RESUMO
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).
Assuntos
Engenharia Genética/métodos , Hipóxia/metabolismo , Receptores de Antígenos Quiméricos/síntese química , Citocinas/metabolismo , Citotoxicidade Imunológica , Humanos , Imunoterapia Adotiva/métodos , Ativação Linfocitária/imunologia , Oxigênio/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Microambiente Tumoral/fisiologiaAssuntos
Humanos , Imunoterapia Adotiva/métodos , Linfoma Difuso de Grandes Células B/terapia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Receptores de Antígenos Quiméricos/uso terapêutico , Imunoterapia Adotiva/instrumentação , Imunoterapia Adotiva/tendências , Linfoma Difuso de Grandes Células B/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Receptores de Antígenos Quiméricos/síntese químicaRESUMO
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.
