Development of ADA against recombinant human interferon beta in immune tolerant mice requires rapid recruitment of CD4⁺ T cells, induces formation of germinal centers but lacks susceptibility for (most) adjuvants.

Immunological processes leading to formation of antidrug antibodies (Abs) against recombinant human proteins remain poorly understood. Animal and clinical studies revealed that immunogenicity shares both T-cell-dependent (requirement of CD4(+) T cells, isotype switching) and T-cell-independent (involvement of Marginal Zone B cells, apparent lack of memory) characteristics. We used immune tolerant mice to study the mechanism underlying immunogenicity in more detail. We found that CD4(+) T cells were crucial at early stages of Ab responses against rhIFNß. In addition, we found a similar number of germinal centers (GCs) in spleen after rhIFNß treatment as after treatment with a foreign protein. However, neither Ab titers nor the number of GCs was increased by adsorption of rhIFNß on aluminum hydroxide. Therefore, we tested the effect of several immune adjuvants in a follow-up study. We found that only conjugation of rhIFNß to a carrier protein (cholera toxin subunit B) was effective in boosting Ab titers. However, these conjugates failed to trigger rhIFNß specific memory formation. Our findings show that early events of the immunogenicity reaction to self-proteins are CD4(+) T-cell dependent. Nevertheless, despite those similarities, immunogenicity of human proteins is clearly not a classical CD4(+) T-cell-dependent response.

Practical considerations for the pharmacokinetic and immunogenic assessment of antibody-drug conjugates.

Currently, the most bioanalytically challenging drugs are antibody-drug conjugates (ADCs), constructs comprising a monoclonal antibody and a cytotoxic drug connected by a linker. The bioanalytical challenges arise from the heterogeneous nature of ADCs and their complex in vivo behavior, resulting in a high number of analytes to be measured. Measuring the concentration of biologics in blood/plasma/serum is a necessity to properly assess their pharmacokinetic (PK)/pharmacodynamic behaviors in vivo. An additional bioanalytical challenge is to monitor the stability of the ADCs, as cytotoxic drugs released from the ADC in blood circulation may pose a potential safety risk because of their high cytotoxic potency. The nature of ADCs does not only complicate bioanalysis, but also immunogenicity assessment. Questions, such as 'Which part of the ADCs is the anti-drug antibodies directed against?' may arise, and their answer normally includes several immunogenicity risk assessment strategies. This review will focus on the bioanalytical challenges of ADCs, current approaches involving ligand-binding assays (LBAs), liquid chromatography and mass spectrometry platforms, and recommendations on which approach to use for which stage of drug development, and will close with immunogenicity assessment. In order to appropriately tackle the bioanalytical and immunogenic challenges of ADCs and consider every angle, the authors of this review have expertise in ligand binding and liquid chromatography-mass spectrometry.

Development of a transgenic mouse model to study the immunogenicity of recombinant human insulin.

Mouse models are commonly used to assess the immunogenicity of therapeutic proteins and to investigate the immunological processes leading to antidrug antibodies. The aim of this work was to develop a transgenic (TG) Balb/c mouse model for evaluating the immunogenicity of recombinant human insulin (insulin) formulations. Validation of the model was performed by measuring the antibody response against plain and particulate insulin in TG and nontransgenic (NTG) mice. Intraperitoneal administration of insulin (20 µg/dose, 12 doses over a period of 4 weeks) did not break the immune tolerance of the TG mice, whereas it did elicit antibodies in NTG mice. The immune tolerance of TG mice could be circumvented, albeit at low titers, by administering insulin covalently bound to 50-nm polystyrene nanoparticles. The TG mouse model was employed to compare the immunogenicity of oxidized aggregated insulin, oxidized nonaggregated insulin, and three commercially available formulations of insulin variants (i.e., Levemir®, Insulatard®, and Actrapid®). Oxidized insulin, aggregated or nonaggregated, was moderately immunogenic in TG mice (50% and 33% responders, respectively), whereas the immunogenicity of the commercial formulations was low. This model can be used to compare the immunogenicity of insulin formulations and to study immune mechanisms of antibody formation against insulin.

Antibody response against Betaferon® in immune tolerant mice: involvement of marginal zone B-cells and CD4+ T-cells and apparent lack of immunological memory.

PURPOSE: The immunological processes underlying immunogenicity of recombinant human therapeutics are poorly understood. Using an immune tolerant mouse model we previously demonstrated that aggregates are a major trigger of the antidrug antibody (ADA) response against recombinant human interferon beta (rhIFNß) products including Betaferon®, and that immunological memory seems to be lacking after a rechallenge with non-aggregated rhIFNß. The apparent absence of immunological memory indicates a CD4+ T-cell independent (Tind) immune response underlying ADA formation against Betaferon®. This hypothesis was tested. METHODS: Using the immune tolerant mouse model we first validated that rechallenge with highly aggregated rhIFNß (Betaferon®) does not lead to a subsequent fast increase in ADA titers, suggesting a lack of immunological memory. Next we assessed whether Betaferon® could act as Tind antigen by inactivation of marginal zone (MZ) B-cells during treatment. MZ B-cells are major effector cells involved in a Tind immune response. In a following experiment we depleted the mice from CD4+ T-cells to test their involvement in the ADA response against Betaferon®. RESULTS: Inactivation of MZ B-cells at the start of Betaferon® treatment drastically lowered ADA levels, suggesting a Tind immune response. However, persistent depletion of CD4+ T-cells before and during Betaferon® treatment abolished the ADA response in almost all mice. CONCLUSION: The immune response against rhIFNß in immune tolerant mice is neither a T-cell independent nor a classical T-cell dependent immune response. Further studies are needed to confirm absence of immunological memory (cells).

Natural antibodies against bone morphogenic proteins and interferons in healthy donors and in patients with infections linked to type-1 cytokine responses.

In patients receiving recombinant therapeutic proteins, the production of antibodies against the therapeutics is a rising problem. The antibodies can neutralize and interfere with the efficacy and safety of drugs and even cause severe side effects if they cross-react against the natural, endogenous protein. Various factors have been identified to influence the immunogenic potential of recombinant human therapeutics, including several patients' characteristics. In recent years, so-called naturally occurring antibodies against cytokines and growth factors have been detected in naive patients before start of treatment with recombinant human therapeutics. The role of naturally occurring antibodies is not well understood and their influence on production of anti-drug antibodies is not known. One might speculate that the presence of naturally occurring antibodies increases the likelihood of eliciting anti-drug antibodies once treatment with the corresponding recombinant therapeutic protein is started. We screened serum samples from 410 healthy controls and patients for auto-antibodies against bone morphogenetic proteins (BMPs) 2 and 7 and interferon (IFN)-α, -ß, and -γ in a new 3-step approach: rough initial screening, followed by competition and protein A/G depletion. Naturally occurring antibodies against these proteins were detected in 2% to 4% of the tested sera. Individuals who are 65 years or older had a slightly higher occurrence of naturally occurring antibodies. Auto-antibodies against BMP-7 and IFN-α were mainly comprised of IgM isotypes, and natural antibodies against BMP-2, IFN-ß, and -γ were mainly IgG. To ensure assay specificity, assays were also used to detect antibodies against BMP-7 in patients being treated with rhBMP-7 before and after surgical procedure. Fifty percent of the treated patients had persistent anti-BMP-7 antibodies over time. The 3-step approach provides an attractive tool to identify naturally occurring antibodies in naive patients.

Oxidized and aggregated recombinant human interferon beta is immunogenic in human interferon beta transgenic mice.

PURPOSE: To study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFNß-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice. METHODS: Untreated rhIFNß-1a was degraded by metal-catalyzed oxidation, H(2)O(2)-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFNß-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15× during 3 weeks. Both binding and neutralizing antibodies were measured. RESULTS: All rhIFNß-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFNß-1a contained high levels of covalent aggregates as compared with untreated rhIFNß-1a. H(2)O(2)-treated rhIFNß-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFNß-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFNß-1a. All degraded samples showed alterations in tertiary protein structure. Wild-type mice showed equally high antibody responses against all preparations. Transgenic mice were discriminative, showing elevated antibody responses against both metal-catalyzed oxidized and H(2)O(2)-treated rhIFNß-1a as compared to untreated and guanidine-treated rhIFNß-1a. CONCLUSIONS: Oxidation-mediated aggregation increased the immunogenicity of rhIFNß-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic.

Aggregated recombinant human interferon Beta induces antibodies but no memory in immune-tolerant transgenic mice.

PURPOSE: To study the influence of protein aggregation on the immunogenicity of recombinant human interferon beta (rhIFNbeta) in wild-type mice and transgenic, immune-tolerant mice, and to evaluate the induction of immunological memory. METHODS: RhIFNbeta-1b and three rhIFNbeta-1a preparations with different aggregate levels were injected intraperitoneally in mice 15x during 3 weeks, and the mice were rechallenged with rhIFNbeta-1a. The formation of binding (BABs) and neutralizing antibodies (NABs) was monitored. RESULTS: Bulk rhIFNbeta-1a contained large, mainly non-covalent aggregates and stressed rhIFNbeta-1a mainly covalent, homogeneous (ca. 100 nm) aggregates. Reformulated rhIFNbeta-1a was essentially aggregate-free. All products induced BABs and NABs in wild-type mice. Immunogenicity in the transgenic mice was product dependent. RhIFNbeta-1b showed the highest and reformulated rhIFNbeta-1a the lowest immunogenicity. In contrast with wild-type mice, transgenic mice did not show NABs, nor did they respond to the rechallenge. CONCLUSIONS: The immunogenicity of the products in transgenic mice, unlike in wild-type mice, varied. In the transgenic mice, neither NABs nor immunological memory developed. The immunogenicity of rhIFNbeta in a model reflecting the human immune system depends on the presence and the characteristics of aggregates.

Hybrid transgenic immune tolerant mouse model for assessing the breaking of B cell tolerance by human interferon beta.

To date, the therapeutic efficacy of recombinant human proteins is limited by their potential to break B cell tolerance in patients. The formation of neutralising antibodies (NABs) directed against recombinant human interferon beta (rhIFNbeta) is associated with a decrease in the therapeutic effect of the protein. For this reason, there is a need to study factors that can cause the immunogenicity of rhIFNbeta. Transgenic C57Bl/6 mice that are immune tolerant for human interferon beta (hIFNbeta) have been employed in a mouse model for assessing the breaking of immune tolerance by rhIFNbeta. In this study, we used the original C57Bl/6 mouse model as well as the hybrid offspring from crossings of transgenic C57Bl/6 mice with wildtype FVB/N mice to study the immunogenicity of three commercial rhIFNbeta products, Rebif, Avonex and Betaferon. As determined by ELISA, wildtype C57Bl/6 mice failed to form binding antibodies (BABs) against Rebif and Avonex formulated with human serum albumin. Because not all interferon beta products induce antibodies in wildtype C57Bl/6 mice, the transgenic C57Bl/6 mice cannot be used to study the breaking of tolerance by these products. However, the crossing of transgenic C57Bl/6 mice with FVB/N mice resulted in wildtype hybrid offspring in which all products were immunogenic and transgenic hybrid offspring that showed immune tolerance for hIFNbeta. Thus, these C57Bl/6 x FVB/N hybrid transgenic mice can be used to study the breaking of immune tolerance for all rhIFNbeta products. Of the three products, only Betaferon was able to break immune tolerance in the transgenic hybrids. With an MxA gene expression inhibition assay, NABs were detected in Betaferon treated wildtype hybrid mice, but not in transgenic hybrid mice, indicating a distinct immune mechanism in wildtype and transgenic mice. A pegylated rhIFNbeta-1a variant, PEG-rhIFNbeta-1a, induced antibodies in wildtype hybrid mice, but did not break the immune tolerance of transgenic hybrid mice. This suggests that pegylation did not affect the potential of rhIFNbeta-1a to break B cell tolerance.

Immunological mechanism underlying the immune response to recombinant human protein therapeutics.

Recombinant human (rhu) protein therapeutics are powerful tools to treat several severe diseases such as multiple sclerosis and diabetes mellitus, among others. A major drawback of these proteins is the production of anti-drug antibodies (ADAs). In some cases, these ADAs have neutralizing capacity and can interfere with the efficacy and safety of the drug. Little is known about the immunological mechanisms underlying the unwanted immune response against human homolog protein therapeutics. This article aims to provide current insights into recent immunological developments and to link this with regard to production of ADAs. A particular focus is given to aggregates being present in a rhu protein formulation and their impact on the immune system, subsequently leading to breakage of tolerance and formation of ADAs. Aggregation is one of the key factors in immunogenicity and by reducing aggregation one can reduce immunogenicity and make drugs safer and more efficient.

B-1 cells and naturally occurring antibodies: influencing the immunogenicity of recombinant human therapeutic proteins?

Recombinant human therapeutic proteins are increasingly being used to treat serious and life-threatening diseases like multiple sclerosis, diabetes mellitus, and cancer. An important side effect of these proteins is the development of antidrug antibodies, which can be neutralizing and thus interfere with the efficacy and safety of the drug. Some biophysical properties, for example, aggregation, also can initiate the immunogenic response to human therapeutics. Many other factors including patients' characteristics may influence this response. Besides induced antibodies, autoantibodies (i.e. naturally occurring antibodies [NAs]) against therapeutic relevant proteins in naïve patients are increasingly being identified. The role of autoreactive B cells and their escape from deletion, production of NAs and their pivotal function in the immune system, the dualistic role of B-1 cells in autoimmunity, and the influence of NAs on disease outcome and their possible impact on the efficacy of human therapeutics will be presented and discussed.