Development of Superagonist Mimics to Epitopes Defined by Cytotoxic and Helper T Cells
Abstract
Human mucin glycoprotein MUC1 is a "self' peptide overexpressed on all surfaces of breast cancers and is an attractive candidate immunogen for a breast cancer vaccine. MUC1 is only weakly immunogenic, eliciting a low frequency of cytolytic T lymphocytes (CTL). We proposed to enhance immunity to MUC1 by using peptide mimics of native MUC1 epitopes. Mimic peptides contain amino acid substitutions; some mimics augment the T cell response to the native epitopes. The activity of mimics takes advantage of the degeneracy of T cell recognition, i.e., on the fact that a single T cell receptor (TCR) can recognize many different peptides. To develop MUC1 specific mimic peptides we planned to: 1) establish MUC1 specific CTL lines, 2) use combinatorial peptide libraries in a positional scanning format to determine which amino acid substitutions in the original peptide were recognized best by the CTL, 3) synthesize those mimics; 4) test them on an index cell line; and 5) identify those that were stronger immunogens for CTL than the native peptide. First we had to choose the best (most immunogenic) native MUC1 epitope and develop a CTL line from a healthy HLA-A*0201+ individual. Such a line had to be: (1) highly specific for chosen peptide, 2) strongly cytotoxic against MUC 1 positive adenocarcinoma cells, and (3) contain at least 10(exp 8) T cells. All of these qualities are required for successful analysis of the peptide library. Because MUC1 is a weak antigen, this first step proved to be very difficult, but we ultimately found a leader sequence LLLLTVLTV that consistently led to CTL and developed the CCM4 cell line. With this index cell line, we screened combinatorial nonamer libraries and identified amino acid substitutions that allowed better recognition of the peptides than the native. We then synthesized 96 peptides containing as many as 8 substitutions. Unfortunately the CCM4 line did not survive frozen storage, so a new CTL line must be established.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 01, 2003
- Accession Number
- ADA424648
Entities
People
- Malcolm S. Mitchell
Organizations
- Wayne State University