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Scientific and regulatory considerations on the immunogenicity of biologics
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Vol.24 No.6 June 2006
Scientific and regulatory considerations on the immunogenicity of biologics Gopi Shankar1, Elizabeth Shores2, Carrie Wagner1 and Anthony Mire-Sluis3 1
Clinical Pharmacology & Experimental Medicine, Centocor Research & Development, Inc., 145 King of Prussia Rd, Radnor, PA 19087, USA 2 Division of Therapeutic Proteins, CDER, FDA, N29A RM2A01 HFM-538, 8800 Rockville Pike, Bethesda, MD 20892, USA 3 Product Quality and External Affairs, MS 92-2-B, One Amgen Center Drive, Amgen Inc., Thousand Oaks, CA 91320, USA
Immune responses against non-vaccine biologics can affect their efficacy and safety, resulting in adverse events that could include administration reactions, hypersensitivity, deficiency syndromes and lack of a clinical response in treated patients. With the relatively recent development of numerous biologics, immunogenicity testing has become a key component in the demonstration of clinical safety and efficacy; in fact, it is highly unlikely that regulatory approval would be granted for a biologic without an assessment of its immunogenicity. However, recommendations from regulatory agencies regarding the requirements for when and how to carry out immunogenicity testing are dispersed among numerous guidance documents. To enable the evaluation of the effects of immunogenicity on safety and efficacy, the authors have consolidated recommendations from the regulatory guidelines, and present current approaches and future directions for the assessment of immunogenicity. Introduction Biotechnology-derived products (biologics) hold a great deal of promise among the therapeutic interventions for a wide range of disorders, including cancer and inflammatory diseases. Millions of people worldwide have benefited from the w190 biologic products and vaccines available today, and more than 370 new products are currently in various stages of clinical testing (http://www.bio.org). Biologics differ from traditional pharmaceutical drugs. They are generally large and complex proteins, which might or might not be glycosylated, and can be of nonhuman origin, contain a partial human-sequence or can comprise a complete human-sequence. All of these types of products have the potential to generate immune responses or immunogenicity through the production of anti-drug antibodies (ADA) or cell-based immune responses [1,2]. Subsets of biological products, namely vaccines, are intended to induce an immune response and are not the subject of this manuscript. This article focuses on the biological products that are used therapeutically, for which an immune response is not an intended outcome. Corresponding author: Shankar, G. ([email protected]). Available online 2 May 2006
Although immune responses to biologics are often not associated with adverse events, there are some examples of immune responses to biologics that have produced serious clinical consequences [3]. In these instances, the ADA can lower bioavailability by eliminating or neutralizing the biologic by binding to its active region such as a receptor-binding site on a protein [4–6]. ADA also have the potential to neutralize the endogenous counterpart of the biologic and worsen the disease that the biologic was intended to treat. Neutralizing ADA that are cross-reactive with the endogenous protein can cause significant safety concerns because they inhibit the inherent physiological pathway and can lead to a deficiency syndrome. For example, pure red-cell aplasia (PRCA) is a rare condition in which the red cell lineage can be specifically depleted by anti-erythropoietin antibodies (anti-EPO) [7,8]. Likewise, recombinant human thrombopoietin has been associated with cross-reactive anti-thrombopoietin antibodies, which cause thrombocytopenia or pancytopenia (depletion of all 3 hematopoietic lineages) [9]. ADA can also cause adverse events, including administration reactions such as systemic infusion reactions, localized injection reactions or acute hypersensitivity reactions [3]. For these reasons, the potential to induce ADA following treatment with biologics is a vital safety issue that is becoming an important consideration in the evaluation of these therapies and a crucial aspect of regulatory filings. Pharmaceutical products are regulated by government agencies, for example, the Food and Drug Administration (FDA) in the USA and the European Medicines Agency (EMEA) in Europe, and these agencies provide written guidelines to assist manufacturers during drug development, approval, and post-marketing. However, there are no comprehensive guidelines on the approaches required for immunogenicity testing during product development. Likewise, there is no single source of the requirements for non-clinical or clinical studies and no specific guidance for performing or interpreting assays. In this article, we will review and discuss the regulatory issues involved in nonclinical and clinical testing during developmental and post-market comparability evaluations.
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Assessing immunogenicity in non-clinical studies Although the immune responses observed in animal studies are not necessarily predictive of immunogenicity in humans, measuring antibody responses can be vital for assessing the validity of toxicology studies [10–12]. ADA can affect pharmacokinetics, pharmacodynamics and/or biological activity; therefore, it is important to confirm that toxicological data in animals is truly reflective of the expected exposure and activity of the drug and is not compromised by ADA. Animal models can be useful for understanding the physiological sequelae that might occur upon the binding of ADA, particularly neutralizing ADA that cross-react with endogenous protein. Such data can reveal the level of redundancy of a natural molecule, such as induction of
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thrombocytopenia in the presence of anti-thrombopoietin antibodies, reduction in red cell production with anti-EPO antibodies or development of pulmonary alveolar proteinosis with anti-granulocyte macrophage colony-stimulating factor (GM-CSF) antibodies [11,13–15]. Although it is a regulatory requirement that nonclinical immunogenicity studies are conducted, it is difficult to determine whether a high rate of immunogenicity in animals should preclude initiating clinical development because this is dependent on several factors, including the manufacturer’s knowledge about the biologic, its mechanism of action and the clinical consequences of immunogenicity. Hence, a strategy where the risk to the patient is taken into account, for example, one which considers the existence of a naturally occurring
Table 1. Regulatory and scientific implications for immunogenicity testing in non-clinical studies Implication Immunogenicity studies are required in repeatdose, non-clinical studies, and antibody responses, including biological consequences of an immune response, should be studied.
Refs [12]
Relevant quote(s) † ‘. the measurement of antibodies associated with administration of these types of products [biotechnology-derived] should be performed when conducting repeated dose toxicity studies to aid in the interpretation of these studies.’ † ‘Antibody responses should be characterized (e.g. titer, number of responding animals, neutralizing or non-neutralizing).’ † ‘Immunotoxicological testing strategies may require screening studies followed by mechanistic studies to clarify such issues.’ † ‘.their appearance [of antibody responses] should be correlated with any pharmacological and/or toxicological changes.’ † ‘.the effects of antibody formation on pharmacokinetic/pharmacodynamic parameters, incidence and/or severity of adverse effects, complement activation, or the emergence of new toxic effects should be considered.’ † ‘Attention should also be paid to the evaluation of possible pathological changes related to immune complex formation and deposition.’
The relevance of the animal models used in non-clinical testing should be understood, particularly with regards to the activity of the test molecule.
[18–20]
† ‘Present regulations (21 CFR 312.23(a)(8)(ii)(a)) require an integrated summary of the toxicologic effects of the drug in animals and in vitro. The particular studies needed depend on the nature of the drug and the phase of human investigation. When species specificity, immunogenicity, or other considerations appear to make many or all toxicological models irrelevant, sponsors are encouraged to contact the agency to discuss toxicological testing.’ † ‘Some classes of therapeutic biologics may have very limited interspecies crossreactivity or pronounced immunogenicity, or may work by mechanisms that are not known to be conserved between (nonhuman) animals and humans; in these cases, safety data from animal studies may be very limited in scope and interpretability.’
The value of animal models in predicting immu nogenicity is product and species specific; how ever, such studies are required by the regulators unless justified otherwise.
[12,21–23]
† ‘Studies of immunogenicity in animal models are generally of limited value. Therefore, we recommend that human clinical data assessing the repeat use of a biological imaging agent be obtained before application for licensure of such an agent.’ † ‘The induction of antibody formation in animals is not predictive of a potential for antibody formation in humans.’ † ‘Although demonstrating immunogenicity in an animal model does not necessarily predict adverse effects in humans, there are other reasons why it might be important to assess antidrug immune responses.’ † [Regarding biotechnology-derived wound products] ‘Although the development of antibodies to antigenic products has generally not been predictive of the clinical response, data on this should be collected to provide a complete preclinical safety assessment.’
The development of antibodies alone should not stop a toxicology study. Continued study might reveal the biological effects of neutralization.
[12]
† ‘The detection of antibodies should not be the sole criterion for the early termination of a preclinical safety study or modification in the duration of the study design unless the immune response neutralizes the pharmacological and/or toxicological effects of the biopharmaceutical in a large proportion of the animals.’
Abbreviation: CFR, Code of Federal Regulations www.sciencedirect.com
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counterpart or whether the disease is life threatening or chronic, should put the utility of non-clinical models into perspective. Such an approach has been encouraged by FDA personnel in scientific publications [11,16,17]. Although the general principles of a risk-based approach apply to both non-clinical and clinical studies, the process can be complex and needs careful consideration. For example, in the case of non-clinical studies, when taking into account the similarity of the sequence of the product to an endogenous protein, the physiological redundancy of the endogenous molecule, the presence of pre-existing antibodies and the biological activity of the drug all have to be considered. The available guidelines describing the need for immunogenicity testing during non-clinical studies, including detecting and characterizing antibodies in repeat-dose studies, are summarized in Table 1.
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Assessing immunogenicity in clinical studies It is an FDA policy to include immunogenicity as part of the review of clinical safety assessments for biologic license applications (BLA) [24], and similar requirements exist for other regulatory agencies. Immune responses to therapeutic proteins have been defined almost exclusively by the detection of circulating antibodies. To understand the true induction of ADA and their potential clinical effects, samples need to be taken before and during the clinical study. Pre-exposure samples are important because some patients might have preexisting antibodies. It is also important to consider the timing of post-exposure sample acquisition carefully, to ensure minimal interference from circulating levels of the biologic. Furthermore, ADA-screening assays should also be capable of detecting all isotypes of the induced antibodies.
Table 2. Regulatory and scientific implications for clinical immunogenicity assessments Implication There is a requirement to measure ADA and characterize the nature of the antibodies – non-neutralizing ADA are also important to consider.
Refs [23,25]
Relevant quote(s) † ‘For biological products and some drugs, immunogenicity is generally addressed by measuring antibody titres before and after the treatment. Further immunologic characterization may be recommended, since the development of an immune response can render the product inactive (neutralizing antibodies), and/or induce acute or chronic immune reactions (e.g. anaphylaxis, contact sensitization, autoimmune disease).’ † ‘Non-neutralizing antibodies may have a profound effect on PK and may therefore be just as important as neutralizing antibodies.’ † ‘.if Phase-2 clinical data suggest that agent-induced neutralizing antibodies could interfere with the efficacy of a biologic agent over time, it may become necessary to formally investigate the possibility in a randomized controlled setting.’ † ‘Potentially important issues for biological products include assessments of immunogenicity, both the incidence and consequences of neutralizing antibody formation and the potential for adverse events related to binding antibody formation.’ † [Regarding the content of the Common Technical Document submission] ‘It is particularly important to summarize analyses of potentially relevant correlates of immunogenicity, e.g. to determine the extent to which the presence of antibodies of a particular type or titer appears to correlate with alterations of PK, changes in PD, loss of efficacy, loss of adverse event profile, or development of adverse events. Particular attention should be paid to events that might be immunologically mediated (e.g. serum sickness) and events that might result from binding of cross-reactive endogenous substances by antibodies to the administered drug.’
The presence or absence of antibodies should be correlated to clinical sequelae, such as effects on PK and/or PD, induction of AEs and clinical studies should be designed to ensure that such correlations can be made.
[25–27]
Patients that do mount an immune response should be monitored closely, both short term and long term, during clinical studies.
[28]
† ‘When a patient is found to have developed an antibody response against the therapeutic or diagnostic mAb, adverse events should be anticipated and appropriate precautions taken.’ † ‘Vital signs should be observed closely for at least one hour after completion of the mAb administration. The possibility of delayed adverse effects from immune responses to mAb should be considered and reflected in the trial design, including appropriate clinical and laboratory testing.’ † ‘The correlation between circulating levels of soluble antigen and immune complex-mediated adverse events such as serum sickness should be explored if such adverse events are observed.’
If neutralizing antibodies are discovered during a clinical study, redesign might be necessary.
[25]
† ‘The occurrence of neutralizing antibodies may call for the reconsideration of doses and dose regimens.’
Samples from phase 3 studies should be retained for potential retrospective investigations.
[26]
† ‘.in circumstances when earlier safety data signal an unusual or important concern, a sponsor should consider reserving blood samples (or any other bodily fluids/tissues that may be collected during clinical trials) for some or all patients in phase 3 studies for possible assessments at a later time. Such later assessments could include pharmacogenomic markers, immunogenicity, or measurements of other biomarkers that might prove helpful clinically.’
Abbreviations: ADA, anti-drug antibody; AE, adverse event; mAb, monoclonal antibody; PD, pharmacodynamics; PK, pharmacokinetics www.sciencedirect.com
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Pre-existing or drug-induced antibodies should be characterized by their ability to neutralize the drug or any other endogenous protein that might be structurally or functionally related to it. Immune responses with severe clinical consequences often involve functionally neutralizing antibodies; therefore, to ensure the safety and efficacy of biologics, the FDA and the biopharmaceutical industry have recognized that assessment of the antibodies’ neutralizing ability should be a routine part of ADA characterization, particularly for biologics that have endogenous counterparts. Furthermore, the regulations stress that non-neutralizing antibodies also can be clinically significant because they can alter the half-life or the biodistribution of the product. This can affect the biological activity of the product by either reducing activity through an increased rate of clearance or increasing product activity by lowering the clearance rate and making the drug available to its target for a longer period. In parallel, increased or decreased activity or altered tissue distribution and/or concentration in certain organs, such as the kidney or liver, could affect drug toxicity. Although immunological characterization, such as individual Ig isotyping, is also recommended, the value of isotyping is generally restricted to the presence of specific anti-drug IgE antibodies that can cause allergic reactions, particularly in allergy-prone patient populations. With biologics there is often some, albeit low, incidence of hypersensitivity reactions, although the relationship of these reactions to the production of IgE has been difficult to assess due to the short half-life of IgE – by the time the allergic reaction occurs, most of the IgE is bound to immune-mediating cells. General isotyping might also provide insight into the immunological mechanisms or consequences underlying a particular immune response. Accordingly, the presence of IgG and
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IgM, indicators of maturity of the immune response, might be a ‘nice to know’ component of immunogenicity evaluations but is generally not a regulatory requirement. Table 2 provides quotes from several regulatory documents, mandating the characterization of ADA immunogenicity in clinical studies. When immune responses are detected and/or characterized, it is important that the results be correlated (positively or negatively) with pharmacological and/or toxicological changes or clinical outcomes, such as adverse events, administration reactions and autoimmunity. It is noteworthy that the FDA recommends the retention of samples from phase 3 studies for potential retrospective analysis if appropriate assays or studies are not undertaken at the time the samples were originally drawn (e.g. if certain correlative events appear during post-study analysis of data or for post-study pharmacogenetic analysis) [22]. In this context, it is advisable to make provisions for such testing in the original patient consent forms, and plan for long-term storage to save subsequent time and effort should the necessity to perform unplanned studies arise. The use of a risk-based approach can also be applied to clinical studies; thus, the FDA is guiding manufacturers to incorporate programs for immunogenicity risk-management into clinical development from the earliest stages [11,16,17]. An immunogenicity-testing strategy needs to consider the nature of the biologic, its target population, the disease being treated and the treatment regimen. For example, patient populations can influence the rate and consequences of immunogenicity, depending on such features as whether they exhibit immunosuppression, have received recent or concomitant immunosuppressive therapy, possess a specific genetic trait or defect or have pre-existing antibodies.
Table 3. Product-specific implications for immunogenicity assessments Product type Monoclonal antibody products
Refs [21,28]
Relevant quote(s) † ‘The specificity of the immune response to the mAb should be identified and characterized in a sample of patients. These studies should establish whether the responses are generated against a heavy-chain isotype determinant, a light-chain constant (C) region, variable (V) region, idiotypic epitopes(s), immunoconjugate, or neoantigen.’ † ‘The assay(s) used to detect the anti-mAb antibody should be standardized to the greatest extent possible. Aliquots of a “reference” preparation of antibody, e.g. anti-mouse antibody for a HAMA assay with defined specificity from a human or primate source should be aliquoted and frozen to facilitate future intra- and inter-study comparisons.’ † [Regarding medical imaging monoclonals] ‘We recommend that studies in which repeat dosing of a biological imaging agent is planned incorporate pharmacokinetic data, human antimouse antibody (HAMA), human anti-humanized antibody (HAHA), or human anti-chimeric antibody (HACA) levels.’
Plant-derived biologicals
[29]
† ‘You should evaluate your product for plant specific modifications that may contribute to unintended immunogenicity.’
Synthetic peptide drug substances
[30]
† ‘To comply with section 505(b) of the Food, Drug, and Cosmetic Act, 21 U.S.C. 355(b), certain information pertaining to the method of preparation of the drug substance and the control testing used to monitor its identity, strength, quality, and purity should be provided in all submissions to the Centers. Certain biological characteristics, such as potency, immunogenicity, or antigenicity, may also be necessary.’
Platelet substitutes
[31]
† ‘Infusion of only subcellular parts of the platelet may induce immunogenic response.’ † ‘It should be demonstrated in test animals that the platelet substitute is no more immunogenic than intact platelets.’
Abbreviations: HACA, human anti-chimeric antibody; HAHA, human anti-humanized antibody; HAMA, human anti-mouse antibody; mAb, monoclonal antibody; USC, United States Code www.sciencedirect.com
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Immunogenicity issues specific to product type Some immunogenicity issues are product specific. Existing FDA guidance relating to these are listed in Table 3. In Europe, product-specific guidance for similar biological medicinal products includes details of the duration and design of immunogenicity studies, the need for appropriate assays and a prelicensing pharmacovigilance plan. However, the EMEA generally adopts a case-by-case approach to the requirements for these types of studies. In fact, the need to provide more guidance on risk management and immunogenicity has been acknowledged by the EMEA, and this agency is planning to develop guidelines on immunogenicity assessment of biological and/or biotechnology-derived proteins. For monoclonal antibodies and Fc fusion proteins, the possibility that a product might have both binding activity and functions mediated by the Fc portion must be considered; consequently, immunogenicity studies will need to focus on both portions of the molecule. In addition, studies might also need to concentrate on areas in the antibody structure that are related to the source of the genetic sequence, that is the species from which it was originally cloned (e.g. anti-mouse for a murine antibody or anti-chimera for a chimeric antibody). There is increasing interest in the manufacture of biologics in plant species, and FDA guidance states that immunogenicity studies need to focus specifically on any modifications to a product that are plant-related, such as carbohydrate structures, to ensure that the assay design enables the detection of antibodies against such modifications. It is also interesting to note that peptide products, although often much smaller and less complex than the other products of biotechnology, still require an assessment of immunogenicity.
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Immunogenicity testing in comparability assessments Following substantial changes in manufacturing, immunogenicity testing is an important aspect of product comparability assessments and has received even more attention with the potential advent of ‘follow-on’ or ‘biosimilar’ biologics. Table 4 provides relevant quotes from European and international documents on product comparability [32,33]. It is interesting to note that these recent guidelines provide some of the most detailed descriptions of what is necessary for the conduct of an immunogenicity study, regardless of whether or not the results are to be used for product comparability. It is necessary to conduct clinical studies if physicochemical tests alone cannot predict whether the molecule has changed in a way that might result in altered immunogenicity or if the potential adverse events for that particular product might be serious in nature. It might be necessary to carry out immunogenicity studies in each clinical indication because immune responses can be generated differently depending on the disease and the seriousness of the clinical consequences of any response. European guidance emphasizes that immunogenicity assessments conducted during clinical studies might not be adequate to assess the rate and consequences of immune responses, particularly rare adverse events; thus, a post-marketing risk management and pharmacovigilance plan must be considered. Such studies can require considerable numbers of patients if the occurrence of an adverse event is rare. Immunogenicity, assay development and validation Although regulatory agencies have not released regulatory documents specifically related to immunogenicity
Table 4. Immunogenicity assessments relative to comparability testing Implication Comparability testing is required through clinical as opposed to non-clinical studies when structural testing might not assure differences in immunogenicity and it is particularly important to assess in repeat-dose products.
Refs [32]
Relevant quote(s) † ‘Immunogenicity must always be addressed by clinical data, unless clinically relevant immunogenicity can be excluded by other means.’ † ‘Immunological studies are expected if physico-chemical characterisation is not sufficient due to the complexity of the molecule and an impact on immunogenicity cannot be excluded with reasonable certainty.’ † ‘The issue of immunogenicity must always be considered when a claim of comparability is made, especially when repeated administration is proposed.’
The extent of immunogenicity studies depends on the nature of the product, the patient population and the possible consequences.
[33]
† [Regarding factors to consider in planning non-clinical and clinical studies] ‘The relationship between the therapeutic protein and endogenous proteins and the consequences for immunogenicity.’ † ‘The impact of possible differences can vary between patient groups, e.g. the risk of unwanted immunogenicity. It may be appropriate to consider the consequences separately for each indication.’
A post-marketing pharmacovigilance plan might be required, depending on the product.
[32]
† ‘In view of the unpredictability of the onset and incidence of immunogenicity, post-marketing monitoring of antibodies at predetermined intervals will be required for at least one year for a new biotechnological product and may be required after a change to an existing product.’
Assays for immunogenicity studies should include validated screening and neutralizing-ADA assays.
[32]
† The assessment of immunogenicity requires validated antibody assays, characterisation of the observed immune response, as well as evaluation of the correlation between antibodies, pharmacokinetics/pharmacodynamics and efficacy and safety.’ † ‘The screening assays should be sensitive enough to detect low titre antibodies as well as antibodies to conformational and linear epitopes. An assay for neutralizing antibodies should be available for further characterisation of antibodies detected by the screening assays.’
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assays, agencies and industry have extensive understanding of the requirements of assays that test for antibody production from vaccines. Although the intent of vaccines is to induce antibodies, the issues relating to an assay detecting either unwanted or desired antibodies have considerable overlap (e.g. the need for appropriate controls, reproducibility and robustness). Good Laboratory Practice guidelines cover the principles for assay development and implementation [34,35] and have some applicability to immunogenicity assays. Some specific statements pertaining to assays have been provided by the FDA and the EMEA [28,32]. Industry and FDA representatives of the American Association for Pharmaceutical Science (AAPS) are currently preparing white papers on the topics of neutralizing antibody assays and immunoassay validation. One such paper, describing the key considerations for immunoassay development and implementation, has already been published [36]. Immunogenicity assessment generally uses an algorithm of immunoassay-based testing. This typically includes a screening assay for detecting potentially ADA-positive samples, which requires sensitive assays that detect as many of the induced antibodies as possible (e.g. low-affinity or low-abundance and different isotypes). Once identified, a specificity assay confirms whether the samples are true- or false-positives, followed by an assay to obtain a relative measure of the antibody concentration in serum, such as titre [36–38]. Following confirmation, ADA-positive samples are often characterized by antibody isotyping and the determination of their neutralizing activity. Method validation refers to the determination of assay reliability and provides criteria for maintaining reliable long-term performance. It establishes the limitations and overall performance expectations of a method with the goal of demonstrating suitability for its intended analytical application [39,40]. Regulatory guidance exists on the fundamental criteria for general assay validation, including the evaluations of specificity, selectivity, range of linearity, detection and/or quantification limits, robustness and system suitability [39,41], in addition to testing the established assay for its accuracy and precision against pre-determined acceptance criteria. Conclusions The safety and efficacy of biologics in patients can be altered, significantly, by the development of immune responses. Manufacturers can address these concerns by including appropriately designed immunogenicity studies during product development and beyond. The need for immunogenicity testing is clear, but consolidated regulatory documents on precisely how to execute such assessments are not available. The development of such guidance requires the partnership between regulatory agencies and industry to ensure that method development, non-clinical testing and the conduct of clinical studies is appropriately planned and leads to insightful and scientifically valid immunogenicity assessments. Acknowledgements The authors thank the following individuals for their review and feedback on this manuscript: Keith Weber and Steven Kozlowski of the US Food www.sciencedirect.com
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and Drug Administration (FDA); Glenn Begley of Amgen; and Mary Whitman and Patti Shirey of Centocor. ICH and European guidelines are available from the European Medicines Agency website (http://www. emea.eu.int). FDA guidelines are available on the FDA website (http:// www.fda.gov/cder).
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20 US Department of Health and Human Services Food and Drug Administration (2002) Draft Guidance for Industry and Reviewers on Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers (http://www.fda.gov/OHRMS/DOCKETS/ 98fr/03-906.htm) 21 US Department of Health and Human Services Food and Drug Administration (2004) Guidance for Industry Developing Medical Imaging Drugs and Biological Products. Part 1: Conducting Safety Assessments (http://www.fda.gov/cber/gdlns/medimagesaf.pdf) 22 US Department of Health and Human Services Food and Drug Administration (2002) Guidance for Industry Immunotoxicology Evaluation of Investigational New Drugs (http://www.fda.gov/cder/ guidance/4945fnl.PDF) 23 US Department of Health and Human Services Food and Drug Administration (2000) Draft Guidance for Industry Chronic Cutaneous Ulcer and Burn Wounds – Developing Products for Treatments (http://www.fda.gov/cder/guidance/3226dft.pdf) 24 Center for Drug Evaluation and Research (2004) Manual of Policies and Procedures (MAPP) (http://www.fda.gov/cder/mapp.htm) 25 US Department of Health and Human Services Food and Drug Administration (1999) Guidance for Industry Clinical Development Programs for Drugs, Devices, and Biological Products for the Treatment of Rheumatoid Arthritis (RA) (http://www.fda.gov/cber/ gdlns/rheumcln.pdf) 26 US Department of Health and Human Services Food and Drug Administration (2004) Guidance for Industry Premarketing Risk Assessment (http://www.fda.gov/cder/guidance/6357fnl.pdf) 27 The European Agency for the Evaluation of Medicinal Products (EMEA) (2003) The Common Technical Document for the Registration of Pharmaceuticals for Human Use. Efficacy – ICH M4E. Clinical Overview and Clinical Summary of Module 2. Module 5: Clinical Study Reports (http://www.tga.gov.au/docs/pdf/euguide/ich/ctdm2efficacy.pdf) 28 US Department of Health and Human Services Food and Drug Administration (1997) Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (http://www. fda.gov/cber/gdlns/ptc_mab.pdf) 29 US Department of Health and Human Services Food and Drug Administration (2002) Draft Guidance for Industry Drugs, Biologics, and Medical Devices Derived from Bioengineered Plants for Use in Humans and Animals (http://www.fda.gov/cber/gdlns/bioplant.pdf) 30 Center for Drug Evaluation and Research Center for Biologics Evaluation and Research (1994) Guidance for Industry for the Submission of Chemistry, Manufacturing, and Controls Information for Synthetic Peptide Substances (http://www.fda.gov/cber/gdlns/ cmcsyn.pdf)
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31 US Department of Health and Human Services Food and Drug Administration (1999) Draft Guidance for Industry for Platelet Testing and Evaluation of Platelet Substitute Products (http://www.fda.gov/ cber/gdlns/platelet.pdf) 32 The European Agency for the Evaluation of Medicinal Products (EMEA) (2003) Guideline on Comparability of Medicinal Products Containing Biotechnology-derived Proteins as Active Substance: Nonclinical and Clinical Issues EMEA/CPMP/3097/02/Final (http:// www.emea.eu.int/pdfs/human/ewp/309702en.pdf) 33 International conference on harmonization (ICH) of technical requirements for registration of pharmaceuticals for human use (2004) ICH Harmonized Tripartite Guideline Q5E Comparability of Biotechnological/Biological Products Subject to Changes in their Manufacturing Process (CPMP/ICH/5721/03) (http://www.emea.eu.int/pdfs/human/ ich/572103en.pdf) 34 Code of Federal Regulations 21 CFR Part 58: Good Laboratory Practice for Non-clinical Laboratory Studies, US Government Printing Office, Washington US (http://www.access.gpo.gov/nara/cfr/ waisidx_00/21cfr58_00.html) 35 Organisation for Economic Co-operation and Development (1997) The OECD Principles on Good Laboratory Practice ENV/MC/CHEM (98)17 (http://www.olis.oecd.org/olis/1998doc.nsf/87fae4004d4fa67ac125685d005300b3/ 3d81d76159dd69e78025675c00409638?OpenDocument) 36 Mire-Sluis, A.R. et al. (2004) Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J. Immunol. Methods 289, 1–16 37 Wadhwa, M. et al. (2003) Strategies for detection, measurement, and characterization of unwanted antibodies induced by therapeutic biologicals. J. Immunol. Methods 278, 1–17 38 Geng, D. et al. (2005) Validation of immunoassays used to assess immunogenicity to therapeutic monoclonal antibodies. J. Pharm. Biomed. Anal. 39, 364–375 39 US Department of Health and Human Services, Food and Drug Administration (CDER and CVM) (2001) Guidance For Industry Bioanalytical Method Validation (http://www.fda.gov/cder/guidance/ 4252fnl.pdf) 40 International conference on harminization (ICH) of technical requirements for registration of pharmaceuticals for human use (1995) ICH Harmonized Tripartite Guideline – Q2A, Text on validation of analytical procedures (http://www.fda.gov/cder/guidance/ichq2a.pdf) 41 International conference on harminization (ICH) of technical requirements for registration of pharmaceuticals for human use (1996) ICH Harmonized Tripartite Guideline – Q2B. Validation of analytical procedures: methodology (http://www.fda.gov/cder/guidance/1320fnl. pdf)
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Scientific and regulatory considerations on the immunogenicity of biologics Gopi Shankar1, Elizabeth Shores2, Carrie Wagner1 and Anthony Mire-Sluis3 1
Clinical Pharmacology & Experimental Medicine, Centocor Research & Development, Inc., 145 King of Prussia Rd, Radnor, PA 19087, USA 2 Division of Therapeutic Proteins, CDER, FDA, N29A RM2A01 HFM-538, 8800 Rockville Pike, Bethesda, MD 20892, USA 3 Product Quality and External Affairs, MS 92-2-B, One Amgen Center Drive, Amgen Inc., Thousand Oaks, CA 91320, USA
Immune responses against non-vaccine biologics can affect their efficacy and safety, resulting in adverse events that could include administration reactions, hypersensitivity, deficiency syndromes and lack of a clinical response in treated patients. With the relatively recent development of numerous biologics, immunogenicity testing has become a key component in the demonstration of clinical safety and efficacy; in fact, it is highly unlikely that regulatory approval would be granted for a biologic without an assessment of its immunogenicity. However, recommendations from regulatory agencies regarding the requirements for when and how to carry out immunogenicity testing are dispersed among numerous guidance documents. To enable the evaluation of the effects of immunogenicity on safety and efficacy, the authors have consolidated recommendations from the regulatory guidelines, and present current approaches and future directions for the assessment of immunogenicity. Introduction Biotechnology-derived products (biologics) hold a great deal of promise among the therapeutic interventions for a wide range of disorders, including cancer and inflammatory diseases. Millions of people worldwide have benefited from the w190 biologic products and vaccines available today, and more than 370 new products are currently in various stages of clinical testing (http://www.bio.org). Biologics differ from traditional pharmaceutical drugs. They are generally large and complex proteins, which might or might not be glycosylated, and can be of nonhuman origin, contain a partial human-sequence or can comprise a complete human-sequence. All of these types of products have the potential to generate immune responses or immunogenicity through the production of anti-drug antibodies (ADA) or cell-based immune responses [1,2]. Subsets of biological products, namely vaccines, are intended to induce an immune response and are not the subject of this manuscript. This article focuses on the biological products that are used therapeutically, for which an immune response is not an intended outcome. Corresponding author: Shankar, G. ([email protected]). Available online 2 May 2006
Although immune responses to biologics are often not associated with adverse events, there are some examples of immune responses to biologics that have produced serious clinical consequences [3]. In these instances, the ADA can lower bioavailability by eliminating or neutralizing the biologic by binding to its active region such as a receptor-binding site on a protein [4–6]. ADA also have the potential to neutralize the endogenous counterpart of the biologic and worsen the disease that the biologic was intended to treat. Neutralizing ADA that are cross-reactive with the endogenous protein can cause significant safety concerns because they inhibit the inherent physiological pathway and can lead to a deficiency syndrome. For example, pure red-cell aplasia (PRCA) is a rare condition in which the red cell lineage can be specifically depleted by anti-erythropoietin antibodies (anti-EPO) [7,8]. Likewise, recombinant human thrombopoietin has been associated with cross-reactive anti-thrombopoietin antibodies, which cause thrombocytopenia or pancytopenia (depletion of all 3 hematopoietic lineages) [9]. ADA can also cause adverse events, including administration reactions such as systemic infusion reactions, localized injection reactions or acute hypersensitivity reactions [3]. For these reasons, the potential to induce ADA following treatment with biologics is a vital safety issue that is becoming an important consideration in the evaluation of these therapies and a crucial aspect of regulatory filings. Pharmaceutical products are regulated by government agencies, for example, the Food and Drug Administration (FDA) in the USA and the European Medicines Agency (EMEA) in Europe, and these agencies provide written guidelines to assist manufacturers during drug development, approval, and post-marketing. However, there are no comprehensive guidelines on the approaches required for immunogenicity testing during product development. Likewise, there is no single source of the requirements for non-clinical or clinical studies and no specific guidance for performing or interpreting assays. In this article, we will review and discuss the regulatory issues involved in nonclinical and clinical testing during developmental and post-market comparability evaluations.
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Assessing immunogenicity in non-clinical studies Although the immune responses observed in animal studies are not necessarily predictive of immunogenicity in humans, measuring antibody responses can be vital for assessing the validity of toxicology studies [10–12]. ADA can affect pharmacokinetics, pharmacodynamics and/or biological activity; therefore, it is important to confirm that toxicological data in animals is truly reflective of the expected exposure and activity of the drug and is not compromised by ADA. Animal models can be useful for understanding the physiological sequelae that might occur upon the binding of ADA, particularly neutralizing ADA that cross-react with endogenous protein. Such data can reveal the level of redundancy of a natural molecule, such as induction of
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thrombocytopenia in the presence of anti-thrombopoietin antibodies, reduction in red cell production with anti-EPO antibodies or development of pulmonary alveolar proteinosis with anti-granulocyte macrophage colony-stimulating factor (GM-CSF) antibodies [11,13–15]. Although it is a regulatory requirement that nonclinical immunogenicity studies are conducted, it is difficult to determine whether a high rate of immunogenicity in animals should preclude initiating clinical development because this is dependent on several factors, including the manufacturer’s knowledge about the biologic, its mechanism of action and the clinical consequences of immunogenicity. Hence, a strategy where the risk to the patient is taken into account, for example, one which considers the existence of a naturally occurring
Table 1. Regulatory and scientific implications for immunogenicity testing in non-clinical studies Implication Immunogenicity studies are required in repeatdose, non-clinical studies, and antibody responses, including biological consequences of an immune response, should be studied.
Refs [12]
Relevant quote(s) † ‘. the measurement of antibodies associated with administration of these types of products [biotechnology-derived] should be performed when conducting repeated dose toxicity studies to aid in the interpretation of these studies.’ † ‘Antibody responses should be characterized (e.g. titer, number of responding animals, neutralizing or non-neutralizing).’ † ‘Immunotoxicological testing strategies may require screening studies followed by mechanistic studies to clarify such issues.’ † ‘.their appearance [of antibody responses] should be correlated with any pharmacological and/or toxicological changes.’ † ‘.the effects of antibody formation on pharmacokinetic/pharmacodynamic parameters, incidence and/or severity of adverse effects, complement activation, or the emergence of new toxic effects should be considered.’ † ‘Attention should also be paid to the evaluation of possible pathological changes related to immune complex formation and deposition.’
The relevance of the animal models used in non-clinical testing should be understood, particularly with regards to the activity of the test molecule.
[18–20]
† ‘Present regulations (21 CFR 312.23(a)(8)(ii)(a)) require an integrated summary of the toxicologic effects of the drug in animals and in vitro. The particular studies needed depend on the nature of the drug and the phase of human investigation. When species specificity, immunogenicity, or other considerations appear to make many or all toxicological models irrelevant, sponsors are encouraged to contact the agency to discuss toxicological testing.’ † ‘Some classes of therapeutic biologics may have very limited interspecies crossreactivity or pronounced immunogenicity, or may work by mechanisms that are not known to be conserved between (nonhuman) animals and humans; in these cases, safety data from animal studies may be very limited in scope and interpretability.’
The value of animal models in predicting immu nogenicity is product and species specific; how ever, such studies are required by the regulators unless justified otherwise.
[12,21–23]
† ‘Studies of immunogenicity in animal models are generally of limited value. Therefore, we recommend that human clinical data assessing the repeat use of a biological imaging agent be obtained before application for licensure of such an agent.’ † ‘The induction of antibody formation in animals is not predictive of a potential for antibody formation in humans.’ † ‘Although demonstrating immunogenicity in an animal model does not necessarily predict adverse effects in humans, there are other reasons why it might be important to assess antidrug immune responses.’ † [Regarding biotechnology-derived wound products] ‘Although the development of antibodies to antigenic products has generally not been predictive of the clinical response, data on this should be collected to provide a complete preclinical safety assessment.’
The development of antibodies alone should not stop a toxicology study. Continued study might reveal the biological effects of neutralization.
[12]
† ‘The detection of antibodies should not be the sole criterion for the early termination of a preclinical safety study or modification in the duration of the study design unless the immune response neutralizes the pharmacological and/or toxicological effects of the biopharmaceutical in a large proportion of the animals.’
Abbreviation: CFR, Code of Federal Regulations www.sciencedirect.com
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counterpart or whether the disease is life threatening or chronic, should put the utility of non-clinical models into perspective. Such an approach has been encouraged by FDA personnel in scientific publications [11,16,17]. Although the general principles of a risk-based approach apply to both non-clinical and clinical studies, the process can be complex and needs careful consideration. For example, in the case of non-clinical studies, when taking into account the similarity of the sequence of the product to an endogenous protein, the physiological redundancy of the endogenous molecule, the presence of pre-existing antibodies and the biological activity of the drug all have to be considered. The available guidelines describing the need for immunogenicity testing during non-clinical studies, including detecting and characterizing antibodies in repeat-dose studies, are summarized in Table 1.
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Assessing immunogenicity in clinical studies It is an FDA policy to include immunogenicity as part of the review of clinical safety assessments for biologic license applications (BLA) [24], and similar requirements exist for other regulatory agencies. Immune responses to therapeutic proteins have been defined almost exclusively by the detection of circulating antibodies. To understand the true induction of ADA and their potential clinical effects, samples need to be taken before and during the clinical study. Pre-exposure samples are important because some patients might have preexisting antibodies. It is also important to consider the timing of post-exposure sample acquisition carefully, to ensure minimal interference from circulating levels of the biologic. Furthermore, ADA-screening assays should also be capable of detecting all isotypes of the induced antibodies.
Table 2. Regulatory and scientific implications for clinical immunogenicity assessments Implication There is a requirement to measure ADA and characterize the nature of the antibodies – non-neutralizing ADA are also important to consider.
Refs [23,25]
Relevant quote(s) † ‘For biological products and some drugs, immunogenicity is generally addressed by measuring antibody titres before and after the treatment. Further immunologic characterization may be recommended, since the development of an immune response can render the product inactive (neutralizing antibodies), and/or induce acute or chronic immune reactions (e.g. anaphylaxis, contact sensitization, autoimmune disease).’ † ‘Non-neutralizing antibodies may have a profound effect on PK and may therefore be just as important as neutralizing antibodies.’ † ‘.if Phase-2 clinical data suggest that agent-induced neutralizing antibodies could interfere with the efficacy of a biologic agent over time, it may become necessary to formally investigate the possibility in a randomized controlled setting.’ † ‘Potentially important issues for biological products include assessments of immunogenicity, both the incidence and consequences of neutralizing antibody formation and the potential for adverse events related to binding antibody formation.’ † [Regarding the content of the Common Technical Document submission] ‘It is particularly important to summarize analyses of potentially relevant correlates of immunogenicity, e.g. to determine the extent to which the presence of antibodies of a particular type or titer appears to correlate with alterations of PK, changes in PD, loss of efficacy, loss of adverse event profile, or development of adverse events. Particular attention should be paid to events that might be immunologically mediated (e.g. serum sickness) and events that might result from binding of cross-reactive endogenous substances by antibodies to the administered drug.’
The presence or absence of antibodies should be correlated to clinical sequelae, such as effects on PK and/or PD, induction of AEs and clinical studies should be designed to ensure that such correlations can be made.
[25–27]
Patients that do mount an immune response should be monitored closely, both short term and long term, during clinical studies.
[28]
† ‘When a patient is found to have developed an antibody response against the therapeutic or diagnostic mAb, adverse events should be anticipated and appropriate precautions taken.’ † ‘Vital signs should be observed closely for at least one hour after completion of the mAb administration. The possibility of delayed adverse effects from immune responses to mAb should be considered and reflected in the trial design, including appropriate clinical and laboratory testing.’ † ‘The correlation between circulating levels of soluble antigen and immune complex-mediated adverse events such as serum sickness should be explored if such adverse events are observed.’
If neutralizing antibodies are discovered during a clinical study, redesign might be necessary.
[25]
† ‘The occurrence of neutralizing antibodies may call for the reconsideration of doses and dose regimens.’
Samples from phase 3 studies should be retained for potential retrospective investigations.
[26]
† ‘.in circumstances when earlier safety data signal an unusual or important concern, a sponsor should consider reserving blood samples (or any other bodily fluids/tissues that may be collected during clinical trials) for some or all patients in phase 3 studies for possible assessments at a later time. Such later assessments could include pharmacogenomic markers, immunogenicity, or measurements of other biomarkers that might prove helpful clinically.’
Abbreviations: ADA, anti-drug antibody; AE, adverse event; mAb, monoclonal antibody; PD, pharmacodynamics; PK, pharmacokinetics www.sciencedirect.com
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Pre-existing or drug-induced antibodies should be characterized by their ability to neutralize the drug or any other endogenous protein that might be structurally or functionally related to it. Immune responses with severe clinical consequences often involve functionally neutralizing antibodies; therefore, to ensure the safety and efficacy of biologics, the FDA and the biopharmaceutical industry have recognized that assessment of the antibodies’ neutralizing ability should be a routine part of ADA characterization, particularly for biologics that have endogenous counterparts. Furthermore, the regulations stress that non-neutralizing antibodies also can be clinically significant because they can alter the half-life or the biodistribution of the product. This can affect the biological activity of the product by either reducing activity through an increased rate of clearance or increasing product activity by lowering the clearance rate and making the drug available to its target for a longer period. In parallel, increased or decreased activity or altered tissue distribution and/or concentration in certain organs, such as the kidney or liver, could affect drug toxicity. Although immunological characterization, such as individual Ig isotyping, is also recommended, the value of isotyping is generally restricted to the presence of specific anti-drug IgE antibodies that can cause allergic reactions, particularly in allergy-prone patient populations. With biologics there is often some, albeit low, incidence of hypersensitivity reactions, although the relationship of these reactions to the production of IgE has been difficult to assess due to the short half-life of IgE – by the time the allergic reaction occurs, most of the IgE is bound to immune-mediating cells. General isotyping might also provide insight into the immunological mechanisms or consequences underlying a particular immune response. Accordingly, the presence of IgG and
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IgM, indicators of maturity of the immune response, might be a ‘nice to know’ component of immunogenicity evaluations but is generally not a regulatory requirement. Table 2 provides quotes from several regulatory documents, mandating the characterization of ADA immunogenicity in clinical studies. When immune responses are detected and/or characterized, it is important that the results be correlated (positively or negatively) with pharmacological and/or toxicological changes or clinical outcomes, such as adverse events, administration reactions and autoimmunity. It is noteworthy that the FDA recommends the retention of samples from phase 3 studies for potential retrospective analysis if appropriate assays or studies are not undertaken at the time the samples were originally drawn (e.g. if certain correlative events appear during post-study analysis of data or for post-study pharmacogenetic analysis) [22]. In this context, it is advisable to make provisions for such testing in the original patient consent forms, and plan for long-term storage to save subsequent time and effort should the necessity to perform unplanned studies arise. The use of a risk-based approach can also be applied to clinical studies; thus, the FDA is guiding manufacturers to incorporate programs for immunogenicity risk-management into clinical development from the earliest stages [11,16,17]. An immunogenicity-testing strategy needs to consider the nature of the biologic, its target population, the disease being treated and the treatment regimen. For example, patient populations can influence the rate and consequences of immunogenicity, depending on such features as whether they exhibit immunosuppression, have received recent or concomitant immunosuppressive therapy, possess a specific genetic trait or defect or have pre-existing antibodies.
Table 3. Product-specific implications for immunogenicity assessments Product type Monoclonal antibody products
Refs [21,28]
Relevant quote(s) † ‘The specificity of the immune response to the mAb should be identified and characterized in a sample of patients. These studies should establish whether the responses are generated against a heavy-chain isotype determinant, a light-chain constant (C) region, variable (V) region, idiotypic epitopes(s), immunoconjugate, or neoantigen.’ † ‘The assay(s) used to detect the anti-mAb antibody should be standardized to the greatest extent possible. Aliquots of a “reference” preparation of antibody, e.g. anti-mouse antibody for a HAMA assay with defined specificity from a human or primate source should be aliquoted and frozen to facilitate future intra- and inter-study comparisons.’ † [Regarding medical imaging monoclonals] ‘We recommend that studies in which repeat dosing of a biological imaging agent is planned incorporate pharmacokinetic data, human antimouse antibody (HAMA), human anti-humanized antibody (HAHA), or human anti-chimeric antibody (HACA) levels.’
Plant-derived biologicals
[29]
† ‘You should evaluate your product for plant specific modifications that may contribute to unintended immunogenicity.’
Synthetic peptide drug substances
[30]
† ‘To comply with section 505(b) of the Food, Drug, and Cosmetic Act, 21 U.S.C. 355(b), certain information pertaining to the method of preparation of the drug substance and the control testing used to monitor its identity, strength, quality, and purity should be provided in all submissions to the Centers. Certain biological characteristics, such as potency, immunogenicity, or antigenicity, may also be necessary.’
Platelet substitutes
[31]
† ‘Infusion of only subcellular parts of the platelet may induce immunogenic response.’ † ‘It should be demonstrated in test animals that the platelet substitute is no more immunogenic than intact platelets.’
Abbreviations: HACA, human anti-chimeric antibody; HAHA, human anti-humanized antibody; HAMA, human anti-mouse antibody; mAb, monoclonal antibody; USC, United States Code www.sciencedirect.com
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Immunogenicity issues specific to product type Some immunogenicity issues are product specific. Existing FDA guidance relating to these are listed in Table 3. In Europe, product-specific guidance for similar biological medicinal products includes details of the duration and design of immunogenicity studies, the need for appropriate assays and a prelicensing pharmacovigilance plan. However, the EMEA generally adopts a case-by-case approach to the requirements for these types of studies. In fact, the need to provide more guidance on risk management and immunogenicity has been acknowledged by the EMEA, and this agency is planning to develop guidelines on immunogenicity assessment of biological and/or biotechnology-derived proteins. For monoclonal antibodies and Fc fusion proteins, the possibility that a product might have both binding activity and functions mediated by the Fc portion must be considered; consequently, immunogenicity studies will need to focus on both portions of the molecule. In addition, studies might also need to concentrate on areas in the antibody structure that are related to the source of the genetic sequence, that is the species from which it was originally cloned (e.g. anti-mouse for a murine antibody or anti-chimera for a chimeric antibody). There is increasing interest in the manufacture of biologics in plant species, and FDA guidance states that immunogenicity studies need to focus specifically on any modifications to a product that are plant-related, such as carbohydrate structures, to ensure that the assay design enables the detection of antibodies against such modifications. It is also interesting to note that peptide products, although often much smaller and less complex than the other products of biotechnology, still require an assessment of immunogenicity.
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Immunogenicity testing in comparability assessments Following substantial changes in manufacturing, immunogenicity testing is an important aspect of product comparability assessments and has received even more attention with the potential advent of ‘follow-on’ or ‘biosimilar’ biologics. Table 4 provides relevant quotes from European and international documents on product comparability [32,33]. It is interesting to note that these recent guidelines provide some of the most detailed descriptions of what is necessary for the conduct of an immunogenicity study, regardless of whether or not the results are to be used for product comparability. It is necessary to conduct clinical studies if physicochemical tests alone cannot predict whether the molecule has changed in a way that might result in altered immunogenicity or if the potential adverse events for that particular product might be serious in nature. It might be necessary to carry out immunogenicity studies in each clinical indication because immune responses can be generated differently depending on the disease and the seriousness of the clinical consequences of any response. European guidance emphasizes that immunogenicity assessments conducted during clinical studies might not be adequate to assess the rate and consequences of immune responses, particularly rare adverse events; thus, a post-marketing risk management and pharmacovigilance plan must be considered. Such studies can require considerable numbers of patients if the occurrence of an adverse event is rare. Immunogenicity, assay development and validation Although regulatory agencies have not released regulatory documents specifically related to immunogenicity
Table 4. Immunogenicity assessments relative to comparability testing Implication Comparability testing is required through clinical as opposed to non-clinical studies when structural testing might not assure differences in immunogenicity and it is particularly important to assess in repeat-dose products.
Refs [32]
Relevant quote(s) † ‘Immunogenicity must always be addressed by clinical data, unless clinically relevant immunogenicity can be excluded by other means.’ † ‘Immunological studies are expected if physico-chemical characterisation is not sufficient due to the complexity of the molecule and an impact on immunogenicity cannot be excluded with reasonable certainty.’ † ‘The issue of immunogenicity must always be considered when a claim of comparability is made, especially when repeated administration is proposed.’
The extent of immunogenicity studies depends on the nature of the product, the patient population and the possible consequences.
[33]
† [Regarding factors to consider in planning non-clinical and clinical studies] ‘The relationship between the therapeutic protein and endogenous proteins and the consequences for immunogenicity.’ † ‘The impact of possible differences can vary between patient groups, e.g. the risk of unwanted immunogenicity. It may be appropriate to consider the consequences separately for each indication.’
A post-marketing pharmacovigilance plan might be required, depending on the product.
[32]
† ‘In view of the unpredictability of the onset and incidence of immunogenicity, post-marketing monitoring of antibodies at predetermined intervals will be required for at least one year for a new biotechnological product and may be required after a change to an existing product.’
Assays for immunogenicity studies should include validated screening and neutralizing-ADA assays.
[32]
† The assessment of immunogenicity requires validated antibody assays, characterisation of the observed immune response, as well as evaluation of the correlation between antibodies, pharmacokinetics/pharmacodynamics and efficacy and safety.’ † ‘The screening assays should be sensitive enough to detect low titre antibodies as well as antibodies to conformational and linear epitopes. An assay for neutralizing antibodies should be available for further characterisation of antibodies detected by the screening assays.’
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assays, agencies and industry have extensive understanding of the requirements of assays that test for antibody production from vaccines. Although the intent of vaccines is to induce antibodies, the issues relating to an assay detecting either unwanted or desired antibodies have considerable overlap (e.g. the need for appropriate controls, reproducibility and robustness). Good Laboratory Practice guidelines cover the principles for assay development and implementation [34,35] and have some applicability to immunogenicity assays. Some specific statements pertaining to assays have been provided by the FDA and the EMEA [28,32]. Industry and FDA representatives of the American Association for Pharmaceutical Science (AAPS) are currently preparing white papers on the topics of neutralizing antibody assays and immunoassay validation. One such paper, describing the key considerations for immunoassay development and implementation, has already been published [36]. Immunogenicity assessment generally uses an algorithm of immunoassay-based testing. This typically includes a screening assay for detecting potentially ADA-positive samples, which requires sensitive assays that detect as many of the induced antibodies as possible (e.g. low-affinity or low-abundance and different isotypes). Once identified, a specificity assay confirms whether the samples are true- or false-positives, followed by an assay to obtain a relative measure of the antibody concentration in serum, such as titre [36–38]. Following confirmation, ADA-positive samples are often characterized by antibody isotyping and the determination of their neutralizing activity. Method validation refers to the determination of assay reliability and provides criteria for maintaining reliable long-term performance. It establishes the limitations and overall performance expectations of a method with the goal of demonstrating suitability for its intended analytical application [39,40]. Regulatory guidance exists on the fundamental criteria for general assay validation, including the evaluations of specificity, selectivity, range of linearity, detection and/or quantification limits, robustness and system suitability [39,41], in addition to testing the established assay for its accuracy and precision against pre-determined acceptance criteria. Conclusions The safety and efficacy of biologics in patients can be altered, significantly, by the development of immune responses. Manufacturers can address these concerns by including appropriately designed immunogenicity studies during product development and beyond. The need for immunogenicity testing is clear, but consolidated regulatory documents on precisely how to execute such assessments are not available. The development of such guidance requires the partnership between regulatory agencies and industry to ensure that method development, non-clinical testing and the conduct of clinical studies is appropriately planned and leads to insightful and scientifically valid immunogenicity assessments. Acknowledgements The authors thank the following individuals for their review and feedback on this manuscript: Keith Weber and Steven Kozlowski of the US Food www.sciencedirect.com
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and Drug Administration (FDA); Glenn Begley of Amgen; and Mary Whitman and Patti Shirey of Centocor. ICH and European guidelines are available from the European Medicines Agency website (http://www. emea.eu.int). FDA guidelines are available on the FDA website (http:// www.fda.gov/cder).
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20 US Department of Health and Human Services Food and Drug Administration (2002) Draft Guidance for Industry and Reviewers on Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers (http://www.fda.gov/OHRMS/DOCKETS/ 98fr/03-906.htm) 21 US Department of Health and Human Services Food and Drug Administration (2004) Guidance for Industry Developing Medical Imaging Drugs and Biological Products. Part 1: Conducting Safety Assessments (http://www.fda.gov/cber/gdlns/medimagesaf.pdf) 22 US Department of Health and Human Services Food and Drug Administration (2002) Guidance for Industry Immunotoxicology Evaluation of Investigational New Drugs (http://www.fda.gov/cder/ guidance/4945fnl.PDF) 23 US Department of Health and Human Services Food and Drug Administration (2000) Draft Guidance for Industry Chronic Cutaneous Ulcer and Burn Wounds – Developing Products for Treatments (http://www.fda.gov/cder/guidance/3226dft.pdf) 24 Center for Drug Evaluation and Research (2004) Manual of Policies and Procedures (MAPP) (http://www.fda.gov/cder/mapp.htm) 25 US Department of Health and Human Services Food and Drug Administration (1999) Guidance for Industry Clinical Development Programs for Drugs, Devices, and Biological Products for the Treatment of Rheumatoid Arthritis (RA) (http://www.fda.gov/cber/ gdlns/rheumcln.pdf) 26 US Department of Health and Human Services Food and Drug Administration (2004) Guidance for Industry Premarketing Risk Assessment (http://www.fda.gov/cder/guidance/6357fnl.pdf) 27 The European Agency for the Evaluation of Medicinal Products (EMEA) (2003) The Common Technical Document for the Registration of Pharmaceuticals for Human Use. Efficacy – ICH M4E. Clinical Overview and Clinical Summary of Module 2. Module 5: Clinical Study Reports (http://www.tga.gov.au/docs/pdf/euguide/ich/ctdm2efficacy.pdf) 28 US Department of Health and Human Services Food and Drug Administration (1997) Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (http://www. fda.gov/cber/gdlns/ptc_mab.pdf) 29 US Department of Health and Human Services Food and Drug Administration (2002) Draft Guidance for Industry Drugs, Biologics, and Medical Devices Derived from Bioengineered Plants for Use in Humans and Animals (http://www.fda.gov/cber/gdlns/bioplant.pdf) 30 Center for Drug Evaluation and Research Center for Biologics Evaluation and Research (1994) Guidance for Industry for the Submission of Chemistry, Manufacturing, and Controls Information for Synthetic Peptide Substances (http://www.fda.gov/cber/gdlns/ cmcsyn.pdf)
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31 US Department of Health and Human Services Food and Drug Administration (1999) Draft Guidance for Industry for Platelet Testing and Evaluation of Platelet Substitute Products (http://www.fda.gov/ cber/gdlns/platelet.pdf) 32 The European Agency for the Evaluation of Medicinal Products (EMEA) (2003) Guideline on Comparability of Medicinal Products Containing Biotechnology-derived Proteins as Active Substance: Nonclinical and Clinical Issues EMEA/CPMP/3097/02/Final (http:// www.emea.eu.int/pdfs/human/ewp/309702en.pdf) 33 International conference on harmonization (ICH) of technical requirements for registration of pharmaceuticals for human use (2004) ICH Harmonized Tripartite Guideline Q5E Comparability of Biotechnological/Biological Products Subject to Changes in their Manufacturing Process (CPMP/ICH/5721/03) (http://www.emea.eu.int/pdfs/human/ ich/572103en.pdf) 34 Code of Federal Regulations 21 CFR Part 58: Good Laboratory Practice for Non-clinical Laboratory Studies, US Government Printing Office, Washington US (http://www.access.gpo.gov/nara/cfr/ waisidx_00/21cfr58_00.html) 35 Organisation for Economic Co-operation and Development (1997) The OECD Principles on Good Laboratory Practice ENV/MC/CHEM (98)17 (http://www.olis.oecd.org/olis/1998doc.nsf/87fae4004d4fa67ac125685d005300b3/ 3d81d76159dd69e78025675c00409638?OpenDocument) 36 Mire-Sluis, A.R. et al. (2004) Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J. Immunol. Methods 289, 1–16 37 Wadhwa, M. et al. (2003) Strategies for detection, measurement, and characterization of unwanted antibodies induced by therapeutic biologicals. J. Immunol. Methods 278, 1–17 38 Geng, D. et al. (2005) Validation of immunoassays used to assess immunogenicity to therapeutic monoclonal antibodies. J. Pharm. Biomed. Anal. 39, 364–375 39 US Department of Health and Human Services, Food and Drug Administration (CDER and CVM) (2001) Guidance For Industry Bioanalytical Method Validation (http://www.fda.gov/cder/guidance/ 4252fnl.pdf) 40 International conference on harminization (ICH) of technical requirements for registration of pharmaceuticals for human use (1995) ICH Harmonized Tripartite Guideline – Q2A, Text on validation of analytical procedures (http://www.fda.gov/cder/guidance/ichq2a.pdf) 41 International conference on harminization (ICH) of technical requirements for registration of pharmaceuticals for human use (1996) ICH Harmonized Tripartite Guideline – Q2B. Validation of analytical procedures: methodology (http://www.fda.gov/cder/guidance/1320fnl. pdf)
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