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Published on: February 27, 2019
Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling
Elisa Landoni1, Giovanni Fucá1, Jian Wang2
1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
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.
Insights
CAR-T cell tonic signaling, caused by unstable antibody fragments, impairs cancer treatment. Computational modeling identified and corrected this instability, enhancing CAR-T cell antitumor effects.
Area of Science:
- Immunology
- Biotechnology
- Molecular Biology
Background:
- Chimeric antigen receptor (CAR)-T cell therapy shows promise in cancer treatment.
- CAR tonic signaling, characterized by spontaneous CAR-T cell activation and cytokine release, is a detrimental factor limiting therapeutic efficacy.
- This tonic signaling is linked to the CAR construct's intrinsic properties.
Purpose of the Study:
- To investigate the underlying cause of CAR tonic signaling.
- To identify strategies for mitigating tonic signaling in CAR-T cells.
- To enhance the antitumor activity of CAR-T cells.
Main Methods:
- Analysis of CAR constructs, specifically the single-chain variable fragment (scFv) derived from monoclonal antibodies (mAbs).
- Utilized computational modeling to identify amino acids within scFv framework regions (FWRs) responsible for instability.
- Employed amino acid substitutions and FWR humanization to correct scFv instability.
Main Results:
- CAR tonic signaling is attributed to the intrinsic molecular instability of the scFv, leading to self-aggregation and aberrant CD3ζ signaling.
- Specific amino acids in the scFv FWRs were identified as drivers of this instability.
- scFv modifications, including amino acid substitutions and humanization, successfully abrogated tonic signaling without affecting antigen specificity.
- Corrected CAR-T cells exhibited enhanced antitumor effects.
Conclusions:
- CAR tonic signaling is a direct consequence of scFv molecular instability.
- Computational analysis of scFv is a viable method for identifying and rectifying instability.
- Optimizing scFv stability can significantly improve CAR-T cell-based cancer immunotherapy.
