Proceeding on the assumption that all cancer cells have equal malignant capacities, current regimens in cancer therapy attempt to eradicate all malignant cells of a tumor lesion. Using in vivo targeting of tumor cell subsets, we demonstrate that selective elimination of a definite, minor tumor cell subpopulation is particularly effective in eradicating established melanoma lesions irrespective of the bulk of cancer cells. Tumor cell subsets were specifically eliminated in a tumor lesion by adoptive transfer of engineered cytotoxic T cells redirected in an antigen-restricted manner via a chimeric antigen receptor. Targeted elimination of less than 2% of the tumor cells that coexpress high molecular weight melanoma-associated antigen (HMW-MAA) (melanomaassociated chondroitin sulfate proteoglycan, MCSP) and CD20 lastingly eradicated melanoma lesions, whereas targeting of any random 10% tumor cell subset was not effective. Our data challenge the biological therapy and current drug development paradigms in the treatment of cancer.

Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less‐differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long‐term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib‐induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib‐based CART cell generation resulted in an enrichment of less‐differentiated naïve‐like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD‐1 and Tim‐3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib‐treated CART cells was validated in a xenograft mouse model. Intracellular TNF‐α and IFN‐γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib‐based CART cell generation protocols are warranted.