ProImmune REVEAL® Class II Rapid Epitope Discovery System

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Mouse Class II MHC-Peptide Binding Assays

HLA-Peptide Binding Assays as part of Immunogenicity Risk Management

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ProImmune REVEAL® Class II Rapid Epitope Discovery System

ProImmune's HLA-peptide binding assay for class II alleles is a powerful cell-free in vitro assay that can rapidly predict CD4+ T cell epitopes in any protein sequence. The assay identifies the most likely immunogenic peptides in a protein sequence, and thus can play a key role in the development of novel immunotherapies. When applied to therapeutic proteins this information can be used to help develop protein engineering strategies for T cell epitope depletion in order to avoid adverse reactions and neutralizing antibodies.

 

Class II HLA-peptide Binding Assay

Flow diagram for the REVEAL Class II HLA-peptide Binding Assay

 

The HLA-peptide binding assay can be combined with on- and off-rate assays or stability assays to give in-depth information about the binding characteristics of individual peptides identified in the epitope discovery process.

The technology is widely applicable across many disease areas such as cancer, infectious diseases, autoimmunity and in transplantation, and is ideally suited to therapeutic programmes with stringent time pressures.

Unlike traditional methods for discovering T cell antigens, such as functional cellular assays, the  assays do not consume precious patient samples, and have the advantage of determining the HLA restriction of each antigen in a single step. The technology therefore brings significant timesaving and helps to reduce the overall cost and risk in epitope discovery projects.

The binding assays are available for 26 HLA-DR, 11 -DQ alleles and 19 -DP alleles (see the table below for a comprehensive list), and mouse class II alleles H-2 IAb and IAd.


Despite there being a wide range of binding prediction algorithms available for peptide binding to class II HLA alleles, the accuracy of these in silico approaches does not always correlate well with physical binding data. The ProImmune REVEAL® Class II HLA-binding assay overcomes these limitations by confirming with certainty whether the peptide sequence binds to the HLA allele, and therefore eliminates the serious problem of the high proportion of false positive and false negative results generated by the algorithm approach.

 

Modules of the ProImmune REVEAL® Class II Rapid Epitope Discovery System in Detail

 

Module 1: PEPscreen® Custom Peptide Library Synthesis

The HLA-peptide binding assay requires a set of synthetic peptides, which are provided as part of the service by ProImmune. The peptide sequences are generated as an overlapping peptide library from a known protein. ProImmune's technical advisors can assist with the design of a suitable peptide library for your protein of interest.

The PEPscreen®: Custom Peptide Library is made using a high-throughput synthesis platform in a turnaround time of 10-15 working days. Peptides with a length of 15 amino acids are generated at 0.5 to 2 mg quantity with high average purity, and 100% are quality checked by Mass Spectrometry. These peptides are then used in the assay. On request, a second set of peptides can be synthesized for use in functional cellular assays, such as ELISpot.

 

Module 2:  ProImmune REVEAL® Class II HLA-Peptide Binding Assay

The high throughput HLA-peptide binding assay is a custom service that determines the ability of each candidate peptide to bind to one or more class II HLA alleles compared with a positive and an intermediate control peptide. The assay is a measure of the ability of each peptide to stabilize the HLA-peptide complex. Detection is based on the presence or absence of the native conformation of this HLA-peptide complex.

There are currently 56 DR, DQ and DP alleles available for the HLA-peptide binding assay.

Table of class II alleles available for REVEAL HLA-peptide binding assays

Table 1: Shows the DR, DQ and DP alleles currently available for the class II HLA-peptide binding assay.

Each peptide is given a score relative to the positive control peptide, which is a known T cell epitope. In addition results for an intermediate control peptide are shown for comparison. The intermediate control is a known T cell epitope, or in the case of certain class II alleles, a known epitope for the beta-chain in the allele alpha and beta pair. The score is reported quantitatively as a percentage of the signal generated by the test peptide versus the positive control peptide.  The customer may select any or all of the peptides that pass the HLA-peptide binding assay for further rate or stability assays.

The binding assay is also available for two mouse alleles, H-2 IAb and H-2 IAd.

 

ProImmune REVEAL® Class II Complete Rate Assay

On- and off-rate assays will give kinetic information about the binding properties of individual peptides. These assays can be particularly important for intermediate affinity peptides, which may exhibit slower binding to a particular HLA allele than high affinity peptides. If a slow off-rate is also seen this indicates that once the peptide is bound it could be presented for a considerable period of time, and it would thus merit further study as a potential epitope. Peptides with a fast on-rate are not guaranteed to be better epitopes. A more detailed study of their kinetics may reveal that they also have a fast off-rate, implying that they may not be good candidate epitopes. If cell samples used for final epitope validation are limited in supply rate assay data can also be used to prioritize downstream work on the most promising candidate peptides. The complete rate assay is available for the following class II alleles:

DR1 (DRB1*01:01), DR2 (DRB1*15:01), DR3 (DRB1*03:01), DR4 (DRB1*04:01), DR5 (DRB1*11:01), DR7 (DRB1*07:01).

See how complete rate assays were used in a study published in the journal Blood

 

ProImmune REVEAL® Class II Quick Check Stability Assay

The Quick Check Stability Assay is available for any of the DR, DQ and DP class II alleles, and must be ordered at the same time as Module 2: HLA-peptide binding assay. The stability assay measures the amount of peptide bound to the allele at time zero and time 24 hours, at 37°C, and gives an indication of the stability of the HLA-peptide complex. Each peptide is given a ‘stability index’, allowing comparison of the relative stability of the different peptide epitopes.

The HLA-peptide binding assay reflects the on-rate properties of a peptide more strongly than the stability of the assembled complex. However, when the outcomes of the stability assay and the binding assay are combined, the subsequent data provide more complete information as to whether the peptide could be presented long enough for it to be a good T cell epitope. This index provides a method for comparing the results of different peptides across the allele being measured. The results can be used as a cost effective way to prioritize which of the peptides could merit further study in functional cellular assays and enables the user to optimize the use of cell samples and the time needed for testing.

 

Epitope Validation

Following identification of binding peptides by the HLA-peptide binding assay those peptides that are functional T cell epitopes can be confirmed using cellular assays, such as ELISpot, intracellular cytokine staining and T cell proliferation assays.  ProImmune offers all these assays as part of a range of cellular analysis services. While final validation on patient cells will always require the resources of relevant cell samples and lab time, the data from the HLA-peptide binding and rate assays helps to put these resources to their optimal use.

Ordering

The ProImmune REVEAL® Class II custom service is tailored to the individual needs of the customer. Contact us for a specific quotation for your project. Request a Quote

 

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