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.
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.
Watch the in-depth FDA study using the ProPresent® MAPPS assay presented by Dr. Zuben Sauna at our Mastering Immunity 2018 Conference in Boston
Key publication by team at CSL Behring investigating reduced immunogenicity of sc-rFVIII AFSTYLA®
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)
Example cell sources
Healthy donor cells
Cell samples from treated patients
Cultured cells, e.g.
DCs, B cells and other cells
DNA / RNA
Viruses / VLPs
(either from known mutation databases or established from exome sequencing)
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): generate 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
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®.
Watch this short clip by Dr. Jad Maamary from Merck describe how they used ProPresent® to analyze naturally processed and presented peptides from ipililumab and nivolumab
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.
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.
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.
Wojciech Jankowski, Yara Park, Joseph McGill, Eugene Maraskovsky, Marco Hofmann, Vincent Diego, Bernadette Luu, Tom Howard, Roberta Kellerman, Nigel Kay and Zuben Sauna
Peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to FVIII products
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
Joost Gouw, Juandy Jo, Laura Meulenbroek, Sam Heijer, Erica Kremer, Elena Sandalova, André Knulst, Prescilla Jeurink, Johan Garssen, Anneke Rijnierse, Léon Knippels LMJ
Identification of Peptides with Tolerogenic Potential in a Hydrolyzed Whey-Based Infant Formula
Clin Exp Allergy 2018
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
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.
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