Cell expressing co-receptor

Abstract

Provided is a genetically engineered cell expressing a co-receptor and a membrane-bound cytokine. The co-receptor comprises a KIR extracellular domain, a transmembrane region, and an intracellular region. When the co-receptor and the membrane-bound cytokine are co-expressed in an immune cell, they can further enhance the cytokine secretion level, proliferation level, and cytotoxicity of the immune cell toward a target cell.

Description

Cell expressing accessory receptor

[0001] This application claims priority to the application with the application number CN202410998498.9, the title of which is "Cell expressing accessory receptor", filed on July 24, 2024, the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, in particular to a cell expressing accessory receptor. BACKGROUND

[0003] Killer-cell Ig-like receptors (KIRs) are a group of germline-encoded receptors with activating or inhibitory functions expressed on the surface of NK cells and a small subset of T cells, including KIR3DL1, 2, 3, KIR3DS1, KIR2DL1, 2, 3, 4, 5A, 5B and KIR2DS1, 2, 3, 4, 5, in addition to two pseudogenes 2DP1 and 3DP1. The ligands of the inhibitory KIRs studied more are mainly various types of HLA class I molecules. There is still a lack of immune effector cells developed based on KIR receptors and cytokines in the art. SUMMARY

[0004] To this end, the present application provides a genetically engineered cell expressing an accessory receptor and a membrane surface cytokine, the accessory receptor comprising a KIR extracellular domain, a transmembrane region and an intracellular region.

[0005] The present application provides an accessory receptor comprising an extracellular domain, a transmembrane region and an intracellular region, the extracellular domain comprising a KIR extracellular domain or a functional fragment or variant thereof retaining the biological function of binding to KIR ligands, and the intracellular region comprising a signal transduction domain and / or a costimulatory domain.

[0006] In one or more embodiments, the accessory receptor further comprises a signal peptide.

[0007] In one or more embodiments, the accessory receptor further comprises a hinge region. Optionally, the hinge region comprises a fragment of the proximal membrane end of the native extracellular domain of the costimulatory signaling molecule. In some embodiments, the hinge region is located at the N-terminal of the transmembrane region.

[0008] 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 selected from any one or more of KIR2DL3, KIR3DL1 and KIR3DL2.

[0009] In one or more embodiments, the co-stimulatory domain is an intracellular domain of a co-stimulatory signaling molecule or a functional fragment or mutant thereof that retains the biological function of the co-stimulatory signaling molecule to transmit a co-stimulatory signal, activating the immune cell.

[0010] In one or more embodiments, the accessory receptor further comprises a membrane surface tag. In one or more embodiments, the membrane surface tag comprises a BCMA extracellular domain or a variant thereof, or a claudin protein extracellular domain or a fragment or variant thereof. Preferably, the membrane surface tag is located at the C-terminus of the extracellular domain of the KIR.

[0011] In one or more embodiments, the accessory receptor further comprises a linker located between the extracellular domain of the KIR or a functional fragment or variant thereof and the membrane surface tag; preferably, the linker is a rigid linker or a flexible linker.

[0012] In one or more embodiments, the membrane surface tag further comprises a linker or hinge located at the N-terminus or C-terminus of the BCMA extracellular domain or a variant thereof, or a claudin protein extracellular domain or a fragment or variant thereof.

[0013] In one or more embodiments, the claudin protein is claudin 18, preferably claudin 18.2. In one or more embodiments, the fragment of the claudin protein extracellular domain is a claudin protein extracellular epitope.

[0014] In one or more embodiments, the BCMA extracellular domain comprises the sequence set forth in SEQ ID NO: 26; the nucleic acid sequence thereof comprises the sequence set forth in SEQ ID NO: 25.

[0015] In one or more embodiments, the linker located at the N-terminus or C-terminus of the BCMA extracellular domain or a variant thereof, or a claudin protein extracellular domain or a fragment or variant thereof is a rigid linker or a flexible linker. In one or more embodiments, the linker is a rigid linker.

[0016] In one or more embodiments, 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. Preferably, the transmembrane region is a CD28 transmembrane region, an IL7Ra transmembrane region, or mutants thereof that retain transmembrane function.

[0017] In one or more embodiments, the costimulatory signaling molecule endodomain includes, but is not limited to, any one or more of the endodomains 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. Preferably, the costimulatory signaling molecule endodomain is a CD28 endodomain and / or an OX40 endodomain.

[0018] In one or more embodiments, the hinge region comprises, but is not limited to, a juxtamembrane fragment of a 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. Preferably, the hinge region is a CD28 extracellular hinge region, a CD8 extracellular hinge region, or an IL7Ra extracellular hinge region.

[0019] In one or more embodiments, the costimulatory receptor comprises a KIR2DL3 extracellular region or a KIR2DL4 extracellular region, an extracellular hinge region, a transmembrane region, and one or more intracellular domains selected from a CD80 intracellular domain, a CD28 intracellular domain, and an OX40 intracellular domain. The transmembrane region can be a CD80 transmembrane region or a CD28 transmembrane region or a mutant thereof. The extracellular hinge region can comprise a CD28 extracellular hinge region or a CD8 extracellular hinge region. In one or more embodiments, the costimulatory receptor comprises a KIR2DL3 extracellular region or a KIR2DL4 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain. In one or more embodiments, the costimulatory receptor comprises a KIR2DL3 extracellular region or a KIR2DL4 extracellular region, a CD28 extracellular hinge region, a CD80 transmembrane region, and a CD80 intracellular domain. In one or more embodiments, the costimulatory receptor comprises a KIR3DL1 extracellular region or a KIR3DL2 extracellular region, an extracellular hinge region, a transmembrane region, and one or more intracellular domains selected from a CD80 intracellular domain, a CD28 intracellular domain, and an OX40 intracellular domain. The transmembrane region can be a CD80 transmembrane region or a CD28 transmembrane region or a mutant thereof. The extracellular hinge region can comprise a CD28 extracellular hinge region or a CD8 extracellular hinge region. In one or more embodiments, the costimulatory receptor comprises a KIR3DL1 extracellular region or a KIR3DL2 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain. In one or more embodiments, the costimulatory receptor comprises a KIR3DL1 extracellular region or a KIR3DL2 extracellular region, a CD28 extracellular hinge region, a CD80 transmembrane region, and a CD80 intracellular domain.

[0020] In one or more embodiments, the costimulatory receptor comprises a KIR2DL3 extracellular region, a linker, a membrane surface tag, a transmembrane region, and one or more intracellular domains selected from a CD28 intracellular domain, an OX40 intracellular domain, and an IL-7Ra intracellular domain, the transmembrane region being a CD28 transmembrane region or an IL7Ra transmembrane region or a mutant thereof. Wherein the membrane surface tag comprises a Claudin 18.2 antigenic epitope or a BCMA extracellular domain; the costimulatory receptor further comprises a CD28 extracellular hinge region or a CD8 extracellular hinge region between the membrane surface tag and the transmembrane region. In one or more embodiments, the costimulatory receptor comprises a KIR2DL3 extracellular region, a Claudin 18.2 antigenic epitope, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain. In one or more embodiments, the costimulatory receptor comprises a KIR2DL3 extracellular region, a BCMA extracellular domain, an IL7Ra transmembrane region or any one of mutants 1-4 thereof, an IL-7Ra intracellular domain. In one or more embodiments, the costimulatory receptor comprises a KIR2DL3 extracellular region, a linker, a BCMA extracellular domain, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain.

[0021] In one or more embodiments, the costimulatory receptor comprises a KIR2DL4 extracellular region, a linker, a membrane surface tag, a transmembrane region, and one or more intracellular domains selected from a CD28 intracellular domain, an OX40 intracellular domain, and an IL-7Ra intracellular domain, the transmembrane region being a CD28 transmembrane region or an IL7Ra transmembrane region or a mutant thereof (e.g. mutant 4). Wherein the membrane surface tag comprises a Claudin 18.2 antigenic epitope or a BCMA extracellular domain; the costimulatory receptor further comprises a CD28 extracellular hinge region, a CD8 extracellular hinge region, or an IL7Ra extracellular hinge region between the membrane surface tag and the transmembrane region. In one or more embodiments, the costimulatory receptor comprises a KIR2DL4 extracellular region, a Claudin 18.2 antigenic epitope, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain, or the costimulatory receptor comprises a KIR2DL4 extracellular region, a BCMA extracellular domain, an IL7Ra extracellular hinge region, an IL7Ra transmembrane region, and an IL-7Ra intracellular domain, or the costimulatory receptor comprises a KIR2DL4 extracellular region, a Claudin 18.2 antigenic epitope, an IL7Ra extracellular hinge region, an IL7Ra transmembrane region mutant 4, and an IL-7Ra intracellular domain. In one or more embodiments, the costimulatory receptor comprises a KIR2DL4 extracellular region, a linker, a BCMA extracellular domain, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain.

[0022] In one or more embodiments, the costimulatory receptor comprises a KIR3DL1 extracellular region or a KIR3DL2 extracellular region, a membrane surface tag, a transmembrane region that is a CD28 transmembrane region, and one or more intracellular domains selected from a CD28 intracellular domain, an OX40 intracellular domain, and an IL-7Ra intracellular domain.

[0023] In one or more embodiments, the amino acid sequence of the CD28 transmembrane region is set forth in SEQ ID NO: 4.

[0024] In one or more embodiments, the amino acid sequence of the CD28 intracellular domain is set forth in SEQ ID NO: 16.

[0025] In one or more embodiments, the amino acid sequence of the OX40 intracellular domain is set forth in SEQ ID NO: 14.

[0026] In one or more embodiments, the amino acid sequence of the KIR2DL3 extracellular region is set forth in SEQ ID NO: 36.

[0027] In one or more embodiments, the amino acid sequence of the KIR2DL4 extracellular region is set forth in SEQ ID NO: 38.

[0028] In one or more embodiments, the amino acid sequence of the KIR3DL1 extracellular region is set forth in SEQ ID NO: 40.

[0029] In one or more embodiments, the amino acid sequence of the KIR3DL2 extracellular region is set forth in SEQ ID NO: 42.

[0030] In one or more embodiments, the amino acid sequence of the CD28 extracellular hinge region is set forth in SEQ ID NO: 8.

[0031] In one or more embodiments, the amino acid sequence of the CD8 extracellular hinge region is set forth in SEQ ID NO: 151.

[0032] A second aspect of the present application provides a costimulatory receptor comprising an extracellular domain comprising an extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to a KIR ligand, a transmembrane region, and an intracellular domain comprising a CD62L intracellular domain.

[0033] In one or more embodiments, the KIR is an activating KIR or an inhibitory KIR,

[0034] In one or more embodiments, the activating KIR is KIR2DL4 and the inhibitory KIR is selected from any one or more of KIR2DL3, KIR3DL1 and KIR3DL2.

[0035] In one or more embodiments, the intracellular domain comprises a signal transduction domain and / or a costimulatory domain,

[0036] In one or more embodiments, the costimulatory domain is an intracellular domain of a costimulatory signaling molecule or a functional fragment or mutant thereof that retains the biological function of the costimulatory signaling molecule to transmit a costimulatory signal, activating an immune cell.

[0037] In one or more embodiments, the costimulatory signaling molecule intracellular domain comprises any one or more selected from the group consisting of 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 mutants thereof.

[0038] In one or more embodiments, the costimulatory signaling molecule intracellular domain is an intracellular domain of CD28 and / or an intracellular domain of OX40.

[0039] In one or more embodiments, the CD62L intracellular domain is a full-length CD62L intracellular domain or a truncated CD62L intracellular domain.

[0040] In one or more embodiments, the costimulatory signaling molecule intracellular domain is an intracellular domain of CD28 and / or an intracellular domain of OX40.

[0041] In one or more embodiments, the hinge region comprises a juxtamembrane fragment of a native extracellular domain of the costimulatory signaling molecule, or, the hinge region comprises but is not limited to any one or more 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.

[0042] In one or more embodiments, the hinge region is a CD28 extracellular hinge region, a CD8 extracellular hinge region, or an IL7Ra extracellular hinge region.

[0043] In one or more embodiments, the costimulatory receptor further comprises a membrane surface tag.

[0044] In one or more embodiments, the membrane surface tag comprises a BCMA extracellular domain or a variant thereof, or a claudin protein extracellular domain or a fragment or variant thereof.

[0045] In one or more embodiments, the membrane surface tag is located at the C-terminus of the extracellular domain of KIR.

[0046] In one or more embodiments, the claudin protein is claudin 18, and the fragment of the claudin protein extracellular domain is a claudin protein extracellular antigenic epitope.

[0047] In one or more embodiments, the transmembrane region comprises one or more selected from the group consisting of a transmembrane region 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-100R, 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, CD80, and CD27, or a mutant thereof that retains transmembrane function.

[0048] In one or more embodiments, the transmembrane region is a CD28 transmembrane region, an IL7R alpha transmembrane region, or a mutant thereof that retains transmembrane function.

[0049] In one or more embodiments, the costimulatory receptor further comprises a signal peptide.

[0050] In one or more embodiments, the costimulatory receptor comprises an extracellular domain comprising a KIR2DL3 extracellular region or a KIR2DL4 extracellular region, a membrane surface tag, a transmembrane region, and a CD62L intracellular domain, optionally further comprising one or more intracellular domains selected from the group consisting of a CD28 intracellular domain, an OX40 intracellular domain, and an IL-7R alpha intracellular domain, the transmembrane region being a CD28 transmembrane region or an IL7R alpha transmembrane region or a mutant thereof.

[0051] In one or more embodiments, the membrane surface tag comprises a Claudin 18.2 antigen epitope or a BCMA extracellular domain or a mutant thereof, and / or the costimulatory receptor further comprises a CD28 hinge region between the membrane surface tag and the transmembrane region.

[0052] In one or more embodiments, the costimulatory receptor further comprises a linker; preferably, the linker is a rigid linker or a flexible linker; preferably, the linker is located between the extracellular ligand binding domain and the membrane surface tag, and / or the linker is located between the membrane surface tag and the transmembrane region.

[0053] In one or more embodiments, the costimulatory receptor comprises:

[0054] (1) KIR2DL3 extracellular region, Claudin 18.2 antigenic epitope, CD28 extracellular hinge region, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain, and OX40 intracellular domain,

[0055] (2) KIR2DL3 extracellular region, BCMA extracellular domain, IL7Ra transmembrane region or mutant thereof, CD62L intracellular domain, IL-7Ra intracellular domain, or

[0056] (3) KIR2DL3 extracellular region, linker, BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain, and OX40 intracellular domain,

[0057] (4) KIR2DL3 extracellular region, BCMA extracellular domain or variant thereof, linker, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain, and OX40 intracellular domain

[0058] (5) KIR2DL4 extracellular region, Claudin 18.2 antigenic epitope, CD28 extracellular hinge region, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain, and OX40 intracellular domain,

[0059] (6) KIR2DL4 extracellular region, BCMA extracellular domain, IL7Ra extracellular hinge region, IL7Ra transmembrane region, CD62L intracellular domain, and IL-7Ra intracellular domain,

[0060] (7) KIR2DL4 extracellular region, Claudin 18.2 antigenic epitope, IL7Ra extracellular hinge region, IL7Ra transmembrane region mutant, CD62L intracellular domain, and IL-7Ra intracellular domain,

[0061] (8) KIR2DL4 extracellular region, linker, BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain, and OX40 intracellular domain,

[0062] (9) KIR2DL4 extracellular region, BCMA extracellular domain mutant, linker, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain, and OX40 intracellular domain,

[0063] (10) KIR2DL3 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain, and OX40 intracellular domain,

[0064] (11) KIR2DL3 extracellular region, CD28 extracellular hinge region, CD80 transmembrane region, CD62L endodomain, and CD80 endodomain,

[0065] (12) KIR2DL4 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD62L endodomain, CD28 endodomain, and OX40 endodomain,

[0066] (13) KIR2DL4 extracellular region, CD28 extracellular hinge region, CD80 transmembrane region, CD62L endodomain, and CD80 endodomain,

[0067] (14) KIR2DL3 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, truncated CD62L endodomain, CD28 endodomain, and OX40 endodomain,

[0068] (15) KIR2DL3 extracellular region, CD28 extracellular hinge region, CD80 transmembrane region, truncated CD62L endodomain, and CD80 endodomain,

[0069] (16) KIR2DL4 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, truncated CD62L endodomain, CD28 endodomain, and OX40 endodomain,

[0070] (17) KIR2DL4 extracellular region, CD28 extracellular hinge region, CD80 transmembrane region, truncated CD62L endodomain, and CD80 endodomain,

[0071] (18) KIR3DL1 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD62L endodomain, CD28 endodomain, and OX40 endodomain,

[0072] (19) KIR3DL2 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD62L endodomain, CD28 endodomain, and OX40 endodomain,

[0073] (20) KIR2DL3 extracellular region, CD28 transmembrane region, CD62L endodomain, and OX40 endodomain,

[0074] (21) KIR2DL3 extracellular region, CD28 transmembrane region, truncated CD62L endodomain, and OX40 endodomain,

[0075] (22) KIR2DL3 extracellular region, BCMA extracellular domain mutant, CD28 transmembrane region, CD62L endodomain, and OX40 endodomain,

[0076] (23) KIR2DL3 ectodomain, BCMA ectodomain mutant, CD28 transmembrane domain, truncated CD62L endodomain, and OX40 endodomain,

[0077] (24) KIR2DL4 ectodomain, CD28 transmembrane domain, CD62L endodomain, and OX40 endodomain,

[0078] (25) KIR2DL4 ectodomain, CD28 transmembrane domain, truncated CD62L endodomain, and OX40 endodomain,

[0079] (26) KIR2DL4 ectodomain, BCMA ectodomain mutant, CD28 transmembrane domain, CD62L endodomain, and OX40 endodomain,

[0080] (27) KIR2DL4 ectodomain, BCMA ectodomain mutant, CD28 transmembrane domain, truncated CD62L endodomain, and OX40 endodomain,

[0081] (28) KIR2DL3 ectodomain, CD8 ectodomain hinge region, CD28 transmembrane domain, CD62L endodomain, and OX40 endodomain,

[0082] (29) KIR2DL3 ectodomain, CD8 ectodomain hinge region, CD28 transmembrane domain, truncated CD62L endodomain, and OX40 endodomain,

[0083] (30) KIR2DL4 ectodomain, CD8 ectodomain hinge region, CD28 transmembrane domain, CD62L endodomain, and OX40 endodomain,

[0084] (31) KIR2DL4 ectodomain, CD8 ectodomain hinge region, CD28 transmembrane domain, truncated CD62L endodomain, and OX40 endodomain.

[0085] In one or more embodiments, the amino acid sequence of the CD28 transmembrane domain is set forth in SEQ ID NO: 4.

[0086] In one or more embodiments, the amino acid sequence of the CD28 ectodomain hinge region is set forth in SEQ ID NO: 8.

[0087] In one or more embodiments, the amino acid sequence of the CD8 ectodomain hinge region is set forth in SEQ ID NO: 151.

[0088] In one or more embodiments, the amino acid sequence of the CD80 transmembrane domain is set forth in SEQ ID NO: 10.

[0089] In one or more embodiments, the amino acid sequence of the CD28 intracellular domain is set forth in SEQ ID NO: 6.

[0090] In one or more embodiments, the amino acid sequence of the OX40 intracellular domain is set forth in SEQ ID NO: 14.

[0091] In one or more embodiments, the amino acid sequence of the CD80 intracellular domain is set forth in SEQ ID NO: 12.

[0092] In one or more embodiments, the amino acid sequence of the KIR2DL3 extracellular region is set forth in SEQ ID NO: 36.

[0093] In one or more embodiments, the amino acid sequence of the KIR2DL4 extracellular region is set forth in SEQ ID NO: 38.

[0094] In one or more embodiments, the amino acid sequence of the KIR3DL1 extracellular region is set forth in SEQ ID NO: 40.

[0095] In one or more embodiments, the amino acid sequence of the KIR3DL2 extracellular region is set forth in SEQ ID NO: 42.

[0096] In one or more embodiments, the amino acid sequence of the CD28 extracellular hinge region is set forth in SEQ ID NO: 8.

[0097] In one or more embodiments, the amino acid sequence of the CD62L intracellular domain is set forth in SEQ ID NO: 32.

[0098] In one or more embodiments, the amino acid sequence of the truncated CD62L intracellular domain is set forth in SEQ ID NO: 34.

[0099] In one or more embodiments, the amino acid sequence of the costimulatory receptor is set forth in any one of SEQ ID NOs: 46, 50, 52, 54, 58, 62, 64, 66, 96, 100, 113, 115, 117, 119, 121, 123, 125, 127, 153, 155, 157, and 159.

[0100] A third aspect of the present application provides a costimulatory receptor, comprising: a KIR extracellular region, an extracellular hinge region, a transmembrane region, and an intracellular domain.

[0101] In one or more embodiments, the KIR extracellular region is a KIR2DL3 extracellular region, a KIR2DL4 extracellular region, a KIR3DL1 extracellular region, or a KIR3DL2 extracellular region.

[0102] In one or more embodiments, the extracellular hinge region is a CD28 extracellular hinge region or a CD8 extracellular hinge region.

[0103] In one or more embodiments, the transmembrane region is a CD28 transmembrane region or a CD80 transmembrane region.

[0104] In one or more embodiments, the intracellular domain comprises a CD28 intracellular domain and an OX40 intracellular domain.

[0105] In one or more embodiments, the intracellular domain comprises a CD80 intracellular domain.

[0106] In one or more embodiments, the KIR2DL3 extracellular region has an amino acid sequence as set forth in SEQ ID NO: 36.

[0107] In one or more embodiments, the KIR2DL4 extracellular region has an amino acid sequence as set forth in SEQ ID NO: 38.

[0108] In one or more embodiments, the KIR3DL1 extracellular region has an amino acid sequence as set forth in SEQ ID NO: 40.

[0109] In one or more embodiments, the KIR3DL2 extracellular region has an amino acid sequence as set forth in SEQ ID NO: 42.

[0110] In one or more embodiments, the CD28 transmembrane region has an amino acid sequence as set forth in SEQ ID NO: 4.

[0111] In one or more embodiments, the CD28 extracellular hinge region has an amino acid sequence as set forth in SEQ ID NO: 8.

[0112] In one or more embodiments, the CD28 intracellular domain has an amino acid sequence as set forth in SEQ ID NO: 6.

[0113] In one or more embodiments, the OX40 intracellular domain has an amino acid sequence as set forth in SEQ ID NO: 14.

[0114] In one or more embodiments, the CD80 transmembrane region has an amino acid sequence as set forth in SEQ ID NO: 10.

[0115] In one or more embodiments, the amino acid sequence of the CD80 endodomain is set forth in SEQ ID NO: 12.

[0116] In one or more embodiments, the amino acid sequence of the costimulatory receptor is set forth in any one of SEQ ID NOs: 44, 48, 56, 60, 94, and 98.

[0117] The fourth aspect of the application also provides a fusion protein comprising the costimulatory receptor and the membrane surface cytokine according to any one of the embodiments herein.

[0118] In one or more embodiments, the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchoring region, either directly linked or linked via a linker.

[0119] In one or more embodiments, the cytokine is IL-7.

[0120] 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.

[0121] In one or more embodiments, the GPI anchoring region is CD52.

[0122] In one or more embodiments, the membrane surface cytokine comprises: a CD52 signal peptide, IL-7, a linker, CD52.

[0123] In one or more embodiments, the membrane surface cytokine further comprises a BCMA extracellular domain or a mutant thereof.

[0124] In one or more embodiments, the amino acid sequence of the cytokine IL-7 is set forth in SEQ ID NO: 24.

[0125] In one or more embodiments, the amino acid sequence of the BCMA extracellular domain mutant is set forth in SEQ ID NO: 26.

[0126] In one or more embodiments, the amino acid sequence of the CD52 signal peptide is set forth in SEQ ID NO: 28.

[0127] In one or more embodiments, the amino acid sequence of the CD52 is set forth in SEQ ID NO: 30.

[0128] In one or more embodiments, the membrane surface cytokine comprises a CD52 signal peptide, IL-7, a linker, and CD52. In one or more embodiments, the amino acid sequence of the linker is set forth in SEQ ID NO: 22. In one or more embodiments, the amino acid sequence of the membrane surface cytokine is set forth in SEQ ID NO: 129.

[0129] 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 linker is set forth in SEQ ID NO: 22. In one or more embodiments, the amino acid sequence of the membrane surface cytokine is set forth in SEQ ID NO: 68.

[0130] In one or more embodiments, the membrane surface cytokine is at the N-terminus and / or C-terminus of the co-receptor.

[0131] In one or more embodiments, the membrane surface cytokine is linked to the co-receptor via a linker; preferably, the amino acid sequence of the linker is set forth in SEQ ID NO: 16, 18, or 20.

[0132] In one or more embodiments, the membrane surface cytokine is linked to the co-receptor via a cleavable sequence; preferably, the cleavable sequence is a viral 2A peptide sequence, such as P2A, T2A, F2A, preferably P2A. Preferably, the cleavable sequence is a non-viral linker sequence. For example, the amino acid sequence of the cleavable sequence is set forth in SEQ ID NO: 110.

[0133] In one or more embodiments, the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 102, 104, 106, 108, 131, 133, 135, 137, 141, 143, 145, 147, 161, 163, 167, and 169.

[0134] The present application also provides a polynucleotide molecule having a nucleic acid sequence encoding the co-receptor or the fusion protein of any one of the embodiments of the present application, or a complement thereof.

[0135] In one or more embodiments, the transmembrane region is a CD28 transmembrane region, the coding sequence of which is set forth in SEQ ID NO: 3.

[0136] In one or more embodiments, the transmembrane region is a CD80 transmembrane region, the coding sequence of which is set forth in SEQ ID NO: 9

[0137] In one or more embodiments, the intracellular domain comprises a CD28 intracellular domain, the coding sequence of which is set forth in SEQ ID NO: 5.

[0138] In one or more embodiments, the intracellular domain comprises an OX40 intracellular domain, the coding sequence of which is set forth in SEQ ID NO: 17.

[0139] In one or more embodiments, the intracellular domain comprises a CD80 intracellular domain, the coding sequence of which is set forth in SEQ ID NO: 11.

[0140] In one or more embodiments, the extracellular domain comprises a CD28 extracellular hinge region, the coding sequence of which is set forth in SEQ ID NO: 5.

[0141] In one or more embodiments, the extracellular domain comprises a CD8 extracellular hinge region, the coding sequence of which is set forth in SEQ ID NO: 150.

[0142] In one or more embodiments, the extracellular domain comprises a KIR2DL3 extracellular region, the coding sequence of which is set forth in SEQ ID NO: 35.

[0143] In one or more embodiments, the extracellular domain comprises a KIR2DL4 extracellular region, the coding sequence of which is set forth in SEQ ID NO: 37.

[0144] In one or more embodiments, the extracellular domain comprises a KIR3DL1 extracellular region, the coding sequence of which is set forth in SEQ ID NO: 39.

[0145] In one or more embodiments, the extracellular domain comprises a KIR3DL2 extracellular region, the coding sequence of which is set forth in SEQ ID NO: 41.

[0146] In one or more embodiments, the nucleic acid sequence of the costimulatory receptor or fusion protein further comprises a UTR. In one or more embodiments, the UTR is a 5’ UTR or a 3’ UTR. In one or more embodiments, the nucleotide sequence of the 5’ UTR is set forth in SEQ ID NO: 111.

[0147] In one or more embodiments, the polynucleotide molecule encoding the accessory receptor comprises a nucleic acid sequence selected from the group consisting of any one of the nucleic acid sequences set forth in SEQ ID NO: 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 93, 95, 97, 99, 112, 114, 116, 118, 120, 122, 124, 126, 152, 154, 156, and 158, or the complement of any one of the nucleic acid sequences.

[0148] In one or more embodiments, the polynucleotide molecule encoding the fusion protein comprises a nucleic acid sequence selected from the group consisting of any one of the nucleic acid sequences set forth in SEQ ID NO: 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 101, 103, 105, 107, 130, 132, 134, 136, 138, 139, 140, 142, 144, 146, 148, 149, 160, 162, 164, 165, 166, 168, 170, and 171, or the complement of any one of the nucleic acid sequences.

[0149] The present application also provides a nucleic acid construct comprising the polynucleotide molecule of any one of the embodiments of the present application.

[0150] In one or more embodiments, the nucleic acid construct comprises an expression cassette of the accessory receptor as described in any one of the embodiments herein and an expression cassette of the membrane surface cytokine as described in any one of the embodiments of the fourth aspect herein; or the nucleic acid construct is an expression cassette in which the coding sequence of the accessory receptor as described in any one of the embodiments herein and the coding sequence of the membrane surface cytokine as described in any one of the embodiments of the fourth aspect herein are within the expression cassette.

[0151] In one or more embodiments, the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchoring region, which are directly linked or linked via a linker.

[0152] In one or more embodiments, the nucleic acid construct is a vector.

[0153] In one or more embodiments, the vector is an expression vector or a cloning vector.

[0154] In one or more embodiments, the vector is a viral vector or a non-viral vector, preferably a non-viral vector. In one or more embodiments, the non-viral vector is an integrating non-viral vector, preferably an integrating non-viral vector based on a transposon system.

[0155] The present application also provides a nucleic acid construct comprising: an expression cassette of a costimulatory receptor and an expression cassette of a membrane surface cytokine; or the nucleic acid construct is an expression cassette, wherein a coding sequence of a costimulatory receptor and a coding sequence of a membrane surface cytokine are in the expression cassette, wherein,

[0156] The costimulatory receptor comprises an extracellular domain comprising an extracellular domain of a KIR or a functional fragment or variant thereof that retains the biological function of binding to a KIR ligand, a transmembrane region, and an intracellular domain comprising a signal transduction domain and / or a costimulatory domain,

[0157] The membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchoring region.

[0158] In one or more embodiments, the costimulatory receptor is as described in any of the second aspect of the present application.

[0159] In one or more embodiments, the amino acid sequence of the costimulatory receptor is as set forth in any one of SEQ ID NOs: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 94, 96, 98, 100, 113, 115, 117, 119, 121, 123, 125, 127, 153, 155, 157, and 159.

[0160] In one or more embodiments, the membrane surface cytokine is as described in any of the fourth aspect of the present application.

[0161] In one or more embodiments, the nucleic acid construct is a vector.

[0162] In one or more embodiments, the vector is an expression vector or a cloning vector.

[0163] In one or more embodiments, the vector is a viral vector or a non-viral vector, preferably a non-viral vector. In one or more embodiments, the non-viral vector is an integration competent non-viral vector, preferably an integration competent non-viral vector based on a transposon system.

[0164] The present application also provides a genetically engineered cell, which:

[0165] (1) expresses the costimulatory receptor as described in any of the second or third aspect of the present application or the fusion protein as described in the fourth aspect, and / or carries a coding sequence of the costimulatory receptor or the fusion protein;

[0166] (2) expresses the costimulatory receptor and the membrane surface cytokine as described in any of the embodiments herein;

[0167] (3) a nucleic acid construct as described in any of the embodiments herein.

[0168] In one or more embodiments, the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchoring region, either directly linked or linked through a linker.

[0169] In one or more embodiments, the membrane surface cytokine is at the N-terminus and / or C-terminus of a co-receptor.

[0170] In one or more embodiments, the cytokine is IL-7.

[0171] 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 at the N-terminus of the cytokine.

[0172] In one or more embodiments, the transmembrane domain is CD52 or its GPI anchoring domain.

[0173] In one or more embodiments, the membrane surface cytokine comprises: a CD52 signal peptide, IL-7, a linker, CD52, or the membrane surface cytokine comprises: a CD52 signal peptide, IL-7, a linker, CD52.

[0174] In one or more embodiments, the membrane surface cytokine further comprises a BCMA extracellular domain or a mutant thereof.

[0175] In one or more embodiments, the BCMA extracellular domain mutant has an amino acid sequence as set forth in SEQ ID NO: 26.

[0176] 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 membrane surface cytokine has an amino acid sequence as set forth in SEQ ID NO: 68.

[0177] In one or more embodiments, the membrane surface cytokine is linked to a co-receptor through a linker; preferably, the linker has an amino acid sequence as set forth in SEQ ID NO: 16, 18, or 20.

[0178] In one or more embodiments, the membrane surface cytokine is linked to a co-receptor through a cleavable sequence; preferably, the cleavable sequence is a viral 2A peptide sequence, such as P2A, T2A, F2A, preferably P2A. Preferably, the cleavable sequence is a non-viral linker sequence. For example, the cleavable sequence has an amino acid sequence as set forth in SEQ ID NO: 110.

[0179] In one or more embodiments, the cell is an immune effector cell.

[0180] In one or more embodiments, the immune effector cell comprises a T cell, an NK cell, a CAR-T, a CAR-NK, a TCR-T, a CIK, a DN T, and a TIL.

[0181] In one or more embodiments, the cell further expresses a CAR, or carries a coding sequence of a CAR.

[0182] In one or more embodiments, the cell further expresses an exogenous TCR, or carries a coding sequence of an exogenous TCR.

[0183] The present application also provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient, and any one or more of the co-receptor of any of the embodiments of the second or third aspect of the present application, the fusion protein of any of the embodiments of the present application, the polynucleotide molecule, the nucleic acid construct, and the genetically engineered cell of any of the embodiments of the present application. The pharmaceutical composition is used for treating or preventing cancer.

[0184] In one or more embodiments, the cancer comprises breast cancer, colon cancer, prostate cancer, melanoma, ovarian cancer, cervical cancer, kidney cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, and thyroid cancer.

[0185] The present application also provides use of the co-receptor of any of the embodiments of the second or third aspect of the present application, the fusion protein of any of the embodiments of the present application, the polynucleotide molecule, the nucleic acid construct, and the genetically engineered cell of any of the embodiments of the present application in the manufacture of a medicament for treating or preventing cancer.

[0186] The present application also provides use of the co-receptor and the membrane surface cytokine in the manufacture of a medicament for treating or preventing cancer, the co-receptor comprising an extracellular domain comprising an extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligand, a transmembrane region, and an intracellular region comprising a signal transduction domain and / or a costimulatory domain, the membrane surface cytokine comprising a cytokine and a transmembrane domain or a GPI anchoring region.

[0187] Advantages of the present application:

[0188] The auxiliary receptor of the present application can strengthen the core connection of the immune synapse while transmitting the signal of HLA binding to the extracellular region of the receptor to the intracellular region, activate the activation signal pathway of the downstream costimulatory molecule, and improve the activation level of the immune effector cell through double action. The CD62L intracellular domain can further enhance the formation of the immune synapse structure on the basis of the existing immune synapse core structure of the auxiliary receptor or chimeric antigen receptor, and further improve the activation, proliferation and target cell killing level of T cells. When the auxiliary receptor (auxiliary receptor) of the present application and the membrane surface cytokine are co-expressed in immune cells, the cytokine secretion level, proliferation level and killing of target cells of immune cells can be further improved. DETAILED DESCRIPTION

[0189] It should be understood that the above technical features of the present application and the technical features specifically described below (such as examples) can be combined with each other to form a preferred technical solution within the scope of the present application.

[0190] The KIR receptor on the surface of the immune effector cell is activated or inhibited by binding to the I-type HLA on the surface of the antigen presenting cell. The extracellular region of KIR is fused with the transmembrane region and intracellular domain of the classical costimulatory signal molecule to form a new receptor, which can bind to different types of I-type HLA and is a new type of auxiliary receptor. The receptor plays an immune activation role in the following ways: 1) when the receptor is expressed in T cells, it strengthens the first signal and second signal connection between T cells and antigen presenting cells by binding to I-type HLA, further physically strengthens the most core component of T cell immune synapse, and enhances the interaction between T cells and antigen presenting cells, creating favorable conditions for antigen recognition and activation of T cells; 2) after the receptor is expressed in immune effector cells including T cells and NK cells, the KIR extracellular region in the extracellular domain binds to the corresponding I-type HLA on the surface of the antigen presenting cell, which in turn activates the intracellular domain of the downstream costimulatory molecule, thereby improving the activation and proliferation level of the immune effector cell, and improving the activation level of the immune effector cell through double action; 3) the extracellular region of KIR is fused with the intracellular domain of CD62L to form a new receptor, which can further enhance the formation of the immune synapse structure on the basis of the existing immune synapse core structure of the auxiliary receptor or chimeric antigen receptor, and further improve the activation, proliferation and target cell killing level of T cells, 4) the membrane surface cytokine and the auxiliary receptor are co-expressed in immune cells, which can further improve the cytokine secretion level, proliferation level and killing of target cells of immune cells.

[0191] DEFINITIONS

[0192] The following terms are used in the present application. Terms not specifically defined herein have their art-recognized meanings.

[0193] In the present application, the term "immune cell" has its art-recognized meaning and refers to a cell involved in or associated with an immune response, including various lymphocytes, dendritic cells, monocyte / macrophages, granulocytes, mast cells, etc. Lymphocytes include, for example, T lymphocytes, tumor infiltrating lymphocytes (TILs), B lymphocytes, NK lymphocytes, and DN T cells. Immune cells suitable for use in the present application include, among others, those commonly used in tumor adoptive cell therapy.

[0194] The term "expression cassette" refers to the complete elements required for expression of a gene, including a promoter, a gene coding sequence, and a polyadenylation signal sequence.

[0195] The term "coding sequence" is defined herein as the portion of a nucleic acid sequence that directly specifies the amino acid sequence of its protein product (e.g., a co-receptor, a CAR). The boundaries of the coding sequence are generally determined by a ribosome binding site (for prokaryotes) just upstream of the open reading frame at the 3' end of the mRNA and a transcription terminator sequence just downstream of the open reading frame at the 5' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0196] The term "costimulatory signaling molecule" refers to a molecule present on the surface of an antigen presenting cell that binds to a costimulatory signaling molecule receptor on a Th cell, resulting in a costimulatory signal. It activates a second signal of the immune cell, enhances the proliferative capacity of the immune cell and the secretion function of cytokines, and prolongs the survival time of the activated immune cell. The proliferation of lymphocytes requires not only the binding of antigen, but also the signal of costimulatory molecules. The costimulatory signal is transmitted to the T cell mainly through the binding of the costimulatory molecules CD80, CD86 expressed on the surface of antigen presenting cells to the CD28 molecule on the surface of T cells. B cells receive a costimulatory signal through general pathogen components such as LPS, or through complement components, or through the CD40L on the surface of activated antigen-specific Th cells.

[0197] The term "linker" or "hinge" is a polypeptide fragment that connects different proteins or polypeptides, with the purpose of allowing the connected proteins or polypeptides to maintain their respective spatial conformation, so as to maintain the function or activity of the proteins or polypeptides. Exemplary linkers include linkers containing G and / or S, rigid linkers or flexible linkers, and, for example, Furin 2A peptide. In one or more embodiments, the amino acid sequence of the linker is set forth in SEQ ID NO: 16, 18, 20, 22, or 110.

[0198] The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is compatible with the subject and the active ingredient in a pharmacological and / or physiological sense that is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ion strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80; ion strength enhancers include, but are not limited to, sodium chloride.

[0199] The term "effective amount" refers to a dosage that can achieve treatment, prevention, alleviation and / or relief of the disease or condition described in the present application in a subject.

[0200] The term "disease and / or condition" refers to a physical state of the subject that is associated with the disease and / or condition described in the present application.

[0201] The term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present application to treat, prevent, alleviate and / or relieve the disease or condition described in the present application.

[0202] The term "extracellular region" refers to the segment of the membrane protein that is located outside the cell.

[0203] The term "domain" refers to a region of a protein that has a specific structure and independent function. The number of amino acid residues in a common domain is between 100 and 400. The smallest domain has 40-50 amino acid residues, and the largest domain can have more than 400 amino acid residues.

[0204] Co-receptor

[0205] The co-receptor of the present application comprises the extracellular region of the KIR extracellular domain (extracellular ligand binding domain), the transmembrane region (transmembrane domain), and the intracellular domain (or cytoplasmic domain). The KIR extracellular domain can be itself or a fragment thereof, as long as the fragment retains the biological function of binding to the KIR ligand.

[0206] In the costimulatory receptor herein, the KIR extracellular domain is used to interact with KIR ligands (mainly various types of HLA molecules) to down-regulate the level of "don't eat me" signaling pathway, and the signaling pathway of the immune effector cell is dominated by activation, so that the immune effector cell enters an activated and proliferative state, attacks the target cell and eliminates abnormal cells. The extracellular domain of an activating KIR or an inhibitory KIR or a fragment thereof retaining the KIR ligand binding ability can be used. An exemplary activating KIR is KIR2DL4; exemplary inhibitory KIRs are selected from KIR2DL3, KIR3DL1 and KIR3DL2. Preferably, the amino acid sequence of the extracellular region of KIR2DL3 is as shown in SEQ ID NO: 36, and the coding sequence thereof is as shown in SEQ ID NO: 35; the amino acid sequence of the extracellular region of KIR2DL4 is as shown in SEQ ID NO: 38, and the coding sequence thereof is as shown in SEQ ID NO: 37; the amino acid sequence of the extracellular region of KIR3DL1 is as shown in SEQ ID NO: 40, and the coding sequence thereof is as shown in SEQ ID NO: 39; the amino acid sequence of the extracellular region of KIR3DL2 is as shown in SEQ ID NO: 42, and the coding sequence thereof is as shown in SEQ ID NO: 41.

[0207] In the costimulatory receptor herein, the intracellular domain can comprise a CD62L intracellular domain. The CD62L intracellular domain can be itself or a fragment thereof, as long as the fragment retains the biological function of the CD62L intracellular domain. The amino acid sequence and the corresponding coding sequence of an exemplary CD62L intracellular domain can be as shown in SEQ ID NO: 32 and 31, respectively. Exemplarily, the fragment of the CD62L intracellular domain can be a truncated CD62L, the amino acid sequence and the corresponding coding sequence of which can be as shown in SEQ ID NO: 34 and 33, respectively.

[0208] In the costimulatory receptor herein, the intracellular domain can comprise a signal transduction domain and / or a costimulatory domain.

[0209] In the present application, the co-stimulatory signaling molecule includes 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, CD80, and CD27. The intracellular domain (intracellular region) of one or more of these co-stimulatory signaling molecules or a functional fragment thereof or a mutant thereof that retains the biological function of the co-stimulatory signaling molecule to transmit a co-stimulatory signal and activate an immune cell can be used to construct the helper receptor of the present application. The amino acid sequence of an exemplary intracellular region of CD28 and the corresponding coding sequence can be shown in SEQ ID NO: 6 and 5, respectively. The amino acid sequence of an exemplary intracellular region of OX40 and the corresponding coding sequence can be shown in SEQ ID NO: 14 and 13, respectively. An exemplary IL-7R can be IL-7R alpha. The amino acid sequence of an exemplary intracellular region of CD80 and the corresponding coding sequence can be shown in SEQ ID NO: 10 and 9, respectively. The intracellular domain of the co-stimulatory signaling molecule can also be the intracellular domain of the co-stimulatory signaling molecule described in WO2021244486, which is incorporated herein by reference in its entirety.

[0210] In the present application, the signal transduction domain can be selected by a person skilled in the art as needed, for example, CD3 zeta intracellular signaling region.

[0211] In the present application, the transmembrane region includes, but is not limited to, a transmembrane region 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, or a mutant thereof that retains the transmembrane function.

[0212] In the present application, the extracellular region comprising KIR extracellular domain can be linked to the transmembrane region via a hinge region. The hinge region includes, but is not limited to, a juxtamembrane 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. Preferably, the hinge region is a CD28 extracellular hinge region, a CD8 extracellular hinge region, or an IL7Rα extracellular hinge region.

[0213] It should be understood that "functional fragment" as used herein refers to a fragment that retains a desired biological function. For example, a functional fragment of an intracellular domain as used herein refers to a fragment that retains the biological function of the costimulatory signaling molecule to deliver a costimulatory signal, to activate an immune cell. Functional fragments of each extracellular domain and functional fragments of each intracellular domain suitable for use in the present application can be readily determined by one of skill in the art in view of the present disclosure.

[0214] The accessory receptor of the present application can also have a membrane surface tag extracellularly. Thus, in some embodiments, the accessory receptor described herein further comprises a membrane surface tag at the C-terminus of the KIR extracellular domain. The membrane surface tag can function as an immune brake element, a recognition element, a linker, an element inducing ADCC, ADCP and / or CDC effect. The membrane surface tag comprises a membrane surface functional domain.

[0215] The membrane surface functional domain can be a claudin protein extracellular domain or a fragment thereof. The claudin protein is preferably claudin 18, such as claudin 18.2. The fragment of the extracellular domain mainly refers to the extracellular antigen epitope of the corresponding protein. Therefore, the fragment of the claudin protein extracellular domain is the extracellular antigen epitope of the claudin protein.

[0216] The membrane surface functional domain can also be a BCMA extracellular domain or a fragment or mutant thereof.

[0217] The membrane surface tag can also have a connecting fragment at the N-terminus or C-terminus of the membrane surface functional domain (BCMA extracellular domain or claudin protein extracellular domain) for connection with other polypeptides or polypeptide parts. The connecting fragment is usually a hinge or a linker. The hinge comprises one or more selected from the group consisting of CD8 extracellular hinge region, IgG1 Fc CH2CH3 hinge region, IgD hinge region, CD28 extracellular hinge region, IgG4 Fc CH2CH3 hinge region and CD4 extracellular hinge region.

[0218] A "mutant" as described herein includes mutants of each of the domains, so long as the mutant retains the respective biological function of the KIR extracellular domain, membrane surface tag, transmembrane region, intracellular domain. For example, mutants of the KIR extracellular domain suitable for use in the application include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a KIR extracellular domain as a comparator; mutants of the membrane surface tag suitable for use in the application include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a membrane surface tag as a comparator; mutants of the transmembrane region suitable for use in the application include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a transmembrane region as a comparator; mutants of the intracellular domain suitable for use in the application include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to an intracellular domain as a comparator. Alternatively, a mutant as described in the application has 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 residue insertions, substitutions, or deletions compared to a comparator sequence. For example, in the art, conservative substitutions that use amino acids with similar or analogous properties are typically not expected to alter the function of a protein or polypeptide. "Amino acids with similar or analogous properties" include, for example, families of amino acid residues with similar side chains, including families with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), uncharged polar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0219] The application also includes mutants of the previously described accessory receptors, 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 to the accessory receptors described previously. More specifically, the application 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 residue insertions, substitutions, or deletions compared to the accessory receptors described previously. Such mutants retain the biological function of the accessory receptors described herein, including but not limited to the function of recognizing KIR ligands and activating immune effector cells into an activated and proliferative state. Mutations can occur in any one, any two, or all three of the extracellular domain, the transmembrane region, and the intracellular domain described herein.

[0220] The polypeptides described herein can be modified polypeptides. Modifications, which generally do not alter the primary structure, include chemical derivatization of the polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides that have been glycosylationally modified during synthesis and processing or further processing steps. Such modifications can be accomplished by exposing the polypeptides to enzymes that glycosylate (such as mammalian glycosylation enzymes or deglycosylation enzymes). Modified forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their proteolytic resistance or to optimize solubility.

[0221] Exemplary accessory receptors of the application include, but are not limited to, accessory receptors comprising or consisting of the extracellular domain, the hinge region, the transmembrane region, and the intracellular domain set forth in each row of Table 1, from N-terminus to C-terminus:

[0222] Table 1, accessory receptors (N-terminus to C-terminus)

[0223] In some embodiments, the accessory receptors described herein further comprise a signal peptide. Preferably, the signal peptide is located at the N-terminus of the accessory receptor. The signal peptide can be any signal peptide known in the art that is capable of directing the polypeptide out of the nucleus, including but not limited to CD8, CD4, CD28, CD137, EGFR, TGFBRI, TGFBRII, TGFBRIII, and antibody light chain signal peptides. In some embodiments, the signal peptide is a CD8 signal peptide, which comprises the amino acid sequence set forth in SEQ ID NO: 2, the coding sequence of which is set forth in SEQ ID NO: 1.

[0224] It should be understood that, as desired, a linker sequence can be used to connect between the extracellular domain described herein and the transmembrane region, and / or the transmembrane region and the intracellular domain. Linker sequences known in the art can be used, such as a linker sequence containing G and S, such as (GSSS)n or (GSSSS)n, wherein n is an integer from 1 to 8. The linker can also be a rigid linker or a flexible linker.

[0225] Preferably, the amino acid sequence of the costimulatory receptor of the present application is as set forth in any one of SEQ ID NOs: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 94, 96, 98, 100, 113, 115, 117, 119, 121, 123, 125, 127, 153, 155, 157, and 159.

[0226] Fusion protein

[0227] The present application also provides a fusion protein comprising a costimulatory receptor and a membrane surface cytokine. The membrane surface cytokine is at the N-terminus and / or C-terminus of the costimulatory receptor. The membrane surface cytokine is connected to the costimulatory receptor via a cleavable sequence; preferably, the cleavable sequence is, for example, P2A, T2A, F2A, preferably P2A. Alternatively, the membrane surface cytokine can be connected to the costimulatory receptor via a linker; preferably, the amino acid sequence of the linker is as set forth in SEQ ID NO: 16, 18, or 20. Alternatively, the membrane surface cytokine is directly connected to the costimulatory receptor.

[0228] The membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchoring region, which are directly connected or connected via a linker. Linker sequences known in the art can be used, such as a linker sequence containing G and S, such as (GSSS)n or (GSSSS)n, wherein 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 a rigid linker as set forth in SEQ ID NO: 16, 18, or 20.

[0229] Among the membrane surface cytokines, the cytokine can be any polypeptide cytokine, including but not limited to interleukins, tumor necrosis factors (TNF), interferons (IFN), colony stimulating factors (CSF), and tumor growth factors (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-alpha and TNF-beta. The interferons include but are not limited to IFN-alpha, IFN-beta, and IFN-gamma. 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-alpha, TGF-beta 1, TGF-beta 2, and TGF-beta 3.

[0230] Among the membrane surface cytokines, the transmembrane domain is as described elsewhere herein, including but not limited to any one or more of the transmembrane regions 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, CD80, and CD27, or a mutant thereof that retains transmembrane function.

[0231] The GPI anchor region in the membrane surface cytokine factor comprises one or more selected from the group consisting of 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, or a GPI anchor domain thereof (also referred to herein as a GPI signal sequence). Preferably, the GPI anchor region is a CD52 protein, a CD48 protein, a CD55 protein, an ALPP protein, a CD90 protein, or a GPI anchor domain thereof. The sequence of a GPI anchor protein or its anchor domain is known in the art. Moreover, one skilled in the art can readily obtain the sequence of the anchor domain from the sequence of a GPI anchor protein.

[0232] The membrane surface cytokine factor can further comprise a signal peptide. Preferably, the signal peptide is located at the N-terminus of the cytokine factor. The signal peptide can be any signal peptide known in the art that is capable of directing the polypeptide out of the nucleus, including but not limited to CD8, CD4, CD28, CD52, CD137, EGFR, TGFBRI, TGFBRII, TGFBRIII, and antibody light chain signal peptide. In some embodiments, the signal peptide is a CD8 signal peptide or a CD52 signal peptide.

[0233] The membrane surface cytokine factor can further comprise a membrane surface tag. The membrane surface tag is preferably a BCMA extracellular domain or a mutant thereof that retains biological function. Preferably, the mutant BCMA extracellular domain has an amino acid sequence as set forth in SEQ ID NO: 26.

[0234] In one or more embodiments, the fusion protein comprises, or comprises in order from N-terminus to C-terminus:

[0235] (1) a CD52 signal peptide, IL-7, a linker 2, CD52, a linker 3, a CD8 signal peptide, a KIR2DL3 extracellular region, a mutant BCMA extracellular domain, a linker 1, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain,

[0236] (2) a CD8 signal peptide, a KIR2DL3 extracellular region, a mutant BCMA extracellular domain, a linker 1, a CD28 transmembrane region, a CD28 intracellular domain, an OX40 intracellular domain, a linker 3, a CD52 signal peptide, IL-7, a linker 2, CD52,

[0237] (3) CD52 signal peptide, IL-7, linker 2, CD52, linker 3, CD8 signal peptide, KIR2DL3 ectodomain, mutant BCMA ectodomain, linker 1, CD28 transmembrane domain, CD62L endodomain, CD28 endodomain, OX40 endodomain,

[0238] (4) CD8 signal peptide, KIR2DL3 ectodomain, mutant BCMA ectodomain, linker 1, CD28 transmembrane domain, CD62L endodomain, CD28 endodomain, OX40 endodomain, P2A, CD52 signal peptide, IL-7, linker 2, CD52,

[0239] (5) CD52 signal peptide, IL-7, linker 2, CD52, linker 3, CD8 signal peptide, KIR2DL4 ectodomain, mutant BCMA ectodomain, linker 1, CD28 transmembrane domain, CD28 endodomain, OX40 endodomain,

[0240] (6) CD8 signal peptide, KIR2DL4 ectodomain, mutant BCMA ectodomain, linker 1, CD28 transmembrane domain, CD28 endodomain, OX40 endodomain, linker 3, CD52 signal peptide, IL-7, linker 2, CD52,

[0241] (7) CD52 signal peptide, IL-7, linker 2, CD52, linker 3, CD8 signal peptide, KIR2DL4 ectodomain, mutant BCMA ectodomain, linker 1, CD28 transmembrane domain, CD62L endodomain, CD28 endodomain, OX40 endodomain,

[0242] (8) CD8 signal peptide, KIR2DL4 ectodomain, mutant BCMA ectodomain, linker 1, CD28 transmembrane domain, CD62L endodomain, CD28 endodomain, OX40 endodomain, linker 3, CD52 signal peptide, IL-7, linker 2, CD52.

[0243] (9) CD52 signal peptide, IL-7, linker 4, mutant BCMA ectodomain, CD52, linker 1, CD8 signal peptide, KIR2DL3 ectodomain, CD28 transmembrane domain, CD28 endodomain, OX40 endodomain,

[0244] (10) CD52 signal peptide, IL-7, linker 4, mutant BCMA ectodomain, CD52, linker 1, CD8 signal peptide, KIR2DL3 ectodomain, CD28 ectodomain, CD28 transmembrane domain, CD62L endodomain, CD28 endodomain, OX40 endodomain,

[0245] (11) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 1, CD8 signal peptide, KIR2DL3 extracellular region, CD28 extracellular hinge region, CD80 transmembrane region, CD80 intracellular domain,

[0246] (12) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 2, CD8 signal peptide, KIR2DL3 extracellular region, CD28 extracellular hinge region, CD80 transmembrane region, CD62L intracellular signaling region, CD80 intracellular domain,

[0247] (13) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 1, CD8 signal peptide, KIR2DL3 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, truncated CD62L intracellular signaling region, CD28 intracellular domain, OX40 intracellular domain,

[0248] (14) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 1, CD8 signal peptide, KIR2DL3 extracellular region, CD28 extracellular hinge region, CD80 transmembrane region, truncated CD62L intracellular signaling region, CD80 intracellular domain

[0249] (15) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 2, CD8 signal peptide, KIR2DL4 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD28 intracellular domain, OX40 intracellular domain,

[0250] (16) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 3, CD8 signal peptide, KIR2DL4 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD62L intracellular signaling region, CD28 intracellular domain, OX40 intracellular domain,

[0251] (17) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 3, CD8 signal peptide, KIR2DL4 extracellular region, CD28 extracellular hinge region, CD80 transmembrane region, CD80 intracellular domain,

[0252] (18) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 3, CD8 signal peptide, KIR2DL4 extracellular region, CD28 extracellular hinge region, CD80 transmembrane region, CD62L intracellular signaling region, CD80 intracellular domain,

[0253] (19) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 2, CD8 signal peptide, KIR2DL4 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, truncated CD62L intracellular signaling region, CD28 intracellular domain, OX40 intracellular domain,

[0254] (20) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 2, CD8 signal peptide, KIR2DL4 extracellular region, CD28 extracellular hinge region, CD80 transmembrane region, truncated CD62L intracellular signaling region, CD80 intracellular domain,

[0255] (21) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 1, CD8 signal peptide, KIR3DL1 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD28 intracellular domain, OX40 intracellular domain,

[0256] (22) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 1, CD8 signal peptide, KIR3DL1 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD62L intracellular signaling region, CD28 intracellular domain, OX40 intracellular domain,

[0257] (23) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 2, CD8 signal peptide, KIR3DL2 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD28 intracellular domain, OX40 intracellular domain,

[0258] (24) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 2, CD8 signal peptide, KIR3DL2 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD62L intracellular signaling region, CD28 intracellular domain, OX40 intracellular domain,

[0259] (25) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 5, CD8 signal peptide, KIR2DL3 extracellular region, CD28 transmembrane region, CD62L intracellular signaling region, OX40 intracellular domain,

[0260] (26) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 5, CD8 signal peptide, KIR2DL3 extracellular region, CD28 transmembrane region, truncated CD62L intracellular signaling region, OX40 intracellular domain,

[0261] (27) CD52 signal peptide, IL-7, linker 4, CD52, linker 5, CD8 signal peptide, KIR2DL3 extracellular region, mutant BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular signaling region, OX40 intracellular domain,

[0262] (28) CD52 signal peptide, IL-7, linker 4, CD52, linker 5, CD8 signal peptide, KIR2DL3 extracellular region, mutant BCMA extracellular domain, CD28 transmembrane region, truncated CD62L intracellular signaling region, OX40 intracellular domain,

[0263] (29) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 5, CD8 signal peptide, KIR2DL4 extracellular region, CD28 transmembrane region, CD62L intracellular signaling region, OX40 intracellular domain,

[0264] (30) CD52 signal peptide, IL-7, linker 4, mutant BCMA extracellular domain, CD52, linker 5, CD8 signal peptide, KIR2DL4 extracellular region, CD28 transmembrane region, truncated CD62L intracellular signaling region, OX40 intracellular domain,

[0265] (31) CD52 signal peptide, IL-7, linker 4, CD52, linker 5, CD8 signal peptide, KIR2DL4 extracellular region, mutant BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular signaling region, OX40 intracellular domain,

[0266] (32) CD52 signal peptide, IL-7, linker 4, CD52, linker 5, CD8 signal peptide, KIR2DL4 extracellular region, mutant BCMA extracellular domain, CD28 transmembrane region, truncated CD62L intracellular signaling region, OX40 intracellular domain,

[0267] (33) CD52 signal peptide, IL-7, linker 4, CD52, linker 5, CD8 signal peptide, KIR2DL3 extracellular region, CD8 extracellular hinge region, CD28 transmembrane region, CD62L intracellular signaling region, OX40 intracellular domain,

[0268] (34) CD52 signal peptide, IL-7, linker 4, CD52, linker 5, CD8 signal peptide, KIR2DL3 extracellular region, CD8 extracellular hinge region, CD28 transmembrane region, truncated CD62L intracellular signaling region, OX40 intracellular domain

[0269] (35) CD52 signal peptide, IL-7, linker 4, CD52, linker 5, CD8 signal peptide, KIR2DL4 extracellular region, CD8 extracellular hinge region, CD28 transmembrane region, CD62L intracellular signaling region, OX40 intracellular domain

[0270] (36) CD52 signal peptide, IL-7, linker 4, CD52, linker 5, CD8 signal peptide, KIR2DL4 extracellular region, CD8 extracellular hinge region, CD28 transmembrane region, truncated CD62L intracellular signaling region, OX40 intracellular domain.

[0271] CAR-T

[0272] The immune cells of the present application can further express a CAR, or a coding sequence of a CAR. The CARs described in the present application can be various CARs known in the art.

[0273] The CAR can sequentially comprise a polypeptide (such as scFv) binding to a tumor cell membrane antigen, a hinge region, a transmembrane region, and an intracellular signaling region. The CARs of the present application can be constructed using the hinge region, the transmembrane region, and the intracellular signaling region for constructing a CAR known in the art. Generally, the polypeptide binding to a tumor cell membrane antigen is capable of binding to a membrane antigen widely expressed by a tumor cell with moderate affinity, and the polypeptide is usually inserted with an antigen epitope at 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 binding 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.

[0274] The chimeric antigen receptors of the present application can be directed to 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 Melan-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family proteins, BAGE (B melanoma antigen family) family proteins, GAGE (growth hormone releasing factor) family proteins, AFP, MUC1 (also known as 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), 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 pro-gastrin-releasing peptide).

[0275] A single cell can express multiple CARs, including CARs targeting different tumor antigens.

[0276] T cell receptor (TCR)-T

[0277] The immune cells of the present application can further express an exogenous TCR or contain a coding sequence expressing an exogenous TCR gene. The TCRs described in the present application can be various TCRs known in the art, for example, TCRs that are HLA typed matched, whose sequences and structures are known, and whose antigenic peptide sequences bound are also known.

[0278] The exogenous TCRs described herein include αβ double chains, which can form a complete TCR complex with the γε, δε and ξξ double chain structures endogenously expressed in immune effector cells such as T cells. The exogenous genes encoding the exogenous TCRs described herein include genes of the αβ double chains, the coding sequences of the α chain and the β chain being covalently linked by a linker sequence that can be cleaved in vivo, such as a P2A, T2A or F2A sequence, or by a DNA fragment encoding an IRES sequence. In addition to the αβ double chains encoding the exogenous TCRs, the genes encoding the exogenous TCRs described herein can also include a tag protein gene expressed in fusion with the αβ gene, such as an EGFP, RFP, YFP gene and the like. The tag protein gene can be covalently linked to the gene of the αβ double chain by a linker sequence that can be cleaved in vivo, such as a 2A sequence, such as a P2A, T2A or F2A coding DNA sequence, or by a DNA sequence encoding an IRES sequence. The tag protein, such as an EGFP, RFP, YFP gene and the like, co-expressed with the TCR αβ double chain, can serve as an identification index for detecting the expression of the exogenous TCR.

[0279] The TCR-Ts of the present application can be directed to 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 Melan-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family proteins, BAGE (B melanoma antigen family) family proteins, GAGE (growth hormone releasing factor) family proteins, 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 pro-gastrin-releasing peptide).

[0280] A single cell can express multiple exogenous TCRs, including exogenous TCRs targeting different tumor antigens.

[0281] Polynucleotide molecules

[0282] The present application provides polynucleotide molecules encoding the costimulatory receptors described herein or the fusion proteins described herein. The present application also provides the complement of the coding sequence of the costimulatory receptors or the fusion proteins. The polynucleotide molecules can be recombinant nucleic acid molecules or synthetic; they can comprise DNA, RNA, and PNA (peptide nucleic acid) and can be hybrids thereof. Exemplarily, the polynucleotide molecules encoding the costimulatory receptors of the present application have the sequence set forth in any one of SEQ ID NOs: 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 93, 95, 97, 99, 112, 114, 116, 118, 120, 122, 124, 126, 152, 154, 156, and 158. Exemplarily, the polynucleotide molecules encoding the fusion proteins of the present application have the sequence set forth in any one of SEQ ID NOs: 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 101, 103, 105, 107, 130, 132, 134, 136, 138, 139, 140, 142, 144, 146, 148, 149, 160, 162, 164, 165, 166, 168, 170, and 171.

[0283] Also provided is an expression cassette of the costimulatory receptors or the fusion proteins of the present application, which is a nucleic acid construct comprising a promoter, a costimulatory receptor coding sequence, and a PolyA tailing signal sequence. The nucleic acid construct can further comprise other elements required for expression, including but not limited to enhancers, etc. The nucleic acid construct can further comprise a UTR sequence. The UTR sequence can be a 5’ UTR or a 3’ UTR, which is capable of enhancing the expression of the costimulatory receptors or the fusion proteins of the present application. Exemplarily, the UTR is a 5’ UTR, which is located at the 5’ end of the expression cassette of the costimulatory receptors or the fusion proteins, and has the nucleotide sequence set forth in SEQ ID NO: 111.

[0284] Also provided is a nucleic acid construct comprising an expression cassette of the costimulatory receptors described herein and an expression cassette of the membrane surface cytokines described herein; or the nucleic acid construct is an expression cassette, wherein the coding sequence of the costimulatory receptors described herein and the coding sequence of the membrane surface cytokines described herein are within the expression cassette.

[0285] Also provided is a vector containing the polynucleotide molecule, expression cassette or nucleic acid construct described herein. The vector can be a plasmid, cosmid, virus and bacteriophage. The vector can be a viral vector or a non-viral vector. The vector can be a cloning vector, an integration vector, and can also be an expression vector. The expression vector can be a transposon vector. In certain embodiments, the expression vector is one or more selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 and Ty. In addition to the polynucleotide molecule described herein, the expression vector usually contains other elements commonly contained in vectors, such as a multiple cloning site, a resistance gene, a replication initiation site, etc. In certain embodiments, the recombinant expression vector uses pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector, pUC57, pMAX or pDC315 series vector as a backbone. In other embodiments, the recombinant expression vector uses pCDNA3 series vector, pCDNA4 series vector, pCDNA5 series vector, pCDNA6 series vector, pRL series vector, pUC57 vector, pMAX vector or pDC315 series vector as a backbone. In certain embodiments, the present application uses pNB vector constructed in CN105154473A. In certain embodiments, the present application uses pKB20 vector described in WO2022078310A1.

[0286] The CAR of the present application can also be expressed in the immune cells described herein by a conventional vector. The vector can be a conventional expression CAR vector, including but not limited to the various types of transposon vectors and recombinant expression vectors described above.

[0287] In some embodiments, the same vector encodes both the helper receptor and the CAR of the present application. The vector can be bicistronic. The coding sequence of the CAR can be placed 5' or 3' to the coding sequence of the helper receptor. The expression of the CAR and the helper receptor can be under the control of the same or different regulatory sequences.

[0288] Where the polynucleotide sequence is known, each polynucleotide molecule can be prepared and the corresponding vector constructed using methods routine in the art. 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 techniques described in other standard textbooks. Alternatively, the nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the nucleic acid molecules of the application can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts, see Sambrook et al. (supra).

[0289] Host cells

[0290] As used herein, "host cell" refers to a eukaryotic cell that is capable of replicating a vector and / or expressing a heterologous gene encoded by a vector. A host cell can serve as a recipient of a vector. A host cell can be "transfected" or "transformed," which refers to a process by which exogenous nucleic acid is transferred into a host cell. A transformed cell includes the primary subject cell and its progeny. The terms "engineered" and "recombinant" cell or host cell as used herein often refer to a cell into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Thus, a recombinant cell can be distinguished from a cell that does not contain the introduced recombinant nucleic acid.

[0291] A host cell of the application (1) expresses an accessory receptor or fusion protein of the application, and / or carries a coding sequence for the accessory receptor or fusion protein; (2) expresses an accessory receptor and a membrane surface cytokine as described herein; (3) comprises a nucleic acid construct as described herein. Thus, a host cell of the application can express an accessory receptor as described herein, such as an accessory receptor described in the "SUMMARY" section, second or third part, alone. A host cell of the application can also express an accessory receptor as described herein and a membrane surface cytokine as described herein, separately, at the same time. A host cell of the application can also express a fusion protein as described herein, comprising an accessory receptor and a membrane surface cytokine.

[0292] The cells of the application are preferably immune cells, useful for tumor adoptive cell therapy. Such cells of the application are also referred to as the helper receptor modified cells of the application. More specifically, the cells of the application are preferably immune effector cells, including T cells, such as cytotoxic T cells (also referred to 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 capable of eliciting effector functions.

[0293] Herein, the cells can be autologous cells, syngeneic cells, allogeneic cells, and even xenograft cells, relative to the individual receiving them.

[0294] The nucleic acid constructs / recombinant expression vectors of the application can be introduced into the cells of interest. The methods of introduction are routine in the art, including but not limited to: viral transduction, microinjection, particle bombardment, biolistic transformation, and electroporation. In certain embodiments, the nucleic acid constructs or recombinant expression vectors are introduced using electroporation. Where multiple expression cassettes are located on different nucleic acid constructs / recombinant expression vectors, these nucleic acid constructs / recombinant expression vectors can be introduced into the cells simultaneously, or sequentially.

[0295] In addition to carrying the helper receptor and / or the coding sequence thereof, the cells of the application can also have one or more other properties useful for cell immunotherapy, such as tumor adoptive cell therapy. Such other properties can be intrinsic to the cells or can be part of the cells after human genetic manipulation. For example, the cells of the application can carry a chimeric antigen receptor, an alpha beta T cell receptor, and / or an antigen-specific receptor, such as a tumor-specific receptor, or the coding sequence thereof.

[0296] Pharmaceutical compositions

[0297] Herein, a "pharmaceutical composition" refers to a composition for administration to an individual and encompasses compositions of cells for immunotherapy. The pharmaceutical compositions of the application can also 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. The compositions containing such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to subjects in a suitable dose.

[0298] The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages will depend on a variety of factors including the type and severity of the disease to be treated, the age, body size, health, and sex of the patient, time and route of administration, and other drugs being administered concurrently.

[0299] The compositions of the present application can be administered topically or systemically. In certain embodiments, the compositions provided by the present application (e.g., cells expressing a costimulatory receptor of the present application) can be administered parenterally, e.g., intravenously, intraarterially, intrathecally, subdermally, or intramuscularly. In certain other embodiments, DNA encoding a construct provided by the present application can be administered directly to a target site, e.g., by gene gun delivery to an internal or external target site or by catheter delivery to an intraarterial site. In preferred embodiments, the pharmaceutical composition is administered subcutaneously, and in more preferred embodiments, intravenously. Parenteral formulations include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic / 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, or fixed oils. Intravenous vehicles include liquids and nutrient

[0300] Compositions for parenteral (e.g., intravenous) administration of the cells described herein can also be stored in lyophilized form or in solution (e.g., a cryopreserved formulation). Cryopreserved formulations can be stored in a ready-to-use form or in a form that is further formulated prior to administration. Cryopreserved formulations can tolerate long distance shipping without damaging the cells. In addition to the cells themselves, cryopreserved formulations typically include components such as a cell cryoprotectant, human serum albumin (HSA), and the like. Cryopreserved pharmaceutical compositions are stored at a low temperature (e.g., in liquid nitrogen) prior to administration (e.g., intravenous infusion). Upon thawing, the cryopreserved formulation can be infused directly to a patient or formulated into an infusion composition. Those of skill in the art are aware of the components and concentrations of conventional cryoprotectants. For example, the cryoprotectant or infusion composition can also include dimethyl sulfoxide, sodium chloride, dextrose, sodium acetate, potassium chloride, or magnesium chloride, among others, in concentrations that can be determined by those of skill in the art (e.g., an experienced physician) based on the cells, the disease, the patient, and the like.

[0301] Methods and uses

[0302] The accessory receptors, fusion proteins, polynucleotide molecules, vectors, host cells, and pharmaceutical compositions comprising the same described herein can be used for preventing, treating or ameliorating cancer, especially cancer in which the cancer cells express the corresponding tumor antigen on their surface, or for the manufacture of a medicament for preventing, treating or ameliorating cancer.

[0303] The present application also provides the use of the accessory receptors described herein comprising an extracellular domain comprising an extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligands, a transmembrane region, and an intracellular region comprising a signal transduction domain and / or a costimulatory domain, and the use of the membrane surface cytokine described in any of the embodiments herein comprising a cytokine and a transmembrane domain or a GPI anchoring region, in the manufacture of a medicament for treating or preventing cancer.

[0304] As used herein, "treatment" or "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and can include even a small reduction in one or more measurable markers of a disease or disorder (e.g., cancer) in therapy. Treatment can optionally include a reduction or alleviation of symptoms of a disease or disorder, or a delay in the progression of the disease or disorder. "Treatment" does not necessarily indicate complete eradication or cure of the disease or disorder, or associated symptoms thereof.

[0305] As used herein, "prevention" refers to a method for preventing, inhibiting or reducing the likelihood of the occurrence or recurrence of a disease or disorder (e.g., cancer). It also refers to delaying the occurrence or recurrence of a disease or disorder, or delaying the appearance or recurrence of symptoms of a disease or disorder. As used herein, "prevention" also includes reducing the intensity, impact, symptoms and / or burden of a disease or disorder prior to the occurrence or recurrence thereof.

[0306] The present application includes administration of cells, polynucleotide molecules and vectors, alone or in any combination, using standard vectors and / or gene delivery systems, optionally with pharmaceutically acceptable carriers or excipients. In certain embodiments, the polynucleotide molecules or vectors can be stably integrated into the genome of the subject after administration.

[0307] In specific embodiments, viral vectors that are specific to certain cells or tissues and persist in the cells can be used. Suitable pharmaceutical carriers and excipients are well known in the art. The compositions prepared according to the present application can be used for the prevention or treatment or delay of the diseases identified above.

[0308] Further, the present application provides a method of preventing, treating or ameliorating cancer comprising the step of administering to a subject in need thereof an effective amount of a cell carrying the accessory receptor, polynucleotide molecule and / or vector of the present application and / or generated by the method of the present application.

[0309] The methods herein can be used to prevent, treat or ameliorate a variety of cancers, including various solid and hematological tumors, including but not limited to lung cancer (e.g., non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythro leukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma and glioma, among others. More specifically, the cancers herein include, but are not limited to, breast, prostate, lung and colon cancers or epithelial cancers, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, melanoma; genito-urinary tract cancers, such as ovarian cancer, endometrial cancer, cervical cancer; kidney cancer, lung cancer, stomach cancer, small intestine cancer, liver cancer, pancreatic cancer, gall bladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, among others. The compositions of the present application can be administered for all stages and types of cancer, including for example, minimal residual disease, early stage cancer, advanced stage cancer and / or metastatic cancer and / or refractory cancer.

[0310] By way of example, a cancer patient or a patient susceptible to cancer or suspected of having cancer is treated as follows. Cells modified as described herein can be administered to an individual and remain for an extended period of time. The individual can receive one or more administrations of the cells, and the administrations can be spaced apart by days, weeks, months or years. In particular embodiments, multiple administrations can occur within weeks or months, e.g., 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 inhibit immune recognition and are located at a tumor site. In cases where the individual is provided with cells after a tumor has recurred following an initial treatment with cells of the present application, the cells can be altered to recognize a different target tumor antigen. For example, when the initial round includes cells carrying an accessory receptor of the present application and another receptor specific for a particular antigen, a subsequent round (including after tumor recurrence) can use a receptor specific for a different particular antigen.

[0311] In some embodiments, an individual in need is provided with an effective amount of therapeutic cells carrying or expressing an accessory receptor and optional CAR or exogenous transgenic TCR of any embodiment of the present application. These cells can be delivered simultaneously or non-simultaneously with one or more other cancer treatments. The cells and other cancer treatments can be delivered in the same or separate formulations. The cells and other cancer treatments can be provided to the individual by separate delivery routes. The cells and / or other cancer treatments can be delivered, e.g., by injection at a tumor site or intravenously or orally. Conventional delivery routes for such compositions are known in the art.

[0312] The number of cells employed will depend on a variety of circumstances, such as the purpose of the introduction, the life span of the cells, the protocol to be used, the number of administrations, the ability of the cells to proliferate, the stability of the recombinant construct, and the like.

[0313] The cells can be administered as desired. In certain embodiments, various protocols can be used to adjust the protocol parameters. In particular embodiments, the route or number or timing of administration, the life span of the cells, and / or the number of cells present can vary. The number of administrations can depend, for example, at least in part, on the factors described above.

[0314] Kits

[0315] Any of the compositions described herein can be included in a kit. In one non-limiting example, a kit can include cells expressing a costimulatory receptor as described in any of the embodiments of the application and / or reagents for generating one or more cells for cell therapy containing a recombinant expression vector. The kit components are provided in suitable container means.

[0316] Some of the components of these kits can be packaged in aqueous medium or in a freeze-dried form. Vessel means in these kits will typically include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component can be placed, and preferably suitably aliquoted. Where

[0317] Where the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, particularly preferably an aqueous, sterile solution. In some cases, the vessel means itself can be a syringe, pipette, and / or other such device.

[0318] The components of the kit can also be provided in dry, for example, in lyophilized, form. Where the reagents and / or components are provided in dry form, the powder can be reconstituted by addition of a suitable solvent. Thus, the kit can further include a second vessel means containing a sterile, pharmaceutically acceptable buffer and / or other diluent.

[0319] The components of the kit can also be provided in the form of a cryopreservation formulation, such as a cryopreservation solution. The cryopreservation formulation, upon thawing, can be directly or formulated into an infusion composition for infusion into a patient. Thus, the kit can further include a cell cryobag, a cell cryovial, a temperature maintenance device, such as a container containing liquid nitrogen, a thawing device, and the like.

[0320] In a specific embodiment of the application, cells to be used in the cell therapy described herein are provided in a kit. In some embodiments, the cells are essentially the only component of the kit. The kit can comprise reagents and materials to prepare the desired cells. In specific embodiments, the reagents and materials comprise primers, nucleotides, appropriate buffers or buffer reagents, salts, etc. for amplification of the desired sequences, and in some cases the reagents include DNA and / or vectors encoding the costimulatory receptor and / or regulatory elements thereof described in any of the embodiments herein.

[0321] Embodiments of the application will be described in detail in the following Examples. Those skilled in the art will appreciate that the following Examples are intended to be illustrative only and are not intended to limit the scope of the application. Unless specific techniques or conditions are mentioned, techniques and conditions described in the literature (e.g., refer to J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, 2001, or corresponding references, or according to the product manual) are used, or unless otherwise indicated. When a reagent or instrument is not specified, it is a conventional product that can be obtained commercially.

[0322] Examples

[0323] Summary of sequences of elements in Examples:

[0324] CD8 signal peptide: nucleotide sequence of SEQ ID NO: 1, amino acid sequence of SEQ ID NO: 2;

[0325] CD28 transmembrane region: nucleotide sequence of SEQ ID NO: 3, amino acid sequence of SEQ ID NO: 4;

[0326] CD28 intracellular domain: nucleotide sequence of SEQ ID NO: 5, amino acid sequence of SEQ ID NO: 6;

[0327] CD28 extracellular hinge region: nucleotide sequence of SEQ ID NO: 7, amino acid sequence of SEQ ID NO: 8;

[0328] CD80 transmembrane region: nucleotide sequence of SEQ ID NO: 9, amino acid sequence of SEQ ID NO: 10;

[0329] CD80 intracellular domain: nucleotide sequence of SEQ ID NO: 11, amino acid sequence of SEQ ID NO: 12;

[0330] OX40 intracellular domain: nucleotide sequence of SEQ ID NO: 13, amino acid sequence of SEQ ID NO: 14;

[0331] Linker 1 : nucleotide sequence of SEQ ID NO: 15, amino acid sequence of SEQ ID NO: 16;

[0332] Linker 2: nucleotide sequence of SEQ ID NO: 17, amino acid sequence of SEQ ID NO: 18;

[0333] Linker 3: nucleotide sequence of SEQ ID NO: 19, amino acid sequence of SEQ ID NO: 20;

[0334] Linker 4: nucleotide sequence of SEQ ID NO: 21, amino acid sequence of SEQ ID NO: 22;

[0335] IL-7: nucleotide sequence of SEQ ID NO: 23, amino acid sequence of SEQ ID NO: 24;

[0336] Mutant BCMA extracellular domain: nucleotide sequence of SEQ ID NO: 25, amino acid sequence of SEQ ID NO: 26;

[0337] CD52 signal peptide: nucleotide sequence of SEQ ID NO: 27, amino acid sequence of SEQ ID NO: 28;

[0338] CD52: nucleotide sequence of SEQ ID NO: 29, amino acid sequence of SEQ ID NO: 30;

[0339] CD62L intracellular signaling region: nucleotide sequence of SEQ ID NO: 31, amino acid sequence of SEQ ID NO: 32;

[0340] Truncated CD62L intracellular signaling region: nucleotide sequence of SEQ ID NO: 33, amino acid sequence of SEQ ID NO: 34;

[0341] KIR2DL3 extracellular region: nucleotide sequence of SEQ ID NO: 35, amino acid sequence of SEQ ID NO: 36;

[0342] KIR2DL4 extracellular region: nucleotide sequence of SEQ ID NO: 37, amino acid sequence of SEQ ID NO: 38;

[0343] KIR3DL1 extracellular region: nucleotide sequence of SEQ ID NO: 39, amino acid sequence of SEQ ID NO: 40;

[0344] KIR3DL2 extracellular region: nucleotide sequence of SEQ ID NO: 41, amino acid sequence of SEQ ID NO: 42;

[0345] K23-9: nucleotide sequence of SEQ ID NO: 43, amino acid sequence of SEQ ID NO: 44;

[0346] K23-10: nucleotide sequence of SEQ ID NO: 45, amino acid sequence of SEQ ID NO: 46;

[0347] K23-11: nucleotide sequence of SEQ ID NO: 47, amino acid sequence of SEQ ID NO: 48;

[0348] K23-12: nucleotide sequence of SEQ ID NO: 49, amino acid sequence of SEQ ID NO: 50;

[0349] K23-13: nucleotide sequence of SEQ ID NO: 51, amino acid sequence of SEQ ID NO: 52;

[0350] K23-14: nucleotide sequence of SEQ ID NO: 53, amino acid sequence of SEQ ID NO: 54;

[0351] K24-6: nucleotide sequence of SEQ ID NO: 55, amino acid sequence of SEQ ID NO: 56;

[0352] K24-7: nucleotide sequence of SEQ ID NO: 57, amino acid sequence of SEQ ID NO: 58;

[0353] K24-8: nucleotide sequence of SEQ ID NO: 59, amino acid sequence of SEQ ID NO: 60;

[0354] K24-9: nucleotide sequence of SEQ ID NO: 61, amino acid sequence of SEQ ID NO: 62;

[0355] K24-10: nucleotide sequence of SEQ ID NO: 63, amino acid sequence of SEQ ID NO: 64;

[0356] K24-11: nucleotide sequence of SEQ ID NO: 65, amino acid sequence of SEQ ID NO: 66;

[0357] IL7-mBCMA-GPI: nucleotide sequence of SEQ ID NO: 67, amino acid sequence of SEQ ID NO: 68;

[0358] IL7-mBCMA-GPI-K23-9: nucleotide sequence of SEQ ID NO: 69, amino acid sequence of SEQ ID NO: 70;

[0359] IL7-mBCMA-GPI-K23-10: nucleotide sequence of SEQ ID NO: 71, amino acid sequence of SEQ ID NO: 72;

[0360] IL7-mBCMA-GPI-K23-11: nucleotide sequence of SEQ ID NO: 73, amino acid sequence of SEQ ID NO: 74;

[0361] IL7-mBCMA-GPI-K23-12: nucleotide sequence of SEQ ID NO: 75, amino acid sequence of SEQ ID NO: 76;

[0362] IL7-mBCMA-GPI-K23-13: nucleotide sequence of SEQ ID NO: 77, amino acid sequence of SEQ ID NO: 78;

[0363] IL7-mBCMA-GPI-K23-14: nucleotide sequence of SEQ ID NO: 79, amino acid sequence of SEQ ID NO: 80;

[0364] IL7-mBCMA-GPI-K24-6: nucleotide sequence of SEQ ID NO: 81, amino acid sequence of SEQ ID NO: 82;

[0365] IL7-mBCMA-GPI-K24-7: nucleotide sequence of SEQ ID NO: 83, amino acid sequence of SEQ ID NO: 84;

[0366] IL7-mBCMA-GPI-K24-8: nucleotide sequence of SEQ ID NO: 85, amino acid sequence of SEQ ID NO: 86;

[0367] IL7-mBCMA-GPI-K24-9: nucleotide sequence of SEQ ID NO: 87, amino acid sequence of SEQ ID NO: 88;

[0368] IL7-mBCMA-GPI-K24-10: nucleotide sequence of SEQ ID NO: 89, amino acid sequence of SEQ ID NO: 90;

[0369] IL7-mBCMA-GPI-K24-11: nucleotide sequence of SEQ ID NO: 91, amino acid sequence of SEQ ID NO: 92;

[0370] K31-1: nucleotide sequence of SEQ ID NO: 93, amino acid sequence of SEQ ID NO: 94;

[0371] K31-2: nucleotide sequence of SEQ ID NO: 95, amino acid sequence of SEQ ID NO: 96;

[0372] K32-1: nucleotide sequence of SEQ ID NO: 97, amino acid sequence of SEQ ID NO: 98;

[0373] K32-2: nucleotide sequence of SEQ ID NO: 99, amino acid sequence of SEQ ID NO: 100;

[0374] IL7-mBCMA-GPI-K31-1: nucleotide sequence of SEQ ID NO: 101, amino acid sequence of SEQ ID NO: 102;

[0375] IL7-mBCMA-GPI-K31-2: nucleotide sequence of SEQ ID NO: 103, amino acid sequence of SEQ ID NO: 104;

[0376] IL7-mBCMA-GPI-K32-1: nucleotide sequence of SEQ ID NO: 105, amino acid sequence of SEQ ID NO: 106;

[0377] IL7-mBCMA-GPI-K32-2: nucleotide sequence of SEQ ID NO: 107, amino acid sequence of SEQ ID NO: 108;

[0378] Linker 5: nucleotide sequence of SEQ ID NO: 109, amino acid sequence of SEQ ID NO: 110;

[0379] 5’UTR: nucleotide sequence of SEQ ID NO: 111;

[0380] K23-15: nucleotide sequence of SEQ ID NO: 112, amino acid sequence of SEQ ID NO: 113;

[0381] K23-16: nucleotide sequence of SEQ ID NO: 114, amino acid sequence of SEQ ID NO: 115;

[0382] K23-17: nucleotide sequence of SEQ ID NO: 116, amino acid sequence of SEQ ID NO: 117;

[0383] K23-18: nucleotide sequence of SEQ ID NO: 118, amino acid sequence of SEQ ID NO: 119;

[0384] K24-12: nucleotide sequence of SEQ ID NO: 120, amino acid sequence of SEQ ID NO: 121;

[0385] K24-13: nucleotide sequence of SEQ ID NO: 122, amino acid sequence of SEQ ID NO: 123;

[0386] K24-14: nucleotide sequence of SEQ ID NO: 124, amino acid sequence of SEQ ID NO: 125;

[0387] K24-15: nucleotide sequence of SEQ ID NO: 126, amino acid sequence of SEQ ID NO: 127;

[0388] IL7-GPI: nucleotide sequence of SEQ ID NO: 128, amino acid sequence of SEQ ID NO: 129;

[0389] IL7-mBCMA-GPI-K23-15: nucleotide sequence of SEQ ID NO: 130, amino acid sequence of SEQ ID NO: 131;

[0390] IL7-mBCMA-GPI-K23-16: nucleotide sequence of SEQ ID NO: 132, amino acid sequence of SEQ ID NO: 133;

[0391] IL7-GPI-K23-17: nucleotide sequence of SEQ ID NO: 134, amino acid sequence of SEQ ID NO: 135;

[0392] IL7-GPI-K23-18: nucleotide sequence of SEQ ID NO: 136, amino acid sequence of SEQ ID NO: 137;

[0393] 5’UTR-IL7-GPI-K23-17: nucleotide sequence of SEQ ID NO: 138;

[0394] 5’UTR-IL7-GPI-K23-18: nucleotide sequence of SEQ ID NO: 139;

[0395] IL7-mBCMA-GPI-K24-12: nucleotide sequence of SEQ ID NO: 140, amino acid sequence of SEQ ID NO: 141;

[0396] IL7-mBCMA-GPI-K24-13: nucleotide sequence of SEQ ID NO: 142, amino acid sequence of SEQ ID NO: 143;

[0397] IL7-GPI-K24-14: nucleotide sequence of SEQ ID NO: 144, amino acid sequence of SEQ ID NO: 145;

[0398] IL7-GPI-K24-15: nucleotide sequence of SEQ ID NO: 146, amino acid sequence of SEQ ID NO: 147;

[0399] 5'UTR-IL7-GPI-K24-14: nucleotide sequence of SEQ ID NO: 148;

[0400] 5'UTR-IL7-GPI-K24-15: nucleotide sequence of SEQ ID NO: 149;

[0401] CD8 extracellular hinge region: nucleotide sequence of SEQ ID NO: 150, amino acid sequence of SEQ ID NO: 151 ;

[0402] K23-19: nucleotide sequence of SEQ ID NO: 152, amino acid sequence of SEQ ID NO: 153;

[0403] K23-20: nucleotide sequence of SEQ ID NO: 154, amino acid sequence of SEQ ID NO: 155;

[0404] K24-16: nucleotide sequence of SEQ ID NO: 156, amino acid sequence of SEQ ID NO: 157;

[0405] K24-17: nucleotide sequence of SEQ ID NO: 158, amino acid sequence of SEQ ID NO: 159;

[0406] IL7-GPI-K23-19: nucleotide sequence of SEQ ID NO: 160, amino acid sequence of SEQ ID NO: 161 ;

[0407] IL7-GPI-K23-20: nucleotide sequence of SEQ ID NO: 162, amino acid sequence of SEQ ID NO: 163;

[0408] 5'UTR-IL7-GPI-K23-19: nucleotide sequence of SEQ ID NO: 164;

[0409] 5'UTR-IL7-GPI-K23-20: nucleotide sequence of SEQ ID NO: 165;

[0410] IL7-GPI-K24-16: nucleotide sequence of SEQ ID NO: 166, amino acid sequence of SEQ ID NO: 167;

[0411] IL7-GPI-K24-17: nucleotide sequence of SEQ ID NO: 168, amino acid sequence of SEQ ID NO: 169;

[0412] 5'UTR-IL7-GPI-K24-16: nucleotide sequence of SEQ ID NO: 170;

[0413] 5'UTR-IL7-GPI-K24-17: nucleotide sequence of SEQ ID NO: 171.

[0414] The helper receptors involved in the examples are shown in Table 2:

[0415] Table 2 Helper receptor structures and sequences

[0416] Example 1: Construction of helper receptor expression vectors

[0417] The pKB20 vector was constructed according to the method described in Example 1 on page 21 of the specification of PCT application WO2022078310A1. The pKB20 vector containing the expression frame of the exogenous gene was constructed according to the method described in the example for constructing pKB20-EGFP. Specifically, the sequences shown in Table 2 SEQ ID NO: 43, 45, 47, 49, 51, 53, 112, 114, 116, 118, 152, 154, 55, 57, 59, 61, 63, 65, 120, 122, 124, 126, 156, 158, 69, 71, 73, 75, 77, 79, 130, 132, 134, 136, 160, 162, 138, 139, 164, 165, 81, 83, 85, 87, 89, 91, 140, 142, 144, 146, 166, 168, 148, 149, 170, 171, 93, 95, 97, 99, 101, 103, 105 and 107 were obtained by synthesis by a commissioned company, and the 2 ends of SEQ ID NO: 43, 45, 47, 49, 51, 53, 112, 114, 116, 118, 55, 57, 59, 61, 63, 65, 120, 122, 124, 126, 69, 71, 73, 75, 77, 79, 130, 132, 134, 136, 138, 139, 81, 83, 85, 87, 89, 91, 140, 142, 144, 146, 148, 149, 93, 95, 97, 99, 101, 103, 105 and 107 were added with a linker containing the corresponding enzyme cutting site by ligase, and then cloned into the prepared pKB20 vector according to the method described in Example 1 on page 21 of the specification of WO2022078310A1, and named pKB20-K23-9, pKB20-K23-10, pKB20-K23-11, pKB20-K23-12, pKB20-K23-13, pKB20-K23-14, pKB20-K23-15, pKB20-K23-16, pKB20-K23-17, pKB20-K23-18, pKB20-K23-19, pKB20-K23-20, pKB20-K24-6, pKB20-K24-7, pKB20-K24-8, pKB20-K24-9, pKB20-K24-10, pKB20-K24-11, pKB20-K24-12, pKB20-K24-13, pKB20-K24-14, pKB20-K24-15, pKB20-K24-16, pKB20-K24-17, pKB20-IL7-mBCMA-GPI-K23-9, pKB20-IL7-mBCMA-GPI-K23-10,pKB20-IL7-mBCMA-GPI-K23-11, pKB20-IL7-mBCMA-GPI-K23-12, pKB20-IL7-mBCMA-GPI-K23-13, pKB20-IL7-mBCMA-GPI-K23-14, pKB20-IL7-mBCMA-GPI-K23-15, pKB20-IL7-mBCMA-GPI-K23-16, pKB20-IL7-GPI-K23-17, pKB20-IL7-GPI-K23-18, pKB20-IL7-GPI-K23-19, pKB20-IL7-GPI-K23-20, pKB20-5'UTR-IL7-GPI-K23-17, pKB20-5'UTR-IL7-GPI-K23-18, pKB20-5'UTR-IL7-GPI-K23-19, pKB20-5'UTR-IL7-GPI-K23-20, pKB20-IL7-mBCMA-GPI-K24-6, pKB20-IL7-GPI-mBCMA-K24-7, pKB20-IL7-mBCMA-GPI-K24-8, pKB20-IL7-mBCMA-GPI-K24-9, pKB20-IL7-mBCMA-GPI-K24-10, pKB20-IL7-mBCMA-GPI-K24-11, pKB20-IL7-mBCMA-GPI-K24-12, pKB20-IL7-mBCMA-GPI-K24-13, pKB20-IL7-GPI-K24-14, pKB20-IL7-GPI-K24-15, pKB20-IL7-GPI-K24-16, pKB20-IL7-GPI-K24-17, pKB20-5'UTR-IL7-GPI-K24-14, pKB20-5'UTR-IL7-GPI-K24-15, pKB20-5'UTR-IL7-GPI-K24-16, pKB20-5'UTR-IL7-GPI-K24-17, pKB20-K31-1, pKB20-K31-2, pKB20-K32-1, pKB20-K32-2, pKB20-IL7-mBCMA-GPI-K31-1, pKB20-IL7-mBCMA-GPI-K31-2, pKB20-IL7-mBCMA-GPI-K32-1, pKB20-IL7-mBCMA-GPI-K32-2.

[0418] Example 2: Isolation and culture of melanoma tissue-derived TIL cells

[0419] Freshly resected melanoma specimens were collected and processed immediately under sterile conditions. The procedure was as follows: normal tissue and necrotic areas surrounding the cancer specimen were removed and small tissue pieces of 2-3 mm in size were taken from different areas of the specimen, 3-4 pieces per well of a 6-well plate. 3 mL of complete medium (AIM-V medium with 5% human AB serum), OKT-3 at a final concentration of 30 ng / mL and IL-2 at 3000 IU / mL were added to each well. The 24-well plates were incubated in a 37°C, 5% CO2incubator. Half of the medium was changed for all wells on day 5-6 after the start of the culture. Thereafter, half of the medium was changed every 1-2 days depending on the growth of the TIL. Once the TIL had overgrown the well and all adherent cells had been removed, the TIL from each overgrown well were collected and pooled until sufficient amounts of TIL cells were available for the subsequent experiments. 3

[0420] Example 3: Genetic modification and expansion of TILs

[0421] 1) AIM-V medium was pre-added to a 12-well plate, 2 mL per well, for a total of 65 wells, and then transferred to a cell culture incubator at 37°C, 5% CO2for 1 hour of pre-warming;

[0422] 2) The single dose of electroporation solution was prepared for each well according to the following table:

[0423] ​Plasmids pKB20-K23-9, pKB20-K23-10, pKB20-K23-11, pKB20-K23-12, pKB20-K23-13, pKB20-K23-14, pKB20-K23-15, pKB20-K23-16, pKB20-K23-17, pKB20-K23-18, pKB20-K23-19, pKB20-K23-20, pKB20-K24-6, pKB20-K24-7, pKB20-K24-8, pKB20-K24-9, pKB20-K24-10, pKB20-K24-11, pKB20-K24-12, pKB20-K24-13, pKB20-K24-14, pKB20-K24-15, pKB20-K24-16, pKB20-K24-17, pKB20-mBCMA-IL7-GPI-K23-9, pKB20-mBCMA-IL7-GPI-K23-10, pKB20-mBCMA-IL7-GPI-K23-11, pKB20-mBCMA-IL7-GPI-K23-12, pKB20-mBCMA-IL7-GPI-K23-13, pKB20-mBCMA-IL7-GPI-K23-14, pKB20-IL7-mBCMA-GPI-K23-15, pKB20-IL7-mBCMA-GPI-K23-16, pKB20-IL7-GPI-K23-17, pKB20-IL7-GPI-K23-18, pKB20-IL7-GPI-K23-19, pKB20-IL7-GPI-K23-20, pKB20-5’UTR-IL7-GPI-K23-17, pKB20-5’UTR-IL7-GPI-K23-18, pKB20-5’UTR-IL7-GPI-K23-19, pKB20-5’UTR-IL7-GPI-K23-20, pKB20-mBCMA-IL7-GPI-K24-6, pKB20-mBCMA-IL7-GPI-K24-7, pKB20-mBCMA-IL7-GPI-K24-8, pKB20-mBCMA-IL7-GPI-K24-9, pKB20-mBCMA-IL7-GPI-K24-10, pKB20-mBCMA-IL7-GPI-K24-11, pKB20-IL7-mBCMA-GPI-K24-12, pKB20-IL7-mBCMA-GPI-K24-13, pKB20-IL7-GPI-K24-14, pKB20-IL7-GPI-K24-15, pKB20-IL7-GPI-K24-16,pKB20-IL7-GPI-K24-17, pKB20-5'UTR-IL7-GPI-K24-14, pKB20-5'UTR-IL7-GPI-K24-15, pKB20-5'UTR-IL7-GPI-K24-16, pKB20-5'UTR-IL7-GPI-K24-17, pKB20-K31-1, pKB20-K31-2, pKB20-K32-1, pKB20-K32-2, pKB20-mBCMA-IL7-GPI-K31-1, pKB20-mBCMA-IL7-GPI-K31-2, pKB20-mBCMA-IL7-GPI-K32-1, pKB20-IL7-mBCMA-GPI-K32-2 and control empty plasmid pKB20, 64 groups of experimental group electroporation system and 1 group of control group were prepared;

[0424] 3) Take the TIL obtained in Example 2 into 65 EP tubes, add 5 x 10 6 cells into each EP tube, centrifuge at 1200 rpm for 5 min, discard the supernatant, then resuspend the cells with 500 μL of normal saline, repeat the centrifugation step to wash the cell pellet;

[0425] 4) To the electrotransformation solution of each different experimental group and control group prepared in 2), add plasmid pKB20-K23-9, pKB20-K23-10, pKB20-K23-11, pKB20-K23-12, pKB20-K23-13, pKB20-K23-14, pKB20-K23-15, pKB20-K23-16, pKB20-K23-17, pKB20-K23-18, pKB20-K23-19, pKB20-K23-20, pKB20-K24-6, pKB20-K24-7, pKB20-K24-8, pKB20-K24-9, pKB20-K24-10, pKB20-K24-11, pKB20-K24-12, pKB20-K24-13, pKB20-K24-14, pKB20-K24-15, pKB20-K24-16, pKB20-K24-17, pKB20-mBCMA-IL7-GPI-K23-9, pKB20-mBCMA-IL7-GPI-K23-10, pKB20-IL7-mBCMA-GPI-K23-11, pKB20-mBCMA-IL7-GPI-K23-12, pKB20-mBCMA-IL7-GPI-K23-13, pKB20-mBCMA-IL7-GPI-K23-14, pKB20-IL7-mBCMA-GPI-K23-15, pKB20-IL7-mBCMA-GPI-K23-16, pKB20-IL7-GPI-K23-17, pKB20-IL7-GPI-K23-18, pKB20-IL7-GPI-K23-19, pKB20-IL7-GPI-K23-20, pKB20-5'UTR-IL7-GPI-K23-17, pKB20-5'UTR-IL7-GPI-K23-18, pKB20-5'UTR-IL7-GPI-K23-19, pKB20-5'UTR-IL7-GPI-K23-20, pKB20-mBCMA-IL7-GPI-K24-6, pKB20-mBCMA-IL7-GPI-K24-7, pKB20-mBCMA-IL7-GPI-K24-8, pKB20-mBCMA-IL7-GPI-K24-9, pKB20-mBCMA-IL7-GPI-K24-10, pKB20-mBCMA-IL7-GPI-K24-11, pKB20-IL7-mBCMA-GPI-K24-12, pKB20-IL7-mBCMA-GPI-K24-13, pKB20-IL7-GPI-K24-14, pKB20-IL7-GPI-K24-15,pKB20-IL7-GPI-K24-16, pKB20-IL7-GPI-K24-17, pKB20-5'UTR-IL7-GPI-K24-14, pKB20-5'UTR-IL7-GPI-K24-15, pKB20-5'UTR-IL7-GPI-K24-16, pKB20-5'UTR-IL7-GPI-K24-17, pKB20-K31-1, pKB20-K31-2, pKB20-K32-1, pKB20-K32-2, pKB20-mBCMA-IL7-GPI-K31-1, pKB20-mBCMA-IL7-GPI-K31-2, pKB20-mBCMA-IL7-GPI-K32-1, pKB20-IL7-mBCMA-GPI-K32-2, and control empty plasmid pKB20 5 μg, followed by incubation at room temperature for 30 min or less;

[0426] 5) Resuspend all tubes with plasmid-containing electroporation solution prepared in 4) with 100 μL per tube, carefully pipette cell resuspension into LONZA 100 μL electroporation cuvette, place the electroporation cuvette into the LONZA Nucleofector TM 2b into the electroporation slot, start the electroporation program, electroporation program selection X001;

[0427] 6) After the end of the electrotransformation, carefully remove the electrotransformation cup and pipette the cell suspension into an EP tube. Add 200 μL of pre-warmed AIM-V medium to each tube and then transfer the contents to the wells of the 12-well plate containing pre-warmed AIM-V medium in 1) above. Incubate at 37°C, 5% CO2. After 5 days of incubation, obtain the following overexpressing helper receptors: K23-9, K23-10, K23-11, K23-12, K23-13, K23-14, K23-15, K23-16, K23-17, K23-18, K23-19, K23-20, K24-6, K24-7, K24-8, K24-9, K24-10, K24-11, K24-12, K24-13, K24-14, K24-15, K24-16, K24-17, IL7-mBCMA-GPI-K23-9, IL7-mBCMA-GPI-K23-10, IL7-mBCMA-GPI-K23-11, IL7-mBCMA-GPI-K23-12, IL7-mBCMA-GPI-K23-13, IL7-mBCMA-GPI-K23-14, IL7-mBCMA-GPI-K23-15, IL7-mBCMA-GPI-K23-16, IL7-GPI-K23-17, IL7-GPI-K23-18, IL7-GPI-K23-19, IL7-GPI-K23-20, 5’UTR-IL7-GPI-K23-17, 5’UTR-IL7-GPI-K23-18, 5’UTR-IL7-GPI-K23-19, 5’UTR-IL7-GPI-K23-20, IL7-mBCMA-GPI-K24-6, IL7-mBCMA-GPI-K24-7, IL7-mBCMA-GPI-K24-8, IL7-mBCMA-GPI-K24-9, IL7-mBCMA-GPI-K24-10, IL7-mBCMA-GPI-K24-11, IL7-mBCMA-GPI-K24-12, IL7-mBCMA-GPI-K24-13, IL7-GPI-K24-14, IL7-GPI-K24-15, IL7-GPI-K24-16, IL7-GPI-K24-17, 5’UTR-IL7-GPI-K24-14, 5’UTR-IL7-GPI-K24-15, 5’UTR-IL7-GPI-K24-16, 5’UTR-IL7-GPI-K24-17, K31-1, K31-2, K32-1, K32-2, IL7-mBCMA-GPI-K31-1, IL7-mBCMA-GPI-K31-2, IL7-mBCMA-GPI-K32-1,TIL cells of IL7-mBCMA-GPI-K32-2 and control TIL cells, respectively, named TIL-K23-9, TIL-K23-10, TIL-K23-11, TIL-K23-12, TIL-K23-13, TIL-K23-14, TIL-K23-15, TIL-K23-16, TIL-K23-17, TIL-K23-18, TIL-K23-19, TIL-K23-20, TIL-K24-6, TIL-K24-7, TIL-K24-8, TIL-K24-9, TIL-K24-10, TIL-K24-11, TIL-K24-12, TIL-K24-13, TIL-K24-14, TIL-K24-15, TIL-K24-16, TIL-K24-17, TIL-IL7-mBCMA-GPI-K23-9, TIL-IL7-mBCMA-GPI-K23-10, TIL-IL7-mBCMA-GPI-K23-11, TIL-IL7-mBCMA-GPI-K23-12, TIL-IL7-mBCMA-GPI-K23-13, TIL-IL7-mBCMA-GPI-K23-14, TIL-IL7-mBCMA-GPI-K23-15, TIL-IL7-mBCMA-GPI-K23-16, TIL-IL7-GPI-K23-17, TIL-IL7-GPI-K23-18, TIL-IL7-GPI-K23-19, TIL-IL7-GPI-K23-20, TIL-5’UTR-IL7-GPI-K23-17, TIL-5’UTR-IL7-GPI-K23-18, TIL-5’UTR-IL7-GPI-K23-19, TIL-5’UTR-IL7-GPI-K23-20, TIL-IL7-mBCMA-GPI-K24-6, TIL-IL7-mBCMA-GPI-K24-7, TIL-IL7-mBCMA-GPI-K24-8, TIL-IL7-mBCMA-GPI-K24-9, TIL-IL7-mBCMA-GPI-K24-10, TIL-IL7-mBCMA-GPI-K24-11, TIL-IL7-mBCMA-GPI-K24-12, TIL-IL7-mBCMA-GPI-K24-13, TIL-IL7-GPI-K24-14, TIL-IL7-GPI-K24-15, TIL-IL7-GPI-K24-16, TIL-IL7-GPI-K24-17, TIL-5’UTR-IL7-GPI-K24-14, TIL-5’UTR-IL7-GPI-K24-15,TIL-5'UTR-IL7-GPI-K24-16, TIL-5'UTR-IL7-GPI-K24-17, TIL-K31-1, TIL-K31-2, TIL-K32-1, TIL-K32-2, TIL-IL7-mBCMA-GPI-K31-1, TIL-IL7-mBCMA-GPI-K31-2, TIL-IL7-mBCMA-GPI-K32-1, TIL-IL7-mBCMA-GPI-K32-2, and TIL-CTRL.

[0428] Example 4: Cell survival rate of TILs of the electroporation-assisted receptors

[0429] The cell survival rate of each group was detected by trypan blue staining and cell counter. The results showed that the cell survival rate of each TIL expressing the auxiliary receptor prepared in Example 3 and the control TIL was more than 95%.

[0430] For TILs of the T cells auxiliary receptors only electroporated, fluorescent antibodies targeting the extracellular domain of KIR were used for flow cytometry detection. For TILs of the IL-7-GPI containing the extracellular domain of BCMA and the T cell auxiliary receptor fusion protein electroporated, fluorescent antibodies targeting the extracellular domain of BCMA and fluorescent antibodies targeting the extracellular domain of KIR were used for flow cytometry detection, respectively, as follows:

[0431] 1) Collect TIL-K23-9, TIL-K23-10, TIL-K23-11, TIL-K23-12, TIL-K23-13, TIL-K23-14, TIL-K23-15, TIL-K23-16, TIL-K23-17, TIL-K23-18, TIL-K23-19, TIL-K23-20, TIL-K24-6, TIL-K24-7, TIL-K24-8, TIL-K24-9, TIL-K24-10, TIL-K24-11, TIL-K24-12, TIL-K24-13, TIL-K24-14, TIL-K24-15, TIL-K24-16, TIL-K24-17, TIL-IL7-mBCMA-GPI-K23-9, TIL-IL7-mBCMA-GPI-K23-10, TIL-IL7-mBCMA-GPI-K23-11, TIL-IL7-mBCMA-GPI-K23-12, TIL-IL7-mBCMA-GPI-K23-13, TIL-IL7-mBCMA-GPI-K23-14, TIL-IL7-mBCMA-GPI-K23-15, TIL-IL7-mBCMA-GPI-K23-16, TIL-IL7-GPI-K23-17, TIL-IL7-GPI-K23-18, TIL-IL7-GPI-K23-19, TIL-IL7-GPI-K23-20, TIL-5’UTR-IL7-GPI-K23-17, TIL-5’UTR-IL7-GPI-K23-18, TIL-5’UTR-IL7-GPI-K23-19, TIL-5’UTR-IL7-GPI-K23-20, TIL-IL7-mBCMA-GPI-K24-6, TIL-IL7-mBCMA-GPI-K24-7, TIL-IL7-mBCMA-GPI-K24-8, TIL-IL7-mBCMA-GPI-K24-9, TIL-IL7-mBCMA-GPI-K24-10, TIL-IL7-mBCMA-GPI-K24-11, TIL-IL7-mBCMA-GPI-K24-12, TIL-IL7-mBCMA-GPI-K24-13, TIL-IL7-GPI-K24-14, TIL-IL7-GPI-K24-15, TIL-IL7-GPI-K24-16, TIL-IL7-GPI-K24-17, TIL-5’UTR-IL7-GPI-K24-14, TIL-5’UTR-IL7-GPI-K24-15, TIL-5’UTR-IL7-GPI-K24-16,TIL-5'UTR-IL7-GPI-K24-17, TIL-K31-1, TIL-K31-2, TIL-K32-1, TIL-K32-2, TIL-IL7-mBCMA-GPI-K31-1, TIL-IL7-mBCMA-GPI-K31-2, TIL-IL7-mBCMA-GPI-K32-1, TIL-IL7-mBCMA-GPI-K32-2 and TIL-CTRL, each group of cells, 1 x 10, 6 800g, centrifugation for 5 min;

[0432] 2) Discard the supernatant, resuspend the cells in physiological saline, 800g, centrifugation for 5 min;

[0433] 3) Discard the supernatant, resuspend the cells in 100 μL physiological saline per sample. For each group of TILs electroporated with IL-7-GPI and T cell helper receptor fusion proteins containing the extracellular domain of BCMA, the cell sample was divided into two parts, one added 2 μL of BCMA flow antibody (Biolegend, Cat#: 357504) per tube, and the other added flow antibody targeting the extracellular region of KIR, incubated at room temperature for 30 minutes; for each group of TILs electroporated with only T cell helper receptor, added flow antibody targeting the extracellular region of KIR, incubated at room temperature for 30 minutes; flow antibody targeting the extracellular region of KIR includes: flow antibody targeting the extracellular region of KIR2DL3 (R&D Systems, Cat# FAB2014P), flow antibody targeting the extracellular region of KIR2DL4 (R&D Systems, Cat# AF647), flow antibody targeting the extracellular region of KIR3DL1 (R&D Systems, Cat# FAB12251P) and flow antibody targeting the extracellular region of KIR3DL2 (R&D Systems, Cat# FAB2878P);

[0434] 4) Centrifuge the cells labeled with antibodies in 3) at 800g for 5 min, add an appropriate amount of physiological saline to each group, centrifuge at 800g for 5 min, wash twice, discard the supernatant;

[0435] 5) Resuspend with 400 μL of physiological saline and detect on a flow cytometer.

[0436] The positive rates of each group of cells are shown in Table 3 below:

[0437] Table 3 Percentage of TILs with helper receptor expression

[0438] The results show that the proportion of TILs expressing the auxiliary receptor in each group of TILs that have been electroporated with the auxiliary receptor is about 30%-60%. Among them, the proportion of TILs expressing the auxiliary receptor containing the intracellular signal region of CD62L in the intracellular domain of the auxiliary receptor is significantly higher than that of TILs expressing the auxiliary receptor without the intracellular signal region of CD62L in the intracellular domain.

[0439] Example 5: Killing effect of TIL cells overexpressing auxiliary receptors on homologous tumor cells

[0440] The fresh melanoma tissue of Example 2 was cut into 3x3x3mm pieces, and the pieces were mixed as evenly as possible, and then primary melanoma cells were obtained by culturing 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 section.

[0441] Agilent's real-time cell functional analyzer (RTCA) to detect TIL-K23-9, TIL-K23-10, TIL-K23-11, TIL-K23-12, TIL-K23-13, TIL-K23-14, TIL-K23-15, TIL-K23-16, TIL-K23-17, TIL-K23-18, TIL-K23-19, TIL-K23-20, TIL-K24-6, TIL-K24-7, TIL-K24-8, TIL-K24-9, TIL-K24-10, TIL-K24-11, TIL-K24-12, TIL-K24-13, TIL-K24-14, TIL-K24-15, TIL-K24-16, TIL-K24-17, TIL-IL7-mBCMA-GPI-K23-9, TIL-IL7-mBCMA-GPI-K23-10, TIL-IL7-mBCMA-GPI-K23-11, TIL-IL7-mBCMA-GPI-K23-12, TIL-IL7-mBCMA-GPI-K23-13, TIL-IL7-mBCMA-GPI-K23-14, TIL-IL7-mBCMA-GPI-K23-15, TIL-IL7-mBCMA-GPI-K23-16, TIL-IL7-GPI-K23-17, TIL-IL7-GPI-K23-18, TIL-IL7-GPI-K23-19, TIL-IL7-GPI-K23-20, TIL-5'UTR-IL7-GPI-K23-17, TIL-5'UTR-IL7-GPI-K23-18, TIL-5'UTR-IL7-GPI-K23-19, TIL-5'UTR-IL7-GPI-K23-20, TIL-IL7-mBCMA-GPI-K24-6, TIL-IL7-mBCMA-GPI-K24-7, TIL-IL7-mBCMA-GPI-K24-8, TIL-IL7-mBCMA-GPI-K24-9, TIL-IL7-mBCMA-GPI-K24-10, TIL-IL7-mBCMA-GPI-K24-11, TIL-IL7-mBCMA-GPI-K24-12, TIL-IL7-mBCMA-GPI-K24-13, TIL-IL7-GPI-K24-14, TIL-IL7-GPI-K24-15, TIL-IL7-GPI-K24-16, TIL-IL7-GPI-K24-17, TIL-5'UTR-IL7-GPI-K24-14, TIL-5'UTR-IL7-GPI-K24-15, obtained in Example 3.TIL-5'UTR-IL7-GPI-K24-16, TIL-5'UTR-IL7-GPI-K24-17, TIL-K31-1, TIL-K31-2, TIL-K32-1, TIL-K32-2, TIL-IL7-mBCMA-GPI-K31-1, TIL-IL7-mBCMA-GPI-K31-2, TIL-IL7-mBCMA-GPI-K32-1, TIL-IL7-mBCMA-GPI-K32-2 and TIL-CTRL cells on their homologous melanoma primary cells in vitro killing activity, the specific steps are as follows:

[0442] (1) Zero: add 50 μL DMEM culture solution to each well, put into the instrument, select step 1, zero;

[0443] (2) Target cell plating: the melanoma primary cells obtained by culture were plated at 10 4 cells / 50 μL per well in a plate containing a detection electrode, placed for a few minutes, and then put into the instrument after the cells were stable, step 2 was started, and the cells were cultured;

[0444] (3) Add effector cells: the positive rate of each effector cell was adjusted to 30% with TIL-CTRL. After the target cells were cultured for 24 h, the cell index was observed, and when the cell index was 1, the effector cells TIL-K23-9, TIL-K23-10, TIL-K23-11, TIL-K23-12, TIL-K23-13, TIL-K23-14, TIL-K23-15, TIL-K23-16, TIL-K23-17, TIL-K23-18, TIL-K23-19, TIL-K23-20, TIL-K24-6, TIL-K24-7, TIL-K24-8, TIL-K24-9, TIL-K24-10, TIL-K24-11, TIL-K24-12, TIL-K24-13, TIL-K24-14, TIL-K24-15, TIL-K24-16, TIL-K24-17, TIL-IL7-mBCMA-GPI-K23-9, TIL-IL7-mBCMA-GPI-K23-10, TIL-IL7-mBCMA-GPI-K23-11, TIL-IL7-mBCMA-GPI-K23-12, TIL-IL7-mBCMA-GPI-K23-13, TIL-IL7-mBCMA-GPI-K23-14, TIL-IL7-mBCMA-GPI-K23-15, TIL-IL7-mBCMA-GPI-K23-16, TIL-IL7-GPI-K23-17, TIL-IL7-GPI-K23-18, TIL-IL7-GPI-K23-19, TIL-IL7-GPI-K23-20, TIL-5'UTR-IL7-GPI-K23-17, TIL-5'UTR-IL7-GPI-K23-18, TIL-5'UTR-IL7-GPI-K23-19, TIL-5'UTR-IL7-GPI-K23-20, TIL-IL7-mBCMA-GPI-K24-6, TIL-IL7-mBCMA-GPI-K24-7, TIL-IL7-mBCMA-GPI-K24-8, TIL-IL7-mBCMA-GPI-K24-9, TIL-IL7-mBCMA-GPI-K24-10, TIL-IL7-mBCMA-GPI-K24-11, TIL-IL7-mBCMA-GPI-K24-12, TIL-IL7-mBCMA-GPI-K24-13, TIL-IL7-GPI-K24-14, TIL-IL7-GPI-K24-15, TIL-IL7-GPI-K24-16, TIL-IL7-GPI-K24-17,TIL-5'UTR-IL7-GPI-K24-14, TIL-5'UTR-IL7-GPI-K24-15, TIL-5'UTR-IL7-GPI-K24-16, TIL-5'UTR-IL7-GPI-K24-17, TIL-K31-1, TIL-K31-2, TIL-K32-1, TIL-K32-2, TIL-IL7-mBCMA-GPI-K31-1, TIL-IL7-mBCMA-GPI-K31-2, TIL-IL7-mBCMA-GPI-K32-1, TIL-IL7-mBCMA-GPI-K32-2 and TIL-CTRL cells, 50 μL per well, the effector-target ratio is 2:1, start step 3, after co-culturing for more than 60 h, observe the cell proliferation curve and calculate the target cell killing rate. The target cell killing rate calculation formula is as follows:

[0445] Wherein A is the cell index of the group without adding any effector cells, i.e., only target cells (i.e., tumor cells), and B is the cell index of the group added with effector cells.

[0446] The results are shown in Table 4.

[0447] Table 4 Killing of target cells by TIL expressing accessory receptors

[0448] The results show that, compared with TIL-CTRL, each group of TIL expressing T cell accessory receptors and expressing a fusion protein comprising membrane surface IL-7 and T cell accessory receptors has a significantly stronger killing effect on the same source matched melanoma primary cells. Compared with each group of TIL expressing T cell accessory receptors comprising KIR3DL1 and KIR3DL2 extracellular domains, each group of TIL expressing T cell accessory receptors comprising KIR2DL3 and KIR2DL4 extracellular domains has a significantly stronger killing effect on target cells. At the same time, compared with TIL expressing only T cell accessory receptors KIR extracellular domains, each group of TIL expressing membrane surface IL-7 (IL-7-GPI, IL7-mBCMA-GPI) on this basis has a further improved killing effect on target cells.

[0449] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details in accordance with all the teachings disclosed herein, and such changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A helper receptor comprising an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic domain, the extracellular domain comprising an extracellular domain of a KIR or a functional fragment or variant thereof that retains the biological function of binding a KIR ligand, preferably the intracellular region comprises a CD62L intracellular domain. Preferably, the KIR is an activating KIR or an inhibitory KIR, More preferably, the activating KIR is KIR2DL4 and the inhibitory KIR is selected from any one or more of KIR2DL3, KIR3DL1 and KIR3DL2.

2. The auxiliary receptor of claim 1, wherein, The cytoplasmic domain comprises a signal transduction domain and / or a costimulatory domain, Preferably, the costimulatory domain is an intracellular domain of a costimulatory signaling molecule or a functional fragment or mutant thereof that retains the biological function of the costimulatory signaling molecule to deliver a costimulatory signal, activating an immune cell, More preferably, the costimulatory signaling molecule intracellular domain comprises any one or more of an intracellular domain 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, CD80 and CD27 or a mutant thereof, Further preferably, the costimulatory signaling molecule intracellular domain is an intracellular domain of CD28 and / or an intracellular domain of OX40, Further preferably, the costimulatory signaling molecule intracellular domain is an intracellular domain of CD80.

3. The auxiliary receptor of claim 1, wherein, The helper receptor further comprises a hinge region, Preferably, the hinge region comprises a juxtamembrane fragment of a native extracellular domain of the costimulatory signaling molecule, or, the hinge region comprises one or more 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 a juxtamembrane fragment of a native extracellular domain of CD27, More preferably, the hinge region is a CD28 extracellular hinge region, a CD8 extracellular hinge region, or an IL7Rα extracellular hinge region.

4. The auxiliary receptor according to claim 1 or 2, wherein The transmembrane region comprises one or more selected from the group consisting of: a transmembrane region 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, CD80, and CD27, or a mutant thereof that retains transmembrane function, Preferably, the transmembrane region is a CD28 transmembrane region, an IL7Rα transmembrane region, a CD80 transmembrane region, or a mutant thereof that retains transmembrane function.

5. The costimulatory receptor of claim 4, wherein The costimulatory receptor comprises an extracellular ligand binding domain comprising a KIR2DL3 extracellular region or a KIR2DL4 extracellular region, a transmembrane region that is a CD28 transmembrane region or an IL7Rα transmembrane region or a mutant thereof, and a CD62L intracellular domain, optionally further comprising one or more intracellular domains selected from the group consisting of a CD28 intracellular domain, an OX40 intracellular domain, and an IL-7Rα intracellular domain, Preferably, the CD62L intracellular domain is a full-length CD62L intracellular domain or a truncated CD62L intracellular domain, Preferably, the costimulatory receptor further comprises a CD28 hinge region between the extracellular ligand-binding domain and the transmembrane region, Preferably, the costimulatory receptor further comprises a Claudin 18.2 antigen epitope or a BCMA extracellular domain or a mutant thereof; Preferably, the costimulatory receptor further comprises a Claudin 18.2 antigen epitope or a BCMA extracellular domain or a mutant thereof; Preferably, the costimulatory receptor further comprises a Claudin 18.2 antigen epitope or a BCMA extracellular domain or a mutant thereof; Preferably, the costimulatory receptor further comprises a Claudin 18.2 antigen epitope or a BCMA extracellular domain or a mutant thereof; More preferably, the costimulatory receptor comprises: (1) a KIR2DL3 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, an OX40 intracellular domain, (2) a KIR2DL3 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD62L intracellular domain, a CD28 intracellular domain, an OX40 intracellular domain, (3) a KIR2DL3 extracellular region, a CD28 extracellular hinge region, a CD80 transmembrane region, a CD80 intracellular domain, (4) a KIR2DL3 extracellular region, a CD28 extracellular hinge region, a CD80 transmembrane region, a CD62L intracellular domain, a CD80 intracellular domain, (5) a KIR2DL3 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a truncated CD62L intracellular domain, a CD28 intracellular domain, an OX40 intracellular domain, (6) a KIR2DL3 extracellular region, a CD28 extracellular hinge region, a CD80 transmembrane region, a truncated CD62L intracellular domain, a CD80 intracellular domain, (7) a KIR2DL4 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, an OX40 intracellular domain, (8) a KIR2DL4 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD62L intracellular domain, a CD28 intracellular domain, an OX40 intracellular domain, (9) a KIR2DL4 extracellular region, a CD28 extracellular hinge region, a CD80 transmembrane region, a CD80 intracellular domain, (10) a KIR2DL4 extracellular region, a CD28 extracellular hinge region, a CD80 transmembrane region, a CD62L intracellular domain, a CD80 intracellular domain, (11) a KIR2DL4 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a truncated CD62L intracellular domain, a CD28 intracellular domain, an OX40 intracellular domain, (12) a KIR2DL4 extracellular region, a CD28 extracellular hinge region, a CD80 transmembrane region, a truncated CD62L intracellular domain, a CD80 intracellular domain, (13) a KIR3DL1 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, an OX40 intracellular domain, (14) a KIR3DL1 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD62L intracellular domain, a CD28 intracellular domain, an OX40 intracellular domain, (15) a KIR3DL2 extracellular region, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, an OX40 intracellular domain, (16) KIR3DL2 extracellular region, CD28 extracellular hinge region, CD28 transmembrane region, CD62L endodomain, CD28 endodomain, OX40 endodomain, (17) KIR2DL3 extracellular region, Claudin 18.2 antigenic epitope, CD28 extracellular hinge region, CD28 transmembrane region, CD62L endodomain, CD28 endodomain, and OX40 endodomain, (18) KIR2DL3 extracellular region, BCMA extracellular domain, IL7Ra transmembrane region or mutant thereof, CD62L endodomain, IL-7Ra endodomain, or (19) KIR2DL3 extracellular region, linker, BCMA extracellular domain, CD28 transmembrane region, CD62L endodomain, CD28 endodomain, and OX40 endodomain, (20) KIR2DL3 extracellular region, BCMA extracellular domain or variant thereof, linker, CD28 transmembrane region, CD62L endodomain, CD28 endodomain, and OX40 endodomain (21) KIR2DL4 extracellular region, Claudin 18.2 antigenic epitope, CD28 extracellular hinge region, CD28 transmembrane region, CD62L endodomain, CD28 endodomain, and OX40 endodomain, (22) KIR2DL4 extracellular region, BCMA extracellular domain, IL7Ra extracellular hinge region, IL7Ra transmembrane region, CD62L endodomain, and IL-7Ra endodomain, (23) KIR2DL4 extracellular region, Claudin 18.2 antigenic epitope, IL7Ra extracellular hinge region, IL7Ra transmembrane region mutant, CD62L endodomain, and IL-7Ra endodomain, (24) KIR2DL4 extracellular region, linker, BCMA extracellular domain, CD28 transmembrane region, CD62L endodomain, CD28 endodomain, and OX40 endodomain, (25) KIR2DL4 extracellular region, BCMA extracellular domain mutant, linker, CD28 transmembrane region, CD62L endodomain, CD28 endodomain, and OX40 endodomain, (26) KIR2DL3 extracellular region, CD28 transmembrane region, CD62L endodomain, and OX40 endodomain, (27) KIR2DL3 extracellular region, CD28 transmembrane region, truncated CD62L endodomain, and OX40 endodomain, (28) KIR2DL3 extracellular region, BCMA extracellular domain mutant, CD28 transmembrane region, CD62L endodomain, and OX40 endodomain, (29) KIR2DL3 extracellular region, BCMA extracellular domain mutant, CD28 transmembrane region, truncated CD62L endodomain, and OX40 endodomain, (30) KIR2DL4 extracellular region, CD28 transmembrane region, CD62L endodomain, and OX40 endodomain, (31) KIR2DL4 extracellular region, CD28 transmembrane region, truncated CD62L intracellular domain, and OX40 intracellular domain, (32) KIR2DL4 extracellular region, BCMA extracellular domain mutant, CD28 transmembrane region, CD62L intracellular domain, and OX40 intracellular domain, (33) KIR2DL4 extracellular region, BCMA extracellular domain mutant, CD28 transmembrane region, truncated CD62L intracellular domain, and OX40 intracellular domain, (34) KIR2DL3 extracellular region, CD8 extracellular hinge region, CD28 transmembrane region, CD62L intracellular domain, and OX40 intracellular domain, (35) KIR2DL3 extracellular region, CD8 extracellular hinge region, CD28 transmembrane region, truncated CD62L intracellular domain, and OX40 intracellular domain, (36) KIR2DL4 extracellular region, CD8 extracellular hinge region, CD28 transmembrane region, CD62L intracellular domain, and OX40 intracellular domain, (37) KIR2DL4 extracellular region, CD8 extracellular hinge region, CD28 transmembrane region, truncated CD62L intracellular domain, and OX40 intracellular domain.

6. The costimulatory receptor of any one of claims 1-5, wherein, the amino acid sequence of the CD28 transmembrane region is set forth in SEQ ID NO: 4, the amino acid sequence of the CD28 intracellular domain is set forth in SEQ ID NO: 6, the amino acid sequence of the CD28 extracellular hinge region is set forth in SEQ ID NO: 8, the amino acid sequence of the CD80 transmembrane region is set forth in SEQ ID NO: 10, the amino acid sequence of the CD80 intracellular domain is set forth in SEQ ID NO: 12, the amino acid sequence of the OX40 intracellular domain is set forth in SEQ ID NO: 14, the CD62L intracellular domain comprises the sequence set forth in SEQ ID NO: 32, the truncated CD62L intracellular domain comprises the sequence set forth in SEQ ID NO: 34, the amino acid sequence of the KIR2DL3 extracellular region is set forth in SEQ ID NO: 36, the amino acid sequence of the KIR2DL4 extracellular region is set forth in SEQ ID NO: 38, the amino acid sequence of the KIR3DL1 extracellular region is set forth in SEQ ID NO: 40, the amino acid sequence of the KIR3DL2 extracellular region is set forth in SEQ ID NO: 42, the amino acid sequence of the linker is set forth in SEQ ID NO: 16, 18, 20, 22, or 110, or the amino acid sequence of the costimulatory receptor is set forth in any one of SEQ ID NOs: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 94, 96, 98, 100, 113, 115, 117, 119, 121, 123, 125, 127, 153, 155, 157, and 159.

7. A fusion protein comprising a helper receptor and a membrane surface cytokine, preferably, the helper receptor comprises an extracellular domain, a transmembrane region and an intracellular region, the extracellular domain comprises an extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding KIR ligand, the intracellular region comprises a signal transduction domain and / or a costimulatory domain, preferably, the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchoring region, preferably, the membrane surface cytokine is at the N-terminus and / or C-terminus of the helper receptor, preferably, the cytokine is IL-7, preferably, the GPI anchoring 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 cytokine comprises: a CD52 signal peptide, an IL-7, a linker, a CD52, preferably, the membrane surface cytokine comprises: a CD52 signal peptide, an IL-7, a linker, a mutant BCMA extracellular domain, a CD52, preferably, the membrane surface cytokine is connected to the helper 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 connected to the helper receptor; preferably, the membrane surface cytokine is at the N-terminus of the helper receptor, preferably, the helper receptor is as described in any one of claims 1-6, preferably, the amino acid sequence of the helper receptor is as set forth in any one of SEQ ID NOs: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 94, 96, 98, 100, 113, 115, 117, 119, 121, 123, 125, 127, 153, 155, 157 and 159, preferably, the amino acid sequence of the fusion protein is as set forth in any one of SEQ ID NOs: 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 80, 92, 102, 104, 106, 108, 131, 133, 135, 137, 141, 143, 145, 147, 161, 163, 167 and 169.

8. A polynucleotide molecule having: a nucleic acid sequence encoding the helper receptor of any one of claims 1-6 or the fusion protein of claim 7, or a complement thereof, preferably, the nucleic acid sequence encoding the helper receptor of any one of claims 1-6 comprises a sequence selected from any one of SEQ ID NOs: 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 93, 95, 97, 99, 112, 114, 116, 118, 120, 122, 124, 126, 152, 154, 156 and 158, or a complement thereof. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Preferably, the nucleic acid sequence encoding the fusion protein of claim 7 comprises a sequence selected from any one of SEQ ID NO: 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 101, 103, 105, 107, 130, 132, 134, 136, 138, 139, 140, 142, 144, 146, 148, 149, 160, 162, 164, 165, 166, 168, 170, and 171, or a complement thereof.

9. A nucleic acid construct comprising the polynucleotide molecule of claim 8; Preferably, the nucleic acid construct further comprises at least one regulatory element operably linked to the polynucleotide for expressing the accessory receptor or fusion protein, 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 transposon system-based non-viral integration vector.

10. A nucleic acid construct comprising: an expression cassette of an accessory receptor and an expression cassette of a membrane surface cytokine; or the nucleic acid construct is an expression cassette, wherein a coding sequence of an accessory receptor and a coding sequence of a membrane surface cytokine are within the expression cassette, the accessory receptor comprises an extracellular domain comprising an extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to a KIR ligand, a transmembrane region, and an intracellular region comprising a signal transduction domain and / or a costimulatory domain; the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchoring region.

11. The nucleic acid construct of claim 10, wherein, the accessory receptor is as described in any one of claims 1-6.

12. The nucleic acid construct of claim 10, wherein the amino acid sequence of the accessory receptor is as set forth in any one of SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 94, 96, 98, 100, 113, 115, 117, 119, 121, 123, 125, 127, 153, 155, 157, and 159.

13. The nucleic acid construct of claim 10, wherein the membrane surface cytokine is as described in claim 7.

14. The nucleic acid construct of claim 10, wherein the nucleic acid construct is an expression vector or a cloning vector, or the nucleic acid construct is a viral vector or a non-viral vector; more preferably, the non-viral vector is a transposon system-based non-viral integration vector.

15. A genetically engineered cell, which: (1) expresses the accessory receptor of any one of claims 1-6 or the fusion protein of claim 7, and / or carries a coding sequence of the accessory receptor or fusion protein, (2) an auxiliary receptor comprising an extracellular domain comprising an extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to a KIR ligand, a transmembrane region, and an intracellular region comprising a signal transduction domain and / or a costimulatory domain, and a membrane surface cytokine comprising a cytokine and a transmembrane domain or a GPI anchoring region, (3) a nucleic acid construct of any one of claims 9-14, Preferably, the cell is an immune effector cell; more preferably, the immune effector cell comprises a T cell, a NK cell, a CAR-T, a CAR-NK, a TCR-T, a CIK, a DN T, and a TIL, the cell further expresses a CAR, or carries a coding sequence of a CAR, the cell further expresses an exogenous TCR, or carries a coding sequence of an exogenous TCR.

16. A pharmaceutical composition comprising a pharmaceutically acceptable excipient, and, any one or more of the auxiliary receptor of any one of claims 1-6, the fusion protein of claim 7, the polynucleotide molecule of claim 8, the nucleic acid construct of any one of claims 9-14, and the genetically engineered cell of claim 15.

17. Use of any one or more of the auxiliary receptor of any one of claims 1-6, the fusion protein of claim 7, the polynucleotide molecule of claim 8, the nucleic acid construct of any one of claims 9-14, and the genetically engineered cell of claim 15 in the manufacture of a medicament for treating or preventing cancer.

18. Use of an auxiliary receptor comprising an extracellular domain comprising an extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to a KIR ligand, a transmembrane region, and an intracellular region comprising a signal transduction domain and / or a costimulatory domain, and a membrane surface cytokine comprising a cytokine and a transmembrane domain or a GPI anchoring region in the manufacture of a medicament for treating or preventing cancer.

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