Generation of a novel pan-cancer chemokine receptor-modified CAR-T cell therapy directed towards nfP2X7

Abstract

CAR-T cell therapy has achieved significant success in the treatment of multiple haematological malignancies. However, solid cancers present challenges such as tumour antigen heterogeneity and complex mechanisms of immune evasion that have limited the translation of CAR-T cell therapies from haematological malignancies to solid cancers. This study details the development of a novel CAR-T cell therapy directed towards the nonfunctional form of the P2X7 receptor (nfP2X7), which is an ATP receptor expressed by a diverse range of malignant cells that is completely absent on healthy tissues. This is the first use of nfP2X7 as a target antigen for CAR-T cell therapy and represents one of the first CAR-T cell therapies developed with the potential to act universally against different solid cancers. To investigate the role of nfP2X7-targeting CAR-T cells in antitumour cytotoxicity, minimally differentiated CAR-T cells were generated, which were highly activated, cytotoxic and displayed minimal expression of co-inhibitory receptors. NfP2X7-targeting CAR-T cells effectively lysed tumour cell lines derived from a broad range of solid and haematological cancers including breast, prostate, ovarian, colorectal, skin, lung, neuroblastoma and B-cell lymphoma in vitro. Furthermore, administration of CAR-T cells significantly inhibited the growth of highly aggressive triple-negative human breast cancer and human prostate cancer xenografts in immunocompromised mice, which was associated with an increased intratumoural accumulation of CAR-T cells compared to untransduced control T cells. After identification of a suitable target antigen, the next major challenge is homing CART cells into hostile and densely barricaded tumours. As many solid tumours are poorly infiltrated by T cells due to a lack of appropriate chemokine receptor signalling, the investigation moved onto the concept of engineering the expression of select chemokine receptors onto CAR-T cells to enhance their trafficking into solid tumours. As the ligands for CXCR2, CXCR6 and CCR10 were found to be highly expressed in human breast and prostate tumours, these chemokine receptors were engineered to be co-expressed with the nfP2X7-specific CAR. In vivo delivery of CXCR2-expressing CAR-T cells resulted in enhanced tumour inhibition compared to control CAR-T cells. This may be attributed to enhanced proliferation and/or cytotoxicity of CAR-T cells mediated by the engineered expression of CXCR2, which is a consequence of CXCR2 expression that has not been previously reported on T cells. In contrast, CXCR6 and CCR10 overexpression on CART cells did not improve their in vivo anti-tumour efficacy. These results demonstrate that engineered CXCR2 expression significantly improves the in vivo anti-tumour efficacy of CAR-T cells in some contexts. Altogether, this study details the preclinical development of a novel CAR-T cell therapy directed towards nfP2X7. These findings highlight the potential of nfP2X7-targeting CAR-T cells as a novel pan-cancer immunotherapy and provides evidence towards a potential role of CXCR2 in improving CAR-T cell anti-tumour efficacy. By addressing the initial challenges related to the identification of a suitable tumour antigen and directed homing of CAR-T cells into solid tumours, it may provide a potential avenue to translate the success of CAR-T cell therapies from haematological malignancies to a broad range of solid cancers.