(685d) Engineering a Novel IL-2-Based Immunocytokine with Enhanced Effector and Memory Function.

The interleukin-2 (IL-2) cytokine has been used as a cancer therapeutic for several decades. However, toxic side effects such as vascular leak syndrome along with its very short serum half-life have largely restricted the clinical use of IL-2. IL-2 signals through a high affinity heterotrimeric receptor (IL-2Rα, IL-2Rβ, and γc subunits) or through an intermediate affinity heterodimeric receptor (IL-2Rβ and γc subunits). Regulatory T (Treg) cells highly express IL-2Ra, whereas naïve immune effector cells (i.e., effector T cells and natural killer cells) express minimal levels of IL-2Rα, which results in IL-2 preferentially activating Treg over immune effector cells, thus impeding immune-mediated tumor clearance. One strategy for improving IL-2 efficacy has been the development of immunocytokine (ICs) that link cytokines to functionally biasing anti-cytokine antibodies in order to extend IL-2 half-life and specifically target it towards desired immune cells. Our group has previously designed and therapeutically validated an IC termed IL-2/F10 IC which biases IL-2 activity towards effector cells over Tregs. We demonstrated that F10 IC potently stimulates immune effector cell expansion and inhibits tumor growth in mouse cancer models. However, in cancer treatment regimens, immune effector cell activity is required early on for tumor clearance, but memory is needed later to maintain disease protection. To achieve both effector and memory responses, we aim to create a variant of IL-2/F10 IC that employs the IL-2 variant H9T, which has been shown to elicit a memory phenotype in targeted T cells. We believe the combination of effector activities and memory induction stimulated by an H9T- and F10-containing IC will result in effective long-term cancer treatment.

Methods

All proteins in this study were expressed and purified from a mammalian cell expression system. The binding and competitive properties of H9T/F10 IC were interrogated via biolayer interferometry, comparing cytokine and receptor interactions to those of the component H9T cytokine and F10 antibody, as well as the parent IL-2/F10 IC molecule. H9T/F10 IC was then characterized through signaling assays on IL-2-responsive cell lines and primary human peripheral blood mononuclear cells. Following in vitro characterization, in vivo immune cell subset expansion studies and syngeneic mouse tumor models were conducted to assess therapeutic potential.

Results/ Conclusion/Discussion

Consistent with the parent F10/IL-2 IC, the engineered H9T/F10 IC did not bind to IL-2Rα, but demonstrated enhanced affinity for IL-2Rβ compared to native IL-2, indicating that immune effector cell-bias was intact. We also observed preferential signaling on IL-2Rα^Low cells for H9T/F10 IC, although the overall signaling extent for our engineered molecule was lower than that of F10 IC due to attenuating mutations in the H9T IL-2 variant. In initial studies in mouse bladder cancer models, we observed a greater abundance of memory cells upon treatment with H9T/F10 IC compared to the parent IL-2/F10 IC and the native IL-2 cytokine.

Development of the H9T/F10 IC combines effector function with promotion of memory phenotype, which can address potential cancer reoccurrence. To this end, we are examining the durability of cures through modified dosing schedules and rechallenge in mouse tumor models, in order to highlight the benefits of our effector cell- and memory cell-activating engineered IC. Overall, we are creating a new IL-2-based fusion protein that will induce robust and durable immunostimulatory activities as a powerful and versatile new weapon in cancer immunotherapy.