ProPresent Antigen Presentation Assay Service | ProImmune

Immunogenicity Management at ProImmune

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MHC-Peptide Binding Assays

CFSE T cell proliferation assays

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ProPresent® Antigen Presentation Assay

Master Immunogenicity, Accelerate Product Development

ProPresent® is a new assay service for the direct identification of the portions of a protein (antigen) that are made visible to T cells by the immune system. The assay identifies antigens that are displayed to T cells by HLA molecules on dendritic cells, which is the most direct way for measuring antigen processing and presentation. ProPresent® is the only commercially available service for this purpose and will accelerate drug and vaccine development programs where understanding detailed immune responses to a new compound is essential.

Drawing of Class II MHC molecule on dendritic cell

  • Identify naturally presented peptides directly

  • Determine which portions of your drug, vaccine or compound are visible to the immune system

  • Compare proteins, protein formulations and the effect of donor HLA types

  • Compatible with fully-formulated biologics

ProImmune’s ProPresent® Antigen Presentation Assay tells you exactly which epitopes from your biotherapeutic drug or other protein of interest are presented by HLA molecules to T cells. It is the only commercially available service that determines the peptides that are actually presented by dendritic cells (DC) following uptake and processing of your protein. Peptides are identified by the classical method of HLA-peptide complex extraction, peptide elution and subsequent peptide epitope identification by sequencing mass spectrometry. ProPresent® can be used to identify the peptides associated with HLA-DR, DP or DQ, and with HLA Class I.  Combined with ProImmune’s REVEAL® HLA-peptide binding assays and functional T cell assays, ProPresent completes the picture in understanding the potential immunogenicity of your compounds.

Knowledge gained from ProPresent® and ProImmune’s REVEAL® Immunogenicity services provides you with the information needed to understand and manage immunogenicity of biotherapeutics or other protein compounds. Our system permits comparison of data from a set of donor samples and can help explain whether patients with particular HLA-types could be at higher or lower risk of an adverse reaction to a biological compound. The assay is compatible with fully formulated biologics and can also be used to compare different proteins, or different formulations of the same protein for an alteration in the pattern of presented epitopes. ProImmune’s whole protein DC-T cell and peptide T cell proliferation assays can be employed to confirm functional relevance of the epitopes identified by ProPresent®.

Applications Process Flow Frequently-Asked Questions
Sample Data ProPresent® in Context  


Applications for the ProPresent® Antigen Presentation Assay

  • Epitope discovery and characterization

    • Epitope discovery and characterization
    • Establish epitopes for monitoring a patient response to a vaccine or biologic
    • Profile known or suspected allergens
    • Identify the immunological impact of sequence variants of the same protein, e.g. in viral proteins, or for tumorigenic point mutations
    • Investigate the population bias of epitope responses using HLA-typed donors
  • Risk Assessment of Biologics

    • Identify presented epitopes in food additives or other consumer goods, e.g. cosmetics
    • Identify presented epitopes in pharmaceuticals and other biological products
    • Compare and contrast the presented epitopes from different batches, formulations, or production methods for the same biologic, to pre-empt safety concerns
  • Profile the responses to biosimilars and biobetters

  • Establish a baseline for safety assessment; compare novel agents to established, safe, comparator proteins

  • Generate data to support a regulatory submission


Process Flow for ProPresent® Antigen Presentation Assay Service

  • Protein is supplied by the customer

  • A panel of HLA-typed, healthy donor peripheral blood mononuclear cell (PBMC) samples are prepared from the ProImmune tissue bank (selected to reflect HLA distribution of choice)

  • Monocytes from donor PBMC are cultured in defined media and differentiated to produce dendritic cells (DC)

  • DC are loaded with the test antigen and induced to mature

  • DC are harvested, HLA molecules are purified and the associated peptides are eluted

  • Peptide samples are analyzed by sequencing mass spectrometry

  • The mass spectrometry data is compared against a protein database library consisting of the sequence of interest and the international protein index (IPI) of the organism of choice

  • Peptides are ranked by significance according to a probability based algorithm

  • Data are verified by searching against a scrambled decoy database to reduce false positives

  • A full data report is compiled, listing all detected epitopes including presentation of nested epitope sets and anchor analysis against characterized HLA alleles with a summary of detected control proteins.

  • Delivery time can be approximately 6 weeks, depending on the scope of the project

ProPresent™ Workflow

Figure 1: ProPresent® workflow. The protein of interest is cultured with dendritic cells. DC take up the protein and process it. HLA molecules (HLA Class II molecules are shown) present epitopes from the protein on the DC surface.
Cells are lyzed and HLA-peptide molecules are then recovered in an immune affinity step. Peptides are recovered from the HLA molecules and analyzed by sequencing mass spectrometry.
Peptides identified by mass spectrometry are subjected to rigorous analysis to identify true positive peptides with high confidence.

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Frequently-Asked Questions

Which HLA alleles can be analyzed using ProPresent®?

We offer analysis of binding to individual HLA Class II alleles (HLA-DR, DP, DQ), and can also analyze cross-presentation by HLA Class I, currently as a pan- Class I analysis. 

Can non-human samples be analyzed by ProPresent®?

ProPresent® can be performed using samples from model animals or cell lines.  Our only requirement is that an appropriate anti-MHC antibody be available, and we can include an optimization step if you ask us to work with an untested antibody

Can ProPresent® be used to investigate antigen presentation by cryopreserved cells?

Yes.  We can extract the MHC-peptide complexes from well-preserved cells to use in ProPresent® and give you a unique insight into the peptides present on their cell surface.

How do I validate the epitopes identified by ProPresent®?

We recommend testing the peptides in our Naïve T cell CFSE proliferation assays, but there are a range of options available - simply ask us which is best for you.


Sample ProPresent® Data for Humira®

Humira® (Adalimumab) is an established anti-human TNFα (human tumor necrosis factor alpha) therapeutic monoclonal antibody used in the treatment of conditions such as Crohn’s disease, rheumatoid arthritis and ankylosing spondylitis. 5-20% of patients repeatedly dosed with Humira® produce anti-drug antibodies, so ProPresent® was applied to identify potentially immunogenic regions.

nested pepttides

20 donors of a range of HLA types -chosen to cover the HLA-DR expression distribution in the global population- were tested in the assay.

From a 122 amino acid residue stretch of the Humira® variable heavy chain sequence (full sequence information for Humira® is not available in the public domain), 3 potential epitopes were identified using ProPresent® analysis of epitopes presented by HLA-DR, including one (DNAKNSLYLYLQMNSLRAEDTA) which was presented by a range of donors of different HLA-DR types.

To read more about this study, conducted by ProImmune, contact us to request a full sample data report.

Figure 2: one of the nested sequence sets identified by mass spectrometry, covering the putative DNAKNSLYLYLQMNSLRAEDTA epitope.


ProPresent® in Context

ProImmune provides several modular tools for understanding immune responses. The in vitro methods we offer can define sequences within an antigen that can bind to HLA molecules, and whether or not these sequences cause T cell responses. However, functional assays do not reflect the many complex internal cellular processes important in the presentation of antigens by HLA Class II. These processes are accounted for using the ProPresent antigen presentation assay, which determines the repertoire of naturally presented peptides in antigen-pulsed DC. The methodology automatically includes natural editing activities, such as protease-based cleavage and peptide editing by HLA-DM and other antigen presentation pathway components.

The following table summarizes key elements that form part of an ideal immunogenicity risk assessment strategy for a biological compound and which of ProImmune’s services is most appropriate for each stage.

ProImmune Services Matrix

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