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).
Figure 2.
mAb CC4 was able to target xenografted tumors, suppress tumor growth in vivo, and raised ADCC reactions in vitro.
Non-small cell lung cancer cells A549 was stained with cy5 labeled CC4 in immunofluorescence (A). Cells were injected to nude mice to form tumors and CC4-cy5 or IgG-cy5 was administrated intravenously. In vivo optical imaging of fluorescent antibodies was performed in the indicated times (B and C). LS174T cells were injected subcutaneously into nude mice. After forming tumor, mice were treated with CC4 or normal IgG control for indicated times. Tumors were taken out for measuring the size and calculating the volume, as well as photography (D, n = 7 in each group, **P<0.01 and *P<0.05). LS174T cells were subjected to ADCC assays using murine spleen cells as effector cells. Results for specific cytotoxicity of cancer cells were presented in bar graphs (E).
Figure 3.
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).
Figure 4.
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).
Figure 5.
mAb CC4 enhanced the killing of HLA-Ilow colorectal cancer cells by CD16- NK cells.
CD56+CD16+ (A) and CD56+CD16- NK cells (B) isolated from human PBMCs were analyzed by flow cytometry. NK cytotoxicity assays were performed against various colorectal cancer cell lines using either CD56+CD16+ (C) or CD56+CD16- (D) NK cells as effector cells. Significant differences between mAb CC4 treatments and controls were indicated by double stars (**P<0.01).
Figure 6.
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).