The stability of an immunogen against enzymatic degradation is considered an important factor for the design of synthetic vaccines. For our studies, we have selected an epitope from the tandemrepeat unit of the high-molecular-weight MUC2 mucin glycoprotein, which can be underglycosylated in case of colon cancer. In this study, we prepared a MUC2 peptide containing the PTGTQ epitope of a MUC2 protein backbone-specific mAb 996 and its derivatives. In these peptides, the N-and C-terminal flanking regions were systematically substituted by up to three D-amino acids. Peptides prepared by solid-phase synthesis were tested for their mAb 996 binding in competitive ELISA experiments, and their stability was studied in serum and lysosomal preparation. Our data show that the epitope function of peptide 15 TPTPTGTQTPT 25 is retained even in the presence of two D-amino acid residues at its N-terminal flanking region and up to three at its C-terminal flanking region (tpTPTGTQtpt). Also, this partly D peptide shows high resistance against proteolytic degradation in diluted human serum and in lysosomal preparation. These findings suggest that, by appropriate combination of structural modifications (namely, D-amino acid substitution) in the flanks of an Ab epitope, it is feasible to construct a synthetic antigen with preserved recognition properties and high stability against enzymatic degradation. Peptides tPTPTGTQTpt and tpTPTGTQTpt derived from this study can be used for immunization experiments and as potential components of synthetic vaccines for tumor therapy.human serum ͉ rat liver lysosome preparation T he design of appropriate immunogen and its delivery, among other factors (e.g., schedule and route of immunization), are essential for the development of an effective peptide-based subunit vaccine. A major problem limiting the use of peptides is their instability, which is mainly due to the rapid degradation in vivo by proteases. There are different approached for protecting biologically active peptides from enzymatic decomposition, such as alteration of the amide bond (1), cyclization, conjugation to carrier molecule (2), and incorporation of nonproteinogenic amino acids such as ␤-Ala (3) or D-amino acids (4, 5).Sela and Zisman (as reviewed in ref. 6) clearly demonstrated that the presence of D-amino acid residues in linear and multichain polypeptide antigens could improve their stability to proteolysis and alter the biological half-life but also influence B or T cell immunrecognition. The D-amino acids were incorporated into epitope peptides mainly to investigate their effect on peptide-specific B cell and͞or T cell immunogenicity and͞or to analyze the fine specificity and cross-reactivity of Abs͞T cells induced by the all-L, all-D peptides (7). Correlation between the characteristics of immune response and extended biological half-life, as well as their stability to proteolysis, were described (8-10). However, most of the available literature reports on peptides in which all L-amino acid residues were replaced by their...

One linear and three cyclic peptides corresponding to the 278-287 ((278)LLEDPVGTVA(287)) sequence of glycoprotein D (gD-1) of herpes simplex virus were synthesized for the analysis of the effect of cyclization on protection against enzymatic degradation. In this design, the turn-forming motif ((281)DPVG(284)) was positioned in the central part of the peptide and elongated by three amino acids at both termini. Cyclopeptide formation was achieved by the introduction of a peptide bond, a disulfide bridge or a thioether link. The stability of these peptides was compared in human serum and also in rat lysosomal preparations. The data obtained in 10% and 50% human serum show that all three types of cyclization enhanced the stability, but at different levels. Complete stability was only achieved by the introduction of a thioether link, while the presence of a disulfide or peptide bond resulted in improved, but partial resistance against hydrolytic decomposition. In lysosomal preparations the presence of cyclic primary structure provided full protection against enzymatic hydrolysis. Taken together, these findings indicate that by appropriate structural modification it is feasible to construct a synthetic antigen with high stability against enzymatic degradation in complex biological fluids. Further studies are in progress to identify enzymes responsible for degradation in diluted human sera as well as in the lysosomal preparations and to gain more detailed information on the mechanism of action.

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