Abstract 736: Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity

Abstract

Although four ADCs have been approved and over sixty others are in development, the majority contain payloads belonging to two classes; tubulin inhibitors and DNA cross-linkers. Challenges in the development of ADCs include managing off-target toxicity and hydrophobicity. Some payload classes (e.g., PBD dimers) are notably hydrophobic leading to problems (e.g., aggregation) during conjugation. Thus, there is interest in developing novel payloads which retain the potency of DNA cross-linkers but have lower hydrophobicity and a wider therapeutic window when part of an ADC. The pyridinobenzodiazepines (PDDs) are a new class of sequence-selective, DNA mono-alkylating ADC payload which contain a polyheterocyclic chain with sufficient span to guide them to specific DNA sequences (e.g., transcription factor binding sites). The lead PDD payload, FGX-2-62, has a different sequence-selectivity profile to other DNA-interactive agents, spanning 8-9 base-pairs compared to 6-7 for a PBD dimer, and DNA footprinting experiments indicate a preference for 5'-XGXWWWWXX-3' sequences (X is any base; W is A/T). Transcription factor array studies have shown that the molecule inhibits DNA-binding of oncogenic transcription factors (e.g., NF-κB and GATA). In in vitro cell line studies, FGX-2-62 has low pM cytotoxicity in a diverse cell line panel, including stem cells, cells from both solid and blood cancers (e.g., 9 pM in HL-60) and MDR-resistant tumours, and arrests the cell cycle at the G0/G1 phase compared to G2-M arrest for PBD dimers. It is compatible with attachment to most linker technologies, and is significantly less hydrophobic than other payload classes. Initial MTD studies were carried out by separately conjugating (with negligible aggregation) FGX-2-62 and the PBD dimer Talirine to a THIOMAB® version of trastuzumab (DAR = 2). In female athymic nude mice, a greater tolerance was observed for the THIOMAB®-(FGX-2-62) ADC compared to the THIOMAB®-PBD dimer (i.e, MTD >8 mg.kg-1 versus 4 mg.kg-1). In an efficacy study, FGX-2-62 was conjugated to a cancer stem cell-targeting IgG1 antibody (Bstrongximab) with DAR 1.9. Initial evaluation afforded IC50 values of 0.67 nM and 0.47 nM in two antigen positive cell-lines, and an MTD of 6 mg.kg-1 in mice. In an antigen-positive embryonal carcinoma stem cell CDX mouse model, complete regression was observed at a dose of 2 mg.kg-1 (Q7Dx3). In a cholangiocarcinoma PDX model, complete tumour regression was observed out to 80 days (when experiment was terminated) at a dose of 5 mg.kg-1 (Q7Dx3), with no observed toxicity. The favourable hydrophobicity profile of the PDDs and ease of conjugation, along with their novel mechanism of action, significant in vitro cytotoxicity, in vivo efficacy and tolerability in MTD studies suggest that they represent a promising new class of ADC payloads. Citation Format: Nicolas Veillard, Paolo Andriollo, Julia Mantaj, Keith R. Fox, K Miraz Rahman, George Procopiou, Francesco Cascio, David B. Corcoran, Ilona Pysz, Patricia A. Cooper, Steven D. Shnyder, Yawen Ju, Edwin Tan, William M. Schopperle, Paul J. Jackson, David E. Thurston. Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 736.

A Novel Antibody-Drug Conjugate (ADC) Delivering a DNA Mono-Alkylating Payload to Chondroitin Sulfate Proteoglycan (CSPG4)-Expressing Melanoma

“…CD30 down-regulation, MMAE resistance, and upregulation of the MDR1 drug exporter protein have all been shown to be associated with resistance to brentuximab vedotin [47]. However, our data are consistent with previous reports of low nM to pM cytotoxicity for PDDs against a panel of tumor cell types, including MDR-resistant tumors [45]. From the antigen-antibody targeting perspective, CSPG4 may promote chemoresistance by activation of integrin-dependent PI3K/Akt signaling in cell lines and patient samples [48].…”

“…Due to the mono-alkylating mechanism of action of the PDDs, there is evidence to suggest that they may enhance the therapeutic window of ADCs by reducing off-target cytotoxic effects. Consistent with this notion, previous work comparing the site-specific THIOMAB conjugation of a PDD and the PBD dimer Talirine to trastuzumab resulted in an MTD of 4mg/kg for the THIOMAB-PBD dimer and an MTD of > 8mg/kg for the THIOMAB-(PDD) ADC [45]. In this treatment setting, the anti-CSPG4-(PDD) administered in two 2 mg/kg doses significantly restricted tumor growth compared with isotype-(PDD) ADC, PDD payload alone, anti-CSPG4 plus unconjugated PDD or buffer control-treated groups.…”

Antibody-drug conjugates (ADCs) delivering pyrrolobenzodiazepine (PBD) dimers for cancer therapy

“…It has been proposed that the formation and persistence of highly potent DNA interstrand cross-links produced by the PBD dimers cause significant systemic toxicities in patients and thereby limit the therapeutic index that can be achieved [75]. This has led to the development of DNA covalent-binding payloads that can only mono-alkylate rather than cross-link DNA such as the indolinobenzodiazepines (IGNs) produced by ImmunoGen Inc [76] and the pyridinobenzodiazepines (PDDs) produced by Femtogenics Ltd [77]. ADCs such as [78], and IMGN632 targeting CD123 [79], showed promising preclinical activity and safety profiles suggesting a potential for increased therapeutic index in the clinic.…”

DNA sequence-selective G-A cross-linking ADC payloads for use in solid tumour therapies

“…We recently reported a class of DNA G-mono-alkylating ADC payloads, the pyridinobenzodiazepine (PDD) monomers 16 , 17 . The core unit of a PDD ( cf .…”