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CN-122012624-A - Novel TCR-CAR plasmid and CAR-T cell for expressing flagellin, preparation method thereof and application thereof in resisting tumor

CN122012624ACN 122012624 ACN122012624 ACN 122012624ACN-122012624-A

Abstract

The invention discloses a novel TCR-CAR plasmid for expressing flagellin, a CAR-T cell, a preparation method thereof and application thereof in resisting tumors, and belongs to the technical field of CAR-T cells. The novel structural design of the invention can start the expression secretion of the flagellin along with the activation of the T cells and release the flagellin to tumor sites after the CAR recognizes the target antigen, and the release of the flagellin gradually subsides after the activation signal disappears, and starts a new round of secretion and release after the CAR recognizes the target antigen again. The invention can effectively solve the technical problems that the release of flagellin polypeptide cannot be controlled accurately in the prior art, the release of engineered immune cells is random, and further, in the application of treating solid tumors, the anti-tumor effect cannot be exerted because the tumor cannot be effectively accessed into the interior of the tumor.

Inventors

  • WAN XIAOCHUN
  • ZHANG GUIZHONG
  • Niu Xiangyun
  • ZHANG PENGCHAO
  • Dai liujiang
  • Peng Xixia

Assignees

  • 中国科学院深圳先进技术研究院

Dates

Publication Date
20260512
Application Date
20241112

Claims (10)

  1. 1. A novel TCR-CAR plasmid expressing flagellin, characterized in that a nucleic acid sequence of a promoter element and codon optimized flagellin FLAGELLIN is added to the second generation CAR-T, which is induced by NFAT.
  2. 2. The novel flagellin-expressing TCR-CAR plasmid of claim 1, wherein the 5' end of the plasmid is linked to an NFAT-inducible expression promoter element and FLAGELLIN gene, the FLAGELLIN gene being independently inducible by the NFAT-inducible expression promoter element to drive its expression.
  3. 3. A novel flagellin-expressing TCR-CAR plasmid as claimed in claim 2 wherein the amino acid sequence of the anti-CD19 CAR, which does not comprise the NFAT-FLAGELLIN sequence, is as shown in SEQ ID No. 1.
  4. 4. A novel TCR-CAR plasmid expressing a flagellin as claimed in any one of claims 1 to 3 wherein the amino acid sequence of the codon optimised flagellin FLAGELLIN is as shown in SEQ ID No. 2.
  5. 5. A novel TCR-CAR plasmid expressing flagellin as claimed in any one of claims 1 to 3 wherein in the second generation CAR-T the CAR structure consists of scFV, CD8 a hinge and transmembrane domain, 4-1BB co-stimulatory domain and cd3ζ signaling activating domain targeting human CD19, whilst introducing a Flag tag at the N-terminus, driven by the EF 1a promoter.
  6. 6. A recombinant lentivirus obtained by transfecting a host cell with the novel flagellin-expressing TCR-CAR plasmid of any one of claims 1-5.
  7. 7. The recombinant lentivirus of claim 6, wherein the host cell comprises a human primary T cell.
  8. 8. A flagellin CAR-T cell expressed from a recombinant lentiviral infected T cell of claim 6 or 7, wherein: The T cells comprise peripheral blood T cells, umbilical cord blood T cells or T cells derived from stem cell induction.
  9. 9. The use of a novel flagellin-expressing TCR-CAR plasmid according to any one of claims 1-5, a recombinant lentivirus according to claim 6 or 7, or a flagellin-expressing CAR-T cell according to claim 8 for the preparation of an anti-solid tumor drug, wherein the drug is a drug capable of controlling expression and secretion of flagellin.
  10. 10. An anti-tumor pharmaceutical composition comprising an effective amount of the flagellin CAR-T cell of claim 8.

Description

Novel TCR-CAR plasmid and CAR-T cell for expressing flagellin, preparation method thereof and application thereof in resisting tumor Technical Field The invention belongs to the technical field of CAR-T cells, and particularly relates to a novel TCR-CAR plasmid for expressing flagellin, a CAR-T cell, a preparation method thereof and application thereof in resisting tumors. Background The CAR-T therapy utilizes the specificity and durability of immune cells to kill tumor cells, provides a more accurate and long-acting treatment mode for tumor patients, and has remarkable anti-tumor activity in various hematopoietic malignant tumors. There are a number of cell products available worldwide that target CD19 and B cell maturation antigens (B cell maturation antigen, BCMA). However, although CAR-T cell therapy is promising for hematological tumors, it is much less effective for solid tumors that constitute the vast majority of cancers. The high antigenic heterogeneity in solid tumors compared to liquid tumors provides them with an efficient mechanism for escaping CAR-T cells, which typically encode a specificity for a single antigenic target, and thus fail to recognize all cancer cells in a tumor. Secondly, solid tumors have complex tumor microenvironments, so that CAR-T cells are difficult to infiltrate, and effective tumor killing cannot be realized. Immunosuppressive ligands, such as PD-L1, that are overexpressed on tumor cells can bind to PD-1 expressed on T cells resulting in a loss of killing capacity of T cells. When the CAR-T cells treat solid tumors, immune checkpoint pathway inhibitors are simultaneously administered to block the starting of a brake system, so that the anti-tumor activity of the CAR-T cells can be enhanced. The CAR-T cells combine or co-express cytokines, such as IL-12, IL-18, IL-15, etc., and studies indicate that CAR-T cell cytotoxicity can be enhanced, TME is remodeled, T cell infiltration is promoted, the number of M1 type macrophages and natural killer (natural killers, NKs) cells is increased, the number of Tregs is reduced, the anti-tumor activity of CAR-T and other immune cells is enhanced, and the persistence of CAR-T cells in vivo can also be increased. Toll-like receptors (Toll like receptors, TLRs), a type I transmembrane protein, a novel pattern recognition receptor (Pattern recognition receptor, PRR) which is thought to be the link between innate immunity and acquired immunity, are involved in immune diseases by distinguishing pathogen-associated molecular patterns (Pathogen-associated molecular pattern, PAMPs) from some endogenous ligands. Mammalian TLRs are partially localized to the cell membrane and include TLRs 4 (recognizing bacterial LPS), TLR5 (recognizing bacterial flagellin) and TLRs 1,2 and 6 (recognizing bacterial lipoproteins), and intracellular localized TLRs such as nucleic acids of TLR3 (recognizing double-stranded RNA), TLR7 and 8 (recognizing single-stranded RNA), TLR9 (recognizing unmethylated CpG containing ssDNA) and TLR13 (recognizing bacterial ribosomal RNA). In addition to TLR3, the signaling pathway of other TLRs is primarily dependent on MYD88 signaling pathway. TLR agonists are capable of activating the immune system to kill tumor cells, thereby eliciting strong anti-tumor activity, and therefore the use of TLR-specific agonists has great potential in tumor therapy. TLR5 can bind to the monomeric form of flagellin of most gram-negative bacteria to induce MyD 88-dependent signal transduction and can activate nuclear transcription factor NF- κb in epithelial cells, monocytes and dendritic cells, thereby activating the innate immune response of the body against flagellin bacteria. TLR5 is present on a variety of cells including monocytes, macrophages, neutrophils, lymphocytes, NK cells, dendritic cells, epithelial cells and lymph node stromal cells, and studies have shown that the use of flagellin in combination with CpG-ODN induces the expression of IL-12 and IL-23 in DCs, inducing a Th1 cellular immune response, producing a synergistic anti-tumor response. The engineering salmonella expression and secretion vibrio vulnificus FlaB flagellin can effectively target TLR5 in tumor microenvironment to play anti-tumor activity, and the increase of M1 phenotype in tumor infiltration macrophages can be obviously observed. The flagellin-Grp 170 fusion protein drives DCs better presentation of tumor antigens to CD8 + T cells, which in turn induces a potent cytotoxic CD8 + T cell response at the tumor site and throughout the body. T cells secreting TLR5 ligands can also improve tumor microenvironment, inducing anti-tumor responses, which also suggests targeting TLR5 in tumors as a novel strategy to remodel tumor microenvironment. The recombinant salmonella flagellin derivative CBLB502 has binding activity with TLR5 to induce activation of NF- κB signaling pathway. CBLB502 can significantly improve the effect of CAR-NK in colon cancer mouse model, and can acti