Nanobody MET CAR T cells show efficacy in solid tumors

Abstract

BackgroundMET overexpression is associated with poor prognosis in many solid tumors due to its central role in tumor survival, invasion, metastasis, and chemoresistance. While targeting MET with antibody-drug conjugates has shown promising results, engineered cellular immunotherapeutic approaches have not been extensively explored. Compared to conventional single-chain variable fragments (scFv), naturally occurring single-domain antibodies consisting of variable heavy chains only (VHH or nanobodies) are smaller, retain high specificity, and exhibit remarkable biochemical stability. In this study, we tested the efficacy of MET-targeting VHH-CAR-T (chimeric antigen receptor T cells). MethodsWe generated a panel of VHH-CAR-Ts using mRNA electroporation. VHH-CAR-T cells were evaluated in functional assays including cell binding avidity, cytokine production profiles, hydrogel microwell-based cellular kinetics, and in vitro cytotoxicity. We also assessed the therapeutic efficacy of VHH-CAR-T in an in vivo mouse model of metastatic triple negative breast cancer (TNBC). ResultsAmong the tested VHH, we identified those with intermediate avidity as most effective for in vitro tumor killing. VHH-CAR-Ts with CD28 costimulatory domains demonstrated augmented cytotoxicity with favorable selectivity, requiring a minimum antigen density threshold for activation. Mechanistically, VHH-CAR-Ts demonstrated low tonic signaling, high avidity, potent cytokine production, and rapid tumor killing kinetics. When administered in an mRNA format, VHH-CAR-Ts exhibited potent and prolonged control of tumor growth in an in vivo metastatic model of TNBC. ConclusionTaken together, these results demonstrate that VHH-CAR-Ts exhibit robust MET specificity and potent therapeutic efficacy both in vitro and in vivo. Thus, VHH-CAR-T cell therapy represents a promising immunotherapeutic strategy for targeting MET-overexpressing solid tumors. What is already known on this topicMET signaling is an important contributor to the aggressiveness of many solid tumors, and targeting MET by antibody-drug conjugates has shown efficacy and safety. Targeting MET by CAR-T cells has been under study, though with limited potency. What this study addsThis study is the first to demonstrate effectiveness of anti-MET VHH-CAR-T cells. Compared with other antigen binding domains, VHH-incorporated CAR-T cells show low tonic signaling, a favorable cytokine profile, and potent tumor killing. How this study might affect research, practice or policyWith the multiple advantages of VHHs including small size, stability, and low potential for tonic signaling, VHH-CAR-T cells represent a promising approach for CAR-T design against solid tumors.