The sequentially repeating nature of the core
mucin polypeptide chain MUC-1 on the surface of malignant cells makes
it an excellent target for cancer immunotherapy. We describe a reliable and efficient method of synthesizing oligomers, up to five tandem repeats and oligomer heterotope derivatives with a 15-amino acid epitope from tetanus toxin using an improved convergent solid-phase peptide synthesis. The different oligomers were easily distinguishable by reverse-phase high pressure liquid chromatography, but they were
poorly fixed and migrated with the same migration rate, irrespective of
size, in electrophoretic studies. In contrast, the oligomer heterotopes
exhibited size-dependent electrophoretic behavior but in
high pressure liquid chromatography chromatograms the different heterotopes were eluted simultaneously in two peaks representing the
L- and D-enantiomers of the derivatives.
The oligomer heterotopes were recognized as antigens in Western
blotting with a murine monoclonal antibody against the epitope APDTR.
In enzyme immunoassay studies with the same antibody an increasing
reactivity was observed against the larger oligomers and confirmed by
inhibition assays as the MUC-1 pentamer was the most efficient
inhibitor. These results support the suggestion that the pentamer
attains a structure closer to the native conformation and is more
immunogenic. In conclusion, large composite peptides can be reliably
synthesized with the convergent solid-phase peptide strategy offering
an attractive option to vaccine designing and development.
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INTRODUCTION |
The implication of the mucin MUC-1 in many human tumors of
epithelial origin has generated great interest in recent years, particularly as a target for immunotherapy (1-3). The core protein of
MUC-1 is composed of a number of tandem repeats, each repeat consisting
of a 20-amino acid (aa)1
peptide (4, 5). Murine monoclonal antibodies raised against MUC-1
appear to react selectively with breast cancer mucins but not with
normal mucin (6, 7) and are targeted principally to the epitope
APDTRP of the 20-mer peptide (8-10).
The exposure of the MUC-1 tandem repeats on the cell surface of
malignant cells combined with the ability of the immune system to
respond specifically to these peptide epitopes offers an ideal opportunity to rationally design appropriate synthetic vaccines to
target this tumor-associated antigen. Recent vaccination studies in
humans using a MUC-1 dimer conjugated with diphtheria toxoid showed
that the formulation used was not sufficiently immunogenic (11). This,
perhaps, is not surprising as one or two repeats do not attain a native
conformation, whereas peptides consisting of three or more repeats take
a rod-shaped ordered structure (12). A five-repeat peptide has been
reported to induce enhanced antigenicity (13), and the results of a
phase I vaccination trial were encouraging (14). In the present study
we exploited the recent advances in convergent solid-phase synthesis
(CSPP) to reliably prepare and evaluate MUC-1 oligomers up to five
repeats and MUC-1 oligomer derivatives with a 15-aa epitope from
tetanus toxin.
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EXPERIMENTAL PROCEDURES |
Materials--
The 20-mer MUC-1 fragment 6 and the
15-mer ttp fragment 7 (Fig. 1) as well as smaller MUC-1
fragments (Table I) were synthesized by an automated solid-phase Fmoc
strategy (430A Peptide Synthesizer, Applied Biosystems) with the
N-methylpyrrolidinone-dicyclohexylcarbodiimide (NMP-DCC)
method according to the manufacturer's instructions, modified to take
2-chlorotrityl chloride resin (CLTR). The solid-phase support was in
all cases CLTR. Protected amino acids and CLTR were obtained from CBL
Patras (Patra, Greece). NMP, dimethylformamide (DMF), dichloromethane
(DCM), acetonitrile, methanol, trifluoroacetic acid, acetic acid, and
diisopropylcarbodiimide (DIC) were obtained from Merck (Germany). DCC,
ethanedithiol, thioanisole, phenol, piperidine, trifluoroethanol (TFE),
and diisopropylethylamine (DIPEA) were obtained from Sigma.
Synthesis of Protected Fragments 6 and
7--
For the synthesis of the required protected fragment
6 and of the protected fragment 7 (Fig. 1) we
used the acid labile Fmoc/t-butyl amino acids. Trityl was
applied for the protection of the side chains of His and Ser. Pmc
(2,2,5,7,8-pentamethylchroman-6-sulfonyl) was applied for Arg. This
protection scheme allowed the mildest possible deprotection conditions
of the final protected peptide. The solid support CLTR was chosen
because it effectively suppresses diketopiperazin formation during the
synthesis of peptides containing Pro (15-17) at the C terminus, and in
addition the protected peptides synthesized on this resin could be
cleaved quantitatively under extremely mild conditions in the absence
of acids (18).
The synthesis of 6 was initiated with the esterification of
Fmoc-Pro-OH 1 on 1 g CLTR 2 with DIPEA in DMF, to yield Fmoc-Pro-O-CLTR 3. Unreacted remaining trityl chloride functions were converted to the corresponding methyl ether by
washing the resin three times with methanol/DIPEA/DCM (15:5:80) for 5 min each. Fmoc removal was performed immediately after the end of the
esterification procedure by treating with 25% piperidine in DMF to
obtain resin 4 with a substitution of 0.3 mmol of Pro/g of
resin. The peptide chain was elongated using DCC/HOBt as the condensing
agent, NMP as the solvent, and 25% piperidine in NMP for the removal
of Fmoc. Finally, the obtained resin peptide ester 5 (Fig.
1) was treated at room temperature for 60 min with a 3:7 mixture of
TFE/DCM. Protected peptide 6 was recovered after solvent
evaporation in vacuum, precipitation with water, filtration and drying
in ether. The protected ttp 7 was synthesized by similar
methods.
Synthesis of MUC-1 Oligomers and ttp-MUC-1 Heterotope Derivatives
by CSPP--
A small scale pilot synthesis was performed by condensing
a 2-fold molar excess of protected fragment 6 over the
resin-bound amino component 5. The protected fragment was
applied as a 0.05 M solution in Me2SO and with
condensing agent DCC/HOBt. After 24 h of condensation, unreacted
remaining amino groups were capped by acetylation using a 10-fold molar
excess of acetic anhydride and DIPEA in NMP. The amino-terminal Fmoc
group was then removed by treatment with 25% piperidine in NMP.
Completion of the Fmoc removal was checked by TLC (19). After each
condensation, the resin-bound peptide was cleaved deprotected and
analyzed by reverse phase-HPLC. Portions of the resins with the
oligomers still attached were used for the synthesis of the ttp-MUC-1
heterotope derivatives 8, 10, 12, 14, 16 (Fig. 2) using the
same strategy. Protected ttp 7 was dissolved in
Me2SO (0.05 M) activated with DCC-HOBt and
reacted with the MUC-1 oligomer resins 5, 9, 11, 13, 15 (Fig. 2) in two-molar excess for 24 h.
In a second CSPP preparation of the 5-mer MUC-1 we started with a new
synthesis of resin 5 that condensed with the protected MUC-1
6 four times in succession, introducing the following
modifications: (i) a lower proline substitution in resin
5 (0.1 mmol/g resin); (ii) a more concentrated solution of 6 (0.1 M); (iii) each
fragment was applied in three-fold molar excess; and (iv)
DCC was replaced by DIC as the dehydrating agent.
Cleavage, Deprotection, and Isolation of Peptides--
After the
final coupling step, all synthesized peptides were treated with 25%
piperidine for removal of the Fmoc, washed with diethyl ether, and
dried. The cleavage and deprotection was performed with a
trifluoroacetic acid:water:phenol:ethanedithiol:thioanisole solution
(82.5:5:5:2.5:5) for 2 h at room temperature. The crude peptides
were precipitated in ice-cold ether, washed several times with ether,
dried, dissolved in water, lyophilized, and stored at
20 °C until
used. The peptides synthesized for the present study are listed in
Table I.
HPLC Analysis, SDS-PAGE Electrophoresis and Western
Blotting--
HPLC was performed in the Waters 600 LC system (Waters)
with a UV 486 detector using a semipreparative C18 column (Synchropack RP-P 250 × 7.8-mm, Synchrom Inc.).
SDS-PAGE electrophoresis was performed with the Phast system (Amersham
Pharmacia Biotech, Sweden), and the gels were stained with silver
nitrate according to the manufacturer's instructions.
Gel transfer to nitrocellulose (Biotrace NT, Gelman Sciences, Germany)
was performed with passive diffusion with transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, containing
20% methanol) for 15 min. The membranes were either silver-stained
directly (20) or blocked with 2% gelatin in 10 mM
Tris-buffered saline, pH 7.4, and used for Western blotting.
For the Western blotting studies, mouse anti-MUC-1 monoclonal antibody
BC-2 was incubated at 1:2000 with the blotted membrane in 10 mM Tris-buffered saline, pH 7.4, containing 0.1% gelatin for 1 h at room temperature. After washing excess reagents, rabbit anti-mouse IgG alkaline phosphatase conjugate was added in the same
buffer and incubated for 1 h at room temperature. The reactions were revealed using 5-bromo-4-chloro-3-indolyl phosphate-nitro blue
tetrazolium as substrate (21).
Mass Spectroscopy--
Mass spectrograms of MUC-1 pentamer were
kindly performed by Prof. Giovanni Sidonna (Calabria, Italy), using a
Fisons EC-VG platform mass spectrometer.
Enzyme-linked Immunosorbent Assay--
The assay was performed
essentially as described previously (22). Briefly, MUC-1 oligomers were
immobilized onto flat-bottomed microtiter wells (NUNC, Denmark) by
overnight incubation in 50 mM carbonate-bicarbonate buffer,
pH 9.6. The wells were blocked with 1% gelatin in 50 mM
PBS, pH 7.0, for 1 h at room temperature and washed with 0.1%
Tween in PBS. Murine monoclonal anti-MUC-1 antibody (BC-2) in PBS with
0.1% bovine serum albumin were added to each well (100 µl) and
incubated for 1 h at room temperature. The BC-2 antibody (IgG1)
with specificity to peptide epitope APDTR of MUC-1 (1) was a gift from
Dr. P.-X. Xing (Austin Research Institute, Heidelberg, Australia).
After washing off excess antibody, a second incubation with a rabbit
anti-mouse antibody-peroxidase conjugate (100 µl) followed for 1 h at room temperature. After four washes with PBS the antigen-antibody
reaction was revealed by adding to each well 100 µl of substrate
(0.25 mM tetramethyl benzidine, 0.03% v/v hydrogen
peroxide in 50 mM sodium acetate buffer, pH 5.2) (23). The
reaction was stopped after 15 min by the addition of 50 µl 2 M sulfuric acid per well. The absorbance was measured at
450 nm (Multiscan, Flow Labs, Finland).
Inhibition Enzyme Immunoassay--
The monoclonal anti-MUC-1
antibody BC-2 was diluted 1:6000 in PBS with 0.2% bovine serum
albumin. 150-µl aliquots were admixed with equal volumes of various
peptides in serial dilutions or with an equal volume of PBS (control).
The mixtures were dispensed (100 µl) into MUC-1 pentamer-coated wells
(2 µg/ml) in duplicate and incubated at 37 °C for 1 h. After
removing unreacted reagents, 100 µl of diluted rabbit anti-mouse
IgG-peroxidase conjugate were dispensed to each well and incubated at
37 °C for a further 1 h. The antigen-antibody reaction was
revealed as described above. Inhibition was measured by the decrease in
binding of the antibody compared with the control.
 |
RESULTS AND DISCUSSION |
The repetition of the MUC-1 20-mer peptide is an attractive model
with which to study the efficiency of the convergent approach for the
solid-phase synthesis of large peptides and small proteins (24). The
method is based on the principle that protected peptide fragments
corresponding to the entire protein sequence can be condensed
sequentially on a suitable solid support. We applied this method for
the synthesis of MUC-1 oligomers using Fmoc amino acids and
2-chlorotrityl resin as the solid support (15).
Our synthesis strategy for the preparation of MUC-1 oligomers utilized
the sequential condensation of the protected peptide 6 (Figs. 1 and
2). The aa sequence of this peptide was designed to contain the immunodominant epitope APDTRP in the interior of the peptide so that each repeated unit would contain one epitope and
prolines at positions 1 and 20. The rationale for our choice was that:
(i) the optical stability of proline at the C-terminal position of the electrophilically activated fragment would ensure the
optical purity of the resulting oligomers; (ii) fragment
6 was soluble in the solvent used (Me2SO) as
required for a successful solid-phase condensation reaction; and
(iii) both the free NZ-function of the resin-bound amino
component as well as the C-terminal function of the applied carboxy
component were exposed and easily reached by the other reactants. This
could not be the case if completely formed
-turns were contained in
one of the reactive terminal positions. We anticipated that the
presence of several
-turn and
-sheet destructive Pro residues in
the peptide chain, especially at both N- and C-terminal positions of
the 20-mer, would impede
-turn formations and enhance peptide
solubility. The selected MUC-1 20-aa protected sequence had the
required reactivity at both terminal positions.

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Fig. 1.
Strategy for Fmoc synthesis of basic peptide
fragments. Fmoc, N-(9-fluorenyl)methoxycarbonyl;
DIPEA, diisopropylethylamine; DCM,
dichloromethane; CLTR, 2-chlorotrityl chloride resin;
Trt, trityl; tBut, t-butyl;
Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl;
Boc, tertiary butyloxycarbonyl.
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Fig. 2.
Fragment condensation strategy for the
synthesis of MUC-1 oligomers and ttp-MUC-1 heterotope
derivatives.
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The protected 20-aa fragment 6 was synthesized starting with
the esterification of Fmoc-Pro-OH 1 with CLTR 2 (Fig. 1) (15). The obtained resin-bound proline derivative
3, with a loading of 0.3 mmol of Pro/g of resin was
deprotected at the amino function soon after the esterification
reaction by treatment with 25% piperidine in DMF. This step was
considered very important in order to avoid premature cleavage of the
amino acid from the resin due to slow hydrolysis of unreacted trityl
chloride residues. This in turn could cause evolution of hydrogen
chloride, which subsequently affects the cleavage of the extremely
acid-sensitive bond of the amino acid to the resin (18). Chain
elongation starting from resin 4 was performed by applying a
three-fold molar excess of the Fmoc amino acids preactivated by
DCC/HOBt. For the side-chain protection of the amino acids,
t-butyl-type groups were applied, trityl for the protection
of the side chains of His (15) and Ser (16) and Pmc for the guanidino
function of Arg (25). Completion of the coupling reactions was
determined by the Kaiser test and of the Fmoc removal by TLC. Due to
the presence of the very sensitive Nim-trityl-histidine
moieties in the sequence of the MUC-1 20-aa peptide, the cleavage of
the protected fragment 5 from the resin after the completion
of the synthesis was effected by TFE/DCM (3:7) and not as in the usual
treatment with acetic acid or 1,1,1,3,3,3-hexafluoro-2-propanol-DCM
mixtures. We obtained by this method a crude protected peptide
6 in 96% yield and 97% purity as revealed by HPLC analysis
of the peptide after deprotection (Fig.
3a). The high purity of the
fragment allowed us to use it directly in the fragment condensation
reactions (Fig. 2) without further purification. In addition, the
absence of acetic acid from the cleavage mixture, which is responsible
for extensive acetylations of the resin-bound amino components, allowed
the use of the fragment 6 without prior reprecipitation from TFE/water (26). Similarly, the protected ttp 7 was
synthesized in 95% yield and 96% purity.

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Fig. 3.
HPLC analysis of crude MUC-1 oligomers using
convergent solid-phase peptide synthesis; (a) monomer;
(b) dimer; (c) trimer; (d)
tetramer; (e) pentamer. HPLC was performed in a
semipreparative C18 column, and the eluent was monitored at 220 nm. For
a-d, an elution gradient from acetonitrile containing 0.1%
trifluoroacetic acid:water (0-60%) was applied for 30 min at a flow
rate of 1 ml/min and for e, a gradient from 13 to 28%
acetonitrile was applied for 30 min with the same flow rate.
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Initially, we commenced the synthesis of MUC-1 oligomers using the
resin 5 with a loading of 0.3 mmol of Pro/g of resin. This
first preparation showed incomplete condensations during the synthesis
of the di-, tri-, tetra- and pentamers 9, 11, 13, 15 (Fig.
3, b-e). HPLC analysis of the final deprotected crude
products indicated that condensation of 6 with the tetramer
to form the pentamer proceeded at room temperature for 24 h and
gave a yield less than 40% (Fig. 3e).
To overcome the difficulties of the condensation reactions, we made the
following modifications in the CSPP. We used a lower proline
substitution (0.1 mmol/g resin). This was important because in
proceeding with the condensation reactions, the peptide/resin weight
ratio increased and considerably altered the swelling properties of the
resin. Resins with a high peptide loading reveal an increased polarity
of the polystyrene matrix. Therefore, movement of large polar molecules
such as the free protected peptide fragments, is restricted through
polar interactions with support and can reach only with difficulty the
reactive NH2-terminal positions of the resin-bound amino
component. We also applied a more concentrated solution of 6 and three-fold molar excess of fragment 6 over the
resin-bound component 5, using the condensing agent DIC/HOBt
(1:1.5) in Me2SO instead of DCC. This aspect was considered
important as we speculated that during the long-lasting condensation
reactions dicyclohexyl urea could precipitate and block the resin pores
hindering the movement of the protected fragments inside the resin. The
improved condensation yields that resulted from the above changes
allowed us to prepare the peptide oligomers on a larger scale. The
starting resin 5 (0.1 mmol/g resin) was condensed with the
protected MUC-1 6 four times in succession thus resulting in
the formation of the pentamer MUC-1. The yield and purity of the crude
MUC-1 pentamer peptide were satisfactory as shown by HPLC analysis
(Fig. 4b) and compared with
the first preparation (Fig. 4c) thus allowing us to obtain
the pentamer in a highly purified form after HPLC purification (Fig.
4a). The molecular weight of the MUC-1 pentamer determined
by mass spectroscopy (Fig. 5) was in
agreement with the expected molecular weight as calculated from the
amino acid content .

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Fig. 4.
Comparison of HPLC chromatograms of MUC-1
pentamer peptides synthesized with CSPP and modified CSPP.
(a), HPLC-purified pentamer from the modified CSPP
preparation; (b), crude pentamer synthesized with modified
CSPP; (c), crude pentamer synthesized with CSPP. HPLC was
performed in a semipreparative C18 column, and the eluent was monitored
at 220 nm. An elution gradient from acetonitrile containing 0.1%
trifluoroacetic acid:water was applied for 30 min at a flow rate of 1 ml/min from 13 to 28% acetonitrile.
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Electrophoretic Analysis of the MUC-1 and ttp-MUC-1
Oligomers--
The synthetic MUC-1 oligomer peptides exhibited unusual
behavior in SDS-PAGE. The peptides could not be visualized directly on
the polyacrylamide gels because they could be not fixed by methanol/acetic acid, glutaraldehyde, or trichloroacetic acid. The
electrophoretic properties were studied when the oligomers were
transferred to a nitrocellulose membrane with passive diffusion immediately after the electrophoretic separation and by the silver staining, which followed (20). The monomer and dimer peptides were not
visualized at all, probably due to insufficient amounts being
transferred onto the nitrocellulose (Fig.
6, lanes 1 and 2).
The other oligomers (3-mer, 4-mer, and 5-mer) became clearly visible.
They appeared to migrate slowly with the same electrophoretic rate
(Fig. 6, lanes 3-5) although the oligomers differed from each other by 20 and 40 aa in length. This anomaly may be attributed to
the high proline content and alanine-proline sequences that have been
reported to alter the charge and conformation of the SDS-polypeptide
complex (27-29).

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Fig. 6.
Electrophoresis analysis of MUC-1 oligomers
(lanes 1-5) and ttp-MUC-1 heterotope derivatives
(lane 6). Lane 1, monomer (20 aa); lane
2, dimer (40 aa); lane 3, trimer (60 aa); lane
4, tetramer (80 aa); lane 5, pentamer (100 aa);
lane 6, mixture of ttp and ttp-(MUC-1)1-5 in
ascending order. The peptides were electrophoresed at constant 500 V
(10 mA) using SDS-PAGE in 20% polyacrylamide containing 30% ethylene
glycol, passively transferred to nitrocellulose, and stained with
silver nitrate.
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In contrast, the ttp-MUC-1 heterotope derivatives showed normal
electrophoretic behavior, probably due to the influencing presence of
the ttp fragment (Fig. 7). These peptides
were easily fixed onto polyacrylamide gels and stained with silver
nitrate. Electrophoretic studies from the first preparation of the
ttp-MUC-1 heterotope derivatives (Table
I) showed the anticipated ascending molecular weight order from ttp to ttp-5-mer (Fig. 7, lanes
1-8) and clearly demonstrated the incompletion of the reactions
during the synthesis of the 3-mer, 4-mer, and 5-mer as had also been observed by HPLC analysis. It was noted that the ttp-MUC-1 heterotope derivatives could not be separated by HPLC as they were all eluted simultaneously from the gradient systems we applied (Fig.
8). This phenomenon may be attributed to
the dominating hydrophobic nature of the ttp peptide, equally affecting
the elution of the different derivatives.

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Fig. 7.
Electrophoresis (lanes 1-8) and
Western blot (lane 9) analysis of the
ttp-(MUC-1)1-5 heterotope peptide derivatives from the
first CSPP preparation. Lane 1, ttp; lane 2,
ttp-(MUC-1)1; lane 3, ttp-(MUC-1)2;
lane 4, ttp-(MUC-1)3; lane 5,
ttp-(MUC-1)4; lane 6,
L-ttp-(MUC-1)5; lane 7,
D-ttp-(MUC-1)5; lane 8, mixture of
ttp-(MUC-1)0-5. The peptides were electrophoresed at
constant 500 V (10 mA) using SDS-PAGE in 20% polyacrylamide containing
30% ethylene glycol, and stained with silver nitrate. Lane
9, immunoelectrophoresis of the ttp-(MUC-1)0-5
mixture, reacted in succession with the monoclonal BC-2 and anti-mouse
IgG-alkaline phosphatase, and stained with 5-bromo-4-chloro-3-indolyl
phosphate-nitro blue tetrazolium.
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Fig. 8.
HPLC analysis of ttp-(MUC-1)1-3
showing increasing racemization as the length of MUC-1 oligomers
increased; (a), ttp-(MUC-1)3; (b),
ttp-(MUC-1)2; (c),
ttp-(MUC-1)1. HPLC was performed in a semipreparative
C18 column, and the eluent was monitored at 220 nm. An elution gradient
from acetonitrile containing 0.1% tri-fluoroacetic acid:water was
applied for 30 min at a flow rate of 1 ml/min, from 15 to 40%
acetonitrile.
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As expected, the condensation of the protected ttp fragment with the
resin-bound MUC-1 oligomers proceeded with the partial racemization of
the C-terminal leucine residue.
In fact, we observed that two distinct peaks were always present in the
reverse phase-HPLC elution profile for each derivative. The two
components corresponding to the L- and
D-enantiomers of the derivatives were separated by
semipreparative HPLC. Both showed identical electrophoretic properties
(Fig. 7, lanes 6 and 7), exactly the same
molecular weight and the same amino acid ratio as determined by aa
analysis of the ttp-MUC-1 5-mer. The percentage of racemization
appeared to increase in proportion to the length of MUC-1 oligomer.
This was due to the slower fragment condensation reactions leading
to a longer exposure of the activated ttp in the racemization prone
benzotriazoyl ester state prior to condensation (Fig. 8).
Immunological Properties of MUC-1 Oligomers--
The murine
monoclonal antibody BC-2 was titrated against equal amounts of MUC-1
oligomers in enzyme-linked immunosorbent assay (Fig.
9). The results showed a characteristic
preference of the antibody for the oligomers and much less for the
monomer in agreement with previous observations (12). This was also
demonstrated in Western blotting using a mixture of the ttp-MUC-1
oligomers (Fig. 7, lane 9) where ttp-(MUC-1)1
did not give any visible immunoreaction, whereas all the other oligomer
derivatives reacted with the monoclonal antibody. Finally, in an
inhibition enzyme immunoassay study of the monoclonal antibody by a
range of peptides (Fig. 10 and Table I), it was demonstrated that free MUC-1 oligomers were more efficient inhibitors than the monomer and the APDTRP epitope. The control peptide
STAPPAHGV, which represented a region of MUC-1 exclusive of the
immunodominant epitope, did not react with the antibody. These results
strongly support the suggestion that as the number of MUC-1 tandem
repeats increases, the epitopes may attain a structure closer to native
conformation of unglycosylated mucin and become more immunogenic
(12).

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Fig. 9.
Enzyme immunoassay reactivity of MUC-1
oligomers against a monoclonal antibody (BC-2), which recognizes the
APDTRP epitope.
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Fig. 10.
Competitive inhibition enzyme immunoassay of
the APDPTRP-recognizing monoclonal antibody by a range of synthetic
peptides. The plates were coated with the MUC-1 pentamer.
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Synthesis of large peptides such as the ones required for
immunotherapeutic and vaccination studies is difficult to achieve with
a sequential extension approach as the reaction efficiency decreases
dramatically with the increasing size of the peptide beyond 30-40 aa,
affecting both purity and yields. Recent advances in peptide chemistry
for cleaving peptides from resins under extremely mild conditions
allowed us to adopt an improved convergent solid-phase peptide
synthesis strategy and prepare MUC-1 oligomers under controlled conditions up to five repeats and derivatives of the same oligomers with a tetanus toxin T cell epitope. HPLC and mass spectroscopy analysis confirmed the purity and correct size of the pentamer MUC-1,
and immunological studies demonstrated that the oligomers were
recognized as specific antigens by anti-mucin antibodies. The
construction of large composite synthetic peptides, such as those
demonstrated here, offers a rational approach to designing and studying
synthetic vaccines with defined structural and accessory epitopes as a
means of evoking precise immune responses, which are required in
immunotherapy.
We thank Prof. Giovanni Sidonna, University
of Calabria, Italy, for the mass spectrograms; Dr. P.-X. Xing,
Immunology and Vaccine Laboratory, the Austin Research Institute,
Heidelberg, Australia, for the supply of the monoclonal antibody BC2;
and George Konidakis, Medical School, University of Crete, Greece and
Maria Sioumpara, Institute of Molecular Biology and Biotechnology, for
kind assistance with the synthesis of the peptides in the study and
Hara Vlata, IMBB, for the enzyme immunoassays.