And CEACAM5 (Carcinoembryonic Antigen)

Research Article

Inhibition of Adhesion, Invasion, and Metastasis by Antibodies Targeting CEACAM6 (NCA-90) and CEACAM5 (Carcinoembryonic Antigen)

Rosalyn D. Blumenthal,1 Hans J. Hansen,2 and David M. Goldenberg1

1Center for Molecular Medicine and Immunology, Garden State Cancer Center, Belleville, New Jersey and 2Immunomedics, Inc., Morris Plains, New Jersey

Abstract Carcinoembryonic antigen (CEA, CEACAM5, and CD66e) is CEACAM5 and CEACAM6 are overexpressed in many cancers expressed on many cancers (2–6) and has been implicated with and are associated with adhesion and invasion. The effects of malignancy, particularly enhanced metastasis (7–9). CEACAM5 three monoclonal antibodies targeting different epitopes functions as a chemoattractant and as an adhesion molecule (10), and has been reported to promote the metastatic potential in some on these antigens (NH2-terminal [MN-3] and A1B1 domains [MN-15] shared by CEACAM5 and CEACAM6 and the A3B3 experimental tumors (11). CEACAM5 has been shown to be domain [MN-14] restricted to CEACAM5) were evaluated in involved in both homophilic (CEACAM5 to CEACAM5) binding migration, invasion, and adhesion assays in vitro using a panel between the N domains of antiparallel CEACAM5 molecules on of human pancreatic, breast, and colonic cancer cell lines, opposite cell surfaces and heterophilic binding (CEACAM5 binding and in the GW-39 human colonic micrometastasis model to non-CEACAM5 molecules; refs. 12–14). Ribozyme-mediated in vivo. MN-3 FabV and MN-15 FabV were both effective at suppression of CEACAM5 expression reduces metastases in a nude inhibiting cell migration. MN-15 FabV treatment inhibited mouse tail vein injection model (15), whereas anti-CEACAM5 invasion, reducing cell penetration through an extracellular intact mAb, MN-14, also delayed death due to metastasis in a matrix (ECM). MN-3 FabValso decreased invasion but was less human colorectal cancer model (16) and reduced growth of the TT effective than MN-15 FabV in four of five cell lines. All three human medullary thyroid cancer xenograft (17). monoclonal antibody (mAb) Fabs decreased adhesion of Nonspecific cross-reacting antigen (NCA-90, CEACAM6, and tumor cells to endothelial cells by 49% to 58%. MN-15 FabV CD66c) is a member of the CEA family (18) that is also expressed but not MN-3 or MN-14 Fabs induced a decrease in adhesion on many human cancers (19–21). CEACAM6 in tumors correlates of three of six cell lines to the ECM protein, fibronectin, but inversely with cellular differentiation (22) and is an independent adhesion to vitronectin, laminin, collagen-I, and collagen-IV prognostic factor associated with a higher risk of colorectal cancer was not affected. In vivo studies showed that treatment with relapse (23). CEACAM6 is also an important protein associated MN-3 FabV or MN-15 FabV of mice implanted with GW-39 with cell adhesion; its expression on neutrophils is involved in human colonic cancer cells increased their survival (P < adhesion to cytokine-activated endothelial cells (24). This molecule 0.025 and P < 0.01, respectively). These studies show that exhibits homotypic and heterotypic interactions with other antibody Fabs that target either CEACAM5 or CEACAM6 members of the CEA family and with integrin receptors (14). affect cell migration, cell invasion, and cell adhesion in vitro, Antibodies targeting the N domain interfere with cell-cell and that MN-15 and MN-3 Fabs have antimetastatic effects interactions (25). Normal cells routinely undergo apoptosis in the in vivo, resulting in improved survival of mice with absence of adhesive interactions with ECM, a phenomenon known metastases. Thus, blocking the N and A1B1 domains of as ‘‘anoikis’’ (26). Resistance to anoikis, a characteristic of tumor CEACAM5/CEACAM6 can impede the metastatic process. cells, facilitates tumorigenesis and metastasis. CEACAM5 and (Cancer Res 2005; 65(19): 8809-17) CEACAM6 both inhibit anoikis (27, 28), and modulation of CEACAM6 expression alters the malignant phenotype of cancer Introduction cells (29). Silencing the CEACAM6 gene by small interfering RNA enhances cell anoikis, increases caspase activation in response to The eradication of metastatic disease is crucial for achieving anchorage-independent conditions, down-regulates the Akt cell survival in most patients with cancer. The metastatic process survival pathway, and inhibits metastasis in vivo (30). Expression of consists of a series of sequential steps, including invasion of this membrane protein is also associated with increased invasive- extracellular matrix (ECM), extravasation into vessels, transport in ness through a c-src-dependent mechanism (31). Thus, CEACAM6 the circulation, adhesion to endothelial cells in a new tissue, extra- may represent a therapeutic target to control malignancy and/or vasation through the vessel wall, and migration and proliferation metastasis. in response to organ-specific factors at the new site (1). This article Antigenic sites on CEACAM5 and CEACAM6 have been deals with the development of an antibody-based therapeutic characterized, and panels of antibodies recognizing specific approach to impede metastasis. epitopes have been generated (32, 33). Three subdomains in the N region that are required for intercellular homotypic adhesion have been identified by site-directed deletions and point mutations Requests for reprints: Rosalyn D. Blumenthal, Garden State Cancer Center, (34). Peptides have been developed to these regions in an effort to CMMI, 520 Belleville Avenue, Belleville, NJ 07109. Phone: 973-844-7014; Fax; 973-844- block adhesion (35). The concentration of peptide required to block 7020; E-mail: [email protected]. I2005 American Association for Cancer Research. cell aggregation was 1 mg/mL or a 25-fold higher molar con- doi:10.1158/0008-5472.CAN-05-0420 centration than the complete antibody, despite the predicted www.aacrjournals.org 8809 Cancer Res 2005; 65: (19). October 1, 2005

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research greater penetration of a smaller peptide. This inefficient dose surface. Monolayers were rinsed several times to remove floating cells and requirement could be due to instability of the small peptide or due 4 mL of fresh medium were added back in the absence or presence of j to its low affinity. We postulated that using monovalent antibody antibody IgG or FabV (10 Ag/mL) and incubated at 37 C. After 18 to fragments would be a more effective approach for targeting these 24 hours, the medium was removed and coverslips stained with 1 mL Wright-Giemsa for 1 minute. The stain was washed off with distilled water, domains, because stability of the mAb will likely be greater and the air-dried, and mounted onto slides with Cytoseal 60. Repopulation of the affinity higher than corresponding peptides. Our studies have wound space was evaluated by counting the number of cells that migrated primarily been done with monovalent fragments of the antibodies into the wound area in 10 representative fields. Regions of migration were of interest, rather than intact IgGs, because the single binding arm photographed for documentation (38, 39). of the Fab will prevent complexation of tumor cells, which might Endothelial cell adhesion assay. Human umbilical vein endothelial occur with a bivalent IgG. In addition, it allowed us to study the cells (HUVEC; Cambrex, San Diego, CA.) were grown in collagen-coated j efficacy of a CEA/NCA-90-targeted antibody without the potential dishes in EGM Media in a humidified atmosphere with 5% CO2 at 37 C. 4 effector cell-inducing activity of the Fc region. We have assessed At passages 2 to 5, cells were plated at a density of 4 Â 10 cells per well in h h the expression of CEACAM5 and CEACAM6 on a panel of tumor 96-well plates 24 hours before the assay. Interleukin-1 (IL-1 , 1 ng/mL; cell lines and have determined the effects of several antibodies Hoffmann-La Roche, Nutley, NJ) was added 4 hours before the assay. At the start of the study, the medium with the IL-1h was removed and fresh targeting different epitopes of CEACAM5 and CEACAM6 for their DMEM with 1% bovine serum albumin (BSA) added and incubated for ability to affect tumor cell migration, invasion, and cell adhesion 30 minutes. Fresh medium without antibodies or with 10 Ag/mL of MN-15 in vitro. We have also evaluated the therapeutic potential of FabV, MN-3 FabV, or Ag8 FabVwas added. Tumor cells (1 Â 106 cells/mL) these antibody Fabs in controlling metastasis and survival of prelabeled overnight with 3H-thymidine (100 AL per well using 1.0 ACi/mL) mice bearing a CEACAM5/CEACAM6-expressing human colonic were added to HUVEC cultures and incubated for 30 minutes at 37jC with carcinoma. rotation in medium with 20% FBS. Samples were washed thrice with PBS to remove unattached cells. Attached cells were solubilized with 0.1 N NaOH and radioactivity was measured in a h scintillation counter. The cpm Materials and Methods attached/total cpm added (attaching potential) was determined. Antibody production. MN-15 binds to the A1B1 domain (Gold group 4) Adhesion to extracellular matrix proteins assay. The assay was done and MN-3 binds to the N domain (Gold group 5) found on both CEACAM5 using the Cytomatrix screening kit from Chemicon (Kit #ECM205, and CEACAM6. MN-14 binds to the A3B3 domain (Gold group 3) only found Temecula, CA). The kit contains 96-well plates with strips coated with on CEACAM5 (ref. 36; Fig. 1). These antibodies and their FabVfragments, fibronectin, vitronectin, laminin, collagen-I, or collagen-IV. Subconfluent including the nonspecific Ag8 FabV and anti-a-fetoprotein IgG, were cell cultures were used for these studies. Cells (1 Â 106 cells/mL) were j supplied by Immunomedics, Inc. (Morris Plains, NJ). MN-3 and MN-15 seeded onto coated substrate and incubated at 37 C for 1 hour in a CO2 were used as murine mAbs, whereas MN-14 was included in its humanized form, hMN-14 or labetuzumab (37). Cell culture. All cell lines were maintained in monolayer culture having RPMI 1640 supplemented with 10% fetal bovine serum (FBS) plus 100 units/ mL penicillin, 100 units/mL streptomycin, 4 mmol/L glutamine, and 1% nonessential amino acids and grown at 37jC in 95% air and 5% CO2. Cells were harvested using 1% trypsin and counted by hemocytometer. Viability was determined by trypan blue exclusion. CEACAM5 and CEACAM6 expression. Cells were harvested from culture and aliquoted into FACScan tubes containing 2 mL Dulbecco’s PBS with 0.2% sodium azide and 1% appropriate blocking serum. Cells were incubated with 10 Ag/mL of hMN-14 anti-CEACAM5 in PBS (+ NaN3 +1% blocking serum) for 1 hour on ice, pelleted (1,440 rpm Â 5 minutes), and supernatant decanted. Some tubes were then incubated with 10 Ag/mL murine MN-15 IgG or MN-3 IgG (to determine CEACAM6 expression) for 1 hour on ice and pelleted. Neither MN-15 nor MN-3 bind CEACAM5 once MN-14 is bound so that only CEACAM6 is detected by MN-15 or MN-3. Second antibody conjugated with FITC (FITC-goat-anti-human for CEACAM5 determination and FITC-goat-anti-mouse for CEACAM6 determination) was added (1:500 secondary antibody in PBS + NaN3 +1% blocking serum) for 30 minutes on ice in the dark. Cells were pelleted, washed twice with 2 mL ice-cold PBS + NaN3, and resuspended in 1.5% paraformaldehyde. Fluorescence-1 was read on a Becton Dickinson (San Jose, CA) flow cytometer. Percentage of cells that were positive and mean channel fluorescence (MCF) were recorded. CEACAM5 and CEACAM6 expression on GW-39 tumor xenografts was determined by immunohistochemistry using a similar approach of hMN-14 followed by biotinylated GAH to detect CEACAM5 and preincubation with hMN-14 followed by mMN-15 or mMN-3 and biotinylated GAM second antibody to detect CEACAM6 on paraffin sections. Spontaneous migration assay. Glass coverslips were placed in 100 Â 15 mm Petri dishes and UV sterilized overnight. Suspensions of cancer cells (2-4 Â 105 cells/mL) were prepared from 80% to 90% confluent monolayers. Cells were plated on each coverslip and incubated for 2 to Figure 1. Schematic structure of domains of CEACAM5 and CEACAM6. 5 days to reach 70% to 80% confluence. Two diagonal cell-free paths Epitopes recognized by MN-15, MN-3, and MN-14 antibodies on CEACAM5 and (‘‘wounds’’) were created by dragging a sterile yellow pipette tip across the CEACAM6 are noted.

Cancer Res 2005; 65: (19). October 1, 2005 8810 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Anti-CEACAM6 Antibodies Inhibit Metastasis incubator in PBS containing Ca2+/Mg2+ (200 AL per well). Adherent cells medium (1 Â 106 cells/mL), and appropriate antibody Fabs were added to were fixed and stained. The plate was washed to remove unadhered cells individual cell aliquots. Cell invasion potential was determined as follows. and stained with 100 AL per well of MTS (Cell Titer Aq 96, Promega, Baseline invasion and invasion in the presence of a chemoattractant Madison, WI) for 5 minutes at room temperature. Excess stain was (10% FBS) were measured after a 72-hour incubation period at 37jCin removed with three PBS washes. Solubilization buffer [100 AL of a 50:50 a5%CO2 incubator. The bottom side of the collagen-coated polycarbonate mixture of 0.1 mol/L NaH2PO4 (pH 4.5) and 50% ethanol] was added to membrane insert of the invasion chamber was placed in 400 AL of cell stain each well. Relative attachment was determined using absorbance for 20 minutes at room temperature and washed. The dye was extracted and readings (A540 nm À A570 nm). transferred into a 96-well microtiter plate for colorimetric measurements Collagen-based invasion assay. The assay was done using Chemicon (A560 nm). (Kit #ECM551). Tumor cells at 80% confluence and were serum-starved for In vivo therapy studies. Female athymic nude mice (6 to 8 weeks old) 18 to 24 hours before the assay were used. Cells were harvested with 5 mL were purchased From Taconic (Germantown, NY). Therapy studies were of 2 mmol/L EDTA/PBS per 100-mm dish and incubated at 37jC for 5 to done using our CEACAM5+/CEACAM6+ GW-39 intrapulmonary micro- 15 minutes. Cells were collected into 10 to 20 mL of quenching medium metastasis model, GW-39iv (40–42). The GW-39 human colonic carcinoma (serum-free DMEM containing 5% BSA) to inactivate trypsin/EDTA from has been maintained as a serially transplanted signet-ring cell cancer line the harvesting buffer. Cells were pelleted, resuspended in quenching since 1966 (42). Stock s.c. GW-39 tumors grown in nude mice were used to

Table 1. CEACAM5 and CEACAM6 expression in tumor cell lines

Background CEACAM5 expression CEACAM6 expression with MN-15 CEACAM6 expression with MN-3

% Positive MCF % Positive MCF % Positive MCF % Positive MCF

Breast BT-20 4.4 12.3 57.1 17.9 34.1 48.7 33.5 50.5 MDA-231 2.3 10.6 17.2 16.5 9.0 37.0 6.1 24.3 MDA-468 2.0 71.6 23.6 93.2 97.1 194.0 85.6 155.5 T47D 6.0 15.4 15.7 15.8 54.8 51.1 52.4 42.4 ZR75-30 3.3 21.0 27.9 28.0 99.1 641.0 79.0 671.6 MCF-7 3.2 12.8 20.6 18.4 98.8 238.2 95.4 256.0 BT-549 6.4 18.1 5.0 26.2 7.9 18.8 8.2 20.7 MDA-435 3.0 27.4 2.9 27.5 46.4 30.1 42.0 23.0 ZR75-1 3.1 10.3 10.6 89.7 35.9 115.5 26.9 98.1 SKBR-3 7.6 264.5 ND ND 29.1 120.1 19.6 248.6 Ovarian OVCAR3 2.05 32.1 3.33 40.6 52.2 51.1 ND ND IGROV-1 16.0 17.1 15.7 13.1 28.2 30.4 ND ND SKOV3nmp2 5.1 34.5 21.1 32.6 40.6 52.9 43.8 40.4 A2780 11.8 22.5 12.4 11.2 15.0 23.8 ND ND Colon HT-29 6.6 43.5 17.0 35.9 98.4 36.4 81.8 30.6 WidR 4.5 45.7 5.1 46.3 ND ND ND ND MOSER 4.5 37.5 56.1 65.1 10.3 18.2 8.4 16.1 LS174T 4.4 61.9 68.5 94.2 94.5 219.0 70.5 163.4 HCT-15 4.1 56.9 0.80 48.8 23.4 48.5 ND ND Pancreatic CAPAN-1 4.4 76.8 33.5 114.7 96.7 476.5 69.1 383.4 HS766T 4.4 248.9 ND ND 30.8 172.3 23.5 142.6 BXPC3 4.9 14.4 10.5 16.8 99.4 702.3 99.0 751.1 PANC-1 5.0 19.7 9.2 22.6 13.9 49.9 ND ND Prostate PC-3 4.7 18.9 15.1 23.3 14.9 25.7 15.5 26.4 ALVA-31 4.6 28.1 3.8 26.0 11.6 52.2 10.2 67.1 Du-145 4.8 19.3 3.5 20.1 9.9 21.1 ND ND LNCAP 3.1 17.5 19.1 27.4 8.4 28.8 ND ND M12 4.7 17.4 10.4 33.9 22.4 24.3 ND ND P69 5.1 53.1 5.9 75.8 8.7 50.0 ND ND NSCLC CALU-1 4.6 13.6 2.4 11.9 8.7 22.0 7.4 26.2 CALU-3 4.4 29.8 1.3 25.0 38.4 110.4 30.5 93.4 A549 4.8 27.6 8.9 31.1 77.3 224.0 57.0 182.3 SKMES-1 4.6 40.3 5.2 41.9 17.0 56.1 16.8 71.7

Abbreviation: ND, not determined.

www.aacrjournals.org 8811 Cancer Res 2005; 65: (19). October 1, 2005

CEACAM6À (Moser and LNCAP), four were CEACAM5+/CEA- CAM6+, and 12 were CEACAM5À/CEANCAM6+. The CEACAM6+ cell lines included 7 of 10 breast cancers, one of four ovarian cancers, three of four colon cancers, three of four pancreatic cancers, one of six prostate cancers, and two of four non–small cell lung cancer lines. Many of these tumor lines had >95% of cells expressing the CEACAM6 antigen and, in some cases, expression was very high (MCF of 641 for ZR75-30, 702 for BXPC3, and 476 for CaPAN-1). In contrast, the most positive CEACAM5-expressing lines had <60% of cells expressing the antigen, with a MCF <120. Effect of anti-CEACAM5 and anti-CEACAM6 antibodies on cell migration. Using the wound-healing assay, we assessed cell migratory activity in vitro in a panel of cell lines. LS174T and HT-29 cells showed the most migratory activity, whereas ZR75-30, MCF-7, and BXPC3 cells showed very little migration (results not shown). Compared with an irrelevant antibody, MN-3 and MN-15 intact IgG and FabV all reduced migration in both cell lines. In HT-29, the number of migrating cells per field decreased from 14.9 F 9.1 to 6.1 F 0.9 and 3.7 F 1.6 cells with MN-3 IgG and FabV, respectively, and to 5.5 F 1.8 and 5.4 F 0.8 with MN-15 IgG and FabV, respectively (P < 0.01 for all comparisons of mAb treatment with untreated cells; n = 10 for each arm; Fig. 2). In LS174T studies, untreated cells had 38.8 F 10.9 migrating cells per field, whereas cells treated with MN-3 IgG or FabVhad 21.4 F 5.2 and 18.1 F 1.6 migrating cells, respectively (P < 0.001 versus untreated), and cells treated with MN-15 IgG or FabV had 20.3 F 0.1 and 21.6 F 1.2 migrating cells, respectively (P < 0.001 versus untreated). Thus, blocking either the N or A1B1 domain of CEACAM6 with either an intact IgG or a monovalent Fab inhibited migration in vitro. Effect of MN-15 and MN-3 on tumor cell adhesion to endothelial cells. Because both CEACAM5 and CEACAM6 are known to have an adhesion role, we evaluated whether blocking these antigens reduces adhesion to endothelial cells. In the Figure 2. In vitro migration assay. Top, visual presentation of HT-29 CEACAM5À/CEACAM6À MCA38 murine colonic cancer line, (CEACAM5À/CEACAM6+) cell migration (arrows) when cells were left untreated neither MN-3 FabV targeting the N domain nor MN-15 FabV (A) or treated with 10 Ag/mL of MN-3 IgG (B). Bottom, migration of HT-29 cells (18 hours) and LS174T (CEACAM5+/CEACAM6+) cells (24 hours) in the targeting the A1B1 domain of CEACAM6 had an effect on tumor absence or presence of 10 Ag/mL of intact antibody IgG or antibody FabV. cell-endothelial cell adhesion (Fig. 3). In contrast, both antibodies Anti-a-fetoprotein IgG was used as a nonspecific antibody in these studies. reduced endothelial cell binding of MCA38cea (a human CEA- The average of 10 fields was recorded in two to four replicate studies for each cell line. MN-3 and MN-15 IgG and FabVreduced migration of HT-29 cells transfected line) from 11.68 F 0.77% to 6.42 F 2.1% (MN-3) and (P < 0.01) and LS174T cells (P < 0.001). 5.53 F 1.15% (MN-15), being significant (P < 0.05) for both Fabs. Both antibodies induced a 49% to 58% adhesion-inhibition in four prepare a 10% cell suspension. Cells (30 AL) were injected i.v. into the caudal other cell lines (P < 0.01 for MN-3 FabV on MCF-7, HT-29, and vein of 5- to 6-week-old female nude mice. This results in f50 to 100 tumor BT-20; P < 0.02 for MN-15 on the same three lines; and P < 0.05 for nodules developing in the lungs and resulting in a median survival of 7 to 9 both Fabs on Moser cell adhesion to endothelial cells). An isotype- weeks (40, 41). Cells were pretreated with antibody in vitro (10 Ag/mL) matched irrelevant antibody FabV (Ag8) did not affect tumor before implantation, and then mice were given one additional dose (100 Ag cell-endothelial cell adhesion nor did the MN-14 anti-CEA FabV. per mouse i.v.) 1 day after implantation. Body weight was monitored weekly The magnitude of the antiadhesion effect did not seem strictly and animal survival recorded. Results were analyzed by the Kaplan-Meier correlated with the amount of CEACAM5 or CEACAM6 expressed, estimated survival curves, and significance was determined with the log- because MN-15 FabVresulted in a greater decrease in adhesion in rank comparison of survival curves. Median survival time for each treatment group also was determined. All studies used 10 mice per HT-29 cells (48%) than with MCF-7 cells (41%) that express much treatment group. Animal studies were done under protocols approved by more CEACAM6. the Institutional Animal Care and Use Committee. Effect of anti-CEACAM5 and anti-CEACAM6 antibodies on tumor cell adhesion to extracellular matrix proteins. In addition to tumor cell binding to endothelial cells, these cells can Results also bind ECM proteins. The extent of tumor cell binding to ECM CEACAM5 and CEACAM6 expression in cell lines. Flow proteins varies among different cell lines. MCF-7 bound well to four F cytometric analysis of CEACAM5 and CEACAM6 expression in a of five proteins (A560 nm > 1.1) except laminin (A560 nm = 0.2 0.04), panel of 31 to 33 commonly used solid tumor cell lines revealed whereas ZR75-30 attached weakly to all five ECM proteins that only 6 of 29 (20.7%) expressed significant amounts of evaluated (A560 nm < 0.45). MDA-468 bound quite well to F F CEACAM5, whereas 16 of 30 (53.3%) lines were positive for collagen-I and collagen-IV (A560 nm = 1.25 0.07 and 0.97 CEACAM6 (Table 1). Two cancer cell lines were CEACAM5+/ 0.03, respectively) but not as well to fibronectin, vitronectin, or

Cancer Res 2005; 65: (19). October 1, 2005 8812 www.aacrjournals.org

F laminin. The reverse pattern was seen with CaPAN-1 (A560 nm =1.78 a continuous exposure to antibody that would be available at any 0.21 for fibronectin, 0.88 F 0.11 for vitronectin, 1.14 F 0.09 for time that a cell from a primary tumor might initiate the metastatic laminin, 0.07 F 0.00 for collagen-I, and 0.13 F 0.08 for collagen-IV; cascade. The results presented in Fig. 6 illustrate that both MN-15 Fig. 4). None of the antibodies (IgG or FabV) modulated adhesion to and MN-3 Fabs increased median survival time (>77 and 77 days, vitronectin, laminin, collagen-I, or collagen-IV (results not shown). respectively; P < 0.001 versus untreated cells) of these mice, However, MN-15 IgG decreased adhesion of MCF-7, MDA-468, and whereas hMN-14 FabV(49 days) did not affect survival significantly CaPAN-1 to fibronectin by 29% (P < 0.01), 51% (P < 0.001), and 47% (42 days for untreated mice). Although the study was continued (P < 0.02), respectively, while not affecting the binding of ZR75-30, until mice were near death, rather than sacrificing them at a BXPC3, or Moser to fibronectin (Table 2). Two of these three mAb- defined time after treatment and counting the number of lung unresponsive cell lines had the lowest baseline adhesion to nodules, median survival time should correlate with amount of fibronectin. lung metastatic disease. The results with hMN-14 FabVin this study Effect of MN-3, MN-15, and MN-14 Fabs on tumor cell are similar to what we reported recently for hMN-14 F(ab)2 in invasion. Specific invasion in response to FBS as a chemo- this metastatic model (16), indicating that targeting the A3B3 attractant was 1.9-fold (LS174T) to 7.4-fold (BXPC3) higher than in domain does not affect median survival in this model, whereas the absence of FBS (Fig. 5). MN-15 FabV was more effective than targeting the N and A1B1 domains shared by CEACAM5 and MN-3 FabVat reducing tumor cell invasion in vitro in five cell lines CEACAM6 do affect metastasis and host survival when the that expressed CEACAM5, CEACAM6, or both antigens. MN-14 respective Fabs are given. anti-CEA (CEACAM5) had no effect on tumor cell invasion in most cell lines. MDA-231 expresses neither antigen, and its invasion was not reduced by either MN-3 or MN-15 IgG or FabV. For the five Discussion antigen-positive lines, both the intact IgG and the monovalent FabV A long-term goal of immunotherapy has been to induce for a given antibody were equally effective. For example, MN-15 antitumor responses against tumor-related antigens. Promising FabVreduced cell invasion of LS174T, MCF-7, ZR75-30, BXPC3, and results of several naked mAbs have been reported for solid tumors CaPAN-1 cells by 30% (P < 0.02), 77% (P < 0.01), 49% (P < 0.01), 44% and for lymphoma (43–46). Antibodies that directly perturb (P < 0.01), and 73% (P < 0.002), respectively. The effect of MN-3 FabV signaling mechanisms have shown clinical benefit, such as those on the same five lines was a reduction in invasion of 3% (P = NS), directed against the extracellular domains of HER-2/neu, epidermal 47% (P < 0.01), 59% (P < 0.01), 0% (P = NS), and 55% (P < 0.05), growth factor receptor, and CD20 (47, 48). The studies in this article respectively. Thus, the A1B1 domain of CEACAM6 seems to be a suggest that instead of being directly therapeutic by immune more important target than the N domain for the process of tumor effector or direct cytostatic mechanisms, mAbs against CEACAM6 cell invasion. inhibit migration, invasion, and adhesion, thereby limiting metas- Effect of MN-3, MN-15, and MN-14 FabVon survival of mice tasis. The monovalent FabVform was used for most of our studies to bearing GW-39 intrapulmonary colonic micrometastases. GW- avoid effector cell function from the Fc region of the mAb and focus 39 expresses both CEACAM5 and CEACAM6, as shown by on mechanisms implicated in the metastatic process. immunohistochemistry (Fig. 6, top). Based on the in vitro results CEACAM5 is overexpressed in a majority of carcinomas, suggesting that anti-CEACAM6 antibodies had limited antiprolifer- including those of the gastrointestinal, respiratory and genitouri- ative effects yet showed significant anti-invasive and antiadhesive nary systems, and the breast (2–6). Our results show that another properties, we pretreated GW-39 tumor cell suspensions with each CEA family member, CEACAM6, may be as good, if not better, as a of the antibody fragments (10 Ag/mL) for 15 minutes before i.v. target for solid tumor antimetastatic therapy. We have shown that injection of 30 AL of cells and then dosed with 100 Ag of antibody CEACAM6 is expressed in a larger percentage of solid tumor cell on the day after transplantation. This design simulates the effect of lines than CEACAM5, and the high MCF on many of these lines

Figure 3. In vitro endothelial cell adhesion assay. Percentage adhesion of various tumor cells with varying amounts of expressed CEACAM5 and CEACAM6 to HUVEC cells in the absence or presence of MN-15 FabV, MN-3 FabV, or Ag8 FabV control. Cells were labeled with 1 ACi/mL of 3H-thymidine and added to HUVEC cultures and incubated for 30 minutes at 37jC. Samples were washed thrice with PBS to remove unattached cells. Attached tumor cells were solubilized with 0.1 N NaOH and radioactivity was measured in a h-scintillation counter. The cpm attached/total cpm added (attaching potential) was determined. Results of a typical study are presented. Cell lines used include BT-20 (CEACAM5+/CEACAM6+), MCF-7 (CEACAM5À/CEACAM6+), HT-29 (CEACAM5À/CEACAM6+), Moser (CEACAM5+/CEACAM6À), MCA38cea (CEACAM5+/CEACAM6À), and MCA38 (CEACAM5À/CEACAM6À). Both MN-15 and MN-3 induced a 49% to 58% inhibition in adhesion in four cell lines (P < 0.01 for MN-3 FabVon MCF-7, HT-29, and BT-20; P < 0.02 for MN-15 on the same three lines; and P < 0.05 for both Fabs on Moser adhesion to endothelial cells).

www.aacrjournals.org 8813 Cancer Res 2005; 65: (19). October 1, 2005

Figure 4. In vitro ECM adhesion assay. Percentage of adhesion of a panel of tumor cell lines with varying expression of CEACAM5 and CEACAM6 to ECM proteins: fibronectin, vitronectin, laminin, collagen-I, and collagen-IV proteins using the Cytomatrix screening kit from Chemicon. Attached cells were stained with MTS and attachment was determined using absorbance readings at A 540 nm and A 570 nm. Cell lines used for these studies include MCF-7 (CEACAM5À/CEACAM6+), MDA468 (CEACAM5+/À/CEACAM6+), ZR75-30 (CEACAM5À/CEACAM6++), CaPAN-1 (CEACAM5+/CEACAM6++), BXPC3 (CEACAM5À/CEACAM6++), MOSER (CEACAM5+/CEACAM6À), MDA-231 (CEACAM5À/CEACAM6À), and LS174T (CEACAM5+/CEACAM6++). MN-15 IgG decreased adhesion of MCF-7, MDA-468, and CaPAN-1 to fibronectin by 29% (P < 0.01), 51% (P < 0.001), and 47% (P < 0.02), respectively, while not affecting the binding of ZR75-30, BXPC3, or Moser to fibronectin. suggests that more anti-CEACAM6 mAb can be delivered to these thelial cells (50) via activation of Kupffer cells and stimulation tumor cells. Thus, the MN-15 and MN-3 mAbs are useful for tumors of IL-1h, tumor necrosis factor a, and IL-6 production. These that express CEACAM6 or CEACAM5, because they target epitopes cytokines then induce the expression of intercellular adhesion that are shared by both antigens. These mAbs may therefore have molecules on endothelial cells, thus increasing adhesion of advantages over mAbs like MN-14, which only target CEACAM5, or tumor cells to endothelium. Our results have shown that MN-3 mAbs used by others (49) that are specific for only CEACAM6 and and MN-15 Fabs are more active than MN-14 FabV at reducing do not cross-react with CEACAM5. Our data are consistent with adhesion of CEACAM5+/CEACAM6+ tumor cells to endothelial previous reports that showed a relatively high level of CEACAM6 in cells. Thus, both the N and the A1B1 domains but not the A3B3 the sera of a large number of lung, liver, pancreatic, breast, and domain are involved in tumor cell to endothelial cell adhesion. colorectal cancer patients (21). Interestingly, some patients were Tumor cells can adhere to other tumor cells, to endothelial CEACAM5À/CEACAM6+, further suggesting that CEACAM6 might cells, as well as to ECM proteins. We have found that the be a useful independent target. amount of adhesion to a panel of these proteins varied between It is known that CEACAM6 is expressed on the surface of cell lines and was not related to the type of tumor (e.g., breast neutrophils, thus modulating adherence to endothelial-leukocyte and pancreatic) or to the amount of CEACAM5 or CEACAM6 adhesion molecule-1 on activated endothelial cells (24). We have expressed. Tumor cell interactions with ECM proteins are shown that mAbs to different epitopes on CEACAM6 (N and important for migration and invasion and therefore metastasis. A1B1 domains) affect cell adhesion with endothelial cells. There For example, tumor cell interactions with fibronectin are is also evidence that CEACAM5 can affect cell adhesion to endo- involved in the development of secondary tumors inside the

Table 2. Adhesion to fibronectin

Cell line No antibody MN-15 IgG (P) MN-15 FabV(P) MN-3 IgG MN-3 FabV MN-14 IgG MN-14 FabV Ag8 IgG Ag8 FabV

MCF-7 1.18 F 0.09* 0.84 F 0.04 (<0.01) 0.83 F 0.00 (<0.02) 1.05 F 0.04 1.05 F 0.00 1.08 F 0.21 1.14 F 0.02 1.28 F 0.01 0.91 F 0.01 MDA-468 0.51 F 0.00 0.25 F 0.02 (<0.001) 0.27 F 0.00 (<0.01) 0.43 F 0.09 0.44 F 0.01 0.32 F 0.01 0.40 F 0.02 0.47 F 0.00 0.49 F 0.12 CaPan-1 1.78 F 0.21 0.95 F 0.26 (<0.02) 1.38 F 0.04 (<0.05) 1.76 F 0.01 1.56 F 0.05 1.69 F 0.00 1.73 F 0.09 1.56 F 0.18 1.81 F 0.13 ZR75-30 0.25 F 0.05 0.18 F 0.03 0.25 F 0.06 0.18 F 0.05 0.17 F 0.01 0.23 F 0.04 0.18 F 0.04 0.21 F 0.04 0.15 F 0.06 BXPC3 0.73 F 0.05 0.74 F 0.05 0.74 F 0.02 0.78 F 0.03 0.70 F 0.03 0.57 F 0.08 0.70 F 0.04 0.72 F 0.09 0.74 F 0.02 MOSER 0.42 F 0.06 0.52 F 0.07 0.50 F 0.02 0.50 F 0.03 0.41 F 0.01 0.51 F 0.01 0.45 F 0.02 0.36 F 0.02 0.41 F 0.02

NOTE: Relative attachment (mean F SD) recorded for triplicate samples. *Cell lines used for these studies include MCF-7 (CEACAM5À/CEACAM6+), MDA468 (CEACAM5+/À/CEACAM6+), CaPAN-1 (CEACAM5+/ CEACAM6++), ZR75-30 (CEACAM5À/CEACAM6++), BXPC3 (CEACAM5À/CEACAM6++), and MOSER (CEACAM5+/CEACAM6À).

Cancer Res 2005; 65: (19). October 1, 2005 8814 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Anti-CEACAM6 Antibodies Inhibit Metastasis bone marrow stroma via the a5h1 integrin (51). Blocking CAM6, with MN-3 > MN-15, decreases the number of migrating cells. adhesion with polypeptide fragments of heparin-binding domains In our in vitro assay, the process of cell invasion, which involves of fibronectin inhibited metastasis (52). Similar results have been adhesion to ECM and migration steps, was inhibited by MN-15 FabV> obtained with a peptide blocking tumor cell-laminin adhesion MN-3 FabV, suggesting that the A1B1 domain of CEACAM6 is more (53). Interestingly, only MN-15 (A1B1 domain) was able to reduce important for this step but that the N domain also plays a role. adhesion to fibronectin in three of six tumor cell lines tested. One of the notable advantages of MN-15 or MN-3 mAb therapy, The percent inhibition in fibronectin adhesion in this panel of compared with our previously reported results with MN-14 anti- cell lines did not correlate with the amount of adhesion in CEA IgG (16), is the ability to target tumors that express either untreated cell samples. mAbs targeting the N or A3B3 domain CEACAM6, CEACAM5, or both, whereas MN-14 can only be used did not affect cell adhesion to fibronectin. In one report by for CEACAM5+ tumors. As shown in Table 1, many solid tumor Duxbury (49), mAb-mediated CEACAM6 cross-linking resulted in lines express CEACAM6 but not CEACAM5 or express more increased ECM adhesion. However, the targeted epitope was CEACAM6 than CEACAM5. These tumor types are candidates for different from the ones studied here. metastasis-directed mAb therapy with CEACAM6 mAbs. Active migration of tumor cells is a prerequisite of tumor cell An important consideration based on the in vivo experiments is invasion and metastasis. Adhesion molecules that increase invasion the availability of mAb when cells first enter the circulation. MN-15 also enhance the migratory process (38). Overexpression of FabV and MN-3 FabV showed therapeutic efficacy if cells were CEACAM6 has been reported to promote cellular invasiveness of exposed to mAb before the initiation of the metastatic process. pancreatic cancer (29). Agents that inhibit metastases often affect However, if mAb was delivered after cancer cells had exited the several steps including migration, adhesion, and invasion (39, 54–56). vasculature and had begun to seed in the lung, mAbs alone were Because our data also suggest that CEACAM6 has a role in adhesion not therapeutic (data not shown). Therefore, to be clinically and invasion, it is important to also assess the ability of these mAbs applicable, anti-CEACAM5/CEACAM6 mAbs would need to be to impede migration. We have shown that in cell lines with strong available continuously, perhaps using implantable pumps, to migratory tendencies, mAb blocking of CEACAM5 and/or CEA- maintain a desired level in the circulation at all times.

Figure 5. In vitro invasion assay. Percentage of invasion of a panel of human tumor cell lines with varying amounts of CEACAM5 and CEACAM6 through a polycarbonate membrane coated with ECM proteins over a 72-hour incubation period (Chemicon kit). Invasion in the absence of antibody FabVor in the presence of MN-3, MN-15, or nonspecific Ag8 FabV(10 Ag/mL) was recorded. FBS in the lower chamber served as the chemoattractant for invasion. Reduced invasion was noted in samples that did not have FBS. Cell lines used for these studies include LS174T (CEACAM5+/CEACAM6++), MCF-7 (CEACAM5À/CEACAM6+), ZR75-30 (CEACAM5À/CEACAM6++), BXPC3 (CEACAM5À/CEACAM6++), CaPAN-1 (CEACAM5+/CEACAM6++), and MDA-231 (CEACAM5À/CEACAM6À). MN-15 FabV reduced cell invasion of LS174T, MCF-7, ZR75-30, BXPC3, and CaPAN-1 cells by 30% (P < 0.02), 77% (P < 0.01), 49% (P < 0.01), 44% (P < 0.01), and 73% (P < 0.002), respectively. The effect of MN-3 FabVon the same five lines was a reduction in invasion of 3% (P = NS), 47% (P < 0.01), 59% (P < 0.01), 0% (P = NS), and 55% (P < 0.05), respectively.

www.aacrjournals.org 8815 Cancer Res 2005; 65: (19). October 1, 2005

Figure 6. In vivo micrometastasis study. Top, immunohistochemistry of GW-39 tumor sections stained with a nonspecific antibody (A), MN-14 anti-CEACAM5 (B), and MN-15 anti-CEACAM6 (C) and photographed at 100Â. Bottom, survival of nude mice implanted with 30 AL of a 10% suspension of GW-39 human colonic cancer cells. Mice were implanted with cells that were preincubated for 30 minutes with MN-3-, MN-15-, or hMN-14-FabV(10 Ag/mL). Mice also received a single 100 Ag dose of the same FabV1 day after cell implantation.

Overall, the antimetastasis and mAb inhibition of adhesion, and A1B1 domains of these antigens block migration, adhesion to invasion, and migration is a technology that should be relatively endothelial cells and ECM, and invasion, and also increase the nontoxic, not limited by issues of drug resistance, and easy to apply median survival of mice with intrapulmonary micrometastases of as an adjuvant with other standard and/or experimental therapy human colonic cancer. approaches. Because CEACAM6 is also expressed in normal lung, spleen, and granulocytes (57), the effect of anti-CEACAM6 mAb on normal tissues remains to be determined. In one report, CEACAM6- Acknowledgments targeted immunotoxin therapy was effective in a tumor-bearing Received 2/7/2005; revised 6/4/2005; accepted 7/18/2005. nude mouse model (58), but this model does not express CEACAM6 Grant support: NIH/USPHS grant R01 99529, New Jersey Commission on Cancer on normal tissues and can therefore not reliably assess whether Research grant 1670, and Immunomedics, Inc. The costs of publication of this article were defrayed in part by the payment of page mAb-toxin conjugates targeting CEACAM6 will be tolerated in charges. This article must therefore be hereby marked advertisement in accordance humans. with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Evelyn Leone, Rosana Michel, Louis Osorio, and Marisol Rodriguez for In summary, we have shown that anti-CEACAM6/CEACAM5 excellent technical assistance and Li Zeng at Immunomedics for purification of intact mAb fragments devoid of effector cell functions and targeting the N and monovalent antibody fragments.

Cancer Res 2005; 65: (19). October 1, 2005 8816 www.aacrjournals.org

References differentially expressed in normal tissues and oppositely metastasis: I. In vitro studies of adhesion, migration deregulated in hyperplastic colorectal polyps and early and invasion of MDA-MB231 human breast cancer cells. 1. Fidler IJ. Critical factors in the biology of human adenomas. Am J Pathol 2000;157:1051–2. Anticancer Res 2003;23:3671–9. cancer metastasis: twenty- eighth G.H.A. Clowes memo- 20. Hinoda Y, Saito T, Takahashi H, Itoh F, Adachi M, 40. Sharkey RM, Weadock KS, Natale A, et al. Successful rial award lecture. Cancer Res 1990;50:6130–8. Imai K. Induction of nonspecific cross-reacting antigen radioimmunotherapy for lung metastasis of human 2. Goldenberg DM, Sharkey RM, Primus FJ. Carcinoem- mRNA by interferon-g and anti-fibronectin receptor colonic cancer in nude mice. J Natl Cancer Inst 1991; bryonic antigen in histopathology: immunoperoxidase antibody in colon cancer cells. J Gastroenterol 1997; 83:627–32. staining of conventional tissue sections. J Natl Cancer 32:200–5. 41. Blumenthal RD, Sharkey RM, Haywood L, et al. Inst 1976;57:11–22. 21. Kuroki M, Matsushita H, Matsumoto H, Hirose Y, Targeted therapy of athymic mice bearing GW-39 human 3. Shively JE, Beatty JD. CEA-related antigens: molecular Senba T, Ymamoto T. Nonspecific cross-reacting antigen colonic cancer micrometastases with 131I-labeled biology and clinical significance. Crit Rev Oncol 50/90 (NCA-50/90) as a new tumor marker. Anticancer monoclonal antibodies. Cancer Res 1992;52:6036–44. Hematol 1985;2:355–99. Res 1999;19:5599–606. 42. Goldenberg D, Witte S, Elster K. GW-39: a new 4. Thompson JA, Grunert F, Zimmermann W. Carci- 22. Ilantzis C, DeMarte L, Screaton RA, Stanners CP. human tumor serially transplantable in the golden noembryonic antigen gene family: molecular biology Deregulated expression of the human marker CEA and hamster. Transplantation 1966;4:760–4. and clinical perspectives. J Clin Lab Anal 1991;5:344–66. CEA family member CEACAM6 disrupts tissue archi- 43. Borden E, Esserman L, Linder D, Campbell M, 5. Gold P, Goldenberg NA. The carcinoembryonic antigen tecture and blocks coloncyte differentiation. Neoplasia Fulton A. Biological therapies for breast carcinoma: (CEA): past present, and future. McGill J Med 1997; 2002;4:151–63. concepts for improvement in survival. Semin Oncol 3:46–66. 23. Jantscheff P, Terracciano L, Lowy A, et al. Expression 1999;26:28–40. 6. Hammarstrom S. The carcinoembryonic antigen of CEACAM6 in resectable colorectal cancer: a factor of 44. Sliwkowski M, Lofgren J, Lewis G, Hotaling T, Fendly (CEA) family: structures, suggested functions and independent prognostic significance. J Clin Oncol 2003; B, Fox J. Nonclinical studies addressing the mechanism expression in normal and malignant tissues. Semin 21:3638–46. of action of trastuzumab (Herceptin). Semin Oncol 1999; Cancer Biol 1999;9:67–81. 24. Kuijpers TW, Hoogerwerf M, van der Laan lJ, et al. 26:60–70. 7. Jessup JM, Thomas P. CEA and metastasis: a facilitator CD66 nonspecific cross-reacting antigens are involved 45. Bodey B, Siegel S, Kaiser H. Human cancer detection of site-specific metastasis. In: Stanners CP, editor. Cell in neutrophil adherence to cytokine-activated endothe- and immunotherapy with conjugated and non-conju- adhesion and communication by the CEA family. Vol. 5. lial cells. J Cell Biol 1992;118:457–66. gated monoclonal antibodies. Anticancer Res 1996; Amsterdam: Harwood Academic Publishers; 1998. p. 25. Yamanka T, Kuroki M, Matsuo Y, Matsuoka Y. 16:661–74. 195–222. Analysis of heterophilic cell adhesion mediated by 46. Riethmuller G, Schneider-Gadicke E, Schlimok G, 8. Yoshioka T, Masuko T, Kotanagi H, et al. Homotypic CD66b and CD66c using their soluble recombinant pro- et al. The German Cancer Aid 17–1A Study Group. adhesion through carcinoembryonic antigen plays a role teins. Biochem Biophys Res Commun 1996;219:842–7. Randomized trial of monoclonal antibody for adjuvant in hepatic metastasis development. Jpn J Cancer Res 26. Frisch SM, Francis H. Disruption of epithelial cell- therapy of resected Dukes’ C colorectal carcinoma. 1998;89:177–85. matrix interactions induces apoptosis. J Cell Biol 1994; Lancet 1994;343:1177–83. 9. Hashino J, Fukuda Y, Oikawa S, Nakazato H, Nakanishi 124:616–26. 47. Weiner L, Adams G. New approaches to antibody T. Metastatic potential of human colorectal carcinoma 27. Ordonez C, Screaton RA, Ilantzis C, Stanners CP. therapy. Oncogene 2000;19:6144–51. SW1222 cells transfected with cDNA encoding carcino- Human carcinoembryonic antigen functions as a 48. Alas S, Bonavida B. Rituximab inactivates signal embryonic antigen. Clin Exp Metastasis 1994;12:324–8. general inhibitor of anoikis. Cancer Res 2000;60:3419–24. transducer and activation of transcription 3 (STAT3) 10. Kim JC, Koo KH, Kim BS, Park KC, Bicknell DC, 28. Duxbury MS, Ito H, Zinner MJ, Ashley SW, Whang EE. activity in B-non-Hodgkin’s lymphoma through inhi- Bodmer WF. Carcino-embryonic antigen may function CEACAM6 gene silencing impairs anoikis resistance and bition of the interleukin 10 autocrine/paracrine loop as a chemoattractant in colorectal carcinoma cell lines. in vivo metastatic ability of pancreatic adenocarcinoma and results in down regulation of bcl-2 and Int J Cancer 1999;82:880–5. cells. Oncogene 2004;23:465–73. sensitization to cytotoxic drugs. Cancer Res 2001;61: 11. Minami S, Furui J, Kanematsu T. Role of carcinoem- 29. Duxbury MS, Ito H, Benoit E, Zinner MJ, Ashley SW, 5137–44. bryonic antigen in the progression of colon cancer cells Whang EE. Overexpression of CEACAM6 promotes 49. Duxbury MS, Ito H, Ashley SW, Whang EE. CEACAM6 that express carbohydrate antigen. Cancer Res 2001; insulin-like growth factor I-induced pancreatic adeno- cross-linking induces caveolin-1-dependent, Src-mediat- 61:2732–5. carcinoma cellular invasiveness. Oncogene 2004. ed focal adhesion kinase phosphorylation in BxPC3 12. Benchimol S, Fuks A, Jothy S, Beauchemin N, Shirota 30. Duxbury MS, Ito H, Benoit E, Waseem T, Ashley SW, pancreatic adenocarcinoma cells. J Biol Chem 2004; K, Stanners CP. Carcinoembryonic antigen, a human Whang EE. A novel role for carcinoembryonic antigen- 279:23176–82. tumor marker, functions as a intercellular adhesion related cell adhesion molecule 6 as a determinant of 50. Gangopadhyay A, Lazerus DA, Thomas P. Adhesion molecule. Cell 1989;57:327–34. gemcitabine chemoresistance in pancreatic adenocarci- of colorectal carcinoma cells to the nedothelium is 13. Oikawa S, Inuzuka C, Kuroki M, Matsuoka Y, Kosaki noma cells. Cancer Res 2004;64:3987–93. mediated by cytokines from CEA stimulated Kupffer G, Nakazato H. Cell adhesion activity of non-specific 31. Duxbury MS, Ito H, Benoit E, Ashley SW, Whang EE. cells. Clin Exp Metastasis 1998;16:703–12. cross-reacting antigen (NCA) and carcinoembryonic CEACAM6 is a determinant of pancreatic adenocarci- 51. Van der Velde-Zimmermann D, Verdaasdonk MA, antigen (CEA) expressed on CHO cell surface: homo- noma cellular invasiveness. Br J Cancer 2004;91:1384–90. Rademakers LH, De Weger RA, Van den Tweel JG, Joling philic and heterophilic adhesion. Biochem Biophys Res 32. Audette M, Buchegger F, Schreyer M, Mach JP. P. Fibronectin distribution in human bone marrow Commun 1989;164:39–45. Monoclonal antibody against carcinoembryonic antigen stroma: matrix assembly and tumor cell adhesion via a5 14. Stanners CP, Fuks A. Properties of adhesion mediated (CEA) identifies two new forms of crossreacting h1 integrin. Exp Cell Res 1997;230:111–20. by the human CEA family. In: Stanners CP, editor. antigens of molecular weight 90,000 and 160,000 in 52. Matsumoto Y, Saiki I, Makabe T, et al. Inhibitory Cell adhesion and communication by the CEA family. normal granulocytes. Mol Immunol 1987;24:1177–86. effect of antimetastatic fusion polypeptide of human Vol. 5. Amsterdam: Harwood Academic Publishers; 1998. 33. Bjerner J, Lebedin Y, Bellanger L, et al. Protein fibronectin on tumor cell adhesion to extracellular p. 57–72. epitopes in carcinoembryonic antigen. Tumour Biol matrices. Jpn J Cancer Res 1991;82:1130–8. 15. Wirth T, Soeth E, Czubayko F, Juhl H. Inhibition of 2002;23:249–62. 53. Islam SM, Whalen GF, Sharif SF. Inhibition of tumor endogenous carcinoembryonic antigen (CEA) increases 34. Taheri M, Saragovi U, Fuks A, Makkerh J, Mort J, cell adhesion to lymph nodes by laminin-related peptide the apoptotic rate of colon cancer cells and inhibits Stanners CP. Self recognition in the Ig superfamily. J Biol and neuraminidase. Surgery 1993;113:676–82. metastatic tumor growth. Clin Exp Metastasis 2002; Chem 2000;275:26935–43. 54. Chen J, Thompson LU. Lignans and tamoxifen, alone 19:155–60. 35. Taheri M, Saragovi HU, Stanners CP. The adhesive or in combinations, reduce human breast cancer cell 16. Blumenthal RD, Osorio L, Hayes MK, Horak ID, and differentiation-inhibitory activities of the immuno- adhesion, invasion and migration in vitro. Breast Cancer Hansen HJ, Goldenberg DM. Carcinoembryonic antigen globulin superfamily member, carcinoembryonic anti- Res Treat 2003;80:163–70. antibody inhibits metastasis and augments chemother- gen, can be independently blocked. J Biol Chem 2003; 55. Zeng G, Gao L, Yu RK. Reduced cell migration, tumor apy in a human colonic carcinoma xenograft. Cancer 278:14632–9. growth and experimental metastasis of rat F-11 cells Immuno Immunother 2005;54:315–27. 36. Jessup JM, Kim JC, Thomas P, et al. Adhesion to whose expression of GD3-synthase is suppressed. Int J 17. Stein R, Goldenberg DM. A humanized monoclonal carcinoembryonic antigen by human colorectal carci- Cancer 2000;88:53–7. antibody to carcinoembryonic antigen, labetuzumab, noma cells involves at least two epitopes. Int J Cancer 56. Jadeski LC, Hum KO, Chakraborty C, Lala PK. Nitric inhibits tumor growth and sensitizes human medullary 1993;55:262–8. oxide promotes murine mammary tumour growth and thyroid cancer xenografts to decarbazine chemotherapy. 37. Sharkey RM, Juweid M, Shevitz J, et al. Evaluation of a metastasis by stimulating tumour cell migration, Mol Cancer Ther 2005;3:1559–64. complementarity-determining region-grafted (human- invasiveness and angiogenesis. Int J Cancer 2000; 18. Kuroki M, Matsuo Y, Kinugasa T, Matsuoka Y. Three ized) anti-carcinoembryonic antigen monoclonal anti- 86:30–9. different NCA species, CGM6/CD67, NCA-95, and NCA- body in preclinical and clinical studies. Cancer Res 1995; 57. Grunert F, Daniel S, Nagel G, von Kleist S, Jantscheff 90 are comprised in the major 90 to 100 kDa band of 55:5935–45. P. CD66b, CD66c and carcinoembryonic antigen (CEA) granulocyte NCA detectable upon SDS-polyacrylamide 38. Hazan RB, Phillips GR, Qiao RF, Norton L, Aaronson are independently regulated markers in sera of tumor gel electrophoresis. Biochem Biophys Res Commun SA. Expgenous expression of N-cadherin in breast patients. Int J Cancer 1995;63:349–55. 1992;182:501–6. cancer cells induces cell migration, invasion, and 58. Duxbury MS, Ito H, Ashley SW, Whang EE. CEACAM6 19. Scholzel S, Zimmermann W, Schwarzkopf G, Grunert metastasis. J Cell Biol 2000;148:779–90. as a novel target for indirect type 1 immunotoxin-based F, Rogaczewski B, Thompson J. Carcinoembryonic 39. Tantivejkul K, Vucenik I, Shamsuddin AM. Inositol therapy in pancreatic adenocarcinoma. Biochem Bio- antigen family members CEACAM6 and CEACAM7 are hexaphosphate (IP6) inhibits key events of cancer phys Res Commun 2004;317:837–43.

www.aacrjournals.org 8817 Cancer Res 2005; 65: (19). October 1, 2005

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Inhibition of Adhesion, Invasion, and Metastasis by Antibodies Targeting CEACAM6 (NCA-90) and CEACAM5 (Carcinoembryonic Antigen)

Rosalyn D. Blumenthal, Hans J. Hansen and David M. Goldenberg

Cancer Res 2005;65:8809-8817.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/65/19/8809

Cited articles This article cites 51 articles, 12 of which you can access for free at: http://cancerres.aacrjournals.org/content/65/19/8809.full#ref-list-1

Citing articles This article has been cited by 12 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/65/19/8809.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/65/19/8809. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.