Poster CC14 - Investigating the mechanism and immunomodulatory effects of IL-7 potentiation by a neutralizing anti-cytokine monoclonal antibody

Introduction: 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 mitogenic in vivo effects of the cytokine. This is partially attributed to half-life extension, but a half-life independent contribution has also been reported. Thus, the molecular mechanism underlying IL-7/M25 activity remains largely unknown. We have conducted studies to elucidate this mechanism and investigate the IL-7/M25 complex’s interaction with the immune system and translation to immune cell activation and expansion.

Materials and

Methods: We have determined the structure of the IL-7/M25 complex using x-ray crystallography and conducted studies investigating its biophysical and pharmacokinetic properties. These experiments include biolayer interferometry binding studies, in vivo half-life quantification, and pSTAT5 signaling assays. Furthermore, the IL-7/M25 complex has been investigated as an immunomodulatory molecule through in vivo immune cell expansion and phenotyping via flow cytometry.

Results, Conclusions, and Discussions: Results
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 vitro, pSTAT5 signaling assays revealed attenuated signaling potency of the IL-7/M25 complex compared to soluble IL-7. In vivo, we discovered that M25 extends the half-life of IL-7 while still allowing dissociation of the cytokine. The IL-7/M25 complex also enhances expansion of total splenocytes and specific immune cell subsets in vivo, including CD8+ T cells and natural killer cells.

Conclusions
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. These studies also confirm that the IL-7/M25 complex neutralizes cytokine bioactivity in vitro while potentiating its effects in vivo. Furthermore, the results of immune cell phenotyping show that the IL-7/M25 complex can be used to enhance the expansion of immune cell subsets.

Discussions
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 adoptive T cell model for cancer treatment.

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