(D-148) Engineering a novel IL-2-based immunocytokine with enhanced effector and memory function.

Introduction:: 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-2Ra, IL-2Rb, and gc subunits) or through an intermediate affinity heterodimeric receptor (IL-2Rb and gc subunits). Regulatory T (Treg) cells highly express IL-2Ra, whereas naïve effector cells (i.e., effector T cells and natural killer cells), 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 immunocytokines (ICs) that link cytokines to 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 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 a tumor-free state. To achieve this, we aim to create an immunocytokine 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 the effector activities and memory induction stimulated by an H9T-containing F10 IC will result in an effective long-term cancer treatment.


Materials and 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 will be interrogated via biolayer interferometry (BLI), comparing cytokine and receptor interactions to those of the component H9T and F10 antibody. 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 will be conducted to assess therapeutic potential.


Results, Conclusions, and Discussions:: Previous work showed that F10 IC does not interact with IL-2Ra but has enhanced affinity for IL-2Rb compared to native IL-2), which leads to its immune effector cell-biased activities. We anticipate that the engineered H9T/F10 IC will exhibit similar behavior. Similar to F10 IC, we expect to observe preferential signaling on IL-2Ra^Low cells for H9T/F10 IC, although the overall signaling extent for our engineered molecule is expected to be lower than that of F10 IC due to attenuating mutations in the H9T IL-2 variant. We also expect to observe greater abundance of memory cells and more effective disease control in mouse cancer models upon treatment with H9T/F10 IC compared to the parent F10 IC and native IL-2.

Furthermore, development of the H9T/F10 IC combines effector function with promotion of memory phenotype, which can address potential cancer reoccurrence. We will examine 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 immunocytokine that will induce robust and durable immunostimulatory activities as a powerful and versatile new weapon in cancer immunotherapy.

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