REACTIVITY OF NATURAL AND INDUCED HUMAN ANTIBODIES TO MUC1MUCIN WITH MUC1 PEPTIDES AND N-ACETYLGALACTOSAMINE(GalNAc) PEPTIDESSilvia VON MENSDORFF-POUILLY ,1* Eftichia PETRAKOU,2 Peter KENEMANS,1 KeesVAN UFFELEN,3 Albert A. VERSTRAETEN,1
Frank G.M. SNIJDEWINT,1 Gerard J.VAN KAMP,3 Dick J. SCHOL,1 Celso A. REIS,4 Michael R. Price,2 Philip O. LIVINGSTON5 and
Joseph HILGERS1
1Department of Obstetrics and Gynaecology, Academic Hospital Vrije Universiteit, Amsterdam, The Netherlands2Cancer Research Laboratories, School of Pharmaceutical Sciences, The University of Nottingham, Nottingham, United Kingdom3Department of Clinical Chemistry, Academic Hospital Vrije Universiteit, Amsterdam, The Netherlands4Department of Oral Diagnostics, School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Denmark5Memorial Sloan-Kettering Cancer Center, New York, New York, USA
Recent years have seen an increase in the number of studiesinvolved in the development of modes of immunotherapy of can-cer directed towards engaging and enhancing the host’s owndefense to the tumor. Active immunotherapy that engenders im-munological memory may provide an effective surveillance mech-anism to protect cancer patients from recurrence of disease, andtreatment with cancer vaccines based on tumor antigens as a modeof therapy of cancer is being increasingly investigated. Polymor-phic epithelial mucin (PEM, MUC1) (for a review see Taylor-Papadimitriouet al., 1999), an epithelial mucin present at theapical surface of normal glandular epithelial cells and overex-pressed by the majority of carcinomas, represents one of thesetarget antigens. Several phase I clinical trials, involving differentMUC1-based vaccine substrates, adjuvants and carrier proteinshave been carried out and phase II trials are in progress (for areview see Miles and Taylor-Papadimitriou, 1999).
MUC1 is a high molecular weight transmembrane moleculewith a large, highly glycosylated extracellular domain mainlyconsisting of multiple peptide repeats of a highly conserved se-quence of 20 amino acids (Gendleret al.,1990). Each repeat hasfive potential sites forO-linked glycosylation (Table I) that arevariably occupied in the mature MUC1 mucin shed into humanmilk (Muller et al., 1997). In cancer cells, MUC1 is not onlyoverexpressed but also deficiently glycosylated, which leads to theincreased exposure of a variable number of immunodominantareas on its peptide core (Burchell and Taylor-Papadimitriou,1993). This potentially immunogenic molecule is present in thecirculation of carcinoma patients and measurement of its concen-tration is used to monitor treatment of breast cancer patients (Bonet al., 1997).
Humoral responses to MUC1 have been described in healthysubjects, benign diseases and carcinoma patients (Hinodaet al.,1993; Rughettiet al., 1993; Koteraet al., 1994; Gourevitchet al.,1995; Richardset al., 1998). We found that the presence ofcirculating immune complexes containing MUC1 at the time ofdiagnosis was related to a favourable disease outcome in breastcancer patients (von Mensdorff-Pouillyet al., 1996). We alsoobserved a benefit in survival in early stage breast cancer patientswith circulating unbound MUC1 Abs at first diagnosis (vonMensdorff-Pouillyet al., 2000). While most murine monoclonalantibodies (MAbs) generated against MUC1 are directed to thePDTRPAP sequence on the MUC1 tandem repeat (Priceet al.,1998), additional sequences of the repeat appear to be involved inthe human humoral response to MUC1. Human Abs produced byimmortalized B-cells obtained from tumour draining negativelymph nodes of breast and ovarian cancer patients recognized theAPPAH sequence of the MUC1 tandem repeat (Petrarcaet al.,1996). This sequence is part of the STAPPAHGV sequence of theMUC1 tandem repeat, which was shown to bind strongly toHLA-A11, and to a lesser extent to HLA-A1, A2.1 and A3molecules (Domenechet al., 1995). Two human single-chain FvAbs to MUC1 core peptide selected from a large naive phageantibody library, which bound to tumor cells and tissues, recog-nized the PAPG(S) and (T)RPAPGSTAPPAH sequences of theMUC1 peptide core (Henderikxet al., 1998). Carcinoma patientsimmunized with a mannan-MUC1 fusion protein developed IgG1
Grant sponsor: Dutch Cancer Society; Grant number: VU96-1318; Grantsponsor: Biocare Foundation; Grant number: 94-23; Grant sponsor: DanishCancer Society.
*Correspondence to: Silvia von Mensdorff-Pouilly, Dept. of Obstetricsand Gynaecology, Academic Hospital Free University, De Boelelaan 1117,1081 HV Amsterdam, The Netherlands. Fax:13120 4444818.E-mail: [email protected]
Received 30 September 1999; Revised 3 January 2000
Publication of the International Union Against Cancer
Abs that reacted mostly to the STAPPAHG and PAPGSTAPsequences of the MUC1 peptide core (Karanikaset al., 1997). Onthe other hand, Adluriet al. (1999) found that the specificity ofMUC1 Abs induced by vaccination of breast cancer patients witha 30-mer MUC1 peptide conjugated to KLH at the N-terminal endwas primarily to the final sequence of the immunogen, APDTRPAat the C-terminal end. Samples from these patients are included inthe present study.
MUC1 expressed by the normal mammary gland is composed of50–60% carbohydrate. Mucin-typeO-linked glycosylation is ini-tiated by a family of enzymes,N-acetylgalactosaminyltransferases(GalNAc-T), which transfer GalNAc to the side chains of serineand threonine residues (Wandallet al., 1997; Bennettet al., 1998).This GalNAc residue and the residue(s) directly linked to it con-stitute core structures to which further monosaccharides can beattached, catalysed by linkage specific glycosyl-transferases, thuselongating and branching the carbohydrate chains. In normal mu-cin, an average of 2.6 of the five sites available per repeat forO-linked glycosylation are occupied (Mu¨ller et al., 1997) and it isassumed that elongation of the carbohydrate chains would have anegative effect on the glycosylation of vicinal sites (Hanischet al.,1999). Breast cancer cell lines show loss of activity ofN-acetyl-glucosaminyltransferases (GlcNAc-T) in addition to a tenfold in-crease ofa2-3-sialyltransferase, which explains in part the shortersugar side chains observed on MUC1 in carcinomas (Whitehouseet al., 1997), as premature sialylation of the side chains preventstheir further extension. Furthermore, the presence of shorter sidechains may allow a higher degree of substituted sites compared tonormal epithelial cells, as demonstrated for the breast cancer cellline T47D, which does not express a functional core2 enzyme andhas been shown to have on average 4.8 of the five putative siteswithin the tandem repeat which are glycosylated (Hanischet al.,1999). These truncated carbohydrate chains are in themselvesimmunogenic (Livingstonet al., 1997) with the consequence thatcryptic epitopes, both peptide and carbohydrate in nature, presentin the carcinoma-associated mucin could be important in inducingan antibody response. Among the carbohydrate tumor antigensexpressed on MUC1 are the blood-group-related antigens TN(GalNAca) and sialyl TN (NeuAca2-6GalNAca) and the Thom-sen-Friedenreich (TF or T) antigen (Galb1-3GalNAca). The re-strictive distribution of these antigens in normal tissues and theirextensive expression in a variety of epithelial cancers (Zhangetal., 1997) make them good targets for immunotherapy as well.Sialyl TN antibody responses induced by a sialyl TN-KLH plusDetox vaccine (Theratope) in breast and colorectal cancer patientswere associated with prolonged survival (MacLeanet al., 1996).
The objective of this study was to define and compare thespecificity of natural MUC1 Abs (von Mensdorff-Pouillyet al.,1998) and MUC1 Abs induced in breast cancer patients aftervaccination with a 30-mer (Gilewskiet al., in press) and with a33-mer MUC1 peptide conjugated to KLH and mixed with QS-21in two phase I clinical trials carried out at the Memorial Sloan-
Kettering Cancer Center to obtain information towards the formu-lation of an optimal MUC1 vaccine for the treatment of patientswith adenocarcinomas.
MATERIAL AND METHODS
Controls, patients, and serum samplesSerum samples with high levels of IgG and /or IgM Abs to
MUC1 were obtained from 55 control subjects (2 healthy men, 26healthy women, 12 pregnant women and 15 benign breast tumourpatients) and from 26 breast cancer patients before primary treat-ment, selected from a screened population of 492 subjects (302controls and 190 breast cancer patients).
A total of 187 serial plasma samples obtained from breast cancerpatients (N5 19) taking part in two phase I vaccination trials withMUC1 peptides (Memorial Sloan-Kettering Cancer Center, NY)were tested for the presence of MUC1 Abs. Breast cancer patientseligible for the study included Stage IV patients who were cur-rently free of disease and Stage I– III patients with rising CEA orCA 15.3 and clinically no evidence of disease. A first group ofpatients (N5 9), was vaccinated with a non-glycosylated 30-merMUC1 peptide with the sequence VTSAPDTRPAPGSTAPPAH-GVTSAPDTRPA (Gilewskiet al., in press), a second group (N510), with a non-glycosylated 33-mer MUC1 peptide with the se-quence VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGS. Thepeptides were conjugated to the carrier protein keyhole limpet hemo-cyanin (KLH) and administered subcutaneously, 100mg MUC1peptide and 100mg of the immunological adjuvant QS-21 perdose, on weeks 1, 2, 3, 7 and 19.
Serum and plasma samples were collected, aliquoted and storedat 280°C until analysed.
Peptides and GalNAc-peptidesThe following peptides and GalNAc-peptides, listed in Table I,
were synthesised for use in the study: (1) twenty overlapping20-mer peptides covering the sequence of a single tandem repeat;(2) a 60-mer peptide corresponding to 3 repeats of the MUC1peptide core; (3) a 60-mer reverse peptide corresponding to 3reverse sequence tandem repeats; (4) a modified 60-mer peptidewith an alanine substituting for the arginine in residue 10 of eachtandem repeat; (5) a second modified 60-mer peptide with twoalanines substituting for the two prolines in residue 18 and 19 ofeach tandem repeat; (6) a 60-mer glycopeptide with 3 mol Gal-NAc, attached to the threonine in the PDTR sequence of eachrepeat; (7) a 60-mer glycopeptide with 6 mol GalNAc, attached tothe threonines to the left and to the right of the PDTR sequence ofeach repeat; (8) a 60-mer glycopeptide with 9 mol GalNAc,attached to the threonine to the left and to the serine and threonineto the right of the PDTR sequence of each repeat; and (9) a 60-merglycopeptide with 15 mol GalNAc occupying all available sites forO-linked glycosylation.
TABLE I – PEPTIDES AND GALNAC-PEPTIDES
Single tandem repeat:
NH2-HGVTSAPDTR10PAPGSTAP18P19A-COOH
Peptides Abbreviation used in the text1) 20 overlapping single tandem repeats 20 mer2) NH2-(HGVTSAPDTRPAPGSTAPPA)3-COOH 60 mer3) NH2-(GHAPPATSGPAPRTDPASTV)3-COOH reverse4) NH2-(HGVTSAPDTAPAPGSTAPPA)3-COOH PDTA5) NH2-(HGVTSAPDTRPAPGSTAAAA)3-COOH STAAAA
1Sites where GalNAc is attached by O-linked glycosylation.–2Synthetically glycosylated.–3Enzymati-cally glycosylated.
703EPITOPE MAPPING OF MUC1 ANTIBODIES
Peptides were synthesized (Dr. L. Vernie, The NetherlandsCancer Institute, Amsterdam, The Netherlands) using a solid-phase procedure by which amino acids are coupled as fluoren-9-yl-methoxycarbonyl (Fmoc) derivatives. The peptides were pu-rified by reverse-phase high pressure liquid chromatography(HPLC) and analysed by mass spectometry. For the synthesis ofglycopeptides GalNAc(3) and GalNAc(9) (Table I), tri-O-acetyl-GalNAc a-FMOC-Thr and tri-O-acetyl-GalNAca-FMOC-Ser(Glycotech Corp., Rockville, MD) were used as building blocks atthe appropriate place in the sequence. In the case of the GalNAc(6)and GalNAc(15) glycopeptides (Table I), the synthetic 60-merpeptide was enzymatically glycosylated employing recombinantGalNAc-transferases (Wandallet al., 1997; Bennettet al., 1998).The GalNAc-peptides were purified by reverse-phase-HPLC andanalysed by MALDI-mass spectometry.
The peptides and GalNAc-peptides were conjugated to bovineserum albumin (BSA) using a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) conjugation kit (Imject™ Im-munogen EDC Conjugation Kit for KLH and BSA, Pierce, Rock-ford, IL).
The human anti-MUC1 antibody HuHMFG-1 was a gift fromDr J. Taylor-Papadimitriou, ICRF, London, UK. HuHMFG-1 is areshaped human IgG1 antibody that recognizes the PDTR se-quence of the MUC1 mucin tandem repeat. The humanized anti-body was obtained by grafting the complementary regions identi-fied in the murine HMFG-1 onto a human immunoglobulinframework (Verhoeyenet al., 1993).
MUC1 Ab assayThe serial plasma samples obtained from the vaccinated patients
were tested for the presence of IgG and IgM MUC1 Abs employ-ing an enzyme-linked immunoassay as described before (vonMensdorff-Pouillyet al., 1998). In short, a 60-mer peptide, corre-sponding to three tandem repeats of the MUC1 peptide core,conjugated to BSA and unconjugated BSA, were adsorbed inalternate columns in 96-well ELISA plates. After overnight incu-bation, the wells were incubated with BSA to block non-specificadsorption sites. Serum samples diluted 1:500 (for IgG determi-nations) and 1:1,000 (for IgM determinations) were incubatedovernight and immunoglobulins bound to the peptide were de-tected with horseradish peroxidase-conjugated rabbit anti-humanIgG or IgM (DAKO A/S, Glostrup, Denmark), diluted 1:10,000.Tetramethylbenzidine (TMB) was used as substrate and the reac-tion was quantified at 450 nm in an ELISA reader. The assay wasperformed for each serum sample in duplicate and results calcu-lated as the mean difference between the readings in OpticalDensity Units (O.D.) in MUC1-coated and BSA-coated wells. Afour-point standard curve was constructed for each plate using apositive serum sample from a healthy control and from a breastcancer patient for the IgG and IgM determinations, respectively,diluted stepwise from 1:250 to 1:2,000. An arbitrary value of ‘1’was ascribed to the serum dilution 1:2,000 and the value of thesamples tested was calculated within each plate in relation to thestandard curve by least square regression analysis and expressed inarbitrary units per mL (arb.U/mL). Samples with MUC1 Ab levelshigher than the limit of the standard curve (8 arb. U/mL) weretested in increasing dilutions until the readings fell within therange of the standard curve and the results extrapolated for sampledilutions of 1:500 for IgG and 1:1,000 for IgM determinations.
Immunoglobulin G subclassesThe IgG subclass of the natural and induced IgG MUC1 Abs
was defined by detecting the immunoglobulins bound in the assayto the BSA-conjugated 60-mer peptide with mouse anti-humanIgG2 (clone HP-6014) and IgG4 (clone HP-6023) MAbs (Sigma-Aldrich, Zwijndrecht, The Netherlands) and with HRP conjugatedmouse anti-human IgG1 (clone MH161-1) and IgG3 (cloneMH163-1) MAbs (CLB, Amsterdam, The Netherlands). The dilu-tions used were 1:5,000 for the IgG1 and IgG2, 1:1,000 for theIgG3 and 1:2,000 for IgG4 conjugate. In the case of IgG2 and
IgG4 determinations, an extra incubation step with horseradishperoxidase labeled rabbit-anti-mouse IgG (dilution 1:1,000, Dako)was incorporated in the assay. Serum and plasma samples weretested at dilutions 1:40 and 1:100 for the natural and inducedimmune response, respectively. A serum sample with no Abs toMUC1 was used as a negative control.
Epitope mappingModified peptides and GalNAc-peptides. Eighty-one serum
samples from control subjects and breast cancer patients withnatural MUC1 IgG and/or IgM Abs and 72 serial plasma samplesfrom 8 patients in the first group and from 10 patients in the secondgroup of vaccinated patients with induced MUC1 IgG and/or IgMAbs were tested for reactivity with the BSA-conjugated modifiedpeptides and GalNAc-peptides employing the assay (vonMensdorff-Pouillyet al., 1998) described above. Sample dilutionsfor IgG determinations ranged from 1:100 to 1:1,000 for thenatural Abs and from 1:500 to 1:8,000 for the induced Abs. IgMpositive samples were diluted in values ranging from 1:500 to1:4,000 for both the natural and induced Abs. The followingmodifications were introduced in the assay: The modified peptidesand the GalNAc-peptides, each conjugated to BSA, were adsorbedin duplicate columns in 96-well ELISA plates. In each plate, thefirst two columns were coated with the BSA-conjugated 60-merpeptide used in the MUC1 Ab assay and the last two with BSA.The samples were incubated one sample per row and the assay wasperformed for each sample and peptide/GalNAc-peptide in dupli-cate. Results were calculated as the mean difference between theOptical Density Units (O.D.) measured in experimental wells(BSA-conjugated peptide/GalNAc-peptide) and BSA-coated con-trol wells. The percentage of reactivity of the samples with themodified peptides and GalNAc-peptides was calculated as the ratioof the result obtained with the corresponding modified peptide/GalNAc-peptide to the result obtained with the 60-mer peptide(100% reactivity) within the same plate.
Overlapping 20-mer peptides.Twenty-one MUC1 IgG positiveserum samples, obtained from 7 control subjects, 4 breast cancerpatients and 10 vaccinated patients, as well as 23 MUC1 IgMpositive serum samples, obtained from 8 control subjects, 7 breastcancer patients and 8 vaccinated patients were tested for reactivitywith overlapping 20-mer peptides following the sequence of thetandem repeat. HuHMFG-1 was used as a positive control. Theoverlapping 20-mer peptides, each conjugated to BSA, were ad-sorbed in duplicate columns in 96-well ELISA plates as describedabove for the modified peptides and GalNAc-peptides and theresults calculated accordingly.
Inhibition of the specific reactivity. Inhibition of the specificreactivity was tested in 9 MUC1 IgG positive samples, obtainedfrom 2 control subjects, 2 breast cancer patients and 5 vaccinatedpatients and in 9 MUC1 IgM positive samples, obtained from 2control subjects, 2 breast cancer patients and 5 vaccinated patients.Serum and plasma samples were diluted appropriately in PBScontaining 1% BSA and 0.02% sodium azide and incubated over-night at 4°C with 0.1 or 1 mg/mL of either one of the two modifiedpeptides or with 1 mg/mL of the reverse sequence 60-mer peptideas a negative control. The pre-incubated samples and a samplewithout peptide were then tested alongside for reactivity to theBSA-conjugated 60-mer peptide and modified peptides with theassay as described above. The samples incubated with the reversepeptide were tested for reactivity to the BSA-conjugated 60-merpeptide only. The percentage of reactivity of the pre-incubatedsamples and the samples without peptide with the different coatingpeptides was calculated as the ratio of the result obtained with eachof the coating peptides to the result obtained with the correspond-ing sample without antigen tested with the 60-mer peptide.
Overlapping heptamer peptides. Thirty-one MUC1 IgG positiveserum samples obtained from 14 control subjects, 15 breast cancerpatients and 2 vaccinated patients and 18 MUC1 IgM positiveserum samples from 8 control subjects, 9 breast cancer patients and1 vaccinated patient, diluted 1:100, were tested in ELISA with the
‘Pepscan’ technique (Priceet al., 1990; Petrakouet al., 1998) forreactivity with overlapping heptamers following the sequence ofthe MUC1 tandem repeat bound at the C-terminus to the head ofpolyethylene pins. The series of 20 heptamer peptides was syn-thesized so that each overlapped its neighbour in all but one aminoacid with the series of peptides covering the entire 20-amino acidMUC1 repeat sequence. Two peptides, TRPAPGS and GLAQ,were synthesized separately to be used as positive and negativeantibody binding controls for the anti-RPAP Mab C595. Sampleswere tested on each peptide in duplicate. The shortest sequence ofamino acid residues, which is common to a series of peptides thatreact positively with the samples, is defined as the minimumbinding sequence for the MUC1 Abs in that sample. Positivity wasdefined as a signal higher than the mean1 2SD of the overallresponse with the various peptides.
Statistical methodsStatistical analysis was performed using SPSS 7.5 software
(SPSS Inc., Chicago, IL.). The percentage of reactivity of theindividual samples with the peptides and GalNAc-peptides wasanalysed using the Wilcoxon Rank Sum W Test. Results in thedifferent groups were analysed using the Mann-Whitney U Test.The correlation among assay results obtained with the variouspeptides and GalNAc-peptides was evaluated by linear regressionanalysis. In all cases, a two-tailedp , 0.05 was consideredsignificant.
RESULTS
MUC1 antibody levels in the study populationMedian levels of natural MUC1 IgG Abs in the control subjects
and breast cancer patients were 4.82 arb. U/mL (range: 2–16.70)and median MUC1 IgM Ab levels were 3.02 arb. U/mL (range:1.44–12.68). Eight out of 9 breast cancer patients vaccinated withthe 30-mer MUC1 peptide and all 10 patients vaccinated with the33-mer MUC1 peptide developed IgG and IgM MUC1 Abs withmedian levels higher than the natural humoral response. Both IgGand IgM antibody levels were notably higher in the patientsvaccinated with the 33-mer MUC1 peptide than in the patientsvaccinated with the 30-mer MUC1 peptide. MUC1 IgG Ab levelsin the vaccinated patients are shown in Table II.Maximal MUC1IgG responses were observed on weeks 5, 7, 9, and 21, i.e., 2weeks following the third, fourth, and fifth immunization. MUC1IgG Ab levels tended to fall rapidly after week 21, but remainedwell above the initial, pre-vaccination levels in all cases (Fig. 1).Median MUC1 IgM Ab levels prior to vaccination and at week 5were 0.33 arb. U/mL (range: 0.17–0.92) and 3.4 arb. U/mL (range:1.3–25.2), respectively, in the patients vaccinated with the 30-merpeptide and 0.27 arb. U/mL (range: 0.09–1.01) and 7.64 arb. U/mL(range: 3.17–38.86), respectively, in the patients vaccinated withthe 33-mer peptide.
Immunoglobulin G subclassesNaturally occurring MUC1 Abs were mostly IgG2 in both
controls and cancer patients. A predominance of IgG1 and IgG3
Abs was found only in four and three cases, respectively. No IgG4Abs were observed. No difference in IgG subclasses was foundbetween controls and breast cancer patients. The vaccinated pa-tients developed strong MUC1 IgG1 and IgG3, but also IgG2responses. IgG4 responses were low.
Epitope mappingOverlapping heptamer peptides. Results obtained with the over-
lapping heptamers are shown in Table IIIand Figure 2. The mostfrequently occurring minimal epitopic sequence was RPAPGS,followed by epitopic regions involving the two prolines in theSTAPPAHGVT sequence. The PDTR sequence formed part of theminimal epitopic sequence only in three of the samples (two IgGand one IgM) with naturally occurring MUC1 Abs but was foundin all three samples with induced MUC1 Abs. Abs directed to morethan one epitope were found in 13 MUC1 IgG (4 controls, 7 breastcancer patients and the 2 vaccinated patients tested) and 5 MUC1IgM (2 controls, 2 breast cancer patients and the vaccinated patienttested) positive samples. No clear minimal epitopic sequence couldbe defined in 7 MUC1 IgG and 8 MUC1 IgM positive samples. Inthese samples the signal was above background but equally reac-tive across peptides, indicating a broad polyclonal response or thepresence of Abs to conformational epitopes that do not recognisethe short peptide sequences attached to the pins.
Modified peptides and GalNAc-peptides. The mean6 SD per-centage of reactivity of the serum and plasma samples with themodified peptides and GalNAc-peptides in the various groupsstudied is shown in Figure 3a (IgG responses) andb (IgM respons-es). Analysis of the results obtained in each of the groups studiedwith the modified peptides and GalNAc-peptides compared to thereactivity with the 60-mer peptide taken as a standard (100%reactivity) is contained in Table IV. A comparison of the resultsobtained with the modified peptides and GalNAc-peptides betweenthe different groups studied is shown in Table V.
TABLE II – SERIAL MUC1 IGG AB LEVELS (ARB. U/ML) IN BREAST CANCER PATIENTS AFTER VACCINATION WITHMUC1 PEPTIDES CONJUGATED TO KLH PLUS QS-21
9/21 0.05 9/21 0.0431Analysis of Ab levels at different weeks (Mann-WhitneyU test).–2Samples from only 8 and 7 patients were available at week 19 and 21,
respectively.–3Samples from only 9 and 7 patients were available at week 19 and 21, respectively.
FIGURE 1 – Serial MUC1 IgG and IgM Ab levels measured with theMUC1 Ab assay in a breast cancer patient vaccinated with a 33-merMUC1 peptide conjugated to KLH plus QS-21 (Memorial Sloan-Kettering Cancer Center, NY). Further results obtained with a samplefrom this patient are shown in Figure 5b1 andb2.
705EPITOPE MAPPING OF MUC1 ANTIBODIES
The reactivity of MUC1 IgG and IgM positive samples with thePDTA peptide was significantly lower than that obtained with the60-mer peptide in both the control subjects and the breast cancerpatients (Table IV).
The reactivity of the naturally occurring MUC1 Abs with theSTAAAA peptide did not differ significantly from that obtainedwith the 60-mer peptide with the exception of MUC1 IgG positivesamples obtained from control subjects, which showed lower re-activity with the STAAAA peptide. MUC1 IgG positive samplesfrom both groups of vaccinated patients showed lower reactivitywith the STAAAA peptide.
A comparison of the reactivity of the MUC1 IgG positivesamples with the PDTA versus the STAAAA peptide showed nosignificant difference for the natural Abs, while the induced Absshowed significantly lower reactivity with the STAAAA peptide.On the other hand, both natural and induced MUC1 IgM Absshowed significantly lower reactivity with the PDTA than with theSTAAAA peptide.
MUC1 IgG positive serum samples from breast cancer patientsreacted more strongly with the GalNAc(3) and with the GalNAc(9)peptide than with the non-glycosylated 60-mer peptide (Table IV).MUC1 IgM positive samples from both control subjects and breastcancer patients showed stronger reactivity with the GalNAc(3)peptide than with the 60-mer peptide. The reactivity of IgG andIgM positive samples with the GalNAc(15) peptide was signifi-cantly lower than with the 60-mer peptide (Table IV).
MUC1 IgG and IgM positive samples from the vaccinatedpatients in group 1 and 2 showed, with one exception, equallysignificant lowered reactivity with each of the glycosylated pep-tides tested (Table IV). A comparison between the different gly-cosylated peptides showed, with one exception, significantly lowerreactivity (p , 0.0001) of both MUC1 IgG and IgM positivesamples with increasing glycosylation of the peptide (Fig. 3a,b).The exception was the reactivity of the IgM positive samples withthe GalNAc(15), which was higher than that obtained with theGalNAc(9) (Fig. 3b).
A comparison between the various groups studied of the resultsobtained with the modified peptides and GalNAc-peptides (TableV) gave no significant difference between control subjects andbreast cancer patients. Induced MUC1 IgG Abs showed a lowerreactivity with the STAAAA but higher with the PDTA peptidethan the natural IgG Abs while no sustained significant differencewas found with the IgM positive samples. The reactivity of MUC1IgG and IgM Abs with each of the GalNAc-peptides was signifi-cantly lower for the induced than for the naturally occurring Abs.Among the vaccinated patients, the reactivity of IgG Abs with eachof the glycosylated peptides was lower in group 2 than in group 1.
A comparison of the reactivity of MUC1 IgG versus IgMpositive samples with the modified peptides gave no significantdifference in breast cancer patients. Both groups of vaccinatedpatients showed a lower reactivity of IgG samples with theSTAAAA peptide (p , 0.0001) but a higher reactivity with thePDTA peptide (p 5 0.001) than IgM positive samples. IgMpositive samples from the vaccinated patients reacted morestongly with the GalNAc(6) peptide than IgG positive sampleswhile no significant difference was found with the GalNAc(9)peptide.
Linear regression analysis gave a strong correlation (p ,0.0001) between the results obtained with the MUC1 IgG or theMUC1 IgM positive samples from the vaccinated patients and thevarious GalNAc-peptides. Analysis of the results obtained with theIgG positive samples showed a strong correlation (p , 0.0001)between the reactivity with the STAAAA-modified 60-mer peptideon the one hand and the glycosylated peptides with 6, 9 and 15 molGalNAc on the other in group 1, but only with the glycosylatedpeptide with 6 mol GalNAc in group 2. No correlation was foundbetween the PDTA peptide and the GalNAc(3) peptide.
Overlapping 20-mer peptides and inhibition of the specific re-activity. While most IgG positive samples tested with the over-lapping 20-mer peptides showed a participation of both thePDTRPAP and the STAPPAHGV regions in the immune re-sponse, definition of the epitopes was less clear-cut in theresults obtained with the IgM positive samples. In general,MUC1 IgM positive samples tended to give a wide, elongatedsequence as the epitopic region. Examples of the results ob-tained with the natural and induced MUC1 Abs are shown,respectively, in Figures 4 and 5. The dip shown in the graphrepresents the binding sequence of the MUC1 Ab contained inthe sample. The minimal binding sequence of HuHMFG-1 (Fig.4a), used as a positive control, is defined by this method asAPDTR, with participation in the epitope of the flanking aminoacids. The influence of arginine in the conformation of theepitope (Briggset al., 1993) is shown by the abolition ofreactivity of HuHMFG-1 with the PDTA peptide. The reactivityof HuHMFG-1 with the STAAAA peptide is conserved. Thesamples depicted in Figures 4b and 5a1 show a dip in reactivitywith the STAPPAHGVT sequence. Inhibition of the reactivityof the sample from the vaccinated patient (Fig. 5a2) is completeexcept in the case of the sample incubated with the STAAAApeptide (no intact STAPPA sequence that can inhibit) tested onthe PDTA peptide (STAPPA sequence conserved). While in thevaccinated patient this STAPPAHGVT response is associated toa diminished reactivity with the GalNAc(6) and GalNAc(9)peptides (Fig. 5a1), recognition of these twoGalNAc-peptides isbarely affected in the sample from the control subject (Fig. 4b).Reactivity with the GalNAc(3) peptide is conserved in bothcases. The MUC1 IgG positive sample from a breast cancerpatient (Fig. 4c) shows fine specificity for the SAPDT as well asfor the STAPPAHG sequences of the tandem repeat togetherwith good recognition of the GalNAc-peptides. Reactivity withthe GalNAc(3) peptide is 113%.
Induced Abs with participation of the PDTRPAP sequence intheir epitope show diminished recognition of the GalNAc(3) pep-tide (Fig. 5b1 andc1). The concomitant presence of Abs directedto the PDTRPAP and the STAPPAHG regions in the sample from
TABLE III – EPITOPE MAPPING OF SERUM MUC1 ANTIBODIES WITHOVERLAPPING HEPTAMERS FOLLOWING THE TANDEM REPEAT
SEQUENCE OF THE MUC1 MUCIN PEPTIDE CORE (‘PEPSCAN’ TECHNIQUE)
IgG IgM
Minimal epitope1 N Minimal epitope1 N
14 8
Control subjects RPAPGS(T) 8 (T)RPAPGS 5(N 5 22) 4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
1Antibodies directed to two diverse sequences were present in 13IgG and in 5 IgM positive samples.
706 VON MENSDORFF-POUILLYET AL.
a vaccinated patient depicted in Figure 5b1 is demonstrated also inthe results obtained with the pre-incubated samples (Fig. 5b2). Thesample incubated with the STAAAA peptide (no intact STAPPAsequence that can inhibit) tested on the PDTA peptide (STAPPAsequence conserved), as well as the sample incubated with thePDTA peptide (no intact PDTRPAP sequence that can inhibit)tested on the STAAAA peptide (PDTRPAP sequence conserved)give, respectively, 60 and 40% reactivity.
Figure 4d shows results obtained with the MUC1 IgM positiveserum sample obtained from a breast cancer patient, which is usedto create the IgM standard curve in the MUC1 Ab assay (vonMensdorff-Pouilly et al., 1998). The dip in reactivity with the20-mer peptides signalises the sequence GSTAP together with theflanking amino acids as the epitope, as well as a participation of thePDTRPAP sequence (diminished reactivity with the PDTA pep-tide) in the immune response. In this and in other cases (Table III),no minimal epitopic sequence could be defined with the overlap-ping heptamers.
DISCUSSION
An optimal MUC1 vaccine should induce high titer Abs thatbind effectively to the mucin on the tumor cell membrane. Only inthis way can Abs, by capping the mucin and thereby exposing cellsurface receptors necessary for cell-cell interaction, restore celladhesion (Wesselinget al., 1995) or allow recognition of the tumorcell by effector cells of the immune system (van de Wiel-vanKemenadeet al., 1993). Our results show that the majority ofnaturally occurring human MUC1 Abs, unlike murine MUC1MAbs generated against the whole mucin molecule, are directed toother sequences on the MUC1 peptide core, rather than to se-quences in the PDTRPAP region of the tandem repeat. The ma-jority of the serum samples reacted, to a greater or lesser extent,with both modified peptides, suggesting that Abs directed to morethan one epitope coexist in one and the same subject. This isconfirmed by the results obtained with the overlapping hepta- and20-mer peptides. The most frequent minimal epitopic sequence to
FIGURE 2 – Binding profiles of two MUC1 IgG positive samples from (a) a breast cancer patient (T1N0M0), minimal epitopic sequencesRPAPGS and STAPPA, and (b) a vaccinated patient in group 1 (the same as in Fig. 5a1 anda2), minimal epitopic sequences PPAHGVT andPDTRP, to synthetic heptamer peptides spanning the MUC1 tandem repeat sequence, determined using the ‘Pepscan’ technique. The anti-RPAPMAb C595 was tested against tethered tetrapeptides TRPAPGS and GLAQ, as positive and negative controls, respectively. Values represent themeans of signals from tethered peptides synthesized on adjacent pins.
which natural MUC1 Abs are directed is RPAPGS. Interestingly,a human scFv antibody to the MUC1 core peptide isolated from alarge naive phage antibody library, which stained best adenocar-cinoma, bound to the PAPG(S) sequence of the tandem repeat(Henderikxet al., 1998).
That the natural Abs bind predominantly to sequences involvingthe arginine, also when using longer peptides, is demonstrated bythe fact that the reactivity of both the IgG and IgM samples wasaffected to a greater extent by the substitution of the arginine in thePDTRPAP sequence than by the substitution of the two prolines inthe STAPPA sequence. While this seems to apply also to the IgMMUC1 Abs induced by vaccination, induced MUC1 IgG Abs seemto be predominantly directed to regions on the MUC1 tandemrepeat that include one or both prolines in the STAPPA sequence.This is in agreement with the results obtained by Karanikaset al.(1997) who found that the reactivity of IgG1 MUC1 Abs inducedafter injection of mannan-MUC1 fusion protein was directed pre-dominantly to the PAPGSTAP and STAPPAGH sequences. On theother hand, Adluriet al.(1999) found that only peptides containingAPDTRPA at the C-terminal end were able to completely inhibitELISA reactivity of MUC1 IgG and IgM positive plasma samplesfrom six patients in group 1 (also included in this study) to the30-mer peptide used as immunogen. They found a weak inhibition(20–40%) of anti-MUC1 IgG reactivity with a peptide containingthe full STAPPAHGV sequence in only three of the six cases. Ourresults with the MUC1 IgM positive samples agree with thoseobtained by Adluriet al., but we also consistently find an addi-tional, in some cases predominant, response to the STAPPAHGVsequence in the MUC1 IgG positive samples from the same groupof patients (see Figs. 2b, 5a1,a2 showing results all obtained withthe same sample from a patient in group 1). Differences in resultsmay be due to a higher sensitivity of our assay indicated by thehigher sample dilutions used by us in the inhibition tests: 1:4,000for both IgG and IgM positive samples against 1:80 and 1:320 forIgM and IgG samples, respectively, by Adluriet al.(1999). Wehave observed that assays based on peptides adsorbed directly tothe plate (Koteraet al., 1994; Richardset al., 1998) have a goodsensitivity for detecting natural MUC1 IgM responses but, incontrast to our assay (von Mensdorff-Pouillyet al., 1998), fail todetect natural IgG responses.
Remarkably, patients vaccinated with the 33-mer peptide, whichhas RPAPGS (the most frequent minimal epitopic sequence rec-ognized by the natural Abs) at the C-terminal end, produced higherantibody titers than the patients vaccinated with the 30-mer pep-tide. The RPAPGS sequence contains a residue that can be glyco-sylated in the tumor-associated mucin and is flanked by two otherpotential glycosylation sites. Naturally occurring MUC1 Abs areable to recognize the GalNAc-peptides just as well as the nakedpeptide. In the case of the IgG and IgM positive samples frombreast cancer patients, recognition of the GalNAc-peptide with aGalNAc attached to the threonine in the PDTR sequence is en-
TABLE IV – REACTIVITY OF THE SERUM SAMPLES WITH THE MODIFIED PEPTIDES AND GALNAC-PEPTIDES,ANALYSED AS RATIOS OF THE RESULT OBTAINED WITH THE 60-MER PEPTIDE (100% REACTIVITY)1
FIGURE 3 – Reactivity of (a) MUC1 IgG positive and (b) MUC1IgM positive samples with the modified peptides and the GalNAc-peptides. The bars show the percentage of reactivity (mean6 SD) ineach group calculated in relation to the reactivity (100%) with the60-mer peptide.
hanced in relation to that of the naked peptide. The same appliesto the IgG positive samples and the GalNAc-peptide withGalNAcs attached to the threonines and serine in the STAPPAH-GVT sequence. From the point of view of a method for screeeningfor MUC1 Abs in breast cancer patients, these results indicate thata MUC1 Ab assay based on GalNAc peptides may have a highersensitivity than assays based on non-glycosylated peptides.Tumor-associated MUC1 has shorter carbohydrate side-chains(Whitehouseet al., 1997) and it is likely that these glycosylatedsequences are involved in eliciting the respective B-cell re-sponse. Karstenet al. (1998) have shown that glycosylationwith GalNAc at the T of the PDTR enhances binding of 12DTR-specif MAbs with the single tandem repeat. While ten ofthese MAbs were generated against cellular immunogens or
mucin preparations,i.e., glycosylated forms of the mucin mostcertainly containing glycosylated DTR sequences, two of themwere generated to non-glycosylated peptide sequences, indicat-ing that a glycosylation-induced change in the peptide confor-mation could be responsible for the enhanced binding. ThePDTRP sequence forms the tip of a protruding ’knob’ andattains its native conformation on synthetic peptides with morethan two repeats (Fontenotet al., 1993). As we employ peptidesand GalNAc-peptides with three repeats, where the role of theGalNAc in stabilizing the ‘knob’ conformation is no longer ofimportance, there is a strong probability that the natural im-mune response is directed to a structure where both the peptidesequence and the glycan contribute to epitope conformation.This probability is strengthened by the fact that naturally oc-
FIGURE 4 – Reactivity with the overlapping 20-mer peptides, modified peptides and GalNAc-peptides of (a) HuHMFG-1 at a concentrationof 0.5 mg/mL, (b) a MUC1 IgG positive serum sample from a pregnant woman diluted 1:500, (c) a MUC1 IgG positive serum sample from abreast cancer patient (pTisN0M0) diluted 1:500 and (d) a MUC1 IgM positive serum sample from a breast cancer patient (pT1N0M0) diluted1:1,000. The amino acids in the x axis indicate the first amino acid in the sequence of each of the 20 overlapping peptides. The bars show thepercentage of reactivity of the sample with each peptide calculated in relation to the reactivity (100%) with the 60-mer peptide. The epitope isdefined by the sequence showing a dip in reactivity.
TABLE V – REACTIVITY OF THE SERUM SAMPLES WITH THE MODIFIED PEPTIDES AND GALNAC-PEPTIDES, ANALYSED AS RATIOS OF THE RESULTOBTAINED WITH THE 60-MER PEPTIDE (100% REACTIVITY)1
curring IgG antibodies prefer the glycosylated to the non-glycosylated STAPPA sequence and by the responses to theglycosylated peptides observed in the vaccinated patients.
The MUC1 IgG and IgM Abs induced by vaccination with theMUC1 peptides recognize predominantly the naked peptide. Ad-dition of GalNAc groups to the peptide mask the peptide epitope
to which the induced Abs are directed. Recognition of theGalNAc-peptides is highly impaired and diminishes further as thenumber of glycosylated sites increases. The stronger Ab responsepresent in the patients vaccinated with the 33-mer peptide isaccompanied by an even lower recognition of the glycosylatedpeptides. The predominance of the STAPPA sequence in the IgG
FIGURE 5 – Reactivity with the overlapping 20-mer peptides, modified peptides and GalNAc-peptides (a1, b1,c1) of (a) a MUC1 IgG positiveplasma sample taken at week 5 from a vaccinated patient in group 1, (b) a MUC1 IgG and (c) a MUC1 IgM positive plasma sample taken atweek 9 from two vaccinated patients in group 2. Reactivity of the same samples with the 60-mer peptide and the modified peptides (a2, b2, c2)after preincubation with the modified peptides and the reverse peptide as negative control. The samples were tested at dilution 1:4,000. The barsshow the percentage of reactivity of the sample with each peptide calculated in relation to the reactivity (100%) with the 60-mer peptide.
710 VON MENSDORFF-POUILLYET AL.
responses is again indicated by the correlation between resultsobtained with the GalNAc-peptides and the STAAAA peptide.While the reactivity of the induced IgG Abs is already stronglyreduced after glycosylation of the threonine in the STAPPA se-quence, the effect on the IgM responses is more obvious when aGalNAc is added to the serine, which is also part of the RPAPGSsequence. This masking of the epitopes by minimal glycosylationexplains the moderate to weak binding of the MUC1 Abs inducedby the 30-mer peptide to tumor cells expressing MUC1 (Adlurietal., 1999). But binding there is, and it may be effective to a certainextent. As the levels of MUC1 Abs induced in the vaccinatedpatients are so much higher than the levels found in the naturalimmune response and because the response is polyclonal, therestill may be enough induced Abs not affected by glycosylation ableto bind to the mucin and capable of mediating a clinical response.Our preliminary results with samples from the vaccinated patientsin group 2 indicate that Abs to MUC1 may mediate cell death by
inducing antibody-dependent cellular cytotoxicity (Snijdewintetal., data not shown).
Our results indicate that the optimal MUC1 vaccine would notbe a peptide but a glycopeptide, possibly the same 33-mer peptideused in the second phase I trial but with GalNAc (or sialyl-T
N)
attached to the threonine in the PDTR and to the serine andthreonine in the STAPPA sequences. It remains to be seen whetherimmunization with MUC1 glycopeptides in an adjuvant setting caninduce antibodies with a binding capacity to cancer mucin highenough to mediate tumor cell killing and to protect patients withadenocarcinoma from recurrence of disease.
ACKNOWLEDGEMENTS
This work was supported in part by the Danish Cancer Society(to C.A.R.). We thank Ms A. Kok for expert technical assistance,Dr J. Taylor-Papadimitriou for the gift of HuHMFG1 antibody andDr H. Clausen for critical reading of the manuscript.
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