Molecular mechanisms of heparin-induced modulation of human interleukin 12 bioactivity.

Human interleukin-12 (hIL-12) is a heparin-binding cytokine whose activity was previously shown to be enhanced by heparin and other sulfated glycosaminoglycans. The current study investigated the mechanisms by which heparin increases hIL-12 activity. Using multiple human cell types, including natural killer cells, an IL-12 indicator cell line, and primary peripheral blood mononuclear and T cells, along with bioactivity, flow cytometry, and isothermal titration calorimetry assays, we found that heparin-dependent modulation of hIL-12 function correlates with several of heparin's biophysical characteristics, including chain length, sulfation level, and concentration. Specifically, only heparin molecules longer than eight saccharide units enhanced hIL-12 activity. Furthermore, heparin molecules with three sulfate groups per disaccharide unit outperformed heparin molecules with one or two sulfate groups per disaccharide unit in terms of enhanced hIL-12 binding and activity. Heparin also significantly reduced the EC50 value of hIL-12 by up to 11.8-fold, depending on the responding cell type. Cytokine-profiling analyses revealed that heparin affected the level, but not the type, of cytokines produced by lymphocytes in response to hIL-12. Interestingly, although murine IL-12 also binds heparin, heparin did not enhance its activity. Using the gathered data, we propose a model of hIL-12 stabilization in which heparin serves as a co-receptor enhancing the interaction between heterodimeric hIL-12 and its receptor subunits. The results of this study provide a foundation for further investigation of heparin's interactions with IL-12 family cytokines and for the use of heparin as an immunomodulatory agent.

Localized Interleukin-12 for Cancer Immunotherapy.

Interleukin-12 (IL-12) is a potent, pro-inflammatory type 1 cytokine that has long been studied as a potential immunotherapy for cancer. Unfortunately, IL-12's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. Early clinical trials in the mid-1990's showed that systemic delivery of IL-12 incurred dose-limiting toxicities. Nevertheless, IL-12's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. The development of strategies which maximize IL-12 delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. Diverse IL-12 delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. Several localized IL-12 delivery approaches have recently reached the clinical stage with several more at the precipice of translation. Taken together, localized delivery systems are supporting an IL-12 renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. This review begins with a brief historical account of cytokine monotherapies and describes how IL-12 went from promising new cure to ostracized black sheep following multiple on-study deaths. The bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for IL-12-based cancer immunotherapies. Advantages and limitations of different delivery technologies are highlighted. Finally, perspectives on how IL-12-based immunotherapies may be utilized for widespread clinical application in the very near future are offered.