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Identification of PAM4 (clivatuzumab)-reactive epitope on MUC5AC: a promising biomarker and therapeutic target for pancreatic cancer.

Liu D, Chang CH, Gold DV, Goldenberg DM - Oncotarget (2015)

Bottom Line: PAM4 is a monoclonal antibody showing high specificity for pancreatic ductal adenocarcinoma (PDAC).In present study, we provide further evidence validating MUC5AC as the PAM4 antigen, and locate PAM4-reactive epitope within the N-terminal cysteine-rich subdomain 2 (Cys2), thus differentiating PAM4 from most anti-MUC5AC antibodies known to-date.These findings shed light on the mechanism of PAM4-based diagnosis and treatment for pancreatic cancer, and guide further exploration of its clinical utility.

View Article: PubMed Central - PubMed

Affiliation: IBC Pharmaceuticals, Inc., Morris Plains, New Jersey 07950, United States of America.

ABSTRACT
PAM4 is a monoclonal antibody showing high specificity for pancreatic ductal adenocarcinoma (PDAC). Humanized PAM4 labeled with 90Y in combination with low-dose gemcitabine has shown promising therapeutic activity, and is being evaluated in a phase III clinical trial. Prior efforts have suggested that PAM4 potentially reacts with MUC5AC, a secretory mucin expressed de novo in early pancreatic neoplasia and retained throughout disease progression. In present study, we provide further evidence validating MUC5AC as the PAM4 antigen, and locate PAM4-reactive epitope within the N-terminal cysteine-rich subdomain 2 (Cys2), thus differentiating PAM4 from most anti-MUC5AC antibodies known to-date. Specifically, we show (i) PAM4-antigen and MUC5AC were co-localized in multiple human cancer cell lines, including Capan-1, BxPC-3, and CFPAC-1; (ii) MUC5AC-specific siRNA prominently reduced the expression of both MUC5AC and PAM4-antigen in CFPAC-1 cells; (iii) PAM4 preferentially binds to the void-volume fractions from Sepharose-CL2B chromatography of Capan-1 culture supernatants, which were revealed by Western blot to display the ladder pattern characteristic of oligomeric MUC5AC; and (iv) the N-terminal Cys2 within several recombinant MUC5AC fragments is essential for binding to PAM4. These findings shed light on the mechanism of PAM4-based diagnosis and treatment for pancreatic cancer, and guide further exploration of its clinical utility.

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Co-localization of PAM4 antigen with MUC5AC by immunofluorescence staining(A) Mucin-expressing cell lines were stained with DAPI, hPAM4, and anti-MUC5AC (2-12M1 for Capan-1 and BxPC-3; 2-11M1 for HT-29 and MCF-7), then examined by immunofluorescence microcopy. (B) BxPC-3 and HT-29 cells were stained with DAPI, hPAM4, and α-MUC1. PAM4 antigen was shown to co-localize with MUC5AC, not MUC1.
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Figure 1: Co-localization of PAM4 antigen with MUC5AC by immunofluorescence staining(A) Mucin-expressing cell lines were stained with DAPI, hPAM4, and anti-MUC5AC (2-12M1 for Capan-1 and BxPC-3; 2-11M1 for HT-29 and MCF-7), then examined by immunofluorescence microcopy. (B) BxPC-3 and HT-29 cells were stained with DAPI, hPAM4, and α-MUC1. PAM4 antigen was shown to co-localize with MUC5AC, not MUC1.

Mentions: Several cell lines were subjected to immunofluorescence microscopy in order to evaluate localization patterns (heterogeneous and/or homogenous) of MUC1, MUC5AC, and/or MUC17, as detected by hPAM4 and other mucin-specific mAbs. The cell lines examined included those derived from human pancreatic (Capan-1, BxPC3, CFPAC-1, and AsPC-1), colorectal (HT-29 and LS174 T), breast (MCF-7), and lung (A549) carcinomas. As shown in Figure 1 and Supplementary Table S1, in each of the cell lines examined, hPAM4 exclusively co-localized with MUC5AC (as identified by two anti-MUC5AC mAbs, 2-11M1 and 2-12M1, Figure 1A), but not with MUC1 (Figure 1B) or MUC17 (data not shown), suggesting that MUC5AC is the hPAM4-reactive antigen.


Identification of PAM4 (clivatuzumab)-reactive epitope on MUC5AC: a promising biomarker and therapeutic target for pancreatic cancer.

Liu D, Chang CH, Gold DV, Goldenberg DM - Oncotarget (2015)

Co-localization of PAM4 antigen with MUC5AC by immunofluorescence staining(A) Mucin-expressing cell lines were stained with DAPI, hPAM4, and anti-MUC5AC (2-12M1 for Capan-1 and BxPC-3; 2-11M1 for HT-29 and MCF-7), then examined by immunofluorescence microcopy. (B) BxPC-3 and HT-29 cells were stained with DAPI, hPAM4, and α-MUC1. PAM4 antigen was shown to co-localize with MUC5AC, not MUC1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414189&req=5

Figure 1: Co-localization of PAM4 antigen with MUC5AC by immunofluorescence staining(A) Mucin-expressing cell lines were stained with DAPI, hPAM4, and anti-MUC5AC (2-12M1 for Capan-1 and BxPC-3; 2-11M1 for HT-29 and MCF-7), then examined by immunofluorescence microcopy. (B) BxPC-3 and HT-29 cells were stained with DAPI, hPAM4, and α-MUC1. PAM4 antigen was shown to co-localize with MUC5AC, not MUC1.
Mentions: Several cell lines were subjected to immunofluorescence microscopy in order to evaluate localization patterns (heterogeneous and/or homogenous) of MUC1, MUC5AC, and/or MUC17, as detected by hPAM4 and other mucin-specific mAbs. The cell lines examined included those derived from human pancreatic (Capan-1, BxPC3, CFPAC-1, and AsPC-1), colorectal (HT-29 and LS174 T), breast (MCF-7), and lung (A549) carcinomas. As shown in Figure 1 and Supplementary Table S1, in each of the cell lines examined, hPAM4 exclusively co-localized with MUC5AC (as identified by two anti-MUC5AC mAbs, 2-11M1 and 2-12M1, Figure 1A), but not with MUC1 (Figure 1B) or MUC17 (data not shown), suggesting that MUC5AC is the hPAM4-reactive antigen.

Bottom Line: PAM4 is a monoclonal antibody showing high specificity for pancreatic ductal adenocarcinoma (PDAC).In present study, we provide further evidence validating MUC5AC as the PAM4 antigen, and locate PAM4-reactive epitope within the N-terminal cysteine-rich subdomain 2 (Cys2), thus differentiating PAM4 from most anti-MUC5AC antibodies known to-date.These findings shed light on the mechanism of PAM4-based diagnosis and treatment for pancreatic cancer, and guide further exploration of its clinical utility.

View Article: PubMed Central - PubMed

Affiliation: IBC Pharmaceuticals, Inc., Morris Plains, New Jersey 07950, United States of America.

ABSTRACT
PAM4 is a monoclonal antibody showing high specificity for pancreatic ductal adenocarcinoma (PDAC). Humanized PAM4 labeled with 90Y in combination with low-dose gemcitabine has shown promising therapeutic activity, and is being evaluated in a phase III clinical trial. Prior efforts have suggested that PAM4 potentially reacts with MUC5AC, a secretory mucin expressed de novo in early pancreatic neoplasia and retained throughout disease progression. In present study, we provide further evidence validating MUC5AC as the PAM4 antigen, and locate PAM4-reactive epitope within the N-terminal cysteine-rich subdomain 2 (Cys2), thus differentiating PAM4 from most anti-MUC5AC antibodies known to-date. Specifically, we show (i) PAM4-antigen and MUC5AC were co-localized in multiple human cancer cell lines, including Capan-1, BxPC-3, and CFPAC-1; (ii) MUC5AC-specific siRNA prominently reduced the expression of both MUC5AC and PAM4-antigen in CFPAC-1 cells; (iii) PAM4 preferentially binds to the void-volume fractions from Sepharose-CL2B chromatography of Capan-1 culture supernatants, which were revealed by Western blot to display the ladder pattern characteristic of oligomeric MUC5AC; and (iv) the N-terminal Cys2 within several recombinant MUC5AC fragments is essential for binding to PAM4. These findings shed light on the mechanism of PAM4-based diagnosis and treatment for pancreatic cancer, and guide further exploration of its clinical utility.

Show MeSH
Related in: MedlinePlus