；
元件B为特异性结合靶细胞表面抗原的部分；和
元件L为柔性接头；所述柔性接头是可选择的；
“-”为肽键；
“…”为二硫键。
在一个实施方案中，在前述的修饰细胞中，A1包含抗体的重链可变区时，A2包含抗体的轻链可变区；或者A2包含抗体的重链可变区时，A1包含抗体的轻链可变区；并且A1和A2之间通过二硫键形成二聚体，优选地，所述二聚体为非免疫效应分子。
在一个实施方案中，在前述的修饰细胞中，所述抗体的重链可变区包含如SEQ ID NO:9所示的序列。
在一个实施方案中，在前述的修饰细胞中，所述抗体的轻链可变区包含如SEQ ID NO:11所示的序列。
在一个实施方案中，在前述的修饰细胞中，所述特异性结合靶细胞表面抗原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
在一个实施方案中，在前述的修饰细胞中，所述TCRβ链氨基酸序列包含如SEQ ID NO:13、17或21所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:15、19或23所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:9所示的序列，轻链可变区包含如SEQ ID NO:11所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:13所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:15所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:9所示的序列，轻链可变区包含如SEQ ID NO:11所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:17所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:19所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:9所示的序列，轻链可变区包含如SEQ ID NO:11所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:21所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:23所示的序列。
组合或试剂盒
另一方面，本公开提供了一种组合或试剂盒，包括a.一种复合蛋白；b.一种细胞；其中所述细胞表达细胞表面标记分子，其中所述复合蛋白包含结合所述细胞的细胞表面标记分子的部分和特异性结合靶细胞表面抗原的部分。
在一个实施方案中，在前述组合或试剂盒中，所述特异性结合靶细胞表面抗 原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
在一个实施方案中，在前述组合或试剂盒中，所述特异性结合所述修饰细胞的细胞表面标记分子的部分为特异性结合所述修饰细胞的细胞表面标记分子的抗体或其功能片段。
在一个实施方案中，在前述组合或试剂盒中，所述细胞的细胞表面标记分子选自CD8、CD4、CD3、NKG2D、CD16、CD2、CD56、CD28或CD26。
在一个实施方案中，在前述组合或试剂盒中，所述细胞为免疫细胞，优选为T细胞、γδT细胞、CD4和CD8双阴性T细胞、CD4+T细胞、CD8+T细胞、NK细胞、NKT细胞或单核细胞。
在一个实施方案中，在前述组合或试剂盒中，所述靶细胞表面抗原选自pMHC。
在一个实施方案中，在前述组合或试剂盒中，所述复合蛋白的结构从N端到C端如式Ia所示：
A-L-B  (Ia)
B-L-A  (Ib)
其中，
元件A包含特异性结合所述修饰细胞的细胞表面标记分子的抗体或其功能片段；
元件B包含特异性结合靶细胞表面抗原的部分；和
元件L为柔性接头；所述柔性接头是可选择的；
“-”为肽键。
在一个实施方案中，在前述组合或试剂盒中，所述元件A是一种免疫效应分子；在一个实施方案中，在前述组合或试剂盒中，所述元件A是一种非免疫效应分子。
在一个实施方案中，在前述组合或试剂盒中，所述元件A包含的抗体为单链抗体scFv。
在一个实施方案中，在前述组合或试剂盒中，所述元件A包含的抗体为抗CD3抗体。
在一个实施方案中，在前述组合或试剂盒中，所述抗体中的重链可变区包含 如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列。
在一个实施方案中，在前述组合或试剂盒中，所述单链抗体包含OKT3、UCHT-1、TR66、BMA031或12F6。
在一个实施方案中，在前述组合或试剂盒中，所述靶细胞表面抗原选自pMHC。
在一个实施方案中，在前述组合或试剂盒中，所述特异性结合靶细胞表面抗原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
在一个实施方案中，在前述的修饰细胞中，所述TCRβ链氨基酸序列包含如SEQ ID NO:5、25或40所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:7、27、42或44所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:5所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:7所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:25所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:27所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:25所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:44所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:40所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:42所示的序列。
在一个实施方案中，在前述组合或试剂盒中，所述复合蛋白的结构从N端到C端如式Ic或Id所示：
A2…A1-L-B  (Ic)；
B-L-A1…A2  (Id)；
其中，
元件A1、A2各自独立地包含非免疫效应分子；优选地，所述非免疫效应分子包含抗体的重链可变区或抗体的轻链可变区；
元件B为特异性结合靶细胞表面抗原的部分；和
元件L为柔性接头；所述柔性接头是可选择的；
“-”为肽键；
“…”为二硫键。
在一个实施方案中，在前述组合或试剂盒中，A1包含抗体的重链可变区时，A2包含抗体的轻链可变区；或者A2包含抗体的重链可变区时，A1包含抗体的轻链可变区；并且A1和A2之间通过二硫键形成二聚体，优选地，所述二聚体为非免疫效应分子。
在一个实施方案中，在前述组合或试剂盒中，所述抗体的重链可变区包含如SEQ ID NO:9所示的序列。
在一个实施方案中，在前述组合或试剂盒中，所述抗体的轻链可变区包含如SEQ ID NO:11所示的序列。
在一个实施方案中，在前述组合或试剂盒中，所述特异性结合靶细胞表面抗原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
在一个实施方案中，在前述组合或试剂盒中，所述TCRβ链氨基酸序列包含如SEQ ID NO:13、17或21所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:15、19或23所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:9所示的序列，轻链可变区包含如SEQ ID NO:11所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:13所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:15所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:9所示的序列，轻链可变区包含如SEQ ID NO:11所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:17所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:19所示的序列。
在一个具体实施方案中，所述重链可变区包含如SEQ ID NO:9所示的序列，轻链可变区包含如SEQ ID NO:11所示的序列；TCRβ链氨基酸序列包含如SEQ ID NO:21所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:23所示的序列。
在一个实施方案中，在前述组合或试剂盒中，所述组合或试剂盒进一步包括可选择性的可药用载体，优选为缓冲液，更优选地，所述缓冲液包含白蛋白或血浆。
治疗方法或用途
再一方面，本公开提供了一种治疗疾病的方法，包含向所需要的受试者施用如前所述的修饰细胞或如前所述的组合或试剂盒。
本公开提供了如前所述的修饰细胞或如前所述的组合或试剂盒在所需要的受试者中治疗疾病的方法中的用途。
本公开提供了如前所述的修饰细胞或如前所述的组合或试剂盒在制备用于在所需要的受试者中治疗疾病的药物中的用途。
在一个具体实施方案中，所述疾病包括癌症、微生物感染性疾病、自身免疫性疾病或衰老性疾病。
制备方法
又一方面，提供了一种制备如前所述的修饰细胞的方法，其中将所述复合蛋白与免疫细胞孵育形成所示修饰细胞。
在一个实施方案中，孵育时间为约1小时以上。
在一个实施方案中，孵育温度约为4℃、室温或37℃。
在一个实施方案中，所述复合蛋白与免疫细胞孵育的缓冲液中含有白蛋白或血浆。
附图说明
参考下述附图，本公开可以被更充分地理解。
图1示出SDS-PAGE法检测复合蛋白的结构和纯度。(a)抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白；(b)抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白；(c)抗CD3抗体(UCHT1)-gp100(HATima)复合蛋白；(d)抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白；(e)抗CD3抗体(UCHT1)-i1G4(ImmTAC)复合蛋白；(f)抗CD3抗体(UCHT1)-igp100(ImmTAC)复合蛋白；(g)抗CD3抗体(UCHT1)-gp100(ImmTAC)复合蛋白。泳道1：分子量标记；泳道2：非还原状态下的多结构域双功能复合分子；泳道3：还原状态下的多结构域双功能融合分子。
图2示出不同浓度的抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的水平。(a)不同浓度的抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的阳性率；(b)不同浓度的抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的平均荧光强度。
图3示出相同浓度抗CD3抗体(UCHT1)-1G4(HATima)分子结合不同数量T细胞的水平。(a)相同浓度抗CD3抗体(UCHT1)-1G4(HATima)分子结合不同数量T细胞的阳性率。
图4示出不同孵育温度下，不同浓度抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的水平。(a)不同浓度抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的阳性率；(b)不同浓度抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的平均荧光强度。
图5示出不同孵育时间下，不同浓度抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的水平。(a)不同浓度抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的阳性率；(b)不同浓度抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的平均荧光强度。
图6示出在不同孵育缓冲液下，抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的水平。(a)抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的阳性率；(b)抗CD3抗体(UCHT1)-1G4(HATima)分子结合T细胞的平均荧光强度。对照组为不含抗CD3抗体(UCHT1)-1G4(HATima)的RPMI-1640培养基组。
图7示出在一定的浓度范围，抗CD3抗体(UCHT1)-1G4(HATima)的结合对T细胞活化的影响。流式细胞术检测共培养系统中CD3阳性细胞群的CD137阳性率。靶细胞为NCI-H1299-A2细胞。
图8示出复合蛋白结合的分子数量对T细胞肿瘤识别功能的影响。(a)相同浓度抗CD3抗体(UCHT1)-1G4(HATima)与不同数量T细胞制备的DCT，流式细胞术检测结合复合蛋白的T细胞的阳性率。(b)乳酸脱氢酶释放实验检测不同方法制备的DCT对肿瘤细胞识别功能的差异。
图9示出对比DCT与TCR-T细胞的肿瘤识别功能差异。(a)流式细胞术检测结合复合蛋白的T细胞和TCR-T细胞的阳性率。(b)乳酸脱氢酶释放实验检测DCT与TCR-T细胞对肿瘤细胞识别功能的差异。
图10示出抗CD3抗体(UCHT1)-1G4(HATima)重定向T细胞杀伤肿瘤细胞。(a)流式细胞术检测T细胞的比例和表型。(b)流式细胞术检测抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白制备的CD3+T-DCT细胞的阳性率。(c)乳酸脱氢酶释放实验，检测CD3+T-DCT细胞对靶细胞的杀伤水平。(d) 流式细胞术检测CD3+T-DCT细胞在不同处境下的CD137表达水平。(e)流式细胞术检测CD3+T-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白结合水平。(f)体外HepG2细胞成瘤模型检测CD3+T-DCT细胞对肿瘤的杀伤作用。
图11示出抗CD3抗体(UCHT1)-1G4(HATima)重定向外周血单个核细胞(PBMC)杀伤肿瘤细胞。(a)流式细胞术检测PBMC中T细胞的比例。(b)流式细胞术检测抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白制备的PBMC-DCT细胞的阳性率。(c)乳酸脱氢酶释放实验，检测PBMC-DCT细胞对靶细胞的杀伤水平。(d)流式细胞术检测PBMC-DCT细胞在不同处境下的CD137表达水平。(e)流式细胞术检测PBMC-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白结合水平。
图12示出抗CD3抗体(UCHT1)-1G4(HATima)重定向外周血淋巴细胞(PBL)杀伤肿瘤细胞。(a)流式细胞术检测PBL中T细胞的比例。(b)流式细胞术检测抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白制备的PBL-DCT细胞的阳性率。(c)乳酸脱氢酶释放实验，检测PBL-DCT细胞对靶细胞的杀伤水平。(d)流式细胞术检测PBL-DCT细胞在不同处境下的CD137表达水平。(e)流式细胞术检测PBL-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白结合水平。
图13示出抗CD3抗体(UCHT1)-1G4(HATima)重定向γδT细胞杀伤肿瘤细胞。(a)流式细胞术检测γδT细胞的比例。(b)流式细胞术检测抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白制备的γδT-DCT细胞的阳性率。(c)乳酸脱氢酶释放实验，检测γδT-DCT细胞和γδT细胞对肿瘤细胞的杀伤水平。(d)流式细胞术检测γδT-DCT细胞和γδT细胞在不同处境下的CD137表达水平。(e)流式细胞术检测γδT-DCT细胞和γδT细胞在不同处境下的抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白结合水平。
图14示出抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向T细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白制备的CD3+T-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测CD3+T-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测CD3+T-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测CD3+T-DCT细胞在不同处境下的抗 CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白结合水平。(e)体外HepG2细胞成瘤模型检测CD3+T-DCT细胞对肿瘤的杀伤作用。
图15示出抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向外周血单个核细胞(PBMC)杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白制备的PBMC-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测PBMC-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测PBMC-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测PBMC-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白结合水平。
图16示出抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向外周血淋巴细胞(PBL)杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白制备的PBL-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测PBL-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测PBL-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测PBL-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白结合水平。
图17示出抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向γδT细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白制备的γδT-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测γδT-DCT细胞和γδT细胞对肿瘤细胞的杀伤水平。(c)流式细胞术检测γδT-DCT细胞和γδT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测γδT-DCT细胞和γδT细胞在不同处境下的抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白结合水平。
图18示出抗CD3抗体(UCHT1)-AFP(HATima)重定向T细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白制备的CD3+T-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测CD3+T-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测CD3+T-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测CD3+T-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白结合水平。
图19示出抗CD3抗体(UCHT1)-AFP(HATima)重定向外周血单个核细胞(PBMC)杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白制备的PBMC-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测PBMC-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测 PBMC-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测PBMC-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白结合水平。
图20示出抗CD3抗体(UCHT1)-AFP(HATima)重定向外周血淋巴细胞(PBL)杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白制备的PBL-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测PBL-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测PBL-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测PBL-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白结合水平。
图21示出抗CD3抗体(UCHT1)-AFP(HATima)重定向γδT细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白制备的γδT-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测γδT-DCT和γδT细胞对靶细胞的杀伤水平。(c)流式细胞术检测γδT-DCT和γδT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测γδT-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白结合水平。
图22示出抗CD3抗体(UCHT1)-gp100(HATima)重定向免疫细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-gp100(HATima)复合蛋白制备的T-DCT、PBMC-DCT、PBL-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测T-DCT、PBMC-DCT、PBL-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测T-DCT、PBMC-DCT、PBL-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测T-DCT、PBMC-DCT、PBL-DCT和CD4+T-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-gp100(HATima)复合蛋白结合水平。
图23示出抗CD3抗体(UCHT1)-gp100(ImmTAC)重定向免疫细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-gp100(ImmTAC)复合蛋白制备的T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT、CD8+T-DCT、PBMC-DCT和PBL-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT、CD8+T-DCT、PBMC-DCT和PBL-DCT细胞对靶细胞 的杀伤水平。(c)流式细胞术检测T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT、CD8+T-DCT、PBMC-DCT和PBL-DCT细胞在不同处境下的CD137表达水平。
图24示出抗CD3抗体(UCHT1)-i1G4(ImmTAC)重定向免疫细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-i1G4(ImmTAC)复合蛋白制备的T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT、CD8+T-DCT、PBMC-DCT和PBL-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT、CD8+T-DCT、PBMC-DCT和PBL-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT、CD8+T-DCT、PBMC-DCT和PBL-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT、CD8+T-DCT、PBMC-DCT和PBL-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-i1G4(ImmTAC)复合蛋白结合水平。
图25示出抗CD3抗体(UCHT1)-igp100(ImmTAC)重定向免疫细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-igp100(ImmTAC)复合蛋白制备的T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-igp100(ImmTAC)复合蛋白结合水平。
图26示出抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向免疫细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白制备的DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测CD4+T-DCT和CD8+T-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白结合水平。
图27示出抗CD3抗体(UCHT1)-1G4(HATima)重定向免疫细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白 制备的DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测CD4+T-DCT和CD8+T-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白结合水平。
图28示出抗CD3抗体(UCHT1)-AFP(HATima)重定向免疫细胞杀伤肿瘤细胞。(a)流式细胞术检测抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白制备的DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞的阳性率。(b)乳酸脱氢酶释放实验，检测DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞对靶细胞的杀伤水平。(c)流式细胞术检测DNT-DCT、CD4+T-DCT和CD8+T-DCT细胞在不同处境下的CD137表达水平。(d)流式细胞术检测CD8+T-DCT细胞在不同处境下的抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白结合水平。
图29示出用于修饰细胞的HATima分子和ImmTAC分子结构中，抗CD3抗体(UCHT1)的生物学活性功能比较。(a)SDS-PAGE法检测VH-VL和scFv蛋白的结构和纯度。泳道1：分子量标记；泳道2：非还原状态下的蛋白；泳道3：还原状态下的蛋白。(b)乳酸脱氢酶释放实验，检测VH-VL和scFv蛋白重定向的T细胞对靶细胞的杀伤水平。(c)流式细胞术检测浓度为1E-9M的VH-VL和scFv蛋白重定向的T细胞在不同处境下的CD137表达水平。(d)流式细胞术检测浓度为1E-7M的VH-VL和scFv蛋白重定向的T细胞在不同处境下的CD137表达水平。CD3抗体(UCHT1)-1G4(HATima)和抗CD3抗体(UCHT1)-1G4(ImmTAC)为阳性对照蛋白。
下面将通过具体描述，对本公开作进一步的说明。
除非另有限定，本文中所使用的所有技术和科学术语具有与本公开所属技术领域的普通技术人员通常理解相同的含义。
本申请中，单数形式“一个”、“该”包括复数对象，除非上下文另外清楚规定。
本文使用的术语“复合蛋白”是包含特异性结合所述修饰细胞的细胞表面标记分子的部分和特异性结合靶细胞表面抗原的部分可选择地以柔性连接肽连接的结构，其中特异性结合所述修饰细胞的细胞表面标记分子的部分可以为特异性结合 所述修饰细胞的细胞表面标记分子的抗体或其片段；所述抗体可以为完整抗体、单链抗体scFv、重链可变区或轻链可变区、或这些抗体的功能性片段。特异性结合靶细胞表面抗原的部分可以为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体。
本文使用的术语“结合”意指可为共价(例如通过化学耦联)或非共价(例如离子相互作用、疏水相互作用、氢键等)的结合或连接。在优选的实施方式中，所述的结合或连接为非共价相互作用。在一个实例中，术语结合意指关联抗原结合部分与其抗原的连接。
本文使用的术语“免疫细胞”包括造血来源并且在免疫反应中起作用的细胞。免疫细胞包括淋巴细胞，如B细胞和T细胞；自然杀手细胞；骨髓细胞，如单核细胞、巨噬细胞、嗜酸性细胞、肥大细胞、嗜碱性细胞及粒细胞。本领域人员应理解，这些免疫细胞可以是任何来源的，包括但不限于原代细胞、传代细胞、或由干细胞诱导后获得得到的细胞。
“疾病”是任何会受益于使用本公开的修饰细胞或组合的医学治疗的症状。病症的非限制性实例包括癌症、微生物感染性疾病、自身免疫性疾病或衰老性疾病。
本文中使用的术语“自身免疫性疾病”通常指的是特征为具有自识别的成分的疾病。自身免疫性疾病的实例包括，但不限于自身免疫性肝炎、多发硬化症、系统性红斑狼疮、特发性血小板减少性紫癜、重症肌无力、I型糖尿病、类风湿性关节炎、银屑病、桥本氏甲状腺炎、格雷夫斯氏病(Grave's disease)、强直性脊柱炎、Sjogrens病(Sjogrens Disease)、CREST综合症、硬皮病、IgA肾病(Neprhopathy)、大疱性类天疱疮、寻常型天疱疮(Pemphigous Vulgaris)、ANCA相关性血管炎、抗磷脂综合症及更多。大多数自身免疫性疾病也是慢性炎性疾病。其被定义为与炎性细胞(白细胞)长期(>6个月)激活相关的疾病过程。慢性炎症导致患者器官或组织的损伤。许多疾病是慢性炎性病症，但已知并不具有自身免疫基础。例如动脉粥样硬化、充血性心力衰竭、克罗恩病(Crohn's disease)、溃疡性结肠炎、结节性多动脉炎、惠普耳氏病(Whipple's Disease)、原发性硬化性胆管炎和更多。
术语“癌症”指哺乳动物中的生理病症，其通常的特征为不受调控的细胞生长/增殖。癌症的实例包括，但不限于：癌(carcinoma)、淋巴瘤、胚细胞瘤和白血病。癌症更具体的实例包括，但不限于：结肠直肠癌、慢性淋巴细胞白血病(CLL)、包 括非小细胞肺癌(NSCLC)的肺癌、乳腺癌、卵巢癌、子宫颈癌、子宫内膜癌、前列腺癌、结肠直肠癌、肠类癌、膀胱癌、胃癌、胰腺癌、肝癌(肝细胞癌)、肝母细胞癌、食道癌、肺腺癌、间皮瘤、滑膜肉瘤、骨肉瘤、头颈鳞状细胞癌、青少年鼻咽纤维血管瘤、脂肪肉瘤、甲状腺癌、黑色素瘤、基底细胞癌(BCC)、髓母细胞瘤和硬纤维瘤。尤其感兴趣的使用本公开的方法治疗的癌症包括神经胶质瘤、髓母细胞瘤、结肠癌、结肠直肠癌、黑素瘤、乳腺癌、肺癌、肝癌和胃癌。
如本文所用，术语“受试者”包括任何人或非人动物。术语“非人类动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，例如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、大鼠、小鼠、两栖动物、爬行动物等。除非另有说明，否则术语“患者”或“受试者”可互换使用。在本公开中，优选的受试者是人类。
如本文所用，术语“治疗”是指向受试者施用有效量的具有根据本文所述的方法离体改变的靶基因的多核苷酸序列的细胞，以使得所述受试者具有所述疾病的至少一种症状的减少或所述疾病的改善，例如，有益的或所需的临床结果。出于本公开的目的，有益的或所需的临床结果包括但不限于一种或多种症状的减轻、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和，以及缓解(无论是部分缓解还是全部缓解)，无论是可检测的或是不可检测的。治疗可指与未接受治疗情况下的预期存活期相比，延长存活期。因此，本领域的技术人员意识到治疗可改善疾病状况，但可能不是疾病的完全治愈。如本文所用，术语“治疗”包括预防。或者，治疗在疾病的进展减少或停止的情况下是“有效的”。“治疗”还可意指与在未接受治疗情况下的预期存活期相比，延长存活期。需要治疗的病人包括已经被诊断具有与多核苷酸序列的表达相关的病症，以及由于遗传易感性或其他因素可能发展这种病症。
“免疫效应分子”免疫效应多肽是通过直接或间接激活免疫系统的体液或细胞组分，例如通过T细胞激活，诱导或刺激免疫应答的分子。免疫效应分子可以是scFv抗体，如抗CD3 scFv。非免疫效应分子，本领域技术人员应理解，其不激活免疫系统的体液或细胞组分，例如不诱导或刺激免疫应答。
实施例
下述实施例中的实验方法，如无特殊说明，均为常规方法。将参照下述非限制性实验实施例进一步理解本公开。
实施例1：材料的制备
1.1复合蛋白的制备
1.1.1载体构建和包涵体表达纯化
将编码复合蛋白基因(参见表1)分别克隆到pET-28a的表达质粒。质粒分别转化到大肠杆菌菌株BL21-DE3中，卡那霉素抗性的单克隆在LB培养基(卡那霉素50μg/mL)中37℃生长至OD600为1.0左右，然后用1mM的IPTG诱导蛋白表达。诱导后3小时，在Thermo Scientific HERAEUS X1R离心机中以4000g离心15分钟收集细胞。使用20mL的BugBuster Master Mix(Merck Millipore)，涡旋震荡重悬菌体，然后在室温振荡处理20分钟后，放入提前预冷好的高速离心机中，6000g，4℃，离心15分钟，然后去除上清。再次加入10mL的BugBuster Master Mix，涡旋震荡重悬沉淀，室温振荡处理5分钟。再次向其中加入30mL的稀释10倍的BugBuster(Merck Millipore)，上下颠倒几次以混匀液体，再次放入高速离心机中，6000g，4℃，离心15分钟。弃上清后，加入30mL的稀释10倍的BugBuster，涡旋震荡重悬沉淀，放入高速离心机中，6000g，4℃，离心15分钟。弃去上清，重复上一步两次。弃去上清，加入30mL的PBS重悬包涵体，并且6000g，4℃，离心15分钟。弃去上清后，加入6M的盐酸胍溶解包涵体。将纯化的包涵体进行倍比稀释，然后取样进行SDS-PAGE检测其纯度和估计其产量，并将所有的包涵体进行蛋白定量，并分装，转入-80℃进行保存。
表1.序列信息




表2.复合蛋白的序列信息






1.1.2蛋白复性
配制TCR的复性缓冲液(5M尿素、100mM Tris pH 8.1、0.4M的L-精氨酸、2mM EDTA、6.5mM半胱胺和1.87mM胱胺)，并提前预冷至4℃。从冻存液中解冻约12mg的TCRα链和11.2mg的抗体重链-TCRβ链，6mg抗体轻链包涵体。分别加入到6mL 6M盐酸胍溶液中，并加入终浓度为15μM的DTT，混匀后，放置在37℃温箱中孵育40分钟。将孵育好的TCRα链和重链-TCRβ链，以及抗体轻链的包涵体分别加入到TCR的复性缓冲液中，并在冷库中反应30分钟。准备10kDa的透析袋，将反应后的复性液加入到透析袋中，然后放入到提前预冷好的去离子水中，在冷库中透析过夜。第二天，将透析袋转移至提前预冷好的10mM Tris-HCl中进行透析。晚上再将透析袋转移至提前预冷好的10mM Tris-HCl中进行透析过夜。
1.1.3蛋白纯化
通过下文所述3步纯化方法将可溶性且正确折叠的多结构域融合分子与错误折叠、降解产物和杂质分离。首先使用阴离子交换纯化。将复性透析好的样品放入提前预冷好的高速离心机中，8000×g，15分钟，4℃以离心去除沉淀，并且再次用0.45μm的滤膜过滤上清。利用阴离子交换柱Q HP(GE Healthcare)来纯化复性的样品。先用A液(10mM Tris-HCl，pH8.0)冲洗4个柱体积后，再以流速为5mL/分钟的流速进行上样。待样品完全上样后，再次用A液冲洗柱子约4个柱体积，当电导和UV280都趋于稳定的时候开始进行梯度洗脱，设置0-100％的B液(1M NaCl+10mM Tris-HCl，PH 8.0)，时间为50分钟，以3mL/分钟的流速洗脱样品，当UV280明显升高时开始收集，各管收集1mL。通过12％SDS-PAGE分析峰组分，然后合并。之后再使用分子筛(Superdex 75，GE Healthcare)纯化。上一步纯化的样品用10kDa的超滤管进行浓缩，以3500×g，4℃浓缩至500 μL，之后进行分子筛纯化。将分子筛先用去离子水进行平衡，然后用PBS再次平衡。清洗500μL的上样环并且进行上样，上样结束后再次用PBS进行平衡及洗脱，流速为1mL/分钟，各管收集0.4mL。通过12％SDS-PAGE分析峰组分，然后合并。最后再进行一次阴离子交换纯化，使用阴离子交换柱Q HP(GE Healthcare)。将上一步纯化的样品使用预冷的10mM Tris稀释20倍，再进行上样。先用A液冲洗4个柱体积后，再以流速为5mL/分钟的流速进行上样。待样品完全上样后，再次用A液冲洗柱子约4个柱体积，当电导和UV280都趋于稳定的时候开始进行梯度洗脱，设置0-100％的B液，时间为50分钟，以3mL/分钟的流速洗脱样品，当UV280明显升高时开始收集，各管收集1mL。通过12％SDS-PAGE分析峰组分，然后合并。用10kDa的超滤管进行浓缩，以3500×g，4℃浓缩至500μL，并使用PBS缓冲液进行缓冲液置换，并测定其浓度，然后分装保存于-80℃。经复性纯化的复合蛋白，采用SDS-PAGE法检测其结构和纯度。图1结果显示：抗CD3抗体(UCHT1)-1G4(HATima)(a)、抗CD3抗体(UCHT1)-AFP(HATima)(b)、抗CD3抗体(UCHT1)-gp100(HATima)(c)和抗CD3抗体(UCHT1)-1G4(HATima)(d)的分子量为70kDa，在还原的状态下分解为3条带，复合蛋白的纯度为80-90％。
1.2外周血单个核细胞(PBMC)的制备
从白膜或外周血中，利用Ficoll密度梯度离心分离法，分离外周血单个核细胞(PBMC)。在50mL离心管中加入20mL淋巴细胞分离液。白膜/外周血与磷酸盐缓冲液(PBS)以1:1的比例混合，稀释血液。用滴管吸取稀释液，沿管壁缓慢叠加于Ficoll层液面上。水平离心(2000rpm，20分钟，室温RT)。吸取云雾层，置入50mL离心管中，加入5倍以上体积的磷酸盐缓冲液(PBS)，离心(1800rpm，10分钟，室温RT)，洗涤细胞。弃上清，加入8mL预热的红细胞裂解液，吹匀，37℃培养箱孵育10分钟。加入30mL的磷酸盐缓冲液(PBS)终止，离心(1200rpm，10分钟，RT)。弃上清，加入完全培养基(RPMI-1640+10％FBS)重悬细胞，Countstar细胞分选仪检测细胞数量和活力。
1.3外周血淋巴细胞(PBL)的制备
收集外周血单个核细胞(PBMC)，离心(300g，5分钟，室温RT)，弃上清。用1mL RPMI-1640培养基重悬细胞，Countstar进行细胞计数，调整细胞密度为2×106个/mL。细胞接种于6孔板，每孔3mL，放置37℃ CO2培养箱1小时。 倒置显微镜下观察细胞贴壁状态，看到孔内铺满“伸展贴壁”的细胞，即可完成单核细胞的贴壁。用移液器吹匀每个角落，重悬外周血淋巴细胞(PBL)。把上清转移至另一15mL离心管。离心(300g，5分钟，RT)，弃上清。用1mL完全RPMI-1640培养基重悬细胞，Countstar进行细胞计数。
1.4 T细胞的制备
采用Stemcell的EasySepTMHuman T Cell Isolation Kit(货号：17951)，从外周血单个核细胞中，分离T细胞。收集PBMC，Countstar进行细胞计数，离心(300g，5分钟，室温RT)，弃上清。用EasySepTMBuffer重悬细胞，并调整细胞密度为5×107个/mL，细胞悬液体积为0.25mL-2mL。细胞悬液转移至聚苯乙烯圆底管(5mL体积)。以50μL/mL的比例，加入Isolation Cocktail。用手指“轻弹”混匀，室温孵育5分钟。振荡Dextran RapidSpheres(微球)30秒，使得微球呈现均匀分散的状态。以40μL/mL的比例，加入RapidSpheresTM，用手指“轻弹”混匀，无需孵育。加入EasySepTMBuffer，至总体积为2.5mL。用移液枪轻吹2-3次混匀。把聚苯乙烯圆底管插入磁铁，室温孵育3分钟。拿起磁铁，以一个连续的运动翻转磁铁和管，将富集的细胞悬浮液倒入一个新的管中。将磁铁和管倒置2-3秒，然后直立返回，不要摇晃或吸掉任何可能挂在管口上的液滴。倒出的细胞为T细胞。计数，离心(300g，5分钟，室温RT)，弃上清。加入含IL-2的完全RPMI-1640培养基(RPMI-1640+10％FBS+100U/mL IL-2，以下简称“培养基”)，重悬，调整细胞密度为1×106个/mL。取一定量样本，用于流式做表型和纯度检测。以1:1的比例，加入Human T-Activator CD3/CD28，混匀，接种于48孔板，每孔500μL，放置37℃ 5％CO2培养箱中培养。4天后，弃磁珠；每2-3天换液一次，更换完全培养基和扩大培养；12天后，取一定量细胞，用于流式做表型和纯度检测；收集细胞，用于冻存和功能实验。流式检测使用的抗体分别为：FITC抗-人CD3抗体(biolegend，货号：317306)；APC抗-人CD4抗体(biolegend，货号：300514)；PE抗-人CD8抗体(biolegend，货号：344706)。
1.5 CD4+T细胞的制备
采用美天旎的CD4 MicroBeads,human(货号：130-045-101)，从T细胞中，阳选CD4+T细胞。配置分离缓冲液，成份为含0.5％牛血清白蛋白和2mM乙二胺四乙酸(EDTA)的磷酸盐缓冲液。收集T细胞，Countstar进行细胞计数，离心(300g，5分钟，室温RT)，弃上清。用分离缓冲液重悬细胞，调整细胞密度 为1×107个/80μL。按照每1×107个细胞需要20μL CD4 MicroBeads的比例，加入CD4 MicroBeads，混匀，4℃放置15分钟。按照每1×107个细胞，加入2mL分离缓冲液，离心(300g，5分钟，室温RT)，弃上清。加入500μL分离缓冲液，重悬细胞，加入已润湿和挂在磁铁的分离柱中，待细胞悬液流过柱子，加入1mL分离缓冲液，冲洗柱子，并重复1次。往柱子中加入2mL分离缓冲液，把柱子移离磁铁，用活塞把2mL分离缓冲液推至15mL离心管中。分离缓冲液中含有CD4+T细胞，计数，离心(300g，5分钟，室温RT)，弃上清。加入含IL-2的完全RPMI-1640培养基(RPMI-1640+10％FBS+100U/mL IL-2，以下简称“培养基”)，重悬，调整细胞密度为1×106个/mL。取一定量样本，用于流式做表型和纯度检测。以1:1的比例，加入Human T-Activator CD3/CD28，混匀，接种于48孔板，每孔500μL，放置37℃ 5％CO2培养箱中培养。4天后，弃磁珠；每2-3天换液一次，更换培养基和扩大培养；12天后，取一定量细胞，用于流式做表型和纯度检测；收集细胞，用于冻存和功能实验。流式检测使用的抗体分别为：FITC抗-人CD3抗体(biolegend，货号：317306)；APC抗-人CD4抗体(biolegend，货号：300514)。
1.6 CD8+T细胞的制备
采用美天旎的CD8 MicroBeads,human(货号：130-045-201)，从T细胞中，阳选CD8+T细胞。配置分离缓冲液，成份为含0.5％牛血清白蛋白和2mM乙二胺四乙酸(EDTA)的磷酸盐缓冲液。收集T细胞，Countstar进行细胞计数，离心(300g，5分钟，RT室温)，弃上清。用分离缓冲液重悬细胞，调整细胞密度为1×107个/80μL。按照每1×107个细胞需要20μL CD8 MicroBeads的比例，加入CD8 MicroBeads，混匀，4度放置15分钟。按照每1×107个细胞，加入2mL分离缓冲液，离心(300g，5分钟，RT室温)，弃上清。加入500μL分离缓冲液，重悬细胞，加入已润湿和挂在磁铁的分离柱中，待细胞悬液流过柱子，加入1mL分离缓冲液，冲洗柱子，并重复1次。往柱子中加入2mL分离缓冲液，把柱子移离磁铁，用活塞把2mL分离缓冲液推至15mL离心管中。分离缓冲液中含有CD8+T细胞，计数，离心(300g，5分钟，RT室温)，弃上清。加入含IL-2的完全RPMI-1640培养基(RPMI-1640+10％FBS+100U/mL IL-2，以下简称“培养基”)，重悬，调整细胞密度为1×106个/mL。取一定量样本，用于流式做表型和 纯度检测。以1:1的比例，加入Human T-Activator CD3/CD28，混匀，接种于48孔板，每孔500μL，放置37℃ 5％CO2培养箱中培养。4天后，弃磁珠；每2-3天换液一次，更换培养基和扩大培养；12天后，取一定量细胞，用于流式做表型和纯度检测；收集细胞，用于冻存和功能实验。流式检测使用的抗体分别为：FITC抗-人CD3抗体(biolegend，货号：317306)；PE抗-人CD8抗体(biolegend，货号：344706)。
1.7双阴性T细胞(DNT)的制备
采用美天旎的CD8 MicroBeads,human(货号：130-045-201)和CD4 MicroBeads,human(货号：130-045-101)，从T细胞中，阴选CD4-CD8-双阴性T细胞，以下简称双阴性T细胞。配置分离缓冲液，成份为含0.5％牛血清白蛋白和2mM乙二胺四乙酸(EDTA)的磷酸盐缓冲液。收集T细胞，Countstar进行细胞计数，离心(300g，5分钟，RT室温)，弃上清。用分离缓冲液重悬细胞，调整细胞密度为1×107个/80μL。按照每1×107个细胞需要20μL CD8 MicroBeads和20μLCD4 MicroBeads的比例，同时加入CD8 MicroBeads和CD4 MicroBeads，混匀，4度放置15分钟。按照每1×107个细胞，加入2mL分离缓冲液，离心(300g，5分钟，RT室温)，弃上清。加入1mL分离缓冲液，重悬细胞，加入已润湿和挂在磁铁的分离柱中，收集流穿柱子的液体，该液体中即含有双阴性T细胞，计数，离心(300g，5分钟，RT室温)，弃上清。加入含IL-2的完全RPMI-1640培养基(RPMI-1640+10％FBS+100U/mL IL-2，以下简称“培养基”)，重悬，调整细胞密度为1×106个/mL。取一定量样本，用于流式做表型和纯度检测。以1:1的比例，加入Human T-Activator CD3/CD28，混匀，接种于48孔板，每孔500μL，放置37℃ 5％CO2培养箱中培养。4天后，弃磁珠；每2-3天换液一次，更换培养基和扩大培养；12天后，取一定量细胞，用于流式做表型和纯度检测；收集细胞，用于冻存和功能实验。流式检测使用的抗体分别为：FITC抗-人CD3抗体(biolegend，货号：317306)；PE抗-人CD8抗体(biolegend，货号：344706)；APC抗-人CD4抗体(biolegend，货号：300514)。
1.8 γδT细胞的制备
收集外周血单个核细胞，计数，离心(300g，5分钟，RT室温)，弃上清。用完全培养基(ImmunoCultTM-XF T cell expansion medium+5％FBS+100U/mL IL-2+10ng/ml IL-15)重悬细胞，调整细胞密度为2×106个/mL，添加唑来膦酸 (Zoledronate，InvivoChem，货号：V1560)，工作浓度为5μM。细胞接种于24孔板，放置37℃ 5％CO2培养箱中培养。每2-3天换液一次，更换完全培养基和扩大培养，但不添加唑来膦酸；12-14天后，取一定量细胞，用于流式做表型和纯度检测；收集细胞，用于冻存和功能实验。流式检测使用的抗体分别为：FITC抗-人CD3抗体(biolegend，货号：317306)；PE抗-人TCR Vδ2抗体(biolegend，货号：331408)。
1.9复合蛋白包被于免疫细胞的表面，即DCT的制备
从复合蛋白的浓度、免疫细胞的数量、缓冲液中血清/血浆蛋白/白蛋白等的浓度、孵育系统体积、孵育温度、孵育时间等参数的不同组合，把复合蛋白分别加入上述各种免疫细胞中孵育，流式检测被复合蛋白结合的免疫细胞的比例和荧光强度，ELISA检测系统中残留的复合蛋白的浓度，并检测和对比其对肿瘤的识别功能。
实施例2：细胞表面结合复合蛋白分子数的可调控性
2.1不同复合蛋白的浓度
复合蛋白以抗CD3抗体(UCHT1)-1G4(HATima)为例子。在3×105个T细胞/100μL RPMI-1640培养基的细胞悬液中，加入复合蛋白，终浓度分别为3×10-7mol/L、3×10-8mol/L、3×10-9mol/L、3×10-10mol/L和0mol/L，4度孵育30分钟。离心(500g，5分钟，4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。加入1μL pHLA-tetramer-APC，4度孵育1小时。加入1mL RPMI-1640培养基，离心(500g，5分钟，4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测pHLA-tetramer-APC的荧光信号。pHLA-tetramer-APC的荧光信号代表结合复合蛋白的T细胞的比例及强度，其与T细胞表面结合的复合蛋白的数量成正相关。附图2所示：工作浓度为3×10-7mol/L、3×10-8mol/L、3×10-9mol/L和3×10-10mol/L的复合蛋白，结合T细胞的阳性率分别为98.9％、98.2％、47.7％和0.62％(图2a)，其荧光强度分别为：15413、14614、6465、827(图2b)。随着复合蛋白浓度的下降，其结合于T细胞表面的分子数量下降。
2.2复合蛋白结合不同数量的T细胞
复合蛋白以抗CD3抗体(UCHT1)-1G4(HATima)为例子。在含1x10-9mol/L复合蛋白的100μL RPMI-1640培养基中，加入不同数量的T细胞，分别为4×106 个、2×106个、1×106个和0.3×106个，4度孵育1小时。离心(500g,5分钟，4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。加入1μL pHLA-tetramer-APC，4度孵育1小时。加入1mL RPMI-1640培养基，离心(500g，5分钟，4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测pHLA-tetramer-APC的荧光信号。pHLA-tetramer-APC的荧光信号代表结合复合蛋白的T细胞的比例及强度，其与T细胞表面结合的复合蛋白的数量成正相关。附图3所示：在100μL RPMI-1640培养基体系中，1×10-9mol/L抗CD3抗体(UCHT1)-1G4(HATima)结合4×106个、2×106个、1×06个和0.3×106个T细胞，阳性率分别是37.9％、48.4％、62.4％和65.9％(图3)。随着T细胞数量的下降，同一浓度复合蛋白结合于T细胞表面的分子数量上升。
2.3在不同孵育温度下，复合蛋白结合T细胞的差异
复合蛋白以抗CD3抗体(UCHT1)1G4(HATima)为例子。在3×105个T细胞/100μL RPMI-1640培养基的细胞悬液中，加入复合蛋白，终浓度分别为3×10-7mol/L、3×10-8mol/L、3×10-9mol/L、2×10-9mol/L、1×10-9mol/L、3×10-10mol/L和0mol/L，分别在4度和37度孵育1小时。离心(500g，5分钟，4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。加入1μL pHLA-tetramer-APC，4度孵育1小时。加入1mL RPMI-1640培养基，离心(500g，5分钟，4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测pHLA-tetramer-APC的荧光信号。pHLA-tetramer-APC的荧光信号代表结合复合蛋白的T细胞的比例及强度，其与T细胞表面结合的复合蛋白的数量成正相关。附图4所示：工作浓度为3×10-7mol/L、3×10-8mol/L、3×10-9mol/L、2×10-9mol/L、1×10-9mol/L和3×10-10mol/L的复合蛋白，在4度下孵育，结合T细胞的阳性率分别为98.9％、98.9％、77.8％、62.9％、40.7％和7.26％；在37度下孵育，结合T细胞的阳性率分别为95.4％、89.8％、27.4％、14.8％、8.13％和0.57％(图4a)。在相同浓度下，4度孵育组的平均荧光强度均高于37度孵育组(图4b)。高的孵育温度会降低结合于T细胞表面的复合蛋白的数量，尤其对低浓度的复合蛋白的影响较为明显，下降率达80％(工作浓度为1×10-9mol/L)。
2.4在不同孵育时间下，复合蛋白结合T细胞的差异
复合蛋白以抗CD3抗体(UCHT1)-1G4(HATima)为例子。在3×105个T细胞/100μL RPMI-1640培养基的细胞悬液中，加入复合蛋白，终浓度分别为3×10-8 mol/L、3×10-9mol/L、2×10-9mol/L和1×10-9mol/L，在37度分别孵育1小时和18小时。离心(500g，5分钟，4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。加入1μL pHLA-tetramer-APC，4度孵育1小时。加入1mL RPMI-1640培养基，离心(500g，5分钟，4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测pHLA-tetramer-APC的荧光信号。pHLA-tetramer-APC的荧光信号代表结合复合蛋白的T细胞的比例及强度，其与T细胞表面结合的复合蛋白的数量成正相关。附图5所示：工作浓度为3×10-8mol/L、3×10-9mol/L、2×10-9mol/L和1×10-9mol/L的复合蛋白，在37度孵育1小时，结合T细胞的阳性率分别为89.8％、27.4％、14.8％和8.13％；在37度下孵育18小时，结合T细胞的阳性率分别为36.5％、1.15％、0.047％和0％(图5a)。在相同浓度和孵育温度下，孵育1小时组的平均荧光强度均高于孵育18小时组(图5b)。在37度下长时间的孵育，显著降低结合于T细胞表面的复合蛋白的数量，尤其对低浓度的复合蛋白的影响较为明显，下降率达95％(工作浓度为3×10-9mol/L)。
2.5孵育缓冲液成份对复合蛋白结合T细胞的影响
复合蛋白以抗CD3抗体(UCHT1)-1G4(HATima)为例子。制备不同的孵育缓冲液，分别是RPMI-1640培养基、磷酸盐缓冲液、含30μg/mL小鼠白蛋白的磷酸盐缓冲液和含10％小鼠血浆的磷酸盐缓冲液，4度保存。不同的孵育缓冲液稀释复合蛋白，至工作浓度为2×10-9mol/L，37度放置1小时，降温至4度待用；重悬T细胞，至细胞密度为3×105个/50μL。相同孵育缓冲液的混合，即50μL T细胞悬液与50μL复合蛋白混合，4度孵育1小时。离心(500g，5分钟，4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。加入1μL pHLA-tetramer-APC，4度孵育1小时。加入1mL RPMI-1640培养基，离心(500g，5分钟，4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测pHLA-tetramer-APC的荧光信号。pHLA-tetramer-APC的荧光信号代表结合复合蛋白的T细胞的比例及强度，其与T细胞表面结合的复合蛋白的数量成正相关。附图6所示：在RPMI-1640培养基、磷酸盐缓冲液、含30μg/mL小鼠白蛋白的磷酸盐缓冲液和含10％小鼠血浆的磷酸盐缓冲液等孵育缓冲液的处理下，1×10-9mol/L的复合蛋白，结合T细胞的阳性率分别为2.12％、1.79％、90.6％和98.4％(图6a)；其平均荧光强度分别为818、897、5890和10455(图6b)。血浆中的成分，如白蛋白，提高复合蛋白与CD3抗原的结合，增加T细胞表面结合的复合蛋白的数量。
2.6在一定的浓度范围，复合蛋白的结合对T细胞活化的影响
复合蛋白以抗CD3抗体(UCHT1)-1G4(HATima)为例子。用HIPP-T009培养基，配置细胞密度为3×105个/100μL的T细胞和NCI-H1299-A2靶细胞悬液。在96孔U底板中，加入100μL T细胞悬液，再加入复合蛋白，混匀，终浓度分别为3×10-8mol/L、3×10-9mol/L、2×10-9mol/L和1×10-9mol/L，4度孵育1小时，制备DCT。分别加入100μL NCI-H1299-A2靶细胞悬液和HIPP-T009培养基。放置37度5％CO2培养箱培养18小时。整块96孔板离心(500g，5分钟，4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)和PEcy7抗-人CD137抗体(biolegend，货号：309818)，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g，5分钟，4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体和PEcy7抗-人CD137的荧光信号，分析CD3阳性细胞群的CD137比例，代表T细胞的活化程度。附图7所示：3×10-8mol/L、3×10-9mol/L、2×10-9mol/L和1×10-9mol/L复合蛋白制备的DCT，在没有接触靶细胞时，18小时的培养，T细胞的活化率分别是17.2％、3.91％、2.01％和1.07％；当DCT与NCI-H1299-A2靶细胞共培养18小时后，T细胞的活化率分别是78.7％、54.2％、69.1％和69.3％。工作浓度低于3×10-9mol/L的抗CD3抗体(UCHT1)-1G4(HATima)制备的DCT，T细胞不因复合蛋白的结合而活化。
2.7复合蛋白结合的分子数量对T细胞肿瘤识别功能的影响
复合蛋白以抗CD3抗体(UCHT1)-1G4(HATima)为例子。DCT制备的方法为：在含1×10-9mol/L复合蛋白的300μL HIPP-T009培养基中，分别加入0.9×106个和6×106个的T细胞，4度孵育1小时。等同于：在含1×10-9mol/L复合蛋白的100μL HIPP-T009培养基中，分别加入0.3×106个和2×106个的T细胞，4度孵育1小时。各取15μL和50μL的细胞悬液，离心(500g，5分钟，4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。加入1μL APC抗-人TCR Vβ13.1抗体(biolegend，货号：362408)，4度孵育1小时。加入1mL RPMI-1640培养基，离心(500g，5分钟，4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测anti-hTCR Vβ13.1-APC的荧光信号。附图8a结果显示：在1x10-9mol/L复合蛋白作用下，0.3×106个T细胞组的结合率为98.4％，2×106个的 T细胞的结合率为75.5％。在相同浓度的复合蛋白作用下，细胞数量少的T细胞结合更多的复合蛋白分子数量。采用HIPP-T009培养基，把已制备好的DCT，制备不同密度的细胞悬液，分别是1.25×103个/75μL、2.5×103个/75μL、5×103个/75μL和1×104个/75μL；用胰酶消化HepG2-NYESO1(NY-ESO-1阳性，A2阳性)、HepG2(NY-ESO-1阴性，A2阳性)、NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为1×104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各有2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，并且加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)×100％。附图8b结果显示：在效靶比为1:4、1:2和1:1下，0.3×106个T细胞制备的DCT对阳性靶细胞(HepG2-NYESO1和NCI-H1299-A2)的特异性杀伤率高于2×106个T细胞制备的DCT，具有效靶比梯度依赖性，尤其是效靶比为1:1时，具有显著性差异。2种方法制备的DCT对阴性靶细胞(HepG2和NCI-H1299)均无杀伤效应。T细胞表面结合的复合蛋白的分子数量与其肿瘤识别功能呈正相关。
实施例3：修饰细胞的肿瘤识别和杀伤功能
3.1 DCT的肿瘤识别功能与TCR-T细胞的差异
TCR-T细胞的制备方法：利用NheI和SalI酶切位点，把TCRα和β链序列插入慢病毒表达载体pGZ178中，与包装质粒(pMDLg/pRRE、pRSV-REV和pMD.2G)混匀，在转染试剂PEI-MAX的作用下，转染293T细胞，培养3天。收集含慢病毒颗粒的培养上清，用浓缩管浓缩，分装后-80℃冻存，并检测其滴度。包被抗CD3/CD28抗体的磁珠联合IL-2(100IU/mL)刺激T细胞，24小时后，加入protamine(10μg/mL)和含TCR基因的慢病毒液，混匀，培养7天。复合 蛋白以抗CD3抗体(UCHT1)-1G4(HATima)为例子。DCT制备的方法为：在含1×10-7mol/L复合蛋白的200μL HIPP-T009培养基中，加入5×106个的T细胞，4度孵育1小时。各取0.3×106个细胞，离心(500g，5分钟，4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。加入1μL pHLA-tetramer-APC，4度孵育1小时。加入1mL RPMI-1640培养基，离心(500g，5分钟，4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测pHLA-tetramer-APC的荧光信号。附图9a结果显示：在1×10-7mol/L复合蛋白作用下，5×106个T细胞制备的DCT的阳性率为97.4％；本批次TCR-T细胞的阳性率为27.3％。往DCT细胞中，加入相同志愿者来源的T细胞，把DCT细胞的阳性率调整为27.3％。采用HIPP-T009培养基，把已调整好的DCT和TCR-T细胞，制备不同密度的细胞悬液，分别是1.25×103个/75μL、2.5×103个/75μL、5×103个/75μL和1×104个/75μL；用胰酶消化HepG2-NYESO1(NY-ESO-1阳性，A2阳性)、HepG2(NY-ESO-1阴性，A2阳性)、NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为1×104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)×100％。附图9b结果显示：在效靶比为1:8、1:4和1:2下，5×106个T细胞制备的DCT对HepG2-NYESO1阳性靶细胞的特异性杀伤率显著性高于TCR-T细胞，具有效靶比梯度依赖性；在效靶比为1:2和1:1下，5×106个T细胞制备的DCT对H1299-A2阳性靶细胞的特异性杀伤率等同于TCR-T细胞，具有效靶比梯度依赖性。DCT和TCR-T细胞对阴性靶细胞(HepG2和NCI-H1299)均无杀伤效应。利用不同因素组合制备的DCT，其对肿瘤的识别功能可以优于TCR-T细胞。
3.2抗CD3抗体(UCHT1)-1G4(HATima)重定向T细胞杀伤肿瘤细胞
利用Human T-Activator CD3/CD28，扩增经磁珠分选的T细胞，培养12天，流式细胞术检测T细胞的纯度，以及CD8和CD4的比例。结果显示：扩增培养后的细胞，CD3阳性率>99％，CD8与CD4的比值为2.5:1，CD4与CD8的总阳性率为95％(图10a)。以“在含1x10-7mol/L复合蛋白抗CD3抗体(UCHT1)-1G4(HATima)的200μL HIPP-T009培养基中，加入5x106个的T细胞，4度孵育1小时”的方案，制备CD3+T-DCT，流式检测DCT的阳性率。结果显示：大于99％的T细胞包被CD3抗体(UCHT1)-1G4(HATima)复合蛋白(图10b)。
采用HIPP-T009培养基，把制备好的CD3+T-DCT，调整至不同密度的细胞悬液，分别是1x103个/75μL、1.25x103个/75μL、2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL和8x104个/75μL；用胰酶消化HepG2-NYESO1(NY-ESO-1阳性，A2阳性)、HepG2(NY-ESO-1阴性，A2阳性)、NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为1x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-1G4(HATima)重定向的CD3+T-DCT高效杀伤HepG2-NYESO1和NCI-H1299-A2等阳性靶肿瘤细胞，并具有效靶比梯度依赖性，对HepG2和NCI-H1299等阴性肿瘤细胞不识别(图10c)。
收集96孔U底板中效靶比为1:2的HepG2-NYESO-1/DCT、HepG2/DCT和仅有DCT组的细胞；4:1的NCI-H1299-A2/DCT、NCI-H1299/DCT和仅有DCT组 的细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表T细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-1G4(HATima)制备的CD3+T-DCT细胞，在遇到阳性靶细胞后，才诱导T细胞上调CD137的表达，即T细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图10d)，尽管T细胞表面结合高水平的抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白(图10e)。
采用B-NDG小鼠(购自百奥赛图)构建HepG2-NYESO-1荷瘤模型，即B-NDG小鼠皮下注射5x106个HepG2-NYESO-1细胞，肿瘤生长至14天，瘤体平均体积为226mm3。小鼠随机分为对照组和CD3+T-DCT组，每组6只。静脉回输2x107个T细胞，1小时后，DCT组回输5x106个CD3+T-DCT细胞，DCT的制备同细胞实验采用的DCT；对照组的T细胞不负载复合蛋白。每隔一天回输1次CD3+T-DCT细胞，共回输7次。在T细胞回输当天起，腹腔注射IL-2(50000U)，连续14天，每天1次。每隔3-4天，用游标卡测量肿瘤的长和宽，采用公式“(长x宽x宽)x0.5”计算肿瘤的体积。当其中一组平均肿瘤体积达到2000mm3，二氧化碳安乐死处死小鼠，实验到达终点。结果显示：在CD3+T-DCT回输后第11天，DCT组肿瘤被清除，观察至实验终点(第53天)，肿瘤仍然不复发；对照组肿瘤正常生长，至第53天，平均肿瘤体积为2229mm3(图10f)。CD3抗体(UCHT1)-1G4(HATima)重定向的T细胞，体内外均能有效杀伤肿瘤细胞，同时不造成T细胞的“自发性”活化。
3.3抗CD3抗体(UCHT1)-1G4(HATima)重定向外周血单个核细胞(PBMC)杀伤肿瘤细胞
通过Ficoll密度梯度离心分离法，分离获得外周血单个核细胞(PBMC)，流式检测结果显示：CD3阳性细胞群为62.3％(图11a)。以“在含1x10-7mol/L复 合蛋白抗CD3抗体(UCHT1)-1G4(HATima)的200μL HIPP-T009培养基中，加入5x106个的PBMC，4度孵育1小时”的方案，制备PBMC-DCT，流式检测DCT的阳性率。结果显示：CD3细胞群与pHLA-tetramer细胞群互为阳性，即复合蛋白仅结合T细胞，结合率为100％(图11b)。
采用HIPP-T009培养基，把制备好的PBMC-DCT，调整至不同密度的细胞悬液，分别是1.25x103个/75μL、2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL和8x104个/75μL；用胰酶消化HepG2-NYESO1(NY-ESO-1阳性，A2阳性)、HepG2(NY-ESO-1阴性，A2阳性)、NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为1x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-1G4(HATima)重定向的PBMC-DCT高效杀伤HepG2-NYESO1和NCI-H1299-A2等阳性靶肿瘤细胞，并具有效靶比梯度依赖性，对HepG2和NCI-H1299等阴性肿瘤细胞不识别(图11c)。
收集96孔U底板中效靶比为2:1的HepG2-NYESO-1/DCT、HepG2/DCT和仅有DCT组的细胞；4:1的NCI-H1299-A2/DCT、NCI-H1299/DCT和仅有DCT组的细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC 的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表T细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-1G4(HATima)制备的PBMC-DCT细胞，在遇到阳性靶细胞后，才诱导PBMC中的T细胞上调CD137的表达，即T细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图11d)，尽管T细胞表面结合高水平的抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白(图11e)。
3.4抗CD3抗体(UCHT1)-1G4(HATima)重定向外周血淋巴细胞(PBL)杀伤肿瘤细胞
通过“贴壁”法，去除外周血单个核细胞(PBMC)中的单核细胞，获得外周血淋巴细胞(PBL)。流式检测结果显示：CD3阳性细胞群为82.7％(图12a)。以“在含1x10-7mol/L复合蛋白抗CD3抗体(UCHT1)-1G4(HATima)的200μL HIPP-T009培养基中，加入5x106个的PBL，4度孵育1小时”的方案，制备PBL-DCT，流式检测DCT的阳性率。结果显示：CD3细胞群与pHLA-tetramer细胞群互为阳性，即复合蛋白仅结合T细胞，结合率为100％(图12b)。
采用HIPP-T009培养基，把制备好的PBL-DCT，调整至不同密度的细胞悬液，分别是1.25x103个/75μL、2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL和8x104个/75μL；用胰酶消化HepG2-NYESO1(NY-ESO-1阳性，A2阳性)、HepG2(NY-ESO-1阴性，A2阳性)、NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为1x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝ (实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-1G4(HATima)重定向的PBL-DCT高效杀伤HepG2-NYESO1和NCI-H1299-A2等阳性靶肿瘤细胞，并具有效靶比梯度依赖性，对HepG2和NCI-H1299等阴性肿瘤细胞不识别(图12c)。
收集96孔U底板中效靶比为2:1的HepG2-NYESO-1/DCT、HepG2/DCT和仅有DCT组的细胞；4:1的NCI-H1299-A2/DCT、NCI-H1299/DCT和仅有DCT组的细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表T细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-1G4(HATima)制备的PBL-DCT细胞，在遇到阳性靶细胞后，才诱导PBL中的T细胞上调CD137的表达，即T细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图12d)，尽管T细胞表面结合高水平的抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白(图12e)。
3.5抗CD3抗体(UCHT1)-1G4(HATima)重定向γδT细胞杀伤肿瘤细胞
采用唑来膦酸(5μM)，扩增外周血单个核细胞中的γδT细胞，在完全培养基(ImmunoCultTM-XF T cell expansion medium+5％FBS+200U/mL IL-2+10ng/mL IL-15)中培养12天，流式检测其表型和纯度。结果显示：87.8％的细胞为TCR Vd2/CD3双阳性，即在扩增培养的细胞中，γδT细胞的比例为87.8％(图13a)。以“在含1x10-8mol/L复合蛋白抗CD3抗体(UCHT1)-1G4(HATima)的200μL HIPP-T009培养基中，加入5x106个的γδT细胞，4度孵育1小时”的方案，制备γδT-DCT，流式检测DCT的阳性率。结果显示：97.8％的γδT细胞包被抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白(图13b)。
采用HIPP-T009培养基，把制备好的γδT-DCT和γδT细胞，调整至不同密度的细胞悬液，分别是5x102个/75μL、1x103个/75μL、2x103个/75μL和4x103个 /75μL；1x104个/75μL、2x104个/75μL、4x104个/75μL和8x104个/75μL；用胰酶消化HepG2-NYESO1(NY-ESO-1阳性，A2阳性)、HepG2(NY-ESO-1阴性，A2阳性)、NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为2x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-1G4(HATima)重定向的γδT-DCT高效杀伤HepG2-NYESO1和NCI-H1299-A2等阳性靶肿瘤细胞，并具有效靶比梯度依赖性；其对HepG2和NCI-H1299等阴性肿瘤细胞的识别，等同于γδT细胞对肿瘤细胞的识别水平(图13c)。
收集96孔U底板中，效靶比为1:5的HepG2-NYESO-1/DCT、HepG2/DCT、仅有DCT、HepG2-NYESO-1/γδT、HepG2/γδT、仅有γδT组的细胞；2:1的NCI-H1299-A2/DCT、NCI-H1299/DCT、仅有DCT、NCI-H1299-A2/γδT、NCI-H1299/γδT、仅有γδT组的细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表γδT-DCT或γδT细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-1G4(HATima)制备的γδT-DCT细胞，在遇到阳性靶细胞后，才诱导T细胞上调CD137的表达，即γδT-DCT细 胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，γδT-DCT细胞不上调CD137的表达，表现为静息状态，与γδT结果一致(图13d)。1x10-8mol/L浓度的复合蛋白CD3抗体(UCHT1)-1G4(HATima)制备的γδT-DCT，在细胞培养18小时后，流式检测不到细胞表面结合的抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白(图13e)。
3.6抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向T细胞杀伤肿瘤细胞
利用Human T-Activator CD3/CD28，扩增经磁珠分选的T细胞，培养12天，流式细胞术检测T细胞的纯度，以及CD8和CD4的比例。结果显示：扩增培养后的细胞，CD3阳性率>99％，CD8与CD4的比值为2.5:1，CD4与CD8的总阳性率为95％(图10a)。以“在含1x10-7mol/L复合蛋白抗CD3抗体(UCHT1)-1G4(ImmTAC)的200μL HIPP-T009培养基中，加入5x106个的T细胞，4度孵育1小时”的方案，制备CD3+T-DCT，流式检测DCT的阳性率。结果显示：大于99％的T细胞包被抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白(图14a)。
采用HIPP-T009培养基，把制备好的CD3+T-DCT，调整至不同密度的细胞悬液，分别是1x103个/75μL、1.25x103个/75μL、2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL和8x104个/75μL；用胰酶消化HepG2-NYESO1(NY-ESO-1阳性，A2阳性)、HepG2(NY-ESO-1阴性，A2阳性)、NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为1x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-1G4 (ImmTAC)重定向的CD3+T-DCT高效杀伤HepG2-NYESO1和NCI-H1299-A2等阳性靶肿瘤细胞，并具有效靶比梯度依赖性，对HepG2和NCI-H1299等阴性肿瘤细胞不识别(图14b)。
收集96孔U底板中效靶比为1:2的HepG2-NYESO-1/DCT、HepG2/DCT和仅有DCT组的细胞；4:1的NCI-H1299-A2/DCT、NCI-H1299/DCT和仅有DCT组的细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表T细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-1G4(ImmTAC)制备的CD3+T-DCT细胞，在遇到阳性靶细胞后，才诱导T细胞上调CD137的表达，即T细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图14c)，尽管T细胞表面结合高水平的抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白(图14d)。
采用B-NDG小鼠(购自百奥赛图)构建HepG2-NYESO-1荷瘤模型，即B-NDG小鼠皮下注射5x106个HepG2-NYESO-1细胞，肿瘤生长至14天，瘤体平均体积为226mm3。小鼠随机分为对照组和CD3+T-DCT组，每组6只。静脉回输2x107个T细胞，1小时后，DCT组回输5x106个CD3+T-DCT细胞，DCT的制备同细胞实验采用的DCT；对照组的T细胞不负载复合蛋白。每隔一天回输1次CD3+T-DCT细胞，共回输7次。在T细胞回输当天起，腹腔注射IL-2(50000U)，连续14天，每天1次。每隔3-4天，用游标卡测量肿瘤的长和宽，采用公式“(长x宽x宽)x0.5”计算肿瘤的体积。当其中一组平均肿瘤体积达到2000mm3，二氧化碳安乐死处死小鼠，实验到达终点。结果显示：在CD3+T-DCT回输后第11天，DCT组肿瘤被清除，观察至实验终点(第53天)，肿瘤仍然不复发；对照组肿瘤正常生长，至第53天，平均肿瘤体积为2229mm3(图14e)。抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向的T细胞，体内外均能有效杀伤肿瘤细胞， 同时不造成T细胞的“自发性”活化。
3.7抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向外周血单个核细胞(PBMC)杀伤肿瘤细胞
通过Ficoll密度梯度离心分离法，分离获得外周血单个核细胞(PBMC)，流式检测结果显示：CD3阳性细胞群为61.4％(图15a)。以“在含1x10-7mol/L复合蛋白抗CD3抗体(UCHT1)-1G4(ImmTAC)的200μL HIPP-T009培养基中，加入5x106个的PBMC，4度孵育1小时”的方案，制备PBMC-DCT，流式检测DCT的阳性率。结果显示：CD3细胞群与pHLA-tetramer细胞群互为阳性，即复合蛋白仅结合T细胞，结合率为100％(图15a)。
采用HIPP-T009培养基，把制备好的PBMC-DCT，调整至不同密度的细胞悬液，分别是1.25x103个/75μL、2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL和8x104个/75μL；用胰酶消化HepG2-NYESO1(NY-ESO-1阳性，A2阳性)、HepG2(NY-ESO-1阴性，A2阳性)、NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为1x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向的PBMC-DCT高效杀伤HepG2-NYESO1和NCI-H1299-A2等阳性靶肿瘤细胞，并具有效靶比梯度依赖性，对HepG2和NCI-H1299等阴性肿瘤细胞不识别(图15b)。
收集96孔U底板中效靶比为2:1的HepG2-NYESO-1/DCT、HepG2/DCT和仅有DCT组的细胞；4:1的NCI-H1299-A2/DCT、NCI-H1299/DCT和仅有DCT 组的细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μl FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表T细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-1G4(ImmTAC)制备的PBMC-DCT细胞，在遇到阳性靶细胞后，才诱导PBMC中的T细胞上调CD137的表达，即T细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图15c)，尽管T细胞表面结合高水平的抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白(图15d)。
3.8抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向外周血淋巴细胞(PBL)杀伤肿瘤细胞
通过“贴壁”法，去除外周血单个核细胞(PBMC)中的单核细胞，获得外周血淋巴细胞(PBL)。流式检测结果显示：CD3阳性细胞群为80.9％(图16a)。以“在含1x10-7mol/L复合蛋白CD3抗体(UCHT1)-1G4(ImmTAC)的200μL HIPP-T009培养基中，加入5x106个的PBL，4度孵育1小时”的方案，制备PBL-DCT，流式检测DCT的阳性率。结果显示：CD3细胞群与pHLA-tetramer细胞群互为阳性，即复合蛋白仅结合T细胞，结合率为100％(图16a)。
采用HIPP-T009培养基，把制备好的PBL-DCT，调整至不同密度的细胞悬液，分别是1.25x103个/75μL、2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL和8x104个/75μL；用胰酶消化HepG2-NYESO1(NY-ESO-1阳性，A2阳性)、HepG2(NY-ESO-1阴性，A2阳性)、NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为1x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加 裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向的PBL-DCT高效杀伤HepG2-NYESO1和NCI-H1299-A2等阳性靶肿瘤细胞，并具有效靶比梯度依赖性，对HepG2和NCI-H1299等阴性肿瘤细胞不识别(图16b)。
收集96孔U底板中效靶比为2:1的HepG2-NYESO-1/DCT、HepG2/DCT和仅有DCT组的细胞；4:1的NCI-H1299-A2/DCT、NCI-H1299/DCT和仅有DCT组的细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表T细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-1G4(ImmTAC)制备的PBL-DCT细胞，在遇到阳性靶细胞后，才诱导PBL中的T细胞上调CD137的表达，即T细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图16c)，尽管T细胞表面结合高水平的抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白(图16d)。
3.9抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向γδT细胞杀伤肿瘤细胞
采用唑来膦酸(5μM)，扩增外周血单个核细胞中的γδT细胞，在完全培养基(ImmunoCultTM-XF T cell expansion medium+5％FBS+200U/mL IL-2+10ng/mL IL-15)中培养12天，流式检测其表型和纯度。结果显示：87.8％的细胞为TCR  Vd2/CD3双阳性，即在扩增培养的细胞中，γδT细胞的比例为87.8％(图13a)。以“在含1x10-8mol/L复合蛋白抗CD3抗体(UCHT1)-1G4(ImmTAC)的200μL HIPP-T009培养基中，加入5x106个的γδT细胞，4度孵育1小时”的方案，制备γδT-DCT，流式检测DCT的阳性率。结果显示：98.8％的γδT细胞包被抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白(图17a)。
采用HIPP-T009培养基，把制备好的γδT-DCT和γδT细胞，调整至不同密度的细胞悬液，分别是5x102个/75μL、1x103个/75μL、2x103个/75μL和4x103个/75μL；1x104个/75μL、2x104个/75μL、4x104个/75μL和8x104个/75μL；用胰酶消化HepG2-NYESO1(NY-ESO-1阳性，A2阳性)、HepG2(NY-ESO-1阴性，A2阳性)、NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为2x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向的γδT-DCT高效杀伤HepG2-NYESO1和NCI-H1299-A2等阳性靶肿瘤细胞，并具有效靶比梯度依赖性；其对HepG2和NCI-H1299等阴性肿瘤细胞的识别，等同于γδT细胞对肿瘤细胞的识别水平(图17b)。
收集96孔U底板中，效靶比为1:5的HepG2-NYESO-1/DCT、HepG2/DCT、仅有DCT、HepG2-NYESO-1/γδT、HepG2/γδT、仅有γδT组的细胞；2:1的NCI-H1299-A2/DCT、NCI-H1299/DCT、仅有DCT、NCI-H1299-A2/γδT、NCI-H1299/γδT、仅有γδT组的细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818) 和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表γδT-DCT或γδT细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-1G4(ImmTAC)制备的γδT-DCT细胞，在遇到阳性靶细胞后，才诱导T细胞上调CD137的表达，即γδT-DCT细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，γδT-DCT细胞不上调CD137的表达，表现为静息状态，与γδT结果一致(图17c)。1x10-8mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-1G4(ImmTAC)制备的γδT-DCT，在细胞培养18小时后，流式检测不到细胞表面结合的抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白(图17d)。
3.10抗CD3抗体(UCHT1)-AFP(HATima)重定向T细胞杀伤肿瘤细胞
利用Human T-Activator CD3/CD28，扩增经磁珠分选的T细胞，培养12天，流式细胞术检测T细胞的纯度。结果显示：扩增培养后的细胞，CD3阳性率>98％(图18a)。以“在含1x10-8mol/L复合蛋白抗CD3抗体(UCHT1)-AFP(HATima)的200μL HIPP-T009培养基中，加入5x106个的T细胞，4度孵育1小时”的方案，制备CD3+T-DCT，流式检测DCT的阳性率。结果显示：大于98％的T细胞包被抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白(图18a)。
采用HIPP-T009培养基，把制备好的CD3+T-DCT，调整至不同密度的细胞悬液，分别是5x103个/75μL、1x104个/75μL、2x104个/75μL和4x104个/75μL；用胰酶消化HepG2(AFP阳性，A2阳性)和NCI-H1299-A2(AFP阴性，A2阳性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为2x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到 96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-AFP(HATima)重定向的CD3+T-DCT高效杀伤HepG2阳性靶肿瘤细胞，并具有效靶比梯度依赖性，对NCI-H1299-A2阴性肿瘤细胞不识别(图18b)。
收集96孔U底板中效靶比为1:1的HepG2/DCT、NCI-H1299-A2/DCT和仅有DCT组的细胞，离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μl RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表T细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-AFP(HATima)制备的CD3+T-DCT细胞，在遇到阳性靶细胞后，才诱导T细胞上调CD137的表达，即T细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图18c)。1x10-8mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-AFP(HATima)制备的CD3+T-DCT，在细胞培养18小时后，流式检测不到细胞表面结合的抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白(图18d)。
3.11抗CD3抗体(UCHT1)-AFP(HATima)重定向外周血单个核细胞(PBMC)杀伤肿瘤细胞
通过Ficoll密度梯度离心分离法，分离获得外周血单个核细胞(PBMC)，流式检测结果显示：CD3阳性细胞群为78.8％(图19a)。以“在含1x10-8mol/L复合蛋白抗CD3抗体(UCHT1)-AFP(HATima)的200μL HIPP-T009培养基中，加入5x106个的PBMC，4度孵育1小时”的方案，制备PBMC-DCT，流式检测DCT的阳性率。结果显示：CD3细胞群与pHLA-tetramer细胞群互为阳性，即复合蛋白仅结合T细胞，结合率为100％(图19a)。
采用HIPP-T009培养基，把制备好的PBMC-DCT，调整至不同密度的细胞悬液，分别是2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL和4x104个/75μL；用胰酶消化HepG2(AFP阳性，A2阳性)和NCI-H1299-A2(AFP阴性，A2阳性)细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为2x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-AFP(HATima)重定向的PBMC-DCT高效杀伤HepG2阳性靶肿瘤细胞，并具有效靶比梯度依赖性，对NCI-H1299-A2阴性肿瘤细胞不识别(图19b)。
收集96孔U底板中效靶比为2:1的HepG2/DCT、NCI-H1299-A2/DCT和仅有DCT组的细胞，离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μl RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表T细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-AFP(HATima)制备的PBMC-DCT细胞，在遇到阳性靶细胞后，才诱导PBMC中的T细胞上调CD137的表达，即T细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图19c)。1x10-8mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-AFP(HATima)制备的PBMC-DCT，在细胞培养18小时后，流式 检测不到细胞表面结合的抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白(图19d)。
3.12抗CD3抗体(UCHT1)-AFP(HATima)重定向外周血淋巴细胞(PBL)杀伤肿瘤细胞
通过“贴壁”法，去除外周血单个核细胞(PBMC)中的单核细胞，获得外周血淋巴细胞(PBL)。流式检测结果显示：CD3阳性细胞群为82.9％(图20a)。以“在含1x10-8mol/L复合蛋白抗CD3抗体(UCHT1)-AFP(HATima)的200μL HIPP-T009培养基中，加入5x106个的PBL，4度孵育1小时”的方案，制备PBL-DCT，流式检测DCT的阳性率。结果显示：CD3细胞群与pHLA-tetramer细胞群互为阳性，即复合蛋白仅结合T细胞，结合率为100％(图20a)。
采用HIPP-T009培养基，把制备好的PBL-DCT，调整至不同密度的细胞悬液，分别是2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL和4x104个/75μL；用胰酶消化HepG2(AFP阳性，A2阳性)和NCI-H1299-A2(AFP阴性，A2阳性)细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为2x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-AFP(HATima)重定向的PBL-DCT高效杀伤HepG2阳性靶肿瘤细胞，并具有效靶比梯度依赖性，对NCI-H1299-A2阴性肿瘤细胞不识别(图20b)。
收集96孔U底板中效靶比为2:1的HepG2/DCT、NCI-H1299-A2/DCT和仅有DCT组的细胞，离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号： 317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μl RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μl RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表T细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-AFP(HATima)制备的PBL-DCT细胞，在遇到阳性靶细胞后，才诱导PBL中的T细胞上调CD137的表达，即T细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图20c)。1x10-8mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-AFP(HATima)制备的PBL-DCT，在细胞培养18小时后，流式检测不到细胞表面结合的抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白(图20d)。
3.13抗CD3抗体(UCHT1)-AFP(HATima)重定向γδT细胞杀伤肿瘤细胞
采用唑来膦酸(5μM)，扩增外周血单个核细胞中的γδT细胞，在完全培养基(ImmunoCultTM-XF T cell expansion medium+5％FBS+200U/mL IL-2+10ng/mL IL-15)中培养12天，流式检测其表型和纯度。结果显示：87.8％的细胞为TCR Vd2/CD3双阳性，即在扩增培养的细胞中，γδT细胞的比例为87.8％(图13a)。以“在含1x10-9mol/L复合蛋白抗CD3抗体(UCHT1)-AFP(HATima)的200μL HIPP-T009培养基中，加入5x106个的γδT细胞，4度孵育1小时”的方案，制备γδT-DCT，流式检测DCT的阳性率。结果显示：100％的γδT细胞包被抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白(图21a)。
采用HIPP-T009培养基，把制备好的γδT-DCT和γδT细胞，调整至不同密度的细胞悬液，分别是5x103个/75μL、1x104个/75μL、2x104个/75μL和4x104个/75μL；用胰酶消化HepG2(AFP阳性，A2阳性)和NCI-H1299-A2(AFP阴性，A2阳性)等细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为2x104个/75μL。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小 时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-AFP(HATima)重定向的γδT-DCT高效杀伤HepG2阳性靶肿瘤细胞，并具有效靶比梯度依赖性；其对NCI-H1299-A2阴性肿瘤细胞的识别，等同于γδT细胞对肿瘤细胞的识别水平(图21b)。
收集96孔U底板中，效靶比为1:1的HepG2/DCT、NCI-H1299-A2/DCT、仅有DCT组的细胞；HepG2/γδT、NCI-H1299-A2/γδT、仅有γδT组的细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表γδT-DCT或γδT细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-AFP(HATima)制备的γδT-DCT细胞，在遇到阳性靶细胞后，才诱导T细胞上调CD137的表达，即γδT-DCT细胞表现为活化状态；对阴性肿瘤细胞和没有肿瘤细胞时，γδT-DCT细胞不上调CD137的表达，表现为静息状态，与γδT结果一致(图21c)。1x10-9mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-AFP(HATima)制备的γδT-DCT，在细胞培养18小时后，流式检测不到细胞表面结合的抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白(图21d)。
3.14抗CD3抗体(UCHT1)-gp100(HATima)重定向免疫细胞杀伤肿瘤细胞
按照实施例1的说明，制备免疫细胞，包括T细胞、γδT细胞、CD4和CD8双阴性T细胞(DNT)、CD4+T细胞、CD8+T细胞、PBMC和PBL，流式检测其表型和纯度。DCT制备的方法：在含1x10-8mol/L复合蛋白抗CD3抗体(UCHT1) -gp100(HATima)的HIPP-T009培养基(200μL)中，加入5x106个免疫细胞，4度孵育1小时，制备DCT，流式检测DCT的阳性率。结果显示：100％的T细胞、γδT细胞、DNT、CD4+T细胞、CD8+T细胞、PBMC和PBL包被抗CD3抗体(UCHT1)-gp100(HATima)复合蛋白(图22a)。
采用HIPP-T009培养基，把制备好的DCT细胞或对照细胞(DNT和γδT细胞)，调整至不同密度的细胞悬液，分别是2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL、8x104个/75μL、16x104个/75μL、32x104个/75μL；收集T2细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，调整细胞密度为2x104个/75μL，分别负载终浓度为1x10-8M的gp100肽(YLEPGPVTA，阳性靶点)和NY-ESO-1肽(SLLMWITQC，阴性对照)。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-gp100(HATima)重定向的T-DCT、PBMC-DCT、PBL-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT和CD8+T-DCT高效杀伤负载gp100肽的T2阳性靶肿瘤细胞，并具有效靶比梯度依赖性；其对负载NY-ESO-1肽的T2阴性肿瘤细胞不识别，或者等同于对照细胞(DNT和γδT细胞)对肿瘤细胞的识别水平(图22b)。
收集96孔U底板中，不同效靶比的实验孔细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、 PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表DCT细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-gp100(HATima)制备的DCT细胞，在遇到阳性靶细胞(T2-gp100肽)后，才诱导T细胞、PBMC、PBL、γδT细胞、DNT、CD4+T细胞和CD8+T细胞上调CD137的表达，即DCT细胞表现为活化状态；对阴性肿瘤细胞(T2-NYESO-1肽)和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图22c)。1x10-8mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-gp100(HATima)制备的T-DCT、PBMC-DCT、PBL-DCT、和CD4+T-DCT，在细胞培养18小时后，与阳性靶细胞(T2-gp100肽)共培养的DCT细胞，其表面结合的抗CD3抗体(UCHT1)-gp100(HATima)复合蛋白显著下降(图22d)。
3.15抗CD3抗体(UCHT1)-gp100(ImmTAC)重定向免疫细胞杀伤肿瘤细胞
按照实施例1的说明，制备免疫细胞，包括T细胞、γδT细胞、CD4和CD8双阴性T细胞(DNT)、CD4+T细胞、CD8+T细胞、PBMC和PBL，流式检测其表型和纯度。DCT制备的方法：在含1x10-8mol/L复合蛋白抗CD3抗体(UCHT1)-gp100(ImmTAC)的HIPP-T009培养基(200μL)中，加入5x106个免疫细胞，4度孵育1小时，制备DCT，流式检测DCT的阳性率。结果显示：100％的T细胞、γδT-DCT、DNT、CD4+T细胞、CD8+T细胞、PBMC和PBL包被抗CD3抗体(UCHT1)-gp100(ImmTAC)复合蛋白(图23a)。
采用HIPP-T009培养基，把制备好的DCT细胞或对照细胞(DNT和γδT细胞)，调整至不同密度的细胞悬液，分别是5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL、8x104个/75μL、16x104个/75μL；收集T2细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为2x104个/75μL，分别负载终浓度为1x10-8M的gp100肽(YLEPGPVTA，阳性靶点)和AFP肽(FMNKFIYEI，阴性对照)。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置 45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-gp100(ImmTAC)重定向的T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT、CD8+T-DCT、PBMC-DCT和PBL-DCT高效杀伤负载gp100肽的T2阳性靶肿瘤细胞，并具有效靶比梯度依赖性；其对负载AFP肽的T2阴性肿瘤细胞不识别，或者等同于对照细胞(DNT和γδT细胞)对肿瘤细胞的识别水平(图23b)。
收集96孔U底板中，不同效靶比的实验孔细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)和PEcy7抗-人CD137抗体(biolegend，货号：309818)，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体和PEcy7抗-人CD137的荧光信号，分析CD3阳性细胞群的CD137的比例，代表DCT细胞的活化程度。结果显示：由抗CD3抗体(UCHT1)-gp100(ImmTAC)制备的DCT细胞，在遇到阳性靶细胞(T2-gp100肽)后，才诱导T细胞、γδT细胞、DNT、CD4+T细胞、CD8+T、PBMC和PBL细胞上调CD137的表达，即DCT细胞表现为活化状态；对阴性肿瘤细胞(T2-AFP肽)和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图23c)。
3.16抗CD3抗体(UCHT1)-i1G4(ImmTAC)重定向免疫细胞杀伤肿瘤细胞
按照实施例1的说明，制备免疫细胞，包括T细胞、γδT细胞、CD4和CD8双阴性T细胞(DNT)、CD4+T细胞、CD8+T细胞、PBMC和PBL，流式检测其表型和纯度。DCT制备的方法：在含1x10-7mol/L至1x10-10mol/L复合蛋白抗CD3抗体(UCHT1)-i1G4(ImmTAC)的HIPP-T009培养基(200μL)中，加入5x106个免疫细胞，4度孵育1小时，制备DCT，流式检测DCT的阳性率。结果显示：100％的T细胞、γδT细胞、DNT、CD4+T细胞、CD8+T细胞、PBMC和 PBL包被抗CD3抗体(UCHT1)-i1G4(ImmTAC)复合蛋白(图24a)。
采用HIPP-T009培养基，把制备好的DCT细胞或对照细胞(DNT)，调整至不同密度的细胞悬液，分别是0.625x103个/75μL、1.25x103个/75μL、2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL、8x104个/75μL；用胰酶消化阳性(HepG2-NYESO-1(NYESO-1+/HLA-A0201+))和阴性(HepG2(NYESO-1-/HLA-A0201+))靶细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，调整细胞密度为2x104个/75μL。96孔U底板中，依次加入75μL效应细胞和75μL靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-i1G4(ImmTAC)重定向的T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT、CD8+T-DCT、PBMC-DCT和PBL-DCT高效杀伤HepG2-NYESO-1(NYESO-1+/HLA-A0201+)细胞(阳性靶肿瘤)，并具有效靶比梯度依赖性；其对HepG2(NYESO-1-/HLA-A0201+)细胞(阴性肿瘤)不识别，或者等同于对照细胞(DNT)对肿瘤细胞的识别水平(图24b)。
收集96孔U底板中，不同效靶比的实验孔细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表DCT细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-i1G4(ImmTAC)制备的DCT 细胞，在遇到阳性靶细胞(HepG2-NYESO-1)后，才诱导T细胞、γδT细胞、DNT、CD4+T细胞、CD8+T、PBMC和PBL细胞上调CD137的表达，即DCT细胞表现为活化状态；对阴性肿瘤细胞(HepG2)和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图24c)。1x10-7mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-i1G4(ImmTAC)制备的T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT、CD8+T-DCT、PBMC-DCT和PBL-DCT，在细胞培养18小时后，其表面结合的抗CD3抗体(UCHT1)-i1G4(ImmTAC)复合蛋白均比较低(图24d)。
3.17抗CD3抗体(UCHT1)-igp100(ImmTAC)重定向免疫细胞杀伤肿瘤细胞
按照实施例1的说明，制备免疫细胞，包括T细胞、γδT细胞、CD4和CD8双阴性T细胞(DNT)、CD4+T细胞和CD8+T细胞，流式检测其表型和纯度。DCT制备的方法：在含1x10-8mol/L复合蛋白抗CD3抗体(UCHT1)-igp100(ImmTAC)的HIPP-T009培养基(200μL)中，加入5x106个免疫细胞，4度孵育1小时，制备DCT，流式检测DCT的阳性率。结果显示：100％的T细胞、γδT细胞、DNT、CD4+T细胞和CD8+T细胞包被抗CD3抗体(UCHT1)-igp100(ImmTAC)复合蛋白(图25a)。
采用HIPP-T009培养基，把制备好的DCT细胞或对照细胞(DNT和γδT细胞)，调整至不同密度的细胞悬液，分别是5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL、8x104个/75μL、16x104个/75μL、32x104个/75μL；收集T2细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，调整细胞密度为2x104个/75μL，分别负载终浓度为1x10-8M的gp100肽(YLEPGPVTA，阳性靶点)和AFP肽(FMNKFIYEI，阴性对照)。96孔U底板中，依次加入75μL的效应细胞和75μL的靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞 自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-igp100(ImmTAC)重定向的T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT和CD8+T-DCT高效杀伤负载gp100肽的T2阳性靶肿瘤细胞，并具有效靶比梯度依赖性；其对负载AFP肽的T2阴性肿瘤细胞不识别，或者等同于对照细胞(DNT和γδT细胞)对肿瘤细胞的识别水平(图25b)。
收集96孔U底板中，不同效靶比的实验孔细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表DCT细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-igp100(ImmTAC)制备的DCT细胞，在遇到阳性靶细胞(T2-gp100肽)后，才诱导T细胞、γδT细胞、DNT、CD4+T细胞和CD8+T细胞上调CD137的表达，即DCT细胞表现为活化状态；对阴性肿瘤细胞(T2-AFP肽)和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图25c)。1x10-8mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-igp100(ImmTAC)制备的T-DCT、γδT-DCT、DNT-DCT、CD4+T-DCT和CD8+T-DCT，在细胞培养18小时后，与阳性靶细胞(T2-gp100肽)共培养的DCT细胞，其表面结合的抗CD3抗体(UCHT1)-igp100(ImmTAC)复合蛋白显著下降(图25d)。
3.18抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向免疫细胞杀伤肿瘤细胞
按照实施例1的说明，制备免疫细胞，包括CD4和CD8双阴性T细胞(DNT)、CD4+T细胞和CD8+T细胞，流式检测其表型和纯度。DCT制备的方法：在含1x10-7mol/L(对于CD4+T细胞和CD8+T细胞)或1x10-8mol/L(对于DNT)复合蛋白抗CD3抗体(UCHT1)-1G4(ImmTAC)的HIPP-T009培养基(200μL)中，加入5x106个免疫细胞，4度孵育1小时，制备DCT，流式检测DCT的阳性率。结果显示：100％的DNT、CD4+T细胞和CD8+T细胞包被抗CD3抗体(UCHT1) -1G4(ImmTAC)复合蛋白(图26a)。
采用HIPP-T009培养基，把制备好的DCT细胞或对照细胞(DNT)，调整至不同密度的细胞悬液，分别是1.25x103个/75μL、2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL、8x104个/75μL、16x104个/75μL；用胰酶消化阳性(HepG2-NYESO-1(NYESO-1+/HLA-A0201+))和阴性(HepG2(NYESO-1-/HLA-A0201+))靶细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，调整细胞密度为2x104个/75μL。96孔U底板中，依次加入75μL效应细胞和75μL靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向的DNT-DCT、CD4+T-DCT和CD8+T-DCT高效杀伤HepG2-NYESO-1(NYESO-1+/HLA-A0201+)细胞(阳性靶肿瘤)，并具有效靶比梯度依赖性；其对HepG2(NYESO-1-/HLA-A0201+)细胞(阴性肿瘤)不识别，或者等同于对照细胞(DNT)对肿瘤细胞的识别水平(图26b)。收集96孔U底板中，不同效靶比的实验孔细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表DCT细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-1G4(ImmTAC)制备的DCT细胞，在遇到阳性靶细胞(HepG2-NYESO-1)后，才诱导DNT、CD4+T细胞和CD8+T 细胞上调CD137的表达，即DCT细胞表现为活化状态；对阴性肿瘤细胞(HepG2)和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图26c)。1x10-7mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-1G4(ImmTAC)制备的CD4+T-DCT和CD8+T-DCT，在细胞培养18小时后，与阳性靶细胞共培养的DCT细胞，其表面结合的抗CD3抗体(UCHT1)-1G4(ImmTAC)复合蛋白显著下降(图26d)。
3.19抗CD3抗体(UCHT1)-1G4(HATima)重定向免疫细胞杀伤肿瘤细胞
按照实施例1的说明，制备免疫细胞，包括CD4和CD8双阴性T细胞(DNT)、CD4+T细胞和CD8+T细胞，流式检测其表型和纯度。DCT制备的方法：在含1x10-7mol/L(对于CD4+T细胞和CD8+T细胞)或1x10-8mol/L(对于DNT)复合蛋白抗CD3抗体(UCHT1)-1G4(HATima)的HIPP-T009培养基(200μL)中，加入5x106个免疫细胞，4度孵育1小时，制备DCT，流式检测DCT的阳性率。结果显示：100％的DNT、CD4+T细胞和CD8+T细胞包被抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白(图27a)。
采用HIPP-T009培养基，把制备好的DCT细胞或对照细胞(DNT)，调整至不同密度的细胞悬液，分别是1.25x103个/75μL、2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL、8x104个/75μL、16x104个/75μL；用胰酶消化阳性(HepG2-NYESO-1(NYESO-1+/HLA-A0201+))和阴性(HepG2(NYESO-1-/HLA-A0201+))靶细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为2x104个/75μL。96孔U底板中，依次加入75μL效应细胞和75μL靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞 自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-1G4(HATima)重定向的DNT-DCT、CD4+T-DCT和CD8+T-DCT高效杀伤HepG2-NYESO-1(NYESO-1+/HLA-A0201+)细胞(阳性靶肿瘤)，并具有效靶比梯度依赖性；其对HepG2(NYESO-1-/HLA-A0201+)细胞(阴性肿瘤)不识别，或者等同于对照细胞(DNT)对肿瘤细胞的识别水平(图27b)。
收集96孔U底板中，不同效靶比的实验孔细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表DCT细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-1G4(HATima)制备的DCT细胞，在遇到阳性靶细胞(HepG2-NYESO-1)后，才诱导DNT、CD4+T细胞和CD8+T细胞上调CD137的表达，即DCT细胞表现为活化状态；对阴性肿瘤细胞(HepG2)和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为静息状态(图27c)。1x10-7mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-1G4(HATima)制备的CD4+T-DCT和CD8+T-DCT，在细胞培养18小时后，与阳性靶细胞共培养的DCT细胞，其表面结合的抗CD3抗体(UCHT1)-1G4(HATima)复合蛋白显著下降(图27d)。
3.20抗CD3抗体(UCHT1)-AFP(HATima)重定向免疫细胞杀伤肿瘤细胞
按照实施例1的说明，制备免疫细胞，包括CD4和CD8双阴性T细胞(DNT)、CD4+T细胞和CD8+T细胞，流式检测其表型和纯度。DCT制备的方法：在含1x10-8mol/L复合蛋白抗CD3抗体(UCHT1)-AFP(HATima)的HIPP-T009培养基(200μL)中，加入5x106个免疫细胞，4度孵育1小时，制备DCT，流式检测DCT的阳性率。结果显示：100％的DNT、CD4+T细胞和CD8+T细胞包被抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白(图28a)。
采用HIPP-T009培养基，把制备好的DCT细胞或对照细胞(DNT)，调整 至不同密度的细胞悬液，分别是2.5x103个/75μL、5x103个/75μL、1x104个/75μL、2x104个/75μL、4x104个/75μL、8x104个/75μL、16x104个/75μL、32x104个/75μL；用胰酶消化阳性(HepG2(AFP+/HLA-A0201+))和阴性(NCI-H1299-A2(AFP-/HLA-A0201+))靶细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为2x104个/75μL。96孔U底板中，依次加入75μL效应细胞和75μL靶肿瘤细胞，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：抗CD3抗体(UCHT1)-AFP(HATima)重定向的DNT-DCT、CD4+T-DCT和CD8+T-DCT高效杀伤HepG2(AFP+/HLA-A0201+)细胞(阳性靶肿瘤)，并具有效靶比梯度依赖性；其对NCI-H1299-A2(AFP-/HLA-A0201+)细胞(阴性肿瘤)不识别，或者等同于对照细胞(DNT)对肿瘤细胞的识别水平(图28b)。
收集96孔U底板中，不同效靶比的实验孔细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD3抗体(biolegend，货号：317306)、PEcy7抗-人CD137抗体(biolegend，货号：309818)和pHLA-tetramer-APC，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD3抗体、PEcy7抗-人CD137和pHLA-tetramer-APC的荧光信号，分析CD3阳性细胞群的CD137和pHLA-tetramer的比例，分别代表DCT细胞的活化程度和表面结合的复合蛋白比例。结果显示：由抗CD3抗体(UCHT1)-AFP(HATima)制备的DCT细胞，在遇到阳性靶细胞(HepG2)后，才诱导DNT、CD4+T细胞和CD8+T细胞上调CD137的表达，即DCT细胞表现为活化状态；对阴性肿瘤细胞(NCI-H1299-A2)和没有肿瘤细胞时，DCT细胞不上调CD137的表达，表现为 静息状态(图28c)。1x10-8mol/L浓度的复合蛋白抗CD3抗体(UCHT1)-AFP(HATima)制备的CD8+T-DCT，在细胞培养18小时后，其表面结合的抗CD3抗体(UCHT1)-AFP(HATima)复合蛋白下降，并组间无差异(图28d)。
实施例4：用于修饰细胞的HATima分子和ImmTAC分子结构中，抗CD3抗体(UCHT1)的生物学活性功能比较
4.1 VH-VL和scFv蛋白的制备
HATima分子结构中的抗CD3抗体(UCHT1)为重链可变区(SEQ ID NO:9和SEQ ID NO:10)与轻链可变区(SEQ ID NO:11和SEQ ID NO:12)，通过链间二硫键连接，即VH-VL；ImmTAC分子结构中的抗CD3抗体(UCHT1)为重链可变区与轻链可变区，通过linker连接，即scFv(SEQ ID NO:51和SEQ ID NO:52)。载体构建和包涵体表达纯化同1.1.1。蛋白的复性同1.1.2，透析缓冲液为10mM PB pH 6.0；VH和VL的包涵体加入的量各为6mg，scFv的包涵体加入的量为6mg。蛋白的纯化同1.1.3。图29a结果显示：在非还原的状态下，VH-VL为单条带，分子量为22kDa；在还原的状态下，VH-VL仍为单条带，但分子量下降为15kDa，说明VH-VL被分解为2条链，蛋白的纯度大于90％。在非还原的状态下，scFv为单条带，分子量为27kDa；在还原的状态下，scFv仍为单条带，分子量仍为27kDa，说明scFv为整体的单条链，蛋白的纯度大于90％。
4.2 VH-VL和scFv蛋白的生物学活性检测
采用HIPP-T009培养基，把T细胞调整为密度1x104个/50μL的细胞悬液。用胰酶消化NCI-H1299-A2(NY-ESO-1阳性，A2阳性)和NCI-H1299(NY-ESO-1阳性，A2阴性)细胞，磷酸盐缓冲液洗一次，HIPP-T009培养基重悬，并调整细胞密度为1x104个/75μL。蛋白药物，分别是抗CD3抗体(UCHT1)-1G4(HATima)、抗CD3抗体(UCHT1)-1G4(ImmTAC)、VH-VL和scFv，用HIPP-T009培养基稀释为不同的摩尔浓度，分别3x10-7M/50μL、3x10-8M/50μL、3x10-9M/50μL、3x10-10M/50μL、3x10-11M/50μL、3x10-12M/50μL、3x10-13M/50μL和0μL。96孔U底板中，依次加入50μL的效应细胞、50μL的靶肿瘤细胞和50μL对应浓度的待测试蛋白，同时设置靶细胞最大裂解孔、靶细胞自发孔、效应细胞自发孔、培养基自发孔以及培养基加裂解液的自发孔，每个孔的终体积均为150μL，并且 各2个复孔。将加完细胞的96孔U底板放入37度5％CO2的细胞培养箱中培养18小时。最大裂解孔加入15μL裂解液，混匀，37度5％CO2的细胞培养箱放置45分钟。把96孔U底细胞培养板放入离心机中，离心(250g，4分钟)。取出50μL上清到96孔平底板中，加入50μL底物液，避光常温反应30分钟。反应结束后，加入50μL的终止液，立即用酶标仪测定490nm处的吸光值。根据产品说明书计算特异性杀伤的效率＝(实验孔-效应细胞自发孔-靶细胞自发孔)/(靶细胞最大裂解孔-靶细胞自发孔)x100％。结果显示：CD3抗体(UCHT1)-1G4(HATima)和抗CD3抗体(UCHT1)-1G4(ImmTAC)重定向的T细胞特异性杀伤NCI-H1299-A2(NY-ESO-1阳性，A2阳性)细胞，并具有浓度梯度依赖性，其对NCI-H1299(NY-ESO-1阳性，A2阴性)细胞不识别；VH-VL重定向的T细胞对NCI-H1299-A2(NY-ESO-1阳性，A2阳性)细胞和NCI-H1299(NY-ESO-1阳性，A2阴性)细胞均不识别；scFv重定向的T细胞对NCI-H1299-A2(NY-ESO-1阳性，A2阳性)细胞和NCI-H1299(NY-ESO-1阳性，A2阴性)细胞均表现出一定程度的识别，并且不依赖于靶点，虽然杀伤率低于CD3抗体(UCHT1)-1G4(HATima)和抗CD3抗体(UCHT1)-1G4(ImmTAC)(图29b)。
收集96孔U底板中1E-7M和1E-9M浓度组的细胞。离心(500g,5min,4℃)，弃上清，加入100μL RPMI-1640培养基，重悬细胞。分别加入1μL FITC抗-人CD45抗体(biolegend，货号：368508)和PEcy7抗-人CD137抗体(biolegend，货号：309818)，同时设置不染色组和同型对照组，4度孵育30分钟。加入100μL RPMI-1640培养基，离心(500g,5min,4℃)，弃上清，重复1次。加入100μL RPMI-1640培养基，流式检测FITC抗-人CD45抗体和PEcy7抗-人CD137的荧光信号，分析CD45阳性细胞群的CD137比例，代表T细胞的活化程度。图29c结果显示：在蛋白浓度为1E-9M时，scFv在NCI-H1299-A2(NY-ESO-1阳性，A2阳性)细胞组、NCI-H1299(NY-ESO-1阳性，A2阴性)组和无肿瘤细胞组中，均显著性诱导T细胞上调CD137的表达，即T细胞表现为活化状态，表现为无靶点依赖性；仅在NCI-H1299-A2(NY-ESO-1阳性，A2阳性)细胞组中，scFv诱导的T细胞的活化程度低于CD3抗体(UCHT1)-1G4(HATima)和抗CD3抗体(UCHT1)-1G4(ImmTAC)。VH-VL均不能诱导T细胞上调CD137的表达。图29d结果显示：当蛋白浓度升高至1E-7M时，在NCI-H1299-A2(NY-ESO-1阳性，A2阳性)细胞组、NCI-H1299(NY-ESO-1阳性，A2阴性)组和无肿瘤细 胞组中，VH-VL依然不能诱导T细胞上调CD137的表达；scFv均表现为不依赖于靶点的T细胞活化，仅在NCI-H1299-A2(NY-ESO-1阳性，A2阳性)细胞组中，scFv诱导的T细胞的活化程度低于CD3抗体(UCHT1)-1G4(HATima)和抗CD3抗体(UCHT1)-1G4(ImmTAC)。结果表明：scFv是免疫效应分子，VH-VL是非免疫效应分子。
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Claims (44)

  1. 一种修饰细胞，其中所述修饰细胞是由一种复合蛋白结合于所述修饰细胞的细胞表面标记分子而成，其中所述复合蛋白包含特异性结合所述修饰细胞的细胞表面标记分子的部分和特异性结合靶细胞表面抗原的部分。
  2. 如权利要求1所述的修饰细胞，其中，所述特异性结合靶细胞表面抗原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
  3. 如权利要求1或2所述的修饰细胞，其中，所述特异性结合所述修饰细胞的细胞表面标记分子的部分为特异性结合所述修饰细胞的细胞表面标记分子的抗体或其功能片段。
  4. 如权利要求1至3任一项所述的修饰细胞，其中，所述修饰细胞的细胞表面标记分子选自CD8、CD4、CD3、NKG2D、CD16、CD2、CD56、CD28或CD26。
  5. 如权利要求1-4中任一项所述的修饰细胞，其中，所述修饰细胞为免疫细胞，优选为T细胞、γδT细胞、CD4和CD8双阴性T细胞、CD4+T细胞、CD8+T细胞、NK细胞、NKT细胞或单核细胞。
  6. 如权利要求1-5中任一项所述的修饰细胞，其中，所述靶细胞表面抗原选自pMHC。
  7. 如权利要求2所述的修饰细胞，其中，所述复合蛋白的结构从N端到C端如式Ia所示：
    A-L-B  (Ia)
    B-L-A  (Ib)
    其中，
    元件A包含特异性结合所述修饰细胞的细胞表面标记分子的抗体或其功能片段；
    元件B包含特异性结合靶细胞表面抗原的部分；和
    元件L为柔性接头；所述柔性接头是可选择的；
    “-”为肽键。
  8. 如权利要求7所述的修饰细胞，其中，所述元件A包含的抗体为单链抗 体scFv。
  9. 如权利要求8所述的修饰细胞，其中，所述元件A包含的抗体为抗CD3抗体。
  10. 如权利要求9所述的修饰细胞，其中，所述抗体中的重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列。
  11. 如权利要求9所述的修饰细胞，其中所述单链抗体包含OKT3、UCHT-1、TR66、BMA031或12F6。
  12. 如权利要求7所述的修饰细胞，其中，所述靶细胞表面抗原选自pMHC。
  13. 如权利要求7所述的修饰细胞，其中，所述特异性结合靶细胞表面抗原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
  14. 如权利要求13所述的修饰细胞，其中，所述TCRβ链氨基酸序列包含如SEQ ID NO:5、25或40所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:7、27、42或44所示的序列。
  15. 如权利要求2所述的修饰细胞，其中，所述复合蛋白的结构从N端到C端如式Ic或Id所示：
    A2…A1-L-B  (Ic)；
    B-L-A1…A2  (Id)；
    其中，
    元件A1、A2各自独立地包含非免疫效应分子；优选地，所述非免疫效应分子包含抗体的重链可变区或抗体的轻链可变区；
    元件B为特异性结合靶细胞表面抗原的部分；和
    元件L为柔性接头；所述柔性接头是可选择的；
    “-”为肽键；
    “…”为二硫键。
  16. 如权利要求15所述的修饰细胞，其中，A1包含抗体的重链可变区时，A2包含抗体的轻链可变区；或者A2包含抗体的重链可变区时，A1包含抗体的轻链可变区；并且A1和A2之间通过二硫键形成二聚体，优选地，所述二聚体为非免疫效应分子。
  17. 如权利要求16所述的修饰细胞，其特征在于，所述抗体的重链可变区 包含如SEQ ID NO:9所示的序列。
  18. 如权利要求16所述的修饰细胞，其特征在于，所述抗体的轻链可变区包含如SEQ ID NO:11所示的序列。
  19. 如权利要求15所述的修饰细胞，其中，所述特异性结合靶细胞表面抗原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
  20. 如权利要求19所述的修饰细胞，其中，所述TCRβ链氨基酸序列包含如SEQ ID NO:13、17或21所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:15、19或23所示的序列。
  21. 一种组合或试剂盒，包括a.一种复合蛋白；b.一种细胞；其中所述细胞表达细胞表面标记分子，其中所述复合蛋白包含结合所述细胞的细胞表面标记分子的部分和特异性结合靶细胞表面抗原的部分。
  22. 如权利要求21所述的组合或试剂盒，其中，所述特异性结合靶细胞表面抗原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
  23. 如权利要求21或22所述的组合或试剂盒，其中，所述特异性结合所述修饰细胞的细胞表面标记分子的部分为特异性结合所述修饰细胞的细胞表面标记分子的抗体或其功能片段。
  24. 如权利要求21至23中任一项所述的组合或试剂盒，其中，所述细胞的细胞表面标记分子选自CD8、CD4、CD3、NKG2D、CD16、CD2、CD56、CD28或CD26。
  25. 如权利要求21至24中任一项所述的组合或试剂盒，其中，所述细胞为免疫细胞，优选为T细胞、γδT细胞、CD4和CD8双阴性T细胞、CD4+T细胞、CD8+T细胞、NK细胞、NKT细胞或单核细胞。
  26. 如权利要求21-25中任一项所述的组合或试剂盒，其中，所述靶细胞表面抗原选自pMHC。
  27. 如权利要求22所述的组合或试剂盒，其中，所述复合蛋白的结构从N端到C端如式Ia所示：
    A-L-B  (Ia)
    B-L-A  (Ib)
    其中，
    元件A包含特异性结合所述修饰细胞的细胞表面标记分子的抗体或其功能片段；
    元件B包含特异性结合靶细胞表面抗原的部分；和
    元件L为柔性接头；所述柔性接头是可选择的；
    “-”为肽键。
  28. 如权利要求27所述的组合或试剂盒，其中，所述元件A包含的抗体为单链抗体scFv。
  29. 如权利要求28所述的组合或试剂盒，其中，所述元件A包含的抗体为抗CD3抗体。
  30. 如权利要求29所述的组合或试剂盒，其中，所述抗体中的重链可变区包含如SEQ ID NO:1所示的序列，轻链可变区包含如SEQ ID NO:3所示的序列。
  31. 如权利要求29所述的组合或试剂盒，其中所述单链抗体包含OKT3、UCHT-1、TR66、BMA031或12F6。
  32. 如权利要求27所述的组合或试剂盒，其中，所述靶细胞表面抗原选自pMHC。
  33. 如权利要求27所述的组合或试剂盒，其中，所述特异性结合靶细胞表面抗原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
  34. 如权利要求33所述的组合或试剂盒，其中，所述TCRβ链氨基酸序列包含如SEQ ID NO:5、25或40所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:7、27、42或44所示的序列。
  35. 如权利要求22所述的组合或试剂盒，其中，所述复合蛋白的结构从N端到C端如式Ic或Id所示：
    A2…A1-L-B  (Ic)；
    B-L-A1…A2  (Id)；
    其中，
    元件A1、A2各自独立地包含非免疫效应分子；优选地，所述非免疫效应分子包含抗体的重链可变区或抗体的轻链可变区；
    元件B为特异性结合靶细胞表面抗原的部分；和
    元件L为柔性接头；所述柔性接头是可选择的；
    “-”为肽键；
    “…”为二硫键。
  36. 如权利要求35所述的组合或试剂盒，其中，A1包含抗体的重链可变区时，A2包含抗体的轻链可变区；或者A2包含抗体的重链可变区时，A1包含抗体的轻链可变区；并且A1和A2之间通过二硫键形成二聚体，优选地，所述二聚体为非免疫效应分子。
  37. 如权利要求36所述的组合或试剂盒，其特征在于，所述抗体的重链可变区包含如SEQ ID NO:9所示的序列。
  38. 如权利要求36所述的组合或试剂盒，其特征在于，所述抗体的轻链可变区包含如SEQ ID NO:11所示的序列。
  39. 如权利要求35所述的组合或试剂盒，其中，所述特异性结合靶细胞表面抗原的部分为TCR分子、单链αβTCR或者TCRα链/TCRβ链异二聚体、TCR样抗体或TCR模拟抗体。
  40. 如权利要求39所述的组合或试剂盒，其中，所述TCRβ链氨基酸序列包含如SEQ ID NO:13、17或21所示的序列，TCRα链氨基酸序列包含如SEQ ID NO:15、19或23所示的序列。
  41. 如权利要求21至25中任一项所述的组合或试剂盒，其中，所述组合或试剂盒进一步包括可选择性的可药用载体，优选为缓冲液，更优选地，所述缓冲液包含白蛋白或血浆。
  42. 一种治疗疾病的方法，包含向所需要的受试者施用如权利要求1至20中任一项所述的修饰细胞或如权利要求21-41中任一项所述的组合或试剂盒。
  43. 如权利要求42所述的方法，其中所述疾病包括癌症、微生物感染性疾病、自身免疫性疾病或衰老性疾病。
  44. 一种制备如权利要求1至20中任一项所述的修饰细胞的方法，其中将所述复合蛋白与免疫细胞孵育形成所示修饰细胞。
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