Modified cell and application thereof

Abstract

Provided is a modified cell wherein the modified cell is formed by binding a compound protein to a cell surface marker molecule of the modified cell wherein the compound 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 invention also provides a method for preparing the modified cell and a method for treating diseases by using the modified cell.

Description

Modified cell and its use

[0001] priority

[0002] This application claims the benefit of and priority to PCT International Application No. PCT / CN2022 / 123635, filed on September 30, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of immunology, and in particular to a modified cell and its use in treating diseases. Background Art

[0004] Currently, viral vector systems are the most commonly used to redirect T cells using T cell receptors (TCRs) or chimeric antigen receptors (CARs). These systems utilize transgenic technologies such as lentivirus to insert exogenous therapeutic genes into the T cell genome, achieving stable gene expression. This complex process results in genetic modification of the therapeutic cells. The altered genes can lead to the retention of cells unrelated to therapeutic efficacy after treatment. Furthermore, the potential risks of genetic modification require further research to ensure safety. Therefore, the biosafety of viral vectors needs to be further improved. The integration of exogenous therapeutic genes into T cells can inadvertently insert into driver genes, posing a risk of tumorigenicity to these genetically modified T cells. Current research attempts to mitigate this problem of cell retention, such as by introducing suicide genes. However, once off-target effects occur in some patients, even with the introduction of suicide genes, it is difficult to eliminate the infused immune cells. Therefore, some studies are looking to achieve T cell redirection using non-viral vector systems, which are considered safer. However, these methods have limited ability to deliver target genes into cells, and their redirection efficiency needs to be further improved.

[0005] Summary of the Invention

[0006] Modified cells

[0007] In one aspect, the present disclosure provides a modified cell, wherein the modified cell is formed by a complex protein binding 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.

[0008] In one embodiment, the portion that specifically binds to a target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

[0009] In one embodiment, 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.

[0010] In one embodiment, the cell surface marker molecule of the modified cell is selected from CD8, CD4, CD3, NKG2D, CD16, CD2, CD56, CD28 or CD26.

[0011] In one embodiment, the modified cells are immune cells, preferably T cells, γδ T cells, CD4 and CD8 double negative T cells, CD4 + T cells, CD8 + T cells, NK cells, NKT cells or monocytes.

[0012] In one embodiment, the target cell surface antigen is selected from pMHC.

[0013] In one embodiment, in the aforementioned modified cell, the structure of the complex protein from N-terminus to C-terminus is as shown in Formula Ia: ALB (Ia) BLA (Ib)

[0014] in,

[0015] Element A comprises an antibody or a functional fragment thereof that specifically binds to a cell surface marker molecule of the modified cell;

[0016] Element B comprises a portion that specifically binds to a target cell surface antigen; and

[0017] Element L is a flexible joint; the flexible joint is optional;

[0018] “-” is a peptide bond.

[0019] In one embodiment, in the aforementioned modified cells, the element A is an immune effector molecule; in one embodiment, in the aforementioned modified cells, the element A may also be a non-immune effector molecule.

[0020] In one embodiment, in the aforementioned modified cell, the antibody comprised by the element A is a single-chain antibody scFv.

[0021] In one embodiment, in the aforementioned modified cell, the antibody comprised by the element A is an anti-CD3 antibody.

[0022] In one embodiment, in the aforementioned modified cell, the heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO: 1, and the light chain variable region comprises the sequence shown in SEQ ID NO: 3.

[0023] In one embodiment, in the aforementioned modified cell, the single-chain antibody comprises OKT3, UCHT-1, TR66, BMA031 or 12F6.

[0024] In one embodiment, in the aforementioned modified cell, the target cell surface antigen is selected from pMHC.

[0025] In one embodiment, in the aforementioned modified cells, 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 αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimicking antibody.

[0026] In one embodiment, in the aforementioned modified cell, the TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO: 5, 25 or 40, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO: 7, 27, 42 or 44.

[0027] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:5, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:7.

[0028] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:25, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:27.

[0029] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:25, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:44.

[0030] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:40, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:42.

[0031] In one embodiment, in the aforementioned modified cell, 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);

[0032] in,

[0033] 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;

[0034] Element B is a portion that specifically binds to a target cell surface antigen; and

[0035] Element L is a flexible joint; the flexible joint is optional;

[0036] “-” is a peptide bond;

[0037] “…” indicates a disulfide bond.

[0038] In one embodiment, in the aforementioned modified cell, when A1 comprises the heavy chain variable region of an antibody, A2 comprises the light chain variable region of an antibody; or when A2 comprises the heavy chain variable region of an antibody, A1 comprises the light chain variable region of an antibody; and A1 and A2 form a dimer through a disulfide bond, preferably, the dimer is a non-immune effector molecule.

[0039] In one embodiment, in the aforementioned modified cell, the heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO:9.

[0040] In one embodiment, in the aforementioned modified cell, the light chain variable region of the antibody comprises the sequence shown in SEQ ID NO:11.

[0041] In one embodiment, in the aforementioned modified cells, the portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

[0042] In one embodiment, in the aforementioned modified cell, the TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO: 13, 17 or 21, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO: 15, 19 or 23.

[0043] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:13, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:15.

[0044] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:17, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:19.

[0045] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:21, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:23.

[0046] Combination or kit

[0047] On the other hand, 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, 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.

[0048] In one embodiment, in the aforementioned combination or kit, the portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

[0049] In one embodiment, in the aforementioned combination or kit, 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.

[0050] In one embodiment, in the aforementioned combination or kit, the cell surface marker molecule of the cell is selected from CD8, CD4, CD3, NKG2D, CD16, CD2, CD56, CD28 or CD26.

[0051] In one embodiment, in the aforementioned combination or kit, the cells are immune cells, preferably T cells, γδ T cells, CD4 and CD8 double negative T cells, CD4 +T cells, CD8 + T cells, NK cells, NKT cells or monocytes.

[0052] In one embodiment, in the aforementioned combination or kit, the target cell surface antigen is selected from pMHC.

[0053] In one embodiment, in the aforementioned combination or kit, the structure of the complex protein from N-terminus to C-terminus is as shown in Formula Ia: ALB (Ia) BLA (Ib)

[0054] in,

[0055] Element A comprises an antibody or a functional fragment thereof that specifically binds to a cell surface marker molecule of the modified cell;

[0056] Element B comprises a portion that specifically binds to a target cell surface antigen; and

[0057] Element L is a flexible joint; the flexible joint is optional;

[0058] “-” is a peptide bond.

[0059] In one embodiment, in the aforementioned combination or kit, the element A is an immune effector molecule; in one embodiment, in the aforementioned combination or kit, the element A is a non-immune effector molecule.

[0060] In one embodiment, in the aforementioned combination or kit, the antibody comprised by the element A is a single-chain antibody scFv.

[0061] In one embodiment, in the aforementioned combination or kit, the antibody comprised by the component A is an anti-CD3 antibody.

[0062] In one embodiment, in the aforementioned combination or kit, the heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO: 1, and the light chain variable region comprises the sequence shown in SEQ ID NO: 3.

[0063] In one embodiment, in the aforementioned combination or kit, the single-chain antibody comprises OKT3, UCHT-1, TR66, BMA031 or 12F6.

[0064] In one embodiment, in the aforementioned combination or kit, the target cell surface antigen is selected from pMHC.

[0065] In one embodiment, in the aforementioned combination or kit, the portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

[0066] In one embodiment, in the aforementioned modified cell, the TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO: 5, 25 or 40, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO: 7, 27, 42 or 44.

[0067] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:5, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:7.

[0068] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:25, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:27.

[0069] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:25, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:44.

[0070] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:40, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:42.

[0071] In one embodiment, in the aforementioned combination or kit, 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);

[0072] in,

[0073] 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;

[0074] Element B is a portion that specifically binds to a target cell surface antigen; and

[0075] Element L is a flexible joint; the flexible joint is optional;

[0076] “-” is a peptide bond;

[0077] “…” indicates a disulfide bond.

[0078] In one embodiment, in the aforementioned combination or kit, when A1 comprises the heavy chain variable region of an antibody, A2 comprises the light chain variable region of an antibody; or when A2 comprises the heavy chain variable region of an antibody, A1 comprises the light chain variable region of an antibody; and A1 and A2 form a dimer through a disulfide bond, preferably, the dimer is a non-immune effector molecule.

[0079] In one embodiment, in the aforementioned combination or kit, the heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO:9.

[0080] In one embodiment, in the aforementioned combination or kit, the light chain variable region of the antibody comprises the sequence shown in SEQ ID NO:11.

[0081] In one embodiment, in the aforementioned combination or kit, the portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

[0082] In one embodiment, in the aforementioned combination or kit, the TCR β chain amino acid sequence comprises the sequence shown in SEQ ID NO: 13, 17 or 21, and the TCR α chain amino acid sequence comprises the sequence shown in SEQ ID NO: 15, 19 or 23.

[0083] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:13, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:15.

[0084] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:17, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:19.

[0085] In a specific embodiment, 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 TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO:21, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO:23.

[0086] In one embodiment, in the aforementioned combination or kit, the combination or kit further comprises an optional pharmaceutically acceptable carrier, preferably a buffer, more preferably, the buffer comprises albumin or plasma.

[0087] Treatment methods or uses

[0088] In yet another aspect, the present disclosure provides a method for treating a disease, comprising administering the modified cell or the combination or kit as described above to a subject in need thereof.

[0089] The present disclosure provides use of a modified cell as described above, or a combination or kit as described above, in a method of treating a disease in a subject in need thereof.

[0090] 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.

[0091] In a specific embodiment, the disease comprises cancer, a microbial infectious disease, an autoimmune disease, or a disease of aging.

[0092] Preparation method

[0093] In another aspect, a method for preparing the modified cells as described above is provided, wherein the complex protein is incubated with immune cells to form the modified cells.

[0094] In one embodiment, the incubation time is about 1 hour or more.

[0095] In one embodiment, the incubation temperature is about 4°C, room temperature, or 37°C.

[0096] In one embodiment, the buffer used to incubate the complex protein with immune cells contains albumin or plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] The present disclosure can be more fully understood with reference to the following drawings.

[0098] Figure 1 shows the structure and purity of the complex proteins as determined by SDS-PAGE. (a) Anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein; (b) Anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein; (c) Anti-CD3 antibody (UCHT1)-gp100 (HATima) complex protein; (d) Anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein; (e) Anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) complex protein; (f) Anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) complex protein; (g) Anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) complex protein. Lane 1: Molecular weight marker; Lane 2: Multi-domain bifunctional complex molecule under non-reducing conditions; Lane 3: Multi-domain bifunctional fusion molecule under reducing conditions.

[0099] Figure 2 shows the levels of T cell binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at different concentrations. (a) The positive rate of T cell binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at different concentrations; (b) The mean fluorescence intensity of T cell binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at different concentrations.

[0100] Figure 3 shows the binding levels of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at the same concentration to different numbers of T cells. (a) The positive rate of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at the same concentration to different numbers of T cells.

[0101] Figure 4 shows the levels of T cell binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at different concentrations at different incubation temperatures. (a) Positive rate of T cell binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at different concentrations; (b) Mean fluorescence intensity of T cell binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at different concentrations.

[0102] Figure 5 shows the levels of T cell binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at different concentrations at different incubation times. (a) Positive rate of T cell binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at different concentrations; (b) Mean fluorescence intensity of T cell binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules at different concentrations.

[0103] Figure 6 shows the level of T cell binding of anti-CD3 antibody (UCHT1)-1G4 (HATima) molecules in different incubation buffers. (a) Positive rate of T cell binding by anti-CD3 antibody (UCHT1)-1G4 (HATima); (b) Mean fluorescence intensity of T cell binding by anti-CD3 antibody (UCHT1)-1G4 (HATima). The control group consisted of RPMI-1640 culture medium without anti-CD3 antibody (UCHT1)-1G4 (HATima).

[0104] Figure 7 shows the effect of the combination of anti-CD3 antibody (UCHT1)-1G4 (HATima) on T cell activation over a range of concentrations. Flow cytometry was used to determine the CD137 positivity rate of CD3-positive cell populations in the co-culture system. The target cells were NCI-H1299-A2 cells.

[0105] Figure 8 shows the effect of the number of complex protein molecules on T cell tumor recognition. (a) DCT prepared with the same concentration of anti-CD3 antibody (UCHT1)-1G4 (HATima) and different numbers of T cells. Flow cytometry was used to determine the positive rate of T cells binding to the complex protein. (b) A lactate dehydrogenase release assay was used to determine the differences in tumor cell recognition ability of DCT prepared by different methods.

[0106] Figure 9 compares the tumor recognition capabilities of DCT and TCR-T cells. (a) Flow cytometry analysis of the positive rates of T cells and TCR-T cells that bind to the complex protein. (b) Lactate dehydrogenase release assays assess the differences in tumor cell recognition between DCT and TCR-T cells.

[0107] Figure 10 shows that anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects T cells to kill tumor cells. (a) Flow cytometry was used to detect the proportion and phenotype of T cells. (b) Flow cytometry was used to detect the CD3 + Positive rate of T-DCT cells. (c) Lactate dehydrogenase release assay to detect CD3 + The level of T-DCT cells killing target cells. (d) Flow cytometry detection of CD3 + CD137 expression levels of T-DCT cells in different situations. (e) Flow cytometry detection of CD3 + The binding level of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein to T-DCT cells under different conditions. (f) In vitro HepG2 cell tumorigenesis model was used to detect the cytotoxic effect of CD3+ T-DCT cells on tumors.

[0108] Figure 11 shows the redirection of peripheral blood mononuclear cells (PBMCs) to kill tumor cells by the anti-CD3 antibody (UCHT1)-1G4 (HATima). (a) Flow cytometry was used to determine the proportion of T cells in PBMCs. (b) Flow cytometry was used to determine the positive rate of PBMC-DCT cells prepared with the anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein. (c) A lactate dehydrogenase release assay was used to determine the cytotoxicity of PBMC-DCT cells against target cells. (d) Flow cytometry was used to determine the expression of CD137 on PBMC-DCT cells under different conditions. (e) Flow cytometry was used to determine the binding level of the anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein to PBMC-DCT cells under different conditions.

[0109] Figure 12 shows the ability of the anti-CD3 antibody (UCHT1)-1G4 (HATima) to redirect peripheral blood lymphocytes (PBLs) to kill tumor cells. (a) Flow cytometry was used to determine the proportion of T cells in PBLs. (b) Flow cytometry was used to determine the positive rate of PBL-DCT cells prepared with the anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein. (c) A lactate dehydrogenase release assay was used to determine the ability of PBL-DCT cells to kill target cells. (d) Flow cytometry was used to determine the expression of CD137 in PBL-DCT cells under different conditions. (e) Flow cytometry was used to determine the binding level of the anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein to PBL-DCT cells under different conditions.

[0110] Figure 13 shows the redirection of γδT cells to kill tumor cells by the anti-CD3 antibody (UCHT1)-1G4 (HATima). (a) Flow cytometry was used to determine the proportion of γδT cells. (b) Flow cytometry was used to determine the positive rate of γδT-DCT cells prepared with the anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein. (c) Lactate dehydrogenase release assay was used to determine the tumor cell killing capacity of γδT-DCT cells and γδT cells. (d) Flow cytometry was used to determine the CD137 expression levels of γδT-DCT cells and γδT cells under different conditions. (e) Flow cytometry was used to determine the binding level of the anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein to γδT-DCT cells and γδT cells under different conditions.

[0111] Figure 14 shows that anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects T cells to kill tumor cells. (a) Flow cytometry detection of CD3 prepared by anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein +Positive rate of T-DCT cells. (b) Lactate dehydrogenase release assay to detect CD3 + The killing level of target cells by T-DCT cells. (c) Flow cytometry detection of CD3 + CD137 expression levels of T-DCT cells under different conditions. (d) Flow cytometry detection of CD3 + The binding level of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein in T-DCT cells under different conditions. (e) In vitro HepG2 cell tumorigenesis model to detect CD3 + The killing effect of T-DCT cells on tumors.

[0112] Figure 15 shows the ability of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) to redirect peripheral blood mononuclear cells (PBMCs) to kill tumor cells. (a) Flow cytometry was used to detect the positive rate of PBMC-DCT cells prepared with the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein. (b) Lactate dehydrogenase release assay was used to detect the cytotoxicity of PBMC-DCT cells against target cells. (c) Flow cytometry was used to detect the expression of CD137 on PBMC-DCT cells under different conditions. (d) Flow cytometry was used to detect the binding level of the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein on PBMC-DCT cells under different conditions.

[0113] Figure 16 shows the ability of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) to redirect peripheral blood lymphocytes (PBLs) to kill tumor cells. (a) Flow cytometry was used to detect the positive rate of PBL-DCT cells prepared with the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein. (b) Lactate dehydrogenase release assay was used to detect the cytotoxicity of PBL-DCT cells against target cells. (c) Flow cytometry was used to detect CD137 expression levels in PBL-DCT cells under different conditions. (d) Flow cytometry was used to detect the binding level of the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein to PBL-DCT cells under different conditions.

[0114] Figure 17 shows the redirection of γδT cells to kill tumor cells by the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC). (a) Flow cytometry was used to detect the positive rate of γδT-DCT cells prepared with the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein. (b) Lactate dehydrogenase release assay was used to detect the tumor cell killing ability of γδT-DCT cells and γδT cells. (c) Flow cytometry was used to detect the CD137 expression levels of γδT-DCT cells and γδT cells under different conditions. (d) Flow cytometry was used to detect the binding level of the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein on γδT-DCT cells and γδT cells under different conditions.

[0115] Figure 18 shows that anti-CD3 antibody (UCHT1)-AFP (HATima) redirects T cells to kill tumor cells. (a) Flow cytometry detection of CD3 prepared from anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein + Positive rate of T-DCT cells. (b) Lactate dehydrogenase release assay to detect CD3 + The killing level of target cells by T-DCT cells. (c) Flow cytometry detection of CD3 + CD137 expression levels of T-DCT cells under different conditions. (d) Flow cytometry detection of CD3 + The binding level of anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein on T-DCT cells under different situations.

[0116] Figure 19 shows the ability of anti-CD3 antibody (UCHT1)-AFP (HATima) to redirect peripheral blood mononuclear cells (PBMCs) to kill tumor cells. (a) Flow cytometry was used to detect the positive rate of PBMC-DCT cells prepared with the anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein. (b) Lactate dehydrogenase release assay was used to detect the cytotoxicity of PBMC-DCT cells against target cells. (c) Flow cytometry was used to detect CD137 expression levels on PBMC-DCT cells under different conditions. (d) Flow cytometry was used to detect the binding level of the anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein on PBMC-DCT cells under different conditions.

[0117] Figure 20 shows the ability of anti-CD3 antibody (UCHT1)-AFP (HATima) to redirect peripheral blood lymphocytes (PBLs) to kill tumor cells. (a) Flow cytometry was used to determine the positive rate of PBL-DCT cells prepared with the anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein. (b) Lactate dehydrogenase release assay was used to determine the cytotoxicity of PBL-DCT cells against target cells. (c) Flow cytometry was used to determine the expression of CD137 in PBL-DCT cells under different conditions. (d) Flow cytometry was used to determine the binding level of the anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein to PBL-DCT cells under different conditions.

[0118] Figure 21 shows the redirection of γδT cells to kill tumor cells using the anti-CD3 antibody (UCHT1)-AFP (HATima) complex. (a) Flow cytometry was used to detect the positive rate of γδT-DCT cells prepared with the anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein. (b) Lactate dehydrogenase release assay was used to detect the cytotoxicity of γδT-DCT and γδT cells against target cells. (c) Flow cytometry was used to detect the expression of CD137 by γδT-DCT and γδT cells under different conditions. (d) Flow cytometry was used to detect the binding level of the anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein to γδT-DCT cells under different conditions.

[0119] Figure 22 shows that anti-CD3 antibody (UCHT1)-gp100 (HATima) redirects immune cells to kill tumor cells. (a) Flow cytometry detection of T-DCT, PBMC-DCT, PBL-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT and CD8 + (b) Lactate dehydrogenase release assay to detect the positive rate of T-DCT, PBMC-DCT, PBL-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT and CD8 + The killing level of T-DCT cells on target cells. (c) Flow cytometry was used to detect the killing level of T-DCT, PBMC-DCT, PBL-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT and CD8 + The expression levels of CD137 in T-DCT cells under different conditions. (d) Flow cytometry was used to detect the expression of CD137 in T-DCT, PBMC-DCT, PBL-DCT and CD4 +The binding level of anti-CD3 antibody (UCHT1)-gp100 (HATima) complex protein on T-DCT cells under different circumstances.

[0120] Figure 23 shows that anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) redirects immune cells to kill tumor cells. (a) Flow cytometry detection of T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT、CD8 + The positive rates of T-DCT, PBMC-DCT and PBL-DCT cells. (b) Lactate dehydrogenase release assay to detect T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT、CD8 + The killing level of target cells by T-DCT, PBMC-DCT and PBL-DCT cells. (c) Flow cytometry was used to detect the killing level of T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT、CD8 + CD137 expression levels of T-DCT, PBMC-DCT and PBL-DCT cells in different situations.

[0121] Figure 24 shows that anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) redirects immune cells to kill tumor cells. (a) Flow cytometry detection of T-DCT, γδT-DCT, DNT-DCT, and CD4 + T-DCT、CD8 + The positive rates of T-DCT, PBMC-DCT and PBL-DCT cells. (b) Lactate dehydrogenase release assay to detect T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT、CD8 + The killing level of target cells by T-DCT, PBMC-DCT and PBL-DCT cells. (c) Flow cytometry was used to detect the killing level of T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT、CD8 + The expression levels of CD137 in T-DCT, PBMC-DCT and PBL-DCT cells under different conditions. (d) Flow cytometry was used to detect the expression of CD137 in T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT、CD8 +The binding levels of anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) complex protein on T-DCT, PBMC-DCT and PBL-DCT cells under different circumstances.

[0122] Figure 25 shows that anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) redirects immune cells to kill tumor cells. (a) Flow cytometry detection of T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT and CD8 + (b) Lactate dehydrogenase release assay to detect T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT and CD8 + The killing level of T-DCT cells on target cells. (c) Flow cytometry detection of T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT and CD8 + The expression levels of CD137 in T-DCT cells under different conditions. (d) Flow cytometry was used to detect the expression of CD137 in T-DCT, γδT-DCT, DNT-DCT, and CD4 + T-DCT and CD8 + The binding level of anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) complex protein on T-DCT cells under different circumstances.

[0123] Figure 26 shows that anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects immune cells to kill tumor cells. (a) Flow cytometry detection of DNT-DCT, CD4 + T-DCT and CD8 + The positive rate of T-DCT cells. (b) Lactate dehydrogenase release assay to detect DNT-DCT, CD4 + T-DCT and CD8 + The killing level of target cells by T-DCT cells. (c) Flow cytometry was used to detect the cytotoxicity of DNT-DCT and CD4 + T-DCT and CD8 + CD137 expression levels of T-DCT cells under different conditions. (d) Flow cytometry detection of CD4 + T-DCT and CD8 + The binding level of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein on T-DCT cells under different circumstances.

[0124] Figure 27 shows that anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects immune cells to kill tumor cells. (a) Flow cytometry detection of DNT-DCT, CD4 + T-DCT and CD8 + The positive rate of T-DCT cells. (b) Lactate dehydrogenase release assay to detect DNT-DCT, CD4 + T-DCT and CD8 + The killing level of target cells by T-DCT cells. (c) Flow cytometry was used to detect the cytotoxicity of DNT-DCT and CD4 + T-DCT and CD8 + CD137 expression levels of T-DCT cells under different conditions. (d) Flow cytometry detection of CD4 + T-DCT and CD8 + The binding level of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein on T-DCT cells under different circumstances.

[0125] Figure 28 shows that anti-CD3 antibody (UCHT1)-AFP (HATima) redirects immune cells to kill tumor cells. (a) Flow cytometry detection of DNT-DCT, CD4 + T-DCT and CD8 + The positive rate of T-DCT cells. (b) Lactate dehydrogenase release assay to detect DNT-DCT, CD4 + T-DCT and CD8 + The killing level of target cells by T-DCT cells. (c) Flow cytometry was used to detect the cytotoxicity of DNT-DCT and CD4 + T-DCT and CD8 + CD137 expression levels of T-DCT cells under different conditions. (d) Flow cytometry detection of CD8 + The binding level of anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein on T-DCT cells under different situations.

[0126] Figure 29 shows a comparison of the biological activities of the anti-CD3 antibody (UCHT1) in the HATima and ImmTAC structures used for cell modification. (a) SDS-PAGE assay to determine the structure and purity of the VH-VL and scFv proteins. Lane 1: Molecular weight marker; Lane 2: Protein in a non-reduced state; Lane 3: Protein in a reduced state. (b) Lactate dehydrogenase release assay to determine the cytotoxicity of T cells redirected by VH-VL and scFv proteins against target cells. (c) Flow cytometry assay to determine the CD137 expression levels of T cells redirected by VH-VL and scFv proteins at a concentration of 1E-9 M under different conditions. (d) Flow cytometry assay to determine the CD137 expression levels of T cells redirected by VH-VL and scFv proteins at a concentration of 1E-7 M under different conditions. CD3 antibody (UCHT1)-1G4 (HATima) and anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) served as positive control proteins.

[0127] The present disclosure will be further illustrated through detailed description below.

[0128] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0129] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0130] The term "complex protein" as used herein 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, optionally connected by a flexible connecting peptide, wherein the portion that specifically binds to a cell surface marker molecule of the modified cell can be an antibody or a fragment thereof that specifically binds to a 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 portion that specifically binds to a target cell surface antigen can be a TCR molecule, a single-chain αβTCR, or a TCRα chain / TCRβ chain heterodimer.

[0131] As used herein, the term "binding" refers to a bond or connection that can be covalent (e.g., by chemical coupling) or non-covalent (e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc.). In preferred embodiments, the bond or connection is a non-covalent interaction. In one example, the term binding refers to the connection of an antigen-binding moiety to its antigen.

[0132] As used herein, the term "immune cell" includes cells of hematopoietic origin that play a role in immune responses. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; and bone marrow cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. Those skilled in the art will appreciate that these immune cells can be of any origin, including but not limited to primary cells, passaged cells, or cells derived from stem cells.

[0133] A "disease" is any condition that would benefit from medical treatment using the modified cells or combinations of the present disclosure. Non-limiting examples of disorders include cancer, microbial infectious diseases, autoimmune diseases, or diseases of aging.

[0134] The term "autoimmune disease" as used herein generally refers to a disease characterized by a composition having self-identification. The example of an autoimmune disease includes, but is 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 (Grave's disease), ankylosing spondylitis, Sjogrens disease (Sjogrens Disease), CREST syndrome, scleroderma, IgA nephropathy (Neprhopathy), bullous pemphigoid, pemphigus vulgaris (Pemphigous Vulgaris), ANCA-associated vasculitis, antiphospholipid syndrome and more. Most autoimmune diseases are also chronic inflammatory diseases. It is defined as a disease process related to the long-term (>6 months) activation of inflammatory cells (leukocytes). Chronic inflammation causes the damage of patient's organs or tissues. Many diseases are chronic inflammatory conditions, but it is known that there is no autoimmune basis. Examples include atherosclerosis, congestive heart failure, Crohn's disease, ulcerative colitis, polyarteritis nodosa, Whipple's disease, primary sclerosing cholangitis, and many more.

[0135] The term "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), lung cancer including non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, colorectal cancer, intestinal carcinoid, bladder cancer, gastric cancer, pancreatic cancer, liver cancer (hepatocellular carcinoma), hepatoblastoma, esophageal cancer, lung adenocarcinoma, mesothelioma, synovial sarcoma, osteosarcoma, head and neck squamous cell carcinoma, juvenile nasopharyngeal angiofibroma, liposarcoma, thyroid cancer, melanoma, basal cell carcinoma (BCC), medulloblastoma, and desmoid tumors. Cancers of particular interest for treatment using the methods of the present disclosure include glioma, medulloblastoma, colon cancer, colorectal cancer, melanoma, breast cancer, lung cancer, liver cancer, and gastric cancer.

[0136] As used herein, 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.

[0137] As used herein, the term "treating" refers to administering to a subject an effective amount of cells having a polynucleotide sequence of a target gene altered ex vivo according to the methods described herein, such that the subject experiences a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, a decrease in the extent of the disease, stabilization of the disease state (i.e., no worsening), a delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Treatment can refer to prolonging survival compared to the expected survival if not receiving treatment. Thus, those skilled in the art recognize that treatment can improve the disease state but may not be a complete cure for the disease. As used herein, the term "treatment" includes prevention. Alternatively, treatment is "effective" if the progression of the disease is reduced or stopped. "Treatment" can also mean prolonging survival compared to the expected survival if not receiving treatment. Patients in need of treatment include those already diagnosed with a condition associated with expression of the polynucleotide sequence, as well as those at risk of developing such a condition due to genetic predisposition or other factors.

[0138] An "immune effector molecule" is an immune effector polypeptide 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, as will be understood by those skilled in the art, do not activate the humoral or cellular components of the immune system, e.g., do not induce or stimulate an immune response. Example

[0139] The experimental methods in the following examples are conventional methods unless otherwise specified. The present disclosure will be further understood with reference to the following non-limiting experimental examples.

[0140] Example 1: Preparation of materials

[0141] 1.1 Preparation of complex protein

[0142] 1.1.1 Vector construction and inclusion body expression purification

[0143] The genes encoding the complex proteins (see Table 1) were cloned into the pET-28a expression plasmid. The plasmids were transformed into Escherichia coli strain BL21-DE3. Kanamycin-resistant colonies were grown in LB medium (50 μg / mL of kanamycin) at 37°C to an OD600 of approximately 1.0. Protein expression was then induced with 1 mM IPTG. Three hours after induction, cells were harvested by centrifugation at 4000 g for 15 minutes in a Thermo Scientific HERAEUS X1R centrifuge. The cells were resuspended in 20 mL of BugBuster Master Mix (Merck Millipore) by vortexing, then shaken at room temperature for 20 minutes. The cells were then centrifuged in a pre-cooled high-speed centrifuge at 6000 g at 4°C for 15 minutes. The supernatant was removed. 10 mL of BugBuster Master Mix was added, the pellet was resuspended by vortexing, and shaken at room temperature for 5 minutes. Add 30 mL of 10-fold diluted BugBuster (Merck Millipore) again, invert several times to mix the liquid, and place it in a high-speed centrifuge again, centrifuge at 6000g, 4°C, and centrifuge for 15 minutes. After discarding the supernatant, add 30 mL of 10-fold diluted BugBuster, vortex to resuspend the pellet, and place it in a high-speed centrifuge, centrifuge at 6000g, 4°C, and centrifuge for 15 minutes. Discard the supernatant and repeat the previous step twice. Discard the supernatant, add 30 mL of PBS to resuspend the inclusion bodies, and centrifuge at 6000g, 4°C, and centrifuge for 15 minutes. After discarding the supernatant, add 6M guanidine hydrochloride to dissolve the inclusion bodies. The purified inclusion bodies are diluted in series, and then samples are taken for SDS-PAGE to detect their purity and estimate their yield. All inclusion bodies are quantified for protein, aliquoted, and stored at -80°C.

[0144] Table 1. Sequence information

[0145] Table 2. Sequence information of complex proteins

[0146] 1.1.2 Protein renaturation

[0147] Prepare TCR refolding buffer (5 M urea, 100 mM Tris pH 8.1, 0.4 M L-arginine, 2 mM EDTA, 6.5 mM cysteamine, and 1.87 mM cystamine) and pre-cool to 4°C. Thaw approximately 12 mg of TCR α chain, 11.2 mg of antibody heavy chain-TCR β chain, and 6 mg of antibody light chain inclusion bodies from cryopreservation. Add each to 6 mL of 6 M guanidine hydrochloride solution and add DTT to a final concentration of 15 μM. Mix thoroughly and incubate at 37°C for 40 minutes. Add the incubated TCR α chain, heavy chain-TCR β chain, and antibody light chain inclusion bodies to TCR refolding buffer and allow to react in the refrigerator for 30 minutes. Prepare a 10kDa dialysis bag. Add the refolding solution to the bag and place it in pre-chilled deionized water. Dialyze overnight in a cold room. The next day, transfer the bag to pre-chilled 10mM Tris-HCl for dialysis. In the evening, transfer the bag to pre-chilled 10mM Tris-HCl for dialysis.

[0148] 1.1.3 Protein purification

[0149] Soluble and correctly folded multi-domain fusion molecules are separated from misfolded, degraded products and impurities by the three-step purification method described below. First, 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, and the supernatant is filtered again with a 0.45μm filter membrane. The renatured sample was purified using a Q HP (GE Healthcare). After rinsing with Solution A (10 mM Tris-HCl, pH 8.0) for 4 column volumes, the sample was loaded at a flow rate of 5 mL / min. After complete sample loading, the column was rinsed with Solution A for approximately 4 column volumes. When both the conductivity and UV280 values ​​stabilized, a gradient elution was initiated using Solution B (1 M NaCl + 10 mM Tris-HCl, pH 8.0) from 0 to 100% over 50 minutes at a flow rate of 3 mL / min. When the UV280 value increased significantly, 1 mL of sample was collected from each tube. Peak fractions were analyzed by 12% SDS-PAGE and pooled. The sample was then purified using molecular sieves (Superdex 75, GE Healthcare). The purified sample was concentrated using a 10 kDa ultrafiltration tube at 3500 × g at 4°C to 500 μL, and then purified using molecular sieves. The molecular sieve was first equilibrated with deionized water and then equilibrated again with PBS. A 500 μL sample loop was washed and loaded. After loading, the sample was equilibrated and eluted again with PBS at a flow rate of 1 mL / min, and 0.4 mL was collected from each tube. The peak fractions were analyzed by 12% SDS-PAGE and then combined. Finally, an anion exchange purification was performed again using an anion exchange column. Q HP (GE Healthcare). The sample purified in the previous step was diluted 20 times with pre-cooled 10mM Tris before loading. After washing with solution A for 4 column volumes, the sample was loaded at a flow rate of 5mL / min. After the sample was completely loaded, the column was washed with solution A for about 4 column volumes again. When the conductivity and UV280 tended to be stable, gradient elution was started, and solution B was set from 0-100% for 50 minutes. The sample was eluted at a flow rate of 3mL / min. When UV280 increased significantly, collection was started, and 1mL was collected in each tube. Peak components were analyzed by 12% SDS-PAGE and then combined. Concentrated using a 10kDa ultrafiltration tube, concentrated to 500μL at 3500×g and 4℃, and buffer exchanged with PBS buffer. The concentration was determined and then aliquoted and stored at -80℃. The structure and purity of the refolded and purified complex protein were detected by SDS-PAGE. The results in Figure 1 show that the molecular weight of anti-CD3 antibody (UCHT1)-1G4 (HATima) (a), anti-CD3 antibody (UCHT1)-AFP (HATima) (b), anti-CD3 antibody (UCHT1)-gp100 (HATima) (c) and anti-CD3 antibody (UCHT1)-1G4 (HATima) (d) is 70 kDa, which is decomposed into 3 bands under the reduced state, and the purity of the complex protein is 80-90%.

[0150] 1.2 Preparation of peripheral blood mononuclear cells (PBMC)

[0151] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat or peripheral blood using Ficoll density gradient centrifugation. Add 20 mL of lymphocyte separation buffer to a 50 mL centrifuge tube. Mix the buffy coat / peripheral blood with phosphate-buffered saline (PBS) at a 1:1 ratio to dilute the blood. Use a pipette to draw the diluent slowly over the Ficoll layer along the tube wall. Centrifuge horizontally (2000 rpm, 20 minutes, room temperature). Aspirate the cloudy layer and place it into a 50 mL centrifuge tube. Add 5 times the volume of phosphate-buffered saline (PBS) and centrifuge (1800 rpm, 10 minutes, room temperature) to wash the cells. Discard the supernatant and add 8 mL of preheated red blood cell lysis buffer, vortex thoroughly, and incubate at 37°C for 10 minutes. Stop the process by adding 30 mL of phosphate-buffered saline (PBS) and centrifuge (1200 rpm, 10 minutes, room temperature). The supernatant was discarded, and complete culture medium (RPMI-1640 + 10% FBS) was added to resuspend the cells. The cell number and viability were detected using a Countstar cell sorter.

[0152] 1.3 Preparation of peripheral blood lymphocytes (PBL)

[0153] Peripheral blood mononuclear cells (PBMCs) were collected and centrifuged (300 g, 5 minutes, room temperature), and the supernatant was discarded. The cells were resuspended in 1 mL of RPMI-1640 medium and counted using Countstar. The cell density was adjusted to 2 × 10 6 Cells were seeded in a 6-well plate, 3 mL per well, and placed in a 37°C CO2 incubator for 1 hour. Observe the cell adhesion status under an inverted microscope. When the wells are filled with "stretched and adherent" cells, the monocyte adhesion is complete. Use a pipette to blow evenly into each corner and resuspend the peripheral blood lymphocytes (PBL). Transfer the supernatant to another 15 mL centrifuge tube. Centrifuge (300 g, 5 minutes, RT) and discard the supernatant. Resuspend the cells in 1 mL of complete RPMI-1640 medium and count the cells using Countstar.

[0154] 1.4 T cell preparation

[0155] EasySep with Stemcell TM Human T Cell Isolation Kit (Cat. No. 17951) was used to isolate T cells from peripheral blood mononuclear cells. PBMCs were collected, cell counted using Countstar, and centrifuged (300 g, 5 minutes, room temperature), and the supernatant was discarded. TM Resuspend the cells in Buffer and adjust the cell density to 5×10 7Cells / mL, cell suspension volume 0.25mL-2mL. Transfer the cell suspension to a polystyrene round-bottom tube (5mL volume). Add Isolation Cocktail at a ratio of 50μL / mL. Mix by flicking with your finger and incubate at room temperature for 5 minutes. Oscillate Dextran RapidSpheres (microspheres) for 30 seconds to make the microspheres uniformly dispersed. Add RapidSpheres at a ratio of 40μL / mL. TM , flick with your finger to mix, no incubation required. Add EasySep TM Buffer, to a total volume of 2.5mL. Mix by blowing gently 2-3 times with a pipette. Insert the polystyrene round-bottom tube into the magnet and incubate at room temperature for 3 minutes. Pick up the magnet, flip the magnet and tube in one continuous motion, and pour the enriched cell suspension into a new tube. Invert the magnet and tube for 2-3 seconds, then return them upright. Do not shake or suck off any droplets that may be hanging on the mouth of the tube. 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 Take a certain amount of sample for flow cytometry phenotype and purity testing. Add Human T-Activator CD3 / CD28 was mixed and seeded into a 48-well plate, 500 μL per well, and cultured in a 37°C, 5% CO2 incubator. After 4 days, the beads were discarded. The culture medium was replaced with complete medium every 2-3 days, and the culture was expanded. After 12 days, a certain amount of cells was collected for phenotypic and purity analysis by flow cytometry. Cells were then collected for cryopreservation and functional experiments. The antibodies used for flow cytometry were: FITC anti-human CD3 antibody (BioLegend, Catalog No. 317306); APC anti-human CD4 antibody (BioLegend, Catalog No. 300514); and PE anti-human CD8 antibody (BioLegend, Catalog No. 344706).

[0156] 1.5 CD4 + Preparation of T cells

[0157] CD4 MicroBeads, human (Cat. No. 130-045-101) from Miltenyi Biotec were used to positively select CD4 +T cells. Prepare separation buffer consisting of phosphate buffered saline containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid (EDTA). Collect T cells, count them using a Countstar, centrifuge (300 g, 5 minutes, room temperature), and discard the supernatant. Resuspend the cells in separation buffer and adjust the cell density to 1 × 10 7 / 80μL. 7 1×10 cells require 20 μL of CD4 MicroBeads. Add CD4 MicroBeads, mix well, and let stand at 4°C for 15 minutes. 7 For each cell, add 2 mL of separation buffer, centrifuge (300 g, 5 minutes, room temperature), and discard the supernatant. Add 500 μL of separation buffer, resuspend the cells, and add them to the separation column that has been wetted 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, remove the column from the magnet, and use the piston to push 2 mL of separation buffer into a 15 mL centrifuge tube. The separation buffer contains CD4 + T cells were counted and centrifuged (300 g, 5 minutes, room temperature), and the supernatant was discarded. Complete RPMI-1640 medium containing IL-2 (RPMI-1640 + 10% FBS + 100 U / mL IL-2, hereinafter referred to as "medium") was added and resuspended to adjust the cell density to 1×10 6 Take a certain amount of sample for flow cytometry phenotype and purity testing. Add Human T-Activator CD3 / CD28 was mixed and seeded into a 48-well plate, 500 μL per well, and cultured in a 37°C, 5% CO2 incubator. After 4 days, the beads were discarded. The culture medium was replaced and the culture was expanded every 2-3 days. After 12 days, a certain amount of cells was collected for phenotypic and purity analysis by flow cytometry. Cells were then collected for cryopreservation and functional experiments. The antibodies used for flow cytometry were: FITC anti-human CD3 antibody (BioLegend, Cat. No. 317306); APC anti-human CD4 antibody (BioLegend, Cat. No. 300514).

[0158] 1.6 CD8 + Preparation of T cells

[0159] CD8 MicroBeads, human (Cat. No. 130-045-201) from Miltenyi Biotec were used to positively select CD8 from T cells. +T cells. Prepare separation buffer consisting of phosphate buffered saline containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid (EDTA). Collect T cells, count them using a Countstar, centrifuge (300 g, 5 minutes, room temperature), and discard the supernatant. Resuspend the cells in separation buffer and adjust the cell density to 1 × 10 7 / 80μL. 7 1×10 cells require 20μL CD8 MicroBeads. Add CD8 MicroBeads, mix well, and let stand at 4°C for 15 minutes. 7 For each cell, add 2 mL of separation buffer, centrifuge (300 g, 5 minutes, RT), and discard the supernatant. Add 500 μL of separation buffer, resuspend the cells, and add them to the separation column that has been wetted 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, remove the column 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 were counted and centrifuged (300 g, 5 minutes, room temperature), and the supernatant was discarded. Complete RPMI-1640 medium containing IL-2 (RPMI-1640 + 10% FBS + 100 U / mL IL-2, hereinafter referred to as "medium") was added and resuspended to adjust the cell density to 1×10 6 Take a certain amount of sample for flow cytometry phenotype and purity testing. Add Human T-Activator CD3 / CD28 was mixed and seeded into a 48-well plate, 500 μL per well, and cultured in a 37°C, 5% CO2 incubator. After 4 days, the beads were discarded. The culture medium was replaced and the culture was expanded every 2-3 days. After 12 days, a certain amount of cells was collected for phenotypic and purity analysis by flow cytometry. Cells were then collected for cryopreservation and functional experiments. The antibodies used for flow cytometry were: FITC anti-human CD3 antibody (BioLegend, Cat. No. 317306); PE anti-human CD8 antibody (BioLegend, Cat. No. 344706).

[0160] 1.7 Preparation of double-negative T cells (DNT)

[0161] CD4 was negatively selected from T cells using Miltenyi Biotec's CD8 MicroBeads, human (Cat. No. 130-045-201) and CD4 MicroBeads, human (Cat. No. 130-045-101). - CD8 -Double-negative T cells, hereinafter referred to as double-negative T cells. Prepare separation buffer, consisting of phosphate buffered saline containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid (EDTA). Collect T cells, count cells using Countstar, centrifuge (300g, 5 minutes, room temperature), and discard the supernatant. Resuspend the cells in separation buffer and adjust the cell density to 1×10 7 / 80μL. 7 Each cell requires 20 μL of CD8 MicroBeads and 20 μL of CD4 MicroBeads. Add CD8 MicroBeads and CD4 MicroBeads at the same time, mix well, and let it stand at 4 degrees for 15 minutes. 7 1×10 cells, add 2 mL of separation buffer, centrifuge (300 g, 5 minutes, RT), and discard the supernatant. Add 1 mL of separation buffer, resuspend the cells, add to the separation column that has been wetted and hung on the magnet, collect the liquid that flows through the column, which contains double-negative T cells, count, centrifuge (300 g, 5 minutes, RT), and discard the supernatant. Add complete RPMI-1640 medium containing IL-2 (RPMI-1640 + 10% FBS + 100 U / mL IL-2, hereinafter referred to as "medium"), resuspend, and adjust the cell density to 1×10 6 Take a certain amount of sample for flow cytometry phenotype and purity testing. Add Human T-Activator CD3 / CD28 was mixed and seeded into a 48-well plate, 500 μL per well, and cultured in a 37°C, 5% CO2 incubator. After 4 days, the beads were discarded. The culture medium was replaced and the culture was expanded every 2-3 days. After 12 days, a certain amount of cells was collected for phenotypic and purity analysis by flow cytometry. Cells were then collected for cryopreservation and functional experiments. The antibodies used for flow cytometry were: FITC anti-human CD3 antibody (BioLegend, Cat. No. 317306); PE anti-human CD8 antibody (BioLegend, Cat. No. 344706); and APC anti-human CD4 antibody (BioLegend, Cat. No. 300514).

[0162] 1.8 Preparation of γδ T cells

[0163] Peripheral blood mononuclear cells were collected, counted, centrifuged (300g, 5 minutes, room temperature), and the supernatant was discarded. TM -XF T cell expansion medium + 5% FBS + 100 U / mL IL-2 + 10 ng / ml IL-15) to resuspend the cells and adjust the cell density to 2 × 106 Cells were plated in 24-well plates at 4% 4% CO2 and cultured at 5 μM. Zoledronic acid (Zoledronate, InvivoChem, Catalog No. V1560) was added at a working concentration of 5 μM. The cells were cultured in a 37°C, 5% CO2 incubator. The culture medium was changed every 2-3 days with complete medium and expanded without zoledronic acid. After 12-14 days, a certain amount of cells was collected for flow cytometry phenotyping and purity analysis. Cells were then collected for cryopreservation and functional analysis. Antibodies used for flow cytometry analysis were: FITC anti-human CD3 antibody (BioLegend, Catalog No. 317306); PE anti-human TCR Vδ2 antibody (BioLegend, Catalog No. 331408).

[0164] 1.9 Preparation of DCT by coating the surface of immune cells with complex proteins

[0165] Based on 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 complex protein was added to the above-mentioned various immune cells for incubation. The proportion and fluorescence intensity of immune cells bound by the complex protein were detected by flow cytometry, and the concentration of the residual complex protein in the system was detected by ELISA. The tumor recognition function was then tested and compared.

[0166] Example 2: Controllability of the number of complex protein molecules bound to the cell surface

[0167] 2.1 Concentrations of different complex proteins

[0168] The complex protein is exemplified by anti-CD3 antibody (UCHT1)-1G4 (HATima). 5 The complex proteins were added to the cell suspension of 10 T cells / 100 μL RPMI-1640 medium to a final concentration of 3×10 -7 mol / L、3×10 -8 mol / L、3×10 -9 mol / L、3×10 -10mol / L and 0mol / L, incubate at 4 degrees for 30 minutes. Centrifuge (500g, 5 minutes, 4°C), discard the supernatant, add 100μL RPMI-1640 medium, and resuspend the cells. Add 1μL 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. Add 100μL RPMI-1640 medium and detect the fluorescence signal of pHLA-tetramer-APC 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. As shown in Figure 2: The working concentration is 3×10 -7 mol / L、3×10 -8 mol / L、3×10 -9 mol / L and 3×10 -10 At 100 mol / L of the complex protein, the positive rates of binding to T cells were 98.9%, 98.2%, 47.7%, and 0.62%, respectively (Figure 2a), and their fluorescence intensities were 15413, 14614, 6465, and 827, respectively (Figure 2b). As the concentration of the complex protein decreased, the number of molecules bound to the T cell surface decreased.

[0169] 2.2 Complex protein binding to different numbers of T cells

[0170] The complex protein is exemplified by anti-CD3 antibody (UCHT1)-1G4 (HATima). -9 mol / L complex protein in 100 μL RPMI-1640 medium, and different numbers of T cells were added, including 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 100μL RPMI-1640 medium, and resuspend the cells. Add 1μL 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. Add 100μL RPMI-1640 medium and detect the fluorescence signal of pHLA-tetramer-APC 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. As shown in Figure 3: In a 100μL RPMI-1640 culture medium system, 1×10 -9mol / L anti-CD3 antibody (UCHT1)-1G4 (HATima) binds to 4×10 6 2×10 6 pcs, 1×0 6 and 0.3×10 6 The positive rates of the three T cells were 37.9%, 48.4%, 62.4% and 65.9% respectively (Figure 3). As the number of T cells decreased, the number of molecules of the same concentration of complex protein bound to the T cell surface increased.

[0171] 2.3 Differences in complex protein binding to T cells at different incubation temperatures

[0172] The complex protein is exemplified by the anti-CD3 antibody (UCHT1) 1G4 (HATima). 5 The complex proteins were added to the cell suspension of 10 T cells / 100 μL RPMI-1640 medium to a final concentration of 3×10 -7 mol / L、3×10 -8 mol / L、3×10 -9 mol / L, 2×10 -9 mol / L, 1×10 -9 mol / L、3×10 -10 mol / L and 0mol / L, incubate at 4 degrees and 37 degrees for 1 hour respectively. Centrifuge (500g, 5 minutes, 4°C), discard the supernatant, add 100μL RPMI-1640 medium, and resuspend the cells. Add 1μL 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. Add 100μL RPMI-1640 medium and detect the fluorescence signal of pHLA-tetramer-APC 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. As shown in Figure 4: The working concentration is 3×10 -7 mol / L、3×10 -8 mol / L、3×10 -9 mol / L, 2×10 -9 mol / L, 1×10 -9 mol / L and 3×10 -10mol / L of complex protein, incubated at 4 degrees, the positive rates of binding to T cells were 98.9%, 98.9%, 77.8%, 62.9%, 40.7% and 7.26% respectively; incubated at 37 degrees, the positive rates of binding to T cells were 95.4%, 89.8%, 27.4%, 14.8%, 8.13% and 0.57% respectively (Figure 4a). At the same concentration, the average fluorescence intensity of the 4-degree incubation group was higher than that of the 37-degree incubation group (Figure 4b). High incubation temperature will reduce the number of complex proteins bound to the surface of T cells, especially the effect on low concentrations of complex proteins is more obvious, with a decrease rate of 80% (working concentration is 1×10 -9 mol / L).

[0173] 2.4 Differences in complex protein binding to T cells at different incubation times

[0174] The complex protein is exemplified by anti-CD3 antibody (UCHT1)-1G4 (HATima). 5 The complex proteins were added to the cell suspension of 10 T cells / 100 μL RPMI-1640 medium to a final concentration of 3×10 -8 mol / L、3×10 -9 mol / L, 2×10 -9 mol / L and 1×10 -9 mol / L, incubate at 37 degrees for 1 hour and 18 hours respectively. Centrifuge (500g, 5 minutes, 4°C), discard the supernatant, add 100μL RPMI-1640 medium, and resuspend the cells. Add 1μL 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. Add 100μL RPMI-1640 medium and detect the fluorescence signal of pHLA-tetramer-APC 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. As shown in Figure 5: The working concentration is 3×10 -8 mol / L、3×10 -9 mol / L, 2×10 -9 mol / L and 1×10 -9mol / L of complex protein, incubated at 37 degrees for 1 hour, the positive rates of binding to T cells were 89.8%, 27.4%, 14.8% and 8.13%, respectively; incubated at 37 degrees for 18 hours, the positive rates of binding to T cells were 36.5%, 1.15%, 0.047% and 0%, respectively (Figure 5a). At the same concentration and incubation temperature, the average fluorescence intensity of the group incubated for 1 hour was higher than that of the group incubated for 18 hours (Figure 5b). Long-term incubation at 37 degrees significantly reduced the number of complex proteins bound to the surface of T cells, especially the effect on low concentrations of complex proteins was more obvious, with a decrease rate of 95% (working concentration was 3×10 -9 mol / L).

[0175] 2.5 Effects of incubation buffer components on complex protein binding to T cells

[0176] The complex protein is an anti-CD3 antibody (UCHT1)-1G4 (HATima) as an example. Different incubation buffers were prepared, including RPMI-1640 medium, phosphate buffered saline, phosphate buffered saline containing 30 μg / mL mouse albumin, and phosphate buffered saline containing 10% mouse plasma, and stored at 4°C. The complex protein was diluted 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 μL of the same incubation buffer was mixed with 50 μL of the complex protein and incubated at 4°C for 1 hour. Centrifuge (500 g, 5 minutes, 4°C), discard the supernatant, add 100 μL of RPMI-1640 medium, and resuspend the cells. Add 1 μL of pHLA-tetramer-APC and incubate at 4°C for 1 hour. Add 1 mL of RPMI-1640 medium, centrifuge (500 g, 5 minutes, 4°C), discard the supernatant, and repeat once. Add 100 μL of RPMI-1640 medium and detect the fluorescence signal of pHLA-tetramer-APC by flow cytometry. The fluorescence signal of pHLA-tetramer-APC represents the proportion and intensity of T cells bound to the complex protein and is positively correlated with the amount of complex protein bound to the T cell surface. As shown in Figure 6: Under the treatment of incubation buffers such as RPMI-1640 medium, phosphate buffer, phosphate buffer containing 30 μg / mL mouse albumin, and phosphate buffer containing 10% mouse plasma, 1×10 -9The positive rates of T cell binding for the complex protein at 100 μmol / L were 2.12%, 1.79%, 90.6%, and 98.4%, respectively (Figure 6a); their mean fluorescence intensities were 818, 897, 5890, and 10455, respectively (Figure 6b). Plasma components, such as albumin, enhance the binding of the complex protein to the CD3 antigen, increasing the amount of the complex protein bound to the T cell surface.

[0177] 2.6 Effects of protein complex binding on T cell activation within a certain concentration range

[0178] The complex protein is an example of anti-CD3 antibody (UCHT1)-1G4 (HATima). HIPP-T009 medium was used and the cell density was set to 3×10 5 In a 96-well U-bottom plate, add 100 μL of T cell suspension and then add the complex protein and mix well. 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, incubate at 4°C for 1 hour to prepare DCT. Add 100 μL of NCI-H1299-A2 target cell suspension and HIPP-T009 culture medium, respectively. Incubate at 37°C in a 5% CO2 incubator for 18 hours. Centrifuge the entire 96-well plate (500g, 5 minutes, 4°C), discard the supernatant, add 100 μL of RPMI-1640 culture medium, and resuspend the cells. Add 1 μL of FITC anti-human CD3 antibody (BioLegend, Catalog No.: 317306) and PEcy7 anti-human CD137 antibody (BioLegend, Catalog No.: 309818), respectively. Set up unstained and isotype controls, and incubate at 4°C for 30 minutes. Add 100 μL of RPMI-1640 culture medium, centrifuge (500g, 5 minutes, 4°C), discard the supernatant, and repeat once. 100 μL RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody and PEcy7 anti-human CD137 were detected by flow cytometry to analyze the CD137 ratio of CD3 positive cell population, which represents the degree of T cell activation. -8 mol / L、3×10 -9 mol / L, 2×10 -9 mol / L and 1×10 -9When DCT was prepared with 100 mol / L complex protein without contact with target cells, the activation rates of T cells were 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 activation rates of T cells were 78.7%, 54.2%, 69.1% and 69.3% respectively. The working concentration was less than 3×10 -9 mol / L anti-CD3 antibody (UCHT1)-1G4 (HATima), T cells are not activated by the binding of complex proteins.

[0179] 2.7 Effect of the number of complex protein-bound molecules on T cell tumor recognition function

[0180] The complex protein is exemplified by anti-CD3 antibody (UCHT1)-1G4 (HATima). The DCT preparation method is as follows: -9 mol / L complex protein in 300 μL HIPP-T009 culture medium, and 0.9×10 6 and 6×10 6 T cells, incubated at 4 degrees for 1 hour. -9 mol / L complex protein in 100 μL HIPP-T009 culture medium, and 0.3×10 6 and 2×10 6 T cells were incubated at 4 degrees for 1 hour. Take 15 μL and 50 μL of cell suspension respectively, centrifuge (500g, 5 minutes, 4°C), discard the supernatant, add 100 μL RPMI-1640 medium, and resuspend the cells. Add 1 μL APC anti-human TCR Vβ13.1 antibody (biolegend, catalog number: 362408) and incubate at 4 degrees for 1 hour. Add 1 mL RPMI-1640 medium, centrifuge (500g, 5 minutes, 4°C), discard the supernatant, and repeat once. Add 100 μL RPMI-1640 medium and detect the fluorescence signal of anti-hTCR Vβ13.1-APC by flow cytometry. The results in Figure 8a show that at 1x10 -9 mol / L compound protein, 0.3×10 6 The binding rate of the T cell group was 98.4%, and the binding rate of 2×10 6 The binding rate of T cells was 75.5%. Under the same concentration of complex protein, T cells with fewer cells bound more complex protein molecules. Using HIPP-T009 culture medium, the prepared DCT was used to prepare cell suspensions of different densities, namely 1.25×10 3 / 75μL, 2.5×10 3 / 75μL, 5×103 / 75μL and 1×10 4 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 trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1×10 4 75 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and each well had two replicates. The 96-well U-bottom plate with cells was placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate, and 50 μL of substrate solution was added. The reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added, and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum lysis wells of target cells - spontaneous pores of target cells) × 100%. The results in Figure 8b show that at effector-target ratios of 1:4, 1:2, and 1:1, 0.3×10 6 The specific killing rate of DCT prepared by T cells against positive target cells (HepG2-NYESO1 and NCI-H1299-A2) was higher than 2×10 6 DCT prepared from T cells showed a gradient-dependent effector-target ratio, with significant differences observed at a 1:1 effector-target ratio. DCT prepared by either method had no cytotoxic effect on negative target cells (HepG2 and NCI-H1299). The number of complex protein molecules bound to the T cell surface was positively correlated with its tumor recognition function.

[0181] Example 3: Modifying tumor recognition and killing functions of cells

[0182] 3.1 Differences between the tumor recognition function of DCT and TCR-T cells

[0183] Preparation method of TCR-T cells: Using the NheI and SalI restriction sites, the TCRα and β chain sequences are inserted into the lentiviral expression vector pGZ178, mixed with the packaging plasmids (pMDLg / pRRE, pRSV-REV and pMD.2G), and transfected into 293T cells under the action of the transfection reagent PEI-MAX, and cultured for 3 days. The culture supernatant containing lentiviral particles is collected, concentrated with a concentrator tube, and frozen at -80°C after aliquoting, and the titer is detected. Magnetic beads coated with anti-CD3 / CD28 antibodies are combined with IL-2 (100 IU / mL) to stimulate T cells. After 24 hours, protamine (10 μg / mL) and lentiviral solution containing TCR genes are added, mixed, and cultured for 7 days. The complex protein takes anti-CD3 antibody (UCHT1)-1G4 (HATima) as an example. The method for preparing DCT is: in a solution containing 1×10 -7 mol / L complex protein in 200 μL HIPP-T009 culture medium, add 5×10 6 T cells were incubated at 4°C for 1 hour. 6 Cells were centrifuged (500g, 5 minutes, 4°C), the supernatant was discarded, and 100μL RPMI-1640 medium was added to resuspend the cells. 1μL pHLA-tetramer-APC was added and incubated at 4°C for 1 hour. 1mL RPMI-1640 medium was added and centrifuged (500g, 5 minutes, 4°C), the supernatant was discarded, and repeated once. 100μL RPMI-1640 medium was added and the fluorescence signal of pHLA-tetramer-APC was detected by flow cytometry. The results in Figure 9a show that at 1×10 -7 mol / L compound protein, 5×10 6 The positive rate of DCT prepared from T cells was 97.4%; the positive rate of TCR-T cells in this batch was 27.3%. T cells from the same volunteer were added to the DCT cells to adjust the positive rate of DCT cells to 27.3%. Using HIPP-T009 medium, the adjusted DCT and TCR-T cells were prepared to prepare cell suspensions of different densities, namely 1.25×10 3 / 75μL, 2.5×10 3 / 75μL, 5×10 3 / 75μL and 1×10 4HepG2-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 trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1×10 4 75 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum lysis wells of target cells - spontaneous pores of target cells) × 100%. The results in Figure 9b show that at effector-target ratios of 1:8, 1:4, and 1:2, 5×10 6 The specific killing rate of DCT prepared by 5×10 T cells on HepG2-NYESO1 positive target cells was significantly higher than that of TCR-T cells, and was dependent on the effector-target ratio gradient. At the effector-target ratio of 1:2 and 1:1, the killing rate of 5×10 6 DCTs prepared from individual T cells demonstrated a specific killing rate against H1299-A2-positive target cells equivalent to that of TCR-T cells, with a gradient-dependent effector-target ratio. Neither DCTs nor TCR-T cells had a killing effect against negative target cells (HepG2 and NCI-H1299). DCTs prepared using different combinations of factors can demonstrate superior tumor recognition capabilities to TCR-T cells.

[0184] 3.2 Anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects T cells to kill tumor cells

[0185] use Human T-Activator CD3 / CD28 was used to expand magnetic bead-sorted T cells and culture them for 12 days. Flow cytometry was then used to assess T cell purity and the ratio of CD8 to CD4. The results showed that after expansion and culture, the CD3 positivity rate of the expanded cells was >99%, the CD8 to CD4 ratio was 2.5:1, and the total CD4 to CD8 positivity rate was 95% (Figure 10a). -7 mol / L complex protein anti-CD3 antibody (UCHT1)-1G4 (HATima) was added to 200 μL HIPP-T009 culture medium, and 5x10 6 T cells, incubated at 4 degrees for 1 hour" to prepare CD3 + T-DCT, flow cytometry detection of the positive rate of DCT showed that more than 99% of T cells were coated with the CD3 antibody (UCHT1)-1G4 (HATima) complex protein ( Figure 10b ).

[0186] The prepared CD3 + T-DCT, cell suspensions adjusted to different densities were 1x10 3 / 75μL, 1.25x10 3 / 75μL, 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 / 75μL and 8x10 4 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 trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 475 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - effector cell spontaneous pore - target cell spontaneous pore) / (target cell maximum lysis well - target cell spontaneous pore) x 100%. The results showed that the anti-CD3 antibody (UCHT1)-1G4 (HATima) redirected CD3 + T-DCT efficiently killed positive target tumor cells such as HepG2-NYESO1 and NCI-H1299-A2 in a gradient-dependent manner, and did not recognize negative tumor cells such as HepG2 and NCI-H1299 ( Figure 10c ).

[0187] Cells were collected from HepG2-NYESO-1 / DCT, HepG2 / DCT, and DCT-only groups at a 1:2 effector-to-target ratio; and from NCI-H1299-A2 / DCT, NCI-H1299 / DCT, and DCT-only groups at a 4:1 effector-to-target ratio in 96-well U-bottom plates. The cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and the cells were resuspended in 100μL of RPMI-1640 medium. 1μL of FITC anti-human CD3 antibody (BioLegend, Catalog No. 317306), PEcy7 anti-human CD137 antibody (BioLegend, Catalog No. 309818), and pHLA-tetramer-APC were added, respectively. Unstained and isotype controls were also included, and the cells were incubated at 4°C for 30 minutes. 100μL of RPMI-1640 medium was added, and the cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and repeated once. 100 μL RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137 and pHLA-tetramer-APC were detected by flow cytometry. 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 complex proteins bound to the surface, respectively. The results showed that CD3 prepared by anti-CD3 antibody (UCHT1)-1G4 (HATima) + T-DCT cells induce T cells to upregulate CD137 expression only after encountering positive target cells, that is, T cells appear to be in an activated state; when facing negative tumor cells or when there are no tumor cells, DCT cells do not upregulate CD137 expression and appear to be in a resting state (Figure 10d), although high levels of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein are bound to the T cell surface (Figure 10e).

[0188] The HepG2-NYESO-1 tumor model was established using B-NDG mice (purchased from Biocytogen). 6 HepG2-NYESO-1 cells were grown, and the average tumor volume was 226 mm on day 14. 3 The mice were randomly divided into control group and CD3 + T-DCT group, 6 rats per group. 2x10 7 T cells, and 1 hour later, the DCT group was re-infused with 5x10 6 CD3 + The preparation of T-DCT cells and DCT was the same as that used in the cell experiment; T cells in the control group were not loaded with complex proteins. CD3 +T-DCT cells were infused a total of 7 times. Starting from the day of T cell infusion, IL-2 (50,000 U) 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 tumor volume was calculated using the formula "(length x width x width) x 0.5". When the average tumor volume of one group reached 2000 mm 3 The mice were killed by carbon dioxide euthanasia and the experiment reached the end point. + On the 11th day after T-DCT infusion, the tumors in the DCT group were cleared and did not recur until the end of the experiment (day 53). The tumors in the control group grew normally, and on day 53, the average tumor volume was 2229 mm. 3 (Figure 10f) T cells redirected by the CD3 antibody (UCHT1)-1G4 (HATima) can effectively kill tumor cells both in vitro and in vivo without causing "spontaneous" activation of T cells.

[0189] 3.3 Anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects peripheral blood mononuclear cells (PBMCs) to kill tumor cells

[0190] Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Flow cytometry analysis showed that the CD3 positive cell population was 62.3% (Figure 11a). -7 mol / L complex protein anti-CD3 antibody (UCHT1)-1G4 (HATima) was added to 200 μL HIPP-T009 culture medium, and 5x10 6 PBMCs were incubated at 4°C for 1 hour to prepare PBMC-DCT, and the DCT positivity rate 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, with a binding rate of 100% (Figure 11b).

[0191] The prepared PBMC-DCT was adjusted to different cell suspension densities using HIPP-T009 culture medium, namely 1.25x10 3 / 75μL, 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 / 75μL and 8x10 4HepG2-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 trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 75 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum target cell lysis well - spontaneous pores of target cells) x 100%. The results showed that PBMC-DCT redirected by the anti-CD3 antibody (UCHT1)-1G4 (HATima) efficiently killed positive target tumor cells, such as HepG2-NYESO1 and NCI-H1299-A2, in a gradient-dependent effector-target ratio. Negative tumor cells, such as HepG2 and NCI-H1299, were not recognized (Figure 11c).

[0192] Cells were collected from HepG2-NYESO-1 / DCT, HepG2 / DCT, and DCT-only groups at a 2:1 effector-to-target ratio; and from NCI-H1299-A2 / DCT, NCI-H1299 / DCT, and DCT-only groups at a 4:1 effector-to-target ratio in 96-well U-bottom plates. The cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and the cells were resuspended in 100μL of RPMI-1640 medium. 1μL of FITC anti-human CD3 antibody (BioLegend, Catalog No. 317306), PEcy7 anti-human CD137 antibody (BioLegend, Catalog No. 309818), and pHLA-tetramer-APC were added, respectively. Unstained and isotype controls were also included, and the cells were incubated at 4°C for 30 minutes. 100μL of RPMI-1640 medium was added, and the cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and repeated once. 100 μL of RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137, and pHLA-tetramer-APC were detected by flow cytometry. The ratios of CD137 and pHLA-tetramer in the CD3-positive cell population were analyzed, representing the degree of T cell activation and the proportion of the complex protein bound to the surface, respectively. The results showed that PBMC-DCT cells prepared with the anti-CD3 antibody (UCHT1)-1G4 (HATima) only induced upregulation of CD137 expression in PBMC T cells after encountering positive target cells, indicating an activated T cell state. However, in the absence of negative tumor cells or tumor cells, DCT cells did not upregulate CD137 expression and remained in a quiescent state (Figure 11d), despite the presence of high levels of the anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein on their T cell surface (Figure 11e).

[0193] 3.4 Anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects peripheral blood lymphocytes (PBL) to kill tumor cells

[0194] By using the "adherence" method, monocytes were removed from peripheral blood mononuclear cells (PBMC) to obtain peripheral blood lymphocytes (PBL). Flow cytometry results showed that the CD3 positive cell population was 82.7% (Figure 12a). -7 mol / L complex protein anti-CD3 antibody (UCHT1)-1G4 (HATima) was added to 200 μL HIPP-T009 culture medium, and 5x10 6PBL-DCT was prepared using the protocol of "incubating PBLs at 4°C for 1 hour" and the DCT positivity was detected by flow cytometry. The results showed that the CD3 cell population and the pHLA-tetramer cell population were mutually positive, meaning that the complex protein only bound to T cells with a binding rate of 100% ( Figure 12b ).

[0195] The prepared PBL-DCT was adjusted to different cell suspension densities using HIPP-T009 culture medium, namely 1.25x10 3 / 75μL, 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 / 75μL and 8x10 4 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 trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 75 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions as (experimental wells - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum target cell lysis wells - spontaneous pores of target cells) x 100%. The results showed that PBL-DCT redirected by the anti-CD3 antibody (UCHT1)-1G4 (HATima) efficiently killed positive target tumor cells, such as HepG2-NYESO1 and NCI-H1299-A2, in a gradient-dependent effector-target ratio. Negative tumor cells, such as HepG2 and NCI-H1299, were not recognized (Figure 12c).

[0196] Cells were collected from HepG2-NYESO-1 / DCT, HepG2 / DCT, and DCT-only groups at a 2:1 effector-to-target ratio; and from NCI-H1299-A2 / DCT, NCI-H1299 / DCT, and DCT-only groups at a 4:1 effector-to-target ratio in 96-well U-bottom plates. The cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and the cells were resuspended in 100μL of RPMI-1640 medium. 1μL of FITC anti-human CD3 antibody (BioLegend, Catalog No. 317306), PEcy7 anti-human CD137 antibody (BioLegend, Catalog No. 309818), and pHLA-tetramer-APC were added, respectively. Unstained and isotype controls were also included, and the cells were incubated at 4°C for 30 minutes. 100μL of RPMI-1640 medium was added, and the cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and repeated once. 100 μL of RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137, and pHLA-tetramer-APC were detected by flow cytometry. The ratios of CD137 and pHLA-tetramer in the CD3-positive cell population were analyzed, representing the degree of T cell activation and the proportion of the complex protein bound to the surface, respectively. The results showed that PBL-DCT cells prepared with the anti-CD3 antibody (UCHT1)-1G4 (HATima) induced T cells in the PBL to upregulate CD137 expression only after encountering positive target cells, indicating that the T cells were in an activated state. However, in the absence of tumor cells or negative tumor cells, DCT cells did not upregulate CD137 expression and appeared to be in a quiescent state (Figure 12d), despite the high level of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein bound to the T cell surface (Figure 12e).

[0197] 3.5 Anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects γδT cells to kill tumor cells

[0198] Zoledronic acid (5 μM) was used to expand γδ T cells from peripheral blood mononuclear cells and culture them in complete culture medium (ImmunoCult TM -XF T cell expansion medium + 5% FBS + 200U / mL IL-2 + 10ng / mL IL-15) for 12 days, and the phenotype and purity were analyzed by flow cytometry. The results showed that 87.8% of the cells were TCR Vd2 / CD3 double positive, that is, the proportion of γδT cells in the expanded cells was 87.8% (Figure 13a). -8mol / L complex protein anti-CD3 antibody (UCHT1)-1G4 (HATima) was added to 200 μL HIPP-T009 culture medium, and 5x10 6 γδT cells were incubated at 4°C for 1 hour to prepare γδT-DCT, and the positive rate of DCT was detected by flow cytometry. The results showed that 97.8% of γδT cells were coated with the anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein ( Figure 13b ).

[0199] Using HIPP-T009 culture medium, the prepared γδT-DCT and γδT cells were adjusted to different cell suspension densities, namely 5x10 2 / 75μL, 1x10 3 / 75μL, 2x10 3 / 75μL and 4x10 3 pcs / 75μL; 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 / 75μL and 8x10 4 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 trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 475 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental wells - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum target cell lysis wells - spontaneous pores of target cells) x 100%. The results showed that γδT-DCT redirected by the anti-CD3 antibody (UCHT1)-1G4 (HATima) efficiently killed positive target tumor cells, such as HepG2-NYESO1 and NCI-H1299-A2, in a gradient-dependent effector-target ratio. Its recognition of negative tumor cells, such as HepG2 and NCI-H1299, was equivalent to that of γδT cells (Figure 13c).

[0200] Cells were collected from 96-well U-bottom plates at an effector-target ratio of 1:5 for HepG2-NYESO-1 / DCT, HepG2 / DCT, DCT alone, HepG2-NYESO-1 / γδT, HepG2 / γδT, and γδT alone; and at a 2:1 ratio for NCI-H1299-A2 / DCT, NCI-H1299 / DCT, DCT alone, NCI-H1299-A2 / γδT, NCI-H1299 / γδT, and γδT alone. The cells were centrifuged (500 g, 5 min, 4°C), the supernatant discarded, and the cells resuspended in 100 μL of RPMI-1640 medium. 1 μL of FITC anti-human CD3 antibody (biolegend, cat. no. 317306), PEcy7 anti-human CD137 antibody (biolegend, cat. no. 309818), and pHLA-tetramer-APC were added, along with unstained and isotype controls. The cells were incubated at 4°C for 30 minutes. 100 μL of RPMI-1640 medium was added, and the cells were centrifuged (500 g, 5 min, 4°C). The supernatant was discarded, and the supernatant was discarded. 100 μL of RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137, and pHLA-tetramer-APC were measured by flow cytometry. The ratios of CD137 and pHLA-tetramer in the CD3-positive cell population were analyzed, representing the degree of activation of γδT-DCT or γδT cells and the proportion of surface-bound complex proteins, respectively. The results showed that γδT-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, γδT-DCT cells showed an activated state; when negative tumor cells or no tumor cells were encountered, γδT-DCT cells did not upregulate CD137 expression and showed a resting state, which was consistent with the results of γδT (Figure 13d). -8 mol / L concentration of the complex protein CD3 antibody (UCHT1)-1G4 (HATima), after 18 hours of cell culture, no anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein bound to the cell surface could be detected by flow cytometry ( Figure 13e ).

[0201] 3.6 Anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects T cells to kill tumor cells

[0202] use Human T-Activator CD3 / CD28 was used to expand magnetic bead-sorted T cells and culture them for 12 days. Flow cytometry was then used to assess T cell purity and the ratio of CD8 to CD4. The results showed that after expansion and culture, the CD3 positivity rate of the expanded cells was >99%, the CD8 to CD4 ratio was 2.5:1, and the total CD4 to CD8 positivity rate was 95% (Figure 10a). -7 mol / L complex protein anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) was added to 200 μL HIPP-T009 culture medium, and 5x10 6 T cells, incubated at 4 degrees for 1 hour" to prepare CD3 + T-DCT, flow cytometry detection of the positive rate of DCT. The results showed that more than 99% of T cells were coated with the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein (Figure 14a).

[0203] The prepared CD3 + T-DCT, cell suspensions adjusted to different densities were 1x10 3 / 75μL, 1.25x10 3 / 75μL, 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 / 75μL and 8x10 4 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 trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 475 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - effector cell spontaneous pore - target cell spontaneous pore) / (target cell maximum lysis well - target cell spontaneous pore) x 100%. The results showed that the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirected CD3 + T-DCT efficiently killed positive target tumor cells such as HepG2-NYESO1 and NCI-H1299-A2 in a gradient-dependent manner, and did not recognize negative tumor cells such as HepG2 and NCI-H1299 ( Figure 14b ).

[0204] Cells were collected from HepG2-NYESO-1 / DCT, HepG2 / DCT, and DCT-only groups at a 1:2 effector-to-target ratio; and from NCI-H1299-A2 / DCT, NCI-H1299 / DCT, and DCT-only groups at a 4:1 effector-to-target ratio in 96-well U-bottom plates. The cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and the cells were resuspended in 100μL of RPMI-1640 medium. 1μL of FITC anti-human CD3 antibody (BioLegend, Catalog No. 317306), PEcy7 anti-human CD137 antibody (BioLegend, Catalog No. 309818), and pHLA-tetramer-APC were added, respectively. Unstained and isotype controls were also included, and the cells were incubated at 4°C for 30 minutes. 100μL of RPMI-1640 medium was added, and the cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and repeated once. 100 μL RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137 and pHLA-tetramer-APC were detected by flow cytometry. 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 complex proteins bound to the surface, respectively. The results showed that CD3 prepared by anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) + T-DCT cells induce T cells to upregulate CD137 expression only after encountering positive target cells, that is, T cells appear to be in an activated state; when facing negative tumor cells or when there are no tumor cells, DCT cells do not upregulate CD137 expression and appear to be in a resting state (Figure 14c), although high levels of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein are bound to the T cell surface (Figure 14d).

[0205] The HepG2-NYESO-1 tumor model was established using B-NDG mice (purchased from Biocytogen). 6 HepG2-NYESO-1 cells were grown, and the average tumor volume was 226 mm on day 14. 3 The mice were randomly divided into control group and CD3 + T-DCT group, 6 rats per group. 2x10 7 T cells, and 1 hour later, the DCT group was re-infused with 5x10 6 CD3 + The preparation of T-DCT cells and DCT was the same as that used in the cell experiment; T cells in the control group were not loaded with complex proteins. CD3 +T-DCT cells were infused a total of 7 times. Starting from the day of T cell infusion, IL-2 (50,000 U) 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 tumor volume was calculated using the formula "(length x width x width) x 0.5". When the average tumor volume of one group reached 2000 mm 3 The mice were killed by carbon dioxide euthanasia and the experiment reached the end point. + On the 11th day after T-DCT infusion, the tumors in the DCT group were cleared and did not recur until the end of the experiment (day 53). The tumors in the control group grew normally, and on day 53, the average tumor volume was 2229 mm. 3 ( Figure 14e ) T cells redirected by the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) can effectively kill tumor cells both in vitro and in vivo without causing "spontaneous" activation of T cells.

[0206] 3.7 Anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects peripheral blood mononuclear cells (PBMCs) to kill tumor cells

[0207] Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Flow cytometry analysis showed that the CD3 positive cell population was 61.4% (Figure 15a). -7 mol / L complex protein anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) was added to 200 μL HIPP-T009 culture medium, and 5x10 6 PBMCs were incubated at 4°C for 1 hour to prepare PBMC-DCT, and the DCT positivity rate 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, with a binding rate of 100% (Figure 15a).

[0208] The prepared PBMC-DCT was adjusted to different cell suspension densities using HIPP-T009 culture medium, namely 1.25x10 3 / 75μL, 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 / 75μL and 8x10 4HepG2-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 trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 75 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental wells - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum target cell lysis wells - spontaneous pores of target cells) x 100%. The results showed that PBMC-DCT redirected by the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) efficiently killed positive target tumor cells, such as HepG2-NYESO1 and NCI-H1299-A2, in a gradient-dependent effector-target ratio. Negative tumor cells, such as HepG2 and NCI-H1299, were not recognized (Figure 15b).

[0209] Cells were collected from HepG2-NYESO-1 / DCT, HepG2 / DCT, and DCT-only groups at a 2:1 effector-to-target ratio; and from NCI-H1299-A2 / DCT, NCI-H1299 / DCT, and DCT-only groups at a 4:1 effector-to-target ratio in 96-well U-bottom plates. The cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and the cells were resuspended in 100μL of RPMI-1640 medium. 1μL of FITC anti-human CD3 antibody (BioLegend, Catalog No. 317306), PEcy7 anti-human CD137 antibody (BioLegend, Catalog No. 309818), and pHLA-tetramer-APC were added, along with unstained and isotype controls. The cells were incubated at 4°C for 30 minutes. 100μL of RPMI-1640 medium was added, the cells were centrifuged (500g, 5 min, 4°C), and the supernatant discarded. This was repeated once. 100 μL of RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137, and pHLA-tetramer-APC were detected by flow cytometry. The ratios of CD137 and pHLA-tetramer in the CD3-positive cell population were analyzed, representing the degree of T cell activation and the proportion of the complex protein bound to the surface, respectively. The results showed that PBMC-DCT cells prepared with the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) induced T cells in the PBMC to upregulate CD137 expression only after encountering positive target cells, indicating that the T cells were in an activated state. However, in the absence of negative tumor cells or tumor cells, DCT cells did not upregulate CD137 expression and remained in a quiescent state (Figure 15c), despite the presence of high levels of the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein on their T cell surface (Figure 15d).

[0210] 3.8 Anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects peripheral blood lymphocytes (PBL) to kill tumor cells

[0211] By using the "adherence" method, monocytes were removed from peripheral blood mononuclear cells (PBMC) to obtain peripheral blood lymphocytes (PBL). Flow cytometry results showed that the CD3 positive cell population was 80.9% (Figure 16a). -7 mol / L complex protein CD3 antibody (UCHT1)-1G4 (ImmTAC) in 200 μL HIPP-T009 culture medium, add 5x10 6PBL-DCT was prepared using the protocol of "incubating PBLs at 4°C for 1 hour" and the DCT positivity was detected by flow cytometry. The results showed that the CD3 cell population and the pHLA-tetramer cell population were mutually positive, meaning that the complex protein only bound to T cells with a binding rate of 100% ( Figure 16a ).

[0212] The prepared PBL-DCT was adjusted to different cell suspension densities using HIPP-T009 culture medium, namely 1.25x10 3 / 75μL, 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 / 75μL and 8x10 4 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 trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 75 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental wells - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum target cell lysis wells - spontaneous pores of target cells) x 100%. The results showed that PBL-DCT redirected by the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) efficiently killed positive target tumor cells, such as HepG2-NYESO1 and NCI-H1299-A2, in a gradient-dependent effector-target ratio. Negative tumor cells, such as HepG2 and NCI-H1299, were not recognized (Figure 16b).

[0213] Cells were collected from HepG2-NYESO-1 / DCT, HepG2 / DCT, and DCT-only groups at a 2:1 effector-to-target ratio; and from NCI-H1299-A2 / DCT, NCI-H1299 / DCT, and DCT-only groups at a 4:1 effector-to-target ratio in 96-well U-bottom plates. The cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and the cells were resuspended in 100μL of RPMI-1640 medium. 1μL of FITC anti-human CD3 antibody (BioLegend, Catalog No. 317306), PEcy7 anti-human CD137 antibody (BioLegend, Catalog No. 309818), and pHLA-tetramer-APC were added, respectively. Unstained and isotype controls were also included, and the cells were incubated at 4°C for 30 minutes. 100μL of RPMI-1640 medium was added, and the cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and repeated once. 100 μL of RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137, and pHLA-tetramer-APC were detected by flow cytometry. The ratios of CD137 and pHLA-tetramer in the CD3-positive cell population were analyzed, representing the degree of T cell activation and the proportion of the complex protein bound to the surface, respectively. The results showed that PBL-DCT cells prepared with the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) induced T cells in the PBL to upregulate CD137 expression only after encountering positive target cells, indicating that the T cells were in an activated state. However, in the absence of negative tumor cells or tumor cells, DCT cells did not upregulate CD137 expression and appeared to be in a quiescent state (Figure 16c), despite the presence of high levels of the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein on their T cell surface (Figure 16d).

[0214] 3.9 Anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects γδT cells to kill tumor cells

[0215] Zoledronic acid (5 μM) was used to expand γδ T cells from peripheral blood mononuclear cells and culture them in complete culture medium (ImmunoCult TM -XF T cell expansion medium + 5% FBS + 200U / mL IL-2 + 10ng / mL IL-15) for 12 days, and the phenotype and purity were analyzed by flow cytometry. The results showed that 87.8% of the cells were TCR Vd2 / CD3 double positive, that is, the proportion of γδT cells in the expanded cells was 87.8% (Figure 13a). -8mol / L complex protein anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) was added to 200 μL HIPP-T009 culture medium, and 5x10 6 γδT cells were incubated at 4°C for 1 hour to prepare γδT-DCT, and the positive rate of DCT was detected by flow cytometry. The results showed that 98.8% of the γδT cells were coated with the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein ( Figure 17a ).

[0216] Using HIPP-T009 culture medium, the prepared γδT-DCT and γδT cells were adjusted to different cell suspension densities, namely 5x10 2 / 75μL, 1x10 3 / 75μL, 2x10 3 / 75μL and 4x10 3 pcs / 75μL; 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 / 75μL and 8x10 4 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 trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 475 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental wells - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum target cell lysis wells - spontaneous pores of target cells) x 100%. The results showed that γδT-DCT redirected by the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) efficiently killed positive target tumor cells, such as HepG2-NYESO1 and NCI-H1299-A2, in a gradient-dependent effector-target ratio. Its recognition of negative tumor cells, such as HepG2 and NCI-H1299, was equivalent to that of γδT cells (Figure 17b).

[0217] Cells were collected from 96-well U-bottom plates at an effector-target ratio of 1:5 for HepG2-NYESO-1 / DCT, HepG2 / DCT, DCT alone, HepG2-NYESO-1 / γδT, HepG2 / γδT, and γδT alone; and at a 2:1 ratio for NCI-H1299-A2 / DCT, NCI-H1299 / DCT, DCT alone, NCI-H1299-A2 / γδT, NCI-H1299 / γδT, and γδT alone. The cells were centrifuged (500 g, 5 min, 4°C), the supernatant discarded, and the cells resuspended in 100 μL of RPMI-1640 medium. 1 μL of FITC anti-human CD3 antibody (biolegend, cat. no. 317306), PEcy7 anti-human CD137 antibody (biolegend, cat. no. 309818), and pHLA-tetramer-APC were added, along with unstained and isotype controls. The cells were incubated at 4°C for 30 minutes. 100 μL of RPMI-1640 medium was added, and the cells were centrifuged (500 g, 5 min, 4°C). The supernatant was discarded, and the mixture was repeated once. 100 μL of RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137, and pHLA-tetramer-APC were measured by flow cytometry. The ratios of CD137 and pHLA-tetramer in the CD3-positive cell population were analyzed, representing the degree of activation of γδT-DCT or γδT cells and the proportion of surface-bound complex proteins, respectively. The results showed that γδT-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, γδT-DCT cells appeared to be in an activated state; when negative tumor cells or no tumor cells were encountered, γδT-DCT cells did not upregulate CD137 expression and appeared to be in a resting state, which was consistent with the results of γδT (Figure 17c). -8 mol / L concentration of the complex protein anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) was used to prepare γδT-DCT. After 18 hours of cell culture, no anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein bound to the cell surface could be detected by flow cytometry ( Figure 17d ).

[0218] 3.10 Anti-CD3 antibody (UCHT1)-AFP (HATima) redirects T cells to kill tumor cells

[0219] use Human T-Activator CD3 / CD28 was used to expand magnetic bead-sorted T cells and culture them for 12 days. The purity of the T cells was determined by flow cytometry. The results showed that the CD3 positive rate of the expanded cells was >98% (Figure 18a). -8 mol / L complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) was added to 200 μL HIPP-T009 culture medium, and 5x10 6 T cells, incubated at 4 degrees for 1 hour" to prepare CD3 + T-DCT, flow cytometry detection of the positive rate of DCT. The results showed that more than 98% of T cells were coated with the anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein (Figure 18a).

[0220] The prepared CD3 + T-DCT, adjusted to different cell suspension densities, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL and 4x10 4 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 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - effector cell spontaneous pore - target cell spontaneous pore) / (target cell maximum lysis well - target cell spontaneous pore) x 100%. The results showed that anti-CD3 antibody (UCHT1)-AFP (HATima) redirected CD3 +T-DCT efficiently killed HepG2-positive target tumor cells in a gradient-dependent manner and had no recognition of NCI-H1299-A2-negative tumor cells ( FIG. 18 b ).

[0221] HepG2 / DCT, NCI-H1299-A2 / DCT, and DCT-only cells were collected from a 96-well U-bottom plate at a 1:1 effector-target ratio. The cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and the cells were resuspended in 100μL of RPMI-1640 medium. 1μL of FITC anti-human CD3 antibody (biolegend, cat. no. 317306), PEcy7 anti-human CD137 antibody (biolegend, cat. no. 309818), and pHLA-tetramer-APC were added, respectively. Unstained and isotype controls were also included. The cells were incubated at 4°C for 30 minutes. 100μL of RPMI-1640 medium was added, the cells were centrifuged (500g, 5 min, 4°C), and the supernatant discarded. Repeat once. 100 μl RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137 and pHLA-tetramer-APC were detected by flow cytometry. 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 complex proteins bound to the surface, respectively. The results showed that CD3 prepared by anti-CD3 antibody (UCHT1)-AFP (HATima) + T-DCT cells induce T cells to upregulate CD137 expression only after encountering positive target cells, that is, T cells appear to be in an activated state; when encountering negative tumor cells or when there are no tumor cells, DCT cells do not regulate CD137 expression and appear to be in a resting state (Figure 18c). 1x10 -8 mol / L concentration of the complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) prepared CD3 + In T-DCT, after 18 hours of cell culture, no anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein bound to the cell surface could be detected by flow cytometry ( FIG. 18 d ).

[0222] 3.11 Anti-CD3 antibody (UCHT1)-AFP (HATima) redirects peripheral blood mononuclear cells (PBMCs) to kill tumor cells

[0223] Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation. Flow cytometry results showed that the CD3 positive cell population was 78.8% (Figure 19a). -8mol / L complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) was added to 200 μL HIPP-T009 culture medium, and 5x10 6 PBMCs were incubated at 4°C for 1 hour to prepare PBMC-DCT, and the DCT positivity rate 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, with a binding rate of 100% (Figure 19a).

[0224] The prepared PBMC-DCT was adjusted to different cell suspension densities using HIPP-T009 culture medium, which were 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL and 4x10 4 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 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum target cell lysis well - spontaneous pores of target cells) x 100%. The results showed that PBMC-DCT redirected by anti-CD3 antibody (UCHT1)-AFP (HATima) efficiently killed HepG2-positive target tumor cells in a gradient-dependent effector-target ratio, and did not recognize NCI-H1299-A2-negative tumor cells (Figure 19b).

[0225] HepG2 / DCT, NCI-H1299-A2 / DCT, and DCT-only cells were collected from a 96-well U-bottom plate at a 2:1 effector-target ratio. The cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and the cells were resuspended in 100μL of RPMI-1640 medium. 1μL of FITC anti-human CD3 antibody (biolegend, cat. no. 317306), PEcy7 anti-human CD137 antibody (biolegend, cat. no. 309818), and pHLA-tetramer-APC were added, respectively. Unstained and isotype controls were also included. The cells were incubated at 4°C for 30 minutes. 100μL of RPMI-1640 medium was added, the cells were centrifuged (500g, 5 min, 4°C), and the supernatant discarded. This was repeated once. 100 μl of RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137, and pHLA-tetramer-APC were detected by flow cytometry. The ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, representing the degree of T cell activation and the ratio of surface-bound complex proteins, respectively. The results showed that PBMC-DCT cells prepared with anti-CD3 antibody (UCHT1)-AFP (HATima) induced T cells in PBMC to upregulate CD137 expression only after encountering positive target cells, that is, T cells appeared in an activated state; in the absence of negative tumor cells or tumor cells, DCT cells did not increase CD137 expression and appeared in a resting state (Figure 19c). 1x10 -8 PBMC-DCT prepared with the complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) at a concentration of 1 mol / L, after 18 hours of cell culture, no anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein bound to the cell surface could be detected by flow cytometry ( Figure 19d ).

[0226] 3.12 Anti-CD3 antibody (UCHT1)-AFP (HATima) redirects peripheral blood lymphocytes (PBL) to kill tumor cells

[0227] By using the "adherence" method, monocytes were removed from peripheral blood mononuclear cells (PBMC) to obtain peripheral blood lymphocytes (PBL). Flow cytometry results showed that the CD3 positive cell population was 82.9% (Figure 20a). -8 mol / L complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) was added to 200 μL HIPP-T009 culture medium, and 5x10 6PBL-DCT was prepared using the protocol of "incubating PBLs at 4°C for 1 hour" and the DCT positivity was detected by flow cytometry. The results showed that the CD3 cell population and the pHLA-tetramer cell population were mutually positive, meaning that the complex protein only bound to T cells with a binding rate of 100% ( Figure 20a ).

[0228] The prepared PBL-DCT was adjusted to different cell suspension densities using HIPP-T009 culture medium, which were 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL and 4x10 4 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 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental wells - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum target cell lysis wells - spontaneous pores of target cells) x 100%. The results showed that PBL-DCT redirected by anti-CD3 antibody (UCHT1)-AFP (HATima) efficiently killed HepG2-positive target tumor cells in a gradient-dependent effector-target ratio, and did not recognize NCI-H1299-A2-negative tumor cells (Figure 20b).

[0229] Cells from HepG2 / DCT, NCI-H1299-A2 / DCT, and DCT-only groups at a 2:1 effector-target ratio in a 96-well U-bottom plate were harvested and centrifuged (500g, 5 min, 4°C). The supernatant was discarded and the cells were resuspended in 100μL of RPMI-1640 medium. 1μL of FITC anti-human CD3 antibody (biolegend, cat. no. 317306), PEcy7 anti-human CD137 antibody (biolegend, cat. no. 309818), and pHLA-tetramer-APC were added, respectively. Unstained and isotype controls were also included. The cells were incubated at 4°C for 30 minutes. 100μL of RPMI-1640 medium was added and the cells were centrifuged (500g, 5 min, 4°C). The supernatant was discarded and repeated once. 100 μl of RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137, and pHLA-tetramer-APC were detected by flow cytometry. The ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, representing the degree of T cell activation and the ratio of surface-bound complex proteins, respectively. The results showed that PBL-DCT cells prepared with anti-CD3 antibody (UCHT1)-AFP (HATima) induced T cells in PBL to upregulate CD137 expression only after encountering positive target cells, that is, T cells appeared in an activated state; in the absence of negative tumor cells or tumor cells, DCT cells did not increase CD137 expression and appeared in a resting state (Figure 20c). 1x10 -8 In the PBL-DCT prepared with the complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) at a concentration of 1 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 ( Figure 20 d ).

[0230] 3.13 Anti-CD3 antibody (UCHT1)-AFP (HATima) redirects γδT cells to kill tumor cells

[0231] Zoledronic acid (5 μM) was used to expand γδ T cells from peripheral blood mononuclear cells and culture them in complete culture medium (ImmunoCult TM -XF T cell expansion medium + 5% FBS + 200U / mL IL-2 + 10ng / mL IL-15) for 12 days, and the phenotype and purity were analyzed by flow cytometry. The results showed that 87.8% of the cells were TCR Vd2 / CD3 double positive, that is, the proportion of γδT cells in the expanded cells was 87.8% (Figure 13a). -9mol / L complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) was added to 200 μL HIPP-T009 culture medium, and 5x10 6 γδT cells were incubated at 4°C for 1 hour to prepare γδT-DCT, and the positive rate of DCT was detected by flow cytometry. The results showed that 100% of the γδT cells were coated with the anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein ( Figure 21a ).

[0232] Using HIPP-T009 culture medium, the prepared γδT-DCT and γδT cells were adjusted to different cell suspension densities, namely 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL and 4x10 4 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 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum target cell lysis well - spontaneous pores of target cells) x 100%. The results showed that γδT-DCT redirected by anti-CD3 antibody (UCHT1)-AFP (HATima) efficiently killed HepG2-positive target tumor cells in a gradient-dependent effector-target ratio. Its recognition of NCI-H1299-A2-negative tumor cells was equivalent to that of γδT cells (Figure 21b).

[0233] Cells from HepG2 / DCT, NCI-H1299-A2 / DCT, and DCT-only groups, as well as HepG2 / γδT, NCI-H1299-A2 / γδT, and γδT-only groups, were collected from 96-well U-bottom plates at a 1:1 effector-target ratio. Cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and the cells resuspended in 100μL of RPMI-1640 medium. 1μL of FITC anti-human CD3 antibody (BioLegend, Catalog No. 317306), PEcy7 anti-human CD137 antibody (BioLegend, Catalog No. 309818), and pHLA-tetramer-APC were added, respectively. Unstained and isotype controls were also included, and the cells were incubated at 4°C for 30 minutes. 100μL of RPMI-1640 medium was added, and the cells were centrifuged (500g, 5 min, 4°C), the supernatant discarded, and repeated once. 100 μL of RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137, and pHLA-tetramer-APC were detected by flow cytometry. The ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, representing the degree of activation of γδT-DCT or γδT cells and the ratio of surface-bound complex proteins, respectively. The results showed that γδT-DCT cells prepared with anti-CD3 antibody (UCHT1)-AFP (HATima) induced T cells to upregulate CD137 expression only after encountering positive target cells, that is, γδT-DCT cells appeared to be in an activated state; in the absence of negative tumor cells or tumor cells, γδT-DCT cells did not increase CD137 expression and appeared to be in a resting state, which was consistent with the results of γδT (Figure 21c). 1x10 -9 mol / L concentration of the complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) was used to prepare γδT-DCT. After 18 hours of cell culture, no anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein bound to the cell surface could be detected by flow cytometry ( Figure 21d ).

[0234] 3.14 Anti-CD3 antibody (UCHT1)-gp100 (HATima) redirects immune cells to kill tumor cells

[0235] According to the description of Example 1, immune cells were prepared, including T cells, γδT cells, CD4 and CD8 double negative T cells (DNT), CD4 + T cells, CD8 + T cells, PBMC and PBL were tested for phenotype and purity by flow cytometry. -8mol / L complex protein anti-CD3 antibody (UCHT1) -gp100 (HATima) in HIPP-T009 culture medium (200 μL), add 5x10 6 The immune cells were 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 T cells, γδT cells, DNTs, CD4 + T cells, CD8 + T cells, PBMCs, and PBLs were coated with anti-CD3 antibody (UCHT1)-gp100 (HATima) complex protein ( FIG. 22 a ).

[0236] The prepared DCT cells or control cells (DNT and γδT cells) were adjusted to different cell suspension densities using HIPP-T009 culture medium, which were 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 pcs / 75μL、8x10 4 / 75μL, 16x10 4 / 75μL, 32x10 4 T2 cells were collected, washed once with phosphate buffer, and resuspended in HIPP-T009 medium to adjust the cell density to 2x10 4 / 75μL, respectively, with a final loading concentration of 1x10 -8M gp100 peptide (YLEPGPVTA, positive target) and NY-ESO-1 peptide (SLLMWITQC, negative control) were added to a 96-well U-bottom plate. 75 μL of effector cells and 75 μL of target tumor cells were added sequentially. A maximum target cell lysis well, spontaneous target cell wells, spontaneous effector cell wells, spontaneous culture medium wells, and spontaneous culture medium plus lysis solution wells were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells was placed in a cell culture incubator at 37°C and 5% CO2 for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37°C and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to react at room temperature in the dark for 30 minutes. After the reaction, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum lysis well of target cells - spontaneous pores of target cells) x 100%. The results showed that the anti-CD3 antibody (UCHT1)-gp100 (HATima) redirected T-DCT, PBMC-DCT, PBL-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT efficiently killed T2-positive target tumor cells loaded with gp100 peptide in a gradient-dependent effector-target ratio; it did not recognize T2-negative tumor cells loaded with NY-ESO-1 peptide, or its recognition level was equivalent to that of control cells (DNT and γδT cells) for tumor cells ( Figure 22b ).

[0237] Collect cells from experimental wells with different effector-target ratios in a 96-well U-bottom plate. Centrifuge (500g, 5 min, 4°C), discard the supernatant, add 100μL RPMI-1640 medium, and resuspend the cells. Add 1μL of FITC anti-human CD3 antibody (biolegend, Catalog No.: 317306), PEcy7 anti-human CD137 antibody (biolegend, Catalog No.: 309818), and pHLA-tetramer-APC, respectively. Set up unstained and isotype controls, and incubate at 4°C for 30 minutes. Add 100μL RPMI-1640 medium, centrifuge (500g, 5 min, 4°C), discard the supernatant, and repeat once. 100 μL RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137 and pHLA-tetramer-APC were detected by flow cytometry. The ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, which represented the degree of activation of DCT cells and the ratio of surface-bound complex proteins, respectively. The results showed that DCT cells prepared by anti-CD3 antibody (UCHT1)-gp100 (HATima) induced T cells, PBMC, PBL, γδT cells, DNT, CD4 + T cells and CD8 + T cells upregulate the expression of CD137, indicating that DCT cells are in an activated state; when negative tumor cells (T2-NYESO-1 peptide) or in the absence of tumor cells are present, DCT cells do not upregulate the expression of CD137 and are in a resting state (Figure 22c). -8 T-DCT, PBMC-DCT, PBL-DCT, and CD4 + In T-DCT, after 18 hours of cell culture, the amount of anti-CD3 antibody (UCHT1)-gp100 (HATima) complex protein bound to the surface of DCT cells co-cultured with positive target cells (T2-gp100 peptide) was significantly reduced ( FIG22 d ).

[0238] 3.15 Anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) redirects immune cells to kill tumor cells

[0239] According to the description of Example 1, immune cells were prepared, including T cells, γδT cells, CD4 and CD8 double negative T cells (DNT), CD4 + T cells, CD8 + T cells, PBMC and PBL were tested for phenotype and purity by flow cytometry.-8 mol / L complex protein anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) was added to HIPP-T009 culture medium (200 μL), and 5x10 6 The immune cells were 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 T cells, γδT-DCT, DNT, CD4 + T cells, CD8 + T cells, PBMCs, and PBLs were coated with anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) complex protein ( FIG. 23 a ).

[0240] Using HIPP-T009 culture medium, the prepared DCT cells or control cells (DNT and γδT cells) were adjusted to different cell suspension densities, namely 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 pcs / 75μL、8x10 4 / 75μL, 16x10 4 T2 cells were collected, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 / 75μL, respectively, with a final loading concentration of 1x10 -8M gp100 peptide (YLEPGPVTA, positive target) and AFP peptide (FMNKFIYEI, negative control) were added to a 96-well U-bottom plate. 75 μL of effector cells and 75 μL of target tumor cells were added sequentially. A maximum target cell lysis well, spontaneous target cell wells, spontaneous effector cell wells, spontaneous culture medium wells, and spontaneous culture medium plus lysis solution wells were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells was placed in a cell culture incubator at 37°C and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37°C and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to react at room temperature in the dark for 30 minutes. After the reaction, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum lysis wells of target cells - spontaneous pores of target cells) x 100%. The results showed that the T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT、CD8 + T-DCT, PBMC-DCT, and PBL-DCT efficiently killed T2-positive target tumor cells loaded with gp100 peptide in a gradient-dependent manner; they did not recognize T2-negative tumor cells loaded with AFP peptide, or their recognition level for tumor cells was equivalent to that of control cells (DNT and γδT cells) ( Figure 23b ).

[0241] Collect cells from experimental wells with different effector-target ratios in a 96-well U-bottom plate. Centrifuge (500g, 5 min, 4°C), discard the supernatant, and resuspend the cells in 100μL of RPMI-1640 medium. Add 1μL of FITC anti-human CD3 antibody (BioLegend, Catalog No. 317306) and PEcy7 anti-human CD137 antibody (BioLegend, Catalog No. 309818), respectively. Set up unstained and isotype controls, and incubate at 4°C for 30 minutes. Add 100μL of RPMI-1640 medium, centrifuge (500g, 5 min, 4°C), discard the supernatant, and repeat once. Add 100μL of RPMI-1640 medium, and analyze the fluorescence signals of FITC anti-human CD3 antibody and PEcy7 anti-human CD137 by flow cytometry. Analyze the proportion of CD137 in the CD3-positive cell population, which represents the degree of DCT cell activation. The results showed that DCT cells prepared by anti-CD3 antibody (UCHT1)-gp100 (ImmTAC) induced T cells, γδT cells, DNTs, CD4 + T cells, CD8 + T, PBMC, and PBL cells upregulated the expression of CD137, indicating that DCT cells were in an activated state. In the presence of negative tumor cells (T2-AFP peptide) or in the absence of tumor cells, DCT cells did not upregulate the expression of CD137 and were in a resting state ( FIG23 c ).

[0242] 3.16 Anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) redirects immune cells to kill tumor cells

[0243] According to the description of Example 1, immune cells were prepared, including T cells, γδT cells, CD4 and CD8 double negative T cells (DNT), CD4 + T cells, CD8 + T cells, PBMC and PBL were tested for phenotype and purity by flow cytometry. -7 mol / L to 1x10 -10 mol / L complex protein anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) was added to HIPP-T009 culture medium (200 μL), and 5x10 6 The immune cells were 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 T cells, γδT cells, DNTs, CD4 + T cells, CD8 + T cells, PBMCs, and PBLs were coated with anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) complex protein ( FIG. 24 a ).

[0244] The prepared DCT cells or control cells (DNT) were adjusted to different cell suspension densities using HIPP-T009 culture medium, which were 0.625x10 3 / 75μL, 1.25x10 3 / 75μL, 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 pcs / 75μL、8x10 4 / 75μL; positive (HepG2-NYESO-1 (NYESO-1 + / HLA-A0201 + )) and negative (HepG2(NYESO-1 - / HLA-A0201 + )) Target cells were washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 4 75 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells was placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - effector cell spontaneous pore - target cell spontaneous pore) / (target cell maximum lysis well - target cell spontaneous pore) x 100%. The results showed that the anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) redirected T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT、CD8 + T-DCT, PBMC-DCT and PBL-DCT efficiently killed HepG2-NYESO-1 (NYESO-1 + / HLA-A0201 +) cells (positive target tumors), and has a gradient dependence on the effector-target ratio; its effect on HepG2 (NYESO-1 - / HLA-A0201 + ) cells (negative tumor) did not recognize, or the recognition level of tumor cells was equal to that of control cells (DNT) ( Figure 24 b ).

[0245] Collect cells from experimental wells with different effector-target ratios in a 96-well U-bottom plate. Centrifuge (500g, 5 min, 4°C), discard the supernatant, add 100μL RPMI-1640 medium, and resuspend the cells. Add 1μL of FITC anti-human CD3 antibody (biolegend, Catalog No.: 317306), PEcy7 anti-human CD137 antibody (biolegend, Catalog No.: 309818), and pHLA-tetramer-APC, respectively. Set up unstained and isotype controls, and incubate at 4°C for 30 minutes. Add 100μL RPMI-1640 medium, centrifuge (500g, 5 min, 4°C), discard the supernatant, and repeat once. 100 μL RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137, and pHLA-tetramer-APC were detected by flow cytometry. The ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, representing the degree of activation of DCT cells and the ratio of surface-bound complex proteins, respectively. The results showed that DCT cells prepared by anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) induced T cells, γδT cells, DNTs, and CD4 T cells only after encountering positive target cells (HepG2-NYESO-1). + T cells, CD8 + T, PBMC and PBL cells upregulate the expression of CD137, indicating that DCT cells are in an activated state; in the absence of negative tumor cells (HepG2) or tumor cells, DCT cells do not upregulate the expression of CD137, indicating a resting state (Figure 24c). -7 T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT、CD8 + After 18 hours of cell culture, the amount of anti-CD3 antibody (UCHT1)-i1G4 (ImmTAC) complex protein bound to the surface of T-DCT, PBMC-DCT, and PBL-DCT was relatively low ( FIG. 24 d ).

[0246] 3.17 Anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) redirects immune cells to kill tumor cells

[0247] According to the description of Example 1, immune cells were prepared, including T cells, γδT cells, CD4 and CD8 double negative T cells (DNT), CD4 + T cells and CD8 + T cells, flow cytometry to detect their phenotype and purity. -8 mol / L complex protein anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) was added to HIPP-T009 culture medium (200 μL), and 5x10 6 The immune cells were 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 T cells, γδT cells, DNTs, CD4 + T cells and CD8 + T cells were coated with anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) complex protein ( FIG. 25 a ).

[0248] Using HIPP-T009 culture medium, the prepared DCT cells or control cells (DNT and γδT cells) were adjusted to different cell suspension densities, namely 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 pcs / 75μL、8x10 4 / 75μL, 16x10 4 / 75μL, 32x10 4 T2 cells were collected, washed once with phosphate buffer, and resuspended in HIPP-T009 medium to adjust the cell density to 2x10 4 / 75μL, respectively, with a final loading concentration of 1x10 -8M gp100 peptide (YLEPGPVTA, positive target) and AFP peptide (FMNKFIYEI, negative control) were added to a 96-well U-bottom plate. 75 μL of effector cells and 75 μL of target tumor cells were added sequentially. A maximum target cell lysis well, spontaneous target cell wells, spontaneous effector cell wells, spontaneous culture medium wells, and spontaneous culture medium plus lysis solution wells were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells was placed in a cell culture incubator at 37°C and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37°C and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to react at room temperature in the dark for 30 minutes. After the reaction, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum lysis well of target cells - spontaneous pores of target cells) x 100%. The results showed that the anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) redirected T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT and CD8 + T-DCT efficiently killed T2-positive target tumor cells loaded with gp100 peptide in a gradient-dependent manner; it did not recognize T2-negative tumor cells loaded with AFP peptide, or its recognition level was equivalent to that of control cells (DNT and γδT cells) for tumor cells ( Figure 25b ).

[0249] Collect cells from experimental wells with different effector-target ratios in a 96-well U-bottom plate. Centrifuge (500g, 5 min, 4°C), discard the supernatant, add 100μL RPMI-1640 medium, and resuspend the cells. Add 1μL of FITC anti-human CD3 antibody (biolegend, Catalog No.: 317306), PEcy7 anti-human CD137 antibody (biolegend, Catalog No.: 309818), and pHLA-tetramer-APC, respectively. Set up unstained and isotype controls, and incubate at 4°C for 30 minutes. Add 100μL RPMI-1640 medium, centrifuge (500g, 5 min, 4°C), discard the supernatant, and repeat once. 100 μL RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137 and pHLA-tetramer-APC were detected by flow cytometry. The ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, which represented the activation degree of DCT cells and the ratio of surface-bound complex proteins, respectively. The results showed that DCT cells prepared by anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) induced T cells, γδT cells, DNTs, and CD4 T cells only after encountering positive target cells (T2-gp100 peptide). + T cells and CD8 + T cells upregulate the expression of CD137, indicating that DCT cells are in an activated state; when negative tumor cells (T2-AFP peptide) or in the absence of tumor cells are present, DCT cells do not upregulate the expression of CD137 and are in a resting state (Figure 25c). 1x10 -8 T-DCT, γδT-DCT, DNT-DCT, CD4 + T-DCT and CD8 + In T-DCT, after 18 hours of cell culture, the amount of anti-CD3 antibody (UCHT1)-igp100 (ImmTAC) complex protein bound to the surface of DCT cells co-cultured with positive target cells (T2-gp100 peptide) was significantly decreased ( Figure 25 d ).

[0250] 3.18 Anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirects immune cells to kill tumor cells

[0251] According to the description of Example 1, immune cells were prepared, including CD4 and CD8 double negative T cells (DNT), CD4 + T cells and CD8 + T cells, flow cytometry to detect their phenotype and purity. -7mol / L (for CD4 + T cells and CD8 + T cells) or 1x10 -8 mol / L (for DNT) complex protein anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) was added to HIPP-T009 culture medium (200 μL), and 5x10 6 The immune cells were 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 ( FIG. 26 a ).

[0252] The prepared DCT cells or control cells (DNT) were adjusted to different cell suspension densities using HIPP-T009 culture medium, which were 1.25x10 3 / 75μL, 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 pcs / 75μL、8x10 4 / 75μL, 16x10 4 / 75μL; positive (HepG2-NYESO-1 (NYESO-1 + / HLA-A0201 + )) and negative (HepG2(NYESO-1 - / HLA-A0201 + )) Target cells were washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 475 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells was placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - effector cell spontaneous pore - target cell spontaneous pore) / (target cell maximum lysis well - target cell spontaneous pore) x 100%. The results showed that anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) redirected DNT-DCT, CD4 + T-DCT and CD8 + T-DCT effectively kills HepG2-NYESO-1 (NYESO-1 + / HLA-A0201 + ) cells (positive target tumors), and has a gradient dependence on the effector-target ratio; its effect on HepG2 (NYESO-1 - / HLA-A0201 +) cells (negative tumor) do not recognize, or the recognition level of tumor cells is equivalent to that of control cells (DNT) (Figure 26b). 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 100μL RPMI-1640 medium, and resuspend the cells. Add 1μL 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. Add 100μL RPMI-1640 medium, centrifuge (500g, 5min, 4°C), discard the supernatant, and repeat once. 100 μL RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137 and pHLA-tetramer-APC were detected by flow cytometry. The ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, which represented the activation degree of DCT cells and the ratio of surface-bound complex proteins, respectively. The results showed that DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) induced DNT and CD4 only after encountering positive target cells (HepG2-NYESO-1). + T cells and CD8 + T cells upregulate the expression of CD137, indicating that DCT cells are in an activated state; when negative tumor cells (HepG2) or when there are no tumor cells, DCT cells do not upregulate the expression of CD137 and are in a resting state (Figure 26c). -7 mol / L concentration of the complex protein anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) prepared CD4 + T-DCT and CD8 + In T-DCT, after 18 hours of cell culture, the amount of anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) complex protein bound to the surface of DCT cells co-cultured with positive target cells was significantly decreased ( Figure 26d ).

[0253] 3.19 Anti-CD3 antibody (UCHT1)-1G4 (HATima) redirects immune cells to kill tumor cells

[0254] According to the description of Example 1, immune cells were prepared, including CD4 and CD8 double negative T cells (DNT), CD4 + T cells and CD8 + T cells, flow cytometry to detect their phenotype and purity. -7mol / L (for CD4 + T cells and CD8 + T cells) or 1x10 -8 mol / L (for DNT) complex protein anti-CD3 antibody (UCHT1)-1G4 (HATima) was added to HIPP-T009 culture medium (200 μL), and 5x10 6 The immune cells were 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 (HATima) complex protein ( FIG. 27 a ).

[0255] The prepared DCT cells or control cells (DNT) were adjusted to different cell suspension densities using HIPP-T009 culture medium, which were 1.25x10 3 / 75μL, 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 pcs / 75μL、8x10 4 / 75μL, 16x10 4 / 75μL; positive (HepG2-NYESO-1 (NYESO-1 + / HLA-A0201 + )) and negative (HepG2(NYESO-1 - / HLA-A0201 + )) Target cells were washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 475 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells was placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - effector cell spontaneous pore - target cell spontaneous pore) / (target cell maximum lysis well - target cell spontaneous pore) x 100%. The results showed that anti-CD3 antibody (UCHT1)-1G4 (HATima) redirected DNT-DCT, CD4 + T-DCT and CD8 + T-DCT effectively kills HepG2-NYESO-1 (NYESO-1 + / HLA-A0201 + ) cells (positive target tumors), and has a gradient dependence on the effector-target ratio; its effect on HepG2 (NYESO-1 - / HLA-A0201 + ) cells (negative tumor) did not recognize, or the recognition level of tumor cells was equal to that of control cells (DNT) ( Figure 27 b ).

[0256] Collect cells from experimental wells with different effector-target ratios in a 96-well U-bottom plate. Centrifuge (500g, 5 min, 4°C), discard the supernatant, add 100μL RPMI-1640 medium, and resuspend the cells. Add 1μL of FITC anti-human CD3 antibody (biolegend, Catalog No.: 317306), PEcy7 anti-human CD137 antibody (biolegend, Catalog No.: 309818), and pHLA-tetramer-APC, respectively. Set up unstained and isotype controls, and incubate at 4°C for 30 minutes. Add 100μL RPMI-1640 medium, centrifuge (500g, 5 min, 4°C), discard the supernatant, and repeat once. 100 μL RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137 and pHLA-tetramer-APC were detected by flow cytometry. The ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, which respectively represented the activation degree of DCT cells and the ratio of complex proteins bound to the surface. The results showed that DCT cells prepared by anti-CD3 antibody (UCHT1)-1G4 (HATima) induced DNT and CD4 only after encountering positive target cells (HepG2-NYESO-1). + T cells and CD8 + T cells upregulate the expression of CD137, indicating that DCT cells are in an activated state; when negative tumor cells (HepG2) or when there are no tumor cells, DCT cells do not upregulate the expression of CD137 and are in a resting state (Figure 27c). -7 mol / L concentration of the complex protein anti-CD3 antibody (UCHT1)-1G4 (HATima) prepared CD4 + T-DCT and CD8 + In T-DCT, after 18 hours of cell culture, the amount of anti-CD3 antibody (UCHT1)-1G4 (HATima) complex protein bound to the surface of DCT cells co-cultured with positive target cells was significantly reduced ( FIG. 27 d ).

[0257] 3.20 Anti-CD3 antibody (UCHT1)-AFP (HATima) redirects immune cells to kill tumor cells

[0258] According to the description of Example 1, immune cells were prepared, including CD4 and CD8 double negative T cells (DNT), CD4 + T cells and CD8 + T cells, flow cytometry to detect their phenotype and purity. -8mol / L complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) was added to HIPP-T009 culture medium (200 μL), and 5x10 6 The immune cells were 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)-AFP (HATima) complex protein ( FIG. 28 a ).

[0259] The prepared DCT cells or control cells (DNT) were adjusted to different cell suspension densities using HIPP-T009 culture medium, which were 2.5x10 3 / 75μL, 5x10 3 / 75μL, 1x10 4 / 75μL, 2x10 4 / 75μL, 4x10 4 pcs / 75μL、8x10 4 / 75μL, 16x10 4 / 75μL, 32x10 4 / 75μL; positive (HepG2 (AFP + / HLA-A0201 + )) and negative (NCI-H1299-A2 (AFP - / HLA-A0201 + )) Target cells were washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 2x10 475 μL of effector cells and 75 μL of target tumor cells were added to a 96-well U-bottom plate in sequence. A target cell maximum lysis well, a target cell spontaneous well, an effector cell spontaneous well, a culture medium spontaneous well, and a culture medium plus lysis solution spontaneous well were set up. The final volume of each well was 150 μL, and two replicates were used for each well. The 96-well U-bottom plate with cells was placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 and cultured for 18 hours. 15 μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees Celsius and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250 g, 4 minutes). 50 μL of supernatant was removed and transferred to a 96-well flat-bottom plate. 50 μL of substrate solution was added and the reaction was allowed to proceed at room temperature in the dark for 30 minutes. After the reaction was completed, 50 μL of stop solution was added and the absorbance at 490 nm was immediately measured using a microplate reader. The specific killing efficiency was calculated according to the product instructions = (experimental well - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum lysis wells of target cells - spontaneous pores of target cells) x 100%. The results showed that anti-CD3 antibody (UCHT1)-AFP (HATima) redirected DNT-DCT, CD4 + T-DCT and CD8 + T-DCT effectively kills HepG2 (AFP + / HLA-A0201 + ) cells (positive target tumors) and has a gradient dependence on the effector-target ratio; its effect on NCI-H1299-A2 (AFP - / HLA-A0201 + ) cells (negative tumor) did not recognize, or the recognition level of tumor cells was equal to that of control cells (DNT) ( Figure 28 b ).

[0260] Collect cells from experimental wells with different effector-target ratios in a 96-well U-bottom plate. Centrifuge (500g, 5 min, 4°C), discard the supernatant, add 100μL RPMI-1640 medium, and resuspend the cells. Add 1μL of FITC anti-human CD3 antibody (biolegend, Catalog No.: 317306), PEcy7 anti-human CD137 antibody (biolegend, Catalog No.: 309818), and pHLA-tetramer-APC, respectively. Set up unstained and isotype controls, and incubate at 4°C for 30 minutes. Add 100μL RPMI-1640 medium, centrifuge (500g, 5 min, 4°C), discard the supernatant, and repeat once. 100 μL RPMI-1640 medium was added, and the fluorescence signals of FITC anti-human CD3 antibody, PEcy7 anti-human CD137 and pHLA-tetramer-APC were detected by flow cytometry. The ratio of CD137 and pHLA-tetramer in the CD3-positive cell population was analyzed, which respectively represented the activation degree of DCT cells and the ratio of complex proteins bound to the surface. The results showed that DCT cells prepared by anti-CD3 antibody (UCHT1)-AFP (HATima) induced DNT and CD4 only after encountering positive target cells (HepG2). + T cells and CD8 + T cells upregulate the expression of CD137, indicating that DCT cells are in an activated state; when negative tumor cells (NCI-H1299-A2) or in the absence of tumor cells are present, DCT cells do not upregulate the expression of CD137 and are in a resting state (Figure 28c). 1x10 -8 mol / L concentration of the complex protein anti-CD3 antibody (UCHT1)-AFP (HATima) prepared CD8 + In T-DCT, after 18 hours of cell culture, the amount of anti-CD3 antibody (UCHT1)-AFP (HATima) complex protein bound to its surface decreased, and there was no difference among the groups ( FIG28 d ).

[0261] Example 4: Comparison of the biological activities of the anti-CD3 antibody (UCHT1) in the HATima and ImmTAC molecular structures used to modify cells

[0262] 4.1 Preparation of VH-VL and scFv Proteins

[0263] The anti-CD3 antibody (UCHT1) in the HATima molecular structure consists of 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), linked by an interchain disulfide bond, i.e., VH-VL. The anti-CD3 antibody (UCHT1) in the ImmTAC molecular structure consists of a heavy chain variable region and a light chain variable region, linked by a linker, i.e., scFv (SEQ ID NO:51 and SEQ ID NO:52). Vector construction and inclusion body expression and purification are the same as in 1.1.1. Protein refolding is the same as in 1.1.2, using 10 mM PB pH 6.0 as the dialysis buffer. The amount of VH and VL inclusion bodies added is 6 mg each, and the amount of scFv inclusion bodies added is 6 mg. Protein purification is the same as in 1.1.3. The results in Figure 29a show that under non-reduced conditions, VH-VL is a single band with a molecular weight of 22 kDa. Under reduced conditions, VH-VL remains a single band, but the molecular weight drops to 15 kDa, indicating that VH-VL is broken into two chains and the protein purity is greater than 90%. Under non-reduced conditions, scFv is a single band with a molecular weight of 27 kDa. Under reduced conditions, scFv remains a single band with a molecular weight of 27 kDa, indicating that the scFv is a single chain and the protein purity is greater than 90%.

[0264] 4.2 Biological Activity Detection of VH-VL and scFv Proteins

[0265] Using HIPP-T009 medium, T cells were adjusted to a density of 1x10 4 NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were trypsinized, washed once with phosphate buffer, resuspended in HIPP-T009 medium, and the cell density was adjusted to 1x10 4 Protein drugs, including anti-CD3 antibody (UCHT1)-1G4 (HATima), anti-CD3 antibody (UCHT1)-1G4 (ImmTAC), VH-VL, and scFv, were diluted to different molar concentrations using HIPP-T009 medium, 3x10 -7 M / 50μL, 3x10 -8 M / 50μL, 3x10 -9 M / 50μL, 3x10 -10 M / 50μL, 3x10 -11 M / 50μL, 3x10 -12 M / 50μL, 3x10 -13M / 50μL and 0μL. In a 96-well U-bottom plate, 50μL of effector cells, 50μL of target tumor cells and 50μL of the corresponding concentration of the test protein were added in sequence. At the same time, 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 150μL, and there were 2 replicates for each well. The 96-well U-bottom plate with cells added was placed in a cell culture incubator at 37 degrees and 5% CO2 and cultured for 18 hours. 15μL of lysis solution was added to the maximum lysis well, mixed, and placed in a cell culture incubator at 37 degrees and 5% CO2 for 45 minutes. The 96-well U-bottom cell culture plate was placed in a centrifuge and centrifuged (250g, 4 minutes). 50μL of supernatant was removed and transferred to a 96-well flat-bottom plate, 50μL of substrate solution was added, and the reaction was carried out at room temperature in the dark for 30 minutes. After the reaction is completed, 50 μL of stop solution is added and the absorbance at 490 nm is immediately measured with a microplate reader. According to the product instructions, the efficiency of specific killing is calculated = (experimental well - spontaneous pores of effector cells - spontaneous pores of target cells) / (maximum lysis wells of target cells - spontaneous pores of target cells) x 100%. The results showed that T cells redirected by CD3 antibody (UCHT1)-1G4 (HATima) and anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) specifically killed NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) cells, and had a concentration gradient dependence. They did not recognize NCI-H1299 (NY-ESO-1 positive, A2 negative) cells; VH-VL redirected T cells specifically killed NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) cells and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells were not recognized; scFv-redirected T cells showed a certain degree of recognition of NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) cells and NCI-H1299 (NY-ESO-1 positive, A2 negative) cells, and were independent of the target, although the killing rate was lower than that of CD3 antibody (UCHT1)-1G4 (HATima) and anti-CD3 antibody (UCHT1)-1G4 (ImmTAC) ( Figure 29b ).

[0266] Collect cells from the 1E-7M and 1E-9M concentration groups in a 96-well U-bottom plate. Centrifuge (500g, 5 min, 4°C), discard the supernatant, and resuspend the cells in 100μL of RPMI-1640 medium. Add 1μL of FITC anti-human CD45 antibody (biolegend, cat. no. 368508) and PEcy7 anti-human CD137 antibody (biolegend, cat. no. 309818), respectively. Set up unstained and isotype controls, and incubate at 4°C for 30 minutes. Add 100μL of RPMI-1640 medium, centrifuge (500g, 5 min, 4°C), discard the supernatant, and repeat once. Add 100μL of RPMI-1640 medium, and analyze the fluorescence signals of FITC anti-human CD45 and PEcy7 anti-human CD137 by flow cytometry. Analyze the proportion of CD137 in the CD45-positive cell population, which represents the degree of T cell activation. The results in Figure 29c show that at a protein concentration of 1E-9M, scFv significantly induced T cells to upregulate CD137 expression in the NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) cell group, the NCI-H1299 (NY-ESO-1 positive, A2 negative) group, and the tumor-free cell group, indicating that the T cells were activated and showed no target dependence; only in the NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) cell group, the degree of T cell activation induced by scFv was lower than that of the CD3 antibody (UCHT1)-1G4 (HATima) and the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC). Neither VH nor VL could induce T cells to upregulate CD137 expression. Figure 29d shows that when the protein concentration was increased to 1E-7M, VH-VL still failed to induce T cells to upregulate CD137 expression in the NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) cell group, the NCI-H1299 (NY-ESO-1 positive, A2 negative) group, and the tumor-free cell group. scFvs all exhibited target-independent T cell activation. Only in the NCI-H1299-A2 (NY-ESO-1 positive, A2 positive) cell group was the degree of T cell activation induced by scFv lower than that of the CD3 antibody (UCHT1)-1G4 (HATima) and the anti-CD3 antibody (UCHT1)-1G4 (ImmTAC). These results indicate that scFvs are immune effector molecules, while VH-VLs are non-immune effector molecules.

[0267] Incorporated by Reference

[0268] Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0269] Equivalence

[0270] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above-described embodiments should be considered in all cases as illustrative rather than limiting of the invention described herein. The scope of the present disclosure is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalents of the claims are intended to be embraced therein.

Claims

1. A modified cell, wherein the modified cell is formed by binding a complex protein 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.

2. The modified cell according to claim 1, wherein The portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

3. The modified cell according to claim 1 or 2, wherein 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.

4. The modified cell according to any one of claims 1 to 3, wherein The cell surface marker molecules of the modified cells are selected from CD8, CD4, CD3, NKG2D, CD16, CD2, CD56, CD28 or CD26.

5. The modified cell according to any one of claims 1 to 4, wherein The modified cells are immune cells, preferably T cells, γδT cells, CD4 and CD8 double negative T cells, CD4 + T cells, CD8 + T cells, NK cells, NKT cells or monocytes.

6. The modified cell according to any one of claims 1 to 5, wherein The target cell surface antigen is selected from pMHC.

7. The modified cell according to claim 2, wherein The structure of the complex protein from N-terminus to C-terminus is shown in Formula Ia: ALB (Ia) BLA (Ib) in, 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; and Element L is a flexible joint; the flexible joint is optional; "-" is a peptide bond.

8. The modified cell according to claim 7, wherein The antibody contained in the element A is a single-chain antibody scFv.

9. The modified cell according to claim 8, wherein The antibody contained in the element A is an anti-CD3 antibody.

10. The modified cell according to claim 9, wherein The heavy chain variable region of the antibody 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 modified cell of claim 9 , wherein the single-chain antibody comprises OKT3, UCHT-1, TR66, BMA031, or 12F6.

12. The modified cell according to claim 7, wherein The target cell surface antigen is selected from pMHC.

13. The modified cell according to claim 7, wherein The portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

14. The modified cell according to claim 13, wherein The TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO: 5, 25 or 40, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO: 7, 27, 42 or 44.

15. The modified cell according to claim 2, wherein 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); in, 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; and Element L is a flexible joint; the flexible joint is optional; "-" is a peptide bond; "…" is a disulfide bond.

16. The modified cell according to claim 15, wherein When A1 comprises the heavy chain variable region of an antibody, A2 comprises the light chain variable region of an antibody; or when A2 comprises the heavy chain variable region of an antibody, A1 comprises the light chain variable region of an antibody; and A1 and A2 form a dimer through a disulfide bond, preferably, the dimer is a non-immune effector molecule.

17. The modified cell according to claim 16, wherein The heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO:

9.

18. The modified cell according to claim 16, wherein The light chain variable region of the antibody comprises the sequence shown in SEQ ID NO:

11.

19. The modified cell according to claim 15, wherein The portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

20. The modified cell according to claim 19, wherein The TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO: 13, 17 or 21, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO: 15, 19 or 23.

21. A combination or kit comprising a. a complex protein; b. a cell; wherein the cell expresses a cell surface marker molecule, 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.

22. The combination or kit of claim 21, wherein The portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

23. The combination or kit of claim 21 or 22, wherein 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.

24. The combination or kit of any one of claims 21 to 23, wherein The cell surface marker molecules of the cells are selected from CD8, CD4, CD3, NKG2D, CD16, CD2, CD56, CD28 or CD26.

25. The combination or kit of any one of claims 21 to 24, wherein The cells are immune cells, preferably T cells, γδT cells, CD4 and CD8 double negative T cells, CD4 + T cells, CD8 + T cells, NK cells, NKT cells or monocytes.

26. The combination or kit of any one of claims 21 to 25, wherein The target cell surface antigen is selected from pMHC.

27. The combination or kit of claim 22, wherein The structure of the complex protein from N-terminus to C-terminus is shown in Formula Ia: ALB (Ia) BLA (Ib) in, 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; and Element L is a flexible joint; the flexible joint is optional; "-" is a peptide bond.

28. The combination or kit of claim 27, wherein The antibody contained in the element A is a single-chain antibody scFv.

29. The combination or kit of claim 28, wherein The antibody contained in the element A is an anti-CD3 antibody.

30. The combination or kit of claim 29, wherein The heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO: 1, and the light chain variable region comprises the sequence shown in SEQ ID NO:

3.

31. The combination or kit of claim 29, wherein the single-chain antibody comprises OKT3, UCHT-1, TR66, BMA031, or 12F6.

32. The combination or kit of claim 27, wherein The target cell surface antigen is selected from pMHC.

33. The combination or kit of claim 27, wherein The portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

34. The combination or kit of claim 33, wherein The TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO: 5, 25 or 40, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO: 7, 27, 42 or 44.

35. The combination or kit of claim 22, wherein 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); in, 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; and Element L is a flexible joint; the flexible joint is optional; "-" is a peptide bond; "…" is a disulfide bond.

36. The combination or kit of claim 35, wherein When A1 comprises the heavy chain variable region of an antibody, A2 comprises the light chain variable region of an antibody; or when A2 comprises the heavy chain variable region of an antibody, A1 comprises the light chain variable region of an antibody; and A1 and A2 form a dimer through a disulfide bond, preferably, the dimer is a non-immune effector molecule.

37. The combination or kit of claim 36, wherein The heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO:

9.

38. The combination or kit of claim 36, wherein The light chain variable region of the antibody comprises the sequence shown in SEQ ID NO:

11.

39. The combination or kit of claim 35, wherein The portion that specifically binds to the target cell surface antigen is a TCR molecule, a single-chain αβTCR or a TCRα chain / TCRβ chain heterodimer, a TCR-like antibody or a TCR mimetic antibody.

40. The combination or kit of claim 39, wherein The TCRβ chain amino acid sequence comprises the sequence shown in SEQ ID NO: 13, 17 or 21, and the TCRα chain amino acid sequence comprises the sequence shown in SEQ ID NO: 15, 19 or 23.

41. The combination or kit of any one of claims 21 to 25, wherein The combination or kit further comprises an optional pharmaceutically acceptable carrier, preferably a buffer, more preferably, the buffer comprises albumin or plasma.

42. A method of treating a disease comprising administering to a subject in need thereof the modified cell of any one of claims 1 to 20 or the combination or kit of any one of claims 21 to 41.

43. The method of claim 42, wherein the disease comprises cancer, a microbial infectious disease, an autoimmune disease, or a disease of aging.

44. A method for preparing the modified cell according to any one of claims 1 to 20, wherein the complex protein is incubated with immune cells to form the modified cell.

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