Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors.

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+CD3ζ) 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 compared with T cells that express traditional linear CARs.
Competing Interests: J.M. is CSO, scientific founder, and shareholder of Leucid Bio. T.M., F.K., M.G., and A.A. are employees of Leucid Bio. L.H. and B.D. undertook PhD studentships funded by Leucid Bio. L.M.W., D.M.D., C.H., and D.L.-Y. have acted as consultants for Leucid Bio, and D.M.D. is currently an employee of Leucid Bio. J.M., D.Y.A., L.M.W., B.D., T.M., F.K., L.H., and M.G. are co-inventors on patent filings in relation to pCAR technology. B.D. and D.Y.A. are shareholders of Autolus Therapeutics. D.Y.A. is an employee of Autolus Therapeutics. The other authors declare no competing interests.
(© 2021 The Author(s).)