Cell surface receptor containing CD62l intracellular domain

Abstract

The present invention relates to a cell surface receptor containing a CD62L intracellular domain. Provided is a cell surface receptor, which contains an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain containing the CD62L intracellular domain. The cell surface receptor can improve the activation, proliferation and target cell killing level of T cells.

Description

Cell surface receptor containing the CD62L intracellular domain

[0001] This application claims priority to Chinese application No. 202410236496.6, filed on March 1, 2024, entitled “Cell surface receptor comprising a CD62L intracellular domain”. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to a cell surface receptor comprising a CD62L intracellular domain. Background Art

[0003] The formation of the immune synapse is a core process in T cell antigen recognition. The structure of the immune synapse crucially influences subsequent T cell activation and signaling. Early in the formation of the immune synapse, specific T cell receptors (TCRs) bind to peptide-major histocompatibility complexes (pMHCs) to form microclusters, the core structures of the immune synapse. TCR-pMHC binding alone is insufficient to maintain the stability of the immune synapse structure. Surrounding the core structure of the immune synapse are numerous synergistic adhesion-mediating mechanisms and complementary receptor-ligand binding-mediated signaling, including integrin complexes formed by LFA-1 and ICAM-1, and costimulatory signaling through CD28 binding to CD80 / CD86. When a T cell localizes to a cell expressing a matching pMHC complex, it must rapidly transition from a loosely adherent, highly motile state to a tightly adherent, sedentary state within seconds. This rapid transition requires crucial changes in the cytoskeleton, primarily through actin and microstructure remodeling. Once activated, TCR signaling induces actin polymerization, which further positively feeds back TCR triggering and amplifies contact between T cells and antigen-presenting cells, allowing the immune synapse to form a stable interface. During the formation of the T cell immune synapse, F-actin, as a component of the cytoskeleton, plays an important role in its function and regulation. The F-actin network determines the positioning of the receptor-ligand complex formed by the co-stimulatory signal CD28 and its ligand CD80 within the immune synapse structure. Strengthening the structure of the immune synapse may facilitate subsequent T cell activation and signaling. Summary of the Invention

[0004] To this end, the present invention provides a cell surface receptor comprising the CD62L intracellular domain, wherein immune cells comprising the cell surface receptor have a higher positive rate, proliferation level and killing effect on target cells.

[0005] The present invention provides a cell surface receptor comprising an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic domain comprising a CD62L intracellular domain.

[0006] In one or more embodiments, the CD62L intracellular domain comprises the sequence shown in SEQ ID NO:10.

[0007] In one or more embodiments, the CD62L intracellular domain is a truncated CD62L intracellular domain. In one or more embodiments, the truncated CD62L intracellular domain comprises the sequence shown in SEQ ID NO: 36.

[0008] In one or more embodiments, the cytoplasmic domain comprises a signal transduction domain and / or a costimulatory domain. In one or more embodiments, the costimulatory domain is an intracellular domain of a costimulatory signal molecule or a functional fragment or mutant thereof that retains the biological function of the costimulatory signal molecule to transmit costimulatory signals and activate immune cells.

[0009] In one or more embodiments, the intracellular domain of the costimulatory signaling molecule includes but is not limited to any one or more of the intracellular domains of CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM and CD27, or their mutants. Preferably, the intracellular domain of the costimulatory signal molecule is the intracellular domain of CD28 and / or the intracellular domain of OX40.

[0010] In one or more embodiments, the signal transduction domain is the CD3ζ intracellular signaling region.

[0011] In one or more embodiments, the cytoplasmic domain further comprises one or more intracellular domains selected from the group consisting of a CD28 intracellular domain, an OX40 intracellular domain, and a CD3ζ intracellular signaling region.

[0012] In one or more embodiments, the CD62L intracellular domain is located N-terminally to the CD28 intracellular domain, the OX40 intracellular domain, or the CD3ζ intracellular signaling region.

[0013] In one or more embodiments, the cytoplasmic domain comprises a CD62L intracellular domain and a CD28 intracellular domain. Preferably, the cytoplasmic domain further comprises an OX40 intracellular domain and / or a CD3ζ intracellular signaling region.

[0014] In one or more embodiments, the cytoplasmic domain comprises a CD62L intracellular domain and an OX40 intracellular domain.

[0015] In one or more embodiments, the cytoplasmic domain comprises a truncated CD62L intracellular domain and an OX40 intracellular domain.

[0016] In one or more embodiments, the cytoplasmic domain comprises, from N-terminus to C-terminus:

[0017] CD62L intracellular domain, CD28 intracellular domain,

[0018] CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain,

[0019] CD62L intracellular domain, CD28 intracellular domain and CD3ζ intracellular signaling region,

[0020] CD62L intracellular domain, OX40 intracellular domain, or

[0021] Truncated CD62L intracellular domain, OX40 intracellular domain.

[0022] In one or more embodiments, the CD28 intracellular domain comprises the amino acid sequence shown in SEQ ID NO:6.

[0023] In one or more embodiments, the OX40 intracellular domain comprises the amino acid sequence set forth in SEQ ID NO:8.

[0024] In one or more embodiments, the CD3ζ intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO:22.

[0025] In one or more embodiments, the transmembrane region is selected from but not limited to CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, Any one or more of the transmembrane regions of CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, CD27, KIR2DL3 and KIR2DL4, or mutants thereof that retain transmembrane function.

[0026] In one or more embodiments, the transmembrane region is the CD28 transmembrane region, which has the amino acid sequence shown in SEQ ID NO: 4.

[0027] In one or more embodiments, the transmembrane region is the KIR2DL3 transmembrane region having the amino acid sequence shown in SEQ ID NO: 42.

[0028] In one or more embodiments, the transmembrane region is the KIR2DL4 transmembrane region having the amino acid sequence shown in SEQ ID NO: 44.

[0029] In one or more embodiments, the cell surface receptor further comprises a hinge region between the extracellular ligand binding domain and the transmembrane domain.

[0030] In one or more embodiments, the hinge region includes, but is not limited to, a membrane-proximal fragment of a native extracellular domain selected from the group consisting of CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, and CD27.

[0031] In one or more embodiments, the hinge region is the extracellular hinge region of CD28 and / or the extracellular hinge region of IL7Rα. Preferably, the hinge region is the extracellular hinge region of CD28, having the amino acid sequence shown in SEQ ID NO: 46.

[0032] In one or more embodiments, the extracellular ligand-binding domain comprises the extracellular domain of a KIR or a functional fragment or variant thereof that retains the biological function of binding a KIR ligand. In one or more embodiments, the KIR is an activating KIR or an inhibitory KIR. In some embodiments, the activating KIR is KIR2DL4; the inhibitory KIR is any one or more selected from KIR2DL3, KIR3DL1, and KIR3DL2.

[0033] In one or more embodiments, the extracellular ligand binding domain comprises a KIR2DL3 extracellular region and / or a KIR2DL4 extracellular region.

[0034] In one or more embodiments, the extracellular ligand binding domain comprises a truncated KIR2DL3 extracellular region and / or a truncated KIR2DL4 extracellular region.

[0035] In one or more embodiments, the extracellular ligand binding domain comprises an antibody or antigen-binding fragment thereof that targets a tumor antigen. In one or more embodiments, the tumor antigen is selected from one or more of the following: HER2, CD19, EpCAM.

[0036] In one or more embodiments, the extracellular ligand-binding domain further comprises the extracellular domain of BCMA or a mutant thereof.

[0037] In one or more embodiments, the extracellular ligand binding domain comprises, from N-terminus to C-terminus:

[0038] KIR2DL3 extracellular region and BCMA extracellular domain or its mutants,

[0039] KIR2DL4 extracellular region and BCMA extracellular domain or mutants thereof, or

[0040] Antibodies or antigen-binding fragments thereof targeting tumor antigens and the BCMA extracellular domain or mutants thereof.

[0041] In one or more embodiments, the KIR2DL3 extracellular region has the amino acid sequence shown in SEQ ID NO:12.

[0042] In one or more embodiments, the KIR2DL4 extracellular region has the amino acid sequence shown in SEQ ID NO:14.

[0043] In one or more embodiments, the truncated KIR2DL3 extracellular region has the amino acid sequence shown in SEQ ID NO:100.

[0044] In one or more embodiments, the truncated KIR2DL4 extracellular region has the amino acid sequence shown in SEQ ID NO:106.

[0045] In one or more embodiments, the antibody or antigen-binding fragment thereof targeting a tumor antigen is a 4D5 single-chain antibody having the amino acid sequence shown in SEQ ID NO:16.

[0046] In one or more embodiments, the BCMA extracellular domain has the amino acid sequence shown in SEQ ID NO:18.

[0047] In one or more embodiments, the BCMA extracellular domain mutant has the amino acid sequence shown in SEQ ID NO: 20.

[0048] In one or more embodiments, the extracellular ligand binding domain further comprises a signal peptide.

[0049] In one or more embodiments, the signal peptide is a CD8 signal peptide, and the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO: 2.

[0050] In one or more embodiments, the signal peptide is a KIR2DL3 signal peptide. The amino acid sequence of the KIR2DL3 signal peptide is shown in SEQ ID NO: 38.

[0051] In one or more embodiments, the signal peptide is a KIR2DL4 signal peptide. The amino acid sequence of the KIR2DL4 signal peptide is shown in SEQ ID NO: 40.

[0052] In one or more embodiments, the cell surface receptor comprises:

[0053] Optional CD8 signal peptide, and KIR2DL3 extracellular region, BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain,

[0054] Optional CD8 signal peptide, and KIR2DL4 extracellular region, BCMA extracellular domain mutant, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain,

[0055] Optional CD8 signal peptide, and 4D5 single-chain antibody, BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and CD3ζ intracellular signaling region,

[0056] Optional CD8 signal peptide, and KIR2DL3 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain,

[0057] Optional CD8 signal peptide, and KIR2DL3 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain,

[0058] an optional CD8 signal peptide, and a KIR2DL3 extracellular region, a KIR2DL3 transmembrane region, a truncated CD62L intracellular domain, and an OX40 intracellular domain,

[0059] an optional KIR2DL3 signal peptide, and a KIR2DL3 extracellular region, a KIR2DL3 transmembrane region, a truncated CD62L intracellular domain, and an OX40 intracellular domain,

[0060] an optional CD8 signal peptide, and the KIR2DL3 extracellular region, the CD28 hinge region, the CD28 transmembrane region, the truncated CD62L intracellular domain, and the OX40 intracellular domain,

[0061] an optional CD8 signal peptide, and a truncated KIR2DL3 extracellular region, a CD28 hinge region, a CD28 transmembrane region, a truncated CD62L intracellular domain, and an OX40 intracellular domain,

[0062] Optional CD8 signal peptide, and KIR2DL4 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain,

[0063] Optional CD8 signal peptide, and KIR2DL4 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain,

[0064] Optional CD8 signal peptide, and KIR2DL4 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain,

[0065] Optional KIR2DL4 signal peptide, and KIR2DL4 extracellular region, KIR2DL4 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, or

[0066] an optional CD8 signal peptide, and the KIR2DL4 extracellular region, the CD28 hinge region, the CD28 transmembrane region, the truncated CD62L intracellular domain, and the OX40 intracellular domain,

[0067] Optional CD8 signal peptide, and truncated KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain.

[0068] In one or more embodiments, the cell surface receptor has any one of the amino acid sequences shown in SEQ ID NOs: 26, 30, 34, 60, 62, 64, 66, 68, 80, 82, 84, 86, 88, 102, and 108.

[0069] The present invention also provides a fusion protein comprising the cell surface receptor and membrane surface cytokine described in any embodiment herein.

[0070] In one or more embodiments, the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region, which are directly linked or linked through a linker.

[0071] In one or more embodiments, the cytokine is IL-7.

[0072] In one or more embodiments, the membrane surface cytokine further comprises a signal peptide. Preferably, the signal peptide is a CD52 signal peptide. Preferably, the signal peptide is located at the N-terminus of the cytokine.

[0073] In one or more embodiments, the GPI anchor is CD52.

[0074] In one or more embodiments, the membrane surface cytokine comprises: CD52 signal peptide, IL-7, linker, CD52.

[0075] In one or more embodiments, the membrane surface cytokine further comprises the extracellular domain of BCMA or a mutant thereof.

[0076] In one or more embodiments, the amino acid sequence of the cytokine IL-7 is shown in SEQ ID NO:49.

[0077] In one or more embodiments, the amino acid sequence of the BCMA extracellular domain is shown in SEQ ID NO: 18.

[0078] In one or more embodiments, the amino acid sequence of the BCMA extracellular domain mutant is shown in SEQ ID NO: 20.

[0079] In one or more embodiments, the amino acid sequence of the CD52 signal peptide is shown in SEQ ID NO:48.

[0080] In one or more embodiments, the amino acid sequence of CD52 is as shown in SEQ ID NO:54.

[0081] In one or more embodiments, the membrane surface cytokine comprises a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain mutant, and CD52. In one or more embodiments, the amino acid sequence of the membrane surface cytokine is shown in SEQ ID NO: 58.

[0082] In one or more embodiments, the membrane surface cytokine is at the N-terminus and / or C-terminus of the cell surface receptor.

[0083] In one or more embodiments, the membrane surface cytokine is connected to the cell surface receptor via a linker; preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 56.

[0084] In one or more embodiments, the membrane surface cytokine is linked to the cell surface receptor via a cleavable sequence; preferably, the cleavable sequence is such as P2A, T2A, F2A, preferably P2A.

[0085] In one or more embodiments, the fusion protein comprises:

[0086] Optionally, the membrane surface cytokine, CD8 signal peptide, KIR2DL3 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain;

[0087] Optionally, the membrane surface cytokine, CD8 signal peptide, KIR2DL3 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0088] Optionally, the membrane surface cytokine, CD8 signal peptide, KIR2DL3 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0089] Optionally, the membrane surface cytokine, KIR2DL3 signal peptide, KIR2DL3 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0090] Optionally, the membrane surface cytokine, CD8 signal peptide, KIR2DL3 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0091] Optionally, the membrane surface cytokine, CD8 signal peptide, KIR2DL3 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0092] Optionally, the membrane surface cytokine, CD8 signal peptide, truncated KIR2DL3 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0093] Optionally, the membrane surface cytokine, CD8 signal peptide, and KIR2DL4 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain;

[0094] Optionally, the membrane surface cytokine, CD8 signal peptide, and KIR2DL4 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0095] Optionally, the membrane surface cytokine, CD8 signal peptide, and KIR2DL4 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0096] Optionally, the membrane surface cytokine, KIR2DL4 signal peptide, and KIR2DL4 extracellular region, KIR2DL4 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0097] Optionally, the membrane surface cytokine, CD8 signal peptide, and KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; or

[0098] Optionally, the membrane surface cytokine, CD8 signal peptide, and truncated KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain.

[0099] In one or more embodiments, the amino acid sequence of the cell surface receptor is as shown in any one of SEQ ID NOs: 26, 30, 34, 60, 62, 64, 66, 68, 80, 82, 84, 86, 88, 102, and 108.

[0100] In one or more embodiments, the amino acid sequence of the fusion protein is as shown in any one of SEQ ID NOs: 70, 72, 74, 76, 78, 90, 92, 94, 96, 98, 104 and 110.

[0101] The present invention also provides a polynucleotide having:

[0102] (1) a sequence encoding the cell surface receptor or the fusion protein described herein, and / or

[0103] (2) The complementary sequence of (1).

[0104] In one or more embodiments, the sequence of the polynucleotide is as shown in any one of SEQ ID NO: 25, 29, 33, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 101, 103, 107 and 109, or a complementary sequence thereof.

[0105] The present invention also provides nucleic acid constructs comprising the polynucleotides described herein.

[0106] In one or more embodiments, the nucleic acid construct further comprises at least one regulatory element for expressing the cell surface receptor and / or the fusion protein operably linked to the polynucleotide.

[0107] In one or more embodiments, the nucleic acid construct is an expression vector or a cloning vector.

[0108] In one or more embodiments, the nucleic acid construct is an expression vector; preferably a viral vector or a non-viral vector.

[0109] In one or more embodiments, the non-viral vector is a non-viral integrating vector based on a transposon system.

[0110] The present invention also provides a host cell, which:

[0111] (1) comprising, expressing or secreting the cell surface receptor and / or the fusion protein described in any embodiment of the present invention,

[0112] (2) A nucleic acid construct comprising a polynucleotide encoding the cell surface receptor and / or the fusion protein described in any embodiment of the present invention or comprising the polynucleotide.

[0113] In one or more embodiments, the host cell is an immune effector cell.

[0114] In one or more embodiments, the host cell is a T cell, a NK cell, a CAR-T cell, a CAR-NK cell, a TCR-T cell, a CIK cell, a DN T cell, or a tumor infiltrating lymphocyte (TIL).

[0115] The present invention also provides a method for treating an individual suffering from cancer, comprising introducing an immune cell into the individual, wherein the immune cell comprises the cell surface receptor and / or the fusion protein described in any embodiment of the present invention.

[0116] In one or more embodiments, the immune cell is a T cell, a NK cell, a CIK cell, or a tumor infiltrating lymphocyte (TIL).

[0117] In one or more embodiments, the individual has a cancer associated with a tumor antigen that is targeted by the extracellular ligand binding domain of the cell surface receptor.

[0118] In one or more embodiments, the individual suffers from a HER2-related cancer, including but not limited to melanoma, breast cancer, gastric cancer, colon cancer, bladder cancer, ovarian cancer, endometrial cancer, or lung cancer.

[0119] In one or more embodiments, the cancer is selected from the group consisting of lung cancer, melanoma, gastric cancer, bladder cancer, endometrial cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma, acute lymphoblastic leukemia, small cell lung cancer, Hodgkin lymphoma, and childhood acute lymphoblastic leukemia.

[0120] The present invention also provides a pharmaceutical composition comprising any one or more of the cell surface receptors, fusion proteins, polynucleotides, nucleic acid constructs and host cells described in any embodiment of the present invention, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0121] The present invention also provides use of any one or more of the cell surface receptors, fusion proteins, polynucleotides, nucleic acid constructs and host cells described in any embodiment of the present invention in the preparation of drugs for treating or preventing cancer.

[0122] In one or more embodiments, the cancer is a tumor antigen-associated cancer, and the tumor antigen is a tumor antigen targeted by the extracellular ligand binding domain of the cell surface receptor.

[0123] In one or more embodiments, the cancer is a HER2-related cancer, including but not limited to melanoma, breast cancer, gastric cancer, colon cancer, bladder cancer, ovarian cancer, endometrial cancer, and lung cancer.

[0124] In one or more embodiments, the cancer is selected from the group consisting of lung cancer, melanoma, gastric cancer, bladder cancer, endometrial cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma, acute lymphoblastic leukemia, small cell lung cancer, Hodgkin lymphoma, and childhood acute lymphoblastic leukemia.

[0125] Advantages of the present invention: The receptor structure of the present invention contains the CD62L intracellular domain, which can further enhance the formation of the immune synapse structure on the basis of the existing immune synapse core structure of the cell surface receptor or chimeric antigen receptor, thereby further improving the activation, proliferation and target cell killing levels of T cells. DETAILED DESCRIPTION

[0126] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0127] The present invention introduces the intracellular region of CD62L into the structure of T cell surface receptors, such as signal transduction receptors or chimeric antigen receptors (CARs), and further enhances TCR signal transduction and T cell activation by improving the stability of the immune synapse structure of T cells expressing the receptor.

[0128] definition

[0129] The present invention uses the following terms: For terms not specifically defined herein, they have the meanings generally known in the art.

[0130] In the present invention, "immune cells" have a well-known meaning in the art and refer to cells involved in or associated with immune responses, including various lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Lymphocytes include, for example, T lymphocytes, tumor infiltrating lymphocytes (TIL), B lymphocytes, NK lymphocytes, and DN T cells. Immune cells suitable for the present invention particularly include those commonly used in adoptive cell therapy for tumors.

[0131] The term "expression cassette" refers to the complete elements required to express a gene, including the promoter, gene coding sequence, and Poly A tailing signal sequence.

[0132] The term "coding sequence" is defined herein as the portion of the nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., cell surface receptor, CAR). The boundaries of the coding sequence are typically determined by the ribosome binding site (for prokaryotes) immediately upstream of the mRNA 5' end open reading frame and the transcription termination sequence immediately downstream of the mRNA 3' end open reading frame. Coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0133] The term "co-stimulatory signal molecule" refers to a molecule that exists on the surface of antigen-presenting cells and can bind to the co-stimulatory signal molecule receptor on Th cells to produce a co-stimulatory signal. It can activate the second signal of immune cells, enhance the proliferation ability of immune cells and the secretion function of cytokines, and prolong the survival time of activated immune cells. The proliferation of lymphocytes requires not only the binding of antigens, but also the reception of co-stimulatory molecule signals. The co-stimulatory signal is transmitted to T cells mainly through the co-stimulatory molecules CD80 and CD86 expressed on the surface of antigen-presenting cells and binding to the CD28 molecules on the surface of T cells. B cells can receive co-stimulatory signals through general pathogen components such as LPS, or through complement components, or through CD40L on the surface of activated antigen-specific Th cells.

[0134] The term "linker" or "hinge" refers to a polypeptide segment that connects different proteins or polypeptides. Its purpose is to maintain the spatial conformation of the connected proteins or polypeptides to maintain their function or activity. Exemplary linkers include linkers containing G and / or S, rigid linkers, flexible linkers, and, for example, the Furin 2A peptide.

[0135] The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0136] The term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and / or relief of the diseases or conditions described in the present invention in a subject.

[0137] The term "disease and / or condition" refers to a physical condition of the subject, which is associated with the diseases and / or conditions described herein.

[0138] The term "subject" may refer to a patient or other animal that receives the pharmaceutical composition of the present invention to treat, prevent, alleviate and / or relieve the diseases or conditions described in the present invention, particularly mammals, such as humans, dogs, monkeys, cows, horses, etc.

[0139] The term "extracellular region" refers to the segment of a membrane protein located outside the cell.

[0140] The term "domain" refers to a region in a protein with a specific structure and independent function. The number of amino acid residues in a common domain ranges from 100 to 400. The smallest domain has only 40 to 50 amino acid residues, while the largest domain can exceed 400 amino acid residues.

[0141] Cell surface receptors

[0142] The cell surface receptor (fusion protein) of the present invention comprises an extracellular ligand-binding domain (extracellular region), a transmembrane domain (transmembrane region), and a cytoplasmic domain (intracellular region) comprising the CD62L intracellular domain. In the cell surface receptor herein, the CD62L intracellular domain can be itself or a fragment, as long as the fragment retains the biological function of the CD62L intracellular domain. Exemplary amino acid sequences and corresponding coding sequences of the intracellular region of CD28 can be shown in SEQ ID NOs: 10 and 9, respectively.

[0143] In one or more embodiments, the extracellular ligand binding domain comprises a KIR extracellular domain. The KIR extracellular domain can be itself or a fragment, as long as the fragment retains the biological function of binding to the KIR ligand. In this case, the cell surface receptor is also referred to herein as a coreceptor.

[0144] In the cell surface receptors herein, the KIR extracellular domain is used to interact with KIR ligands (mainly various types of class I HLA molecules), thereby downregulating the level of the "don't eat me" signaling pathway, activating the signaling pathway of immune effector cells to dominate, and then causing the immune effector cells to enter an activated and proliferative state, attacking target cells and thus eliminating abnormal cells. The extracellular domain of an activating KIR or an inhibitory KIR or a fragment thereof that retains the ability to bind to a KIR ligand can be used. An exemplary activating KIR is KIR2DL4; an exemplary inhibitory KIR is selected from KIR2DL3, KIR3DL1 and KIR3DL2. Preferably, the amino acid sequence of the KIR2DL3 extracellular region is shown in SEQ ID NO: 12, and its coding sequence is shown in SEQ ID NO: 11; the amino acid sequence of the KIR2DL4 extracellular region is shown in SEQ ID NO: 14, and its coding sequence is shown in SEQ ID NO: 13. Preferably, the fragment of the KIR2DL3 extracellular region that retains its ligand binding ability can be a truncated KIR2DL3 extracellular region. Preferably, the amino acid sequence of the truncated KIR2DL3 extracellular region is shown in SEQ ID NO: 100, and its coding sequence is shown in SEQ ID NO: 99. Preferably, the fragment of the KIR2DL4 extracellular region that retains its ligand binding ability can be a truncated KIR2DL4 extracellular region. Preferably, the amino acid sequence of the truncated KIR2DL4 extracellular region is shown in SEQ ID NO: 106, and its coding sequence is shown in SEQ ID NO: 105.

[0145] In one or more embodiments, the extracellular ligand binding domain comprises an antibody or antigen binding fragment thereof that targets a tumor antigen. In this case, the cell surface receptor is also referred to herein as a chimeric antigen receptor. Antibodies targeting tumor antigens can be selected from any antibodies known in the art as needed. Herein, tumor antigens include but are not limited to: CD19, HER2, CD20, CEA, GD2, FR, PSMA, PMEL, CA9, CD171 / L1-CAM, IL-13RL1, MART-1, ERBB2, NY-ESO-1, MAGE family proteins, BAGE family proteins, GAGE ​​family proteins, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR 2, IL-11R / , EGP-2, EGP-40, FBP, GD3, PSCA, FSA, PSA, HMGA2, fetal acetylcholine receptor, LeY, EpCAM, MSLN, IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC, AFU, EBV-VCA, POA and PROGRP. Preferably, the antibody is an anti-HER2 antibody.

[0146] In one or more embodiments, the extracellular ligand-binding domain further comprises a BCMA extracellular domain or a mutant thereof (e.g., a point mutant). For example, the amino acid sequence of the BCMA extracellular domain is shown in SEQ ID NO: 18, and its coding sequence is shown in SEQ ID NO: 17; the amino acid sequence of the BCMA extracellular domain mutant is shown in SEQ ID NO: 20, and its coding sequence is shown in SEQ ID NO: 19.

[0147] In the cell surface receptors herein, the intracellular region also comprises a costimulatory domain. In the present invention, co-stimulatory signal molecules include CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM and CD27. The receptor of the present invention can be constructed using one or more of the intracellular domains (intracellular regions) of these costimulatory signal molecules or their functional fragments or mutants that retain the biological functions of the costimulatory signal molecules to transmit costimulatory signals and activate immune cells. The amino acid sequence and the corresponding coding sequence of the intracellular region of exemplary CD28 can be shown as SEQ ID NO: 6 and 5, respectively. The amino acid sequence and the corresponding coding sequence of the intracellular region of exemplary OX40 can be shown as SEQ ID NO: 8 and 7, respectively. The intracellular domain of the costimulatory signal molecule can also be the intracellular domain of the costimulatory signal molecule described in WO2021244486, which is incorporated herein by reference in its entirety.

[0148] In the cell surface receptors herein, the intracellular region may further comprise a signal transduction domain. The amino acid sequence and corresponding coding sequence of an exemplary CD3ζ intracellular signaling region may be shown in SEQ ID NOs: 22 and 21, respectively.

[0149] Herein, the transmembrane region includes but is not limited to any one or more of the transmembrane regions selected from CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10.CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM and CD27, or mutants thereof that retain transmembrane function. Illustratively, the amino acid sequence and nucleotide sequence of the CD28 transmembrane region are shown in SEQ ID NOs: 4 and 3, respectively.

[0150] In the present invention, the extracellular domain can be connected to the transmembrane region via a hinge region. The hinge region includes, but is not limited to, a membrane-proximal fragment of the native extracellular domain selected from CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, and CD27.

[0151] It should be understood that the "functional fragment" described herein refers to a fragment that retains the desired biological function. For example, the functional fragment of the intracellular domain described herein refers to a fragment that retains the biological function of the costimulatory signal molecule to transmit costimulatory signals and activate immune cells. Functional fragments of each extracellular domain and each intracellular domain suitable for use in the present invention can be easily determined by those skilled in the art in combination with existing technical means in the art.

[0152] The "mutants" described herein include mutants of each domain, as long as the mutant retains the corresponding biological function of the extracellular domain, transmembrane region, and intracellular domain. For example, mutants of the CD62L intracellular domain suitable for the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the CD62L intracellular domain used as a comparison; mutants of the transmembrane region suitable for the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the transmembrane region used as a comparison; mutants of the extracellular domain suitable for the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the extracellular domain used as a comparison. Alternatively, compared to the sequence used for comparison, the mutants of the present invention have one or more (e.g., within 20, within 15, within 10, within 8, within 5, or within 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted, or deleted. For example, in the art, conservative substitutions with amino acids having similar or similar properties generally do not alter the function of a protein or polypeptide. "Amino acids with similar or similar properties" include, for example, families of amino acid residues having similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0153] The present invention also includes mutants of the cell surface receptors described above, such as mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the cell surface receptors. More specifically, the present invention includes mutants having one or more (such as within 20, within 15, within 10, within 8, within 5 or within 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted or deleted compared to the cell surface receptors described above. Such mutants retain the biological functions of the cell surface receptors described herein, including but not limited to the function of enhancing the formation of immune synapse structures. Mutations may occur in any one, any two or all three of the extracellular domain, transmembrane region and intracellular domain described herein.

[0154] The polypeptides described herein may be modified polypeptides. Modifications (generally without altering the primary structure) include chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation during polypeptide synthesis and processing or during further processing steps. Such modifications can be accomplished by exposing the polypeptide to a glycosylation enzyme (e.g., a mammalian glycosylase or deglycosylase). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or optimize their solubility.

[0155] Exemplary cell surface receptors of the present invention include, but are not limited to, cell surface receptors comprising, from N-terminus to C-terminus, an extracellular domain, a hinge region, a transmembrane region, and an intracellular region as shown in each row of Table 1 below, or consisting of an extracellular domain, a hinge region, a transmembrane region, and an intracellular region as shown in each row of the following table:

[0156] Cell surface receptors (N-terminus to C-terminus)

[0157] In some embodiments, the cell surface receptor described herein further comprises a signal peptide. Preferably, the signal peptide is located at the N-terminus of the cell surface receptor. The signal peptide can be any signal peptide conventional in the art that can guide the nuclear export of a polypeptide, including but not limited to CD8, CD4, CD28, CD137, EGFR, TGFBRI, TGFBRII, TGFBRIII, KIR2DL3, KIR2DL4, and antibody light chain signal peptides. In some embodiments, the signal peptide is a CD8 signal peptide, comprising the amino acid sequence of SEQ ID NO: 2, and its coding sequence is shown in SEQ ID NO: 1. In some embodiments, the signal peptide is a KIR2DL3 signal peptide, comprising the amino acid sequence of SEQ ID NO: 38, and its coding sequence is shown in SEQ ID NO: 37. In some embodiments, the signal peptide is a KIR2DL4 signal peptide, comprising the amino acid sequence of SEQ ID NO: 40, and its coding sequence is shown in SEQ ID NO: 39.

[0158] It should be understood that, as needed, the extracellular domain and the transmembrane region, and / or the transmembrane region and the intracellular domain described herein can be connected by a linker sequence. Linker sequences well known in the art can be used, such as linker sequences containing G and S, such as (GSSS)n or (GSSSS)n, where n is an integer from 1 to 8. The linker can also be a rigid linker or a flexible linker.

[0159] Preferably, the amino acid sequence of the cell surface receptor of the present invention is shown in any one of SEQ ID NOs: 26, 30, 34, 60, 62, 64, 66, 68, 80, 82, 84, 86 and 88.

[0160] Fusion protein

[0161] The present invention also provides a fusion protein comprising the cell surface receptor and the membrane surface cytokine. The membrane surface cytokine is at the N-terminus and / or C-terminus of the cell surface receptor. The membrane surface cytokine is linked to the cell surface receptor via a cleavable sequence; preferably, the cleavable sequence is, for example, P2A, T2A, or F2A, preferably P2A. Alternatively, the membrane surface cytokine and the cell surface receptor can be linked via a linker; preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 56. Alternatively, the membrane surface cytokine is directly linked to the cell surface receptor.

[0162] The membrane-surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region, which are directly connected or connected via a linker. Linker sequences known in the art can be used, such as linker sequences containing G and S, such as (GSSS)n or (GSSSS)n, where n is an integer from 1 to 8. The linker can also be a rigid linker or a flexible linker. For example, the linker is the linker shown in SEQ ID NO: 52.

[0163] Among the membrane surface cytokines, the cytokines may be any polypeptide cytokines, including but not limited to interleukins, tumor necrosis factor (TNF), interferon (IFN), colony stimulating factor (CSF) and tumor growth factor (TGF). The interleukins include but are not limited to IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18 and IL-21. The tumor necrosis factors include but are not limited to TNF-α and TNF-β. The interferons include but are not limited to IFN-α, IFN-β and IFN-γ. The colony stimulating factors include but are not limited to M-CSF, G-CSF and GM-CSF. The tumor growth factors include but are not limited to TGF-α, TGF-β1, TGF-β2 and TGF-β3.

[0164] The transmembrane domains in the membrane surface cytokines are as described elsewhere herein, including but not limited to CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, C Any one or more of the transmembrane regions of D27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, CD80 and CD27, or mutants thereof that retain their transmembrane function.

[0165] The GPI anchor region in the membrane surface cytokine includes one or more selected from the following or its GPI anchor domain (also referred to herein as GPI signal sequence): CD44, CD56, CD73, CD55, Thy1, AchE, IAP, ALPP, CD59, CD14, CD16, CD24, CD28, CD48, CD52, CD58, CD66a, CD66c, CD66d, CD66e, CD67, CD87, CD108, CD157, uPAR, JMH protein, GDNFR, CNTFR, TAG-1, PrP, phosphatidylinositol protein, semaphorin 7, CEA, GFR, Ly6G, transferrin receptor, contactin (F3) and T-cadherin; preferably, the GPI anchor region is CD52 protein, CD48 protein, CD55 protein, ALPP protein, CD90 protein or their GPI anchor domain. GPI anchor proteins or their anchoring domain sequences are known in the art. Moreover, those skilled in the art can easily obtain the anchoring domain sequence thereof based on the sequence of the GPI anchor protein.

[0166] The membrane surface cytokine may further include a signal peptide. Preferably, the signal peptide is located at the N-terminus of the cytokine. The signal peptide can be any signal peptide conventional in the art that can guide the polypeptide to exit the nucleus, including but not limited to CD8, CD4, CD28, CD52, CD137, EGFR, TGFBRI, TGFBRII, TGFBRIII and antibody light chain signal peptides. In some embodiments, the signal peptide is a CD8 signal peptide or a CD52 signal peptide.

[0167] The membrane surface cytokine may further include a membrane surface tag. The membrane surface tag is preferably the BCMA extracellular domain or a mutant thereof that retains biological function. Preferably, the amino acid sequence of the BCMA extracellular domain is as shown in SEQ ID NO: 18. Preferably, the amino acid sequence of the mutant BCMA extracellular domain is as shown in SEQ ID NO: 20.

[0168] In one or more embodiments, the fusion protein comprises or comprises, in order from N-terminus to C-terminus:

[0169] (1) CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region, CD8 signal peptide, KIR2DL3 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain;

[0170] (2) CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region, CD8 signal peptide, KIR2DL3 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain, and OX40 intracellular domain;

[0171] (3) CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region, CD8 signal peptide, KIR2DL3 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain, and OX40 intracellular domain;

[0172] (4) CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor, KIR2DL3 signal peptide, KIR2DL3 extracellular domain, KIR2DL3 transmembrane domain, truncated CD62L intracellular domain, and OX40 intracellular domain;

[0173] (5) CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region, CD8 signal peptide, KIR2DL3 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0174] (6) CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region, CD8 signal peptide, and KIR2DL4 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain;

[0175] (7) CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region, CD8 signal peptide, and KIR2DL4 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0176] (8) CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region, CD8 signal peptide, and KIR2DL4 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0177] (9) CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region, KIR2DL4 signal peptide, and KIR2DL4 extracellular region, KIR2DL4 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0178] (10) CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region, CD8 signal peptide, and KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain;

[0179] (11) CD8 signal peptide, KIR2DL3 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0180] (12) CD8 signal peptide, KIR2DL3 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0181] (13) CD8 signal peptide, KIR2DL3 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0182] (14) KIR2DL3 signal peptide, KIR2DL3 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0183] (15) CD8 signal peptide, KIR2DL3 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0184] (16) CD8 signal peptide, truncated KIR2DL3 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0185] (17) CD8 signal peptide, KIR2DL4 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0186] (18) CD8 signal peptide, KIR2DL4 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0187] (19) CD8 signal peptide, KIR2DL4 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0188] (20) KIR2DL4 signal peptide, KIR2DL4 extracellular region, KIR2DL4 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0189] (21) CD8 signal peptide, KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region;

[0190] (22) CD8 signal peptide, truncated KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, CD52 signal peptide, IL-7, linker 1, mutant BCMA extracellular domain, CD52 GPI anchor region.

[0191] CAR-T

[0192] The immune cells of the present invention may express other CARs, or contain coding sequences of these CARs, and the CARs may be various CARs known in the art.

[0193] CAR may comprise a polypeptide (such as scFv) that binds to a tumor cell membrane antigen, a hinge region, a transmembrane region, and an intracellular signaling region in sequence. The hinge region, transmembrane region, and intracellular signaling region known in the art for constructing CAR can be used to construct the CAR of the present invention. Typically, a polypeptide that binds to a tumor cell membrane antigen is able to bind to a membrane antigen widely expressed by tumor cells with moderate affinity. The polypeptide is usually inserted with an antigen epitope, and the insertion position is selected from any one, two, or three of the following three positions: the N-terminus of the polypeptide, between the polypeptide and the hinge region, and inside the polypeptide. The polypeptide that binds to a tumor cell membrane antigen is a natural polypeptide or an artificially synthesized polypeptide; preferably, the artificially synthesized polypeptide is a single-chain antibody or a Fab fragment.

[0194] Other CARs further expressed by the immune cells of the present invention may target one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT1), FR (Flavin reductase), PSMA (prostate-specific membrane antigen), PMEL premelanosome protein), CA9 (carbonic anhydrase IX), CD171 / L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family protein, BAGE (B melanoma antigen family) family protein, GAGE ​​(growth hormone releasing factor) family protein, AFP, MUC1 (also known as mucin1), CD22, CD23, CD30, CD33, CD44v7 / 8, CD 70, VEGFR1, VEGFR2, IL-11R / , EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR), and PROGRP (GRP gastrin-releasing peptide).

[0195] A single cell can express multiple CARs, including CARs targeting different tumor antigens.

[0196] T cell receptor (TCR)-T

[0197] The immune cells of the present invention may further express exogenous TCRs or contain coding sequences for expressing exogenous TCR genes. The TCRs of the present invention may be any TCR known in the art, such as TCRs with HLA typing matching, known sequence and structure, and with known antigenic peptide sequences.

[0198] The exogenous TCR described in the present invention includes an αβ double chain, which can form a complete TCR complex with the double-chain structure of γε, δε and ξξ endogenously expressed by immune effector cells such as T cells. The exogenous gene encoding the exogenous TCR described in the present invention includes an αβ double chain gene, and the α chain and β chain coding sequences are covalently linked by a linker sequence that can be cut in vivo, such as a DNA sequence encoding a P2A, T2A or F2A sequence, or covalently linked by a DNA fragment encoding an IRES sequence. In addition to the αβ double chain encoding the exogenous TCR, the gene encoding the exogenous TCR described in the present invention can also include a tag protein gene expressed in fusion with the αβ gene, such as EGFP, RFP, YFP genes, etc. The tag protein gene can be covalently linked to the αβ double chain gene through a linker sequence that can be cut in vivo, such as a 2A sequence, a DNA sequence encoding a P2A, T2A or F2A sequence, or a DNA sequence encoding an IRES sequence. The tag protein, such as EGFP, RFP, YFP gene, etc., is co-expressed with the TCRαβ double chain and can be used as an identification indicator for detecting the expression of exogenous TCR.

[0199] The TCR-T of the present invention may target one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT1), FR (Flavin reductase), PSMA (prostate-specific membrane antigen), PMEL premelanosome protein), CA9 (carbonic anhydrase IX), CD171 / L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family protein, BAGE (B melanoma antigen family) family protein, GAGE ​​(growth hormone releasing factor) family protein, AFP, MUC1 (mucin 1), CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, IL-11R / , EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16, mucin 16), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR), microglobulin) and PROGRP (GRP gastrin-releasing peptide).

[0200] A single cell can express multiple exogenous TCRs, including those targeting different tumor antigens.

[0201] polynucleotide molecules

[0202] The present invention provides polynucleotide molecules encoding the cell surface receptors described herein. The present invention also provides a complementary sequence to the coding sequence for the cell surface receptor. The polynucleotide molecules can be recombinant or synthetic; they can comprise DNA, RNA, and PNA (peptide nucleic acid), and can be hybrids thereof. Exemplarily, the polynucleotide molecules of the present invention have the sequence shown in any one of SEQ ID NOs: 25, 29, and 33.

[0203] Also provided is an expression cassette for the cell surface receptor of the present invention, which is a nucleic acid construct comprising a promoter, a cell surface receptor coding sequence, and a poly A tailing signal sequence. The nucleic acid construct may also contain other elements required for expression, including but not limited to enhancers.

[0204] Also provided is a vector containing the polynucleotide molecules, expression cassettes, or nucleic acid constructs described herein. The vector can be a plasmid, cosmid, virus, or phage. The vector can be a viral vector or a non-viral vector. The vector can be a cloning vector, an integrating vector, or an expression vector. The expression vector can be a transposon vector. In certain embodiments, the expression vector is one or more of the following transposon vectors: piggybac, sleeping beauty, frog prince, Tn5, and Ty. In addition to the polynucleotide molecules described herein, the expression vector typically contains other elements typically found in vectors, such as a multiple cloning site, resistance genes, and an origin of replication. In certain embodiments, the recombinant expression vector utilizes a pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector, pUC57, pMAX, or pDC315 series vector as its backbone. In other embodiments, the recombinant expression vector uses a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector, a pUC57 vector, a pMAX vector, or a pDC315 series vector as a backbone. In certain embodiments, the present invention uses the pNB vector constructed in CN105154473A. In certain embodiments, the present invention uses the pKB20 vector described in WO2022078310A1.

[0205] The CAR of the present invention can also be expressed in the immune cells of the present invention through conventional vectors. The vector can be a conventional CAR expression vector, including but not limited to the various transposon vectors and recombinant expression vectors described above.

[0206] In some embodiments, the same vector encodes both the cell surface receptor and the CAR of the present invention. The vector can be bicistronic. The coding sequence of the CAR can be set at the 5' or 3' end of the cell surface receptor coding sequence. The expression of the CAR and the cell surface receptor can be under the guidance of the same or different regulatory sequences.

[0207] In the case where the polynucleotide sequence is known, each polynucleotide molecule can be prepared using a method conventional in the art, and a corresponding vector can be constructed. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory), Ausubel et al. (1989, Short Protocols in Molecular Biology, Wiley) or the technology described in other standard textbooks. Alternatively, the nucleic acid molecule and vector can be reconstructed into a liposome for delivery to a target cell. The vector containing the nucleic acid molecule of the present invention can be transferred to a host cell by a well-known method, which varies according to the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al. (see above).

[0208] host cells

[0209] As used herein, "host cell" refers to a eukaryotic cell capable of replicating a vector and / or expressing a heterologous gene encoded by a vector when expressing a heterologous nucleic acid sequence. A host cell can serve as a recipient for a vector. A host cell can be "transfected" or "transformed," which refers to a process by which an exogenous nucleic acid is transfected or transduced into a host cell. Transformed cells include the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells often refer to cells into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Thus, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acid.

[0210] As used herein, host cells include cells carrying the polynucleotide molecules and / or polypeptides described herein. Specifically, the present invention provides cells carrying the cell surface receptors described herein and / or their coding sequences. The cells of the present invention are preferably immune cells and can be used for adoptive cell therapy of tumors. Such cells of the present invention are also referred to as cells modified with cell surface receptors of the present invention.

[0211] More specifically, the cells of the present invention are preferably immune effector cells, including T cells, such as cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells), NK cells, NKT cells, CAR-T, CAR-NK, TCR-T, CIK, TIL, DN T cells; and other immune cells that can trigger effector functions.

[0212] As used herein, cells can be autologous, syngeneic, allogeneic, and in some cases even xenogeneic, relative to the individual receiving them.

[0213] The nucleic acid construct / recombinant expression vector of the present invention can be transferred into the cells of interest. The methods of transfer are conventional in the art, including but not limited to viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation. In certain embodiments, electroporation is used to transfer the nucleic acid construct or recombinant expression vector.

[0214] In addition to carrying the cell surface receptors and / or their coding sequences described herein, the cells of the present invention may also have one or more other properties that can be used for cellular immunotherapy (e.g., adoptive cell therapy for tumors). Such other properties may be inherent to the cells or may be part of the cells after genetic manipulation by humans. For example, the cells of the present invention may carry other chimeric antigen receptors, αβ T cell receptors, and / or antigen-specific receptors, such as tumor-specific receptors, or their coding sequences.

[0215] Pharmaceutical composition

[0216] As used herein, a "pharmaceutical composition" refers to a composition for administration to an individual and encompasses compositions of cells for use in immunotherapy. The pharmaceutical compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject at an appropriate dose.

[0217] The dosage regimen can be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on a variety of factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and other drugs administered concurrently.

[0218] The compositions of the present invention can be given topically or systemically. In certain embodiments, compositions provided by the invention (e.g., cells expressing cell surface receptors according to the present invention) can be given parenterally, such as intravenously, intra-arterially, intrathecally, subdermally or intramuscularly. In certain other embodiments, the DNA encoding the construct provided by the invention can be directly given to the target site, such as by gene gun delivery to an internal or external target site or by catheter delivery to an intra-arterial site. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in a more preferred embodiment, intravenously. Parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic solutions / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous carriers include fluid and nutrient supplements, electrolyte supplements (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases. In addition, the pharmaceutical compositions of the present invention may include proteinaceous carriers, such as serum albumin or immunoglobulins, preferably of human origin. In addition to the proteinaceous chimeric cytokine receptor constructs or nucleic acid molecules or vectors encoding the same, it is contemplated that the pharmaceutical compositions of the present invention may also include biologically active agents, depending on the intended use of the pharmaceutical composition.

[0219] Compositions for parenteral (e.g., intravenous) administration of the cells described herein can also be stored in a lyophilized form or in a solution (e.g., a cryopreserved formulation). The cryopreserved formulation can be stored in a ready-to-use form or in a form that is further formulated prior to administration. The cryopreserved formulation can withstand long-distance transportation without damaging the cells. In addition to the cells themselves, the cryopreserved formulation typically includes components such as a cell freezing solution and human serum albumin (HSA). Prior to administration (e.g., intravenous infusion), the frozen pharmaceutical composition must be stored at low temperatures (e.g., in liquid nitrogen). After thawing, the cryopreserved formulation can be infused directly into the patient or formulated as an infusion composition. Those skilled in the art are aware of the components and concentrations of conventional freezing solutions. For example, the freezing solution or infusion composition may also include dimethyl sulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride, or magnesium chloride, and the concentrations thereof can be determined by those skilled in the art (e.g., an experienced physician) based on the conditions of the cells, disease, and patient.

[0220] Methods and Applications

[0221] The cell surface receptors, polynucleotide molecules, vectors, host cells and pharmaceutical compositions comprising these substances described in the present invention can be used to prevent, treat or alleviate cancer, especially cancers in which corresponding tumor antigens are expressed on the surface of cancer cells, or to prepare drugs for preventing, treating or alleviating cancer.

[0222] As used herein, "treat" or "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of the disease or condition (e.g., cancer) being treated. Treatment may optionally include a reduction or alleviation of symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily mean complete eradication or cure of the disease or condition or its associated symptoms.

[0223] As used herein, "prevention" refers to methods for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" also includes reducing the intensity, impact, symptoms, and / or burden of a disease or condition before it occurs or recurs.

[0224] The present invention includes administering cells, polynucleotide molecules, and vectors, alone or in any combination, using standard vectors and / or gene delivery systems, optionally with a pharmaceutically acceptable carrier or excipient. In certain embodiments, following administration, the polynucleotide molecule or vector can stably integrate into the subject's genome.

[0225] In specific embodiments, viral vectors can be used that are specific for certain cells or tissues and persist in said cells. Suitable pharmaceutical carriers and excipients are well known in the art. Compositions prepared according to the present invention can be used to prevent, treat, or delay the above-identified diseases.

[0226] In addition, the present invention provides a method for preventing, treating or alleviating cancer, comprising the following steps: administering to a subject in need thereof an effective amount of cells, wherein the cells carry the cell surface receptors, polynucleotide molecules and / or vectors described in the present invention and / or produced by the method of the present invention.

[0227] The methods herein can be used to prevent, treat or alleviate various cancers, including various solid tumors and hematological tumors, including but not limited to lung cancer (such as non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma and brain glioma, etc. More specifically, the cancers herein include but are not limited to breast, prostate, lung and colon cancer or epithelial cancers, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, melanoma; genital-urinary tract cancers, such as ovarian cancer, endometrial cancer, cervical cancer; kidney cancer, lung cancer, stomach cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, etc. Administration of the compositions of the present invention can be used for all stages and types of cancer, including, for example, minimal residual disease, early cancer, advanced cancer and / or metastatic cancer and / or cancer that is difficult to treat.

[0228] By way of example, a cancer patient or a patient susceptible to cancer or a patient suspected of having cancer is treated as follows. Cells modified as described herein can be given to an individual and stay for an extended period of time. The individual can receive one or more administrations of cells, and the intervals between administrations can be several days, weeks, months or years. In a specific embodiment, multiple administrations can occur over several weeks or months, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks or months. In some embodiments, the genetically modified cells are encapsulated to suppress immune recognition and are located at the tumor site. In the case of providing cells to an individual after tumor recurrence after initial treatment using the cells of the present invention, these cells can be changed to recognize different target tumor antigens. For example, when the initial round includes cells carrying a cell surface receptor of the present invention and another receptor specific for a specific antigen, receptors for different specific antigens can be used after subsequent rounds (including after tumor recurrence).

[0229] In some embodiments, an effective amount of therapeutic cells is provided to an individual in need, wherein the therapeutic cells carry or express the cell surface receptors and optional CAR or exogenous transgenic TCR described in any embodiment of the present invention. These cells can be delivered simultaneously or simultaneously with one or more other cancer treatments. These cells and other cancer therapeutics can be delivered in the same or separate formulations. Cells and other cancer therapeutics can be provided to an individual by a separate delivery route. Cells and / or other cancer therapeutics can be delivered, for example, by injection at the tumor site or intravenously or orally. Conventional delivery routes for such compositions are known in the art.

[0230] The number of cells employed will depend on various circumstances, such as the purpose of introduction, the lifespan of the cells, the regimen to be used, the number of administrations, the ability of the cells to reproduce, the stability of the recombinant construct, and the like.

[0231] Cells can be administered as needed. In certain embodiments, a variety of regimens can be used to adjust regimen parameters. In specific embodiments, the route or number or timing of administration, the lifespan of the cells, and / or the number of cells present can vary. The number of administrations can, for example, depend at least in part on the above factors.

[0232] Reagent test kit

[0233] Any of the compositions described herein may be included in a kit. In one non-limiting example, a kit may include cells expressing a cell surface receptor described in any embodiment of the present invention for use in cell therapy and / or reagents for generating one or more cells for use in cell therapy containing a recombinant expression vector. The kit components are provided in a suitable container.

[0234] Some components of these test kits can be packaged in aqueous media or packaged into lyophilized forms. The container apparatus of these test kits generally includes at least one vial, test tube, flask, bottle, syringe or other container apparatus, wherein the component can be placed, and preferably suitably subpackaged therein. In the case where there is more than one component in the test kit, the test kit generally also contains a second, third or other container, wherein the other components can be placed separately. However, various combinations of components can be included in the vial. The test kit of the present invention generally also includes an apparatus for containing the component in a commercially available closed constraint form. Such containers may include injection-molded or blow-molded plastic containers, wherein the required vial is retained.

[0235] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, particularly preferably a sterile aqueous solution. In some cases, the container means itself can be a syringe, pipette and / or other such device.

[0236] The components of the kit may also be provided in dry powder form. When the reagents and / or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent. Thus, the kit may also include a second container containing a sterile, pharmaceutically acceptable buffer and / or other diluent.

[0237] The components of the kit can also be provided in the form of a cryopreservation preparation (e.g., a cryopreservation solution). After thawing, the cryopreservation preparation can be infused into the patient directly or formulated into an infusion composition. Therefore, the kit can also include cell freezing bags, cell freezing tubes, a temperature maintenance device (e.g., a container containing liquid nitrogen), a thawing device, etc.

[0238] In a specific embodiment of the present invention, cells to be used for cell therapy as described herein are provided in a kit. In some embodiments, the cells are essentially the only component of the kit. The kit may contain reagents and materials for preparing the desired cells. In a specific embodiment, the reagents and materials include primers, nucleotides, suitable buffers or buffering agents, salts, etc. for amplifying the desired sequence, and in some cases, the reagents include DNA and / or vectors encoding the cell surface receptors and / or their regulatory elements as described in any embodiment of the present invention.

[0239] The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will appreciate that the following examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the examples, if specific techniques or conditions are not indicated, the techniques or conditions described in the literature in this area (e.g., with reference to "Molecular Cloning Experiment Guide" by J. Sambrook et al., translated by Huang Peitang et al., 3rd edition, Science Press), corresponding references, or product specifications are used. Reagents or instruments used that do not indicate manufacturers are all conventional products that can be obtained commercially.

[0240] Example

[0241] The sequence of the elements in the embodiment is summarized as follows:

[0242] CD8 signal peptide: nucleotide sequence SEQ ID NO: 1, amino acid sequence SEQ ID NO: 2;

[0243] CD28 transmembrane region: nucleotide sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4;

[0244] CD28 intracellular domain: nucleotide sequence SEQ ID NO: 5, amino acid sequence SEQ ID NO: 6;

[0245] OX40 intracellular domain: nucleotide sequence SEQ ID NO: 7, amino acid sequence SEQ ID NO: 8;

[0246] CD62L intracellular domain: nucleotide sequence SEQ ID NO: 9, amino acid sequence SEQ ID NO: 10;

[0247] KIR2DL3 extracellular region: nucleotide sequence SEQ ID NO: 11, amino acid sequence SEQ ID NO: 12;

[0248] Truncated KIR2DL3 extracellular region: nucleotide sequence SEQ ID NO: 99, amino acid sequence SEQ ID NO: 100;

[0249] KIR2DL4 extracellular region: nucleotide sequence SEQ ID NO: 13, amino acid sequence SEQ ID NO: 14;

[0250] Truncated KIR2DL4 extracellular region: nucleotide sequence SEQ ID NO: 105, amino acid sequence SEQ ID NO: 106;

[0251] 4D5 scFv: nucleotide sequence SEQ ID NO: 15, amino acid sequence SEQ ID NO: 16;

[0252] BCMA extracellular domain: nucleotide sequence SEQ ID NO: 17, amino acid sequence SEQ ID NO: 18;

[0253] Point mutation BCMA extracellular domain: nucleotide sequence SEQ ID NO: 19, amino acid sequence SEQ ID NO: 20;

[0254] CD3ζ intracellular signaling domain: nucleotide sequence SEQ ID NO: 21, amino acid sequence SEQ ID NO: 22;

[0255] KR23: nucleotide sequence SEQ ID NO: 23, amino acid sequence SEQ ID NO: 24;

[0256] KR23-62LIC: nucleotide sequence SEQ ID NO: 25, amino acid sequence SEQ ID NO: 26;

[0257] KR24: nucleotide sequence SEQ ID NO: 27, amino acid sequence SEQ ID NO: 28;

[0258] KR24-62LIC: nucleotide sequence SEQ ID NO: 29, amino acid sequence SEQ ID NO: 30;

[0259] 4D5: nucleotide sequence SEQ ID NO: 31, amino acid sequence SEQ ID NO: 32;

[0260] 4D5-62LIC: nucleotide sequence SEQ ID NO: 33, amino acid sequence SEQ ID NO: 34;

[0261] Truncated CD62L intracellular domain: nucleotide sequence SEQ ID NO: 35, amino acid sequence SEQ ID NO: 36;

[0262] KIR2DL3 signal peptide: nucleotide sequence SEQ ID NO: 37, amino acid sequence SEQ ID NO: 38

[0263] KIR2DL4 signal peptide: nucleotide sequence SEQ ID NO: 39, amino acid sequence SEQ ID NO: 40

[0264] KIR2DL3 transmembrane region: nucleotide sequence SEQ ID NO: 41, amino acid sequence SEQ ID NO: 42

[0265] KIR2DL4 transmembrane region: nucleotide sequence SEQ ID NO: 43, amino acid sequence SEQ ID NO: 44

[0266] CD28 hinge region: nucleotide sequence SEQ ID NO: 45, amino acid sequence SEQ ID NO: 46

[0267] CD52 signal peptide: nucleotide sequence SEQ ID NO: 47, amino acid sequence SEQ ID NO: 48

[0268] IL-7: nucleotide sequence SEQ ID NO: 49, amino acid sequence SEQ ID NO: 50

[0269] Linker 1: nucleotide sequence SEQ ID NO: 51, amino acid sequence SEQ ID NO: 52

[0270] CD52GPI: nucleotide sequence SEQ ID NO: 53, amino acid sequence SEQ ID NO: 54

[0271] Linker 2: nucleotide sequence SEQ ID NO: 55, amino acid sequence SEQ ID NO: 56

[0272] mbIL-7: nucleotide sequence SEQ ID NO: 57, amino acid sequence SEQ ID NO: 58

[0273] KR23-62LIC-2: nucleotide sequence SEQ ID NO: 59, amino acid sequence SEQ ID NO: 60

[0274] KR23-62LIC-3: nucleotide sequence SEQ ID NO: 61, amino acid sequence SEQ ID NO: 62

[0275] KR23-62LIC-4: nucleotide sequence SEQ ID NO: 63, amino acid sequence SEQ ID NO: 64

[0276] KR23-62LIC-5: nucleotide sequence SEQ ID NO: 65, amino acid sequence SEQ ID NO: 66

[0277] KR23-62LIC-6: nucleotide sequence SEQ ID NO: 67, amino acid sequence SEQ ID NO: 68

[0278] KR23-62LIC-7: nucleotide sequence SEQ ID NO: 101, amino acid sequence SEQ ID NO: 102

[0279] mbIL-7-KR23-62LIC-2: nucleotide sequence SEQ ID NO: 69, amino acid sequence SEQ ID NO: 70

[0280] mbIL-7-KR23-62LIC-3: nucleotide sequence SEQ ID NO: 71, amino acid sequence SEQ ID NO: 72

[0281] mbIL-7-KR23-62LIC-4: nucleotide sequence SEQ ID NO: 73, amino acid sequence SEQ ID NO: 74

[0282] mbIL-7-KR23-62LIC-5: nucleotide sequence SEQ ID NO: 75, amino acid sequence SEQ ID NO: 76

[0283] mbIL-7-KR23-62LIC-6: nucleotide sequence SEQ ID NO: 77, amino acid sequence SEQ ID NO: 78

[0284] mbIL-7-KR23-62LIC-7: nucleotide sequence SEQ ID NO: 103, amino acid sequence SEQ ID NO: 104

[0285] KR24-62LIC-2: nucleotide sequence SEQ ID NO: 79, amino acid sequence SEQ ID NO: 80

[0286] KR24-62LIC-3: nucleotide sequence SEQ ID NO: 81, amino acid sequence SEQ ID NO: 82

[0287] KR24-62LIC-4: nucleotide sequence SEQ ID NO: 83, amino acid sequence SEQ ID NO: 84

[0288] KR24-62LIC-5: nucleotide sequence SEQ ID NO: 85, amino acid sequence SEQ ID NO: 86

[0289] KR24-62LIC-6: nucleotide sequence SEQ ID NO: 87, amino acid sequence SEQ ID NO: 88

[0290] KR24-62LIC-7: nucleotide sequence SEQ ID NO: 107, amino acid sequence SEQ ID NO: 108

[0291] mbIL-7-KR24-62LIC-2: nucleotide sequence SEQ ID NO: 89, amino acid sequence SEQ ID NO: 90

[0292] mbIL-7-KR24-62LIC-3: nucleotide sequence SEQ ID NO: 91, amino acid sequence SEQ ID NO: 92

[0293] mbIL-7-KR24-62LIC-4: nucleotide sequence SEQ ID NO: 93, amino acid sequence SEQ ID NO: 94

[0294] mbIL-7-KR24-62LIC-5: nucleotide sequence SEQ ID NO: 95, amino acid sequence SEQ ID NO: 96

[0295] mbIL-7-KR24-62LIC-6: nucleotide sequence SEQ ID NO: 97, amino acid sequence SEQ ID NO: 98

[0296] mbIL-7-KR24-62LIC-7: nucleotide sequence SEQ ID NO: 109, amino acid sequence SEQ ID NO: 110

[0297] The signal transduction receptors involved in the examples are shown in Table 1:

[0298] Table 1 Cell surface receptor structures and sequences

[0299] Example 1: Construction of receptor expression vector

[0300] The pKB20 vector was constructed according to the method described in Example 1 on page 21 of PCT application WO2022078310A1. The pKB20 vector containing the exogenous gene expression cassette was constructed according to the method for constructing pKB20-EGFP described in the same example.Specifically, the commissioned company synthesized the sequences shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 101, 103, 107 and 109 in Table 1, respectively. The two ends of NO: 23, 25, 27, 29, 31, 33, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 101, 103, 107 and 109 were ligated with linkers containing corresponding restriction sites and cloned into the prepared pKB20 vector according to the method described in Example 1 on page 21 of WO2022078310A1 specification, and named as pKB20-KR23, pKB20-KR23-62LIC, respectively. , pKB20-KR24, pKB20-KR24-62LIC, pKB20-4D5, pKB20-4D5-62LIC, pKB20-KR23-62LIC-2, pKB20-KR23-62LIC-3, pKB20- KR23-62LIC-4, pKB20-KR23-62LIC-5, pKB20-KR23-62LIC-6, pKB20-mbIL-7-KR23-62LIC-2, pKB20-mbIL-7-KR23-62LI C-3, pKB20-mbIL-7-KR23-62LIC-4, pKB20-mbIL-7-KR23-62LIC-5, pKB20-mbIL-7-KR23-62LIC-6, pKB20-KR24-62LIC- 2. pKB20-KR24-62LIC-3, pKB20-KR24-62LIC-4, pKB20-KR24-62LIC-5, pKB20-KR24-62LIC-6, pKB20-mbIL-7-KR24-62L IC-2, pKB20-mbIL-7-KR24-62LIC-3, pKB20-mbIL-7-KR24-62LIC-4, pKB20-mbIL-7-KR24-62LIC-5, pKB20-mbIL-7-KR24-62LIC-6, pKB20-KR23-62LIC-7, pKB20-mbIL-7-KR23-62LIC-7, pKB20-KR24-62LIC-7, and pKB20-mbIL-7-KR24-62LIC-7. The recombinant plasmids obtained above were transformed into E. coli (DH5c). After correct sequencing, the plasmids were extracted and purified using Qiagen's plasmid purification kit to obtain high-quality plasmids of each recombinant expression vector.

[0301] Example 2: Isolation and culture of melanoma tissue-derived TIL cells

[0302] Freshly resected melanoma specimens that are positive for Her2, HLA-G, and HLA-C are collected and immediately processed under sterile conditions. The specific method is as follows: remove the normal tissue and necrotic areas around the cancer specimen, and remove 2-3mm thick pieces of tissue from different areas of the specimen. 3 Place 3-4 small tissue pieces in each well of a 6-well plate. Add 3 mL of complete culture medium (AIM-V medium containing 5% human AB serum), OKT-3 at a final concentration of 30 ng / mL, and IL-2 at 3000 IU / mL to each well. Culture the 24-well plate in a 37°C, 5% CO2 incubator. On the 5th to 6th day after the start of culture, half of the medium was replaced for all wells. After that, half of the medium was replaced every 1-2 days depending on the growth of TIL. Once the TIL in the well is full and all adherent cells have been removed, the TIL in each full well is collected, and after collecting enough TIL cells for subsequent experiments, prepare for the next experiment.

[0303] Example 3: Genetic modification and proliferation of TIL

[0304] 1) Add AIM-V medium to 31 wells of a 12-well plate in advance, 2 mL per well, and then transfer to a cell culture incubator at 37°C with 5% CO2 for 1 hour.

[0305] 2) Prepare the electroporation solution ratio for each well according to the following table:

[0306] Plasmids tested as needed: pKB20-KR23, pKB20-KR23-62LIC, pKB20-KR24, pKB20-KR24-62LIC, pKB20-4D5, pKB20-4D5-62LIC, pKB20-KR23-62LIC-2, pKB20-KR23-62LIC-3, pKB20-KR23-62LIC-4, pKB20-KR23-62LIC- 5. pKB20-KR23-62LIC-6, pKB20-KR23-62LIC-7, pKB20-mbIL-7-KR23-62LIC-2, pKB20-mbIL-7-KR23 -62LIC-3, pKB20-mbIL-7-KR23-62LIC-4, pKB20-mbIL-7-KR23-62LIC-5, pKB20-mbIL-7-KR23-62LI C-6, pKB20-mbIL-7-KR23-62LIC-7, pKB20-KR24-62LIC-2, pKB20-KR24-62LIC-3, pKB20-KR24-62LI C-4, pKB20-KR24-62LIC-5, pKB20-KR24-62LIC-6, pKB20-KR24-62LIC-7, pKB20-mbIL-7-KR24-62LI C-2, pKB20-mbIL-7-KR24-62LIC-3, pKB20-mbIL-7-KR24-62LIC-4, pKB20-mbIL-7-KR24-62LIC-5, pKB20-mbIL-7-KR24-62LIC-6, pKB20-mbIL-7-KR24-62LIC-7, and the control empty plasmid pKB20, to prepare 26 electroporation systems for the experimental groups and 1 group for the control group;

[0307] 3) The TIL obtained in Example 2 was transferred into 27 EP tubes, and 5×10 6 The cells were centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL of normal saline and the cell pellet was washed by repeating the centrifugation step;

[0308] 4) Add plasmids pKB20-KR23, pKB20-KR23-62LIC, pKB20-KR24, pKB20-KR24-62LIC, pKB20-4D5, pKB20-4D5-62LIC, pKB20-KR23-62LIC-2, pKB20-KR23-62LIC-3, and pKB20-KR23-62LIC to the electroporation solution of each experimental group and control group prepared in 2). C-4, pKB20-KR23-62LIC-5, pKB20-KR23-62LIC-6, pKB20-KR23-62LIC-7, pKB20-mbIL-7-KR23-62LI C-2, pKB20-mbIL-7-KR23-62LIC-3, pKB20-mbIL-7-KR23-62LIC-4, pKB20-mbIL-7-KR23-62LIC-5, p KB20-mbIL-7-KR23-62LIC-6, pKB20-mbIL-7-KR23-62LIC-7, pKB20-KR24-62LIC-2, pKB20-KR24-62 LIC-3, pKB20-KR24-62LIC-4, pKB20-KR24-62LIC-5, pKB20-KR24-62LIC-6, pKB20-KR24-62LIC-7, p 5 μg of pKB20-mbIL-7-KR24-62LIC-2, pKB20-mbIL-7-KR24-62LIC-3, pKB20-mbIL-7-KR24-62LIC-4, pKB20-mbIL-7-KR24-62LIC-5, pKB20-mbIL-7-KR24-62LIC-6, pKB20-mbIL-7-KR24-62LIC-7, and control empty plasmid pKB20 were added, and then allowed to stand at room temperature for less than 30 min;

[0309] 5) Resuspend all tubes with the plasmid electroporation solution prepared in 4), 100 μL per tube. Carefully pipette the cell resuspension into a LONZA 100 μL electroporation cup and place the cup into the LONZA Nucleofector. TM 2b In the electroporation tank, start the electroporation program and select X001;

[0310] 6) After electroporation is completed, carefully remove the electroporation cuvette, aspirate the cell suspension and transfer it to an EP tube, add 200 μL of preheated AIM-V medium to each tube, and then transfer it to the wells of the 12-well plate containing preheated AIM-V medium in 1) and culture at 37°C and 5% CO2; after 5 days of culture, overexpression of auxiliary receptors or chimeric antigen receptors KR23, KR23-62LIC, KR24, KR24-62LIC, 4D5, 4D5-62LIC, KR23-62LIC-2, KR23-62LIC-3, KR23-62LIC-4, KR23-62LIC-5, KR23-62LIC-6, KR23-62LIC-7, mbIL-7-KR23-62LIC-2, mbIL-7-KR23-62LIC-3, mbIL-7-KR The TIL cells of KR23-62LIC-4, mbIL-7-KR23-62LIC-5, mbIL-7-KR23-62LIC-6, mbIL-7-KR23-62LIC-7, KR24-62LIC-2, KR24-62LIC-3, KR24-62LIC-4, KR24-62LIC-5, KR24-62LIC-6, KR24-62LIC-7, mbIL-7-KR24-62LIC-2, mbIL-7-KR24-62LIC-3, mbIL-7-KR24-62LIC-4, mbIL-7-KR24-62LIC-5, mbIL-7-KR24-62LIC-6, mbIL-7-KR24-62LIC-7 were compared with the TIL cells of the control group.They are named TIL-KR23, TIL-KR23-62LIC, TIL-KR24, TIL-KR24-62LIC, TIL-4D5, TIL-4D5-62LIC, TIL-KR23-62LIC-2, TIL-KR23-62LIC-3, TIL-KR23-62LIC-4, TIL-KR23-62LIC-5, T IL-KR23-62LIC-6, TIL-KR23-62LIC-7, TIL-mbIL-7-KR23-62LIC-2, TIL-mbIL-7-KR23-62LIC-3, TIL-mbIL-7-KR23-62LIC-4, TIL-mbIL-7-KR23-62LIC-5, TIL-mbIL-7-KR23 -62LIC-6, TIL-mbIL-7-KR23-62LIC-7, TIL-KR24-62LIC-2, TIL-KR24-62LIC-3, TIL-KR24-62LIC-4, TIL-KR24-62LIC-5, TIL-KR24-62LIC-6, TIL-KR24-62LIC-7, TIL-mbIL- 7-KR24-62LIC-2, TIL-mbIL-7-KR24-62LIC-3, TIL-mbIL-7-KR24-62LIC-4, TIL-mbIL-7-KR24-62LIC-5, TIL-mbIL-7-KR24-62LIC-6, TIL-mbIL-7-KR24-62LIC-7 and TIL-CTRL. ,

[0311] Example 4: Cell survival rate and auxiliary receptor expression positive rate of TIL electroporated with cell surface receptors

[0312] The cell survival rates of each group were detected by trypan blue staining and cell counting. The results showed that the cell survival rates of the TILs expressing coreceptors or chimeric antigen receptors prepared in Example 3 and the control group TILs were all above 95%.

[0313] For each receptor containing the BCMA extracellular domain, the BCMA extracellular domain is fused to the extracellular region of each receptor and can serve as a tag for the exogenous transgene. The proportion of BCMA-positive cells is detected using a fluorescent antibody targeting the BCMA extracellular domain; the method is as follows:

[0314] 1) Collect cells from each group of TIL-KR23, TIL-KR23-62LIC, TIL-KR24, TIL-KR24-62LIC, TIL-4D5, TIL-4D5-62LIC and TIL-CTRL, and collect 1×10 cells from each group. 6, 800g, centrifugation for 5min;

[0315] 2) Discard the supernatant, add physiological saline to resuspend the cells, and centrifuge at 800g for 5 minutes;

[0316] 3) Discard the supernatant and resuspend the cells in 100 μL of saline per sample. Add 2 μL of BCMA flow cytometry antibody (Biolegend, Cat#: 357504) to each tube and incubate at room temperature for 30 minutes. Centrifuge for 5 minutes, resuspend and wash with saline, centrifuge again at 800g for 5 minutes, repeat twice, resuspend with 100 μL of saline, and add 2 μL of fluorescent secondary antibody targeting the primary antibody (Abcam, Cat#: ab72465) and incubate at room temperature for 30 minutes.

[0317] 4) Centrifuge the antibody-labeled cells from each group in 3) at 800 g for 5 min, add appropriate amount of physiological saline, centrifuge at 800 g for 5 min, wash twice, and discard the supernatant;

[0318] 5) Resuspend in 400 μL of physiological saline and analyze by flow cytometry.

[0319] For each receptor that does not contain the BCMA extracellular domain, the percentage of positive cells was detected using fluorescent antibodies targeting the extracellular domain of KIR2DL3 or KIR2DL4; the method is as follows:

[0320] 1) Collect TIL-KR23-62LIC-2, TIL-KR23-62LIC-3, TIL-KR23-62LIC-4, TIL-KR23-62LIC-5, TIL-KR23-62LIC-6, TIL-KR23-62LIC-7, TIL-mbIL-7-KR23-62LIC-2, TIL-mbIL-7-KR23-62LIC-3, TIL-mbIL-7-KR23-62LIC-4, TIL-mbIL-7-KR23-62LIC-5, TIL-mbIL-7-KR23-62LIC-6, TIL-mbIL-7-KR23-62LIC-7, TIL-KR24-6 The cells of each group were collected as 1 × 10 cells per group (TIL-2LIC-2, TIL-KR24-62LIC-3, TIL-KR24-62LIC-4, TIL-KR24-62LIC-5, TIL-KR24-62LIC-6, TIL-KR24-62LIC-7, TIL-mbIL-7-KR24-62LIC-2, TIL-mbIL-7-KR24-62LIC-3, TIL-mbIL-7-KR24-62LIC-4, TIL-mbIL-7-KR24-62LIC-5, TIL-mbIL-7-KR24-62LIC-6, TIL-mbIL-7-KR24-62LIC-7, and TIL-CTRL. 6 , 800g, centrifugation for 5min;

[0321] 2) Discard the supernatant, add physiological saline to resuspend the cells, and centrifuge at 800g for 5 minutes;

[0322] 3) Discard the supernatant and resuspend the cells in 100 μL of saline per sample. For cells containing the KIR2DL3 extracellular domain receptor, add 2 μL of KIR2DL3 flow cytometry antibody (Human KIR2DL3 / CD158b2 PE-conjugated Antibody, R&D Systems Cat#: FAB2014P) to each tube; for cells containing the KIR2DL4 extracellular domain receptor, add 2 μL of KIR2DL4 flow cytometry antibody (Human KIR2DL4 / CD158d PE-conjugated Antibody, R&D Systems Cat#: FAB2238P) to each tube. For TIL-CTRL, add both KIR2DL3 and KIR2DL4 flow cytometry antibodies. Incubate at room temperature for 30 minutes; centrifuge for 5 minutes, resuspend and wash with saline, and centrifuge again at 800g for 5 minutes.

[0323] 4) Resuspend in 400 μL of physiological saline and analyze by flow cytometry.

[0324] The positive rates of cells in each group containing receptors with the BCMA extracellular domain are shown in Table 2 below:

[0325] Table 2

[0326] The positive rates of cells in each group that do not contain the BCMA extracellular domain receptor are shown in Table 3 below:

[0327] Table 3

[0328] The results showed that the proportion of positive TILs in the TILs expressing receptors containing the CD62L intracellular signaling region in the intracellular domain was significantly higher than that in the TILs expressing the corresponding receptors without the CD62L intracellular signaling region in the intracellular domain.

[0329] Example 5: Cytotoxicity of TILs Overexpressing Cell Surface Receptors on Syngeneic Tumor Cells

[0330] The fresh melanoma tissue of Example 2 was cut into 3×3×3 mm pieces. The pieces were mixed as evenly as possible and then cultured to obtain primary melanoma cells according to the method described in Robert Suriano et al. Ex Vivo Derived Primary Melanoma Cells: Implications for Immunotherapeutic Vaccines J Cancer 2013; 4(5): 371-382. Materials and Methods.

[0331] Agilent's real-time label-free cell function analyzer (RTCA) was used to detect the TIL-KR23, TIL-KR23-62LIC, TIL-KR24, TIL-KR24-62LIC, TIL-4D5, TIL-4D5-62LIC, TIL-KR23-62LIC-2, TIL-KR23-62LIC-3, TIL-KR23-62LIC obtained in Example 3. IC-4, TIL-KR23-62LIC-5, TIL-KR23-62LIC-6, TIL-KR23-62LIC-7, TIL-mbIL-7-KR23-62LIC-2, TIL-mbIL-7-KR23-62LIC-3, TIL-mbIL-7-KR23-62LIC-4, TIL-mbIL-7-KR23-62LIC-5, TIL-mbIL- 7-KR23-62LIC-6, TIL-mbIL-7-KR23-62LIC-7, TIL-KR24-62LIC-2, TIL-KR24-62LIC-3, TIL-KR2 4-62LIC-4, TIL-KR24-62LIC-5, TIL-KR24-62LIC-6, TIL-KR24-62LIC-7, TIL-mbIL-7-KR24-62L The in vitro cytotoxicity of IC-2, TIL-mbIL-7-KR24-62LIC-3, TIL-mbIL-7-KR24-62LIC-4, TIL-mbIL-7-KR24-62LIC-5, TIL-mbIL-7-KR24-62LIC-6, TIL-mbIL-7-KR24-62LIC-7, and TIL-CTRL to their homologous primary melanoma cells was evaluated as follows:

[0332] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place it in the instrument, select step 1, and adjust to zero;

[0333] (2) Target cell plating: The primary melanoma cells obtained by culture were plated at 10 per well. 4 Spread 50 μL of cells on a plate containing detection electrodes and leave it for a few minutes. After the cells are stable, place them in the instrument and start step 2 to culture the cells.

[0334] (3) Adding effector cells: After culturing target cells for 18 h to 24 h, observe the cell index. When the cell index is 1, add effector cells TIL-KR23, TIL-KR23-62LIC, TIL-KR24, TIL-KR24-62LIC, TIL-4D5, TIL-4D5-62LIC, TIL-KR23-62LIC-2, TIL-KR23-62LIC-3, TIL-KR23-62LIC-4, TIL-KR23 -62LIC-5, TIL-KR23-62LIC-6, TIL-KR23-62LIC-7, TIL-mbIL-7-KR23-62LIC-2, TIL-mbIL-7-KR23-62LIC- 3. TIL-mbIL-7-KR23-62LIC-4, TIL-mbIL-7-KR23-62LIC-5, TIL-mbIL-7-KR23-62LIC-6, TIL-mbIL-7-KR23 -62LIC-7, TIL-KR24-62LIC-2, TIL-KR24-62LIC-3, TIL-KR24-62LIC-4, TIL-KR24-62LIC-5, TIL-KR24-62L IC-6, TIL-KR24-62LIC-7, TIL-mbIL-7-KR24-62LIC-2, TIL-mbIL-7-KR24-62LIC-3, TIL-mbIL-7-KR24-62L IC-4, TIL-mbIL-7-KR24-62LIC-5, TIL-mbIL-7-KR24-62LIC-6, TIL-mbIL-7-KR24-62LIC-7, and TIL-CTRL. All effector cells except TIL-CTRL were adjusted to a 34% positive cell ratio with TIL-CTRL. 50 μL was added to each well with an effector-target ratio of 1:1. Step 3 was started. After 72 hours of co-culture, the cell proliferation curve and killing level were observed, and the target cell killing rate was calculated. The target cell killing rate was calculated as follows:

[0335] A is the cell index of the group with only target cells (ie, tumor cells) but no effector cells added, and B is the cell index of the groups with effector cells added.

[0336] The results are shown in Table 4:

[0337] Table 4

[0338] The results in Table 4 show that compared with TIL-CTRL, TIL-KR23, TIL-KR23-62LIC, TIL-KR24, TIL-KR24-62LIC, TIL-4D5, TIL-4D5-62LIC, TIL-KR23-62LIC-2, TIL-KR23-62LIC-3, TIL-KR23-62LIC-4, TIL-KR23-62 LIC-5, TIL-KR23-62LIC-6, TIL-KR23-62LIC-7, TIL-mbIL-7-KR23-62LIC-2, TIL-mbIL-7-KR23-62LIC-3, TIL-mbIL-7-KR23-62LIC-4, TIL-mbIL-7-KR23-62LIC-5, TIL-mbIL-7-KR23- 62LIC-6, TIL-mbIL-7-KR23-62LIC-7, TIL-KR24-62LIC-2, TIL-KR24-62LIC-3, TIL-KR24-62LIC-4, TIL-KR24-62LIC-5, TIL-KR24-62LIC-6, TIL-KR24-62LIC-7, TIL-mbIL-7-KR24-62LIC-2, TIL-mbIL-7-KR24-62LIC-3, TIL-mbIL-7-KR24-62LIC-4, TIL-mbIL-7-KR24-62LIC-5, TIL-mbIL-7-KR24-62LIC-6 and TIL-mbIL-7-KR24-62LIC-7 had significantly stronger killing effects on homologous melanoma primary tumor cells. Moreover, compared with the corresponding TILs (TIL-KR23, TIL-KR24, TIL-4D5) that express receptors that do not contain the CD62L intracellular domain, TILs that express receptors that contain the CD62L intracellular domain have significantly improved killing ability against target cells.

[0339] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

[0340] Part of this article

Claims

1. A cell surface receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytoplasmic domain comprising a CD62L intracellular domain, Preferably, the CD62L intracellular domain comprises the sequence shown in SEQ ID NO: 10; Preferably, the CD62L intracellular domain is a truncated CD62L intracellular domain, and more preferably, the truncated CD62L intracellular domain comprises the sequence shown in SEQ ID NO:

36.

2. The cell surface receptor according to claim 1, wherein The cytoplasmic domain comprises a signal transduction domain and / or a costimulatory domain, Preferably, the costimulatory domain is the intracellular domain of the costimulatory signal molecule or a functional fragment or mutant thereof that retains the biological function of the costimulatory signal molecule to transmit costimulatory signals and activate immune cells.

3. The cell surface receptor according to claim 2, wherein The intracellular domain of the costimulatory signal molecule is the intracellular domain of CD28 and / or the intracellular domain of OX40, and the signal transduction domain is the CD3ζ intracellular signaling region.

4. The cell surface receptor according to claim 3, wherein The CD62L intracellular domain is located at the N-terminus of the CD28 intracellular domain, the OX40 intracellular domain or the CD3ζ intracellular signaling region. Preferably, the cytoplasmic domain comprises the CD62L intracellular domain and the CD28 intracellular domain, and optionally further comprises the OX40 intracellular domain and / or the CD3ζ intracellular signaling region; Preferably, the cytoplasmic domain comprises the CD62L intracellular domain and the OX40 intracellular domain; Preferably, the cytoplasmic domain comprises a truncated CD62L intracellular domain and an OX40 intracellular domain.

5. The cell surface receptor according to claim 1, wherein The extracellular ligand-binding domain comprises the extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligand. Preferably, the KIR is an activating KIR or an inhibitory KIR, More preferably, the activating KIR is KIR2DL4, and the inhibitory KIR is any one or more selected from KIR2DL3, KIR3DL1 and KIR3DL2.

6. The cell surface receptor according to claim 1, wherein The extracellular ligand binding domain comprises an antibody or an antigen-binding fragment thereof that targets a tumor antigen.

7. The cell surface receptor according to claim 5 or 6, wherein The extracellular ligand-binding domain further comprises the BCMA extracellular domain or a mutant thereof.

8. The cell surface receptor according to claim 1, wherein The transmembrane region is any one selected from the group consisting of the CD28 transmembrane region, the KIR2DL3 transmembrane region and the KIR2DI4 transmembrane region.

9. The cell surface receptor according to any one of claims 1 to 8, wherein The extracellular ligand binding domain also comprises a signal peptide.

10. The cell surface receptor according to any one of claims 1 to 8, wherein The cell surface receptors include: Optional CD8 signal peptide, and KIR2DL3 extracellular region, BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain, Optional CD8 signal peptide, and KIR2DL4 extracellular region, BCMA extracellular domain mutant, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain, Optional CD8 signal peptide, and 4D5 single-chain antibody, BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and CD3ζ intracellular signaling region, Optional CD8 signal peptide, and KIR2DL3 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain, Optional CD8 signal peptide, and KIR2DL3 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, an optional CD8 signal peptide, and a KIR2DL3 extracellular region, a KIR2DL3 transmembrane region, a truncated CD62L intracellular domain, and an OX40 intracellular domain, an optional KIR2DL3 signal peptide, and a KIR2DL3 extracellular region, a KIR2DL3 transmembrane region, a truncated CD62L intracellular domain, and an OX40 intracellular domain, an optional CD8 signal peptide, and the KIR2DL3 extracellular region, the CD28 hinge region, the CD28 transmembrane region, the truncated CD62L intracellular domain, and the OX40 intracellular domain, an optional CD8 signal peptide, and the KIR2DL3 extracellular region, the CD28 hinge region, the CD28 transmembrane region, the truncated CD62L intracellular domain, and the OX40 intracellular domain, an optional CD8 signal peptide, and a truncated KIR2DL3 extracellular region, a CD28 hinge region, a CD28 transmembrane region, a truncated CD62L intracellular domain, and an OX40 intracellular domain, Optional CD8 signal peptide, and KIR2DL4 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain, Optional CD8 signal peptide, and KIR2DL4 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, Optional CD8 signal peptide, and KIR2DL4 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, an optional KIR2DL4 signal peptide, and a KIR2DL4 extracellular region, a KIR2DL4 transmembrane region, a truncated CD62L intracellular domain, and an OX40 intracellular domain, Optional CD8 signal peptide, and KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain, or Optional CD8 signal peptide, and truncated KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain.

11. The cell surface receptor according to claim 10, wherein The CD28 transmembrane region has an amino acid sequence as shown in SEQ ID NO: 4, The KIR2DL3 transmembrane region has an amino acid sequence as shown in SEQ ID NO: 42, The KIR2DL4 transmembrane region has an amino acid sequence as shown in SEQ ID NO: 44, The CD28 intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 6, The OX40 intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 8, The CD3ζ intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO: 22, The KIR2DL3 extracellular region has an amino acid sequence as shown in SEQ ID NO: 12, The KIR2DL4 extracellular region has an amino acid sequence as shown in SEQ ID NO: 14, The truncated KIR2DL3 extracellular region has an amino acid sequence as shown in SEQ ID NO: 100, The truncated KIR2DL4 extracellular region has an amino acid sequence as shown in SEQ ID NO: 106, and the antibody or antigen-binding fragment thereof targeting a tumor antigen is a 4D5 single-chain antibody having an amino acid sequence as shown in SEQ ID NO:

16. The BCMA extracellular domain has the amino acid sequence shown in SEQ ID NO: 18, and / or The BCMA extracellular domain mutant has an amino acid sequence as shown in SEQ ID NO: 20, Preferably, the cell surface receptor has any one of the amino acid sequences shown in SEQ ID NO: 26, 30, 34, 60, 62, 64, 66, 68, 80, 82, 84, 86, 88, 102 and 108.

12. A fusion protein comprising the cell surface receptor according to any one of claims 1 to 11 and a membrane surface cytokine, Preferably, the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region, Preferably, the membrane surface cytokine is located at the N-terminus and / or C-terminus of the cell surface receptor. Preferably, the cytokine is IL-7, Preferably, the GPI anchor region is CD52, Preferably, the membrane surface cytokine further comprises a BCMA extracellular domain; preferably, the BCMA extracellular domain is a mutant BCMA extracellular domain, Preferably, the membrane surface cytokines include: CD52 signal peptide, IL-7, linker, CD52, Preferably, the membrane surface cytokine comprises: CD52 signal peptide, IL-7, linker, mutant BCMA extracellular domain, CD52, Preferably, the membrane surface cytokine is connected to the cell surface receptor via a linker; preferably, the linker comprises a cleavable sequence; preferably, the cleavable sequence is one or more selected from P2A, T2A, F2A and E2A, Preferably, the membrane surface cytokine is directly linked to the cell surface receptor; preferably, the membrane surface cytokine is at the N-terminus of the cell surface receptor. Preferably, the fusion protein comprises: Optionally, the membrane surface cytokine, CD8 signal peptide, KIR2DL3 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, CD8 signal peptide, KIR2DL3 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, CD8 signal peptide, KIR2DL3 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, KIR2DL3 signal peptide, KIR2DL3 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, CD8 signal peptide, KIR2DL3 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, CD8 signal peptide, truncated KIR2DL3 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, CD8 signal peptide, and KIR2DL4 extracellular region, CD28 transmembrane region, CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, CD8 signal peptide, and KIR2DL4 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, CD8 signal peptide, and KIR2DL4 extracellular region, KIR2DL3 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, KIR2DL4 signal peptide, and KIR2DL4 extracellular region, KIR2DL4 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; or Optionally, the membrane surface cytokine, CD8 signal peptide, and KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, CD8 signal peptide, and KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; Optionally, the membrane surface cytokine, CD8 signal peptide, and truncated KIR2DL4 extracellular region, CD28 hinge region, CD28 transmembrane region, truncated CD62L intracellular domain and OX40 intracellular domain; Preferably, the amino acid sequence of the cell surface receptor is as shown in any one of SEQ ID NOs: 26, 30, 34, 60, 62, 64, 66, 68, 80, 82, 84, 86, 88, 102 and 108, Preferably, the amino acid sequence of the fusion protein is as shown in any one of SEQ ID NOs: 70, 72, 74, 76, 78, 90, 92, 94, 96, 988, 104 and 110.

13. A polynucleotide comprising: (1) a sequence encoding the cell surface receptor according to any one of claims 1 to 11 or the fusion protein according to claim 12, and / or (2) The complementary sequence of (1). Preferably, the sequence of the polynucleotide is as shown in any one of SEQ ID NO: 25, 29, 33, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 101, 103, 107 and 109, or a complementary sequence thereof.

14. A nucleic acid construct comprising the polynucleotide according to claim 13, Preferably, The nucleic acid construct further comprises at least one regulatory element for expressing the cell surface receptor according to any one of claims 1 to 11 or the fusion protein according to claim 12, which is operably linked to the polynucleotide. The nucleic acid construct is an expression vector or a cloning vector, The nucleic acid construct is a viral vector or a non-viral vector; more preferably, the non-viral vector is a non-viral integration vector based on a transposon system.

15. A host cell, which: (1) containing, expressing or secreting the cell surface receptor according to any one of claims 1 to 11 and / or the fusion protein according to claim 12, (2) a polynucleotide encoding the cell surface receptor according to any one of claims 1 to 11 and / or the fusion protein according to claim 12, or a nucleic acid construct comprising the polynucleotide, Preferably, The host cell is an immune effector cell; more preferably, the host cell is a T cell, NK cell, CIK cell, DN T cell or tumor infiltrating lymphocyte, and / or The cells also express CAR, or carry a coding sequence for CAR, and / or The cells also express exogenous TCR, or carry the coding sequence of an exogenous TCR.

16. A pharmaceutical composition comprising any one or more of the cell surface receptor according to any one of claims 1 to 11, the fusion protein according to claim 12, the polynucleotide according to claim 13, the nucleic acid construct according to claim 14, and the host cell according to claim 15. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

17. Use of any one or more of the cell surface receptor according to any one of claims 1 to 11, the fusion protein according to claim 12, the polynucleotide according to claim 13, the nucleic acid construct according to claim 14, and the host cell according to claim 15 in the preparation of a drug for treating or preventing cancer. Preferably, The cancer is a tumor antigen-related cancer, and the tumor antigen is a tumor antigen targeted by the extracellular ligand binding domain of the cell surface receptor; preferably, the cancer is a HER2-related cancer.

18. The use according to claim 17, characterized in that The cancer is selected from the group consisting of lung cancer, melanoma, breast cancer, gastric cancer, bladder cancer, endometrial cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma, acute lymphoblastic leukemia, small cell lung cancer, Hodgkin lymphoma and childhood acute lymphoblastic leukemia.

Images