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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-knockdown of PAM4 antigen and MUC5AC by MUC5AC-specific siRNA(A) CFPAC-1 cells were treated with a MUC5AC-specific siRNA, followed by staining with DAPI, hPAM4, and 2-11M1. (B) CFPAC-1 cells were treated with a MUC5AC-specific siRNA, followed by staining with DAPI, hPAM4, α-MUC1. Untreated Cells or cells treated with only the transfection agent (mock) served as controls. Cells treated with MUC5AC-specific siRNA lost the binding to anti-MUC5AC and hPAM4 concurrently, with little effect on the binding to anti-MUC1.
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Figure 2: Co-knockdown of PAM4 antigen and MUC5AC by MUC5AC-specific siRNA(A) CFPAC-1 cells were treated with a MUC5AC-specific siRNA, followed by staining with DAPI, hPAM4, and 2-11M1. (B) CFPAC-1 cells were treated with a MUC5AC-specific siRNA, followed by staining with DAPI, hPAM4, α-MUC1. Untreated Cells or cells treated with only the transfection agent (mock) served as controls. Cells treated with MUC5AC-specific siRNA lost the binding to anti-MUC5AC and hPAM4 concurrently, with little effect on the binding to anti-MUC1.

Mentions: The disparate localization between PAM4 and anti-MUC1 or anti-MUC17 indicates that PAM4 reacts with neither MUC1 nor MUC17. On the other hand, the co-localization of PAM4 and the two anti-MUC5AC mAbs (2-11M1 and 2-12M1) is consistent with PAM4 being specific for MUC5AC [19]. To investigate if hPAM4 associates with MUC5AC, we employed the RNAi method to specifically knockdown MUC5AC. As shown in Figure 2A, hPAM4 and 2-11M1 are co-localized in untreated CFPAC-1 cells, as well as the mock-treated (transfection agent alone) cells. In contrast, treatment with MUC5AC-specific siRNA resulted in substantially reduced immunostaining for both 2-11M1 and hPAM4. Moreover, as shown in Figure 2B, siRNA knockdown of MUC5AC did not alter the anti-MUC1 immunostaining, providing further evidence that hPAM4 is not reactive with MUC1.


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-knockdown of PAM4 antigen and MUC5AC by MUC5AC-specific siRNA(A) CFPAC-1 cells were treated with a MUC5AC-specific siRNA, followed by staining with DAPI, hPAM4, and 2-11M1. (B) CFPAC-1 cells were treated with a MUC5AC-specific siRNA, followed by staining with DAPI, hPAM4, α-MUC1. Untreated Cells or cells treated with only the transfection agent (mock) served as controls. Cells treated with MUC5AC-specific siRNA lost the binding to anti-MUC5AC and hPAM4 concurrently, with little effect on the binding to anti-MUC1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Co-knockdown of PAM4 antigen and MUC5AC by MUC5AC-specific siRNA(A) CFPAC-1 cells were treated with a MUC5AC-specific siRNA, followed by staining with DAPI, hPAM4, and 2-11M1. (B) CFPAC-1 cells were treated with a MUC5AC-specific siRNA, followed by staining with DAPI, hPAM4, α-MUC1. Untreated Cells or cells treated with only the transfection agent (mock) served as controls. Cells treated with MUC5AC-specific siRNA lost the binding to anti-MUC5AC and hPAM4 concurrently, with little effect on the binding to anti-MUC1.
Mentions: The disparate localization between PAM4 and anti-MUC1 or anti-MUC17 indicates that PAM4 reacts with neither MUC1 nor MUC17. On the other hand, the co-localization of PAM4 and the two anti-MUC5AC mAbs (2-11M1 and 2-12M1) is consistent with PAM4 being specific for MUC5AC [19]. To investigate if hPAM4 associates with MUC5AC, we employed the RNAi method to specifically knockdown MUC5AC. As shown in Figure 2A, hPAM4 and 2-11M1 are co-localized in untreated CFPAC-1 cells, as well as the mock-treated (transfection agent alone) cells. In contrast, treatment with MUC5AC-specific siRNA resulted in substantially reduced immunostaining for both 2-11M1 and hPAM4. Moreover, as shown in Figure 2B, siRNA knockdown of MUC5AC did not alter the anti-MUC1 immunostaining, providing further evidence that hPAM4 is not reactive with MUC1.

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