Abstract A005: Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223
Authors: Sharma Aanchal , To Minh D , Kataria Hardeep , Liu Qian , Merchant Rosemina , Merchant Fahar 
Published: December 2023

Abstract
Introduction: Combination treatment with a long-acting IL-2 superkine (MDNA109FEAA-Fc; aka MDNA19) and anti-PD1 or anti-CTLA4 has shown clear evidence of synergy in blunting tumor growth. To further leverage this synergism, we designed MDNA223, a BiSKIT (Bifunctional SuperKine for ImmunoTherapy) comprising MDNA109FEAA with enhanced affinity for IL-2Rb and no binding to IL-2Ra, fused to either human or mouse anti-PD1. We present in vitro and in vivo characterization of MDNA223 and demonstrate its enhanced IL-2R agonism and effective PD1/PDL-1 blockade in multiple “hot” and “cold” syngeneic tumor models.
Experimental Procedure: In vitro studies included human PBMCs and cell-based reporter assays. In vivo studies were conducted in CT26, B16F10, E0771 and TRAMP-C1 tumor models by intraperiotonneal (IP) administration. Intratumoral treatment was performed in a CT26 bilateral tumor model with or without a STING agonist. Analysis of tumor infiltrating lymphocytes (TILs) was performed using flow cytometry.
Results: In studies with human PBMCs, MDNA223 showed increased p-STAT5 signaling in CD8+ T cells and reduced activity in Tregs compared to rhIL-2, consistent with its enhanced receptor selectivity. MDNA223 and anti-PD1 were similarly potent at PD1/PDL-1 blockade in a cell-based reporter assay. In mice, MDNA223 induced proliferation (based on Ki67 expression) and expansion of peripheral CD8+ T cells for over 7 days. IP treatment with MDNA223 resulted in significantly greater inhibition of CT26, B16F10, E0771 and TRAMP-C1 tumor growth when compared to combination of MDNA19 with anti-PD1 at equimolar dosage. This observation is consistent with the proposed cis-binding mechanism of MDNA223 to both, IL-2R and PD1 on the same effector immune cell. In a bilateral CT26 tumor model, IT treatment with MDNA223 not only inhibited the treated tumor but also exhibited an abscopal effect by slowing growth of the untreated tumor implanted on the opposite flank. TILs analysis of B16F10 melanoma following a single dose of MDNA223 showed increased infiltration of CD8+ T cells over Tregs compared to single agent treatment with MDNA19 or anti-PD1 as well as their combination. Treatment with MDNA223 also resulted in a larger population of functional effector CD8+ T cells expressing low levels of the Tim-3 exhaustion marker and high levels of the cytotoxic Granzyme-B protease.
Conclusion: MDNA223 demonstrated superior therapeutic activity over the combination of MDNA19 and anti-PD1 in both immunologically ‘hot’ (CT26) and ‘cold’ (B16F10, E0771 and TRAMP-C1) tumor models. Additional studies with variants of MDNA223 capable of tumor selective targeting and/or conditional activation within the tumor micro-environment are in progress to maximize activity at the tumor site with tunable peripheral stimulation.
Citation Format: Aanchal Sharma, Minh D To, Hardeep Kataria, Qian Liu, Rosemina Merchant, Fahar Merchant. Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A005.