Heteroclitic recognition of combinatorial TX1TX2T peptide mixtures by mucin-2 protein specific monoclonal antibody.

The mucin-2 (MUC2) glycoprotein secreted by the epithelial cells of human colon may be abnormally under-glycosylated in the case of cancer. Monoclonal antibody (mAb) 994 raised against the immunogenic part of the protein core, recognizes malignant human colon tissues as well as pentapeptides with TX1TX2T motif present in MUC2. Using a combinatorial approach and ELISA experiments it was found that mAb 994 is able to recognize peptides of the sub-library TQTX2T very strongly, and to some extent also peptides from TETX2T, TLTX2T and TVTX2T sub-libraries. Binding studies with peptides corresponding to the TQTX2T and TETX2T sub-libraries showed that mAb 994 recognized only six peptides (IC50 = 9-208 micromol dm(-3)) from the 19 compounds of the TQTX2T sub-library and only three peptides (IC50 = 3500-16700 micromol dm(-3)) from the 'second-best' TETX2T sub-library. The most pronounced mAb binding occurred when Gln was in position X1 and it was much weaker in the case of Glu, Val or Leu. As for X2 amino acids, the presence of Pro, Ala can provide a strong, while Tyr, Trp, Phe and Ser a weaker, peptide-antibody interaction. Data from this study suggest that pentapeptide TQTPT, whose sequence is present in the native protein, is bound most strongly. However, almost identical binding properties were observed with peptide TQTAT, whose sequence is not present in the protein. Apart from this, some other 'heteroclitic' peptides were found with a different rank in the binding-hierarchy. Based on these peptides artificial compounds can be prepared as potential candidates for vaccine development. Results of this study also provide a rationale for understanding the molecular background of the heteroclitic nature of the MUC2 protein core specific mAb 994.

Identification and solution conformation of multiple epitopes recognized by a MUC2 mucin-specific monoclonal antibody.

We have identified the optimal epitope, 21TQTPT25, in the tandem repeat of mucin 2 (MUC2) glycoprotein by using glycoprotein-specific monoclonal antibody, MAb 994, and synthetic, overlapping and truncated oligopeptides corresponding to the sequence 13TPTPTPTGTQTPTT26. We found that peptides containing the 21TQTPT25 sequence were able to inhibit the 994 antibody binding and also peptides 21TQTPT25 and 17TPTGTQTPT25 were the most inhibitory compounds with the lowest IC50 value (IC50=4 and 3 microM, respectively) tested. Interestingly, 21TQTPT25 peptide adopts an unordered structure even in TFE, a solvent that promotes an ordered conformation, as detected by circular dichroism and Fourier-transform infrared spectroscopy. However, Thr at position 26 or amidation of Thr25 at the C-terminus results in a much weaker (3 orders of magnitude) MAb interaction, which can be due to the presence of a turn conformation in peptides with a T26 or an amide C-terminus. We have also observed that MAb 994 recognized two other pentapeptides with the TX1TX2T motif, like 13TPTPT17 (IC50=180 microM) and 19TGTQP23 (IC50=65 microM), whose sequences are present in the native glycoprotein. These findings might suggest that in the MUC2 tandem repeat unit there are multiple antigenic sites available for recognition in underglycosylated tumor tissue and also explain the heteroclitic nature of MAb 994.

Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library.

A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.

Immune Responses to the MUC1 Mucin.

MUC1 mucins are highly glycosylated glycoproteins expressed on the luminal surfaces of glandular epithelia. In breast and ovarian carcinomas, their expression is frequently upregulated and they may be secreted into the circulation of cancer patients. Early studies aimed at the production of anti-MUC1 monoclonal antibodies revealed that MUC1 was a potent immunogen in mice with many monoclonal antibodies raised defining epitopes within the protein core of MUC1. The immunogenicity of MUC1 has now been extended to human studies and it is apparent that patients with breast and ovarian malignant disease are able to mount immune responses against MUC1. These findings provide information on the mechanisms involved in the recognition of MUC1 expressing tumours. The utilisation of MUC1 related immunogens to stimulate immune responses to tumours could lead to the improved management of patients and the development of new immunotherapeutic strategies aimed at the eradication of MUC1 mucin expressing cancers.