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/fisiologiaRESUMO
Utilizing T cells expressing chimeric antigen receptors (CARs) to identify and attack solid tumors has proven challenging, in large part because of the lack of tumor-specific targets to direct CAR binding. Tumor selectivity is crucial because on-target, off-tumor activation of CAR T cells can result in potentially lethal toxicities. This study presents a stringent hypoxia-sensing CAR T cell system that achieves selective expression of a pan-ErbB-targeted CAR within a solid tumor, a microenvironment characterized by inadequate oxygen supply. Using murine xenograft models, we demonstrate that, despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumor efficacy without off-tumor toxicity. This dynamic on/off oxygen-sensing safety switch has the potential to facilitate unlimited expansion of the CAR T cell target repertoire for treating solid malignancies.
Assuntos
Hipóxia/metabolismo , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/genética , Linfócitos T/metabolismo , Microambiente Tumoral/imunologia , Animais , Linhagem Celular Tumoral/metabolismo , Modelos Animais de Doenças , Genes erbB/genética , Humanos , Hipóxia/genética , Imunoterapia Adotiva/métodos , Camundongos Transgênicos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Background: Histamine is widely used as a pharmacological tool for the evaluation of airway responsiveness. Nevertheless, undesirable and contradictory effects have been described after histamine provocation tests. In previous evaluations of airway responsiveness in a guinea pig asthma model, the control groups consistently showed high neutrophil counts in bronchoalveolar lavage fluid (BALF) immediately after the histamine challenge. The changes in cytokine and chemokine levels in guinea pig lung associated with histamine induced-neutrophilia are described in this paper. Methods: Immediately and 24 h after histamine challenge, airway wall and BALF eosinophil and neutrophil counts as well as lung cytokines (IL-5, IL-10, IL-17A, TNFα and TGFβ) and chemokines (CCL11 and CXCL8) levels were evaluated. Results: Histamine inhalation generated an all-or-none bronchial response, and the dose inducing airway obstruction was similar in all guinea pigs. Immediate increases in neutrophil counts in airway wall and BALF and in IL-5, IL-10 and IL-17A levels in the lung homogenate were observed after histamine challenge. Significant correlations were found between neutrophil counts from airway wall and IL-5, IL-10 and IL-17A levels in the lung homogenate. Conclusions: Histamine inhalation induced rapid neutrophil LBA and airway wall infiltration that was not associated with CXCL8 expression but with a Th2 and Th17 cytokines that probably are involved in the recruitment and activation of neutrophils.