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Final Summer Repor_ChristopheLanglois

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					Christophe Langlois                                        Dr.Beauchemin’s laboratory



Inhibition of mouse melanoma and colon
carcinoma using anti-CEACAM1 antibodies
Abstract
CEACAM1 is a protein involved in various contact-mediated cellular processes such as cell
adhesion and signaling. It is also implicated in metabolism, has an important role in the
immune system and regulates angiogenesis. Level of CEACAM1 expression also varies in
certain cancers. Indeed, CEACAM1 is downregulated in colon carcinoma whereas it is
overexpressed in melanoma. Our laboratory already development a model of CEACAM -/-
melanoma tumors with the use of an sh-RNA sequence that effectively targets and
downregulate CEACAM1. This model showed that CEACAM1 has an important role in the
proliferation, migration and invasion of melanoma tumors. These results led us to develop
another approach to investigate the role of CEACAM1 in various cancers. In this project, we
produced and purified Fab fragments from two anti-CEACAM1 antibodies: AgB10 and CC1.
We then investigated whether the Fab fragments or the IgG would have an effect on the
migration and proliferation of two cell lines (B16F10 and MC38). Our results show that the
CC1 IgG effectively inhibits the proliferation and migration of MC38 cells in vitro.


Introduction
Cell adhesion molecules, or CAMs, play a critical role in cell-cell and cell-matrix
interactions. Indeed, these cell surface proteins mediate different cellular interactions such
as adhesion, recognition, communication and signaling. In particular, CEACAMs, a type of
surface glycoproteins specifically related to carcinoembryonic antigen are cell adhesion
molecules that play an important role on these contact-mediated cellular processes.
CEACAMs are also members of the mammalian immunoglobulin family. CEACAM genes are
located as a cluster of genes on chromosome 19q13.1-19q13.2 in humans. So far, 29 human
CEACAM genes have been discovered. In mice, Ceacam genes are clustered on chromosome
7. CEACAM1 plays a crucial role in cell surface adhesion and also participates in important
signal transduction pathways. Indeed, CEACAM1 has an imperative function in insulin
metabolism (Poy, Yang et al. 2002) and it also mediates essential processes involved in the
cytoskeletal network of the cell (Fournes, Farrah et al. 2003). Furthermore, CEACAM1 has
an important role in angiogenesis (Nouvion, Oubaha et al. 2010). Previous work
determined that this cell adhesion molecule stimulates blood vessel growth and



                          McGill-Oncology summer scholarship award 2011
Christophe Langlois                                       Dr.Beauchemin’s laboratory


proliferation. CEACAM1 expression also has different effects on the immune system (Gray-
Owen and Blumberg). It can increase the adhesion of neutrophils (Pan and Shively) but it
can also inhibit T-cell function (Nagaishi, Chen et al. 2008). All of these CEACAM1
physiological implications are closely related to tumor formation and proliferation, thus
mis-expression of this protein has an effect on many physiological functions and could
easily lead to the formation of a tumor. However, CEACAM1 expression in cancer is
bimodal. In many cases such as liver, colon, breast, prostate, endometrium and bladder
cancers, it has been shown that CEACAM1 expression is downregulated whereas it is
overexpressed in stomach cancers, primary lung tissues tumors and malignant melanoma
(Muller, Klaile et al. 2009). Overexpression in the latter tumors correlates with degree of
invasion. The implication of CEACAM1 in melanoma and colon carcinoma is the main
subject of this project as the significance of CEACAM1 expression in these tumors is not
fully understood. Dr. Anne-Laure Nouvion, a post-doctoral fellow in our laboratory showed
that a shRNA of 21 nucleotide targeting the N-terminal IgV-like domain of CEACAM1 can
effectively shut down the expression of this protein in B16F10 mouse melanoma cells.
Stable CEACAM1-deficient melanoma tumor cells have already been generated and were
tested for xenograft tumor development in wild-type and Ceacam1-/- mice to study their
effect on tumorigenesis. It was shown that CEACAM1 downregulation in B16F10 cells
decreased tumor proliferation, migration and invasion in vivo (Nouvion et al; manuscript
currently under review at Experiment Cell Research). The next step in the study of the role
of CEACAM1 in melanoma and colon carcinoma is to use an antibody approach to inhibit
the function of CEACAM1 in both of these tumors. The goal of this project was then to
produce and purify two kwown monovalent IgG monoclonal antibodies specific for
CEACAM1: CC1 and AgB10. We then used the papain enzyme to cleave the two IgG and
produce Fab fragments. Both the Fab fragments and the whole IgG were used in
proliferation and migration assays to assess the effect of inhibiting CEACAM in vitro in
MC38 and B16F10 cells.




                         McGill-Oncology summer scholarship award 2011
Christophe Langlois                                      Dr.Beauchemin’s laboratory


Materials and methods
Cell culture
The mouse melanoma cell line B16F10 was obtained from the American Type Culture
Collection. The MC38 cell line was kindly provided by Dr. John E. Shively. Both cell lines
were cultured in Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamine
adjusted to contain 1.5 g/l sodium bicarbonate and 4.5 g/l glucose supplemented with 10%
fetal bovine serum (FBS, Gibco) Hybridoma cell lines producing the AgB10 and CC1
monoclonal antibodies were already generated by collaborators. Both cell lines were
cultured in Protein-Free Hybridoma Medium (Gibco).


Proliferation assays and migration assays
For proliferation assays, MC38 and B16F10 cells were seeded into 100 μL of media in
microtiter plates (E-Plate, Roche). Cells were incubated with either an IgG1 control (CT)
mAb, the anti-CC1 IgG1 mAb or the AgB10 IgG1 mAb. Real-time cell proliferation was
monitored every 30 min for 60 h using the xCELLigence System (Roche). Cell sensor
impedance was expressed as an arbitrary Cell Index unit. The Cell Index at each time point
is defined as (Rn-Rb)/15, where Rn is the cell-electrode impedance of the cell-containing
well and Rb is the background impedance of the well with the media alone. Results were
analyzed using the RTCA analyzer software (Roche) or Microsoft Excel. Migration assays
were performed in the presence of 10% FBS media at the bottom of a Transwell plate.


Fab production and protein analysis
Hybridoma cell lines producing the AgB10 and CC1 antibody were passed twice and were
allowed to incubate for 7 days after which the media was collected. The antibodies were
then precipitated using a saturated ammonium sulfate solution. The antibody solutions
were dialyzed against PBS and quantified. Next, both IgG were purified on a protein G-
agarose column to eliminate any impurities that were precipitated with the antibodies. A
solution of glycine (pH 2.7) was used to elute the column. The monovalent antibodies were
then digested using immobilized papain on beads. The antibody solutions were incubated
with papain for 4 hours at 37°C and papain was inactivated with Iodoacetamide. The Fab
fragments were isolated by adsorbing the solution containing the Fab fragments, the Fc


                        McGill-Oncology summer scholarship award 2011
Christophe Langlois                                       Dr.Beauchemin’s laboratory


portion and the undigested IgGs on a protein A-agarose column where the flow-through of
the column would contain the purified Fab fragments. Each Fab production step was
analyzed by running samples in a 15% SDS-PAGE gel, stained with Coomassie blue for 45
minutes and de-stained overnight with a solution of methanol and acetic acid.



Results

Production of Fab fragments
Two known CEACAM1 specific antibodies were used to produce Fab fragments. CC1 is an
anti-mouse monovalent IgG1 raised against CEACAM1 purified from BALB/c liver
membranes(Smith, Cardellichio et al. 1991). The epitope recognized by the antibody lies
within the CEACAM1 N-domain and its binding is dependent on the glycosylation of the Ig-
like domains of the protein(Daniels, Letourneau et al. 1996).
On the other hand, AgB10 is an anti-mouse blocking monovalent IgG raised against ghosts
of hepatocytes in C57Bl/6 mice(Kuprina, Baranov et al. 1990; Fujita, Otsuka et al. 2009). It
recognizes a conformational epitope in the A1 domain of the protein. Similarly to the CC1
mAb, its binding depends on the glycosylation of the protein(Daniels, Letourneau et al.
1996).




                         McGill-Oncology summer scholarship award 2011
Christophe Langlois                                        Dr.Beauchemin’s laboratory

                Summary of CC1 and AgB10 reactivity




Figure 1: Representations of different constructs used to test CC1 and AgB10 reactivity (Daniels,
Letourneau et al. 1996).

Since CEACAM1 is activated by homophilic interaction and due to the double binding
capacity of IgG antibodies, we hypothesized that incubation with whole IgGs would lead to
CEACAM1 activation. Therefore, it was necessary to develop Fab fragments with a single
binding site for the protein which would prevent the activation of CEACAM1 upon
attachment, according to our hypothesis. Prior to digestion, both antibodies were
precipitated from hybridoma media and were purified using a protein G-agarose column
(Fig 2). Then, antibodies were digested using the enzyme papain, which cleaves Fc region
from the two Fab fragments. The Fab fragments were then purified using a protein A-
agarose column (refer to Materials and Methods).




                          McGill-Oncology summer scholarship award 2011
Christophe Langlois                                               Dr.Beauchemin’s laboratory

              Production and purification of Fab fragments




                                                                    Fc portion




                                                                      Fc portion




Figure 2.. A: digestion of an IgG with the enzyme papain. B: Steps of production and purification of the CC1
Fab fragments. C: Steps of production and purification of the AgB10 Fab fragments. Legend: Pre-G= IgG
precipitated and concentrated before protein G-agarose purification; Post-G= IgG after passage on protein G-
agarose column; IgG digested= IgG digested with immobilized papain; Fab= Fab fragment purified after
passage on protein A-agarose column

Proliferation assays
The Fab fragments as well as the whole IgG were tested on two different strains of cells:
B16F10 (melanoma) and MC38 cells (colon carcinoma). First, we investigated the effect of
AgB10 and CC1 Fab fragments on proliferation of B16F10 cells and Figure 3a-b shows that
neither of the Fab fragments had a significant effect on melanoma proliferation. When the
experiment was repeated on MC38 cells, we found that CC1 Fab induced a small decrease in




                             McGill-Oncology summer scholarship award 2011
Christophe Langlois                                       Dr.Beauchemin’s laboratory


the proliferation of colon carcinoma cells between 24 and 48 hours at a concentration of 30
ug. However, the cell index for this concentration reached the level of the control. The
AgB10 Fab fragment did not induce any effect on the proliferation of MC38 Cells (data not
shown).
                      Effects of CC1 and AgB10 Fab fragments
                      on the proliferation B16F10 and MC38
                      cells




 A




 B




 C



                         McGill-Oncology summer scholarship award 2011
Christophe Langlois                                                Dr.Beauchemin’s laboratory

Figure 3. A: Proliferation of B16F10 cells in presence of CC1 Fab fragment. B: Proliferation of B16F10 cells
in presence of AgB10 Fab fragment. C: Proliferation of MC38 cells in presence of CC1 Fab fragment. Control=
PBS

Next, we looked at the proliferation of melanoma cells in the presence of the whole IgGs.
We found out that CC1 slightly decrease the proliferation of B16F10 cells in a dose-
dependent manner. The effect was small but statistically significant. Finally, the AgB10 IgG
did not have any significant effect.


                        Effects of CC1 and AgB10 whole-IgG on
                        the proliferation B16F10 cells




 A




 B

Figure 4 A: Proliferation of B16F10 cells in the presence of different concentrations of CC1 IgG. This IgG
induced a small non-significant decrease in proliferation in a dose-dependent manner. B: Proliferation of
B16F10 cells in the presence of the AgB10 IgG. There is no significant difference in the proliferation of the
melanoma in presence of the IgG. Control=PBS

Interestignly, the CC1 IgG significantly decreased the MC38 cell proliferation cells in a dose-
dependent manner (Figure 5). Furthermore, a concentration of CC1 IgG above 60 ug/100 ul



                              McGill-Oncology summer scholarship award 2011
Christophe Langlois                                       Dr.Beauchemin’s laboratory


completely inhibited the MC38 cell proliferation. Once again, the AgB10 IgG did not have
any effect on proliferation. Until then, we only used PBS as a control for both the Fab
fragments and the IgGs. However, to test if the result obtained with CC1 and the MC38 cells
was due to the effect of CC1 itself or a generic effect of IgGs we investigated the
proliferation of MC38 cells in the presence of a non-target control IgGs. Figure 5A shows
that there is no significant effect of the non-target IgG on the proliferation of colon
carcinoma cells.

                        Effects of CC1 IgG on the proliferation of
                        MC38 cells




A




B




                         McGill-Oncology summer scholarship award 2011
Christophe Langlois                                                Dr.Beauchemin’s laboratory



Figure 5: A. Proliferation of MC38 cells in the presence of a non-target control IgG. B: Proliferation of MC38
cells in the presence of increasing doses of CC1 IgG


                     Effects of CC1 IgG on the migration of
                     MC38 cells




 A




 B




                               McGill-Oncology summer scholarship award 2011
Christophe Langlois                                                Dr.Beauchemin’s laboratory




Figure 6: A. Migration of MC38 cells in the presence of a non-target control IgG1. B: Migration of MC38 cells
in the presence of increasing doses of CC1 IgG1 mAb.


Migration assays
Following the proliferation assays, we investigated whether or not the CC1 and AgB10
mAbs would also modify MC38 cell migration. It should be noted that since B16F10 cells
do not migrate in vitro, no migration assays were undertaken with these cells. As for
proliferation assays, colon carcinoma cell migration was also inhibited in a dose-dependent
manner in the presence of the CC1 mAb (Figure 6). The control IgG1 monoclonal antibody
also significantly inhibited migration after 15 hours at a concentration of 30 ug. However,
the effect was much less than what was observed with the CC1 IgG. The AgB10 IgG did not
have a significant effect on migration (data not shown)



Discussion
Fab fragments and IgGs had previously been used to investigate different properties of the
CEACAM1 molecule. For example, treatment of mice with the AgB10 antibody increased the
occurrence of experimental autoimmune encephalomyelitis (EAE) in these animals,
suggesting that CEACAM1 has a protective role in this disease(Fujita, Otsuka et al. 2009).
Until now, monovalent antibodies and their Fab fragments have primarily been used to
demonstrate the role of CEACAM1 in immunity. Indeed, the AgB10 monovalent IgG was
used to show that CEACAM1 has a regulatory role in the development and maturation of
dentritic cells(Kammerer, Stober et al. 2001) while other CEACAM1 monovalent antibodies
were used to investigate the role of CEACAM1 in neutrophils adhesion in vitro(Nair and
Zingde 2001). It was also shown with the use of Fab fragments that CEACAM1 has a role in
FAS-ligand induced apoptosis(Singer, Klaile et al. 2005). Finally, the CC1 IgG was used to
study the role of CEACAM1 in T cell development and proliferation(Gray-Owen and
Blumberg 2006). Furthermore, both AgB10 and CC1 IgGs had been shown to block the
activity of CEACAM1 in vitro and in vivo (Smith, Cardellichio et al. 1991; Fujita, Otsuka et al.
2009)
The CC1 mAb induced a decrease in both the proliferation and migration of MC38 cells in a



                              McGill-Oncology summer scholarship award 2011
Christophe Langlois                                        Dr.Beauchemin’s laboratory


dose-dependent manner, whereas the control IgGs did not have a significant impact on
both of these characteristics, suggesting that the effect is specific to the CC1 mAb. The
proliferation decrease was surprisingly more important in a concentration of 60 ug of IgG
or more, but this is essentially non-physiological concentrations. Further experiments
would be necessary to investigate whether this data results from total inhibition of
proliferation caused by the inhibition of CEACAM1 activity or if it is the result of cell death
provoked by the large concentration of CC1 mAb. However, the same IgG did not have any
significant effect on B16F10 cell proliferation. Differences in signaling pathways and level
of CEACAM1 expression could explain this discrepancy. Further experiments would be
necessary to investigate the difference between these two cell lines. Interestingly, neither
the AgB10 mAb nor its Fab fragments induced any significant differences in the B16F10
and MC38 cell proliferation and migration. This antibody recognizes a very particular
conformation of CEACAM1 that may not be relevant within the context of these
experiments (Daniels, Letourneau et al. 1996).Furthermore, even if the CC1 IgG decreased
the MC38 cell proliferation, its Fab fragments did not produce any significant changes in
similar proliferation assays. This could be explained by the fact that CEACAM1 is activated
by homophilic interaction. Indeed, the Fab fragments would bind to the N-terminal domain
of the CEACAM1 molecules and prevent their binding. However, the CC1 IgG can bind two
CEACAM1 molecules, bringing these in close proximity and activating them by the same
process. If this model were found to be true, it would mean that activation of CEACAM1
leads to a decrease in proliferation and migration capacity in MC38 cells, reinforcing the
CEACAM1 tumor-suppressor function in these tumors. Finally, the next step would be to
evaluate the CC1 IgG effect by injecting mice that develop colon carcinoma tumors with the
monovalent antibody. If this approach were successful in a mouse model, a similar
approach    will   eventually     also   be   attempted    in   combination   with   different
chemotherapeutic regimens utilized to treat cancer to determine if this antibody increases
the efficiency of chemotherapy.




                          McGill-Oncology summer scholarship award 2011
Christophe Langlois                                        Dr.Beauchemin’s laboratory


Acknowledgements
I would like to thank Dr. Nicole Beauchemin for giving me the opportunity to work on this
project in her laboratory, Dr. Anne-Laure Nouvion for her support and knowledge, Ms.
Valérie Breton for her technical assistance and the McGill Department of Oncology for
funding my summer research project.



References

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        embryogenesis: correlation with events of epithelial differentiation, mesenchymal-
        epithelial interactions, absorption, and myogenesis." Developmental dynamics : an
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Fournes, B., J. Farrah, et al. (2003). "Distinct Rho GTPase activities regulate epithelial cell
        localization of the adhesion molecule CEACAM1: involvement of the CEACAM1
        transmembrane domain." Molecular and cellular biology 23(20): 7291-7304.
Fujita, M., T. Otsuka, et al. (2009). "Carcinoembryonic antigen-related cell adhesion
        molecule 1 modulates experimental autoimmune encephalomyelitis via an iNKT
        cell-dependent mechanism." The American journal of pathology 175(3): 1116-1123.
Gray-Owen, S. D. and R. S. Blumberg (2006). "CEACAM1: contact-dependent control of
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Kammerer, R., D. Stober, et al. (2001). "Carcinoembryonic antigen-related cell adhesion
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Kuprina, N. I., V. N. Baranov, et al. (1990). "The antigen of bile canaliculi of the mouse
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Muller, M. M., E. Klaile, et al. (2009). "Homophilic adhesion and CEACAM1-S regulate
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        biology 187(4): 569-581.
Nagaishi, T., Z. Chen, et al. (2008). "CEACAM1 and the regulation of mucosal inflammation."
        Mucosal immunology 1 Suppl 1: S39-42.
Nair, K. S. and S. M. Zingde (2001). "Adhesion of neutrophils to fibronectin: role of the cd66
        antigens." Cellular immunology 208(2): 96-106.
Nouvion, A. L., M. Oubaha, et al. (2010). "CEACAM1: a key regulator of vascular
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Pan, H. and J. E. Shively (2010). "Carcinoembryonic antigen-related cell adhesion molecule-
        1 regulates granulopoiesis by inhibition of granulocyte colony-stimulating factor
        receptor." Immunity 33(4): 620-631.
Poy, M. N., Y. Yang, et al. (2002). "CEACAM1 regulates insulin clearance in liver." Nature
        genetics 30(3): 270-276.




                          McGill-Oncology summer scholarship award 2011
Christophe Langlois                                       Dr.Beauchemin’s laboratory


Singer, B. B., E. Klaile, et al. (2005). "CEACAM1 (CD66a) mediates delay of spontaneous and
       Fas ligand-induced apoptosis in granulocytes." European journal of immunology
       35(6): 1949-1959.
Smith, A. L., C. B. Cardellichio, et al. (1991). "Monoclonal antibody to the receptor for
       murine coronavirus MHV-A59 inhibits viral replication in vivo." The Journal of
       infectious diseases 163(4): 879-882.




                         McGill-Oncology summer scholarship award 2011

				
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