Abstract 688: Characterization and circumvention of drug resistance mechanisms in SGN-35-resistant HL and ALCL clonal cell lines
Authors: Lewis Timothy S. , Gordon Kristine , Li Fu , Weimann Allana , Bruders Rebecca , Miyamoto Jamie , Chace Dana , Law Che-Leung 
Published: May 2015

Abstract
SGN-35 (cAC10-vcMMAE, brentuximab vedotin) is an auristatin antibody-drug conjugate (ADC), which targets CD30, for the treatment of relapsed Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). We developed three SGN-35 resistant (BVR) cell line models to identify mechanisms by which HL and ALCL cells may become resistant to SGN-35 therapy. Resistant DEL and Karpas-299 (ALCL) and L540cy (HL) cell lines were generated by continuous exposure to increasing dose levels of SGN-35 over time. BVR cell lines with &gt;10-fold decrease in sensitivity to SGN-35 versus parental cell lines were then made clonal. Multiple parental and BVR cell clones for each cell type were then characterized by cytotoxicity assay for sensitivity to SGN-35 and other anti-CD30 ADCs conjugated to alternate auristatin or pyrrolobenzodiazepine (PBD) dimer chemotypes. CD30 expression was analyzed by flow cytometry to monitor downregulation of the ADC targeted cell surface antigen. Changes in multidrug resistance efflux activity were evaluated by rhodamine 123 efflux, flow cytometry of transporter protein expression, and cytotoxicity measurements in the presence of the ABC1 transporter inhibitor verapamil. Gene expression changes between parental and BVR clones were measured using RNA sequencing, and confirmed by qPCR and western blotting. The L540cy-BVR, DEL-BVR, and Karpas-299-BVR clonal cell lines displayed unique paths to resistance. The L540cy-BVR resistance was due to upregulated transporter activity, which was reversible with verapamil. A minor increase in P-gp level was detected, and an increase in other transporter gene mRNA levels was also observed. CD30 expression on the surface of L540cy-BVR cell lines was unaffected. In contrast, the DEL-BVR resistant lines had moderate decrease in CD30 expression levels combined with increased P-gp expression. These resistant lines also had increased expression of NNMT, an enzyme involved in the metabolism of xenobiotic compounds. Finally, resistance in Karpas-299-BVR cells was due entirely to loss of CD30 protein expression. While no longer sensitive to SGN-35, the L540cy-BVR and DEL-BVR clones were sensitive to new anti-CD30 ADCs in vitro. Anti-CD30 mAb cAC10 conjugated to mcMMAF and other related auristatins efficiently killed L540-BVR and DEL-BVR cells. In addition, CD30-directed PBD dimer conjugates were highly potent on these resistant cell lines. These new anti-CD30 auristatin and PBD dimer ADCs also showed significantly improved activity over SGN-35 in a subcutaneous DEL-BVR mouse xenograft model. Our data suggest that downregulation of CD30 cell surface expression and upregulation of transporter activity are potential mechanisms for SGN-35 acquired resistance in systemic ALCL and HL. Newly developed antibody-drug conjugates utilizing MMAF-like and DNA-damaging PBD dimer drug-linkers are highly effective at overcoming SGN-35 resistance in vitro.
Citation Format: Timothy S. Lewis, Kristine Gordon, Fu Li, Allana Weimann, Rebecca Bruders, Jamie Miyamoto, Dana Chace, Che-Leung Law. Characterization and circumvention of drug resistance mechanisms in SGN-35-resistant HL and ALCL clonal cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 688. doi:10.1158/1538-7445.AM2014-688