(314b) Structural Characterization of an Effector-Biasing Interleukin-2 Immunocytokine
AIChE Annual Meeting
Tuesday, November 15, 2022 - 12:48pm to 1:06pm
To address shortcomings of IL-2 therapy, previous studies have explored linking cytokines such as IL-2 to antibodies to improve both pharmacokinetics and targeting, in molecules called immunocytokines (ICs). Taking these ideas a step further, we have developed ICs which fuse antibodies against IL-2 to the cytokine itself. One of these molecules, denoted F10 IC, biases the IL-2 response toward immunostimulatory activity. Encouragingly, F10 IC has proven to be safe and effective as a cancer therapeutic in mouse models of melanoma. However, numerous molecular and mechanistic features of F10 IC have yet to be elucidated. Improved understanding of IL-2 ICs promises to advance their therapeutic design and development. Here, we present a structure-based characterization of the F10 IC molecule, and propose a model in which interactions between the antibody and cytokine result in a functionally relevant conformational change to IL-2.
Methods: High-resolution characterization of the interaction between the F10 antibody fragment and IL-2 was carried out using x-ray crystallography. Structural features of the full-length ICs were elucidated using cryogenic electron microscopy (cryo-EM). Single particle reconstruction methods were employed to generate intermediate-resolution maps into which IC models were built. Structural studies were supplemented with immune cell subset expansion profiling in mice following administration of our immunostimulatory IC versus IL-2 alone.
Results: Structural analysis revealed F10 (present as an scFv for crystallographic studies) as a direct steric inhibitor of the alpha subunit of the receptor complex. Further analysis demonstrated that F10 scFv binding resulted in a conformational change to IL-2, resulting in a conformation more similar to the receptor bound state. This feature was also observed in an immunostimulatory anti-IL-2 antibody that recognizes the mouse cytokine. Cellular subset and animal studies demonstrated the superiority of F10 IC over IL-2 alone in the selective expansion of CD8+ T cells and NK cells.
Implications: Our study demonstrates the mechanism by which the F10 IC biases IL-2 signaling in an immunostimulatory manner. The structural and functional insights our studies revealed will provide actionable data for further rational design of cytokine therapies. Future studies will focus on structure-informed improvements to the ICs, and therapeutic evaluation in animal models of cancer. Continued development of IL-2-containing ICs represents a fundamental advance for cytokine therapy, with improved pharmacokinetic properties and signaling specificity compared to unconjugated IL-2.
Acknowledgments: This research was funded by NIH R01EB029455, DoD Concept Award W81XWH-18-1-0735, and a Melanoma Research Alliance Young Investigator Award to Dr. Jamie Spangler.