691 MDK-271: A dual function molecule consisting of empirically-designed peptidyl agonists of IL-2/15Rβγc and IL-7Rαγc, unrelated to IL-2, IL-15, or IL-7, incorporated into a bispecific Fc fusion protein

Background Activation of IL-2/15Rβγc or IL-7R on immune cells using modified versions of IL-2 or IL-7 is under investigation as monotherapy, or in combination with checkpoint inhibitors, engineered T or NK cells, or neo-antigen vaccines. We previously described small synthetic peptides, unrelated to IL-2, IL-15, or IL-7, that selectively activate either IL-2/15Rβγc or IL-7R. IL-2/15Rβγc and IL-7R activation exhibit complementary effects on immune cells that when combined may offer benefits over each independent mechanism. We now report the creation of an Fc-fusion protein that incorporates both IL-2/15Rβγc and IL-7R agonist peptides, and characterize its properties in cell lines and human (PBMC) lymphocyte subpopulations. Methods Peptide agonists of IL-2/15Rβγc (MDK1169) and IL-7R (MDK1319) were separately fused to each chain of obligate heterodimeric (asymmetric) Fc molecules. The Fc-fusion was purified by protein-A and size exclusion chromatography, and characterized by LC-MS. Receptor-mediated signaling, proliferation, and cell-surface receptor expression in cell lines, PBMCs, mixed and isolated lymphocyte subpopulations were determined by flow cytometry and ELISA to evaluate effects of IL-2, IL-2v, IL-7, MDK-202 or MDK-701 (Fc-fusions of MDK1169 and MDK1319, respectively), and combinations (mixtures) of these molecules, in comparison with the dual agonist MDK-271. Results LC-MS analysis indicates MDK-271 is a heterodimeric molecule containing one copy each of MDK1169 (IL-2/15Rβγc-biased agonist) and MDK1319 (IL-7R agonist) fused to individual Fc-chains. Cell-based assay of MDK-271 demonstrates potent, fully efficacious phosphorylation of STAT5 in TF-1 cells expressing Rγc, and engineered to express either IL-2/15Rβ or IL-7Rα. PBMCs exposed ex vivo to MDK-271 exhibit additive, complementary, and synergistic effects among various lymphocyte subpopulations: CD4+Tn, Teff, Treg, Tmem; C8+Tn, Teff, Tmem; and NK cells, in this analysis. The mono-specific agonists MDK-202 and MDK-701 produce proliferative effects and signaling patterns in responsive cell lines and lymphocyte subsets similar to those induced by IL-2v (an IL-2/15Rβγc-biased mutant of IL-2) and IL-7, respectively. Combining both activities in MDK-271 induces response profiles that differ in some T-cell subsets from those of mono-specific agonists of the two receptors. Animal studies designed to understand the effects of these differences are underway. Conclusions IL-2/15Rβγc and IL-7R are both currently undergoing extensive scrutiny as potential immuno-oncology therapeutic targets. The biology of these cytokines is both overlapping and complementary in stimulating and supporting T-cell populations; and some recent evidence suggests possible superiority of the combination. Based on in vitro properties, the Fc-peptide fusion reported here, exhibiting both IL-2/15Rβγc-biased agonist and Il-7Rαγc agonist activities, could be valuable in anti-tumor therapeutic applications. Ethics Approval The use of human PBMC in this study was authorized under Minimal Risk Research Related Activities at Stanford Blood Center (SQL 79075)