Antibody–drug conjugates (ADCs) selectively deliver chemotherapeutic agents to target cells and are important cancer therapeutics. However, the mechanisms by which ADCs are internalized and activated remain unclear. Using CRISPR-Cas9 screens, we uncover many known and novel endolysosomal regulators as modulators of ADC toxicity. We identify and characterize C18ORF8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of screens with ADCs bearing different linkers, we show that a subset of late endolysosomal regulators selectively influence toxicity of noncleavable linker ADCs. Surprisingly, we find cleavable valine–citrulline linkers can be processed rapidly after internalization without lysosomal delivery. Lastly, we show that sialic acid depletion enhances ADC lysosomal delivery and killing in diverse cancer cell types, including with FDA (US Food and Drug Administration)-approved trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal new regulators of endolysosomal trafficking, provide important insights for ADC design and identify candidate combination therapy targets.
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We thank G. Hess, P. Drake, A. Lu and S. Pfeffer for assistance and discussions, A. Gupta for help with LC–MS/MS and M. Dubreuil and E. Jeng for comments on the manuscript. We also thank M. Pegram and W. Liang for generously providing the T-DM1 used in our experiments. This work was funded by grants from NIH (grant nos. F31 GM126688-01A1 to C.K.T., NIH 1DP2HD084069-01 to M.C.B. and NIH R01 CA227942 to C.R.B.).
R.M.B. and D.R. are employees of Catalent Biologics. C.R.B. is a cofounder and Scientific Advisory Board member of Redwood Bioscience, which generated antibody–drug conjugates used in this work. Stanford University has filed a patent application based on the findings in this article.
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Supplementary Table 1, Supplementary Figures 1–8.
Genome-wide CRISPR screen results with anti-CD22-maytansine ADC in Ramos cells.
ADC/endolysosomal sublibrary screen results with anti-CD22-maytansine (noncleavable ADC), anti-CD22-VC–maytansine (cleavable ADC) and free maytansine in Ramos cells.
Drug target, kinases and phosphatases sublibrary screen results with anti-CD22-maytansine (noncleavable ADC) and free maytansine in Ramos cells.
Count files for all screens in Ramos cells.
ADC/endolysosomal sublibrary sgRNA composition.
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Tsui, C.K., Barfield, R.M., Fischer, C.R. et al. CRISPR-Cas9 screens identify regulators of antibody–drug conjugate toxicity. Nat Chem Biol 15, 949–958 (2019). https://doi.org/10.1038/s41589-019-0342-2
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