Modifications to the framework regions eliminate chimeric antigen receptor tonic signaling

Elisa Landoni, Giovanni Fuca, Jian Wang, Venkat R. Chirasani, Zhiyuan Yao, Elena Dukhovlinova, Soldano Ferrone, Barbara Savoldo, Lee K. Hong, Peishun Shou, Silvia Musio, Francesco Padelli, Gaetano Finocchiaro, Miriam Droste, Brian Kuhlman, Abdijapar Shamshiev, Serena Pellegatta, Nikolay V. Dokholyan, Gianpietro Dotti

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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 CD3z 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.

Original languageEnglish (US)
Pages (from-to)441-453
Number of pages13
JournalCancer Immunology Research
Volume9
Issue number4
DOIs
StatePublished - Apr 2021

All Science Journal Classification (ASJC) codes

  • Immunology
  • Cancer Research

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