Abstract 1749: A new class of DNA sequence-selective G-A cross-linking antibody-drug conjugate (ADC) payloads

Abstract Antibody-Drug Conjugates (ADCs) are rapidly growing in importance as a targeted cancer therapy, with over ten approved, and many currently undergoing clinical trials. ADCs comprise of a cytotoxic agent (i.e., the "payload") conjugated via a chemical linker to a tumor-targeting antibody. There is a demand for novel ADC payloads with unique mechanisms of action, enhanced tolerability profiles and improved physicochemical properties. Such payloads could lead to ADCs with therapeutic efficacy in patients resistant to other therapies, and payloads with reduced hydrophobicity should lead to improved conjugation, minimal aggregation, and higher Drug-Antibody Ratios (DARs). We report here, studies on a novel class of Cyclopropabenzindole-Pyridinobenzodiazepine (CBI-PDD) payloads, designed through molecular modeling, which form cross-links between Guanine and Adenine bases within the DNA minor groove with defined sequence specificity. Eleven novel analogs will be described which have been structurally modified with various functional groups to allow tunable hydrophobicity, and new vectors through which antibodies may be attached. Most of these analogs are highly cytotoxic, with some exhibiting IC50 values in tumor cells down to the femtomolar range (e.g., FGX37-140, 0.9 pM in both RAJI and Jurkat cells, 72 h incubation). The DNA interactivity of these analogs has been studied using a range of methods such as gel-based DNA footprinting and cross-linking, FRET melting and Transcription Factor (TF) Array assays, the results of which will be described. Overall, the analogs appear to favor the formation of intrastrand rather than interstrand DNA cross-links and can inhibit the DNA binding of several key cancer-related transcription factors such as NFκB. To explore the potential of these analogs as ADC payloads, one member, FGX8-46, was conjugated to the EGFR-targeting antibody Cetuximab in stochastic fashion (DAR2) to produce Cetuximab-(FGX16-11). This ADC had an unexpectedly high Maximum Tolerated Dose (MTD) in a standard mouse model of at least 45 mg/kg, perhaps reflecting the novel mechanism of action of the payload. It was then evaluated in a Human Tumor Xenograft study based on BALB/c mice transplanted with the EGFR-expressing human colon cancer cell line SW-48 using single doses ranging from 1 mg/kg to 40 mg/kg. Cetuximab-(FGX16-11) was active at all dose levels including 1 mg/kg which provided tumor suppression out to approximately three weeks. Complete tumor suppression out to 50 days was seen with 20 mg/kg. Overall, the unique mechanism of action, potent cytotoxicity, and excellent in vivo tolerability and significant efficacy in ADC format, make the CBI-PDDs a promising new class of ADC payloads suitable for further development. Citation Format: George Procopiou, Paul J. Jackson, Daniella M. di Mascio, Jennifer L. Auer, Paolo Andriollo, Ilona Pysz, Khondaker M. Rahman, Keith R. Fox, David E. Thurston. A new class of DNA sequence-selective G-A cross-linking antibody-drug conjugate (ADC) payloads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1749.