Abstract 587: A half-life extended, tumor-activated IL-12 increased the infiltration of effector immune cells into the tumor microenvironment and demonstrated anti-tumor activity in syngeneic mouse models
Authors: Malkova Natalia , Patel Ekta , Vu Sallyann , Fantini Damiano , O'Donnell Rebekah , Fanny Manoussa , Greene Justin , Guzman Wilson , Crowe David , Hsiao Stephanie , Johnson Parker , McLaughlin Megan , Yerov Oleg , Jenkins Kurt , Halpin-Veszeleiova Katarina , Madala Hanumantha Rao , O'Toole Caitlin , Taylor Jake , Pederzoli-Ribeil Magali , Nicholson Benjamin , Bialucha Carl Uli , O'Neil Jennifer E. 
Published: April 2023

Abstract
Interleukin-12 (IL-12) is a proinflammatory cytokine, which bridges innate and adaptive immunity via induction of T helper 1 differentiation and promotes cytolytic activity of natural killer and T cells. IL-12 has demonstrated potent antitumor activity in syngeneic mouse models and promising anti-tumor efficacy in humans. However, development of IL-12-based treatments has been limited by severe systemic toxicities in the clinical setting. To overcome toxicity and potentially improve the therapeutic index of IL-12 in a clinical setting, XTX301 was engineered as a half-life extended and masked IL-12. The masking domain of XTX301 is designed to pharmacologically inactivate IL-12 in circulation and non-tumor tissue, while enabling generation of an active IL-12 moiety upon cleavage of a linker sequence by matrix metalloproteinases that are enriched in the tumor microenvironment. To confirm the ability of human tumor associated proteases to activate XTX301, cleavage was assessed in primary human tumor samples and in plasma from cancer patients. We observed cleavage of XTX301 in the majority of human tumors samples tested, but no activation was observed in plasma from cancer patients. Human IL-12 does not bind and signal through the mouse IL-12 receptors, hence three murine surrogates were created for in vivo studies: mXTX301, a non-cleavable control, and an unmasked control molecule. Tumor growth inhibition was observed after a single dose of mXTX301 as low as 0.039 mg/kg. The non-cleavable form of mXTX301 was less potent than mXTX301, demonstrating that the anti-tumor activity of mXTX301 is dependent on protease activation. The unmasked control was not well tolerated, with a &gt; 20% body weight loss observed by Day 6, resulting in 75% of animals being euthanized by Day 11. Unlike the unmasked control, mXTX301 was well-tolerated at all tested doses, as evidenced by no loss in animals’ body weights, and demonstrated minimal pharmacodynamic activity in non-tumor tissues. In tumors, mXTX301 stimulated the infiltration of cytotoxic CD8+ T cells and induced the expression of several immune-related genes including those associated with IFN-γ cell signaling, antigen processing and presentation, and defense response as well as enrichment of gene signatures for T cells, natural killer (NK) cells, macrophages and dendritic cells as determined by RNA sequencing. In non-human primates, the highest non-severely toxic dose of XTX301 was 2mg/kg dosed weekly for a total of 4 doses. In summary, a half-life extended tumor-activated IL-12 molecule, mXTX301, demonstrated anti-tumor activity in preclinical mouse models with improved tolerability compared to a systemically active IL-12 molecule suggesting that XTX301 has potential for exerting potent anti-tumor activity while widening the therapeutic index of IL-12 treatment.
Citation Format: Natalia Malkova, Ekta Patel, Sallyann Vu, Damiano Fantini, Rebekah O'Donnell, Manoussa Fanny, Justin Greene, Wilson Guzman, David Crowe, Stephanie Hsiao, Parker Johnson, Megan McLaughlin, Oleg Yerov, Kurt Jenkins, Katarina Halpin-Veszeleiova, Hanumantha Rao Madala, Caitlin O'Toole, Jake Taylor, Magali Pederzoli-Ribeil, Benjamin Nicholson, Carl Uli Bialucha, Jennifer E. O'Neil. A half-life extended, tumor-activated IL-12 increased the infiltration of effector immune cells into the tumor microenvironment and demonstrated anti-tumor activity in syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 587.