HYBRIDOMAVolume 19 Number 3 2000Mary Ann Liebert Inc

A Mouse IgG1 Monoclonal Antibody Specific for N-GlycolylGM3 Ganglioside Recognized Breast and Melanoma Tumors

ADRIANA CARR AILETTE MULLET ZAIMA MAZORRA ANA MARIA VAacuteZQUEZMAURO ALFONSO CIRCE MESA ENRIQUE RENGIFO ROLANDO PEacuteREZ

and LUIS ENRIQUE FERNAacuteNDEZ

ABSTRACT

14F7 murine monoclonal antibody (MAb) is an IgG1 immunoglobulin that is generated by immunizing Balbcmice with GM3(NeuGc) ganglioside hydrophobically conjugated with human very-low-density lipoproteinsand in the presence of Freundrsquos adjuvants 14F7 MAb binds specifically to GM3(NeuGc) whereas neither N-glycolyl or N-acetyl gangliosides nor a sulfated glycolipid are recognized as assessed by enzyme-linked im-munosorbent assay or immunostaining on thin layer chromatograms Immunohistochemical studies in freshtumor tissues showed that 14F7 MAb strongly recognized in antigen expressed in human breast and melanomatumors

241

INTRODUCTION

GANG LIO SIDES are sialic acidndashcontaining glycosphingolipi dsthat are present in the plasma membranes of vertebrates (1)

Some of these molecules have been reported as tumor-associ -ated antigens or tumor markers(2) leading to the use of anti-ganglioside monoclonal antibodies (MAbs) in the diagnosis andtherapy of cancer(34) N-acetyl (NeuAc) and N-glycolyl(NeuGc) neuraminic acids are the most common types of sialicacids found in animals(5) In general the NeuGc residue is notexpressed in human and chicken normal tissues but is widelypresent in other vertebrate (67) In contrast it has been reportedthat Anti-NeuGcndashcontaining ganglioside antibodies(89) recog-nize some human tumors and cancerous cell lines These stud-ies have been carried out using polyclonal or monoclonal anti-bodies of chicken(10) human(11) or murine (12) origin but in allthese tumors minimal levels of NeuGc were reported On theother hand recently we found increased levels of GM3(NeuGc)ganglioside in human breast cancer (13) a finding that certainlymakes this molecule an attractive target for cancer therapy

Several MAbs recognizing NeuGc-containing ganglioside sshowing different binding specificity have been obtained whilesome of them react with an epitope shared by more than oneof these gangliosides as GMR8 or GMR3(14) YK3(15) andP3(1617) other MAbs are highly specific against a particula rganglioside as Y-2-HD1(18) MK-2-34(19) or SHS1(20) A com-mon characteristic of all these MAbs is that they are IgM anti-bodies

Here we describe for the first time the generation and char-acterization of an IgG1 highly specific anti-GM3(NeuGc) MAbthat has been designated as 14F7 This MAb was generated byimmunization of mice with a vaccine formulation containingGM3(NeuGc) hydrophobically conjugated with human very-low-density lipoproteins (VLDL)(21) Noteworthy is the fact thata preliminary immunohistochemical study with 14F7 MAbshowed a strong recognition of breast and melanoma tumor tis-sues

MATERIALS AND METHODS

Animals

Six- to 8-week-old female Balbc mice were purchased fromCENPALAB Havana Cuba

Glycolipids

GM3(NeuAc) and GM3(NeuGc) were obtained from dogand horse erythrocytes using a modification of the Folchmethod(22) GM1 GM2 GD1a GD1b and GT1b were purifiedfrom bovine brain(23) and GD3 was purchased from SigmaGM2(NeuGc) was obtained from the liver of Balbc mice(24)

and (NeuGc-NeuGc)GD3 from bear erythrocytes was kindlysupplied by Y Hashimoto and S Suzuki (Tokyo MetropolitanInstitute of Medical Science Japan) S Sonnino (Milan Uni-versity Italy) and J Portoukalian (Faculty of Medicine Lyon-

Center of Molecular Immunology Havana Cuba

Sud France) kindly supplied ISO3-Gal-Cer and IV3NeuGc a -nLc4Cer respectively

Generation of Mabs

Balbc mice were immunized intramuscularly four times at2-weeks interval with 200 m g of GM3(NeuGc) coupled hy-drophobically with human VLDL and emulsified for the firstinjection in complete Freundrsquos adjuvant and incomplete Fre-undrsquos adjuvant in the subsequent doses Three days after thelast boost the animals were sacrificed and the spleen cells wereharvested and fused with mouse myeloma cell lineP3X63Ag653 using polyethylenglyco l 3000ndash3600 (Sigma StLouis MO) The antibodies produced by the hybridoma weretested by ELISA for their binding to GM3(NeuGc) andGM3(NeuAc) as described below The selected hybridomaswere cloned twice by the limiting dilution method

Enzyme-linked immunosorbent assay (ELISA)

ELISA experiments using gangliosides were carried out aspreviously described (25) Briefly gangliosides (200 ngwell) in50 m L of methanol were dried in 96-well flexible polyviny lchloride-activated microtiter plates (ICNndashFlow Laboratories Oxford Shire UK) for 90 min at 37degC Then the plates wereblocked with 1 bovine serum albumin (BSA) in 005 M TrisHCl buffer pH 78 for 30 min at 37degC Serum samples or hy-bridoma supernatants were incubated for 2 h at 37degC Afterwashing with phosphate-buffere d saline (PBS) the second an-tibody consisting of alkaline phosphatase conjugated goat antimouse IgM 1 IgG (Jackson Immunoresearch Laboratories IncWest Grove PA) diluted 15000 was added and incubated for1 h at 37degC Afterwards the plates were washed again and asubstrate solution of 1 mgmL of p-nitrophenylphos phate(Sigma) in diethanolamin buffer pH 98 was added After 30min the absorbance was measured at 405 nm with an ELISAreader (Organon Teknika Salzburg Austria)

Isotype and subclass determination

MAb isotype was determined by ELISA using biotinylatedgoat anti-mouse IgM ( m chain specific) and goat anti-mouseIgG ( d chain specific) (Jackson Immunoresearch Laboratories) and for subclass analysis biotinylated MAbs specific for mouseIgG1 IgG2a IgG2b and IgG3 (PharMingen San Diego CA)were used

Thin layer chromatography

HPTLC-aluminium sheets (Merck Darmstadt Germany)were used for the glycolipids fractionation The solvent systemused for chromatographic development chromatography waschloroformmethanol025 KCL and 25 M NH3 (541) (vv)TLC plates were stained either with orcinol (neutral glycolipids )or resorcinol reagent(26) (gangliosides) Additionally sulfatedglycolipid was visualized by the Azure A method(27)

Enzyme immunostaining on HPTLC plates

Immunostaining on HPTLC plates was performed as previ-ously reported (28) Briefly after chromatography of glycolipids the plates were soaked for 75 s on hexane containing 01 poly-

isobutylmethacr ylate (Aldrich Chemical Company Inc Mil-waukee WI) After drying the plates were incubated with PBScontaining 1 BSA for 30 min Then plates were incubatedwith 14F7 MAb solution for 1 h at room temperature Afterwashing with PBS the plates were incubated with horseradishperoxidasendashconjugated goat anti-mouse IgG (Jackson Im-munoresearch Laboratories) for 1 h at room temperature Theplates were washed again and incubated with the substrate so-lution consisting of 40 m gmL o-phenylendiami n (Sigma) in 80mM citrate phosphate buffer pH 50 containing 012 H2O2Dipping the plates in PBS stopped the reaction

Immunohistochemical technique

Normal adult tissues were obtained at autopsy from traumavictims within a few hours after death Tumor samples weretaken at surgery frozen in liquid nitrogen and stored at 2 70degCImmunostaining of cryostat sections was performed by theavidin-biotin-per oxidase complex method as previously de-scribed (29) Staining of all tissues was performed with the 33di-Amino Bencidine (Sigma) and H2O2 peroxidase substrate except for melanoma tissues which were stained with the 3-amino-9-ethyl-c arbazole (Sigma) and H2O2 peroxidase sub-strates

RESULTS

Specificity of MAb 14F7 against different glycolipids

One mouse showing the highest IgG antibody titer againstGM3(NeuGc) was selected for the fusion experiment A totalof 1536 culture supernatants from growing hybridomas werescreened by ELISA against GM3(NeuGc) and GM3(NeuAc)and only 55 of the supernatants contained specific antibod-

CARR ET AL242

FIG 1 Specificity of 14F7 MAb against different purifiedgangliosides by ELISA Each well of a 96-well plate was coatedwith 200 ng of gangliosides of 50 m L in methanol Reactivitywas determined with serial dilutions of the purified antibody

ies to GM3(NeuGc) ganglioside and one hybridoma designatedas 14F7 was selected and cloned The MAb produced by thishybridoma belongs to the IgG1 subclass

Specificity test of 14F7 MAb against several gangliosides performed by ELISA showed strong and exclusive reactivitywith GM3(NeuGc) No reactivity was observed with the othermonosialoganglioside s tested (GM3 GM2 GM1 andGM2(NeuGc)) (Fig 1) Antibody binding to different purifiedglycolipids separated on HPTLC plates were also tested (Fig2) 14F7 MAb reacted with GM3(NeuGc) ganglioside but notwith other N-glycolylated gangliosides tested such asGM2(NeuGc) GD3(NeuGc-NeuGc) and IV3NeuGc a -nLc4CerReactivity of 14F7 MAb was not observed with all the NeuAc-containing gangliosides and with the sulfated glycolipid testedThe reactivity of 14F7 MAb against glycolipids was summa-rized in Table 1

Tissue specificity of 14F7 MAb

A preliminary immunohistochemical study was performedwith fresh tissue sections of human benign and malignant tu-mors (Table 2) 14F7 MAb reacted with all ductal infiltratingbreast carcinoma 1818 and melanoma 1010 tissues tested Pos-itive cells showed a cytoplasmatic staining None of the sam-ples from lung carcinoma of different histologycal types andnervous system tumors analyzed were stained with this MAb

However some benign lesions of the breast (such as breast fi-brocystic and breast fibroadenoma) showed a positive stainingto extracellular secretion Figure 3 shows the recognition by14F7 MAb of a breast carcinoma while Figure 4 shows thestrong staining with 14F7 of a melanoma tumor and not of theskin tissue The results of the immunohistochemical analysis ofadult fresh normal tissues are shown in Table 3 14F7 MAbstained the extracellular secretion of breast glands and mucuscells from small intestine and large intestine

DISCUSSION

Some MAbs specific against NeuGc-containing gangliosideshave been reported previously Three murine MAbsmdashY-2-HD-1(18) MK2-34(19) and GMR14(30)mdashwere reported to reactspecifically with GM2(NeuGc) On the other hand one murineand one human MAbs were generated by Ozawa et al(14) andFurokawa et al(11) respectively which recognized NeuGc a 2 R3Gal sequences internal position Also they have obtainedmurine and human MAbs that recognize NeuGc-containingdisialoganglioside s but these MAbs did not react with NeuGc-containing monosialyl derivates YK3 MAb was described toidentify the NeuGc a 2 R 8NeuGc2 R 3Gal b 1 structure in dif-ferent disialo ganglioside s(15) and SHS1 is a very specific MAb

GENERATION OF AN ANTI-GM3(NeuGc)ANTIBODY 243

FIG 2 TLC immunostaining of standard monosialogangl iosides with 14F7 MAb Standard monosialogangl iosides were de-veloped in chloroform methanol 025 KCL in 25 M ammonium solution (AB) Resorcinol staining (A) and immunostain -ing with 14F7 MAb (B)

TA BL E 1 ST RU CT URE OF GLY CO LIPIDS USED IN T HIS STUD Y

Abbreviation Structure Binding TLCi

LacCer Gal b 1-4GlcCer 2IV3 NeuAc a -Gc3Cer GalNeuAc b 4-Gal b 1-4GlcCer 2IV3 NeuAc a -Gc4Cer Gal b 3-GalNeuAc b 4-Gal b 1-4GlcCer 2GM3 NeuAc a 2-3 Gal b 1-4GlcCer 2GM2 GalNAcb 1-4(NeuAc a 2-3) Gal b 1-4GlcCer 2GM1a Gal b 1ndash3 GalNAcb 1-4(NeuAc a 2-3) Gal b 1-4GlcCer 2GD3 NeuAc a 2-8NeuAc a 2-3Gal b 1-4GlcCer 1 1 1GD2 Gal b 1ndash4(NeuAc a 2-8NeuAc a 2-3)Gal b 1-4GlcCer 2GD1a NeuAc a 2-3 Gal b 1ndash3 GalNAcb 1-4(NeuAc a 2-3) Gal b 1-4GlcCer 2GD1b Gal b 1-3GalNAc b 1-4(NeuAc a 2-8Neuac a 2-3)Gal b 1-4GlcCer 2GT1b NeuAc a 2-3Gal b 1-3GalNAc b 1-4(NeuAc a 2-8Neuac a 2-3)Gal b 1-4GlcCer 2GM3(NeuGc) NeuGc a 2-3 Gal b 1-4GlcCer 2GM2(NeuGc) GalNAcb 1-4(NeuGc a 2-3) Gal b 1-4GlcCer 2GD3(NeuGc-NeuGc) NeuGc a 2-8NeuGc a 2-3Gal b 1-4GlcCer 2IV3 NeuGc a -nLc4Cer NeuGc a 2-3Gal b 1-4GlcNeuAc b 1-33Gal b 1-4GlcCer 2I SO3-GalCer HSO3-3GalCer 2

to i-active ganglioside (NeuGc) One common characteristic ofall these MAbs is that they are IgM antibodies

To our knowledge this is the first description of a murineIgG1 MAb specific for N-glycolyl neuraminic acid containinggangliosides The binding specificity of 14F7 MAb was strictlyrestricted to N-glycolyl function since this MAb was able todiscriminate between NeuGc and NeuAc-variant s of GM3 gan-glioside On the other hand the antibody was completely un-reactive with GM2(NeuGc) having NeuGc a 2 R 3Gal structurein an internal position indicating that the addition of one N-acetyl-galactosa mine to the galactose residue affected MAb

binding The finding that 14F7 did not react with GD3 (NeuGc-NeuGc) containing NeuGc a 2 R 8NeuGc2 R 3Gal b 1 terminalstructure suggests that MAb binding is dependent not only onthe external position of the N-glycolyl neuraminic acid groupbut also on the sialic acid linkage These evidence together withthe fact that 14F7 MAb did not bind to IV3NeuGc a nLc4-Cerdiffering from GM3 (NeuGc) in that the terminal NeuGc a 2 R3Gal-structure is linked to GlcNAc suggest that the epitoperecognized by 14F7 comprises the tri-saccharide structureNeuGc a 2 R 3Gal b 1 R 4Glc- Ganglioside recognition patternof 14F7 MAb is different from the previously reported for othermurine and human MAbs which bind not only to GM3 (NeuGc)but also to several gangliosides having NeuGc a 2 R 3Gal-ter-minal structure(111416)

Positive evidences of NeuGc-containing gangliosides in hu-man tumors are still controversial between different groupsHowever it is generally agreed that normal human tissues onlyexpress NeuAc-gangliosides Some articles have described N-glycolylated neuraminic acid in human tumors stained with amurine monoclonal antibody(20) or chicken polyclonal anti-bodies (10) Additionally there are some reports where NeuGc-containing ganglioside composition has been determined bybiochemical analysis in human tumors(3132) and cell lines(8)

We used a preliminary immunohistochemical study with14F7 in normal and tumor sections to clarify if human tumorsexpress NeuGc antigen or not Previous evidences of NeuGcantigen have been shown in breast cancer Breast cancer cellswere detected by membrane immunofluorescence(33) with anti-Hanganutziu Deicher (HD) antibodies where the NeuGc is theimmunogenic residue of the HD antigen Furthermore recentlyour group reported for the first time the chemical quantifica -tion of NeuGc-containing gangliosides in breast cancer On theother hand immunohistochemical analysis in these tissues with3E12 MAb which is specific to the mucin determinant ex-pressed and secreted in breast tumor N-glycolyl sialyl Tn gly-coprotein (34) showed a positive staining in more than 90 of

CARR ET AL244

TABLE 2 REC OGN ITION PA TTER N O F 14F7 MAB

W ITH DIFFERE NT HU M AN TU M ORS

Localization Positive casestotal

BreastFibrocystic disease 59a

Fibroadenoma 55a

Infiltrating ductal carcinom a 1818Skin

Epidermoid carcinoma 09Melanoma 1010

LungNon small cell lung cancer 020Small cell lung carcinom a 010

Central nervous systemOligodendroglioma grade II 03Meningioma 03Multiple glioblastoma 01Meningeal sarcoma 02

Immune systemB cell lymphoma (lymph node) 05T cell lymphoma (lymph node) 03Sezary syndrome 02

aPositive staining of extracellular secretion

FIG 3 Intense immunoperoxidase staining of infiltrating ductal breast carcinoma showing membrane and cytoplasmatic recog-nition with 14F7 MAb ( 3 500)

the breast tumor samples Another MAb (P3) reported by ourgroup binds to several NeuGc containing gangliosides and alsoreacts with sulfated glycolipids showing a positive staining ofthe breast tumors and lymph node metastases samples How-ever 14F7 which is a more specific anti-GM3(NeuGc) gan-glioside MAb is also capable of showing a strong cytoplas -matic and membrane staining in breast malignant tumor cells(Ductal infiltrating carcinoma) This finding allows us to pre-clude that the structure recognized in breast cancer tissues couldbe the oligosaccharide core of GM3(NeuGc) ganglioside inglyco-lipids glycoproteins or mimicries of this antigen On theother hand benign disease or normal tissues showed positivestaining only in the extracellular secretions

Immunohistochemical experiments showed that melanomatumors were stained by 14F7 MAb but no reactivity was foundwith normal skin The expression of NeuGc-containing gan-gliosides in melanoma cells has been contradictory Hirabayash iet al(35) reported the presence of different HD gangliosides inmelanoma tumors in small amount by enzyme TLC immunos-taining Furthermore Kawachi and Saida(36) described that theglycoprotein fractions of human melanoma tissues were recog-nized by chicken anti-HD (anti-NeuGc lactosylceramid e) anti-body by Western Blotting assay Saida et al(37) has concludedthat only melanoma tumors were clearly positive after com-paring the NeuGc ganglioside expression in melanoma tumorsmelanocytic nevus and melanocytes from normal skin Fur-thermore we have recently demonstrated that the 14F7 MAbbehaves like P3 MAb also recognizing melanoma tumors (per-sonal communication)

Although different biochemistry studies have shown eitherthe absence or low level expression of these gangliosides inmelanoma Doreacute et al(38) and Nakarai et al(39) reported two pre-vious evidences about the HD antigen as a possible target forimmunotherapy in melanoma patients Doreacute et al(38) demon-strated the best prognosis in patients with IgG anti-GM3(NeuGc)gangliosides after the immunization with vaccinia virus human

GENERATION OF AN ANTI-GM3(NeuGc)ANTIBODY 245

FIG 4 Strong immunoperoxidase reactivity with 14F7 MAb in melanoma tumors and not with normal skin tissue ( 3 250)

TA BLE 3 REC OGN ITIO N PATT ER N OF 14F7 MAB W ITH

DIFFER ENT NORM AL AD ULT HUM AN TISSU ES

Tissues Positive casestotal

Urogenital tractKidney 07Prostate 05Ovary 05Breast gland (secretion) 34

Endocrine systemAdrenal gland 03

Immune systemTonsils 05Spleen 03

Central nervous systemBrain

Neuron 05Glias 05

Cerebellum 05Skin

Keratinocytes 07Melanocytes 07

Respiratory tractBronchial epithelium 07Neumocytes 07

Gastrointestinal tractEsophagus 07Stomach 07Small intestine (mucus cell) 55Large intestine (mucus cell) 77Pancreas 07Liver 07

melanoma oncolysate while Nakarai et al(39) reported anti-HDantibodies in the sera of melanoma patients with strong recog-nition of HD antigen Additionally higher levels of IgG and IgManti-HD antibodies were found in patients who were disease-

free for more than 5 years after surgery in comparison with thosewho relapsed within 2 years The immunohistochemistry studywith 14F7 MAb showed strong staining of tumor cells (morethan 90 of positive cells) This evidence together with the re-sults of the P3 staining in melanoma tumors could be an addi-tional support to the hypothesis about the role of N-glycolylatedGM3 as target for cancer immunotherapy

The heterophilic characteristics of HD antigen on glycolipidsor glycoproteins as well as the nonexpression in normal tissuesin human and chickens have been reported Although some anti-HD antibodies have recognized colon tumors by immunohisto-chemistry we have not yet tested 14F7 MAb in these tumorstissues However the staining of mucus cells but not secretionof normal small and large intestine was observed Studies withother fresh human tumor samples from different localizationsare in progress in our laboratory

Our results indicate that the 14F7 MAb could be a possiblecandidate for diagnosis used as a therapeutic agent in melanomaand breast tumors

ACKNOWLEDGMENTS

We thank Dr Francisco Estevez for photography assistance We also thank Dr Blanca Tormo and Ernesto Moreno for theirhelpful advice

REFERENCES

1 Stults CLM Sweeley CC and Matcher BA Glycosphingolipi dsstructure biological source and properties Methods Enzymol1989179167 ndash214

2 Hakomori SH Possible functions of tumor-associated carbohydrateantigens Curr Opin Immunol 19913646 ndash653

3 Hougton AN Mintzer D Cordon-Cardo C Welt S Fliegel B Vad-han S Carswell E Melamed MR Oettgen HF Old LJ Mouse mon-oclonal antibody IgG3 antibody detecting GD3 ganglioside a phaseI trial in patients with malignant melanoma Proc Natl Acad SciUSA 1985821242 ndash1246

4 Zhang S Cordon Cardo C Zhang HS Reuter VE Adluri S Hamil-ton WB Lloyd KO and Livingston PO Selection of carbohydratetumor antigens as targets for immune attack using immunohisto -chemistry I Focus on gangliosides Int J Cancer 19977342 ndash49

5 Corfield AP and Schauer R Occurrence of sialic acids Cell BiolMonogr 1982105ndash50

6 Leeden RW and Yu RK Chemistry and analysis of sialic acid InBiological Role of Sialic Acid Rosemberg A and Schengtrund C-L (Eds) Plenum Press New York 1976 pp 1ndash48

7 Kawai T Kato A Higashi H Kato S and Naiki M Quantitativedetermination of N-glycolylneura minic acid expression in humancancerous tissues and avian lymphoma cell lines as a tumor-asso-ciated sialic acid by gas chromatographyndashmass spectometry Can-cer Res 1991511242 ndash1246

8 Higashi H Sasabe T Fukui Y Maru M and Kato S Detection ofgangliosides as N-glycolylneuram inic acidndashspecific tumor-associ-ated Hanganutziu-D eicher antigen in human retinoblastoma cellsJpn J Cancer Res 198879952 ndash956

9 Fukui Y Maru M Ohkawara KI Miyake T Osada Y Wang DIto T Higashi H Naiki M Wakamiya N and Kato S Detectionof glycoproteins as tumor-associated Hanganutziu-D eicher antigenin human gastric cancer cell line NUGC4 Biochem Biophys ResCommun 19891601149 ndash1154

10 Koda T Shimosakoda T Asaoka H Nishinaka S Tamura I NakabaH and Matsuda H Detection of the Hanganutziu-D eicher antigenin patients with hepatocellular carcinoma Int Hepatol Commun19942310 ndash315

11 Furukawa K Yamaguchi H Oettgen HF Old LJ and Lloyd KOAnalysis of the expression of N-glycolylneuraminic acidndashcontain-ing gangliosides in cell and tissues using two human monoclona lantibodies J Biol Chem 198826318507 ndash18512

12 Miyake M Hashimoto K Ito M Ogawa O Arai E Hitomi S andKannagi R The abnormal occurrence and the differentiation-d e-pendent distribution of N-acetyl and N-glycolyl species of the gan-glioside GM2 in human germ cell tumors A study with specificmonoclonal antibodies Cancer 199065499 ndash505

13 Marquina G Waki H Fernaacutendez LE Kon K Carr A Valiente OPeacuterez R and Ando S Gangliosides expressed in human breast can-cer Cancer Res 1996565165 ndash5171

14 Ozawa H Kawashima Y and Tai T Generation of murine mono-clonal antibodies specific for N-glycolylneuram inic acid contain-ing gangliosides Arch Biochem Biophys 1992294427 ndash433

15 Nakamura K Suzuki H Hirabayashi Y and Suzuki A IV3

a (NeuGc a 2-8NeuGc)-Gg4Cer is restricted to CD4 1 T cells pro-ducing interleukin 2 and small population of mature thymocytes inmice J Biol Chem 19952703876 ndash3881

16 Vazquez AM Alfonso M Lanne B Karlsson K-A Carr A Bar-roso O Fernaacutendez LE Rengifo E Lanio ME Alvarez C ZeuthenJ and Peacuterez R Generation of murine monoclonal antibody spe-cific for N-glycolylneuraminic acid containing gangliosides thatalso recognizes sulfated glycolipids Hybridoma 199514551 ndash556

17 Moreno E Lanne B Vazquez AM Kawashima I Tai T Fernaacuten-dez LE Karlsson K-A Angstrom J and Peacuterez R Delineation ofepitope recognized by an antibody specific for N-glycolylneu-raminic acidndashcontaining gangliosides Glycobiology 19988695 ndash705

18 Sanai Y Yamasaki M and Nagai Y Monoclonal antibody directedto Hanganutziu-D eicher active ganglioside GM2 (NeuGc) BiochimBiophys Acta 1988958368 ndash374

19 Miyake M Ito M Hitomi SH Ikeda S Taki T Kurata M Ino AMiyake N and Kannagi R Generation of murine monoclonal an-tibodies that can discriminate N-glycolyl and N-acetyl neuraminicacid residues of GM2 ganglioside Cancer Res 198886154 ndash6160

20 Watarai S Kushi Y Shigito R Misawa N Eishi Y Handa S andYasuda T Production of monoclonal antibodies directed toHanganutziu Deicher active ganglioside N-glycolylneuram inicacidndashcontaining gangliosides J Biochem 19951171062 ndash1069

21 Dumontet C Rebbaa A and Pourtokalian J Very low densitylipoproteins and interleukin 2 enhance the immunogenicity of 9-O-acetyl-GD3 ganglioside in Balbc mice J Immunol Methods1997206115 ndash123

22 Folch PJ Arsove S and Meath JA Isolation of brain stradin a newtype of large molecular tissue component J Biol Chem 1951191 819ndash831

23 Svenerholm L Ganglioside isolation Methods Carbohydr Chem19766464 ndash474

24 Hashimoto Y Otsuka H Sudo K Susuki K and Yamakawa T Ge-netic regulation of GM2 expression in liver mouse J Biochem198393895 ndash901

25 Alfonso M Vazquez A Carr A Haerslev T Fernaacutendez LE LanioME Alvarez C Zeuthen J and Peacuterez R T cellndashindependent B cellresponse to self-monosialo gangliosides primary response mono-clonal antibodies Hybridoma 199514209 ndash216

26 Svenerholm L Quantitative estimation of sialic acid II A colori-metric resorcinol-hydrochoric acid methods Biochem BiophysActa 195724604 ndash611

27 Kean EL Rapid sensitive spectrofotometric methods for quantita -tive determination of sulfatide J Lipid Res 19689319 ndash327

28 Kawashima I Ozawa H Kotami M Susuki M Kawano T Go-

CARR ET AL246

ies to GM3(NeuGc) ganglioside and one hybridoma designatedas 14F7 was selected and cloned The MAb produced by thishybridoma belongs to the IgG1 subclass

Specificity test of 14F7 MAb against several gangliosides performed by ELISA showed strong and exclusive reactivitywith GM3(NeuGc) No reactivity was observed with the othermonosialoganglioside s tested (GM3 GM2 GM1 andGM2(NeuGc)) (Fig 1) Antibody binding to different purifiedglycolipids separated on HPTLC plates were also tested (Fig2) 14F7 MAb reacted with GM3(NeuGc) ganglioside but notwith other N-glycolylated gangliosides tested such asGM2(NeuGc) GD3(NeuGc-NeuGc) and IV3NeuGc a -nLc4CerReactivity of 14F7 MAb was not observed with all the NeuAc-containing gangliosides and with the sulfated glycolipid testedThe reactivity of 14F7 MAb against glycolipids was summa-rized in Table 1

Tissue specificity of 14F7 MAb

A preliminary immunohistochemical study was performedwith fresh tissue sections of human benign and malignant tu-mors (Table 2) 14F7 MAb reacted with all ductal infiltratingbreast carcinoma 1818 and melanoma 1010 tissues tested Pos-itive cells showed a cytoplasmatic staining None of the sam-ples from lung carcinoma of different histologycal types andnervous system tumors analyzed were stained with this MAb

However some benign lesions of the breast (such as breast fi-brocystic and breast fibroadenoma) showed a positive stainingto extracellular secretion Figure 3 shows the recognition by14F7 MAb of a breast carcinoma while Figure 4 shows thestrong staining with 14F7 of a melanoma tumor and not of theskin tissue The results of the immunohistochemical analysis ofadult fresh normal tissues are shown in Table 3 14F7 MAbstained the extracellular secretion of breast glands and mucuscells from small intestine and large intestine

DISCUSSION

Some MAbs specific against NeuGc-containing gangliosideshave been reported previously Three murine MAbsmdashY-2-HD-1(18) MK2-34(19) and GMR14(30)mdashwere reported to reactspecifically with GM2(NeuGc) On the other hand one murineand one human MAbs were generated by Ozawa et al(14) andFurokawa et al(11) respectively which recognized NeuGc a 2 R3Gal sequences internal position Also they have obtainedmurine and human MAbs that recognize NeuGc-containingdisialoganglioside s but these MAbs did not react with NeuGc-containing monosialyl derivates YK3 MAb was described toidentify the NeuGc a 2 R 8NeuGc2 R 3Gal b 1 structure in dif-ferent disialo ganglioside s(15) and SHS1 is a very specific MAb

GENERATION OF AN ANTI-GM3(NeuGc)ANTIBODY 243

FIG 2 TLC immunostaining of standard monosialogangl iosides with 14F7 MAb Standard monosialogangl iosides were de-veloped in chloroform methanol 025 KCL in 25 M ammonium solution (AB) Resorcinol staining (A) and immunostain -ing with 14F7 MAb (B)

TA BL E 1 ST RU CT URE OF GLY CO LIPIDS USED IN T HIS STUD Y

Abbreviation Structure Binding TLCi

LacCer Gal b 1-4GlcCer 2IV3 NeuAc a -Gc3Cer GalNeuAc b 4-Gal b 1-4GlcCer 2IV3 NeuAc a -Gc4Cer Gal b 3-GalNeuAc b 4-Gal b 1-4GlcCer 2GM3 NeuAc a 2-3 Gal b 1-4GlcCer 2GM2 GalNAcb 1-4(NeuAc a 2-3) Gal b 1-4GlcCer 2GM1a Gal b 1ndash3 GalNAcb 1-4(NeuAc a 2-3) Gal b 1-4GlcCer 2GD3 NeuAc a 2-8NeuAc a 2-3Gal b 1-4GlcCer 1 1 1GD2 Gal b 1ndash4(NeuAc a 2-8NeuAc a 2-3)Gal b 1-4GlcCer 2GD1a NeuAc a 2-3 Gal b 1ndash3 GalNAcb 1-4(NeuAc a 2-3) Gal b 1-4GlcCer 2GD1b Gal b 1-3GalNAc b 1-4(NeuAc a 2-8Neuac a 2-3)Gal b 1-4GlcCer 2GT1b NeuAc a 2-3Gal b 1-3GalNAc b 1-4(NeuAc a 2-8Neuac a 2-3)Gal b 1-4GlcCer 2GM3(NeuGc) NeuGc a 2-3 Gal b 1-4GlcCer 2GM2(NeuGc) GalNAcb 1-4(NeuGc a 2-3) Gal b 1-4GlcCer 2GD3(NeuGc-NeuGc) NeuGc a 2-8NeuGc a 2-3Gal b 1-4GlcCer 2IV3 NeuGc a -nLc4Cer NeuGc a 2-3Gal b 1-4GlcNeuAc b 1-33Gal b 1-4GlcCer 2I SO3-GalCer HSO3-3GalCer 2

to i-active ganglioside (NeuGc) One common characteristic ofall these MAbs is that they are IgM antibodies

To our knowledge this is the first description of a murineIgG1 MAb specific for N-glycolyl neuraminic acid containinggangliosides The binding specificity of 14F7 MAb was strictlyrestricted to N-glycolyl function since this MAb was able todiscriminate between NeuGc and NeuAc-variant s of GM3 gan-glioside On the other hand the antibody was completely un-reactive with GM2(NeuGc) having NeuGc a 2 R 3Gal structurein an internal position indicating that the addition of one N-acetyl-galactosa mine to the galactose residue affected MAb

binding The finding that 14F7 did not react with GD3 (NeuGc-NeuGc) containing NeuGc a 2 R 8NeuGc2 R 3Gal b 1 terminalstructure suggests that MAb binding is dependent not only onthe external position of the N-glycolyl neuraminic acid groupbut also on the sialic acid linkage These evidence together withthe fact that 14F7 MAb did not bind to IV3NeuGc a nLc4-Cerdiffering from GM3 (NeuGc) in that the terminal NeuGc a 2 R3Gal-structure is linked to GlcNAc suggest that the epitoperecognized by 14F7 comprises the tri-saccharide structureNeuGc a 2 R 3Gal b 1 R 4Glc- Ganglioside recognition patternof 14F7 MAb is different from the previously reported for othermurine and human MAbs which bind not only to GM3 (NeuGc)but also to several gangliosides having NeuGc a 2 R 3Gal-ter-minal structure(111416)

Positive evidences of NeuGc-containing gangliosides in hu-man tumors are still controversial between different groupsHowever it is generally agreed that normal human tissues onlyexpress NeuAc-gangliosides Some articles have described N-glycolylated neuraminic acid in human tumors stained with amurine monoclonal antibody(20) or chicken polyclonal anti-bodies (10) Additionally there are some reports where NeuGc-containing ganglioside composition has been determined bybiochemical analysis in human tumors(3132) and cell lines(8)

We used a preliminary immunohistochemical study with14F7 in normal and tumor sections to clarify if human tumorsexpress NeuGc antigen or not Previous evidences of NeuGcantigen have been shown in breast cancer Breast cancer cellswere detected by membrane immunofluorescence(33) with anti-Hanganutziu Deicher (HD) antibodies where the NeuGc is theimmunogenic residue of the HD antigen Furthermore recentlyour group reported for the first time the chemical quantifica -tion of NeuGc-containing gangliosides in breast cancer On theother hand immunohistochemical analysis in these tissues with3E12 MAb which is specific to the mucin determinant ex-pressed and secreted in breast tumor N-glycolyl sialyl Tn gly-coprotein (34) showed a positive staining in more than 90 of

CARR ET AL244

TABLE 2 REC OGN ITION PA TTER N O F 14F7 MAB

W ITH DIFFERE NT HU M AN TU M ORS

Localization Positive casestotal

BreastFibrocystic disease 59a

Fibroadenoma 55a

Infiltrating ductal carcinom a 1818Skin

Epidermoid carcinoma 09Melanoma 1010

LungNon small cell lung cancer 020Small cell lung carcinom a 010

Central nervous systemOligodendroglioma grade II 03Meningioma 03Multiple glioblastoma 01Meningeal sarcoma 02

Immune systemB cell lymphoma (lymph node) 05T cell lymphoma (lymph node) 03Sezary syndrome 02

aPositive staining of extracellular secretion

FIG 3 Intense immunoperoxidase staining of infiltrating ductal breast carcinoma showing membrane and cytoplasmatic recog-nition with 14F7 MAb ( 3 500)

the breast tumor samples Another MAb (P3) reported by ourgroup binds to several NeuGc containing gangliosides and alsoreacts with sulfated glycolipids showing a positive staining ofthe breast tumors and lymph node metastases samples How-ever 14F7 which is a more specific anti-GM3(NeuGc) gan-glioside MAb is also capable of showing a strong cytoplas -matic and membrane staining in breast malignant tumor cells(Ductal infiltrating carcinoma) This finding allows us to pre-clude that the structure recognized in breast cancer tissues couldbe the oligosaccharide core of GM3(NeuGc) ganglioside inglyco-lipids glycoproteins or mimicries of this antigen On theother hand benign disease or normal tissues showed positivestaining only in the extracellular secretions

Immunohistochemical experiments showed that melanomatumors were stained by 14F7 MAb but no reactivity was foundwith normal skin The expression of NeuGc-containing gan-gliosides in melanoma cells has been contradictory Hirabayash iet al(35) reported the presence of different HD gangliosides inmelanoma tumors in small amount by enzyme TLC immunos-taining Furthermore Kawachi and Saida(36) described that theglycoprotein fractions of human melanoma tissues were recog-nized by chicken anti-HD (anti-NeuGc lactosylceramid e) anti-body by Western Blotting assay Saida et al(37) has concludedthat only melanoma tumors were clearly positive after com-paring the NeuGc ganglioside expression in melanoma tumorsmelanocytic nevus and melanocytes from normal skin Fur-thermore we have recently demonstrated that the 14F7 MAbbehaves like P3 MAb also recognizing melanoma tumors (per-sonal communication)

Although different biochemistry studies have shown eitherthe absence or low level expression of these gangliosides inmelanoma Doreacute et al(38) and Nakarai et al(39) reported two pre-vious evidences about the HD antigen as a possible target forimmunotherapy in melanoma patients Doreacute et al(38) demon-strated the best prognosis in patients with IgG anti-GM3(NeuGc)gangliosides after the immunization with vaccinia virus human

GENERATION OF AN ANTI-GM3(NeuGc)ANTIBODY 245

FIG 4 Strong immunoperoxidase reactivity with 14F7 MAb in melanoma tumors and not with normal skin tissue ( 3 250)

TA BLE 3 REC OGN ITIO N PATT ER N OF 14F7 MAB W ITH

DIFFER ENT NORM AL AD ULT HUM AN TISSU ES

Tissues Positive casestotal

Urogenital tractKidney 07Prostate 05Ovary 05Breast gland (secretion) 34

Endocrine systemAdrenal gland 03

Immune systemTonsils 05Spleen 03

Central nervous systemBrain

Neuron 05Glias 05

Cerebellum 05Skin

Keratinocytes 07Melanocytes 07

Respiratory tractBronchial epithelium 07Neumocytes 07

Gastrointestinal tractEsophagus 07Stomach 07Small intestine (mucus cell) 55Large intestine (mucus cell) 77Pancreas 07Liver 07

melanoma oncolysate while Nakarai et al(39) reported anti-HDantibodies in the sera of melanoma patients with strong recog-nition of HD antigen Additionally higher levels of IgG and IgManti-HD antibodies were found in patients who were disease-

free for more than 5 years after surgery in comparison with thosewho relapsed within 2 years The immunohistochemistry studywith 14F7 MAb showed strong staining of tumor cells (morethan 90 of positive cells) This evidence together with the re-sults of the P3 staining in melanoma tumors could be an addi-tional support to the hypothesis about the role of N-glycolylatedGM3 as target for cancer immunotherapy

The heterophilic characteristics of HD antigen on glycolipidsor glycoproteins as well as the nonexpression in normal tissuesin human and chickens have been reported Although some anti-HD antibodies have recognized colon tumors by immunohisto-chemistry we have not yet tested 14F7 MAb in these tumorstissues However the staining of mucus cells but not secretionof normal small and large intestine was observed Studies withother fresh human tumor samples from different localizationsare in progress in our laboratory

Our results indicate that the 14F7 MAb could be a possiblecandidate for diagnosis used as a therapeutic agent in melanomaand breast tumors

ACKNOWLEDGMENTS

We thank Dr Francisco Estevez for photography assistance We also thank Dr Blanca Tormo and Ernesto Moreno for theirhelpful advice

REFERENCES

1 Stults CLM Sweeley CC and Matcher BA Glycosphingolipi dsstructure biological source and properties Methods Enzymol1989179167 ndash214

2 Hakomori SH Possible functions of tumor-associated carbohydrateantigens Curr Opin Immunol 19913646 ndash653

3 Hougton AN Mintzer D Cordon-Cardo C Welt S Fliegel B Vad-han S Carswell E Melamed MR Oettgen HF Old LJ Mouse mon-oclonal antibody IgG3 antibody detecting GD3 ganglioside a phaseI trial in patients with malignant melanoma Proc Natl Acad SciUSA 1985821242 ndash1246

4 Zhang S Cordon Cardo C Zhang HS Reuter VE Adluri S Hamil-ton WB Lloyd KO and Livingston PO Selection of carbohydratetumor antigens as targets for immune attack using immunohisto -chemistry I Focus on gangliosides Int J Cancer 19977342 ndash49

5 Corfield AP and Schauer R Occurrence of sialic acids Cell BiolMonogr 1982105ndash50

6 Leeden RW and Yu RK Chemistry and analysis of sialic acid InBiological Role of Sialic Acid Rosemberg A and Schengtrund C-L (Eds) Plenum Press New York 1976 pp 1ndash48

7 Kawai T Kato A Higashi H Kato S and Naiki M Quantitativedetermination of N-glycolylneura minic acid expression in humancancerous tissues and avian lymphoma cell lines as a tumor-asso-ciated sialic acid by gas chromatographyndashmass spectometry Can-cer Res 1991511242 ndash1246

8 Higashi H Sasabe T Fukui Y Maru M and Kato S Detection ofgangliosides as N-glycolylneuram inic acidndashspecific tumor-associ-ated Hanganutziu-D eicher antigen in human retinoblastoma cellsJpn J Cancer Res 198879952 ndash956

9 Fukui Y Maru M Ohkawara KI Miyake T Osada Y Wang DIto T Higashi H Naiki M Wakamiya N and Kato S Detectionof glycoproteins as tumor-associated Hanganutziu-D eicher antigenin human gastric cancer cell line NUGC4 Biochem Biophys ResCommun 19891601149 ndash1154

10 Koda T Shimosakoda T Asaoka H Nishinaka S Tamura I NakabaH and Matsuda H Detection of the Hanganutziu-D eicher antigenin patients with hepatocellular carcinoma Int Hepatol Commun19942310 ndash315

11 Furukawa K Yamaguchi H Oettgen HF Old LJ and Lloyd KOAnalysis of the expression of N-glycolylneuraminic acidndashcontain-ing gangliosides in cell and tissues using two human monoclona lantibodies J Biol Chem 198826318507 ndash18512

12 Miyake M Hashimoto K Ito M Ogawa O Arai E Hitomi S andKannagi R The abnormal occurrence and the differentiation-d e-pendent distribution of N-acetyl and N-glycolyl species of the gan-glioside GM2 in human germ cell tumors A study with specificmonoclonal antibodies Cancer 199065499 ndash505

13 Marquina G Waki H Fernaacutendez LE Kon K Carr A Valiente OPeacuterez R and Ando S Gangliosides expressed in human breast can-cer Cancer Res 1996565165 ndash5171

14 Ozawa H Kawashima Y and Tai T Generation of murine mono-clonal antibodies specific for N-glycolylneuram inic acid contain-ing gangliosides Arch Biochem Biophys 1992294427 ndash433

15 Nakamura K Suzuki H Hirabayashi Y and Suzuki A IV3

a (NeuGc a 2-8NeuGc)-Gg4Cer is restricted to CD4 1 T cells pro-ducing interleukin 2 and small population of mature thymocytes inmice J Biol Chem 19952703876 ndash3881

16 Vazquez AM Alfonso M Lanne B Karlsson K-A Carr A Bar-roso O Fernaacutendez LE Rengifo E Lanio ME Alvarez C ZeuthenJ and Peacuterez R Generation of murine monoclonal antibody spe-cific for N-glycolylneuraminic acid containing gangliosides thatalso recognizes sulfated glycolipids Hybridoma 199514551 ndash556

17 Moreno E Lanne B Vazquez AM Kawashima I Tai T Fernaacuten-dez LE Karlsson K-A Angstrom J and Peacuterez R Delineation ofepitope recognized by an antibody specific for N-glycolylneu-raminic acidndashcontaining gangliosides Glycobiology 19988695 ndash705

18 Sanai Y Yamasaki M and Nagai Y Monoclonal antibody directedto Hanganutziu-D eicher active ganglioside GM2 (NeuGc) BiochimBiophys Acta 1988958368 ndash374

19 Miyake M Ito M Hitomi SH Ikeda S Taki T Kurata M Ino AMiyake N and Kannagi R Generation of murine monoclonal an-tibodies that can discriminate N-glycolyl and N-acetyl neuraminicacid residues of GM2 ganglioside Cancer Res 198886154 ndash6160

20 Watarai S Kushi Y Shigito R Misawa N Eishi Y Handa S andYasuda T Production of monoclonal antibodies directed toHanganutziu Deicher active ganglioside N-glycolylneuram inicacidndashcontaining gangliosides J Biochem 19951171062 ndash1069

21 Dumontet C Rebbaa A and Pourtokalian J Very low densitylipoproteins and interleukin 2 enhance the immunogenicity of 9-O-acetyl-GD3 ganglioside in Balbc mice J Immunol Methods1997206115 ndash123

22 Folch PJ Arsove S and Meath JA Isolation of brain stradin a newtype of large molecular tissue component J Biol Chem 1951191 819ndash831

23 Svenerholm L Ganglioside isolation Methods Carbohydr Chem19766464 ndash474

24 Hashimoto Y Otsuka H Sudo K Susuki K and Yamakawa T Ge-netic regulation of GM2 expression in liver mouse J Biochem198393895 ndash901

25 Alfonso M Vazquez A Carr A Haerslev T Fernaacutendez LE LanioME Alvarez C Zeuthen J and Peacuterez R T cellndashindependent B cellresponse to self-monosialo gangliosides primary response mono-clonal antibodies Hybridoma 199514209 ndash216

26 Svenerholm L Quantitative estimation of sialic acid II A colori-metric resorcinol-hydrochoric acid methods Biochem BiophysActa 195724604 ndash611

27 Kean EL Rapid sensitive spectrofotometric methods for quantita -tive determination of sulfatide J Lipid Res 19689319 ndash327

28 Kawashima I Ozawa H Kotami M Susuki M Kawano T Go-

CARR ET AL246

to i-active ganglioside (NeuGc) One common characteristic ofall these MAbs is that they are IgM antibodies

To our knowledge this is the first description of a murineIgG1 MAb specific for N-glycolyl neuraminic acid containinggangliosides The binding specificity of 14F7 MAb was strictlyrestricted to N-glycolyl function since this MAb was able todiscriminate between NeuGc and NeuAc-variant s of GM3 gan-glioside On the other hand the antibody was completely un-reactive with GM2(NeuGc) having NeuGc a 2 R 3Gal structurein an internal position indicating that the addition of one N-acetyl-galactosa mine to the galactose residue affected MAb

binding The finding that 14F7 did not react with GD3 (NeuGc-NeuGc) containing NeuGc a 2 R 8NeuGc2 R 3Gal b 1 terminalstructure suggests that MAb binding is dependent not only onthe external position of the N-glycolyl neuraminic acid groupbut also on the sialic acid linkage These evidence together withthe fact that 14F7 MAb did not bind to IV3NeuGc a nLc4-Cerdiffering from GM3 (NeuGc) in that the terminal NeuGc a 2 R3Gal-structure is linked to GlcNAc suggest that the epitoperecognized by 14F7 comprises the tri-saccharide structureNeuGc a 2 R 3Gal b 1 R 4Glc- Ganglioside recognition patternof 14F7 MAb is different from the previously reported for othermurine and human MAbs which bind not only to GM3 (NeuGc)but also to several gangliosides having NeuGc a 2 R 3Gal-ter-minal structure(111416)

Positive evidences of NeuGc-containing gangliosides in hu-man tumors are still controversial between different groupsHowever it is generally agreed that normal human tissues onlyexpress NeuAc-gangliosides Some articles have described N-glycolylated neuraminic acid in human tumors stained with amurine monoclonal antibody(20) or chicken polyclonal anti-bodies (10) Additionally there are some reports where NeuGc-containing ganglioside composition has been determined bybiochemical analysis in human tumors(3132) and cell lines(8)

We used a preliminary immunohistochemical study with14F7 in normal and tumor sections to clarify if human tumorsexpress NeuGc antigen or not Previous evidences of NeuGcantigen have been shown in breast cancer Breast cancer cellswere detected by membrane immunofluorescence(33) with anti-Hanganutziu Deicher (HD) antibodies where the NeuGc is theimmunogenic residue of the HD antigen Furthermore recentlyour group reported for the first time the chemical quantifica -tion of NeuGc-containing gangliosides in breast cancer On theother hand immunohistochemical analysis in these tissues with3E12 MAb which is specific to the mucin determinant ex-pressed and secreted in breast tumor N-glycolyl sialyl Tn gly-coprotein (34) showed a positive staining in more than 90 of

CARR ET AL244

TABLE 2 REC OGN ITION PA TTER N O F 14F7 MAB

W ITH DIFFERE NT HU M AN TU M ORS

Localization Positive casestotal

BreastFibrocystic disease 59a

Fibroadenoma 55a

Infiltrating ductal carcinom a 1818Skin

Epidermoid carcinoma 09Melanoma 1010

LungNon small cell lung cancer 020Small cell lung carcinom a 010

Central nervous systemOligodendroglioma grade II 03Meningioma 03Multiple glioblastoma 01Meningeal sarcoma 02

Immune systemB cell lymphoma (lymph node) 05T cell lymphoma (lymph node) 03Sezary syndrome 02

aPositive staining of extracellular secretion

FIG 3 Intense immunoperoxidase staining of infiltrating ductal breast carcinoma showing membrane and cytoplasmatic recog-nition with 14F7 MAb ( 3 500)

the breast tumor samples Another MAb (P3) reported by ourgroup binds to several NeuGc containing gangliosides and alsoreacts with sulfated glycolipids showing a positive staining ofthe breast tumors and lymph node metastases samples How-ever 14F7 which is a more specific anti-GM3(NeuGc) gan-glioside MAb is also capable of showing a strong cytoplas -matic and membrane staining in breast malignant tumor cells(Ductal infiltrating carcinoma) This finding allows us to pre-clude that the structure recognized in breast cancer tissues couldbe the oligosaccharide core of GM3(NeuGc) ganglioside inglyco-lipids glycoproteins or mimicries of this antigen On theother hand benign disease or normal tissues showed positivestaining only in the extracellular secretions

Immunohistochemical experiments showed that melanomatumors were stained by 14F7 MAb but no reactivity was foundwith normal skin The expression of NeuGc-containing gan-gliosides in melanoma cells has been contradictory Hirabayash iet al(35) reported the presence of different HD gangliosides inmelanoma tumors in small amount by enzyme TLC immunos-taining Furthermore Kawachi and Saida(36) described that theglycoprotein fractions of human melanoma tissues were recog-nized by chicken anti-HD (anti-NeuGc lactosylceramid e) anti-body by Western Blotting assay Saida et al(37) has concludedthat only melanoma tumors were clearly positive after com-paring the NeuGc ganglioside expression in melanoma tumorsmelanocytic nevus and melanocytes from normal skin Fur-thermore we have recently demonstrated that the 14F7 MAbbehaves like P3 MAb also recognizing melanoma tumors (per-sonal communication)

Although different biochemistry studies have shown eitherthe absence or low level expression of these gangliosides inmelanoma Doreacute et al(38) and Nakarai et al(39) reported two pre-vious evidences about the HD antigen as a possible target forimmunotherapy in melanoma patients Doreacute et al(38) demon-strated the best prognosis in patients with IgG anti-GM3(NeuGc)gangliosides after the immunization with vaccinia virus human

GENERATION OF AN ANTI-GM3(NeuGc)ANTIBODY 245

FIG 4 Strong immunoperoxidase reactivity with 14F7 MAb in melanoma tumors and not with normal skin tissue ( 3 250)

TA BLE 3 REC OGN ITIO N PATT ER N OF 14F7 MAB W ITH

DIFFER ENT NORM AL AD ULT HUM AN TISSU ES

Tissues Positive casestotal

Urogenital tractKidney 07Prostate 05Ovary 05Breast gland (secretion) 34

Endocrine systemAdrenal gland 03

Immune systemTonsils 05Spleen 03

Central nervous systemBrain

Neuron 05Glias 05

Cerebellum 05Skin

Keratinocytes 07Melanocytes 07

Respiratory tractBronchial epithelium 07Neumocytes 07

Gastrointestinal tractEsophagus 07Stomach 07Small intestine (mucus cell) 55Large intestine (mucus cell) 77Pancreas 07Liver 07

melanoma oncolysate while Nakarai et al(39) reported anti-HDantibodies in the sera of melanoma patients with strong recog-nition of HD antigen Additionally higher levels of IgG and IgManti-HD antibodies were found in patients who were disease-

free for more than 5 years after surgery in comparison with thosewho relapsed within 2 years The immunohistochemistry studywith 14F7 MAb showed strong staining of tumor cells (morethan 90 of positive cells) This evidence together with the re-sults of the P3 staining in melanoma tumors could be an addi-tional support to the hypothesis about the role of N-glycolylatedGM3 as target for cancer immunotherapy

The heterophilic characteristics of HD antigen on glycolipidsor glycoproteins as well as the nonexpression in normal tissuesin human and chickens have been reported Although some anti-HD antibodies have recognized colon tumors by immunohisto-chemistry we have not yet tested 14F7 MAb in these tumorstissues However the staining of mucus cells but not secretionof normal small and large intestine was observed Studies withother fresh human tumor samples from different localizationsare in progress in our laboratory

Our results indicate that the 14F7 MAb could be a possiblecandidate for diagnosis used as a therapeutic agent in melanomaand breast tumors

ACKNOWLEDGMENTS

We thank Dr Francisco Estevez for photography assistance We also thank Dr Blanca Tormo and Ernesto Moreno for theirhelpful advice

REFERENCES

1 Stults CLM Sweeley CC and Matcher BA Glycosphingolipi dsstructure biological source and properties Methods Enzymol1989179167 ndash214

2 Hakomori SH Possible functions of tumor-associated carbohydrateantigens Curr Opin Immunol 19913646 ndash653

3 Hougton AN Mintzer D Cordon-Cardo C Welt S Fliegel B Vad-han S Carswell E Melamed MR Oettgen HF Old LJ Mouse mon-oclonal antibody IgG3 antibody detecting GD3 ganglioside a phaseI trial in patients with malignant melanoma Proc Natl Acad SciUSA 1985821242 ndash1246

4 Zhang S Cordon Cardo C Zhang HS Reuter VE Adluri S Hamil-ton WB Lloyd KO and Livingston PO Selection of carbohydratetumor antigens as targets for immune attack using immunohisto -chemistry I Focus on gangliosides Int J Cancer 19977342 ndash49

5 Corfield AP and Schauer R Occurrence of sialic acids Cell BiolMonogr 1982105ndash50

6 Leeden RW and Yu RK Chemistry and analysis of sialic acid InBiological Role of Sialic Acid Rosemberg A and Schengtrund C-L (Eds) Plenum Press New York 1976 pp 1ndash48

7 Kawai T Kato A Higashi H Kato S and Naiki M Quantitativedetermination of N-glycolylneura minic acid expression in humancancerous tissues and avian lymphoma cell lines as a tumor-asso-ciated sialic acid by gas chromatographyndashmass spectometry Can-cer Res 1991511242 ndash1246

8 Higashi H Sasabe T Fukui Y Maru M and Kato S Detection ofgangliosides as N-glycolylneuram inic acidndashspecific tumor-associ-ated Hanganutziu-D eicher antigen in human retinoblastoma cellsJpn J Cancer Res 198879952 ndash956

9 Fukui Y Maru M Ohkawara KI Miyake T Osada Y Wang DIto T Higashi H Naiki M Wakamiya N and Kato S Detectionof glycoproteins as tumor-associated Hanganutziu-D eicher antigenin human gastric cancer cell line NUGC4 Biochem Biophys ResCommun 19891601149 ndash1154

10 Koda T Shimosakoda T Asaoka H Nishinaka S Tamura I NakabaH and Matsuda H Detection of the Hanganutziu-D eicher antigenin patients with hepatocellular carcinoma Int Hepatol Commun19942310 ndash315

11 Furukawa K Yamaguchi H Oettgen HF Old LJ and Lloyd KOAnalysis of the expression of N-glycolylneuraminic acidndashcontain-ing gangliosides in cell and tissues using two human monoclona lantibodies J Biol Chem 198826318507 ndash18512

12 Miyake M Hashimoto K Ito M Ogawa O Arai E Hitomi S andKannagi R The abnormal occurrence and the differentiation-d e-pendent distribution of N-acetyl and N-glycolyl species of the gan-glioside GM2 in human germ cell tumors A study with specificmonoclonal antibodies Cancer 199065499 ndash505

13 Marquina G Waki H Fernaacutendez LE Kon K Carr A Valiente OPeacuterez R and Ando S Gangliosides expressed in human breast can-cer Cancer Res 1996565165 ndash5171

14 Ozawa H Kawashima Y and Tai T Generation of murine mono-clonal antibodies specific for N-glycolylneuram inic acid contain-ing gangliosides Arch Biochem Biophys 1992294427 ndash433

15 Nakamura K Suzuki H Hirabayashi Y and Suzuki A IV3

a (NeuGc a 2-8NeuGc)-Gg4Cer is restricted to CD4 1 T cells pro-ducing interleukin 2 and small population of mature thymocytes inmice J Biol Chem 19952703876 ndash3881

16 Vazquez AM Alfonso M Lanne B Karlsson K-A Carr A Bar-roso O Fernaacutendez LE Rengifo E Lanio ME Alvarez C ZeuthenJ and Peacuterez R Generation of murine monoclonal antibody spe-cific for N-glycolylneuraminic acid containing gangliosides thatalso recognizes sulfated glycolipids Hybridoma 199514551 ndash556

17 Moreno E Lanne B Vazquez AM Kawashima I Tai T Fernaacuten-dez LE Karlsson K-A Angstrom J and Peacuterez R Delineation ofepitope recognized by an antibody specific for N-glycolylneu-raminic acidndashcontaining gangliosides Glycobiology 19988695 ndash705

18 Sanai Y Yamasaki M and Nagai Y Monoclonal antibody directedto Hanganutziu-D eicher active ganglioside GM2 (NeuGc) BiochimBiophys Acta 1988958368 ndash374

19 Miyake M Ito M Hitomi SH Ikeda S Taki T Kurata M Ino AMiyake N and Kannagi R Generation of murine monoclonal an-tibodies that can discriminate N-glycolyl and N-acetyl neuraminicacid residues of GM2 ganglioside Cancer Res 198886154 ndash6160

20 Watarai S Kushi Y Shigito R Misawa N Eishi Y Handa S andYasuda T Production of monoclonal antibodies directed toHanganutziu Deicher active ganglioside N-glycolylneuram inicacidndashcontaining gangliosides J Biochem 19951171062 ndash1069

21 Dumontet C Rebbaa A and Pourtokalian J Very low densitylipoproteins and interleukin 2 enhance the immunogenicity of 9-O-acetyl-GD3 ganglioside in Balbc mice J Immunol Methods1997206115 ndash123

22 Folch PJ Arsove S and Meath JA Isolation of brain stradin a newtype of large molecular tissue component J Biol Chem 1951191 819ndash831

23 Svenerholm L Ganglioside isolation Methods Carbohydr Chem19766464 ndash474

24 Hashimoto Y Otsuka H Sudo K Susuki K and Yamakawa T Ge-netic regulation of GM2 expression in liver mouse J Biochem198393895 ndash901

25 Alfonso M Vazquez A Carr A Haerslev T Fernaacutendez LE LanioME Alvarez C Zeuthen J and Peacuterez R T cellndashindependent B cellresponse to self-monosialo gangliosides primary response mono-clonal antibodies Hybridoma 199514209 ndash216

26 Svenerholm L Quantitative estimation of sialic acid II A colori-metric resorcinol-hydrochoric acid methods Biochem BiophysActa 195724604 ndash611

27 Kean EL Rapid sensitive spectrofotometric methods for quantita -tive determination of sulfatide J Lipid Res 19689319 ndash327

28 Kawashima I Ozawa H Kotami M Susuki M Kawano T Go-

CARR ET AL246

the breast tumor samples Another MAb (P3) reported by ourgroup binds to several NeuGc containing gangliosides and alsoreacts with sulfated glycolipids showing a positive staining ofthe breast tumors and lymph node metastases samples How-ever 14F7 which is a more specific anti-GM3(NeuGc) gan-glioside MAb is also capable of showing a strong cytoplas -matic and membrane staining in breast malignant tumor cells(Ductal infiltrating carcinoma) This finding allows us to pre-clude that the structure recognized in breast cancer tissues couldbe the oligosaccharide core of GM3(NeuGc) ganglioside inglyco-lipids glycoproteins or mimicries of this antigen On theother hand benign disease or normal tissues showed positivestaining only in the extracellular secretions

Immunohistochemical experiments showed that melanomatumors were stained by 14F7 MAb but no reactivity was foundwith normal skin The expression of NeuGc-containing gan-gliosides in melanoma cells has been contradictory Hirabayash iet al(35) reported the presence of different HD gangliosides inmelanoma tumors in small amount by enzyme TLC immunos-taining Furthermore Kawachi and Saida(36) described that theglycoprotein fractions of human melanoma tissues were recog-nized by chicken anti-HD (anti-NeuGc lactosylceramid e) anti-body by Western Blotting assay Saida et al(37) has concludedthat only melanoma tumors were clearly positive after com-paring the NeuGc ganglioside expression in melanoma tumorsmelanocytic nevus and melanocytes from normal skin Fur-thermore we have recently demonstrated that the 14F7 MAbbehaves like P3 MAb also recognizing melanoma tumors (per-sonal communication)

Although different biochemistry studies have shown eitherthe absence or low level expression of these gangliosides inmelanoma Doreacute et al(38) and Nakarai et al(39) reported two pre-vious evidences about the HD antigen as a possible target forimmunotherapy in melanoma patients Doreacute et al(38) demon-strated the best prognosis in patients with IgG anti-GM3(NeuGc)gangliosides after the immunization with vaccinia virus human

GENERATION OF AN ANTI-GM3(NeuGc)ANTIBODY 245

FIG 4 Strong immunoperoxidase reactivity with 14F7 MAb in melanoma tumors and not with normal skin tissue ( 3 250)

TA BLE 3 REC OGN ITIO N PATT ER N OF 14F7 MAB W ITH

DIFFER ENT NORM AL AD ULT HUM AN TISSU ES

Tissues Positive casestotal

Urogenital tractKidney 07Prostate 05Ovary 05Breast gland (secretion) 34

Endocrine systemAdrenal gland 03

Immune systemTonsils 05Spleen 03

Central nervous systemBrain

Neuron 05Glias 05

Cerebellum 05Skin

Keratinocytes 07Melanocytes 07

Respiratory tractBronchial epithelium 07Neumocytes 07

Gastrointestinal tractEsophagus 07Stomach 07Small intestine (mucus cell) 55Large intestine (mucus cell) 77Pancreas 07Liver 07

melanoma oncolysate while Nakarai et al(39) reported anti-HDantibodies in the sera of melanoma patients with strong recog-nition of HD antigen Additionally higher levels of IgG and IgManti-HD antibodies were found in patients who were disease-

free for more than 5 years after surgery in comparison with thosewho relapsed within 2 years The immunohistochemistry studywith 14F7 MAb showed strong staining of tumor cells (morethan 90 of positive cells) This evidence together with the re-sults of the P3 staining in melanoma tumors could be an addi-tional support to the hypothesis about the role of N-glycolylatedGM3 as target for cancer immunotherapy

The heterophilic characteristics of HD antigen on glycolipidsor glycoproteins as well as the nonexpression in normal tissuesin human and chickens have been reported Although some anti-HD antibodies have recognized colon tumors by immunohisto-chemistry we have not yet tested 14F7 MAb in these tumorstissues However the staining of mucus cells but not secretionof normal small and large intestine was observed Studies withother fresh human tumor samples from different localizationsare in progress in our laboratory

Our results indicate that the 14F7 MAb could be a possiblecandidate for diagnosis used as a therapeutic agent in melanomaand breast tumors

ACKNOWLEDGMENTS

We thank Dr Francisco Estevez for photography assistance We also thank Dr Blanca Tormo and Ernesto Moreno for theirhelpful advice

REFERENCES

1 Stults CLM Sweeley CC and Matcher BA Glycosphingolipi dsstructure biological source and properties Methods Enzymol1989179167 ndash214

2 Hakomori SH Possible functions of tumor-associated carbohydrateantigens Curr Opin Immunol 19913646 ndash653

3 Hougton AN Mintzer D Cordon-Cardo C Welt S Fliegel B Vad-han S Carswell E Melamed MR Oettgen HF Old LJ Mouse mon-oclonal antibody IgG3 antibody detecting GD3 ganglioside a phaseI trial in patients with malignant melanoma Proc Natl Acad SciUSA 1985821242 ndash1246

4 Zhang S Cordon Cardo C Zhang HS Reuter VE Adluri S Hamil-ton WB Lloyd KO and Livingston PO Selection of carbohydratetumor antigens as targets for immune attack using immunohisto -chemistry I Focus on gangliosides Int J Cancer 19977342 ndash49

5 Corfield AP and Schauer R Occurrence of sialic acids Cell BiolMonogr 1982105ndash50

6 Leeden RW and Yu RK Chemistry and analysis of sialic acid InBiological Role of Sialic Acid Rosemberg A and Schengtrund C-L (Eds) Plenum Press New York 1976 pp 1ndash48

7 Kawai T Kato A Higashi H Kato S and Naiki M Quantitativedetermination of N-glycolylneura minic acid expression in humancancerous tissues and avian lymphoma cell lines as a tumor-asso-ciated sialic acid by gas chromatographyndashmass spectometry Can-cer Res 1991511242 ndash1246

8 Higashi H Sasabe T Fukui Y Maru M and Kato S Detection ofgangliosides as N-glycolylneuram inic acidndashspecific tumor-associ-ated Hanganutziu-D eicher antigen in human retinoblastoma cellsJpn J Cancer Res 198879952 ndash956

9 Fukui Y Maru M Ohkawara KI Miyake T Osada Y Wang DIto T Higashi H Naiki M Wakamiya N and Kato S Detectionof glycoproteins as tumor-associated Hanganutziu-D eicher antigenin human gastric cancer cell line NUGC4 Biochem Biophys ResCommun 19891601149 ndash1154

10 Koda T Shimosakoda T Asaoka H Nishinaka S Tamura I NakabaH and Matsuda H Detection of the Hanganutziu-D eicher antigenin patients with hepatocellular carcinoma Int Hepatol Commun19942310 ndash315

11 Furukawa K Yamaguchi H Oettgen HF Old LJ and Lloyd KOAnalysis of the expression of N-glycolylneuraminic acidndashcontain-ing gangliosides in cell and tissues using two human monoclona lantibodies J Biol Chem 198826318507 ndash18512

12 Miyake M Hashimoto K Ito M Ogawa O Arai E Hitomi S andKannagi R The abnormal occurrence and the differentiation-d e-pendent distribution of N-acetyl and N-glycolyl species of the gan-glioside GM2 in human germ cell tumors A study with specificmonoclonal antibodies Cancer 199065499 ndash505

13 Marquina G Waki H Fernaacutendez LE Kon K Carr A Valiente OPeacuterez R and Ando S Gangliosides expressed in human breast can-cer Cancer Res 1996565165 ndash5171

14 Ozawa H Kawashima Y and Tai T Generation of murine mono-clonal antibodies specific for N-glycolylneuram inic acid contain-ing gangliosides Arch Biochem Biophys 1992294427 ndash433

15 Nakamura K Suzuki H Hirabayashi Y and Suzuki A IV3

a (NeuGc a 2-8NeuGc)-Gg4Cer is restricted to CD4 1 T cells pro-ducing interleukin 2 and small population of mature thymocytes inmice J Biol Chem 19952703876 ndash3881

16 Vazquez AM Alfonso M Lanne B Karlsson K-A Carr A Bar-roso O Fernaacutendez LE Rengifo E Lanio ME Alvarez C ZeuthenJ and Peacuterez R Generation of murine monoclonal antibody spe-cific for N-glycolylneuraminic acid containing gangliosides thatalso recognizes sulfated glycolipids Hybridoma 199514551 ndash556

17 Moreno E Lanne B Vazquez AM Kawashima I Tai T Fernaacuten-dez LE Karlsson K-A Angstrom J and Peacuterez R Delineation ofepitope recognized by an antibody specific for N-glycolylneu-raminic acidndashcontaining gangliosides Glycobiology 19988695 ndash705

18 Sanai Y Yamasaki M and Nagai Y Monoclonal antibody directedto Hanganutziu-D eicher active ganglioside GM2 (NeuGc) BiochimBiophys Acta 1988958368 ndash374

19 Miyake M Ito M Hitomi SH Ikeda S Taki T Kurata M Ino AMiyake N and Kannagi R Generation of murine monoclonal an-tibodies that can discriminate N-glycolyl and N-acetyl neuraminicacid residues of GM2 ganglioside Cancer Res 198886154 ndash6160

20 Watarai S Kushi Y Shigito R Misawa N Eishi Y Handa S andYasuda T Production of monoclonal antibodies directed toHanganutziu Deicher active ganglioside N-glycolylneuram inicacidndashcontaining gangliosides J Biochem 19951171062 ndash1069

21 Dumontet C Rebbaa A and Pourtokalian J Very low densitylipoproteins and interleukin 2 enhance the immunogenicity of 9-O-acetyl-GD3 ganglioside in Balbc mice J Immunol Methods1997206115 ndash123

22 Folch PJ Arsove S and Meath JA Isolation of brain stradin a newtype of large molecular tissue component J Biol Chem 1951191 819ndash831

23 Svenerholm L Ganglioside isolation Methods Carbohydr Chem19766464 ndash474

24 Hashimoto Y Otsuka H Sudo K Susuki K and Yamakawa T Ge-netic regulation of GM2 expression in liver mouse J Biochem198393895 ndash901

25 Alfonso M Vazquez A Carr A Haerslev T Fernaacutendez LE LanioME Alvarez C Zeuthen J and Peacuterez R T cellndashindependent B cellresponse to self-monosialo gangliosides primary response mono-clonal antibodies Hybridoma 199514209 ndash216

26 Svenerholm L Quantitative estimation of sialic acid II A colori-metric resorcinol-hydrochoric acid methods Biochem BiophysActa 195724604 ndash611

27 Kean EL Rapid sensitive spectrofotometric methods for quantita -tive determination of sulfatide J Lipid Res 19689319 ndash327

28 Kawashima I Ozawa H Kotami M Susuki M Kawano T Go-

CARR ET AL246

free for more than 5 years after surgery in comparison with thosewho relapsed within 2 years The immunohistochemistry studywith 14F7 MAb showed strong staining of tumor cells (morethan 90 of positive cells) This evidence together with the re-sults of the P3 staining in melanoma tumors could be an addi-tional support to the hypothesis about the role of N-glycolylatedGM3 as target for cancer immunotherapy

The heterophilic characteristics of HD antigen on glycolipidsor glycoproteins as well as the nonexpression in normal tissuesin human and chickens have been reported Although some anti-HD antibodies have recognized colon tumors by immunohisto-chemistry we have not yet tested 14F7 MAb in these tumorstissues However the staining of mucus cells but not secretionof normal small and large intestine was observed Studies withother fresh human tumor samples from different localizationsare in progress in our laboratory

Our results indicate that the 14F7 MAb could be a possiblecandidate for diagnosis used as a therapeutic agent in melanomaand breast tumors

ACKNOWLEDGMENTS

We thank Dr Francisco Estevez for photography assistance We also thank Dr Blanca Tormo and Ernesto Moreno for theirhelpful advice

REFERENCES

1 Stults CLM Sweeley CC and Matcher BA Glycosphingolipi dsstructure biological source and properties Methods Enzymol1989179167 ndash214

2 Hakomori SH Possible functions of tumor-associated carbohydrateantigens Curr Opin Immunol 19913646 ndash653

3 Hougton AN Mintzer D Cordon-Cardo C Welt S Fliegel B Vad-han S Carswell E Melamed MR Oettgen HF Old LJ Mouse mon-oclonal antibody IgG3 antibody detecting GD3 ganglioside a phaseI trial in patients with malignant melanoma Proc Natl Acad SciUSA 1985821242 ndash1246

4 Zhang S Cordon Cardo C Zhang HS Reuter VE Adluri S Hamil-ton WB Lloyd KO and Livingston PO Selection of carbohydratetumor antigens as targets for immune attack using immunohisto -chemistry I Focus on gangliosides Int J Cancer 19977342 ndash49

5 Corfield AP and Schauer R Occurrence of sialic acids Cell BiolMonogr 1982105ndash50

6 Leeden RW and Yu RK Chemistry and analysis of sialic acid InBiological Role of Sialic Acid Rosemberg A and Schengtrund C-L (Eds) Plenum Press New York 1976 pp 1ndash48

7 Kawai T Kato A Higashi H Kato S and Naiki M Quantitativedetermination of N-glycolylneura minic acid expression in humancancerous tissues and avian lymphoma cell lines as a tumor-asso-ciated sialic acid by gas chromatographyndashmass spectometry Can-cer Res 1991511242 ndash1246

8 Higashi H Sasabe T Fukui Y Maru M and Kato S Detection ofgangliosides as N-glycolylneuram inic acidndashspecific tumor-associ-ated Hanganutziu-D eicher antigen in human retinoblastoma cellsJpn J Cancer Res 198879952 ndash956

9 Fukui Y Maru M Ohkawara KI Miyake T Osada Y Wang DIto T Higashi H Naiki M Wakamiya N and Kato S Detectionof glycoproteins as tumor-associated Hanganutziu-D eicher antigenin human gastric cancer cell line NUGC4 Biochem Biophys ResCommun 19891601149 ndash1154

10 Koda T Shimosakoda T Asaoka H Nishinaka S Tamura I NakabaH and Matsuda H Detection of the Hanganutziu-D eicher antigenin patients with hepatocellular carcinoma Int Hepatol Commun19942310 ndash315

11 Furukawa K Yamaguchi H Oettgen HF Old LJ and Lloyd KOAnalysis of the expression of N-glycolylneuraminic acidndashcontain-ing gangliosides in cell and tissues using two human monoclona lantibodies J Biol Chem 198826318507 ndash18512

12 Miyake M Hashimoto K Ito M Ogawa O Arai E Hitomi S andKannagi R The abnormal occurrence and the differentiation-d e-pendent distribution of N-acetyl and N-glycolyl species of the gan-glioside GM2 in human germ cell tumors A study with specificmonoclonal antibodies Cancer 199065499 ndash505

13 Marquina G Waki H Fernaacutendez LE Kon K Carr A Valiente OPeacuterez R and Ando S Gangliosides expressed in human breast can-cer Cancer Res 1996565165 ndash5171

14 Ozawa H Kawashima Y and Tai T Generation of murine mono-clonal antibodies specific for N-glycolylneuram inic acid contain-ing gangliosides Arch Biochem Biophys 1992294427 ndash433

15 Nakamura K Suzuki H Hirabayashi Y and Suzuki A IV3

a (NeuGc a 2-8NeuGc)-Gg4Cer is restricted to CD4 1 T cells pro-ducing interleukin 2 and small population of mature thymocytes inmice J Biol Chem 19952703876 ndash3881

16 Vazquez AM Alfonso M Lanne B Karlsson K-A Carr A Bar-roso O Fernaacutendez LE Rengifo E Lanio ME Alvarez C ZeuthenJ and Peacuterez R Generation of murine monoclonal antibody spe-cific for N-glycolylneuraminic acid containing gangliosides thatalso recognizes sulfated glycolipids Hybridoma 199514551 ndash556

17 Moreno E Lanne B Vazquez AM Kawashima I Tai T Fernaacuten-dez LE Karlsson K-A Angstrom J and Peacuterez R Delineation ofepitope recognized by an antibody specific for N-glycolylneu-raminic acidndashcontaining gangliosides Glycobiology 19988695 ndash705

18 Sanai Y Yamasaki M and Nagai Y Monoclonal antibody directedto Hanganutziu-D eicher active ganglioside GM2 (NeuGc) BiochimBiophys Acta 1988958368 ndash374

19 Miyake M Ito M Hitomi SH Ikeda S Taki T Kurata M Ino AMiyake N and Kannagi R Generation of murine monoclonal an-tibodies that can discriminate N-glycolyl and N-acetyl neuraminicacid residues of GM2 ganglioside Cancer Res 198886154 ndash6160

20 Watarai S Kushi Y Shigito R Misawa N Eishi Y Handa S andYasuda T Production of monoclonal antibodies directed toHanganutziu Deicher active ganglioside N-glycolylneuram inicacidndashcontaining gangliosides J Biochem 19951171062 ndash1069

21 Dumontet C Rebbaa A and Pourtokalian J Very low densitylipoproteins and interleukin 2 enhance the immunogenicity of 9-O-acetyl-GD3 ganglioside in Balbc mice J Immunol Methods1997206115 ndash123

22 Folch PJ Arsove S and Meath JA Isolation of brain stradin a newtype of large molecular tissue component J Biol Chem 1951191 819ndash831

23 Svenerholm L Ganglioside isolation Methods Carbohydr Chem19766464 ndash474

24 Hashimoto Y Otsuka H Sudo K Susuki K and Yamakawa T Ge-netic regulation of GM2 expression in liver mouse J Biochem198393895 ndash901

25 Alfonso M Vazquez A Carr A Haerslev T Fernaacutendez LE LanioME Alvarez C Zeuthen J and Peacuterez R T cellndashindependent B cellresponse to self-monosialo gangliosides primary response mono-clonal antibodies Hybridoma 199514209 ndash216

26 Svenerholm L Quantitative estimation of sialic acid II A colori-metric resorcinol-hydrochoric acid methods Biochem BiophysActa 195724604 ndash611

27 Kean EL Rapid sensitive spectrofotometric methods for quantita -tive determination of sulfatide J Lipid Res 19689319 ndash327

28 Kawashima I Ozawa H Kotami M Susuki M Kawano T Go-

CARR ET AL246

mobuchi M and Tai T Characterization of ganglioside expressionin human melanoma cell immunological and biochemical analy-sis J Biochem 1993114186 ndash193

29 Hsu SM Raine L and Fanger H Use the avidin-biotin ndashperoxidasecomplex (ABC) in immunoperoxidase technique a comparison be-tween ABC and unlabelled antibody PAP procedures J HistochemCytochem 198929577 ndash580

30 Kawashima I Ozawa H Kotani M Susuki M Kawano T Go-mobushi M and Tai T Characterization of ganglioside expressionin human melanoma cells immunological and biochemical analy-sis J Biochem 1993114186 ndash193

31 Higashi H Hirabayashi Y Fukui Y Naiki M Matsumoto M UedaS and Kato S Characterization of N-glycolylneuram inic acidndashcon-taining gangliosides as tumor-associated Hanganutziu-D eicherantigen in human colon cancer Cancer Res 1985453796 ndash3802

32 Hirabayashi Y Kasakura H Matsumoto M Higashi H Kato S Ka-sai N and Naiki M Specific expression of unusual GM2 ganglio -side with Hanganutziu-D eicher antigen activity on human coloncancer Jpn J Cancer Res 198778251 ndash260

33 Ikuta K Nishi Y Simizu Y Higashi H Kitamoto N Kato S Fu-jita M Nakano Y Taguchi T and Naiki M Hanganutziu-D eichertype heterophyle antigen-positive cells in human cancer tissuesdemonstrated by membrane immunofluorescence Biken J19822547 ndash50

34 Devine PL Clark BA Birrel GW Layton GT Ward BG AlewoodPF and McKenzie IAC The breast tumorndashassociated epitope de-fined by monoclonal antibody 3E12 is an O-linked mucin carbo-hydrate containing N-glycolyl neuraminic acid Cancer Res1991515826 ndash5836

35 Hirabayashi Y Higashi H Kato S Taniguchi M and MatsumotoM Occurrence of tumor associated ganglioside antigens with

Hanganutziu Deicher antigenic activity in human melanoma JpnJ Cancer Res 198778251 ndash260

36 Kawachi S and Saida T Analysis of the expression ofHanganutziu-Deicher (HD) antigen in human malignant melanomaJ Dermatol 199219827 ndash830

37 Saida T Ikegawa S Takizawa Y and Kawachi S Immunohisto-chemical detection of heterophile Hanganutziu-D eicher (HD) anti-gen in human malignant melanoma Arch Dermatol Res 1990282 179ndash182

38 Dore JD Portoukalian J Berthier Vergnes O Jacubovich R Gen-eve J Bailly M Leftheriotis E Weissbrod A and Mayer MReacuteponse de malades atteints de meacutelanome aacute lrsquoimmunisation paroncolisats de meacutelanome au virus de la vaccine Bull Cancer199077881 ndash891

39 Nakarai H Chandler PJ Kano K Morton DL and Irie RFHanganutziu-Deicher (HD) antigen as a possible target for im-munotherapy of melanoma Int Arch Allerg Appl Immunol 199091323ndash328

Address reprint requests toAdriana Carr

Center of Molecular ImmunologyPO Box 16040

Havana 11600 Cuba

E-mail adrianaictcimsldcu

Received for publication April 2 1999 Accepted after revi-sions March 6 2000

GENERATION OF AN ANTI-GM3(NeuGc)ANTIBODY 247