Anti-Human CEACAM5 Recombinant Antibody (CC4) (CAT#: TAB-435LC)

Figure 1 Characterization of mAb CC4 and identification of mAb CC4 antigen.

MAb CC4 was used to stain LS174T cells in flow cytometry assay (A) and immunofluorescence (B). Isotype-matched murine normal IgG served as negative control. Frozen sections of normal colon and colorectal tumor tissues were stained by mAb CC4 in immunohistochemical analysis (C); and extracts from these tissues and whole cell extracts of LS174T and SW1116 were subjected to immunoblot using mAb CC4 (D, lower panels). LS174T cell lysate were immunoblotted with CC4 under reduced or non-reduced conditions (D, upper panels). Immunoprecipitation was performed using mAb CC4 or mIgG to accumulate the antigen protein and immunoprecipitates were subjected to Western blot assays (E). Four peptides were obtained from LC-MS analysis using CC4 precipitates and the antigen was identified as CEACAM5, of which partial amino acid sequence and these identified peptides were illustrated (F). Bladder cancer cells T2-4 were transfected with CEACAM5 expression plasmids and subjected to flow cytometry analysis with CC4 (G).

Zheng, C., Feng, J., Lu, D., Wang, P., Xing, S., Coll, J. L.,... & Yan, X. (2011). A novel anti-CEACAM5 monoclonal antibody, CC4, suppresses colorectal tumor growth and enhances NK cells-mediated tumor immunity. PloS one, 6(6), e21146.

Figure 2 mAb CC4 suppressed proliferation, migration and aggregation of colorectal cancer cells.

LS174T cells treated with indicated concentrations of mAb CC4 were subjected to proliferation assays using CFSE staining (A). The same assay was also performed using Lovo cells under the treatment of 25 µg/ml mAb CC4 (B). LS174T cells were used in migration assays using transwell system (C) and aggragation assays in the presence of indicated concentrations of mAb CC4 or normal mIgG (Di) with Ca2+ or EGTA (Dii). Statistically significant differences between mAb CC4 treated groups and control groups were indicated by double stars (**P<0.01).

Zheng, C., Feng, J., Lu, D., Wang, P., Xing, S., Coll, J. L.,... & Yan, X. (2011). A novel anti-CEACAM5 monoclonal antibody, CC4, suppresses colorectal tumor growth and enhances NK cells-mediated tumor immunity. PloS one, 6(6), e21146.

Figure 3 Epitope mapping of mAb CC4.

Recombinant CEACAM5 fractions and full-length deletion mutants were illustrated in (A). These recombinant fractions were expressed in E. Coli and whole cell lysates were separated by SDS-PAGE and analyzed by immunoblot with anti-His-tag (positive control) and mAb CC4 (B and C). Plasmid expressing full-length CEACAM5 wild-type or mutants were transfected into 293T cells and these cells were then subjected to flow cytometry assays using mAb CC4 or rabbit polyclonal anti-CEA (RACEA) or mIgG (D). Amino acid sequences of region aa42–61 of CEACAM1 and CEACAM5 were shown; and 293T cells were transfected with plasmids expressing CEACAM1 wt or mutant and analyzed by flow cytometry with indicated antibodies (E).

Zheng, C., Feng, J., Lu, D., Wang, P., Xing, S., Coll, J. L.,... & Yan, X. (2011). A novel anti-CEACAM5 monoclonal antibody, CC4, suppresses colorectal tumor growth and enhances NK cells-mediated tumor immunity. PloS one, 6(6), e21146.

Figure 4 mAb CC4 blocked the suppression of NK killings induced by over-expression of CEACAM5 in colorectal cancer cells.

Forced expressions of CEACAM1 and CEACAM5 in HCT-15 cells were confirmed by flow cytometry (A). Stable transfectants were then subjected to NK cytotoxicity assays using CD56+CD16+ NK cells (B) and CD56+CD16-NK cells under different ratios of numbers of target cells to that of effector cells (C and D). Significant differences between mAb CC4 treated or RACEA-treated groups and the corresponding control groups were indicated by double stars (**P<0.01).

Zheng, C., Feng, J., Lu, D., Wang, P., Xing, S., Coll, J. L.,... & Yan, X. (2011). A novel anti-CEACAM5 monoclonal antibody, CC4, suppresses colorectal tumor growth and enhances NK cells-mediated tumor immunity. PloS one, 6(6), e21146.