2 NEMOD GmbH & Co. KG, Robert-Rössle-Str. 10, D-13125 Berlin, Germany; 3 Max Delbrück Centre for Molecular Medicine, D-13125 Berlin-Buch, Germany; 4 Institute of Organic Chemistry, University of Hamburg, D-20146 Hamburg, Germany; and 5 Glycotope GmbH, D-13125 Berlin-Buch, Germany
Received on February 6, 2004; revised on April 20, 2004; accepted on April 20, 2004
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Abstract |
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Key words: conformation / glycosylation / MUC1 antibodies / tumor epitope / tumor vaccine
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Introduction |
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In the mid-1990s a plethora of monoclonal anti-MUC1 antibodies became available that required comparison and standardization. Thus the ISOBM TD-4 International Workshop on Monoclonal Antibodies against MUC1 in 1996 became a landmark in the field. Fifty-six supposedly MUC1-specific monoclonal antibodies (mAbs) were compared and their epitopes mapped (Rye and Price, 1998).
At that time it was well established that tumor MUC1similar to other membrane glycoproteinsdiffers from normal MUC1 by modified (essentially truncated) glycan side chains, which results in a better accessibility of peptide epitopes (Brockhausen, 1999; Burchell et al., 2001
; Lloyd et al., 1996
; Taylor-Papadimitriou et al., 1999
). In addition, it was assumed that the immunodominant DTR motif was generally not glycosylated (Finn et al., 1995
). Thus the tumor specificity of anti-MUC1 mabs was interpreted solely in terms of better access to peptide epitopes due to reduced interference by glycans located in the vicinity of the epitope. This picture changed when we found that the DTR sequence is actually glycosylated (Müller et al., 1997
) and that short glycans typical for tumor MUC1 (GalNAc
1- or Galß1-3GalNAc
1-) at this site may even improve antibody binding to the DTR epitope (Karsten et al., 1998
). Our hypothesis was that glycosylation at this site might induce a knoblike structure similar to that described for repetitive non-glycosylated tandem repeat peptides (Fontenot et al., 1995
). Employing a large panel of monoclonal anti-MUC1 antibodies and three sets of specially devised antigenic structures, among them a unique set of novel synthetic glycopeptides, we continued our studies with the aim of a more refined epitope characterization. As a result, we were able to (1) dissect the glycosylation effect from the length effect, (2) describe subgroups of anti-MUC1 antibodies recognizing the DTR motif, and (3) define a novel tumor-specific MUC1 epitope.
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Results |
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Glycosylation dependency
The unexpected observation made by us in 1997 that many antibodies specific for the immunodominant DTR region of MUC1 bound better to the glycosylated than to the unglycosylated epitope (Karsten et al., 1998) was reexamined in this study with a specially devised 30-mer peptide/ glycopeptide pair of test antigens with the DTR motif situated in the middle of the peptide, and its threonine O-glycosylated with GalNAc
in the glycopeptide variant. Absorbance ratios Aglycosylated:Aunglycosylated were calculated for each antibody and are presented in Table II. These ratios were taken from one experiment but confirmed in repeated experiments of the same kind and also in different experiments employing the same but N-terminally biotinylated peptide and glycopeptide, respectively. No qualitative differences were observed between direct coating to the polystyrene surface and coating via biotin/streptavidin, confirming that the glycosylation effect was not due to a different degree of binding of glycopeptides as compared to their unglycosylated counterparts on the solid phase. Differences in absorbance seen between individual experiments did not change in any case the classification of the antibodies, which was done as follows. GD-1: binding strongly dependent on DTR glycosylation (ratio > 6); GD-2: moderately influenced (ratio 1.35.9); GI: independent of glycosylation (ratio 0.81.2); iGD: inversely influenced (binding inhibited) by glycosylation (ratio < 0.8).
The results showed that about half of the antibodies were glycosylation-influenced, that is, revealed better binding to the glycosylated epitope. Only in one case (VU-4H5) was inhibition of antibody binding observed after glycosylation of the epitope with GalNAc.
Both control antibodies recognizing non-DTR epitopes (BCP9, MF11) were found not influenced by DTR glycosylation in their binding, as could have been anticipated.
Combined glycosylation and length effects
Both the length effect and the glycosylation effect suggest a modification of the conformation of the DTR region as the reason for improved antibody binding, which for the length effect has indeed been demonstrated (Fontenot et al., 1993). Therefore, we were interested to examine whether increasing peptide length and glycosylation at the DTR lead to the same assumed conformation as indicated by an identical maximum absorbance or whether both effects are additive and can be distinguished. This question could be resolved with a novel series of glycopeptides (O-glycosylated with GalNAc at the threonine of each DTR motif) of different length (one to five tandem repeats); see Table III.
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Periodate oxidation
Periodate treatment according to Woodward et al. (1985) is a valuable tool to confirm carbohydrate involvement in immunological recognition. This treatment cleaves sugar rings between vicinal OH groups and renders the structure unrecognizable for a specific antibody. It was therefore of interest to examine to what extent the observed effects were actually carbohydrate-induced, and if so, whether they were reversible.
The results shown in Figure 4 confirmed that the binding of mAb A76-A/C7 is glycosylation-conditioned (Figure 4A). However, the length effect partially compensates for the decrease in binding caused by periodate oxidation. This confirms the conclusion that both effects may overlap but are not identical. A different binding pattern was provided by mAb HMFG-1, whose binding is strongly influenced by the number of tandem repeats but not by DTR glycosylation (Figure 4B). An antibody classified as being neither glycosylation- nor length-dependent, E29, revealed a binding pattern as expected in this case and was not influenced by periodate oxidation of the glycopeptide in its binding (Figure 4C). This is also proof that the peptide was not damaged under these conditions. Interestingly, binding of antibody VU-4H5, which is suppressed by DTR glycosylation, was not restored after periodate oxidation but increased moderately with increased peptide length (Figure 4D). The unique behavior of mAb VU-4H5 suggests steric hindrance of antibody binding by the glycan even after oxidative ring cleavage and again demonstrates the length effect as an independent phenomenon.
Search for possible correlations
The fact that about half of the antibodies bound better to the glycosylated epitope initiated a search for possible correlations to other known properties of the antibodies, such as length dependency, isotype, epitope characteristics, immunogen, or the efficiency to detect carcinoma-specific MUC1. Except length and DTR-glycosylation influence, which were based on our own data (Table II), the other parameters were taken from the reports of the 1996 workshop (Rye and Price, 1998). In detail, isotypes, peptide sequences agreed on as epitopes and immunogens were taken from the Summary Report (Price et al., 1998
), whereas the efficiency data were from Table 3 in Norum et al. (1998)
. The latter were arbitrary points based on the efficiency with which solid phase-coated antibodies presented shed tumor MUC1 (from a pool of patients' sera) for a given tracer antibody in immunoradiometric sandwich assays.
Among the examined parameters, the strongest correlation was found between glycosylation dependency and the type of immunogen used for generating the antibody (Tables IV and V). In fact, among the antibodies included in the study, all mAbs generated with immunogens from human tumor sources (e.g., breast cancer cells or MUC1 prepared from tumor ascites) bound better to the DTR-glycosylated peptide than to the naked peptide. The extent of this phenomenon varied from strong to weak. On the other hand, almost all mAbs generated with immunogens from nontumor sources (e.g., naked peptides or milk fat globule membranes) were glycosylation-independent. The only exception was SM3, which was generated with partially deglycosylated HMFG as immunogen, and was slightly glycosylation-influenced. It should be mentioned, however, that this preparation was treated with anhydrous hydrogen fluoride (Burchell et al., 1987), which, under mild conditions (1 h at 0°C), leaves GalNAc O-glycosidically bound to the peptide backbone intact (Mort and Lamport, 1977
), resulting in structures similar to the tumor epitope defined by us (see Discussion).
Among the other parameters, some interesting correlations to the two subgroups (glycosylation-dependent [A, Table IV] versus glycosylation-independent mabs [B, Table V]) were also found. First, the length dependency is more pronounced in subgroup A compared to B. Second, in subgroup A the mean peptide length of the epitopes (8.4 amino acids) was longer than in subgroup B (5.6 amino acids). Both differences may be interpreted as indicators of the prevalence of conformation epitopes rather than sequence epitopes in glycosylation-effected MUC1 antibodies. Efficiency points in recognizing shed tumor MUC1 according to Norum et al. (1998) were also differing between groups A and B. The numerical sum of all points of group A was 435 as compared to 218 in group B, although individual scores varied considerably. This can be explained by the fact that shed tumor MUC1 is obviously different from cell-bound tumor MUC1 because it is only weakly detected by some mAbs (unpublished data). No correlation was found to isotypes.
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Discussion |
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Our conclusion is supported by a recent study in which the fine specificities of anti-MUC1 antibodies in human sera were reported. Anti-MUC1 antibodies from breast cancer patients revealed a preference for glycosylated peptides (Von Mensdorff-Pouilly et al., 2000a).
The present study was done with GalNAc1-substituted peptides; from our earlier data (Karsten et al., 1998
) it can be inferred that Galß1-3GalNAc
1- instead of GalNAc
1- is equally effective with respect to an enhanced binding of antibodies of the respective group. Both are typical glycans of tumor MUC1 (Goletz et al., 2003
; Lloyd et al., 1996
). However the glycan moiety itself is not directly recognized by the MUC1 antibodies. This can be inferred from the following: (1) all mAbs included in this study recognize defined peptide sequences (Price et al., 1998
) and bind significantly to unglycosylated oligomeric MUC1 tandem repeats; (2) binding increments by glycosylation are similar with either GalNAc
1- or Galß1-3GalNAc
1-, which are two immunologically completely different entities; (3) the mAbs do not bind to either of the glycans in enzyme-linked immunosorbent assay (ELISA) (data not shown); and (4) structural studies with mAb SM3 (glycosylation-dependent, Table II) revealed no evidence for carbohydrate involvement in binding (Möller et al., 2002
).
A number of nuclear magnetic resonance (NMR) studies have dealt with the secondary structure of the DTR motif (Fontenot et al., 1993, 1995
; Grinstead et al., 2002
; Kirnarsky et al., 2000
; Scanlon et al., 1992
; Schuman et al., 2003
; Tendler, 1990
). In one case, the crystal structure of an antibody-peptide complex has been accomplished (Dokurno et al., 1998
). The influence of peptide elongation (from one to three nonglycosylated tandem repeats) was described as leading to a knoblike, type I ß-turn conformation of the region around DTR (Fontenot et al., 1993
, 1995
). The effect of DTR glycosylation on its conformation has recently been investigated (Kirnarsky et al., 2000
; Möller et al., 2002
), albeit only with short glycopeptides. Attachment of GalNAc
1-O- shifts the conformation of the DTR motif from the type I ß-turn toward a more rigid and extended state (Schuman et al., 2003
). This supports our suggestion that length and glycosylation effects lead to different conformations of this region.
O-glycosylation with GalNAc has also been shown to result in a more rigid conformation in case of peptides other than MUC1 (Huang et al., 1997; Live et al., 1999
).
Whereas a ß-turn within the DTR motif may explain the immunodominance of this region in case of the unglycosylated peptide (Fontenot et al., 1993, 1995
; Schuman et al., 2003
), our data suggest that this is not the tumor-specific MUC1 epitope. The correlations found by us imply that tumor MUC1 contains the glycosylated DTR, which is in agreement with biochemical data (Müller et al., 1999
), and indicate the prevalence of conformation epitopes rather than sequence epitopes among DTR-specific antibodies generated against tumor MUC1. We hypothesize that the tumor-specific MUC1 epitope is a certain conformation of the ...PDTRP... epitope. Its exact structure, of course, cannot be deduced from our data. It should have similarity to that described by Schuman et al. (2003)
, but may be modified by the length effect. In any case, the tumor-specific MUC1 epitope is clearly different from what it hitherto was believed to be. The oligomeric MUC1 glycopeptides described here could provide an excellent subject for structural studies, which may be able to elucidate the complex interplay of glycosylation and oligomerization on the conformation of this immunologically important site of tumor MUC1.
Our results should be relevant to MUC1-based immunotherapies. Antibodies that specifically recognize the epitope described here are less prone to bind to normal MUC1. This explains, at least in part, the already described more or less tumor-specific histological staining pattern of some MUC1 antibodies, for example, SM3 (Burchell et al., 1987) or A76-A/C7 (Cao et al., 1997a
). However, it is evident from this and earlier studies (Cao and Karsten, 2001
; Norum et al., 1998
) that even nominally similar antibodies reveal astonishing differences in their fine specificity. In addition, the selection of an individual antibody for adjuvant immunotherapy depends on a number of other parameters, too. The case for cancer vaccines is more straightforward. On the one hand, the DTR motif is well known as a B cell epitope. The presence of anti-MUC1 antibodies in the serum has been found beneficial for breast cancer patients (Von Mensdorff-Pouilly et al., 2000b
). On the other hand, among known MUC1 T cell epitopes (Brossart et al., 1999
; Feuerer et al., 2001
), the DTR region has also been reported (Gad et al., 2003
; Mukherjee et al., 2000
). Most recently, Apostolopoulos et al. (2003)
have actually shown that glycosylation with GalNAc at the DTR motif leads to a high-affinity binding to a murine major histocompatibility class I molecule, H-2Kb, and that the glycosylated MUC1 peptide is able to evoke strong specific T cell responses in vitro and in vivo. Similar results have been obtained with other proteins (e.g., a lysozyme peptide; Harding et al., 1993
). In conclusion, we propose that MUC1-based cancer vaccines should contain the ...PDTRP... sequence O-glycosylated at the threonine with Tn or TF (i.e., in a conformation corresponding to tumour MUC1).
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Materials and methods |
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Peptides and glycopeptides
Three sets of synthetic peptides/glycopeptides, all based on the MUC1 tandem repeat sequence, were employed for three different series of experiments.
Series I
Unglycosylated 20-mers and their oligomers of the type [VTSAPDTRPAPGSTAPPAHG]n, with n=1, 3, 4, 5, and 6, were obtained from Dr. J. Hilgers (Department of Obstetrics and Gynaecology, Free University, Amsterdam, The Netherlands). In the peptides with n=36, R was missing in the second repeat.
Series II
A glycosylated 30-mer with the sequence APPAHGVTSAPDT[GalNAc]RPAPGSTAPPAHGVTSA and its unglycosylated counterpart were synthesized by Biosyntan GmbH (Berlin-Buch). In addition, the same pair of glycosylated versus unglycosylated peptide was prepared in an N-terminally biotinylated form, which then could be coated more quantitatively on streptavidin microtiter plates (see later description). Biotinylation (with 6-aminohexanoic acid as N-terminal spacer) was performed as follows. Biotin was preactivated with 2-(1H-benzotriazole-1-yl)-1,1,3, 3-tetramethyluronium tetrafluoroborate and N-methylmorpholine in N-methylpyrrolidone for 10 min, and this mixture together with the solid phase containing the peptide/glycopeptide was stirred for 2 h.
Series III
A novel series of glycosylated MUC1 tandem repeat peptides of different length of the sequence A[HGVTSAPDT(GalNAc)RPAPGSTAPPA]n, with n=15 (TR1aTR5a), was synthesized (Table III).
The synthesis of the glycopeptides was conducted as a glycopeptide solid phase synthesis on Wang resin via the 9-fluorenyl-methoxycarbonyl (FMOC)3 technique. The amino acids were coupled as FMOC-fluorophenylesters, and the FMOC-ThrGalNAc building blocks with the help of O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate. The synthesis was started with a larger amount of resin (2.5 g) until TR1a was reached. One-fifth of the resin was then removed, and the synthesis was continued with the remaining resin via TR2a, TR3a, TR4a, and TR5a. After each of the intermediates had been achieved, a portion of the resin was removed, and the majority of the resin was used for the next step. The deacetylation of the saccharide was conducted with methanol/hydrazine 5:1 except for TR4a and TR5a, where 2-propanol/hydrazine 5:1 was used. Cleavage from the resin was achieved with 0.1% aqueous trifluoroacetic acid (95%). The purification of the substances was carried out by high-pressure liquid chromatography on reversed phase columns (RP18) in a gradient mode with water/acetonitrile as mobile phase. Structural elucidation was performed with NMR spectroscopy, and the molecular weights were determined by matrix-assisted laser desorption and ionization mass spectrometry.
Enzyme immunoassays
ELISAs were performed as follows.
Experimental series I (length effect)
Polystyrene microtiter plates of tissue culture type (Nunc, Wiesbaden, Germany) were coated with 50 µl per well of a solution of 10 µg/ml of peptide (from series I) in 0.1 M carbonate buffer, pH 9.6, overnight at 37°C to dryness. Antigens to be compared were always coated on the same plate. After three washes with phosphate buffered saline (PBS)/0.05% Tween 20, the purified antibodies were added (50 µl, 10 µg/ml) in PBS/Tween containing 1% bovine serum albumin (BSA), and incubated for 2 h in a moist chamber at 37°C. After three washings as before, the plates were incubated with peroxidase-labeled rabbit anti-mouse immunoglobulin serum (P260, Dako, Hamburg, Germany) diluted 1:2000 in PBS/BSA for 1.5 h at 37°C. After three final washings, color development was accomplished with o-phenylenediamine, and the absorbance (A) measured with a Spectra plate reader (SLT Labinstruments, Salzburg, Austria) at 492 nm. Blank values were subtracted, and means were calculated. From the means, ratio values A100-mer:A20-mer were calculated.
Experimental series II (glycosylation effect)
Experimental details were similar to series I except that the 30-mer peptide and glycopeptide from series II were employed, and water was used for coating instead of carbonate buffer to avoid ß-elimination during the drying process. To exclude the possibility that glycopeptides and peptides sticked differently to the plastic, a parallel series of experiments with biotinylated (glyco)peptides was performed. In this case, the protocol was modified as follows. Streptavidin-precoated microtiter plates (BioTeZ, Berlin-Buch) were coated with 100 µl per well containing 0.5 µg/ml of the biotinylated antigen in PBS/BSA (1 h at room temperature). The following steps were as described except that 100 µl volumes were used throughout and the concentrations of the primary and second antibodies were halved. Blank values were subtracted, and means were calculated. From the means, ratio values Aglycosylated peptide:Aunglycosylated peptide were calculated.
Experimental series III (combined effects)
Experimental details were similar to series I except that glycopeptides of series III were employed and water was used for coating instead of carbonate buffer. The concentration of the primary antibodies was varied between 0.1 and 5 µg/ml. Evaluation of binding patterns was generally based on data obtained at 5 µg/ml, although in most cases the overall patterns were found consistent for a given mAb within the whole concentration range. In some experiments the antigen concentration was also varied. Best results were obtained at 10 µg/ml. In some experiments, 3,3',5,5'-tetramethylbenzidine instead of o-phenylenediamine was used as substrate for peroxidase, and A was measured at 450 nm.
In all ELISA experiments, coating was done on a weight per ml basis to approximate equimolar concentrations of tandem repeats.
In some cases, carbohydrate-selective periodate oxidation of the coated antigens (10 mM NaIO4 in 50 mM sodium acetate buffer, pH 4.5, for 1 h at 25°C, followed by reduction of aldehydes by 50 mM NaBH4) was performed as described (Woodward et al., 1985).
Values were generated in duplicates and repeated at least three times. Graphs were prepared with the GraphPad Prism 3.0 program (GraphPad Software, San Diego, CA). Error bars indicate SE.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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