Immunogenicity Assay Services | ProImmune

Immunogenicity of Biotherapeutics

ProPresent® Antigen Presentation Assays

CFSE T Cell Proliferation Assays

ProImmune REVEAL® Class II HLA-peptide Binding Assays

B Cell Linear Epitope Mapping

HLA Tissue Typing Service

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Immunogenicity Services from ProImmune

ProImmune provides a suite of services for understanding immune responses. These can be used together as part of your immunogenicity risk management programme, or as stand-alone assays.  The in vitro assays we offer can define sequences within an antigen that 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 assessed using the ProPresent® antigen presentation assay, which determines the repertoire of naturally presented peptides in antigen-pulsed DC.

ProPresent® Antigen Presentation Assays

The ProPresent® Antigen Presentation Assay is a mass spectrometry based method for identifying the presented epitopes from your protein of interest.

 ProPresent Antigen Presentation Assay

Figure 1: A panel of donor PBMC samples are prepared and induced to differentiate into dendritic cells (DC), and these are loaded with your protein of interest.  The DC are matured in culture. Once fully mature, the HLA-DR molecules are purified and the associated peptides are eluted.  These peptides are analyzed by sequencing mass spectrometry, and epitopes are identified and ranked using a combination of algorithms and sequence data.

The methodology automatically includes natural editing activities, such as protease-based cleavage and peptide editing by HLA-DM and other antigen presentation pathway components.

This assay offers a fast track to identifying with confidence the naturally presented epitopes from your protein. You supply us with the purified protein and your sequence data, and we do the rest.

 

CFSE T Cell Proliferation Assays

We have developed ultra-sensitive T cell proliferation assays to measure proliferation of CD4+ T cells in response to either whole-protein antigens, or individual peptides.

Unlike traditional T cell assays, which are based on radioactive thymidine incorporation, ProImmune’s assay utilizes 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) -based flow cytometry, which has the following advantages:

  • Live gating reduces assay noise from irrelevant cells and improves sensitivity;
  • The analysis is carried out only on CD4+ T cells which are clearly identified as a subset;
  • The percentage of proliferating CD4+ T cells is actually determined by gating;
  • Proliferation is measured for the full assay time, and during a time window at the end when relevant responses could be missed;
  • Further phenotyping of responses is possible, e.g. to distinguish true naïve from memory responses that may exist to a particular antigen.

Our T cell assays are carried out on a representative donor group, which includes 20-50 individuals chosen to reflect a broad HLA background representing the target population group for the drug. Critically all donors used are HLA-genotyped on DQ, DP and DR loci, enabling a more accurate mapping of responses to four digit HLA sub-type. In our experience, a relevant number of responses can be attributed to DP and DQ alleles whose relevance has often been neglected in the past by other groups.

ProImmune offers two types of T cell proliferation assay:

Naïve Primary T Cell Assay for Peptide Epitope Screening

DC-T cell Assay for Whole Protein Screening

While our peptide T cell assays can give a structural source map of T cell antigenicity in any drug lead, our DC-T cell assays for whole proteins can be used to compare many important features of formulated drugs, such as structural and post-translational modifications, as well as the impact of excipients, conjugates and degradation products. Visit the information page for these assays for more in-depth information (links above).

DC T Cell Proliferation Assay

Figure 2: The proliferative response of CD4+ T cells to drug-loaded DC. (1) CFSE-labeled T cells incubated with DCs that were not co-cultured with antigen, (2) CFSE-labeled T cells incubated with DCs that had been previously co-cultured with whole protein antigen (Drug 1), (3) CFSE-labeled T cells incubated with DCs that had been previously co-cultured with control antigen (Tuberculin PPD).

 

Class II HLA-Peptide Binding and Stability Assays

Our ProImmune REVEAL® class II HLA-peptide binding and stability assays have been developed specifically to enable you to identify candidate epitopes in vitro in a high-throughput, comprehensive system.  Your protein sequence is broken down into an overlapping peptide library, and the ability of these peptides to bind to HLA molecules is assayed in vitro.

We offer analysis of peptide binding to 56 HLA DR, DQ and DP alleles;  table of available alleles: the widest allele coverage available.  The assays can be further customized - in addition to investigating binding, we can also measure the stability of that binding in either our quick-check or full rate assays. 

The HLA-peptide binding assays can determine the exact binding sequence in a binding peptide and the HLA restriction of that sequence, and provide key information for epitope ranking and possible modification. Critically therefore, the assays can show peptides that bind to many class II alleles, implicating them as pan-DR, pan-DQ or pan-DP binding sequences that could be immunogenic over a significant percentage of the population.

Further information about the Class II HLA-peptide binding assay.

The Class II MHC-peptide binding assay is also available for mouse alleles H-2 IAb and IAd.

 

Combining HLA-Peptide Binding and T Cell Proliferation Assays

There is a substantial advantage to combining peptide T cell proliferation assays with in vitro HLA-peptide binding assays.

Epitopes that are seen in the T cell assays can be compared with the profile of HLA binding. Given the high resolution of our donor panel and the wide range of alleles used, we often identify very clear hits where several donors for a hit peptide are positive for the HLA sub-type, and the same profile is seen for HLA binding. Such high-confidence results usually also give a clear guide to the key residues involved in HLA binding, which would be targets for further protein engineering if required.

 

HLA Tissue Type and Immunogenicity

An individual’s immune response to a foreign agent, such as a vaccine, drug or allergen, will be strongly influenced by their HLA tissue type. In addition there are numerous examples of diseases with known HLA association – for instance, Type 1 Diabetes is associated with HLA-DRB1*04:01, Multiple Sclerosis with HLA-DRB1*15:01 and HIV infection with B*57:01 and B*35:01. It seems absurd, but HLA type is ignored in most clinical trials where the presence of a novel therapeutic is known to influence the immune response.

One of the most relevant examples is recombinant factor VIII, used to treat suffers of hemophilia.  There is clear bias towards an anti-drug response in a subset of recipients, with common HLA alleles.  Efforts are underway to understand this bias fully, and to develop variants of Factor VIII that will be less immunogenic for these individuals.

Autoimmune diseases including Rheumatoid Arthritis (RA) show a strong association with HLA DRB1*01:01 and HLA-DRB1*04:01.  A therapeutic target in RA is CD20, and the anti-CD20 antibody  Rituxan® (Rituximab), (a chimeric mouse/human monoclonal antibody developed by IDEC Pharmaceuticals) is used in RA treatment.  However, Rituxan® contains DR1 and DR4 restricted T cell epitopes, which in the RA population contribute to a high rate of reported immunogenicity.

These examples illustrate the case for understanding your patient population in terms of HLA type, and thus knowing at an early stage how your biologic may perform in different populations.

ProImmune's REVEAL® Immunogenicity System (using the HLA binding assays and T cell proliferation assays in combination) can clearly identify how drug antigenicity is distributed across the HLA background and the system can be used to re-weight the potential impact of drug antigenicity in relation to an HLA  biased disease-state population.  Whole-protein antigenicity with respect to HLA type can also be evaluated, using our DC T cell assays.  Alternatively,  ProPresent® Antigen Presentation Assay can be used to give you the HLA-association of presented epitopes.

ProImmune offers HLA Tissue Typing as a service, using PCR-SSOP, PCR-SSP and PCR sequencing.  Whether you are engaged in a clinical trial or experiments for discovery research, in advance of starting functional cellular assays (such as ELISpot or flow cytometry testing), we highly recommend you find out the tissue type of your donor population, to add value and greater depth to your results.

 

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