RESUMEN
B-cell maturation antigen (BCMA) is a promising therapeutic target for multiple myeloma (MM), but expression is variable, and early reports of BCMA targeting chimeric antigen receptors (CARs) suggest antigen downregulation at relapse. Dual-antigen targeting increases targetable tumor antigens and reduces the risk of antigen-negative disease escape. "A proliferation-inducing ligand" (APRIL) is a natural high-affinity ligand for BCMA and transmembrane activator and calcium-modulator and cyclophilin ligand (TACI). We quantified surface tumor expression of BCMA and TACI on primary MM cells (n = 50). All cases tested expressed BCMA, and 39 (78%) of them also expressed TACI. We engineered a third-generation APRIL-based CAR (ACAR), which killed targets expressing either BCMA or TACI (P < .01 and P < .05, respectively, cf. control, effector-to-target [E:T] ratio 16:1). We confirmed cytolysis at antigen levels similar to those on primary MM, at low E:T ratios (56.2% ± 3.9% killing of MM.1s at 48 h, E:T ratio 1:32; P < .01) and of primary MM cells (72.9% ± 12.2% killing at 3 days, E:T ratio 1:1; P < .05, n = 5). Demonstrating tumor control in the absence of BCMA, we maintained cytolysis of primary tumor expressing both BCMA and TACI in the presence of a BCMA-targeting antibody. Furthermore, using an intramedullary myeloma model, ACAR T cells caused regression of an established tumor within 2 days. Finally, in an in vivo model of tumor escape, there was complete ACAR-mediated tumor clearance of BCMA+TACI- and BCMA-TACI+ cells, and a single-chain variable fragment CAR targeting BCMA alone resulted in outgrowth of a BCMA-negative tumor. These results support the clinical potential of this approach.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Antígeno de Maduración de Linfocitos B/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Receptores Quiméricos de Antígenos/uso terapéutico , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Animales , Antineoplásicos Inmunológicos/síntesis química , Antineoplásicos Inmunológicos/química , Línea Celular Tumoral , Citotoxicidad Inmunológica , Humanos , Ligandos , Ratones , Terapia Molecular Dirigida , Receptores Quiméricos de Antígenos/síntesis química , Receptores Quiméricos de Antígenos/química , Proteína Activadora Transmembrana y Interactiva del CAML/química , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/químicaRESUMEN
Exploiting hypoxia in solid malignancies to restrict expression of chimeric antigen receptors (CARs) on engineered T cells to the tumor microenvironment overcomes the risk of on-target off-tumor toxicity and minimizes tonic signaling, which promotes CAR T cell exhaustion. This protocol summarizes the synthetic biology underlying the development of a stringent oxygen-sensitive CAR for in vitro and in vivo preclinical characterization. For complete details on the use and execution of this protocol, please refer to Kosti et al. (2021).