Membrane-expressed il-10 and use thereof

Abstract

Provided is a fusion protein, comprising IL-10 and a transmembrane domain, wherein the fusion protein is expressed on an immune cell, and the immune cell does not comprise a regulatory T cell. Also provided is a modified immune cell, comprising a fusion protein and an antigen recognition receptor, wherein the fusion protein comprises IL-10 and a transmembrane domain, and the immune cell does not comprise a regulatory T cell. Also provided is a use of the fusion protein and the modified immune cell in prevention and / or treatment of diseases.

Description

Membrane-expressed IL-10 and its uses Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a fusion protein comprising IL-10 and a transmembrane domain and its application. Background Art

[0002] Diseases such as tumors can be controlled, developed, or eliminated by the immune system. Cytokines, as powerful regulators of the immune response, have the potential to significantly influence the outcomes of immuno-oncology treatments. Interleukin 10 (IL-10) is a multifunctional cytokine that plays an important role in anti-tumor immunity. Research results have shown that IL-10-dependent CD8+ T cell stimulation can induce tumor-specific immunity and mediate tumor regression. However, the anti-tumor effects and sustainability of existing IL-10 therapies are poor, and T cell depletion limits anti-tumor immunity and responses to immunotherapy. Especially in solid tumors, the depletion of immune cells weakens the cytotoxic killing effect, which subsequently promotes tumor development and requires multiple treatments.

[0003] Currently, the effectiveness of existing IL-10 anti-tumor therapies needs to be improved. Therefore, it is urgent to develop a method that can better enhance the ability of immune cells to kill tumor cells and increase the duration of killing to solve related problems. Summary of the Invention

[0004] The present application provides a fusion protein comprising IL-10 and a transmembrane domain, which can be expressed on the cell membrane surface. The expression of the fusion protein on the surface of cytotoxic immune cells can improve their activation efficacy and promote the secretion of IL-2 and IFN-γ. The fusion protein can also improve the lethality and persistence of immune cells against tumor cells. The fusion protein of the present application is co-expressed with ROR1 CAR, GPC3 CAR and NKG2D CAR on T cells, and can maintain a long-term effective killing effect on tumor cells.

[0005] In one aspect, the present application provides a fusion protein comprising IL-10 and a transmembrane domain, wherein the fusion protein is expressed on immune cells, excluding regulatory T cells.

[0006] In certain embodiments, the IL-10 is human IL-10.

[0007] In certain embodiments, the amino acid sequence of IL-10 is as shown in SEQ ID NO:9.

[0008] In certain embodiments, the transmembrane domain is selected from the transmembrane domain of the following protein: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM.

[0009] In certain embodiments, the transmembrane domain is the transmembrane domain of CD8.

[0010] In certain embodiments, the amino acid sequence of the transmembrane domain is shown in SEQ ID NO:11.

[0011] In certain embodiments, the fusion protein comprises IL-10 and a CD8 transmembrane domain.

[0012] In certain embodiments, the IL-10 and the transmembrane domain are indirectly linked.

[0013] In certain embodiments, the fusion protein further comprises a hinge region.

[0014] In certain embodiments, the IL-10 and transmembrane domain are connected by a hinge region.

[0015] In certain embodiments, the hinge region is selected from the hinge region of the following group of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 or LIGHT.

[0016] In certain embodiments, the hinge region is the hinge region of CD8.

[0017] In certain embodiments, the amino acid sequence of the hinge region is shown in SEQ ID NO:11.

[0018] In certain embodiments, the fusion protein comprises IL-10, a CD8 hinge region, and a CD8 transmembrane domain.

[0019] In certain embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:16.

[0020] In certain embodiments, the immune cells promote an immune response.

[0021] In certain embodiments, the immune cell is a T cell, a NK cell, an iNKT cell, a CIK cell, a γδ T cell, or a double negative T (DNT) cell.

[0022] In certain embodiments, the fusion protein is used to treat tumors, autoimmune diseases, severe infections, chronic aging, or cardiac fibrosis.

[0023] In certain embodiments, the fusion protein is used in adoptive cell therapy (ACT).

[0024] In certain embodiments, the ACT therapy is CAR-T therapy, TCR-T therapy, TIL therapy, NK therapy, iNKT therapy, CAR-NK therapy, CAR-iNKT therapy, γδT therapy, DNT therapy, or CAR-DNT therapy.

[0025] In certain embodiments, the fusion protein is co-expressed with an antigen recognition receptor.

[0026] In certain embodiments, the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0027] In certain embodiments, the antigen recognition receptor specifically binds to a target antigen.

[0028] In certain embodiments, the target antigen is an antigen associated with tumors, autoimmune diseases, severe infections, chronic aging, or cardiac fibrosis.

[0029] In certain embodiments, the fusion protein is co-expressed with the CAR.

[0030] In certain embodiments, the CAR targets ROR1, mesothelin, CD19, CD22, DLL3, GPC3, GPA33, B7-H3, EGFR, IL-13Rα2, FAP, TREM, Clauding 18.2, CDH17, B7H3, MUC1, GD2, CEA, CLL1, c-Met, or HER2.

[0031] In certain embodiments, the fusion protein is co-expressed with a CAR targeting ROR1.

[0032] In certain embodiments, the fusion protein is co-expressed with a CAR targeting GPC3.

[0033] In certain embodiments, the fusion protein is co-expressed with the NKG2D CAR.

[0034] On the other hand, the present application provides a modified immune cell comprising the fusion protein and an antigen recognition receptor, wherein the immune cell does not include regulatory T cells.

[0035] In certain embodiments, the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0036] In certain embodiments, the antigen recognition receptor specifically binds to a target antigen.

[0037] In certain embodiments, the target antigen is an antigen associated with tumors, autoimmune diseases, severe infections, chronic aging, or cardiac fibrosis.

[0038] In certain embodiments, the antigen recognition receptor is a CAR.

[0039] In certain embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0040] In certain embodiments, the antigen binding domain is an antibody or an antigen binding fragment thereof.

[0041] In certain embodiments, the antigen-binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment.

[0042] In certain embodiments, the antigen binding domain is the extracellular domain (ECD) of NKG2D.

[0043] In certain embodiments, the antigen binding domain specifically binds to a target antigen.

[0044] In certain embodiments, the target antigen is ROR1, mesothelin, CD19, CD22, DLL3, GPC3, GPA33, B7-H3, EGFR, IL-13Rα2, FAP, TREM, Clauding 18.2, CDH17, B7H3, MUC1, GD2, CEA, CLL1, c-Met, or HER2.

[0045] In certain embodiments, the target antigen is ROR1.

[0046] In certain embodiments, the target antigen is GPC3.

[0047] In certain embodiments, the antigen binding domain comprises a VHH, wherein the VHH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 19.

[0048] In certain embodiments, the antigen binding domain comprises a VHH, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0049] In certain embodiments, the antigen binding domain comprises an scFv, the scFv comprising VH and VL, the VH comprising HCDR1, HCDR2 and HCDR3, the VL comprising LCDR1, LCDR2 and LCDR3, the amino acid sequence of the HCDR1 being shown in SEQ ID NO:24, the amino acid sequence of the HCDR2 being shown in SEQ ID NO:25, the amino acid sequence of the HCDR3 being shown in SEQ ID NO:26, the amino acid sequence of the LCDR1 being shown in SEQ ID NO:31, the amino acid sequence of the LCDR2 being shown in SEQ ID NO:32, and the amino acid sequence of the LCDR3 being shown in SEQ ID NO:33.

[0050] In certain embodiments, the antigen binding domain comprises an scFv, the scFv comprises VH and VL, the amino acid sequence of the VH is shown in SEQ ID NO: 30, and the amino acid sequence of the VL is shown in SEQ ID NO: 38.

[0051] In certain embodiments, the antigen binding domain comprises a scFv, the amino acid sequence of which is shown in SEQ ID NO:12.

[0052] In certain embodiments, the antigen binding domain comprises NKG2D ECD, and the amino acid sequence of the NKG2D ECD is shown in SEQ ID NO:14.

[0053] In certain embodiments, the transmembrane domain in the chimeric antigen receptor is selected from the transmembrane domain of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM.

[0054] In certain embodiments, the transmembrane domain in the chimeric antigen receptor is the transmembrane domain of CD28.

[0055] In certain embodiments, the intracellular signaling domain is selected from the intracellular signaling domain of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 or a domain containing at least one ITAM.

[0056] In certain embodiments, the intracellular signaling domain is the intracellular signaling domain of CD3ζ.

[0057] In certain embodiments, the chimeric antigen receptor further comprises an intracellular costimulatory domain.

[0058] In certain embodiments, the intracellular costimulatory domain is selected from the intracellular costimulatory domain of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0059] In certain embodiments, the intracellular costimulatory domain is the intracellular costimulatory domain of 4-1BB.

[0060] In certain embodiments, the chimeric antigen receptor further comprises a hinge region.

[0061] In certain embodiments, the hinge region in the chimeric antigen receptor is selected from the hinge region of the following group of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.

[0062] In certain embodiments, the hinge region in the chimeric antigen receptor is the hinge region of CD28, CD8, or IgG4.

[0063] In certain embodiments, the modified immune cell comprises a fusion protein comprising IL-10, a hinge region, and a transmembrane domain, and a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0064] In certain embodiments, the modified immune cell comprises a fusion protein comprising IL-10, a CD8 hinge region, and a CD8 transmembrane domain, and a chimeric antigen receptor comprising an antigen binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0065] In certain embodiments, the immune cells promote an immune response.

[0066] In certain embodiments, the immune cell is a T cell, a NK cell, an iNKT cell, a CIK cell, a γδT cell, or a DN T cell.

[0067] In certain embodiments, the immune cell is a T cell.

[0068] In another aspect, the present application provides an isolated nucleic acid molecule encoding the fusion protein.

[0069] In certain embodiments, the nucleic acid molecule further encodes an antigen recognition receptor.

[0070] In certain embodiments, the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0071] In certain embodiments, the antigen recognition receptor specifically binds to a target antigen.

[0072] In certain embodiments, the target antigen is an antigen associated with tumors, autoimmune diseases, severe infections, chronic aging, or cardiac fibrosis.

[0073] In certain embodiments, the antigen recognition receptor is a CAR.

[0074] In certain embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0075] In certain embodiments, the antigen binding domain is an antibody or an antigen binding fragment thereof

[0076] In certain embodiments, the antigen-binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment.

[0077] In certain embodiments, the antigen binding domain is the extracellular domain (ECD) of NKG2D.

[0078] In certain embodiments, the antigen binding domain specifically binds to a target antigen.

[0079] In certain embodiments, the target antigen is ROR1, mesothelin, CD19, CD22, DLL3, GPC3, GPA33, B7-H3, EGFR, IL-13Rα2, FAP, TREM, Clauding 18.2, CDH17, B7H3, MUC1, GD2, CEA, CLL1, c-Met, or HER2.

[0080] In certain embodiments, the target antigen is ROR1.

[0081] In certain embodiments, the target antigen is GPC3.

[0082] In certain embodiments, the antigen binding domain comprises a VHH, wherein the VHH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 19.

[0083] In certain embodiments, the antigen binding domain comprises a VHH, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0084] In certain embodiments, the antigen binding domain comprises an scFv, wherein the scFv comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3.

[0085] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 24, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 25, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 26, and the amino acid sequence of LCDR1 is shown in

[0086] The amino acid sequence of LCDR2 is shown in SEQ ID NO: 31, the amino acid sequence of LCDR3 is shown in SEQ ID NO: 32, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 33.

[0087] In certain embodiments, the antigen binding domain comprises an scFv, the scFv comprises VH and VL, the amino acid sequence of the VH is shown in SEQ ID NO: 30, and the amino acid sequence of the VL is shown in SEQ ID NO: 38.

[0088] In certain embodiments, the antigen binding domain comprises a scFv, the amino acid sequence of which is shown in SEQ ID NO:12.

[0089] In certain embodiments, the antigen binding domain comprises NKG2D ECD, and the amino acid sequence of the NKG2D ECD is shown in SEQ ID NO:14.

[0090] In certain embodiments, the transmembrane domain in the chimeric antigen receptor is selected from the transmembrane domain of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM.

[0091] In certain embodiments, the transmembrane domain in the chimeric antigen receptor is the transmembrane domain of CD28.

[0092] In certain embodiments, the intracellular signaling domain is selected from the intracellular signaling domain of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 or a domain containing at least one ITAM.

[0093] In certain embodiments, the intracellular signaling domain is the intracellular signaling domain of CD3ζ.

[0094] In certain embodiments, the chimeric antigen receptor further comprises an intracellular costimulatory domain.

[0095] In certain embodiments, the intracellular costimulatory domain is selected from the intracellular costimulatory domain of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0096] In certain embodiments, the intracellular costimulatory domain is the intracellular costimulatory domain of 4-1BB.

[0097] In certain embodiments, the chimeric antigen receptor further comprises a hinge region.

[0098] In certain embodiments, the hinge region in the chimeric antigen receptor is selected from the hinge region of the following group of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.

[0099] In certain embodiments, the hinge region in the chimeric antigen receptor is the hinge region of CD28, CD8, or IgG4.

[0100] In certain embodiments, the nucleic acid molecule comprises a sequence encoding a self-cleaving peptide.

[0101] In certain embodiments, the sequence encoding the self-cleaving peptide is located between the sequences encoding the fusion protein and the antigen recognition receptor.

[0102] In certain embodiments, the self-cleaving peptide comprises a 2A peptide.

[0103] In certain embodiments, the self-cleaving peptide is P2A, T2A, E2A, or F2A.

[0104] In certain embodiments, the self-cleaving peptide is P2A.

[0105] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0106] In another aspect, the present application provides a cell comprising the nucleic acid molecule or the vector.

[0107] On the other hand, the present application provides a pharmaceutical composition comprising the fusion protein, the modified immune cell, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0108] On the other hand, the present application provides a method for preparing the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition.

[0109] On the other hand, the present application provides the use of the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a drug for preventing and / or treating tumors, autoimmune diseases, severe infections, chronic aging or cardiac fibrosis.

[0110] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0111] In some embodiments, the tumor is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and skin cancer.

[0112] On the other hand, the present application provides a method for preventing and / or treating tumors, autoimmune diseases, severe infections, chronic aging or cardiac fibrosis, which comprises administering the fusion protein, the modified immune cells, the nucleic acid molecule, the vector, the cells and / or the pharmaceutical composition to a subject in need.

[0113] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0114] In some embodiments, the tumor is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and skin cancer.

[0115] On the other hand, the present application provides the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition, which are used to prevent and / or treat tumors, autoimmune diseases, severe infections, chronic aging or cardiac fibrosis.

[0116] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0117] In some embodiments, the tumor is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and skin cancer.

[0118] 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

[0119] 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:

[0120] Figure 1 shows the structural design of ROR1 VHH CAR, ROR1 VHH CAR-membrane-bound IL-10 and ROR1 VHH CAR-secreted IL-10 described in this application.

[0121] Figure 2 shows the flow cytometry experimental results of the CAR structure expression described in this application.

[0122] FIG3 shows the activation effect of the membrane-bound IL-10 described in the present application on ROR1 VHH-CAR-membrane-bound IL-10 T cells.

[0123] FIG4 shows the killing effect of the membrane-bound IL-10 described in the present application on tumor target cells.

[0124] FIG5 shows the activation effect of the membrane-bound IL-10 described in the present application on ROR1 VHH-CAR-membrane-bound IL-10 T cells.

[0125] FIG6 shows the killing effect of the membrane-bound IL-10 described in the present application on A375 tumor target cells.

[0126] FIG7 shows the killing effect of the membrane-bound IL-10 described in the present application on HCC827 tumor target cells.

[0127] FIG8 shows the killing effect of the membrane-bound IL-10 described in the present application on 293T tumor target cells.

[0128] FIG9 shows the structural design of GPC3 scfv CAR and GPC3 scfv CAR-membrane-bound IL-10 described in this application.

[0129] Figure 10 shows the flow cytometry experimental results of the CAR structure expression described in this application.

[0130] FIG11 shows the activation effect of the membrane-bound IL-10 described in the present application on GPC3 scfv-CAR-membrane-bound IL-10 T cells.

[0131] FIG12 shows the killing effect of the membrane-bound IL-10 described in the present application on Huh7 tumor target cells.

[0132] FIG13 shows the killing effect of the membrane-bound IL-10 described in the present application on Hep3b tumor target cells.

[0133] FIG14 shows the killing effect of the membrane-bound IL-10 described in the present application on MB231 tumor target cells.

[0134] Figure 15 shows the structural design of NKG2D ECD CAR and NKG2D ECD CAR-membrane-bound IL-10 described in this application.

[0135] Figure 16 shows the flow cytometry experimental results of the CAR structure expression described in this application.

[0136] FIG17 shows the activation effect of the membrane-bound IL-10 described in the present application on NKG2D ECD-CAR-membrane-bound IL-10 T cells.

[0137] FIG18 shows the killing effect of the membrane-bound IL-10 described in the present application on MB231 tumor target cells.

[0138] FIG19 shows the killing effect of the membrane-bound IL-10 described in the present application on A375 tumor target cells.

[0139] FIG20 shows the killing effect of the membrane-bound IL-10 described in the present application on Huh7 tumor target cells. DETAILED DESCRIPTION

[0140] 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.

[0141] Definition of terms

[0142] In this application, the terms "IL-10" and "interleukin-10" are generally used interchangeably and generally refer to an inflammatory and immunosuppressive factor. In this application, the IL-10 may be a monomer or a multimer. For example, the IL-10 may be a dimer. For example, the IL-10 may be a homodimer. In this application, the IL-10 may include IL-10 of any species. For example, the IL-10 may be human or non-human. For example, the IL-10 may be murine. For example, the IL-10 may be human. In this application, the IL-10 may be full-length IL-10 or a truncated IL-10. For example, the IL-10 may be a functionally active fragment that retains the function of the full-length IL-10. In this application, the IL-10 may be wild-type IL-10 or artificially modified IL-10. For example, the IL-10 is a modified IL-10.

[0143] In this application, the term "transmembrane domain" generally refers to a domain that can cross the plasma membrane of a cell, and these domains are anchored on the cell membrane. In this application, the transmembrane domain can be a transmembrane domain of a peptide, polypeptide or protein. For example, the membrane-spanning domain can include the membrane-spanning domain of one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the membrane-spanning domain can include the membrane-spanning domain of CD8. For example, the membrane-spanning domain can include the membrane-spanning domain of CD28. In the present application, the transmembrane domain may be natural or synthetic. In the present application, the transmembrane domain may be of any species. For example, the transmembrane domain may be human. For example, the transmembrane domain may be the transmembrane domain of human CD8. For example, the transmembrane domain may be the transmembrane domain of human CD28.

[0144] In this application, the term "hinge region" generally refers to a dimer molecule composed of two polypeptides having the same amino acid sequence, generally comprising about 25 amino acid residues. In this application, the hinge region is flexible. In this application, the hinge region can be the hinge region of a peptide, polypeptide or protein. For example, the hinge region can include a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT. For example, the hinge region can be derived from the hinge region of CD8. For example, the hinge region can be derived from the hinge region of CD28. For example, the hinge region can be derived from the hinge region of IgG4. In the present application, the hinge region may be natural or synthetic. In the present application, the hinge region may be of any species. For example, the hinge region may be human. For example, the hinge region may be the hinge region of human CD8. For example, the hinge region may be the hinge region of human CD28. For example, the hinge region may be the hinge region of human IgG4.

[0145] As used herein, the term "fusion protein" generally refers to a protein composed of two or more polypeptides that are typically not naturally bound together but are linked together at the amino and carboxyl termini via peptide bonds to form a continuous polypeptide. As used herein, "binding" refers to direct or indirect binding of the two or more polypeptide components. Indirect binding, as used herein, may be through a linker.

[0146] The proteins and / or amino acid sequences involved in this application should also be understood to include at least the following scope: variants or homologs with the same or similar functions as the protein. In this application, the variant can be a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein. For example, the functional variant may include a protein or polypeptide that has been subjected to amino acid changes by at least 1, such as 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant can substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant can retain at least about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitutions may be conservative substitutions.

[0147] In the present application, a portion of the amino acid sequence of the fusion protein may be homologous to a corresponding amino acid sequence in an antibody from a specific species, or belong to a specific class.

[0148] In the present application, the homolog can be a protein or polypeptide 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 protein and / or the polypeptide.

[0149] In this application, the term "immune cell" generally refers to an immune cell that participates in an immune response and performs effector functions. For example, the immune cell can clear foreign antigens or promote immune effector responses. In this application, the immune cell can be a natural immune cell or a modified immune cell. In this application, the natural immune cell generally refers to a naturally occurring cell that participates in an immune response or is associated with an immune response. For example, a natural immune cell can be a T cell, NK cell, NKT cell, dendritic cell, macrophage, TIL cell, iNKT cell, CIK cell, γδT cell or double negative T (DNT) cell. In this application, the modified immune cell is also referred to as an engineered immune cell, and generally refers to an immune cell that is genetically modified by transcribing additional genetic material in the form of DNA or RNA. For example, the modified immune cell can express a chimeric antigen receptor (CAR), a T cell receptor (TCR) or an NKG2D CAR. In this application, the modified immune cell can express a CAR. For example, the modified immune cell can be a CAR-T cell, a CAR-iNKT cell or a CAR-DNT cell. In the present application, the modified immune cell may express a TCR. For example, the modified immune cell may be a TCR-T cell.

[0150] In this application, the term "antigen recognition receptor" generally refers to a protein present on the cell surface that causes a response in the immune system by binding to a target antigen. In this application, the antigen recognition receptor may be a B cell receptor, a T cell receptor, or an NKG2D CAR. In this application, the antigen recognition receptor may be natural or synthetic. For example, the antigen recognition receptor may be a chimeric antigen receptor.

[0151] In this application, the term "chimeric antigen receptor" is generally used interchangeably with "CAR" and generally refers to a recombinant polypeptide that can specifically bind to a corresponding antigen. In this application, the chimeric antigen receptor may include an extracellular domain, a transmembrane domain, and an intracellular domain. For example, the extracellular domain may include an antigen binding domain. For example, the extracellular domain may include a signal peptide. For example, the intracellular domain may include an intracellular signaling domain. For example, the intracellular domain may include an intracellular costimulatory domain. In this application, a hinge region may be included between the extracellular domain and the transmembrane domain.

[0152] In this application, the term "target antigen" generally refers to any molecule that can elicit an immune response or can be bound by an antibody or antigen-binding molecule. In this application, the target antigen can be a macromolecule, a peptide, a polypeptide, or an amino acid. In this application, the target antigen can be endogenously expressed or recombinantly expressed. For example, the target antigen can be expressed by genomic DNA. For example, the target antigen can be produced by mutation of a normal protein. For example, the target antigen can be produced by protein shearing or hydrolysis. In this application, the target antigen can be an antigen associated with a tumor, an autoimmune disease, a severe infection, chronic aging, or cardiac fibrosis. For example, the target antigen can be a tumor antigen. For example, the target antigen can be a tumor-associated antigen. For example, the target antigen can be a tumor-specific antigen. In the present application, the target antigen can be ROR1, mesothelin, CD19, CD22, DLL3, GPC3, GPA33, B7-H3, EGFR, IL-13Rα2, FAP, TREM, Clauding 18.2, CDH17, B7H3, MUC1, GD2, CEA, CLL1, c-Met, HER2, NY-ESO-1, MAGEA4, HPV E6, HPV E7, KRAS G12C, or KRAS G12D. For example, the target antigen can be ROR1. For example, the target antigen can be GPC3. For example, the target antigen can be an NKG2D ligand.

[0153] In this application, the term "self-cleaving peptide" generally refers to a class of polypeptides that can achieve the function of cleaving proteins. For example, the self-cleaving peptide can achieve protein cleavage via ribosome skipping. In this application, the self-cleaving peptide can be a 2A peptide. For example, the self-cleaving peptide can be T2A, F2A, P2A and / or E2A. For example, the self-cleaving peptide can be P2A.

[0154] As used herein, the term "pharmaceutically acceptable carrier" generally refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. For example, the pharmaceutically acceptable carrier includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the dose and concentration employed. For example, a physiologically acceptable carrier can be water, salt, protein, polysaccharide, lipid, or inactive viral particles.

[0155] In this application, the term "prevention and / or treatment" generally refers to the prevention and / or treatment of a disease. For example, the prevention and / or treatment may be preventing the onset of the disease, slowing or reversing the progression of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing further increase in the severity of the disease and any symptoms associated therewith. In this application, the disease may be a tumor, an autoimmune disease, a severe infection, chronic aging, or cardiac fibrosis. For example, the disease may be a tumor.

[0156] Detailed Description of the Invention

[0157] The present application provides a fusion protein comprising IL-10 and a transmembrane domain, which can be expressed on the cell membrane surface. Expression of the fusion protein on the surface of cytotoxic immune cells can enhance their activation efficacy, as well as the immune cells' ability to kill and sustain tumor cells. The present application also provides a modified immune cell comprising the fusion protein and an antigen-recognizing receptor. The present application also provides nucleic acid molecules, vectors, cells, pharmaceutical compositions, and the use of the fusion protein and the modified immune cell in preventing and / or treating diseases.

[0158] Fusion protein

[0159] In one aspect, the present application provides a fusion protein comprising IL-10, wherein the fusion protein is expressed on immune cells, and the immune cells do not include regulatory T cells.

[0160] In the present application, the IL-10 may be IL-10 from a different species. For example, the IL-10 may be human IL-10. For example, the amino acid sequence of the IL-10 may be as shown in SEQ ID NO:9.

[0161] In the present application, the fusion protein can also include a transmembrane domain. In the present application, the transmembrane domain can be selected from the transmembrane domain of the following group of proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM. For example, the transmembrane domain can be the transmembrane domain of CD8. For example, the transmembrane domain can be the transmembrane domain of CD28. For example, the transmembrane domain can be the transmembrane domain of 2B4.

[0162] For example, the amino acid sequence of the transmembrane domain may be as shown in SEQ ID NO: 11. For example, the amino acid sequence of the transmembrane domain may be as shown in SEQ ID NO: 4.

[0163] In the present application, the fusion protein may comprise IL-10 and a transmembrane domain. For example, the fusion protein may comprise IL-10 and a CD8 transmembrane domain. For example, the fusion protein may comprise IL-10 and a CD28 transmembrane domain. For example, the fusion protein may comprise IL-10 and a 2B4 transmembrane domain.

[0164] In the present application, the IL-10 and transmembrane domain can be directly or indirectly connected. For example, the IL-10 and transmembrane domain can be directly connected. For example, the IL-10 and transmembrane domain can be indirectly connected. For example, the IL-10 and transmembrane domain can be indirectly connected via a hinge region.

[0165] In the present application, the fusion protein may further comprise a hinge region. In the present application, the hinge region may be selected from the hinge region of the following protein groups: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 or LIGHT.

[0166] For example, the hinge region may be the hinge region of CD8. For example, the hinge region may be the hinge region of CD28. For example, the hinge region may be the hinge region of IgG1. For example, the hinge region may be the hinge region of IgG4.

[0167] For example, the amino acid sequence of the hinge region may be as shown in SEQ ID NO: 10. For example, the amino acid sequence of the hinge region may be as shown in SEQ ID NO: 3. For example, the amino acid sequence of the hinge region may be as shown in SEQ ID NO: 13.

[0168] In the present application, the fusion protein may comprise IL-10, a hinge region and a transmembrane domain. In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain. In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD28 transmembrane domain. In the present application, the fusion protein may comprise IL-10, a CD28 hinge region and a CD8 transmembrane domain. In the present application, the fusion protein may comprise IL-10, a CD28 hinge region and a CD28 transmembrane domain.

[0169] In the present application, the fusion protein may comprise the amino acid sequence shown in SEQ ID NO:16.

[0170] In the present application, the immune cell may be an immune cell that promotes an immune response. For example, the immune cell may be an immune cell that kills target cells. In the present application, the immune cell may be a natural immune cell or a modified immune cell.

[0171] For example, the immune cell can be a T cell, an NK cell, an NKT cell, a dendritic cell, a macrophage, a TIL cell, an iNKT cell, a CIK cell, a γδT cell, or a DN T cell. For example, the immune cell can be a T cell. For example, the immune cell can be a DN T cell.

[0172] In the present application, the immune cell can be a modified immune cell. For example, the immune cell can express a chimeric antigen receptor (CAR), a T cell receptor (TCR), an NKG2D CAR or a Toll-like receptor. For example, the immune cell can be a CAR-T cell, a CAR-NK cell, a CAR-iNKT cell, a CAR-DNT cell, a TCR-T cell or a TCR-NK cell.

[0173] In the present application, the fusion protein can be used to treat tumors, autoimmune diseases, severe infections, chronic aging, or cardiac fibrosis. For example, the fusion protein can be used to treat solid tumors. For example, the fusion protein can be used to treat hematological tumors. For example, the fusion protein can be used to treat lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and skin cancer.

[0174] In the present application, the fusion protein can be used for adoptive cell therapy. In the present application, the ACT therapy can be CAR-T therapy, TCR-T therapy, TIL therapy, NK therapy, iNKT therapy, CAR-NK therapy, CAR-iNKT therapy, γδT therapy, DNT therapy or CAR-DNT therapy. For example, the ACT therapy can be CAR-T therapy. For example, the ACT therapy can be TCR-T therapy.

[0175] In the present application, the fusion protein can be co-expressed with an antigen recognition receptor. In the present application, the antigen recognition receptor can be a natural receptor or an artificially synthesized receptor. For example, the antigen recognition receptor can be a B cell receptor, a T cell receptor, a chimeric antigen receptor, an NKG2D CAR or a Toll-like receptor. For example, the antigen recognition receptor can be a chimeric antigen receptor. For example, the antigen recognition receptor can be an NKG2D CAR.

[0176] In the present application, the fusion protein can be co-expressed with a chimeric antigen receptor (CAR), a T cell receptor (TCR), an NKG2D CAR or a Toll-like receptor.

[0177] In the present application, the antigen recognition receptor can specifically bind to a target antigen. In the present application, the target antigen can be an antigen associated with a tumor, autoimmune disease, severe infection, chronic aging, or cardiac fibrosis. For example, the target antigen can be a tumor antigen. For example, the target antigen can be a tumor-associated antigen. For example, the target antigen can be a tumor-specific antigen.

[0178] In the present application, the fusion protein can be co-expressed with the CAR. In the present application, the CAR can specifically bind to the target antigen. For example, the CAR can specifically bind to A33, B7-H3, BCMA, CA125, CD1, CD10, CD102, CD11a, CD11b, CD123, CD13, CD133, CD134, CD137, CD138, CD14, CD15, CD19, CD2, CD20, CD200, CD21, CD22, CD23, CD25, CD27, CD28, CD3, CD30, CD33, CD 34. CD36, CD37, CD38, CD4, CD40, CD41, CD42, CD43, CD44, CD45, CD5, CD56, CD58, CD65, CD66c, CD7, CD70, C D73, CD74, CD8, CD80, CD81, CD86, CD9, CD94, CD97, CD99, CEA, CEACAM6, CLL1, CS1, DLL1, DLL3, EGFR, EGFR VIII, ErbB2, FGF19, GD2, GD3, HER3, IL3Ra, NCAM, NG2, NKG2A, NTBA, PD-1, PDL-1, PSGL1, PSMA, ROR1, VEGF, 5T4, AFP, BCMA, CTLA-4, ELF2M, FLT3, PSA, HPV-specific antigens, glioma-associated antigens, EBV-specific antigens, mesothelin, GPC3, GPA33, IL-11Rα, IL-13Rα2, FAP, Clauding 18.2, CDH17, MUC1, c-Met, HER2 or TREM. For example, the CAR can specifically bind to ROR1. For example, the CAR can specifically bind to GPC3. In the present application, the CAR can specifically bind to NKG2D ligands (NKG2DLs). For example, the ligands may include six cytomegalovirus UL16 binding proteins 1-6 (ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6) and MHC I chain-associated molecules A and B (MICA, MICB).

[0179] In the present application, the antigen-binding domain may comprise an antigen-binding protein. For example, the antigen-binding protein may be an antibody or an antigen-binding fragment thereof. For example, the antigen-binding protein may include, but is not limited to, a recombinant antibody, a monoclonal antibody, a human antibody, a murine antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, a multi-antibody, an Fv fragment, a scFv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a camelized single-domain antibody. In the present application, the antigen-binding protein may be a chimeric antibody, a humanized antibody, or a fully human antibody. For example, the antigen-binding protein may be mutated / optimized in the FR and constant regions without reducing the binding activity / affinity of the antigen-binding protein to the target antigen. For example, the antigen-binding protein may have lower immunogenicity without reducing the binding activity / affinity of the antigen-binding protein to the target antigen.

[0180] In the present application, the antigen-binding fragment can be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH or dAb fragment of the antibody. For example, the antigen-binding fragment can be an scFv. For example, the antigen-binding fragment can be a VHH.

[0181] In the present application, the antigen-binding protein may comprise an antibody heavy chain variable region VH, wherein the VH comprises a heavy chain complementary determining region HCDR1, HCDR2 and / or HCDR3. In the present application, the VH may comprise H-FR1, H-FR2, H-FR3 and / or H-FR4. In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and / or H-FR4, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3.

[0182] In the present application, the antigen-binding protein may further comprise an antibody light chain variable region VL, wherein the VL comprises light chain complementary determining regions LCDR1, LCDR2, and / or LCDR3. In the present application, the VL may comprise L-FR1, L-FR2, L-FR3, and / or L-FR4. In the present application, the isolated antigen-binding protein may comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and / or L-FR4, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, the L-FR2 is located between the LCDR1 and the LCDR2, the L-FR3 is located between the LCDR2 and the LCDR3, and the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3.

[0183] In the present application, the antigen-binding protein may comprise an scFv, wherein the scFv comprises VH and VL, wherein the VH comprises HCDR1, HCDR2 and HCDR3, and the VL comprises LCDR1, LCDR2 and LCDR3. In the present application, the antigen-binding protein may comprise an scFv, wherein the scFv comprises VH and VL. In the present application, the scFv may further comprise a linker.

[0184] In the present application, the antigen-binding protein may comprise a VHH, wherein the VHH comprises a heavy chain complementary determining region HCDR1, HCDR2 and / or HCDR3. In the present application, the VHH may comprise an H-FR1, H-FR2, H-FR3 and / or H-FR4. In the present application, the isolated antigen-binding protein may comprise an HCDR1, an HCDR2, an HCDR3, an H-FR1, an H-FR2, an H-FR3 and / or an H-FR4, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3.

[0185] In the present application, the antigen binding protein may comprise a VHH targeting ROR1. In the present application, the antigen binding protein may comprise a scFv targeting GPC3.

[0186] In the present application, the antigen binding domain may comprise an antigen binding protein. For example, the antigen binding protein may be a receptor on the surface of an immune cell or a portion thereof. For example, the antigen binding protein may be a portion of a NK cell surface receptor. For example, the antigen binding protein may be a portion of NKG2D, for example, the antigen binding domain may comprise an NKG2D extracellular domain (ECD).

[0187] In the present application, the fusion protein can be co-expressed with a CAR targeting ROR1. In the present application, the fusion protein can be co-expressed with a CAR targeting GPC3.

[0188] In the present application, the fusion protein can be co-expressed with NKG2D CAR. In the present application, the NKG2D CAR can specifically bind to the target antigen. For example, the NKG2D CAR can recognize and bind to NKG2D ligands (NKG2DLs). For example, the ligand can include six cytomegalovirus UL16 binding proteins 1-6 (ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6) and MHC I chain related molecules A and B (MICA, MICB).

[0189] In the present application, the NKG2D CAR may comprise an NKG2D extracellular domain (ECD).

[0190] In the present application, the fusion protein can be co-expressed with a TCR. In the present application, the TCR can specifically bind to a target antigen. For example, the TCR can specifically bind to NY-ESO-1, MAGEA1, MAGEA3, MAGEA4, MAGEA10, HPV E6, HPV E7, KRAS G12C, KRAS G12D, AFP, HBsAg, mesothelin, PD-1, MART-1, CD19, CD28, CD8, CT83, F8, GPC3, H3 K27M, HLA-A, LMP-2, MC2R, or PRAME. For example, the TCR can specifically bind to NY-ESO-1. In the present application, the fusion protein can be co-expressed with a TCR targeting NY-ESO-1.

[0191] Modified immune cells

[0192] On the other hand, the present application provides a modified immune cell comprising a fusion protein and an antigen recognition receptor, wherein the fusion protein comprises IL-10, and the immune cell does not comprise a regulatory T cell. In the present application, the fusion protein may be as described above.

[0193] In the present application, the antigen recognition receptor can be a natural receptor or an artificially synthesized receptor. For example, the antigen recognition receptor can be a B cell receptor, a T cell receptor, a chimeric antigen receptor (CAR), an NKG2D CAR or a Toll-like receptor. For example, the antigen recognition receptor can be a chimeric antigen receptor. For example, the antigen recognition receptor can be an NKG2D CAR.

[0194] In the present application, the fusion protein can be co-expressed with a chimeric antigen receptor, a T cell receptor (TCR), an NKG2D CAR or a Toll-like receptor.

[0195] In the present application, the antigen recognition receptor can specifically bind to a target antigen. In the present application, the target antigen can be an antigen associated with a tumor, autoimmune disease, severe infection, chronic aging, or cardiac fibrosis. For example, the target antigen can be a tumor antigen. For example, the target antigen can be a tumor-associated antigen. For example, the target antigen can be a tumor-specific antigen.

[0196] In the present application, the antigen recognition receptor can be a CAR. In the present application, the CAR can specifically bind to a target antigen. For example, the CAR can specifically bind to A33, B7-H3, BCMA, CA125, CD1, CD10, CD102, CD11a, CD11b, CD123, CD13, CD133, CD134, CD137, CD138, CD14, CD15, CD19, CD2, CD20, CD200, CD21, CD22, CD23, CD25, CD27, CD28, CD3, CD30, CD33, CD 34. CD36, CD37, CD38, CD4, CD40, CD41, CD42, CD43, CD44, CD45, CD5, CD56, CD58, CD65, CD66c, CD7, CD70, C D73, CD74, CD8, CD80, CD81, CD86, CD9, CD94, CD97, CD99, CEA, CEACAM6, CLL1, CS1, DLL1, DLL3, EGFR, EGFR VIII, ErbB2, FGF19, GD2, GD3, HER3, IL3Ra, NCAM, NG2, NKG2A, NTBA, PD-1, PDL-1, PSGL1, PSMA, ROR1, VEGF, 5T4, AFP, BCMA, CTLA-4, ELF2M, FLT3, PSA, HPV-specific antigens, glioma-associated antigens, EBV-specific antigens, mesothelin, GPC3, GPA33, IL-11Rα, IL-13Rα2, FAP, Clauding 18.2, CDH17, MUC1, c-Met, HER2 or TREM. For example, the CAR can specifically bind to ROR1. For example, the CAR can specifically bind to GPC3.

[0197] In the present application, the CAR may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.

[0198] In the present application, the antigen-binding domain may comprise an antigen-binding protein. For example, the antigen-binding protein may be an antibody or an antigen-binding fragment thereof. For example, the antigen-binding protein may include, but is not limited to, a recombinant antibody, a monoclonal antibody, a human antibody, a murine antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, a multi-antibody, an Fv fragment, a scFv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a camelized single-domain antibody. In the present application, the antigen-binding protein may be a chimeric antibody, a humanized antibody, or a fully human antibody. For example, the antigen-binding protein may be mutated / optimized in the FR and constant regions without reducing the binding activity / affinity of the antigen-binding protein to the target antigen. For example, the antigen-binding protein may have lower immunogenicity without reducing the binding activity / affinity of the antigen-binding protein to the target antigen.

[0199] In the present application, the antigen-binding fragment can be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH or dAb fragment of the antibody. For example, the antigen-binding fragment can be an scFv. For example, the antigen-binding fragment can be a VHH.

[0200] In the present application, the antigen-binding protein may comprise an antibody heavy chain variable region VH, wherein the VH comprises a heavy chain complementary determining region HCDR1, HCDR2 and / or HCDR3. In the present application, the VH may comprise H-FR1, H-FR2, H-FR3 and / or H-FR4. In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and / or H-FR4, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3.

[0201] In the present application, the antigen-binding protein may further comprise an antibody light chain variable region VL, wherein the VL comprises light chain complementary determining regions LCDR1, LCDR2, and / or LCDR3. In the present application, the VL may comprise L-FR1, L-FR2, L-FR3, and / or L-FR4. In the present application, the isolated antigen-binding protein may comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and / or L-FR4, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, the L-FR2 is located between the LCDR1 and the LCDR2, the L-FR3 is located between the LCDR2 and the LCDR3, and the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3.

[0202] In the present application, the antigen-binding protein may comprise an scFv comprising HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3. In the present application, the antigen-binding protein may comprise an scFv comprising VH and VL. In the present application, the scFv may further comprise a linker.

[0203] In the present application, the antigen-binding protein may comprise a VHH, wherein the VHH comprises a heavy chain complementary determining region HCDR1, HCDR2 and / or HCDR3. In the present application, the VHH may comprise an H-FR1, H-FR2, H-FR3 and / or H-FR4. In the present application, the isolated antigen-binding protein may comprise an HCDR1, an HCDR2, an HCDR3, an H-FR1, an H-FR2, an H-FR3 and / or an H-FR4, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3.

[0204] In the present application, the antigen-binding domain may comprise a constant region. For example, the antigen-binding domain may be a mouse constant region. For example, the antigen-binding domain may be a human constant region.

[0205] In the present application, the antibody may be a monoclonal antibody, a bispecific antibody or a multispecific antibody.

[0206] In the present application, the antibody may be a murine antibody, a camelid antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0207] For example, the antigen binding domain can specifically bind to A33, B7-H3, BCMA, CA125, CD1, CD10, CD102, CD11a, CD11b, CD123, CD13, CD133, CD134, CD137, CD138, CD14, CD15, CD19, CD2, CD20, CD200, CD21, CD22, CD23, CD25, CD27, CD28, CD3, CD30, CD33, CD34, CD36, CD37, CD38, CD4, CD40, CD41, CD42, CD43, CD44, CD45, CD5, CD56, CD58, CD65, CD66c, CD7, CD70, CD73, CD74, CD8, CD80, CD81, CD86, CD9, CD94, CD97, CD99, CEA, CEACAM6, CLL1, CS1, DLL1, DLL3, EGFR, EGFR VIII, ErbB2, FGF19, GD2, GD3, HER3, IL3Ra, NCAM, NG2, NKG2A, NTBA, PD-1, PDL-1, PSGL1, PSMA, ROR1, VEGF, 5T4, AFP, BCMA, CTLA-4, ELF2M, FLT3, PSA, HPV-specific antigens, glioma-associated antigens, EBV-specific antigens, mesothelin, GPC3, GPA33, IL-11Rα, IL-13Rα2, FAP, or TREM.

[0208] In the present application, the antigen binding domain can specifically bind to ROR1. For example, the antigen binding domain can comprise ROR1 VHH.

[0209] In the present application, the antigen-binding domain may comprise a VHH, and the VHH may comprise HCDR1-3. For example, the VHH comprises HCDR1, HCDR2, and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 19. In the present application, the antigen-binding domain may comprise a VHH, and the amino acid sequence of the VHH may be shown in SEQ ID NO: 1. In the present application, the antigen-binding domain may comprise the amino acid sequence shown in SEQ ID NO: 1.

[0210] In the present application, the antigen-binding domain can specifically bind to GPC3. For example, the antigen-binding domain can comprise a GPC3 scFv.

[0211] In the present application, the antigen-binding domain may comprise an scFv, and the scFv may comprise a VH and a VL, wherein the VH comprises HCDR1-3, and the VL comprises LCDR1-3. For example, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 24, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 25, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 26, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 31, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 32, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 33. In the present application, the antigen-binding domain may comprise an scFv, and the scFv may comprise a VH and a VL, wherein the amino acid sequence of the VH is shown in SEQ ID NO: 30, and the amino acid sequence of the VL is shown in SEQ ID NO: 38. In the present application, the antigen-binding domain may comprise an scFv, and the amino acid sequence of the scFv may be shown in SEQ ID NO: 12. In the present application, the antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO:12.

[0212] In the present application, the antigen recognition receptor can be NKG2D CAR. In the present application, the fusion protein can be co-expressed with NKG2D CAR. In the present application, the NKG2D CAR can specifically bind to the target antigen. For example, the NKG2D CAR can recognize and bind to NKG2D ligands (NKG2DLs). For example, the ligand can include six cytomegalovirus UL16 binding proteins 1-6 (ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6) and MHC I chain-related molecules A and B (MICA, MICB).

[0213] In the present application, the antigen binding domain may include NKG2D ECD. In the present application, the NKG2DCAR may include NKG2D ECD. For example, the amino acid sequence of the NKG2D ECD may be as shown in SEQ ID NO: 14.

[0214] In the present application, the transmembrane domain in the chimeric antigen receptor can be selected from the transmembrane domain of the following group of proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM. For example, the transmembrane domain in the chimeric antigen receptor can be the transmembrane domain of CD8. For example, the transmembrane domain in the chimeric antigen receptor can be the transmembrane domain of CD28.

[0215] For example, the amino acid sequence of the transmembrane domain may be as shown in SEQ ID NO: 11. For example, the amino acid sequence of the transmembrane domain may be as shown in SEQ ID NO: 4.

[0216] In the present application, the intracellular signaling domain can be selected from the intracellular signaling domain of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, or a domain comprising at least one ITAM. For example, the intracellular signaling domain can be the intracellular signaling domain of CD3ζ.

[0217] For example, the amino acid sequence of the intracellular signaling domain may be as shown in SEQ ID NO:6.

[0218] In the present application, the chimeric antigen receptor can also include an intracellular costimulatory domain. In the present application, the intracellular costimulatory domain can be selected from the intracellular costimulatory domain of the following group of proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligand, CD40 and MyD88. For example, the intracellular costimulatory domain can be the intracellular costimulatory domain of 4-1BB. For example, the intracellular costimulatory domain can be the intracellular costimulatory domain of CD28.

[0219] For example, the amino acid sequence of the intracellular costimulatory domain may be as shown in SEQ ID NO:5.

[0220] In the present application, the chimeric antigen receptor may further comprise a hinge region. In the present application, the hinge region in the chimeric antigen receptor may be selected from the hinge region of the following group of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region in the chimeric antigen receptor may be the hinge region of CD8. For example, the hinge region in the chimeric antigen receptor may be the hinge region of CD28. For example, the hinge region in the chimeric antigen receptor may be the hinge region of IgG4.

[0221] For example, the amino acid sequence of the hinge region in the chimeric antigen receptor can be as shown in SEQ ID NO: 10. For example, the amino acid sequence of the hinge region in the fusion protein can be as shown in SEQ ID NO: 3. For example, the amino acid sequence of the hinge region can be as shown in SEQ ID NO: 13.

[0222] In the present application, the fusion protein may comprise IL-10, a hinge region and a transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.

[0223] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.

[0224] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD28 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.

[0225] In the present application, the fusion protein may comprise IL-10, a CD28 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.

[0226] In the present application, the fusion protein may comprise IL-10, a CD28 hinge region and a CD28 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.

[0227] In the present application, the fusion protein may comprise IL-10, a hinge region and a transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, a hinge region, a transmembrane domain, an intracellular costimulatory domain and an intracellular signaling domain.

[0228] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain.

[0229] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain.

[0230] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, an IgG4 hinge region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain.

[0231] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain.

[0232] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, a CD28 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain.

[0233] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain, an IgG4 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain.

[0234] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain targeting ROR1, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain.

[0235] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor may comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain.

[0236] In the present application, the fusion protein may comprise IL-10, CD8 hinge region and CD8 transmembrane domain, and the chimeric antigen receptor may comprise NKG2D ECD, IgG4 hinge region, CD28 transmembrane domain, 4-1BB intracellular co-stimulatory domain and CD3ζ intracellular signaling domain.

[0237] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, the chimeric antigen receptor may comprise an antigen binding domain targeting ROR1, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain, the antigen binding domain targeting ROR1 comprises VHH, the VHH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 19.

[0238] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, the chimeric antigen receptor may comprise an antigen binding domain targeting GPC3, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain, the antigen binding domain targeting GPC3 comprises an scFv, the scFv comprises VH and VL, the VH comprises HCDR1, HCDR2 and HCDR3, the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 24, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 25, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 26, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 31, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 32, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 33.

[0239] In the present application, the fusion protein may comprise IL-10, CD8 hinge region and CD8 transmembrane domain, and the chimeric antigen receptor may comprise NKG2D ECD, IgG4 hinge region, CD28 transmembrane domain, 4-1BB intracellular co-stimulatory domain and CD3ζ intracellular signaling domain.

[0240] In the present application, the antigen recognition receptor may be TCR.

[0241] In the present application, the TCR may be a modified TCR.

[0242] In the present application, the TCR may be a heterodimer.

[0243] In the present application, the TCR may comprise an α chain and a β chain, wherein the α chain comprises an α chain variable region (TRAV) and an α chain constant region (TRAC), and the β chain comprises a β chain variable region (TRBV) and a β chain constant region (TRBC).

[0244] For example, the TRAV and / or TRBV may comprise three hypervariable regions CDR1, CDR2 and CDR3.

[0245] In the present application, the TRAC can be a mouse or human constant region.

[0246] In the present application, the TRBC can be a mouse or human constant region.

[0247] For example, the TRAC and TRBC can be mouse constant regions.

[0248] For example, the TRAC and TRBC can be human constant regions.

[0249] In the present application, the TCR can specifically bind to the target antigen.

[0250] For example, the TCR can specifically bind NY-ESO-1, MAGEA1, MAGEA3, MAGEA4, MAGEA10, HPV E6, HPV E7, KRAS G12C, KRAS G12D, AFP, HBsAg, mesothelin, PD-1, MART-1, CD19, CD28, CD8, CT83, F8, GPC3, H3 K27M, HLA-A, LMP-2, MC2R, or PRAME.

[0251] For example, the TCR can specifically bind to NY-ESO-1.

[0252] In the present application, the α chain and β chain may include a transmembrane region and a cytoplasmic region.

[0253] In the present application, the fusion protein may comprise IL-10, a hinge region and a transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and TRAC, and the β chain may comprise TRBV and TRBC.

[0254] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and TRAC, and the β chain may comprise TRBV and TRBC.

[0255] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region and a CD28 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and TRAC, and the β chain may comprise TRBV and TRBC.

[0256] In the present application, the fusion protein may comprise IL-10, CD28 hinge region and CD8 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and TRAC, and the β chain may comprise TRBV and TRBC.

[0257] In the present application, the fusion protein may comprise IL-10, CD28 hinge region and CD28 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and TRAC, and the β chain may comprise TRBV and TRBC.

[0258] In the present application, the fusion protein may comprise IL-10, CD8 hinge region and CD8 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and hTRAC, and the β chain may comprise TRBV and hTRBC.

[0259] In the present application, the fusion protein may comprise IL-10, CD8 hinge region and CD8 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and mTRAC, and the β chain may comprise TRBV and mTRBC.

[0260] In the present application, the fusion protein may comprise IL-10, CD8 hinge region and CD8 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and hTRAC, and the β chain may comprise TRBV and mTRBC.

[0261] In the present application, the fusion protein may comprise IL-10, CD8 hinge region and CD8 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and mTRAC, and the β chain may comprise TRBV and hTRBC.

[0262] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region, and a CD8 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and TRAC targeting NY-ESO-1, and the β chain may comprise TRBV and TRBC targeting NY-ESO-1.

[0263] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region, and a CD8 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and hTRAC targeting NY-ESO-1, and the β chain may comprise TRBV and hTRBC targeting NY-ESO-1.

[0264] In the present application, the fusion protein may comprise IL-10, a CD8 hinge region, and a CD8 transmembrane domain, the T cell receptor may comprise an α chain and a β chain, the α chain may comprise TRAV and mTRAC targeting NY-ESO-1, and the β chain may comprise TRBV and mTRBC targeting NY-ESO-1.

[0265] In the present application, the immune cells can promote an immune response.

[0266] For example, the immune cells can kill target cells.

[0267] In the present application, the immune cells may be T cells, NK cells, NKT cells, dendritic cells, macrophages, TIL cells, iNKT cells, CIK cells, γδT cells or DNT cells.

[0268] In the present application, the immune cells are T cells.

[0269] For example, the immune cell may be a T cell.

[0270] For example, the immune cell may be a DN T cell.

[0271] Nucleic acid molecules, vectors and host cells

[0272] On the other hand, the present application provides an isolated nucleic acid molecule encoding the fusion protein. For example, the isolated nucleic acid molecule can encode a complete fusion protein or a portion thereof.

[0273] In another aspect, the present application provides an isolated nucleic acid molecule encoding the fusion protein and the antigen recognition receptor.

[0274] For example, the isolated nucleic acid molecule can encode a complete fusion protein and / or antigen recognition receptor, or a portion thereof. For example, the isolated nucleic acid molecule can encode a fusion protein and an antigen recognition receptor separately. For example, the isolated nucleic acid molecule can encode a fusion protein and an antigen recognition receptor simultaneously.

[0275] For example, the nucleic acid molecule encoding the fusion protein can be located before the nucleic acid molecule encoding the antigen recognition receptor.For example, the nucleic acid molecule encoding the fusion protein can be located after the nucleic acid molecule encoding the antigen recognition receptor.

[0276] In the present application, the nucleic acid molecule may comprise a sequence encoding a self-cleaving peptide.

[0277] In the present application, the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the antigen recognition receptor. For example, the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the CAR. For example, the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the ROR1 CAR. For example, the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the GPC3 CAR. For example, the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the NKG2D CAR. For example, the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the TCR.

[0278] In the present application, the sequence encoding the self-cleavage peptide may be located between the sequences encoding the TCR α chain and β chain.

[0279] In the present application, the self-cleaving peptide may comprise a 2A peptide. For example, the self-cleaving peptide may be P2A, T2A, E2A, or F2A. For example, the self-cleaving peptide may be T2A. For example, the self-cleaving peptide may be GSGP2A.

[0280] In the present application, the nucleic acid molecule may comprise a sequence encoding a signal peptide. For example, the signal peptide may be a CD8a signal peptide. For example, the signal peptide may be an IL-2 signal peptide.

[0281] In the present application, the nucleic acid molecule may comprise the following nucleotide sequences from the 5' end to the 3' end: a gene encoding IL-10, a gene encoding a hinge region, and a gene encoding a transmembrane domain.

[0282] In the present application, the nucleic acid molecule may comprise the following nucleotide sequences from the 5' end to the 3' end: a gene encoding IL-10, a gene encoding the CD8 hinge region, and a gene encoding the CD8 transmembrane domain.

[0283] In the present application, the nucleic acid molecule may comprise the following nucleotide sequences from the 5' end to the 3' end: a gene encoding IL-10, a gene encoding the CD8 hinge region, and a gene encoding the CD28 transmembrane domain.

[0284] In the present application, the nucleic acid molecule may comprise the following nucleotide sequences from the 5' end to the 3' end: a gene encoding IL-10, a gene encoding the CD28 hinge region, and a gene encoding the CD8 transmembrane domain.

[0285] In the present application, the nucleic acid molecule may comprise the following nucleotide sequences from the 5' end to the 3' end: a gene encoding IL-10, a gene encoding the CD28 hinge region, and a gene encoding the CD28 transmembrane domain.

[0286] In the present application, the nucleic acid molecule may contain the following nucleotide sequences from the 5' end to the 3' end: a gene encoding a signal peptide, a gene encoding an antigen recognition receptor, a gene encoding a hinge region, a gene encoding a transmembrane domain, a gene encoding an intracellular co-stimulatory domain, a gene encoding an intracellular signaling domain, a gene encoding a self-cleavage peptide, a gene encoding a signal peptide, a gene encoding IL-10, a gene encoding a hinge region, and a gene encoding a transmembrane domain.

[0287] In the present application, the nucleic acid molecule may comprise the following nucleotide sequences from the 5' end to the 3' end: a gene encoding a signal peptide, a gene encoding IL-10, a gene encoding a hinge region, a gene encoding a transmembrane domain, a gene encoding a self-cleavage peptide, a gene encoding a signal peptide, a gene encoding an antigen recognition receptor, a gene encoding a hinge region, a gene encoding a transmembrane domain, a gene encoding an intracellular co-stimulatory domain, and a gene encoding an intracellular signaling domain.

[0288] In the present application, the nucleic acid molecule may comprise the following nucleotide sequences from the 5' end to the 3' end: a gene encoding a CD8a signal peptide, a gene encoding a ROR1 VHH, a gene encoding a CD28 hinge region, a gene encoding a CD28 transmembrane domain, a gene encoding 4-1BB, a gene encoding CD3ζ, a gene encoding a P2A self-cleavage peptide, a gene encoding a CD8a signal peptide, a gene encoding IL-10, a gene encoding a CD8 hinge region, and a gene encoding a CD8 transmembrane domain.

[0289] In the present application, the nucleic acid molecule may comprise the following nucleotide sequences from the 5' end to the 3' end: a gene encoding a CD8a signal peptide, a gene encoding a GPC3 scFv, a gene encoding a CD8 hinge region, a gene encoding a CD28 transmembrane domain, a gene encoding 4-1BB, a gene encoding CD3ζ, a gene encoding a P2A self-cleavage peptide, a gene encoding a CD8a signal peptide, a gene encoding IL-10, a gene encoding a CD8 hinge region, and a gene encoding a CD8 transmembrane domain.

[0290] In the present application, the nucleic acid molecule may comprise the following nucleotide sequences from the 5' end to the 3' end: a gene encoding a CD8a signal peptide, a gene encoding an NKG2D ECD, a gene encoding an IgG4 hinge region, a gene encoding a CD28 transmembrane domain, a gene encoding 4-1BB, a gene encoding CD3ζ, a gene encoding a P2A self-cleavage peptide, a gene encoding a CD8a signal peptide, a gene encoding IL-10, a gene encoding a CD8 hinge region, and a gene encoding a CD8 transmembrane domain.

[0291] In the present application, the nucleic acid molecule can be produced or synthesized by the following methods: (i) in vitro amplification, such as production by polymerase chain reaction (PCR) amplification, (ii) production by cloning and recombination, (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesis, such as by chemical synthesis. In the present application, the nucleic acid molecule can be DNA and / or RNA. In the present application, the nucleic acid molecule can be an artificially synthesized nucleic acid analog. In the present application, the nucleic acid molecule can be a modified nucleic acid molecule.

[0292] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0293] In the present application, the vector may comprise one or more of the nucleic acid molecules. In the present application, the vector may comprise one or more of the nucleic acid molecules. In the present application, the vector may be an expression vector or a cloning vector. In the present application, the vector may be a viral vector or a non-viral vector. In the present application, the vector may be a viral vector, a plasmid vector, a phage vector or other vectors commonly used in, for example, genetic engineering. For example, the viral vector may be an adenovirus, an adeno-associated virus, a retrovirus (including a lentivirus). In the present application, the vector may be a fusion vector or a non-fusion vector.

[0294] In the present application, the vector may further comprise other genes. For example, the other genes may be marker genes.

[0295] In another aspect, the present application provides a cell comprising the nucleic acid molecule or the vector.

[0296] In the present application, the cell can be a prokaryotic cell (eg, a bacterial cell), a CHO cell, a NS / 0 cell, a HEK293T cell or a HEK293A cell, or other eukaryotic cells, such as a fungal or yeast cell.

[0297] In the present application, the cells may be immune cells.

[0298] For example, the immune cell can be a T cell, an NK cell, an NKT cell, a dendritic cell, a macrophage, a TIL cell, an iNKT cell, a CIK cell, a γδT cell, or a DNT cell.

[0299] In the present application, the cell may comprise one or more of the nucleic acid molecules and / or one or more vectors. In the present application, the vector may comprise one or more of the nucleic acid molecules and / or one or more vectors.

[0300] In the present application, the vector can be introduced into the cell by methods known in the art. For example, the method can be electroporation, lipofectine transfection or lipofectamin transfection.

[0301] In another aspect, the present application provides a method for preparing the fusion protein, comprising transfecting or transducing the nucleic acid molecule into a cell. For example, the DNA sequence of the fusion protein is cloned into the vector and then transfected into an immune cell for expression.

[0302] On the other hand, the present application provides a method for preparing the fusion protein and the antigen recognition receptor, which comprises transfecting or transducing the nucleic acid molecule into a cell.

[0303] In the present application, the DNA sequences of the fusion protein and the antigen recognition receptor are cloned into the vector and then transfected into immune cells for expression.

[0304] Pharmaceutical composition

[0305] On the other hand, the present application provides a pharmaceutical composition comprising the fusion protein, the modified immune cell, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0306] In the present application, the pharmaceutical composition can 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 nontoxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention can include liquid, frozen and lyophilized compositions.

[0307] In this application, the pharmaceutically acceptable carrier 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.

[0308] In the present application, the pharmaceutical composition can include parenteral, percutaneous, intracavitary, intra-arterial, 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.

[0309] In another aspect, the present application provides a method for preparing the fusion protein.

[0310] In another aspect, the present application provides a method for preparing the modified immune cells.

[0311] In another aspect, the present application provides a method for preparing the pharmaceutical composition.

[0312] use

[0313] On the other hand, the present application provides the use of the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease.

[0314] On the other hand, the present application provides a method for preventing and / or treating a disease, comprising administering the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition to a subject in need.

[0315] On the other hand, the present application provides the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition, which are used for preventing and / or treating diseases.

[0316] In the present application, the prevention and / or treatment may be preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing further increase in the severity of the disease and any symptoms associated therewith.

[0317] In the present application, the disease may be a tumor, an autoimmune disease, a severe infection, chronic aging or cardiac fibrosis.

[0318] In the present application, the tumor may be a solid tumor. In the present application, the tumor may be a hematological tumor.

[0319] For example, the tumor can be a ROR1-positive tumor. For example, the tumor can be a GPC3-positive tumor. For example, the tumor can be a tumor that expresses an NKG2D ligand.

[0320] For example, the tumor can be lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, or skin cancer.

[0321] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, modified immune cells, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

[0322] Example

[0323] Experimental Materials

[0324] Example 1 Preparation of cells

[0325] 1.1 Cell culture

[0326] HEK293T, MEC1, and MDA-MB-231 cell lines were obtained from ATCC. The MEC1-ROR1 cell line was generated in-house. The HEK293T cell line was maintained in full culture medium (DMEM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine). The MDA-MB-231, MEC1, and MEC1-ROR1 cell lines were maintained in full culture medium (IMDM containing 10% heat-inactivated FBS, 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 FBS supplemented with 10% DMSO (vol / vol).

[0327] 1.2 Construction of clones

[0328] All relevant gene constructs were constructed and synthesized using GeneWiz. Gene constructs encoding the ROR1 VHH CAR, ROR1 VHH CAR-secreted IL-10, and ROR1 VHH CAR-membrane-bound IL-10 were inserted into the pALD expression plasmid via the SalI and SpeI cloning sites. The structures are shown in Figure 1.

[0329] The structure of ROR1 VHH CAR is: CD8a signal peptide (SEQ ID NO: 2), ROR1 VHH (SEQ ID NO: 1), CD28 hinge region (SEQ ID NO: 3), CD28 transmembrane domain (SEQ ID NO: 4), 4-1BB (SEQ ID NO: 5) and CD3ζ (SEQ ID NO: 6).

[0330] The structure of ROR1 VHH CAR-secretory IL-10 is as follows: GSGP2A (SEQ ID NO: 7) is connected to the IL-2 signal peptide (SEQ ID NO: 8) sequence to induce expression, followed by the IL-10 (SEQ ID NO: 9) sequence.

[0331] The structure of ROR1 VHH CAR-membrane-bound IL-10 is as follows: the CD8a signal peptide sequence is connected to the ROR1 VHH CAR through GSGP2A to induce expression, followed by the IL-10 sequence, and finally the CD8 hinge region (SEQ ID NO: 10) and CD8 transmembrane domain (SEQ ID NO: 11) to localize the expression of IL-10 on the cell membrane.

[0332] 1.3 Preparation of Lentivirus

[0333] All lentiviruses were produced using HEK293T cells. Freshly resuspended HEK293T cells were washed in ice-cold PBS and seeded into 10 cm culture dishes at a cell density of 90%-95% of the dish area. A mixture of lentiviral packaging plasmid and transfection plasmid was mixed with Lipofectamine 2000 / 3000 and transiently transfected into the seeded HEK293T cells. After 48 hours of culture, the supernatant was collected, filtered through a 0.45 μm filter, and aliquoted into 1 ml tubes for storage at -80°C.

[0334] 1.4 T cell activation and transduction

[0335] To transduce PBMCs with lentivirus encoding the corresponding constructs, frozen PBMCs were thawed and placed in 24-well plates. CD3- and CD28-coupled magnetic beads and IL-2 (500 IU / ml) were added for 24 hours of activation. The activated PBMCs were then distributed across multiple wells of the 24-well plates. The corresponding lentivirus and polybrene (8 g / ml) were added and centrifuged at 2000 g for 2 hours in a benchtop centrifuge. The cells were then transferred to a 37°C incubator with 5% CO2. The transfected cells were then cultured in X-VIVO-15 cell culture medium supplemented with IL-2 (500 IU / ml) for 48 hours before subsequent experiments and analysis.

[0336] 1.5 Flow cytometry

[0337] Flow cytometry was performed using a flow cytometer (Beckman Coulter) in plate mode, and data were analyzed using FlowJo software. 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 were plated at 400 cells / mL and placed on ice before staining. Fluorescently conjugated antibodies were added for staining and distributed into the wells of a 96-well round-bottom tissue culture plate. After incubation at 4°C for 45 minutes, 150 μL of FACS buffer was added to each well to wash the cells. After centrifugation at 300 x g for 3 minutes, the supernatant was removed. After washing and resuspending in FACS buffer, the cells were analyzed for ROR1 CAR expression by flow cytometry.

[0338] Experimental results:

[0339] The flow cytometry validation results are shown in Figure 2A. The positive rates for the ROR1 VHH CAR T cell group were 29.8%, the ROR1 VHH CAR-secreted IL-10 group were 15.5%, and the ROR1 VHH CAR-membrane-bound IL-10 group were 20.7%, compared with 2.21% for the untransduced virus T cell group. This indicates that the CAR positive expression rates in the membrane-bound IL-10 and secreted IL-10 groups were higher than those in the untransduced group, and that the CAR positive expression rate in the membrane-bound IL-10 group was higher than that in the secreted IL-10 group.

[0340] The above results show that the membrane-bound IL-10 group has a better CAR positive expression rate, better transfection effect, and higher application potential.

[0341] Example 2 Membrane-bound IL-10 can enhance the activation efficacy of ROR1-CAR-T cells

[0342] 2.1 Co-culture of CAR-T cells and tumor cells

[0343] The activation efficacy of ROR1 VHH CAR T cells, ROR1 VHH CAR-secreting IL-10 T cells, and ROR1 VHH CAR-membrane-bound IL-10 T cells were determined by co-culturing them with tumor cells. PBMCs (1×10 4 ) and negative expression cells (MEC(ROR1-), 10×10 4 ), or with target tumor cells (MEC-ROR1 (expressing ROR1+), MB231 (expressing ROR1+), 10×10 4 ) were co-cultured in a round-bottom 96-well tissue culture plate at a 1:10 ratio of effector cells to target cells. IMDM medium was added to a final volume of 200 μL per well. After centrifugation at 300 x g for 5 minutes, the tissue culture 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.

[0344] 2.2 Enzyme-linked immunosorbent assay

[0345] The IFN-γ (Biolegend, CAT#430104) and IL-2 concentrations (Biolegend, CAT#431804) in the culture supernatant were determined using the ELISA kit. The experimental method was performed according to the instructions provided by the reagent manufacturer. Briefly, after incubation at 37°C for 1 hour using assay diluent buffer, 20 μl of culture supernatant (1:5 dilution concentration) or different concentrations of IFN-γ / IL-2 standard solution were mixed and added to an ELISA plate coated with capture antibody. After incubation with streptavidin-conjugated peroxidase, TMB substrate, and stop solution, the absorbance at 450 nm was measured using a microplate reader. The concentration of IFN-γ / IL-2 in the culture supernatant was calculated based on a standard curve of known standards.

[0346] Experimental results:

[0347] The results of the enzyme-linked immunosorbent assay are shown in Figure 3. As shown in Figure 3A, compared with the negative cell group (CAR-T cells + MEC-1), the concentration of IL-2 secreted by PBMCs in the cell culture medium increased after co-culture of lentiviral-transduced PBMCs with target tumor cells (CAR T cells + MEC-1-ROR1-GFP / MB231-GFP). Among them, the IL-2 secretion concentration of ROR1 VHH CAR-membrane-bound IL-10 T cells co-cultured with target tumor cells was much higher than that of the ROR1 VHH CAR T cell group and the ROR1 VHH CAR-secreting IL-10 T cell group.

[0348] As shown in Figure 3B, compared with the negative cell group (CAR-T cells + MEC-1), the concentration of IFN-γ secreted by PBMC in the cell culture medium increased after co-culture of PBMCs transduced with lentivirus and target tumor cells (CAR T cells + MEC-1-ROR1-GFP / MB231-GFP). Among them, the secretion concentration of IFN-γ by ROR1 VHH CAR-membrane-bound IL-10 T cells and target tumor cells was much higher than that of the ROR1 VHH CAR T cell group and the ROR1 VHH CAR-secreting IL-10 T cell group.

[0349] The above results showed that after co-culture with the two target tumor cells, ROR1 VHH CAR T cells, ROR1 VHH CAR-secreting IL-10T cells and ROR1 VHH CAR-membrane-bound IL-10T cells were activated, and the activation effect of ROR1 VHH CAR-membrane-bound IL-10T cells was the strongest, far higher than that of ROR1 VHH CAR-secreting IL-10T cells.

[0350] Example 3: Membrane-expressed IL-10ROR1 CAR T cells have the strongest cell-killing effect

[0351] 3.1 Co-culture of CAR-T cells and tumor cells

[0352] The tumor killing efficacy of ROR1 VHH CAR T cells, ROR1 VHH CAR-secreting IL-10 T cells, and ROR1 VHH CAR-membrane-bound IL-10 T cells were determined by co-culturing them with tumor cells. PBMC (1×10 4 ) and negative expression cells (MEC-1(ROR1-), 10×10 4 ), or with target tumor cells (MEC-1-ROR1 (expressing ROR1+), MB231 (expressing ROR1+), 10×10 4 ) were co-cultured in a flat-bottom 96-well tissue culture plate at a ratio of effector cells to target cells of 1:10, and IMDM culture medium was added to a final volume of 200 μL per well.

[0353] 3.2 Re-challenge experiment of CAR-T cells and tumor cells

[0354] Incucyte instrument detection method: In the SX1 Live-Cell Analysis System, after incubation at 37°C and 5% CO2 for 48 hours, T cells were challenged after drug resistance. The cell culture plate was centrifuged at 300g for 5 minutes, 100μl of supernatant was taken, and 50k tumor cells were added to the final volume of 200μl. Incubation was continued for 24 hours in the SX1 Live-Cell Analysis System. The cytotoxicity of lentiviral-transfected PBMCs on target tumor cells was analyzed using Incucyte 2022A Rev1 software.

[0355] Experimental results:

[0356] The killing effect on target cells is shown in Figure 4. Figure 4A shows that in the group co-cultured with CAR-T cells and MB231-GFP (expressing ROR1+), compared with the untransduced T cell group, ROR1 VHH CAR T cells, ROR1 VHH CAR-secreting IL-10 T cells, and ROR1 VHH CAR-membrane-bound IL-10 T cells all produced significant tumor killing effects before the re-challenge experiment, and the tumor killing effect of ROR1 VHH CAR-membrane-bound IL-10 T cells was superior to that of ROR1 VHH CAR T cells and ROR1 VHH CAR-secreting IL-10 T cells. After the re-challenge experiment, only ROR1 VHH CAR-membrane-bound IL-10 T cells could still maintain an effective killing effect, and the number of tumor cells continued to decrease.

[0357] Figure 4B shows that in the positive control group (CAR-T cells + MEC-1-ROR1-GFP cells), ROR1 VHH CAR T cells, ROR1 VHH CAR-secreting IL-10 T cells, and ROR1 VHH CAR-membrane-bound IL-10 T cells can all effectively kill target tumor cells, and the tumor killing effect of ROR1 VHH CAR-membrane-bound IL-10 T cells is better than that of ROR1 VHH CAR T cells and ROR1 VHH CAR-secreting IL-10 T cells. After the re-challenge test, there was no significant difference in the killing effect of the three CAR-T cells.

[0358] Figure 4C shows that in the negative cell group (CAR-T cells + MEC-1-GFP cells), the number of tumor cells in the transduced PBMC group did not change significantly compared with the non-transduced group, and the CAR-T cells failed to produce a significant cell-killing effect. After the re-challenge test, the three CAR-T cells also did not produce a significant killing effect.

[0359] The above results show that 48 hours after the initial culture, ROR1 VHH CAR-membrane-bound IL-10T cells can still maintain an effective killing effect, indicating that the efficacy of membrane-bound IL-10 is much higher than that of secreted IL-10. Membrane-bound IL-10 can give T cells stronger anti-tumor ability, greatly improving the T cells' ability to kill tumor cells and their ability to continuously kill tumor cells.

[0360] Example 4 Activation and killing efficacy of membrane-expressed IL-10ROR1 CAR-T cells

[0361] 4.1 Co-culture of CAR-T cells with HCC827 and A375 tumor cells

[0362] The activation efficacy of ROR1 VHH CAR T cells and membrane-bound IL-10 T cells was determined by co-culturing them with HCC827 and A375 tumor cells. PBMCs (0.25×10 4 ) and negative expression cells (293T (ROR1-), 1×10 4 ), or with target tumor cells (HCC827 (expressing ROR1+), A375 (expressing ROR1+), 1×10 4 ) were co-cultured in a round-bottom 96-well tissue culture plate at a 1:4 ratio of effector cells to target cells. IMDM medium was added to a final volume of 200 μL per well. After centrifugation at 300 x g for 5 minutes, the tissue culture plate was incubated at 3°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.

[0363] 4.2 Enzyme-linked immunosorbent assay

[0364] IFN-γ in culture supernatants was determined using an EILSA kit (Biolegend, CAT#430104). The experimental method was carried out according to the instructions provided by the reagent manufacturer. Briefly, after incubation at 37°C for 1 hour using assay diluent buffer, 20 μl of culture supernatant (1:5 dilution concentration) or IFN-γ standard solution of different concentrations were mixed and added to an ELISA plate coated with capture antibody. After incubation with streptavidin-conjugated peroxidase, TMB substrate and stop solution, the absorbance at 450 nm was measured using a microplate reader. The concentration of IFN-γ in the culture supernatant was calculated based on a standard curve of known standards.

[0365] Experimental results:

[0366] The results of the enzyme-linked immunosorbent assay are shown in Figure 5. As shown in Figure 5, compared with the negative cell group (CAR-T cells + Hek-293T), the concentration of IFN-γ secreted by PBMCs in the cell culture medium increased after co-culture of PBMCs transduced with lentivirus and target tumor cells (CAR-T cells + A375-GFP / HCC827-GFP). Among them, the secretion concentration of IFN-γ after co-culture of ROR1 VHH-CAR-membrane-bound IL-10-T cells and target tumor cells was much higher than that of the ROR1 VHH-CAR-T cell group.

[0367] The experimental results showed that after co-culture with the two target tumor cells, ROR1 VHH CAR T cells and ROR1 VHH CAR-membrane-bound IL-10T cells were activated. The activation effect of ROR1 VHH CAR-membrane-bound IL-10T cells was the strongest, far higher than that of ROR1 VHH CAR T cells. The membrane-expressed IL-10ROR1 CAR described in this application has strong activation and killing efficacy after being used for T cells, and can effectively kill ROR1+ cell lines such as A549 and HCC827.

[0368] Example 5: Membrane-expressed IL-10ROR1 CAR-T cells have the strongest cell-killing effect on HCC827 / A375 cell lines

[0369] 5.1 Co-culture of CAR-T cells and tumor cells

[0370] The tumor killing efficacy of ROR1 VHH CAR T cells and ROR1 VHH CAR-membrane-bound IL-10 T cells was determined by co-culturing them with tumor cells. PBMC (0.25×10 4 ) and negative expression cells (293T (ROR1-), 1×10 4 ), or with target tumor cells (HCC827 (expressing ROR1+), A375 (expressing ROR1+), 1×10 4 ) were co-cultured in a flat-bottom 96-well tissue culture plate at a ratio of effector cells to target cells of 1:4, and IMDM culture medium was added to a final volume of 200 μL per well.

[0371] 5.2 Re-challenge experiment of CAR-T cells and tumor cells

[0372] Incucyte instrument detection method: In the SX1 Live-Cell Analysis System, after incubation at 37°C and 5% CO2 for 48 hours, T cells were challenged after drug resistance. The cell culture plate was centrifuged at 300g for 5 minutes, 100μl of supernatant was taken, and 5k tumor cells were added to a final volume of 200μl. Incubation was continued for 24 hours in the SX1 Live-Cell Analysis System. The cytotoxicity of lentiviral-transfected PBMCs on target tumor cells was analyzed using Incucyte 2022A Rev1 software.

[0373] Experimental results:

[0374] The killing effect on target cells is shown in Figures 6-8. Figure 6 shows that in the group co-cultured with CAR-T cells and A375-GFP (expressing ROR1+), compared with the untransduced T cell group, only the ROR1 VHH CAR-membrane-bound IL-10 T cells produced a significant tumor killing effect before the re-challenge experiment. After the re-challenge experiment, the ROR1 VHH CAR was able to produce an inhibitory effect on A375-GFP cells, while the ROR1 VHH CAR-membrane-bound IL-10 T cells still maintained a killing effect far superior to that of ROR1 VHH CAR T cells, and the number of tumor cells continued to decrease.

[0375] Figure 7 shows that in the positive control group (CAR-T cells + HCC827-GFP cells), in the co-culture group, compared with the untransduced T cell group, only the ROR1 VHH CAR-membrane-bound IL-10 T cells produced a significant tumor killing effect before the re-challenge experiment. After the re-challenge experiment, the ROR1 VHH CAR was able to inhibit HCC827-GFP cells, and the ROR1 VHH CAR-membrane-bound IL-10 T cells still maintained a killing effect far higher than that of ROR1 VHH CAR T cells, and the number of tumor cells continued to decline.

[0376] Figure 8 shows that in the negative cell group (CAR-T cells + HEK-293T cells), the number of tumor cells in the transduced PBMC group did not change significantly compared with the non-transduced group, and the CAR-T cells failed to produce a significant cell-killing effect. After the re-challenge test, the two CAR-T cells also did not produce a significant killing effect.

[0377] The above results show that 48 hours after the initial culture, ROR1 VHH CAR-membrane-bound IL-10T cells can still maintain an effective killing effect, indicating that membrane-bound IL-10 can give ROR1 VHH CAR-T cells stronger anti-tumor ability, greatly improving the T cells' ability to kill tumor cells and their ability to continuously kill tumor cells.

[0378] Example 6 Functional Comparison of GPC3 scFv CAR T Cells and GPC3 scFv CAR-Membrane-Bound IL-10 T Cells

[0379] All relevant gene constructs were constructed and synthesized using GeneWiz. The gene constructs encoding GPC3 scfv CAR and GPC3 scfv CAR-membrane-bound IL-10 were inserted into the expression plasmid via the Xho1 and SpeI cloning sites. The structures are shown in Figure 9.

[0380] The structure of GPC3 scFv CAR is: CD8a signal peptide, GPC3scFv (SEQ ID NO: 12), CD8 hinge region, CD28 transmembrane domain, 4-1BB and CD3ζ.

[0381] The structure of GPC3 scfv CAR-membrane-bound IL-10 is as follows: the CD8a signal peptide sequence is connected to GSGP2A after GPC3 scfv CAR to induce expression, followed by the IL-10 sequence, and finally the CD8 hinge region and CD8 transmembrane domain are connected to localize the expression of IL-10 on the cell membrane.

[0382] 6.1. Flow cytometry assay to test the expression of GPC3 scFv CAR and GPC3 scFv CAR-membrane-bound IL-10 on PBMC cells

[0383] PBMCs that had been preactivated 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 6 12-well tissue culture plates were filled with GPC3 scFv CAR, GPC3 scFv CAR-membrane-bound IL-10 encoding, or control lentivirus. After 24 hours of incubation, the culture supernatant was removed and 2.0 mL of fresh X-VIVO culture medium was added to each well to resuspend the lentiviral-transduced PBMC cells. After an additional 72 hours of incubation at 37°C and 5% CO2, 0.3 × 10 6 The cells were then centrifuged at 1300 rpm for five minutes and resuspended in 100 μl of FASC buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2 mM EDTA, pH 7.0). Anti-CD3-PB antibody staining was used to isolate T cells, and GPC3-FITC protein was used to detect the expression of GPC3 CAR. Flow cytometry was then performed using a flow cytometer (Beckman Coulter) in plate mode, and the data were analyzed using FlowJo software.

[0384] Experimental results:

[0385] The flow cytometry results are shown in FIG10 . Compared with the non-transduced virus T cell group, the positive rate of the GPC3 CAR group was 60%, and the positive rate of the GPC3 scfv CAR-membrane-bound IL-10 group was 82.8%.

[0386] The above results show that membrane-bound IL-10 has a good CAR positive expression rate after combining with different CARs, has a good transfection effect, and has high application potential.

[0387] 6.2. Membrane-bound IL-10 can enhance T cell activation by GPC3 scFv CAR

[0388] 6.2.1 Co-culture of CAR-T cells and tumor cells

[0389] The activation efficacy of GPC3 scFv CAR T cells, GPC3 scFv CAR-membrane-bound IL-10 and tumor cells was determined by co-culturing them. PBMC (0.3×10 4 ) and negative expression cells (MB231(GPC3-HBSAG-), 1.5×10 4 , or with target tumor cells (Huh7 (expressing GPC3+HBSAG+), Hep3B (expressing GPC3+HBSAG+), 1.5×10 4 ) were co-cultured in a round-bottom 96-well tissue culture plate at a 1:5 ratio of effector cells to target cells. IMDM medium was added to a final volume of 200 μL per well. After centrifugation at 300 x g for 5 minutes, the tissue culture 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.

[0390] 6.2.2 Enzyme-linked immunosorbent assay

[0391] IFN-γ in culture supernatants was determined using the EILSA kit (Biolegend, CAT#430104). The experimental method was carried out according to the instructions provided by the reagent manufacturer. Briefly, after incubation at 37°C for 1 hour using assay diluent buffer, 20 μl of culture supernatant (1:5 dilution concentration) or different concentrations of IFN-γ / IL-2 standard solution were mixed and added to an ELISA plate coated with capture antibody. After incubation with streptavidin-conjugated peroxidase, TMB substrate and stop solution, the absorbance at 450 nm was measured using a microplate reader. The concentration of IFN-γ in the culture supernatant was calculated based on a standard curve of known standards.

[0392] Experimental results:

[0393] The results of the enzyme-linked immunosorbent assay are shown in Figure 11. Compared with the negative cell group (CAR-T cells + mb231), the concentration of IFN-γ secreted by PBMC in the cell culture medium increased after co-culture of PBMCs transduced with lentivirus and target tumor cells (CAR T cells + Huh7 / hep3b). Among them, the secretion concentration of IFN-γ by GPC3 scfv CAR-membrane-bound IL-10 cells and target tumor cells was higher than that of the GPC3 scfv CAR T cell group.

[0394] The above results show that after co-culture with target tumor cells, T cells in the GPC3 scfv CAR-membrane-bound IL-10 group can be effectively activated, and membrane-bound IL-10 has a strong effect of activating T cells after combining with different CARs.

[0395] 6.3. Membrane-bound IL-10 can enhance the tumor killing effect of GPC3 scFv CAR

[0396] 6.3.1 Co-culture of CAR-T cells and tumor cells

[0397] The tumor killing efficacy of GPC3 scFv CAR T cells and GPC3 scFv CAR-membrane-bound IL-10 T cells was determined by co-culturing them with tumor cells. PBMC (0.3×10 4 ) and negative expression cells (MB231(GPC3-HBSAG-), 1.5×10 4 , or with target tumor cells (Huh7 (expressing GPC3+HBSAG+), Hep3B (expressing GPC3+HBSAG+), 1.5×10 4 ) were co-cultured in a round-bottom 96-well tissue culture plate with a ratio of effector cells to target cells of 1:5, and IMDM culture medium was added to a final volume of 200 μL per well.

[0398] 6.3.2 Rechallenge Experiment of CAR-T Cells and Tumor Cells (Incucyte Instrument Detection Method)

[0399] Insert In the SX1 Live-Cell Analysis System, T cells were incubated at 37°C and 5% CO2 for 48 hours before being challenged. The cell culture plate was centrifuged at 300g for 5 minutes, 100μl of the supernatant was collected, and 5k tumor cells were added to a final volume of 200μl. Incubation was continued for 24 hours in the SX1 Live-Cell Analysis System. The cytotoxicity of lentiviral-transfected PBMCs on target tumor cells was analyzed using Incucyte 2022A Rev1 software.

[0400] Experimental results:

[0401] The killing effect on target cells is shown in Figures 12-14. Figures 12-13 show that in the co-culture group of CAR-T cells and Huh7-GFP (expressing GPC3+HBSAG+) / Hep3B-GFP (expressing GPC3+HBSAG+), compared with the untransduced T cell group, the GPC3 scfv CAR T cells and GPC3 scfv CAR-membrane-bound IL-10 T cell groups both produced significant tumor killing effects before the re-challenge experiment. After two tumor cell re-challenges (5×10 3 After 10 min of treatment (10 min / 10 min), both the GPC3 scfv CAR and GPC3 scfv CAR-membrane-bound IL-10 cell groups showed effective killing of tumor cells, among which the GPC3 scfv CAR-membrane-bound IL-10 cell group had a faster killing rate than the GPC3 scfv CAR T cell group.

[0402] Figure 14 shows that in the negative cell group (CAR-T cells + MB231-GFP cells), the number of tumor cells in the transduced PBMC group did not change significantly compared with the non-transduced group, and the CAR-T cells failed to produce a significant cell-killing effect. After the re-challenge test, the two CAR-T cells also did not produce a significant killing effect.

[0403] The above results show that GPC3 scfv CAR-membrane-bound IL-10T cells can still maintain an effective killing effect, indicating that membrane-bound IL-10 has stronger anti-tumor ability after combining with different CARs, greatly improving the T cell's ability to kill tumor cells and its ability to continuously kill tumor cells.

[0404] Example 7 Functional Comparison of NKG2D ECD CAR T Cells and NKG2D ECD CAR-Membrane-Bound IL-10 T Cells

[0405] All relevant gene constructs were constructed and synthesized using GeneWiz. The gene constructs encoding the NKG2DECD CAR and NKG2D ECD CAR-membrane-bound IL-10 were inserted into the expression plasmid via the Xho1 and SpeI cloning sites. The structure is shown in Figure 15.

[0406] The structure of NKG2D ECD CAR is: CD8a signal peptide, NKG2D ECD (SEQ ID NO: 14), IgG4 hinge region (SEQ ID NO: 13), CD28 transmembrane domain, 4-1BB and CD3ζ.

[0407] The structure of NKG2D ECD CAR-membrane-bound IL-10 is as follows: the CD8a signal peptide sequence is connected to GSGP2A after NKG2D ECD CAR to induce expression, followed by the IL-10 sequence, and finally the CD8 hinge region and CD8 transmembrane domain are connected to localize the expression of IL-10 on the cell membrane.

[0408] 7.1. Flow cytometry experiments were used to test the expression of NKG2D ECD CAR and NKG2D ECD CAR-membrane-bound IL-10 on PBMC cells.

[0409] 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 6 12-well tissue culture plates were filled with NKG2D ECD CAR, NKG2D ECD CAR-membrane-bound IL-10 encoding, or control lentivirus were added. After 24 hours of incubation, the culture supernatant was removed and 2.0 mL of fresh X-VIVO culture medium was added to each well to resuspend the lentiviral-transduced PBMC cells. After another 72 hours of incubation at 37°C and 5% CO2, 0.3×10 6 The cells were then centrifuged at 1300 rpm for five minutes and resuspended in 100 μl of FASC buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2 mM EDTA, pH 7.0). Anti-CD3-PB antibody was used to isolate T cells, and Flag-FITC protein was used to detect NKG2D ECD CAR expression. Flow cytometry was then performed using a flow cytometer (Beckman Coulter) in plate mode, and data were analyzed using FlowJo software.

[0410] Experimental results:

[0411] The flow cytometry results are shown in FIG16 . Compared with the non-virus-transduced T cell group, the positive rate of the NKG2D ECD CAR group was 51%, and the positive rate of the NKG2D ECD CAR-membrane-bound IL-10 group was 38.8%.

[0412] The above results show that membrane-bound IL-10 has a good positive expression rate after binding to different antigen recognition receptors, has a good transfection effect, and has high application potential.

[0413] 7.2. Membrane-bound IL-10 can enhance the activation of NKG2D ECD CAR-T cells

[0414] 7.2.1 Co-culture of CAR-T cells and tumor cells

[0415] The activation efficacy of NKG2D ECD CAR-T cells, NKG2D ECD CAR-membrane-bound IL-10 and tumor cells was determined by co-culturing them. PBMC (0.3×10 4 ) and negative expression cells (Huh7 (MIC A / B-), 1.5×10 4 , or with target tumor cells (MB231-GFP (expressing Huh7 (MIC A / B+), A375-GFP (expressing Huh7 (MIC A / B-), 1.5×10 4 ) were co-cultured in a round-bottom 96-well tissue culture plate at a 1:5 ratio of effector cells to target cells. IMDM medium was added to a final volume of 200 μL per well. After centrifugation at 300 x g for 5 minutes, the tissue culture 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.

[0416] 7.2.2 Enzyme-linked immunosorbent assay

[0417] IFN-γ in culture supernatants was determined using the EILSA kit (Biolegend, CAT#430104). The experimental method was carried out according to the instructions provided by the reagent manufacturer. Briefly, after incubation at 37°C for 1 hour using assay diluent buffer, 20 μl of culture supernatant (1:5 dilution concentration) or different concentrations of IFN-γ / IL-2 standard solution were mixed and added to an ELISA plate coated with capture antibody. After incubation with streptavidin-conjugated peroxidase, TMB substrate and stop solution, the absorbance at 450 nm was measured using a microplate reader. The concentration of IFN-γ in the culture supernatant was calculated based on a standard curve of known standards.

[0418] Experimental results:

[0419] The results of the enzyme-linked immunosorbent assay are shown in Figure 17. Compared with the negative cell group (CAR-T cells + Huh7), the concentration of IFN-γ secreted by PBMCs in the cell culture medium increased after co-culture of lentiviral-transduced PBMCs with target tumor cells (CAR T cells + MB231 / A375). Among them, the secretion concentration of IFN-γ by NKG2D ECD CAR-membrane-bound IL-10 cells and target tumor cells was higher than that in the NKG2D ECD CAR T cell group.

[0420] The above results show that after co-culture with target tumor cells, the NKG2D ECD CAR-membrane-bound IL-10 group T cells can be effectively activated, among which membrane-bound IL-10 has a strong effect of activating T cells after binding to different antigen recognition receptors.

[0421] 7.3. Membrane-expressed interleukin-10 enhances the tumor-killing effect of NKG2D ECD CAR

[0422] 7.3.1 Co-culture of CAR-T cells and tumor cells

[0423] The tumor killing efficacy of NKG2D ECD CAR T cells and NKG2D ECD CAR-membrane-bound IL-10 T cells was determined by co-culturing them with tumor cells. PBMC (0.3×10 4 ) and negative expression cells (Huh7 (MIC A / B-), 1.5×10 4 , or with target tumor cells (MB231-GFP (expressing Huh7 (MIC A / B+), A375-GFP (expressing Huh7 (MIC A / B-), 1.5×10 4 ) were co-cultured in a round-bottom 96-well tissue culture plate with a ratio of effector cells to target cells of 1:5, and IMDM culture medium was added to a final volume of 200 μL per well.

[0424] 7.3.2 Rechallenge Experiment of CAR-T Cells and Tumor Cells (Incucyte Instrument Detection Method)

[0425] Insert In the SX1 Live-Cell Analysis System, T cells were incubated at 37°C and 5% CO2 for 48 hours before being challenged. The cell culture plate was centrifuged at 300g for 5 minutes, 100μl of the supernatant was collected, and 5k tumor cells were added to a final volume of 200μl. Incubation was continued for 24 hours in the SX1 Live-Cell Analysis System. The cytotoxicity of lentiviral-transfected PBMCs on target tumor cells was analyzed using Incucyte 2022A Rev1 software.

[0426] Experimental results:

[0427] The killing effect on target cells is shown in Figures 18-20. Figure 18 shows that in the co-culture group of CAR-T cells and MB231-GFP (expressing MICA / B+), compared with the untransduced T cell group, only the NKG2D ECD CAR-membrane-bound IL-10T cell group produced a significant tumor killing effect.

[0428] Figure 19 shows that in the CAR-T cell and A375-GFP (expressing MICA / B+) co-culture group (Figure 18), compared with the non-transduced T cell group, the NKG2D ECD CAR T cell and NKG2D ECD CAR-membrane-bound IL-10 T cell groups produced significant tumor suppression effects before the re-challenge experiment. 3 After 48 hours of treatment (10 min / 10 min), both NKG2D ECD CAR T cells and NKG2D ECD CAR-membrane-bound IL-10T cells showed a tumor cell-killing effect, among which NKG2D ECD CAR-membrane-bound IL-10T had a stronger cell-killing ability.

[0429] Figure 20 shows that in the negative cell group (CAR-T cells + Huh7-GFP cells), the number of tumor cells in the transduced PBMC group did not change significantly compared with the non-transduced group, and the CAR-T cells failed to produce obvious cell killing effects. After the re-challenge test, the two CAR-T cells also did not produce obvious killing effects.

[0430] The above results show that T cells in the NKG2D ECD CAR-membrane-bound IL-10 group can still maintain an effective killing effect, indicating that membrane-bound IL-10 has stronger anti-tumor ability after combining with different CARs, greatly improving the T cell's ability to kill tumor cells and its ability to continuously kill tumor cells.

Claims

1. A fusion protein comprising IL-10 and a transmembrane domain, wherein the fusion protein is expressed on immune cells, and the immune cells do not include regulatory T cells.

2. The fusion protein according to claim 1, wherein the IL-10 is human IL-10.

3. The fusion protein according to any one of claims 1-2, wherein the amino acid sequence of the IL-10 is as shown in SEQ ID NO:

9.

4. The fusion protein according to any one of claims 1-3, wherein the transmembrane domain is selected from the transmembrane domains of the following group of proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM.

5. The fusion protein according to any one of claims 1-4, wherein the transmembrane domain is the transmembrane domain of CD8.

6. The fusion protein according to any one of claims 1-5, wherein the amino acid sequence of the transmembrane domain is as shown in SEQ ID NO:

11.

7. The fusion protein according to any one of claims 1-6, wherein the fusion protein comprises IL-10 and the CD8 transmembrane domain.

8. The fusion protein according to any one of claims 1-7, wherein the IL-10 and the transmembrane domain are indirectly linked.

9. The fusion protein according to any one of claims 1-8, wherein the fusion protein further comprises a hinge region.

10. The fusion protein according to claim 9, wherein the IL-10 and the transmembrane domain are linked by a hinge region.

11. The fusion protein according to any one of claims 9-10, wherein the hinge region is selected from the hinge regions of the following group of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 or LIGHT.

12. The fusion protein according to any one of claims 9-11, wherein the hinge region is the hinge region of CD8.

13. The fusion protein according to any one of claims 9-12, wherein the amino acid sequence of the hinge region is as shown in SEQ ID NO:

10.

14. The fusion protein according to any one of claims 9-13, wherein the fusion protein comprises IL-10, a CD8 hinge region, and a CD8 transmembrane domain.

15. The fusion protein according to any one of claims 1-14, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

16.

16. The fusion protein according to any one of claims 1-15, wherein the immune cells promote an immune response.

17. The fusion protein according to any one of claims 1-16, wherein the immune cells are T cells, NK cells, iNKT cells, CIK cells, γδT cells, or double-negative T (DNT) cells.

18. The fusion protein according to any one of claims 1-17, wherein the fusion protein is used for treating tumors, autoimmune diseases, severe infections, chronic aging, or cardiac fibrosis.

19. The fusion protein according to any one of claims 1-18, wherein the fusion protein is used for adoptive cell therapy (ACT).

20. The fusion protein according to claim 19, wherein the ACT therapy is CAR-T therapy, TCR-T therapy, TIL therapy, NK therapy, iNKT therapy, CAR-NK therapy, CAR-iNKT therapy, γδT therapy, DNT therapy, or CAR-DNT therapy.

21. The fusion protein according to any one of claims 1-20, wherein the fusion protein is co-expressed with an antigen recognition receptor.

22. The fusion protein according to claim 21, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

23. The fusion protein according to any one of claims 21-22, wherein the antigen recognition receptor specifically binds to a target antigen.

24. The fusion protein according to claim 23, wherein the target antigen is an antigen related to tumors, autoimmune diseases, severe infections, chronic aging, or cardiac fibrosis.

25. The fusion protein according to any one of claims 1-24, wherein the fusion protein is co-expressed with a CAR.

26. The fusion protein according to claim 25, wherein the CAR targets ROR1, mesothelin, CD19, CD22, DLL3, GPC3, GPA33, B7-H3, EGFR, IL-13Rα2, FAP, TREM, Claudin 18.2, CDH17, B7H3, MUC1, GD2, CEA, CLL1, c-Met, or HER2.

27. The fusion protein according to any one of claims 1-26, wherein the fusion protein is co-expressed with a CAR targeting ROR1.

28. The fusion protein according to any one of claims 1-27, wherein the fusion protein is co-expressed with a CAR targeting GPC3.

29. The fusion protein according to any one of claims 1-28, wherein the fusion protein is co-expressed with an NKG2D CAR.

30. A modified immune cell, comprising the fusion protein according to any one of claims 1-29 and an antigen recognition receptor, wherein the immune cell does not include regulatory T cells.

31. The modified immune cell according to claim 30, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

32. The modified immune cell according to any one of claims 30-31, wherein the antigen recognition receptor specifically binds to a target antigen.

33. The modified immune cell according to claim 32, wherein the target antigen is an antigen related to tumor, autoimmune disease, severe infection, chronic aging or cardiac fibrosis.

34. The modified immune cell according to any one of claims 30-33, wherein the antigen recognition receptor is a CAR.

35. The modified immune cell according to claim 34, wherein the CAR comprises an antigen-binding domain, a transmembrane domain and an intracellular signaling domain.

36. The modified immune cell according to claim 35, wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof.

37. The modified immune cell according to claim 36, wherein the antigen-binding fragment is a Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH or dAb fragment.

38. The modified immune cell according to any one of claims 35-17, wherein the antigen-binding domain is the extracellular domain (ECD) of NKG2D.

39. The modified immune cell according to any one of claims 35-38, wherein the antigen-binding domain specifically binds to a target antigen.

40. The modified immune cell according to claim 39, wherein the target antigen is ROR1, mesothelin, CD19, CD22, DLL3, GPC3, GPA33, B7-H3, EGFR, IL-13Rα2, FAP, TREM, Claudin 18.2, CDH17, B7H3, MUC1, GD2, CEA, CLL1, c-Met or HER2.

41. The modified immune cell according to any one of claims 39-40, wherein the target antigen is ROR1.

42. The modified immune cell according to any one of claims 39-41, wherein the target antigen is GPC3.

43. The modified immune cell according to any one of claims 35-42, wherein the antigen-binding domain comprises a VHH, the VHH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:17, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:18, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:

19.

44. The modified immune cell according to any one of claims 35-43, wherein the antigen-binding domain comprises a VHH, and the amino acid sequence of the VHH is as shown in SEQ ID NO:

1.

45. The modified immune cell according to any one of claims 35-44, wherein the antigen-binding domain comprises a scFv, the scFv comprises a VH and a VL, the VH comprises HCDR1, HCDR2 and HCDR3, the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:24, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:25, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:26, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:31, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:32, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:

33.

46. The modified immune cell according to any one of claims 35-45, wherein the antigen-binding domain comprises a scFv, the scFv comprises a VH and a VL, the amino acid sequence of the VH is as shown in SEQ ID NO:30, and the amino acid sequence of the VL is as shown in SEQ ID NO:

38.

47. The modified immune cell according to any one of claims 35-46, wherein the antigen-binding domain comprises a scFv, and the amino acid sequence of the scFv is as shown in SEQ ID NO:

12.

48. The modified immune cell according to any one of claims 35-47, wherein the antigen-binding domain comprises an NKG2D ECD, and the amino acid sequence of the NKG2D ECD is as shown in SEQ ID NO:

14.

49. The modified immune cell according to any one of claims 35-48, wherein the transmembrane domain in the chimeric antigen receptor is selected from the transmembrane domains of the following group of proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM.

50. The modified immune cell according to any one of claims 35-49, wherein the transmembrane domain in the chimeric antigen receptor is the transmembrane domain of CD28.

51. The modified immune cell according to any one of claims 35-50, wherein the intracellular signaling domain is selected from the intracellular signaling domains of the following group of proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, or a domain comprising at least one ITAM.

52. The fusion protein according to any one of claims 35-51, wherein the intracellular signaling domain is the intracellular signaling domain of CD3ζ.

53. The modified immune cell according to any one of claims 35-52, wherein the chimeric antigen receptor further comprises an intracellular co-stimulatory domain.

54. The modified immune cell according to claim 53, wherein the intracellular co-stimulatory domain is selected from the intracellular co-stimulatory domains of the following group of proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, the ligand of CD83, CD40, and MyD88.

55. The modified immune cell according to any one of claims 53-54, wherein the intracellular co-stimulatory domain is the intracellular co-stimulatory domain of 4-1BB.

56. The modified immune cell according to any one of claims 35-55, wherein the chimeric antigen receptor further comprises a hinge region.

57. The modified immune cell according to claim 56, wherein the hinge region in the chimeric antigen receptor is selected from the hinge regions of the following group of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

58. The modified immune cell according to any one of claims 56-57, wherein the hinge region in the chimeric antigen receptor is the hinge region of CD28, CD8, or IgG4.

59. The modified immune cell according to any one of claims 30-58, wherein the modified immune cell comprises a fusion protein and a chimeric antigen receptor, the fusion protein comprises IL-10, a hinge region and a transmembrane domain, and the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane domain and an intracellular signaling domain.

60. The modified immune cell according to any one of claims 30-59, wherein the modified immune cell comprises a fusion protein and a chimeric antigen receptor, the fusion protein comprises IL-10, a CD8 hinge region and a CD8 transmembrane domain, and the chimeric antigen receptor comprises an antigen-binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signaling domain.

61. The modified immune cell according to any one of claims 30-60, wherein the immune cell promotes an immune response.

62. The modified immune cell according to any one of claims 30-61, wherein the immune cell is a T cell, a NK cell, an iNKT cell, a CIK cell, a γδT cell or a DNT cell.

63. The modified immune cell according to any one of claims 30-62, wherein the immune cell is a T cell.

64. An isolated nucleic acid molecule encoding the fusion protein according to any one of claims 1-29.

65. The nucleic acid molecule according to claim 64, which further encodes an antigen recognition receptor.

66. The nucleic acid molecule according to claim 65, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

67. The nucleic acid molecule according to any one of claims 65-66, wherein the antigen recognition receptor specifically binds to a target antigen.

68. The nucleic acid molecule according to claim 67, wherein the target antigen is an antigen associated with a tumor, an autoimmune disease, a severe infection, chronic aging or cardiac fibrosis.

69. The nucleic acid molecule according to any one of claims 65-68, wherein the antigen recognition receptor is a CAR.

70. The nucleic acid molecule according to claim 69, wherein the CAR comprises an antigen-binding domain, a transmembrane domain and an intracellular signaling domain.

71. The nucleic acid molecule according to claim 70, wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof 72. The nucleic acid molecule according to claim 71, wherein the antigen-binding fragment is a Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH or dAb fragment.

73. The nucleic acid molecule according to any one of claims 70-72, wherein the antigen-binding domain is the extracellular domain (ECD) of NKG2D.

74. The nucleic acid molecule according to any one of claims 70-63, wherein the antigen-binding domain specifically binds to a target antigen.

75. The nucleic acid molecule according to claim 74, wherein the target antigen is ROR1, mesothelin, CD19, CD22, DLL3, GPC3, GPA33, B7-H3, EGFR, IL-13Rα2, FAP, TREM, Claudin 18.2, CDH17, B7H3, MUC1, GD2, CEA, CLL1, c-Met or HER2.

76. The nucleic acid molecule according to any one of claims 74-75, wherein the target antigen is ROR1.

77. The nucleic acid molecule according to any one of claims 74-76, wherein the target antigen is GPC3.

78. The nucleic acid molecule according to any one of claims 70-77, wherein the antigen-binding domain comprises a VHH, the VHH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:17, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:18, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:

19.

79. The nucleic acid molecule according to any one of claims 70-78, wherein the antigen-binding domain comprises a VHH, and the amino acid sequence of the VHH is as shown in SEQ ID NO:

1.

80. The nucleic acid molecule according to any one of claims 70-79, wherein the antigen-binding domain comprises a scFv, the scFv comprises VH and VL, the VH comprises HCDR1, HCDR2 and HCDR3, the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:24, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:25, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:26, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:31, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:32, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:

33.

81. The nucleic acid molecule according to any one of claims 70-80, wherein the antigen-binding domain comprises a scFv, the scFv comprises VH and VL, the amino acid sequence of VH is as shown in SEQ ID NO:30, and the amino acid sequence of VL is as shown in SEQ ID NO:

38.

82. The nucleic acid molecule according to any one of claims 70-81, wherein the antigen-binding domain comprises a scFv, and the amino acid sequence of the scFv is as shown in SEQ ID NO:

12.

83. The nucleic acid molecule according to any one of claims 70-82, wherein the antigen-binding domain comprises NKG2D ECD, and the amino acid sequence of the NKG2D ECD is as shown in SEQ ID NO:

14.

84. The nucleic acid molecule according to any one of claims 70-83, wherein the transmembrane domain in the chimeric antigen receptor is selected from the transmembrane domains of the following group of proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM.

85. The nucleic acid molecule according to any one of claims 70-84, wherein the transmembrane domain in the chimeric antigen receptor is the transmembrane domain of CD28.

86. The nucleic acid molecule according to any one of claims 70-85, wherein the intracellular signaling domain is selected from the intracellular signaling domains of the following group of proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12 or a domain comprising at least one ITAM.

87. The nucleic acid molecule according to any one of claims 70-86, wherein the intracellular signaling domain is the intracellular signaling domain of CD3ζ.

88. The nucleic acid molecule according to any one of claims 70-87, wherein the chimeric antigen receptor further comprises an intracellular co-stimulatory domain.

89. The nucleic acid molecule according to claim 88, wherein the intracellular co-stimulatory domain is selected from the intracellular co-stimulatory domains of the following group of proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, the ligand of CD83, CD40 and MyD88.

90. The nucleic acid molecule according to any one of claims 88-89, wherein the intracellular co-stimulatory domain is the intracellular co-stimulatory domain of 4-1BB.

91. The nucleic acid molecule according to any one of claims 70-65, wherein the chimeric antigen receptor further comprises a hinge region.

92. The nucleic acid molecule according to claim 91, wherein the hinge region in the chimeric antigen receptor is selected from the hinge regions of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.

93. The nucleic acid molecule according to any one of claims 91-92, wherein the hinge region in the chimeric antigen receptor is the hinge region of CD28, CD8 or IgG4. The nucleic acid molecule according to any one of claims 64-92 comprises a sequence encoding a self-cleaving peptide.

94. The nucleic acid molecule according to claim 93, wherein the sequence encoding the self-cleaving peptide is located between the sequences encoding the fusion protein and the antigen recognition receptor.

95. The nucleic acid molecule according to any one of claims 93-94, wherein the self-cleaving peptide comprises a 2A peptide.

96. The nucleic acid molecule according to any one of claims 93-95, wherein the self-cleaving peptide is P2A, T2A, E2A or F2A.

97. The nucleic acid molecule according to any one of claims 93-96, wherein the self-cleaving peptide is P2A.

98. A vector comprising the nucleic acid molecule according to any one of claims 64-97.

99. A cell comprising the nucleic acid molecule according to any one of claims 64-97 or the vector according to claim 98.

100. A pharmaceutical composition comprising the fusion protein according to any one of claims 1-29, the modified immune cell according to any one of claims 30-63, the nucleic acid molecule according to any one of claims 64-97, the vector according to claim 98 and / or the cell according to claim 99, and optionally a pharmaceutically acceptable carrier.

101. A method for preparing the fusion protein according to any one of claims 1-29, the modified immune cell according to any one of claims 30-63, the nucleic acid molecule according to any one of claims 64-97, the vector according to claim 98, the cell according to claim 99 and / or the pharmaceutical composition according to claim 100.

102. Use of the fusion protein according to any one of claims 1-29, the modified immune cell according to any one of claims 30-63, the nucleic acid molecule according to any one of claims 64-97, the vector according to claim 98, the cell according to claim 99 and / or the pharmaceutical composition according to claim 100 in the preparation of a drug for preventing and / or treating tumors, autoimmune diseases, severe infections, chronic aging or cardiac fibrosis.

103. The use according to claim 102, wherein the tumor is a solid tumor and / or a hematological tumor.

104. The use according to any one of claims 102 - 103, wherein the tumor is selected from the group consisting of: lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, skin cancer.

105. A method for preventing and / or treating a tumor, an autoimmune disease, a severe infection, chronic aging or cardiac fibrosis, which comprises administering to a subject in need thereof the fusion protein according to any one of claims 1 - 29, the modified immune cell according to any one of claims 30 - 63, the nucleic acid molecule according to any one of claims 64 - 97, the vector according to claim 98, the cell according to claim 99 and / or the pharmaceutical composition according to claim 100.

106. The method according to claim 105, wherein the tumor is a solid tumor and / or a hematological tumor.

107. The method according to any one of claims 105 - 106, wherein the tumor is selected from the group consisting of: lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, skin cancer.

108. The fusion protein according to any one of claims 1 - 29 and / or the modified immune cell according to any one of claims 30 - 63, the nucleic acid molecule according to any one of claims 64 - 97, the vector according to claim 98, the cell according to claim 99 and / or the pharmaceutical composition according to claim 100, which are used for preventing and / or treating a tumor, an autoimmune disease, a severe infection, chronic aging or cardiac fibrosis.

109. The fusion protein and the modified immune cell according to claim 108, wherein the tumor is a solid tumor and / or a hematological tumor.

110. The fusion protein and the modified immune cell according to any one of claims 108 - 109, wherein the tumor is selected from the group consisting of: lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, skin cancer.

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