Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling
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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Feb 05, 2021
- Source ID
- 10.1158/2326-6066.cir-20-0451
Entities
People
- Abdijapar Shamshiev
- Barbara Savoldo
- Brian Kuhlman
- Elena Dukhovlinova
- Elisa Landoni
- Francesco Padelli
- Gaetano Finocchiaro
- Gianpietro Dotti
- Giovanni Fucà
- Jian Wang
- Lee K. Hong
- Miriam Droste
- Nikolay V. Dokholyan
- Peishun Shou
- Serena Pellegatta
- Silvia Musio
- Soldano Ferrone
- Venkat R. Chirasani
- Zhiyuan Yao
Organizations
- Harvard Medical School
- National Institutes of Health
- Pennsylvania State University
- United States Department of Defense
- University of North Carolina at Chapel Hill