cancer therapy: preclinical clinical cancer monoclonal ......cancer therapy: preclinical monoclonal...

Cancer Therapy: Preclinical

Monoclonal Antibodies Targeting LecLex-RelatedGlycans with Potent Antitumor ActivityJia Xin Chua1, Mireille Vankemmelbeke1, Richard S. McIntosh1, Philip A. Clarke2,Robert Moss1, Tina Parsons1, Ian Spendlove1, Abid M. Zaitoun3,Srinivasan Madhusudan1, and Lindy G. Durrant1

Abstract

Purpose: To produce antitumor monoclonal antibodies(mAbs) targeting glycans as they are aberrantly expressed intumors and are coaccessory molecules for key survival pathways.

Experimental Design: Two mAbs (FG88.2 and FG88.7) rec-ognizing novel tumor-associated Lewis (Le) glycans were pro-duced by immunizations with plasmamembrane lipid extracts ofthe COLO205 cell line.

Results: Glycan array analysis showed that both mAbs boundLecLex, di-Lea, and LeaLex, as well as Lea-containing glycans. Theseglycans are expressed on both lipids and proteins. Both mAbsshowed strong tumor reactivity, binding to 71% (147 of 208) ofcolorectal, 81% (155 of 192) of pancreatic, 54% (52 of 96) ofgastric, 23% (62 of 274) of non–small cell lung, and 31% (66 of217) of ovarian tumor tissue in combination with a restrictednormal tissue distribution. In colorectal cancer, high FG88 glyco-epitope expressionwas significantly associatedwithpoor survival.

The mAbs demonstrated excellent antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity(CDC), in addition to direct tumor cell killing via a caspase-independent mechanism. Scanning electron microscopy revealedantibody-induced pore formation. In addition, the mAbs inter-nalized, colocalized with lysosomes, and delivered saporin thatkilled cells with subnanomolar potency. In vivo, the mAbs dem-onstrated potent antitumor efficacy in a metastatic colorectaltumor model, leading to significant long-term survival.

Conclusions: The mAbs direct and immune-assisted tumorcell killing, pan-tumor reactivity, and potent in vivo antitumorefficacy indicate their potential as therapeutic agents for thetreatment of multiple solid tumors. In addition, internalizationof saporin conjugates and associated tumor cell killing suggeststheir potential as antibody drug carriers. Clin Cancer Res; 21(13);2963–74. �2015 AACR.

IntroductionSuccessful cancer immunotherapy is dependent on the gen-

eration of monoclonal antibodies (mAbs) with good specific-ity and potent killing. The complexity of the glycome andaltered expression of glycosyl transferases associated withmalignant transformation make cancer cell-associated carbo-hydrates excellent targets (1–4). Glycolipids are particularlyattractive due to their dense cell-surface distribution, mobility,and association with membrane microdomains, all of whichcontribute to their participation in a wide range of cellularsignaling and adhesion processes (4–6). Generating antigly-colipid antibodies, however, is a challenging task as they do

not provide T-cell help and the mAbs are usually low-affinityIgMs.

Lewis (Le) carbohydrate antigens are formed by the sequentialaddition of fucose onto oligosaccharide precursor chains onglycoproteins and glycolipids through the concerted action of aset of glycosyltransferases (7). Type I chains (containingGalb(1!3)GlcNAc) form Lea and Leb, whereas type II chains(containing Galb(1!4)GlcNAc) form Lex and Ley. Lea and Leb

antigens are regarded as blood group antigens, yet many humancancers express Lea or Leb antigens regardless of Lewis blood groupstatus (8–10). In addition, Lea and Leb antigen frequently coexistin human tumor cells (11). Dimeric Lea and Lex have also beenidentified as tumor-associated antigens in breast and gastrointes-tinal carcinomas (9, 10, 12).

Only a limited number of mAbs recognizing glycans have beendescribed (13, 14). GNX-8 (human IgG1) bound the extendedtype I chain epitope Leb-Lea (15) and FC-215 (murine IgM), ananti-Lex mAb that induced transient antitumor responses,but caused profound neutropenia in phase I trials (16, 17).NCC-ST-421 (ST-421, murine IgG3) recognized gastric cancer-associated dimeric Lea and demonstrated significant antitumoreffects in a human tumor xenograft model (18). The murine IgMmAb 43-9F, targeted Lea/Lex epitopes, cross-reacting with simpleand extended Lea epitopes, but had no in vivo antitumor reactivity(19). ThemAb 504/4 (SC104,murine IgG1), recognized sialyl (S)Le-related glycans, induced antibody-dependent cellular cytotox-icity (ADCC) and CDC, as well as direct tumor cell death andimportantly, demonstrated tumor growth inhibition in vivo (20).

1Division of Cancer and Stem Cells, School of Medicine, City HospitalCampus, University of Nottingham, Nottingham, United Kingdom.2Division of Cancer and Stem Cells, School of Medicine, QueensMedical Centre, University of Nottingham, Nottingham, United King-dom. 3Section of Surgery, School of Medicine,QueensMedical Centre,University of Nottingham, Nottingham, United Kingdom.

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

Corresponding Author: Lindy G. Durrant, University of Nottingham, AcademicClinical Oncology, City Hospital Campus, Hucknall Road, Nottingham NG5 1PB,United Kingdom. Phone: 44-115-8231863; Fax: 44-115-8231863; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-3030

�2015 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 2963

on November 1, 2020. © 2015 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 16, 2015; DOI: 10.1158/1078-0432.CCR-14-3030

http://clincancerres.aacrjournals.org/

It is currently in phase I/II clinical trials in gastrointestinalcancer (NCT01447732). More recently, human SLea mAbs wereproduced using a patient vaccination strategy that showed specificbinding to SLea and exhibitedADCC,CDC, and antitumor activityin a xenograft model (21).

In this study, we describe the characterization of two mAbs,FG88.2 and FG88.7, targeting the novel galb1-3GlcNacb1-3Galb1-4(Fuca1-3)GlcNAc (LecLex) glycan as well as di-Lea andLeaLex glyco-epitopes that are expressed by awide range of tumorsbut have a restricted normal tissue expression. BothmAbs displayeffective immune-mediated and direct cytotoxicity that translatedinto potent in vivo antitumor activity. Furthermore, their efficientinternalization suggests potential as antibody drug carriers(ADC).

Materials and MethodsMaterials, cells, and antibodies

Colorectal (COLO205, HCT-15, and HT29), ovarian(OVCAR3), gastric (AGS), lung (DMS79), T-cell leukemia (Jur-kat), andmouse (Balb/c) lymphoblastoidmyeloma (NS0) cancercell lines were all obtained from the ATCC. Colorectal cell lineHCT-15HM2 is a high-metastasizing variant of HCT-15 (22). Allcell lines were regularly authenticated using short tandem repeatprofiling. mAbs, 7-Le and 225-Le, were from Abcam, anti-CD40mAb from R&D Systems, anti-Fas mAb from Upstate (Millipore),anti-CEACAM mAb from eBioscience, OKT3 (anti-CD3),FG27.10 (anti-Ley), FG27.18 (anti-Ley/x), 791T/36 (anti-CD55),and 505/4 (SC104; anti-Sdi-Lea) were produced in house. Alpha-galactosylceramide was from Alexis Biochemical, glycan–humanserum albumin (HSA) conjugates from IsoSep AB and lipids fromSigma.

Generation of mAbsPlasma membrane lipid extract [0.36 mole%] from 5 � 107

COLO205 cells incorporated into liposomes was used to immu-

nize BALB/c mice at two-weekly intervals over a 2-month period,with a-galactosylceramide and anti-CD40mAb as adjuvants. Fivedays after the final immunization, the splenocytes were harvestedand fusedwithNS0myeloma cells. Stable clones were establishedby repeated limiting dilutions and mAbs purified using standardprotein G affinity chromatography.

Flow cytometryCells (1� 105) were incubated with primary mAbs at 4�C for 1

hour followed by FITC-conjugated secondary mAb and fixing, asdescribed previously (23) and in Supplementary Materials andMethods. For the propidium iodide (PI) uptake, cells (5 � 104)were incubated with mAbs for 2 hours at 37�C followed by theaddition of 1 mg of PI for 30 minutes. Cells were resuspended inPBS and run on a Beckman Coulter FC-500 with WinMDI 2.9 foranalysis.

Thin-layer chromatography analysis of glycolipid bindingGlycolipid extract from 2 � 106 COLO205 cells (20) spotted

onto Merck high-performance thin-layer chromatography(HPTLC) silica plates and developed twice in chloroform:methanol:H2O (60:30:5) followed by twice hexane:diethylether:acetic acid (80:20:1.5). The dried plates were sprayedwith 0.1% (w/v) polyisobutylmethacrylate (Sigma) in acetoneand blocked with PBS 2% (w/v) BSA (PBS/BSA) for 1 hour atroom temperature. The plates were then incubated overnight at4�C with primary mAbs followed by two 1-hour incubationswith biotinylated anti-mouse IgG (Sigma) and IRDye 800CWstreptavidin (LICOR Biosciences), respectively. Plates were air-dried and lipid bands visualized using a LICOR Odysseyscanner.

SDS-PAGE and Western blot analysisBriefly, 1� 105 or 1� 106 cell equivalents of cancer cell lysate,

total lipid extract, and plasma membrane lipid extract weresubjected to SDS-PAGE (4%–12% Bis–Tris NOVEX; Invitrogen),and transferred to Immobilon-FL PVDF membranes (EMDMilli-pore). Membranes were blocked for 1 hour followed by incuba-tion with primary mAbs. mAb binding was detected using bio-tinylated anti-mouse IgG (Sigma) and visualized using IRDye800CW streptavidin (LICOR Biosciences).

Lewis antigen and saliva sandwich ELISAELISA plates were coated overnight at 4�C and processed as

described in Supplementary Materials and Methods.

Glycan array analysismAbs were screened for binding to�600 natural and synthetic

glycans (core H group, version 5.1) by the Consortium forFunctional Glycomics (CFG). Slides were incubated with 1 mg/mLof antibody for 1 hour, before detectionwith Alexa Fluor 488–conjugated secondary mAb.

Immunohistochemistry assessmentNormal and tumor tissue binding was analyzed by im-

munohistochemistry (IHC) as described previously (20).Briefly, after antigen-retrieval, blocking of endogenous per-oxidase activity and nonspecific binding sites, the sectionswere incubated with primary mAbs at room temperature for1 hour. Primary mAb binding was detected by biotinylatedsecondary mAb (Vector Labs) followed by preformed

Translational Relevance

Differentially expressed tumor-associated carbohydrateantigens constitute excellent targets for therapeutic antibodydevelopment. This report describes the discovery of twomonoclonal antibodies (mAbs) that recognize extended non-sialylated Le-containing glycans on tumor glycolipid andglycoproteins with high functional affinity. The FG88 mAbsexhibit comprehensive tumor tissue reactivity combined withlimited normal tissue distribution and excellent immune-mediated anddirect tumor cell killing via a uniquemechanismthat may elicit further immune-mediated tumor regression.Furthermore, the mAbs have independent prognostic value inpatients with colorectal tumors. These favorable attributes,combined with their potent antitumor activity in a mousexenograft model suggest promising clinical potential. In addi-tion, the internalization efficiency of the mAbs and theirlysosomal colocalization make them attractive candidates fordrug conjugation. We anticipate that the excellent preclinicaleffectiveness of our murine mAbs will translate, followinghumanization, into therapeutic antitumor efficacy in theclinic.

Chua et al.

Clin Cancer Res; 21(13) July 1, 2015 Clinical Cancer Research2964




streptavidin-biotin/HRPO (Dako Ltd.) and 3,30-diaminoben-zidine as the substrate. Finally, sections were counterstainedwith hematoxylin. Staining was analyzed via New Viewersoftware 2010 and scored with a semiquantitative histologicscore (H-score, 0–300) taking into account the staining inten-sity: negative (0), weak (1), medium (2), and strong (3) andthe percentage of positive cells. The stained slides were ana-lyzed by two independent observers and a consensus wasagreed. Statistical analysis was performed using SPSS 13.0(SPSS Inc.). Stratification cutoff points for the survival analysiswere determined using X-Tile software (24) and P < 0.05 wereconsidered significant.

Patient cohortsThe study populations include cohorts of consecutive series

of 462 archived colorectal cancer (25) specimens (1994–2000;median follow-up, 42 months; censored December 2003;patients with lymph node–positive disease routinely receivedadjuvant chemotherapy with 5-flurouracil/folinic acid), 350ovarian cancer (26) samples (1982–1997; median follow-up,192 months; censored November 2005; patients with stage IIto IV disease received standard adjuvant chemotherapythat in later years was platinum based), 142 gastric cancer(27) samples (2001–2006; median follow-up, 66 months;censored Jan 2009; no chemotherapy), 68 pancreatic and

Figure 1.A, cell-surface binding of FG88hybridoma supernatants. Binding wasassessed by flow cytometry onCOLO205 cells by (i) FG88.2 and (ii)FG88.7 hybridoma supernatants. mAbSC104 (iii) and mouse IgG (iv), both at3.3 � 10�8 mol/L were positive andnegative control, respectively. B, FG88mAbs recognize Lea, c, x-containingglycans on glycolipid andglycoproteins. i, Western blot analysisof FG88.2 mAb (3.3 � 10�8 mol/L).Lane 1, COLO205 cell lysates (1 � 105

cells); lane 2, COLO205 plasmamembrane (1 � 106 cells); lane 3,COLO205 total lipid extract (1 � 106

cells), and lane 4, COLO205 plasmamembrane lipid extract (1 � 106 cells).ii, HPTLC analysis of FG88.2 binding(3.3 � 10�8 mol/L) to cancer cell(2� 106 cells) glycolipid extracts. Lane1, AGS; lane 2, COLO205. C, ELISAanalysis of binding of FG88 mAbs toLewis glycan–HSA conjugates. The7-Le, 225-Le, FG27.18, and FG27.10mAbs were included as positivecontrols for Lea, Leb, Lex, and Ley,respectively. The negative controlconsisted of omission of the primarymAb. Error bars represent the mean�STDof quadruplicatewells (� ,P<0.05;�� , P < 0.01; and �� , P < 0.001 versuscontrol, ANOVA followed by theBonferroni multiple comparisons test,GraphPad Prism 6. D, Glycan arrayscreening of FG88.2 and FG88.7 (CFG,core H, version 5.1). The top fivebinding glycans are shown as apercentage of the highest bindingglycan with glucosylamine (&),galactose (*), fucose (~), andmannose (*). Sp denotes the lengthof spacer between glycan and slide.The complete results for FG88.2 (42)and FG88.7 (43) have been depositedon the CFG website.

Antitumor Activity of Novel Glycan Monoclonal Antibodies

www.aacrjournals.org Clin Cancer Res; 21(13) July 1, 2015 2965




Chua et al.





120 billary/ampullary cancer (28) samples (1993–2010: medi-an follow-up, 45 months; censored 2012; 25%–46% of pati-ents received adjuvant chemotherapy with 5-flurouracil/folinicacid and gemcitabine), 220 non–small cell lung cancers (Jan-uary 1996–July 2006; median follow-up, 36 months; censoredMay 2013; none of the patients received chemotherapy prior tosurgery but 11 patients received radiotherapy and 9 patientsreceived at least 1 cycle of adjuvant chemotherapy postsurgery)obtained from patients undergoing elective surgical resectionof a histologically proven cancer at Nottingham or DerbyUniversity Hospitals (Derby, United Kingdom). No cases wereexcluded unless the relevant clinicopathologic material/datawere unavailable.

Cell viability analysisCells (1� 103/well) were allowed to adhere prior to incubation

for 72 hours with increasing concentrations of mAbs in thepresence or absence of 20 mmol/L of a pan-caspase inhibitor:carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylke-tone (Z-FMK-VAD), after which cell viability was analyzed usingeither 3H-thymidine (0.5 mCi/well) during the final 24 hours andits incorporation measured using Microscint 0 liquid scintillanton a Topcount NXT (PerkinElmer) or a WST-8 assay (CCK-8;Sigma-Aldrich). Phase-contrast images of mAb-treated HCT-15cells were obtainedwith�10magnification using aNikon EclipseTS100.

Confocal microscopyFG88 mAbs, labeled with Alexa Fluor-488 (495/519 nm)

according to the manufacturer's protocol (Invitrogen), wereadded to HCT-15 cells (1.5 � 105) on coverslips and incubatedfor 1hour at 37�Cbefore removing excessmAbwithHEPES buffer(Invitrogen). Hoechst 33258 (350/461 nm) nucleic acid stain(1 mg/mL; Invitrogen) and LysoTracker deep red (647/668 nm)lysosomal stain (50 nmol/L; Invitrogen) were added during thelast 30 minutes, with Cell Mask Orange (554/567 nm) plasmamembrane stain (2.5 mg/mL; Invitrogen) added during the final10 minutes. Localization of the mAbs was visualized using con-focalmicroscopy through a63� 1.4NAoil objective (ZEISSAX10Observer Z1; Carl Zeiss) with Zen 2009 image acquisitionsoftware.

ADC assayADC was evaluated by measuring the cytotoxicity of

immune-complexed mAbs with a mouse Fab–ZAP secondaryconjugate (Advanced Targeting Systems; ref. 29). Cells wereplated in triplicates overnight into 96-well plates (2,000 cells,90 mL/well). After preincubaton (30 minutes at room temper-ature) of a concentration range of FG88 mAbs with 50 ng of the

Fab–ZAP conjugate, 10 mL of conjugate or free mAb was addedto the wells and incubated for 72 hours. Control wells, con-sisted of cells incubated without conjugate, incubated withsecondary Fab–ZAP without primary mAb and incubated witha control mAb in the presence of Fab–ZAP. Cell viability wasmeasured by 3H-thymidine incorporation during the final 24hours. Results are expressed as a percentage of 3H-thymidineincorporation by cells incubated with conjugate compared withprimary mAb only.

ADCC and CDCADCC and CDC were performed as described previously (23).

51Cr-labeled target cells (5 � 103) were coincubated with 100 mLof peripheral blood mononuclear cells (PBMC), 10% (v/v) auto-logous serum or media alone or with mAbs at a range of con-centrations (E:T ratio of 100:1). Spontaneous and maximumrelease [counts per minute (cpm)] was evaluated by incubatingthe labeled cells with medium or with 10% (v/v) Triton X-100,respectively. The mean percentage lysis was calculated asfollows:mean% lysis¼ (experimental cpm� spontaneous cpm)/maximum cpm � spontaneous cpm) � 100.

Scanning electron microscopyHCT-15 cells (1 � 105) were grown on sterile coverslips for 24

hours prior tomAb (30mg/mL) addition for 2 or 20hours at 37�C.Controls included medium alone and 0.5% (v/v) hydrogenperoxide (H2O2; Sigma). Cells were washed with prewarmed0.1 mol/L sodium cacodylate buffer (SDB) pH 7.4 and fixed with12.5% (v/v) glutaraldehyde for 24 hours. Fixed cells were washedtwice with SDB and postfixed with 1% (v/v) osmium tetroxide(pH 7.4) for 45 minutes. After a final wash with H2O, the cellswere dehydrated in increasing concentrations of ethanol andexposed to critical point drying, before sputtering with gold, priorto scanning electron microscopy (SEM) analysis (JSM-840 SEM,JEOL).

In vivo modelThe study was conducted under a UK Home Office Licence in

accordance with National Cancer Research Institute, LaboratoryAnimal Science Association, and Federation of European Labo-ratory Animal Science Associations guidelines. Age-matched maleMF-1 nude mice (n � 8 for each treatment group; Harlan Labo-ratories) were implanted intraperitoneally with HCT-15HM2DLuX cells and tumor establishment monitored by biolumines-cent imaging. Mice (bioluminescent signal � 1 � 107 p/s) weredosed intravenously (i.v.) biweekly withmAbs (0.1mg) or vehicle(PBS, 100 mL) until day 120. Bioluminescent intensity (BLI) wasevaluated weekly. Briefly, 60 mg/kg D-luciferin substrate wasadministered to anesthetized mice subcutaneously (s.c.), and BLI

Figure 2.Human tissue distribution of FG88.2. A, FG88.2 (2 � 10�9 mol/L) tumor tissue binding. Colorectal, gastric, pancreatic, non–small cell lung, and ovarian tumorTMAs were stained with FG88.2 mAb as described in Materials and Methods. Representative images of (i) negative, (ii) weak, (iii) moderate, and (iv) strongFG88.2 staining are shown (magnification,�20). B, the Kaplan–Meier analysis of progression-free survival in the colorectal cancer TMA cohort showing the impact ofFG88.2 glyco-epitope expression. Significance was determined using the log-rank test. C, FG88.2 (2 � 10�9 mol/L) normal human tissue (AMSBIO) binding.Placenta, 1; bladder, 2; adipose, 3; brain, 4; esophagus, 5; colon, 6; cervix,7; diaphragm, 8; skin, 9; gall bladder, 10; heart, 11; ileum, 12; rectum, 13; skeletal muscle, 14;spleen, 15; stomach, 16; breast, 17; cerebellum, 18; thyroid, 19; uterus, 20; duodenum, 21; pancreas, 22; ovary, 23; tonsil, 24; jejunum, 25; kidney, 26; liver, 27;lung, 28; testis, 29; thymus, 30; and small intestine, 31 (magnification, �20). D, FG88.2 tumor cell binding by flow cytometry. i, FG88.2 binding in the concentrationrange: 1.1 � 10�12

–2 � 10�7 mol/L, is presented as geometric mean (Gm). ii, flow cytometry histograms of isotype control or FG88.2 (2 � 10�7 mol/L) bindingto a subset of cancer cells (COLO205, HCT-15, HT29, and OVCAR3) for SABC determination as described in Supplementary Materials and Methods.






readings were taken 15 minutes after substrate administration. Inthis study, weight loss alone was an unreliable indicator of clinicalendpoint due to increasing tumor burden. Thus, mouse survivalwas determined by morbidity and bioluminescent assessment oftumor burden that provided tumor mass to body weight infor-mation. Data processing and integrity checking occurred in SPSSv21.0, statistical analysis using GraphPad Prism 6. Significantdifferences in tumor sizes were assessed by the Mann–WhitneyU test and survival (Kaplan–Meier) analysis was done using thelog-rank (Mantel–Cox) test.

ResultsIdentification and characterization of two mAbs followingCOLO205 lipid immunizations

Initial screening of hybridoma supernatants for COLO205 cellsurface and lipid reactivity led to the identification of two mAbs,FG88.2 and FG88.7. BothmAbs weremouse IgG3 isotypes with klight chains and strong COLO205 cell-surface reactivity (Fig. 1A).FG88.2 recognized glyco-epitopes on glycolipids and glycopro-teins (molecular weight ranging from 10 to >230 kDa) in lipidextracts and cell lysates fromCOLO205 (Fig. 1B, i). Confirmationof glycolipid reactivity came from HPTLC analysis in whichFG88.2 reacted with distinct glycolipid species extracted fromCOLO205, but not AGS (Fig. 1B, ii). FG88.7 displayed similarglyco-epitope recognition (data not shown). DNA sequencingrevealed that FG88.2 and FG88.7 belonged to the IGHV6-6�01heavy chain and IGKV12-41�01 gene families where they have10 and eight mutations from IGHV6-6�01, and 11 and 12mutations from IGKV12-41�01, respectively. The nature andpattern of the mutations suggests affinity maturation. The twoFG88 mAbs differ by only three residues, two in the CDR andone in the FR region.

FG88mAbs recognize Lec- andLea-containing glycanswith highfunctional affinity

Preliminary characterization of the glycan specificity of bothmAbs, using ELISA on Lewis blood group HSA conjugates,revealed a strong preference for the Lea–HSA conjugate withminimal cross-reactivity against Lex–HSA and no binding toLey-, or Leb–HSA (Fig. 1C). Both FG88 mAbs were then screenedin more detail for reactivity with �600 natural and syntheticglycans by the CFG. The mAbs showed a strong preference forextended type I Lea-related glycans with LecLex, LeaLex, and di-Lea

being the strongest binders (Fig. 1D). In addition, the mAbsbound simple Lea on the array but not Lec or Lex. This wascorroborated by competition experiments in which preincuba-tion of both mAbs with the Lea–HSA conjugate, fully inhibitedCOLO205 binding (Supplementary Fig. S1A), with Lex–HSAbeing less effective. We next examined the Lea–HSA bindingkinetics of our mAbs using SPR (Biacore X). Fitting of thebinding curves revealed strong apparent functional affinity(Kd � 10�10 mol/L) with fast association (�105 1/Ms) and slowdissociation (�10�5 1/s) rates for both mAbs (SupplementaryTable S1). This was in line with EC50 values from Lea–HSA ELISA(Supplementary Table S1 and Supplementary Fig. S1B). Cellularfunctional affinity was lower (Kd � 10�9 mol/L; SupplementaryTable S1 and Supplementary Fig. S1C), reflecting the complexmAb binding at the cell surface with mixed glycolipid/glycopro-tein recognition and the possibility of secondary events (inter-nalization or cell killing).

FG88 mAbs exhibit strong differential tumor/normal tissuereactivity, with colorectal staining being an independentprognostic marker, associated with reduced patient survival

We assessed the tumor-binding potential of FG88.2 by IHCscreening for binding to pancreatic, colorectal, gastric, non–small cell lung (NSCLC), and ovarian TMAs. FG88.2 mAbstained 81% (155 of 192) of pancreatic, 71% (147 of 208) ofcolorectal, 54% (52 of 96) of gastric, 23% (62 of 274) ofNSCLC, and 31% (66 of 217) of ovarian tumor tissuesand examples of FG88.2 staining level of tumor tissues areshown (Fig. 2A). No significant association between FG88.2glyco-epitope expression and clinicopathologic variables wasobserved in any of the tumor TMAs. In the colorectal cancercohort (25), however, FG88.2 glyco-epitope expression wassignificantly associated with Bcl-2 (x2 ¼ 6.607; P ¼ 0.013) andwith fucosyl transferase 3 (FUT3) expression (x2 ¼ 26.712; P ¼0.031). The Kaplan–Meier analysis of disease-free survival ofcolorectal cancer patients revealed a significantly lower meansurvival time in the high FG88.2 glyco-epitope expressinggroup [mean survival, 36 months (high; n ¼ 27) versus 79months (low; n ¼ 184); P ¼ 0.01, log-rank test; Fig. 2B]. Onmultivariate analysis using Cox regression, high FG88.2 glyco-epitope expression in colorectal cancer was a marker of poorprognosis that was independent of stage and vascular invasion(P < 0.001).

The strong tumor reactivity of FG88.2 was compared with itsnormal human tissue distribution (Fig. 2C and SupplementaryTable S2). FG88.2 did not bind most normal tissues, includinglung, liver (parenchyma), heart, brain, and kidney. Positive nor-mal tissue staining included columnar epithelium of gall bladder(weak to moderate), bile duct (moderate), thymus (weak), glan-dular epithelium of colon (moderate), squamous epithelium oftonsil (moderate), and pancreas (weak). Normal human tissuebinding by FG88.7 was similar to FG88.2, with the exception ofrectum (Supplementary Table S2). On a normal cynomegalousmonkey (CN) TMA, FG88.2 exhibited weak staining of skin,colon, ovary, liver, and thymus combined with stronger stainingof glandular epithelium of stomach and small intestine (Supple-mentary Fig. S2A).

As previous studies had shown that Lex was expressed onneutrophils, the binding of the FG88 mAbs to granulocytes andpolymorphonuclear cells (PBMCs) from normal donors wasanalyzed by FACS, where no binding was observed (Supple-mentary Fig. S2B). In addition, Lea and Leb antigens found intissue secretions can adsorb to erythrocytes. We determinedsecretor status of 9 healthy human donors by saliva sandwichELISA (Supplementary Fig. S2C), followed by binding analysisof the FG88 mAbs to erythrocytes from a Lea-positive donor.Neither FG88 mAb bound to erythrocytes (SupplementaryFig. S2D).

As it is difficult to use primary tumors for cytotoxic experimentsdue to their high rate of spontaneous apoptosis, we screenedcancer cell lines as models for FG88 mAb binding and killing.High FG88.2-binding cancer cells comprised COLO205 andHCT-15 [geometric mean (Gm) � 1,000 at saturating mAbconcentration]. The antigen density (SABC) was calculated to be618,000 and 902,000 for HCT-15 and COLO205, respectively.Moderately binding cells (Gm � 100) included HT29 andOVCAR3 (SABC: 88,000 and 23,000, respectively) and low bind-ing cells (Gm < 100) encompassed AGS and DMS79 (SABC:12,000 and 10,000, respectively; Fig. 2D).

Chua et al.





FG88 mAbs mediate direct tumor cytotoxicity via a uniquecaspase-independent mechanism

In order to ascertain whether cancer cell binding by the FG88mAbs affected cell viability, we analyzed mAb-induced PI uptakethat reflects compromisedmembrane integrity andensuing loss ofcell viability (Fig. 3A and Supplementary Fig. S3A). FG88.2induced dose-dependent PI uptake, with strong binding cells,such as HCT-15 and COLO205, being more susceptible than themoderate to weak FG88-binding cells. In addition, proliferationanalysis (WST8-based) of mAb-treated HCT-15 cells revealed adose-dependent growth inhibition with an IC50 of 1.04 � 10�8

mol/L (Supplementary Fig. S3B).

Classical extrinsic apoptotic cell death involves activation ofeffector caspases. We thus evaluated caspase activity in FG88.2-induced cytotoxicity via cellular proliferation analysis in thepresence and absence of the cell permeant pan-caspase inhibitorZ-FMK-VAD. FG88.2 binding to HCT-15 cells resulted in a dose-dependent decrease in cellular proliferation that was not affectedby the presence of Z-FMK-VAD, suggesting no involvement ofcaspase activity (Fig. 3B). In contrast, Z-FMK-VAD prevented FasmAb-mediated apoptosis of Jurkat cells under similar conditions(data not shown). Another hallmark of apoptotic cell death isendonuclease-induced DNA fragmentation. FG88.2 did notinduce DNA fragmentation in HCT-15 cells, in contrast to the

Figure 3.Direct cytotoxic activity of FG88mAbs.A, dose-dependent increase in PIuptake by a range of tumor cell linesas a result of FG88.2 binding (7.4 �10�9

–2 � 10�7 mol/L). B, dose-dependent growth inhibition (3H-thymidine incorporation) of HCT-15cells by FG88.2 (2.2� 10�8

–2.7� 10�10

mol/L) in the presence or absence ofZ-FMK-VAD (20 mmol/L). Nonlinearregression was performed usingGraphPad Prism 6. C, phase-contrastimaging of FG88 mAb-treated HCT-15cells. Images (magnification, �10)showing HCT-15 cells after incubationwith FG88.2 and FG88.7, both at1.3 � 10�7 mol/L, and RPMI medium(negative control). D, SEM analysis ofFG88.2-induced ultrastructural cellularchanges. HCT-15 cellswere treatedwithFG88.2 mAb (2 � 10�7 mol/L) for 2 or20 hours. Medium alone (RPMI) and0.5% (v/v) H2O2 were used as negativeand positive controls, respectively.Arrows indicate mAb-induced pores.






anti-Fas mAb that resulted in Z-FMK-VAD–sensitive DNA frag-mentation in Jurkat cells (Supplementary Fig. S3C). The absenceof caspase involvement combined with a lack of DNA fragmen-tation suggests that FG88.2 induces a direct cytotoxic effect via adistinct nonapoptotic mechanism.

Next, light microscopy and SEMwere used to evaluate FG88.2-induced ultrastructural changes. Light microscopy evaluation ofmAb-treated HCT-15 cells showed evidence of monolayer dis-ruption, cell rounding, and clumping within 24 hours of mAbaddition and a decrease in cell numbers between 24 and 48 hoursafter mAb addition. This was maintained over the 72-hour incu-bation period (Fig. 3C). FG88.2-treated HCT-15 cellular aggre-gates displayed a loss of surface microvilli and the formation ofmembrane blebs and surfacewrinkles (Fig. 3D). Importantly, cell-surface pore formation suggests a cell death mechanism reminis-cent of oncosis. Heterogeneous pore sizes with diameters rangingfrom 0.2 to 1 mmwere observed after 2 hours as well as 20 hours.FG88.7 displayed similar characteristics to FG88.2 with respect tocell binding and cytotoxicity (data not shown).

FG88 mAbs internalize efficientlyConfocal microscopy of Alexa Fluor 488–labeled FG88.2 bind-

ing to HCT-15 cells over a 2-hour period showed efficient inter-nalization of a proportion of the mAbs (Fig. 4A). In addition, theFG88 membrane staining pattern suggests heterogeneous distri-bution of the glyco-epitope in the HCT-15 plasma membrane.Over time, internalized FG88 mAbs colocalized with lysosomalcompartments (Fig. 4A). Similar results were obtained withFG88.7 (data not shown). Importantly, internalization was val-idated through toxicity of Fab–ZAP–FG88 immune complexes

containing saporin (29). Internalization of the Fab–ZAP–FG88.2and Fab–ZAP–FG88.7 complexes, but not the Fab–ZAP aloneor the Fab–ZAP preincubated with a control mAb (data notshown), led to a dose-dependent (IC50 10�11 mol/L) decreasein cell viability of the high glyco-epitope–expressing HCT-15 cells(Fig. 4B). The moderately binding HT29 cells were morerefractory.

FG88 mAbs exhibit excellent immune-mediated cancer cellkilling (ADCC and CDC) in vitro

The ability of the FG88 mAbs to induce COLO205 tumor celldeath in the presence of human PBMCs through ADCC wasinvestigated. Both FG88 mAbs induced potent cell lysis of thehigh-binding COLO205 cells in a concentration-dependent man-ner with EC50 values of 10

�9 mol/L and near 100% lysis at 7.4 �10�9mol/L (Fig. 5A).Next, we analyzed a range of tumor cell linesfor their susceptibility to FG88-mediated ADCC. The FG88mAbssignificantly lysed the high glyco-epitope–expressing COLO205and HCT-15 above the killing observed with PBMCs alone(Fig. 5B). The mAb 791T/36, a murine IgG2b that cannot bindhuman CD16 (30), showed no significant killing over the back-ground observed with PBMCs alone, in the majority of cell lines.PBMC killing in the absence of FG88 mAbs was highest for celllines lacking MHC-I, such as HCT-15 and AGS, and probablyreflects NK killing. Noticeably, less immune-mediated killing wasseen with the FG88mAbs on themoderate to weak bindingHT29and DMS79 cells even at high mAb concentration 6.7 � 10�8 M.The low-binding OVCAR3 and AGS cells were refractory.

In addition, the FG88 mAbs were very efficient at inducingCDC of high-binding HCT-15 cells in the presence of human

120 min100 min80 min60 min

i ii HT29HCT15

–8–9–10–11–12–13–14

0

25

50

75

100

log conc (mol/L) log conc (mol/L)

FG88.7FG88.2

–8–9–10–11–12–13–140

25

50

75

100

% C

ontr

ol

% C

ontr

ol

FG88.7FG88.2

B

A

Figure 4.Cellular internalization of FG88mAbs. A, time-lapse confocal microscopy of Alexa Fluor 488–labeled FG88 mAbs internalizing into live HCT-15 cells. Merged imagestaken every 20 minutes demonstrate FG88 internalization and colocalization with lysosomal compartments (arrows). The nucleus was labeled with Hoechst33258, lysosomal compartments with LysoTracker Deep Red and the plasma membrane with CellMask Orange. Magnification, �60. B, targeted toxin (saporin)delivery by internalized FG88 mAbs in two cancer cell lines [HCT-15 (i) and HT29 (ii)]. The antiproliferative effect of internalized FG88 mAbs preincubatedwith a saporin-linked anti-mouse IgG Fab fragment was evaluated using 3H-thymidine incorporation. Concentrations refer to the FG88 mAbs. Results (mean� STDfrom three independent experiments) arepresented as apercentage of proliferation of cells treatedwith primarymAbsonly; normalized values are shown forHCT-15.Nonlinear regression was performed using GraphPad Prism 6.

Chua et al.





complement, with nanomolar EC50 and over 80% lysis at 7.4 �10�9 mol/L mAb (Fig. 5C). The FG88 mAbs displayed significantCDCactivity against COLO205 cells and to a lesser degreeDMS79cells (Fig. 5D).Noor little CDCwas seen on the low- tomoderate-binding cell lines HT29, OVCAR3, and AGS (data not shown).

In order to relate tumor staining by IHC to the antigen densityrequirement for cell killing, HCT-15 xenograft tumor tissue wasstained with FG88.2 (data not shown). From this analysis, theantigen density required for cell killing by our mAbs was com-parable with a score of 2 to 3 (moderate to strong) in the IHCanalysis of the tumor tissues and accounts for 39% to 53% ofgastrointestinal cancers (Supplementary Table S3). Some of thenormal gastrointestinal tissues also showed weak to moderatestaining but this is mainly apical cells and it is unclear if mAb willbe accessible to these cells.

Potent in vivo antitumor activity by the FG88 mAbs in a humanhepatic metastasis xenograft model

The mouse HCT-15HM2 DLuX human hepatic metastasistumor model was used to investigate the antitumor activity ofthe FG88 mAbs. The HCT-15HM2 DLuX cell line is a biolumi-nescent variant of a liver metastasizing HCT-15 cell line. Carci-noma establishment, growth, and metastasis were assessed non-invasively via optical imaging. FG88mAb treatment was initiated10 days following tumor cell implantation and metastasis to theliver. The mAbs (100 mg) or vehicle (PBS) was administered i.v.twice weekly, for 120 days. FG88.2 and FG88.7 significantlyreduced tumor growth compared with vehicle control, by day59 (P ¼ 0.016, Mann–Whitney U test) and day 65 (P ¼ 0.046,Mann–Whitney U test), respectively (Fig. 6A). FG88.7 mAb treat-

ment led to a significant survival benefit [P ¼ 0.0037; HR, 3.06;95% confidence interval (95% CI), 2.26–17.39, log-rank test]compared with vehicle (Fig. 6B).

DiscussionWe have generated two antiglycolipid IgG3 mAbs through

immunization of mice with COLO205-derived membrane lipidsthat cross-react with glyco-epitopes expressed on a range ofglycoproteins. Glycan array and ELISA analysis showed that theoverall glycoreactivity of FG88.2 and FG88.7 was similar. BothmAbs showed a preference for extended type I chain nonsialylatedLea-containing carbohydrates with LecLex (galb1-3GLcNacb1-3Galb1-4(Fuca1-3)GlcNAc) not previously described as a targetfor cancer therapy. Circulating Lea-containing glycolipids can beadsorbed by erythrocytes depending on the secretor status ofindividuals. Importantly, we found no reactivity of the FG88mAbs with erythrocytes from Lea-positive human donors, sug-gesting that the FG88 preference formore complex Lea-containingglycans precludes erythrocyte reactivity. By relating antigen den-sity by IHC to the density required for cell killing, it was observedthat tumors needed to stain moderately to strongly (2–3) to betargets for FG88.2. This would include 39% to 53% of gastroin-testinal cancers. Importantly, the significant association of strongFG88.2 binding with poor outcome in our colorectal cancercohort, independent of stage and vascular invasion, suggests thatthe most aggressive cancers would benefit from FG88.2 therapy.Earlier work has demonstrated the prognostic value of Lex, SLex,and SLea expression in colorectal carcinomas and the associationof SLea expression with increased metastatic potential (31–34),but our study is the first to demonstrate independent prognostic

COLO205 HCT-15 DMS790

25

50

75

100 FG88.2FG88.7791T/36Serum

FG88.2

ADCC

FG88.7791T/36PBMC

COLO205

HCT-15HT29

DMS79AGS

OVCAR3

% C

ell l

ysis

BA

DC

***

*********

******

*********

***

***********

–8–9–10–110

25

50

75

100

0

25

50

75

100

log conc (mol/L)

log conc (mol/L)

% C

ell l

ysis

% C

ell l

ysis

% C

ell l

ysis

FG88.2FG88.7

–8–9–10–11

0

25

50

75

100

FG88.7FG88.2

Figure 5.In vitro immune effector activity ofFG88 mAbs. A, dose-dependentADCC activity of FG88 mAbs onCOLO205 cells. 51Cr-labeledCOLO205 cells were coincubatedwith increasing concentrations ofFG88 mAbs and human PBMCs (E:Tof 100:1). B, ADCC activity of FG88mAbs on a panel of tumor cell lines.FG88 mAbs and control (791T/36)were used at 6.7 � 10�8 mol/L.C, dose-dependent CDC activity ofFG88mAbs onHCT-15 cells. The 51Cr-labeled HCT-15 cells were incubatedin the presence of increasingconcentrations of FG88 mAbs in thepresence of human serum. D, CDCactivity of FG88 mAbs on a panel oftumor cell lines. FG88 mAbs andcontrol (791T/36) were used6.7� 10�8mol/L. Significance (B andD) versus PBMC or serum control,respectively, was established byANOVA followed by the Bonferronimultiple comparisons test, GraphPadPrism 6.






value for Lea/c expression in colorectal cancer as defined by ourFG88.2 mAb.

Some of the normal gastrointestinal tissues also showed weaktomoderate staining but this ismainly apical cells and it is unclearifmabwill be accessible to these cells. Indeed, SC104mab showedsimilar staining but showednot toxicity in primatemodels andnotoxicity in clinical trials (NCT01447732, SC104/CEP-37250/KHK2804). Lewis antigens are only synthesized by primates dueto FUT3/4 expression. In this context, it was of interest that therewas a significant association between FG88.2 binding and FUT3expression in the colorectal cancer cohort. Formal toxicity studieswould thus need to be done in CN monkeys. Staining of a CNmonkey normal TMA with FG88.2 showed a similar bindingpattern to the normal human tissues.

The FG88 mAbs exhibited a direct growth-inhibitory effect(IC50 �10�8 mol/L) on high glyco-epitope–expressing tumorcells. This was not evidenced on low or moderately expressingtumor cells, suggesting a contribution of the high functionalaffinity with which the FG88 mAbs bind to tumor cell surfaces.The lack of cell killing of moderate to low glyco-epitope–expres-sing cells offers a further level of protection for normal tissues. Thegrowth-inhibitory effect of the FG88 mAbs was characterized bycellular aggregation followed by an effect on cell membraneintegrity and pore formation. More detailed characterization ofthe cytotoxic effect revealed absence of DNA fragmentation andlimited involvement of effector caspases, suggesting a nonapop-totic mechanism. A number of other antiglycan mAbs have beenshown to induce nonapoptotic oncosis-like cell death (35–38).Anti-CD20 mAbs in the treatment of B-cell lymphoma have beenclassified functionally in type I and II mAbs. Type II anti-CD20mAbs, such as tositumomab, are effective at mediating direct cellkilling in a caspase-independent manner, which was associatedwith homotypic cell adhesion and exhibitsmany similarities withthe FG88 mAbs direct cell killing (39). In both cases, release ofcellular content into the extracellular space viamAb-induced poreformation, may constitute a form of immunogenic cancer celldeath (ICD) through the release of danger-associated molecularpatterns ("DAMPS"; ref. 40). This ICD may reinitiate immuneresponses in the immunosuppressive tumor microenvironment.

In contrast, SC104, which recognizes sialylated Lea-related gly-cans, was also cytotoxic to high-binding cells, such asHCT-15 andCOLO205, but with a higher IC50 (2 � 10�7 mol/L) and viaclassical apoptosis with evidence of caspase activation (20).

Two independent approaches demonstrated efficient internal-ization and lysosomal localization of a proportion of the FG88mAbs in a glyco-epitope density-dependent manner. This indi-cates their potential clinical usefulness for ADC development.Furthermore, a fraction of the FG88 mAbs remains at the cellsurface where their slow dissociation kinetics enables excellentimmune-effector functions (ADCCandCDC). Importantly,mod-erate- to weak-binding cells were less susceptible to ADCC andCDC, offering a further level of protection.

Carbohydrate-recognizing mAbs generally exhibit weak glycanaffinity. In contrast, the FG88 mAbs displayed strong functionalaffinity for a high-density Lea–HSA glycoconjugate but nanomo-lar functional affinity for tumor cell lines overexpressing the Lea

-containing glyco-epitope such as COLO205 and HCT-15. Thisprobably reflects themore complex binding behavior of themAbsat the cell surface where the mixed glyco-epitope (glycolipid andglycoprotein) recognition, the fluid membrane, or the potentialfor internalization and cell killing can explain the higher cell-surface Kd. Preliminary studies suggest that the glycolipids areresponsible for the direct cell killing and ADCC/CDC, requiringhigh levels ofmAb, 10�8 and10�9mol/L, respectively. In contrast,internalization occurs predominantly via glycoproteins and atsubnanomolar (10�11 mol/L) concentrations of mAb. This sug-gests that targeting glycans shared between glycoproteins andglycolipids may be ideal for ADC and direct/immune effectorcell killing, respectively; potentially debulking the tumor via ADCfollowed by removing residual disease with mAb alone.

The excellent in vitro cytotoxicity of both mAbs translated inpotent antitumor efficacy and significant survival improvement ina colorectal hepatic metastasis xenograft model in which mAbtreatment was initiated 10 days after tumor initiation and devel-opment of liver metastasis. The FG88 mAbs cured both intraper-itoneal disease and liver metastases in 30% of animals. Postmor-tem imaging analysis of those animals showed very limitedevidence of tumor regrowth. Previous xenografts studies have

020,00040,00060,00080,000

100,000120,000140,000160,000

0 20 40 60

Mea

n %

gro

wth

% S

urvi

val

Days after tumor implantation

FG88.2 (1 mg/mL)

FG88.7 (1 mg/mL)

PBS vehicle

BA

**

0 25 50 75 10012

515

0175 20

0225

250 275

3000

25

50

75

100

Day

FG88.2FG88.7Vehicle

Figure 6.In vivo activity of FG88 mAbs in the mouse colorectal hepatic metastasis model. A, reduction in percentage tumor growth (ventral bioluminescent signal)by FG88 mAbs. FG88.2 and FG88.7 significantly reduced tumor growth by day 59 (¼ 0.016) and day 65 (P¼ 0.046), respectively, compared with the vehicle (PBS)control (Mann–Whitney U test). B, survival benefit due to antitumor activity of FG88.7 and FG88.2. The HCT-15HM2 DLuX mouse xenograft model wasused to compare the antitumor effect of the FG88 mAbs to vehicle control (group size n > 8). MF-1 nude mice received mAbs (100 mg) i.v. biweekly, treatmentwas initiated 10 days after tumor inoculation and the final dose was administered on day 120 (arrow).


Chua et al.




shown good antitumor efficacy when treatment was initiated atthe time of tumor implantation or shortly afterwards (18, 41).No studies, to date, have shown tumor eradication of 10-dayestablished tumors and liver metastases. SC104 inhibited tumorgrowth in vivo, but was more effective in the tumor preventionmodel and only worked in the therapeutic models in combina-tion with 5-FU/leucovorin (21). The humanized SC104(NCT01447732) currently in phase I/II trials (SC104/CEP-37250/KHK2804) has been defucosylated for enhanced effectorfunctions. Further studies have been initiated to evaluate thetherapeutic potential of human IgG1 chimeric versions of theFG88 mAbs and the initial results are encouraging.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.X. Chua, T. Parsons, I. Spendlove, L.G. DurrantDevelopment of methodology: J.X. Chua, M. Vankemmelbeke, R.S. McIntosh,P.A. Clarke, R. Moss, T. Parsons, I. Spendlove, L.G. DurrantAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.X. Chua, M. Vankemmelbeke, R.S. McIntosh,P.A. Clarke, R. Moss, A.M. Zaitoun

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.X. Chua, M. Vankemmelbeke, R.S. McIntosh,P.A. Clarke, L.G. DurrantWriting, review, and/or revision of the manuscript: J.X. Chua, M. Vankem-melbeke, R.S. McIntosh, T. Parsons, I. Spendlove, S. Madhusudan, L.G. DurrantAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.X. Chua, R. Moss, A.M. Zaitoun, S. Madhusu-dan, L.G. DurrantStudy supervision: I. Spendlove, L.G. Durrant

AcknowledgmentsThe authors acknowledge the CFG for glycan array analysis and Tim Self

(University of Nottingham) for confocal microscopy.

Grant SupportThis work was supported by the University of Nottingham Strategic Devel-

opment Fund (to L.G. Durrant).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 24, 2014; revised February 27, 2015; accepted February28, 2015; published OnlineFirst March 16, 2015.

References1. Christiansen MN, Chik J, Lee L, Anugraham M, Abrahams JL, Packer NH.

Cell surface protein glycosylation in cancer. Proteomics 2014;14:525–46.2. Dalziel M, Crispin M, Scanlan CN, Zitzmann N, Dwek RA. Emerging

principles for the therapeutic exploitation of glycosylation. Science 2014;343:1235681.

3. Daniotti JL, Vilcaes AA, Torres Demichelis V, Ruggiero FM, Rodriguez-Walker M. Glycosylation of glycolipids in cancer: basis for development ofnovel therapeutic approaches. Front Oncol 2013;3:306.

4. Hakomori S.Glycosylation defining cancermalignancy: newwine in anoldbottle. Proc Natl Acad Sci U S A 2002;99:10231–3.

5. Fuster MM, Esko JD. The sweet and sour of cancer: glycans as noveltherapeutic targets. Nat Rev Cancer 2005;5:526–42.

6. Hakomori SI, Cummings RD. Glycosylation effects on cancer develop-ment. Glycoconj J 2012;29:565–6.

7. Stanley P, Cummings RD. Structures common to different glycans. In:VarkiA, Cummings RD, Esko JD, et al., editors. Essentials of glycobiology. ColdSpring Harbor Laboratory Press: Cold Spring Harbor, New York; 2009.

8. Hakomori S, Andrews HD. Sphingoglycolipids with Leb activity, and theco-presence of Lea-, Leb-glycolipids in human tumor tissue. BiochimBiophys Acta 1970;202:225–8.

9. Inagaki H, Sakamoto J, Nakazato H, Bishop AE, Yura J. Expression of Lewis(a), Lewis(b), and sialated Lewis(a) antigens in early and advanced humangastric cancers. J Surg Oncol 1990;44:208–13.

10. Sakamoto J, Furukawa K, Cordon-Cardo C, Yin BW, RettigWJ, OettgenHF,et al. Expression of Lewisa, Lewisb, X, andYblood group antigens inhumancolonic tumors and normal tissue and in human tumor-derived cell lines.Cancer Res 1986;46:1553–61.

11. BlaszczykM, Pak KY,HerlynM, Sears HF, Steplewski Z. Characterization ofLewis antigens in normal colon and gastrointestinal adenocarcinomas.Proc Natl Acad Sci U S A 1985;82:3552–6.

12. Cazet A, Julien S, Bobowski M, Burchell J, Delannoy P. Tumour-associated carbohydrate antigens in breast cancer. Breast Cancer Res2010;12:204.

13. Durrant LG, Noble P, Spendlove I. Immunology in the clinic review series;focus on cancer: glycolipids as targets for tumour immunotherapy.Clin Exp Immunol 2012;167:206–15.

14. Rabu C, McIntosh R, Jurasova Z, Durrant L. Glycans as targets for thera-peutic antitumor antibodies. Future Oncol 2012;8:943–60.

15. Yang MC, Hsu FL, Handa K, Hakomori S, Lee MH, Liu LY, et al. Humanmonoclonal antibody GNX-8 directed to extended type 1 chain: specificbinding to human colorectal cancer. Int J Cancer. 2009 Dec 21;doi:10.1002/ijc.25131.

16. Capurro M, Ballare C, Bover L, Portela P, Mordoh J. Differential lyticand agglutinating activity of the anti-Lewis(x) monoclonal antibody FC-2.15 on human polymorphonuclear neutrophils and MCF-7 breasttumor cells. In vitro and ex vivo studies. Cancer Immunol Immunother1999;48:100–8.

17. Ballare C, Portela P, Schiaffi J, Yomha R, Mordoh J. Reactivity of mono-clonal antibody FC-2.15 against drug resistant breast cancer cells. Additivecytotoxicity of adriamycin and taxol with FC-2.15. Breast Cancer Res Treat1998;47:163–70.

18. Watanabe M, Ohishi T, Kuzuoka M, Nudelman ED, Stroud MR, Kubota T,et al. In vitro and in vivo antitumor effects of murine monoclonal antibodyNCC-ST-421 reacting with dimeric Le(a) (Le(a)/Le(a)) epitope. Cancer Res1991;51:2199–204.

19. Stroud MR, Levery SB, Martensson S, Salyan ME, Clausen H, Hakomori S.Human tumor-associated Le(a)-Le(x) hybrid carbohydrate antigen IV3(Gal beta 1–>3[Fuc alpha 1–>4]GlcNAc)III3FucnLc4 defined by mono-clonal antibody 43-9F: enzymatic synthesis, structural characterization,and comparative reactivity with various antibodies. Biochemistry 1994;33:10672–80.

20. Durrant LG, Harding SJ, Green NH, Buckberry LD, Parsons T. A newanticancer glycolipidmonoclonal antibody, SC104, which directly inducestumor cell apoptosis. Cancer Res 2006;66:5901–9.

21. Sawada R, Sun SM, Wu X, Hong F, Ragupathi G, Livingston PO,et al. Human monoclonal antibodies to sialyl-Lewis (CA19.9) withpotent CDC, ADCC, and antitumor activity. Clin Cancer Res 2011;17:1024–32.

22. Watson SA, Morris TM, Crosbee DM, Hardcastle JD. A hepatic invasivehuman colorectal xenograft model. Eur J Cancer 1993;29A:1740–5.

23. Noble P, Spendlove I, Harding S, Parsons T, Durrant LG. Therapeutictargeting of Lewis(y) and Lewis(b)with a novelmonoclonal antibody 692/29. PLoS One 2013;8:e54892.

24. Camp RL, Dolled-Filhart M, Rimm DL. X-tile: a new bio-informatics toolfor biomarker assessment and outcome-based cut-point optimization.Clin Cancer Res 2004;10:7252–9.

25. Watson NF, Madjd Z, Scrimegour D, Spendlove I, Ellis IO, ScholefieldJH, et al. Evidence that the p53 negative/Bcl-2 positive phenotype isan independent indicator of good prognosis in colorectal cancer: atissue microarray study of 460 patients. World J Surg Oncol 2005;3:47.

26. Duncan TJ, Rolland P, Deen S, Scott IV, Liu DT, Spendlove I, et al. Loss ofIFN gamma receptor is an independent prognostic factor in ovarian cancer.Clin Cancer Res 2007;13:4139–45.






27. Abdel-Fatah T, Arora A, Gorguc I, Abbotts R, Beebeejaun S, Storr S, et al. AreDNA repair factors promising biomarkers for personalized therapy ingastric cancer? Antioxid Redox Signal 2013;18:2392–8.

28. Storr SJ, Zaitoun AM, Arora A, Durrant LG, Lobo DN,Madhusudan S, et al.Calpain system protein expression in carcinomas of the pancreas, bile ductand ampulla. BMC Cancer 2012;12:511.

29. Kohls MD, Lappi DA. Mab-ZAP: a tool for evaluating antibody efficacy foruse in an immunotoxin. BioTechniques 2000;28:162–5.

30. Price MR, PimmMV, Page CM, Armitage NC, Hardcastle JD, Baldwin RW.Immunolocalization of themurinemonoclonal antibody, 791T/36withinprimary human colorectal carcinomas and identification of the targetantigen. Br J Cancer 1984;49:809–12.

31. Nakagoe T, Fukushima K, Nanashima A, Sawai T, Tsuji T, Jibiki M, et al.Expression of Lewis(a), sialyl Lewis(a), Lewis(x) and sialyl Lewis(x) anti-gens as prognostic factors in patients with colorectal cancer. Can J Gastro-enterol 2000;14:753–60.

32. Kannagi R. Carbohydrate antigen sialyl Lewis a—its pathophysiologicalsignificance and inductionmechanism in cancer progression. Chang GungMed J 2007;30:189–209.

33. Weston BW, Hiller KM, Mayben JP, Manousos GA, Bendt KM, Liu R, et al.Expression of human alpha(1,3)fucosyltransferase antisense sequencesinhibits selectin-mediated adhesion and liver metastasis of colon carcino-ma cells. Cancer Res 1999;59:2127–35.

34. Inagaki Y, Gao J, Song P, Kokudo N, Nakata M, Tang W. Clinicopatho-logical utility of sialoglycoconjugates in diagnosing and treating colorectalcancer. World J Gastroenterol 2014;20:6123–32.

35. Loo D, Pryer N, Young P, Liang T, Coberly S, King KL, et al. The glycotope-specific RAV12 monoclonal antibody induces oncosis in vitro and hasantitumor activity against gastrointestinal adenocarcinoma tumor xeno-grafts in vivo. Mol Cancer Ther 2007;6:856–65.

36. Tan HL, Fong WJ, Lee EH, Yap M, Choo A. mAb 84, a cytotoxic antibodythat kills undifferentiated human embryonic stem cells via oncosis. StemCells 2009;27:1792–801.

37. Zhang C, Xu Y, Gu J, Schlossman SF. A cell surface receptor defined by amAbmediates a unique type of cell death similar to oncosis. ProcNatl AcadSci U S A 1998;95:6290–5.

38. Hernandez AM, Rodriguez N, Gonzalez JE, Reyes E, Rondon T,Grinan T, et al. Anti-NeuGcGM3 antibodies, actively elicited byidiotypic vaccination in nonsmall cell lung cancer patients, inducetumor cell death by an oncosis-like mechanism. J Immunol 2011;186:3735–44.

39. Alduaij W, Ivanov A, Honeychurch J, Cheadle EJ, Potluri S, Lim SH, et al.Novel type II anti-CD20monoclonal antibody (GA101) evokes homotypicadhesion and actin-dependent, lysosome-mediated cell death in B-cellmalignancies. Blood 2011;117:4519–29.

40. Krysko O, Love Aaes T, Bachert C, Vandenabeele P, Krysko DV. Many facesof DAMPs in cancer therapy. Cell Death Dis 2013;4:e631.

41. Schreiber GJ, Hellstrom KE, Hellstrom I. An unmodified anticarcinomaantibody, BR96, localizes to and inhibits the outgrowth of human tumorsin nude mice. Cancer Res 1992;52:3262–6.

42. Consortium for Functional Glycomics FGG. FG88.2 on core H, version5.1 glycan array. [Internet]. Functional Glycomics Gateway; c2010.Available from: http://www.functionalglycomics.org/glycomics/HServlet?operation¼view&sideMenu¼no&psId¼primscreen_5844. Accessed April8, 2015.

43. Consortium for Functional Glycomics FGG. FG88.7 on core H, version5.1 glycan array. [Internet]. Functional Glycomics Gateway; c2010.Available from: http://www.functionalglycomics.org/glycomics/HServlet?operation¼view&sideMenu¼no&psId=primscreen_5847. Accessed April8, 2015.


Chua et al.




2015;21:2963-2974. Published OnlineFirst March 16, 2015.Clin Cancer Res Jia Xin Chua, Mireille Vankemmelbeke, Richard S. McIntosh, et al. Potent Antitumor Activity

-Related Glycans withxLecMonoclonal Antibodies Targeting Le

Updated version

10.1158/1078-0432.CCR-14-3030doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2015/03/18/1078-0432.CCR-14-3030.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/21/13/2963.full#ref-list-1

This article cites 39 articles, 15 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/21/13/2963.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

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

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/21/13/2963To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-14-3030

http://clincancerres.aacrjournals.org/content/suppl/2015/03/18/1078-0432.CCR-14-3030.DC1

http://clincancerres.aacrjournals.org/content/21/13/2963.full#ref-list-1

http://clincancerres.aacrjournals.org/content/21/13/2963.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/21/13/2963


cancer therapy: preclinical clinical cancer monoclonal ......cancer therapy: preclinical monoclonal...

Documents