Abstract 5549: Potency-reduced IL12 heterodimeric Fc-fusions exhibit strong anti-tumor activity
Authors: Varma Rajat , Liu Ke , Bonzon Christine , Rashid Rumana , Rodriguez Nicole , Hassanzadeh-Kiabi Nargess , Ardila Connie , Chu Seung Y. , Muchhal Umesh S. , Desjarlais John R. , Bernett Matthew J. 
Published: September 2020

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 are promising for cancer treatment and may help to convert immunologically suppressed “cold” tumors into inflamed “hot” tumors. Preclinical studies in mice revealed that IL12 can have a dramatic effect on shrinking syngeneic tumors, however clinical studies in humans have 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 various IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency in order to improve tolerability, slow receptor-mediated clearance, and prolong half-life.
IL12 is a heterodimeric protein consisting of two subunits, and thus 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 normal human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity was assessed by engrafting pp65-MCF-7 cells into human PBMC engrafted NSG MHC class I and II double-knockout mice and by measuring tumor volume, lymphocyte activation/proliferation, and IFNγ production over time.
IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and weaker variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to a wild-type IL12-Fc. In vivo anti-tumor activity in the huPBMC-pp65-MCF7 model was achieved with reduced-potency 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, as evidenced by upregulation of CD25. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum cytokine indicated up to 200-fold increases in IFNγ levels. Combined, these data indicate that reduced-potency IL12-Fc retain strong anti-tumor activity, with potential for improvement of therapeutic index.
Citation Format: Rajat Varma, Ke Liu, Christine Bonzon, Rumana Rashid, Nicole Rodriguez, Nargess Hassanzadeh-Kiabi, Connie Ardila, Seung Y. Chu, Umesh S. Muchhal, John R. Desjarlais, Matthew J. Bernett. Potency-reduced IL12 heterodimeric Fc-fusions exhibit strong anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5549.