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Robert Raynor, Ph.D., Beckman Coulter, Inc. There is increasing regulatory pressure on pharmaceutical and biotechnology companies to identify and characterize the specific mechanisms of action for a submitted vaccine.1 Required information may include elucidation of the peptide sequences involved in generation of protective immunity, proof of population coverage, and the characterization of resultant T cell responses. A great deal of attention is now being focused on the evaluation of vaccines inducing a cellular or T cell response to fight disease. A new and proprietary technology developed by Beckman Coulter allows vaccine developers working on T cell mediated vaccines to map and characterize binding of epitopes to MHC complexes. This process provides the tools to better understand the mechanism of action of a target protein, predict patient response, and later measure that response using assays such as MHC tetramer analyses. As part of the body's initial immune response, immunogenic proteins are transported to, and internalized by, antigen presenting cells. Fragments, or peptides, of the original protein may then bind with the MHC molecule and be expressed on the cell's surface. A subset of these peptide-MHC complexes, known as epitopes, will stimulate a T cell response. With the universe of possible peptide sequences, coupled with the genetic variability of MHC molecules among individuals, the challenge of selecting the "right" immunogenic proteins presents a major obstacle to the development of cell-mediated vaccines. Some progress has been made using algorithmic identification, peptide elution and cell-based binding assay techniques, but success has been impeded by method-specific issues such as laborious lab work, low throughput, difficult reproducibility or limitation to only the most commonly studied MHC types. Using the new epitope discovery technology, assays are performed in vitro applying processes that are capable of high throughput, and that not only identify peptides that bind to MHC, but also determine binding affinity and rate of dissociation, or off-rate, for each peptide. This new process may considerably shorten the route to clinical development by allowing a systematic ranking of these candidate epitopes for subsequent functional studies. The epitope discovery process includes peptide synthesis, binding characterization assays and multi-parametric data analysis. The centerpiece of the process is the assembly of properly folded complexes of MHC molecules, beta 2 microglobulin and placeholder peptides, which are bound to a streptavidin-coated microplate wells. These MHC peptide complexes include eight specific Class I alleles, representing close to 90 percent of the human population.2 The process begins with identification of a target protein thought to play a role in a disease process. A complete library of overlapping amino acid sequences spanning the entire length of the protein is then synthesized. A binding assay is performed for each of the test peptides by introducing a buffer designed to unfold and disassociate the MHC and placeholder peptide in the microtitre well. The placeholder peptide and beta 2 microglobulin are washed away, leaving the unfolded MHC bound to the reaction well. A peptide from the synthesized library and additional beta 2 microglobulin are added to each well and incubated in a buffer designed to promote refolding of the complex. A fluorescent-labeled antibody designed to recognize only a properly folded peptide/MHC complex is added to each well. This step provides the identification of those test peptides which bind to the MHC and warrant additional analysis to characterize their binding affinity and rate of dissociation. Peptides that do not bind to the MHC are clearly identified and eliminated from further study. High affinity binding and low dissociation rates are critical factors controlling immunogenicity of peptides.3,4 Within this epitope discovery process, characterization of peptide and MHC interactions can be achieved using the same plate based assay formats with slightly different assay conditions.
Further studies using T cells can be performed to confirm the biological relevance of the identified epitope candidates. With this clear understanding of a protein's characterization profile, an opportunity may exist to re-engineer its amino acid sequence to increase immunogenicity. The epitope discovery system provides a comprehensive set of tools designed to increase efficiency and productivity in the vaccine discovery process by identifying and ranking the most viable epitope candidates based on actual affinity and off-rate determinations. This approach eliminates bias, complexity, and reproducibility issues associated with cell-based methods, allowing clinical development decisions to proceed based on experimental results. References:
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