PepSet™ Peptide Libraries for Immunological Applications

Nicholas J. Ede (PhD)
Chief Scientific Office
Mimotopes Pty Ltd

PepSets peptide libraries are a rapid and cost effective solution for an immense range of bioactivity screening purposes. Mimotopes PepSets (more information) are custom-synthesized peptide libraries supplied for fast, efficient screening work. These sets of chemically synthesized individual peptides are made on a small scale using Mimotopes’ unique proprietary parallel array synthesis platform. PepSets comprising hundreds, or even thousands, of peptides are rapidly synthesized and shipped within a few weeks of ordering.

Peptide Libraries are used for diverse purposes, from peptide epitope mapping and structure-activity studies to the search for peptide drugs. Understanding the basis of clinical disease related to the immune system, whether arising from protective immunity, immunopathology, transplant rejection or reaction to mutated cancer cells, may require testing of large numbers of individuals over many proteins or epitopes. This may be possible only with peptide libraries. To achieve the broad surveys needed, it is critical to use peptide reagents with the right characteristics.

The most common use of PepSets in immunology is for B- and T-cell epitope mapping. An overview of the design guidelines for epitope mapping is shown in Table 1. PepSets are ideal for T cell epitope searching, because T cell epitopes are by nature short linear peptides from the primary protein sequence. They are also appropriate for scanning the primary sequence of proteins for linear, or “continuous”, antibody-defined epitopes.

Linear Antibody Defined Epitope Mapping

Linear antibody epitopes are readily mapped with PepSets. Sets of overlapping peptides from potential target vaccine proteins are screened with immune sera or cells to discover vaccine or diagnostic test candidates. This saves years of expensive “trial and error” research based on predictive methods. Antibody epitope mapping by ELISA is simple to perform using biotinylated peptides because the peptides can be captured onto Streptavidin™ or NeutrAvidin™ coated plates through strong noncovalent avidin-biotin binding. Probing the wells with antibody, and washing away unbound antibodies, quickly reveals those peptides to which the antibody has bound. For simplicity of reagents, an indirect ELISA is usually preferred so that a common enzyme labeled reagent can be used for all tests (Figure 1).

In a typical example, a library of monoclonal antibodies missing word? to a small protein, thought to recognize linear epitopes, was screened using a PepSet of biotinylated peptides. The set consisted of short overlapping peptides derived from the protein being investigated. The peptides were captured onto plate wells coated with streptavidin before completing an ELISA with each monoclonal antibody (Figure 1). Using this approach, the linear epitopes of several monoclonal antibodies were quickly and clearly elucidated. The monoclonal antibodies fell into groups recognizing one of three linear sequences of the protein. One example from each group is shown in Figure 2.

Figure 1: An ELISA using biotinylated peptides and coated plates

Figure 2: Elisa results with 3 monoclonal antibodies

Cytotoxic T-Cell Epitope Mapping

T cell epitope mapping has emerged as one of the most powerful new drug discovery tools for a range of biomedical applications. Initially, T-cell-epitope discovery was applied to the development of vaccines for infectious diseases and cancer. T-cell-epitope-mapping applications now have expanded to include reengineering of protein therapeutics, as well as the fields of autoimmunity, endocrinology, allergy, transplantation and diagnostics. A major challenge is finding the right immunogenic element that can achieve an efficient and long-lasting immunization effect. Considerable effort has been made to find suitable fragments (protein epitopes) that can be included in vaccines to offer protection against serious life-threatening diseases. With this growing interest, a clear need for integration of complementary technologies has emerged, aimed at identifying and selecting peptides capable of generating adequate immunomobilization.

In a collaborative alliance with Mimotopes, Pure Protein (www.pureproteinllc.com) has developed a comprehensive, novel antigen-discovery program to detect cancer and viral T-cell epitopes useful in vaccine and therapeutic/diagnostic modalities. A central feature of this program is direct antigen and epitope discovery. Mimotopes’ and Pure Protein's epitope discovery and validation package offers quick and economical means to determine which targets have the greatest potential for a successful vaccine. The package consists of a standardized fluorescence polarization-based peptide-binding assay approach combined with the use of Truncated Pepset peptide libraries which provides a uniquely efficient means of identifying and evaluating immune target molecules that are recognized by cytotoxic T cells (see Figure 3). This assay’s capacity for high-throughput evaluation of peptide/HLA interactions, coupled with the ability of the immune system to recognize virus-infected or cancerous cells, opens new possibilities to identify an ever-burgeoning number of immunologically active peptide epitopes for the development of vaccines to treat or prevent various types of infectious diseases or cancers.

Figure 3: Peptide Bound to Class I MHC: Only a peptide of the appropriate length, specifically a 9-mer in this case, will bind to the MHC molecule. PepSetsTM-Tc are an ideal way to perform screening with a wide range of peptides for major Histocompatability Antigen binding or T-cell activation

Conclusion

The field of peptide-based vaccine development holds clear promise not only for the development of new approaches to cancer and other viral diseases, but also for providing fundamental new insights into the human immune response. Despite the long list of diseases that can be controlled by vaccination, there are still urgent unmet medical needs, including vaccines for pathogens such as HIV, malaria, and new strains of influenza. Mimotopes has been synthesizing PepSets for immunological applications since 1982 and their products and services enable them to assist their clients respond to the major challenges currently faced in vaccine development.