TY - JOUR
T1 - Modifications to the framework regions eliminate chimeric antigen receptor tonic signaling
AU - Landoni, Elisa
AU - Fuca, Giovanni
AU - Wang, Jian
AU - Chirasani, Venkat R.
AU - Yao, Zhiyuan
AU - Dukhovlinova, Elena
AU - Ferrone, Soldano
AU - Savoldo, Barbara
AU - Hong, Lee K.
AU - Shou, Peishun
AU - Musio, Silvia
AU - Padelli, Francesco
AU - Finocchiaro, Gaetano
AU - Droste, Miriam
AU - Kuhlman, Brian
AU - Shamshiev, Abdijapar
AU - Pellegatta, Serena
AU - Dokholyan, Nikolay V.
AU - Dotti, Gianpietro
N1 - Funding Information:
The Microscopy Services Laboratory, Department of Pathology and Laboratory Medicine, is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center. This work was supported by R01 CA193140 (G. Dotti), 1R35 GM134864 (N.V. Dokholyan), UL1 TR002014 (N.V. Dokholyan), R35 GM131923 (B. Kuhlman), RO1DE028172 (S. Ferrone), RO3CA239193 (S. Ferrone), and RO3CA216114 (S. Ferrone) from NIH; Passan
Funding Information:
Foundation (N.V. Dokholyan); U.S. Department of Defense W81XWH-16-1-0500 (S. Ferrone); and Il Fondo di Gio Onlus (S. Pellegatta). G. Dotti also received support from Cell Medica. Sequences of the 763.74 mAb and its humanized form have been submitted as provisional patent.
Funding Information:
E. Landoni reports a patent for CSPG4.CAR pending. B. Savoldo reports grants from Cell Medica during the conduct of the study, as well as personal fees and other from Tessa Therapeutics (consultant) and grants from bluebird bio and NIH outside the submitted work. B. Kuhlman reports grants from NIH during the conduct of the study. G. Dotti reports grants from Cell Medica during the conduct of the study; grants from Bellicum Pharmaceuticals and bluebird bio and personal fees from Bellicum Pharmaceuticals, Catamaran, and Tessa Therapeutics outside the submitted work; as well as a patent for CSPG4.CAR pending. No disclosures were reported by the other authors.
Publisher Copyright:
2021 American Association for Cancer Research.
PY - 2021/4
Y1 - 2021/4
N2 - 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.
AB - 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.
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U2 - 10.1158/2326-6066.CIR-20-0451
DO - 10.1158/2326-6066.CIR-20-0451
M3 - Article
C2 - 33547226
AN - SCOPUS:85103706785
VL - 9
SP - 441
EP - 453
JO - Cancer immunology research
JF - Cancer immunology research
SN - 2326-6066
IS - 4
ER -