He started by pointing out that for monoclonal antibodies against the CD3 antigen the murine product induces neutralising antibodies after a single dose in 100 % of the patients. The humanized version of the antibody still leads to antibody induction after repeated injection in 25 % of the patients (Fig. 1).

Dr. Bruyns addressed a major challenge in immunogenicity assay development, i.e. the availability of appropriate standards as pointed out in the relevant guideline. According to the EMA guidelines antibody-positive reference material should be “… a human preparation with a significant antibody content which is available in sufficient quantity for continued use…” Given the fact that in many cases such material is not readily available, animal sera will have to be used.

Anyhow, – according to Dr. Bruyns – assays for anti-drug antibodies should be rather called “semi-quantitative”, instead of “quantitative” since reference standards are lacking and individual antisera are distinct with respect to affinity and avidity of antibodies contained therein.

Dr. Bruyns provided detailed information on validation parameters that need to be addressed in assay development, with the definition of “sensitivity” as suggested by Mire-Sluis et al., J. Immunol. Meth. 289: 1 (Fig. 2).

In the second part of his presentation Dr. Bruyns provided details on testing for cell-mediated immune responses, an aspect, which is usually not necessarily incorporated in standard immunogenicity programs. This is inspired by the EMA’s viewpoint, that “… cell-mediated responses could play an important role and their assessment may be considered by applicants on a case by case basis …”.

He presented examples from his practical work on cell-mediated immunity testing with two assay formats. First, EliSpot technology to measure cytokine secretion after antigen-specific stimulation  (Fig. 3). And second, flow cytometry for antigen-specific T cell proliferation, based on the fluorogen carboxyfluorescein diacetate succinimidyl ester (CFSE assay; Fig. 4). Dr.Bruyns pointed out that the selection of the antigen for use in such sensitive assays is critical, since using the “wrong” antigen results in false negative findings. In other words: care should be taken in clearly defining the T cell stimulatory moiety of a given new compound.

In agreement with the conclusions of his preceeding speakers Dr. Liedert and Dr. Buettel, Dr. Bruyns closed his presentation by stating that “assay selection is dependent on the biological in question” and that there is “no best assay for all purposes”.

He started out with the fundamental 2004 citation of the FDA’s Dr. Amy Rosenberg that “All proteins are potentially immunogenic”. Immunogenicity results from two mechanisms, a reaction to neo-antigens and/or the breakdown of immune tolerance, whereby the latter is very much dependent on aggregate formation of the antigen. Aggregate formation allows direct B lymphocyte activation through cross-linking of surface-bound IgM / IgD containing B-cell receptors without the need for antigen-specific help by T lymphocytes.

Dr. Liedert put the topic into perspective by summarizing the potential consequences of anti-drug antibodies (ADAs): Although formation of these ADAs has no clinical consequences in most cases, a sometimes even dramatic impact on safety and/or efficacy cannot be ruled out (Fig. 1).

In the first major part of his presentation Dr. Liedert reviewed the current regulatory framework in Europe (EMA; Fig 2) and the US, whereby in the US a series of four white papers provided basic advices, which were recently compiled in a first FDA Guidance on assay development (Fig. 3). Dr. Liedert emphasized that analysis of immunogenicity is one essential pillar in the assessment of biosimilars and therefore part of corresponding regulations.

In the second part Dr. Liedert addressed the tiered concept of “screening” and “confirmatory” assays for immunogenicity testing and outlined some technical aspects and challenges of current anti-drug antibody assay formats (Figs. 4 and 5).

A major issue in the development process of a given therapeutic protein is assessment of its immunogenicity risk. To this end, Dr. Liedert provided his view on the evaluation of primary and secondary immunogenicity risk factors, which describe and rate clinical consequences and probability of anti-drug immune responses (Fig. 6 and 7). This evaluation then leads to allocation of the drug into a low / medium or high risk category.

The classification helps to define a risk-adapted immunogenicity testing strategy tailored to the specific compound (Fig. 8). Extent and frequency of testing has to be defined as well as degree of assay validation. Concomitant analysis of pharmacokinetics and monitoring of appropriate safety/efficacy marker might support association of positive findings with potential clinical consequences.

In most cases it is acceptable to analyze samples, collected for immunogenicity testing, after the end of a clinical trial. For therapeutic proteins, where immunogenicity-induced depletion of an unique endogenous counterpart is a serious concern, testing may be performed in real time and NAb-positivity may be defined as withdrawal criterion. In this case it should be warranted that the applied assays are capable to measure ADAs in the presence of drug (especially for protein-drugs with a long half-life).

Taken together, Dr. Liedert provided very valuable information derived from his hands-on experience in immunogenicity testing with a clear focus on decision making in specific development projects. He concluded by stating that “any immunogenicity strategy will always have to be adapted individually for each project” and that sponsors should not hesitate to seek Scientific Advice at the Health Authorities.

Disclaimer: The views expressed in this summary are the personal views of Dr. Bernd Liedert and may not be understood or quoted as being made on behalf of, or reflecting the position of Merck Serono.

She addressed the role of the Paul-Ehrlich-Institute and the CHMP guideline on “Immunogenicty assessment of biotechnology derived therapeutic proteins” and the Concept Paper on “Immunogenicity assessment of Monoclonal antibodies intended for in vivo clinical use”. Biotechnological products are more than the drug substance, since e.g. small changes in the production process can have a high impact on the resulting product, in particular on its immunogenicity.

In other words: for biotechnological products “The process is the product” and the complete production process needs to be considered when designing an immunogenicity testing strategy.

Dr. Buettel also provided advice on factors influencing the immunogenicity of biotechnology products and the risk factors to be considered when developing an immunogenicity testing strategy (Fig. 1), the technical key principles of an assay strategy (Fig. 2), and the key principles of immunogenicity testing in clinical trials (Fig. 3) and in pharmacovigilance. For the latter, immunogenicity aspects should always be included into the risk management plan.

Dr. Buettel stressed that the EMEA sees biotechnology derived products as individuals and “that standard algorithms cannot be used for immunogenicity testing of a given compound”.

Dr. Buettel concluded with an overview on a recent workshop held at the Paul-Ehrlich-Institute on immunogencity testing (“Taking immunogenicity assessment of therapeutic proteins to the next level”), the summary of which will be published soon.

Key elements of the EMEA recommendations in brief: Immunogenicity should be evaluated in all study participants and not only in symptomatic individuals. Bioassays for neutralizing antibodies need to be designed in a product-dependent manner. Clear criteria have to be defined to distinguish positive from negative findings and to confirm positive results.

FOCUS Immunology supports its clients to integrate these recommendations into their repsective clinical trial protocols. FOCUS’ services comprise assay development and validation for preclinical and clinical studies, testing under GLP conditions, implementation of confirmatory assays, development of bioassays for testing of neutralising antibodies and sample logistics in international multicenter trials.

If you have questions or want to discuss immunogenicity testing in your specific development project, please do not hesitate to contact our Head of the Immunology Laboratory Dr. Eddy Bruyns via email (eddy.bruyns@focus-cdd.com) or via telephone (+49 6221 649 35 124).

The current EMEA guideline on immunogenicity testing can be found here.