Abstract 1743: IL12 heterodimeric Fc-fusions engineered for reduced potency exhibit strong anti-tumor activity and improved therapeutic index compared to native IL12 agents
Authors: Bernett Matthew J. , Liu Ke , Bonzon Christine , Rashid Rumana , Rodriguez Nicole , Hassanzadeh-Kiabi Nargess , Ardila Connie , Bykova Katrina , Hackett Michael , Barlow Norm , Leung Irene , Nguyen Duc-Hanh , Eivazi Araz , Chu Seung Y. , Varma Rajat , Muchhal Umesh S. , Desjarlais John R. 
Published: July 2021

Abstract
Interleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 may help to establish an inflammatory tumor microenvironment critical for anti-tumor responses. Preclinical studies in mice revealed that native IL12 can dramatically shrink syngeneic tumors, however clinical studies in humans resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and extend half-life.
IL12 is a heterodimeric protein consisting of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity of human IL12-Fc were assessed in a human PBMC engrafted mouse MCF7 tumor model. Surrogate mouse IL12-Fc were evaluated in additional murine tumor models. Tolerability and pharmacodynamic activity were assessed in non-human primates.
IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to native IL12-Fc. Anti-tumor activity was achieved with potency-reduced IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Combined, these data indicate that potency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to narrow therapeutic index associated with recombinant native IL12 or IL12-Fc agents.
Citation Format: Matthew J. Bernett, Ke Liu, Christine Bonzon, Rumana Rashid, Nicole Rodriguez, Nargess Hassanzadeh-Kiabi, Connie Ardila, Katrina Bykova, Michael Hackett, Norm Barlow, Irene Leung, Duc-Hanh Nguyen, Araz Eivazi, Seung Y. Chu, Rajat Varma, Umesh S. Muchhal, John R. Desjarlais. IL12 heterodimeric Fc-fusions engineered for reduced potency exhibit strong anti-tumor activity and improved therapeutic index compared to native IL12 agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1743.