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 conference
Key publication by team at FDA investigating immunogenicity of Cas9 protein
Figure 2: Peptides identiﬁed in the ProPresent® assay. a. Peptides are shown with their positions on the SaCas9 protein (depicted on the X-axis). The peptides identified are shown individually for each of 18 donors. The HLA-DRB1 alleles associated with the donors are depicted on the Y-axis. The peptides are stacked to show multiple peptides detected at each position on the Cas9 sequence for each donor. b. The number of unique, continuous SaCas9 peptides detected on DCs from each donor. c. Results of the ProPresent Assay (colored lines) are overlayed on the results of the ﬂow cytometry-based T-cell proliferation assay (pink areas). We assumed that donors were a match if they shared at least one HLA allele.
Short summary: The objective of this study was to map immunogenic T cell epitopes from Staphylococcus Aureus Cas9 enzyme (SaCas9) epitope mapping was carried out for the full length SaCas9 sequence based on several cell-based assays including ProPresent® to directly measure peptides that are processed and presented by multiple donors. Overall, 22 SaCas9 peptides were identified that are both presented by MHC-II proteins and stimulate CD4+ T cells. While there are no adverse events related to Cas-protein immunogenicity reported up to date, the clinical assessment of safety and efficacy of CRISPR-Cas-mediated gene therapy would benefit from the improved immunogenicity risk assessments using the tools reported in this study. As more clinical data emerges on immune responses to CRISPR-Cas proteins, these tools can be used to better profile and understand the impact of immunogenicity on clinical safety and efficacy.
Key publication by team at CSL Behring investigating reduced immunogenicity of sc-rFVIII AFSTYLA®
Figure 3: 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
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): 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®.
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.
Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4+ T-cells Simhadri (2021) Nature Communications 12: 5090
Peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to FVIII products Jankowski et al (2019) Blood Advances 3:1429
Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools Lamberth et al (2017) Science Transl. Med. 9:372
Identification of Peptides with Tolerogenic Potential in a Hydrolyzed Whey-Based Infant Formula Gouw et al (2018) Clin Exp Allergy 48: 1345
Immunogenicity of Antibody Drug Conjugates: Bioanalytical Methods and Monitoring Strategy for a Novel Therapeutic Modality Hock et al (2015) AAPS Journal 17: 35
Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human IL-21 receptor‐blocking therapeutic antibody. Xue et al (2016) Clin Exp Immunol 183: 102
HLA-DR and HLA-DP Restricted Epitopes from Human Cytomegalovirus Glycoprotein B Recognized by CD4+ T-Cell Clones from Chronically Infected Individuals Ventura et al (2012) J Clin Immunol 32: 1305