P. Ryan1, E. Clarke2, G. dos Santos2, T. Kell2, Y. Huang2, J. Borrok1, V. Balakumar3, S. Gregson3, P. Ryan1, P. Howard3, and R. Dixit1. 1MedImmune LLC, Gaithersburg, MD; 2ReachBio Research Labs, Seattle, WA; and 3Spirogen Ltd, London, United Kingdom.
ADC design involves a tumor antigen-specific antibody, a linker and a cytotoxic drug. In some clinical trials, ADCs caused neutropenia in patients despite having targets that are not present on hematopoietic stem cells (HSC) or neutrophils. The CFU-GM assay, predictive of clinical neutropenia and validated by Pessina et al (2003), evaluated compound effects on primitive myeloid precursors, whereas the platform described by Zhao et al (2017) suggested the extracellular cleavage of the ADC linker was responsible for cytotoxicity to differentiating mature neutrophils.
We tested 2 naked antibodies, 1 cytotoxic drug and multiple ADCs with different cleavable linker sites (valine-alanine, valine-citrulline and maleimidomethyl cyclohexane-1-carboxylate) on both platforms to compare their utility to identify potential off-target bone marrow (or hematologic) effects of ADCs. For the CFU-GM assay, compounds were added to cells in a liquid medium for 72 hours, then the treated cells were transferred to a methylcellulose-based medium for 14 days. To assess neutrophil toxicity using the Zhao platform, CD34+ cells were cultured for 3 days initially to expand the HSC followed by 4 days in a differentiation media and then another 4 days with various cytokines. The various MAbs, payload and ADCs were added to the culture for the final 6 days (G-CSF only) and assessed for CD15, CD66b and CD32 by flow cytometry.
The naked antibodies, 1C1-Maia and trastuzumab, had no effect on CFU-GM or neutrophil numbers. The payload (drug plus linker) demonstrated significant toxicity to the CFU-GM (IC50 of 0.0006 nM), but much less potency to mature neutrophils (IC50 > 0.3 nM). Kadcyla (FDA approved trastuzumab-based ADC) was 3 times more inhibitory to the CFU-GM assay compared to mature neutrophils, whereas trastuzumab-vc-MMAE ADC was 5 times more inhibitory to mature neutrophils compared to primitive progenitors. There was more than a 10-fold difference in the IC50 values of the ADCs R347-Maia-SG3294, CS1-Maia-SG3294 and 1C1-Maia-SG2349 between assays, with more toxicity towards the CFU-GM than the differentiating neutrophils. With many ADCs in clinical trials, these assay platforms may be useful during preclinical development to identify potential off-target effects of antibodies and payloads or susceptibility for single or dual cleavage sites to release the cytotoxic drug.