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).

Effect of treatment regimen on the immunogenicity of human interferon Beta in immune tolerant mice.

PURPOSE: Interferon beta is commonly used as therapeutic in the first line of therapy for multiple sclerosis. However, depending on the product, it induces an antibody response in up to 60% of patients. This study evaluated the impact of therapy related factors like dose, route of administration and administration frequency on the immunogenicity of one of the originator interferon beta drugs (Betaferon®) in an immune tolerant transgenic mouse model. METHODS: Immune tolerant transgenic mice received injections with Betaferon® via different routes, doses and injection frequencies. Anti-drug antibody (ADA) production was measured by ELISA to assess immunogenicity. RESULTS: A single injection of Betaferon® was found to be sufficient for the induction of ADAs. The antibody titer was enhanced with increasing dose and treatment frequency. Among the tested administration routes, the intravenous route was the most immunogenic one, which is in contradiction with one of the dogma in immunogenicity research according to which subcutaneous administration is the most immunogenic route. Intramuscular, intraperitoneal and subcutaneous injections resulted in comparable immunogenicity. CONCLUSION: This study shows that treatment related factors affect significantly immunogenicity of Betaseron® and therefore substantiate the need for further studies on these factors in patients.

Monoclonal Antibody Dimers Induced by Low pH, Heat, or Light Exposure Are Not Immunogenic Upon Subcutaneous Administration in a Mouse Model.

The presence of protein aggregates is commonly believed to be an important risk factor for immunogenicity of therapeutic proteins. Among all types of aggregates, dimers are relatively abundant in most commercialized monoclonal antibody (mAb) products. The aim of this study was to investigate the immunogenicity of artificially created mAb dimers relative to that of unstressed and stressed mAb monomers. A monoclonal murine IgG1 (mIgG1) antibody was exposed to low pH, elevated temperature, or UV irradiation to induce dimerization. Dimers and monomers were purified via size-exclusion chromatography. Physicochemical analysis revealed that upon all stress conditions, new deamidation or oxidation or both of amino acids occurred. Nevertheless, the secondary and tertiary structures of all obtained dimers were similar to those of unstressed mIgG1. Isolated dimers were administered subcutaneously in Balb/c mice, and development of antidrug antibodies and accumulation of follicular T helper cells in draining lymph nodes and spleens were determined. None of the tested dimers or stressed monomers were found to be more immunogenic than the unstressed control in our mouse model. In conclusion, both dimers and monomers generated by using 3 different stress factors have a low immunogenicity similar to that of the unstressed monomers.

[Transformations of erythrocytes shape and its regulation]. / Zmiany ksztaltu erytrocytów i czynniki je wywolujace.

Erythrocytes can occur in many different shapes. Most of them are pathological and can be involved in diseases such a hemolytic anemia's and sickle cell anemia. Only three kinds of red blood cells are no pathological. Echinocytes, stomatocytes and discocytes can occure in blood stream of healthy organism. The echinocyte-dyscocyte-stomatocyte transformation protects red blood cells from lysis caused by echinocytogenic agents (hypertonic saline, basic pH, vanadate, anionic amphiphiles, ATP depletion etc.) or stomatocytogenic agents (hypotonic saline, acidic pH, cationic amphiphiles etc.). Mechanisms of these transformations can be classified in three group: redistribution of bilayer's lipid, modification Donnan's equilibrium and interaction of band 3 protein with different type of external factors.

Submicron Size Particles of a Murine Monoclonal Antibody Are More Immunogenic Than Soluble Oligomers or Micron Size Particles Upon Subcutaneous Administration in Mice.

Protein aggregates are one of the several risk factors for undesired immunogenicity of biopharmaceuticals. However, it remains unclear which features determine whether aggregates will trigger an unwanted immune response. The aim of this study was to determine the effect of aggregates' size on their relative immunogenicity. A monoclonal murine IgG1 was stressed by exposure to low pH and elevated temperature followed by stirring to obtain aggregates widely differing in size. Aggregate fractions enriched in soluble oligomers, submicron size particles and micron size particles were isolated via centrifugation or size-exclusion chromatography and characterized physicochemically. The secondary and tertiary structures of aggregates were altered in a similar way for all the fractions, while no substantial chemical degradation was observed. Development of anti-drug antibodies was measured after subcutaneous administration of each enriched fraction to BALB/c mice. Among all tested fractions, the most immunogenic was the one highly enriched in submicron size particles (∼100-1000 nm). Fractions composed of micron size (>1-100 µm) particles or soluble oligomers (<100 nm) were not immunogenic under the dosing regimen studied in this work. These results show that aggregate size is an important factor for protein immunogenicity.

Perspectives on Subcutaneous Route of Administration as an Immunogenicity Risk Factor for Therapeutic Proteins.

An increasing number of therapeutic proteins are being developed for delivery through the subcutaneous (SC) route of administration. Relative to intravenous (IV) administration, the SC route offers more convenience to patients, flexibility in dosing, and potential to reduce health care costs. There is a perception that SC administration can pose a higher immunogenicity risk than IV administration for a given protein. To evaluate whether there is a difference in therapeutic protein immunogenicity associated with administration routes, a more detailed understanding of the interactions with the immune system by each route is needed. Few approved therapeutic proteins have available clinical immunogenicity data sets in the public domain that represent both IV and SC administration routes. This has prevented a direct comparison of the 2 routes of administration across a large sample size. Of the 6 marketed products where SC and IV route-related incidences of anti-drug antibody (ADA) were available, 4 were associated with higher immunogenicity incidence with SC. In other cases, there was no apparent difference between the SC and IV routes. Overall, the ADA incidence was low (<15%) with no impact on safety or efficacy. The challenges associated with identifying specific risk factors unique to SC administration are discussed.

Fate of Multimeric Oligomers, Submicron, and Micron Size Aggregates of Monoclonal Antibodies Upon Subcutaneous Injection in Mice.

The aim of this study was to examine the fate of differently sized protein aggregates upon subcutaneous injection in mice. A murine and a human monoclonal immunoglobulin G 1 (IgG1) antibody were labeled with a fluorescent dye and subjected to stress conditions to create aggregates. Aggregates fractionated by centrifugation or gel permeation chromatography were administered subcutaneously into SKH1 mice. The biodistribution was measured by in vivo fluorescence imaging for up to 1 week post injection. At several time points, mice were sacrificed and selected organs and tissues were collected for ex vivo analysis. Part of injected aggregated IgGs persisted much longer at the injection site than unstressed controls. Aggregate fractions containing submicron (0.1-1 µm) or micron (1-100 µm) particles were retained to a similar extent. Highly fluorescent "hot-spots" were detected 24 h post injection in spleens of mice injected with submicron aggregates of murine IgG. Submicron aggregates of human IgG showed higher accumulation in draining lymph nodes 1 h post injection than unstressed controls or micron size aggregates. For both tested proteins, aggregated fractions seemed to be eliminated from circulation more rapidly than monomeric fractions. The biodistribution of monomers isolated from solutions subjected to stress conditions was similar to that of unstressed control.

Mouse Models for Assessing Protein Immunogenicity: Lessons and Challenges.

The success of clinical and commercial therapeutic proteins is rapidly increasing, but their potential immunogenicity is an ongoing concern. Most of the studies that have been conducted over the past few years to examine the importance of various product-related attributes (in particular several types of aggregates and particles) and treatment regimen (such as dose, dosing schedule, and route of administration) in the development of unwanted immune responses have utilized one of a variety of mouse models. In this review, we discuss the utility and drawbacks of different mouse models that have been used for this purpose. Moreover, we summarize the lessons these models have taught us and some of the challenges they present. Finally, we provide recommendations for future research utilizing mouse models to improve our understanding of critical factors that may contribute to protein immunogenicity.

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.

Influence of aggregation and route of injection on the biodistribution of mouse serum albumin.

Protein aggregates are a major risk factor for immunogenicity. Until now most studies on aggregate-driven immunogenicity have focused on linking physicochemical features of the aggregates to the formation of anti-drug antibodies. Lacking is however, basic knowledge on the effect of aggregation on the biodistribution and clearance of therapeutic proteins in vivo. The aim of current study was to get insight into the effect of aggregation on biodistribution in mice using different routes of administration. Fluorescently labeled stressed and unstressed mouse serum albumin was injected via different routes in mice and detected via in vivo fluorescence imaging up to 48 hrs post-injection. We found that biodistribution of stressed MSA significantly differed from its unstressed counterpart. Subcutaneous and intramuscular administration resulted in accumulation of protein at the site of injection, from which clearance of stressed MSA was considerably slower than clearance of unstressed MSA. Upon intravenous and intraperitoneal injection of stressed MSA, fluorescent "hotspots" were observed in the spleens, livers and lungs. Further and more detailed examination of biodistribution after intraperitoneal injection showed higher fluorescence in most of tested organs suggesting more efficient diffusion and/or lymphatic uptake from peritoneum of unstressed MSA than the stressed formulation.

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.