Cell in-situ secretion antibodies and uses thereof

Abstract

A cell comprising a vector comprising a nucleic acid molecule encoding an antigen binding protein comprising a constant region of scFv and TCR alpha chains targeting a first antigen, and a constant region of scFv and TCR beta chains targeting a second antigen. The cell can further express a chemokine receptor. The invention also provides a preparation method and application of the cell.

Description

Cells secreting antibodies in situ and their uses Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a cell capable of in situ production and / or secretion of antibodies, as well as a preparation method and use of the cell. Background Art

[0002] Currently, the majority of bispecific antibodies (BsAbs) in clinical trials are mediated by the T cell receptor CD3, which primarily engages immune cells. As of the end of 2020, 111 such tumor-associated antibodies were reported on ClinicalTrials.gov. The anti-tumor mechanism of BsAb is that one arm targets a receptor on the surface of tumor cells, while the other arm recruits and activates T cells, thereby inducing efficient and selective cytotoxic killing. When both T cells and tumor cells are simultaneously bound by BsAbs, an immune synapse is formed between the T cells and the tumor cells. This synapse contains perforin and cytotoxic granzyme B released by the T cells, leading to tumor cell killing.

[0003] Despite the tremendous success of BsAbs, the therapeutic potential of exogenous drug delivery is hampered by short circulation dynamics, poor tumor microenvironment (TME) penetration, and toxicity to non-tumor tissues. Therefore, researchers abroad have proposed the concept of in situ BsAb generation to overcome the immunosuppressive TME and avoid the toxic side effects of continuous drug infusion. Currently, the main methods for generating BsAbs in tumor tissue include engineered oncolytic viruses (OVs), transfection of T cells, and transfection of mesenchymal stem cells (MSCs). Over a decade ago, it was reported that human peripheral blood lymphocytes, particularly CD3+ T cells, could serve as BsAb carriers and successfully produce anti-CEA×CD3 bispecific antibodies in vivo via transduction with an engineered HIV-1-based lentiviral vector. Mechanistically, engineered T cells, through antigen-dependent in situ production and secretion of BsAbs, redirect and activate unmodified bystander T cells to the tumor site, thereby synergistically generating a potent anti-tumor immune response. In situ BsAb generation eliminates the time-consuming and expensive preparation, manufacturing, and storage processes. There is no need to face a series of problems after antibody production, such as antibody degradation, aggregation, denaturation, fragmentation and oxidation, etc. At the same time, it also saves many time-consuming clinical trials to explore the best route of administration and optimal dosage.

[0004] However, the in situ BsAb generation technology currently faces some challenges, such as the expression efficiency of BsAb, the ability to recruit bystander T cells, and the tumor killing efficiency, which still need to be improved. Therefore, it is necessary to design more effective solutions to enhance the therapeutic effect of in situ BsAb generation technology.

[0005] Summary of the Invention

[0006] The present application provides a cell comprising a vector comprising a nucleic acid molecule encoding an antigen-binding protein, wherein the antigen-binding protein comprises a scFv targeting a first antigen and a constant region of a TCRα chain, and a scFv targeting a second antigen and a constant region of a TCRβ chain.

[0007] In certain embodiments, the vector is a viral vector.

[0008] In certain embodiments, the vector is selected from the group consisting of a lentiviral, adenoviral, retroviral, and adeno-associated viral vector.

[0009] In certain embodiments, the vector is a non-viral vector.

[0010] In certain embodiments, the vector is selected from the group consisting of a plasmid, a minicircle DNA vector, an SB plasmid, and a piggybac plasmid.

[0011] In certain embodiments, the cell is an immune cell.

[0012] In certain embodiments, the cell is a T cell or a NK cell.

[0013] In certain embodiments, the antigen binding protein does not comprise the variable region of a TCR alpha chain.

[0014] In certain embodiments, the antigen binding protein does not comprise the variable region of a TCR β chain.

[0015] In certain embodiments, the scFv targeting the first antigen and the constant region of the TCR α chain in the antigen binding protein are connected by a hinge region.

[0016] In certain embodiments, the scFv targeting the second antigen and the constant region of the TCR β chain in the antigen binding protein are connected by a hinge region.

[0017] In certain embodiments, the hinge region comprises an IgG hinge region.

[0018] In certain embodiments, the hinge region comprises an IgG4 hinge region.

[0019] In certain embodiments, the constant region of the TCR α chain and the constant region of the TCR β chain in the antigen binding protein are linked by a disulfide bond.

[0020] In certain embodiments, the constant region of the TCR α chain and the constant region of the TCR β chain in the antigen binding protein are connected by a linker.

[0021] In certain embodiments, the linker comprises a peptide linker.

[0022] In certain embodiments, the amino acid sequence of the linker is SEQ ID NO: 49: GGGGS.

[0023] In certain embodiments, the first antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19, and PD-1.

[0024] In certain embodiments, the second antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19, and PD-1.

[0025] In certain embodiments, the first antigen and the second antigen are different antigen-binding epitopes of the same antigen.

[0026] In certain embodiments, the first antigen and the second antigen are different.

[0027] In certain embodiments, the scFv targeting the first antigen in the antigen binding protein comprises an antibody light chain variable region VL targeting the first antigen and an antibody heavy chain variable region VH targeting the first antigen.

[0028] In certain embodiments, the N-terminus of the VH targeting the first antigen and the C-terminus of the VL targeting the first antigen in the antigen binding protein are linked.

[0029] In certain embodiments, the C-terminus of the VH targeting the first antigen and the N-terminus of the VL targeting the first antigen in the antigen binding protein are linked.

[0030] In certain embodiments, the C-terminus of the VH targeting the first antigen in the antigen binding protein is linked to the N-terminus of the constant region of the TCR α chain.

[0031] In certain embodiments, the C-terminus of the VL targeting the first antigen in the antigen binding protein is linked to the N-terminus of the constant region of the TCR α chain.

[0032] In certain embodiments, the N-terminus of the VH targeting the first antigen in the antigen binding protein is linked to the C-terminus of the constant region of the TCR α chain.

[0033] In certain embodiments, the N-terminus of the VL targeting the first antigen in the antigen binding protein is linked to the C-terminus of the constant region of the TCR α chain.

[0034] In certain embodiments, the scFv targeting the second antigen in the antigen binding protein comprises an antibody light chain variable region VL targeting the second antigen and an antibody heavy chain variable region VH targeting the second antigen.

[0035] In certain embodiments, the N-terminus of the VH targeting the second antigen and the C-terminus of the VL targeting the second antigen in the antigen binding protein are linked.

[0036] In certain embodiments, the C-terminus of the VH targeting the second antigen and the N-terminus of the VL targeting the second antigen in the antigen binding protein are linked.

[0037] In certain embodiments, the C-terminus of the VH targeting the second antigen in the antigen binding protein is linked to the N-terminus of the constant region of the TCRβ chain.

[0038] In certain embodiments, the C-terminus of the VL targeting the second antigen in the antigen binding protein is linked to the N-terminus of the constant region of the TCRβ chain.

[0039] In certain embodiments, the N-terminus of the VH targeting the second antigen in the antigen binding protein is linked to the C-terminus of the constant region of the TCRβ chain.

[0040] In certain embodiments, the N-terminus of the VL targeting the second antigen in the antigen binding protein is linked to the N-terminus of the constant region of the TCRβ chain.

[0041] In certain embodiments, the antigen binding protein further comprises a binding domain that targets a third antigen.

[0042] In certain embodiments, the binding domain targeting a third antigen is a scFv.

[0043] In certain embodiments, the antigen binding protein further comprises a binding domain that targets a fourth antigen.

[0044] In certain embodiments, the binding domain targeting the fourth antigen is a scFv.

[0045] In certain embodiments, the scFv targeting the third antigen in the antigen binding protein is directly or indirectly linked to the constant region of the TCR α chain.

[0046] In certain embodiments, the scFv targeting the third antigen in the antigen binding protein is connected to the constant region of the TCRα chain via an IgG4 hinge region.

[0047] In certain embodiments, the scFv targeting the fourth antigen in the antigen binding protein is directly or indirectly linked to the constant region of the TCR β chain.

[0048] In certain embodiments, the scFv targeting the fourth antigen in the antigen binding protein is connected to the constant region of the TCRβ chain via an IgG4 hinge region.

[0049] In certain embodiments, the scFv targeting a third antigen in the antigen binding protein comprises a VH targeting a third antigen and a VL targeting a third antigen.

[0050] In certain embodiments, the N-terminus of the VH targeting the third antigen and the C-terminus of the VL targeting the third antigen in the antigen-binding protein are directly or indirectly linked.

[0051] In certain embodiments, the C-terminus of the VH targeting the third antigen and the N-terminus of the VL targeting the third antigen in the antigen-binding protein are directly or indirectly linked.

[0052] In certain embodiments, the N-terminus of the VH targeting the third antigen in the antigen binding protein is directly or indirectly linked to the C-terminus of the constant region of the TCRα chain.

[0053] In certain embodiments, the C-terminus of the VH targeting the third antigen in the antigen binding protein is directly or indirectly linked to the N-terminus of the constant region of the TCRα chain.

[0054] In certain embodiments, the N-terminus of the VL targeting the third antigen in the antigen binding protein is directly or indirectly linked to the C-terminus of the constant region of the TCRα chain.

[0055] In certain embodiments, the C-terminus of the VL targeting the third antigen in the antigen binding protein is directly or indirectly linked to the N-terminus of the constant region of the TCR α chain.

[0056] In certain embodiments, the scFv targeting the fourth antigen in the antigen binding protein comprises a VH targeting the fourth antigen and a VL targeting the fourth antigen.

[0057] In certain embodiments, the N-terminus of the VH targeting the fourth antigen and the C-terminus of the VL targeting the fourth antigen in the antigen-binding protein are directly or indirectly linked.

[0058] In certain embodiments, the C-terminus of the VH targeting the fourth antigen and the N-terminus of the VL targeting the fourth antigen in the antigen-binding protein are directly or indirectly linked.

[0059] In certain embodiments, the N-terminus of the VH targeting the fourth antigen in the antigen binding protein is directly or indirectly linked to the C-terminus of the constant region of the TCRβ chain.

[0060] In certain embodiments, the C-terminus of the VH targeting the fourth antigen in the antigen binding protein is directly or indirectly linked to the N-terminus of the constant region of the TCRβ chain.

[0061] In certain embodiments, the N-terminus of the VL targeting the fourth antigen in the antigen binding protein is directly or indirectly linked to the C-terminus of the constant region of the TCRβ chain.

[0062] In certain embodiments, the C-terminus of the VL targeting the fourth antigen in the antigen binding protein is directly or indirectly linked to the N-terminus of the constant region of the TCRβ chain.

[0063] In certain embodiments, the antigen binding protein comprises two polypeptide chains, wherein the first polypeptide chain comprises an scFv targeting a first antigen and a constant region of a TCRα chain; wherein the second polypeptide chain comprises an scFv targeting a second antigen and a constant region of a TCRβ chain.

[0064] In certain embodiments, the first polypeptide chain and the second polypeptide chain in the antigen binding protein are linked by a disulfide bond.

[0065] In certain embodiments, in the first polypeptide chain of the antigen binding protein, the scFv targeting the first antigen is directly or indirectly linked to the constant region of the TCR α chain.

[0066] In certain embodiments, the scFv targeting the first antigen and the constant region of the TCR α chain in the antigen binding protein are connected by a hinge region.

[0067] In certain embodiments, the scFv targeting the first antigen in the antigen binding protein is connected to the constant region of the TCR α chain via an IgG hinge region.

[0068] In certain embodiments, the scFv targeting the first antigen in the antigen binding protein is connected to the constant region of the TCR α chain via the hinge region of IgG4.

[0069] In certain embodiments, the scFv targeting the first antigen in the antigen binding protein comprises a VH targeting the first antigen and a VL targeting the first antigen.

[0070] In certain embodiments, the first polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting the first antigen, a VH targeting the first antigen, and a constant region of the TCRα chain.

[0071] In certain embodiments, the first polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting the first antigen, a linker, a VH targeting the first antigen, an IgG4 hinge region, and a constant region of a TCRα chain.

[0072] In certain embodiments, the antigen binding protein further comprises a binding domain that targets a third antigen.

[0073] In certain embodiments, the binding domain targeting a third antigen comprises a scFv.

[0074] In certain embodiments, the third antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19, and PD-1.

[0075] In certain embodiments, the scFv targeting a third antigen comprises a VH targeting a third antigen and a VL targeting a third antigen.

[0076] In certain embodiments, the first polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, a VL targeting a first antigen, a VH targeting a first antigen, a hinge region of IgG4, a constant region of a TCRα chain, a hinge region of IgG4, a VL targeting a third antigen, and a VH targeting a third antigen.

[0077] In certain embodiments, the first polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, VL targeting a first antigen, VH targeting a first antigen, a hinge region of IgG4, a constant region of a TCRα chain, a hinge region of IgG4, VH targeting a third antigen, and VL targeting a third antigen.

[0078] In certain embodiments, the first polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VH targeting the first antigen, a VL targeting the first antigen, and a constant region of the TCRα chain.

[0079] In certain embodiments, the first polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, VH targeting the first antigen, a linker, VL targeting the first antigen, a hinge region of IgG4, and a constant region of the TCRα chain.

[0080] In certain embodiments, the first polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, VH targeting a first antigen, a linker, VL targeting a first antigen, a hinge region of IgG4, a constant region of a TCRα chain, a hinge region of IgG4, VL targeting a third antigen, a linker, and VH targeting a third antigen.

[0081] In certain embodiments, the first polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, VH targeting a first antigen, a linker, VL targeting a first antigen, a hinge region of IgG4, a constant region of a TCRα chain, a hinge region of IgG4, VH targeting a third antigen, a linker, and VL targeting a third antigen.

[0082] In certain embodiments, the first polypeptide chain of the antigen binding protein comprises a scFv targeting a first antigen, a constant region of a TCR α chain, and a scFv targeting a third antigen.

[0083] In certain embodiments, in the second polypeptide chain of the antigen binding protein, the scFv targeting the second antigen is directly or indirectly linked to the constant region of the TCRβ chain.

[0084] In certain embodiments, the scFv targeting a second antigen and the constant region of the TCR α chain of the antigen binding protein are connected by a hinge region.

[0085] In certain embodiments, the scFv targeting a second antigen and the constant region of the TCR β chain of the antigen binding protein are connected by an IgG hinge region.

[0086] In certain embodiments, the scFv targeting the second antigen and the constant region of the TCR β chain of the antigen binding protein are connected by the hinge region of IgG4.

[0087] In certain embodiments, the scFv targeting a second antigen of the antigen binding protein comprises a VH targeting a second antigen and a VL targeting a second antigen.

[0088] In certain embodiments, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting a second antigen, a VH targeting a second antigen, and a constant region of a TCRβ chain.

[0089] In certain embodiments, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting a second antigen, a linker, a VH targeting a second antigen, an IgG4 hinge region, and a constant region of a TCRβ chain.

[0090] In certain embodiments, the antigen binding protein further comprises a binding domain that targets a fourth antigen.

[0091] In certain embodiments, the binding domain targeting a fourth antigen comprises a scFv.

[0092] In certain embodiments, the fourth antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19, and PD-1.

[0093] In certain embodiments, the scFv targeting a fourth antigen comprises a VH targeting a fourth antigen and a VL targeting a fourth antigen.

[0094] In certain embodiments, the second polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, a VL targeting a second antigen, a linker, a VH targeting a second antigen, a hinge region of IgG4, a constant region of a TCRβ chain, a hinge region of IgG4, a VH targeting a fourth antigen, a linker, and a VL targeting a fourth antigen.

[0095] In certain embodiments, the second polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, a VL targeting a second antigen, a linker, a VH targeting a second antigen, a hinge region of IgG4, a constant region of a TCRβ chain, a hinge region of IgG4, a VL targeting a fourth antigen, a linker, and a VH targeting a fourth antigen.

[0096] In certain embodiments, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VH targeting a second antigen, a VL targeting a second antigen, and a constant region of a TCRβ chain.

[0097] In certain embodiments, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, VH targeting a second antigen, a linker, VL targeting a second antigen, a hinge region of IgG4, and a constant region of a TCRβ chain.

[0098] In certain embodiments, the second polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, VH targeting a second antigen, VL targeting a second antigen, a hinge region of IgG4, a constant region of a TCRβ chain, a hinge region of IgG4, VH targeting a fourth antigen, and VL targeting a fourth antigen.

[0099] In certain embodiments, the second polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, VH targeting a second antigen, VL targeting a second antigen, a hinge region of IgG4, and a constant region of a TCRβ chain, a hinge region of IgG4, a VL targeting a fourth antigen, and a VH targeting a fourth antigen.

[0100] In certain embodiments, the first antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19, and PD-1.

[0101] In certain embodiments, the second antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19, and PD-1.

[0102] In certain embodiments, the first antigen is different from the second antigen.

[0103] In certain embodiments, the first antigen and the second antigen are different antigen-binding epitopes of the same antigen.

[0104] In certain embodiments, the antigen binding protein comprises one polypeptide chain comprising an scFv targeting a first antigen and a constant region of a TCR alpha chain; and an scFv targeting a second antigen and a constant region of a TCR beta chain.

[0105] In certain embodiments, the polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, an scFv targeting a first antigen, a constant region of a TCRα chain, a linker, a constant region of a TCRβ chain, and an scFv targeting a second antigen.

[0106] In certain embodiments, the polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a scFv targeting a first antigen, a hinge region, a constant region of a TCRβ chain, a linker, a constant region of a TCRα chain, a hinge region, and a scFv targeting a second antigen.

[0107] In certain embodiments, the scFv targeting a first antigen comprises a VH targeting a first antigen and a VL targeting a first antigen.

[0108] In certain embodiments, the scFv targeting a second antigen comprises a VH targeting a second antigen and a VL targeting a second antigen.

[0109] In certain embodiments, the polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, VL targeting a first antigen, VH targeting a first antigen, a hinge region, a constant region of a TCRα chain, a linker, a constant region of a TCRβ chain, a hinge region, VH targeting a second antigen, and VL targeting a second antigen.

[0110] In certain embodiments, the polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, VL targeting a first antigen, a linker, VH targeting a first antigen, a hinge region, a constant region of a TCRβ chain, a linker, a constant region of a TCRα chain, a hinge region, VH targeting a second antigen, a linker, and VL targeting a second antigen.

[0111] In certain embodiments, the constant region of the TCR alpha chain is derived from the constant region of a human TCR alpha chain.

[0112] In certain embodiments, the constant region of the TCR α chain comprises the amino acid sequence shown in SEQ ID NO:47.

[0113] In certain embodiments, the constant region of the TCR β chain is derived from the constant region of a human TCR β chain.

[0114] In certain embodiments, the constant region of the TCR β chain comprises the amino acid sequence shown in SEQ ID NO:48.

[0115] In certain embodiments, the scFv targeting the first antigen of the antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:36 and SEQ ID NO:45.

[0116] In certain embodiments, the scFv targeting the second antigen of the antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:36 and SEQ ID NO:45.

[0117] In certain embodiments, the cells are further capable of expressing a chemokine receptor.

[0118] In certain embodiments, the chemokine receptor is selected from the group consisting of: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7.

[0119] In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a scFv targeting a first antigen, a nucleic acid sequence encoding a scFv targeting a second antigen, a nucleic acid sequence encoding the constant region of a TCR α chain, and a nucleic acid sequence encoding a constant region sequence of a TCR β chain / encoding a reverse amino acid sequence of the constant region of a TCR β chain.

[0120] In certain embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding an IgG4 hinge region.

[0121] In certain embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding a cleavage peptide.

[0122] In certain embodiments, the cleavage peptide comprises a 2A peptide.

[0123] In certain embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding a linker.

[0124] In certain embodiments, the amino acid sequence of the linker is SEQ ID NO: 49: GGGGS.

[0125] In certain embodiments, the nucleic acid molecule comprises, from the 5' end to the 3' end, a nucleic acid sequence encoding a scFv targeting a first antigen, a nucleic acid sequence encoding an IgG4 hinge region, a nucleic acid sequence encoding a constant region of a TCR α chain, a nucleic acid sequence encoding a cleavage peptide, a nucleic acid sequence encoding a scFv targeting a second antigen, a nucleic acid sequence encoding an IgG4 hinge region, and a constant region sequence encoding a TCR β chain.

[0126] In certain embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding a scFv targeting a third antigen / a nucleic acid sequence encoding a reverse amino acid sequence of a scFv targeting a third antigen.

[0127] In certain embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding a scFv targeting a fourth antigen / a nucleic acid sequence encoding a reverse amino acid sequence of a scFv targeting a fourth antigen.

[0128] In certain embodiments, the nucleic acid molecule comprises, from the 5' end to the 3' end, the following: a nucleic acid sequence encoding a scFv targeting a first antigen, a nucleic acid sequence encoding an IgG4 hinge region, a nucleic acid sequence encoding a constant region of a TCR α chain, a nucleic acid sequence encoding an IgG4 hinge region, a nucleic acid sequence encoding a reverse amino acid sequence of a scFv targeting a third antigen, a nucleic acid sequence encoding a cleavage peptide, a nucleic acid sequence encoding a scFv targeting a second antigen, a nucleic acid sequence encoding an IgG4 hinge region, a constant region sequence encoding a TCR β chain, a nucleic acid sequence encoding an IgG4 hinge region, and a nucleic acid sequence encoding a reverse amino acid sequence of a scFv targeting a fourth antigen.

[0129] In certain embodiments, the nucleic acid molecule comprises, from the 5' end to the 3' end, a nucleic acid sequence encoding a scFv targeting a first antigen, a nucleic acid sequence encoding an IgG4 hinge region, a nucleic acid sequence encoding a constant region of a TCR α chain, a nucleic acid sequence encoding a linker, a nucleic acid sequence encoding a reverse amino acid sequence of the constant region of a TCR β chain, a nucleic acid sequence encoding an IgG4 hinge region, and a nucleic acid sequence encoding a scFv targeting a second antigen.

[0130] In certain embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding a chemokine receptor.

[0131] In certain embodiments, the chemokine receptor is selected from the group consisting of: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7.

[0132] In certain embodiments, the chemokine is CXCR3.

[0133] In certain embodiments, the nucleic acid sequence encoding the chemokine receptor is directly or indirectly linked to the nucleic acid sequence encoding the antigen binding protein.

[0134] In certain embodiments, a sequence encoding a cleavage peptide is further included between the nucleic acid sequence encoding the chemokine receptor and the nucleic acid sequence encoding the antigen binding protein.

[0135] In certain embodiments, the cleavage peptide is a 2A peptide.

[0136] In certain embodiments, the cleavage peptide is selected from the group consisting of: T2A and P2A.

[0137] In certain embodiments, the cells are capable of secreting the antigen binding protein in situ.

[0138] In certain embodiments, the cells are further capable of expressing the chemokine receptor.

[0139] On the other hand, the present application also provides a pharmaceutical composition comprising the cells and, optionally, a pharmaceutically acceptable carrier.

[0140] On the other hand, the present application also provides the use of the cell or the pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease and / or condition.

[0141] In certain embodiments, the disease and / or condition comprises a tumor.

[0142] In certain embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0143] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0145] Figures 1A-1B show schematic diagrams of secondary and quadruple antibodies developed against TCR constant regions. (Figure 1A) Secondary antibody structure. (Left: Trans format, right: Cis format) (Figure 1B) Quadruple antibody structure.

[0146] Figures 2A-2C show the production of four secondary antibody formats stably expressed in HEK293T cells, and the detection of their binding effects on corresponding ROR1-positive tumors and T cells.

[0147] Figure 3 shows a T cell redirection assay testing four secondary antibody formats.

[0148] Figures 4A-4B show an anti-tumor assay testing four formats of secondary antibodies against the CD19 target.

[0149] Figure 5 shows a schematic diagram of the ROR1 / TIM3 / PD1 / CD3 tetraclonal antibody structure.

[0150] Figure 6 shows the binding to the corresponding target through flow cytometry verification.

[0151] FIG7A shows the results of flow cytometric analysis of the binding ability of secreted BsAb in the supernatant to cells, and FIG7B shows the number of fluorescently labeled tumor cells and the concentration of IFN-γ secreted by T cells.

[0152] Figure 8 shows schematic diagrams of two formats, namely, Trans format TCRC-based BsAb and Cis format TCRC-based BsAb.

[0153] Figure 9A shows flow cytometry validation of the binding of in situ secreted CD19-UCHT1-TCRC BsAbs to the corresponding cell lines MEC-1 (expressing CD19+) and PBMC (expressing CD3+). Figure 9B shows the concentration of IFN-γ secreted by PBMC in cell culture medium. Figure 9C shows the cytotoxic effect of lentiviral-transfected PBMC on target tumor cells. DETAILED DESCRIPTION

[0154] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0155] Definition of terms

[0156] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that can be or has been a recipient of a subject's plasmid or vector, including a nucleic acid molecule of the present invention or a vector of the present invention. In this application, the cell can include a T cell, a NK cell, a NKT cell, a mesenchymal stem cell, a neuronal stem cell, a hematopoietic stem cell, or a mixture thereof.

[0157] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. A vector can be used to transform, transduce, or transfect host cells, allowing the genetic material elements it carries to be expressed in host cells. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site.

[0158] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from their natural environment or artificially synthesized.

[0159] In this application, the term "antigen binding protein" generally refers to a protein with antigen binding ability. The "antigen binding protein" may comprise a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes the antigen binding portion to bind to the antigen. The antigen binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold having a transplanted CDR or CDR derivative. Such scaffolds include, but are not limited to, antibody-derived scaffolds that include mutations introduced, for example, to stabilize the three-dimensional structure of the antigen binding protein and fully synthetic scaffolds that include, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). In addition, peptide antibody mimics ("PAMs") and scaffolds based on antibody mimics using a fibronectin component can be used as scaffolds.

[0160] In this application, the term "variable region" generally refers to the amino-terminal domain of an antibody heavy or light chain. The variable regions of the heavy and light chains can be referred to as "VH" and "VL," respectively (or "VH" and "VL," respectively). These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0161] In this application, the term "variable" generally refers to the fact that certain segments of the variable domains differ greatly in sequence between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, most of which adopt a β-sheet configuration and are connected by three CDRs, which form a loop connection and, in some cases, form part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR region, and the CDRs from the other chain together contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0162] In this application, the term "scFv" generally refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used in this application, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0163] In this application, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigens. TCR is responsible for recognizing antigens that are bound to major histocompatibility complex molecules. TCR is usually composed of a heterodimer of alpha (α) and beta (β) chains, but in some cells, TCR is composed of gamma and delta (γ / δ) chains. TCR can exist in α / β and γ / δ forms, which are similar in structure but have unique anatomical positions and functions. Each chain can be composed of two domains - a variable domain (variable region) and a constant domain (constant region). In some embodiments, TCR can be modified on any cell comprising TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells and γδT cells.

[0164] In this application, the term "constant region" generally refers to a region having a more conserved amino acid sequence relative to the variable region containing the antigen binding site of a TCR. For example, the constant region may be of human origin.

[0165] In this application, the term "directly connected" is contrasted with the term "indirectly connected." The term "directly connected" generally refers to a direct connection. For example, the direct connection may be a case where the substances are directly connected without a spacer. The spacer may be a linker. For example, the linker may be a peptide linker. The term "indirectly connected" generally refers to a case where the substances are not directly connected. For example, the indirect connection may be a case where the substances are connected via a spacer.

[0166] In this application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise the cells described herein, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may also comprise a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present application includes, but is not limited to, liquid, frozen and lyophilized compositions.

[0167] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium; and / or nonionic surfactants.

[0168] In this application, the term "tumor" generally refers to any new pathological tissue proliferation. Tumor cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. In this application, the tumor can include benign tumors and malignant tumors. In this application, the tumor can include solid tumors and / or hematological tumors. In this application, the tumor can include cancer.

[0169] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0170] Detailed Description of the Invention

[0171] Modified cells

[0172] In one aspect, the present application provides a cell comprising a vector comprising a nucleic acid molecule encoding an antigen binding protein comprising a scFv targeting a first antigen and a constant region of a TCRα chain, and a scFv targeting a second antigen and a constant region of a TCRβ chain.

[0173] In the present application, the modified cells are capable of secreting and / or producing the antigen binding protein in situ.

[0174] In the present application, the cells are also capable of expressing chemokine receptors.

[0175] In the present application, the chemokine receptor may be an exogenous chemokine receptor.

[0176] In the present application, the chemokine receptor may be selected from the group consisting of: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and CXCR7.

[0177] In the present application, the chemokine receptor may be CXCR3.

[0178] In the present application, the CXCR3 may be human CXCR3. In the present application, the CXCR3 may comprise the amino acid sequence shown in SEQ ID NO:52.

[0179] In the present application, the cells may be immune cells. For example, the cells may be T cells or NK cells. In the present application, the cells may also be other cells, for example, the cells may be mesenchymal stem cells. For example, the cells may be hematopoietic stem cells. For example, the cells may be PBMC cells.

[0180] In the present application, modified cells can be obtained by transfecting vectors carrying nucleic acid molecules encoding chemokine receptors and antigen binding proteins into cells, so that the cells can secrete chemokine receptors and antigen binding proteins in situ.

[0181] In the present application, the vector may be a viral vector. For example, the vector may be selected from the group consisting of a lentivirus, an adenovirus, a retrovirus, and an adeno-associated virus vector.

[0182] In the present application, the vector may be a non-viral vector.

[0183] In this application, a novel bispecific antibody / polyspecific antibody combination is provided, which is produced and secreted in situ by T cells. Furthermore, the T cells can carry and express the CXCR3 chemokine receptor, allowing these transgenic T cells to be enriched in solid tumor sites, allowing these T cells to produce and secrete BsAbs in situ, redirecting and killing tumors, and improving anti-tumor effects.

[0184] Antigen binding proteins

[0185] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined using a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art, and for details, see http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs can be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity with the amino acid sequence of the CDR.

[0186] In the present application, the antigen binding protein comprises a scFv targeting a first antigen and a constant region of a TCR α chain, and a scFv targeting a second antigen and a constant region of a TCR β chain.

[0187] In the present application, the antigen binding protein may not comprise the variable region of the TCR α chain.

[0188] In the present application, the antigen binding protein may not comprise the variable region of the TCR β chain.

[0189] In the present application, the scFv targeting the first antigen and the constant region of the TCRα chain may be linked via a hinge region.

[0190] In the present application, the scFv targeting the second antigen and the constant region of the TCRβ chain may be linked via a hinge region.

[0191] In the present application, the hinge region may comprise the hinge region of IgG.

[0192] In the present application, the hinge region may comprise the hinge region of IgG4. For example, the hinge region may comprise the amino acid sequence shown in SEQ ID NO: 46.

[0193] In the present application, the constant region of the TCRα chain and the constant region of the TCRβ chain in the antigen binding protein may be directly or indirectly linked. For example, the linking may include linking in one or more ways.

[0194] In the present application, the constant region of the TCR α chain and the constant region of the TCR β chain in the antigen binding protein may be linked by a disulfide bond.

[0195] In the present application, the constant region of the TCRα chain and the constant region of the TCRβ chain of the antigen binding protein can be connected by a linker. For example, the linker can comprise a peptide linker. For example, the amino acid sequence of the linker can be GGGGS (SEQ ID NO: 49).

[0196] In the present application, the constant region of the TCR α chain and the constant region of the TCR β chain in the antigen binding protein may be linked via a disulfide bond and a linker.

[0197] In the present application, the first antigen can be selected from the following group: ROR1, CD3, TIM3, CD19 and PD-1.

[0198] In the present application, the second antigen can be selected from the following group: ROR1, CD3, TIM3, CD19 and PD-1.

[0199] For example, in the present application, the first antigen and the second antigen may be different antigen-binding epitopes of the same antigen. For example, in the present application, the first antigen and the second antigen may be the same antigen-binding epitope of the same antigen. For example, in the present application, the first antigen and the second antigen may be different antigens.

[0200] In the present application, the scFv targeting a first antigen may comprise a light chain variable region VL of an antibody targeting the first antigen and a heavy chain variable region VH of an antibody targeting the first antigen. For example, the N-terminus of the VH targeting the first antigen may be directly or indirectly linked to the C-terminus of the VL targeting the first antigen. For another example, the C-terminus of the VH targeting the first antigen may be directly or indirectly linked to the N-terminus of the VL targeting the first antigen.

[0201] In the present application, the C-terminus of the VH targeting the first antigen is directly or indirectly connected to the N-terminus of the constant region of the TCRα chain, for example, via the hinge region of IgG4.

[0202] In the present application, the C-terminus of the VL targeting the first antigen can be directly or indirectly connected to the N-terminus of the constant region of the TCRα chain, for example, through the hinge region of IgG4.

[0203] In the present application, the N-terminus of the VH targeting the first antigen can be directly or indirectly connected to the C-terminus of the constant region of the TCRα chain, for example, through the hinge region of IgG4.

[0204] In the present application, the N-terminus of the VL targeting the first antigen can be directly or indirectly connected to the C-terminus of the constant region of the TCRα chain, for example, through the hinge region of IgG4.

[0205] In the present application, the scFv targeting the second antigen comprises an antibody light chain variable region VL targeting the second antigen and an antibody heavy chain variable region VH targeting the second antigen.

[0206] In the present application, the N-terminus of the VH targeting the second antigen can be directly or indirectly connected to the C-terminus of the VL targeting the second antigen. For example, they can be connected via a linker. For example, the linker can be a peptide linker.

[0207] In the present application, the C-terminus of the VH targeting the second antigen can be directly or indirectly connected to the N-terminus of the VL targeting the second antigen. For example, they can be connected via a linker. For example, the linker can be a peptide linker.

[0208] In the present application, the C-terminus of the VH targeting the second antigen may be directly or indirectly connected to the N-terminus of the constant region of the TCRβ chain, for example, via a hinge region.

[0209] In the present application, the C-terminus of the VL targeting the second antigen may be directly or indirectly connected to the N-terminus of the constant region of the TCRβ chain, for example, via a hinge region.

[0210] In the present application, the N-terminus of the VH targeting the second antigen may be directly or indirectly connected to the C-terminus of the constant region of the TCRβ chain, for example, via a hinge region.

[0211] In the present application, the N-terminus of the VL targeting the second antigen can be directly or indirectly connected to the N-terminus of the constant region of the TCRβ chain, for example, through a hinge region.

[0212] In the present application, the antigen-binding protein may further comprise a binding domain that targets a third antigen. For example, the binding domain that targets the third antigen may be an antigen-binding fragment. For example, the binding domain that targets the third antigen may be an scFv that targets the third antigen.

[0213] In the present application, the antigen-binding protein may further comprise a binding domain that targets a fourth antigen. For example, the binding domain that targets the fourth antigen may be an antigen-binding fragment. For example, the binding domain that targets the fourth antigen may be an scFv that targets the fourth antigen.

[0214] In the present application, the scFv targeting the third antigen can be directly or indirectly linked to the constant region of the TCRα chain.

[0215] In the present application, the scFv targeting the third antigen is connected to the constant region of the TCRα chain via the IgG4 hinge region.

[0216] In the present application, the scFv targeting the fourth antigen can be directly or indirectly linked to the constant region of the TCRβ chain.

[0217] In the present application, the scFv targeting the fourth antigen can be linked to the constant region of the TCRβ chain via the IgG4 hinge region.

[0218] In the present application, the scFv targeting a third antigen may comprise a VH targeting a third antigen and a VL targeting a third antigen.

[0219] In the present application, the N-terminus of the VH targeting the third antigen can be directly or indirectly connected to the C-terminus of the VL targeting the third antigen, for example, via a linker, for example, a peptide linker.

[0220] In the present application, the C-terminus of the VH targeting the third antigen can be directly or indirectly connected to the N-terminus of the VL targeting the third antigen, for example, via a linker, for example, a peptide linker.

[0221] In the present application, the N-terminus of the VH targeting the third antigen may be directly or indirectly connected to the C-terminus of the constant region of the TCRα chain, for example, via a hinge region.

[0222] In the present application, the C-terminus of the VH targeting the third antigen may be directly or indirectly connected to the N-terminus of the constant region of the TCRα chain, for example, via a hinge region.

[0223] In the present application, the N-terminus of the VL targeting the third antigen may be directly or indirectly connected to the C-terminus of the constant region of the TCRα chain, for example, via a hinge region.

[0224] In the present application, the C-terminus of the VL targeting the third antigen may be directly or indirectly connected to the N-terminus of the constant region of the TCRα chain, for example, via a hinge region.

[0225] In the present application, the scFv targeting the fourth antigen may comprise VH targeting the fourth antigen and VL targeting the fourth antigen.

[0226] In the present application, the N-terminus of the VH targeting the fourth antigen may be directly or indirectly connected to the C-terminus of the VL targeting the fourth antigen, for example, via a linker.

[0227] In the present application, the C-terminus of the VH targeting the fourth antigen may be directly or indirectly connected to the N-terminus of the VL targeting the fourth antigen, for example, via a linker.

[0228] In the present application, the N-terminus of the VH targeting the fourth antigen may be directly or indirectly connected to the C-terminus of the constant region of the TCRβ chain, for example, via a hinge region.

[0229] In the present application, the C-terminus of the VH targeting the fourth antigen may be directly or indirectly connected to the N-terminus of the constant region of the TCRβ chain, for example, via a hinge region.

[0230] In the present application, the N-terminus of the VL targeting the fourth antigen may be directly or indirectly connected to the C-terminus of the constant region of the TCRβ chain, for example, via a hinge region.

[0231] In the present application, the C-terminus of the VL targeting the fourth antigen may be directly or indirectly connected to the N-terminus of the constant region of the TCRβ chain, for example, via a hinge region.

[0232] In the present application, the antigen binding protein may comprise two polypeptide chains, wherein the first polypeptide chain comprises an scFv targeting a first antigen and a constant region of a TCRα chain; wherein the second polypeptide chain comprises an scFv targeting a second antigen and a constant region of a TCRβ chain.

[0233] In the present application, the first polypeptide chain and the second polypeptide chain may be linked via a disulfide bond.

[0234] In the present application, in the first polypeptide chain of the antigen-binding protein, the scFv targeting the first antigen and the constant region of the TCR α chain are directly or indirectly connected. For example, they are connected via a hinge region. For example, the hinge region can be an IgG hinge region. For example, the hinge region can be an IgG4 hinge region.

[0235] In the present application, the scFv targeting the first antigen may comprise VH targeting the first antigen and VL targeting the first antigen.

[0236] In the present application, the first polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, a VL domain targeting the first antigen, a VH domain targeting the first antigen, and a constant region of the TCRα chain, wherein these three domains may be directly or indirectly connected.

[0237] In the present application, the first polypeptide chain of the antigen binding protein may comprise, from N-terminus to C-terminus, VL targeting the first antigen, a linker, VH targeting the first antigen, a hinge region of IgG4, and a constant region of the TCRα chain.

[0238] In the present application, the antigen-binding protein may further comprise a binding domain that targets a third antigen. For example, the binding domain that targets the third antigen may be an antigen-binding fragment. For example, the binding domain that targets the third antigen may be an scFv that targets the third antigen.

[0239] In the present application, the scFv targeting a third antigen comprises VH targeting a third antigen and VL targeting a third antigen.

[0240] In the present application, the first polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, a VL targeting a first antigen, a VH targeting a first antigen, an IgG4 hinge region, a TCRα chain constant region, an IgG4 hinge region, a VL targeting a third antigen, and a VH targeting a third antigen. The various components of the polypeptide chain may be connected directly or indirectly, for example, via a linker.

[0241] In the present application, the first polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, a VL targeting a first antigen, a VH targeting a first antigen, an IgG4 hinge region, a TCRα chain constant region, an IgG4 hinge region, a VH targeting a third antigen, and a VL targeting a third antigen. The various components of the polypeptide chain may be connected directly or indirectly, for example, via a linker.

[0242] In the present application, the first polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, a VH polypeptide targeting the first antigen, a VL polypeptide targeting the first antigen, and a constant region of a TCR α chain. The various components of the polypeptide chain may be connected directly or indirectly, for example, via a linker.

[0243] In the present application, the first polypeptide chain of the antigen binding protein may comprise, from N-terminus to C-terminus, VH targeting the first antigen, VL targeting the first antigen, the hinge region of IgG4, and the constant region of the TCRα chain.

[0244] In the present application, the first polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, VH targeting the first antigen, a linker, VL targeting the first antigen, a hinge region of IgG4, a constant region of a TCRα chain, a hinge region of IgG4, VL targeting a third antigen, a linker, and VH targeting the third antigen.

[0245] In the present application, the first polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, VH targeting the first antigen, a linker, VL targeting the first antigen, a hinge region of IgG4, a constant region of a TCRα chain, a hinge region of IgG4, VH targeting a third antigen, a linker, and VL targeting the third antigen.

[0246] In the present application, the first polypeptide chain of the antigen-binding protein may comprise a scFv targeting a first antigen, a constant region of a TCR α chain, and a scFv targeting a third antigen. The various components of the polypeptide chain may be connected directly or indirectly, for example, via a linker. For example, the linker may be a peptide linker. For example, the linker may be a hinge region.

[0247] In the present application, in the second polypeptide chain of the antigen binding protein, the scFv targeting the second antigen can be directly or indirectly linked to the constant region of the TCRβ chain.

[0248] In the present application, the scFv targeting the second antigen of the antigen binding protein can be connected to the constant region of the TCR α chain through a hinge region.

[0249] In the present application, the scFv targeting the second antigen of the antigen binding protein can be linked to the constant region of the TCRβ chain via the IgG hinge region.

[0250] In the present application, the scFv targeting the second antigen of the antigen binding protein can be connected to the constant region of the TCRβ chain through the hinge region of IgG4.

[0251] In the present application, the scFv targeting the second antigen of the antigen binding protein may comprise a VH targeting the second antigen and a VL targeting the second antigen, wherein the VH and the VL may be connected via a linker.

[0252] In the present application, the second polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, a VL targeting a second antigen, a VH targeting a second antigen, and the constant region of a TCR β chain. The various components of the polypeptide chain may be connected directly or indirectly, for example, via a linker. For example, the linker may be a peptide linker. For example, the linker may be a hinge region.

[0253] In the present application, the second polypeptide chain of the antigen binding protein may comprise, from N-terminus to C-terminus, VL targeting the second antigen, a linker, VH targeting the second antigen, a hinge region of IgG4, and a constant region of the TCRβ chain.

[0254] In the present application, the antigen-binding protein may further comprise a binding domain that targets a fourth antigen. For example, the binding domain that targets the fourth antigen may be an antigen-binding fragment. For example, the binding domain that targets the fourth antigen may be an scFv that targets the fourth antigen.

[0255] In the present application, the scFv targeting the fourth antigen may comprise VH targeting the fourth antigen and VL targeting the fourth antigen.

[0256] In the present application, the second polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, the following sequence: VL targeting the second antigen, a linker, VH targeting the second antigen, a hinge region of IgG4, a constant region of the TCRβ chain, a hinge region of IgG4, VH targeting the fourth antigen, a linker, and VL targeting the fourth antigen.

[0257] In the present application, the second polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, the following sequence: a VL targeting a second antigen, a linker, a VH targeting a second antigen, a hinge region of IgG4, a constant region of a TCRβ chain, a hinge region of IgG4, a VL targeting a fourth antigen, a linker, and a VH targeting a fourth antigen.

[0258] In the present application, the second polypeptide chain of the antigen binding protein may comprise, from N-terminus to C-terminus, VH targeting the second antigen, VL targeting the second antigen, and the constant region of the TCRβ chain.

[0259] In the present application, the second polypeptide chain of the antigen binding protein may comprise, from N-terminus to C-terminus, VH targeting the second antigen, a linker, VL targeting the second antigen, a hinge region of IgG4, and a constant region of the TCRβ chain.

[0260] In the present application, the second polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, VH targeting the second antigen, a linker, VL targeting the second antigen, a hinge region of IgG4, a constant region of the TCRβ chain, a hinge region of IgG4, VH targeting the fourth antigen, and VL targeting the fourth antigen.

[0261] In the present application, the second polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, VH targeting the second antigen, VL targeting the second antigen, the hinge region of IgG4, and the constant region of the TCRβ chain, the hinge region of IgG4, VL targeting the fourth antigen, and VH targeting the fourth antigen.

[0262] In the present application, the first antigen of the antigen binding protein can be selected from the following group: ROR1, CD3, TIM3, CD19 and PD-1.

[0263] In the present application, the second antigen of the antigen binding protein can be selected from the following group: ROR1, CD3, TIM3, CD19 and PD-1.

[0264] In the present application, the first antigen and the second antigen of the antigen-binding protein are the same. In the present application, the first antigen and the second antigen of the isolated antigen-binding protein are different binding epitopes of the same antigen.

[0265] In the present application, the first antigen and the second antigen of the antigen binding protein are different.

[0266] In the present application, the antigen binding protein may comprise one polypeptide chain comprising an scFv targeting a first antigen and a constant region of a TCR α chain; and an scFv targeting a second antigen and a constant region of a TCR β chain.

[0267] In the present application, the polypeptide chain comprises, from N-terminus to C-terminus, an scFv targeting a first antigen, a constant region of a TCRα chain, a linker, a constant region of a TCRβ chain, and an scFv targeting a second antigen.

[0268] In the present application, the polypeptide chain may comprise, from N-terminus to C-terminus, an scFv targeting a first antigen, a hinge region, a constant region of a TCR β chain, a linker, a constant region of a TCR α chain, a hinge region, and an scFv targeting a second antigen. For example, the scFv targeting a first antigen comprises a VH targeting the first antigen and a VL targeting the first antigen. For example, the scFv targeting a second antigen comprises a VH targeting the second antigen and a VL targeting the second antigen. For example, the VH and VL may be directly or indirectly linked. For example, the VH and VL may be linked via a linker. For example, the linker may comprise a peptide linker.

[0269] In the present application, the polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, in sequence: VL targeting a first antigen, a linker, VH targeting a first antigen, a hinge region, a constant region of a TCRα chain, a linker, a constant region of a TCRβ chain, a hinge region, VH targeting a second antigen, a linker, and VL targeting a second antigen.

[0270] In the present application, the polypeptide chain of the antigen-binding protein may comprise, from N-terminus to C-terminus, in sequence: VL targeting a first antigen, a linker, VH targeting a first antigen, a hinge region, a constant region of a TCRβ chain, a linker, a constant region of a TCRα chain, a hinge region, VH targeting a second antigen, a linker, and VL targeting a second antigen.

[0271] In the present application, the constant region of the TCR α chain may be derived from the constant region of a human TCR α chain. For example, the constant region of the TCR α chain may comprise the amino acid sequence shown in SEQ ID NO: 47.

[0272] In the present application, the constant region of the TCR β chain may be derived from the constant region of a human TCR β chain. For example, the constant region of the TCR β chain may comprise the amino acid sequence shown in SEQ ID NO: 48.

[0273] In the present application, the scFv targeting ROR1 may comprise HCDR1, HCDR2, and HCDR3. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 20, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 21. The scFv targeting ROR1 may comprise LCDR1, LCDR2, and LCDR3. For example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 24 (SGS), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 25.

[0274] In the present application, the scFv targeting ROR1 may comprise a VH and a VL. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 22. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the scFv targeting ROR1 may comprise the amino acid sequence shown in SEQ ID NO: 27.

[0275] In the present application, the CD3-targeting scFv may comprise HCDR1, HCDR2, and HCDR3. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 12. The CD3-targeting scFv may comprise LCDR1, LCDR2, and LCDR3. For example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 14, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 15 (YTS), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 16.

[0276] In the present application, the CD3-targeting scFv may comprise a VH and a VL. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO: 17. For example, the CD3-targeting scFv may comprise the amino acid sequence shown in SEQ ID NO: 18.

[0277] In the present application, the TIM3-targeting scFv may comprise HCDR1, HCDR2, and HCDR3. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 38, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 39. The TIM3-targeting scFv may comprise LCDR1, LCDR2, and LCDR3. For example, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 41, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 42 (KVS), and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 43.

[0278] In the present application, the TIM3-targeting scFv may comprise a VH and a VL. For example, the VH may comprise the amino acid sequence set forth in SEQ ID NO: 40. For example, the VL may comprise the amino acid sequence set forth in SEQ ID NO: 44. For example, the TIM3-targeting scFv may comprise the amino acid sequence set forth in SEQ ID NO: 45.

[0279] In the present application, the scFv targeting PD-1 may comprise HCDR1, HCDR2, and HCDR3. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 28, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 29, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 30. The scFv targeting PD-1 may comprise LCDR1, LCDR2, and LCDR3. For example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 32, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 33 (RDS), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 34.

[0280] In the present application, the scFv targeting PD-1 may comprise a VH and a VL. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 31. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO: 35. For example, the scFv targeting PD-1 may comprise the amino acid sequence shown in SEQ ID NO: 36.

[0281] In the present application, the scFv targeting CD19 may comprise HCDR1, HCDR2, and HCDR3. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3. The scFv targeting CD19 may comprise LCDR1, LCDR2, and LCDR3. For example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 6 (HTS), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 7.

[0282] In the present application, the CD19-targeting scFv may comprise a VH and a VL. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 4. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO: 8. For example, the CD19-targeting scFv may comprise the amino acid sequence shown in SEQ ID NO: 9.

[0283] In the present application, the same isolated antigen-binding protein may contain one or more linkers. For example, the linkers may be identical. For example, the linkers may be different. For example, the linkers may be partially identical and partially different.

[0284] In the present application, the hinge region of IgG4 may comprise the amino acid sequence shown in SEQ ID NO: 46. In the present application, the linker may comprise the amino acid sequence shown in SEQ ID NO: 49.

[0285] Pharmaceutical composition, preparation method, and use

[0286] In another aspect, the present application provides a method for preparing the cells described herein.

[0287] On the other hand, the present application also provides a pharmaceutical composition comprising the cells and, optionally, a pharmaceutically acceptable carrier.

[0288] In certain embodiments, the pharmaceutical composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present application includes but is not limited to liquid, frozen and lyophilized compositions.

[0289] In certain embodiments, the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0290] In certain embodiments, the pharmaceutical composition can include parenteral, percutaneous, intracavitary, intraarterial, intravenous, intratumoral, intrathecal and / or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition can be administered uninterruptedly. The uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient's body, as described in WO2015 / 036583.

[0291] On the other hand, the present application also provides use of the cell or the pharmaceutical composition in preparing a drug for preventing and / or treating a disease and / or condition.

[0292] On the other hand, the present application also provides a method for preventing and / or treating a disease and / or condition, comprising administering the cell or the pharmaceutical composition to a subject in need thereof. In the present application, the administration can be carried out in different ways, such as intravenously, intratumorally, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally.

[0293] On the other hand, the present application also provides the cell or the pharmaceutical composition for preventing and / or treating a disease and / or disorder.

[0294] In the present application, the disease and / or disorder may include a tumor.

[0295] In the present application, the tumor may include solid tumors and / or non-solid tumors.

[0296] For example, the tumor may include a ROR1-positive tumor. For example, the tumor may include a PD-1-positive tumor. For example, the tumor may include a TIM3-positive tumor. For example, the tumor may include a CD19-positive tumor.

[0297] Without intending to be bound by any theory, the following examples are merely intended to illustrate the cells, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0298] Example

[0299] Experimental Materials and Methods

[0300] Cell lines and culture

[0301] HEK293T, Jurkat E6, Jurkat-Luc, Raji, JeKo-1, MEC, MEC-ROR1, A549, HCC827, H1975 and MDA-MB-231, HL60, K562 cell lines were obtained from ATCC. HEK293T-PD1 cell line was generated in-house. All these cell lines were maintained in full culture medium (IDMEM containing 10% heat-inactivated FCS, 100 U / mL penicillin / streptomycin and 2 mM L-glutamine). PBMCs were isolated from whole blood of healthy donors using Ficoll-Paque and plated at 2×10 7 1 mL of sample was aliquoted at a concentration of 10 cells / mL and stored frozen in a liquid nitrogen tank. The culture medium was heat-inactivated FCS supplemented with 10% DMSO (vol / vol).

[0302] Amino acid sequence used

[0303] In the technical solutions given in the examples, the exemplary amino acid sequences used are as follows:

[0304] CD19 scFv: SEQ ID NO: 9; CD3 (UCHT1) scFv: SEQ ID NO: 18; ROR1 scFv: SEQ ID NO: 27; PD-1 scFv: SEQ ID NO: 36; TIM3 scFv: SEQ ID NO: 45; IgG4 hinge region: SEQ ID NO: 46.

[0305] Example 1 Construction, cloning, and expression of bispecific / quadruspecific recombinant antibodies

[0306] All gene constructs were synthesized using GeneWiz. Gene domains encoding ROR1 scFvxUCHT1 scFv and ROR1 scFv and UCHT1 scFv single-arm controls were inserted into the expression plasmid pALD with the sffv promoter via the Apa1 / Nhe1 cloning sites. The eGFP marker was removed from this cloning process. ROR1 scFv consists of VL and VH fused to a 28-amino acid linker. To generate cis bispecific antibodies, ROR1 scFv and UCHT1 scFv were connected via a Gly4Ser linker. An N-terminal Kozac sequence, IL2 signal peptide (SPIL2, SEQ ID NO: 51), and C-terminal V5 tag were added.

[0307] To produce trans bispecific antibodies based on TCRC, the gene domains encoding ROR1 scFv-IgG4hinge-TCRCa and UCHT1 scFv-IgG4hinge-TCRCb were connected by a P2A sequence. To produce bispecific antibodies based on cis TCRC, the gene domains encoding ROR1 scFv-IgG4hinge-TCRCa and UCHT1 scFv-IgG4-TCRCb (in reverse sequence) were connected by a GGGGS (SEQ ID NO: 49) sequence. Each trans or cis format has two subformats, in which TCRCα and TCRCβ are exchanged between the two chains. Four bispecific antibody formats based on CD19scFcxUCHT1 scFv TCRC were similarly prepared.

[0308] Additionally, to generate a TCRCαβ-based trans tetraclonal antibody, the gene domains encoding the ROR1 scFv-IgG4hinge-TCRCa-IgG4hinge-UCHT1 scFv and the TIM3 scFv-IgG4hinge-TCRCb-IgG4hinge-PD1 scFv were linked via a P2A sequence. Both the TCRC-based bispecific antibody and the TCRC-based tetraclonal antibody were cloned into the expression plasmid pCDH using the EcoR1 / Not1 cloning sites.

[0309] Example 2 Production of lysate-derived bispecific antibodies / quadruspecific antibodies

[0310] Recombinant antibodies were produced from transiently transfected (lipofectamine-2000-promoted) or stably established HEK293T and Jurkat cell lines (lentiviral vectors). After washing the cells in ice-cold PBS, the cells were resuspended in ice-cold PBS containing 2 mM PMSF at a cell density of 5 x 10 8 / mL. The cell suspension was subjected to three freeze (-80°C) / thaw (37°C) cycles and centrifuged at 14800 RPM for 10 minutes at 4°C using a benchtop centrifuge. The clear supernatant was aspirated, loaded with glycerol to a final concentration of 5%, and stored in 10 μL aliquots at -20°C until use.

[0311] The design of the bispecific antibody / quadruplex antibody constructed in this application is shown in Table 1. The schematic diagram of the bispecific antibody / quadruplex antibody structure described in this application is shown in Figure 1.

[0312] Table 1 Design of secondary and quadruple antibodies developed based on TCR constant regions

[0313] Flow cytometry

[0314] Flow cytometry was performed using a flow cytometer (Beckman Coulter) in plate format, and data were analyzed using FlowJo software. Cells were washed once with FACS buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2 mM EDTA, pH 7.0) and resuspended to 5 × 10 7 cells / mL and placed on ice before staining. For primary antibody staining, 1.5 μL of BsAb and control were dispensed into the wells of a 96-well round-bottom tissue culture plate. Then, 4.5 μL of prepared cells were added to the wells. After incubating the plate at 4°C for 45 minutes, 150 μL of FACS buffer was added to each well for washing the cells. After centrifugation at 300xg for 3 minutes, the supernatant was removed. For secondary antibody staining, the cell pellet was incubated with 20 μL of diluted antibody conjugate at 4°C for 45 minutes. After washing and suspension with FACS buffer, the cells were analyzed by flow cytometry.

[0315] Immunoblotting

[0316] Cell lysates and 4- to 10-fold concentrated supernatants were dissolved in lysis buffer (50 mM Hepes (pH 7.4), 150 mM NaCl, 1% NP-40, and 1 mM EDTA) containing 5% mercaptoethanol and electrophoresed on a 12.5% ​​SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Pall) using a semi-dry transfer system (Bio-Rad). The membrane was blocked with 2% BSA in PBS and probed with horseradish peroxidase-conjugated anti-V5 or anti-HA antibodies (room temperature for 2 hours, antibody dilution 1:500). Specific bands on the membrane were detected using an iBright imaging system (Thermo Scientific) with an enhanced chemiluminescent substrate (Bio-Rad).

[0317] Immunofluorescence

[0318] 5 μL transiently transfected or stably established HEK293T cells (1×10 5 ) were fixed on slides by air drying (room temperature, 30 minutes) and fixed and permeabilized in 100% methanol (4 ° C, 15 minutes). After blocking with 2% BSA in PBS, cells were stained with FITC-coupled antibodies (37 ° C, 45 minutes). The antibodies used included isotype IgG1-FITC control, anti-V5-FITC antibody and anti-HA-FITC antibody. The slides were washed twice and immersed in PBS. The stained cells were visualized and images were captured using a 40X objective lens by fluorescence microscopy (Bio-Rad).

[0319] The expression of the four secondary antibodies in HEK293T cells was determined by immunofluorescence using anti-HA-FITC, as shown in Figure 2A. The expression of the four secondary antibodies in HEK293T cell lysates was determined by immunoblotting using anti-HA-HRP under reducing conditions, as shown in Figure 2B. Flow cytometry was used to determine the binding of the four secondary antibodies to the corresponding ROR1-positive tumors and T cells, using a fluorescently labeled anti-HA-APC antibody, as shown in Figure 2C.

[0320] The results showed that HEK293T cells could stably express and produce four formats of secondary antibodies, and their secondary antibody proteins could bind to corresponding ROR1-positive tumors and T cells.

[0321] Example 3 Redirection test of the dual antibody described in this application

[0322] The efficacy of cell lysate-derived bispecific / quadruplex antibodies in redirecting T cell activation was determined by incubating a mixture of T cells and tumor cells with the lysate-derived bispecific / quadruplex antibodies. Briefly, 100 μL of effector cells (1x10 5cells, untransduced Jurkat T cells, or freshly thawed PBMCs) with 100 μL of target tumor cells (2x10 5 Cells (e.g., Raji, JeKo-1, MEC, MEC-ROR1, A549, HCC827, H1975, or MDA-MB-231) were plated in a round-bottom 96-well tissue culture plate in a final volume of 200 μL per well at a 1:2 effector-to-target ratio. The cell mixture was then loaded with 2 μL of cell lysate-derived BsAb at final dilutions ranging from 1:100 to 1:1600. After centrifugation at 300 x g for 3 minutes, the plate was incubated at 37°C and 5% CO₂ for 16 hours. 150 μL of supernatant was transferred to a new round-bottom 96-well tissue culture plate. The supernatant was used to measure IL-2 concentration using a commercial human IL-2 ELISA kit, and IFN-g concentration was measured using an in-house developed IFN-g ELISA kit, in which 2G1 monoclonal antibody (Cat. No.: M700A, ThermoFisher Scientific) was used as the capture antibody and biotinylated B133.5 antibody (Cat. No. M701B, ThermoFisher Scientific) was used as the developing antibody.

[0323] Lysates containing Trans-TCRCab, Trans-TCRCba, Cis-TCRCab, and Cis-TCRCba secondary antibodies were added to a mixture of PBMC and MEC-ROR1 tumor cells at a 1:2 effector-target ratio. After 16 hours of incubation, supernatants were harvested and assayed for IL-2 and IFN-γ secretion. Lysates from non-transfected HEK293T cells were used as negative controls. MEC (ROR1-) served as negative tumor cell controls. PBMCs from four individuals were tested: numbered 1840, 5583, 1753, and 1142. The results of the redirection assay are shown in Figure 3.

[0324] Four formats of CD19-specific secondary antibodies were designed based on trans and cis structures. Secondary antibody proteins were produced by transiently transfecting HEK293T cells. Lysates containing Trans-TCRCab, Trans-TCRCba, Cis-TCRCab, and Cis-TCRCba secondary antibodies were then added to a mixture of Jurkat cells (Figure 4A) or healthy human 1840 PMBC (Figure 4B) and CD19+ tumor cells (MEC, MEC-ROR1) at an effector-target ratio of 1:2. After 16 hours of incubation, supernatants were harvested and assayed for IL-2 secretion. HL60 (CD19-) tumor cells served as a negative control. ROR1xUCHT-Cis-TCRCab secondary antibody served as a positive control; it produced IL-2 secretion when incubated with MEC-ROR1 cells. Lysates from non-transfected HEK293T cells served as a negative control.

[0325] Example 4 Detection of the effect of the four-antibody structure described in this application binding to the target

[0326] The structure of the ROR1 / TIM3 / PD1 / CD3 tetraclonal antibody is shown in Figure 5. Anti-HA-APC fluorescent antibody and TIM3-His-tag-PE were used to test the binding of the tetraclonal antibody to the corresponding cell lines MEC-ROR1 (expressing ROR1+), Jurkat (expressing CD3+), and HEK-PD1 (expressing PD1+), while MEC served as a negative control cell line. Flow cytometry validation results (Figure 6A) showed that the tetraclonal antibody described in this application binds to cell surface antigens and fusion proteins through four arms, respectively.

[0327] Example 5 Construction, cloning, and expression of target cells that secrete bispecific antibodies in situ

[0328] For protein expression, the expression vector pCDNA3.1-V5 / His B was used. The vector backbone contains a CMV promoter. Primary T cells derived from Jurkat E6 and PBMCs were transduced using the lentiviral vectors pCDH-EF1-IRES-puro and pALD-sffv. The pCDH-EF1-IRES-puro plasmid backbone contains an EF1 promoter and a puromycin selectable marker. The pALD-sffv plasmid backbone contains an sffv promoter and an IRES-linker GFP reporter gene; the GFP gene was removed during cloning.

[0329] All gene constructs were synthesized by GeneWiz. The gene domains of the scFvs are: ROR1 (VL-28aa-linker-VH), CD19 (VL-28aa-linker-VH), UCHT1 (VH-15aa-linker-VL), UCHT1 (VL-28aa-linker-VH), TIM3 (VL-28aa-linker-VH), CD16a (VL-28aa-linker-VH), and PD-1 (VL-28aa-linker-VH). The 15aa-linker is (Gly4Ser)3 (SEQ ID NO: 53). The 28aa-linker is GGGGSGSTSGSGKPGSGEGSTKGGGGGS (SEQ ID NO: 54). The gene domains of the TCR constant region are the mouse TCR α chain constant region (SEQ ID NO: 47) and the mouse TCR β chain constant region (SEQ ID NO: 48).

[0330] There are two formats of TCRC-based BsAb, namely Trans format TCRC-based BsAb and Cis format TCRC-based BsAb.

[0331] In order to produce a trans BsAb encoding construct based on TCRC, the gene domain encoding ROR1scFv-IgG4hinge-TCRC and the gene domain encoding UCHT1scFv-IgG4hinge-TCRC are connected by a P2A sequence. In order to produce a TCRC-based BsAb in a cis format, the gene structure encoding ROR1scFv-IgG4hinge-TCRC and the gene structure encoding UCHT1scFv-IgG4-TCRC (in the reverse amino acid sequence) are connected by a GGGGS (SEQ ID NO: 49) sequence. Each Trans or Cis format has two sub-formats, in which TCR and TCRC are exchanged between the two chains. Four forms of BsAb based on CD19scFvxUCHT1scFv TCRC were similarly prepared. These TCRC-based BsAb encoding constructs were cloned into the pCDNA3.1-V5 / His B vector via the EcoR1 / Not1 cloning sites for protein expression and into the pCDH-EF1-IRES-puro vector for lentiviral production.

[0332] To generate a TCRCab-based Trans-formatted QsAb encoding construct, the first set of gene domains ROR1scFv-IgG4hinge-TCRC-IgG4hinge-UCHT1scFv was constructed, and the second set of gene domains encoding TIM3scFv (or CD16ascFv)-IgG4hinge-TCRC-IgG4hinge-PD1scFv was connected by the P2A sequence.

[0333] To generate 41BBL-specific QsAb encoding constructs, the same first set of gene domains encoding ROR1scFv-IgG4hinge-TCRC-IgG4hinge-UCHT1scFv and the novel second set of gene domains encoding PD1scFv-IgG4hinge-TCRC-IgG4hinge-41BBL were connected by P2A. These TCRC-based QsAb encoding constructs were cloned into the pCDNA3.1-V5 / His B vector via the EcoR1 / Not1 cloning sites for protein expression and into the pCDH-EF1-IRES-puro vector for lentiviral production.

[0334] Production of recombinant antibodies

[0335] Recombinant antibodies were produced from transiently transfected (using lipofectamine-2000) HEK293T cell lines. After washing the cells in ice-cold PBS, the cells were resuspended in ice-cold PBS containing 2 mM PMSF at a cell density of 5 x 10 8 The cell suspension was subjected to three freeze (-80°C) / thaw (37°C) cycles and centrifuged at 14800 RPM for 10 minutes at 4°C using a tabletop centrifuge. The clear supernatant was aspirated, loaded with glycerol at a final concentration of 5, and stored in 10 μL aliquots at -20°C before use.

[0336] T cell transduction

[0337] To transduce Jurkat E6 with LV encoding BsAb, Jurkat E6 (0.5 mL, 0.3 x 10 6 / mL) was added to multiple wells of a 24-well tissue culture plate, and then 0.5mL of BsAb encoding and control lentivirus was added. The cells were stored at 37°C and 5% CO2 for 72 hours. As much culture supernatant as possible was aspirated from each well and stored at -20°C for subsequent Western blot (WB) analysis of secreted BsAb. Then, 2.0mL of fresh IDMEM was immediately added to each well to resuspend the LV-infected Jurkat cells. 0.4mL of cell suspension was collected and washed once with ice-cold PBS. After centrifugation, the cell pellet was stored at -80°C for WB analysis of BsAb. The cell concentration of the remaining 1.6mL cell suspension was counted and adjusted to 1×10 6 / mL and used immediately in T cell redirection experiments.

[0338] To transduce PBMCs with LV encoding BsAb, activated PBMCs that had been preactivated for 72 hours with CD3- and CD28-coupled magnetic beads and IL2 (500 IU / mL) were distributed into multiple wells (1.0 mL, 1.0 x 10 6 / mL) in a 12-well tissue culture plate, and then add 1.0mL of BsAb encoding or control LV and polybrene (8g / mL). After incubation for 16 hours, the culture supernatant was removed and 2.0mL of fresh IDMEM was added to each well to resuspend the LV-infected PBMC cells. After incubation for another 48 hours at 37°C and 5% CO2, the culture supernatant was removed and stored at -20°C for WB analysis of secreted BsAb. The LV-infected PBMCs were then resuspended in IDMEM containing IL2 (500IU / mL), cultured at 37°C and 5% CO2, and used in T cell redirection experiments within 3 days. Aliquots of cells (4x10 6 / mL), washed once with ice-cold PBS, and stored at -80 °C for WB analysis of intracellular BsAb.

[0339] T cell redirection assay and cytokine analysis

[0340] The efficacy of cell lysate-derived BsAbs in redirecting T cell activation was determined by co-incubating lysate-derived BsAbs with a mixture of T cells and tumor cells. Briefly, 100 μL of effector cells (1×10 5 cells, untransduced Jurkat T cells, or freshly thawed PBMCs) with 100 μL of target tumor cells (2x10 5 Cells (e.g., Raji, JeKo-1, MEC, MEC-ROR1, A549, HCC827, H1975, or MDA-MB-231) were plated in a round-bottom 96-well tissue culture plate at a 1:2 effector-to-target ratio in a final volume of 200 μL per well. 2 μL of cell lysate-derived BsAb was then added to the cell mixture, resulting in final dilutions ranging from 1:100 to 1:1600. After centrifugation at 300 x g for 3 minutes, the plate was incubated at 37°C and 5% CO₂ for 16 hours. 150 μL of supernatant was transferred to a new round-bottom 96-well tissue culture plate. The supernatant was used to measure IL-2 concentration using a commercial human IL-2 ELISA kit, and IFN-γ concentration was measured using an in-house developed IFN-γ-ELISA kit, in which 2G1 monoclonal antibody (Cat. No. M700A, ThermoFisher Scientific) was used as the capture antibody and biotinylated 133.5 antibody (Cat. No. M701B, ThermoFisher Scientific) was used as the developing antibody.

[0341] The efficacy of in situ secreted BsAbs in redirecting T cell activation was determined by co-incubating BsAb-secreting T cells with tumor cells. BsAb-secreting T cells were generated by lentiviral transduction as described in the “T cell transduction” section. To set up the T cell redirection assay, 100 μL of lentivirally transduced Jurkat T cells (2x10 5 cells) or transduced PBMCs (2x10 5 cells) and 100 μL of target tumor cells (1x10 5Cells (Raji, JeKo-1, MEC, MEC-ROR1, A549, HCC827, H1975, and MDA-MB-231) were plated in a round-bottom 96-well tissue culture dish at a 2:1 effector-to-target ratio in a final volume of 200 μL per well. After centrifugation at 300 x g for 3 minutes, the plates were incubated at 37°C and 5% CO₂ for 16–60 hours. 150 μL of supernatant was transferred to a new round-bottom 96-well tissue culture plate. IL-2 and IFN secretion by T cells was measured as previously described. Killing of target cells by redirected PBMCs was visualized and images were captured using a fluorescence microscope (Bio-Rad) with a 40X objective, and the frequency of targeted tumor cells was analyzed by flow cytometry.

[0342] Flow cytometry

[0343] Flow cytometry was performed using a flow cytometer (Beckman Coulter) in plate format, and data were analyzed using FlowJo software. Cells were washed once with FACS buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2 mM EDTA, pH 7.0) and resuspended to 5 × 10 7 cells / mL and placed on ice before staining. For primary antibody staining, 1.5 μL of BsAb and control were dispensed into the wells of a 96-well round-bottom tissue culture plate. Then, 4.5 μL of prepared cells were added to the wells. After incubating the plate at 4°C for 45 minutes, 150 μL of FACS buffer was added to each well for washing the cells. After centrifugation at 300xg for 3 minutes, the supernatant was removed. For secondary antibody staining, the cell pellet was incubated with 20 μL of diluted antibody conjugate at 4°C for 45 minutes. After washing and suspension with FACS buffer, the cells were analyzed by flow cytometry.

[0344] Immunoblotting

[0345] Cell lysates and 4- to 10-fold concentrated supernatants were dissolved in lysis buffer (50 mM Hepes (pH 7.4), 150 mM NaCl, 1% NP-40, and 1 mM EDTA) containing 5% mercaptoethanol and electrophoresed on a 12.5% ​​SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Pall) using a semi-dry transfer system (Bio-Rad). The membrane was blocked with 2% BSA in PBS and probed with horseradish peroxidase-conjugated anti-V5 or anti-HA antibodies (room temperature for 2 hours, antibody dilution 1:500). Specific bands on the membrane were detected using an iBright imaging system (Thermo Scientific) with an enhanced chemiluminescent substrate (Bio-Rad).

[0346] Immunofluorescence

[0347] 5 μL transiently transfected or stably established HEK293T cells (1×10 5 ) were fixed on slides by air drying (room temperature, 30 minutes) and fixed and permeabilized in 100% methanol (4 ° C, 15 minutes). After blocking with 2% BSA in PBS, cells were stained with FITC-coupled antibodies (37 ° C, 45 minutes). The antibodies used included isotype IgG1-FITC control, anti-V5-FITC antibody and anti-HA-FITC antibody. The slides were washed twice and immersed in PBS. The stained cells were visualized and images were captured using a 40X objective lens by fluorescence microscopy (Bio-Rad).

[0348] Enzyme-linked immunosorbent assay

[0349] According to the manufacturer's instructions, ELISA Max (Biolegend; 4030104) is used to measure the IL-2 concentration in the culture supernatant. The ELISA developed internally is used to determine the IFN-γ concentration in the culture supernatant. For the latter, in brief, 50L of culture supernatant or different concentrations of IFN-γ standard are mixed with 50L of biotin 133.5 monoclonal antibody (Art. No. M701B, ThermoFisher Scientific) (as development antibody) and added to the ELISA plate coated with 2G1 monoclonal antibody (Art. No.: M700A, ThermoFisher Scientific) (as capture antibody). After incubation with peroxidase, TMB substrate and stop solution conjugated with streptavidin, the absorbance at 450nm is measured using a microplate reader. The concentration of IFN-γ in the culture supernatant is calculated according to the standard curve of known standards.

[0350] Example 6 Target cell killing effect of T cells secreting bispecific antibodies in situ

[0351] The Incucyte cell imaging scanner was used to observe the cytotoxicity of primary T cells against GFP-labeled target cells. Healthy human PBMCs, transduced with a dual-antibody (cis-antibody) virus, were co-cultured with a panel of different target cells for 40 hours, and target cell cytotoxicity was monitored by fluorescence changes (E:T ratio = 2:1).

[0352] Lentiviral transfer vector production and viral packaging

[0353] To transduce primary T cells from PBMCs, the lentiviral vectors pCDH-EF1α-IRES-puro and pALD-sffv were used. The pCDH-EF1α-IRES-puro plasmid backbone contains an EF1α promoter and a puromycin selection marker. The pALD-sffv plasmid backbone contains an sffv promoter and an IRES-linker GFP reporter gene; the GFP gene was removed during cloning. All gene domains were synthesized using GeneWiz. The gene domains for the scFvs are: ROR1 (VL-28aa-linker-VH) and UCHT1 (VL-28aa-linker-VH). The 15aa linker is (Gly4Ser)3 (SEQ ID NO:53), and the 28aa linker is GGGGSGSTSGSGKPGSGEGSTKGGGGGS (SEQ ID NO:54). To generate a cis-form TCRC-based BsAb, the gene domain encoding ROR1scFv-IgG4hinge-TCRα and the gene domain encoding UCHT1scFv-IgG4hinge-TCRβ (in reverse amino acid sequence) were connected by a GGGGS sequence. Both the cis and trans structures have two substructures, in which TCRα and TCRβ swap positions between the two chains (Figure 1A). These TCRC-based BsAb encoding constructs were cloned into the pCDH-EF1α-IRES-puro vector via the EcoRI / NotI cloning sites for lentiviral production.

[0354] Primary T cell transduction

[0355] To transduce PBMCs with lentivirus encoding BsAb, activated PBMCs that had been preactivated for 72 hours with CD3- and CD28-coupled magnetic beads and IL2 (500 IU / mL) were distributed into multiple wells (1.0 mL, 1×10 6 / mL) in a 24-well tissue culture plate, and then add 1.0mL of BsAb encoding or control lentivirus and polybrene (8μg / mL). After incubation for 16 hours, the culture supernatant was removed and 2.0mL of new complete X-Vivo15 was added to each well to resuspend the lentivirus-infected PBMC cells. After incubation for another 48 hours at 37°C and 5% CO2, the culture supernatant was removed and stored at -20°C for flow cytometric analysis of secreted BsAb. The lentivirus-infected PBMCs were then resuspended in complete X-Vivo15 containing IL2 (500IU / mL), cultured at 37°C and 5% CO2, and used in situ secreting T cell tumor killing experiments within 3 days.

[0356] Flow cytometry detection of BsAb secretion

[0357] Flow cytometry was performed using a cell culture plate using a flow cytometer (Beckman Coulter), and the data were analyzed using FlowJo software. MEC-ROR1 cells were washed once with FACS buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2mM EDTA, pH 7.0) and resuspended to 5×10 7 cells / mL and placed on ice for subsequent staining. For primary antibody staining, 5 μL of culture supernatant of primary T cells (TCRαβ 8ab.1 and TCRβα9.7) transduced with BsAb lentivirus and controls were added to different wells of a 96-well round-bottom tissue culture plate. Then, 100 uL of prepared cells were added to the wells. After incubating the plate at 4°C for 25 minutes, 150 μL of FACS buffer was added to each well to wash the cells. After centrifugation at 300 × g for 5 minutes, the supernatant was removed. For secondary antibody staining, the cell pellet was incubated with 100 μL of diluted (1:100) fluorescently labeled secondary antibody anti-HA APC (BioLegend, Cat. 901524) at 4°C for 25 minutes. After washing and resuspension with FACS buffer, the binding of secreted BsAb to cells in the supernatant was analyzed by flow cytometry (Figure 7A).

[0358] In situ secreted BsAb-mediated tumor killing

[0359] The efficacy of in situ secreted BsAbs in re-directing T cell activation was determined by co-incubating BsAb-secreting T cells with tumor cells. As described in the “Primary T Cell Transduction” section, primary T cells secreting BsAbs were constructed by lentiviral transduction. To set up the T cell killing experiment, 100 μL of lentivirally transduced primary T cells (1.5×10 5 cells) and 100 μL target tumor cells (7.5×10 4 MEC, MEC-ROR1, and MB231 cells were cultured in flat-bottom 96-well tissue culture plates (Corning 3596) at a 2:1 effector to target ratio in a final volume of 200 μL per well. After centrifugation at 300 × g for 3 minutes, the plates were placed in an Incucyte cell imaging scanner and scanned in the GFP channel every 2 hours. After incubation for 40–60 hours at 37°C and 5% CO₂, fluorescently labeled target cells were analyzed using the Incucyte's built-in analysis tool to assess the tumor cell-killing efficacy of in situ BsAb-secreting T cells (Figure 7B). 50 μL of the supernatant was transferred to a new round-bottom 96-well tissue culture plate for measurement of T cell secretion of IFN-γ (Figure 7B, lower right).

[0360] Example 7: Killing effect of T cells in situ secreting dual antibodies replacing CD19 as a target on target cells

[0361] As shown in Figure 8, there are two formats for in situ secretion CD19-UCHT1-BsAb based on TCRC, namely Trans format TCRC-based BsAb and Cis format TCRC-based BsAb. In order to produce a trans BsAb encoding construct based on TCRC, the gene domain encoding CD19scFv-IgG4hinge-TCRC and the gene domain encoding UCHT1scFv-IgG4hinge-TCRC are connected by a P2A sequence. In order to produce a TCRC-based BsAb in a cis format, the gene structure encoding CD19scFv-IgG4hinge-TCRC and the gene structure encoding UCHT1scFv-IgG4-TCRC (in the reverse amino acid sequence) are connected by a GGGGS (SEQ ID NO: 49) sequence. Each Trans or Cis format has two sub-formats, in which TCR and TCRC are exchanged between the two chains. Four forms of BsAb based on CD19scFvxUCHT1scFv TCRC were similarly prepared. These TCRC-based BsAb encoding constructs were cloned into the pCDNA3.1-V5 / His B vector via the Ecor1 / Bstb1 cloning sites for protein expression and into the pCDH-EF1-IRES-puro vector for lentiviral production.

[0362] CD19-UCHT1-TCRC BsAbs effectively bind to T cells and CD19+ target cells

[0363] Flow cytometry was used to test the binding of in situ secreted CD19-UCHT1-TCRC BsAbs to the corresponding cell lines MEC-1 (expressing CD19+) and PBMC (expressing CD3+). Activated PBMCs that had been pre-activated for 24 hours with CD3- and CD28-coupled magnetic beads and IL-2 (500 IU / mL) were distributed into multiple wells (1.0 mL, 1.0×10 612-well tissue culture plates were plated with 1.0 mL of lentivirus encoding CD19-UCHT1 TCRC BsAbs or a control were then added. After a 24-hour incubation, the culture supernatant was removed and 2.0 mL of fresh X-VIVO medium was added to each well to resuspend the lentiviral-transduced PBMCs. After incubation for another 24 hours at 37°C and 5% CO2, the culture supernatant was removed and stored at -20°C. 100 μl of the supernatant was incubated with MEC-1 (1×105 cells) or freshly thawed PBMC (1×105 cells) at room temperature for 30 minutes, followed by centrifugation at 1300 rpm for five minutes. The cells were resuspended in 100 μl of FASC buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2 mM EDTA, pH 7.0) and stained with anti-HA-APC antibody as described above. Flow cytometry was then performed using a flow cytometer (Beckman Coulter) in plate mode, and the data were analyzed using FlowJo software.

[0364] The flow cytometry verification results ( FIG9A ) showed that the in situ secretory bispecific antibody described in the present application effectively binds to target tumor cells and T cells through its two arms, respectively.

[0365] CD19-UCHT1 TCRC BsAbs can effectively enhance T cell activation

[0366] The efficacy of in situ secreted BsAbs in T cell activation was determined by co-culturing T cells secreting CD19-UCHT1 TCRC BsAbs with tumor cells. Lentivirally transduced PBMCs (2x104) were co-cultured with negative expressing cells (K562 (expressing CD19-), 1x104) or with target tumor cells (Raji (expressing CD19+) / MEC-1 (expressing CD19+), 1x10 4 ) were co-cultured in a round-bottom 96-well tissue culture plate at a 2:1 ratio of effector cells to target cells. IMDM medium was added to a final volume of 200 μL per well. After centrifugation at 300 × g for 5 minutes, the plate was incubated at 37°C and 5% CO₂ for 24 hours. 100 μL of supernatant was transferred to a new round-bottom 96-well tissue culture plate. Untransduced lentivirus-transduced PBMCs co-cultured with target tumor cells served as a negative control.

[0367] The IFN-γ concentration in the culture supernatant was determined using an ELISA kit developed in-house. In short, 50 L of culture supernatant or different concentrations of IFN-γ standard solution were mixed with 50 L of biotin 133.5 monoclonal antibody (Art. No. M701B, ThermoFisher Scientific) (as a development antibody) and added to an ELISA plate coated with 2G1 monoclonal antibody (Art. No.: M700A, ThermoFisher Scientific) (as a capture antibody). After incubation with peroxidase, TMB substrate and stop solution conjugated with streptavidin, the absorbance at 450 nm was measured using a microplate reader. The concentration of IFN-γ in the culture supernatant was calculated according to a standard curve of known standards.

[0368] As shown in Figure 9B , compared with the negative cell group (PBMC+K562), the concentration of IFN-γ secreted by PBMC in the cell culture medium increased after lentiviral-transduced PBMCs were co-cultured with target tumor cells (PBMC+MEC-1 / PBMC+Raji), indicating that in situ secretion of CD19-UCHT1 TCRC BsAbs can effectively enhance T cell activation.

[0369] T cells secreting CD19-UCHT1 TCRC BsAbs in situ can effectively kill CD19+ target tumor cells

[0370] The killing efficacy of T cells secreting CD19-UCHT1 TCRC BsAbs on target tumor cells was determined by co-culturing BsAbs-secreting T cells with tumor cells. PBMCs (2x10 4 ) and negative control cells (K562 (expressing CD19-), 1×104), or target tumor cells (Raji (expressing CD19+) / MEC-1 (expressing CD19+), 1×10 4 ) were co-cultured in a round-bottom 96-well tissue culture plate with a ratio of effector cells to target cells of 2:1. IMDM culture medium was added to a final volume of 200 μL per well. In the SX1 Live-Cell Analysis System, the cells were incubated at 37°C and 5% CO2 for 48 hours, and the killing effect of lentiviral-transfected PBMCs on target tumor cells was analyzed using Incucyte 2022A Rev1 software. As shown in Figure 9C, in the negative cell group (PBMC+K562-GFP cells), the number of tumor cells in the transduced PBMC group did not change significantly compared with the untransduced group, and the PBMCs failed to produce a significant cytotoxic effect. However, after the transduced PBMCs were co-cultured with target tumor cells (MEC-1-GFP / Raji-GFP cells), the number of tumor cells decreased significantly compared with the untransduced group, resulting in a significant cytotoxic effect.

Claims

1. A cell comprising a vector comprising a nucleic acid molecule encoding an antigen binding protein comprising a scFv targeting a first antigen and a constant region of a TCR alpha chain, and a scFv targeting a second antigen and a constant region of a TCR beta chain.

2. The cell according to claim 1, wherein the vector is a viral vector.

3. The cell according to any one of claims 1-2, wherein the vector is selected from the group consisting of: a lentiviral, adenoviral, retroviral and adeno-associated viral vectors.

4. The cell according to any one of claims 1-3, wherein the vector is a non-viral vector.

5. The cell according to any one of claims 1-4, wherein the vector is selected from the group consisting of: a plasmid, a minicircle DNA vector, a SB plasmid, and a piggybac plasmid. The cell according to any one of claims 1 to 5, which is an immune cell.

7. The cell according to any one of claims 1 to 6, which is a T cell or a NK cell.

8. The cell according to any one of claims 1-7, wherein the antigen binding protein does not comprise the variable region of the TCR alpha chain.

9. The cell according to any one of claims 1-8, wherein the antigen binding protein does not comprise the variable region of the TCR β chain.

10. The cell according to any one of claims 1 to 9, wherein the scFv targeting the first antigen and the constant region of the TCR α chain in the antigen binding protein are connected by a hinge region.

11. The cell according to any one of claims 1 to 10, wherein the scFv targeting a second antigen and the constant region of the TCR β chain in the antigen binding protein are connected by a hinge region.

12. The cell of any one of claims 10-11, wherein the hinge region comprises an IgG hinge region.

13. The cell of any one of claims 10-12, wherein the hinge region comprises an IgG4 hinge region. 14 . The cell according to claim 1 , wherein the constant region of the TCR α chain and the constant region of the TCR β chain in the antigen binding protein are linked by a disulfide bond. 15 . The cell according to any one of claims 1 to 13 , wherein the constant region of the TCR α chain and the constant region of the TCR β chain in the antigen binding protein are connected by a linker.

16. The cell of claim 15, wherein the linker comprises a peptide linker.

17. The cell according to any one of claims 15-16, wherein the amino acid sequence of the linker is SEQ ID NO: 49 (GGGGS).

18. The cell according to any one of claims 1-17, wherein the first antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19 and PD-1.

19. The cell according to any one of claims 1-18, wherein the second antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19 and PD-1.

20. The cell of any one of claims 1-19, wherein the first antigen and the second antigen are different antigen-binding epitopes of the same antigen.

21. The cell of any one of claims 1-19, wherein the first antigen and the second antigen are different.

22. The cell according to any one of claims 1-21, wherein the scFv targeting the first antigen in the antigen binding protein comprises an antibody light chain variable region VL targeting the first antigen and an antibody heavy chain variable region VH targeting the first antigen.

23. The cell according to claim 22, wherein the N-terminus of the VH targeting the first antigen and the C-terminus of the VL targeting the first antigen in the antigen binding protein are linked.

24. The cell according to claim 22, wherein the C-terminus of the VH targeting the first antigen and the N-terminus of the VL targeting the first antigen in the antigen binding protein are linked.

25. The cell according to any one of claims 22-23, wherein the C-terminus of the VH targeting the first antigen in the antigen binding protein is linked to the N-terminus of the constant region of the TCR alpha chain.

26. The cell according to any one of claims 22 and 24, wherein the C-terminus of the VL targeting the first antigen in the antigen binding protein is linked to the N-terminus of the constant region of the TCR alpha chain.

27. The cell according to any one of claims 22 and 24, wherein the N-terminus of the VH targeting the first antigen in the antigen binding protein is linked to the C-terminus of the constant region of the TCR alpha chain.

28. The cell according to any one of claims 22-23, wherein the N-terminus of the VL targeting the first antigen in the antigen binding protein is linked to the C-terminus of the constant region of the TCR alpha chain.

29. The cell according to any one of claims 1-28, wherein the scFv targeting a second antigen in the antigen binding protein comprises an antibody light chain variable region VL targeting a second antigen and an antibody heavy chain variable region VH targeting a second antigen.

30. The cell according to claim 29, wherein the N-terminus of the VH targeting the second antigen and the C-terminus of the VL targeting the second antigen in the antigen binding protein are linked.

31. The cell of claim 29, wherein the C-terminus of the VH targeting the second antigen and the N-terminus of the VL targeting the second antigen in the antigen binding protein are linked.

32. The cell according to any one of claims 29-30, wherein the C-terminus of the VH targeting the second antigen in the antigen binding protein is connected to the N-terminus of the constant region of the TCR β chain.

33. The cell of claim 29 or claim 31, wherein the C-terminus of the VL targeting a second antigen in the antigen binding protein is linked to the N-terminus of the constant region of the TCR β chain.

34. The cell of claim 29 or claim 31, wherein the N-terminus of the VH targeting a second antigen in the antigen binding protein is linked to the C-terminus of the constant region of the TCR β chain.

35. The cell according to any one of claims 29-30, wherein the N-terminus of the VL targeting the second antigen in the antigen binding protein is linked to the C-terminus of the constant region of the TCR β chain.

36. The cell of any one of claims 1-35, wherein the antigen binding protein further comprises a binding domain that targets a third antigen.

37. The cell of claim 36, wherein the binding domain targeting a third antigen is a scFv.

38. The cell of any one of claims 1-37, wherein the antigen binding protein further comprises a binding domain that targets a fourth antigen.

39. The cell of claim 38, wherein the binding domain targeting a fourth antigen is a scFv.

40. The cell according to any one of claims 36-39, wherein the scFv targeting a third antigen in the antigen binding protein is directly or indirectly linked to the constant region of the TCR alpha chain.

41. The cell according to any one of claims 36-40, wherein the scFv targeting the third antigen in the antigen binding protein is connected to the constant region of the TCR α chain through an IgG4 hinge region.

42. The cell according to any one of claims 38-41, wherein the scFv targeting the fourth antigen in the antigen binding protein is directly or indirectly linked to the constant region of the TCR β chain.

43. The cell according to any one of claims 38-42, wherein the scFv targeting the fourth antigen in the antigen binding protein is connected to the constant region of the TCR β chain through an IgG4 hinge region.

44. The cell according to any one of claims 37-43, wherein the scFv targeting a third antigen in the antigen binding protein comprises a VH targeting a third antigen and a VL targeting a third antigen.

45. The cell according to claim 44, wherein the N-terminus of the VH targeting the third antigen and the C-terminus of the VL targeting the third antigen in the antigen binding protein are directly or indirectly linked.

46. ​​The cell according to claim 44, wherein the C-terminus of the VH targeting the third antigen and the N-terminus of the VL targeting the third antigen in the antigen binding protein are directly or indirectly linked.

47. The cell according to any one of claims 44 and claim 46, wherein the N-terminus of the VH targeting the third antigen in the antigen binding protein is directly or indirectly connected to the C-terminus of the constant region of the TCR alpha chain.

48. The cell according to any one of claims 44-45, wherein the C-terminus of the VH targeting the third antigen in the antigen binding protein is directly or indirectly connected to the N-terminus of the constant region of the TCR alpha chain.

49. The cell according to any one of claims 44-45, wherein the N-terminus of the VL targeting the third antigen in the antigen binding protein is directly or indirectly connected to the C-terminus of the constant region of the TCR alpha chain.

50. The cell according to any one of claims 44 and 46, wherein the C-terminus of the VL targeting the third antigen in the antigen binding protein is directly or indirectly linked to the N-terminus of the constant region of the TCR alpha chain.

51. The cell according to any one of claims 39-50, wherein the scFv targeting the fourth antigen in the antigen binding protein comprises a VH targeting the fourth antigen and a VL targeting the fourth antigen.

52. The cell according to claim 51, wherein the N-terminus of the VH targeting the fourth antigen and the C-terminus of the VL targeting the fourth antigen in the antigen binding protein are directly or indirectly connected.

53. The cell according to claim 51, wherein the C-terminus of the VH targeting the fourth antigen and the N-terminus of the VL targeting the fourth antigen in the antigen binding protein are directly or indirectly connected.

54. The cell according to any one of claims 51 and claim 53, wherein the N-terminus of the VH targeting the fourth antigen in the antigen binding protein is directly or indirectly connected to the C-terminus of the constant region of the TCRβ chain.

55. The cell according to any one of claims 51-52, wherein the C-terminus of the VH targeting the fourth antigen in the antigen binding protein is directly or indirectly connected to the N-terminus of the constant region of the TCRβ chain.

56. The cell according to any one of claims 51-52, wherein the N-terminus of the VL targeting the fourth antigen in the antigen binding protein is directly or indirectly connected to the C-terminus of the constant region of the TCRβ chain.

57. The cell according to any one of claims 51 and claim 53, wherein the C-terminus of the VL targeting the fourth antigen in the antigen binding protein is directly or indirectly connected to the N-terminus of the constant region of the TCR β chain.

58. The cell of any one of claims 1-57, wherein the antigen binding protein comprises two polypeptide chains, wherein the first polypeptide chain comprises a scFv targeting a first antigen and a constant region of a TCR alpha chain; wherein the second polypeptide chain comprises a scFv targeting a second antigen and a constant region of a TCR beta chain.

59. The cell of claim 58, wherein the first polypeptide chain and the second polypeptide chain in the antigen binding protein are linked by a disulfide bond.

60. The cell according to any one of claims 58-59, wherein in the first polypeptide chain of the antigen binding protein, the scFv targeting the first antigen is directly or indirectly linked to the constant region of the TCR alpha chain.

61. The cell according to claim 60, wherein the scFv targeting the first antigen and the constant region of the TCR alpha chain in the antigen binding protein are connected by a hinge region.

62. The cell according to any one of claims 60-61, wherein the scFv targeting the first antigen and the constant region of the TCR alpha chain in the antigen binding protein are connected by an IgG hinge region.

63. The cell according to any one of claims 60-62, wherein the scFv targeting the first antigen in the antigen binding protein and the constant region of the TCR α chain are connected by the hinge region of IgG4.

64. The cell of any one of claims 58-63, wherein the scFv targeting a first antigen in the antigen binding protein comprises a VH targeting a first antigen and a VL targeting a first antigen.

65. The cell according to any one of claims 58-64, wherein the first polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting a first antigen, a VH targeting a first antigen, and a constant region of a TCR α chain.

66. According to the cell of any one of claims 58-65, the first polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting a first antigen, a linker, a VH targeting a first antigen, a hinge region of IgG4, and a constant region of a TCRα chain.

67. The cell of any one of claims 1-66, wherein the antigen binding protein further comprises a binding domain that targets a third antigen.

68. The cell of claim 67, wherein the binding domain targeting a third antigen comprises a scFv.

69. The cell according to any one of claims 67-68, wherein the third antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19 and PD-1.

70. The cell of any one of claims 68-69, wherein the scFv targeting a third antigen comprises a VH targeting a third antigen and a VL targeting a third antigen.

71. According to the cell described in any one of claims 58-70, the first polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, a VL targeting a first antigen, a VH targeting a first antigen, a hinge region of IgG4, a constant region of a TCRα chain, a hinge region of IgG4, a VL targeting a third antigen, and a VH targeting a third antigen.

72. According to the cell described in any one of claims 58-71, the first polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting a first antigen, a VH targeting a first antigen, a hinge region of IgG4, a constant region of a TCRα chain, a hinge region of IgG4, a VH targeting a third antigen, and a VL targeting a third antigen.

73. The cell according to any one of claims 58-70, wherein the first polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VH targeting a first antigen, a VL targeting a first antigen, and a constant region of a TCR α chain.

74. According to the cell of any one of claims 58-70, the first polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VH targeting a first antigen, a linker, a VL targeting a first antigen, a hinge region of IgG4, and a constant region of a TCRα chain.

75. According to the cell of any one of claims 58-70, the first polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, a VH targeting a first antigen, a linker, a VL targeting the first antigen, a hinge region of IgG4, a constant region of a TCRα chain, a hinge region of IgG4, a VL targeting a third antigen, a linker, and a VH targeting a third antigen.

76. According to the cell of any one of claims 58-70, the first polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VH targeting a first antigen, a linker, a VL targeting the first antigen, a hinge region of IgG4, a constant region of a TCRα chain, a hinge region of IgG4, a VH targeting a third antigen, a linker, and a VL targeting a third antigen.

77. The cell of any one of claims 58-76, wherein the first polypeptide chain of the antigen binding protein comprises a scFv targeting a first antigen, a constant region of a TCR alpha chain, and a scFv targeting a third antigen.

78. The cell according to any one of claims 58-77, wherein in the second polypeptide chain of the antigen binding protein, the scFv targeting the second antigen is directly or indirectly linked to the constant region of the TCR β chain.

79. The cell of any one of claims 58-78, wherein the scFv targeting a second antigen and the constant region of the TCR β chain of the antigen binding protein are connected by a hinge region.

80. The cell of any one of claims 58-79, wherein the scFv targeting a second antigen and the constant region of the TCR β chain of the antigen binding protein are connected by an IgG hinge region.

81. The cell according to any one of claims 58-80, wherein the scFv targeting a second antigen and the constant region of the TCR β chain of the antigen binding protein are connected by a hinge region of IgG4.

82. The cell of any one of claims 58-81, wherein the scFv targeting a second antigen of the antigen binding protein comprises a VH targeting a second antigen and a VL targeting a second antigen.

83. According to the cell of any one of claims 58-82, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus: a VL targeting a second antigen, a VH targeting a second antigen, and a constant region of a TCRβ chain.

84. According to the cell described in any one of claims 58-82, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting a second antigen, a linker, a VH targeting a second antigen, a hinge region of IgG4, and a constant region of a TCRβ chain.

85. The cell of any one of claims 1-82, wherein the antigen binding protein further comprises a binding domain that targets a fourth antigen.

86. The cell of claim 85, wherein the binding domain targeting a fourth antigen comprises a scFv.

87. The cell of any one of claims 85-86, wherein the fourth antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19 and PD-1.

88. The cell of any one of claims 86-87, wherein the scFv targeting a fourth antigen comprises a VH targeting a fourth antigen and a VL targeting a fourth antigen.

89. According to the cell described in any one of claims 58-89, the second polypeptide chain of the antigen-binding protein comprises, from N-terminus to C-terminus, a VL targeting a second antigen, a linker, a VH targeting a second antigen, a hinge region of IgG4, a constant region of a TCRβ chain, a hinge region of IgG4, a VH targeting a fourth antigen, a linker, and a VL targeting a fourth antigen.

90. According to the cell described in any one of claims 58-89, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting a second antigen, a linker, a VH targeting a second antigen, a hinge region of IgG4, a constant region of a TCRβ chain, a hinge region of IgG4, a VL targeting a fourth antigen, a linker, and a VH targeting a fourth antigen.

91. According to the cell of any one of claims 58-89, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VH targeting a second antigen, a VL targeting a second antigen, and a constant region of a TCRβ chain.

92. According to the cell described in any one of claims 58-89, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VH targeting a second antigen, a linker, a VL targeting a second antigen, a hinge region of IgG4, and a constant region of a TCRβ chain.

93. According to the cell described in any one of claims 58-89, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VH targeting a second antigen, a VL targeting a second antigen, a hinge region of IgG4, a constant region of a TCRβ chain, a hinge region of IgG4, a VH targeting a fourth antigen, and a VL targeting a fourth antigen.

94. According to the cell described in any one of claims 58-89, the second polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VH targeting a second antigen, a VL targeting a second antigen, a hinge region of IgG4, and a constant region of a TCRβ chain, a hinge region of IgG4, a VL targeting a fourth antigen, and a VH targeting a fourth antigen.

95. The cell of any one of claims 1-94, wherein the first antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19, and PD-1.

96. The cell of any one of claims 1-95, wherein the second antigen is selected from the group consisting of ROR1, CD3, TIM3, CD19, and PD-1.

97. The cell of any one of claims 1-96, wherein the first antigen is different from the second antigen.

98. The cell of any one of claims 1-96, wherein the first antigen and the second antigen are different antigen binding epitopes of the same antigen.

99. The cell of any one of claims 1-98, wherein the antigen binding protein comprises one polypeptide chain comprising a scFv targeting a first antigen and a constant region of a TCR alpha chain; and a scFv targeting a second antigen and a constant region of a TCR beta chain.

100. According to the cell according to claim 99, the polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a scFv targeting a first antigen, a constant region of a TCRα chain, a linker, a constant region of a TCRβ chain, and a scFv targeting a second antigen.

101. A cell according to any one of claims 99-100, wherein the polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a scFv targeting a first antigen, a hinge region, a constant region of a TCRβ chain, a linker, a constant region of a TCRα chain, a hinge region, and a scFv targeting a second antigen.

102. The cell of any one of claims 99-101, wherein the scFv targeting a first antigen comprises a VH targeting a first antigen and a VL targeting a first antigen.

103. The cell of any one of claims 99-102, wherein the scFv targeting a second antigen comprises a VH targeting a second antigen and a VL targeting a second antigen.

104. A cell according to any one of claims 99-103, wherein the polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting a first antigen, a VH targeting a first antigen, a hinge region, a constant region of a TCR α chain, a linker, a constant region of a TCR β chain, a hinge region, a VH targeting a second antigen, and a VL targeting a second antigen.

105. A cell according to any one of claims 99-104, wherein the polypeptide chain of the antigen binding protein comprises, from N-terminus to C-terminus, a VL targeting a first antigen, a linker, a VH targeting a first antigen, a hinge region, a constant region of a TCRβ chain, a linker, a constant region of a TCRα chain, a hinge region, a VH targeting a second antigen, a linker, and a VL targeting a second antigen.

106. The cell according to any one of claims 1-105, wherein the constant region of the TCR alpha chain is derived from the constant region of a human TCR alpha chain.

107. The cell according to any one of claims 1-106, wherein the constant region of the TCR α chain comprises the amino acid sequence shown in SEQ ID NO:

47.

108. The cell according to any one of claims 1-107, wherein the constant region of the TCR β chain is derived from the constant region of the human TCR β chain.

109. The cell according to any one of claims 1-108, wherein the constant region of the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO:

48.

110. The cell according to any one of claims 1-109, wherein the scFv targeting the first antigen of the antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:36 and SEQ ID NO:

45.

111. The cell of any one of claims 1-110, wherein the scFv targeting a second antigen of the antigen binding protein comprises an amino acid sequence as shown in any one of SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:36 and SEQ ID NO:

45.

112. The cell of any one of claims 1-111, further capable of expressing a chemokine receptor.

113. The cell of claim 112, wherein the chemokine receptor is selected from the group consisting of: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7.

114. A cell according to any one of claims 1-113, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a scFv targeting a first antigen, a nucleic acid sequence encoding a scFv targeting a second antigen, a nucleic acid sequence encoding a constant region of a TCR α chain, and a nucleic acid sequence encoding a constant region sequence of a TCR β chain / encoding a reverse amino acid sequence of the constant region of a TCR β chain.

115. The cell of any one of claims 1-114, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding an IgG4 hinge region.

116. The cell of any one of claims 1-115, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding a cleavage peptide.

117. The cell of claim 116, wherein the cleavage peptide comprises a 2A peptide.

118. The cell of any one of claims 1-117, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding a linker.

119. The cell of claim 118, wherein the amino acid sequence of the linker is SEQ ID NO: 49 (GGGGS).

120. A cell according to any one of claims 1-119, wherein the nucleic acid molecule comprises, from the 5' end to the 3' end, in sequence: a nucleic acid sequence encoding a scFv targeting a first antigen, a nucleic acid sequence encoding an IgG4 hinge region, a nucleic acid sequence encoding a constant region of a TCR α chain, a nucleic acid sequence encoding a cleavage peptide, a nucleic acid sequence encoding a scFv targeting a second antigen, a nucleic acid sequence encoding an IgG4 hinge region, and a constant region sequence encoding a TCR β chain.

121. The cell according to any one of claims 1-120, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding a scFv targeting a third antigen / a nucleic acid sequence encoding a reverse amino acid sequence of a scFv targeting a third antigen.

122. The cell according to any one of claims 1-121, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding a scFv targeting a fourth antigen / a nucleic acid sequence encoding a reverse amino acid sequence of a scFv targeting a fourth antigen.

123. A cell according to any one of claims 1-122, wherein the nucleic acid molecule comprises, from the 5' end to the 3' end, in sequence: a nucleic acid sequence encoding a scFv targeting a first antigen, a nucleic acid sequence encoding an IgG4 hinge region, a nucleic acid sequence encoding a constant region of a TCR α chain, a nucleic acid sequence encoding an IgG4 hinge region, a nucleic acid sequence encoding a reverse amino acid sequence of a scFv targeting a third antigen, a nucleic acid sequence encoding a cleavage peptide, a nucleic acid sequence encoding a scFv targeting a second antigen, a nucleic acid sequence encoding an IgG4 hinge region, a constant region sequence encoding a TCR β chain, a nucleic acid sequence encoding an IgG4 hinge region, and a nucleic acid sequence encoding a reverse amino acid sequence of a scFv targeting a fourth antigen.

124. A cell according to any one of claims 1-123, wherein the nucleic acid molecule comprises, in order from the 5' end to the 3' end: a nucleic acid sequence encoding a scFv targeting a first antigen, a nucleic acid sequence encoding an IgG4 hinge region, a nucleic acid sequence encoding a constant region of a TCR α chain, a nucleic acid sequence encoding a linker, a nucleic acid sequence encoding a reverse amino acid sequence of the constant region of a TCR β chain, a nucleic acid sequence encoding an IgG4 hinge region, and a nucleic acid sequence encoding a scFv targeting a second antigen.

125. The cell of any one of claims 1-124, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding a chemokine receptor.

126. The cell of claim 125, wherein the chemokine receptor is selected from the group consisting of: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7.

127. The cell of any one of claims 125-126, wherein the chemokine receptor is CXCR3.

128. The cell according to any one of claims 125-127, wherein the nucleic acid sequence encoding the chemokine receptor is directly or indirectly linked to the nucleic acid sequence encoding the antigen binding protein.

129. The cell according to any one of claims 125-128, wherein a sequence encoding a cleavage peptide is further included between the nucleic acid sequence encoding the chemokine receptor and the nucleic acid sequence encoding the antigen binding protein.

130. The cell of claim 129, wherein the cleavage peptide is a 2A peptide.

131. The cell of any one of claims 129-130, wherein the cleavage peptide is selected from: T2A and P2A.

132. The cell of any one of claims 1-132, which is capable of secreting the antigen binding protein in situ.

133. The cell of any one of claims 1-132, further capable of expressing the chemokine receptor.

134. A pharmaceutical composition comprising the cell of any one of claims 1-133, and optionally a pharmaceutically acceptable carrier.

135. Use of the cell of any one of claims 1-133, or the pharmaceutical composition of claim 134, in the preparation of a medicament for preventing and / or treating a disease and / or condition.

136. The use according to claim 135, wherein the disease and / or disorder comprises a tumor.

137. The use according to claim 136, wherein the tumor comprises a solid tumor and / or a hematological tumor.

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