Abstract A203: CD3-EGFR bispecific Probody™ therapeutics induced tumor regressions and increased therapeutic window in preclinical studies

T cell-engaging bispecific antibodies (TCBs) represent a highly potent modality to direct the activity of cytotoxic T cells to tumors. TCBs are designed to bind both a surface tumor antigen and the CD3e subunit of the T cell receptor, thereby enabling T cells to kill tumor cells. Because T cell bispecifics bypass TCR antigen recognition and co-stimulatory requirements for T cell activation, they obviate the need for tumor-specific immunity and overcome many impediments faced by T cells in the tumor-microenvironment. TCBs have shown clinical activity in hematologic malignancies as evidenced by the recent approval of blinatumomab for the treatment of relapsed refractory acute lymphoblastic leukemia based on its ability to direct T cells against CD19+ cells. Development of TCBs for non-hematologic cancers is proving more challenging, however, due to the high potency of the modality and its inability to discriminate between cells expressing target antigen in the tumor and those in healthy tissue. Because antigens with highly restricted tumor expression are rare, toxicity directed against healthy tissues has limited the therapeutic utility of TCBs for most solid tumor targets. In addition, because TCBs are administered at very low doses, preferential distribution to circulating T cells or to healthy tissues can limit drug exposure in tumors. Therefore new methods are needed that enable the potent anti-tumor activity of TCBs without on-target damage to normal tissues. Here we describe a T cell-engaging Bispecific Probody therapeutic (TCBP) targeting CD3 and Epidermal Growth Factor Receptor (EGFR). Probody therapeutics are recombinant, proteolytically-activated antibody prodrugs designed to widen therapeutic window by minimizing interaction with normal tissues and maximizing interactions with tumors. A Probody therapeutic is an antibody in which the antigen-binding site is blocked by a masking peptide extension from the amino terminus of the light chain. The peptide mask, joined to the antibody by a protease-cleavable linker, can be removed by proteases that are more active in the tumor microenvironment than in normal tissue, allowing for tumor-localized activity. We have previously demonstrated that an EGFR-targeted Probody therapeutic limits interaction with EGFR-expressing tissue while preserving anti-tumor activity. To demonstrate that Probody technology can address the limitations of bispecific antibodies, we compared CD3-EGFR TCBPs to the corresponding unmasked TCB for their ability to direct T cell-mediated cytotoxicity of EGFR+ tumor cells in vitro. While the unmasked CD3-EGFR TCB demonstrated potent T cell- and EGFR-dependent killing at single-digit pM concentrations, the CD3-EGFR TCBP exhibited reduced targeted cytotoxicity by up to 3000-fold. In a tumor model utilizing engraftment of human T cells in NSG mice, both TCB Probody therapeutic and its parental TCB effectively eliminated established EGFR+ HT-29 colorectal tumors at equivalent doses. In contrast, an ongoing study in non-human primates has shown at least a 10-fold higher maximum tolerated dose for the CD3-EGFR TCBP relative to its corresponding CD3-EGFR TCB. As a result, Probody technology has the potential to increase therapeutic window and may enable potent bispecific therapies for solid tumors that are limited by on-target toxicities in healthy tissue. Citation Format: Sherry L. LaPorte, Daniel R. Hostetter, Laurie Wong, Jennifer Razo, Linnea Diep, Clayton W. White, Jennifer H. Richardson, W. Michael Kavanaugh, Bryan A. Irving. CD3-EGFR bispecific Probody™ therapeutics induced tumor regressions and increased therapeutic window in preclinical studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A203.