REVEAL™ Immunogenicity System (REVEAL-IS)

ProImmune’s REVEAL™ Immunogenicity System offers a powerful approach to enable developers of biological therapies to minimize the risk of immunogenicity related adverse drug reactions, and aids selection of the best candidates for lead optimization.

REVEAL-IS is the only available system that combines unique assays that are best-in-class to manage immunogenicity risk at a pre-clinical stage: ultra-sensitive CFSE T cell proliferation assays, and the most comprehensive range of cell-free HLA-peptide binding assays covering the entire human population. Both assays are set up on optimized high throughput platforms to run in parallel and provide answers in a time-efficient manner.

Taken together REVEAL-IS helps to establish the most in-depth profile of  the helper T cell immune response to one or more drug leads. If desired, these leads can then be re-engineered to remove unwanted helper T cell epitopes. The best lead from this process can then be tested to validate that its ability to cause T cell proliferation has been reduced.

Immunogenicity in drug development

Disease associated bias of immunogenicity

Prediction of relative antigenicity

CFSE T cell proliferation assays

REVEAL Class II HLA-peptide binding assays

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Working with ProImmune

ProImmune provides the REVEAL™ Immunogenicity System as a highly customizable service that is delivered on a pure ‘fee for service’ basis. ProImmune is independently owned and free of any restrictions or tie-ins as a service provider. We do not develop our own therapeutic pipeline and we have not entered into any strategic alliances that would restrict our capacity to work in specific target areas. In practise this means that our customers do not have to worry about potential conflicts of interest or complex downstream milestone and royalty payment structures.

Our teams of experienced scientists have developed sensitive CFSE T cell assays for the prediction of immunogenicity, and leading recombinant MHC technologies, including physical MHC-peptide binding assays for antigenicity prediction. REVEAL-IS is a modular system and can be tailored to specific project requirements, providing a flexible and cost effective solution as part of the drug development process.

Immunogenicity in Drug Development

The immunogenicity of biological drugs has the potential to be a significant obstacle in the development of successful new therapies. Unwanted immunogenicity can manifest itself particularly through anti-drug antibody (ADA) responses. ADA responses can lead to allergic reactions, reduction or neutralization of the activity of the drug and in some cases cross-reactive immune responses, which could lead to serious adverse events.

Immunogenicity is caused or influenced by a multitude of factors:

Intrinsic antigenicity of T and B cell epitopes in a drug can cause cellular and humoral anti-drug immune responses;

Extrinsic factors that influence immunogenicity include the following:

• The disease setting can pre-dispose for a higher or lower immune response, for example, in many autoimmune settings the immune system is already over-active, whereas in some diseases such as some cancers, patients may be immune compromised or suppressed;
• The drug itself modulates the immune system, e.g. by killing or modifying the function of immune cells;
• The effector function of the drug, such as complement-dependent cytotoxicity opsonization, may lead to immune stimulation;
• Effective valency of the drug, for example due to process and storage dependent aggregation;
• Formulation based effects, such as unwanted adjuvant effects;
• Amount, frequency and route of administration, for example, a drug administered subcutaneously may have a higher potential for immunogenicity than if it is given intravenously.

For a drug to be immunogenic it needs to have T cell epitopes, but extrinsic factors such as those mentioned above will also have a very significant effect on immunogenicity, and may outweigh intrinsic antigenicity. However, understanding antigenicity plays an important part in defining an optimal strategy for the development of drugs that are optimized for the avoidance of unwanted immunogenicity.

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Disease-associated Bias of Immunogenicity

Many diseases with major unmet medical need such as autoimmune diseases, show a pre-dominant association with certain tissue types, especially HLA class II alleles. For example rheumatoid arthritis (RA) is strongly associated with HLA DR1 and HLA DR4. A significant therapeutic target in RA is the B cell surface antigen CD20. Monoclonal anti-CD20 antibodies have been developed for B-cell non-Hodgkin’s lymphoma and B cell leukemia. The most successful anti-CD20 antibody is Rituxan® (Rituximab), a chimeric mouse/human monoclonal antibody developed by IDEC Pharmaceuticals and co-marketed by Biogen Idec, Genentech and Roche. While the implications of the immunogenicity of Rituxan® have not been significant in the original leukemia application, the drug causes significant immunogenicity in the RA indication, where it was also shown to have efficacy. It is thought that DR1 and DR4 restricted T cell epitopes in Rituxan® contribute over proportionally to this observed immunogenicity due to the high incidence of theses alleles in the RA patient population. As one of its key attributes, REVEAL-IS can clearly identify how the 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.

Traditional Animal Models are Insufficient for Predicting Immunogenicity in Many Biological Drugs

New biological drugs are often based on a human background or are humanized (in the case of antibody drugs). This means that they will appear foreign in an animal host and could elicit uncharacteristically high immune responses. The challenge is therefore to find a set of physical testing methods that can be used at a pre-clinical stage to compare the antigenicity of drugs leads that take account for the fact that the human immune system is 'uniquely human'. REVEAL-IS addresses this though replicating a substantial portion of the human T cell immune pathway in comprehensive in vitro assays that span all major human ethnicities. Our T cell assays are carried out on samples derived from a large bank of blood donors, which are fully characterized for their tissue type. Our HLA binding assays replicate the pathway of antigen presentation through recombinant HLA molecules in a cell-free assay.

Evaluating the Relative Antigenicity of a Drug

Evaluating B Cell Epitopes

ADA responses will be directed towards B cell epitopes on a drug. Although it is possible to identify B cell epitopes in any protein through a variety of epitope mapping approaches it can be difficult to quantify the impact of key conformational epitopes that may contribute the majority of relevant B cell antigenicity in a drug lead.

Evaluating T Cell Epitopes

Antibody responses that are of high affinity and sufficient titer require the help of CD4+ T cell immune responses for their maturation. Contrary to the complex three-dimensional B cell epitopes that often depend on secondary and tertiary protein structure, CD4+ T cell epitopes are relatively short linear peptide stretches (typically 11-20 amino acids long). Identifying all potential CD4+ T cell epitopes in the protein content of a drug is therefore a powerful rational approach for assessing and comparing antigenicity of drug leads. The information on such epitopes can be used to identify the best lead from a group of candidates for a new drug. It also raises the possibility that T cell epitopes can be removed in order to avoid unwanted immunogenicity, by mutating key amino acid residues that do not affect drug potency.

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The REVEAL™ Immunogenicity System in Detail

CFSE T Cell Proliferation Assays

The first component in the system is an ultra sensitive flow cytometric T cell proliferation assay. Unlike traditional T cell assays, which are based on radioactive thymidine incorporation, ProImmune’s new assay utilises powerful flow cytometry methods that have 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 collections of donor samples that are untreated with the drug of interest and are therefore presumed to be naïve to the drug antigens encountered. The assay is 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. Please visit the information page for these assays for more in-depth information (link above).

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REVEAL™ Class II HLA-Peptide Binding and Stability Assays

In the second component, the class II HLA binding specificity of putative epitope sequences is screened in comprehensive, high throughput, in vitro class II HLA-peptide binding and stability assays, covering 56 HLA DR, DQ and DP alleles;  table of available alleles. These assays are the only such comprehensive assays offered by any group world-wide.

The REVEAL™ 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 REVEAL™ Class II HLA-peptide binding assay.

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

The Powerful Combination of HLA-Peptide Binding and T Cell Proliferation Assays

While each of the two components of the process has its own features and advantages the system gains its significant predictive power from the combined interpretation of these two complementary 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.