Development of protease activated interleukin-12 cytokine fusion proteins for tumor immunotherapy (TUM7P.946)

Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor responses. However, achieving local expression of cytokines at disseminated sites is challenging and systemic delivery has significant toxicity. We describe a novel approach employing an activatable fusion protein (FP) to reduce the negative side-effects of systemic delivery of the cytokine but which can preferentially affect all tumor sites. This FP consists of a cytokine linked to an inhibitory single chain antibody fragment (scFv) separated by a specific protease cleavage sequence. Importantly, the scFv does not bind to the tumor but rather binds to the cytokine keeping it inactive. However, at the tumor site, the FP can be cleaved by proteases such as Matrix metalloproteases (MMPs) that are over-expressed at tumor sites. This cleavage allows the release of the cytokine from the scFv and its binding to higher affinity receptors on immune cells. We will present data on the construction, expression, and characterization of a panel of murine and human IL-12 FPs. Using phage display, we have identified a panel of 15 scFv that bind to epitopes of murine IL-12 on either the P35 or P40 subunit as well as scFv that bind to human IL-12. Functional and biochemical data on baculovirus expressed IL-12 FPs will be presented. This strategy has the potential to reduce the negative side effects associated with systemic delivery of constitutively active cytokines yet retain their efficacy.