Abstract C160: New class of DNA-alkylating agents with a suitable tolerability profile created for use in antibody-drug conjugates (ADCs). (2013)

Abstract The recent FDA approvals of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla) has spurred tremendous interest in ADCs for the treatment of cancer. Most clinical-stage ADCs, including the two approved ones, utilize a tubulin-acting small molecule as the cytotoxic payload. There is strong interest in preparing DNA-acting payload agents because some cancers are more responsive to DNA-acting agents than to tubulin-acting ones. The key challenge has been developing a DNA-acting payload agent with the necessary tolerability profile as well as potency. We have developed a new class of DNA-acting agents for use in ADCs, IGNs, which demonstrate the desired profile. Initially, our IGNs were indolino-benzodiazepine dimers that were highly potent in vitro (IC50 ∼10−12 M) by virtue of their ability to both alkylate and cross-link DNA. However, a resultant IGN-ADC did not have the desired in vivo tolerability, a known problem with highly potent DNA-acting agents. This IGN-ADC was found to be intolerable in mice (4.4 mg/kg, weekly x 3), resulting in delayed toxicity and mortality. To address this, we tested modifying the saturation level of the di-imine cross-linking groups by generating a mono-imine compound that could still alkylate DNA, but no longer act as a cross-linker. This new, mono-imine IGN, 2m (alkylator only), was highly potent, but with at least 2-fold better tolerability (9.1 mg/kg, weekly x 3) and, most importantly, not associated with delayed toxicity. To assess its potency in an ADC format, 2m and the lead IGN di-imine compound, 2d (crosslinker/alkylator) were conjugated to the folate receptor-α (FRα)-binding antibody, anti-FRα, and the CD33-binding antibody, anti-CD33, via amide bonds. In spite of their different imine saturation levels, anti-FRα-2d and anti-FRα-2m were both highly potent against the FRα-expressing KB cell line (IC50 values of 15 pM and 20 pM, respectively). The addition of excess unconjugated antibody abolished the cytotoxic effect (&amp;gt;3 nM), thus demonstrating that the activity of the conjugate is antigen specific. Similarly, anti-CD33-2d and anti-CD33-2m displayed comparable potency against a panel of CD33-expressing cell lines. This included the human promyelocytic leukemia cell line, NB4, where these two IGN-ADCs had IC50 values in the picomolar range despite the low level of antigen expression on the target cells (∼10,000 molecules/cell). In vivo, the anti-FRα-2m conjugate was found to be effective against KB tumors in mouse xenograft models at doses that were well below the MTD. The mechanism of action, potency, tolerability, and lack of delayed toxicity make IGNs a promising new class of cytotoxic small molecules for use in ADCs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C160. Citation Format: Michael Miller, Nathan Fishkin, Wei Li, Emily Reid, Katie Archer, Erin Maloney, Yelena Kovtun, Gregory Jones, Megan Ellis, Rajeeva Singh, Kathleen Whiteman, Jan Pinkas, Ravi Chari. New class of DNA-alkylating agents with a suitable tolerability profile created for use in antibody-drug conjugates (ADCs). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C160.