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Monoclonal Antibodies Directed against CadherinRGD Exhibit Therapeutic Activity againstMelanoma and Colorectal Cancer MetastasisRub�en A. Bartolom�e1, Carmen Aizpurua2, Marta Ja�en1, Sofía Torres1, Eva Calvi~no1,Juan I. Imbaud2, and J. Ignacio Casal1

Abstract

Purpose: New targets are required for the control of advancedmetastatic disease. We investigated the use of cadherin RGDmotifs, which activate the a2b1integrin pathway, as targets forthe development of therapeutic monoclonal antibodies (mAb).

Experimental Design: Cadherin 17 (CDH17) fragments andpeptides were prepared and used for immunization and antibodydevelopment. Antibodies were tested for inhibition of b1 integrinand cell adhesion, proliferation, and invasion assays using celllines from different cancer types (colorectal, pancreatic, melanoma,and breast cancer). Effects of the mAbs on cell signaling weredetermined by Western blot analysis. Nude mice were used forsurvival analysis after treatmentwith RGD-specificmAbs andmetas-tasis development.

Results: Antibodies against full-length CDH17 failed to blockthe binding to a2b1 integrin. However, CDH17 RGD peptidesgenerated highly selective RGD mAbs that blocked CDH17 and

vascular-endothelial (VE)-cadherin–mediated b1 integrin activa-tion in melanoma and breast, pancreatic, and colorectal cancercells. Antibodies provoked a significant reduction in cell adhesionand proliferation of metastatic cancer cells. Treatment with mAbsimpaired the integrin signaling pathway activation of FAK incolorectal cancer, of JNK and ERK kinases in colorectal andpancreatic cancers, and of JNK, ERK, Src, and AKT in melanomaand breast cancer. In vivo, RGD-specific mAbs increased mousesurvival after inoculation of melanoma and colorectal cancer celllines to cause lung and liver metastasis, respectively.

Conclusions: Blocking the interaction between RGD cadher-ins and a2b1 integrin with highly selective mAbs constitutes apromising therapy against advanced metastatic disease in coloncancer, melanoma, and, potentially, other cancers. Clin CancerRes; 24(2); 433–44. �2017 AACR.

See related commentary by Marshall, p. 253

IntroductionThere is a necessity to find new therapeutic targets to control

metastatic spread in cancer. Therapeutic monoclonal antibo-dies (mAb) against EGFR or VEGF(R) are in clinical use for thetreatment of advanced metastatic colorectal cancer. However,their impact on the final outcome of patients with metastasis isstill limited (1), probably due to the difficulty of assessing theEGFR status of the patients (2, 3), adverse effects related to thewide distribution of these molecules in healthy tissues, and thelack of response in patients with KRAS mutations (4).

Cadherin 17 (CDH17), also known as liver–intestine (LI)-cadherin, is present in fetal liver and the gastrointestinal tract,exhibiting elevated expression during embryogenesis (5).CDH17 localizes to the basolateral domain of hepatocytes andenterocytes, where it mediates intercellular adhesion in a Ca(2þ)-dependent manner to maintain tissue integrity in epithelia (6).

In colon cancer, CDH17 is expressed at low levels in primarytumors or in regional lymph node metastases, as well as inpoorly differentiated colon cancer tumors (7). However,CDH17 is overexpressed in advanced colorectal cancer livermetastasis (8), where it correlates with poor prognosis (9). Itis also highly expressed in gastric cancer, esophagus carcinoma,pancreatic cancer (10), and hepatocarcinoma (11). CDH17facilitates liver colonization and metastasis in orthotopic mousecolorectal cancer models after intrasplenic injection (9, 12).CDH17 binds a2b1 integrin through an RGDmotif and inducesb1 integrin activation, which leads to increased cell adhesion toMatrigel and type IV collagen, and increased proliferation (12).The CDH17 RAD mutant does not induce integrin signaling butrather leads to a reduction in tumor growth and liver coloni-zation (12). In colorectal cancer metastatic cells, we havedescribed a nonconventional situation in which a2b1 integrinbinds to cadherins in a RGD-dependent manner, making itconformationally activatable and allowing it to bind to typeIV collagen. This finding modifies the classical notion that a2b1integrin binds type I collagen using the GFOGER motifs in aRGD- and conformation-independent way (13, 14). Previousdata support a preferential trans activation model for cadherin/integrin interaction, although cis interaction cannot be ruled out(12). No activation effects were observed for the RGD motif onav or a6b4 integrins.

RGD motifs are also present in vascular-endothelial (VE)-cadherin, CDH6 [fetal kidney (K)-cadherin], and CDH20.VE-cadherin is expressed in aggressive human melanoma celllines and cutaneous melanomas (15), but not in poorly

1Department of Cellular and Molecular Medicine, Centro de InvestigacionesBiol�ogicas, CSIC, Madrid, Spain. 2Protein Alternatives SL, Tres Cantos, Madrid,Spain.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: J. Ignacio Casal, Centro de Investigaciones Biol�ogicas(CIB-CSIC), Ramiro deMaeztu 9, Madrid 28040, Spain. Phone: 34 918373112; Fax:34 915360432; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-1444

�2017 American Association for Cancer Research.

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aggressive cell lines isolated from the same tumors (16). VE-cadherin has been involved in vasculogenic mimicry (theability to form novel blood vessel–like structures) in uvealmelanomas (16). It is also expressed in Ewing sarcoma (17)and promotes breast cancer progression (18). Recently, wehave demonstrated that knocking down VE-cadherin sup-presses the lung colonization capacity of melanoma or breastcancer cells inoculated in mice, whereas preincubation withVE-cadherin RGD peptides promotes lung metastasis for bothcancer types (19). Like CDH17 RGD peptides, VE-cadherinRGD peptides cause b1 integrin activation, suggesting that themechanisms of action for both cadherins are similar (12).

We hypothesized that blocking the cadherin RGD motifswould provoke an inhibition of liver and lung metastasisthrough a2b1 integrin inhibition. Here, we investigated theuse of 9-mer peptides containing the CDH17 RGD motif andtheir flanking sequences to retrieve highly selective mAbs withantimetastatic activity in different cancer cell types expressingCDH17 and VE-cadherin. We developed a panel of CDH17RGD-specific mAbs that inhibited b1 integrin activation, celladhesion, and proliferation in colorectal and pancreatic cancercells. This blocking effect was also effective in VE-cadherin–mediated b1 integrin activation of melanoma and breast cancercells. RGD-specific mAbs were able to induce a significantincrease in mouse survival after intravenous and intrasplenicinjection of highly metastatic cells from melanoma and colo-rectal cancer causing lung and liver metastasis, respectively.Consequently, blocking the interaction between RGD cadher-ins and a2b1 integrin represents a promising therapy fordistinct cancer metastases.

Materials and MethodsCell lines, peptides, antibodies, and siRNAs

Metastatic KM12SM human colon cancer cells were obtaineddirectly from Dr. I. Fidler (The University of Texas MD AndersonCancer Center, Houston, TX), whereas MDA-MB-468 triple-neg-ative human breast cancer cells and metastatic BLM humanmelanoma cells were kindly provided by Dr. J. Teixid�o (CIB-CSIC, Madrid, Spain). KM12SM cells were authenticated by shorttandem repeat analysis. SKBR3 breast cancer cells were obtained

from Dr. A Villalobos (IIB-CSIC Madrid, Spain). RKO, Colo320,and HT29 human colon cancer cells; A375 human melanomacells; and the pancreatic cancer cell line PANC1 were purchasedfrom the ATCC and passaged for all of the experiments fewerthan 6 months after purchase. BLM, SKBR3, and MDA-MB-468were not authenticated in our laboratory. All cell lines weretested regularly for Mycoplasma contamination and cultured inDMEM (Invitrogen) containing 10% FCS (Invitrogen) and anti-biotics at 37�C in a 5% CO2 humidified atmosphere.

CDH17 polyclonal antibodies (H-167 and C-17), RhoGDIa(G-2), VE-cadherin (BV9), and FAK (A-17) were purchasedfrom Santa Cruz Biotechnology. CDH17 antibody (#141713)and Src (AF3389) were from R&D Systems. Blocking anti-b1(P5D2), a4 (ALC 1/1), and a5 (P1D6) integrin antibodies werefrom Abcam. b1 integrin antibody specific for high-affinityconformation (HUTS-21) and pY397-FAK (#611722) werefrom BD Transduction Laboratories. Antibodies against phos-pho-Src (#2101), JNK (56G8), phospho-JNK (G9), ERK1/2(L24F12), and phospho-ERK1/2 (#9101) were from Cell Sig-naling Technology. Anti-a3 integrin (AB1920) was from EMDMillipore. The antibody against a1 (TS 2/7) integrin was a kindgift from Dr. C. Bernabeu (CIB-CSIC).

CDH17-derived peptides (VSLRGDTRG, SLRGDTR, andLRGDT), VE-cadherin domain 2 (QGLRGDSGT), VE-cadherindomain 3 (SILRGDYQD), CDH6 (DQDRGDGSL), CDH16(RAIRGDTEG), and CDH20 (DMDRGDGSI) peptides were syn-thesized using solid phase chemistry with a Focus XC instrument(AAPPTec). In the CDH17 9-mer VSLRGDTRG, Tyr at position600 was replaced with a Val to facilitate synthesis and hydrophi-licity. The cyclic RGD peptide Cilengitide was from SelleckChemicals.

siRNAs against human a1 (SASI_Hs01_00067020), a3(SASI_Hs01_00196571), and a2 integrin (19) subunits werepurchased form Sigma-Aldrich and transfected using jetPRIMEreagent (Polyplus Transfection).

Immunization and preparation of mouse mAbsAll animal experiments in this study were conducted according

to the national RD 53/2013 and European UnionDirective 2010/63/EU. All animal protocols were approved by the ethics com-mittee of the Instituto de Salud Carlos III (CBA22_2014-v2) andCommunity ofMadrid (PROEX278/14). Four female Balb/cmicewere immunized 3� intraperitoneally using ovalbumin (OVA)-conjugated CDH17 peptide (VSLRGDTRG) as antigen—the firsttime together with 50 mg of peptide-OVA emulsified in Freund'scomplete adjuvant and the next two times with 25 mg of peptide-OVA emulsified in Freund's incomplete adjuvant. Then, mAbswere prepared according to standard procedures (20). The selec-tion of clones was carried out using an indirect ELISA against theCDH17 protein expressed in E. coli and the peptide VSLRGDTRGcoupled to BSA as described (20), flow cytometry againstKM12SM cells, and b1 integrin activation assays. These antibodieswere used at 10 mg/mL in the different experiments. mAbs weregrown in HAT media, purified by Protein G, and dialyzed againstPBS for final testing and characterization.

Flow cytometryFor flow cytometry, 2.5 � 105 KM12SM cells in 100 mL were

incubated with immune sera or control serum (diluted 1:50) orwith the different antibodies (10 mg/mL) for 30 min at 4�C.After incubation, the cells were washed with 2 mL of PBS and

Translational Relevance

Many unsuccessful attempts have been made in the lastdecades to target integrins with RGD peptides for cancertherapy. Here, we have demonstrated that cellular cadherinRGDmotifs are equally efficient and more selective targets forintegrin inhibition inmetastatic cells than RGD peptides fromthe extracellularmatrix proteins.Wedescribe the developmentand functional characterization of cadherin RGD-specificmonoclonal antibodies that have demonstrated an extraordi-nary efficiency in blocking b1 integrin activation and protect-ing mice against liver and lung metastasis from colorectalcancer and melanoma, respectively. Our data support thatthese highly promising results also can be extended to breast,pancreatic, and other tumors. In summary, these highly selec-tive monoclonal antibodies open a new avenue for the treat-ment of metastasis in different types of cancer.

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Clin Cancer Res; 24(2) January 15, 2018 Clinical Cancer Research434




incubated in the dark at 37�C for 30 minutes in the presence ofAlexa Fluor 488 goat anti-mouse IgG antibodies (ThermoFisher Scientific). Fluorescence was analyzed in a COULTEREPICS XL cytofluorometer. At least 10,000 events per samplewere acquired, and cells were identified on the basis of theirspecific forward (FSC) and side (SSC) light–scattering proper-ties. Mean fluorescence intensities (MFI) for the indicatedantibodies are shown inside each panel.

Analysis of high-affinity conformation for b1 integrinCells were detached with 2 mmol/L EDTA in PBS, washed

with PBS, resuspended in DMEM, and incubated with a CDH17nine-amino acid peptide (VSLRGDTRG) containing the RGDmotif for 25 minutes at 37�C in the presence of immune orcontrol serum (diluted 1:50) or the indicated antibodies (10mg/mL). Peptides were used at 1 mg/mL in the different experi-ments (at approximately 1 mmol/L). After incubation, cells weresubjected to flow cytometry assays using antibodies specific forb1 integrin in high-affinity conformation and Alexa Fluor 488goat anti-mouse IgG antibodies. Fluorescence was analyzed in aCOULTER EPICS XL cytofluorometer. This experiment wasrepeated for the other CDH17 RGD peptides (seven and fiveamino acids), VE-cadherin domains 2 and 3 [VE-cad (D2) andVE-cad (D3)], and CDH6.

Antibody confocal microscopy and internalization assayKM12SM cells were cultured on Matrigel-coated cover slides.

Then, cells were fixed with 4% paraformaldehyde in PBS andpermeabilized with 1% Triton X-100. After washing, cells wereincubated for 40 minutes with primary antibodies (H167 and25.4.1 at 10 mg/mL; 6.5.2, 6.6.1, 12.4.1, and control antibody at30 mg/mL) in PBS with human gamma-globulin (40 mg/mL) atroom temperature. Cells were then incubated 25 minutes withsecondary antibodies coupled with Alexa Fluor 488 and 4,6-diamidino-2-phenylindole (DAPI). Samples were mounted withFluorescence Mounting Medium (Dako), and images were cap-tured using a TCS-SP5-AOBS confocal microscope with 63� oilimmersion objective.

For internalization assays, KM12SM cells were cultured onMatrigel-coated cover slides and treated with the indicatedmouseCDH17 antibodies (10 mg/mL) for 1 hour. Then, cells were fixed,permeabilized, and stained with rabbit anti-LAMP1 antibody(MyBioSource), followed by incubation with secondary antibo-dies (anti-mouse IgG coupled with Alexa Fluor 488 and anti-rabbit IgG coupled with Alexa Fluor 568). Zoom images weretaken with 100� oil immersion objective.

Tubule formationCancer cells (5 � 103) were resuspended in serum-free DMEM

and added upon 96-well plates previously coated with Matrigel(50 mL). After 24-hour incubation, images of the wells were takenunder a microscope with 10� phase contrast and tubules fromfive different wells were counted by two different observers (21).Tubules were defined as tubes when formed by multiple singlecells arranged in a row (one-cell thickness) or a thick bundle(thickness of several cells) connecting two cell "islands."

Cell signaling analysis by Western blot analysisCells were incubated 3 hours in serum-free DMEM, detached

with 2 mmol/L EDTA, washed, and treated with anti-RGD mAbs(10 mg/mL) for 40 minutes. Then, cells were added to plates

previously coated with Matrigel (0.4 mg/mm2) for 30 minutes inthe presence of either control IgG or the mAbs. Finally, cells weredetached as before and lysed with lysis buffer [1% Igepal, 100mmol/L NaCl, 2 mmol/L MgCl2, 10% glycerol, protease inhibi-tors (cOmpleteMini, Roche), and phosphatase inhibitor cocktails2 and 3 (Sigma-Aldrich) in 50 mmol/L Tris-HCl]. Protein extractswere separated in SDS-PAGE and transferred to nitrocellulosemembranes, incubated with primary antibodies, washed, andincubated with HRP-conjugated secondary antibodies (Sigma-Aldrich).Membraneswere visualized using SuperSignalWest PicoChemiluminescent Substrate (Thermo Fisher Scientific).

MTT assaysCancer cells were seeded at 1� 104 cells/well on 96-well plates

and incubated for 48 hours at 37�C inDMEMwith 0.5% serum inthe presence of the indicated antibodies, followed by 1-hourincubation with thiazolyl blue tetrazolium bromide (MTT;0.6 mg/mL; Sigma-Aldrich). Cell viability was determined byabsorbance at 560 nm and comparison with control cells collec-ted ad initium. MTT assays were used as a surrogate of cellproliferation, as no increase in cell detachment or cell death wasobserved after mAb treatments.

Cell adhesion and invasion assaysAdhesion and invasion assays were performed as previously

described (22). Briefly, cancer cells were labeled with BCECF-AM(Sigma-Aldrich), detached with EDTA/PBS, and incubated inserum-free DMEM in the presence of the indicated antibodies(10 mg/mL) for 10 minutes at 37�C. Then, 6� 104 cells in 100 mLwere added to 96-well plates previously coated with Matrigel(0.4 mg/mm2) or type I collagen (Millipore; 0.3 mg/mm2), and theplates were incubated for 25 or 30 minutes at 37 �C, respectively.Subsequently, nonadhered cells were removed by three washeswith DMEM. Bound cells were lysed with 1% SDS in PBS, and theextent of the adhesion was quantified using a fluorescence ana-lyzer, POLARstar Galaxy (BMG LABTECH).

For invasion assays, 6� 104 cells were loaded onto 8-mmpore-size filters coated with 35 mL of Matrigel (1:3 dilution; BDBiosciences) in Transwell plates (Sigma-Aldrich) in the presenceof antibodies. The lower compartment of the invasion chamberwas filled with 5% serum DMEM. After 24 hours, noninvadingcells were removed from the upper surface of the filter, andcells that migrated through the filter were fixed with 4% parafor-maldehyde, stained with crystal violet, and counted under amicroscope.

Metastasis experiments in nude miceThe ethics committees of the Consejo Superior de Investi-

gaciones Científicas and Community of Madrid approved allprotocols used. Swiss nude mice (Charles River; n ¼ 4–6 percondition) were inoculated in the spleen with 1.5 � 106

KM12SM cells in 0.1 mL PBS. A day after inoculation, micewere subjected to removal of the spleen to avoid local growth ofthe tumors. Then, these mice were inoculated intravenouslywith control antibodies or with anti-RGD antibodies 25.4.1and 6.6.1 (50 mg/kg of weight, divided in seven doses) starting2 days after inoculation and for 2 weeks. Mice were inspecteddaily for signs of disease, such as abdominal distension, loco-motive deficit, or tumor detectable by palpation. When signswere visible, mice were euthanized, subjected to necropsy, andinspected for metastasis in liver.

CDH17 RGD Antibodies Inhibit Cancer Metastasis

www.aacrjournals.org Clin Cancer Res; 24(2) January 15, 2018 435




For liver and lung colonization assessment, mice were inocu-lated in the spleen or tail vein respectively, with 1� 106 KM12SM,BLM, or MDA-MB-468 cells, and euthanized 96 hours afterinoculation. RNAwas isolated from the liver using Trizol (ThermoFisher Scientific) and retrotranscribed, and 0.3 mg cDNA wassubjected to PCR with Taq DNA polymerase (Thermo FisherScientific) to amplify human GAPDH as previously described(19). As a control, a 20-cycle amplification of murine b-actin wasperformed.

Statistical analysesDatawere analysedby Student t test (two conditions) or byone-

way ANOVA followed by Tukey–Kramer multiple comparisontest (more than two conditions). Histograms showed the averageof the assessed values, and the error bars showed the SD. Each

experiment was carried out at least three times. Survival curveswere plotted with Kaplan–Meier technique and compared withthe log-rank test. Theminimum acceptable level of significance inall tests was P < 0.05.

ResultsCDH17 RGD peptide induces b1 integrin blocking antibodies

Different commercial CDH17-specific antibodies were firsttested for their capacity to inhibit b1 integrin activation andcell proliferation in metastatic colon cancer cells. CDH17polyclonal antibodies (H167, C-17, or 141713) raised againstthe ectodomain or the carboxy-terminal domain of CDH17failed to inhibit b1 integrin activation induced by CDH17 RGDpeptides (Fig. 1A). We therefore prepared mouse polyclonal

Figure 1.

Testing and production of CDH17-specific antibodies (Ab) to inhibit the activation of b1 integrin. Different strategies were followed for the generation andtesting of inhibitory Abs. A, RKO cells were exposed to a nine-amino acid (9 aa) RGD peptide (1 mg/mL) from CDH17 and treated with the indicatedAbs. Then, cells were subjected to flow cytometry using a specific Ab for testing activated b1 integrin. B, KM12SM cells were treated with the indicatedAbs and subjected to cell adhesion assays to Matrigel. C, KM12SM cells were subjected to MTT assays in the presence of the indicated Abs.D, KM12SM cells were incubated for 1 hour with the indicated concentrations of Cilengitide and subjected to flow cytometry assays as in A. Activation of b1integrin, cell adhesion, or number of viable cells was significantly inhibited by the presence of the indicated anti-cadherin RGD Ab (� , P < 0.05; �� , P < 0.01;�� , P < 0.001).

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antibodies and mAbs against the domain six polypeptide ofCDH17, which contains the RGD domain. These antibodiesinhibited b1 integrin activation to a limited extent (<50%;Fig. 1A). In contrast, mouse polyclonal antibodies from miceimmunized with the CDH17 peptide VSLRGDTRG inhibited b1integrin activation at about 80% to 90%. These data confirmedthe hypothesis that RGD-specific antibodies inhibited CDH17RGD peptide-induced activation of b1 integrin. In addition,RGD peptide-specific antibodies significantly inhibited celladhesion (Fig. 1B) and proliferation (Fig. 1C), whereas full-length CDH17 or domain six antibodies did not affect celladhesion or proliferation of CDH17þ colon cancer cells. Theadhesion to Matrigel was b1 integrin dependent, as confirmedusing b1 integrin blocking antibodies. In summary, a selectiveimmune response against the CDH17 RGD motif displayed b1integrin blocking activity together with a clear reduction in celladhesion and proliferation.

Cilengitide, a cyclic RGD peptide, fails to bind and activatea2b1 integrin

From our previous data, it seemed counterintuitive to useRGD peptides for cancer therapy, as they might activate a2b1integrin in cancer epithelial cells. Cilengitide, a cyclic RGDpentapeptide targeting integrins avb3 and avb5, was unsuc-cessfully tested in clinical trials for glioblastoma (23). We didnot observe any effect of Cilengitide on b1 integrin activationeither as a ligand, probably due to the different flanking se-quences and conformation, or as an inhibitory drug (Fig. 1D).Therefore, not all RGD sequences are appropriate for a2b1integrin activation.

Development of RGD-specific mAbs for inhibition of celladhesion and proliferation in colorectal and pancreatic cancer

Mouse mAbs were prepared against the CDH17 RGD 9-merpeptide and functionally tested for their inhibitory capacity ofb1 integrin activation. After initial anti-peptide ELISA, fourclones, 6.5.2, 6.6.1, 12.4.1, and 25.4.1, were selected for char-acterization (Supplementary Table S1). Only mAbs 6.6.1 and6.5.2 were positive for immunoprecipitation (SupplementaryFig. S1A). mAbs did not work in Western blot analysis (data notshown) and were only weakly positive for flow cytometryanalysis of surface CDH17, except for antibodies 6.6.1 and12.4.1 (Supplementary Fig. S1B). By confocal microscopy, thestaining capacity of the RGD antibodies was good except forantibody 12.4.1 (Fig. 2A). We thus tested antibody internali-zation after CDH17 binding as a potential tool for toxinpayload delivery to tumors. We observed a similar internali-zation in lysosomes (assessed by colocalization with the lyso-somal marker LAMP1) for the anti-RGD mAb 25.4.1 and thecontrol anti-CDH17 antibodies (Fig. 2B), suggesting an endo-cytosis of CDH17 after antibody binding.

The four purified mAbs were functionally active and inhib-ited the activation of b1 integrin (Fig. 2C), cell adhesion (55%–

68%; Fig. 2D), and cell proliferation (Fig. 2E) in KM12SMcolon cancer cells and in PANC1, a CDH17þ pancreatic cancercell line (12). We obtained similar results with HT-29, adifferent CDH17þ colorectal cancer cell line (SupplementaryFig. S2A). In contrast, colorectal Colo320 cells that are negativefor CDH17 expression were not affected by the RGD mAbs(Supplementary Fig. S2B). Collectively, these results supportthat the RGDmAbs preferentially recognize the conformational

epitopes in the native CDH17 and underscore their value for inblocking cell adhesion and proliferation in colorectal andpancreatic cancer cell lines that express CDH17.

Blocking effect of mAbs on b1 integrin activation by differentcadherin RGD peptides

Next, we investigated the minimal length of the RGD motifnecessary to induce b1 integrin activation in combination withthe peptide-blocking capacity of the specificmAbs. In RKOcoloncancer cells, we compared the 7-mer SLRGDTR and the 5-merLRGDT with the positive control 9-mer peptide VSLRGDTRG.Peptides were used at 1 mg/mL in the different experiments. Inthe 9-mer peptide–treated cells,mAbs caused a strong inhibitionof b1 integrin: antibody 25.4.1 caused a complete inhibition,followed by 6.6.1 (90% inhibition), and then 12.4.1 and 6.5.2to a lesser degree (Fig. 3A). Shorter peptides induced integrinactivation but to a lesser extent (50%). The 7-mer peptide waspartially inhibited by mAbs 6.6.1 and 25.4.1 and was totallyblocked by 12.4.1 (Fig. 3B and C). Regarding the 5-mer, mAbshad aminor inhibitory effect, likely due to the short length of thepeptide supporting only a weak interaction with the binding siteof the antibody (Fig. 3B and C).

To study the applicability of our mAbs to other RGD cadher-ins in other tumors, we examined the capacity of VE-cadherinand the CDH6, CDH16, and CDH20 RGD peptides to activateb1 integrin in metastatic melanoma and breast cancer cell linesthat did not express CDH17 (Supplementary Fig. S3). Incuba-tion with 9-mer RGD peptides from VE-cad (D2), VE-cad (D3),CDH6, and CDH20 (but not CDH16) caused b1 integrinactivation in all cancer cell lines (Fig. 3D). We therefore inves-tigated whether mAbs could block the b1 integrin activationcaused by VE-cadherin and CDH6 RGD peptides in RKO cells.VE-cad (D2) was partially blocked by the mAbs 6.6.1 and12.4.1, whereas the VE-cad (D3) site was fully blocked by 6.6.1,12.4.1, and 25.4.1 (Fig. 3E and F). For CDH6 RGD, we observeda complete b1 integrin activation inhibition by mAb 12.4.1, apartial blocking effect for mAbs 6.6.1 and 6.5.2, and no effectfor 25.4.1 in RKO cells (Fig. 3G). Overall, the mAb blockingeffects were more significant for the VE-cad (D3) motif, whichdisplays a flanking sequence YSI/L (RGD) similar to CDH17.

CDH17 RGD mAbs inhibit VE-cadherin–triggereda2b1 integrin activation in melanoma andbreast cancer cells

To confirm the broad applicability of cadherin RGD-specificantibodies for other cancer metastatic cell lines, we tested twomelanoma (BLM and A375) and two breast (MDA-MB-468, atriple negative cell line, and SKBR3) cancer cells expressingVE-cadherin, but not CDH17 (Supplementary Fig. S3; ref. 19).RGD mAbs inhibited the high-affinity conformation of b1integrin (Fig. 4A and Supplementary Fig. S4A), causing a sig-nificant reduction in cell adhesion (Fig. 4B and SupplementaryFig. S4B), proliferation (Fig. 4C and Supplementary Fig. S4C),and invasion capacity through the extracellular matrix (ECM),particularly in breast cancer cells (Fig. 4D and SupplementaryFig. S4D). A blocking anti-b1 integrin antibody was used as apositive control, and antibodies against full-length VE-cadherinand control antibody were used as as negative controls (Fig. 4).Interestingly, the antibody effects on adhesion, proliferation,and invasion were not associated to the reduction of cadherinexpression in the cell surface (Fig. 4E), interference with tubule






formation (Fig. 4F and Supplementary Fig. S5A), or alterationsin endothelial permeability (Supplementary Fig. S5B). Theseresults suggest that the RGD antibodies inhibit b1 integrinactivation in VE-cadherinþ cancer cell lines without interferingwith cadherin homotypic cell–cell interactions, similar to RGD-independent cell aggregation mechanisms observed in CDH17þ

cells (12).To confirm that the blocking capacity of the RGD mAbs was

specific for a2b1 integrin, we investigated for other a integrinsubunits in the cancer cells. We detected the presence of asignificant expression of a1, a2, and a3 on the surface of bothKM12SM and BLM cells (Supplementary Fig. S6A). Then, weperformed siRNA silencing experiments for a1, a2, and a3integrins in both cell lines (Supplementary Fig. S6B and S6C).Although RGD antibodies caused a significant reduction ofadhesion to Matrigel and type I collagen in KM12SM and BLMcells silenced for a1 and a3 integrins, they did not cause any

alteration in a2-silenced cells (Supplementary Fig. S6D).Together, these results confirm the specific blocking capacityof the RGD mAbs on a2b1 integrin.

CDH17 RGD antibodies arrest integrin signaling in melanomaand colorectal, pancreatic, and breast cancers

To investigate whether the integrin signaling pathway wasaffected by the blocking RGD mAbs, representative KM12SM,PANC1, BLM, and MDA-MB-468 cell lines were cultured onMatrigel in the presence of the four RGD mAbs or a controlIgG. In KM12SM colon cancer cells, the four RGD mAbsdiminished the activation of FAK, JNK, and ERK kinases,which correlate with a decrease in cell adhesion and prolif-eration, but they did not affect Src or AKT activation (Fig. 5).In PANC1 pancreatic cancer cells, the mAbs provoked a cleardecrease in JNK, ERK, and Src activation but had no effect onFAK or AKT. The mAbs (except for 6.5.2) inhibited JNK, ERK,

Figure 2.

Phenotypic and functional characterization of CDH17 RGD-specific antibodies. KM12SM cells were subjected to (A) confocal microscopy or (B) antibodyinternalization assays using the CDH17 RGD-specific antibodies or the indicated commercial antibodies. mAbs recognized surface CDH17 at differentextent and are further internalized in the lysosomal compartment. Then, KM12SM and PANC1 cells were treated with the indicated antibodies andsubjected to (C) flow cytometry assays to study b1 integrin in high-affinity conformation, (D) cell adhesion assays to Matrigel, and (E) MTT assays.Activation of b1 integrin, cell adhesion, and number of viable cells were significantly inhibited by the presence of the indicated RGD-specific antibodiesin colorectal and, at a lower extent, pancreatic cancer cells (� , P < 0.05; �� , P < 0.01; �� , P < 0.001). Ctrl ab, control antibody.

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and Src activation in BLM (melanoma) and MDA-MB-468(breast) cancer cell lines, without affecting FAK activation.AKT activation was only reduced in melanoma. Src and AKTactivities have been correlated with cell invasion (19). In

summary, RGD mAbs caused different type-specific effects oncell signaling after blocking the integrin pathway activationin the four tested metastatic cell lines. These results confirmcell type–specific differences observed in VE-cadherin cell

Figure 3

Effect of RGD peptide size and sequence on the activation of b1 integrin. RKO cells were exposed to different CDH17 RGD peptides, ranging in lengthfrom (A) nine amino acids (9 aa), (B) seven amino acids (7 aa), and (C) five amino acids (5 aa), or to peptide-free medium and treated with theindicated antibodies (Ab). Cells were subjected to flow cytometry assays to detect b1 integrin in high–affinity conformation. D, RKO, BLM, and MDA-MB-468 cells were exposed to 9-mers containing the RGD motif from VE-cad (D2), VE-cad (D3), CDH6, CDH16, CDH17, or CDH20 and subjected to flowcytometry assays as above. RKO cells were exposed to a 9 aa peptide containing the RGD motif and the flanking sequences of VE-cad (D2) (E), VE-cad (D3)(F), and CDH6 (G); treated with the indicated antibodies; and subjected to flow cytometry assays as in A. Activation of b1 integrin was significantly inhibitedby the presence of the indicated RGD Ab (� , P < 0.05; ��, P < 0.01; �� , P < 0.001). Peptides were used at 1 mg/mL in the different experiments.






signaling on migration and invasion with respect to CDH17colon cancer cells on cell adhesion.

RGD-specific mAbs showed antimetastatic capacity in vivoFor "in vivo" metastasis experiments, we first examined the

effect of the antibodies on liver and lung homing capacitycaused by colon and melanoma cancer cells, respectively.Tumor cells were inoculated in the spleen (for liver metastasis)or tail vein (for lung metastasis) of nude mice together with theRGD mAbs. Mice were euthanized at 96-hours postinoculationto avoid unspecific binding to target organs. mRNA was thenextracted from liver and lung. In melanoma and breast cancercells treated with mAb 12.4.1, no human GAPDH was detectedin lung or liver (Fig. 6A). Furthermore, in colon cancer cells,liver homing was completely inhibited after tumor cells weretreated with the RGD antibodies (Fig. 6A).

We next investigated the effects of RGD-specific mAbs onmouse survival after liver (colon) or lung metastasis (melano-ma). For colon cancer, highly metastatic KM12SM cells wereinoculated in the spleen to induce liver colonization throughthe hepatic portal vein. After 48 hours, mice were treated onalternate days for 2 weeks, with a total dose of 50 mg ofantibody per kg of mouse weight. When signs of disease wereevident, animals were euthanized and livers were removed forvisual inspection of metastatic nodules. Otherwise, survivinganimals were sacrificed at days 80 and 90 for lung and livermetastasis, respectively. Kaplan–Meier survival curves showed

that all control mice died by day 50, although those inoculatedwith mAb 6.6.1 were still alive at this time point; further,only half of them developed liver metastasis by 90 days, andthe rest survived to the experimental endpoint (Fig. 6B). mAb25.4.1 caused an intermediate survival (50 days), whereasthe half-life of mice inoculated with control antibody was27 days. The number of metastatic nodules observed in liverwas substantially reduced or abolished in half of the treatedmice (Fig. 6C). For lung metastasis in melanoma, mice inoc-ulated with mAbs 6.6.1 and 25.4.1 presented a prolongedsurvival in most of the treated animals. Lung inspectionrevealed a large number of metastases in control mice but veryfew or none in treated mice at the experimental endpoint(Fig. 6C). In summary, blocking the cadherin RGD-inducedactivation of a2b1 integrin constitutes a promising strategy fortreating lung and liver metastasis in melanoma and colorectalcancer.

DiscussionAfter many unsuccessful attempts in the last decades to target

integrins with RGDpeptides for cancer therapy, we propose here anovel strategy. Rather than using ECM RGD motifs, we havedemonstrated that cadherin RGDmotifs are efficient and selectivetargets for a2b1 integrin inhibition in metastatic cells of fourdifferent types of tumors. We have shown the protective effects ofcadherin RGD-specific antibodies using two different cancer cell

Figure 4.

RGD-specific antibodies (Ab) inhibit activation of b1 integrin, cell adhesion, proliferation, and invasion of melanoma and breast cancer cells. BLM andMDA-MB-468 cells were treated with the indicated Abs and subjected to (A) flow cytometry assays to detect b1 integrin in high-affinity conformation,(B) cell adhesion assays to Matrigel, (C) proliferation assays (MTT), (D) cell invasion assays through Matrigel, (E) flow cytometry assays to detectVE-cadherin expression, and (F) tube formation assays. Activation of b1 integrin, cell adhesion, number of viable cells, and number of invasive cellswere significantly inhibited by the presence of the indicated anti-cadherin RGD Ab (� , P < 0.05; �� , P < 0.01; �� , P < 0.001).

Bartolom�e et al.





types (colorectal and melanoma) and two metastatic settings(lung and liver metastasis). In addition, RGDmAbs were effectivein blocking the integrin pathway activation in pancreatic andbreast cancer cell lines. The capacity to inhibit the metastaticcapacity of different cancer cell lines enhances the value andpromise of these mAbs as potential therapeutic agents to controlmetastatic spread in different solid tumors.

We proved that (i) peptide sequences containing the CDH17RGDmotif elicited blocking antibodies for b1 integrin activationin metastatic cells, (ii) the RGD peptide length required forintegrin activation was as short as seven amino acids, (iii) mAbsraised against the CDH17 RGD motif were equally effectiveagainst b1 integrin activation with VE-cadherin and CDH6 RGDpeptides, (iv) RGDmAbs blocked the integrin signaling pathwaycascades in a cancer-dependent manner, and (v) RGD-specificmAbs significantly delayed anddecreased liver and/or lungmetas-tasis in colorectal and melanoma cancers. Moreover, becausethese mAbs did not affect the cell–cell homotypic interactionsof cadherins (12, 19), their side effects on endothelial cells andvascular integrity might be avoided. The selectivity conferred bythe cadherin RGD flanking sequences might avoid unspecificbinding of the mAbs to the RGD motifs of the ECM proteins(fibronectin, collagen, etc.).

Integrins are key molecules in multiple cellular processesrequired for cancer progression and metastasis (see ref. 24for a review). Integrin-targeted treatments for cancer therapyhave been the focus of intense research efforts in the lastdecades, and integrin-targeted antibodies are under clinical

evaluation for different diseases (24, 25). Historically, avb3,a4b1, and a5b1 integrins have been preferentially associatedwith cancer metastasis therapy (26). However, growing evi-dence underscores the critical relevance of a2b1 integrin as akey regulator of cancer metastasis (12, 19, 27, 28) despitesome conflicting results with a mouse mammary tumor model(29, 30). Note that mouse CDH17 and VE-cadherins do nothave RGD motifs (12), suggesting that alternative signalingpathways are used for metastasis progression in mice. A keyrole for a2b1 integrin has been well established in melanoma;rhabdomyosarcoma; and gastric, prostate, and colon cancermetastasis (9, 31–35). Therefore, we speculate that RGD mAbsmight theoretically be applicable for all of these humantumors.

a2b1 integrin has been therapeutically targeted using block-ing antibodies against a2 integrin subunit (GRB-500), which iscurrently in clinical trials for multiple sclerosis and ulcerativecolitis (27). Another a2 integrin blocking mAb demonstratedhigh value in the inhibition of breast carcinoma cell growth(36). Regarding b1 integrin, volociximab (M-200), a monoclo-nal targeting a5b1 integrin is currently in clinical trials forsolid tumors (37). In general, a2b1 integrin targeting is con-sidered safe and tolerable (27). The RGD cadherin selectivity ofour antibodies avoids the adverse effects caused by indiscrim-inate targeting of integrins using other therapeutic approaches,such as cyclic RGD molecules (38). A cyclic RGD peptide(Cilengitide) targeting the ligand binding site of avb3 andavb5 integrins reached clinical trials for glioblastoma but did

Figure 5.

Anti-cadherin RGD antibodies inhibit the integrin pathway cell signaling. KM12SM, PANC1, BLM, and MDA-MB-468 cells were treated with the indicatedantibodies (Ab) and added to Matrigel-coated plates for 30 minutes. Then, cells were lysed and the extracts analyzed by Western blot analysis usingAbs against FAK, JNK, ERK1/2, Src, AKT, or their phosphorylated forms. RhoGDI was used as a loading control.






not reach significant disease outcome improvement (39). Inour hands, Cilengitide was completely ineffective in inducingor blocking a2b1 integrin activation (Fig. 1D).

Metastasis development has been associated with enrichedcancer stem cell populations (40). b1 integrin is critical topreserve stem cell populations (41). Indeed, b1 and a6 integ-rins are enriched in cancer stem cells (24, 40). Interestingly,these two integrins are abundantly present in KM12SM colo-rectal metastatic cancer cells (8). Other stem cell markerspresent in KM12SM cells are ALDH1, ALCAM, and CD44(9). These data suggest that the KM12SM metastatic cells havesome stemness features.

Cancer cell types activate different integrin pathways formetas-tasis; therefore, it was not surprising to observe differences in theblocking effects on downstream signaling caused by the mAbs inthe different cancer types. Cadherin RGD-specific antibodiesblocked the integrin signaling pathways in a cancer cell–depen-dent manner. As a general rule, proliferation pathways werealways inhibited, whereas adhesion and invasion were cell typedependent. These data support that b1 integrin is a critical

molecule for the activation of multiple pathways required formetastatic colonization.

In summary, our data support that antibodies against thecadherin RGD motifs reduced the proliferation, adhesion, andinvasion capacity of metastatic cancer cell lines by inhibitingthe activation of a2b1 integrin. The extended mice survivaldemonstrates the potential therapeutic effect in cancer metas-tasis, specifically in colorectal cancer and melanoma. Moreover,it is likely that this capacity might be extended to otherneoplasias such as pancreatic and breast cancer. Based on thedifferent biochemical properties (antigen recognition by ELISA,immunoprecipitation, confocal microscopy, and yield andmetastatic inhibition capacity), mAb 6.6.1 seems to be themost convenient antibody for further therapeutic develop-ments. The next steps should include the humanization processof the most effective mAbs, particularly 6.6.1, for potentialclinical application. Finally, the crucial role of a2b1 integrin infibrosis, platelet-mediated thrombosis, and angiogenesis (29)might increase considerably the application range of cadherinRGD mAbs to other diseases.

Figure 6.

RGD mAbs increase mouse survival to liver and lung metastasis. A, KM12SM, BLM, and MDA-MB-468 cells treated with control or anti-RGD 6.6.1 mAb wereinoculated intra-tail vein or spleen in Swiss nude mice. At 96 hours after inoculation, RNA was isolated from lungs (for intra-tail inoculation) or liver(for spleen inoculated) and subjected to RT-PCR to amplify human GAPDH for homing detection. Anti-RGD antibodies (Ab) specifically inhibited the homingin lung and liver for melanoma and breast and colorectal cancer. B, Kaplan–Meier survival results for nude mice inoculated with KM12SM or BLM cellsin the spleen or intravenously, respectively. Starting at 48 hours after inoculation, the indicated Abs (50 mg/kg of weight, divided in seven doses) wereadministered intravenously for 2 weeks. When signs of illness were detected, mice were euthanized and examined for macroscopic metastases in liver.Survival was significantly enhanced by the indicated mAb (� , P < 0.05; �� , P < 0.001). C, Representative pictures of livers and lungs from theinoculated mice after necropsy.

Bartolom�e et al.





Disclosure of Potential Conflicts of InterestJ.I. Casal holds ownership interest (including patents) in Protein Alternatives

SL. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: R.A. Bartolom�e, J.I. CasalDevelopment of methodology: R.A. Bartolom�e, J.I. ImbaudAcquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): R.A. Bartolom�e, C. Aizpurua, M. Ja�en, S. Torres, J.I. ImbaudAnalysis and interpretation of data (e.g., statistical analysis, biostati-stics, computational analysis): R.A. Bartolom�e, C. Aizpurua, M. Ja�en,S. Torres, J.I. CasalWriting, review, and/or revision of the manuscript: R.A. Bartolom�e,J.I. Imbaud, J.I. Casal

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. Calvi~noStudy supervision: J.I. Imbaud, J.I. Casal

AcknowledgmentsThe authors appreciate the technical assistance of Mercedes Dominguez

(Centro Nacional de Microbiología, ISCIII, Majadahonda, Madrid) for thepreparation of the monoclonal antibodies.

This research was supported by grant BIO2015-66489-R from theMINECO, Foundation Ram�on Areces, and PRB2 (IPT13/0001-ISCIII-SGEFI/FEDER).

Received May 19, 2017; revised August 2, 2017; accepted September 8, 2017;published OnlineFirst September 15, 2017.

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2018;24:433-444. Published OnlineFirst September 15, 2017.Clin Cancer Res Rubén A. Bartolomé, Carmen Aizpurua, Marta Jaén, et al. MetastasisTherapeutic Activity against Melanoma and Colorectal Cancer Monoclonal Antibodies Directed against Cadherin RGD Exhibit

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