Abstract 2071: Probody-interferon-alpha 2b combines antitumor activity with improved tolerability
Authors: Berezhnoy Alexey , Wang Hsin , Lapuyade Nicole , Cai Na , LePage Carol , Winter Michael B. , Ye Ivan , Lu Hong , Krimm Michael , Wong Ken , Dunn Robert T. , Boustany Leila , Paidhungat Madan , Belvin Marcia , Scolan Erwan Le , Daniel Dylan 
Published: June 2022

Abstract
Type I interferons can exert direct antitumor effects, modulate tumor stroma, and induce de novo antitumor immune responses. They have demonstrated combination activity with PD-(L)1 blockade to potentially expand the benefit to patients with unresponsive tumors. Despite its potential, the toxicity of interferon alpha has limited its clinical use. Here we applied CytomX proprietary Probody® Therapeutics (Pb-Tx) technology to create a conditionally active IFN-α2b (Pb-IFN-α2b) with minimal activity in its prodrug form. The prodrug is activated in the tumor microenvironment (TME), leading to preferential activity in the TME but not in healthy tissues. Pb-IFN-α2b demonstrated an enhanced tolerability profile compared to standard IFN therapy without compromising its antitumor effects. The Pb-Tx platform technology attenuates activity of a molecule by blocking its active regions through affinity or steric interference. Such blockade, termed masking, is reversed upon proteolytic cleavage of a substrate-containing linker between the molecule and the mask by tumor associated proteases. Pb-IFN molecules were engineered with a dual masking approach combining the effects of steric inhibition by Fc fusion and affinity interference by a peptide mask. Pb-IFN-α2b demonstrated significant reduction (1000-fold or more) of its specific activity in vitro, including antiproliferative effects and immune cell activation. Treatment with tumor-associated proteases or exposure to viable tumor tissues fully restored its activity. Activated but not masked Pb-IFN-α2b induced a gene expression profile consistent with interferon signaling in primary human immune cells. In vitro studies with dissociated human tumors demonstrated the ability of Pb-IFN to activate tumor immune infiltrate, which could be further enhanced by concomitant PD-L1 blockade. Antitumor activity of the Pb-IFN-α2b in xenograft studies is equal to or greater than Peg-IFN-α2b. Pb-IFN-α2b demonstrated significant antitumor activity in syngeneic mouse tumor models without evidence of toxicity. Consistent with in vitro observations, this anti-tumor activity was further enhanced by PD-(L)1 blockade. Toxicology studies performed in hamsters demonstrated enhanced tolerability of the molecule compared to its unmasked control. Pb-IFN-α2b did not cause hematological changes or body weight loss associated with unmasked interferon. In cynomolgus monkey Pb-IFN-α2b demonstrated linear pharmacokinetics, extended half-life, and was well tolerated at doses up to 15 mg/kg. Pb-IFN-α2b shows improved tolerability and antitumor activity in preclinical studies compared to traditional IFN treatment. These data support Probody cytokine therapeutics as a promising addition to current immunotherapy regimens, potentially expanding their benefits to patients with typically unresponsive tumors.
Citation Format: Alexey Berezhnoy, Hsin Wang, Nicole Lapuyade, Na Cai, Carol LePage, Michael B. Winter, Ivan Ye, Hong Lu, Michael Krimm, Ken Wong, Robert T. Dunn, Leila Boustany, Madan Paidhungat, Marcia Belvin, Erwan Le Scolan, Dylan Daniel. Probody-interferon-alpha 2b combines antitumor activity with improved tolerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2071.