Abstract 946: SGN-CD70A, a pyrrolobenzodiazepine (PBD) dimer linked ADC, mediates DNA damage pathway activation and G2 cell cycle arrest leading to cell death
Authors: Sandall Sharsti L. , McCormick Renee , Miyamoto Jamie , Biechele Travis , Law Che-Leung , Lewis Timothy S. 
Published: August 2015

Abstract
Seattle Genetics is utilizing the highly potent DNA cross-linking molecule, pyrrolobenzodiazepine (PBD) dimer, as the payload across multiple antibody drug conjugate (ADC) platforms. Previous studies have demonstrated that PBD dimers bind to the minor groove of DNA causing an interstrand crosslink; however, it is poorly understood how the cells recognize and respond to this insult. The DNA damage pathway exists to ensure genomic fidelity by either repairing damaged DNA or forcing programmed cell death if the damage is too extensive. Cells do this in an orchestrated manner that involves sensing the damage and eliciting a signaling cascade to induce repair and cell cycle arrest and ultimately either cell cycle re-start, death, or senescence. Using SGN-CD70A, an ADC consisting of a CD70-directed engineered cysteine antibody linked to two PBD dimers, we have examined the DNA damage response and cell death kinetics in a panel of renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines after treatment with free PBD and SGN-CD70A.
We examined the central DNA damage sensing protein kinases, ATM, ATR, and DNA-PK and find that all three are phosphorylated on activation residues within 24 hours at EC50 doses in both RCC and NHL cell lines. Similarly, the downstream checkpoint kinases, Chk1 and Chk2 are also activated, within 24 hours after treatment with SGN-CD70A. The kinetics of pChk2 activation mirror Caspase 3/7 activation, with both peaking 48-72 hours after treatment. To confirm the involvement of the DNA damage pathway in the PBD mechanism, we used small molecules to inhibit ATM (KU-60019), ATR (VE-821), DNA-PK (NU-7441), and Chk1/2 (AZD7762, LY2603618) and found that all were synergistic with SGN-CD70A and accelerated cell death kinetics. We also conducted a small molecule screen to identify other processes involved in SGN-CD70A's activity. The Mps1 inhibitor (AZ 3146), which shortens mitosis and abrogates the mitotic checkpoint, was strongly synergistic. Conversely, molecules that cause mitotic arrest, such as microtubule inhibitors and Plk1 inhibitors did not show the same degree of synergy. To follow up on this finding we looked at cell cycle kinetics. We found that SGN-CD70A treatment alone causes a G2 arrest and a modest increase in polyploid cells whereas synergistic combinations with SGN-CD70A and DNA damage kinase and Mps1 inhibitors decrease the number of cells in G2 while markedly increasing polyploid cells. These data suggest induction of mitotic catastrophe may be part of the cellular response to SGN-CD70A treatment. Based on these results, we are currently testing whether sequencing SGN-CD70A and various mitotic inhibitors can increase synergy.
Citation Format: Sharsti L. Sandall, Renee McCormick, Jamie Miyamoto, Travis Biechele, Che-Leung Law, Timothy S. Lewis. SGN-CD70A, a pyrrolobenzodiazepine (PBD) dimer linked ADC, mediates DNA damage pathway activation and G2 cell cycle arrest leading to cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 946. doi:10.1158/1538-7445.AM2015-946