Abstract 4608: A 5T4 x CD3 bispecific DART® molecule with extended half-life for T-cell immunotherapy of cancers

Abstract

Abstract Introduction: 5T4 (trophoblast glycoprotein), an oncofetal antigen involved in embryo development, is expressed on the cell surface of multiple cancers. Several 5T4-directed interventions have been reported, including a cancer vaccine and an antibody-drug conjugate. A superantigen-driven redirected cell killing modality was also developed, but obstacles, such as immunogenicity, remain to a successful 5T4 therapeutic molecule. We have developed an Fc-bearing 5T4 x CD3 DART® bispecific protein designed to redirect T cells to target 5T4-expressing tumors. Methods: 5T4 x CD3, a humanized Fc-bearing DART molecule, was stably expressed in CHO cells and purified to homogeneity via a standard antibody-purification platform. In vitro characterization and functional studies were performed with 5T4-positive tumor cell lines and human T cells. In vivo studies were performed in immune-deficient tumor-bearing mice co-implanted with activated human T cells or reconstituted with human peripheral blood mononuclear cells (PBMCs). Pharmacokinetic studies were performed in both human FcRn transgenic mice and cynomolgus monkeys. Results: 5T4 x CD3 demonstrated bispecific binding properties to both human and cynomolgus monkey antigens. In redirected cytolysis studies, 5T4 x CD3 mediated lysis of 5T4-positive pancreatic, lung, renal, triple-negative breast, and ovarian cancer cell lines with EC50 values ranging between 0.03 and 0.08 ng/mL. 5T4 x CD3 displayed favorable pharmacokinetics with a prolonged circulating half-life in human FcRn transgenic mice and cynomolgus monkeys. Tumor clearance studies in NOD/SCID mice implanted subcutaneously with activated human T cells and tumor cells demonstrated robust inhibition of tumor growth upon intravenous administration of 5T4 x CD3 at doses as low as 0.8 μg/kg, but not with a CD3-binding control DART protein. In addition, human PBMC-reconstituted NOD/SCID/IL2 gamma-chain null mice were implanted intradermally with renal and pancreatic tumor cell lines or orthotopically with a triple-negative breast cancer line and, after tumor establishment, were treated with 5T4 x CD3. Anti-tumor activity was observed at doses as low as 4 μg/kg. Immunohistochemical analysis of tumor xenografts confirmed tumor clearance was associated with T-cell recruitment into the tumor mass. Conclusions: In summary, 5T4 x CD3 displays robust antitumor activity against several cancer cell lines in vitro and in vivo together with a favorable pharmacokinetic profile and merits further consideration as a potential treatment for 5T4-positive cancers. Citation Format: Ling Huang, Gurunadh Chichili, Ralph Alderson, Francine Chen, Jennifer Brown, Hua Li, Valentina Ciccarone, Jim Tamura, Daorong Liu, Liqin Liu, Syd Johnson, Ezio Bonvini, Paul Moore. A 5T4 x CD3 bispecific DART® molecule with extended half-life for T-cell immunotherapy of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4608. doi:10.1158/1538-7445.AM2017-4608