Abstract 2012: A humanized anaplastic lymphoma kinase (ALK)-directed antibody-drug conjugate (ADC) demonstrates potent cytotoxicity in neuroblastoma (NBL) and fusion positive rhabdomyosarcoma (FP RMS)

Abstract

Abstract Introduction: ALK is a lineage restricted oncoprotein expressed on the cell surface of NBL as wild-type (WT), mutated, or amplified, and recently shown to be overexpressed in FP RMS. Small molecule inhibitors have shown potent efficacy in NBL with ALK mutations or amplifications, but is limited to a minority of the high-risk population and have demonstrated conferred resistance. We have previously shown the potential of an ADC approach for WT ALK-expressing NBL with a chimeric ALK-directed ADC CDX-0125. Methods: A highly specific humanized ALK antibody KTN0239 was developed with site-specific conjugation of an amine-azido linker conjugated to pyrrolobenzodiazepine (PBD) dimers or duocarmycin using click chemistry. ALK surface expression in ALK amplified (NB-1), WT non-amplified (IMR-32), mutant (Felix, SH-SY5Y, Kelly, NB1-643), and WT null (SK-N-AS) cell lines were quantified with flow cytometry using the primary KTN0239 antibody. Internalization kinetics of KTN0239 were measured using Incucyte® Fabflour-pH labeling. In vitro ADC IC50 values and viability rescue were assessed with a CellTiter-Glo® assay with ADC concentrations ranging from 10 fM to 10 nM. Western blot was used to elucidate cytotoxic mechanism of the ADC. Results: ALK surface protein molecule expression was 21,820 in NB-1 cells, 3,708 in Felix, and 1,426 in SK-N-AS cells. KTN0239 was internalized into NB1 cells with a logarithmic trend, with >65% of antibody internalized within 8 hours. NB-1 cell line viability was rescued to >80% when 2.5 nM of KTN0239 was in competition with the PBD-conjugated ADC at the IC50 concentration. The ADC induced activation of PARP, caspase-3, and γH2AX without attenuation of ALK signaling in NB-1 cells. Conclusions: ADCs directed to ALK-expressing cancers are a promising therapeutic approach. The efficacy of the ADCs are currently being tested in NBL and FP RMS mouse models, and will be reported. Mean IC50 values (pM) of ADCs with a drug antibody ratio (DAR) of 6 in NBL cell lines ADC: PBD-conjugated Duocarmycin-conjugated NB-1 14 71 IMR-32 12 741 Felix 51 110 NB-1643 6.6 1,196 Kelly 517 34,700 SH-SY5Y 693 3,024 SK-N-AS 533 1,420 Citation Format: Alberto D. Guerra, Annie Kennedy, Cynthia Adams, Rawan Shraim, Amber K. Weiner, Chuan Chen, Kateryna Krytska, Colleen Larmour, Smita Matkar, Dimiter Dimitrov, Dontcho V. Jelev, Yael P. Mossé. A humanized anaplastic lymphoma kinase (ALK)-directed antibody-drug conjugate (ADC) demonstrates potent cytotoxicity in neuroblastoma (NBL) and fusion positive rhabdomyosarcoma (FP RMS) [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 2012.