Anti-Human MUC1 Recombinant Antibody Fab Fragment (Clivatuzumab Tetraxetan) (CAT#: TAB-037-F(E))

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.

Liu, D., Chang, C. H., Gold, D. V., & Goldenberg, D. M. (2015). Identification of PAM4 (clivatuzumab)-reactive epitope on MUC5AC: A promising biomarker and therapeutic target for pancreatic cancer. Oncotarget, 6(6), 4274.

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 MUC5ACspecific 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.

Liu, D., Chang, C. H., Gold, D. V., & Goldenberg, D. M. (2015). Identification of PAM4 (clivatuzumab)-reactive epitope on MUC5AC: A promising biomarker and therapeutic target for pancreatic cancer. Oncotarget, 6(6), 4274.

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.

Liu, D., Chang, C. H., Gold, D. V., & Goldenberg, D. M. (2015). Identification of PAM4 (clivatuzumab)-reactive epitope on MUC5AC: A promising biomarker and therapeutic target for pancreatic cancer. Oncotarget, 6(6), 4274.

Figure 4 Agarose gel electrophoresis.

(A) The Capan-1 void-volume peak displayed the characteristic banding pattern of MUC5AC as revealed by Western blot analysis with hPAM4, 45M1, and MAN-5ACI. In the left panel, samples in the lanes marked as 1, 1/2, 1/4, and 1/8 were tested undiluted, 2-, 4- and 8-fold diluted, respectively. In the far right panel, the monomeric and dimeric MUC5AC were indicated as M and D, respectively. (B) The serum from a pancreatic cancer patient (PS) tested positive for hPAM4-reactive substance was differentially detected by hPAM4 and three anti-MUC5AC antibodies (2-11M1, 45M1, and H-160). The Capan-1 void-volume peak (Vo) and normal serum sample (NS) were included as controls.

Liu, D., Chang, C. H., Gold, D. V., & Goldenberg, D. M. (2015). Identification of PAM4 (clivatuzumab)-reactive epitope on MUC5AC: A promising biomarker and therapeutic target for pancreatic cancer. Oncotarget, 6(6), 4274.

Figure 5 Mapping the PAM4-reactive epitope on human MUC5AC.

(A) Schematic diagram of different MUC5AC recombinant fragments (a–h) generated in PANC-1 cells for mapping PAM4 epitope; Numbers are AA positions in the MUC5AC protein sequence (UniProtKB/Swiss-Prot: P98088). (B to E) Western blot of various MUC5AC recombinant fragments by hPAM4, anti-MUC5AC, or antiGFP antibodies, as indicated. See text for details. Lane m indicates samples from untransfected cells.

Liu, D., Chang, C. H., Gold, D. V., & Goldenberg, D. M. (2015). Identification of PAM4 (clivatuzumab)-reactive epitope on MUC5AC: A promising biomarker and therapeutic target for pancreatic cancer. Oncotarget, 6(6), 4274.

Figure 6 Reactivity of several anti-mucin MAbs with a high molecular weight mucin containing fraction (CPM1) isolated from the Capan-1 human pancreatic adenocarcinoma.

MAbs are identified by clone name with reactive species of mucin indicated by horizontal bars beneath MAb clone names (MUC1, etc.). In addition to PAM4, substantial reactions were observed for anti-MUC1, -MUC5AC, and -CEACAM6 antibodies. All MAbs were employed at a concentration of 10 μg/mL.

Gold, D. V., Newsome, G., Liu, D., & Goldenberg, D. M. (2013). Mapping PAM4 (clivatuzumab), a monoclonal antibody in clinical trials for early detection and therapy of pancreatic ductal adenocarcinoma, to MUC5AC mucin. Molecular cancer, 12(1), 143.

Figure 7 Reaction of several anti-mucin MAbs with PAM4-captured antigen.

Mucin antigens were captured on hPAM4 coated plates, and then probed with several murine anti-mucin MAbs for reaction signal. Both anti-MUC5AC MAbs (2-11 M1 and 45 M1) bound to the hPAM4-captured mucin, whereas the anti-MUC1 MAbs (MA5 and KC4) did not bind. The homologous hPAM4/mPAM4, capture/probe immunoassay gave no signal, suggesting the density of PAM4 epitopes within the mucin may be low, possibly only a single site. A rabbit polyclonal anti-CPM1 IgG was used as a positive control for reaction with hPAM4-captured antigen.

Gold, D. V., Newsome, G., Liu, D., & Goldenberg, D. M. (2013). Mapping PAM4 (clivatuzumab), a monoclonal antibody in clinical trials for early detection and therapy of pancreatic ductal adenocarcinoma, to MUC5AC mucin. Molecular cancer, 12(1), 143.

Figure 8 Inhibition of hPAM4/antigen binding reaction by murine anti-mucin MAbs.

A) Anti-mucin mMAbs (purified IgG) were added to CPM1-coated plates as potential inhibitors prior to addition of hPAM4. mPAM4 provided almost complete inhibition of the reaction between hPAM4 and antigen with the 45 M1 anti-MUC5AC providing limited inhibitory effect (ICmax = 25.5%). Neither 2-11 M1, anti-MUC5AC nor MA5 and KC4, anti-MUC1 MAbs were able to inhibit the specific hPAM4/antigen reaction. B) A similar inhibition study was performed with several anti-MUC5AC MAbs obtained as ascites fluids. MAbs 21 M1, 62 M1, and 463 M1, anti-MUC5AC provided substantial inhibitory effect, similar to that observed with mPAM4 IgG self-inhibition.

Gold, D. V., Newsome, G., Liu, D., & Goldenberg, D. M. (2013). Mapping PAM4 (clivatuzumab), a monoclonal antibody in clinical trials for early detection and therapy of pancreatic ductal adenocarcinoma, to MUC5AC mucin. Molecular cancer, 12(1), 143.