Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T-cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity and specificity, pCAR T-cells consistently elicit superior anti-tumor activity both in vitro and in vivo, warranting clinical development.
Overall design
In order to understand the mechanisms underlying the enhanced anti-tumor function of pCAR T-cells, we performed gene expression analysis using the NanoString CAR-T Characterization Panel of M-CSF receptor-targeting pCAR T-cells and compared it to second and third generation CAR-T cells with either 41BB, CD28 or both co-stimulatory domains, as well as to a first generation CAR paired with a tandem dual chimeric cytokine receptor which provided both 41BB and CD28 co-stimulation. A pCAR in which the endodomain of the CAR was truncated was used as signalling-defective control. 4 samples from 4 individual donors were used for each construct. All T-cells were analyzed 24h after stimulation on either target cells either expressing M-CSF receptor or lacking M-CSF receptor expression (negative control).
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