Abstract
BackgroundChimeric antigen receptors (CARs) presented on T cell surfaces enable redirection of T cell specificity, which has enormous promise in antitumor therapy. However, excessive activity and poor control over such engineered T cells cause significant safety challenges, such as cytokine release syndrome and organ toxicities. To enhance the specificity and controllable activity of CAR-T cells, we report a novel switchable dual-receptor CAR-engineered T (sdCAR-T) cell and a new switch molecule of FITC-HM-3 bifunctional molecule (FHBM) in this study.MethodsWe designed a fusion molecule comprising FITC and HM-3. HM-3, an antitumor peptide including an Arg-Gly-Asp sequence, can specifically target integrin αvβ3 that is presented on some tumor cells. Moreover, to improve the specificity of CAR-T cells, we also generated the sdCAR-T cell line against cognate tumor cells expressing human mesothelin (MSLN) and integrin αvβ3. Finally, the activity of sdCAR-T cell and FHBM is verified via in vitro and in vivo experiments.ResultsIn the presence of FHBM, the designed sdCAR-T cells exerted high activity including activation and proliferation and had specific cytotoxicity in a time- and dose-dependent manner in vitro. Furthermore, using a combination of FHBM in nude mice, sdCAR-T cells significantly inhibited the growth of MSLN+ K562 cells and released lower levels of the cytokines (e.g., interleukin-2, interferon γ, interleukin-6, and tumor necrosis factor α) relative to conventional CAR-T cells, obtaining specific, controllable, and enhanced cytotoxicity.ConclusionsOur data indicate that FHBM can accurately control timing and dose of injected CAR-T cells, and sdCAR-T cells exert significant antitumor activity while releasing lower levels of cytokines for the cognate tumor cells expressing both MSLN and integrin αvβ3. Therefore, combination therapies using sdCAR-T cells and the switch molecule FHBM have significant potential to treat malignancies.
Highlights
Chimeric antigen receptors (CARs) presented on T cell surfaces enable redirection of T cell specificity, which has enormous promise in antitumor therapy
Our data indicate that FITC-HM-3 bifunctional molecule (FHBM) can accurately control timing and dose of injected CAR-T cells, and switchable dual-receptor CAR-engineered T (sdCAR-T) cells exert significant antitumor activity while releasing lower levels of cytokines for the cognate tumor cells expressing both MSLN and integrin αvβ3
Design features of the sdCAR structure To engineer an sdCAR that simultaneously interacts with both a tumor antigen and an exogenous molecule for cell activation and proliferation, a dual-receptor CAR model was designed based on the signal pathways of natural immune T cell receptor (TCR). sdCAR consisted of two receptors to control downstream signaling elements, including the immunoreceptor tyrosine-based activation motifs from the TCR/CD3ζ subunit and a costimulatory signal element 4-1BB (Fig. 1a)
Summary
Chimeric antigen receptors (CARs) presented on T cell surfaces enable redirection of T cell specificity, which has enormous promise in antitumor therapy. The adoptive transfer of genetically engineered T cells expressing a chimeric antigen receptor (CAR) specific for the tumor antigen CD19 has been highly successful in the treatment of some human malignancies, including leukemia, lymphoma, and other hematological cancers [1,2,3,4,5,6]. Kloss et al created dual-receptor-engineered T cells that have specific activity against the prostate tumors expressing both prostate stem cell antigen and prostate-specific membrane antigen [17] Another approach is to engineer a switch molecule to control the activity of CAR-T cells. We propose a new CAR method using a dual-receptor model to enhance the efficacy and safety of T cells by introducing a novel bifunctional molecule. Compared with using a single molecule as a switch, a bifunctional molecule significantly improves the therapeutic effects of CAR-T cells by constitutive antitumor activity
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