Background Antigen escape and tumor heterogeneity remain significant hurdles to the development of curative treatments in many cancers. To address tumor heterogeneity, the introduction of pairs of chimeric antigen receptors (CARs) in donor T cells has been demonstrated, however, this adds complexity to a manufacturing process already challenged by cellular product consistency. To tackle tumor heterogeneity while maintaining product purity, we applied our induced pluripotent stem cell (iPSC)-derived T cell (iT) platform to design an off-the-shelf cell therapy capable of targeting multiple tumor antigens through complementary activation pathways, including targeting of both cell-surface antigens as well as intracellular/neoantigens.

Methods A CAR construct targeting BCMA or MICA/B, a T-cell receptor (TCR)αβ targeting NYESO1 (1G4) or tumor-associated metabolite presented by MR1 (MC.7.G5), and a high-affinity non-cleavable (hnCD16) to promote antibody-dependent cellular cytotoxicity (ADCC) were engineered into iPSCs for use as a renewable starting source in deriving uniformly-engineered T cells. Resulting multiplexed-engineered CAR⁺TCR⁺hnCD16⁺ (tri-modal) iPSCs were differentiated into T cells and the function of each individual edit was evaluated by preclinical models designed to represent tumor heterogeneity. In addition to in vitro mix-culture assays, a mixed cell disseminated in vivo model was used to mimic cancer heterogeneity and to evaluate the in vivo potency of tri-modal iT cells at mitigating tumor heterogeneity and antigen escape.

Results Assessment of individual edits in tri-modal iT cells demonstrated independent functionality by exhibiting increased antigen-mediated IFNγ and TNFα production, and degranulation compared to the control group (p<0.0001). Using 9-day daily restimulation assay, each edit produced significant tumor reduction compared to tumor only control (p<0.0001). By stimulating tri-modal iT cells with multiple antigens simultaneously using various solid tumor lines (A549, Caski and MDA-MB-231), we found that co-activation by two or three targeting edits significantly enhanced tumor killing (p<0.0001). Furthermore, when challenged with in vivo heterogenous tumor models, we found that the co-activation of all three targeting moieties in tri-modal iT cells achieved nearly complete tumor clearance (p<0.0001). Ex vivo bone marrow analysis further confirmed antigen-specific target elimination, reenforcing the specificity and potency of the tri-modal iT cells.

Conclusions Our data highlight the potency and broad applicability of tri-modal iT cell expressing CAR, TCR, and hnCD16. This consistent and scalable approach to multiplex-engineered T-cell therapy is an ideal strategy to mitigate antigen escape and combat difficult to treat heterogeneous solid tumors.