Optimization of a parallel CAR for B-cell lymphoma via ITAM attenuation and target specificity validation.

Second-generation CAR T-cells have transformed the management of B-cell malignancy. However, in vivo functional persistence is often limited, highlighting a key mechanism of treatment failure. We have engineered a parallel (p)CAR platform that delivers dual CD28 and 4-1BB co-stimulation via a co-expressed CAR and chimeric co-stimulatory receptor (CCR). To target CD19, we employed an avidity-optimized FMC63 scFv to direct CAR specificity while utilizing an unmodified FMC63 scFv to target the CCR. Here, we describe the late-stage optimization of this system for Phase 1 clinical evaluation. First, we confirmed that the avidity-optimized scFv lacked off-target specificity. To minimize risk of insertional mutagenesis and immunogenicity, respectively, we transitioned from a gammaretrovirus to a third-generation lentiviral expression vector and removed epitope tags used to discriminate between CAR and CCR expression. Most strikingly, we found that inactivation of immune tyrosine activation motif 2 and 3 within our pCAR prototype markedly potentiated efficacy in xenograft-bearing NSG mice. Mechanistically, this resulted from increased pCAR T-cell functional persistence and organ infiltration, with enhanced local clearance of malignant B-cells. These data set the scene for evaluation of this iteratively honed pCAR candidate in a clinical trial in relapsed/refractory B-cell non-Hodgkin's lymphoma.