An immunoproteomic approach to characterize the CAR interactome and signalosome

Area of Science:

• Immunotherapy
• Cellular immunology
• Cancer biology

Background:

• Chimeric antigen receptor (CAR)-T cell therapy is a successful immunotherapy for hematological cancers.
• Understanding the precise signaling mechanisms of CARs is crucial for optimizing their efficacy.

Purpose of the Study:

• To systematically analyze chimeric antigen receptor (CAR) signaling in human primary T cells.
• To compare signaling pathways activated by different CAR designs with varying intracellular domains and antitumor efficacy.

Main Methods:

• Mass spectrometry-based interactome and phosphoproteome analysis of primary human T cells engineered with distinct CARs.
• Comparison of signaling events triggered by second-generation and third-generation CAR designs.

Main Results:

• Second-generation CARs, but not third-generation, induced constitutively phosphorylated CD3ζ, resembling endogenous species.
• This CD3ζ phosphorylation was dependent on the size of the intracellular domains, not costimulatory or hinge regions.
• Second-generation CARs showed stronger downstream secondary messenger phosphorylation, indicating more intense signaling.

Conclusions:

• Second-generation CAR designs activate additional CD3ζ signaling sources, leading to enhanced signaling and superior antitumor efficacy compared to third-generation CARs.
• These findings provide insights into CAR-T cell physical and functional interactions with endogenous T cell molecules.
• This knowledge will guide the development of novel, optimized immune receptors for improved cancer immunotherapy.