Abstract B32: A tumor microenvironment specific EGFR targeting antibody-drug conjugate promotes regression in KRAS or BRAF mutant tumors

The epidermal growth factor (EGF) signaling pathway relies on recognition by its receptor, EGFR, and subsequent downstream signaling by the KRAS and BRAF proteins to relay proper proliferative, migratory, and angiogenic functions. Cancers with activating KRAS or BRAF mutations are resistant to EGFR targeting agents and correspond to a significant unmet medical need. We hypothesized that an anti-EGFR antibody-drug conjugate (ADC) could be active against KRAS or BRAF mutated tumors, due to the cytotoxic mechanism of the ADC warhead. In an effort to eliminate the known dermal toxicity associated with anti-EGFR therapy, and to mitigate potential toxicities associated with treatment by an anti-EGFR ADC, we wished to engineer an antibody with enhanced specificity towards EGFR in the tumor microenvironment (TME) and attenuated binding to EGFR in normal tissue. This was achieved by screening a library of antibody variants (based on cetuximab) in a spatially addressed manner for binding to a recombinant version of the EGFR extracellular domain (EGFR ECD ) in two separate ELISA reaction conditions. High affinity binding to the EGFR ECD was desired in the first condition, which approximated the physicochemical properties of the TME (acidic pH, high lactic acid concentration, 25% human serum). In the second assay condition, which approximated mAb binding to EGFR ECD in normal tissue (neutral pH, low lactic acid concentration, 25% human serum), attenuated binding affinity was desired. We identified a lead mAb variant, cMab-1501, which possessed several fold reduced binding to EGFR ECD in the neutral pH, low lactic acid condition, when compared to EGFR ECD binding in the low-pH, high lactic acid, assay condition. To evaluate enhanced specificity for binding to EGFR in vivo, cMab-1501 was compared to cetuximab for binding to both human donor foreskin xenografts and human A431 tumor xenografts, using a DyLight 755 conjugated version of each antibody, and subsequent fluorescence detection with a Caliper IVIS system. cMab-1501 and cetuximab demonstrated relatively comparable binding towards human A431 tumor xenografts in vivo. In addition, cetuximab bound relatively equally between human tumor xenografts and human skin grafts. However, no binding to EGFR in the human skin graft was detected for cMab-1501 over all days measured; suggesting that cMab-1501 was highly specific for binding to EGFR in the TME. We next generated an cMab-1501 based ADC (antibody-drug conjugate), via maleimide chemistry carrying a protease cleavable valine-citrulline-p-aminobenzyloxycarbonyl monomethylauristatin E (vcPAB-MMAE) cytotoxic moiety, forming a cMab-1501-vcPAB-MMAE conjugate. Both the conjugated and un-conjugated versions of cMab-1501 were rapidly internalized by EGFR positive MDA-MB-231M tumor cells over several hours. In tumor xenograft models, the TME-specific anti-EGFR ADC demonstrated complete tumor regressions against two human EGFR overexpressing tumor types, MDA-MB-231M (TNBC, KRAS G13D) and HT-29 (CRC, BRAF V600E). In both in vivo models, tumors were resistant to treatment by cetuximab. These data suggest that it is possible to engineer a monoclonal antibody with enhanced specificity for its target within the TME and that an ADC-based approach could be utilized as potential treatment of EGFR overexpressing tumors with KRAS or BRAF mutations. Citation Format: Bob Veneziale, Lei Huang, Xiaoming Li, Qiping Zhao, Chunmei Zhao, Ryan Osgood, Jessica Cowell, Sanna Rosengren, Jason Parise, Ge Wei, Kim Phan, Robert Connor, Steve Rowe, Gilbert Keller, Gregory Frost, Dan Maneval, Curtis Thompson, Michael Shepard, Christopher Thanos. A tumor microenvironment specific EGFR targeting antibody-drug conjugate promotes regression in KRAS or BRAF mutant tumors. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B32.