“Boosted” CAR T cell for improved safety and efficacy against low antigen density targets in neuroblastoma

Standard treatment for neuroblastoma, the most common extracranial cancer in the paediatric population, is associated with high relapse rates and toxicity, including detrimental effects on development. In haematological disease CAR T cell therapy, specifically targeting CD19, has resulted in complete remission in various leukaemias and lymphomas, however translation to the solid tumour context has been significantly more challenging. One of the limiting factors is the lack of suitable target antigens that are expressed in tumour cells but lack expression in healthy tissue to reduce on-target off- tumour toxicity. Anaplastic lymphoma kinase (ALK) is expressed in multiple paediatric and adult cancers, including neuroblastoma, retinoblastomas, rhabdomyosarcomas and non-small cell lung cancer, whilst being absent from normal tissue, making it an ideal target for CAR T cell therapy. However, expression levels on tumour cells have been reported to be below the threshold required to appropriately initiate a sustained activation response by CAR T cells. Using a novel anti-ALK binder (ALK8), which in CAR-T format has enhanced sensitivity to low antigen density, I made a number of modifications to augment the CAR T cell response. Firstly, to reduce the antigen threshold required for a sustained response, a second generation ALK8 CAR was modified to enhance signalling through a CD28 hinge/transmembrane (h/tm), as well as boosted through addition of a second CD3z ITAM previously shown to improve CAR function. A second CD3z ITAM failed to convincingly enhance CAR function with marginal 3 improvements limited to CD8h/tm containing CARs. The CD28h/tm however showed marked improvements in in vitro co-cultures with low ALK expressing neuroblastoma cell lines with increases improvements in cytotoxicity, proliferation and cytokine production. However, in long-term repetitive stimulation settings the augmented CARs failed to retain function past 3 stimulations. To further augment the CAR T cell response to low antigen density, a chimeric costimulatory receptor (CCR) responsive to a highly expressed antigen, namely B7H3, was co-expressed. This pCAR, or parallel chimeric antigen receptor, design allows simultaneous co-stimulatory signalling through two distinct receptors. Interestingly, when a matched h/tm was incorporated into the CAR and CCR component, heterodimerisation caused cytotoxicity against B7H3 targets in the absence of ALK expression. Both matched and mismatched h/tm pCARs increased proliferation and cytokine production through engagement of the B7H3 CCR, leading to function being maintained in repetitive stimulation settings. Overall, combination of the ALK8 binder with a boosted CAR design leads to augmentation of a proliferation and cytokine release signal whilst retaining specificity, defining an ALK-CAR capable of sensing naturally occurring low antigen density of ALK, allowing specific targeting of a multitude of solid cancers whilst limiting the likelihood of toxicity to healthy tissue. Combination with a matched h/tm CCR further allows targeting of potentially emerging ALK-loss variants.