Senior Scientist A2 Biotherapeutics, Inc., United States
Disclosure(s):
Alexander Partin: No relevant disclosure to display
Introduction/Rationale: MyD88 is a scaffold protein that plays a key role in propagating innate immune signals. Recent work has shown that a chemically induced dimerization (CID) platform comprising MyD88 fused to the intracellular domain of CD40 can enhance CAR-T cell fitness via multiple mechanisms. However, this system was associated with toxicity in the clinic due to excessive pro-inflammatory signaling. We sought to better understand the mechanistic levers by which engineered MyD88 assembles and propagates pro-inflammatory signals, with the goal of developing next-generation variants with improved safety profiles.
Methods: Using structural analysis and bioinformatics, a series of engineered MyD88 fusion proteins were designed, cloned, and expressed in Jurkat cells and primary human T cells in combination with the Tmod™ NOT logic-gated CAR-T platform. Effects on NF-kB induction, cytokine production and cell expansion were evaluated.
Results: Consistent with previous reports, we observe that Myd88-based CID systems synergize with TCR signaling pathways in human T cells. We demonstrate that engineered MyD88 assembly can be modulated by either raising or lowering the stability of an initial dimeric “seed.” We then characterize how the integration of either natural or synthetic TRAF recruitment motif(s) impart distinct cytokine profiles, enabling the construction of variants with reduced pro-inflammatory signaling.
Conclusion: Our results shed light on the mechanisms underlying inducible Myddosome assembly and offer insight into why MyD88 is particularly amenable to CID. We show that functional outputs can be modulated via altering assembly thresholds as well as tuning signaling motifs, two parameters which help to define design goals for next-generation immunotherapies integrating innate and adaptive signals.
