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.
This is a preview of subscription content, access via your institution
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
casTLE v.1.0 is available at https://bitbucket.org/dmorgens/castle.
Beck, A., Goetsch, L., Dumontet, C. & Corvaïa, N. Strategies and challenges for the next generation of antibody–drug conjugates. Nat. Rev. Drug Discov. 16, 315–337 (2017).
Lyon, R. Drawing lessons from the clinical development of antibody-drug conjugates. Drug Discov. Today Technol. 30, 105–109 (2018).
St Pierre, C. A., Leonard, D., Corvera, S., Kurt-Jones, E. A. & Finberg, R. W. Antibodies to cell surface proteins redirect intracellular trafficking pathways. Exp. Mol. Pathol. 91, 723–732 (2011).
Ritchie, M., Tchistiakova, L. & Scott, N. Implications of receptor-mediated endocytosis and intracellular trafficking dynamics in the development of antibody drug conjugates. MAbs 5, 13–21 (2013).
Loganzo, F. et al. Tumor cells chronically treated with a trastuzumab-maytansinoid antibody-drug conjugate develop varied resistance mechanisms but respond to alternate treatments. Mol. Cancer Ther. 14, 952–963 (2015).
Donaghy, H. Effects of antibody, drug and linker on the preclinical and clinical toxicities of antibody-drug conjugates. MAbs 8, 659–671 (2016).
Lewis Phillips, G. D. et al. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res. 68, 9280–9290 (2008).
Polson, A. G. et al. Antibody-drug conjugates for the treatment of non-Hodgkin’s lymphoma: target and linker-drug selection. Cancer Res. 69, 2358–2364 (2009).
Rabuka, D., Rush, J. S., deHart, G. W., Wu, P. & Bertozzi, C. R. Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nat. Protoc. 7, 1052–1067 (2012).
Agarwal, P. et al. Hydrazino–Pictet–Spengler ligation as a biocompatible method for the generation of stable protein conjugates. Bioconjug. Chem. 24, 846–851 (2013).
Lopus, M. et al. Maytansine and cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule dynamics by binding to microtubules. Mol. Cancer Ther. 9, 2689–2699 (2010).
Morgens, D. W. et al. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nat. Commun. 8, 15178 (2017).
Morgens, D. W., Deans, R. M., Li, A. & Bassik, M. C. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nat. Biotechnol. 34, 634–636 (2016).
Hamblett, K. J. et al. SLC46A3 is required to transport catabolites of noncleavable antibody maytansine conjugates from the lysosome to the cytoplasm. Cancer Res. 75, 5329–5340 (2015).
Hyttinen, J. M. T., Niittykoski, M., Salminen, A. & Kaarniranta, K. Maturation of autophagosomes and endosomes: a key role for Rab7. Biochim. Biophys. Acta 1833, 503–510 (2013).
Poteryaev, D., Datta, S., Ackema, K., Zerial, M. & Spang, A. Identification of the switch in early-to-late endosome transition. Cell 141, 497–508 (2010).
Liu, K. et al. Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion. J. Cell Biol. 212, 181–198 (2016).
Rapiteanu, R. et al. A genetic screen identifies a critical role for the WDR81–WDR91 complex in the trafficking and degradation of tetherin. Traffic 17, 940–958 (2016).
Burger, J. A. & Wiestner, A. Targeting B cell receptor signalling in cancer: preclinical and clinical advances. Nat. Rev. Cancer 18, 148–167 (2018).
Mould, A. W. et al. Global expression profiling of murine MEN1-associated tumors reveals a regulatory role for menin in transcription, cell cycle and chromatin remodelling. Int. J. Cancer 121, 776–783 (2007).
Wood, K., Tellier, M. & Murphy, S. DOT1L and H3K79 methylation in transcription and genomic stability. Biomolecules 8, 11 (2018).
Schröder, B. et al. Integral and associated lysosomal membrane proteins. Traffic 8, 1676–1686 (2007).
Dubowchik, G. M. et al. Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug. Chem. 13, 855–869 (2002).
Caculitan, N. G. et al. Caculitan, N. G. et al. Cathepsin B is dispensable for cellular processing of cathepsin B-cleavable antibody-drug conjugates. Cancer Res. 77, 7027–7037 (2017).
Zmolek, W., Bañas, S., Barfield, R. M., Rabuka, D. & Drake, P. M. A simple LC/MRM-MS-based method to quantify free linker-payload in antibody-drug conjugate preparations. J. Chromatogr. B 1032, 144–148 (2016).
Yoshimori, T., Yamamoto, A., Moriyama, Y., Futai, M. & Tashiro, Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J. Biol. Chem. 266, 17707–17712 (1991).
Pontano Vaites, L., Paulo, J. A., Huttlin, E. L. & Harper, J. W. Systematic analysis of human cells lacking ATG8 proteins uncovers roles for GABARAPs and the CCZ1/MON1 regulator C18orf8/RMC1 in macro and selective autophagic flux. Mol. Cell. Biol. 38, e00392-17 (2017).
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Rillahan, C. D. et al. Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome. Nat. Chem. Biol. 8, 661–668 (2012).
Xiao, H., Woods, E. C., Vukojicic, P. & Bertozzi, C. R. Precision glycocalyx editing as a strategy for cancer immunotherapy. Proc. Natl Acad. Sci. USA 113, 10304–10309 (2016).
Wu, A. M. et al. Differential affinities of erythrina cristagalli lectin (ECL) toward monosaccharides and polyvalent mammalian structural units. Glycoconj. J. 24, 591–604 (2007).
Polson, A. G. et al. Antibody-drug conjugates targeted to CD79 for the treatment of non-Hodgkin lymphoma. Blood 110, 616–623 (2007).
Li, L. et al. The effect of the size of fluorescent dextran on its endocytic pathway. Cell Biol. Int. 39, 531–539 (2015).
Liu, Y. et al. LC-MS/MS method for the simultaneous determination of Lys-MCC-DM1, MCC-DM1 and DM1 as potential intracellular catabolites of the antibody-drug conjugate trastuzumab emtansine (T-DM1). J. Pharm. Biomed. Anal. 137, 170–177 (2017).
Lu, J., Jiang, F., Lu, A. & Zhang, G. Linkers having a crucial role in antibody-drug conjugates. Int. J. Mol. Sci. 17, 561 (2016).
Abdollahpour-Alitappeh, M. et al. Antibody-drug conjugates (ADCs) for cancer therapy: strategies, challenges, and successes. J. Cell Physiol. 234, 5628–5642 (2018).
Lee, B.-C. et al. FRET reagent reveals the intracellular processing of peptide-linked antibody-drug conjugates. Bioconjug. Chem. 29, 2468–2477 (2018).
Wang, H. et al. Aberrant intracellular metabolism of T-DM1 confers T-DM1 resistance in human epidermal growth factor receptor 2-positive gastric cancer cells. Cancer Sci. 108, 1458–1468 (2017).
Li, G. et al. Mechanisms of acquired resistance to trastuzumab emtansine in breast cancer cells. Mol. Cancer Ther. 17, 1441–1453 (2018).
Liu, K. et al. WDR91 is a Rab7 effector required for neuronal development. J. Cell Biol. 216, 3307–3321 (2017).
Kinneer, K. et al. SLC46A3 as a potential predictive biomarker for antibody-drug conjugates bearing non-cleavable linked maytansinoid and pyrrolobenzodiazepine warheads. Clin. Cancer Res. 24, 6570–6582 (2018).
Ríos-Luci, C. et al. Resistance to the antibody-drug conjugate T-DM1 Is based in a reduction in lysosomal proteolytic activity. Cancer Res. 77, 4639–4651 (2017).
Sung, M. et al. Caveolae-mediated endocytosis as a novel mechanism of resistance to trastuzumab emtansine (T-DM1). Mol. Cancer Ther. 17, 243–253 (2018).
Zhang, M. & Varki, A. Cell surface sialic acids do not affect primary CD22 interactions with CD45 and surface IgM nor the rate of constitutive CD22 endocytosis. Glycobiology 14, 939–949 (2004).
Mathew, M. P. et al. Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells. Oncotarget 7, 66491–66511 (2016).
Nabi, I. R., Shankar, J. & Dennis, J. W. The galectin lattice at a glance. J. Cell Sci. 128, 2213–2219 (2015).
Pearce, O. M. T. & Läubli, H. Sialic acids in cancer biology and immunity. Glycobiology 26, 111–128 (2016).
Wang, L., Liu, Y., Wu, L. & Sun, X.-L. Sialyltransferase inhibition and recent advances. Biochim. Biophys. Acta 1864, 143–153 (2016).
Macauley, M. S. et al. Systemic blockade of sialylation in mice with a global inhibitor of sialyltransferases. J. Biol. Chem. 289, 35149–35158 (2014).
Büll, C. et al. Sialic acid blockade suppresses tumor growth by enhancing T-cell-mediated tumor immunity. Cancer Res. 78, 3574–3588 (2018).
Deans, R. M. et al. Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification. Nat. Chem. Biol. 12, 361–366 (2016).
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.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
About this article
Cite this article
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
This article is cited by
Drug Safety (2021)
Nature Chemical Biology (2020)
Nature Chemical Biology (2019)