Background Small cell lung cancer (SCLC) is an aggressively lethal malignancy with a high unmet medical need. Delta-like ligand 3 (DLL3) has been identified as a promising cancer antigen for target immunotherapy in treating SCLC. DLL3-targeting therapies such as T cell engagers and chimeric antigen receptor T cells (CAR-T), so far have shown favorable safety profiles and encouraging therapeutic results.

Methods A diverse panel of single-chain variable fragment (scFv) with high binding affinity for human DLL3 were generated and was compared in a second-generation CAR-T format for target-dependent T cell activation and cytotoxicity. A construct encoding the selected scFv CAR and an armored CD70 was generated and the effector functions of the engineered CAR-T cells were evaluated in vitro. The antitumor efficacies were evaluated against multiple xenograft models in immunodeficient mice. The potential toxicity was characterized in a mouse tumor model with mouse cross-reactive DLL3 CAR-T cells.

Results It was notable to find that a class of membrane-proximal (MPR) epitope targeted DLL3 CARs revealed potent cytotoxicity in treating low DLL3 density tumor cells, in contrast to membrane-distal region (DSL) targeted DLL3 CARs. Consistently, the MPR targeted CAR-T cells was superior in mediating cytokine secretion in response to tumor recognitions. Using transgenic mouse models, we observed that MPR targeted CAR-T cells achieved better tumor control than DSL targeted CAR-T cells. The long-lasting antitumor activities of MPR targeted CAR-T therapy was further evidenced by tumor re-challenge study, where 100% of the cured mice showed tumor rejection. No potential organ toxicity was found in normal tissues and CAR-T infiltration was only observed in tumors and spleens, indicating that MPR targeted CAR-T cells are of safety with no serious off-target toxicity. To further enhance the expansions and persistence, we armored the CAR with a CD70 construct. Using serial killing assays, we demonstrated that CD70 armored CAR-T promoted durable cytotoxicity following serial rounds engagement of low DLL3-expressed tumor cells. Furthermore, the tumor lysing capacity of CD70 armored CAR-T cells was not compromised to TGF-β (transforming growth factor β) inhibition compared to the conventional CAR-T cells.

Conclusions Collectively, our study indicates a combine of a proximal targeting CAR and an armored CD70 construct improve CAR-T persistence and antitumor activity in vitro and in vivo, and result in a potential DLL3 CAR candidate for further preclinical and clinical studies.