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In vivo anti-MUC1+ tumor activity and sequences of high-affinity anti-MUC1-SEA antibodies

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Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha–beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1+ malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1+ Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.

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  1. Nath S, Mukherjee P (2014) MUC1: a multifaceted oncoprotein with a key role in cancer progression. Trends Mol Med 20:332–342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Deng J, Wang L, Chen H, Li L, Ma Y, Ni J, Li Y (2013) The role of tumour-associated MUC1 in epithelial ovarian cancer metastasis and progression. Cancer Metastasis Rev 32:535–551

    Article  CAS  PubMed  Google Scholar 

  3. Rahn JJ, Dabbagh L, Pasdar M, Hugh JC (2001) The importance of MUC1 cellular localization in patients with breast carcinoma: an immunohistologic study of 71 patients and review of the literature. Cancer 91:1973–1982

    Article  CAS  PubMed  Google Scholar 

  4. Remmers N, Anderson JM, Linde EM, DiMaio DJ, Lazenby AJ, Wandall HH, Mandel U, Clausen H, Yu F, Hollingsworth MA (2013) Aberrant expression of mucin core proteins and o-linked glycans associated with progression of pancreatic cancer. Clin Cancer Res 19:1981–1993

    Article  CAS  PubMed  Google Scholar 

  5. Krishn SR, Kaur S, Smith LM, Johansson SL, Jain M, Patel A, Gautam SK, Hollingsworth MA, Mandel U, Clausen H, Lo WC, Fan WT, Manne U, Batra SK (2016) Mucins and associated glycan signatures in colon adenoma-carcinoma sequence: prospective pathological implication(s) for early diagnosis of colon cancer. Cancer Lett 374:304–314

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Xu F, Liu F, Zhao H, An G, Feng G (2015) Prognostic significance of mucin antigen MUC1 in various human epithelial cancers: a meta-analysis. Medicine (Baltimore) 94:e2286

    Article  CAS  Google Scholar 

  7. Cloosen S, Gratama J, van Leeuwen EB, Senden-Gijsbers BL, Oving EB, von Mensdorff-Pouilly S, Tarp MA, Mandel U, Clausen H, Germeraad WT, Bos GM (2006) Cancer specific Mucin-1 glycoforms are expressed on multiple myeloma. Br J Haematol 135:513–516

    Article  CAS  PubMed  Google Scholar 

  8. Guillaume T, Dehame V, Chevallier P, Peterlin P, Garnier A, Gregoire M, Pichinuk E, Rubinstein DB, Wreschner DH (2019) Targeting cell-bound MUC1 on myelomonocytic, monocytic leukemias and phenotypically defined leukemic stem cells with anti-SEA module antibodies. Exp Hematol 70:97–108

    Article  CAS  PubMed  Google Scholar 

  9. Rivalland G, Loveland B, Mitchell P (2015) Update on Mucin-1 immunotherapy in cancer: a clinical perspective. Exp Opin Biol Ther 15:1773–1787

    Article  CAS  Google Scholar 

  10. Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, Duhig T, Peat N, Burchell J, Pemberton L, Lalani EN, Wilson D (1990) Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem 265:15286–15293

    CAS  PubMed  Google Scholar 

  11. Ligtenberg MJ, Vos HL, Gennissen AM, Hilkens J (1990) Episialin, a carcinoma-associated mucin, is generated by a polymorphic gene encoding splice variants with alternative amino termini. J Biol Chem 265:5573–5578

    CAS  PubMed  Google Scholar 

  12. Levitin F, Stern O, Weiss M, Gil-Henn C, Ziv R, Prokocimer Z, Smorodinsky NI, Rubinstein DB, Wreschner DH (2005) The MUC1 SEA module is a self-cleaving domain. J Biol Chem 280:33374–33386

    Article  CAS  PubMed  Google Scholar 

  13. Zrihan-Licht S, Baruch A, Elroy-Stein O, Keydar I, Wreschner DH (1994) Tyrosine phosphorylation of the MUC1 breast cancer membrane proteins. Cytokine receptor-like molecules. FEBS Lett 356:130–136

    Article  CAS  PubMed  Google Scholar 

  14. Wreschner DH, Hareuveni M, Tsarfaty I, Smorodinsky N, Horev J, Zaretsky J, Kotkes P, Weiss M, Lathe R, Dion A et al (1990) Human epithelial tumor antigen cDNA sequences. Differential splicing may generate multiple protein forms. Eur J Biochem 189:463–473

    Article  CAS  PubMed  Google Scholar 

  15. Macao B, Johansson DG, Hansson GC, Hard T (2006) Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin. Nat Struct Mol Biol 13:71–76

    Article  CAS  PubMed  Google Scholar 

  16. Fiedler W, DeDosso S, Cresta S, Weidmann J, Tessari A, Salzberg M, Dietrich B, Baumeister H, Goletz S, Gianni L, Sessa C (2016) A phase I study of PankoMab-GEX, a humanised glyco-optimised monoclonal antibody to a novel tumour-specific MUC1 glycopeptide epitope in patients with advanced carcinomas. Eur J Cancer 63:55–63

    Article  CAS  PubMed  Google Scholar 

  17. Ryuko K, Schol DJ, Snijdewint FG, von Mensdorff-Pouilly S, Poort-Keesom RJ, Karuntu-Wanamarta YA, Verstraeten RA, Miyazaki K, Kenemans P, Hilgers J (2000) Characterization of a new MUC1 monoclonal antibody (VU-2-G7) directed to the glycosylated PDTR sequence of MUC1. Tumour Biol 21:197–210

    Article  CAS  PubMed  Google Scholar 

  18. Zhou D, Xu L, Huang W, Tonn T (2018) Epitopes of MUC1 tandem repeats in cancer as revealed by antibody crystallography: toward glycopeptide signature-guided therapy. Molecules 23:1326

    Article  PubMed Central  CAS  Google Scholar 

  19. Kimura T, Finn OJ (2013) MUC1 immunotherapy is here to stay. Exp Opin Biol Ther 13:35–49

    Article  CAS  Google Scholar 

  20. Ibrahim NK, Yariz KO, Bondarenko I, Manikhas A, Semiglazov V, Alyasova A, Komisarenko V, Shparyk Y, Murray JL, Jones D, Senderovich S, Chau A, Erlandsson F, Acton G, Pegram M (2011) Randomized phase II trial of letrozole plus anti-MUC1 antibody AS1402 in hormone receptor-positive locally advanced or metastatic breast cancer. Clin Cancer Res 17:6822–6830

    Article  CAS  PubMed  Google Scholar 

  21. Rubinstein DB, Karmely M, Ziv R, Benhar I, Leitner O, Baron S, Katz BZ, Wreschner DH (2006) MUC1/X protein immunization enhances cDNA immunization in generating anti-MUC1 alpha/beta junction antibodies that target malignant cells. Cancer Res 66:11247–11253

    Article  CAS  PubMed  Google Scholar 

  22. Pichinuk E, Benhar I, Jacobi O, Chalik M, Weiss L, Ziv R, Sympson C, Karwa A, Smorodinsky NI, Rubinstein DB, Wreschner DH (2012) Antibody targeting of cell-bound MUC1 SEA domain kills tumor cells. Cancer Res 72:3324–3336

    Article  CAS  PubMed  Google Scholar 

  23. Rubinstein DB, Karmely M, Pichinuk E, Ziv R, Benhar I, Feng N, Smorodinsky NI, Wreschner DH (2009) The MUC1 oncoprotein as a functional target: immunotoxin binding to alpha/beta junction mediates cell killing. Int J Cancer 124:46–54

    Article  CAS  PubMed  Google Scholar 

  24. Mazor Y, Barnea I, Keydar I, Benhar I (2007) Antibody internalization studied using a novel IgG binding toxin fusion. J Immunol Methods 321:41–59

    Article  CAS  PubMed  Google Scholar 

  25. Tomayko MM, Reynolds CP (1989) Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 24:148–154

    Article  CAS  PubMed  Google Scholar 

  26. Chalick M, Jacobi O, Pichinuk E, Garbar C, Bensussan A, Meeker A, Ziv R, Zehavi T, Smorodinsky NI, Hilkens J, Hanisch FG, Rubinstein DB, Wreschner DH (2016) MUC1-ARF-A novel MUC1 protein that resides in the nucleus and is expressed by alternate reading frame translation of MUC1 mRNA. PLoS ONE 11:e0165031

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Gold DV, Karanjawala Z, Modrak DE, Goldenberg DM, Hruban RH (2007) PAM4-reactive MUC1 is a biomarker for early pancreatic adenocarcinoma. Clin Cancer Res 13:7380–7387

    Article  CAS  PubMed  Google Scholar 

  28. Sharkey RM, Goldenberg DM (2006) Targeted therapy of cancer: new prospects for antibodies and immunoconjugates. CA Cancer J Clin 56:226–243

    Article  PubMed  Google Scholar 

  29. Guo R, Guo W, Cao L, Liu H, Liu J, Xu H, Huang W, Wang F, Hong Z (2016) Fusion of an albumin-binding domain extends the half-life of immunotoxins. Int J Pharm 511:538–549

    Article  CAS  PubMed  Google Scholar 

  30. Tarp MA, Sorensen AL, Mandel U, Paulsen H, Burchell J, Taylor-Papadimitriou J, Clausen H (2007) Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat. Glycobiology 17:197–209

    Article  CAS  PubMed  Google Scholar 

  31. Panchamoorthy G, Jin C, Raina D, Bharti A, Yamamoto M, Adeebge D, Zhao Q, Bronson R, Jiang S, Li L, Suzuki Y, Tagde A, Ghoroghchian PP, Wong KK, Kharbanda S, Kufe D (2018) Targeting the human MUC1-C oncoprotein with an antibody-drug conjugate. JCI Insight.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Scott AM, Allison JP, Wolchok JD (2012) Monoclonal antibodies in cancer therapy. Cancer Immun 12:14

    PubMed  PubMed Central  Google Scholar 

  33. Finn OJ (2017) Human tumor antigens yesterday, today, and tomorrow, cancer. Immunol Res 5:347–354

    CAS  Google Scholar 

  34. Horm TM, Schroeder JA (2013) MUC1 and metastatic cancer: expression, function and therapeutic targeting. Cell Adhes Migr 7:187–198

    Article  Google Scholar 

  35. Wong N, Major P, Kapoor A, Wei F, Yan J, Aziz T, Zheng M, Jayasekera D, Cutz JC, Chow MJ, Tang D (2016) Amplification of MUC1 in prostate cancer metastasis and CRPC development. Oncotarget 7:83115–83133

    Article  PubMed  PubMed Central  Google Scholar 

  36. Jarantow SW, Bushey BS, Pardinas JR, Boakye K, Lacy ER, Sanders R, Sepulveda MA, Moores SL, Chiu ML (2015) Impact of cell-surface antigen expression on target engagement and function of an epidermal growth factor receptor x c-MET bispecific antibody. J Biol Chem 290:24689–24704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Peat N, Gendler SJ, Lalani N, Duhig T, Taylor-Papadimitriou J (1992) Tissue-specific expression of a human polymorphic epithelial mucin (MUC1) in transgenic mice. Cancer Res 52:1954–1960

    CAS  PubMed  Google Scholar 

  38. Xu H, Gan L, Han Y, Da Y, Xiong J, Hong S, Zhao Q, Song N, Cai X, Jiang X (2019) Site-specific labeling of an anti-MUC1 antibody: probing the effects of conjugation and linker chemistry on the internalization process. RSC Adv 9:1909–1917

    Article  CAS  Google Scholar 

  39. Janus A, Robak T (2019) Moxetumomab pasudotox for the treatment of hairy cell leukemia. Exp Opin Biol Ther 19:501–508

    Article  CAS  Google Scholar 

  40. de Goeij BE, Lambert JM (2016) New developments for antibody-drug conjugate-based therapeutic approaches. Curr Opin Immunol 40:14–23

    Article  PubMed  CAS  Google Scholar 

  41. Advani A, Coiffier B, Czuczman MS, Dreyling M, Foran J, Gine E, Gisselbrecht C, Ketterer N, Nasta S, Rohatiner A, Schmidt-Wolf IG, Schuler M, Sierra J, Smith MR, Verhoef G, Winter JN, Boni J, Vandendries E, Shapiro M, Fayad L (2010) Safety, pharmacokinetics, and preliminary clinical activity of inotuzumab ozogamicin, a novel immunoconjugate for the treatment of B-cell non-Hodgkin’s lymphoma: results of a phase I study. J Clin Oncol 28:2085–2093

    Article  CAS  PubMed  Google Scholar 

  42. Strop P, Delaria K, Foletti D, Witt JM, Hasa-Moreno A, Poulsen K, Casas MG, Dorywalska M, Farias S, Pios A, Lui V, Dushin R, Zhou D, Navaratnam T, Tran TT, Sutton J, Lindquist KC, Han B, Liu SH, Shelton DL, Pons J, Rajpal A (2015) Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading. Nat Biotechnol 33:694–696

    Article  CAS  PubMed  Google Scholar 

  43. Younes A, Bartlett NL, Leonard JP, Kennedy DA, Lynch CM, Sievers EL, Forero-Torres A (2010) Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med 363:1812–1821

    Article  CAS  PubMed  Google Scholar 

  44. Neoptolemos JP, Kleeff J, Michl P, Costello E, Greenhalf W, Palmer DH (2018) Therapeutic developments in pancreatic cancer: current and future perspectives. Nat Rev Gastroenterol Hepatol 15:333–348

    Article  PubMed  Google Scholar 

  45. Grigoriadis A, Mackay A, Noel E, Wu PJ, Natrajan R, Frankum J, Reis-Filho JS, Tutt A (2012) Molecular characterisation of cell line models for triple-negative breast cancers. BMC Genom 13:619

    Article  CAS  Google Scholar 

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Funded by Israel Cancer Association (Project 20112024) and Israel Science Foundation (Project 1167/10).

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Authors and Affiliations



EP, MC, IB, RGK, RZ, NIS, GH, CG, AB, AM, TG, DBR, and DHW contributed to the study conception and design. Material preparation, data collection, and analysis were performed by authors DHW, EP, MC, DBR, MC, AM, CG, AB, and NIS. The first draft of the manuscript was written by DHW, DBR and EP. EP, MC, IB, NIS, DBR, and DHW commented on the various versions of the manuscript and contributed to the actual writing and preparation of its final version. EP, MC, IB, RGK, RZ, NIS, GH, CG, AB, AM, TG, DBR, and DHW read and approved the final manuscript.

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Correspondence to Daniel H. Wreschner.

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Conflict of interest

Daniel H. Wreschner and Daniel B. Rubinstein have ownership interest in BioModifying, LLC. All other authors declare that they have no conflict of interest.

Ethical Approval

Animal care and use Use of mice was done under supervision of the Tel Aviv University Institutional Animal Care and Use Committee (TAU-IACUC), Study Approval License number L-04-12-003. To ameliorate suffering including methods of killing, animal welfare and steps were all performed in accordance with regulations stipulated by TAU-IACUC. Samples used for immunohistochemical stainings These were procured from the Biomax tissue bank, see With all required approvals therein. Cell line authentication Short Tandem Repeat (STR) analysis (PowerPlexW 1.2 System, Promega, Fitchburg, WI), as described [45], was used to validate the human cell lines. STR profiles were matched with the German Collection of Microorganisms and Cell Cultures (DSMZ) database. Cell lines were obtained as gifts from Prof. I. Keydar.

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Pichinuk, E., Chalik, M., Benhar, I. et al. In vivo anti-MUC1+ tumor activity and sequences of high-affinity anti-MUC1-SEA antibodies. Cancer Immunol Immunother 69, 1337–1352 (2020).

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