Neutralizing Anti-drug Antibodies Research Articles

Persistency of Neutralizing Anti-Interferon-β Antibodies in Patients with Multiple Sclerosis Treated with Subcutaneous Interferon-β Depends on Antibody Titers, IgG Subclasses, and Affinity Maturation.

Neutralizing antibodies may affect interferon (IFN)-β treatment efficacy, but mechanisms of neutralizing anti-drug antibody (ADA) evolution are not fully elucidated. We investigated the relationship between ADA titers, IgG subclass profile, and binding affinity with the development and persistency of neutralizing ADA in relapsing-remitting multiple sclerosis (MS) patients treated with subcutaneous IFN-β. A total of 94 patients, who had blood sampling at months 6, 12, 24, and 36 during IFN-β therapy, were included into this retrospective study and stratified to the following: non-neutralizing, transient, and persistent neutralizing ADA status. Patients without or with transient neutralizing ADA displayed predominantly IgG1 and IgG3 subclasses, lower ADA titers, and antibody binding affinity compared with patients having persistent neutralizing ADA, in whom the predominant IgG subclasses were IgG2 and IgG4. Overall, ADA binding affinity positively correlated with IgG4 and neutralizing ADA titers, but negatively with IgG3 titers. Persistency of neutralizing ADA was predicted by their titers at month 24 and month 36 of treatment and by an increase of antibody affinity within the second year of IFN-β treatment. The humoral immune response to IFN-β observed in MS patients as a result of IFN-β therapy is a multifactorial process that is influenced by ADA titers, affinity maturation, and IgG subclass switching.

A randomized, single-blind, single-dose study evaluating the pharmacokinetic equivalence of proposed biosimilar ABP 980 and trastuzumab in healthy male subjects

PurposeThis study compared the pharmacokinetic (PK) profiles of the proposed biosimilar ABP 980 and trastuzumab in healthy males.MethodsIn this single-blind study, 157 healthy males were randomized 1:1:1 to a single 6 mg/kg intravenous infusion of ABP 980, FDA-licensed trastuzumab [trastuzumab (US)], or EU-authorized trastuzumab [trastuzumab (EU)]. Primary endpoints were area under the serum concentration–time curve from time 0 to infinity (AUCinf) and maximum observed serum concentration (Cmax). To establish equivalence, the geometric mean ratio (GMR) and 90% confidence interval (CI) for Cmax and AUCinf had to be within the equivalence criteria of 0.80–1.25.ResultsThe GMRs and 90% CIs for Cmax and AUCinf, respectively, were: 1.04 (0.99–1.08) and 1.06 (1.00–1.12) for ABP 980 versus trastuzumab (US); 0.99 (0.95–1.03) and 1.00 (0.95–1.06) for ABP 980 versus trastuzumab (EU); and 0.96 (0.92–1.00) and 0.95 (0.90–1.01) for trastuzumab (US) versus trastuzumab (EU). All comparisons were within the equivalence criteria of 0.80–1.25. Treatment-emergent adverse events (TEAEs) were reported in 84.0, 75.0, and 78.2 of subjects in the ABP 980, trastuzumab (US), and trastuzumab (EU) groups, respectively. There were no deaths or TEAEs leading to study discontinuation and no binding or neutralizing anti-drug anti-bodies were detected.ConclusionsThis study demonstrated the PK similarity of ABP 980 to both trastuzumab (US) and trastuzumab (EU), and of trastuzumab (US) to trastuzumab (EU). No differences in safety and tolerability between treatments were noted; no subject tested positive for binding anti-bodies.

An immunoinhibition approach to overcome the impact of pre-existing antibodies on cut point establishment for immunogenicity assessment of moxetumomab pasudotox

Immunogenicity can impact PK, PD, efficacy and safety of biopharmaceuticals, and is often evaluated as a secondary objective in clinical studies. Methods to detect anti-drug antibodies (ADA) and neutralizing ADA (NAb) are semi-quantitative and utilize cut points to determine positive or negative samples. Assay cut points are established by the statistical analysis of treatment-naïve subject specimens that are assumed ADA and NAb-negative. Pre-existing antibodies to various biopharmaceuticals have been observed in treatment-naïve subjects and may artificially elevate the cut point, resulting in compromised assay sensitivities, inaccuracy in immunogenicity reporting and ultimately misleading assessment of the impact of immunogenicity on clinical outcomes. Although several approaches such as removal of pre-existing antibody samples or increasing the sample dilution could be used for cut point establishment to mitigate impact of pre-existing antibodies, they each have limitations, especially when a high prevalence of pre-existing antibodies is observed. Here we describe an innovative approach used to establish cut points for ADA and NAb assays of moxetumomab pasudotox (moxetumomab), a recombinant anti-CD22 immunotoxin, to which a high prevalence of pre-existing antibodies was observed. In order to overcome the challenges associated with this high prevalence and prevent establishment of an artificially elevated cut point, we developed an immunoinhibition approach that allowed generation of pseudo ADA and NAb-negative populations for cut point determination. Immunoinhibition was performed by adding excess moxetumomab (for ADA) or a non-CD22 binding PE38-containing immunotoxin, CAT-5001 (for NAb), to treatment-naive samples prior to evaluating samples for cut point establishment. This approach successfully eliminated pre-existing antibody activity in treatment-naive samples, enabling establishment of more accurate ADA and NAb assay cut points. A comparative analysis of the clinical immunogenicity results using cut points derived with immunoinhibition and without immunoinhibition (conventional method) demonstrated that the immunoinhibition approach markedly improved detection sensitivity and accuracy of immunogenicity characterization in patient samples. This innovative approach provides an alternative, practical solution for immunogenicity assay cut point establishment when biopharmaceuticals have a high prevalence of pre-existing antibodies.

Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta

Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the “Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk” (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro–Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519ng/mL for LUCIMU, 814ng/mL for LUCRH, and 320ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low positive titers needs to be further evaluated.

Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins

Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor.We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs.

Abstract CT230: Pediatric phase 1 trial of moxetumomab pasudotox: Activity in chemotherapy refractory acute lymphoblastic leukemia (ALL)

Abstract Background: Novel therapies are needed to overcome chemotherapy resistance and reduce toxicities of treatment for childhood ALL. CD22 is an antigen expressed on B-lineage ALL blasts. Moxetumomab pasudotox (MP) is a recombinant immunoconjugate composed of an anti-CD22 immunoglobulin variable domain genetically fused to a truncated form of Pseudomonas exotoxin. Methods: This is a multicenter, open-label, phase 1, dose-escalation study of MP in pediatric patients (pts) with relapsed/refractory ALL and NHL with CD22 expression. MP is administered as a 30-min IV infusion at doses 5-50 µg/kg every other day for 6-10 doses every 21 days. Cohort A (n=7) consisted of an accelerated dose-escalation phase followed by standard 3+3 dose-escalation. To reduce the incidence of capillary leak syndrome (CLS), subsequent cohorts (B, n=23; C, n=14) received dexamethasone (2.5 mg/m2 every 12 hours) around the first 6 doses of MP in cycle 1. In Cohort C, doses were increased to 10/cycle and an expansion phase at 50 µg/kg was added. Results: 44 pts with ALL 1-23 years of age have been treated. 35/44 had treatment-refractory disease; 20/44 had relapsed after stem cell transplant (SCT). Pts received a median of 1 treatment cycle (range 1-4). The majority (55%) of treatment-related adverse events (AEs) were mild and reversible. The most common treatment-related AEs were increased AST and ALT and weight gain. There were 2 treatment-related deaths and 7 pts discontinued therapy due to a treatment-related AE. Four DLTs included CLS in 2/7 pts in Cohort A (30 µg/kg; 1 reported as pleural effusion); hypercalcemia in 1 pt treated at 40 µg/kg (Cohort B) who died of a cardiac arrhythmia during attempted venous catheter placement; and grade 4 hemolytic uremic syndrome (HUS) in 1 pt at 50 µg/kg (Cohort B). HUS was noted in 6 pts, 5 treated at the 50 µg/kg dose. Two events were grade 4 with remaining cases ≤grade 2. All but 1 patient recovered from HUS. Based on HUS, the 50 µg/kg dose was determined to exceed the maximum tolerated dose and accrual at a lower dose is ongoing. Of the 37 (84%) pts evaluable for response, objective responses were achieved in 11 (30%), including 9 (24%) complete responses (CR). Per investigator report, 4/9 CRs were minimal residual disease negative, and 3 of these pts underwent a second SCT. Hematological activity (≥50% reduction in blasts and/or improvement in neutrophil and/or platelet counts) was observed in 11 (30%) pts. Anti-drug neutralizing antibodies (≥50% neutralization) developed in 7/44 (16%) pts. Conclusions: MP is active in pediatric pts with relapsed/refractory ALL. The observed antileukemic activity and safety profile warrant further investigation; phase 2 trials are in development. Study sponsored by MedImmune, and supported in part by the Intramural Research Program of the NIH, NCI, CCR. ClinicalTrials.gov NCT00659425. Citation Format: Alan S. Wayne, Nirali N. Shah, Deepa Bhojwani, Lewis B. Silverman, James A. Whitlock, Maryalice Stetler-Stevenson, Robert J. Kreitman, Trishna Goswami, Ramy Ibrahim, Ira Pastan. Pediatric phase 1 trial of moxetumomab pasudotox: Activity in chemotherapy refractory acute lymphoblastic leukemia (ALL). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT230. doi:10.1158/1538-7445.AM2014-CT230

Abstract 4505: Anti-tumor activity in pancreatic cancer of a low immunogenic and clinically optimized anti-mesothelin immunotoxin RG7787

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and new therapies are needed. SS1P is a recombinant immunotoxin (RIT) containing an anti-mesothelin dsFv fused to a cytotoxic fragment of Pseudomonas Exotoxin A (PE). In clinical trials, patients developed dose-limiting neutralizing anti-drug antibodies within the first cycle of treatment. RG7787 is a next generation, clinically optimized RIT with decreased immunogenicity and increased resistance to lysosomal proteases. In this study, we evaluated the in vitro and in vivo activity of RG7787 in PDAC. Method Five established PDAC cell lines (KLM-1, AsPC-1, BxPC-1, BxpC3, Panc 3.014, and PK-1) and one primary PDAC line directly obtained from a patient tumor (GUMC108) were used. Cell viability and proliferation were calculated by measuring ATP levels with Cell Titer-Glo assays. In vivo activity was evaluated in a subcutaneous KLM-1 mouse tumor model. Delivery of fluor-labeled RG7787 to mouse tumors was assessed by flow cytometry. Results RG7787 had significant in vitro activity in the PDAC cell lines tested with most IC50 values in the subnanomolar range. It was significantly more active than SS1P in KLM-1, Panc 3.014 and GUMC108, with similar activity in the other cell lines. GUMC108 (IC50 = 18.9 pM) was the most sensitive cell line. In KLM-1 cells, SS1P and RG7787 had similar internalization rates. RG7787 could be safely administered to mice at a dose of 2.5 mg/ kg IV QOD x3, which is more than five times the maximum tolerated murine dose of SS1P. In vivo uptake studies in a KLM-1 subcutaneous mouse xenograft model demonstrated intracellular delivery of RG7787-Alexa647 to 50% of tumor cells after a single 2.5 mg/kg IV dose. Tumor volume was reduced by 13% after a single cycle of RG7787 treatment (n=6). Two cycles of combination treatment with paclitaxel (50 mg/kg IP) and RG7787 shrank tumors to <10 mm3 in 100% of mice by 30 days (n = 11 over 2 experiments). Two single doses of paclitaxel alone (n=6) induced stable disease without significant tumor regression. Complete remission persisted through day 100 in 2/11 combination treated animals. Conclusions RG7787 has superior or similar activity compared to SS1P in established PDAC cell lines and a primary PDAC cell line in vitro. In a KLM-1 mouse xenograft model, the combination of paclitaxel with RG7787 cured 18.1% of mice and reduced tumor size by more than 90% in all other mice treated. RG7787 merits further development for the treatment of pancreatic cancer. Citation Format: Ira Pastan, Kevin Hollevoet, Emily Mason-Osann, Christine Alewine, Xiu-Fen Liu. Anti-tumor activity in pancreatic cancer of a low immunogenic and clinically optimized anti-mesothelin immunotoxin RG7787. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4505. doi:10.1158/1538-7445.AM2014-4505

A method to quantitate the neutralizing capacity of anti-therapeutic protein antibodies in serum and their correlation to clinical impact

A robust, quantitative method for assessing the neutralizing capacity of anti-therapeutic protein antibodies was developed and tested using 4 analytical assay platforms typically used for detection of anti-drug neutralizing antibodies. The method described here utilized titration of increasing concentrations of therapeutic protein into serum containing anti-therapeutic protein antibodies, either positive control antibodies or clinical samples. Neutralizing capacities were calculated by determining the EC50 from the titration curves. The neutralizing capacity of purified anti therapeutic protein antibodies was expressed in terms of “μg of drug neutralized per μg of anti-TP antibody” present. In the case of serum originating from clinical study subjects, the neutralizing capacity of the samples was expressed as “μg of drug neutralized per mL of serum”. A relative shift in EC50 values was observed as the amount of serum or antibody was changed resulting in a proportional shift of the calculated neutralizing capacity. Application of this approach using different assay platforms was consistent providing evidence for its potential to be a useful approach to characterize, qualify and compare neutralizing positive control antibody preparations or clinical samples. Using this methodology, we were able to draw a clear correlation between the neutralizing capacity and the effect of these antibodies on a clinical pharmacodynamic (PD) marker. Determination of the neutralizing capacity of antibody positive samples from subjects in a clinical study indicated direct correlation of pharmacodynamic results with non-response, partial response and response to high, mid and low neutralizing capacities respectively.

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.

Applications of cell-based bioassays measuring the induced expression of endogenous genes.

Cell-based bioassays are used to determine the biological activity of complex biotherapeutic products, to assign potency and to assure the quality and consistency of the manufacturing process. Clinically, these assays are used to assess bioactivity in patient samples, particularly for the detection of antidrug neutralizing antibodies. Owing to their versatility, cellular assays that measure endogenous gene expression by quantitative reverse transcription PCR offer a rapid and automatable alternative to assays measuring functional, late-stage responses. Notably, detection of immediate early gene expression represents a direct response of the cell to receptor ligation by the biotherapeutic. We review current developments in the use of this approach and demonstrate its application to the detection of receptor-binding autoantibodies using, as a case study, the detection of autoantibodies to the thyroid-stimulating hormone receptor.

Understanding the Immunogenicity Concept

Immunogenicity is just one of the many benefits of our immune system and is defined as the ability of different substances to trigger an adaptive cellular and humoral cell immune response that is longterm and leads to immunological memory. In rheumatology and other medical specialties such as dermatology, gastroenterology, neurology and immunology, biological proteins derived from biotechnology are increasingly used as therapeutic agents. These proteins, like many other exogenous agents can induce humoral and cellular immune responses. However, in recent years we have noticed how these concepts have been interpreted haphazardly, and sometimes in the wrong way from the point of view of their clinical significance. The immune response or immunogenicity which can be triggered by a therapeutic protein has been proposed as a cause of potential clinical events, including hypersensitivity reactions, decreased effectiveness of the therapeutic molecule and induction of immune processes, including the formation of antibodies against the protein in question.1,2 The most dreaded immunogenicity in clinical terms occurs due to the so-called neutralizing anti-drug antibodies which, as their name implies, have the ability to bind the agent (drug) and neutralize it, preventing its biological or therapeutic function.3 Many factors can influence the immunogenicity of therapeutic proteins, including those inherent to the patient, the disease itself or those related to the product itself. The determinants of patient-related immunogenicity that may predispose unwanted immune responses include the underlying disease, genetic factors and baseline immune status, including associated immunomodulatory therapy. Furthermore, product related factors also influence the probability of inducing an immune response; for example, the route of administration, the type of protein, aspects of the manufacturing process, impurity profile or excipients and formulation characteristics, stability, degradation products or aggregates, dosage, the dosing interval and treatment duration.4–6 The immune response mechanism directed against therapeutic biological molecules seems to be due to “transient” loss of

The effect of neutralizing antibodies on the sustainable efficacy of biologic therapies: what’s in it for African and Middle Eastern rheumatologists

Over the last decade, biologic therapeutic proteins have advanced the treatment of diseases such as rheumatoid arthritis (RA). Therapeutic antibodies such as infliximab, adalimumab, rituximab, tocilizumab, golimumab, certolizumab pegol, the receptor construct etanercept, and abatacept, an anticluster of differentiation (CD)80/anti-CD86 fusion protein, are used as treatment for RA and ankylosing spondylitis (AS). Infliximab, adalimumab, golimumab, certolizumab pegol, and etanercept are inhibitors of tumor necrosis factor (TNF), a key regulator of inflammation. Left untreated, progression of rheumatic diseases due to inflammation can lead to irreversible joint damage and serious disability. One limitation for the use of therapeutic antibodies is immunogenicity, the induction of antibodies by the adaptive immune system in response to foreign substances. The development of antidrug antibodies (ADAs) has a varying impact on the clinical efficacy of biologic agents for the treatment of RA and AS, depending on whether the ADAs are neutralizing or non-neutralizing. Studies have indicated that neutralizing ADAs are associated with a reduced efficacy, decreased drug survival, increased instances of dose escalation, and adverse events. Comparison studies of anti-TNF biologics have demonstrated that each drug has a different sustained efficacy profile depending on immunogenicity. The purpose of this review is to provide rheumatologists with information regarding the effect of neutralizing antibodies on the sustainable efficacy of anti-TNF biologic therapies. This information will be of value to practicing rheumatologists in Africa and the Middle East who should take into account the potential for changes in the efficacy and safety of biologic therapies and closely monitor patients under their care.

Implementation of design of experiments (DOE) in the development and validation of a cell-based bioassay for the detection of anti-drug neutralizing antibodies in human serum

The administration of biological therapeutics can potentially elicit the development of neutralizing antibodies (NAbs) to the therapeutic drug in patients, which could have a significant impact on drug efficacy and safety. A rigorous in vitro cell-based assay for the detection of NAbs is critical for the assessment of the immunogenicity profile of the therapeutic drug. Conatumumab is a fully human monoclonal agonist antibody directed against the extracellular domain of human TRAIL receptor 2 (TR-2). It is being investigated as a cancer treatment because it is able to induce apoptosis in sensitive tumor cells. This report demonstrates how statistically designed experiments could be employed effectively in different stages of a NAb bioassay life cycle in order to characterize, optimize and stabilize the assay with added benefit of resource efficiency. By combining the approach of design of experiments (DOE) with subject matter expertise and experience, we were able to understand thoroughly how assay parameters affect the performance of the assay individually and interactively, identify the key assay parameters, define assay operating ranges and finally achieve a robust and sensitive cell-based assay for the detection of NAbs to Conatumumab. With the goal of developing a cell-based bioassay that is highly optimized for sensitivity, specificity, precision, and robustness, we performed 2 DOE experiments for assay optimization and 1 DOE experiment to validate assay robustness. We evaluated key operating parameters of the assay such as cell number, percentage of serum matrix, concentration of the therapeutic drug, concentration of the cross-linker, length of various incubation steps, cell age, interval between cell subculture and bioassay time, and detection equipment.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means for correcting for serum matrix effects and allows residual drug levels or anti-drug neutralizing antibodies to be quantified even in serum samples with a relatively high degree of cytotoxicity.

Comparison of competitive ligand-binding assay and bioassay formats for the measurement of neutralizing antibodies to protein therapeutics

Administration of biological therapeutic proteins can lead to unwanted immunogenicity in recipients of these products. The assessment and characterization of such immune reactions can be helpful to better understand their clinical relevance and how they relate to patient safety and therefore, have become an integral part of a product development program for biological therapeutics. Testing for anti-drug antibodies (ADA) to biological/biotechnology-derived therapeutic proteins generally follows a tiered approach. Samples are initially screened for binding antibodies; presumptive positives are then confirmed in a confirmatory assay; subsequently, confirmed-positive samples may be further characterized by titration and with a neutralizing antibody (NAb) assay. Regulatory guidances on immunogenicity state that assessing the neutralizing capacity of antibodies should preferably be done using functional bioassays, while recognizing that competitive ligand-binding (CLB) assays may be substituted when neutralizing bioassays are inadequate or not feasible. This manuscript describes case studies from four companies in which CLB assays and functional bioassays were compared for their ability to detect neutralizing ADA against a variety of biotechnology-derived therapeutic proteins. Our findings indicate that CLB assays are comparable to bioassays for the detection of NAbs, in some cases offering better detection sensitivity, lower variability, and less matrix interference.

A bioactive drug quantitation based approach for the detection of anti-drug neutralizing antibodies in human serum

We describe in this paper a novel approach for the detection of neutralizing antibodies (NAbs) and the assessment of their impact on drug exposure. This approach is based on an in-vitro cell-based bioassay utilizing receptor tyrosine phosphorylation as a cellular endpoint and comprises of two assay formats: a quantitative assay for measurement of bioactive drug concentrations in a serum sample, and a neutralizing antibody detection assay. The bioassay utilizes PC-3 cells that respond to treatment with hepatocyte growth factor (HGF) with phosphorylation of tyrosine residues on c-Met, the receptor for HGF. The drug, a fully human monoclonal antibody to human HGF, inhibits the HGF-induced response in a dose-dependent manner that can be used for quantitative determination of circulating drug levels in serum samples. The approach described here consists of testing a sample with and without an exogenously added concentration of drug. Testing the sample for its inherent bioactivity allows a determination of the circulating levels of bioactive drug. Testing the same sample with an exogenously added concentration of drug and an assessment of its analytical recovery allows a determination of NAb presence. Neutralizing antibodies, if present, are expected to impair the recovery of the exogenously added concentration of drug. This approach provides utility in gaining clinically relevant information when NAb detection is hindered by high levels of circulating drug levels using conventional NAb assay formats. Validation of this method is described in detail along with discussions on the utility of this approach.

Neutralizing antibodies to biological therapies

It is almost a given that when a foreign biologically active molecule is injected into humans it will incite immune reactions leading to antibody formation against the molecule, which at high levels of titer and affinity can interfere with its pharmacokinetic properties and block the molecule from binding to its receptor, rendering it biologically inert. Though the degree of foreign-ness tends to be the strongest driving force for the production of antibodies, the mode of administration (sc, IM, IV), dose, and frequency may also impact their development. The development of monoclonal antibody (MAb) technology has allowed for the production of very specific target-directed immunotherapies that have revolutionized the treatment of autoimmune diseases in particular, such as rheumatoid arthritis, systemic lupus erythematosus, and even multiple sclerosis (MS). Though technology has also made it possible to “humanize” the MAb as much as possible, by nature the MAb must be at least a little foreign, since the specific part of the antibody that binds to the human receptor (referred to as the idiotype) is most likely generated in a different species (figure). Assessing what impact anti-drug neutralizing antibodies (NAbs) have on drug efficacy requires attention to two different categories of drug effect: that on drug measures, such as pharmacokinetics and bioactivity, and the effect on specific disease outcome measures. Drug measures in some cases can be readily quantitated (e.g., natalizumab levels) or inferred from bioactivity measures (e.g., MxA protein expression in response to interferon-β) (IFNβ). Presuming that during the development of these agents, both the sustainable levels and their subsequent bioactivity have been optimized, neurologists assume that once administered, agents are fully capable of maximizing their effects on disease measures, which can be assessed in clinical trials. However, anti-drug antibodies can profoundly affect these measures, …

Overview of Cell-Based Tools for Pre-Clinical Assessment of Immunogenicity of Biotherapeutics

With the increasing number of biotherapeutic drugs entering clinical trials, drug-induced immunogenicity becomes more and more a topic in immunotoxicology of drug development. Immunogenicity relies on the induction or presence of antibodies recognizing a biotherapeutic protein after its administration. Anti-drug antibodies (ADA) that may either bind to a protein drug and/ or neutralize its potency may modulate the pharmacokinetics of therapeutic proteins or evoke adverse events, ranging from hypersensitivity to autoimmune reactions. Screening for binding and neutralizing ADA is integral part of the monitoring program of biotherapeutics in clinical studies. In light of the availability of powerful in silico and in vitro tools for immunogenicity risk assessment, de-risking possibilities during pre-clinical development have become worth being considered. This review, which summarizes a presentation from the Conference on Immunogenicity for Biologics, gives an overview on novel cell-based approaches in immunogenicity risk assessment in the lead optimization phase of biotherapeutics. In particular, a strategy combining a human dendritic cell and a mass spectrometry analysis is compared with in silico algorithms as to its suitability to identify T-cell epitopes conferring immunogenicity. Moreover, the possibility of utilizing T-cell activation assays to rank lead candidates according to their immunogenicity potential is discussed. Finally, a strategy is outlined as to how the results of cell-based risk assessment tools can be exploited to reduce the immunogenicity of biotherapeutic proteins in the future.