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707 IL12 Fc-fusions engineered for reduced potency and extended half-life exhibit strong anti-tumor activity and improved therapeutic index compared to wild-type IL12 agents
  1. Matthew Bernett1,
  2. Ke Liu1,
  3. Christine Bonzon1,
  4. Michael Hackett1,
  5. Katrina Bykova1,
  6. Rumana Rashid1,
  7. Nicole Rodriguez1,
  8. Nargess Hassanzadeh-Kiabi1,
  9. Connie Ardila1,
  10. Norman Barlow1,
  11. Irene Leung1,
  12. Hanh Nguyen1,
  13. Araz Eivazi1,
  14. Kendra Avery1,
  15. Rajat Varma2,
  16. Umesh Muchhal1 and
  17. John Desjarlais1
  1. 1Xencor, Inc., MONROVIA, CA, United States
  2. 2Independent Consultant, Monrovia, CA, United States


Background Interleukin-12 (IL12) is a proinflammatory cytokine that induces differentiation of Th1 cells and increased cytotoxicity of T and NK cells. Stimulation by IL12 leads to production of IFNγ and an inflammatory tumor microenvironment critical for anti-tumor responses. Studies in mice revealed IL12 can dramatically shrink syngeneic tumors, however human clinical studies 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 therapeutic index (TI) in non-human primates (NHP) by reducing receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve TI.

Methods IL12 is a heterodimer 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 IL12p40 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 was assessed in huPBMC-NSG-DKO and huCD34+ MCF7 xenograft models. Surrogate mouse potency-reduced IL12-Fc were evaluated in syngeneic tumor models. Tolerability and pharmacodynamic activity were assessed in NHP.

Results An IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling and IFNγ production in an MLR assay compared to wild-type IL12-Fc. Anti-tumor activity was achieved with potency-reduced IL12-Fc as single-agents 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. Surrogate potency-reduced IL12-Fc had improved tolerability and greater selectivity of IFNγ production in tumors compared to spleen and less production of IL10 compared to wild-type IL12-Fc. In NHP, potency-reduced IL12-Fc had superior exposure with slower, more sustained accumulation of IFNγ and IP10, and a more gradual dose-dependent peak response, as well as more sustained margination of T and NK cells compared to wild-type IL12-Fc.

Conclusions Potency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to narrow TI associated with wild-type IL12 or IL12-Fc agents.

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