Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunoreactivity of fractions eluted from Sepharose CL-2B(A) Capan-1 cell culture supernatant was separated on a Sepharose CL2B column with the eluted fractions analyzed by hPAM4 and α-MUC1. (B) The void-volume (Vo) fractions of Capan-1 reacted positively with three anti-MUC5AC antibodies (45M1, 1-13M1 and H-160), but not with 2Q445, which recognizes the unglycosylated tandem repeat region of MUC5AC. (C) The Capan-1 void-volume peak, following capture by 2-11M1, could be detected directly by HRP-hPAM4, or indirectly by biotin-45M1 plus SA-HRP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4414189&req=5

Figure 3: Immunoreactivity of fractions eluted from Sepharose CL-2B(A) Capan-1 cell culture supernatant was separated on a Sepharose CL2B column with the eluted fractions analyzed by hPAM4 and α-MUC1. (B) The void-volume (Vo) fractions of Capan-1 reacted positively with three anti-MUC5AC antibodies (45M1, 1-13M1 and H-160), but not with 2Q445, which recognizes the unglycosylated tandem repeat region of MUC5AC. (C) The Capan-1 void-volume peak, following capture by 2-11M1, could be detected directly by HRP-hPAM4, or indirectly by biotin-45M1 plus SA-HRP.

Mentions: MUC5AC is a highly oligomeric secretory mucin that has been isolated from cell culture and in vivo mucous secretions [21–22]. Our early studies showed that hPAM4 reacts with mucin derived from the Capan-1 xenografted human PDAC [9]. In the current study, we used Sepharose® CL-2B molecular sieve chromatography to separate the mucin species secreted into the supernatant of Capan-1. The eluted fractions were then examined for immunoreactivity with hPAM4 and α-MUC1. As shown in Figure 3A, PAM4-reactive substance was present predominantly in the void-volume peak, whereas only subsequently eluted fractions were found reactive with α-MUC1. When the Capan-1 void-volume peak was probed with anti-MUC5AC antibodies, we found a positive response with 45M1, 1-13M1, and H-160, but not 2Q445, as shown in Figure 3B. It is noted that the void-volume peaks obtained from other cancer cell lines known to secret MUC5AC, such as HT-29 [21], LS 174T [23], SW1990 [24], CFPAC-1 [25], and Calu-3 [26], were all tested positive for reactivity with hPAM4 (Supplementary Figure S1).


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)

Immunoreactivity of fractions eluted from Sepharose CL-2B(A) Capan-1 cell culture supernatant was separated on a Sepharose CL2B column with the eluted fractions analyzed by hPAM4 and α-MUC1. (B) The void-volume (Vo) fractions of Capan-1 reacted positively with three anti-MUC5AC antibodies (45M1, 1-13M1 and H-160), but not with 2Q445, which recognizes the unglycosylated tandem repeat region of MUC5AC. (C) The Capan-1 void-volume peak, following capture by 2-11M1, could be detected directly by HRP-hPAM4, or indirectly by biotin-45M1 plus SA-HRP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Immunoreactivity of fractions eluted from Sepharose CL-2B(A) Capan-1 cell culture supernatant was separated on a Sepharose CL2B column with the eluted fractions analyzed by hPAM4 and α-MUC1. (B) The void-volume (Vo) fractions of Capan-1 reacted positively with three anti-MUC5AC antibodies (45M1, 1-13M1 and H-160), but not with 2Q445, which recognizes the unglycosylated tandem repeat region of MUC5AC. (C) The Capan-1 void-volume peak, following capture by 2-11M1, could be detected directly by HRP-hPAM4, or indirectly by biotin-45M1 plus SA-HRP.
Mentions: MUC5AC is a highly oligomeric secretory mucin that has been isolated from cell culture and in vivo mucous secretions [21–22]. Our early studies showed that hPAM4 reacts with mucin derived from the Capan-1 xenografted human PDAC [9]. In the current study, we used Sepharose® CL-2B molecular sieve chromatography to separate the mucin species secreted into the supernatant of Capan-1. The eluted fractions were then examined for immunoreactivity with hPAM4 and α-MUC1. As shown in Figure 3A, PAM4-reactive substance was present predominantly in the void-volume peak, whereas only subsequently eluted fractions were found reactive with α-MUC1. When the Capan-1 void-volume peak was probed with anti-MUC5AC antibodies, we found a positive response with 45M1, 1-13M1, and H-160, but not 2Q445, as shown in Figure 3B. It is noted that the void-volume peaks obtained from other cancer cell lines known to secret MUC5AC, such as HT-29 [21], LS 174T [23], SW1990 [24], CFPAC-1 [25], and Calu-3 [26], were all tested positive for reactivity with hPAM4 (Supplementary Figure S1).

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