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1333 FG-3165 is a novel galectin-9 neutralizing antibody that inhibits galectin-9-mediated dimerization of TIM-3 and galectin-9-induced apoptosis of CD4+ and CD8+ T cells
  1. Prachi Bagadia,
  2. Selene Li,
  3. Mitch Brenner,
  4. Veronica Rodriguez,
  5. Mark Sternlicht,
  6. Todd Seeley,
  7. John Hunter,
  8. Dave Olsen and
  9. Gail Walkinshaw
  1. FibrGen, San Francisco, CA, USA

Abstract

Background Galectin-9 (Gal-9) is a β-galactoside binding lectin that contains two conserved carbohydrate-recognition domains. Gal-9 is produced by a variety of cell types and binds to cell-surface receptors on immune cells to promote an immunosuppressive phenotype within the tumor microenvironment. One well characterized Gal-9 receptor that is thought to play a key role in mediating the effects of Gal-9 on T cells, is T-cell immunoglobulin mucin-3 (TIM-3). Here we describe the in vitro characterization of FG-3165, a humanized monoclonal anti-Gal-9 antibody that is being developed for the treatment of solid tumors.

Methods The KD of FG-3165 for recombinant human Gal-9 was determined using a solution equilibrium assay based on a method described previously.1 Effects of Gal-9 +/- FG-3165 on TIM-3 dimerization on the cell surface were assessed using a PathHunter® TIM-3 dimerization assay from Eurofins-DiscoverX. For assessment of T cell apoptosis, human CD4+ or CD8+ T cells were activated with CD3/CD28 beads and treated with 1 µg/mL human Gal-9 +/- FG-3165 for 48 hours. Cells were then stained with annexin V and propidium iodide (PI) and analyzed by flow cytometry. RNAseq was also performed to evaluate the transcriptional changes occurring in activated CD8+ T cells treated with Gal-9 +/- FG-3165 for 3 hours and 6 hours.

Results The observed KD of FG-3165 for human Gal-9 was 0.4 ± 0.2 nM (n = 25). Dimerization of TIM-3 was shown to occur upon treatment of cells with Gal-9 and FG-3165 inhibited this dimerization with an IC50 of ~25 nM. As reported by others, Gal-9 induced apoptosis of both CD4+ and CD8+ T cells. FG-3165 inhibited Gal-9-mediated apoptosis with IC50s of 6.1 nM and 1.3 nM for CD4+ and CD8+ T cells, respectively. RNA-seq analysis revealed that an early transcriptional response to Gal-9 in CD8+ T cells, including regulation of interleukin, proliferation and apoptosis signaling genes, was normalized by FG-3165.

Conclusions FG-3165 binds with sub-nanomolar affinity to human Gal-9, preventing Gal-9-induced dimerization of TIM-3, and inhibiting apoptosis of both cytotoxic T cells and T helper cells. These data indicate that neutralization of Gal-9 by FG-3165 may overcome an important immunosuppressive mechanism in the tumor microenvironment, positioning FG-3165 as a promising candidate for the treatment of solid tumors.

Reference

  1. Ducata, et al. ‘Solution Equilibrium Titration for High-Throughput Affinity Estimation of Unpurified Antibodies and Antibody Fragments’ J. Biomolecular Screening, 2015;20(10):1256–1267.

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