Abstract 1498: Development of an IL13Ralpha2 x CD3 bispecific DART® protein for redirected T-cell killing of solid tumors
Authors: Rillema Jill , Licea Monica , Saini-Lal Shereen , Smith Doug , Chen Francine , Lam Annie , Yang Yinhua , Liu Liqin , Tamura James , Alderson Ralph , Bonvini Ezio , Johnson Syd , Moore Paul 
Published: July 2016

Abstract
Introduction. IL13Rα2 is a membrane-bound protein expressed on a number of malignant tumors, but not at significant levels in most normal tissues. Clinically tested therapies to treat cancer by targeting IL13Rα2 to date have included a ligand toxin conjugate (IL13-PE) and IL13Rα2 directed CAR-T cells. Both strategies appear to be well tolerated, although with mixed results in tumor eradication. An alternative strategy for targeting IL13Rα2 is an IL13Rα2 x CD3 Dual-Affinity Re-Targeting (DART®) bispecific molecule designed to co-engage IL13Rα2 on tumor cells and cytotoxic T cells through CD3, resulting in killing of tumor cells.
Methods. Mouse monoclonal antibodies (mAbs) were generated using standard immunization protocols followed by binding and epitope binning analyses. Cell binding was performed by flow cytometry and frozen normal and tumor tissues analyses by immunohistochemistry (IHC). In vitro studies were performed with cancer cell lines and primary human T cells or peripheral blood mononuclear cells (PBMCs). In vivo studies were performed in immune-deficient tumor-bearing mice co-implanted with activated human T cells or reconstituted with human PBMCs.
Results. A panel of mAbs that bind human and cynomolgus monkey IL13Rα2 proteins were selected, representing a range of binding characteristics and epitope diversity. IHC showed favorable normal vs tumor tissue reactivity, with high levels of IL13Rα2 expression in glioblastoma and melanoma. The mAb panel was converted to DART molecules with an anti-CD3 arm, assessed for the ability to mediate cytotoxic T lymphocyte (CTL) activity and cytokine release and a lead selected for humanization and engineering into an IL13Rα2 x CD3 DART molecule incorporating an Fc domain to prolong circulating half-life. The IL13Rα2 x CD3 DART protein bound human and cynomolgus IL13Rα2 (KD = 0.64 nM and 0.5 nM, respectively) and mediated CTL activity against LOX-IMVI (melanoma) and SK-MES-1 (lung adenocarcinoma) target cells. Cytokines (IFN-γ, IL-10, and TNF-α) were released in the presence of IL13Rα2-expressing target cells and human PBMC, but not with PBMCs alone. Administration of the IL13Rα2 x CD3 DART molecule (≤50 μg/kg IV for 4 consecutive days) prevented tumor growth when tumor cells (A375 and LOX-IMVI melanoma, U87 glioblastoma, or DAOY medulloblastoma cell lines) were co-mixed with activated human T cells and implanted subcutaneously in NOD/SCID/IL2gamma-chain null mice. Tumor eradication was also observed in NOD/SCID/IL2gamma-chain/MHCI KO mice reconstituted with human PBMCs and bearing established LOX-IMVI melanoma tumors following 2 weekly IV doses of 50 μg/kg IL13Rα2 x CD3 DART molecule.
Conclusions: Robust in vitro and in vivo antitumor activity for an IL13Rα2 x CD3 DART protein was demonstrated. Additional studies are underway to further characterize the molecule as a development candidate for treatment of IL13Rα2-positive cancers.
Citation Format: Jill Rillema, Monica Licea, Shereen Saini-Lal, Doug Smith, Francine Chen, Annie Lam, Yinhua Yang, Liqin Liu, James Tamura, Ralph Alderson, Ezio Bonvini, Syd Johnson, Paul Moore. Development of an IL13Ralpha2 x CD3 bispecific DART® protein for redirected T-cell killing of solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1498.