Ralf D. Hess

Immunogenicity can either be wanted or unwanted. Since the first biopharmaceuticals were placed on the market more than 20 years ago, unwanted immunological reactions have been observed in man and, thus, postmarket surveillance contributed to coining this expression. In this context, immunogenicity is the 'unwanted' immune response (IR) that can lead to clinically adverse consequences.

  It can analytically be shown by the detection of neutralizing antibodies (NAbs) and non-neutralizing antibodies (NNAbs) to therapeutic proteins. On the other hand, immunogenicity is also the 'wanted' IR directed against proteins intended for the induction of a protective immunity, such as vaccines.

There are multiple causes for unwanted immunogenicity including: 					

a patient's characteristics and/or genetic background.

The 'wanted' immunogenicity requires computational analysis of protein sequences giving rise to potential B-cell and T-cell epitopes, which are the prerequisites for the induction of long-lasting protective immunity, the major goal of designing vaccines.

This article briefly describes the basic principles of immunity and the nature of antibodies, the different concepts of biopharmaceuticals, regulatory considerations on immunogenicity issues, the impact of the different

 diagnostic methods used for detection of NAbs and NNAbs, and the information obtained through postmarketing surveillance.

Immunogenicity assessment of biopharmaceuticals is a master of disciplines because it requires knowledge of protein/antibody structures and developmental design, as well as knowledge of the immune system paired with understanding of technological and analytical procedures, methods and techniques and their implementation in monitoring clinical and preclinical studies.

Testing for immunogenicity requires the analysis of NAbs and NNAbs to therapeutic proteins and potential cross-reactivity to endogenous protein (when applicable). Major factors causing immunogenicity are: treatment with proteins of nonhuman origin, the presence of impurities, the formation of aggregates, the immune status of the individual patients in addition to protein/antibody formats, post-translational modifications, degradation of products, storage conditions and dose, route, frequency, and duration of therapeutic protein administration.

The immune system can be divided into the innate and adaptive IR. The innate IR is the 

 IR and consists of nonspecific defence mechanisms (e.g., inflammatory response). The induction of toll-like-receptor-mediated signalling, and production of reactive oxygen intermediates through stimulation of macrophages and monocytes in combination with the production of antiviral polypeptides, such as interferons, provide protection within minutes to hours after invasion of pathogens. The adaptive IR takes days to build up pathogen-specific immunocompetent B-and T-cells. B-cells are the major constituents of the humoral response and T-cells are the major components of the cellular immune response. B-and T-cells cooperate through lymphokines, cytokines and through direct interaction, and guide the IR either towards B-cell response (humoral IR) or towards T-cell response (cellular IR), which is dependent on the nature of the antigen, its presentation on antigen-presenting cells and the relative amount of 'polarizing' cytokines.

 The initial ability of an antigen to bind to a complementary receptor on a naive B-cell or to be recognized by a specific T-cell as a peptide presented in the context of a major histocompatibility complex molecule by an antigen-presenting cell (i.e., dendritic cells, macrophage or B-cell) is dependent on the individual's B- or T-cell repertoire, respectively.

  After activation of adaptive immunity, which includes clonal expansion and maturation of antigen-specific effector B-and T-cells, the binding strength will enhance with time because of somatic hypermutations in the encoded variable region genes, resulting in antibodies or T-cell receptors with increased affinity. The IR itself is controlled by mechanisms such as production of anti-idiotype antibodies, regulatory T-cell function, clonal selection processes, and tolerogenic and anergic T-cells, resulting in unresponsiveness, but also in targeted and nontargeted apoptosis.

Biopharmaceuticals comprise therapeutic proteins, antibodies, vaccines, cells and tissues intended for curative and preventive therapeutic use. The development and market of recombinant biopharmaceuticals, such as recombinant therapeutic proteins (e.g., interferon (IFN)-a 2a) or recombinant humanized antibodies (e.g., Humira, anti-TNF-a) has expanded during the past 20 years.

  Recombinant biopharmaceuticals can be produced by recombinant DNA technology through fermentation processes in either yeast, 

 (either by 'phage display' or transformation methods), baculovirus cells (insect cells, e.g., SF9) or eukaryotic cells. The new generations of therapeutic vaccines are primarily produced in eukaryotic cell lines, such as Vero cells (African Green Monkey Kidney cells), Madin Darby Canine Kidney cells or Chinese Hamster Ovary cells. Recombinant therapeutic proteins may be used to directly treat infectious disease (e.g., IFN-a 2b in the treatment of chronic hepatitis C infection) or as replacement proteins (e.g., for in-born protein deficiencies). Recombinant therapeutic antibodies are being developed to treat a wide range of diseases and may act through a variety of pharmacologic mechanisms. One mechanism of therapeutic antibodies targeting tumour cells is the cross-linking of specific proteins (which could be ligands or receptors) that subsequently result in induced cell signalling.

  Direct anti-tumour specific antibody application for solid tumours may only be successful if tumours provide tumour-specific transplantation antigens.

  The pharmacologic action of Rituximab, anti-CD20 monoclonal antibody, in the treatment of Epstein-Barr Virus-associated Post Transplantation Lymphoproliferative Disease

  or non-Hodgkin's Lymphoma has been determined.

  The Fab domain of Rituximab binds to the CD20 antigen on B-lymphocytes and the Fc domain recruits immune effector functions to mediate B-cell lysis 

. Evaluation of safety, efficacy and pharmacokinetics during premarket development of recombinant biopharmaceuticals must include an assessment of immunogenicity.

Most recombinant therapeutic proteins, including antibodies, are homologous of human proteins. European regulatory requirements for chemistry, manufacturing and controls are set forth in the European Pharmacopoeia on the fermentation process,

  and the new European Pharmacopoeia guideline on control of recombinant protein expression,

  as published in July 2007. Additional basic aspects of the pharmaceutical quality system are given in the new ICH Q10 guideline,

  and nonclinical regulatory guidance is given in Snodin and Ryle.

EMEA recently published an updated guideline, 

Immunogenicity assessment of biotechnology-derived therapeutic proteins

  which is much more thorough and complex than the formerly published, but now obsolete, concept paper dated 22 February 2006.

  The new and updated guideline considers not only B-cell immunity assessing methods and analyses, but also T-cell relevant methods, such as the enzyme-linked immunosorbent spot (ELISPOT) assay and the tetramer technology. While ELISPOT is a versatile tool to specifically and sensitively analyse T-cell-function/interaction, T-cell frequencies and T-cell-derived cytokines, tetramer technology provides a tool for the analysis of T-cell receptor specificity, which may also be used to assess T-cell receptor affinities, as well as frequencies.

NAbs or NNabs to therapeutic proteins may be detected in plasma or serum from previously treated individuals by ELISA methodologies, western blot or immunoprecipitation. Biological assays or 'bioassays' provide evidence for detection of NAbs. Determining NAbs to therapeutic antibodies takes the analysis of anti-idiotype, and also anti-allotype antibodies, by competitive ELISA methods with subsequent characterization in neutralizing assays or bioassays.

Bioassays may be divided into two categories:					

the indirect cell-based assay to measure biologic functions.

The direct bioassay can be used to assess proliferative/inhibitory responses on cell lines to biologics by measuring either DNA replication through incorporation of labelled nucleotides (e.g., 3H-Thymidine or Bromodesoxyuridine) or by staining the cells with dyes indicative for vital and metabolic active cells. Neutralizing activity can be shown by the parallel addition of plasma/sera/antibodies of the questioned function, resulting in inhibition of proliferative effects. Indirect biologic function assays may use reverse transcriptase-polymerase chain reaction (RT-PCR) methodology to measure signalling effects through crosslinking of proteins/antibodies to cell membrane molecules. For example, RT-PCR may be used to measure induced mRNA molecules known to be specific to a stimulatory effect on cells, such as increased 2'-5'-oligoadenylatesynthase m-RNA, after virus infection of cells.

Detection of NNAbs can be conducted with classical ELISA technology. Analysis of antibody affinity or avidity can be performed by ELISA technology, immunofluorescence assay or western blot analysis. Surface plasmon resonance may easily allow discrimination between 'early' (lower affinity) 

 'late' (higher affinity) antibodies. Impurities and protein degradation, which may potentially elicit an immune response, may be detected using HPLC methods, polyacrlymide electrophoresis with or without subsequent western blot analysis, or other more sensitive electrophoretic methods.

Twenty years experience from postmarketing surveillance and vigilance of individuals previously treated with recombinant biopharmaceuticals have provided considerable information pertaining to clinically adverse consequences of and potential mechanisms for immunogenicity. The application of the first therapeutic antibodies of murine origin in man in the early to mid 1980s and the administration of xenogeneic therapeutic proteins, such as porcine or bovine insulin in the treatment of diabetes, paired with still ongoing postmarketing surveillance, provide evidence for immunogenicity. These biopharmaceuticals were for curative/supportive use. Recombinant Hirudin (Refludan), an anticoagulant of nonhuman origin (from the medicinal leech), induced antibodies of the NAb-type in some patients, although no clinical phase study (I–III) provided evidence for this complication. Even using 100% homologous human proteins, immunogenicity-related issues (although minimized) may occur with unpredictable consequences. As an example, the application of 100% human erythropoietin led to the dramatic clinical adverse consequence of pure red cell aplasia in patients with chronic kidney failure because of the adjuvant effect of leachables from the container closure that led to development of antibodies that cross-reacted with endogenous erythropoietin. For immunosuppressed patients or patients suffering from chronic diseases, predicting an individual's immune response is not possible because of the complex presentation of the individual's disease state.

  In addition to this, even among healthy individuals there are differences concerning the individual B-and T-cell repertoires that give rise to the vast heterogeneity of immune response outcomes.

  Therefore, clinical assessment of immunogenicity for each new recombinant biopharmaceutical will continue to be required as part of premarket demonstration of safety and efficacy. 

The authors gratefully acknowledge the contributions by Nancy Kirschbaum, Melanie Kaltenbach, Hannah Jahn and Irmgard Scherer of PAREXEL Consulting in support of this article.

is a Principal Consultant at PAREXEL Consulting (Germany).

is Vice President of PAREXEL Consulting (Germany).

Articles

A discussion of the basic principles of immunity and the nature of antibodies.