REVIEWS

JOURNAL OF INTERFERON & CYTOKINE RESEARCH Volume 34, Number 12, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/jir.2013.0011

Mechanisms Underlying the Immunogenicity of Therapeutic Proteins: Risk Assessment and Management Strategies Susan L. Kirshner

Antibodies to therapeutic proteins have caused serious adverse events and loss of efficacy in patients. Therefore, it is critical to manage the risk of antitherapeutic antibodies (ATA) during drug development and in the postmarketing environment. Risk assessments are an important tool for managing immunogenicity risk because they provide a format for considering the consequences and likelihood of ATA development. Because many factors influence both the severity of the consequences and likelihood of ATA development, successful risk assessments require input from all relevant disciplines, including product quality, manufacturing, nonclinical, pharmacology, and clinical. The results of risk assessments are used to develop appropriate risk reduction strategies, which may include product quality and manufacturing controls and elements of clinical trial design. This article discusses considerations for immunogenicity risk assessments and management.

Introduction

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egulatory agencies and industries broadly agree that it is appropriate to use risk-based approaches to manage unwanted immunogenicity of therapeutic proteins (Rosenberg 2003; Rosenberg and Worobec 2004; Shankar and others 2007; European Medicines Agency CHMP Guideline 2008; Koren and others 2008; Hayakawa and Ishii-Watabe 2011; Kirshner 2011; Koren and others 2011; Tovey and Lallemand 2011; Wadhwa and Thorpe 2011; European Medicines Agency CHMP Guideline 2012; CDER, Draft Guidance 2013). Undesirable clinical outcomes due to unwanted immunogenicity of therapeutic proteins are usually directly caused by the presence of antibodies that bind the therapeutic proteins, also known as antitherapeutic antibodies (ATA) or antidrug antibodies. For that reason, to date, industries and regulatory agencies have focused most of their efforts on managing unwanted immunogenicity by reducing ATA development. ATA cause many unwanted clinical outcomes including: inducing life-threatening deficiency syndromes when ATA cross-react with and inhibit the effect of endogenous proteins (Li and others 2001; Basser and others 2002; Gershon and others 2002; Rossert and others 2004; Fotiou and others 2009; Kuter 2009; Shin and others 2012); reducing or preventing the clinical efficacy of the therapeutic (Ponce and others 1997; Brackmann and others 1999; Hay and others 2000; Brooks and others 2003); changing the pharmacokinetic profile of the therapeutic (Schernthaner and others 1983; Pringle and oth-

ers 1989; Calabresi and others 2007; Krystexxa, U.S. Prescribing information 2010; Lyseng-Williamson 2011; Sundy and others 2011); changing the biodistribution of the therapeutic (Brooks and others 2003); and inducing anaphylaxis, hypersensitivity, and infusion reactions (Schernthaner 1993; Rosenberg and others 1999; Foss and others 2001). Although immunogenicity assessment is expected for all therapeutic proteins, the clinical impact and lack of predictability of immunogenicity of biologics is of such great concern that the Biologics Price Competition and Innovation Act explicitly states the need to assess immunogenicity of biosimilar candidates (Patient Protection and Affordable Care Act 2009).

Discussion Risk management involves the ‘‘identification, evaluation, and control of risks’’ (Cox and Tait 1998). Identification and evaluation of risk are performed during the risk assessment. Immunogenicity risk assessments usually use quasiquantitative risk analyses. To this end, numerical scales are developed to describe the qualitative estimates of risk, such as low to high, mild to severe, and rare to frequent. The development of these scales will not be discussed here. The results of the risk assessment are used to inform risk reduction strategies. A key component to successful risk management is to ensure high-quality information is used during the risk assessment process. For that reason, all appropriate subject matter experts should participate in the risk management process.

Division of Therapeutic Proteins, U.S. Food and Drug Administration, Bethesda, Maryland.

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924 Immunogenicity risk assessments generally have two components: estimating the consequences and the likelihood of ATA development. These estimates are combined and used to characterize and categorize the risk of ATA to subjects. Importantly, in risk assessments, severity of the consequences of ATA to subject health is weighted so that very harmful, although rare, consequences of ATA result in the therapeutic being categorized as high risk. Another important consideration is how to weight uncertainty about the knowledge being used in the assessment. In risk assessments, lack of knowledge or uncertainty about the reliability of knowledge is usually factored as increasing risk, as this is more protective of subjects’ health (Rosenberg and others 2012). Assessing the severity of physiological consequences of ATA development is a critical component of a risk assessment and, in the absence of clinical data, requires synthesizing information from a variety of sources. A critical component of the assessment is to understand the protein’s physiological function(s). Such understanding can be gained from studying human conditions associated with loss of function of the protein, basic scientific research on the physiological function(s) of the protein, and studies in animal models. There is frequently uncertainty about how well we understand the physiological function(s) of a protein, and this should be factored into the risk assessment. Furthermore, the severity of the physiological consequences may differ between the populations. So, severity scores may be population or disease specific. Antibodies can bind to any protein expressing the epitope for which they are specific. Therefore, ATA may bind to endogenous human counterparts of the therapeutic. Potential cross-reactivity to an endogenous protein heightens concerns that ATA could cause a human deficiency syndrome (Li and others 2001; Basser and others 2002; Gershon and others 2002; Rossert and others 2004; Fotiou and others 2009; Kuter 2009; Shin and others 2012). In a severity assessment, the cross-reactivity to endogenous proteins with nonredundant functions, such as some growth and differentiation factors (eg, erythropoietin, megakaryocyte growth, and differentiation factor), where loss of function leads to life-threatening conditions, would be rated as high for consequence severity. In contrast, the cross-reactivity to proteins with redundant functions such as type 1 interferons might be rated as low for consequence severity, although the full spectrum of activities of such proteins may not be understood or characterized (Siconolfi and Seeds 2001). ATA that do not cross-react with endogenous proteins may also be rated as high for severity if loss of efficacy of the therapeutic will have severe consequences to human health. For example, loss of function of enzyme replacement therapies for metabolic disorders can result in disease progression and death (Ponce and others 1997; Brooks and others 2003). Loss of function of clotting factor replacement therapies greatly complicates the clinical management of hemophilia (Brackmann and others 1999; Hay and others 2000). In contrast, loss of efficacy when the condition is not life threatening or there are equally good alternative therapies may be rated as low for consequence severity. Another component of immunogenicity risk assessments is predicting the likelihood of ATA development. This is usually expressed as incidence. Multiple factors impact the likelihood of ATA development. These include the protein’s

KIRSHNER species of origin; product attributes including those pertaining to container closure and cold chain considerations; patient attributes; and trial design attributes, and will be discussed briefly below. When evaluating the contribution of species of origin to the incidence of ATA development, products derived from nonhuman species are predicted to elicit a high incidence of ATA because the human immune system is not tolerized to nonhuman proteins. These products are given high species of origin risk scores for ATA incidence. Consistent with this, there is a high incidence of ATA development to most therapeutic proteins of bacterial origin, sometimes after a single therapeutic exposure (Xiaflex, U.S. Prescribing Information 2010; Voraxase, U.S. Prescribing Information 2012). Products of human origin with native human sequences are given low species of origin risk scores, as there is likely to be some level of immune tolerance to these proteins. It is generally harder to break tolerance to proteins with low serum abundance than high serum abundance (Weigle 1981). Therefore, serum abundance should be a factor in risk assessments. Proteins with altered native human sequences or nonhuman proteins that have high homology to human proteins are given intermediate species of origin risk scores, as sequence variation can introduce new epitopes the immune system is not tolerized to. When assessing the contribution of product attributes to the likelihood of ATA development, attributes that should be considered include, but are not limited to, molecular structure, purity, product mechanism of action, formulation, and container closure. There are many aspects of molecular structure that can impact product immunogenicity. For example, it has long been known that aggregated proteins are frequently more immunogenic than their nonaggregated counterparts (Ellis and Henney 1969; Moore and Leppert 1980; Rosenberg 2006; Filipe and others 2010; Weinberg and others 2010). Multiple mechanisms by which aggregated proteins may increase immunogenicity have been described (Rosenberg 2006; Filipe and others 2010; Weinberg and others 2010), but will not be reviewed here. Nevertheless, there is still much we do not know about the role of aggregates in eliciting unwanted immunogenicity. For example, we do not know how aggregate content and size impact immunogenicity. Because of uncertainty, the risk score for aggregates generally increases with aggregate content and size (Rosenberg and others 2012). Another aspect of molecular structure that can impact immunogenicity is glycosylation. Nonnative glycosylation patterns, nonhuman sugars, and nonhuman linkages can all increase the risk of ATA development. Many therapeutic proteins are made in cell lines that introduce nonhuman glycoforms to the protein. N-linked glycans from lower eukaryotes such as yeast are higher in mannose content and have simpler structures than many human glycans. In an animal model of systemic lupus erythematosus, simple N-glycans were found to elicit innate immune responses that resulted in tissue damage (Green and others 2007; Van Dyken and Locksley 2007). Similarly, plant carbohydrates incorporate sugars such as xylose that are not found in human carbohydrates. They also have carbohydrate linkages that are not found in humans. In one study evaluating antibodies against plant sugars, 50% of healthy nonallergic volunteers had antibodies to the core xylose and 25% had antibodies to core alfa(1,3)-fucose (Bardor and others 2003). Thus, using

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plant cell substrates may increase the risk of ATA development or boost responses in subjects who have pre-existing antiplant sugar antibodies. To date, only one product manufactured using a plant cell substrate, Elelyso (taliglucerase alfa), is approved in the United States. Elelyso is a glucocerebrosidase and is used as a replacement therapy in patients with type 1 Gaucher disease, an inborn error of metabolism. Approximately 53% of treated patients developed antibodies to Elelyso (Elelyso, U.S. Prescribing Information 2012). It is not clear whether the antibodies were directed to the sugar moieties, protein moiety, or both. Although direct comparison of immunogenicity rates between products can be misleading because of differences in assay sensitivity and specificity, two other replacement therapies for type 1 Gaucher’s disease, both made in mammalian cell cultures, had immunogenicity incidences of *2%–15% (Cerezyme, U.S. Prescribing Information 2003; VPRIV, U.S. Prescribing Information 2010). This suggests that the presence of plant sugars may indeed have increased the incidence of anti-Elelyso antibodies. Anti-Elelyso antibodies did not impact efficacy during the course of the clinical trial. As discussed above, nonnative sequences are considered higher risk for inducing immunogenicity. Nonnative sequences may be generated by amino acid substitutions, use of modified amino acids, or fusing proteins (Hayakawa and Ishii-Watabe 2011). It was once thought that PEGylation of proteins reduced their immunogenicity. Although this may be true for some proteins, there are now many examples of ATA that bind the PEG moiety of the therapeutic (Armstrong and others 2007; Garay and others 2012). Anti-PEG antibodies can be detected in up to 25% of the general population, depending on the study (Garay and others 2012). This is likely because humans are frequently exposed to PEG through a variety of sources, such as pharmaceuticals, cosmetics, and processed foods (Garay and others 2012). The presence of anti-PEG antibodies in Krystexxa (pegloticase)-treated patients was associated with loss of efficacy and infusion-related reactions (Krystexxa, U.S. Prescribing Information 2010). Therefore, PEGylation should not be considered necessarily reducing the risk of ATA formation, and the development of anti-PEG antibodies should be monitored during clinical trials. Process-related impurities may contribute to the risk of ATA development. Host cell proteins and DNA, particularly bacterial DNA rich in unmethylated CpGs, can increase a product’s immunogenicity. These impurities are known to trigger innate immune responses and so may act as adjuvants to increase the immune response of the co-delivered therapeutic protein. Verthelyi and Wang (2010) reported that trace levels of two impurities, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODN), synergized to increase anti-ovalbumin antibodies in an animal model. In another animal model, they found that administering trace levels of LPS and CpG ODN along with erythropoietin resulted in long-lasting anemia in animals. In the absence of these trace impurities, only transient anemia was observed (Verthelyi and Wang 2010). With modern manufacturing processes, host cell impurities are generally very low in marketed products. However, during development, particularly early development, we have found that host cell impurity levels can be high enough to increase product immunogenicity. They may also trigger anaphylaxis and other hypersensitivity responses. This is usually remedied by better purification

of the product. So the contribution of product purity to the risk of ATA induction may change during development. Product-related impurities may contribute to the risk of ATA development. For example, we have seen that oxidation of cysteines in products can lead to the formation of covalent intermolecular disulfide bonds. These aggregates may increase product immunogenicity. Denatured product is also prone to aggregation, which may increase immunogenicity (Rosenberg and others 2012). Denaturation and aggregation may also expose cryptic epitopes that are usually unavailable to antibodies, thereby increasing product immunogenicity. For example, antibodies to intravenous immunoglobulin (IVIG) were specific to a cryptic epitope that was only exposed in aggregated IVIG (Barandun and others 1962; Ellis and Henney 1969). Similarly, clipping and post-translational modification may alter tertiary structure and reveal cryptic epitopes. In risk assessments, increased levels of productrelated impurities are scored as increasing the risk of ATA development unless there are data showing this to be untrue for that specific protein. Product-related impurities may increase over time. Therefore, the risk of developing ATA may change with product age. Forced degradation, stress temperature, and accelerated temperature stability studies can be used to elucidate the degradation pathways of a protein to help inform the risk assessment. The risk that aged product will be more immunogenic may be reduced by appropriate formulation development, container closure selection, storage conditions, and handling conditions. Container closures can contribute impurities to the product that may impact immunogenicity because they can modify the product or nucleate aggregation (Markovic 2007; Sharma 2007). Impurities from container closures include leachates from glass containers and stoppers (Markovic 2007), including tungsten oxides ( Jiang and others 2009; Liu and others 2010), silicone oil droplets (Thirumangalathu and others 2009), glass chips (Chi and others 2005), and metal shavings (Tyagi and others 2009). These should be considered in a risk assessment. The product’s mechanism of action may also be a risk factor. Products that stimulate immune responses may be more immunogenic than products that suppress immune responses. However, the incidence of ATA to the immunosuppressive monoclonal antibody Tysabri, although not very high, is *9%. Anti-tysabri antibodies can lead to changes in pharmacokinetics, increased incidence of infusion-related reactions, and loss of efficacy (Calabresi and others 2007; Tysabri, U.S. Prescribing Information 2012). Therefore, the weight given to an immunosuppressive mechanism of action in a risk assessment should be carefully considered. Neutralizing anti-tysabri antibodies are reported to have led to the death of a 32-year-old multiple sclerosis patient in Sweden (Svenningsson and others 2013). The patient had high titers of IgG3, which fixes complement. The authors propose that anti-tysabri antibody–lymphocyte complexes led to break down of the blood–brain barrier, resulting in neuroinflammation and disease progression. This case seems distinct from other reports of neuroinflammatory rebound or immune reconstitution inflammatory syndrome (IRIS) in Tysabri-treated patients as this patient had not stopped Tysabri treatment when the neuroinflammation developed (Lenhard and others 2010; Gheuens and others 2012; Svenningsson and others 2013). Anti-tysabri antibody status was

926 not reported by Lenhard and others or Gheuens and others, so it is not known whether there is a role for anti-tysabri antibodies in patients who developed IRIS. Patient attributes are an important component of an immunogenicity risk assessment. Patient attributes include genetics, disease state, immune competency, age, gender, and the presence of pre-existing antibodies. Genetics may impact the likelihood of ATA development in two different ways. Many of the proteins that regulate the immune system are polymorphic. The presence or absence of certain polymorphisms can impact the risk of developing ATA. For example, human leukocyte antigen (HLA) DRB1*0701 is associated with the development of antibodies to interferon beta (Barbosa and others 2006). ATA to Humira (adalimumab) were over twice as prevalent in Japan (40.2%) compared to the Western countries (17.9%) (Hayakawa and Ishii-Watabe 2011). Unfortunately, information on which polymorphisms are associated with increased or decreased risk for developing an immune response is usually not available, particularly early in development. These observations provide a caution against extrapolating immunogenicity risk between the groups with known genetic differences. It may be easier to assess the genetic contribution to the risk of ATA development for products designed to replace deficits in endogenous proteins. It has been observed that patients with more severe genetic lesions that lead to loss of protein expression or large deletions in expressed proteins are more likely to develop ATA (Fakharzadeh and Kazazian 2000). However, missense mutations that result in expressed but inactive or less active proteins have reduced risk for ATA development, or at least persistent ATA development (Kishnani and others 2010). The disease state should also be considered in a risk assessment. Immune-compromised patients are less likely than immune-competent subjects to develop ATA (Li and others 2001; Basser and others 2002; Kuter 2009). Similarly, ATA development may be reduced in patients taking immunosuppressive medications (Maini and others 1998; Pozzilli and others 2002). Patients with autoimmune or chronic inflammatory conditions may be more likely than immunecompromised or immune-competent subjects to develop ATA. There is evidence that delivering proteins in a surgical setting or in the context of a solid matrix, such as a bone filler, may increase the risk of developing ATA. Comorbid conditions may also impact patient’s immune competency. These factors should be considered when assessing the risk of ATA development. Separate risk assessments should be performed for each indication for which the product is being developed. The immune system is known to change with age and be influenced by sex. Changes in hormonal status, such as those women undergo during pregnancy, also impact the immune system. Although we do not have enough knowledge to assess the risk of these factors on ATA development, we do know that risk should not be extrapolated between the groups. Therefore, the results showing that a product was not immunogenic in one group, for example, adults, should not be extrapolated to another group, for example, children. Pre-existing antibodies that cross-react with the therapeutic may be present in some or all subjects. As noted, there is a relatively high incidence of anti-PEG antibodies and antiplant sugar antibodies in the general population (Bardor and others 2003; Garay and others 2012). It was found that

KIRSHNER pre-existing IgE antibodies to the oligosaccharide, galactose-a1,3-galactose, present on the Erbitux (cetuxemab) heavy chain resulted in a higher incidence of anaphylactic reactions to Erbitux in specific regions of the United States (Chung and others 2008). For patients with pre-existing ATA, the relevant question is not whether they will develop ATA but whether the titer of these antibodies will increase after patients have been exposed to the therapeutic. Although we have observed that pre-existing ATA are not always boosted by exposure to the therapeutic, it nevertheless seems prudent to consider preexisting ATA as increasing risk for boosting ATA to ensure appropriate monitoring during the clinical trial is implemented. Trial design attributes may contribute to the risk of ATA development. These attributes include route of delivery, posology, and concomitant medications. It is widely accepted that some routes of administration have more immunogenicity risk than others. Ranking from highest to lowest risk, the routes of administration are subcutaneous (s.c.) ‡ inhalation > intramuscular (i.m.) > intravenous (i.v.) > oral. We have observed a number of examples where there was no difference in the immunogenicity of s.c. and i.v. routes of administration. Nevertheless, unless there are data to support that this is true for a specific protein, the risk of a proposed route of administration should be ranked in the order listed above. The relationship between the dose and risk of developing ATA is very complicated. Both high and low doses of proteins can induce immune tolerance. However, it is unclear what the immune system considers a high or low dose for any given protein therapeutic. Inducing tolerance in humans to an exogenously delivered protein requires controlled conditions and frequently, concomitant immune suppressive medications (Brady and others 1997; Brackmann and others 1999; Rosenberg and others 1999; Hay and others 2000). We have observed both high and low rates of immunogenicity with proteins given in microgram and milligram quantities. This suggests that other factors, such as species of origin of the protein, subject genetics, immune status, concomitant medications, number and frequency of doses, and various product quality attributes discussed above, contribute more than dose to the likelihood that ATA will develop. This should be considered when weighting the risk score for dose. Frequency of administration may also contribute to the risk of ATA development. Generally, a single administration is considered less likely than multiple administrations to elicit ATA. However, single administrations of bacterial proteins or proteins with very long half-lives such as Fc fusion proteins or PEGylated proteins have been found to elicit immune responses. OP-1 (human bone morphogenic protein-7) delivered in a collagen matrix for spinal fusion (OP-1 Putty, U.S. Prescribing Information, 2012) elicited ATA in 92% of treated patients. Indeed, OP-1 ATA were detected up to 2 years after treatment in some patients (OP-1 Putty). We have observed incidences of 15%–20% with other recombinant human bone growth and differentiation factors given with solid matrixes in surgical settings, although the proteins were not covalently linked to the matrix. This suggests that the risk attributed to a single exposure of a therapeutic should be weighted such that factors like duration of exposure, route of exposure, inflammatory setting, and product attributes such as aggregation, and foreign origin would supersede a low-risk score given because a patient receives only a single administration of the therapeutic.

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Another source of uncertainty in an immunogenicity risk assessment is how reliably ATA will be detected. Because the relationship between ATA and safety and efficacy cannot be established unless ATA can reliably be measured, reduced ability to detect ATA should be scored as increasing risk to the patient. This will allow for the implementation of appropriate monitoring and mitigation strategies, such as obtaining samples after a washout period. ATA detection may be unreliable if assays are insensitive. Therefore, it is critical to develop sensitive, specific, and precise ATA detection assays (Mire-Sluis and others 2004; Gupta and others 2007; European Medicines Agency CHMP Guideline 2008; Shankar and others 2008; CDER, Draft Guidance 2009; Gupta and others 2011; Hamilton and others 2011; Smith and others 2011; European Medicines Agency CHMP Guideline 2012). ATA detection may also be reduced by interference from free therapeutic in the serum. Therefore, ATA sampling should occur at trough therapeutic levels, and analytical strategies to overcome interference should be used. In silico models are being developed to predict the immunogenicity of proteins (De Groot and Scott 2007; Bryson and others 2010). Most models focus on predicting T-cell epitopes within the protein. T-cell epitopes are linear and constrained by major histocompatibility complex/HLA molecules, which provides physical parameters that can be used for modeling. T-cell epitopes predicted by the in silico models can be confirmed by testing their ability to activate T cells or peripheral blood lymphocytes using in vitro assays. Models to predict B-cell epitopes are also available. However, because B cells are conformational and can be derived from noncontiguous sequences in the protein, it has been difficult to develop accurate models. Many polymorphic genes are involved in the regulation of immune responses. There is a large gap in our understanding of how these genes interact to promote or reduce unwanted immunogenicity, which complicates predictive model development. In addition, the relationship between the presence of a particular T-cell epitope and unwanted ATA development is unknown. However, even if we could accurately predict the incidence and frequency of ATA development, the clinical consequences of ATA may still not be predicted. Therefore, while predictive immunogenicity models may increase the accuracy of incidence assessments and can help inform risk reduction activities for clinical trials, they cannot replace clinical immunogenicity programs or be used to reduce testing at this time. The purpose of immunogenicity risk assessments is to inform risk management programs. Immunogenicity risk management includes appropriate control of product quality and risk reduction through clinical trial design (Kirshner 2011; Rosenberg and others 2012). To control risk from product quality attributes, it is important to elucidate those attributes that contribute to increase the therapeutics immunogenicity potential (Rosenberg and others 2012). Such knowledge allows for those attributes to be monitored by testing and controlled by the manufacturing process or specifications. It also allows for the development of suitable formulations and selection of appropriate container closures, storage conditions, and handling procedures. Risk can also be reduced by clinical trail design. For lowrisk products, the clinical program mostly will focus on monitoring. Samples for ATA testing should be obtained at appropriate time points and at trough therapeutic concen-

trations. If a product is given once, then samples should be obtained at baseline, one sample between day 7 and 14 to detect early immune responses, particularly IgM responses, and one sample between day 21 and 28 for IgG responses. If a product is given for a longer period of time, then ATA samples should be obtained every 3 months during the first year and every 3–6 months thereafter. A sample should be obtained at the last visit as well. Samples should be obtained for the determination of serum levels of the therapeutic at ATA sampling time points, to allow for evaluating the impact of ATA on pharmacokinetics. Furthermore, changes to trough therapeutic concentrations may signal the presence of ATA in samples where ATA are hard to detect. Any subject who develops ATA or has boosted ATA during the course of the clinical trial should be monitored till they revert to baseline ATA levels. This monitoring is especially important if the product has an endogenous counterpart to ensure that autoimmunity has not developed. All samples should be obtained even for low-risk products because of the potential for errors in the risk assessment. Subjects can be monitored for clinical signs of ATA development. If the product has an endogenous counterpart that mediates a unique function (eg, erythropoietin and production of red blood cells), patients should be monitored for predicted symptoms of a deficiency syndrome. When good pharmacodynamic markers exist, then patients can be monitored for changes in that marker. Sometimes loss of efficacy can also be monitored during the clinical trial. Risk reduction strategies should be implemented for products with intermediate and high immunogenicity risk scores. Some frequently encountered risk reduction strategies include selecting posology, route of administration, target population, and clinical setting to minimize risk. Performing single-dose first in human studies through the least immunogenic route of administration being considered is the most commonly used risk reduction strategy in early clinical development. Risk may also be reduced by subject selection for phase 1 and 2 studies. For example, if the target population is immune suppressed, it may be prudent to perform first in human studies in that population rather than healthy subjects. If subjects are at risk for anaphylaxis or infusion reactions, then the therapeutic should be administered in a clinical setting where adverse events can be treated immediately. Longer observation of patients after dosing may also be warranted as anaphylactic or severe hypersensitivity reactions may not develop immediately. When there is known risk of severe consequences such as developing a deficiency syndrome, sponsors may establish stopping rules based on the detection of ATA. For example, erythropoietin treatment may be stopped in a subject who tests positive for neutralizing anti-erythropoietin ATA to prevent the development of pure red cell aplasia. Critically, risk reduction strategies should be tailored to address the specific issues elucidated by the risk assessment.

Summary All therapeutic proteins have risk for eliciting an immune response in treated subjects. Antitherapeutic immune responses have caused life-threatening deficiency syndromes in some subjects. In other subjects, they have resulted in loss of efficacy of therapeutics that are life-saving or substantially improve patients’ quality of life. Therefore, it is critical to

928 establish immunogenicity risk management programs that include control and monitoring of product quality and aspect clinical trial design. Risk assessments are an important tool for understanding the factors that increase the risk for developing anti-therapeutic immune responses. The results of immunogenicity risk assessments should be used to reduce risk where possible.

Acknowledgment Many thanks to Dr. Amy Rosenberg for her helpful critical reading of this article.

Author Disclosure Statement No competing financial interests exist.

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Address correspondence to: Dr. Susan L. Kirshner Division of Therapeutic Proteins U.S. Food and Drug Administration 29 Lincoln Drive Bldg 29A, Room 2D, 16 HFD-120 Bethesda, MD 20892 E-mail: [email protected] Received 1 February 2013/Accepted 30 August 2013