Each MHC allele has a distinct peptide binding motif which favours certain amino acids at particular points in a sequence, known as anchor residues. Functional assays such as ELISpot or intracellular cytokine staining (ICS) can determine whether a cell responds to a particular peptide, but do not fully answer the question of which MHC allele is involved in a response. Once potential epitopes have been identified by functional assays, it may be necessary to determine to which allele a peptide is binding (MHC restriction). This is especially relevant if these epitopes are to be used for immunotherapeutic purposes or as a biomarkers for immune monitoring.
If the peptide does not bind well, it is unlikely to be a strong epitope. The ideal T cell epitope binds quickly to the MHC complex and remains stable in this complex for a long period of time. Peptides with ‘off-rates’ of greater than 3 hours are more likely to be good T cell epitopes 1,2,3.
There are a number of approaches that can be used to identify peptide binding.
MHC class I antigen presentation and cell surface expression depend primarily on peptide transport into the endoplasmic reticulum or Golgi by the transporter associated with antigen transport (TAP). T2 cell lines are deficient in TAP but still express low amounts of MHC class I on the surface of the cells. The T2 binding assay is based upon the ability of peptides to stabilize the MHC class I complex on the surface of the T2 cell line.
T2 cells are incubated with a specific peptide
Stabilized MHC class I complex is detected using a pan-HLA class I antibody
Analysis is carried out by flow cytometry and binding is assessed in relation to a non-binding negative control
This assay is a useful tool for assessing the binding ability of peptides, for example, those that have given a positive ELISpot result. The T2 binding assay can confirm which MHC allele a peptide is binding to and can give an indication of how strongly a peptide will bind. The main drawback to carrying out T2 binding assays is that determining which allele the peptide is binding to can be a laborious process. Many different alleles may need to be tested requiring a panel of T2 cells transfected with different MHC molecules.
The T2 binding assay is only applicable for MHC class I epitopes and cannot be used to determine MHC class II binding.
There are fewer well described peptide binding assays for CD4+ epitopes. The main protocols described in the literature are inhibition based assays with peptide binding expressed as inhibitory concentration (IC50) values relative to an indicator peptide 4,5.
The ProImmune REVEAL® & ProVE® Rapid Epitope Discovery System is a novel technology from ProImmune for analyzing proteins for potential T cell epitopes within a matter of weeks. The in vitro MHC-peptide binding assay screens individual peptides, or peptide libraries, for their ability to stabilize the MHC complex for a wide range of both class I 6 and class II 7 alleles. This unique approach will measure both the ‘on-rate’ and ‘off-rate’ of each peptide for multiple alleles providing a more in depth understanding of the binding kinetics. In general, good T cell epitopes tend to have rapid on-rates and slow off-rates. The assay assigns each peptide a score relative to a known T cell epitope that has a borderline affinity to the allele of interest and in this way the most immunogenic peptides can be identified.
This strategy can narrow down potential candidate epitopes for further investigation and as the assays are cell free, they do not waste patient samples on testing peptides that are unlikely to be antigenic. The technology is widely applicable across many disease areas including all areas of cancer and infectious diseases. New CD4+ and CD8+ T cell epitopes identified with the help of this technology can be used as core building blocks for vaccine development or as targets for new immunotherapy. Alternatively when applied to therapeutic proteins, the information from the report can be used in conjunction with cellular assays to measure the immunogenicity of proteins in order to avoid adverse reactions or neutralizing antibody titres. The MHC-peptide binding assay can also identify peptides that bind a wide range of DR alleles, thus allowing a therapy to be targeted to particular ethnic populations expressing specific HLA molecules.
Peptides identified as positive in ProImmune REVEAL® assays, with good binding properties, can be further validated as T cell epitopes using ProImmune’s MHC Class I Pentamer technology or using functional cellular assays, such as ELISpot analysis.
1Burshtyn D and Barber B (1993). Dynamics of peptide binding to purified antibody-bound H-2Db and H-2Db beta 2m complexes. J Immunology 151: 3082-3093 [PubMedID: 7690793]
2van der Burg SH et al. (1996). Immunogenicity of peptides bound to MHC class I molecules depends on the MHC-peptide complex stability. J Immunology 156: 3308-3314 [PubMedID: 8617954]
3Peter K et al. (2001). Induction of a cytotoxic T-cell response to HIV-1 proteins with short synthetic peptides and human compatible adjuvants. Vaccine 19: 4121-4129 [PubMedID: 11457536]
4Southwood S et al. (1998). Several common HLA-DR types share largely overlapping peptide binding repertoires. J Immunology, 160: 3363-3373 [PubMedID: 9531296]
5Manici S et al. (1999). Melanoma Cells Present a MAGE-3 Epitope to CD4+ Cytotoxic T Cells in Association with Histocompatibility Leukocyte Antigen DR11. J Exp Med 189: 871-876 [PubMedID: 10049951]
6Blancou P et al. (2007). Immunization of HLA class I transgenic mice identifies autoantigenic epitopes eliciting dominant responses in type 1 diabetes patients. J Immunology 178: 7458-66[PubMedID: 17513797]
7Muixi L et al. (2008). Thyroglobulin peptides associate in vivo to HLA-DR in autoimmune thyroid glands. J Immunology 181: 795-807 [PubMedID: 18566446]
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