Strategic characterization of anti-drug antibody responses for the assessment of clinical relevance and impact.

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Strategic characterization of anti-drug antibody responses for the assessment of clinical relevance and impact

All therapeutic proteins have the potential to induce anti-drug antibodies (ADA). Clinically relevant ADA can impact efficacy and/or safety of a biological therapeutic. Immunogenicity assessment strategy evaluates binding and neutralizing ADA, and the need for additional characterization (e.g., epitope, titer and so on) is determined using a risk-based approach. The choice of characterization assays depends on the type, application and immunogenicity of the therapeutic. ADA characterization can impact the interpretation of the risk profile of a given therapeutic, and offers insight into opportunities for risk mitigation and management. This article describes common ADA characterization methods. Strategic assessment and characterization of clinically relevant ADA are discussed, in order to support clinical options for safe and effective patient care and disease management.

Therapeutic proteins can provide uniquely specific, sensitive and effective treatments for many grievous illnesses. For most people, the use of therapeutic proteins is well tolerated because they are usually similar to ‘self’. However, the normal human immune system has a potent ability to detect and defend ‘self’ from ‘non-self’, and if an immune response develops against a therapeutic protein it can present a significant challenge to its use. The immune response is measured by the development of anti-drug antibodies (ADA). ADA development is dependent on the interaction of numerous factors directly related to the patient (e.g., human leukocyte antigen type, immune competence, disease, concomitant medications and so on), the treatment (e.g., dose, frequency, route of administration and so on) and product (e.g., primary sequence, T-cell epitopes, structural features including glycosylation and aggregation, formulation and so on) [1,2] . Development of ADA after administration of a human protein therapeutic can be seen as a break in tolerance, and has the potential to impact the pharmacokinetics (PK), pharmacodynamics, efficacy and/or safety of the therapeutic. For this reason, immunogenicity assessment of

10.4155/BIO.14.114 © 2014 Future Science Ltd

Suzanna M Tatarewicz*,1, Daniel T Mytych1, Marta Starcevic Manning1, Steven J Swanson1, Michael S Moxness1 & Narendra Chirmule1 Clinical Immunology, Amgen, Thousand Oaks, CA 91320, USA *Author for correspondence: Tel.: +1 805 447 1680 Fax: +1 805 480 1306 [email protected]

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therapeutic proteins remains a required aspect of regulatory filings for a licensing application [3,4] . This requirement is fulfilled by determining ADA binding and neutralizing incidence. A systematic assay validation strategy and statistical criteria for measuring ADA have been standardized through collaborative efforts [5,6] . Additional characterization follows a risk-based approach that considers clinical consequences (e.g., neutralization of endogenous counterpart, anaphylaxis, delayed hypersensitivity, cytokine release and so on), as well as evaluation of patient and product risk factors [4,7,8] . If a robust immune response occurs, characterization of the timing, strength, type and specificity of the response provides a means to differentiate the nature of the ADA that can impact efficacy or safety. Some techniques used to measure binding ADA include radioimmunoassay, radioimmune precipitation, plate- or bead-based bridging and sandwich immunoassays using colorimetric (ELISA) or electrochemiluminescence (ECL) detection, and surface plasmon resonance immunoassay (SPRIA). Each of these techniques has varying sensitivity to detect ADA of different affinities and each technique

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Review Tatarewicz, Mytych, Manning, Swanson, Moxness & Chirmule has inherent limitations for accurate detection of ADA in the presence of drug [9–11] . Circulating therapeutic that is present when samples are collected can form immune complexes with ADA and lead to false-negative results [12] . The impact of drug interference is especially high for most monoclonal antibody (mAb) therapeutics that are dosed in high concentrations and generally have long half-lives. Techniques to disrupt these complexes with pH changes (acid dissociation and neutralization) have dramatically improved the drug tolerance and are used primarily with ELISA or ECL platforms [13,14] . During clinical trials, drug interference is also minimized by timing sample collection immediately prior to the next dose and at the end of the study, after a period that allows for clearance of the drug. In addition, the assessment of sensitivity and drug tolerance in any platform or method is based on a positive control antibody that may not be representative of the actual ADA measured in patient samples. As a result of the variability in ADA detection due to platform and methodology, identification of clinically relevant ADA and interpretation of the impact on therapeutic efficacy and safety is different for each therapeutic. Consideration of this point is emphasized in a required statement associated with the US product label of every licensed biological therapeutic. Therapeutic proteins can vary in structural and functional homology to endogenous proteins. Each unique therapeutic protein can induce diverse polyclonal antibody responses, which vary in time to onset, magnitude, affinity, isotype, subclass and epitope specificity across individuals. Thus, the ability of ADA to have an impact on PK, pharmacodynamics, efficacy and/or safety is variable across molecules and between Key terms Anti-drug antibodies: Antibodies produced against a biological therapeutic after administration. Epitope: The portion of the therapeutic that contains the binding site for the B-cell and its corresponding anti-drug antibodies. The epitope(s) may be comprised of a linear sequence, a specific domain (e.g., glycosylation) or a particular conformation. Immunogenicity: The potential to induce an immune response in humans or animals. During drug development, immunogenicity is assessed primarily by the measurement of binding and neutralizing anti-drug antibodies. Isotype: Specific immunoglobulin forms characterized by sequence, structure and function (IgM, IgG, IgA and IgE). Anti-drug antibody kinetics: Describes the onset, magnitude and persistence of the immune response over time, as a measure of anti-drug antibody levels (individual) or incidence (population). Titer: A means to quantify a specific antibody response through a process of serial dilution.

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individuals. ADA that bind to the therapeutic protein and are present in high levels and/or have a high affinity for the therapeutic protein can impact therapeutic exposure, due to increased clearance of the therapeutic or sustained half-life of the therapeutic. ADA that affect exposure may or may not be neutralizing, but they may be clinically relevant. ADA that bind the ‘active site’ of the therapeutic are usually neutralizing antibodies (NAb) that directly interfere with the mechanism of action and result in a loss of function of the therapeutic. These antibodies can represent significant safety concerns for protein replacement therapeutics as they can cross-react with the endogenous protein as a result of a break in immune tolerance to the endogenous (self) protein. Administration of recombinant human megakaryocyte growth and development factor (MGDF) is one of the classic examples of endogenous protein inactivation. During different clinical studies, some healthy subjects receiving a single injection of recombinant MGDF and some cancer subjects receiving multiple doses of pegylated recombinant human MGDF developed NAb to the respective therapeutic that cross-reacted with endogenous MGDF (aka thrombopoietin). Some of these subjects progressed to develop drug-induced antibody-mediated thrombocytopenia [15,16] . There are numerous other examples (e.g., clotting factors VIII and IX, erythropoiesis-stimulating agents [ESAs] and enzyme replacement therapies), in which serious and potentially life-threatening adverse events have occurred as a result of NAb cross-reactivity to endogenous proteins [17] . Cell-based bioassays are the preferred method for measuring NAb. These methods tend to be significantly different in sensitivity compared with the immunoassays, and typically do not demonstrate the presence of neutralizing activity until the antibody response has sufficiently matured and the therapeutic concentration is below the assay interference level. The methods and respective limitations of cellbased NAb assays are discussed in other papers and are outside the scope of this discussion [18] . Another safety concern for ADA that affects all therapeutic proteins is the development of hypersensitivity reactions (types I–IV) after therapeutic administration [19] . These reactions can be mild (e.g., skin rashes, fever, serum sickness) or severe (e.g., anaphylaxis, cytokine storm); they can be immediate (type I) or delayed (type IV) [20] ; they can involve immune complex mediated disease (type III) [21] ; or ADA can be involved in complement- and antibody-mediated lysis of cells (type II) [22] . This article describes the strategic rationale and methods for characterization of ADA that may develop in response to a therapeutic protein. It illustrates the value of ADA characterization to describe the nature

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Strategic characterization of anti-drug antibody responses for the assessment of clinical relevance & impact

of the immune response that can aid in the interpretation of the ADA impact on the efficacy and safety of the therapeutic. This information can be used to understand and mitigate risks associated with the therapeutic. Ultimately, it can provide objective means to support the clinicians’ options for safe and effective patient care and disease management. Kinetics of ADA development ADA kinetics describe the onset, magnitude and persistence of the immune response over time, as a measure of ADA levels (individual) or incidence (population). The change in ADA levels over time can be related to changes in other attributes of the ADA (e.g., affinity, isotype and specificity). Characterization of the kinetics and ADA attributes provides a context in which to evaluate the development of ADA, and the impact on efficacy and safety. During early clinical development of a therapeutic, early (week 2–4 after dosing) and frequent (monthly) assessment of ADA is recommended and typically needed, to observe a transient (i.e., tolerized) response and should be used to correlate early ADA-related adverse safety events. Induction of an immune response after administration of an immunogenic non-tolerant protein is predominantly a T-cell dependent (TD) process. The kinetics of the immune response after first exposure to theprotein (i.e., primary response) is qualitatively and quantitatively different from the response after reexposure to the protein (i.e., secondary response). A primary TD ADA response can typically be observed, 2–4 weeks after dosing. The nature of the primary response results in predominantly low levels (compared with secondary response) of low affinity IgM and IgG antibodies, which may be weakly neutralizing. The primary response kinetics may or may not be enhanced by the presence of pre-existing cross-reactive antibodies. The levels and persistence of a primary TD ADA response is reflective of the relative immunogenicity of the protein; it is generally of short duration as the B-cell has not received sufficient signals to mature into ADA-producing plasma cells. Repeat exposure to the antigen (i.e., therapeutic protein) subsequently results in the development of a secondary antibody response. This TD B-cell response provides the necessary signals to drive B cells to undergo isotype switching, somatic hypermutation, affinity maturation and differentiation into ADA IgG-producing plasma cells. This secondary IgG response may be comprised of all IgG subclasses (IgG1, IgG2 IgG3 and IgG4). IgG1, IgG2 and IgG3 responses precede the IgG4 subclass response, ultimately resulting in a mixed IgG response. Chronic administration of the antigenic therapeutic protein results in an increase in the presence of IgG4


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ADA, which is representative of a mature immune response. In some subjects, the development of ADA may be transient, suggesting a tolerization process; however, if the immune response is robust, increasing quantities of ADA can be measured around 2–6 weeks after exposure. Affinity maturation of ADA will result in a preponderance of high affinity IgG ADA that may also become NAb over time. In general, neutralizing ADA that develop typically reach sufficient levels to be detected in most in vitro or cell-based functional assays between 2 and 4 months after dosing. Chronic administration of a therapeutic protein can result in the development of IgG4 ADA, which occurs later in comparison to the other IgG subclasses. Persistence of ADA after secondary exposure can continue for more than a year but is often dependent on continued administration of the therapeutic protein. A summary of the kinetics and characteristics of ADA development and detection after chronic dosing are described in Table 1. In a careful analysis of the kinetics and persistence of ADA development after continued administration of natalizumab, persistent ADA were detected in 6% of the patients and transient ADA were detected in 3% [24] . Over 90% of the persistently positive patients developed ADA by 12 weeks. Patients with transient ADA were also detectable by 12 weeks but subsequently became negative and the decrease in circulating drug levels in these patients was completely reversed between 6 and 12 months. In this study, for subjects with transient ADA, therapeutic efficacy was completely restored and was comparable to patients with no detectable ADA, whereas persistent ADA positive subjects lost efficacy. In contrast, ADA to adalimumab developed in 67% of all subjects treated for rheumatoid arthritis (RA) within 28 weeks of treatment and persisted after end of dosing [25] . In an assessment of the decline in ADA titers to infliximab and adalimumab after treatment was terminated, 71% patients that received infliximab (t1/2 = 9.5 days) had undetectable levels of ADA by 12 months, whereas 66% patients that received adalimumab (t1/2 = 15–19 days) had ADA that persisted after 12 months [26] . Kinetic analysis of ADA development is impacted by the ability to detect ADA in the presence of the therapeutic protein. Current EMA and US FDA draft guidelines on immunogenicity testing include recommendations for frequent sample collection and measurement of therapeutic concentrations at all time points collected for ADA analysis [3,4] . ADA and the therapeutic protein will form immune complexes of different sizes in serum that are dependent on the affinity, ADA isotype and number of antigenic epitopes on the therapeutic protein. Immune complexes

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Table 1. Summary of the kinetic characteristics of T-cell dependent anti-drug antibody development, detection and potential impact that is dependent on dose and frequency after chronic dosing. Timing

Nature of ADA

ADA detection and impact

2–4 weeks

Earliest detection of ADA responses Typically low affinity IgM binding antibody Some IgG, may be weakly neutralizing if strong antigen IgE ADA possible

Pre-existing cross-reactive antibodies may result in early detection of IgG (primary response) 2–4 weeks usually not sufficient time for affinity maturation ADA detection and assessment of levels can be impacted by circulating drug Acute hypersensitivity in connection with primary response: IgE ADA possible due to cross-reactive antibodies Delayed hypersensitivity in connection with IgE ADA possible after secondary exposure

4–6 weeks

Binding antibody; potential for strong neutralizing antibody; IgG (IgG1, IgG2)

ADA affinity and specificity variable due to epitope spreading and affinity maturation Often peak ADA levels for primary response Isotype switching ADA detection and assessment of levels can be impacted by circulating drug IgG levels increase IgM levels decrease

2–4 months

Binding and neutralizing antibody; IgG (IgG1, IgG2, IgG3, IgG4 isotypes)

ADA affinity and specificity variable High ADA levels and potential impact on PK, safety and efficacy Chronic dosing increases production of IgG4

6–12 months Binding and neutralizing antibody

ADA levels typically decrease without continued dosing of therapeutic In vitro detection of neutralizing ADA may drop below detectable limits

>1 year

ADA levels typically decrease without continued dosing of therapeutic Persistent ADA response reflect long term B- and T-cell memory [23]

Binding and neutralizing antibody

ADA: Anti-drug antibody; PK: Pharmacokinetics.

interfere with detection of ADA due to the presence of therapeutic. Conversely, accurate measures of drug levels are confounded by the presence of ADA in samples. The measure of ADA and drug levels in the same sample will distinguish the reliability of the ADA and drug determinations; it will provide better accuracy for interpretation of ADA kinetics and impact on PK, efficacy and safety [27] . Determination of ADA levels Determination of ADA levels measures the relative quantity of ADA that is produced after administration. It is important to distinguish differences in the developKey term Relative antibody concentration: A mass-based means to quantify a specific antibody response relative to a standard.

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ment of an ADA response that are related to an impact on therapeutic exposure or safety. The measured level of ADA is highly dependent on the variability over time of the individual response (e.g., affinity, isotype) and the measurement constraints pertinent to the specific technology platform and method (e.g., matrix effects of the serum, specificity and quality of the detecting reagents, wash steps and so on). Because of these differences, the level of ADA can only be described relative to a standard. In contrast, PK assays typically have homogenous analytes that can be replicated for standard curve creation and reliable quantification with a given methodology. There are two primary techniques to measure ADA levels. One is relative antibody concentration. It utilizes a relative standard, with a known mass unit of ADA, to establish a reference standard curve to ascribe the level of ADA in a sample (in mass units). The use of mass units is recommended for mass-based



detection methods such as SPRIA technology. The challenge in using a standard, generated in an animal species, is lack of similarity with the patient’s ADA. Thus, affinity of the ADA could impact the accuracy of the measurement. The use of a relatively high affinity reference antibody to the therapeutic can model a high affinity ADA in the patient, with maximum assay sensitivity, but may reflect the worst-case drug tolerance of the assay. In contrast, the use of a low affinity reference ADA will represent the worst-case assay sensitivity but will better reflect the maximum drug tolerance. Relative inaccuracy of the measurement can be determined by measuring the sample at multiple dilutions to check if the slope is equivalent to the standard curve. Another source of error in the measurement of ADA is the presence of circulating therapeutic, which interferes with the detection. In this case, the use of a standard curve model to quantify ADA levels should be reserved for samples that are drawn after the therapeutic has cleared. A simpler version of the standard curve model is to use assay signal as a surrogate. This value can be the raw result (optical density, ECL value, response unit and so on) or normalized result (S/N, signal minus noise and so on) of the standard at a known concentration. This simple version requires an assay that is relatively linear with respect to signal and is controlled over time with performance acceptance criteria of positive controls that maintain consistency. The precision and accuracy is usually 35 units/ml) were determined for loss of clinical response [44] . Similarly, the prescribing information for Fabrazyme states that subjects with an antibody titer of >12,800 had a higher increase in clearance by 50% (range 5–90%) from week 1 to 12. Identification of isotype & IgG subclass Characterization of isotype and subclass is informative for any ADA response that can be correlated with an impact on efficacy or safety, particularly hypersensitivity reactions. ADA isotype determination can provide information regarding a TD or independent mechanism of ADA development, antigenic attributes of the therapeutic (e.g., T-cell epitopes, glycan residues) and the maturity of the response (immunoglobulin [Ig] class switching). Recommendations and rationale for the use of isotype characterization are outlined in Table 2. The initial immune response involves induction of an antigen-specific low affinity, high avidity IgM. The IgMADA is rapidly produced by activated B cells as it does not involve istoype switching or affinity maturation. For an immunogenic therapeutic that lacks T-cell epitopes, IgM and IgG3 ADA can also be produced in a T-cell independent (TI) mechanism. In a TI response, the activated B cells do not receive the appropriate


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Table 2. Rationale for isotype and subclass characterization of anti-drug antibodies during clinical development of a therapeutic. Characterization rationale (impact on efficacy and safety)

Explanation/justification/observation

Possible ADA by isotype and IgG subclass

Decreased trough concentration of therapeutic: reduced efficacy

High levels of binding or neutralizing ADA

IgM, IgG1–4, IgA1–2

Loss of clinical efficacy

Increased clearance of therapeutic by binding ADA Functional neutralization of therapeutic by ADA

IgM, IgG1–4, IgA1–2

Loss of endogenous protein function

ADA cross-reactive with endogenous protein

Neutralizing IgG1–4, IgA1–2

Pre-existing antibodies

Potential for: epitope spreading, complement activation; hypersensitivity

IgM, IgG, IgA, IgE

Therapeutic has known or suspect repetitive structural features (e.g., addition of PEG moieties, antibody–drug conjugates, aggregation)

T-cell-independent immune response; potential to activate complement

Postdose IgM and IgG3

Evaluate maturity of immune response

Explain observed induction of tolerance Explain apparent reduction in ADA titers detected in bridging assay

IgG4

Type I hypersensitivity

Anaphylaxis

IgE

Type 2 hypersensitivity

Antibody or complement-mediated cytotoxicity

IgM, IgG1 and IgG3


Immune complex

IgG and IgM


Delayed T-cell-dependent response: development of ADA

All isotypes

ADA: Anti-drug antibody; PEG: Polyethylene glycol.

signals to switch isotype by class switch recombination. The IgM antibodies produced in a TI response are broadly specific and have a preference for structural features found in immunoglobulins (Igs), self antigens and common bacterial polysaccharides [45] . When B cells are activated in a TD manner, cytokines are secreted from the interaction of T-helper and dendritic cells. According to the specific cytokines secreted, the antigen specific B cell will undergo affinity maturation and switch isotype from IgM to IgG (1–4), IgA(1–2) and/or IgE [46] . When isotype switching occurs, the constant region of the heavy chain is switched. Each isotype differs in ability to activate complement, specificity to Fc receptors and effector cells. Each antibody isotype functions in different environments (e.g., IgA in mucosal epithelium, IgG in blood and extravascular space and IgE in skin) [47] . If the therapeutic protein induces a Th2 T-cell response, an IgE ADA may result in eliciting an immediate hypersensitivity response (e.g., anaphylaxis, urticaria and so on) between 2–4 h after exposure. An IgE-mediated hypersensitivity reaction becomes more severe when the patient is exposed

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to a subsequent dose of the therapeutic, resulting in IgE-sensitized mast cells binding the therapeutic protein and causing rapid mast cell degranulation. In a TD response, the predominant ADA isotype produced after multiple exposures is usually IgG. Antibody isotype assays determine the nature of the ADA bound to the therapeutic by individual detection with antibodies specific for the human isotype or IgG subclass of interest. With the exception of IgE detection, it is recommended to maintain the same detection platform for isotype and IgG subclass characterization as that used for ADA screening. This approach maintains similarity with respect to serum interference factors, assay sensitivity and assay specificity. Specific methodologies are used to capture and detect IgE ADA because concentrations in serum are extremely low (0.04% of the total Ig) and have a short half-life. Recent regulatory guidelines recommend drug-specific IgE testing when immediate-type reactions occur that are associated with drug administration. The Clinical and Laboratory Standards Institute has published recommendations and guidelines for detection of IgE [48] .



Due to the high sequence homology between isotypes, any isotype or subclass-specific antibody reagent considered for use in an assay should be evaluated for sensitivity and cross-reactivity with all other Ig isotypes and IgG subclasses. In addition, the sensitivity of the isotyping reagents should be similar in order to determine the predominant isotype of the response. If the therapeutic is in whole or part an antibody, the specific isotype and IgG subclass of the therapeutic will interfere with the ability to detect ADA of the same subclass or IgG isotype. For example, if the therapeutic drug is an IgG2 antibody, confirmation with a polyclonal or monoclonal anti-human IgG or anti-human IgG2 will result in binding to the therapeutic as well as any IgG2 ADA. Therefore, the specific isotype and subclass of the therapeutic mAb must either be excluded from analysis or alternative methods of confirmation should be explored. This is often approached by detection of ADA that bind to the target binding portion such as the therapeutic Fab or F(ab´)2 instead of the whole molecule, and confirmation with an isotype Fc or subclass specific antibody reagent. Pre-existing antibodies

Predose samples from therapeutic naive subjects may have detectable pre-existing IgM or IgG ADA. These pre-existing antibodies may be due to an autoimmune disease state, or represent prior exposure to an antigen with similar features. They represent a potential risk factor to enhance the kinetics of the immune response and for hypersensitivity. In some cases, preexisting IgM and IgG ADA may be due to natural serum antibodies produced by B1 cells of the humoral response. In particular, B1 B-cell subsets produce high levels of secreted IgM and lower levels of IgG3. These antibodies show polyspecificity and auto-reactivity for antigens with Ig features, repetitive structural features and common bacterial polysaccharides [49–51] . In the case of erythropoiesis-stimulating agents, pre-existing non-neutralizing IgM and IgG were identified in some subjects from clinical studies. For these subjects, the presence of IgM and IgG ADA were detectable at end of study with no significant difference in concentration or neutralizing activity. The IgM ADA was shown to be specific to the glycosylation moieties of the therapeutic and not the protein itself [52] . In a clinical trial for the treatment of Parkinson’s with recombinant human Glial-derived neurotrophic factor, 17% of subjects (6/34) had pre-existing IgM ADA, one of which also had measurable IgA ADA and another with IgG ADA. Four of these six subjects had subsequent timepoints that were positive for binding IgG ADA, and one continued to develop increasing levels of IgG that matured to neutralizing IgG ADA. Subclass character-


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ization demonstrated a temporal change in ADA levels with IgG1 and IgG2 increasing before high levels of IgG4 and no measurable IgG3, indicating a mature T-cell-dependent immune response [53] . Thus, isotype characterization of an early response can be used to demonstrate isotype switching of ADA and discern potential clinical impact of pre-existing ADA for highrisk therapeutics such as replacement proteins or those with an endogenous counterpart. IgE responses

Although the majority of adverse events associated with biological therapeutics are target related, the occurrence of off-target toxicities related to hypersensitivity reactions and the development of ADA can have potentially severe consequences. In particular, type I (immediate) hypersensitivity results in symptoms of anaphylaxis from minutes to hours after exposure and can result in death. This type of response requires prior sensitization to the antigen (i.e., therapeutic protein) or some other cross-reactive attribute, and is specifically related to IgE ADA-mediated release of histamine and tryptase by mast cells or basophils in the skin or mucous membranes. Immunoassays developed specifically for the detection of IgE ADA are used to determine whether the symptoms associated with anaphylaxis are IgEmediated. Determination of IgE-mediated hypersensitivity can represent criteria for termination of treatment in a patient, as well as criteria for medical intervention in order to continue therapeutic administration. In the case of cetuximab, pre-existing IgE specific to the nonhuman galactose-alpha-1,3-galactose glycosylation in the Fab caused hypersensitivity in 20% of subjects after the first dose [33] . These subjects were all from Tennessee and North Carolina, where it has since been shown that pre-existing IgE specific to the same glycosylation is also found in about 20% of the normal population and is related to tick bites [54,55] . The presence of drugspecific IgE is an indication of sensitization and a risk factor for allergic symptoms. It has been suggested that consideration should be given to the value of preliminary skin prick tests to observe potential hypersensitivity reactions prior to systemic exposure of a biological therapeutic [56] . IgG4 responses

Development of IgG4 ADA to ESAs and anti-TNF‑α therapeutic proteins demonstrate maturation of an ADA response that correlates with loss of efficacy after Key term Pre-existing antibodies: Antibodies present in a patient prior to administration of a therapeutic and have the potential to cross-react with the therapeutic.


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Review Tatarewicz, Mytych, Manning, Swanson, Moxness & Chirmule chronic therapeutic administration. In a study of antiESA IgG4 antibodies, a strong association was demonstrated between the detection of anti-ESA IgG4 associated with loss of ESA effect and the severe consequence of antibody-mediated pure red cell aplasia [57] . In a study of the anti-TNF‑α theraputic, adalimumab, RA patients had a 54% immunogenicity rate. After the first 6 months, 94% of ADA positive patients developed ADA and 71% of those were positive for IgG4 ADA [58] . For the anti-TNF‑α therapeutic, infliximab, IgG4 ADA comprised between 8–89% of the total ADA found in RA patients treated for 3–6 months [59] . The development of IgG4 ADA may prove to be an early indicator of a clinically meaningful ADA response that could be useful to develop criteria that influence treatment decisions. In the use of anti-TNF‑α inhibitors, it is common practice to ‘dose-through’ loss of efficacy. In some dose escalation studies with infliximab, lower ADA responses and improved efficacy have been reported, while others have reported increased adverse events [60,61] . Loss of efficacy at initial minimal doses may lead clinicians to increase the dose and/or frequency or change the therapeutic in non-responders. In most cases, changes to the dose and frequency are made in the absence of objective criteria specific to the patient, such as the presence of ADA. Characterization of ADA responses such as IgE detection, quantitative level of ADA and determination of IgG4 isotype may provide useful immunogenicity biomarkers to identify patients that are at risk of adverse events upon dose escalation or who might benefit from a therapeutic switch due to inefficacy [26,62] . A case study for kinetics & isotype reponses

Lundqvist et al. have shown in a retrospective analysis of Swedish patients treated with natalizumab between 2006 and 2011 (n = 1391), the incidence of developing ADA was only 4% (57/1379) [63] . Nevertheless, there was a statistically significant difference in the level of ADA (0.5–3.0 μg/ml) in subjects that were transiently positive compared with those that were persistently positive (>3.0 μg/ml). This dADA was used to establish a predictive cut-off value for assessing patients at risk for developing persistent ADA to natalizumab. In addition, characterization of the levels of different ADA isotypes (IgG 1, 2, 3, 4 and IgM) in all positive patients demonstrated that patients with persistent ADA had significantly higher levels of IgM ADA (p = 0.0073) and IgG4 ADA (p = 0.011) and lower levels of IgG1 ADA (p = 0.021) compared with transiently positive patients [63] . This study demonstrates that characterization of ADA can enable the establishment of levels and isotypes that may be diagnostic for identification of non-responders.

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A case study of the isotype characteristics of antibodies against FVIII in healthy individuals and different hemophilia A populations: using well-controlled, validated assays, Whelan et al. characterized the qualitative and quantitative differences of antibodies against FVIII in a population of 600 healthy individuals, 77 patients with only binding ADA against FVIII, 20 patients with neutralizing FVIII ADA, 23 patients with FVIII ADA that had undergone successful immune tolerance induction therapy and nine patients with acquired hemophilia A [64] . Subjects with a titer ≥1:80 were also characterized for isotype, subclass, epitope (binding domain) and neutralizing activity. The prevalence of pre-existing FVIII antibodies in healthy individuals was 19% with 2% having titers ≥1:80. The prevalence of ADA in the different therapeutic treated populations was 34% (5% for titers ≥1:80) for patients with only binding antibodies against FVIII; 39% (4% for titers ≥1:80) in patients with successful immune tolerance induction and 100% (all titers ≥1:80) for patients with Nab and acquired hemophilia A. In all but one patient with only binding antibodies to FVIII, the specificity was determined to be to the nonenzymatic ‘B’ domain of FVIII. In addition, the isotype and subclass distribution of FVIII antibodies in healthy individuals and patients with only binding antibodies were distinctly different. IgG4 antibodies were not detected in any samples. Isotypes in healthy individuals were primarily IgG1, IgG3 and IgA; in patients with only binding antibodies, IgG1 and IgG3 were prominent, and in patients with successful immune tolerance induction, IgG1 was dominant. In contrast, almost all patients with Nab ADA to FVIII and those with acquired hemophilia A demonstrated multiple isotypes and IgG subclasses of anti-FVIII antibodies, with a significant correlation of IgG1 and IgG4 ADA in patients with Nab. In healthy individuals, a statistically significant contribution of age and geography was associated with the development of IgA antibodies of low affinity. These antibodies could be produced by either a TI or TD mechanism. Monomeric IgA is associated with the prevention of inflammation and development of autoimmunity [65] . This kind of analysis continues to provide insight into the antibody characteristics that may indicate differences in the regulatory pathways of the immune system and the cellular subsets that are involved. Epitope characterization In the context of ADA assessment, epitope characterization involves determination of the therapeutic domain that contains the binding sites (epitopes) for the B-cell and its corresponding ADA. There are several situations outlined in Table 3 where this type of characterization can contribute to the immunogenicity risk assessment



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Table 3. Rationale for epitope characterization of anti-drug antibodies during clinical development. Epitope characterization rationale

Explanation/justification/observation

Absence of sensitive cell-based neutralizing antibody assay

Provides an indirect assessment of neutralization capability. May contribute to assessment of impact on PK, efficacy and safety

High incidence of pre-existing antibodies

For ADA to mAb or Fc therapeutics, pre-existing cross-reactivity may be due to allotype specificity

Therapeutic shares a domain with endogenous protein or other therapeutics (e.g., PEG, ADC cytotoxin, other post-translational modifications)

Provides risk assessment to determine if ADA pose a risk of depleting endogenous protein Potential consequences for future administration of therapeutics with shared domain or cross-reactivity with similar molecules (e.g., small molecule drugs related to toxin in ADC therapeutic) May provide determination of immunogenic ‘hot spot’ for engineering improved next generation therapeutic May provide understanding of pre-existing cross-reactivity (e.g., PEG)

Protein has multiple unique domains, feature or safety concerns (e.g., ADC, BiTEs, bi-specific Abs, immunotoxins)

Anti-cytotoxin antibodies more likely to form large immune complexes due to repetitive nature of epitope (ADC) Potential for antibodies against tumor targeting region to crosslink T-cells and Fc-γ receptor-expressing cells (BiTEs) May provide differentiation of impact of ADA against various domains on PK, PD, efficacy and/or safety

Protein has known sequence or other attribute change (e.g., posttranslational modification)

Used to characterize immunogenicity risk due to modification May contribute to assessment of potential ADA impact on PK, PD, efficacy and/or safety

Ab: Antibody; ADA: Anti-drug antibody; ADC: Antibody–drug conjugate; BiTE: Bi-specific T-cell engager; mAb: Monoclonal antibody; PD: Pharmacodynamics; PEG: Polyethylene glycol; PK: Pharmacokinetics.

of a therapeutic. Epitope characterization is explored to understand potential safety risks associated with ADA development against a unique sequence, structural feature or domain within a therapeutic or to try and elucidate whether discrete changes in product quality attributes that may occur during manufacturing (e.g., adducts, clips and so on) relate to differences in immunogenicity. In addition, epitope characterization may be used to determine antigenic sites on a therapeutic candidate in order to engineer a less immunogenic drug by manipulation of potential B-cell epitope ‘hot spots’ [66] . B-cell epitopes can be linear, conformational (in terms of a distinct structural feature such as glycosylation) or discontinuous [67] . This lack of commonality between the antigenic epitopes that B cells encounter has hampered the development of predictive algorithms in comparison to those developed for T-cell epitope prediction. However, development of more accurate in silico B-cell prediction methods continues to improve [68,69] . Removal of B-cell epitopes is an attractive complement for de-immunization compared to removal of T-cell agretopes, as the polymorphic nature of the human leukocyte antigen makes it more unlikely to remove all T-cell binding [70] . Nevertheless, the removal of B-cell epitopes is inherently more difficult as many are associated with functional activity of the therapeutic. Thus, a change in the B-cell epitope can potentially affect the biological activity or create new epitopes.


De-immunization of T-cell epitopes, which are all linear epitopes that bind MHC II and drive the TD response, is more effective and less likely to affect the biological activity. In general, deimmunization is most useful when performed in early phase studies. Epitope characterization methods take either a functional or structural approach to determine the site of interaction. Structural characterization methods include x-ray crystallography of antigen–antibody complexes or variations of MS analyses to determine more precise physical and sequence attributes of the interactions [71,72] . Functional characterization methods typically involve an immunoassay that generally identifies the domains containing the epitopes (and not the specific amino acids involved) through competitive inhibition or direct detection using protein fragments, peptides derived from the linear sequence of the antigen, peptide libraries or antigen-derived hybridoma antibodies [73] . Recombinant techniques can be used to make amino acid substitutions within an epitope in order to determine the critical residues required for ADA binding [74] . When evaluated for immunogenicity assessments, epitope characterization methods are easily adapted from the validated screening and specificity immunoassays already used to identify ADA by using therapeutic fragments representing different epitope domains. Rather than using a single domain for epitope


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Review Tatarewicz, Mytych, Manning, Swanson, Moxness & Chirmule characterization and inferring an alternate epitope from a negative result, it is recommended to test all potential domains. In addition, it is important to consider that immunoassays that require passive absorption or chemical cross-linking of the capture antigen to a surface, or conjugation of a detection moiety, may be susceptible to changes in conformational epitopes or exposure of cryptic epitopes. Screening immunoassay designs usually use the whole therapeutic for ADA capture and may use the whole therapeutic conjugated to a detector (i.e., bridging immunoassay) and/or some other secondary reagent conjugated to a detector (i.e., sandwich immunoassay), which can then be measured. Specificity assays designs commonly use competitive inhibition of ADA capture and detection by addition of an excess of soluble whole therapeutic that results in a decrease of the signal. Functional epitope characterization is similar, but adds an excess of an epitope-containing fragment to inhibit the signal. In a similar manner, it is possible to group hybridoma antibodies against a therapeutic into epitope specific bins by mutual competition of all possible antibody pairs in a matrix assessment, where one epitope specific antibody is used as therapeutic detector and the other is used to compete out the signal [75] . This type of competitive inhibition assay design is satisfactory for distinguishing an epitope that binds a predominant proportion of the ADA that are generated, and which is readily observed by a dramatic loss of signal. However, competitive inhibition of signal derived from an epitope that binds a minor proportion of ADA is often too small of a decrease compared with the predominant response and leads to a false-negative result. In the typical polyclonal antibody response, some epitopes may be less immunogenic and represent a minority of the overall response. For these epitopes, an immunoassay design based on detection of the epitope of interest is a better approach. A detection-based assay also uses the whole therapeutic to capture the ADA but signal is only measured when an epitope-specific fragment, peptide or antibody that is conjugated with a measurable detector (e.g., horseradish peroxidase, ruthenium and so on) also binds the ADA. This method has the advantage of being able to measure low prevalence ADA in a polyclonal response. Complex biotherapeutics such as antibody–drug conjugates, bi-specific antibodies, bi-specific T-cell engagers and immunotoxins present unique circumstances where a specific functional epitope may be significantly more immunogenic than another. In a recent study that compared assay design for epitope characterization of an antibody–drug conjugate by competitive inhibition versus detection, the competitive inhibition design was not able to accurately determine the presence of low levels of epitope-specific ADA (e.g.,

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anti-cytotoxin ADA) in the presence of high levels of an ADA of different epitope specificity (e.g., anti-mAb fragment ADA). Being able to accurately determine the specific ADA epitope domain for these kinds of biotherapeutics can provide insight into the mechanisms of any observed safety events and contribute to the design of future therapeutics [76] . Epitope characterization of pre-existing ADA can be used to understand the relevant specificity versus crossreactivity of the ADA and the potential for epitope spreading. Antibodies to allotypes of Ig molecules may account for the presence of some pre-existing ADA. Allotypes are specific amino acid polymorphisms that occur in the constant regions of IgG1, IgG2, IgG3, IgA and the kappa light chain. The Ig allotypes are a determinant of self and represent an immunogenic epitope to an individual that is not of the same allotype. It has been estimated that anti-allotype antibodies persist in about 1% of the population as a result of maternal–fetal transfer of Igs, or blood or platelet transfusions. We have previously reported evidence of anti-allotype antibodies in a Phase 1 single ascending dose clinical study of an aglycosylated IgG1 mAb therapeutic in healthy volunteers. Epitope characterization of the ADA was initiated due to a rapid development of neutralizing ADA and grade 2 hypersensitivity reactions in two subjects. Using a competitive inhibition immunoassay design, ADAs that developed after treatment were determined to be specific for the Fab´2 and were neutralizing. Additional characterization excluded the presence of a neo-epitope due to the lack of glycosylation. Two subjects had pre-existing anti-allotype antibodies with specificity to the G1m1 allotype determinant in the Fc of the therapeutic. Anti-allotype antibodies in these two subjects persisted at similarly low levels, at all timepoints after dosing and were non-neutralizing. It is interesting to note that there was a lack of a secondary immune response for the two subjects with pre-existing anti-allotype antibodies. No specific correlation of ADA and anti-allotype antibodies to hypersensitivity could be concluded [77] . Recently, epitope binning of hybridoma antibodies generated from Xenomouse animals immunized with erythropoietin (EPO) was used to create standardized ADA controls for immunoassays designed to detect ADAs to any EPO-based stimulating agents. Epitope mapping experiments were conducted to select antiEPO ADA that represent NAb specific to the mutually exclusive high and low affinity receptor binding sites that are important for biological activity. In collaboration with the National Institute for Biological Standards and Controls, the reference panel of epitope specific ADA is currently under evaluation by the WHO for use as an internationally accessible reference



panel. This reference panel assures consistent detection of ADA to all ESAs, allows for determination of epitope specificity and enables comparison of assay performance using different platforms. These reagents can be especially useful for immunogenicity comparison of current and upcoming biosimilars for EPO [74] . This approach could also be applied to other biotherapeutics entering the biosimilar arena and provide a means by which to compare biosimilar products. Concluding remarks Here we have summarized strategies and methods for assessment and characterization of ADA responses using common methodologies in order to discern clinically relevant ADA. Characterization of ADA kinetics, levels, isotype, IgG subclass and epitope can provide the quantitative and qualitative information

Review

needed to develop predictive tools and specific, potentially diagnostic, assays that could predict the efficacy and/or safety of a therapeutic protein [78] . An important strategic decision that must be addressed during drug development is to determine when full characterization of ADA is warranted. In cases where the therapeutic protein is a replacement protein with an endogenous counterpart, especially if the protein exerts a critical non-redundant function, full characterization of ADA is certainly justified. Early information on ADA to a high-risk molecule may provide opportunities to mitigate patient risks that outweigh the burden of additional characterization. However, the ability to perform additional characterization will be dependent on the magnitude of ADA in Phase 1 studies. In cases where the therapeutic protein has no endogenous counterpart and any ADA would only impact

Strategy for clinical ADA characterization assays

Is the therapeutic program high-risk? (e.g., endogenous counterpart, potential safety concern) No

Yes

Characterizations implemented in Phase 1 • Level • Isotype/subclass • Binding domain (epitope)

Phase 1, 2, 3, postmarketing • Impact on efficacy, PK, PD, or safety • Regulatory request No

Yes

No

Is program Phase 3?

Characterizations implemented if warranted

Continue to monitor for impact on PK, efficacy, safety

Yes

No

Is incidence of postdose binding antibodies ≥10% and/or predose binding antibodies ≥5%? Yes

Reassess impact on efficacy, PK/PD and safety, and characterization value

Figure 1. Strategy for clinical anti-drug antibody characterization. This decision tree describes a useful starting strategy for a risk-based approach to ADA characterization. For high-risk therapeutics, characterization of ADA is recommended beginning in Phase 1. Phase 1 studies are generally single dose and have few subjects. Understanding the nature of the ADA early in development may provide insight into potential development and safety risks due to immunogenicity prior to entering a larger study. If the therapeutic program is not high risk, then characterization is recommended when there is a known impact to PK, pharmacodynamics, efficacy or safety. For late phase programs, characterization may be warranted if the incidence is high even if no apparent impact on efficacy or safety is readily evident. ADA: Anti-drug antibody; PD: Pharmacodynamics; PK: Pharmacokinetics.



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Review Tatarewicz, Mytych, Manning, Swanson, Moxness & Chirmule the efficacy of the therapeutic, characterization might not provide significant value. The risk-based decision for when to use characterization assays is centered on the clinical impact of the ADA response. When ADA have an impact on the program, either through where they bind on the therapeutic or by the magnitude of the immune response, it is valuable to better understand the antibodies contributing to that immune response by performing characterization assays. It is also important to consider causes other than ADA development that could explain changes in the clearance, exposure, efficacy and safety of a therapeutic. During early clinical development, the frequency and duration of ADA sampling is an important consideration because the onset of the immune response is unpredictable. Careful analysis of the kinetics during early phase clinical studies may be useful to justify less frequent sampling in later phase studies depending on the product and the specific circumstances for its use. In early clinical studies, it is appropriate to obtain the first ADA samples at 2–4 weeks after dosing and continue with frequent intervals (e.g., monthly) such that development of transient, persistent or delayed

ADA can be clearly differentiated. For the physician, ADA characterization can provide perspective on the biological impact of a therapeutic and objective rationale to manage patient care, such as the right choice of therapeutic based on patient profile, changes in dosing regimens, use of concomitant medications to manage the immune response and the decision to terminate or switch treatment [79] . Future perspective The decision to implement ADA characterization should be based on a risk assessment that takes into consideration both patient and product factors, and should be a part of the immunogenicity assessment strategy during clinical development. Characterization of ADA response kinetics and levels are best suited to discern an impact of ADA on PK and efficacy. Additional characterization of ADA isotype, IgG subclass and epitopes are useful to address issues related to safety. A recommended strategy for characterization of ADA responses during clinical development is shown in Figure 1. Antibody characterization of ADA positive samples may be triggered when there are aberrant PK

Executive summary • This article describes the strategic rationale and methods for characterization of anti-drug antibodies (ADA) that may develop in response to a therapeutic protein. • Characterization of the timing, strength, type and specificity of the immune response provides a means to differentiate and describe the nature of the ADA that can impact efficacy or safety. • Kinetics of ADA development describes the onset, magnitude and persistence of the immune response over time, as a measure of ADA levels (individual) or incidence (population). • Measurement of ADA levels and incidence can be used to evaluate potential cut-off levels that correlate to loss of efficacy or adverse safety events. • Identification of ADA isotype and subclass can provide information regarding the mechanism of ADA development, antigenic attributes of the therapeutic (e.g., T-cell epitopes, glycan residues) and the maturity of the immune response. • ADA isotype and subclass can be correlated with an impact on efficacy or safety, particularly hypersensitivity reactions involving IgE ADA. • Isotype and subclass may indicate differences in the regulatory pathways of the immune system and the cellular subsets that are involved. • Epitope characterization can be used to understand potential safety risks associated with ADA development against a unique sequence, structural feature or domain within a therapeutic. • May be used to distinguish binding ADA from potentially neutralizing ADA. • May be used to evaluate pre-existing antibodies for the presence of anti-allotype antibodies. • May be used to determine antigenic sites on a therapeutic candidate in order to engineer a less immunogenic drug. • May be used to try and elucidate whether discrete changes in product quality attributes that may occur during manufacturing (e.g., adducts, clips and so on) relate to differences in immunogenicity. • The strategy for implementation of ADA characterization assays during therapeutic development should follow a risk-based decision process. • High-risk therapeutics (e.g., non-redundant endogenous counterpart, potential safety concern and so on) are recommended to implement ADA characterization beginning in Phase 1. • Therapeutics that are not high risk are recommended to implement ADA characterization based on an associated impact to PK, efficacy or safety, in all phases of development. • Regardless of impact, ADA characterization should be considered during late-phase development when there is a high-incidence of pre-existing ADA (>5%) or developing postdose ADA (>10%).

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profiles, hypersensitivity reactions and/or when high rates or levels of ADA are observed in pre- or post-dose samples. In circumstances where clinical trials are powered for efficacy over safety, it may be prudent to characterize samples from studies with a low incidence of ADA. When there is an impact on the efficacy or safety of a biological therapeutic, characterization of ADA can be used to describe clinically relevant antibodies. References

Financial & competing interests disclosure All authors are employees and shareholders of Amgen, Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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Understanding the Supersensitive Anti-Drug Antibody Assay: Unexpected High Anti-Drug Antibody Incidence and Its Clinical Relevance.

The relevance of work-related learning for vulnerable groups. Dutch case study of a Health Impact Assessment with equity focus.

Assessment of erectile dysfunction in diabetic men: the clinical relevance of self-reported sexual functioning.

Assessment and non-clinical impact of medical devices.

Anti-TNF drug and antidrug antibody level monitoring in IBD: a practical guide.

Preclerkship assessment of clinical skills and clinical reasoning: the longitudinal impact on student performance.

Clinical significance of immunopathological findings in patients with post-pericardiotomy syndrome. I. Relevance of antibody pattern.

26. A critical assessment after 119 studies].

Characterization of guinea pig antibody responses to salivary proteins of Triatoma infestans for the development of a triatomine exposure marker.

Characterization of serum antibody responses to recombinant HIV-1 gp160 vaccine by enzyme immunoassay. NIAID AIDS Vaccine Clinical Trials Network.

Strategic approaches for assessment and minimization of matrix effect in ligand-binding assays.

Regulation of antibody responses by antibody towards the immunogen.