Development of cancer vaccines from fully synthetic mucin-based glycopeptide antigens. A vision on mucins from the bioorganic chemistry perspective - Carbohydrate Chemistry: Chemical and Biological Approaches - page 551

shown that excess levels of carrier protein may lead to a drop in the
immunogenicity of the carbohydrate antigen.

3.3 Unimolecular multiantigenic vaccines
The most recent and attractive approach that we have pursued in the
last years involves the design of unimolecular multiantigenic vaccine
constructs. In this strategy, several synthetic carbohydrate antigens
associated with a particular cancer type are incorporated into a single
peptide backbone and conjugated to the carrier protein. Thus, the in-
clusion of multiple tumor-associated glycolipid glycans (Globo-H, GB5,
GM2)andmucinglycans(Tn,STn,TF)willhopefullyaddresstheissue
of heterogeneity of tumor cell carbohydrate expression. Moreover, the
synthesis of this unimolecular multivalent vaccine structure only re-
quires a single bioconjugation to append the fully elaborated antigenic
glycopeptide to the carrier protein, which could minimize the potential
for adverse immune suppression caused by the carrier protein 34 as well
as simplify regulatory approval. Finally, it is feasible that antigen syn-
ergy could be achieved through the installation of multiple, different
tumor-associated carbohydrate antigens upon a single molecular
scaffold.
The synthesis of a series of unimolecular constructs for evaluation
in preclinical and clinical settings followed the logic of the previously
described ''cassette approach'' for the assembly of the glycopeptide-
based multiantigenic vaccine from the corresponding glycosyl
amino acid precursors. In addition to the native serine or threonine
linkage at the reducing end of the carbohydrate antigen, the primary
building blocks for this vaccine type included also new non-natural
amino acid linkers with a three or five-carbon spacer. While the influ-
ence of these non-natural amino acid residues on the folding of the
peptide was not examined, their use avoids the problems associated
with the instability of O-glycosyl serine/threonine linkages. Moreover, it
was conceived that synthetic compounds mimicking the natural linkage
may prove to be more immunogenic, presumably because of the higher
likelihood that they would be recognized as ''nonself'' by the immune
system.

3.3.1 Unimolecular trivalent vaccines. With the successful develop-
ment of the requisite methods for the synthesis of the glycosyl amino
acids and their further assembly, 35 we initially prepared and evaluated
two unimolecular trivalent structures that contained three known
tumor-associated carbohydrate antigens, a TF-Le y -Tn bearing construct
with native mucin architecture (59) and a Globo-H-Le y -Tn glycopeptide
with a non-native structure (61)(Fig.4). 36 It was indeed interesting to
note that, in preliminary ELISA analysis of the two conjugate vaccines,
the non-natural KLH-conjugated construct 62 appeared to be con-
siderably more antigenic than the naturally inspired mucin-based
conjugate 60.Infact,miceimmunizedwith62 produced antibodies
against each of the three component antigens. 37 Moreover, the