Recommendations for the design, optimization, and qualification of cell-based assays used for the detection of neutralizing antibody responses elicited to biological therapeutics

The administration of biological therapeutics can evoke some level of immune response to the drug product in the receiving subjects. An immune response comprised of neutralizing antibodies can lead to loss of efficacy or potentially more serious clinical sequelae. Therefore, it is important to monitor the immunogenicity of biological therapeutics throughout the drug product development cycle. Immunoassays are typically used to screen for the presence and development of anti-drug product antibodies. However, in-vitro cell-based assays prove extremely useful for the characterization of immunoassay-positive samples to determine if the detected antibodies have neutralizing properties. This document provides scientific recommendations based on the experience of the authors for the development of cell-based assays for the detection of neutralizing antibodies in non-clinical and clinical studies.     

A novel method for detecting neutralizing antibodies against therapeutic proteins by measuring gene expression

The presence of neutralizing antibodies against protein therapeutics is a concern in the biomedical field. Such antibodies not only reduce the efficacy of protein therapeutics, but also impose potential dangers to the patients receiving them. To date, a small number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed. Most of the existing assays, however, either involve the use of radioactive materials or have limited sensitivities and/or poor specificities. With advances in mRNA profiling and detection techniques, we have established a novel and non-radioactive bioassay system using branched DNA (bDNA) technology for detecting protein-therapeutic neutralizing antibodies in patient serum. Our assay measures the variations of target gene expression that reflect the biologic effect of the therapeutic agent and the capability of the antibodies, if present, to neutralize the therapeutics. Compared with most existing assays, the new assay is more sensitive and specific, and completely eliminates the use of radioactive materials. Application of the new assay system can be widely expanded if new target genes and responding cell lines for other therapeutics are identified or engineered.     

Advances in Predicting and Manipulating the Immunogenicity of Biotherapeutics and Vaccines

Therapeutic proteins are vital to the future of human health provision and the survival and profitability of the global pharmaceutical industry. Returns from protein therapeutics are experiencing unprecedented growth: both their number and their economic dividend have increased by an order of magnitude in the last 10 years. The potential immunogenicity of protein therapeutics raises many clinical and safety concerns. Many poorly understood factors relating to both product and host affect immune responses. Available laboratory measurement of immunogenicity is of little utility for predicting the clinical properties of biotherapeutics. Coupled with assay variability and standardization issues, this precludes adequate prediction of the biological or clinical responses of therapeutic proteins, arguing for the utilization of informatic strategies in the analysis and prediction of protein immunogenicity. Currently, many unresolved issues must be addressed and thus circumvented before effective prediction can become routine.     

Prediction of Immunogenicity of Therapeutic Proteins

Most protein therapeutics have the potential to induce undesirable immune responses in patients. Many patients develop anti-therapeutic antibodies, which can affect the safety and efficacy of the therapeutic protein, particularly if the response is neutralizing. There are a variety of factors that influence the immunogenicity of protein therapeutics and, in particular, the presence of B- and T-cell epitopes is considered to be of importance. In silico tools to identify the location of both B- and T-cell epitopes and to assess the potential for immunogenicity have been developed, and such tools provide an alternative to more complex in vitro or in vivo immunogenicity assays. This article reviews computational epitope prediction methods and also the use of manually curated databases containing experimentally derived epitope data. However, due to the complexities of the molecular interactions involved in epitope recognition by the immune system, the heterogeneity of key proteins in human populations and the adaptive nature of the immune response, in silico methods have not yet achieved a level of accuracy that enables them to be used as stand-alone tools for predicting clinical immunogenicity. Computational methods, particularly with regard to T-cell epitopes, only consider a limited number of events in the process of epitope formation and therefore routinely over-predict the number of epitopes within a molecule. Epitope databases such as the Immune Epitope Database (IEDB) and the proprietary T Cell Epitope Database? (TCED?) have reached a size and level of organization that increases their utility; however, they are not exhaustive. These methods have greatest utility as an adjunct to in vitro assays where they can be used either to reduce the amount and complexity of the in vitro screening, or they can be used as tools to analyze the sequence of the identified epitope in order to locate amino acids critical for its properties.     

Detection of neutralizing anti-therapeutic protein antibodies in serum or plasma samples containing high levels of the therapeutic protein

Neutralizing antibodies against therapeutic proteins can be potentially harmful if the antibody blocks not only the therapeutic activities of the therapeutic protein but also the normal functions of the endogenous counterpart. Detection of the neutralizing anti-therapeutic protein antibodies generally relies on bioassays measuring changes in the biologic activity of the therapeutic protein triggered by the presence of the antibody. Most of the bioassays, particularly the cell-based in vitro assays, fail to detect neutralizing anti-therapeutic protein antibodies when the remaining therapeutic protein level in the assay samples is high. The remaining therapeutic protein, either a free molecule or an immune complex with anti-therapeutic protein antibodies, can inhibit the neutralizing activity of the antibody and prevent detection. We describe the development of a procedure that uses acid dissociation and affinity adsorption to remove therapeutic protein from assay samples. With this procedure, we can detect the presence of neutralizing anti-therapeutic protein antibodies from samples containing high levels of therapeutic protein.     

Development and characterization of a human antibody reference panel against erythropoietin suitable for the standardization of ESA immunogenicity testing

Recombinant human erythropoietin (EPO) has been used therapeutically for more than two decades in the treatment of anemia. Although EPO is generally well tolerated, in rare cases, patients have developed anti-EPO antibodies that can negatively impact safety and efficacy. Therefore, the detection of antibodies against EPO is a regulatory requirement during clinical development and post-approval. Although it is a rare phenomenon, antibody-mediated pure red cell aplasia (PRCA) is a serious complication than can result from antibodies that develop and neutralize EPO as well as endogenous erythropoietin. Currently, there are no universally accepted analytical methods to detect the full repertoire of binding and neutralizing anti-EPO antibodies. A number of different methods that differ in terms of antibodies detected and assay sensitivities are used by different manufacturers. There is also a lack of antibody reference reagents, and therefore no consistent basis for detecting and measuring anti-EPO antibodies. Reference reagents, with established ranges, are essential to monitor the safety and efficacy of all erythropoiesis-stimulating agents (ESAs) structurally related to human erythropoietin. This is the first report of the development and characterization of a panel of fully human antibodies against EPO suitable as reference reagents. The characteristics of antibodies within the panel were selected based on the prevalence of non-neutralizing IgG and IgM antibodies in non-PRCA patients and neutralizing IgG antibodies, including IgG1 and IgG4, in antibody-mediated PRCA subjects. The reference panel includes antibodies of high- and low-affinity with binding specificity to neutralizing and non-neutralizing erythropoietin epitopes. The subclass of human antibodies in this reference panel includes an IgG1, IgG2, and IgG4, as well as an IgM isotype. This antibody panel could help select appropriate immunogenicity assays, guide validation, and monitor assay performance. Further, this human anti-ESA antibody panel may help set the limits of each assay platform in terms of the full repertoire of the anti-ESA antibodies, and may facilitate standardization of ESA immunogenicity reporting across assay platforms.     

Immunogenicity of protein therapeutics

Protein therapeutics, such as monoclonal antibodies, enzymes and toxins, hold significant promise for improving human health. However, repeated administration of protein therapeutics, whether natural or recombinant, often leads to the induction of undesirable anti-drug antibodies (ADAs), which interfere with or neutralize the effect of the drug. Although an immune response to foreign proteins can be expected and is well understood, the basis for the development of responses to therapeutic autologous proteins is the subject of some debate. Inflammatory components of the drug delivery vehicle, T cell responses, T and B cell epitopes in the protein drug, and the associated B cell response are all targets for interventions aimed at reducing ADA responses. Here, we review some theories put forward to explain the immunogenicity of therapeutic proteins and describe some emerging protein-engineering approaches that might prevent the development of anti-drug antibodies.     

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.     

Challenges related to the immunogenicity of parenteral recombinant proteins: Underlying mechanisms and new approaches to overcome it

Immune response elicited by therapeutic proteins is an important safety and efficacy issue for regulatory agencies, drug manufacturers, clinicians, and patients. Administration of therapeutic proteins can potentially induce the production of anti-drug antibodies or cell-mediated immune responses. At first, it was speculated that the immunogenicity is related to the non-human origin of these proteins. Later on, it was confirmed that the human proteins may also show immunogenicity. In this review article, we will focus on a number of factors, which play crucial roles in the human protein immunogenicity. These factors are related to the patient's status (or intrinsic properties) and molecular characteristics of the therapeutic protein's (or extrinsic properties). Furthermore, we will discuss available in silico, in vitro, and in vivo methods for the prediction of sequences, which may generate an immune response following parenteral administration of these proteins. In summary, nowadays, it is possible for drug manufacturers to evaluate the risk of immunogenicity of therapeutic proteins and implement a management plan to overcome the problems prior to proceeding to human clinical trials.     

Immune responses in mice vaccinated with a suicidal DNA vaccine expressing the hemagglutinin glycoprotein from the peste des petits ruminants virus

Peste des petits ruminants (PPR), an acute and highly contagious disease, affects sheep, goats, and some small ruminants. The hemagglutinin (H) glycoprotein of the PPR virus (PPRV) is considered important for inducing protective immune responses. In this study, a suicidal DNA vaccine based on the Semliki Forest virus (SFV) replicon was constructed and tested for its ability to induce immunogenicity in a mouse model. For this, the H gene of PPRV was cloned and inserted into pSCA1, an SFV replicon vector. The resultant plasmid named pSCA1-H was then transfected into BHK-21 cells following which the antigenicity of the expressed protein was confirmed by Western blotting and immunofluorescence. The pSCA1-H plasmid was then injected intramuscularly into BALB/c mice thrice at 2-week intervals. To evaluate the immunogenicity of pSCA1-H, specific antibodies and neutralizing antibodies against PPRV-H were measured using an indirect enzyme-linked immunosorbent assay and a microneutralization test, respectively. Cell-mediated immune responses were also examined using a lymphocyte proliferation assay. The results showed that pSCA1-H could express the H protein in BHK-21 cells. Specific antibodies, neutralizing antibodies, and lymphocyte proliferation responses were all induced in mice. Thus, this suicidal DNA vaccine could be a promising new approach for vaccine development against PPR.     

Differential reactivity of anti-primate and anti-human secondary antibodies against human and monkey immunoglobulins: implications for determining the sensitivity of immunogenicity assays

Development of immunogenicity assays for assessment of human antibodies to therapeutic proteins requires a quantitative determination of assay sensitivity. In the absence of true human positive controls, this is usually accomplished by utilizing affinity-purified antibodies from non-human primates or monoclonal antibodies. In the former case, it is generally considered that non-human primate antibodies will be recognized equally to human antibodies by secondary anti-human immunoglobulin reagents used in immunogenicity assays. We present results here demonstrating that this is not the case. In reality, anti-human immunoglobulin secondary antibodies do not recognize primate immunoglobulins as well as human immunoglobulins. As a result, the use of affinity purified primate antibodies to determine the sensitivity of an immunogenicity assay will likely result in the true sensitivity of the assay being underestimated.     

Measurement of absolute T cell receptor rearrangement diversity

Anti-drug antibodies are elicited by virtually all therapeutic proteins, and standardized assays are required for clinical monitoring of patients as well as for comparing antibody response to different therapeutic proteins in clinical trials. Velaglucerase alfa and imiglucerase are enzyme replacement therapies for the long-term treatment of type 1 Gaucher disease, a lysosomal storage disease resulting from an inherited deficiency of the enzyme glucocerebrosidase. We used state-of-the-art tools to develop a panel of assays for detection and characterization of antibody responses to velaglucerase alfa and imiglucerase. Highly-sensitive, direct bridging electrochemiluminescence screening assays were developed using samples from treatment-na?ve individuals with type 1 Gaucher disease to set cut points. A mouse anti-glucocerebrosidase monoclonal antibody used as a calibrator was shown to have similar affinity and binding kinetics for anti-velaglucerase alfa and anti-imiglucerase antibodies. A quantitative radioimmunoprecipitation assay for IgG antibodies was developed to eliminate false-positives from the highly sensitive screening assay. Using 59 samples from treatment-na?ve individuals with type 1 Gaucher disease, the confirmatory cut points were calculated to be 1.42 ng/mL for anti-velaglucerase alfa antibodies and 3.23 ng/mL for anti-imiglucerase antibodies. Isotype-specific indirect electrochemiluminescence assays were developed for IgE, IgA, and IgM subclasses. The IgE subclass assay was shown to be more sensitive than the confirmatory assay using sheep anti-glucocerebrosidase polyclonal antibody cross-linked with fragments specific to human IgE, with cut points for anti-velaglucerase alfa or anti-imiglucerase antibodies determined to be 0.53 and 0.55 ng/mL, respectively. An assay that detects inhibition in vitro of velaglucerase alfa and imiglucerase hydrolysis of a synthetic substrate in the presence of antibodies was developed to test for neutralizing antibodies. Using 52 individual healthy human donor samples and 35 samples from treatment-na?ve individuals with type 1 Gaucher disease, cut points for the velaglucerase alfa and imiglucerase neutralizing antibody assays were determined to be 20%, such that a sample with greater than 20% inhibition of enzyme activity in the presence of antibodies was considered positive for neutralizing antibodies. In conclusion, highly sensitive and equivalent methods were developed and validated to directly compare antibody response to velaglucerase alfa and imiglucerase treatments in patients with Gaucher disease, and may contribute to future internationally standardized assays for antibody detection in patients with Gaucher disease.     

Identification of a critical neutralization determinant of severe acute respiratory syndrome (SARS)-associated coronavirus: importance for designing SARS vaccines

The spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is not only responsible for receptor binding, but also a major antigenic determinant capable of inducing protective immunity. In this study, we demonstrated that the receptor-binding domain (RBD) of S protein is an important immunogenic site in patients with SARS and rabbits immunized with inactivated SARS-CoV. Serum samples from convalescent SARS patients and immunized rabbits had potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein. Depletion of RBD-specific antibodies from patient or rabbit immune sera by immunoadsorption significantly reduced serum-mediated neutralizing activity, while affinity-purified anti-RBD antibodies had relatively higher potency neutralizing infectivity of SARS pseudovirus, indicating that the RBD of S protein is a critical neutralization determinant of SARS-CoV during viral infection and immunization. Two monoclonal antibodies (1A5 and 2C5) targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS-CoV. Both 1A5 and 2C5 possessed potent neutralizing activities, although they directed against distinct conformation-dependant epitopes as shown by ELISA and binding competition assay. We further demonstrated that 2C5, but not 1A5, was able to block binding of the RBD to angiotensin-converting enzyme 2 (ACE2), the functional receptor on targeted cells. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV and for designing SARS vaccines.     

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.     

Conserved and variant epitopes of Plasmodium vivax Duffy binding protein as targets of inhibitory monoclonal antibodies

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains.     

Epitope characterization of pre-existing and developing antibodies to an aglycosylated monoclonal antibody therapeutic of G1m17,1 allotype

Allotypes of IgG1 molecules can influence the immunogenicity of therapeutic monoclonal antibodies and may account for the presence of some pre-existing antibodies. An electrochemiluminescent (ECL) bridging immunoassay was used to characterize the binding epitopes of anti-therapeutic antibodies (ATAs) in a Phase 1 single ascending dose clinical trial of a therapeutic aglycosylated IgG1monoclonal antibody (mAb). There was no evidence for ATAs specific for a possible neo-epitope created due to the lack of glycosylation. ATAs that developed post-treatment were specific for the F(ab')2, whereas, pre-existing ATAs were specific to the Fc region. Further characterization of the pre-existing ATAs identified the specific epitope to be the G1m1 allotype determinant in the Fc of the therapeutic. A novel competitive bridging assay was developed to verify that serum IgG1 from subjects with pre-existing anti-G1m1 antibodies was homozygous for the antithetical allotype (G1m3). The endogenous G1m allotype of all subjects was assessed and correlation to ATA incidence and adverse events was evaluated. Interestingly, the pre-existing anti-allotype antibody in subjects persisted but was not augmented after dosing, indicating the lack of a secondary immune response to this epitope. These studies indicate the relationship of the therapeutic allotype and the corresponding allotype of subjects is an important component to further understand the impact of immunogenicity on the safety and efficacy of therapeutic antibodies.