Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling
Open Access
- 5 February 2021
- journal article
- research article
- Published by American Association for Cancer Research (AACR) in Cancer Immunology Research
- Vol. 9 (4), 441-453
- https://doi.org/10.1158/2326-6066.cir-20-0451
Abstract
Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.Keywords
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Funding Information
- Department of Pathology and Laboratory Medicine (P30 CA016086)
- NIH (R01 CA193140)
- NIH (1R35 GM134864)
- NIH (UL1 TR002014)
- NIH (R35 GM131923)
- NIH (RO1DE028172)
- NIH (RO3CA239193)
- NIH (RO3CA216114)
- U.S. Department of Defense (W81XWH-16-1-0500)
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