ProPresent Antigen Presentation Assay Service | ProImmune

Pfizer Case Study

Sanofi Pasteur Case Study

MHC-Peptide Binding Assays

CFSE T cell proliferation assays


Watch our ProPresent® Podcast

 

ProPresent® Antigen Presentation Assay

discover more with the world's leading MHC-associated peptide proteomics (MAPPS) assay

ProPresent® is ProImmune's MHC-associated peptide proteomics (MAPPS) assay. It lets you directly identify the peptides which are presented by antigen-presenting cells to T cells. Our core assay has a turnaround time of just 3-4 weeks.

 

ProPresent™ Workflow

Figure 1: ProPresent® example workflow when measuring presentation from protein or protein mixture. Antigen-presenting cells take up the protein and process/present peptide fragments bound in MHC molecules on the cell surface.  Cells are lyzed and HLA-peptide molecules recovered in an immune affinity step. Peptides are recovered from the HLA molecules and analyzed by LC-MS/MS.  Identified sequences are subjected to rigorous analysis to identify true positive peptides with high confidence.

ProPresent® tells you exactly which epitopes from your biotherapeutic drug or other protein of interest are presented by HLA molecules to T cells. 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.

 

 

Key publication by team at CSL Behring investigating reduced immunogenicity of sc-rFVIII AFSTYLA®

M. Hofmann, et al. (2017) "Analysis of the Novel Recombinant Factor VIII-SingleChain Protein Predicts A Lower Immunogenic Potential as Compared to Full-length Recombinant FVII"

(read the full abstract here)

Figure 2: ProPresent® assay to investigate modulation of antigen presentation of FVIII by VWF. FL-rFVIII expressed more unique epitopes than AFSTYLA® in the absence of pdVWF due to B domain truncation. Complexing with VWF (in a physiologic relevant ratio) dramatically reduced the generation of T cell epitopes for AFSTYLA® to that observed with pdFVIII in presence of VWF. In contrast, T cell epitopes generated from FL-rFVIII were poorly inhibited by VWF *. Five (magenta boxes, top lower panel) out of  43 uniquely or more frequently presented FL-rFVIII-derived epitopes (identified in presence / absence of VWF) triggered T cell proliferation in a ProMap® study**. A previous ProPresent®/ ProMap® identified 27 FL-rFVIII-B domain derived and one T cell epitope common for FL-rFVIII and BDD-rFVIII, not present in AFSTYLA® (data not shown).

*Healthy donors (n=12) were selected as source of moDCs with HLA-DR/DP/DQ covering ~80% of global population. One row of each data set shows HLA-DR/DP/DQ presented T Cell epitopes from donor 1-12 plotted against FVIII sequence with allele specific color-code

** Healthy donors (n=40), <98% population coverage, n=57 synthetic overlapping peptides tested

Short summary:  AFSTYLA® developed by CSL Behring is the first and only single chain recombinant FVIII molecule designed to increase dosing intervals while providing high clinical efficacy. CSL Behring used ProImmune's ProPresent® antigen presentation assay to investigate AFSTYLA® for antigen presentation by dendritic cells via HLA DR, DQ and DP. It was found that antigen presentation for AFSTYLA® is reduced compared to other commercial rFVIII products. Follow-on ProImmune ProMap® T cell assays also showed a reduced T cell epitope response profile for AFSTYLA®. Overall the findings from such pre-clinical assays are consistent with clinical results for AFSTYLA® that are already available, where no inhibitors to AFSTYLA® were found (read the full abstract here)

 

ProPresent® example applications

  • Determine protein antigenicity

  • Identify which antigens from an oncogene, virus, bacteria, pathogen, vaccine or other vector are actually presented on cells

  • Discovery of new biomarkers

  • Discover new peptide targets for cancer and infectious disease interventions

  • Understand the impact of protein modifications on immune responses

  • Separate analysis for HLA-DR, DP, DQ, pan HLA-ABC, HLA-A2, mouse H-2 available

 

Example cell sources

  • Tumor tissue

  • Cell lines

  • Healthy donor cells

  • Cell samples from treated patients

  • Cultured cells, e.g. DCs, B cells and other cells

Antigen source

  • DNA / RNA

  • Exosomes

  • Viruses

  • VLPs

  • Bacteria

  • Lysates

  • Purified proteins

  • Protein mixtures

 

Detection options

  • Mutated sequences (either from known mutation databases or established from exome sequencing)

  • Peptide modifications, including phosphorylation, chemical modifications, so long as they are well defined

 

Flexible, separable 2 step process

  • Step 1 (carried out by Client or at ProImmune):  Generated cells presenting MHC-peptide molecules, then pellet and freeze cells.

  • Step 2  (carried out at ProImmune): cell lysis and recovery of peptide from MHC followed by LC-MS/MS sequencing of peptides

 

 

ProPresent® and immunogenicity

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 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

 

Detailed Process Flow for ProPresent® Antigen Presentation Assay Service where ProImmune Provides Dendritic Cells

  • 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 Swiss-Prot database 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

 

 

Frequently-Asked Questions

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.

 

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

 

 

Key publications for ProPresent®:

 

Team at Novo Nordisk and FDA investigate the immunogenicity of engineered F-VIIa

Kasper Lamberth, Stine Louise Reedtz-Runge, Jonathan Simon, Ksenia Klementyeva, Gouri Shankar Pandey, Søren Berg Padkjær, Véronique Pascal, Ileana R. León, Charlotte Nini Gudme, Søren Buus and Zuben E. Sauna

Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools

Science Transl. Med. 2017 9:372

 

AAPS Meeting report published by authors from Amgen, Novartis, Genentech and ProImmune:

M. Benjamin Hock, Karen E. Thudium, Monserrat-Carasco-Tiguero and Nikolai F. Schwabe (2015)

Immunogenicity of Antibody Drug Conjugates: Bioanalytical Mehtods and Monitoring Strategy for a Novel Therapeutic Modality

The AAPS Journal, Vol. 17, No 1, January 2015, 35-43

Read full text here

 

Team at Pfizer explores antigen presentation for therapeutic monoclonal antibodies

Xue, L.1, Hickling, T.1, Song, R.2, Nowak, J.3, & Rup, B.1 (2015) Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human IL-21 receptor‐blocking therapeutic antibody. Clinical Experimental Immunology; (Accepted Article); 24 September 2015

1 Pharmacokinetics, Dynamics,& Metabolism -NBE, Pfizer, Inc.

2 Drug Safety R&D, Pfizer, Inc.

3 Clinical Pharmacology, Pfizer, Inc.

Link to publication

Team at Sanofi Pasteur confirms CMV epitope with ProPresent®

Ventura C. et al J. Clin. Immunol. (2012) 32:6 1305-1316

HLA-DR and HLA-DP Restricted Epitopes from Human Cytomegalovirus Glycoprotein B Recognized by CD4+ T-Cell Clones from Chronically Infected Individuals

Read our case study  

Read full-text publication