EBE comments on EMEA concept paper on Guideline on Immunogenicity Assessment of Therapeutic Proteins
Comments on the EMEA/CHMP/BMWP/246511/2005
Concept Paper on Guideline on Immunogenicity Assessment
of Therapeutic Proteins (EMEA/CHMP/BMWP/246511/2005)
EBE and EuropaBio members welcome the EMEA initiative in developing a Concept Paper for the release of a Guideline on Immunogenicity Assessment of Therapeutic Proteins.
EBE and EuropaBio members would like to share with the EMEA the experience they have gained in the field particularly with regard:
- Mechanism and factors influencing immunogenicity
- Impact of immunogenicity on efficacy and safety
- Consequences for biopharmaceuticals manufacturers
- Need of a consistent manufacturing process
- Early risk assessment and de-risking of immunogenicity
- Lack of predictability of animal models
- Lack of predictability of other pre-clinical models
- Monitoring immunogenicity in clinical trials
- Need of post-approval risk management
- Immunogenicity assessment of biosimilars
Immunogenicity is the property of a substance to provoke an immune response when brought into a human or animal organism. This includes the formation of antibodies and may result in the development of immunity, hypersensitivity, or tolerance.
Therapeutic proteins are usually applied to the patients in a parenteral manner, thus entering the bloodstream either directly by intravenous application, or via diffusion to the next blood vessel upon subcutaneous, intramuscular or inhaled application. It is well known that foreign proteins (e.g., proteins obtained from non-human sources) are immunogenic in man. However, experience from the last decade has shown that even recombinant human proteins derived from non-human sources may be immunogenic, and in fact, for most approved biopharmaceuticals immunogenicity has been observed although the ratio of patients developing antibodies upon treatment with protein-based drugs varies widely from only few reported cases (in the 1:1,000,000-range) up to > 80%.
MECHANISM AND FACTORS INFLUENCING IMMUNOGENICITY
Immunogenicity may be based on two mechanisms which can also be distinguished according to the key immune cells that are preferentially involved in mounting the immune response.
The first is the reaction of the body against neo-antigens, i.e. antigenic epitopes unknown to the immune system. In humans, this occurs especially with proteins of animal or microbial origin, but may also be relevant for those recombinant human proteins which are modified in comparison to their native molecular structure.
In the majority of reported immunogenicity cases (> 85%), in particular when foreign antigenic sequences are the target of the immune system, the immune response is mediated by CD4+
T?helper cells that need to be available for recognition of respective T-cell epitopes. These T-cell epitopes are linear stretches within the respective biological and are presented as peptides in the context of HLA class II molecules expressed on antigen presenting cells.
A second mechanism is breaking of self-tolerance which may occur with human homologue (or even human) proteins and is thought to be mainly provoked by impurities or protein aggregates present in the preparation (which may mimic viral or bacterial protein arrays), or maybe brought about by carbohydrate-residues decorating those proteins (Reference: Castelli et al., 2000). Here, B cells turned out to be the principal mediators. Importantly, for breakage of B-cell tolerance
T-helper cells are not required.
Factors influencing immunogenicity of proteins are manifold and may include the protein characteristics:
- Amino acid sequence variations, e.g. in derivative versions of a given protein;
- Variations in glycosylation: missing glycosylation, or the presence of sugar residues uncommon to the human organism, may reveal neo-epitopes. However, minor variances in glycosylation which are within the range of natural variation of the inherently heterogeneous glycosylation are evidently not immunogenic;
- Modification of amino acid side chains, e.g. oxidation, deamidation, isoaspartate formation, disulfide exchange, glycation, hapten-like adducts etc., which may be induced under the conditions used in downstream processing or storage;
- Disturbance of the native three-dimensional structure of the protein, e.g. by proteolysis, partial denaturation, precipitation or surface adsorption, which may lead to aggregation;
- Presence of contaminants and impurities, e.g. host-cell protein, endotoxins, protein fragments;
- Immunogenicity may be influenced when proteins are chemically modified.
The native structure of proteins is potentially influenced by environmental conditions. Therefore, there can be an impact on immunogenicity by formulation additives which may lead to aggregation or micelle formation which possibly can be induced by a minute part of the protein preparation.
The immunogenic potential is dependent on the route of administration, the dosing regimen and length of treatment, and the target patient population. Patient characteristics may have a genetic basis (e.g., polymorphism of HLA molecules that present antigenic T cell epitopes), and may be related to the disease type and status.
IMPACT OF IMMUNOGENICITY ON EFFICACY AND SAFETY
Nearly all approved biopharmaceuticals are inherently immunogenic, albeit to a highly variable extent, with anti-drug antibody incidences in clinical trials ranging from <1-> 80%. Thus immunogenicity in general is not a barrier to therapeutic use of recombinant proteins. However, the consequences of immunogenicity may vary considerably, ranging from neutral, i.e. irrelevant for therapy, to serious and life-threatening. Therefore, the immunogenicity issue has become a subject of concern in the development and approval of biopharmaceuticals.
In many cases, the formation of antibodies against the therapeutic protein has been found to have a significant impact on its clinical safety and efficacy. This effect may be a composite of pharmacokinetic and pharmacodynamic effects. It can result in loss of efficacy due to neutralization of the drug by antibodies. This may impact the necessary dose required to achieve a therapeutic effect, (i.e. for insulin), in other situations change to a different product or discontinuation of therapy may be warranted. In addition, many patients may tolerate to chronic therapies over time. On the other hand, in some cases an enhancement of the drug efficacy has been reported due to decreased clearance which has to be considered more serious due to potential safety consequences if not monitored adequately. There are also examples of general immune effects, such as allergy, anaphylaxis, serum sickness, etc. which usually can be controlled by therapy monitoring and adaptation.
However, in some cases immunogenicity of a protein drug has led to neutralization of the
endogenous native human protein - this may even lead to fatal outcomes. One example is the sudden increase of cases of pure red cell aplasia (PRCA) associated with treatment of patients with one particular brand of erythropoietin.
CONSEQUENCES FOR BIOPHARMACEUTICALS MANUFACTURERS
Due to the unforeseeable, and potentially serious, consequences of immunogenicity, there is the need for manufacturers of therapeutic proteins (innovators as well as manufacturers of biosimilars):
- to assess the immunogenic properties of their protein products as early as possible during development, as well as
- to monitor clinical sequelae possibly due to product immunogenicity in patients during clinical trials and after approval.
Assessment of immunogenicity is even more important since of all recombinant therapeutic proteins approved for marketing until now, about 50% have a modified sequence and therefore possess structural differences as compared to the native human protein, and this percentage is expected to rise further in future. Such modified sequence stretches may be recognized as non-self by the immune system and give rise to immunogenicity.
With biosimilars, the strategy for evaluating immunogenicity should
- take the available history of the innovator product into account and
- include product-specific immunogenicity studies for the respective biosimilar in appropriate clinical trial designs as well as via post-marketing surveillance.
NEED OF A CONSISTENT MANUFACTURING PROCESS
Immunogenicity can be affected dramatically by seemingly subtle changes in the manufacturing process of a protein which may not be detectable by analytical methods. Therefore, one of the indispensable requirements for any protein manufacturer is to produce the product using a consistent, highly reproducible and validated manufacturing process, starting from the raw materials until final formulation, in order to guarantee high purity and consistent quality of his product. This process must be monitored and closely accompanied by sophisticated analytical programs which have to be set up during early development of the drug.
EARLY RISK ASSESSMENT AND DE-RISKING OF IMMUNOGENICITY
Minimizing the immunogenic potential of new therapeutic proteins includes attempts to optimize the amino acid sequence recombinantly introduced modifications or post-translational modifications, such as glycosylation, in order to remove known or expected antigenic epitopes. This is routinely done in the field of monoclonal antibodies where murine antibodies have been replaced by chimeric, humanized, or fully human antibodies, thereby reducing the incidence of immunogenicity. If appropriate, the manufacturer should make attempts during the lead optimization phase to avoid the introduction or trace the presence of neo-epitopes by state-of-the-art methods.
In addition, in vitro approaches based on human antigen presenting cells, in particular dendritic cells, and autologous T-cells have been suggested to rank lead candidates according to their relative immunogenicity potential, as determined in qualified T cell activation assays.
LACK OF PREDICTABILITY OF ANIMAL MODELS
Besides the various methods to eliminate T-cell specific epitopes in proteins and to apply extensive physicochemical and biological testing to ensure integrity of the therapeutic protein, animal models (conventional animals, studies in non-human primates, and more recently, studies in immune-tolerant transgenic mice) have been suggested for preclinical assessment of immunogenicity of therapeutic proteins.
If at all, animal models, such as immune-tolerant transgenic mice, may be used for purposes of assessing relative immunogenicity.
However, immune responses observed during toxicological studies in animal species, such as rodents or non-human primates, are mainly due to species-specific differences in the protein sequence, epitope recognition and processing of antigens between species.
Thus, assays performed in animals are not predictive for an immune response in humans and thus cannot be used to prognosticate immunogenic effects of a new protein in patients or healthy controls.
LACK OF PREDICTABILITY OF OTHER PRE-CLINICAL MODELS
Other pre-clinical approaches to address immunogenicity, such as cell-based ex vivo or in vitro assays or in silico tools aiming at predicting T- or B-cell epitopes, may be valuable for pre-clinical risk assessment and de-risking in the lead optimization phase, as described above. However, at this early stage all these approaches can only insufficiently address the complexity of immunogenic responses observed in the clinical context.
Thus, none of the pre-clinical assays available are qualified for reliably predicting immunogenicity of biotherapeutics to be expected in clinical trials and are therefore unsuitable to replace any clinical study as to determining the incidence of immunogenicity in man.
MONITORING IMMUNOGENICITY IN CLINICAL TRIALS
Because of the lack of predictive and validated pre-clinical methods for the assessment of immunogenicity, clinical trials are indispensable to reveal immunogenicity. The correlation between antibody formation and pharmacokinetics or pharmacodynamics has to be evaluated concerning their relevance for efficacy and safety. In most cases, the risk of immunogenicity will have to be considered separately in different therapeutic indications.
During clinical studies, the formation of anti-product antibodies has to be analyzed carefully with sensitive and compound-specific methods, e.g. immunoassays, and their correlation with clinical findings and/or neutralizing antibodies have to be investigated. The timely availability of these assays to monitor, confirm and characterize immunogenicity in clinical studies is a prerequisite for any clinical program. Assays for binding antibodies are considered to be state-of-the-art for screening purpose. Screening assays should be designed in an appropriate way to be able to detect low-titer and low-affinity antibodies. For further information, please see Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products; Anthony R. Mire-Sluis, Journal of Immunological Methods 289 (2004) 1 – 16.
In cases where antibody formation is observed and confirmed by additional assays, the antibodies have to be characterized carefully in order to assess the impact on the therapy scheme, patient selection, etc. Usually functional assays with the capacity to detect neutralizing effects of the anti-product antibodies need to be available. The nature of these functional assays will depend on the actual drug and the type of drug target intervention. In any case they have to be developed separately for each product.
We recommend that specific details regarding appropriate strategies be included in the guidance. Specific advice regarding assay design, optimisation, and qualification would also be helpful.
- Requirement that the assays for immunogenicity assessment must be capable of detecting all classes and subclasses of immunoglobulins, including IgM (the likely first antibody formed in an immune response)
- Instruction that assays should have a demonstrated ability to detect low affinity (rapidly dissociating) antibodies since these can be clinically relevant but difficult to detect with traditional methodologies
- Recommendation that a sufficient number of subjects should be monitored for antibodies for a period of at least 1 year (only necessary for proteins used for chronic treatment)
- Requirement that, in cases where there are serious clinical consequences of an immune response, significant testing should be performed prior to approval.
NEED OF POST-APPROVAL RISK MANAGEMENT
Because there is considerable inter-individual variability in antibody responses, current regulatory guidelines request data to be collected from a sufficient number of patients to characterize the variability in antibody response.
Rare immunogenic events, e.g. the PRCA case, will not be detectable in pre-approval clinical trials due to the lack of sufficient patient numbers which would be required for adequate statistical evaluation. Therefore, for any therapeutic protein (including biosimilars), a post-approval surveillance program will be necessary which includes immunogenicity testing, pharmacovigilance and relevant epidemiological data.
IMMUNOGENICITY ASSESSMENT OF BIOSIMILARS
The fact that all the currently available pre-clinical assays are not sufficiently predictive for the immunogenicity of innovative biotherapeutics in man, have also a direct bearing on assessing the immunocomparability of biosimilars: comparability or non-comparability of biologicals as to their immunogenic potential can only be determined in appropriately designed clinical studies.
To conclude, a comparison of immunogenicity of innovative and biosimilar protein products can only be made in conducting comparative clinical trials. In this circumstance, comparable immunogenic potential of a biosimilar to the innovative product cannot be the base hypothesis for approval. In the interests of patient safety, no protein drug should be approved without data on its immunogenic properties being obtained in clinical studies of adequate size and duration (e.g., in the case of chronic administration, one-year follow-up data will be required). We recommend that exclusion of a specified rate of immunogenicity should be required before approval. Furthermore the immune system may be influenced by disease and / or by co-medication; this may lead to the observation that the incidence and clinical phenotype is different for the same product when used in different indications.
EBE, European Biopharmaceutical Enterprises is a specialized group within EFPIA. It directly represents 65 European biopharmaceutical companies which are engaged in research and development of new biotech healthcare products. For more information:
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- EBE comments on EMEA concept paper on Guideline on Immunogenicity Assessment of Therapeutic Proteins, 31 May 2006