Anti-Human CEACAM6 Recombinant Antibody (TAB-004LC-VHH) (CAT#: TAB-004LC-VHH)

Figure 1 Expression level of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) by BxPC3 and human umbilical vein endothelial cells (HUVECs) and in their conditioned media.

We used 2A3-Fc antibody conjugated with Alexa Fluor 594 for immunocytochemistry to visualise CEACAM6 expression on BxPC3 cell surface (red, right). An isotype control VTI1-Fc was used as a negative control (left). 40 ,6-diamidino-2-phenylindole (DAPI) was used as nuclear stain (Blue).

Cheng, T. M., Murad, Y. M., Chang, C. C., Yang, M. C., Baral, T. N., Cowan, A., ... & Zhang, J. (2014). Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation, migration, invasion and angiogenesis of pancreatic cancer cells. European Journal of Cancer, 50(4), 713-721.

Figure 2 Inhibition of BxPC3 cell proliferation and MMP-9 and MMP-2 activities by single domain antibodies (sdAbs).

Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) antibodies were tested in MTT assays for their effect on BxPC3 cells proliferation. About 50 lg/ml of sdAb or 360 lg/ml of heavy chain antibody (HCAb) were used to treat the tumour cells. VTI1-Fc, an isotype control HCAb that does not bind to CEACAM6 was used as a negative control. The cells were allowed to grow for 72 h, after which the MTT assays were performed.

Cheng, T. M., Murad, Y. M., Chang, C. C., Yang, M. C., Baral, T. N., Cowan, A., ... & Zhang, J. (2014). Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation, migration, invasion and angiogenesis of pancreatic cancer cells. European Journal of Cancer, 50(4), 713-721.

Figure 3 Inhibition of BxPC3 cell proliferation and MMP-9 and MMP-2 activities by single domain antibodies (sdAbs).

MMP-9 and MMP-2 activities were examined by gelatin zymography. The 2A3 and 2A3-Fc antibodies showed significant reduction in MMP-9 activity in the media from BxPC3 cells. The zymography assays were performed on medium aliquots taken from BxPC3 cultures at the time the proliferation assays were started.

Cheng, T. M., Murad, Y. M., Chang, C. C., Yang, M. C., Baral, T. N., Cowan, A., ... & Zhang, J. (2014). Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation, migration, invasion and angiogenesis of pancreatic cancer cells. European Journal of Cancer, 50(4), 713-721.

Figure 4 In vitro proliferation and gelatinase activity assays.

Percentage of proliferating BxPC3 cells after being treated with various amounts of 2A3 or 2A3-Fc and gemcitabine for a 72 h. Cell proliferation in starvation medium is used as control.

Cheng, T. M., Murad, Y. M., Chang, C. C., Yang, M. C., Baral, T. N., Cowan, A., ... & Zhang, J. (2014). Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation, migration, invasion and angiogenesis of pancreatic cancer cells. European Journal of Cancer, 50(4), 713-721.

Figure 5 In vitro proliferation and gelatinase activity assays.

Top panel, MMP-2 and MMP-9 activities were examined by gelatin zymography. 2A3/2A3Fc, but not gemcitabine, inhibited MMP-9 activity. MMP-2 activity was not affected by the treatment. Bottom panel, The scale of MMP-9 activity reduction caused by 2A3/2A3-Fc treatment.

Cheng, T. M., Murad, Y. M., Chang, C. C., Yang, M. C., Baral, T. N., Cowan, A., ... & Zhang, J. (2014). Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation, migration, invasion and angiogenesis of pancreatic cancer cells. European Journal of Cancer, 50(4), 713-721.

Figure 6 Inhibition of BxPC3 invasion by single domain antibodies (sdAbs).

Top panel, BxPC3 invasion activity was inhibited by 2A3/2A3-Fctreatment. Cells from three random selected fields were counted and normalised against a starvation medium control. The invasive cells on the bottom surface were stained with Calcein AM and photos were taken at 100 magnification. Bottom panel, scale of the reduction of BxPC3 cells by 2A3 and 2A3-Fc. The invasive cells on the bottom surface were stained with Calcein AM and counted by fluorescence microscopy. All experiments were performed in triplicate. ** P < 0.01.

Cheng, T. M., Murad, Y. M., Chang, C. C., Yang, M. C., Baral, T. N., Cowan, A., ... & Zhang, J. (2014). Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation, migration, invasion and angiogenesis of pancreatic cancer cells. European Journal of Cancer, 50(4), 713-721.

Figure 7 Inhibition of angiogenesis by 2A3 and 2A3-Fc.

Top panel, conditioned media from BxPC3 cells that were treated with 2A3 or 2A3-Fc inhibited tubule formation by human umbilical vein endothelial cells (HUVECS) during a tubule formation assay. Bottom panel, capillary-like tube formation (angiogenesis) was inhibited by 2A3 or 2A3-Fc. Single domain antibody (sdAb) 2A3 inhibited tubule formation by 21% and 2A3-Fc inhibited tubule formation by 49%. Three fields that were randomly chosen were captured and the length of the capillary in each field was measured. ** P < 0.01 in 2A3 and *** P < 0.001 in 2A3-Fc.

Cheng, T. M., Murad, Y. M., Chang, C. C., Yang, M. C., Baral, T. N., Cowan, A., ... & Zhang, J. (2014). Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation, migration, invasion and angiogenesis of pancreatic cancer cells. European Journal of Cancer, 50(4), 713-721.

Figure 8 SPR of 2A3-mFc and 9A6.

The binding affinities of 9A6 and 2A3-mFc antibodies to CEACAM6 were determined through Surface Plasmon Resonance (SPR) measurement by using a BIAcore 3000 (GE Healthcare).

Niu, G., Murad, Y. M., Gao, H., Hu, S., Guo, N., Jacobson, O., ... & Chen, X. (2012). Molecular targeting of CEACAM6 using antibody probes of different sizes. Journal of controlled release, 161(1), 18-24.

Figure 9 Cell uptake and efflux assay of ⁶⁴Cu-DOTA-2A3- mFc and ⁶⁴Cu-DOTA-IgG on BxPC3 or UM-22B cells.

The cell uptake and efflux was expressed as percentage of decay-corrected total input radioactivity.

Niu, G., Murad, Y. M., Gao, H., Hu, S., Guo, N., Jacobson, O., ... & Chen, X. (2012). Molecular targeting of CEACAM6 using antibody probes of different sizes. Journal of controlled release, 161(1), 18-24.