High efficiency cell-specific targeting of cytokine activity

Abstract

Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This 'activity-bytargeting' concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution

“…Antigen choice also impacts targeting efficacy: in contrast to our results, several studies have shown that immunocytokines specific for antigens expressed by tumor vasculature or liquid/disseminated tumors perform better than irrelevant immunocytokines (4,7,34), likely because these antigens are in closer contact with the blood and thus, more accessible than solid tumor antigens. Alternatively, the affinity of the cytokine for its receptor can be weakened, so that cytokine function is potentially restored only on high-avidity binding to tumor cells (35). Finally, our mathematical model suggests that small-format immunocytokines may intrinsically exhibit better antigen-targeting specificity than large-format immunocytokines because of size-dependent transport properties (rapid systemic clearance and diffusion by smaller molecules).…”

Delivering Type I Interferon to Dendritic Cells Empowers Tumor Eradication and Immune Combination Treatments

“…The mutation Q124R was introduced into the IFNa2 sequence by site-directed mutagenesis using the QuikChange II-E Site-Directed Mutagenesis Kit (Agilent Technologies) and sdAb were generated at the VIB Protein Service Facility, as described previously (17). AFNs (hIFNa2Q124R or hIFNaR149A coupled via a 20xGGS-linker to an N-terminal targeting sdAb) were constructed in pHen6 vectors, large-scale productions of His-tagged AFNs were performed in E. coli.…”

“…16). To improve the therapeutic index of toxic cytokines, we recently protein-engineered AcTakines (Activated-by-Targeting cytokines), optimized immunocytokines that use mutated cytokines with strongly reduced affinity for their receptor complex instead of WT cytokines (17). Fusing the mutated cytokine to cell-specific targeting domains specifically targets them to the selected cell population, restoring the AcTakine activity at that particular cell population with an up to 3-log targeting efficiency.…”

Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes

“…For example, AcCS might be used to increase the activity of natural IFNs produced locally at the site of a viral infection. In addition, several recent studies have demonstrated targeting IFNs to specific cells can be accomplished using IFN fusion proteins, which target IFNs to various cell surface markers by a variety of binding proteins (9,20,45). Improved targeting efficiency of IFN fusion proteins was recently demonstrated by reducing IFN-IFNAR2 binding affinity (9).…”

“…In addition, several recent studies have demonstrated targeting IFNs to specific cells can be accomplished using IFN fusion proteins, which target IFNs to various cell surface markers by a variety of binding proteins (9,20,45). Improved targeting efficiency of IFN fusion proteins was recently demonstrated by reducing IFN-IFNAR2 binding affinity (9). Combining IFN fusion proteins with AcCS may allow the targeted IFNs to be further inactivated to further improve targeting specificity but allow them to be re-activated using AcCS.…”