(174ah) Investigating the Mechanism and Immunomodulatory Effects of IL-7 Potentiation By a Neutralizing Anti-Cytokine Monoclonal Antibody

IL-7 is a cytokine that plays a critical role in immune cell proliferation, survival, function, and homeostasis. Anti-cytokine antibodies have been identified for many common cytokines such as IL-2, IL-4, and IL-7. It has been discovered that the anti-IL-7 antibody M25 inhibits the in vitro bioactivity while paradoxically potentiating the in vivo bioactivity of the cytokine, but the mechanism underlying this effect remains largely unknown. To elucidate this mechanism and investigate the interactions of IL-7/M25 complexes with the immune system, we have determined the crystallographic structure of the IL-7/M25 complex and conducted studies investigating its biophysical, pharmacokinetic, and immunomodulatory properties. These experiments include biolayer interferometry binding studies, in vivo half-life quantification, pSTAT5 signaling assays, and in vivo immune cell expansion and phenotyping. Elucidating the crystal structure of the IL-7/M25 scFv complex revealed that M25 overlaps with a receptor-binding epitope of IL-7 and that the IL-7/M25 interface is composed of strong electrostatic interactions. Through biolayer interferometry, we found that M25 competes with one of the IL-7 receptor subunits, which results in decreased engagement of IL-7 with its cognate receptor complex. In vivo, we discovered that M25 extends the half-life of IL-7 while still allowing dissociation of the cytokine, and the IL-7/M25 complex enhances expansion of total splenocytes and specific immune cell subsets, including B cells, CD8⁺ T cells, and natural killer cells. Our results indicate that M25 potentiates the activity of IL-7 through multiple distinct mechanisms, including half-life extension and sequestration of the cytokine through receptor competition. This work provides unprecedented insight into the mechanism behind antibody-mediated IL-7 potentiation, which will contribute to a better understanding of other potentiating anti-cytokine antibodies and create new opportunities for biotherapeutic development. Furthermore, this research will inform the future translational and therapeutic applications of IL-7-based immunotherapies. Future work will include studies investigating the complex in an engineered T cell model and as a potential therapeutic for sepsis treatment.