Design of a mucin-selective protease for targeted degradation of cancer-associated mucins.
Journal
Nature biotechnology
ISSN: 1546-1696
Titre abrégé: Nat Biotechnol
Pays: United States
ID NLM: 9604648
Informations de publication
Date de publication:
03 Aug 2023
03 Aug 2023
Historique:
received:
15
06
2022
accepted:
22
05
2023
pubmed:
4
8
2023
medline:
4
8
2023
entrez:
3
8
2023
Statut:
aheadofprint
Résumé
Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.
Identifiants
pubmed: 37537499
doi: 10.1038/s41587-023-01840-6
pii: 10.1038/s41587-023-01840-6
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : NCI NIH HHS
ID : R01 CA227942
Pays : United States
Informations de copyright
© 2023. The Author(s).
Références
Bakshani, C. R. et al. Evolutionary conservation of the antimicrobial function of mucus: a first defence against infection. NPJ Biofilms Microbiomes 4, 14 (2018).
pubmed: 30002868
pmcid: 6031612
Paszek, M. J. et al. The cancer glycocalyx mechanically primes integrin-mediated growth and survival. Nature 511, 319–325 (2014).
pubmed: 25030168
pmcid: 4487551
Freeman, S. A. et al. Integrins form an expanding diffusional barrier that coordinates phagocytosis. Cell 164, 128–140 (2016).
pubmed: 26771488
pmcid: 4715264
Smith, B. A. H. & Bertozzi, C. R. The clinical impact of glycobiology: targeting selectins, Siglecs and mammalian glycans. Nat. Rev. Drug Discov. 20, 217–243 (2021).
pubmed: 33462432
pmcid: 7812346
Wisnovsky, S. et al. Genome-wide CRISPR screens reveal a specific ligand for the glycan-binding immune checkpoint receptor Siglec-7. Proc. Natl Acad. Sci. USA 118, e2015024118 (2021).
pubmed: 33495350
pmcid: 7865165
Kufe, D. W. Functional targeting of the MUC1 oncogene in human cancers. Cancer Biol. Ther. 8, 1197–1203 (2009).
pubmed: 19556858
Xie, G.-D., Liu, Y.-R., Jiang, Y.-Z. & Shao, Z.-M. Epidemiology and survival outcomes of mucinous adenocarcinomas: a SEER population-based study. Sci. Rep. 8, 6117 (2018).
pubmed: 29666453
pmcid: 5904162
Bose, M. & Mukherjee, P. Potential of anti-MUC1 antibodies as a targeted therapy for gastrointestinal cancers. Vaccines 8, 659 (2020).
pubmed: 33167508
pmcid: 7712407
Panchamoorthy, G. et al. Targeting the human MUC1-C oncoprotein with an antibody–drug conjugate. JCI Insight 3, e99880 (2018).
pubmed: 29925694
pmcid: 6124453
Posey, A. D. Jr et al. Engineered CAR T cells targeting the cancer-associated Tn-glycoform of the membrane mucin MUC1 control adenocarcinoma. Immunity 44, 1444–1454 (2016).
pubmed: 27332733
pmcid: 5358667
Zhou, Y., Rajabi, H. & Kufe, D. Mucin 1 C-terminal subunit oncoprotein is a target for small-molecule inhibitors. Mol. Pharmacol. 79, 886–893 (2011).
pubmed: 21346142
pmcid: 3082937
Dvela-Levitt, M. et al. Small molecule targets TMED9 and promotes lysosomal degradation to reverse proteinopathy. Cell 178, 521–535 (2019).
pubmed: 31348885
Burslem, G. M. et al. The advantages of targeted protein degradation over inhibition: an RTK case study. Cell Chem. Biol. 25, 67–77 (2018).
pubmed: 29129716
Blum, T. R. et al. Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity. Science 371, 803–810 (2021).
pubmed: 33602850
pmcid: 8175023
Banik, S. M. et al. Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature 584, 291–297 (2020).
pubmed: 32728216
pmcid: 7727926
Steentoft, C. et al. Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology. EMBO J. 32, 1478–1488 (2013).
pubmed: 23584533
pmcid: 3655468
Noach, I. et al. Recognition of protein-linked glycans as a determinant of peptidase activity. Proc. Natl Acad. Sci. USA 114, E679–E688 (2017).
pubmed: 28096352
pmcid: 5293097
Malaker, S. A. et al. The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins. Proc. Natl Acad. Sci. USA 116, 7278–7287 (2019).
pubmed: 30910957
pmcid: 6462054
Shon, D. J. et al. An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins. Proc. Natl Acad. Sci. USA 117, 21299–21307 (2020).
pubmed: 32817557
pmcid: 7474620
Nason, R. et al. Display of the human mucinome with defined O-glycans by gene engineered cells. Nat. Commun. 12, 4070 (2021).
pubmed: 34210959
pmcid: 8249670
Konstantinidi, A. et al. Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells. J. Biol. Chem. 298, 101784 (2022).
pubmed: 35247390
pmcid: 8980628
Shurer, C. R. et al. Genetically encoded toolbox for glycocalyx engineering: tunable control of cell adhesion, survival, and cancer cell behaviors. ACS Biomater. Sci. Eng. 4, 388–399 (2018).
pubmed: 29805991
Woods, E. C. et al. A bulky glycocalyx fosters metastasis formation by promoting G1 cell cycle progression. eLife 6, e25752 (2017).
pubmed: 29266001
pmcid: 5739539
Park, S. et al. Mucins form a nanoscale material barrier against immune cell attack. Preprint at bioRxiv https://doi.org/10.1101/2022.01.28.478211 (2022).
Jonckheere, N., Skrypek, N. & Van Seuningen, I. Mucins and tumor resistance to chemotherapeutic drugs. Biochim. Biophys. Acta 1846, 142–151 (2014).
pubmed: 24785432
Forcina, G. C., Conlon, M., Wells, A., Cao, J. Y. & Dixon, S. J. Systematic quantification of population cell death kinetics in mammalian cells. Cell Syst. 4, 600–610 (2017).
pubmed: 28601558
pmcid: 5509363
Forcina, G. C. et al. Ferroptosis regulation by the NGLY1/NFE2L1 pathway. Proc. Natl Acad. Sci. USA 119, e2118646119 (2022).
pubmed: 35271393
pmcid: 8931371
Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012).
pubmed: 22632970
pmcid: 3367386
Yang, W. S. et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331 (2014).
pubmed: 24439385
pmcid: 4076414
Egler, R. A., Ahuja, S. P. & Matloub, Y. L-Asparaginase in the treatment of patients with acute lymphoblastic leukemia. J. Pharmacol. Pharmacother. 7, 62–71 (2016).
pubmed: 27440950
pmcid: 4936081
van Putten, J. P. M. & Strijbis, K. Transmembrane mucins: signaling receptors at the intersection of inflammation and cancer. J. Innate Immun. 9, 281–299 (2017).
pubmed: 28052300
pmcid: 5516414
Drago, J. Z., Modi, S. & Chandarlapaty, S. Unlocking the potential of antibody–drug conjugates for cancer therapy. Nat. Rev. Clin. Oncol. 18, 327–344 (2021).
pubmed: 33558752
pmcid: 8287784
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).
pubmed: 27551071
pmcid: 5027407
Gray, M. A. et al. Targeted glycan degradation potentiates the anticancer immune response in vivo. Nat. Chem. Biol. 16, 1376–1384 (2020).
pubmed: 32807964
pmcid: 7727925
Yu, A. C. Y., Worrall, L. J. & Strynadka, N. C. J. Structural insight into the bacterial mucinase StcE essential to adhesion and immune evasion during enterohemorrhagic E. coli infection. Structure 20, 707–717 (2012).
pubmed: 22483117
Czajkowsky, D. M., Hu, J., Shao, Z. & Pleass, R. J. Fc-fusion proteins: new developments and future perspectives. EMBO Mol. Med. 4, 1015–1028 (2012).
pubmed: 22837174
pmcid: 3491832
Pruszynski, M. et al. Targeting breast carcinoma with radioiodinated anti-HER2 nanobody. Nucl. Med. Biol. 40, 52–59 (2013).
pubmed: 23159171
Kleifeld, O. et al. Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat. Biotechnol. 28, 281–288 (2010).
pubmed: 20208520
Pleiner, T., Bates, M. & Görlich, D. A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies. J. Cell Biol. 217, 1143–1154 (2017).
pubmed: 29263082
Malaker, S. A. et al. Revealing the human mucinome. Nat. Commun. 13, 3542 (2022).
pubmed: 35725833
pmcid: 9209528
Imbert, P. R. C. et al. An acquired and endogenous glycocalyx forms a bidirectional ‘don’t eat’ and ‘don’t eat me’ barrier to phagocytosis. Curr. Biol. 31, 77–89 (2021).
pubmed: 33096038
Aslakson, C. J. & Miller, F. R. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 52, 1399–1405 (1992).
pubmed: 1540948
Pimental, R. A., Julian, J., Gendler, S. J. & Carson, D. D. Synthesis and intracellular trafficking of Muc-1 and mucins by polarized mouse uterine epithelial cells. J. Biol. Chem. 271, 28128–28137 (1996).
pubmed: 8910427
Kubala, M. H., Kovtun, O., Alexandrov, K. & Collins, B. M. Structural and thermodynamic analysis of the GFP:GFP–nanobody complex. Protein Sci. 19, 2389–2401 (2010).
pubmed: 20945358
pmcid: 3009406
Rockwell, S. C., Kallman, R. F. & Fajardo, L. F. Characteristics of a serially transplanted mouse mammary tumor and its tissue-culture-adapted derivative. J. Natl Cancer Inst. 49, 735–749 (1972).
pubmed: 4647494
Kim, I. S. et al. Immuno-subtyping of breast cancer reveals distinct myeloid cell profiles and immunotherapy resistance mechanisms. Nat. Cell Biol. 21, 1113–1126 (2019).
pubmed: 31451770
pmcid: 6726554
Boivin, W. A., Cooper, D. M., Hiebert, P. R. & Granville, D. J. Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma. Lab. Invest. 89, 1195–1220 (2009).
pubmed: 19770840
pmcid: 7102238
Wang, J. et al. Siglec receptors modulate dendritic cell activation and antigen presentation to T cells in cancer. Front. Cell Dev. Biol. 10, 828916 (2022).
pubmed: 35309936
pmcid: 8927547
Stanczak, M. A. et al. Self-associated molecular patterns mediate cancer immune evasion by engaging Siglecs on T cells. J. Clin. Invest. 128, 4912–4923 (2018).
pubmed: 30130255
pmcid: 6205408
Beatson, R. et al. The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9. Nat. Immunol. 17, 1273–1281 (2016).
pubmed: 27595232
pmcid: 5257269
Senter, P. D. et al. Anti-tumor effects of antibody–alkaline phosphatase conjugates in combination with etoposide phosphate. Proc. Natl Acad. Sci. USA 85, 4842–4846 (1988).
pubmed: 3387440
pmcid: 280532
Davis, B. G. et al. Selective protein degradation by ligand-targeted enzymes: towards the creation of catalytic antagonists. ChemBioChem 4, 533–537 (2003).
pubmed: 12794865
Békés, M., Langley, D. R. & Crews, C. M. PROTAC targeted protein degraders: the past is prologue. Nat. Rev. Drug Discov. 21, 181–200 (2022).
pubmed: 35042991
pmcid: 8765495
Ge, Y. et al. Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase. Nat. Chem. Biol. 17, 593–600 (2021).
pubmed: 33686291
pmcid: 8085020
Gerry, C. J. & Schreiber, S. L. Unifying principles of bifunctional, proximity-inducing small molecules. Nat. Chem. Biol. 16, 369–378 (2020).
pubmed: 32198490
pmcid: 7312755
Delaveris, C. S., Webster, E. R., Banik, S. M., Boxer, S. G. & Bertozzi, C. R. Membrane-tethered mucin-like polypeptides sterically inhibit binding and slow fusion kinetics of influenza A virus. Proc. Natl Acad. Sci. USA 117, 12643–12650 (2020).
pubmed: 32457151
pmcid: 7293601
Chatterjee, M., van Putten, J. P. M. & Strijbis, K. Defensive properties of mucin glycoproteins during respiratory infections-relevance for SARS-CoV-2. mBio 11, e02374-20 (2020).
pubmed: 33184103
pmcid: 7663010
Kreda, S. M., Davis, C. W. & Rose, M. C. CFTR, mucins, and mucus obstruction in cystic fibrosis. Cold Spring Harb. Perspect. Med. 2, a009589 (2012).
pubmed: 22951447
pmcid: 3426818
Bensing, B. A. et al. Recognition of specific sialoglycan structures by oral streptococci impacts the severity of endocardial infection. PLoS Pathog. 15, e1007896 (2019).
pubmed: 31233555
pmcid: 6611644
Hansson, G. C. Mucins and the microbiome. Annu. Rev. Biochem. 89, 769–793 (2020).
pubmed: 32243763
pmcid: 8442341
Pedram, K. et al. Lysosomal cathepsin D mediates endogenous mucin glycodomain catabolism in mammals. Proc. Natl Acad. Sci. USA 119, e2117105119 (2022).
pubmed: 36122205
pmcid: 9522329
Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods 19, 679–682 (2022).
pubmed: 35637307
pmcid: 9184281
Conlon, M. et al. A compendium of kinetic modulatory profiles identifies ferroptosis regulators. Nat. Chem. Biol. 17, 665–674 (2021).
pubmed: 33686292
pmcid: 8159879
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
pubmed: 34265844
pmcid: 8371605
Weiner, S. J. et al. A new force field for molecular mechanical simulation of nucleic acids and proteins. J. Am. Chem. Soc. 106, 765–784 (1984).
Gray, M. A., Tao, R. N., DePorter, S. M., Spiegel, D. A. & McNaughton, B. R. A nanobody activation immunotherapeutic that selectively destroys HER2-positive breast cancer cells. ChemBioChem 17, 155–158 (2016).
pubmed: 26556305
Malyala, P. & Singh, M. Endotoxin limits in formulations for preclinical research. J. Pharm. Sci. 97, 2041–2044 (2008).
pubmed: 17847072
Lu, L., Riley, N. M., Shortreed, M. R., Bertozzi, C. R. & Smith, L. M. O-Pair search with MetaMorpheus for O-glycopeptide characterization. Nat. Methods 17, 1133–1138 (2020).
pubmed: 33106676
pmcid: 7606753
Madzharova, E., Sabino, F. & auf dem Keller, U. in Collagen: Methods and Protocols (eds Sagi, I. & Afratis, N. A.) 115–126 (Springer, 2019).
Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).
pubmed: 27809316
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
pubmed: 21254760
Rookyard, A. W. et al. A global profile of reversible and irreversible cysteine redox post-translational modifications during myocardial ischemia/reperfusion injury and antioxidant intervention. Antioxid. Redox Signal. 34, 11–31 (2021).
pubmed: 32729339
Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).
pubmed: 27348712
Hornbeck, P. V. et al. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 43, D512–D520 (2015).
pubmed: 25514926
Barnes, J. M. et al. A tension-mediated glycocalyx–integrin feedback loop promotes mesenchymal-like glioblastoma. Nat. Cell Biol. 20, 1203–1214 (2018).
pubmed: 30202050
pmcid: 6932748
Varghese, F., Bukhari, A. B., Malhotra, R. & De, A. IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS ONE 9, e96801 (2014).
pubmed: 24802416
pmcid: 4011881
Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 50, D543–D552 (2022).
pubmed: 34723319