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CN-121991898-A - Enhanced chimeric antigen receptor-T cell and preparation method and application thereof

CN121991898ACN 121991898 ACN121991898 ACN 121991898ACN-121991898-A

Abstract

The present invention relates to an enhanced chimeric antigen receptor-T cell, in which a nucleotide sequence encoding a chimeric antigen receptor is exogenously inserted into the genome of the T cell, the nucleotide sequence comprising at least one of the following genes, NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene or NKp46 gene, such that the expression level of the NKG2D protein, DNAM1 protein, NKp30 protein, NKp44 protein or NKp46 protein of the chimeric antigen receptor-T cell is higher than the expression level of the corresponding protein in the original T cell, and a method for preparing the same. The enhanced chimeric antigen receptor-T cell can effectively target and attack tumor cells, has high killing rate on tumors, does not depend on specific tumor antigens and specific tumor mutations, hopefully breaks through antigen escape, and can be used for preparing anti-tumor products.

Inventors

  • WANG HANLU
  • LIU LI
  • ZHANG CHAO
  • ZHOU LI
  • GAO JUNXIAO
  • Ding Shengzhen

Assignees

  • 合源康华医药科技(北京)有限公司

Dates

Publication Date
20260508
Application Date
20241107

Claims (20)

  1. 1. An enhanced chimeric antigen receptor-T cell, wherein the genome of the T cell has exogenously inserted therein a nucleotide sequence encoding a chimeric antigen receptor, said nucleotide sequence comprising at least one of the following genes: NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene or NKp46 gene, Such that the expression level of the NKG2D protein, DNAM1 protein, NKp30 protein, NKp44 protein or NKp46 protein of the chimeric antigen receptor-T cell is higher than the expression level of the corresponding protein in the original T cell.
  2. 2. The enhanced chimeric antigen receptor-T cell according to claim 1, wherein sequences of NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene and NKp46 gene are shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, respectively.
  3. 3. The enhanced chimeric antigen receptor-T cell according to claim 1 or 2, wherein the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell comprises TRAC site, PD1 site, regnase-1 site, roquin-1 site, CISH site and BCOR site; Alternatively, the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell is a TRAC site; More optionally, the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell is the TRAC site, and the Regnase-1 site in the genome of the T cell is knocked out.
  4. 4. The enhanced chimeric antigen receptor-T cell of any one of claims 1-3, wherein the chimeric antigen receptor comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain; Alternatively, the extracellular antigen-recognition domain is one of a NKG2D protein, DNAM1 protein, NKp30 protein, NKp44 protein, or NKp46 protein.
  5. 5. The enhanced chimeric antigen receptor-T cell of claim 4, wherein the amino acid sequences of the NKG2D protein, DNAM1 protein, NKp30 protein, NKp44 protein, and NKp46 protein are shown in SEQ ID No.6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No.10, respectively.
  6. 6. The enhanced chimeric antigen receptor-T cell according to claim 4, wherein the hinge region is derived from one or more of IgG1, igG4, CD7, CD28, CD84, CD8 a, optionally the hinge region is derived from CD8 a, more optionally the amino acid sequence of the hinge region is as shown in SEQ ID NO:11, and/or Wherein the transmembrane region is derived from one or more of CD3z, CD4, CD7, CD8 alpha, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, fc70, optionally the transmembrane region is derived from CD8 alpha or CD3z, more optionally the amino acid sequence of the transmembrane region is shown as SEQ ID NO:12 or SEQ ID NO: 13.
  7. 7. The enhanced chimeric antigen receptor-T cell of claim 4, wherein the intracellular domain comprises an intracellular signaling region, optionally further comprising a costimulatory signaling region; Further alternatively, wherein the intracellular signaling region is derived from one or more of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CCD5, CD22, CD79a, CD79b, fcRgamma, fcRbeta, CD66d, DAP10, DAP12, syk, optionally the intracellular signaling region is derived from CD3 zeta, more optionally the amino acid sequence of the intracellular signaling region is as shown in SEQ ID NO:14, and/or Further alternatively, wherein the costimulatory signaling region is derived from one, two or more of CD2、CD3、CD7、CD27、CD28、CD30、CD40、CD83、CD244、4-1BB、OX40、LFA-1、ICOS、LIGHT、NKG2C、NKG2D、DAP10、B7-H3、MyD88, alternatively the costimulatory signaling region is derived from 4-1BB, CD28 or OX40, and more alternatively the costimulatory signaling region has the amino acid sequence shown as SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO: 17.
  8. 8. The enhanced chimeric antigen receptor-T cell according to any one of claims 1-7, further comprising a leader peptide at the N-terminus of the chimeric antigen receptor amino acid sequence, optionally wherein the leader peptide is derived from CD8 a, more optionally wherein the leader peptide has the amino acid sequence shown in SEQ ID No. 18.
  9. 9. The enhanced chimeric antigen receptor-T cell of any one of claims 1-8, wherein the chimeric antigen receptor has an amino acid sequence as set forth in SEQ ID NOs 19-30.
  10. 10. The enhanced chimeric antigen receptor-T cell according to claim 9, wherein the nucleotide sequences encoding the chimeric antigen receptor are shown in SEQ ID NOs 31-42, respectively.
  11. 11. The enhanced chimeric antigen receptor-T cell according to any one of claims 1-10, further comprising a knockout of CTLA-4 site, tigit site or NKG2A site in the genome of the T cell.
  12. 12. The enhanced chimeric antigen receptor-T cell of any one of claims 1-11, wherein the chimeric antigen receptor-T cell is a chimeric antigen receptor- γδ T cell.
  13. 13. A method of making an enhanced chimeric antigen receptor-T cell comprising: 1. Preparing T cells; 2. introducing a gene editing tool and a donor template into a T cell, wherein the gene editing tool and the donor template are capable of inserting into the genome of the T cell a nucleotide sequence encoding a chimeric antigen receptor, the nucleotide sequence comprising at least one of the following genes: NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene or NKp46 gene, Such that the expression level of the NKG2D protein, DNAM1 protein, NKp30 protein, NKp44 protein or NKp46 protein of the chimeric antigen receptor-T cell is higher than the expression level of the corresponding protein in the original T cell.
  14. 14. The method of preparation of claim 13, wherein the gene editing tool is selected from one of a CRISPR/Cas system, a zinc finger nuclease system, a transcription activator-like effector nuclease system; Optionally, the gene editing tool is selected from a CRISPR/Cas system; more optionally, the CRISPR/Cas system comprises a Cas protein and a sgRNA.
  15. 15. The method of preparation of claim 14, wherein the Cas protein comprises any one of spCas9, asCas a, or LbCas a; The sgRNA comprises any one of TRAC site, PD1 site, regnase-1 site, roquin-1 site, CISH site and BCOR site; Alternatively, the sgRNA sequence for the TRAC site is: TRAC:5’-AGAGTCTCTCAGCTGGTACACGG-3’(SEQ ID NO:43); The sgRNA sequence for the PD1 site is: PD1:5’-CGACTGGCCAGGGCGCCTGT-3’(SEQ ID NO:44); the sgRNA sequence for position Regnase-1 is: Regnase-1:5’-AAGGAGGTCTTCTCCTGCCG-3’(SEQ ID NO:45); the sgRNA sequence for position Roquin-1 is: Roquin-1:5’-TGAAGACACAAAGCATTATG-3’(SEQ ID NO:46); the sgRNA sequence for CISH site is: CISH:5’-GGCGCATCCTCCTTAGGCAT-3’(SEQ ID NO:47); the sgRNA sequence for BCOR site is: BcoR:5’-AGCACGGCCCATAGGATCGA-3’(SEQ ID NO:48)。
  16. 16. The method of any one of claims 13-15, wherein the donor template is plasmid DNA, dsDNA, or ssDNA; Alternatively, the donor template is dsDNA comprising an NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene, or NKp46 gene; More optionally, the donor template comprises in the 5'-3' direction a left homology arm sequence, a polyA, a marker gene sequence, a nucleotide sequence encoding an intracellular domain, a nucleotide sequence encoding a transmembrane region, a nucleotide sequence encoding a hinge region, a nucleotide sequence encoding an extracellular antigen recognition domain, a promoter sequence, and a right homology arm sequence; further optionally, the donor template further comprises a nucleotide sequence encoding a signal peptide, and the nucleotide sequence encoding a signal peptide is located between the promoter sequence and the nucleotide sequence encoding the extracellular antigen recognition domain.
  17. 17. The method according to claim 16, wherein the promoter is selected from SFFV promoter, CMV promoter or EF 1. Alpha. Promoter, optionally the nucleotide sequence of the EF 1. Alpha. Promoter is shown in SEQ ID NO. 49; the marker gene sequence is an EGFP fluorescent protein gene or a GFP fluorescent protein gene, and optionally, the nucleotide sequence of the GFP fluorescent protein gene is shown as SEQ ID NO. 50; the polyA is a BGH polyA signal sequence, and optionally, the nucleotide sequence of the BGH polyA signal sequence is shown as SEQ ID NO. 51.
  18. 18. The method according to claim 16, wherein the nucleotide sequence encoding the extracellular antigen-recognition domain comprises a nucleotide sequence encoding a NKG2D protein, a nucleotide sequence encoding a DNAM1 protein, a nucleotide sequence encoding a NKp30 protein, a nucleotide sequence encoding a NKp44 protein or a nucleotide sequence encoding a NKp46 protein, and/or The nucleotide sequence encoding the hinge region is a nucleotide sequence encoding CD8 alpha, and/or The nucleotide sequence encoding the transmembrane region is a nucleotide sequence encoding CD8 alpha or CD3, and/or, The nucleotide sequence encoding the intracellular domain includes the nucleotide sequence encoding CD3 zeta, and the nucleotide sequence encoding 4-1BB, CD28 or OX 40.
  19. 19. The method of any one of claims 13-18, wherein the left and right homology arm sequences in the donor template are set forth in SEQ ID No. 52 and SEQ ID No. 53, respectively.
  20. 20. The method of any one of claims 13-19, wherein in step 1, the T cells are activated with a lighting, antibody or α -Glacer.

Description

Enhanced chimeric antigen receptor-T cell and preparation method and application thereof Technical Field The invention relates to the field of biological medicine, in particular to an enhanced chimeric antigen receptor-T cell, a preparation method and application thereof. Background Chimeric antigen Receptor T cell therapy (CHIMERIC ANTIGEN Receptor T CELL THERAPY, CAR-T) was most widely studied in the treatment of B cell-derived malignancies and achieved encouraging therapeutic effects. The clinical experiment result of the phase I shows that the CR of the CD19 CAR-T treatment on the recurrent refractory Acute Lymphoblastic Leukemia (ALL) can reach 69% -90%. However, the success of CD19 CAR-T on ALL has not been replicated in Acute Myelogenous Leukemia (AML) and solid tumors. The autologous CAR-T cells need to be subjected to a series of transformation, amplification and quality inspection in vitro, have the problems of high production difficulty, high cost, long patient waiting time and the like, and limit the large-scale clinical use. This is an urgent need for the advancement of off-the-shelf allogeneic CAR-T therapies. γδt is a subset of T cells, mainly present in tissues, accounting for 1% to 5% of circulating T cells, with TCRs consisting of gamma and delta subunits. Unlike αβt cells, γδ T cell activation is not affected by patient-specific MHC molecules, i.e. the tumor-associated antigen is recognized in an MHC-independent manner. Thus, allogeneic γδ T cells do not induce graft versus host disease (graft versus host disease, GVHD). Studies have shown that CD20 CAR γδ T cells do not induce GVHD, whereas CD20 CAR αβ T cell therapy produces GVHD, resulting in increased mortality. In 2015, the transcriptional patterns of 5872 patient tumor samples in 25 malignant tumors are analyzed, and the research of the existence correlation of gamma delta T cells and the total survival rate is found in [Gentles,A.J.,et al.,The prognostic landscape of genes and infiltrating immune cells across human cancers.Nat Med,2015.21(8):p.938-945.].2007 years, KT Godder et al, and the research shows that the patients with higher gamma delta T cell level after hematopoietic stem cell transplantation have 54.4 percent of five-year survival rate, 70.8 percent of total survival rate and lower gamma delta T cell level, and the five-year survival rate is 19.1 percent and the total survival rate is 19.6％[Godder,K.T.,et al.,Long term disease-free survival in acute leukemia patients recovering with increased gammadelta T cells after partially mismatched related donor bone marrow transplantation.Bone Marrow Transplant,2007.39(12):p.751-7]., so that the gamma delta T plays an important role in disease control and overall survival. The existing gamma delta T cells have good safety, but the curative effect is required to be improved. However, the existing CAR-gamma delta T depends on targets, and the escape of antigens cannot be avoided along with treatment, so that the recurrence rate of tumors is high, and the escape of antigens needs to be overcome. Disclosure of Invention The invention aims to provide an enhanced chimeric antigen receptor-T cell, a preparation method and application thereof, wherein the enhanced chimeric antigen receptor-T cell can effectively target and attack tumor cells, has high killing rate on tumors, does not depend on specific tumor antigens and specific tumor mutations, hopefully breaks through antigen escape, and can be used for preparing anti-tumor products. To achieve the above object, the present invention provides an enhanced chimeric antigen receptor-T cell in which a nucleotide sequence encoding a chimeric antigen receptor is exogenously inserted into the genome of the T cell, said nucleotide sequence comprising at least one of the following genes: NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene or NKp46 gene, Such that the expression level of the NKG2D protein, DNAM1 protein, NKp30 protein, NKp44 protein or NKp46 protein of the chimeric antigen receptor-T cell is higher than the expression level of the corresponding protein in the original T cell. In certain embodiments, the sequences of the NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene and NKp46 gene of the enhanced chimeric antigen receptor-T cell are shown as SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively. In certain embodiments, the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell comprises a TRAC site, a PD1 site, a Regnase-1 site, a Roquin-1 site, a CISH site and a BCOR site; Alternatively, the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell is a TRAC site; More optionally, the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell is the TRAC site, and the Regnase-1 site in the genome of the T cell is knocked out. In certain embodiments,