Targeted chain-exchange-mediated reconstitution of a split type-I cytokine for conditional immunotherapy

Antibody-cytokine fusions targeted against tumor-associated antigens (TAAs) are promising cancer immunotherapy agents, with many such molecules currently undergoing clinical trials. However, due to the limited number of tumor-specific targets, on-target off-tumor effects can lead to systemic toxicity. Additionally, targeted cytokines can be scavenged by cytokine receptors on peripheral cells, decreasing tumor penetration. This study aims at overcoming these issues by engineering a platform for targeted conditionally active type I cytokines. Building on our previously reported PACE (<u>P</u>rodrug-<u>A</u>ctivating <u>C</u>hain <u>E</u>xchange) platform, we split the type I cytokine interleukin-4 (IL-4) to create two inactive IL-4 prodrugs, and fused these split IL-4 counterparts to the C-termini of antibody-like molecules that undergo proximity-induced chain exchange. In doing so, we developed IL-4 prodrugs that preferentially reconstitute into active IL-4 on target cells. We demonstrate that pre-assembled split IL-4 (without additional inactivation) retains activity and present two different strategies of splitting and inactivating IL-4. Using an IL-4 responsive cell-line, we show that IL-4 prodrugs are targeted to TAAs on target cells and regain activity upon chain exchange, primarily in a cis-activation setting. Furthermore, we demonstrate that split IL-4 complementation is also possible in a trans-activation setting, which opens up the possibility for activation of immune cells in the tumor vicinity. We demonstrate that targeted on-cell prodrug conversion is more efficient than nonspecific activation in-solution. Due to the structural similarity between IL-4 and other type I cytokines relevant in cancer immunotherapy such as IL-2, IL-15, and IL-21, cytokine-PACE may be expanded to develop a variety of targeted conditionally active cytokines for cancer immunotherapy.