Cell expressing co-receptor

Abstract

Provided is a genetically engineered cell, which expresses a co-receptor and a membrane surface cytokine. The co-receptor contains a KIR extracellular domain, a transmembrane region and an intracellular region. When the co-receptor and the membrane surface cytokine are co-expressed in an immune cell, the cytokine secretion level, proliferation level and killing ability for a target cell of the immune cell can be further improved.

Description

Cells expressing coreceptors

[0001] This application claims priority to Chinese application CN202410236684.9, filed on March 1, 2024, entitled “Cells expressing auxiliary receptors.” Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to a cell expressing a co-receptor. Background Art

[0003] Killer-cell immunoglobulin-like receptors (KIRs) are a group of germline-encoded receptors expressed on the surface of NK cells and a small number of T cells, with either activating or inhibitory functions. They include KIR3DL1, 2, 3, KIR3DS1, KIR2DL1, 2, 3, 4, 5A, 5B, and KIR2DS1, 2, 3, 4, 5. In addition, there are two pseudogenes, 2DP1 and 3DP1. The ligands of the most studied inhibitory KIRs are primarily various class I HLA molecules. However, there is still a lack of immune effector cells developed based on the combination of KIR receptors and cytokines. Summary of the Invention

[0004] To this end, the present invention provides a genetically engineered cell that expresses a co-receptor and a membrane surface cytokine, wherein the co-receptor comprises a KIR extracellular domain, a transmembrane region and an intracellular region.

[0005] The first aspect of the present invention provides an auxiliary receptor comprising an extracellular domain, a transmembrane region and an intracellular region, wherein the extracellular domain comprises the extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligand, and the intracellular region comprises a signal transduction domain and / or a co-stimulatory domain.

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

[0007] In one or more embodiments, the co-receptor further comprises a hinge region. Optionally, the hinge region comprises a proximal membrane-terminal fragment of the native extracellular domain of the co-stimulatory signal molecule. In some embodiments, the hinge region is located at the N-terminus 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 any one or more selected from KIR2DL3, KIR3DL1, and KIR3DL2.

[0009] In one or more embodiments, the costimulatory domain is the intracellular domain of a costimulatory signal molecule or a functional fragment or mutant thereof that retains the biological function of the costimulatory signal molecule in transmitting costimulatory signals and activating immune cells.

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

[0011] In one or more embodiments, the coreceptor further comprises a linker located between the KIR extracellular domain 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 variant thereof, or the claudin protein extracellular domain or 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 extracellular domain of the claudin protein is an extracellular antigenic epitope of the claudin protein. In one or more embodiments, the fragment of the extracellular domain of the claudin 18.2 protein comprises the sequence set forth in SEQ ID NO: 30; and its nucleic acid sequence comprises the sequence set forth in SEQ ID NO: 29.

[0014] In one or more embodiments, the BCMA extracellular domain comprises the sequence shown in SEQ ID NO: 32; and its nucleic acid sequence comprises the sequence shown in SEQ ID NO: 31.

[0015] In one or more embodiments, the linker located at the N-terminus or C-terminus of the BCMA extracellular domain or variant thereof, or the claudin protein extracellular domain or fragment or variant thereof, is a rigid linker or a flexible linker. In one or more embodiments, the linker is a rigid linker. In one or more embodiments, the sequence of the rigid linker comprises the sequence shown in SEQ ID NO: 60; and the nucleic acid sequence encoding the rigid linker comprises the sequence shown in SEQ ID NO: 59.

[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 the CD28 transmembrane region, the IL7Rα transmembrane region or a mutant thereof that retains the transmembrane function.

[0017] In one or more embodiments, the intracellular domain of the costimulatory signaling molecule includes but is not limited to any one or more of the intracellular domains of CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM and CD27, or their mutants. Preferably, the intracellular domain of the costimulatory signal molecule is the intracellular domain of CD28 and / or the intracellular domain of OX40.

[0018] In one or more embodiments, the hinge region includes, but is not limited to, a membrane-proximal fragment of a native extracellular domain selected from the group consisting of CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, and CD27. Preferably, the hinge region is the extracellular hinge region of CD28 and / or the extracellular hinge region of IL7Rα.

[0019] In one or more embodiments, the auxiliary receptor comprises a KIR2DL3 extracellular region, a linker, a membrane surface tag, a transmembrane region, and 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, wherein the transmembrane region is a CD28 transmembrane region or an IL7Rα transmembrane region or a mutant thereof. Wherein, the membrane surface tag comprises a Claudin18.2 antigen epitope or a BCMA extracellular domain; the auxiliary receptor further comprises a CD28 hinge region between the membrane surface tag and the transmembrane region. In one or more embodiments, the auxiliary receptor comprises a KIR2DL3 extracellular region, a Claudin18.2 antigen 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 auxiliary receptor comprises a KIR2DL3 extracellular region, a BCMA extracellular domain, any one of an IL7Rα transmembrane region or mutants 1-4 thereof, and an IL-7Rα intracellular domain. In one or more embodiments, the coreceptor comprises a KIR2DL3 extracellular region, a linker, a BCMA extracellular domain, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain.

[0020] In one or more embodiments, the auxiliary receptor comprises a KIR2DL4 extracellular region, a linker, a membrane surface tag, a transmembrane region, and 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, wherein the transmembrane region is a CD28 transmembrane region or an IL7Rα transmembrane region or a mutant thereof (e.g., mutant 4). The membrane surface tag comprises a Claudin18.2 antigen epitope or a BCMA extracellular domain; the auxiliary receptor further comprises a CD28 hinge region or an IL7Rα extracellular hinge region between the membrane surface tag and the transmembrane region. In one or more embodiments, the auxiliary receptor comprises a KIR2DL4 extracellular region, a Claudin18.2 antigen epitope, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain, or the auxiliary receptor comprises a KIR2DL4 extracellular region, a BCMA extracellular domain, an IL7Rα extracellular hinge region, an IL7Rα transmembrane region, and an IL-7Rα intracellular domain, or the auxiliary receptor comprises a KIR2DL4 extracellular region, a Claudin18.2 antigen epitope, an IL7Rα extracellular hinge region, an IL7Rα transmembrane region mutant 4, and an IL-7Rα intracellular domain. In one or more embodiments, the auxiliary 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.

[0021] In one or more embodiments, the auxiliary receptor comprises a KIR3DL1 extracellular region or a KIR3DL2 extracellular region, a membrane surface tag, a transmembrane region, and 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, wherein the transmembrane region is a CD28 transmembrane region. In one or more embodiments, the auxiliary receptor comprises a KIR3DL1 extracellular region, a KIR3 or DL2 extracellular region, a Claudin18.2 antigen epitope, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain.

[0022] In one or more embodiments, the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO:4.

[0023] In one or more embodiments, the amino acid sequence of the IL7Rα transmembrane region is shown in SEQ ID NO:6.

[0024] In one or more embodiments, the amino acid sequences of the IL7Rα transmembrane region mutants 1-4 are shown in SEQ ID NOs: 8, 10, 12 and 14.

[0025] In one or more embodiments, the amino acid sequence of the CD28 intracellular domain is shown in SEQ ID NO:16.

[0026] In one or more embodiments, the amino acid sequence of the OX40 intracellular domain is shown in SEQ ID NO:18.

[0027] In one or more embodiments, the amino acid sequence of the IL-7Rα intracellular domain is shown in SEQ ID NO:20.

[0028] In one or more embodiments, the amino acid sequence of the KIR2DL3 extracellular region is shown in SEQ ID NO:22.

[0029] In one or more embodiments, the amino acid sequence of the KIR2DL4 extracellular region is shown in SEQ ID NO:24.

[0030] In one or more embodiments, the amino acid sequence of the KIR3DL1 extracellular region is shown in SEQ ID NO:26.

[0031] In one or more embodiments, the amino acid sequence of the KIR3DL2 extracellular region is shown in SEQ ID NO:28.

[0032] In one or more embodiments, the amino acid sequence of the Claudin 18.2 antigen epitope is shown in SEQ ID NO:30.

[0033] In one or more embodiments, the amino acid sequence of the BCMA extracellular domain is shown in SEQ ID NO: 32.

[0034] In one or more embodiments, the amino acid sequence of the CD28 extracellular hinge region is shown in SEQ ID NO:34.

[0035] In one or more embodiments, the amino acid sequence of the extracellular hinge region of IL7Rα is shown in SEQ ID NO:36.

[0036] In one or more embodiments, the amino acid sequence of the coreceptor is as shown in any one of SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58.

[0037] The second aspect of the present invention provides an auxiliary receptor comprising an extracellular ligand binding domain, a transmembrane domain and an intracellular region, wherein the extracellular domain comprises the extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligand, and the intracellular region comprises the CD62L intracellular domain.

[0038] In one or more embodiments, the KIR is an activating KIR or an inhibitory KIR,

[0039] In one or more embodiments, the activating KIR is KIR2DL4, and the inhibitory KIR is any one or more selected from KIR2DL3, KIR3DL1, and KIR3DL2.

[0040] In one or more embodiments, the intracellular region comprises a signal transduction domain and / or a costimulatory domain,

[0041] In one or more embodiments, the costimulatory domain is the intracellular domain of a costimulatory signal molecule or a functional fragment or mutant thereof that retains the biological function of the costimulatory signal molecule in transmitting costimulatory signals and activating immune cells.

[0042] In one or more embodiments, the intracellular domain of the costimulatory signaling molecule includes 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 the intracellular domain of CD27 or their mutants.

[0043] In one or more embodiments, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28 and / or the intracellular domain of OX40.

[0044] In one or more embodiments, the coreceptor further comprises a hinge region.

[0045] In one or more embodiments, the hinge region includes a proximal membrane fragment of the natural extracellular domain of the co-stimulatory signaling molecule, or the hinge region includes 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-1 Membrane-proximal fragments of the native extracellular domains of OR, 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.

[0046] In one or more embodiments, the hinge region is the extracellular hinge region of CD28 and / or the extracellular hinge region of IL7Rα.

[0047] In one or more embodiments, the coreceptor further comprises a membrane surface tag.

[0048] In one or more embodiments, the membrane surface tag comprises the extracellular domain of BCMA or a variant thereof, or the extracellular domain of claudin protein or a fragment or variant thereof.

[0049] In one or more embodiments, the membrane surface tag is located at the C-terminus of the extracellular domain of the KIR.

[0050] In one or more embodiments, the claudin protein is claudin 18, and the fragment of the extracellular domain of the claudin protein is an extracellular antigen epitope of the claudin protein.

[0051] In one or more embodiments, the transmembrane 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 CD27 transmembrane regions, or mutants thereof that retain transmembrane function.

[0052] In one or more embodiments, the transmembrane region is the CD28 transmembrane region, the IL7Rα transmembrane region, or a mutant thereof that retains transmembrane function.

[0053] In one or more embodiments, the coreceptor further comprises a signal peptide.

[0054] In one or more embodiments, the auxiliary receptor comprises an extracellular ligand binding domain, a membrane surface tag, a transmembrane region and a CD62L intracellular domain, and optionally further comprises 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, the extracellular ligand binding domain comprises a KIR2DL3 extracellular region or a KIR2DL4 extracellular region, and the transmembrane region is a CD28 transmembrane region or an IL7Rα transmembrane region or a mutant thereof.

[0055] In one or more embodiments, the membrane surface tag comprises a Claudin18.2 antigen epitope or a BCMA extracellular domain or a mutant thereof, and / or the auxiliary receptor further comprises a CD28 hinge region between the membrane surface tag and the transmembrane region.

[0056] In one or more embodiments, the auxiliary 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.

[0057] In one or more embodiments, the coreceptor comprises:

[0058] (1) KIR2DL3 extracellular region, Claudin18.2 antigen epitope, CD28 extracellular hinge region, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain,

[0059] (2) KIR2DL3 extracellular domain, BCMA extracellular domain, IL7Rα transmembrane region or its mutant, CD62L intracellular domain, IL-7Rα intracellular domain, or

[0060] (3) KIR2DL3 extracellular region, linker, BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain,

[0061] (4) KIR2DL3 extracellular region, BCMA extracellular domain or its variant, linker, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain

[0062] (5) KIR2DL4 extracellular region, Claudin18.2 antigen epitope, CD28 extracellular hinge region, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain,

[0063] (6) KIR2DL4 extracellular region, BCMA extracellular domain, IL7Rα extracellular hinge region, IL7Rα transmembrane region, CD62L intracellular domain, and IL-7Rα intracellular domain,

[0064] (7) KIR2DL4 extracellular region, Claudin18.2 antigen epitope, IL7Rα extracellular hinge region, IL7Rα transmembrane region mutant, CD62L intracellular domain, and IL-7Rα intracellular domain,

[0065] (8) KIR2DL4 extracellular region, linker, BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain,

[0066] (9) KIR2DL4 extracellular region, BCMA extracellular domain mutant, linker, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain.

[0067] In one or more embodiments, the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO:4.

[0068] In one or more embodiments, the amino acid sequence of the IL7Rα transmembrane region is shown in SEQ ID NO:6.

[0069] In one or more embodiments, the amino acid sequence of the IL7Rα transmembrane region mutant is shown in SEQ ID NOs: 8, 10, 12 and 14.

[0070] In one or more embodiments, the amino acid sequence of the CD28 intracellular domain is shown in SEQ ID NO:16.

[0071] In one or more embodiments, the amino acid sequence of the OX40 intracellular domain is shown in SEQ ID NO:18.

[0072] In one or more embodiments, the amino acid sequence of the IL-7Rα intracellular domain is shown in SEQ ID NO:20.

[0073] In one or more embodiments, the amino acid sequence of the KIR2DL3 extracellular region is shown in SEQ ID NO:22.

[0074] In one or more embodiments, the amino acid sequence of the KIR2DL4 extracellular region is shown in SEQ ID NO:24.

[0075] In one or more embodiments, the amino acid sequence of the KIR3DL1 extracellular region is shown in SEQ ID NO:26.

[0076] In one or more embodiments, the amino acid sequence of the KIR3DL2 extracellular region is shown in SEQ ID NO:28.

[0077] In one or more embodiments, the amino acid sequence of the Claudin 18.2 antigen epitope is shown in SEQ ID NO:30.

[0078] In one or more embodiments, the amino acid sequence of the BCMA extracellular domain is shown in SEQ ID NO: 32.

[0079] In one or more embodiments, the amino acid sequence of the BCMA extracellular domain mutant is shown in SEQ ID NO: 66.

[0080] In one or more embodiments, the amino acid sequence of the CD28 extracellular hinge region is shown in SEQ ID NO:34.

[0081] In one or more embodiments, the amino acid sequence of the extracellular hinge region of IL7Rα is shown in SEQ ID NO:36.

[0082] In one or more embodiments, the CD62L intracellular domain comprises the sequence shown in SEQ ID NO:76.

[0083] In one or more embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 60, 62 or 64.

[0084] In one or more embodiments, the amino acid sequence of the coreceptor is as shown in SEQ ID NO: 82 or 84.

[0085] The third aspect of the present invention provides an auxiliary receptor comprising: a KIR2DL3 extracellular region or a KIR2DL4 extracellular region, a BCMA extracellular domain or a mutant thereof, a linker, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain.

[0086] In one or more embodiments, the amino acid sequence of the KIR2DL3 extracellular region is shown in SEQ ID NO:22.

[0087] In one or more embodiments, the amino acid sequence of the KIR2DL4 extracellular region is shown in SEQ ID NO:24.

[0088] In one or more embodiments, the amino acid sequence of the BCMA extracellular domain is shown in SEQ ID NO: 32.

[0089] In one or more embodiments, the amino acid sequence of the BCMA extracellular domain mutant is shown in SEQ ID NO: 66.

[0090] In one or more embodiments, the linker amino acid sequence is shown in SEQ ID NO: 60, 62 or 64.

[0091] In one or more embodiments, the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO:4.

[0092] In one or more embodiments, the amino acid sequence of the CD28 intracellular domain is shown in SEQ ID NO:16.

[0093] In one or more embodiments, the amino acid sequence of the OX40 intracellular domain is shown in SEQ ID NO:18.

[0094] In one or more embodiments, the amino acid sequence of the coreceptor is as shown in any one of SEQ ID NOs: 78 and 80.

[0095] The fourth aspect of the present invention further provides a fusion protein comprising the auxiliary receptor described in any embodiment herein and a membrane surface cytokine.

[0096] In one or more embodiments, the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region, which are directly connected or connected through a linker; preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 60, 62 or 64, preferably SEQ ID NO: 62.

[0097] In one or more embodiments, the membrane surface cytokine is at the N-terminus and / or C-terminus of the coreceptor.

[0098] In one or more embodiments, the cytokine is IL-7.

[0099] 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.

[0100] In one or more embodiments, the GPI anchor is CD52.

[0101] In one or more embodiments, the membrane surface cytokine comprises: CD52 signal peptide, IL-7, linker, CD52.

[0102] In one or more embodiments, the membrane surface cytokine is linked to the co-receptor via a cleavable sequence; preferably, the cleavable sequence is such as P2A, T2A, F2A, preferably P2A.

[0103] In one or more embodiments, the membrane surface cytokine is connected to the auxiliary receptor via a linker; preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 60, 62 or 64, preferably SEQ ID NO: 64.

[0104] The present invention also provides a polynucleotide molecule comprising: a nucleic acid sequence encoding the auxiliary receptor or fusion protein according to any embodiment of the present invention or a complementary sequence thereof.

[0105] In one or more embodiments, the transmembrane region is the CD28 transmembrane region, the coding sequence of which is shown in SEQ ID NO:3.

[0106] In one or more embodiments, the transmembrane region is the IL7Rα transmembrane region, the coding sequence of which is shown in SEQ ID NO:5.

[0107] In one or more embodiments, the transmembrane region is IL7Rα transmembrane region mutants 1-4, whose coding sequences are shown in SEQ ID NOs: 7, 9, 11 and 13, respectively.

[0108] In one or more embodiments, the intracellular domain comprises a CD28 intracellular domain, the coding sequence of which is shown in SEQ ID NO:15.

[0109] 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.

[0110] In one or more embodiments, the intracellular domain comprises an IL-7Rα intracellular domain, the encoding sequence of which is shown in SEQ ID NO:19.

[0111] In one or more embodiments, the extracellular domain comprises a KIR2DL3 extracellular region, the coding sequence of which is shown in SEQ ID NO:21.

[0112] In one or more embodiments, the extracellular domain comprises the KIR2DL4 extracellular region, the coding sequence of which is shown in SEQ ID NO:23.

[0113] In one or more embodiments, the extracellular domain comprises the KIR3DL1 extracellular region, the coding sequence of which is shown in SEQ ID NO:25.

[0114] In one or more embodiments, the extracellular domain comprises the KIR3DL2 extracellular region, the coding sequence of which is shown in SEQ ID NO:27.

[0115] In one or more embodiments, the coreceptor comprises a membrane surface tag comprising a Claudin 18.2 antigen epitope encoding a sequence as shown in SEQ ID NO: 29 or a BCMA extracellular domain encoding a sequence as shown in SEQ ID NO: 31.

[0116] In one or more embodiments, the hinge region is the CD28 extracellular hinge region, the encoding sequence of which is shown in SEQ ID NO:33.

[0117] In one or more embodiments, the hinge region is the IL7Rα extracellular hinge region, the encoding sequence of which is shown in SEQ ID NO:35.

[0118] In one or more embodiments, the polynucleotide molecule comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97 and 99, or a complementary sequence of any one of the nucleic acid sequences.

[0119] The present invention also provides a nucleic acid construct comprising the polynucleotide molecule according to any embodiment of the present invention.

[0120] In one or more embodiments, the nucleic acid construct contains an expression frame for the auxiliary receptor described in any embodiment herein and an expression frame for the membrane surface cytokine described in any embodiment of the fourth aspect herein; or the nucleic acid construct is an expression frame, wherein the coding sequence of the auxiliary receptor described in any embodiment herein and the coding sequence of the membrane surface cytokine described in any embodiment of the fourth aspect herein are within the expression frame.

[0121] In one or more embodiments, the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region, which are directly linked or linked through a linker.

[0122] In one or more embodiments, the nucleic acid construct is a vector.

[0123] In one or more embodiments, the vector is an expression vector or a cloning vector.

[0124] 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.

[0125] The present invention also provides a nucleic acid construct comprising: an expression frame for an auxiliary receptor and an expression frame for a membrane surface cytokine; or the nucleic acid construct is an expression frame, wherein the coding sequence for the auxiliary receptor and the coding sequence for the membrane surface cytokine are within the expression frame, wherein,

[0126] The auxiliary receptor comprises an extracellular domain, a transmembrane region and an intracellular region, wherein the extracellular domain comprises the extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligand, and the intracellular region comprises a signal transduction domain and / or a costimulatory domain.

[0127] The membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region.

[0128] In one or more embodiments, the coreceptor is as described in any one of claims 1-7.

[0129] In one or more embodiments, the amino acid sequence of the coreceptor is as shown in SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 78, 80, 82 or 84.

[0130] In one or more embodiments, the membrane surface cytokine is as described in any embodiment of the fourth aspect herein.

[0131] In one or more embodiments, the nucleic acid construct is a vector.

[0132] In one or more embodiments, the vector is an expression vector or a cloning vector.

[0133] 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.

[0134] The present invention also provides a genetically engineered cell, which:

[0135] (1) expressing the coreceptor according to any embodiment of the second or third aspect of the present invention or the fusion protein according to the fourth aspect, and / or carrying the coding sequence of the coreceptor or fusion protein;

[0136] (2) expressing the coreceptors and membrane surface cytokines described in any of the embodiments herein;

[0137] (3) comprising a nucleic acid construct as described in any of the embodiments herein.

[0138] In one or more embodiments, the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region, which are directly connected or connected through a linker; preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 60, 62 or 64, preferably SEQ ID NO: 62.

[0139] In one or more embodiments, the membrane surface cytokine is at the N-terminus and / or C-terminus of the coreceptor.

[0140] In one or more embodiments, the cytokine is IL-7.

[0141] 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.

[0142] In one or more embodiments, the transmembrane domain is CD52 or its GPI anchor domain.

[0143] In one or more embodiments, the membrane surface cytokine comprises: CD52 signal peptide, IL-7, linker, CD52.

[0144] In one or more embodiments, the membrane surface cytokine is linked to the co-receptor via a cleavable sequence; preferably, the cleavable sequence is such as P2A, T2A, F2A, preferably P2A.

[0145] In one or more embodiments, the membrane surface cytokine is connected to the auxiliary receptor via a linker; preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 60, 62 or 64, preferably SEQ ID NO: 64.

[0146] In one or more embodiments, the cell is an immune effector cell.

[0147] In one or more embodiments, the immune effector cells include T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DN T and TIL.

[0148] In one or more embodiments, the cells further express CAR, or carry a coding sequence for CAR.

[0149] In one or more embodiments, the cells further express an exogenous TCR, or carry the coding sequence of an exogenous TCR.

[0150] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and any one or more of the coreceptor according to any embodiment of the second or third aspect of the present invention, the fusion protein, polynucleotide molecule, nucleic acid construct, and genetically engineered cell according to any embodiment of the present invention. The pharmaceutical composition is used to treat or prevent cancer.

[0151] In one or more embodiments, the cancer is melanoma.

[0152] The present invention also provides the use of the auxiliary receptor described in any embodiment of the second or third aspect of the present invention, the fusion protein, polynucleotide molecule, nucleic acid construct and genetically engineered cell described in any embodiment of the present invention in the preparation of a drug for treating or preventing cancer.

[0153] The present invention also provides the use of auxiliary receptors and membrane surface cytokines in the preparation of drugs for treating or preventing cancer, wherein the auxiliary receptor comprises an extracellular domain, a transmembrane region and an intracellular region, the extracellular domain comprises the extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligands, the intracellular region comprises a signal transduction domain and / or a co-stimulatory domain, and the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region.

[0154] Advantages of the present invention:

[0155] The auxiliary receptor of the present invention can transmit the signal of HLA and receptor extracellular region binding to intracellular while strengthening the connection of immune synapse core, activate the activation signal pathway of downstream co-stimulatory molecules, and promote the activation level of immune effector cells by dual action. The CD62L intracellular domain can further enhance the formation of immune synapse structure on the basis of the existing immune synapse core structure of auxiliary receptor or chimeric antigen receptor, and then further promote the activation, proliferation and target cell killing level of T cells. When auxiliary receptor of the present invention (auxiliary receptor) is expressed in immune cells together with membrane surface cytokines, the cytokine secretion level, proliferation level and the killing of target cells of immune cells can be further improved. DETAILED DESCRIPTION

[0156] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0157] The KIR receptors on the surface of immune effector cells are activated or inhibited by binding to type I HLA on the surface of antigen-presenting cells. The present invention fuses the extracellular region of KIR with the transmembrane region and intracellular domain of the classic co-stimulatory signal molecule to form a new receptor that can bind to different categories of type I HLA and is a new type of auxiliary receptor. The receptor exerts its immune activation effect in the following ways: 1) When the receptor is expressed in T cells, it strengthens the first signal and second signal connection already existing between T cells and antigen-presenting cells by binding to type I HLA, further strengthening the most core component of the T cell immune synapse from a physical structure, enhancing the interaction between T cells and antigen-presenting cells, and 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 receptor extracellular region in its extracellular domain binds to the corresponding type I HLA on the surface of antigen-presenting cells, thereby activating Its downstream intracellular domain of co-stimulatory molecules, thereby enhancing the activation and proliferation levels of immune effector cells, and enhancing the activation level of immune effector cells through dual effects; 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 immune synapse structure on the basis of the existing immune synapse core structure of auxiliary receptors or chimeric antigen receptors, thereby further enhancing T cell activation, proliferation and target cell killing levels; 4) Membrane surface cytokines and auxiliary receptors are co-expressed in immune cells, which can further enhance the cytokine secretion level, proliferation level and target cell killing of immune cells.

[0158] definition

[0159] The present invention uses the following terms: For terms not specifically defined herein, they have the meanings generally known in the art.

[0160] In the present invention, immune cells have a well-known meaning in the art and refer to cells involved in or associated with an immune response, including various lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Lymphocytes include, for example, T lymphocytes, tumor infiltrating lymphocytes (TIL), B lymphocytes, NK lymphocytes, and DN T cells. Immune cells suitable for the present invention particularly include those commonly used in adoptive cell therapy for tumors.

[0161] The term "expression cassette" refers to the complete elements required to express a gene, including the promoter, gene coding sequence, and Poly A tailing signal sequence.

[0162] The term "coding sequence" is defined herein as the portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., co-receptor, CAR). The boundaries of the coding sequence are typically determined by the ribosome binding site (for prokaryotes) immediately upstream of the mRNA 5' end open reading frame and the transcription termination sequence immediately downstream of the mRNA 3' end open reading frame. Coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0163] The term "co-stimulatory signal molecule" refers to a molecule that exists on the surface of antigen-presenting cells and can bind to the co-stimulatory signal molecule receptor on Th cells to produce a co-stimulatory signal. It can activate the second signal of immune cells, enhance the proliferation ability of immune cells and the secretion function of cytokines, and prolong the survival time of activated immune cells. The proliferation of lymphocytes requires not only the binding of antigens, but also the reception of co-stimulatory molecule signals. The co-stimulatory signal is transmitted to T cells mainly through the co-stimulatory molecules CD80 and CD86 expressed on the surface of antigen-presenting cells and binding to the CD28 molecules on the surface of T cells. B cells can receive co-stimulatory signals through general pathogen components such as LPS, or through complement components, or through CD40L on the surface of activated antigen-specific Th cells.

[0164] The term "linker" or "hinge" refers to a polypeptide segment that connects different proteins or polypeptides. Its purpose is to maintain the spatial conformation of the connected proteins or polypeptides to maintain their function or activity. Exemplary linkers include linkers containing G and / or S, rigid linkers or flexible linkers, and, for example, Furin 2A peptide. In one or more embodiments, the linker is as shown in SEQ ID NOs: 60, 62, or 64.

[0165] The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0166] The term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and / or relief of the diseases or conditions described in the present invention in a subject.

[0167] The term "disease and / or condition" refers to a physical condition of the subject, which is associated with the diseases and / or conditions described herein.

[0168] The term "subject" may refer to a patient or other animal that receives the pharmaceutical composition of the present invention to treat, prevent, alleviate and / or relieve the diseases or conditions described in the present invention, particularly mammals, such as humans, dogs, monkeys, cows, horses, etc.

[0169] The term "extracellular region" refers to the segment of a membrane protein located outside the cell.

[0170] The term "domain" refers to a region in a protein with a specific structure and independent function. The number of amino acid residues in a common domain ranges from 100 to 400. The smallest domain has only 40 to 50 amino acid residues, while the largest domain can exceed 400 amino acid residues.

[0171] Coreceptors

[0172] The co-receptors of the present invention comprise the extracellular region (extracellular ligand binding domain), the transmembrane region (transmembrane domain), and the intracellular region (or cytoplasmic domain) of the KIR extracellular domain. The KIR extracellular domain can be itself or a fragment, as long as the fragment retains the biological function of binding to the KIR ligand.

[0173] In the co-receptors herein, the KIR extracellular domain is used to interact with KIR ligands (mainly various types of class I HLA molecules), thereby downregulating the level of the "don't eat me" signaling pathway, activating the signaling pathway of immune effector cells to dominate, and then causing the immune effector cells to enter an activated and proliferative state, attacking target cells and thus eliminating abnormal cells. The extracellular domain of an activating KIR or an inhibitory KIR or a fragment thereof that retains the KIR ligand binding ability can be used. An exemplary activating KIR is KIR2DL4; an exemplary inhibitory KIR is selected from KIR2DL3, KIR3DL1 and KIR3DL2. Preferably, the amino acid sequence of the KIR2DL3 extracellular region is shown in SEQ ID NO: 22, and its coding sequence is shown in SEQ ID NO: 21; the amino acid sequence of the KIR2DL4 extracellular region is shown in SEQ ID NO: 24, and its coding sequence is shown in SEQ ID NO: 23; the amino acid sequence of the KIR3DL1 extracellular region is shown in SEQ ID NO: 26, and its coding sequence is shown in SEQ ID NO: 25; the amino acid sequence of the KIR3DL2 extracellular region is shown in SEQ ID NO: 28, and its coding sequence is shown in SEQ ID NO: 27.

[0174] In the coreceptors herein, the intracellular region may comprise the CD62L intracellular domain. The CD62L intracellular domain may be itself or a fragment thereof, as long as the fragment retains the biological function of the CD62L intracellular domain. Exemplary amino acid sequences and corresponding coding sequences of the CD62L intracellular domain may be shown in SEQ ID NOs: 76 and 75, respectively.

[0175] In the coreceptors herein, the intracellular region may comprise a signal transduction domain and / or a co-stimulatory domain.

[0176] In the present invention, co-stimulatory signal molecules include CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM and CD27. The auxiliary receptor of the present invention can be constructed using one or more intracellular domains (intracellular regions) of these costimulatory signal molecules or their functional fragments or mutants that retain the biological functions of the costimulatory signal molecules to transmit costimulatory signals and activate immune cells. The amino acid sequence of the exemplary intracellular region of CD28 and the corresponding coding sequence can be shown as SEQ ID NO: 16 and 15, respectively. The amino acid sequence of the exemplary intracellular region of OX40 and the corresponding coding sequence can be shown as SEQ ID NO: 18 and 17, respectively. An exemplary IL-7R can be IL-7Rα, and the exemplary amino acid sequence of its intracellular region and the corresponding coding sequence can be shown as SEQ ID NO: 20 and 19, respectively. The intracellular domain of the costimulatory signal molecule can also be the intracellular domain of the costimulatory signal molecule described in WO2021244486, which is incorporated herein by reference in its entirety.

[0177] In the present invention, the signal transduction domain can be selected by those skilled in the art as needed, such as the CD3ζ intracellular signal transduction region.

[0178] Herein, the transmembrane region includes but is not limited to any one or more of the transmembrane regions selected from CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM and CD27, or mutants thereof that retain transmembrane function. Exemplarily, the amino acid sequence and nucleotide sequence of the CD28 transmembrane region are shown in SEQ ID NOs: 4 and 3, respectively; the amino acid sequence and nucleotide sequence of the IL7Rα transmembrane region are shown in SEQ ID NOs: 6 and 5, respectively; the amino acid sequence and nucleotide sequence of IL7Rα transmembrane region mutant 1 are shown in SEQ ID NOs: 8 and 7, respectively; the amino acid sequence and nucleotide sequence of IL7Rα transmembrane region mutant 2 are shown in SEQ ID NOs: 10 and 9, respectively; the amino acid sequence and nucleotide sequence of IL7Rα transmembrane region mutant 3 are shown in SEQ ID NOs: 12 and 11, respectively; and the amino acid sequence and nucleotide sequence of IL7Rα transmembrane region mutant 4 are shown in SEQ ID NOs: 14 and 13, respectively.

[0179] In the present invention, the extracellular region containing the KIR extracellular domain may be connected to the transmembrane region via a hinge region. The hinge region includes, but is not limited to, a membrane-proximal fragment of the native extracellular domain selected from CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, and CD27. Preferably, the hinge region is the extracellular hinge region of CD28 and / or the extracellular hinge region of IL7Rα.

[0180] It should be understood that the "functional fragment" described herein refers to a fragment that retains the desired biological function. For example, the functional fragment of the intracellular domain described herein refers to a fragment that retains the biological function of the costimulatory signal molecule to transmit costimulatory signals and activate immune cells. Functional fragments of each extracellular domain and each intracellular domain suitable for use in the present invention can be easily determined by those skilled in the art in combination with existing technical means in the art.

[0181] The coreceptors of the present invention may also have an extracellular membrane surface tag. Thus, in some embodiments, the coreceptors described herein further comprise 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, or an element that induces ADCC, ADCP, and / or CDC effects. The membrane surface tag includes a membrane surface functional domain.

[0182] The membrane surface functional domain can be the extracellular domain of the claudin protein or a fragment thereof. The claudin protein is preferably claudin18, such as claudin18.2. The fragment of the extracellular domain mainly refers to the extracellular antigenic epitope of the corresponding protein. Therefore, the fragment of the extracellular domain of the claudin protein is the extracellular antigenic epitope of the claudin protein. Preferably, the fragment of the extracellular domain of the claudin18.2 protein (i.e., the extracellular antigenic epitope of the claudin18.2 protein) comprises the sequence shown in SEQ ID NO: 30; its nucleic acid sequence comprises the sequence shown in SEQ ID NO: 29.

[0183] The membrane surface functional domain may also be the BCMA extracellular domain or a fragment thereof. Preferably, the BCMA extracellular domain comprises the sequence shown in SEQ ID NO: 32; and its nucleic acid sequence comprises the sequence shown in SEQ ID NO: 31.

[0184] The membrane surface tag may also have a connecting segment at the N-terminus or C-terminus of the membrane surface functional domain (BCMA extracellular domain or claudin protein extracellular domain) for connection to other polypeptides or polypeptide portions. The connecting segment is typically a hinge or linker. The hinge includes one or more selected from the following: the extracellular hinge region of CD8, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the CD28 extracellular hinge region, the IgG4 Fc CH2CH3 hinge region, and the CD4 extracellular hinge region.

[0185] The "mutants" described herein include mutants of each domain, as long as the mutant retains the corresponding biological functions of the KIR extracellular domain, membrane surface tag, transmembrane region, and intracellular domain. For example, mutants of the KIR extracellular domain suitable for use in the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the KIR extracellular domain as a comparison; mutants of the membrane surface tag suitable for use in the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the membrane surface tag as a comparison; mutants of the transmembrane region suitable for use in the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the transmembrane region as a comparison; mutants of the intracellular domain suitable for use in the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the intracellular domain as a comparison. Alternatively, compared to the sequence used for comparison, the mutants of the present invention have one or more (e.g., within 20, within 15, within 10, within 8, within 5, or within 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted, or deleted. For example, in the art, conservative substitutions with amino acids having similar or similar properties generally do not alter the function of a protein or polypeptide. "Amino acids with similar or similar properties" include, for example, families of amino acid residues having similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0186] The present invention also includes mutants of the auxiliary receptors described above, such as mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the auxiliary receptor. More specifically, the present invention includes mutants having one or more (such as within 20, within 15, within 10, within 8, within 5 or within 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted or deleted compared to the auxiliary receptors described above. Such mutants retain the biological functions of the auxiliary receptors described in the present invention, including but not limited to the function of recognizing KIR ligands and activating immune effector cells to enter an activated and proliferative state. The mutation may occur in any one, any two or all three of the extracellular domain, transmembrane region and intracellular domain described herein.

[0187] The polypeptides described herein may be modified polypeptides. Modifications (generally without altering the primary structure) include chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation during polypeptide synthesis and processing or during further processing steps. Such modifications can be accomplished by exposing the polypeptide to a glycosylation enzyme (e.g., a mammalian glycosylase or deglycosylase). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or optimize their solubility.

[0188] Exemplary coreceptors of the present invention include, but are not limited to, coreceptors comprising, from N-terminus to C-terminus, the extracellular domain, hinge region, transmembrane region, and intracellular region shown in each row of Table 1 below, or consisting of the extracellular domain, hinge region, transmembrane region, and intracellular region shown in each row of Table 1 below:

[0189] Table 1. Coreceptors (N-terminus to C-terminus)

[0190] In some embodiments, the co-receptor described herein further comprises a signal peptide. Preferably, the signal peptide is located at the N-terminus of the co-receptor. The signal peptide can be any signal peptide conventional in the art that can guide the polypeptide to exit the nucleus, including but not limited to CD8, CD4, CD28, CD137, EGFR, TGFBRI, TGFBRII, TGFBRIII, and antibody light chain signal peptides. In some embodiments, the signal peptide is a CD8 signal peptide, and the CD8 signal peptide comprises the amino acid sequence of SEQ ID NO: 2, and its coding sequence is shown in SEQ ID NO: 1.

[0191] It should be understood that, if desired, the extracellular domain and the transmembrane region, and / or the transmembrane region and the intracellular domain described herein can be connected by a linker sequence. Linker sequences well known in the art can be used, such as linker sequences containing G and S, such as (GSSS)n or (GSSSS)n, where n is an integer from 1 to 8. The linker can also be a rigid linker or a flexible linker. For example, the linker is a rigid linker as shown in SEQ ID NO: 60, 62, or 64.

[0192] Preferably, the amino acid sequence of the auxiliary receptor of the present invention is shown in any one of SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 62 and 64.

[0193] Fusion protein

[0194] The present invention also provides a fusion protein comprising a co-receptor and a membrane surface cytokine. The membrane surface cytokine is at the N-terminus and / or C-terminus of the co-receptor. The membrane surface cytokine and the co-receptor are linked via a cleavable sequence; preferably, the cleavable sequence is, for example, P2A, T2A, or F2A, preferably P2A. Alternatively, the membrane surface cytokine and the co-receptor may be linked via a linker; preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 60, 62, or 64, preferably SEQ ID NO: 64. Alternatively, the membrane surface cytokine is directly linked to the co-receptor.

[0195] The membrane-surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region, the two being directly connected or connected via a linker. Linker sequences known in the art can be used, such as linker sequences containing G and S, such as (GSSS)n or (GSSSS)n, where n is an integer from 1 to 8. The linker can also be a rigid linker or a flexible linker. For example, the linker is a rigid linker as shown in SEQ ID NO: 60, 62, or 64.

[0196] Among the membrane surface cytokines, the cytokines may be any polypeptide cytokines, including but not limited to interleukins, tumor necrosis factor (TNF), interferon (IFN), colony stimulating factor (CSF) and tumor growth factor (TGF). The interleukins include but are not limited to IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18 and IL-21. The tumor necrosis factors include but are not limited to TNF-α and TNF-β. The interferons include but are not limited to IFN-α, IFN-β and IFN-γ. The colony stimulating factors include but are not limited to M-CSF, G-CSF and GM-CSF. The tumor growth factors include but are not limited to TGF-α, TGF-β1, TGF-β2 and TGF-β3.

[0197] The transmembrane domains in the membrane surface cytokines are as described elsewhere herein, including but not limited to CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21 R, 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 any one or more of the transmembrane regions of CD27 or mutants thereof that retain their transmembrane function.

[0198] The GPI anchor region in the membrane surface cytokine includes one or more selected from the following or its GPI anchor domain (also referred to herein as GPI signal sequence): CD44, CD56, CD73, CD55, Thy1, AchE, IAP, ALPP, CD59, CD14, CD16, CD24, CD28, CD48, CD52, CD58, CD66a, CD66c, CD66d, CD66e, CD67, CD87, CD108, CD157, uPAR, JMH protein, GDNFR, CNTFR, TAG-1, PrP, phosphatidylinositol protein, semaphorin 7, CEA, GFR, Ly6G, transferrin receptor, contactin (F3) and T-cadherin; preferably, the GPI anchor region is CD52 protein, CD48 protein, CD55 protein, ALPP protein, CD90 protein or their GPI anchor domain. GPI anchor proteins or their anchoring domain sequences are known in the art. Moreover, those skilled in the art can easily obtain the anchoring domain sequence thereof based on the sequence of the GPI anchor protein.

[0199] The membrane surface cytokine may further include a signal peptide. Preferably, the signal peptide is located at the N-terminus of the cytokine. The signal peptide can be any signal peptide conventional in the art that can guide the polypeptide to exit the nucleus, including but not limited to CD8, CD4, CD28, CD52, CD137, EGFR, TGFBRI, TGFBRII, TGFBRIII and antibody light chain signal peptides. In some embodiments, the signal peptide is a CD8 signal peptide or a CD52 signal peptide.

[0200] In one or more embodiments, the fusion protein comprises or comprises, in order from N-terminus to C-terminus:

[0201] (1) CD52 signal peptide, IL-7, linker 2, CD52, linker 3, CD8 signal peptide, KIR2DL3 extracellular domain, point mutation BCMA extracellular domain, linker 1, CD28 transmembrane region, CD28 intracellular domain and OX40 intracellular domain,

[0202] (2) CD8 signal peptide, KIR2DL3 extracellular region, point mutation BCMA extracellular domain, linker 1, CD28 transmembrane region, CD28 intracellular domain, OX40 intracellular domain, linker 3, CD52 signal peptide, IL, 7, linker 2, CD52,

[0203] (3) CD52 signal peptide, IL, 7, linker 2, CD52, linker 3, CD8 signal peptide, KIR2DL3 extracellular domain, point mutation BCMA extracellular domain, linker 1, CD28 transmembrane region, CD62L intracellular signal region, CD28 intracellular signal region, OX40 intracellular signal region,

[0204] (4) CD8 signal peptide, KIR2DL3 extracellular region, point mutation BCMA extracellular domain, linker 1, CD28 transmembrane region, CD62L intracellular signal region, CD28 intracellular domain, OX40 intracellular domain, P2A, CD52 signal peptide, IL, 7, linker 2, CD52,

[0205] (5) CD52 signal peptide, IL, 7, linker 2, CD52, linker 3, CD8 signal peptide, KIR2DL4 extracellular domain, point mutation BCMA extracellular domain, linker 1, CD28 transmembrane region, CD28 intracellular domain, OX40 intracellular domain,

[0206] (6) CD8 signal peptide, KIR2DL4 extracellular region, point mutation BCMA extracellular domain, linker 1, CD28 transmembrane region, CD28 intracellular domain, OX40 intracellular domain, linker 3, CD52 signal peptide, IL, 7, linker 2, CD52,

[0207] (7) CD52 signal peptide, IL, 7, linker 2, CD52, linker 3, CD8 signal peptide, KIR2DL4 extracellular domain, point mutation BCMA extracellular domain, linker 1, CD28 transmembrane region, CD62L intracellular signal region, CD28 intracellular signal region, OX40 intracellular signal region,

[0208] (8) CD8 signal peptide, KIR2DL4 extracellular region, point mutation BCMA extracellular domain, linker 1, CD28 transmembrane region, CD62L intracellular signal region, CD28 intracellular domain, OX40 intracellular domain, linker 3, CD52 signal peptide, IL, 7, linker 2, CD52.

[0209] CAR-T

[0210] The immune cells of the present invention may further express CAR, or contain a coding sequence for CAR. The CAR of the present invention may be any of various CARs known in the art.

[0211] CAR may comprise a polypeptide (such as scFv) that binds to a tumor cell membrane antigen, a hinge region, a transmembrane region, and an intracellular signaling region in sequence. The hinge region, transmembrane region, and intracellular signaling region known in the art for constructing CAR can be used to construct the CAR of the present invention. Typically, a polypeptide that binds to a tumor cell membrane antigen is able to bind to a membrane antigen widely expressed by tumor cells with moderate affinity. The polypeptide is usually inserted with an antigen epitope, and the insertion position is selected from any one, two, or three of the following three positions: the N-terminus of the polypeptide, between the polypeptide and the hinge region, and inside the polypeptide. The polypeptide that binds to a tumor cell membrane antigen is a natural polypeptide or an artificially synthesized polypeptide; preferably, the artificially synthesized polypeptide is a single-chain antibody or a Fab fragment.

[0212] The chimeric antigen receptor of the present invention can be directed against one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT1), FR (Flavin reductase), PSMA (prostate-specific membrane antigen), PMEL premelanosome protein), CA9 (carbonic anhydrase IX), CD171 / L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family protein, BAGE (B melanoma antigen family) family protein, GAGE ​​(growth hormone releasing factor) family protein, AFP, MUC1 (also known as mucin1), CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, V EGFR1, VEGFR2, IL-11R / , EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR), and PROGRP (GRP gastrin-releasing peptide).

[0213] A single cell can express multiple CARs, including CARs targeting different tumor antigens.

[0214] T cell receptor (TCR)-T

[0215] The immune cells of the present invention may further express exogenous TCRs or contain coding sequences for expressing exogenous TCR genes. The TCRs of the present invention may be any TCR known in the art, such as TCRs with HLA typing matching, known sequence and structure, and with known antigenic peptide sequences.

[0216] The exogenous TCR described in the present invention includes an αβ double chain, which can form a complete TCR complex with the double-chain structure of γε, δε and ξξ endogenously expressed by immune effector cells such as T cells. The exogenous gene encoding the exogenous TCR described in the present invention includes a gene for the αβ double chain, and the α chain and β chain coding sequences are covalently linked by a linker sequence that can be cut in vivo, such as a DNA sequence encoding a P2A, T2A or F2A sequence, or can be covalently linked by a DNA fragment encoding an IRES sequence. In addition to the αβ double chain encoding the exogenous TCR, the gene encoding the exogenous TCR described in the present invention can also include a tag protein gene expressed in fusion with the αβ gene, such as EGFP, RFP, YFP gene, etc. The tag protein gene can be covalently linked to the gene for the αβ double chain through a linker sequence that can be cut in vivo, such as a 2A sequence, a DNA sequence encoding a P2A, T2A or F2A, or a DNA sequence encoding an IRES sequence. The tag protein, such as EGFP, RFP, YFP gene, etc., is co-expressed with the TCRαβ double chain and can be used as an identification indicator for detecting the expression of exogenous TCR.

[0217] The TCR-T of the present invention may target one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT1), FR (Flavin reductase), PSMA (prostate-specific membrane antigen), PMEL premelanosome protein), CA9 (carbonic anhydrase IX), CD171 / L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family protein, BAGE (B melanoma antigen family) family protein, GAGE ​​(growth hormone releasing factor) family protein, AFP, MUC1 (mucin 1), CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, IL-11R / , EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16, mucin 16), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR), microglobulin) and PROGRP (GRP gastrin-releasing peptide).

[0218] A single cell can express multiple exogenous TCRs, including those targeting different tumor antigens.

[0219] polynucleotide molecules

[0220] The present invention provides polynucleotide molecules encoding the coreceptors described herein. The present invention also provides a complementary sequence to the coding sequence of the coreceptors. The polynucleotide molecules can be recombinant or synthetic; they can comprise DNA, RNA, and PNA (peptide nucleic acid), and can be hybrids thereof. Exemplarily, the polynucleotide molecules of the present invention have the sequence set forth in any one of SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61, and 63.

[0221] Also provided is an expression cassette for the coreceptor or fusion protein of the present invention, which is a nucleic acid construct comprising a promoter, a coreceptor coding sequence, and a poly A tailing signal sequence. The nucleic acid construct may also contain other elements required for expression, including but not limited to enhancers.

[0222] Also provided is a nucleic acid construct containing an expression cassette for the auxiliary receptor described herein and an expression cassette for the membrane surface cytokine described herein; or the nucleic acid construct is an expression cassette, wherein the coding sequence for the auxiliary receptor described herein and the coding sequence for the membrane surface cytokine described herein are within the expression frame.

[0223] Also provided is a vector containing the polynucleotide molecules, expression cassettes, or nucleic acid constructs described herein. The vector can be a plasmid, cosmid, virus, or phage. The vector can be a viral vector or a non-viral vector. The vector can be a cloning vector, an integrating vector, or an expression vector. The expression vector can be a transposon vector. In certain embodiments, the expression vector is one or more of the following transposon vectors: piggybac, sleeping beauty, frog prince, Tn5, and Ty. In addition to the polynucleotide molecules described herein, the expression vector typically contains other elements typically found in vectors, such as a multiple cloning site, resistance genes, and an origin of replication. In certain embodiments, the recombinant expression vector utilizes a pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector, pUC57, pMAX, or pDC315 series vector as its backbone. In other embodiments, the recombinant expression vector uses a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector, a pUC57 vector, a pMAX vector, or a pDC315 series vector as a backbone. In certain embodiments, the present invention uses the pNB vector constructed in CN105154473A. In certain embodiments, the present invention uses the pKB20 vector described in WO2022078310A1.

[0224] The CAR of the present invention can also be expressed in the immune cells of the present invention through conventional vectors. The vector can be a conventional CAR expression vector, including but not limited to the various transposon vectors and recombinant expression vectors described above.

[0225] In some embodiments, the same vector encodes both the coreceptor and the CAR of the present invention. The vector can be bicistronic. The CAR coding sequence can be placed 5' or 3' to the coreceptor coding sequence. Expression of the CAR and coreceptor can be under the direction of the same or different regulatory sequences.

[0226] In the case where the polynucleotide sequence is known, each polynucleotide molecule can be prepared using a method conventional in the art, and a corresponding vector can be constructed. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory), Ausubel et al. (1989, Short Protocols in Molecular Biology, Wiley) or the technology described in other standard textbooks. Alternatively, the nucleic acid molecule and vector can be reconstructed into a liposome for delivery to a target cell. The vector containing the nucleic acid molecule of the present invention can be transferred to a host cell by a well-known method, which varies according to the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al. (see above).

[0227] host cells

[0228] As used herein, "host cell" refers to a eukaryotic cell capable of replicating a vector and / or expressing a heterologous gene encoded by a vector when expressing a heterologous nucleic acid sequence. A host cell can serve as a recipient for a vector. A host cell can be "transfected" or "transformed," which refers to a process by which an exogenous nucleic acid is transfected or transduced into a host cell. Transformed cells include the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells often refer to cells into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Thus, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acid.

[0229] The host cells of the present invention (1) express the auxiliary receptors or fusion proteins described herein, and / or carry the coding sequences of the auxiliary receptors or fusion proteins; (2) express the auxiliary receptors and membrane surface cytokines described herein; and (3) contain the nucleic acid constructs described herein. Therefore, the host cells of the present invention can express the auxiliary receptors described herein alone, such as the auxiliary receptors described in Sections 2 or 3 of the "Summary of the Invention". The host cells of the present invention can also express the auxiliary receptors described herein and the membrane surface cytokines described herein simultaneously. The host cells of the present invention can also express the fusion proteins containing the auxiliary receptors and membrane surface cytokines described herein.

[0230] The cells of the present invention are preferably immune cells and can be used for adoptive cell therapy of tumors. Such cells of the present invention are also referred to as cells modified with auxiliary receptors of the present invention. More specifically, the cells of the present invention 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 that can induce effector functions.

[0231] As used herein, cells can be autologous, syngeneic, allogeneic, and in some cases even xenogeneic, relative to the individual receiving them.

[0232] The nucleic acid construct / recombinant expression vector of the present invention can be transferred into the cells of interest. Transfer methods are conventional in the art, including but not limited to viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation. In certain embodiments, electroporation is used to transfer the nucleic acid construct or recombinant expression vector. When multiple expression cassettes are located in different nucleic acid constructs / recombinant expression vectors, these nucleic acid constructs / recombinant expression vectors can be transferred into the cells simultaneously or sequentially.

[0233] In addition to carrying the auxiliary receptors described herein and / or their coding sequences, the cells of the present invention may also have one or more other properties useful for cellular immunotherapy (e.g., adoptive cell therapy for tumors). Such other properties may be intrinsic to the cells or may be part of the cells after genetic manipulation by humans. For example, the cells of the present invention may carry chimeric antigen receptors, αβ T cell receptors, and / or antigen-specific receptors, such as tumor-specific receptors, or their coding sequences.

[0234] Pharmaceutical composition

[0235] As used herein, a "pharmaceutical composition" refers to a composition for administration to an individual and encompasses compositions of cells for use in immunotherapy. The pharmaceutical compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject at an appropriate dose.

[0236] The dosage regimen can be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on a variety of factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and other drugs administered concurrently.

[0237] The compositions of the present invention can be administered topically or systemically. In certain embodiments, the compositions provided by the present invention (e.g., cells expressing the coreceptors described herein) can be administered parenterally, such as intravenously, intra-arterially, intrathecally, subdermally, or intramuscularly. In certain other embodiments, the DNA encoding the constructs provided by the present invention can be administered directly to the target site, such as by gene gun delivery to an internal or external target site or by catheter delivery to an intra-arterial site. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in a more preferred embodiment, intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous 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 solution, or fixed oils. Intravenous carriers include fluid and nutrient supplements, electrolyte supplements (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases. In addition, the pharmaceutical compositions of the present invention may include proteinaceous carriers, such as serum albumin or immunoglobulins, preferably of human origin. In addition to the proteinaceous chimeric cytokine receptor constructs or nucleic acid molecules or vectors encoding the same, it is contemplated that the pharmaceutical compositions of the present invention may also include biologically active agents, depending on the intended use of the pharmaceutical composition.

[0238] Compositions for parenteral (e.g., intravenous) administration of the cells described herein can also be stored in a lyophilized form or in a solution (e.g., a cryopreserved formulation). The cryopreserved formulation can be stored in a ready-to-use form or in a form that is further formulated prior to administration. The cryopreserved formulation can withstand long-distance transportation without damaging the cells. In addition to the cells themselves, the cryopreserved formulation typically includes components such as a cell freezing solution and human serum albumin (HSA). Prior to administration (e.g., intravenous infusion), the frozen pharmaceutical composition must be stored at low temperatures (e.g., in liquid nitrogen). After thawing, the cryopreserved formulation can be infused directly into the patient or formulated as an infusion composition. Those skilled in the art are aware of the components and concentrations of conventional freezing solutions. For example, the freezing solution or infusion composition may also include dimethyl sulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride, or magnesium chloride, and the concentrations thereof can be determined by those skilled in the art (e.g., an experienced physician) based on the conditions of the cells, disease, and patient.

[0239] Methods and Applications

[0240] The auxiliary receptors, fusion proteins, polynucleotide molecules, vectors, host cells and pharmaceutical compositions containing these substances described in the present invention can be used to prevent, treat or alleviate cancer, especially cancers in which corresponding tumor antigens are expressed on the surface of cancer cells, or to prepare drugs for preventing, treating or alleviating cancer.

[0241] The present invention also provides the use of the auxiliary receptor described in any embodiment herein and the membrane surface cytokine described in any embodiment herein in the preparation of a drug for treating or preventing cancer, wherein the auxiliary receptor comprises an extracellular domain, a transmembrane region and an intracellular region, the extracellular domain comprises the extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligand, the intracellular region comprises a signal transduction domain and / or a co-stimulatory domain, and the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region.

[0242] As used herein, "treat" or "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of the disease or condition (e.g., cancer) being treated. Treatment may optionally include a reduction or alleviation of symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily mean complete eradication or cure of the disease or condition or its associated symptoms.

[0243] As used herein, "prevention" refers to methods for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" also includes reducing the intensity, impact, symptoms, and / or burden of a disease or condition before it occurs or recurs.

[0244] The present invention includes administering cells, polynucleotide molecules, and vectors, alone or in any combination, using standard vectors and / or gene delivery systems, optionally with a pharmaceutically acceptable carrier or excipient. In certain embodiments, following administration, the polynucleotide molecule or vector can stably integrate into the subject's genome.

[0245] In specific embodiments, viral vectors can be used that are specific for certain cells or tissues and persist in said cells. Suitable pharmaceutical carriers and excipients are well known in the art. Compositions prepared according to the present invention can be used to prevent, treat, or delay the above-identified diseases.

[0246] In addition, the present invention provides a method for preventing, treating or alleviating cancer, comprising the following steps: administering to a subject in need thereof an effective amount of cells, wherein the cells carry the auxiliary receptor, polynucleotide molecule and / or vector described in the present invention and / or produced by the method described in the present invention.

[0247] The methods herein can be used to prevent, treat or alleviate various cancers, including various solid tumors and hematological tumors, including but not limited to lung cancer (such as non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma and brain glioma, etc. More specifically, the cancers herein include but are not limited to breast, prostate, lung and colon cancer or epithelial cancers, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, melanoma; genital-urinary tract cancers, such as ovarian cancer, endometrial cancer, cervical cancer; kidney cancer, lung cancer, stomach cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, etc. Administration of the compositions of the present invention can be used for all stages and types of cancer, including, for example, minimal residual disease, early cancer, advanced cancer and / or metastatic cancer and / or cancer that is difficult to treat.

[0248] By way of example, a cancer patient or a patient susceptible to cancer or a patient suspected of having cancer is treated as follows. Cells modified as described herein can be given to an individual and stay for an extended period of time. The individual can receive one or more administrations of cells, and the intervals between administrations can be several days, weeks, months or years. In a specific embodiment, multiple administrations can occur over several weeks or months, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks or months. In some embodiments, the genetically modified cells are encapsulated to suppress immune recognition and are located at the tumor site. In the case of providing cells to an individual after tumor recurrence after initial treatment using the cells of the present invention, these cells can be altered to recognize different target tumor antigens. For example, when the initial round includes cells carrying a co-receptor of the present invention and another receptor specific for a specific antigen, receptors for different specific antigens can be used after subsequent rounds (including after tumor recurrence).

[0249] In some embodiments, an effective amount of therapeutic cells is provided to an individual in need, wherein the therapeutic cells carry or express the auxiliary receptors and optional CAR or exogenous transgenic TCR described in any embodiment of the present invention. These cells can be delivered simultaneously or simultaneously with one or more other cancer treatments. These cells and other cancer therapeutics can be delivered in the same or separate formulations. Cells and other cancer therapeutics can be provided to an individual by a separate delivery route. Cells and / or other cancer therapeutics can be delivered, for example, by injection at the tumor site or intravenously or orally. Conventional delivery routes for such compositions are known in the art.

[0250] The number of cells employed will depend on various circumstances, such as the purpose of introduction, the lifespan of the cells, the regimen to be used, the number of administrations, the ability of the cells to reproduce, the stability of the recombinant construct, and the like.

[0251] Cells can be administered as needed. In certain embodiments, a variety of regimens can be used to adjust regimen parameters. In specific embodiments, the route or number or timing of administration, the lifespan of the cells, and / or the number of cells present can vary. The number of administrations can, for example, depend at least in part on the above factors.

[0252] Reagent test kit

[0253] Any of the compositions described herein can be included in a kit. In one non-limiting example, a kit can include cells expressing a coreceptor according to any embodiment of the present invention for use in a cell therapy and / or reagents for generating one or more cells for use in a cell therapy comprising a recombinant expression vector. The kit components can be provided in a suitable container.

[0254] Some components of these test kits can be packaged in aqueous media or packaged into lyophilized forms. The container apparatus of these test kits generally includes at least one vial, test tube, flask, bottle, syringe or other container apparatus, wherein the component can be placed, and preferably suitably subpackaged therein. In the case where there is more than one component in the test kit, the test kit generally also contains a second, third or other container, wherein the other components can be placed separately. However, various combinations of components can be included in the vial. The test kit of the present invention generally also includes an apparatus for containing the component in a commercially available closed constraint form. Such containers may include injection-molded or blow-molded plastic containers, wherein the required vial is retained.

[0255] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, particularly preferably a sterile aqueous solution. In some cases, the container means itself can be a syringe, pipette and / or other such device.

[0256] The components of the kit may also be provided in dry powder form. When the reagents and / or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent. Thus, the kit may also include a second container containing a sterile, pharmaceutically acceptable buffer and / or other diluent.

[0257] The components of the kit can also be provided in the form of a cryopreservation preparation (e.g., a cryopreservation solution). After thawing, the cryopreservation preparation can be infused into the patient directly or formulated into an infusion composition. Therefore, the kit can also include cell freezing bags, cell freezing tubes, a temperature maintenance device (e.g., a container containing liquid nitrogen), a thawing device, etc.

[0258] In a specific embodiment of the present invention, 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 may contain reagents and materials for preparing the desired cells. In a specific embodiment, the reagents and materials include primers, nucleotides, suitable buffers or buffering agents, salts, etc. for amplifying the desired sequence, and in some cases, the reagents include DNA and / or vectors encoding the coreceptors and / or their regulatory elements as described in any embodiment of the present invention.

[0259] The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will appreciate that the following examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the examples, if specific techniques or conditions are not indicated, the techniques or conditions described in the literature in this area (e.g., with reference to "Molecular Cloning Experiment Guide" by J. Sambrook et al., translated by Huang Peitang et al., 3rd edition, Science Press), corresponding references, or product specifications are used. Reagents or instruments used that do not indicate manufacturers are all conventional products that can be obtained commercially.

[0260] Example

[0261] The sequence of the elements in the embodiment is summarized as follows:

[0262] CD8 signal peptide: nucleotide sequence SEQ ID NO: 1, amino acid sequence SEQ ID NO: 2;

[0263] CD28 transmembrane region: nucleotide sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4;

[0264] IL7Rα transmembrane region: nucleotide sequence SEQ ID NO: 5, amino acid sequence SEQ ID NO: 6;

[0265] IL7Rα transmembrane region mutant 1: nucleotide sequence SEQ ID NO: 7, amino acid sequence SEQ ID NO: 8;

[0266] IL7Rα transmembrane region mutant 2: nucleotide sequence SEQ ID NO: 9, amino acid sequence SEQ ID NO: 10;

[0267] IL7Rα transmembrane region mutant 3: nucleotide sequence SEQ ID NO: 11, amino acid sequence SEQ ID NO: 12;

[0268] IL7Rα transmembrane region mutant 4: nucleotide sequence SEQ ID NO: 13, amino acid sequence SEQ ID NO: 14;

[0269] CD28 intracellular domain: nucleotide sequence SEQ ID NO: 15, amino acid sequence SEQ ID NO: 16;

[0270] OX40 intracellular domain: nucleotide sequence SEQ ID NO: 17, amino acid sequence SEQ ID NO: 18;

[0271] IL-7Rα intracellular domain: nucleotide sequence SEQ ID NO: 19, amino acid sequence SEQ ID NO: 20;

[0272] KIR2DL3 extracellular region: nucleotide sequence SEQ ID NO: 21, amino acid sequence SEQ ID NO: 22;

[0273] KIR2DL4 extracellular region: nucleotide sequence SEQ ID NO: 23, amino acid sequence SEQ ID NO: 24;

[0274] KIR3DL1 extracellular region: nucleotide sequence SEQ ID NO: 25, amino acid sequence SEQ ID NO: 26;

[0275] KIR3DL2 extracellular region: nucleotide sequence SEQ ID NO: 27, amino acid sequence SEQ ID NO: 28;

[0276] Claudin 18.2 antigen epitope: nucleotide sequence SEQ ID NO: 29, amino acid sequence SEQ ID NO: 30;

[0277] BCMA extracellular domain: nucleotide sequence SEQ ID NO: 31, amino acid sequence SEQ ID NO: 32;

[0278] CD28 extracellular hinge region: nucleotide sequence SEQ ID NO: 33, amino acid sequence SEQ ID NO: 34;

[0279] IL7Rα extracellular hinge region: nucleotide sequence SEQ ID NO: 35, amino acid sequence SEQ ID NO: 36;

[0280] K23-1: nucleotide sequence SEQ ID NO: 37, amino acid sequence SEQ ID NO: 38;

[0281] K23-2: nucleotide sequence SEQ ID NO: 39, amino acid sequence SEQ ID NO: 40;

[0282] K23-3: nucleotide sequence SEQ ID NO: 41, amino acid sequence SEQ ID NO: 42;

[0283] K23-4: nucleotide sequence SEQ ID NO: 43, amino acid sequence SEQ ID NO: 44;

[0284] K23-5: nucleotide sequence SEQ ID NO: 45, amino acid sequence SEQ ID NO: 46;

[0285] K23-6: nucleotide sequence SEQ ID NO: 47, amino acid sequence SEQ ID NO: 48;

[0286] K24-1: nucleotide sequence SEQ ID NO: 49, amino acid sequence SEQ ID NO: 50;

[0287] K24-2: nucleotide sequence SEQ ID NO: 51, amino acid sequence SEQ ID NO: 52;

[0288] K24-3: nucleotide sequence SEQ ID NO: 53, amino acid sequence SEQ ID NO: 54;

[0289] K31-1: nucleotide sequence SEQ ID NO: 55, amino acid sequence SEQ ID NO: 56;

[0290] K32-1: nucleotide sequence SEQ ID NO: 57, amino acid sequence SEQ ID NO: 58;

[0291] Linker 1: nucleotide sequence SEQ ID NO: 59, amino acid sequence SEQ ID NO: 60;

[0292] Linker 2: nucleotide sequence SEQ ID NO: 61, amino acid sequence SEQ ID NO: 62;

[0293] Linker 3: nucleotide sequence SEQ ID NO: 63, amino acid sequence SEQ ID NO: 64;

[0294] Point mutation BCMA extracellular region: nucleotide sequence SEQ ID NO: 65, amino acid sequence SEQ ID NO: 66;

[0295] P2A: nucleotide sequence SEQ ID NO: 67, amino acid sequence SEQ ID NO: 68;

[0296] CD52 signal peptide: nucleotide sequence SEQ ID NO: 69, amino acid sequence SEQ ID NO: 70;

[0297] IL-7: nucleotide sequence SEQ ID NO: 71, amino acid sequence SEQ ID NO: 72;

[0298] CD52: nucleotide sequence SEQ ID NO: 73, amino acid sequence SEQ ID NO: 74;

[0299] CD62L intracellular signaling region: nucleotide sequence SEQ ID NO: 75, amino acid sequence SEQ ID NO: 76;

[0300] K23-7: nucleotide sequence SEQ ID NO: 77, amino acid sequence SEQ ID NO: 78;

[0301] K23-8: nucleotide sequence SEQ ID NO: 79, amino acid sequence SEQ ID NO: 80;

[0302] K24-4: nucleotide sequence SEQ ID NO: 81, amino acid sequence SEQ ID NO: 82;

[0303] K24-5: nucleotide sequence SEQ ID NO: 83, amino acid sequence SEQ ID NO: 84;

[0304] IL-7-GPI-K23-7: nucleotide sequence SEQ ID NO: 85, amino acid sequence SEQ ID NO: 86;

[0305] K23-7-IL-7-GPI: nucleotide sequence SEQ ID NO: 87, amino acid sequence SEQ ID NO: 88;

[0306] IL-7-GPI-K23-8: nucleotide sequence SEQ ID NO: 89, amino acid sequence SEQ ID NO: 90;

[0307] K23-8-2A-IL-7-GPI: nucleotide sequence SEQ ID NO: 91, amino acid sequence SEQ ID NO: 92;

[0308] IL-7-GPI-K24-4: nucleotide sequence SEQ ID NO: 93, amino acid sequence SEQ ID NO: 94;

[0309] K24-4-IL-7-GPI: nucleotide sequence SEQ ID NO: 95, amino acid sequence SEQ ID NO: 96;

[0310] IL-7-GPI-K24-5: nucleotide sequence SEQ ID NO: 97, amino acid sequence SEQ ID NO: 98;

[0311] K24-5-IL-7-GPI: nucleotide sequence SEQ ID NO: 99, amino acid sequence SEQ ID NO: 100;

[0312] The auxiliary receptors involved in the examples are shown in Table 2:

[0313] Table 2 Coreceptor structures and sequences

[0314] Example 1: Construction of co-receptor expression vector

[0315] According to the method described in Example 1 on page 21 of PCT application WO2022078310A1, a pKB20 vector was constructed. According to the method for constructing pKB20-EGFP described in the embodiment, a pKB20 vector containing an exogenous gene expression cassette was constructed. Specifically, the commissioned company synthesized the sequences shown in SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 77, 79, 81, 83, 85, 87, 89, 94, 93, 95, 97 and 99 in Table 2, and replaced them with SEQ ID NOs: The two ends of NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 77, 79, 81, 83, 85, 87, 89, 94, 93, 95, 97 and 99 were ligated with linkers containing corresponding restriction sites and cloned into the prepared pKB20 vector according to the method described in Example 1 on page 21 of WO2022078310A1 specification, and named as pKB20-K23-1, pKB20-K23-2, pKB20-K23-3, pKB20-K23-4, pKB20-K23-5, pKB20-K23-6, pKB20-K24-1, pKB20-K24-2, pKB20- K24-3, pKB20-K31-1, pKB20-K32-1, pKB20-K23-7, pKB20-K23-8, pKB20-K2 4-4, pKB20-K24-5, pKB20-IL-7-GPI-K23-7, pKB20-K23-7-IL-7-GPI, pKB2 0-IL-7-GPI-K23-8, pKB20-K23-8-2A-IL-7-GPI, pKB20-IL-7-GPI-K24-4, pKB20-K24-4-IL-7-GPI, pKB20-IL-7-GPI-K24-5, and pKB20-K24-5-IL-7-GPI. The recombinant plasmids obtained above were transformed into E. coli (DH5c). After correct sequencing, the plasmids were extracted and purified using a plasmid purification kit from Qiagen to obtain high-quality plasmids of each recombinant expression vector.

[0316] Example 2: Isolation and culture of melanoma tissue-derived TIL cells

[0317] Freshly resected melanoma specimens expressing HLA-G and HLA-C were collected and immediately processed under sterile conditions. The specific method was as follows: normal tissue and necrotic areas surrounding the cancer specimen were removed, and 2-3 mm sized smears were removed from different areas of the specimen. 3Place 3-4 small tissue pieces in each well of a 6-well plate. Add 3 mL of complete culture medium (AIM-V medium containing 5% human AB serum), OKT-3 at a final concentration of 30 ng / mL, and IL-2 at 3000 IU / mL to each well. Culture the 24-well plate in a 37°C, 5% CO2 incubator. On the 5th to 6th day after the start of culture, half of the medium was replaced for all wells. After that, half of the medium was replaced every 1-2 days depending on the growth of TIL. Once the TIL in the well is full and all adherent cells have been removed, the TIL in each full well is collected, and after collecting enough TIL cells for subsequent experiments, prepare for the next experiment.

[0318] Example 3: Genetic modification and proliferation of TIL

[0319] 1) Add AIM-V medium to 24 wells of a 12-well plate in advance, 2 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for 1 hour.

[0320] 2) Prepare the electroporation solution ratio for each well according to the following table:

[0321] Plasmids tested as needed: pKB20-K23-1, pKB20-K23-2, pKB20-K23-3, pKB20-K23-4, pKB20-K23-5, pKB20-K23-6, pKB20-K24-1, pKB20-K24-2, pKB20-K24-3, pKB20-K31-1, pKB20-K32-1, pKB20-K23-7, pKB20-K23-8, pKB20-K24-4, pKB20-K24-5, pKB20-I L-7-GPI-K23-7, pKB20-K23-7-IL-7-GPI, pKB20-IL-7-GPI-K23-8, pKB20-K23-8-2A-IL-7-GPI, pKB20-IL-7-GPI-K24-4, pKB20-K24-4-IL-7-GPI, pKB20-IL-7-GPI-K24-5, and pKB20-K24-5-IL-7-GPI, as well as the control empty plasmid pKB20, were prepared as electroporation systems for the experimental group (group 23) and the control group (group 1);

[0322] 3) Take the TIL obtained in Example 2 and put it into 24 EP tubes. Add 5×10 6 The cells were centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL of normal saline and the cell pellet was washed by repeating the centrifugation step;

[0323] 4) Add plasmids pKB20-K23-1, pKB20-K23-2, pKB20-K23-3, pKB20-K23-4, pKB20-K23-5, pKB20-K23-6, pKB20-K24-1, pKB20-K24-2, pKB20-K24-3, pKB20-K31-1, pKB20-K32-1, pKB20-K23-7, pKB20-K23-8, pKB20-K24-9 to the electroporation solution of each experimental group and control group prepared in 2). -4, pKB20-K24-5, pKB20-IL-7-GPI-K23-7, pKB20-K23-7-IL-7-GPI, pKB20-IL-7-GPI-K23-8, pKB20-K23-8-2A-IL-7-GPI, pKB20-IL-7-GPI-K24-4, pKB20-K24-4-IL-7-GPI, pKB20-IL-7-GPI-K24-5 and pKB20-K24-5-IL-7-GPI and the control empty plasmid pKB20 5gg, and then let it stand at room temperature for less than 30 min;

[0324] 5) Resuspend all tubes with the plasmid electroporation solution prepared in 4), 100 μL per tube. Carefully pipette the cell resuspension into a LONZA 100 μL electroporation cup and place the cup into the LONZA Nucleofector. TM 2b In the electroporation tank, start the electroporation program and select X001;

[0325] 6) After electroporation, carefully remove the electroporation cuvette, aspirate the cell suspension and transfer it to an EP tube. Add 200 μL of preheated AIM-V medium to each tube, and then transfer it to the wells of the 12-well plate containing preheated AIM-V medium in 1) and culture at 37°C and 5% CO2. After 5 days of culture, cells overexpressing the auxiliary receptors K23-1, K23-2, K23-3, K23-4, K23-5, K23-6, K24-1, K24-2, and K24- 3. K31-1, K32-1, K23-7, K23-8, K24-4, K24-5, IL-7-GPI-K23-7, K23-7-IL-7-GPI, IL-7-GPI-K 23-8, K23-8-2A-IL-7-GPI, IL-7-GPI-K24-4, K24-4-IL-7-GPI, IL-7-GPI-K24-5 and K24-5-IL-7- The TIL cells of GPI and the control group were named TIL-K23-1, TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K24-1, TIL-K24-2, TIL-K24-3, TIL-K31-1, TIL-K32-1, TIL-K23-7, TIL-K23-8, TIL-K31-1, TIL-K32-1, TIL-K31-7, TIL-K31-8, TIL-K31-1 ... L-K24-4, TIL-K24-5, TIL-IL-7-GPI-K23-7, TIL-K23-7-IL-7-GPI, TIL-IL-7-GPI-K23-8, TIL -K23-8-2A-IL-7-GPI, TIL-IL-7-GPI-K24-4, TIL-K24-4-IL-7-GPI, TIL-IL-7-GPI-K24-5, TIL - K24-5-IL-7-GPI and TIL-CTRL.

[0326] Example 4: Cell survival rate and co-receptor expression positive rate of TIL electroporated with co-receptors

[0327] The cell survival rates of each group were detected by trypan blue staining and cell counting. The results showed that the cell survival rates of the coreceptor-expressing TILs prepared in Example 3 and the control group TILs were all above 94%.

[0328] The BCMA extracellular domain or Claudin18.2 antigen epitope is expressed in the extracellular region of each auxiliary receptor and fused with the KIR extracellular region, which can serve as a tag for exogenous transferred genes. They were respectively directed against TILs expressing coreceptors containing the BCMA extracellular domain (TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K24-2, TIL-K23-7, TIL-K24-4, TIL-K23-7, TIL-K23-8, TIL-K24-4, TIL-K24-5, TIL-IL-7-GPI-K23-7, TIL-K23-7-IL-7-GPI, TIL-IL-7-GPI-K23-8, TIL-K23-8-2A-IL-7-GPI, TIL-IL-7-GPI-K24- 4. For TIL-K24-4-IL-7-GPI, TIL-IL-7-GPI-K24-5, TIL-K24-5-IL-7-GPI) and TIL expressing coreceptors containing the Claudin18.2 epitope (TIL-K23-1, TIL-K24-1, TIL-K24-3, TIL-K31-1, TIL-K32-1), the BCMA extracellular domain or Claudin18.2 was used as a label. Fluorescent antibodies targeting the BCMA extracellular domain or fluorescent antibodies targeting the Claudin18.2 epitope were used to detect the proportion of BCMA and Claudin18.2 epitope-positive cells, respectively, as follows:

[0329] 1) Collect TIL-K23-1, TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K24-1, TIL-K24-2 , TIL-K24-3, TIL-K31-1, TIL-K32-1, TIL-K23-7, TIL-K23-8, TIL-K24-4, TIL-K24-5, TIL-IL-7-GPI- The cells of each group were collected at 1 × 10 for each of the following: K23-7, TIL-K23-7-IL-7-GPI, TIL-IL-7-GPI-K23-8, TIL-K23-8-2A-IL-7-GPI, TIL-IL-7-GPI-K24-4, TIL-K24-4-IL-7-GPI, TIL-IL-7-GPI-K24-5, TIL-K24-5-IL-7-GPI, and TIL-CTRL. 6 , 800g, centrifugation for 5min;

[0330] 2) Discard the supernatant, add physiological saline to resuspend the cells, and centrifuge at 800g for 5 minutes;

[0331] 3) Discard the supernatant and resuspend the cells in 100 μL of saline for each sample. For each group of TILs electroporated with a coreceptor containing the BCMA extracellular domain, add 2 μL of BCMA flow cytometry antibody (Biolegend, Cat#: 357504) to each tube and incubate at room temperature for 30 minutes. For TILs electroporated with a coreceptor containing the Claudin18.2 epitope, first add 2 μL of a primary antibody targeting the Claudin18.2 epitope without a fluorescent group (Abcam, Cat#: ab222512) to each tube and incubate at room temperature for 30 minutes. Centrifuge at 800g for 5 minutes, resuspend and wash with saline, centrifuge at 800g for 5 minutes, repeat twice, resuspend with 100 μL of saline, and then add 2 μL of a fluorescent secondary antibody targeting the primary antibody (Abcam, Cat#: ab72465) and incubate at room temperature for 30 minutes. TIL-CTRL is detected by BCMA flow cytometry to determine the background value.

[0332] 4) Centrifuge the antibody-labeled cells from each group in 3) at 800 g for 5 min, add appropriate amount of physiological saline, centrifuge at 800 g for 5 min, wash twice, and discard the supernatant;

[0333] 5) Resuspend in 400 μL of physiological saline and analyze by flow cytometry.

[0334] The positive rates of cells in each group are shown in Table 3 below:

[0335] Table 3 Proportion of TILs with positive co-receptor expression

[0336] The results showed that the proportion of TILs with positive coreceptor expression in each group of TILs electroporated with coreceptors ranged from 34.4% to 66.2%. Specifically, TILs expressing coreceptors with intracellular domains containing the CD62L intracellular signaling region had a significantly higher proportion of coreceptor-positive TILs than TILs expressing coreceptors without the CD62L intracellular signaling region.

[0337] Example 5: Cytotoxicity of TIL cells overexpressing coreceptors against homologous tumor cells

[0338] The fresh melanoma tissue of Example 2 was cut into 3×3×3 mm pieces. The pieces were mixed as evenly as possible and then cultured to obtain primary melanoma cells according to the method described in Robert Suriano et al. Ex Vivo Derived Primary Melanoma Cells: Implications for Immunotherapeutic Vaccines J Cancer 2013; 4(5): 371-382. Materials and Methods.

[0339] The real-time label-free cell function analyzer (RTCA) of Aisen Company was used to detect the TIL-K23-1, TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K24-1, TIL-K24-2, TIL-K24-3, TIL-K23-7, TIL-K23-8, TIL-K24-4, TIL-K24-5, TIL-IL-7-GPI-K obtained in Example 3. The in vitro cytotoxicity of TIL-23-7, TIL-K23-7-IL-7-GPI, TIL-IL-7-GPI-K23-8, TIL-K23-8-2A-IL-7-GPI, TIL-IL-7-GPI-K24-4, TIL-K24-4-IL-7-GPI, TIL-IL-7-GPI-K24-5, TIL-K24-5-IL-7-GPI and TIL-CTRL cells against their homologous primary melanoma cells was evaluated as follows:

[0340] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place it in the instrument, select step 1, and adjust to zero;

[0341] (2) Target cell plating: The primary melanoma cells obtained by culture were plated at 10 per well. 4 Spread 50 μL of cells on a plate containing detection electrodes and leave it for a few minutes. After the cells are stable, place them in the instrument and start step 2 to culture the cells.

[0342] (3) Adding effector cells: Use TIL-CTRL to uniformly adjust the positive rate of all effector cells involved in the killing test to 32%. After the target cells are cultured for 18h-24h, the cell index is observed. When the cell index is 1, effector cells TIL-K23-1, TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K24-1, TIL-K24-2, TIL-K24-3, TIL-K23-7, TIL-K23-8, TIL-K24-4, TIL-K24-5, TIL-I TIL-7-GPI-K23-7, TIL-K23-7-IL-7-GPI, TIL-IL-7-GPI-K23-8, TIL-K23-8-2A-IL-7-GPI, TIL-IL-7-GPI-K24-4, TIL-K24-4-IL-7-GPI, TIL-IL-7-GPI-K24-5, TIL-K24-5-IL-7-GPI, and TIL-CTRL were added in 50 μL per well with an effector-target ratio of 1:1. Step 3 was started. After co-culture for more than 48-72 hours, the cell proliferation curve was observed and the killing rate was calculated. The target cell killing rate was calculated as follows:

[0343] A is the cell index of the group with only target cells (ie, tumor cells) but no effector cells added, and B is the cell index of the groups with effector cells added.

[0344] The results are shown in Table 4:

[0345] Table 4 Killing rate of target cells by TIL expressing different co-receptors

[0346] The results showed that compared with TIL-CTRL, TIL-K23-1, TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K 24-1, TIL-K24-2, TIL-K24-3, TIL-K23-7, TIL-K23-8, TIL-K24-4, TIL-K24-5, TIL-IL-7-GPI-K23- 7. TIL-K23-7-IL-7-GPI, TIL-IL-7-GPI-K23-8, TIL-K23-8-2A-IL-7-GPI, TIL-IL-7-GPI-K24-4, TIL-K24-4-IL-7-GPI, TIL-IL-7-GPI-K24-5, and TIL-K24-5-IL-7-GPI cells have significantly stronger killing effects on homologous melanoma primary tumor cells. Moreover, compared with TILs without fusion-expressed auxiliary receptors for mbIL-7-GPI (TIL-K23-7, TIL-K23-8, TIL-K24-4, and TIL-K24-5), their corresponding TILs with fusion-expressed auxiliary receptors for mbIL-7-GPI (TIL-IL-7-GPI-K23-7, TIL-K23-7-IL-7-GPI, TIL-IL-7-GPI-K23-8, TIL-K23-8-2A-IL-7-GPI, TIL-IL-7-GPI-K24-4, TIL-K24-4-IL-7-GPI, TIL-IL-7-GPI-K24-5, and TIL-K24-5-IL-7-GPI) all showed significantly enhanced killing ability against target cells.

[0347] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

[0348] Part of this article

Claims

1. A co-receptor comprising an extracellular ligand binding domain, a transmembrane domain and an intracellular region, wherein the extracellular domain comprises the extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligand, preferably, the intracellular region comprises the 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 any one or more selected from KIR2DL3, KIR3DL1 and KIR3DL2.

2. The coreceptor according to claim 1, wherein The intracellular region comprises a signal transduction domain and / or a costimulatory domain, Preferably, the costimulatory domain is the intracellular domain of the costimulatory signal molecule or a functional fragment or mutant thereof that retains the biological function of the costimulatory signal molecule to transmit costimulatory signals and activate immune cells. More preferably, the intracellular domain of the costimulatory signal molecule includes any one or more selected from the following: 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 the intracellular domains of CD27, or mutants thereof, Further preferably, the intracellular domain of the costimulatory signal molecule is the intracellular domain of CD28 and / or the intracellular domain of OX40.

3. The auxiliary receptor according to claim 1 or 2, characterized in that The coreceptor further comprises a hinge region, Preferably, the hinge region includes a proximal membrane fragment of the natural extracellular domain of the co-stimulatory signal molecule, or the hinge region includes but is not limited to any one or more selected from the following: 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, I Membrane-proximal fragments of the native extracellular domains of 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, More preferably, the hinge region is the extracellular hinge region of CD28 and / or the extracellular hinge region of IL7Rα.

4. The auxiliary receptor according to claim 1 or 2, characterized in that The coreceptor also includes a membrane surface tag, Preferably, The membrane surface tag includes the BCMA extracellular domain or a variant thereof, or the claudin protein extracellular domain or a fragment or variant thereof, and / or, The membrane surface tag is located at the C-terminus of the extracellular domain of KIR. More preferably, the claudin protein is claudin18, and the fragment of the extracellular domain of the claudin protein is an extracellular antigen epitope of the claudin protein.

5. The auxiliary receptor according to claim 1 or 2, wherein The transmembrane 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 CD27 transmembrane regions, or mutants thereof that retain transmembrane function. Preferably, the transmembrane region is the CD28 transmembrane region, the IL7Rα transmembrane region or a mutant thereof that retains the transmembrane function.

6. The coreceptor according to claim 4, wherein The auxiliary receptor comprises an extracellular ligand-binding domain, a membrane surface tag, a transmembrane region and a CD62L intracellular domain, and optionally further comprises 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, wherein the extracellular ligand-binding domain comprises a KIR2DL3 extracellular domain or a KIR2DL4 extracellular domain, and the transmembrane region is a CD28 transmembrane region or an IL7Rα transmembrane region or a mutant thereof. Preferably, the membrane surface tag comprises a Claudin18.2 antigen epitope or a BCMA extracellular domain or a mutant thereof, and / or the auxiliary receptor further comprises a CD28 hinge region between the membrane surface tag and the transmembrane region. Preferably, the auxiliary 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, More preferably, the coreceptor comprises: (1) KIR2DL3 extracellular region, Claudin18.2 antigen epitope, CD28 extracellular hinge region, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain, (2) KIR2DL3 extracellular domain, BCMA extracellular domain, IL7Rα transmembrane region or its mutant, CD62L intracellular domain, IL-7Rα intracellular domain, or (3) KIR2DL3 extracellular region, linker, BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain, (4) KIR2DL3 extracellular region, BCMA extracellular domain or its variant, linker, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain (5) KIR2DL4 extracellular region, Claudin18.2 antigen epitope, CD28 extracellular hinge region, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain, (6) KIR2DL4 extracellular region, BCMA extracellular domain, IL7Rα extracellular hinge region, IL7Rα transmembrane region, CD62L intracellular domain, and IL-7Rα intracellular domain, (7) KIR2DL4 extracellular region, Claudin18.2 antigen epitope, IL7Rα extracellular hinge region, IL7Rα transmembrane region mutant, CD62L intracellular domain, and IL-7Rα intracellular domain, (8) KIR2DL4 extracellular region, linker, BCMA extracellular domain, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain, (9) KIR2DL4 extracellular region, BCMA extracellular domain mutant, linker, CD28 transmembrane region, CD62L intracellular domain, CD28 intracellular domain and OX40 intracellular domain.

7. The coreceptor according to any one of claims 1 to 6, wherein The amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO:

4. The amino acid sequence of the IL7Rα transmembrane region is shown in SEQ ID NO:

6. The amino acid sequences of the IL7Rα transmembrane region mutants are shown in SEQ ID NOs: 8, 10, 12 and 14. The amino acid sequence of the CD28 intracellular domain is shown in SEQ ID NO: 16, The amino acid sequence of the OX40 intracellular domain is shown in SEQ ID NO: 18, The amino acid sequence of the IL-7Rα intracellular domain is shown in SEQ ID NO:

20. The amino acid sequence of the KIR2DL3 extracellular region is shown in SEQ ID NO:

22. The amino acid sequence of the KIR2DL4 extracellular region is shown in SEQ ID NO:

24. The amino acid sequence of the KIR3DL1 extracellular region is shown in SEQ ID NO:

26. The amino acid sequence of the KIR3DL2 extracellular region is shown in SEQ ID NO:

28. The amino acid sequence of the Claudin 18.2 antigen epitope is shown in SEQ ID NO:

30. The amino acid sequence of the BCMA extracellular domain is shown in SEQ ID NO:

32. The amino acid sequence of the BCMA extracellular domain mutant is shown in SEQ ID NO:

66. The amino acid sequence of the CD28 extracellular hinge region is shown in SEQ ID NO:

34. The amino acid sequence of the IL7Rα extracellular hinge region is shown in SEQ ID NO:

36. The CD62L intracellular domain comprises the sequence shown in SEQ ID NO: 76, The amino acid sequence of the linker is shown in SEQ ID NO: 60, 62 or 64, or The amino acid sequence of the auxiliary receptor is shown in SEQ ID NO: 82 or 84.

8. A coreceptor comprising: a KIR2DL3 extracellular region or a KIR2DL4 extracellular region, a BCMA extracellular domain or a mutant thereof, a linker, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain, Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 60, 62 or 64, More preferably, the amino acid sequence of the auxiliary receptor is as shown in any one of SEQ ID NOs: 78 and 80.

9. A fusion protein comprising a coreceptor and a membrane surface cytokine, preferably, the coreceptor comprises an extracellular domain, a transmembrane region and an intracellular region, the extracellular domain comprises the extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to a KIR ligand, and 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 anchor region, Preferably, the membrane surface cytokine is at the N-terminus and / or C-terminus of the auxiliary receptor, Preferably, the cytokine is IL-7, Preferably, the GPI anchor region is CD52, Preferably, the membrane surface cytokines include: CD52 signal peptide, IL-7, linker, CD52, Preferably, the membrane surface cytokine is linked to the auxiliary receptor via a cleavable sequence; preferably, the cleavable sequence is, for example, P2A, T2A, or F2A. Preferably, the membrane surface cytokine is connected to the auxiliary receptor via a linker. Preferably, the auxiliary receptor is as described in any one of claims 1 to 8, Preferably, the amino acid sequence of the auxiliary receptor is shown in SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 78, 80, 82 or 84.

10. A polynucleotide molecule comprising: a nucleic acid sequence encoding the coreceptor according to any one of claims 1 to 8 or the fusion protein according to claim 9 or a complementary sequence thereof, Preferably, the polynucleotide molecule comprises a sequence selected from any one of SEQ ID NOs: 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97 and 99, or a complementary sequence thereof.

11. A nucleic acid construct comprising the polynucleotide molecule according to claim 10; Preferably, The nucleic acid construct further comprises at least one regulatory element for expressing the co-receptor or fusion protein operably linked to the polynucleotide, The nucleic acid construct is an expression vector or a cloning vector, The nucleic acid construct is a viral vector or a non-viral vector; more preferably, the non-viral vector is a non-viral integration vector based on a transposon system.

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

13. The nucleic acid construct according to claim 12, wherein The auxiliary receptor is as described in any one of claims 1-8.

14. The nucleic acid construct according to claim 12, wherein The amino acid sequence of the auxiliary receptor is shown in SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 78, 80, 82 or 84.

15. The nucleic acid construct according to claim 12, wherein The membrane surface cytokine is as described in claim 9.

16. The nucleic acid construct according to claim 12, 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 non-viral integration vector based on a transposon system.

17. A genetically engineered cell, which: (1) expressing the coreceptor according to any one of claims 1 to 8 or the fusion protein according to claim 9, and / or carrying the coding sequence of the coreceptor or fusion protein, (2) expressing auxiliary receptors and membrane surface cytokines, wherein the auxiliary receptor comprises an extracellular domain, a transmembrane region and an intracellular region, wherein the extracellular domain comprises the extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligands, wherein the intracellular region comprises a signal transduction domain and / or a co-stimulatory domain, and wherein the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region, (3) comprising the nucleic acid construct according to any one of claims 11 to 16, Preferably, The cells are immune effector cells; more preferably, the immune effector cells include T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DN T and TIL, The cells also express CAR, or carry a coding sequence for CAR, The cells also express exogenous TCR, or carry the coding sequence of an exogenous TCR.

18. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and any one or more of the co-receptor according to any one of claims 1-8, the fusion protein according to claim 9, the polynucleotide molecule according to claim 10, the nucleic acid construct according to any one of claims 11-16, and the genetically engineered cell according to claim 17.

19. Use of any one or more of the auxiliary receptor according to any one of claims 1 to 8, the fusion protein according to claim 9, the polynucleotide molecule according to claim 10, the nucleic acid construct according to any one of claims 11 to 16, and the genetically engineered cell according to claim 17 in the preparation of a medicament for treating or preventing cancer.

20. Use of auxiliary receptors and membrane surface cytokines in the preparation of drugs for treating or preventing cancer, wherein the auxiliary receptor comprises an extracellular domain, a transmembrane region and an intracellular region, the extracellular domain comprises the extracellular domain of KIR or a functional fragment or variant thereof that retains the biological function of binding to KIR ligands, the intracellular region comprises a signal transduction domain and / or a co-stimulatory domain, and the membrane surface cytokine comprises a cytokine and a transmembrane domain or a GPI anchor region.

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