566 MDK-202: An empirically-designed peptidyl agonist of the IL-2/15βγc receptor, devoid of Rα interaction, unrelated to IL-2 or IL-15, and fused to an Fc-domain for PK enhancement

Background Efforts to modify IL-2 for immuno-oncology applications focus on modifying the receptor selectivity of IL-2 to bias effects on immune cells; in particular, to reduce Rα interaction via mutation, chemical modification, complexation with antibodies, or fusion to the Rα-ectodomain. IL-2/15Rβγc-biased agonists also incorporate PK enhancement to extend duration of action, and reduce side effects associated with peak drug levels. We previously reported discovery of small synthetic peptides, unrelated to IL-2 or IL-15, that simultaneously bind IL-2Rβ and γc subunits to induce IL-2/15R signaling. These peptides do not bind IL-2Rα, and are therefore IL-2/15Rβγc-selective agonists with MW less than 5000D. We now describe properties of an IL-2/15Rβγc agonist peptide fused to an Fc-domain (MDK-202). Methods Peptides were selected from recombinant peptide libraries to identify molecules binding simultaneously to the β and γc subunits of IL-2/15R. Active peptides were fused to Fc-domains to evaluate efficacy, potency, and quality of signaling upon activating IL-2/15Rβγc in cell lines and human lymphocytes. PK and PD properties in mice and NHP were also determined. Results MDK-202 exhibits in vitro potency similar to the synthetic peptide (MDK1169). MDK-202 does not bind IL-2Rα, activates the major IL-2/15R signaling pathways: JAK-STAT(pSTAT5), MAPK (pERK1/2), PI3K (pAKT), and induces proliferation (Ki-67) in human PBMCs, with kinetics and efficacy similar to IL-2. Agonism is attributable to direct activation of IL-2/15Rβγc as shown by dependence on Rβ expression in test cells, and insensitivity to blockade by neutralizing antibodies against IL-2 and IL-15. At concentrations greatly exceeding that required for maximum IL-2/15R activation in vitro, MDK-202 does not interfere with the activities of other Rγc family receptors. The predicted immunogenicity potential for MDK-202 is very low, and in the unlikely event of MDK-202-induced ADA, neutralization of endogenous IL-2 or IL-15 would not be expected. MDK-202 is highly stable in human serum, showing no significant degradation after 4 days at 37C. In ex vivo human PBMC and in vivo studies in normal mice, hPBMC-engrafted NCG mice, and non-human primates, MDK-202 exhibited extended half-life, and activation, proliferation, and population dynamics of lymphocytes similar to those induced by ‘non-Rα’ variants of IL-2. Conclusions MDK-202 is an attractive alternative to IL-2/15 variants for use in immuno-oncology therapy. Constructed without reference or similarity to cytokine or receptor structures or contacts, the peptidyl agonist component (MDK1169) is completely unique, and shown to be active when fused to other proteins such as anti-PD-1 antibodies and other cytokine receptor agonists. Ethics Approval Animal studies were performed by Charles Rivers Laboratories, as approved by the CRL Instuition Ethics Board with the following study and approval numbers:CRL-220007; 20222440 : PK Cynomolgus Monkeys: BA-e451;BA-e451: PD NCG mice BA-e411; BA-e411:PD NCG miceKey 2152; US19001: PK mice: The use of human PBMC in this study was authorized under Minimal Risk Research Related Activities at Stanford Blood Center (SQL 79075)