Construction and functional evaluation of cyclic peptide-based CAR T cells in tumor models
摘要
Cyclic peptide-based chimeric antigen receptor (CAR) T cells provide a compact, engineerable recognition modality that can mediate antigen-dependent cytotoxicity while exhibiting an attenuated cytokine secretion profile, supporting the development of potentially safer immunotherapies for solid tumors. Here, we present a comprehensive workflow spanning CAR construct design and generation through in vitro and in vivo functional evaluation. The protocol includes the generation of Jurkat NFAT reporter cell lines and luciferase-expressing tumor target lines, which are widely used in different assays. Together, these standardized readouts enable rigorous, objective comparison of CAR T cell efficacy and safety across tumor models.
参考文献
Q. Xiao, X. Su, Anti-tumor Efficacy of CD19 CAR-T in a Raji B Cell Xenografted Mouse Model. Bio Protoc. 2023, 13, e4655.
2. S. Prommersberger, M. Hudecek, T. Nerreter, Antibody-Based CAR T Cells Produced by Lentiviral Transduction. Curr. Protoc. Immunol. 2020, 128, e93.
3. M. M. Safont, et al., Protocol for the development of a bioluminescent AML-PDX mouse model for the evaluation of CAR T cell therapy. STAR Protoc. 2024, 5, 103522.
4. D. Zhang, et al., Protocol to generate traceable CAR T cells for syngeneic mouse cancer models. STAR Protoc. 2024, 5, 102898.
5. M. E. Selli, et al., Inducing T cell dysfunction by chronic stimulation of CAR-engineered T cells targeting cancer cells in suspension cultures. STAR Protoc. 2023, 4, 101954.
6. N. Tatari, et al., Preclinical Testing of CAR T Cells in a Patient-Derived Xenograft Model of Glioblastoma. STAR Protoc. 2020, 1, 100174.
7. C. L. Wu, J. C. Leroux, M. A. Gauthier, Twin disulfides for orthogonal disulfide pairing and the directed folding of multicyclic peptides. Nat. Chem. 2012, 4, 1045–1050.
8. C. Wu, Motif-Directed Oxidative Folding to Design and Discover Multicyclic Peptides for Protein Recognition. Acc. Chem. Res. 2025, 58, 1620–1631.
9. C. Xu, et al., Directed Evolution of Multicyclic Peptides Using Yeast Display for Sensitive and Selective Fluorescent Analysis of CD28 on the Cell Surface. Anal. Chem. 2025, 97, 4031–4040.
10. Z. P. Duan, et al., Triscysteine disulfide-directing motifs enabling design and discovery of multicyclic peptide binders. Nat. Commun. 2024, 15, 7799.
11. S. M. Lu, et al., Disulfide-directed multicyclic peptide libraries for the discovery of peptide ligands and drugs. J. Am. Chem. Soc. 2023, 145, 1964–1972.
12. J. Li, et al., De Novo Discovery of Cysteine Frameworks for Developing Multicyclic Peptide Libraries for Ligand Discovery. J. Am. Chem. Soc. 2023, 145, 28264–28275.
13. S. Yao, et al., De novo design and directed folding of disulfide-bridged peptide heterodimers. Nat. Commun. 2022, 13.
14. Y. P. Wu, et al., Structure-guided design of CPPC-paired disulfide-rich peptide libraries for ligand and drug discovery. Chem. Sci. 2022, 13, 7780–7789.
15. S. M. Lu, et al., Directed disulfide pairing and folding of peptides for the de novo development of multicyclic peptide libraries. J. Am. Chem. Soc. 2020, 142, 16285–16291.
16. Y. Zheng, et al., Artificial disulfide-rich peptide scaffolds with precisely defined disulfide patterns and a minimized number of isomers. Chem. Sci. 2017, 8, 2547–2552.
17. X. Meng, et al., Disulfide-Directed Multicyclic Peptides for Chimeric Antigen Receptors Targeting Solid Tumors. J. Am. Chem. Soc. 2026, 148, 400–413.
18. H. Liu, et al., Proline-mediated enhancement in evolvability of disulfide-rich peptides for discovering protein binders. J. Am. Chem. Soc. 2025, 147, 24870–24883.
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