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CN-121975739-A - CAR-T cell for fusion expression of LCK protein kinase domain and application thereof

CN121975739ACN 121975739 ACN121975739 ACN 121975739ACN-121975739-A

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a CAR-T cell for fusion expression of an LCK protein kinase domain and application thereof. The invention discloses an engineering T cell which expresses fusion protein of chimeric antigen receptor CAR and LCK protein kinase domain (LCK-KD) and application of the engineering T cell in preparing antitumor drugs. By virus transduction technology, three constructed different CARs and LCK-KD fusion expression retroviral vectors are respectively introduced into T cells, so that engineering T cells capable of stably expressing CAR-LCK-KD fusion proteins are successfully obtained. The method significantly reduces the size of the vector insert and the protein expression is stable. And the obtained engineering T cells have a strong killing effect on tumor cells, and simultaneously the release amount of interleukin-2 and interferon-gamma is obviously reduced, so that the risk of inducing cytokine storm is greatly reduced.

Inventors

  • ZHAO BIN
  • GUO LIWEI
  • ZHOU QI

Assignees

  • 浙江大学

Dates

Publication Date
20260505
Application Date
20251224

Claims (10)

  1. 1. An engineered T cell capable of expressing a CAR-LCK-KD fusion protein of a chimeric antigen receptor CAR and an LCK protein kinase domain.
  2. 2. The engineered T-cell of claim 1, wherein the CAR targets any one of B7-H3, CEA, HER2, MSLN, MUC1, CLDN18.2, epCAM, EGFR, EGFR variant III、CD19、CD20、CD30、CD40、CLL-1、ELANE、EFNB2、GD2/GD3、β-HCG、TG、hTERT、EDA/EDB、TnC A1、AFP、PSA、PAP、NY-ESO-1、LAGA-1a、p53、PSMA、PCTA-1、IGF1、IGF-II、IGFI、MHC、5T4、ROR1、Nkp30、NKG2D、MCSP、LRP6、CD38/CS1、MART1、WT1、MUC1、LMP2、EphA2、ML-TAP、ERG、NA17、PAX3、ALK、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD70、CD79、CD116、CD117、CD135、CD123、CD133、CD138、CTLA-4、B7-1、B7-2、BCMA、FLT-3.
  3. 3. The engineered T-cell of claim 1, wherein the CAR-LCK-KD fusion protein comprises a fusion protein of three different LCK-KD fusion positions of CAR8-LCK-KD, CAR8-LCK-KD-CD3, and CAR8-linker-LCK-KD-CD3, having amino acid sequences as shown in SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8, respectively.
  4. 4. An engineered nucleic acid molecule encoding the CAR-LCK-KD fusion protein of claim 3, comprising engineered nucleic acid molecule sequences encoding CAR8-LCK-KD, CAR8-LCK-KD-CD3, and CAR8-linker-LCK-KD-CD3 fusion proteins, having nucleotide sequences set forth in SEQ ID NO 3, SEQ ID NO 5, and SEQ ID NO 7, respectively.
  5. 5. A viral vector comprising a retroviral vector and a lentiviral vector comprising the engineered nucleic acid molecule sequence of claim 4.
  6. 6. A method of preparing an engineered T-cell according to claim 1, comprising the steps of: s.1, constructing a viral vector, wherein the viral vector comprises an engineering nucleic acid molecule sequence encoding CAR8-LCK-KD, CAR8-LCK-KD-CD3 and CAR8-linker-LCK-KD-CD3 fusion proteins; s.2, co-transfecting the virus vector and auxiliary plasmid in the S.1 into 293T cells, and packaging by a transient transfection three-plasmid system to obtain recombinant viruses; s.3, adopting the T cells after transduction and activation of the recombinant virus in S.2, and screening to obtain engineering T cells which integrate exogenous genes in genome and can continuously express CAR-LCK-KD fusion proteins.
  7. 7. The method of claim 6, wherein in step S.1, the nucleotide sequence of the CAR8-LCK-KD is shown in SEQ ID NO. 3, the nucleotide sequence of the CAR8-LCK-KD-CD3 is shown in SEQ ID NO. 5, and the nucleotide sequence of the CAR8-linker-LCK-KD-CD3 is shown in SEQ ID NO. 7.
  8. 8. A pharmaceutical composition with tumor resistance for treating tumor diseases is characterized by comprising at least one of the engineering T cells disclosed in any one of claims 1-3, or the engineering nucleic acid molecules disclosed in claim 4, or the viral vectors disclosed in claim 5, or the engineering T cells prepared by the preparation method disclosed in claim 6.
  9. 9. Use of an engineered T cell according to any one of claims 1-3, an engineered nucleic acid molecule according to claim 4, a viral vector according to claim 5 or a pharmaceutical composition according to claim 8 for the preparation of a medicament for the diagnosis, treatment or prevention of a tumor.
  10. 10. The use according to claim 9, wherein the tumour comprises a B7-H3 expressing tumour, the B7-H3 expressing tumour comprises cervical cancer and the tumour cells of the cervical cancer comprise HeLa cells.

Description

CAR-T cell for fusion expression of LCK protein kinase domain and application thereof Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a CAR-T cell for fusion expression of an LCK protein kinase domain and application thereof. Background Chimeric antigen Receptor T cell therapy (CHIMERIC ANTIGEN Receptor T cell, CAR-T) is a very promising approach to anti-tumor immune cell therapy. A CAR is an artificially constructed cell surface receptor whose protein structure includes an extracellular single-chain antibody variable region capable of specifically recognizing a tumor antigen, a transmembrane region, and an intracellular signaling region. The heart of CAR-T therapy is the engineering of CARs. The first generation CAR increases CD3 zeta chain in cells to realize basic connection of antigen recognition and T cell activation, but has weak activation signals, T cells cannot be effectively amplified and survive for a long time, clinical curative effect is limited, the second generation CAR can obviously enhance killing power and durability of the T cells by increasing CD3 zeta and an intracellular co-stimulation domain (such as CD28 or 4-1 BB), is the basis of most of current batch therapies, but faces adverse reactions such as cell exhaustion, cytokine storm and the like, the third generation CAR can further enhance activation signals by increasing CD3 zeta and two co-stimulation domains, but CAR-T cells secrete a large amount of cytokines due to excessive activation, and the novel generation CAR is combined with modular design and multifunctional integration concept to try to improve safety and accurate regulation capability so as to break through complex tumor environment, but has more complex design and needs support and verification of more clinical data. While CAR-T cell therapy has achieved several success in hematological tumor clinical treatment, it is also accompanied by a series of common and serious adverse reactions including cytokine release syndrome (cytokine release syndrome, CRS), immune effector cell-related neurotoxicity (immune effector cell-associated neurotoxicity syndrome, ICANS), hematological toxicity, B cell deficiency, and the like. The key reason is that genetically engineered T cells are overactivated in vivo, eliciting a severe immune inflammatory response and even attacking normal cells that are not tumor. LCK is a non-receptor tyrosine kinase belonging to Src family members and serves as a core participant in T cell antigen receptor signaling pathway, regulating the overall process of T cells from development, maturation to activation and function. LCK proteins comprise a membrane-contacting region, a substrate-acting region, a kinase domain and a regulatory region, and initiate downstream cascades by phosphorylating tyrosine residues on the immunoreceptor tyrosine-activating motif in the TCR-CD3 complex. Our previous studies found that co-expression of CAR proteins and LCK proteins in T cells can enhance the persistence and killing capacity of CAR-T cells. On the basis of the invention, the LCK kinase domain (LCK KINASE domain, LCK-KD) and the CAR protein are fused and expressed in the T cells, so that the release of cytokines is obviously reduced and the risk of inducing cytokine storm is reduced while the effective killing of the LCK kinase domain is ensured. Disclosure of Invention Aiming at the problems existing in the prior art, the invention aims to provide a CAR-T cell for fusion expression of LCK protein kinase domain and application thereof. In a first aspect the invention provides an engineered T cell capable of expressing a CAR-LCK-KD fusion protein of a chimeric antigen receptor CAR and an LCK protein kinase domain. Further, the CAR targets B7-H3, CEA (carcinoembryonic antigen), HER2 (human epidermal growth factor receptor 2), MSLN (mesothelin), MUC1 (mucin 1), CLDN18.2 (fibronectin 18.2), epCAM (epithelial cell adhesion molecule), EGFR (epidermal growth factor receptor), EGFR variant III, CD19, CD20, CD30, CD40, CLL-1 (C-type lectin-like molecule-1), ELANE (neutrophil elastase), EFNB2 (hepadnexin B2), GD2/GD3 (bisialoganglioside), β -HCG (β -human chorionic gonadotropin), TG (thyroglobulin), hTERT (human telomerase reverse transcriptase), EDA/EDB (extra domain a and extra domain B of fibronectin), tnC A1 (A1 domain of tenascin-C), AFP (alpha-fetoprotein), PSA (prostate specific antigen), NY-ESO 1, p-34 a, p-34, p-37-24, IGF-1, p-35B (p-35), IGF-3, p-35, 3, and insulin-related histones (insulin-35, 3, or the like factor 1, or the like). Furthermore, the CAR-LCK-KD fusion proteins comprise fusion proteins of three different LCK-KD fusion positions of CAR8-LCK-KD, CAR8-LCK-KD-CD3 and CAR8-linker-LCK-KD-CD3, the amino acid sequences of the fusion proteins are respectively shown as SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 8, and the linker is a flexible connecting molecule for connecting the CAR and the LCK