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.     

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.     

Immunogenicity issues in drug development

Immunogenicity is an important factor that manufacturers must consider as they develop new protein therapeutics. It is important to understand the immunogenicity of new proteins both at the preclinical phase and in the clinical phase of development. This paper provides an overview of the issues that manufacturers should consider including some of the potential reasons that some proteins induce an immune response, a discussion regarding current methodology used to understand immunogenicity, and some examples of marketed protein therapeutics with immunogenicity issues. Given the increasing scrutiny from regulatory agencies around the way immunogenicity is assessed by manufacturers, the strategy of detecting and characterizing antibodies that are formed against protein therapeutics is becoming an important topic. Screening assays are typically performed first on all serum samples collected in the course of a trial to detect the presence of antibodies that can bind to the protein therapeutic. There are several platforms in use: radioimmune precipitation assays (RIP), enzyme linked immunosorbent assays (ELISA), electrochemiluminescent assays (ECL), and biosensor-based assays. Each has its advantages and disadvantages, and needs to be evaluated to identify the optimal platform for a specific therapeutic protein. Once antibodies are identified, a confirmatory assay is performed to verify and characterize the antibodies. A biological assay should be used next to test if these antibodies are capable of neutralizing the biological effect of the drug. Any sample that is positive for neutralizing antibodies, indicates that the antibody is probably having an impact on the patient's ability to derive full benefit from the therapeutic protein, and may be critical for patient safety.     

Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products

The appropriate evaluation of the immunogenicity of biopharmaceuticals is of major importance for their successful development and licensure. Antibodies elicited by these products in many cases cause no detectable clinical effects in humans. However, antibodies to some therapeutic proteins have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In addition, antibodies can affect drug efficacy. In non-clinical studies, anti-drug antibodies (ADA) can complicate interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, it is important to develop testing strategies that provide valid assessments of antibody responses in both non-clinical and clinical studies. This document provides recommendations for antibody testing strategies stemming from the experience of contributing authors. The recommendations are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry. The strategies proposed are also expected to contribute to better understanding of antibody responses and to further advance immunogenicity evaluation.     

Immunogenicity of biotherapeutics-an overview

With the recent increase in the approval and use of biotherapeutics in clinical practice, management of the development of anti-drug antibodies (ADA) has become a key issue for effective long-term use of these drugs. In most instances, the clinical benefit derived from the use of the therapeutics outweighs the risk of developing ADA. In rare instances, however, safety issues accompany development of ADA. Although it is unclear why certain individuals generate an immune response while others tolerate the drug, growing experience from the clinic has facilitated a better appreciation of many patient-, disease- and product-related factors that contribute to immunogenicity. Furthermore, improvements in protein production, purification and delivery methods along with use of humanized or fully human recombinant proteins have helped to reduce the rates of immunogenicity considerably. This document provides an overview of the scientific reasons for developing an immunogenic response, factors that contribute to the immunogenicity of biotherapeutics, clinical impact of immunogenicity and general strategies used to manage this risk.     

ADAM metallopeptidase domain 17 (ADAM17) is naturally processed through major histocompatibility complex (MHC) class I molecules and is a potential immunotherapeutic target in breast, ovarian and prostate cancers

Selection of suitable antigens is critical for the development of cancer vaccines. Most desirable are over-expressed cell surface proteins that may serve as targets for both antibodies and T cells, thus maximizing a concerted immune response. Towards this goal, we characterized the relevance of tumour necrosis factor-α-converting enzyme (ADAM17) for such targeted therapeutics. ADAM17 is one of the several metalloproteinases that play a key role in epidermal growth factor receptor (EGFR) signalling and has recently emerged as a new therapeutic target in several tumour types. In the present study, we analysed the expression profile of ADAM17 in a variety of normal and cancer cells of human origin and found that this protein is over-expressed on the surface of several types of cancer cells compared to the normal counterparts. Furthermore, we analysed the presentation of a human leucocyte antigen (HLA)-A2-restricted epitope from ADAM17 protein to specific T cells established from normal donors as well as ovarian cancer patients. Our analysis revealed that the HLA-A2-restricted epitope is processed efficiently and presented by various cancer cells and not by normal cells. Tumour-specific T cell activation results in the secretion of both interferon-γ and granzyme B that can be blocked by HLA-A2 specific antibodies. Collectively, our data present evidence that ADAM17 can be a potential target antigen to devise novel immunotherapeutic strategies against ovarian, breast and prostate cancer.     

Thyrotrophin receptor-specific memory T cell responses require normal B cells in a murine model of Graves' disease

The role of B cells as antigen-presenting cells is being recognized increasingly in immune responses to infections and autoimmunity. We compared T cell responses in wild-type and B cell-deficient mice immunized with the thyrotrophin receptor (TSHR), the major autoantigen in Graves' disease. Three B cell-deficient mouse strains were studied: JHD (no B cells), mIgM (membrane-bound monoclonal IgM+ B cells) and (m + s)IgM (membrane-bound and secreted monoclonal IgM). Wild-type and B cell-deficient mice (BALB/c background) were studied 8 weeks after three injections of TSHR or control adenovirus. Only wild-type mice developed IgG class TSHR antibodies and hyperthyroidism. After challenge with TSHR antigen, splenocyte cultures were tested for cytokine production. Splenocytes from TSHR adenovirus injected wild-type and mIgM-mice, but not from JHD- or (m + s)IgM- mice, produced interferon (IFN)-gamma in response to TSHR protein. Concanavalin A and pokeweed mitogen induced comparable IFN-gamma secretion in all groups of mice except in the JHD strain in which responses were reduced. The absence in (m + s)IgM mice and presence in mIgM mice of an anamnestic response to TSHR antigen was unrelated to lymphoid cell types. Surprisingly, although TSHR-specific antibodies were undetectable, low levels of serum IgG were present in mIgM- but not (m + s)IgM mice. Moreover, IFN-gamma production by antigen-stimulated splenocytes correlated with IgG levels. In conclusion, T cell responses to TSHR antigen developed only in mice with IgG-secreting B cells. Consequently, in the TSHR-adenovirus model of Graves' disease, some normal B cells appear to be required for the development of memory T cells.     

Statistical considerations for calculation of immunogenicity screening assay cut points

Most therapeutic proteins induce an unwanted immune response. Antibodies elicited by these therapeutic proteins may significantly alter drug safety and efficacy, highlighting the need for the strategic assessment of immunogenicity at various stages of clinical development. Immunogenicity testing is generally conducted by a multi-tiered approach whereby patient samples are initially screened for the presence of anti-drug antibodies in a screening assay. The screening assay cut point is statistically determined by evaluation of drug-na?ve samples and is typically chosen to correspond to a false positive rate of 5%. While various statistical approaches for determination of this screening cut point have been commonly adopted and described in the immunogenicity literature, the performance of these approaches has not been fully evaluated. This paper reviews various statistical approaches for cut point calculation, evaluates the impact of sampling design and variability on the performance of each statistical approach, and highlights the difference between an 'average' or 'confidence-level' cut point in order to develop more specific recommendations regarding the statistical calculation of immunogenicity screening cut points.     

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.     

Pneumococcal surface protein A (PspA) is effective at eliciting T cell-mediated responses during invasive pneumococcal disease in adults

Humoral immune response is essential for protection against invasive pneumococcal disease and this property is the basis of the polysaccharide-based anti-pneumococcal vaccines. Pneumococcal surface protein A (PspA), a cell-wall-associated surface protein, is a promising component for the next generation of pneumococcal vaccines. This PspA antigen has been shown to stimulate an antibody-based immunity. In the present study, we evaluated the capacity of PspA to stimulate CD4+ T cells which are needed for the correct development of a B cell based immune response in humans. Cellular immunity to PspA was evaluated by whole-blood culture with different pneumococcal antigens, followed by flow cytometric detection of activated CD4+CD25+ T cells. T cell-mediated immune responses to recombinant PspA proteins were assessed in acute-phase and convalescent blood from adults with invasive pneumococcal disease and in blood from healthy subjects. All cases had detectable antibodies against PspA on admission. We found that invasive pneumococcal disease induced transient T cell depletion but adaptive immune responses strengthened markedly during convalescence. The increased production of both interleukin (IL)-10 and interferon (IFN)-gamma during convalescence suggests that these cytokines may be involved in modulating antibody-based immunity to pneumococcal disease. We demonstrated that PspA is efficient at eliciting T cell immune responses and antibodies to PspA. This study broadens the applicability of recombinant PspA as potent pneumococcal antigen for vaccination against S. pneumoniae.     

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.     

Selective CD4+ T cell help for antibody responses to a large viral pathogen: deterministic linkage of specificities

Antibody responses are critical components of protective immune responses to many pathogens, but parameters determining which proteins are targeted remain unclear. Vaccination with individual MHC-II-restricted vaccinia virus (VACV, smallpox vaccine) epitopes revealed that CD4(+) T cell help to B cells was surprisingly nontransferable to other virion protein specificities. Many VACV CD4(+) T cell responses identified in an unbiased screen targeted antibody virion protein targets, consistent with deterministic linkage between specificities. We tested the deterministic linkage model by efficiently predicting new vaccinia MHC II epitopes (830% improved efficiency). Finally, we showed CD4(+) T cell help was limiting for neutralizing antibody development and protective immunity in vivo. In contrast to the standard model, these data indicate individual proteins are the unit of B cell-T cell recognition for a large virus. Therefore, MHC restriction is a key selective event for the antiviral antibody response and is probably important for vaccine development to large pathogens.     

Isolation of human anti-idiotypic antibodies by phage display for clinical immune response assays

The clinical development of therapeutic proteins requires assays that measure the pharmacokinetic (PK) profile of, and the potential immune response (IR) to, the protein agent. Each assay requires reagents that are highly specific for the therapeutic protein. For therapeutic monoclonal antibodies, anti-CDR-specific, or anti-idiotypic (anti-id), antibodies are an ideal class of reagents suitable for these assays because of their high specificity and affinity to the drug antibody. We generated anti-ids to two human antibodies by antibody phage display using the MorphoSys HuCAL GOLD Fab library. To selectively target the CDR regions, serum and a framework-matched mAb were included as competitors during the phage selection process. Panels of CDR-specific Fabs, with low to sub-nM affinities, were isolated against both targets. The CDR specificity of these Fabs was shown by their lack of binding to a framework-matched control mAb and by competition of this binding with the soluble antigens of the respective therapeutic mAb targets. The candidate anti-id Fabs were able to detect both immobilized and soluble target Ab without being affected by serum, a requirement for both PK assay and the IR bridging assay format. Combinations of the Fabs for PK detection assays were identified by pairwise binding studies, although the pair for one target mAb lacks the desired sensitivity for PK assays. To evaluate their potential as anti-drug antibodies (ADAs), the best Fabs for one of the targets were converted and produced as the required bivalent human mAbs. In comparison to rodent mAbs and primate polyclonal serum, the phage display derived human mAbs were equally effective as reference standards. Our results demonstrate that competition-based phage selection can be an effective method for the isolation of anti-idiotypic antibodies for PK and IR assay development, and in this latter case, overcome limitations of current methods using rodent derived anti-ids.     

Immunogenicity and autoimmunity during anti-TNF therapy

The introduction of anti-tumour necrosis factor (TNF) agents for the treatment of rheumatoid arthritis (RA), Crohn's disease (CD) or spondyloarthritis (SpA) has revolutionised the therapeutic approach to patients with active disease failing to respond to conventional therapy. However, some of the patients treated with selective TNF inhibitors may develop autoantibodies, such as antinuclear antibodies (ANAs) and anti-double-stranded DNA (anti-dsDNA) antibodies. Furthermore, anti-phospholipid antibodies, which are mainly detected by means of anti-cardiolipin assays, have been found in RA patients receiving TNF blockers. There have also been a number of reports of the development of anti-drug antibodies, of which those against infliximab can interfere with the drug's pharmacokinetics (and therefore its effects), and may also cause acute and delayed infusion and injection site reactions. The onset of autoimmune diseases during biological treatment is rare, but it needs to be promptly recognised in order to plan appropriate patient management. The addition of an immunosuppressive drug can reduce the induction of anti-TNF antibodies.     

TNFRSF13B in B cell responses to organ transplantation

Antibodies directed against organ transplants are thought to pose the most vexing hurdle to enduring function and survival of the transplants, particularly organ xenotransplants, and accordingly basic and clinical investigation has focused on elucidating the specificity and pathogenicity of graft-specific antibodies. While much has been learned about these matters, far less is known about the B cells producing graft-specific antibodies and why these antibodies appear to injure some grafts but not others. With the goal of addressing those questions, we have investigated the properties of tumor necrosis factor receptor super family-13B (TNFRSF13B), which regulates various aspects of B cell responses. A full understanding of the functions of TNFRSF13B however is hindered by extreme polymorphism and by diversity of interactions of the protein. Nevertheless, TNFRSF13B variants have been found to exert distinct impact on natural and elicited antibody responses and host defense and mutations of TNFRSF13B have been found to influence the propensity for development of antibody-mediated rejection of organ transplants. Because B cell responses potentially limit application of xenotransplantation, understanding how TNFRSF13B diversity and TNFRSF13B variants govern immunity in xenotransplantation could inspire development of novel therapeutics that could in turn accelerate clinical implementation of xenotransplantation.     

Immune inhibitory proteins and their pathogenic and therapeutic implications in autoimmunity and autoimmune hepatitis

Abstract

Key inhibitory proteins can blunt immune responses to self-antigens, and deficiencies in this repertoire may promote autoimmunity. The goals of this review are to describe the key immune inhibitory proteins, indicate their possible impact on the development of autoimmune disease, especially autoimmune hepatitis, and encourage studies to clarify their pathogenic role and candidacy as therapeutic targets. English abstracts were identified in PubMed by multiple search terms. Full length articles were selected for review, and secondary and tertiary bibliographies were developed. Cytotoxic T lymphocyte antigen-4 impairs ligation of CD28 to B7 ligands on antigen presenting cells and inhibits the adaptive immune response by increasing anti-inflammatory cytokines, generating regulatory T cells, and reducing T cell activation and proliferation. Programed cell death antigen-1 inhibits T cell selection, activation, and proliferation by binding with two ligands at different phases and locations of the immune response. A soluble alternatively spliced variant of this protein can dampen the inhibitory signal. Autoimmune hepatitis has been associated with polymorphisms of the cytotoxic T lymphocyte antigen-4 gene, reduced hepatic expression of a ligand of programed cell death antigen-1, an interfering soluble variant of this key inhibitory protein, and antibodies against it. Findings have been associated with laboratory indices of liver injury and suboptimal treatment response. Abatacept, belatacept, CD28 blockade, and induction of T cell exhaustion are management considerations that require scrutiny. In conclusion, deficiencies in key immune inhibitory proteins may promote the occurrence of autoimmune diseases, such as autoimmune hepatitis, and emerging interventions may overcome these deficiencies. Investigations should define the nature, impact and management of these inhibitory disturbances in autoimmune hepatitis.     

ELISA methods for the analysis of antibody responses induced in multiple sclerosis patients treated with recombinant interferon-beta.

Upon treatment with protein therapeutics, a subset of patients will typically develop antibodies against the drug. These anti-drug antibodies can be of concern because they have the potential to alter the drug's therapeutic activity. In the case of relapsing-remitting multiple sclerosis (RRMS) patients receiving recombinant interferon-beta (IFN-beta), those receiving BETASERON (IFN-beta-1b; E. coli expressed, non-glycosylated, des-Met-1, Cys17Ser recombinant IFN-beta) have a higher incidence of IFN-beta specific antibodies compared to those receiving AVONEX (IFN-beta-1a; mammalian cell-expressed, natural sequence, glycosylated recombinant IFN-beta). The current study reports the development and characterization of ELISAs that detect distinct components of the anti-IFN-beta response in patients' sera, and therefore can potentially be used to characterize the composition of the anti-IFN-beta antibody response. ELISAs were developed using a constant detecting reagent but a variety of IFN-beta-derived test antigens (e.g., native IFN-beta, biotinylated IFN-beta, IFN-beta peptides) and capture methods. Assays were characterized using serum samples from a small number of patients treated with recombinant IFN-beta (either BETASERON or AVONEX). Assays in which IFN-beta was captured via a specific mAb, or in which biotinylated IFN-beta was captured via streptavidin, detected serum antibodies that recognize IFN-beta in its native structural state. In contrast, assays in which IFN-beta was coated directly onto the assay plates detected antibodies that recognize forms of IFN-beta possessing a folded structure distinct from the native structure. Certain epitopes present on native IFN-beta were not represented in these assays in which the test antigen was directly coated on plastic. Antibodies specific for linear epitopes could be detected using linear peptides as test antigens; the locations of these epitopes were mapped by reference to the X-ray crystal structure of IFN-beta-1a. Together, these data show that the mode of antigen presentation employed in IFN-beta ELISAs determines which antibody specificities are detected, and can affect whether or not a given serum sample is identified as positive for anti-IFN-beta antibodies. As a consequence, screening samples in a single ELISA format presenting IFN-beta in a non-native form may lead to underestimation of the incidence of IFN-beta treated MS patients that have generated antibodies specific to the native, active form of the drug.     

Immunogenicity of therapeutic proteins: Influence of aggregation

Abstract

The elicitation of anti-drug antibodies (ADA) against biotherapeutics can have detrimental effects on drug safety, efficacy, and pharmacokinetics. The immunogenicity of biotherapeutics is, therefore, an important issue. There is evidence that protein aggregation can result in enhanced immunogenicity; however, the precise immunological and biochemical mechanisms responsible are poorly defined. In the context of biotherapeutic drug development and safety assessment, understanding the mechanisms underlying aggregate immunogenicity is of considerable interest. This review provides an overview of the phenomenon of protein aggregation, the production of unwanted aggregates during bioprocessing, and how the immune response to aggregated protein differs from that provoked by non-aggregated protein. Of particular interest is the nature of the interaction of aggregates with the immune system and how subsequent ADA responses are induced. Pathways considered here include ‘classical’ activation of the immune system involving antigen presenting cells and, alternatively, the breakdown of B-cell tolerance. Additionally, methods available to screen for aggregation and immunogenicity will be described. With an increased understanding of aggregation-enhanced immune responses, it may be possible to develop improved manufacturing and screening processes to avoid, or at least reduce, the problems associated with ADA.