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KR-20260068106-A - Transformed recombinant immune cells that specifically target tumors and their applications

KR20260068106AKR 20260068106 AKR20260068106 AKR 20260068106AKR-20260068106-A

Abstract

The present invention provides a transformed recombinant immune cell that specifically targets tumors. The recombinant immune cell comprises a chimeric polypeptide and a chimeric antigen receptor; the chimeric polypeptide comprises a first extracellular region, a first transmembrane region, and a first intracellular region having HLA-G protein binding activity, wherein the N-terminus of the first transmembrane region is connected to the C-terminus of the first extracellular region, the N-terminus of the first intracellular region is connected to the C-terminus of the first transmembrane region, and the first transmembrane region comprises the transmembrane region of the Notch receptor protein of the Western clawed frog; and the antigen chimeric receptor does not have HLA-G protein binding activity. The recombinant immune cell significantly improves the accurate recognition of tumor cells by immune cells, reduces detargeting effects of cell therapy, accurately and effectively distinguishes between tumor cells and normal cells, and provides an effective targeting and targeting method for the treatment of a wide range of tumors (particularly solid tumors).

Inventors

  • 왕, 팅
  • 조우, 잉리
  • 장, 차이
  • 첸, 민화

Assignees

  • 상하이 엔케이 쎌테크 씨오., 엘티디.

Dates

Publication Date
20260513
Application Date
20241227
Priority Date
20231228

Claims (18)

  1. As a chimeric polypeptide, It includes a first extracellular region, a first transmembrane region and a first intracellular region, and The first extracellular region has HLA-G protein binding activity; The first transmembrane region comprises the transmembrane region of the Notch receptor protein of the Western clawed frog or an amino acid sequence having at least 80% identity therewith, and the N-terminus of the first transmembrane region is connected to the C-terminus of the first extracellular region; A chimeric polypeptide characterized in that the N-terminus of the first intracellular region is connected to the C-terminus of the first transmembrane region.
  2. In paragraph 1, The transmembrane region of the Notch receptor protein of the above-mentioned clawed frog has the amino acid sequence represented by SEQ ID NO. 1; Optionally, the transmembrane region further comprises an epidermal growth factor-like repeat sequence and/or a RAM sequence; Optionally, the C-terminus of the epidermal growth factor-like repeat sequence is connected to the N-terminus of the transmembrane region of the Notch receptor protein of the clawed frog; and/or, the C-terminus of the transmembrane region of the Notch receptor protein of the clawed frog is connected to the N-terminus of the RAM sequence; Optionally, the first transmembrane region has an amino acid sequence represented by SEQ ID NO. 1 or 25 or an amino acid sequence having at least 90% identity therewith; Optionally, the chimeric polypeptide is characterized in that the first transmembrane region has an amino acid sequence represented by SEQ ID NO. 1 or 25.
  3. In paragraph 1, The first extracellular region comprises a first binding protein or a fragment thereof that binds to an HLA-G protein; Optionally, the first binding protein or a fragment thereof comprises at least one of an antibody or a functional fragment thereof, a receptor; Optionally, the first binding protein or a fragment thereof comprises a first binding fragment and/or a second binding fragment; The first binding fragment is the extracellular domain of the ILT2 protein or its active fragment; The second binding fragment is the extracellular domain of the ILT4 protein or its active fragment; Optionally, the first binding protein or a fragment thereof comprises a first binding fragment and a second binding fragment, and the first binding fragment is connected to the second binding fragment; Optionally, the extracellular domain of the ILT2 protein is composed of an ILT2-D1 fragment, an ILT2-D2 fragment, an ILT2-D3 fragment, and an ILT2-D4 fragment; The above ILT2-D1 fragment has an amino acid sequence represented by SEQ ID NO. 2; The above ILT2-D2 fragment has an amino acid sequence represented by SEQ ID NO. 3; The above ILT2-D3 fragment has an amino acid sequence represented by SEQ ID NO. 4; The above ILT2-D4 fragment has an amino acid sequence represented by SEQ ID NO. 5; Optionally, the extracellular domain of the ILT2 protein has an amino acid sequence represented by SEQ ID NO. 6; Optionally, the first binding fragment is, At least one of the above ILT2-D1 fragment, ILT2-D2 fragment, ILT2-D3 fragment and ILT2-D4 fragment; or Selected from the extracellular region of the above ILT2 protein; Optionally, the first coupling fragment is selected from the ILT2-D1 fragment and the ILT2-D2 fragment, and the ILT2-D1 fragment is connected to the ILT2-D2 fragment; Optionally, the C end of the ILT2-D1 fragment is connected to the N end of the ILT2-D2 fragment, or the N end of the ILT2-D1 fragment is connected to the C end of the ILT2-D2 fragment; Optionally, the first binding fragment is selected from the extracellular region of the ILT2 protein; Optionally, the extracellular domain of the ILT4 protein is composed of an ILT4-D1 fragment, an ILT4-D2 fragment, an ILT4-D3 fragment, and an ILT4-D4 fragment; The above ILT4-D1 fragment has an amino acid sequence represented by SEQ ID NO. 7; The above ILT4-D2 fragment has an amino acid sequence represented by SEQ ID NO. 8; The above ILT4-D3 fragment has an amino acid sequence represented by SEQ ID NO. 9; The above ILT4-D4 fragment has an amino acid sequence represented by SEQ ID NO. 10; Optionally, the extracellular domain of the ILT4 protein has an amino acid sequence represented by SEQ ID NO. 11; Optionally, the second bonding fragment is, At least one of the above ILT4-D1 fragment, ILT4-D2 fragment, ILT4-D3 fragment and ILT4-D4 fragment; or Selected from the extracellular region of the above ILT4 protein; Optionally, the second coupling fragment is selected from the ILT4-D1 fragment and the ILT4-D2 fragment, and the ILT4-D1 fragment is connected to the ILT4-D2 fragment; Optionally, the C end of the ILT4-D1 fragment is connected to the N end of the ILT4-D2 fragment, or the N end of the ILT4-D1 fragment is connected to the C end of the ILT4-D2 fragment; Optionally, the second binding fragment is selected from the extracellular region of the ILT4 protein; Optionally, if the first extracellular region comprises a plurality of the first binding proteins or fragments thereof, the first extracellular region further comprises a first linking peptide, and any two of the first binding proteins or fragments thereof may or may not be connected through the first linking peptide; Optionally, the first binding protein or a fragment thereof comprises a first binding fragment and a second binding fragment, and the first binding fragment and the second binding fragment are connected through the first linking peptide; Optionally, the C-terminus of the first binding fragment is connected to the N-terminus of the first linking peptide, and the C-terminus of the first linking peptide is connected to the N-terminus of the second binding fragment, or the C-terminus of the second binding fragment is connected to the N-terminus of the first linking peptide, and the C-terminus of the first linking peptide is connected to the N-terminus of the first binding fragment; Optionally, the amino acid sequence of the first linking peptide is (GGGGS)n, where n is any integer between 1 and 10; Optionally, n is 1, 2, 3, or 4; Optionally, the amino acid sequence of the first linking peptide is GGGGS; Optionally, the first extracellular region is, ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linkage peptide + ILT2-D1 fragment + ILT2-D2 fragment; Extracellular domain of ILT2 protein + first linkage peptide + extracellular domain of ILT4 protein; Extracellular domain of ILT4 protein + first linkage peptide + extracellular domain of ILT2 protein; ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + extracellular domain of ILT4 protein; ILT4-D1 fragment + ILT4-D2 fragment + first linkage peptide + extracellular domain of ILT2 protein; ILT2-D1 fragment + first linkage peptide + ILT4-D1 fragment; ILT2-D1 fragment + first linkage peptide + ILT4-D1 fragment; ILT2-D1 fragment + first linkage peptide + ILT4-D2 fragment; ILT2-D1 fragment + 1st linkage peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D1 fragment + 1st linkage peptide + extracellular domain of ILT4 protein; ILT2-D1 fragment + 1st linkage peptide + ILT2-D1 fragment; ILT2-D1 fragment + 1st linkage peptide + ILT2-D2 fragment; ILT2-D1 fragment + 1st linkage peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT2-D1 fragment + 1st linkage peptide + extracellular domain of ILT2 protein; ILT4-D1 fragment + 1st linkage peptide + ILT4-D1 fragment; ILT4-D1 fragment + 1st linkage peptide + ILT4-D2 fragment; ILT4-D1 fragment + 1st linkage peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D1 fragment + 1st linkage peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D1 fragment + 1st linkage peptide + extracellular domain of ILT4 protein; ILT4-D1 fragment + 1st linkage peptide + ILT2-D1 fragment; ILT4-D1 fragment + 1st linkage peptide + ILT2-D2 fragment; ILT4-D1 fragment + 1st linkage peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT4-D1 fragment + 1st linkage peptide + extracellular domain of ILT2 protein; ILT2-D2 fragment + 1st linkage peptide + ILT4-D1 fragment; ILT2-D2 fragment + 1st linkage peptide + ILT4-D2 fragment; ILT2-D2 fragment + 1st linkage peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D2 fragment + 1st linkage peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D2 fragment + 1st linkage peptide + extracellular domain of ILT4 protein; ILT2-D2 fragment + 1st linkage peptide + ILT2-D1 fragment; ILT2-D2 fragment + 1st linkage peptide + ILT2-D2 fragment; ILT2-D2 fragment + 1st linkage peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT2-D2 fragment + 1st linkage peptide + extracellular domain of ILT2 protein; ILT4-D2 fragment + 1st linkage peptide + ILT4-D1 fragment; ILT4-D2 fragment + 1st linkage peptide + ILT4-D2 fragment; ILT4-D2 fragment + 1st linkage peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D2 fragment + 1st linkage peptide + extracellular domain of ILT4 protein; ILT4-D2 fragment + 1st linkage peptide + ILT2-D1 fragment; ILT4-D2 fragment + 1st linkage peptide + ILT2-D2 fragment; ILT4-D2 fragment + 1st linkage peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT4-D2 fragment + 1st linkage peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT4-D2 fragment + 1st linkage peptide + extracellular domain of ILT2 protein; ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + ILT4-D1 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + ILT4-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + extracellular domain of ILT4 protein; ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + ILT2-D1 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + ILT2-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + ILT2-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linkage peptide + extracellular domain of ILT2 protein; ILT4-D1 fragment + ILT4-D2 fragment + first linkage peptide + ILT4-D1 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linkage peptide + ILT4-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linkage peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linkage peptide + extracellular domain of ILT4 protein; ILT4-D1 fragment + ILT4-D2 fragment + first linkage peptide + ILT2-D1 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linkage peptide + ILT2-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + I-linking peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + I-linking peptide + extracellular domain of ILT2 protein; Extracellular domain of ILT2 protein + I-linking peptide + ILT4-D1 fragment; Extracellular domain of ILT2 protein + I-linking peptide + ILT4-D2 fragment; Extracellular domain of ILT2 protein + I-linking peptide + ILT4-D1 fragment + ILT4-D2 fragment; Extracellular domain of ILT2 protein + I-linking peptide + extracellular domain of ILT4 protein; Extracellular domain of ILT2 protein + I-linking peptide + ILT2-D1 fragment; Extracellular domain of ILT2 protein + I-linking peptide + ILT2-D2 fragment; Extracellular domain of ILT2 protein + I-linking peptide + ILT2-D1 fragment + ILT2-D2 fragment; Extracellular domain of ILT2 protein + I-linking peptide + ILT2-D1 fragment + ILT2-D2 fragment; Selected from one of the following: extracellular domain of ILT2 protein + first linking peptide + extracellular domain of ILT2 protein; extracellular domain of ILT4 protein + first linking peptide + ILT4-D1 fragment; extracellular domain of ILT4 protein + first linking peptide + ILT4-D2 fragment; extracellular domain of ILT4 protein + first linking peptide + ILT4-D1 fragment + ILT4-D2 fragment; extracellular domain of ILT4 protein + first linking peptide + extracellular domain of ILT4 protein; extracellular domain of ILT4 protein + first linking peptide + ILT2-D1 fragment; extracellular domain of ILT4 protein + first linking peptide + ILT2-D2 fragment; extracellular domain of ILT4 protein + first linking peptide + ILT2-D1 fragment + ILT2-D2 fragment; extracellular domain of ILT4 protein + first linking peptide + ILT2-D1 fragment + ILT2-D2 fragment; extracellular domain of ILT4 protein + first linking peptide + extracellular domain of ILT2 protein; and Optionally, the first extracellular region is, 1) Extracellular domain of ILT2 protein; 2) Extracellular domain of ILT4 protein; 3) ILT2-D1 fragment and ILT2-D2 fragment sequentially from the N-terminus to the C-terminus; 4) ILT4-D1 fragment and ILT4-D2 fragment sequentially from the N-terminus to the C-terminus; 5) ILT2-D1 fragment, ILT2-D2 fragment, first linking peptide, ILT4-D1 fragment and ILT4-D2 fragment sequentially from N-terminus to C-terminus; 6) A chimeric polypeptide characterized by being sequentially selected from one of the ILT4-D1 fragment, ILT4-D2 fragment, first linking peptide, ILT2-D1 fragment, and ILT2-D2 fragment from the N-terminus to the C-terminus.
  4. In paragraph 1, The first intracellular region comprises at least one of a transcription activating protein, a transcription repressing protein, a transcription factor, a site-specific nuclease, a recombinant enzyme, an activating immune receptor intracellular domain, and an inhibiting immune receptor intracellular domain; Optionally, the first intracellular region comprises at least one of GaL4-VP64, GaL4-VP16, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, HAP1-VP16, and LexA-VP64; Optionally, the chimeric polypeptide is characterized in that the Gal4-VP64 comprises an amino acid sequence represented by SEQ ID NO. 12.
  5. As a first nucleic acid molecule, The first nucleic acid molecule above codes for a chimeric polypeptide according to any one of claims 1 to 4; Optionally, the first nucleic acid molecule is characterized in that the first nucleic acid molecule is DNA.
  6. As a first expression vector, Carrying the first nucleic acid molecule according to paragraph 5; Optionally, the first expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage; Preferably, the first expression vector is characterized in that the first expression vector is a plasmid expression vector.
  7. As a second nucleic acid molecule, It comprises a first nucleic acid fragment and a second nucleic acid fragment, wherein the 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment; The second nucleic acid fragment is used to code for an antigen chimeric receptor targeting a first molecule, and the first nucleic acid fragment is used to bind to a first intracellular region and induce the expression of the antigen chimeric receptor; A second nucleic acid molecule characterized in that the first intracellular region above corresponds to the first intracellular region defined in the chimeric polypeptide according to any one of claims 1 to 4.
  8. In Paragraph 7, The above antigen chimeric receptor includes a second extracellular region, a second transmembrane region, and a second transmembrane region, and The second extracellular region has first molecule binding activity, and the first molecule is not an HLA-G protein; The N-terminus of the second transmembrane region is connected to the C-terminus of the second extracellular region; The N-terminus of the second intracellular region is connected to the C-terminus of the second transmembrane region; Optionally, the first molecule comprises at least one of a tumor antigen, a virus, a bacterium, an endotoxin, an antibody, a cell receptor, and a ligand of a cell receptor; Optionally, the tumor antigen comprises at least one of a tumor-associated antigen and a tumor-specific antigen, preferably a tumor-specific antigen; Optionally, the first molecule is MICA, MICB, ULBPs, B7H6, B7H3, GFP, eGFP, CD19, ALPPL2, BCMA, SIRPα, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44, CD44v6, CD45, CD48, CD51, CD52, CD56, CD59, CD66, CD70, CD71, CD72, CD73, CD74, CD79A, CD79B, CD80, CD86, CD94, CD95, CD123, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD181, CD182, CD183, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD262, CD269, CD273, CD274, CD276, CD279, CD295, CD339, CD340, EGFR, EGFR VIII, HER2, FGFR2, AFP, CA125, MSLN, GPC3, CEA, CLDN1, CLDN3, CLDN6, CLDN18.1, CLDN18.2, EpCAM, PSCA, GD2, GD3, IL-13, IL-13RA2, ROR1, MUC-1, PSMA, MAGEA1, 4-1BB, It includes at least one of 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, EPHA2, VEGF-A, VEGFR-1, and VEGFR-2; Optionally, the second extracellular region comprises a second binding protein or a fragment thereof that binds to the first molecule; Optionally, the second binding protein or a fragment thereof comprises at least one of an antibody or a functional fragment thereof, a receptor, a ligand of the receptor, and a cell adhesion molecule; Optionally, the second binding protein or a fragment thereof is an antibody or a fragment thereof that binds to the extracellular region of an activating receptor expressed on the surface of an immune cell or to the first molecule; Optionally, the antibody or fragment thereof that binds to the first molecule is a single-chain antibody; Optionally, the activation receptor is selected from receptors on the surface of NK cells; Optionally, the activating receptor is selected from at least one of NKG2D, NKp30, NKp44, NKp46, DNAM-1, PD-1, TIGIT, NKG2A, NKG2B, NKG2C, NKG2E, NKG2H, CD16, NKp80, CD226, CD160, CD161, CD96, PVRIG, SLAM, CD200R, CD49a, TIM-3, LAG-3, CD112R, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, LIR1, LIR2, SIGLE3, SIGLE7, SIGLE9, KLRG1; Optionally, the activation receptor is selected from NKG2D and/or NKp30; Optionally, the second binding protein or a fragment thereof comprises a third binding fragment and/or a fourth binding fragment; The above third binding fragment is the extracellular region of NKG2D or its active fragment; The above-mentioned fourth binding fragment is the extracellular domain of NKp30 or its active fragment; Optionally, the extracellular region of the NKG2D has an amino acid sequence represented by SEQ ID NO. 13; Optionally, the extracellular region of the NKp30 has the amino acid sequence represented by SEQ ID NO. 14; Optionally, the third binding fragment has an amino acid sequence represented by SEQ ID NO. 13 or an amino acid sequence having at least 90% identity therewith; Optionally, the fourth binding fragment has an amino acid sequence represented by SEQ ID NO. 14 or an amino acid sequence having at least 90% identity therewith; Optionally, the second extracellular region further comprises a second linking peptide, and the third linking fragment and the fourth linking fragment are connected through the second linking peptide; Optionally, the C-terminus of the third binding fragment is connected to the N-terminus of the second linking peptide, and the C-terminus of the second linking peptide is connected to the N-terminus of the fourth binding fragment, or the C-terminus of the fourth binding fragment is connected to the N-terminus of the second linking peptide, and the C-terminus of the second linking peptide is connected to the N-terminus of the third binding fragment; Optionally, the amino acid sequence of the second linking peptide is (GGGGS)n, where n is any integer between 0 and 10; Optionally, n is 0, 1, 2, 3, or 4; Optionally, the amino acid sequence of the second linking peptide is GGGGS; Optionally, the second extracellular region further comprises a hinge region; The C-terminus of the third binding fragment is connected to the N-terminus of the second linking peptide, the C-terminus of the second linking peptide is connected to the N-terminus of the fourth binding fragment, and the C-terminus of the fourth binding fragment is connected to the N-terminus of the hinge region, or the C-terminus of the fourth binding fragment is connected to the N-terminus of the second linking peptide, the C-terminus of the second linking peptide is connected to the N-terminus of the third binding fragment, and the C-terminus of the third binding fragment is connected to the N-terminus of the hinge region; Optionally, the hinge region comprises at least one of a hinge region of a CD8α molecule or a variant thereof, a hinge region of an immunoglobulin or a variant thereof; Optionally, a second nucleic acid molecule characterized in that the hinge region has an amino acid sequence represented by SEQ ID NO. 15.
  9. In paragraph 8, The second transmembrane region is selected from at least one of the transmembrane region of a CD8α molecule, the transmembrane region of a CD28 molecule, the transmembrane region of a CD3ζ molecule, the transmembrane region of a CD4 molecule, the transmembrane region of a CD16 molecule, the transmembrane region of a 4-1BB molecule, the transmembrane region of an OX40 molecule, the transmembrane region of an ICOS molecule, the transmembrane region of a CTLA-4 molecule, the transmembrane region of a PD-1 molecule, the transmembrane region of a LAG-3 molecule, the transmembrane region of a 2B4 molecule, the transmembrane region of an NKG2D molecule, the transmembrane region of a DNAM-1 molecule, the transmembrane region of an NKp44 molecule, the transmembrane region of an NKp46 molecule, the transmembrane region of a KIR2DS1 molecule, the transmembrane region of a KIR2DS2 molecule, the transmembrane region of a KIR2DS4 molecule, and the transmembrane region of a BTLA molecule; Optionally, the second transmembrane region is selected from the transmembrane region of the CD8α molecule; Optionally, the second transmembrane region has an amino acid sequence represented by SEQ ID NO. 16; Optionally, the second intracellular region comprises an intracellular signaling domain and a co-stimulation domain; Optionally, the intracellular signaling domain is selected from at least one of the intracellular signaling domain of a CD3ζ molecule and the intracellular signaling domain of an FcεRIγ molecule; Optionally, the co-stimulation domain is selected from at least one of the intracellular signaling domain of a 4-1BB molecule, the intracellular signaling domain of a CD28 molecule, the intracellular signaling domain of a CD27 molecule, the intracellular signaling domain of a CD40 molecule, the intracellular signaling domain of an OX40 molecule, the intracellular signaling domain of an ICOS molecule, the intracellular signaling domain of a DAP10 molecule, the intracellular signaling domain of a DAP12 molecule, and the intracellular signaling domain of a DNAM-1 molecule; Optionally, the second intracellular region has an amino acid sequence represented by SEQ ID NO. 17; Optionally, a second nucleic acid molecule characterized in that the second nucleic acid fragment has a nucleotide sequence represented by SEQ ID NO. 18.
  10. In any one of paragraphs 7 through 9, The second nucleic acid molecule further comprises a third nucleic acid fragment, and the third nucleic acid fragment is used to code for a signal peptide; The 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment; Optionally, the signal peptide is selected from at least one of the signal peptide of a CD8α molecule, the signal peptide of an IgG molecule, and the signal peptide of a CD28 molecule; Optionally, the signal peptide has an amino acid sequence represented by SEQ ID NO. 19; Optionally, the third nucleic acid fragment has a nucleotide sequence represented by SEQ ID NO. 20; Optionally, a second nucleic acid molecule characterized in that the first nucleic acid fragment has a nucleotide sequence represented by SEQ ID NO. 21.
  11. As a second expression vector, Carrying a second nucleic acid molecule according to any one of paragraphs 7 to 10; Optionally, the second expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage; Preferably, the second expression vector is characterized in that the second expression vector is a plasmid expression vector.
  12. As a recombinant immune cell, Carrying the first nucleic acid molecule according to paragraph 5 or the first expression vector according to paragraph 6; or, A recombinant immune cell characterized by expressing a chimeric polypeptide according to any one of claims 1 to 4.
  13. In Paragraph 12, The expression of the chimeric polypeptide in the above-mentioned recombinant immune cells is obtained by introducing the first expression vector according to claim 6 into a host cell; Optionally, the recombinant immune cell further comprises a chimeric antigen receptor, said chimeric antigen receptor corresponding to an antigen chimeric receptor encoded by a second nucleic acid fragment limited in a second nucleic acid molecule according to any one of claims 7 to 10; Optionally, a recombinant immune cell characterized in that the expression of the chimeric antigen receptor in the recombinant immune cell is obtained by introducing the second expression vector according to claim 11 into a host cell.
  14. In Paragraph 13, The above host cell comprises at least one of an immune cell, a neuron, a progenitor cell or precursor cell, an epithelial cell, an endothelial cell, and a stem cell; Optionally, the recombinant immune cell is characterized in that the host cell comprises at least one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, and γδ T cells.
  15. As a pharmaceutical composition, Comprising recombinant immune cells according to any one of claims 12 to 14; A pharmaceutical composition characterized by optionally additionally comprising a pharmaceutically acceptable auxiliary substance.
  16. As a use of a recombinant immune cell according to any one of paragraphs 12 to 14 or a pharmaceutical composition according to paragraph 15, The above use is, Manufacturing of drugs for the prevention and/or treatment of diseases, Used for the prevention and/or treatment of diseases; Optionally, the above diseases include cancer or tumors, autoimmune diseases, inflammation, and related diseases caused by cellular aging.
  17. As a recombinant immune cell according to any one of claims 12 to 14 or a pharmaceutical composition according to claim 15 for the prevention and/or treatment of disease, Optionally, the disease comprises a recombinant immune cell or pharmaceutical composition including cancer or tumor, autoimmune disease, inflammation and related disease due to cellular senescence.
  18. As a method of treating or preventing disease, A method comprising the step of administering to a subject a pharmaceutically acceptable amount of recombinant immune cells according to any one of claims 12 to 14 or a pharmaceutical composition according to claim 15.

Description

Transformed recombinant immune cells that specifically target tumors and their applications The present invention relates to the field of biopharmaceuticals, specifically to a transformed recombinant immune cell that specifically targets a tumor, a method for producing the same, and applications. More specifically, the present invention relates to a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a second nucleic acid molecule, a second expression vector, a recombinant immune cell, a pharmaceutical composition, and uses thereof. With the rapid advancement of biotechnology, immunotherapy has established itself as one of the major therapies in the field of cancer treatment. Cancer immunotherapy primarily includes adoptive cell therapy, immunomodulators, tumor vaccines, and immune checkpoint blocking therapies. In the field of adoptive cell therapy, chimeric antigen receptor modified immune cell therapy, particularly chimeric antigen receptor modified T-cell (CAR-T) therapy, is currently receiving significant attention and is emerging as a popular therapy in this field. CAR-T is a representative immunotherapy, the principle of which involves forming CAR-T cells by performing chimeric antigen receptor modification on T cells extracted from the patient themselves through genetic engineering. Through the modified chimeric antigen receptor, these CAR-T cells can target and kill tumors by specifically recognizing tumor surface-associated antigens (tumor cell markers). Compared to general immune cells, CAR-T cells exhibit higher targetability, apoptotic activity, and persistence. Currently, modified immunotherapy using CAR-T cells targeting CD19 and BCMA has shown significant efficacy in the treatment of hematological malignancies such as B-cell lymphoma and is considered one of the most promising tumor treatment methods. However, due to the lack of targets capable of effectively distinguishing between normal cells and tumor cells, CAR-T cell therapy inevitably causes the death of some normal cells expressing target proteins, resulting in damage to normal tissues. Furthermore, the widespread presence of targets induces sustained activation of CAR-T cells, leading to the release of massive amounts of cytokines and triggering a cytokine storm. At the same time, since CAR-T cell therapy typically recognizes only a single antigen, tumor cells may achieve evasion by losing the antigens recognizable by CAR-T cells through genetic mutations, and subsequently exhibit drug resistance. Additionally, due to the high heterogeneity of tumors, the single target loaded onto CAR-T cells alone cannot fully target tumor cells, leading to incomplete or ineffective tumor treatment. The aforementioned core issues have been major obstacles to the development and expansion of the field of cell therapy. According to recent research, it can be very difficult to solve the aforementioned problems by finding a single molecule as a target for CAR therapy that is both broader and more specific, while simultaneously being more efficient and safer. Broadness and specificity are two sides of the same coin; a single target cannot possess both of these characteristics simultaneously, and to date, no target molecule possessing both of these characteristics has been discovered in tumor treatment research. Therefore, attempts to realize the broadness and specificity of immunotherapy by combining specific target combinations with tools that recognize and activate those target combinations are currently one of the most promising new directions in cell therapy. The above-described aspects and advantages of the present invention and/or additional aspects and advantages will be clearly and easily understood through the description of the embodiments together with the drawings below. Herein, Figure 1 is a schematic diagram of the structural configuration of the SynNotch receptor of Example 1 of the present invention. Figure 2 is a schematic diagram of the structural configuration of the CAR receptor of Example 2 of the present invention. Figure 3 is a diagram showing the results of Syn-CAR-NK cell death against dual-target positive K562 cells of Example 3 of the present invention. Figure 4 is a diagram showing the results of the death of Syn-CAR-NK cells against single-target positive Aspc-1 cells of Example 3 of the present invention. Figure 5 is a diagram showing the apoptosis result of Syn-CAR-NK on untargeted THLE3 cells of Example 3 of the present invention. Figure 6 is a diagram showing the treatment results of Syn-CAR-NK cells on a mouse subcutaneous implant tumor of dual-target positive colon cancer according to Example 4 of the present invention. Figure 7 is a diagram showing the treatment results of Syn-CAR-NK cells on a single-target positive pancreatic cancer mouse subcutaneous transplant tumor of Example 4 of the present invention. Embodiments of the present invention are described in detail below. The embodiments