Construction and Functional Evaluation of Cyclic Peptide-Based CAR T Cells in Tumor Models

Cyclic peptides are emerging as a promising class of recognition modules for chimeric antigen receptor (CAR) engineering. Compared with single-chain variable fragment (scFv)-based CARs, disulfide-directed multicyclic peptides (DDMPs) represent a novel alternative, offering a markedly smaller molecular size (<5 kDa), enhanced structural stability through disulfide-directed cyclization, and broad tolerance to sequence diversification that supports systematic affinity and specificity optimization. DDMP-based CAR T cells leverage these properties to 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. While DDMPs are used as the exemplar recognition module, sections A and C-L of the protocol are directly applicable to any CAR format, including scFv- and nanobody-based designs with minimal modifications, making the workflow accessible to the broader CAR T-cell research community. 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. Key features • DDMPs (<5 kDa) are compact, disulfide-cyclized antigen recognition modules that tolerate extensive sequence diversification, enabling affinity and specificity optimization beyond conventional scFv-based CARs. • An integrated pipeline normalizes all functional comparisons to CAR-positive cell numbers, eliminating transduction efficiency as a confounding variable across construct designs. • Complementary readouts cross-validate efficacy and specificity: NFAT activation, luminescence-based killing, flow cytometry-based cytolysis, and ELISA-based cytokine secretion. • Xenograft imaging via the in vivo imaging system (IVIS) validates DDMP-CAR T-cell antitumor activity, extending cross-validation to preclinical tumor models.