Search

Background Biosimilars have been used for 15 years in the European Union (EU), and have been shown to reduce costs and increase access to important biological medicines. In spite of their considerable exposure and excellent safety record, many prescribers still have doubts on the safety and interchangeability of biosimilars, especially monoclonal antibodies (mAbs) and fusion proteins. Objectives The aim of this study was to analyse the short- and long-term safety and interchangeability data of biosimilar mAbs and fusion proteins to provide unbiased information to prescribers and policy makers. Methods Data on the safety, immunogenicity and interchangeability of EU-licensed mAbs and fusion proteins were examined using European Public Assessment Reports (EPARs) and postmarketing safety surveillance reports from the European Medicines Agency (EMA). As recent biosimilar approvals allow self-administration by patients by the subcutaneous route, the administration devices were also analyzed. Results Prelicensing data of EPARs (six different biosimilar adalimumabs, three infliximabs, three etanercepts, three rituximabs, two bevacizumabs, and six trastuzumabs) revealed that the frequency of fatal treatment-emergent adverse events (TEAEs), TEAEs leading to discontinuation of treatment, serious adverse events (SAEs), and main immune-mediated adverse events (AEs) were comparable between the biosimilars and their reference products. The availability of new biosimilar presentations and administration devices may add to patient choice and be an emerging factor in the decision to switch patients. Analysis of postmarketing surveillance data covering up to 7 years of follow-up did not reveal any biosimilar-specific adverse effects. No product was withdrawn for safety reasons. This is in spite of considerable exposure to biosimilars in treatment-naïve patients and in patients switched from the reference medicinal product to the biosimilar. Analysis of data from switching studies provided in regulatory submissions showed that single or multiple switches between the originator and its biosimilar versions had no negative impact on efficacy, safety or immunogenicity. Conclusions In line with previous reports of prelicensing studies of biosimilar mAbs and etanercepts, this study demonstrated comparable efficacy, safety, and immunogenicity compared with the reference products. This is the first study to comprehensively analyze postmarketing surveillance data of the biosimilar mAbs and etanercept. An analysis of more than 1 million patient-treatment years of safety data raised no safety concerns. Based on these data, we argue that biosimilars approved in the EU are highly similar to and interchangeable with their reference products. Thus, additional systematic switch studies are not required to support the switching of patients. Supplementary Information The online version contains supplementary material available at 10.1007/s40265-021-01601-2.

Background Biosimilars are expected to decrease growing health care expenditures. Given that uptake of biosimilars has been modest, automatic substitution has been suggested to increase their use, but the practice is not yet allowed or implemented in many jurisdictions. Methods A systematic review was performed by searching databases Scopus, Medline (Ovid), CINAHL, and Web of Science. Peer-reviewed, original studies written in English and published during the period January 1, 2006 to April 24, 2021 reporting any interventions, pilots or any other studies including experiences or perceptions of any relevant stakeholders on automatic substitution of biologics were included without limitation by setting or geography. The quality of the included studies were evaluated by pre-determined criteria. Results Altogether, 27 studies fulfilled the inclusion criteria, of which 23 were surveys, and four semi-structured interviews reporting mainly stakeholders’ perceptions on automatic substitution. Most of the studies (56%, 15/27) were from Europe. Studies were conducted among prescribers (n = 12), pharmacists (n = 5), patients (n = 4), payers (n = 1), and mixed stakeholders (n = 5). The primary objective of the majority (81%, 22/27) of the studies was to investigate some other biosimilar topic than automatic substitution. The reported perceptions of substitution were mainly negative. Studies evaluating risks, safety or effectiveness, or reporting real-life experiences of biologic substitution were lacking except one intervention and two prospective risk management studies. The overall quality of the studies was low to moderate, and the results were not generalizable due to convenience sampling not representing the populations of interest, and low response rates. Conclusions The current research evidence on the automatic substitution of biologics is scarce and of low to moderate quality, reflecting low stakeholder knowledge and their cautious attitude towards biosimilars. The safe and efficient implementation of automatic substitution requires well-designed practices, pilot studies, and evolving legislation. Supplementary Information The online version contains supplementary material available at 10.1007/s40259-021-00493-8.

The World Health Organization (WHO) guidelines on evaluation of similar biotherapeutic products (SBPs; also called biosimilars) were adopted by the WHO Expert Committee on Biological Standardization (ECBS) in 2009. In 2019, the ECBS considered that a more tailored and potentially reduced clinical data package may be acceptable in cases where this was clearly supported by the available scientific evidence. The goal of this publication is to review the current clinical experience and scientific evidence and to provide an expert perspective for updating the WHO guidelines to provide more flexibility and clarity. As the first step, the relevant guidelines by other regulatory bodies were reviewed in order to identify issues that might help with updating the WHO guidelines. Next, a literature search was conducted for information on the long-term efficacy, safety, and immunogenicity of biosimilars to identify possible long-term problems. Finally, a search for articles concerning the role of clinical studies in the benefit-risk evaluation of biosimilars was conducted. The analysis of other guidelines suggested that the WHO guidelines may need more emphasis on the importance of the state-of-the-art physicochemical and structural comparability exercise and in vitro functional testing. The use of "foreign" reference product will also need clarifications. The value of in vivo toxicological tests in the development of biosimilars is questionable, and the non-clinical part needs revisions accordingly. The concepts of "totality of evidence," "stepwise development," and "residual uncertainty" were applied in the evaluation of the clinical sections of the guideline. The review of long-term safety and efficacy demonstrated the robustness of the current biosimilar development concept. The analysis of the roles of different development phases suggested that the large efficacy, safety, and immunogenicity studies are, in most cases, redundant. The residual uncertainty of safety, immunogenicity, and efficacy of biosimilars that has shaped the current regulatory guidelines is now substantially reduced. This will allow the re-evaluation of the non-clinical and clinical requirements of the current WHO main guideline. The shift of the relative impact of the development phases towards physico-chemical and in vitro functional testing will provide a relief to the manufacturers and new challenges to the regulators.

Biosimilar versions of original therapeutic proteins and peptides are approved on the basis of an extensive demonstration of analytical similarity to the originator product, including in-vitro tests for function. Comparative clinical evaluation is also performed in the pre-authorisation phase to confirm that the analytical similarity has led to comparable safety and efficacy, including immunogenicity. This article reviews the regulatory standards applicable to the assessment of relative immunogenicity of biosimilar candidates for marketing authorisation in the European Union and USA. The most critical elements for the design of an effective product-specific strategy to exclude potential incremental immunogenicity are discussed, and case examples are presented to illustrate how data are assessed by regulatory authorities.

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California on 3–7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event – a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid ligand binding assay (LBA)/LCMS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for biotherapeutics, biomarkers and immunogenicity assays using hybrid LBA/LCMS and regulatory agencies’ inputs. Part 1 (LCMS for small molecules, peptides and small molecule biomarkers) and Part 3 (LBA: immunogenicity, biomarkers and pharmacokinetic assays) are published in Volume 9 of Bioanalysis, issues 22 and 24 (2017), respectively.

Many of the best-selling 'blockbuster' biological medicinal products are, or will soon be, facing competition from similar biological medicinal products (biosimilars) in the EU. Biosimilarity is based on the comparability concept, which has been used successfully for several decades to ensure close similarity of a biological product before and after a manufacturing change. Over the last 10 years, experience with biosimilars has shown that even complex biotechnology-derived proteins can be copied successfully. Most best-selling biologicals are used for chronic treatment. This has triggered intensive discussion on the interchangeability of a biosimilar with its reference product, with the main concern being immunogenicity. We explore the theoretical basis of the presumed risks of switching between a biosimilar and its reference product and the available data on switches. Our conclusion is that a switch between comparable versions of the same active substance approved in accordance with EU legislation is not expected to trigger or enhance immunogenicity. On the basis of current knowledge, it is unlikely and very difficult to substantiate that two products, comparable on a population level, would have different safety or efficacy in individual patients upon a switch. Our conclusion is that biosimilars licensed in the EU are interchangeable.

The guidelines for immunogenicity studies by the European Medicines Agency and the US FDA are based on different legislations and regulatory philosophies. In spite of the different background, the main guidelines are compatible on the scientific level, especially for new innovative therapeutic protein products. The importance of sensitive and drug-tolerant antidrug antibody assays and multidisciplinary approach to development and assessment are highlighted by both agencies. The main differences are in the field of biosimilars. The nonclinical in vivo immunogenicity studies are seen more useful by the FDA than by the European Medicines Agency. The draft FDA guidance on interchangeability will complicate global biosimilar development by requiring clinical switch studies with US sourced reference product.

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9–13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event – a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies’ input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis, issues 22 and 24 (2018), respectively.

Evaluation of the relative immunogenicity of a biosimilar candidate in direct comparison to the reference product is a general regulatory requirement, with the main weight of evidence deriving from head-to-head clinical studies in populations that are adequately sensitive to reveal clinically meaningful differences across the proposed conditions of use.

Biosimilars are regulated by the centralized marketing authorization procedure co-ordinated by the European Medicines Agency (EMA). Marketing authorization is granted by the Commission of the European Union (EU). Thus, each biosimilar has one regulatory assessment as well as the same product information and conditions of use in the EU. The current regulatory framework is a result of 20 years evolution of legislation and regulatory guidelines. The concept of biosimilarity is based on the long experience of comparability studies of individual biological products after manufacturing changes. Therefore, the development of a biosimilar is an extensive comparability exercise with head-to-head comparisons to its reference product that must have a full dossier of quality, safety and efficacy. For the sake of global development, the current guidance allows the use of a reference sourced from a non-EU country in certain non-clinical and clinical studies provided that it can be shown to be a relevant as a comparator. The high similarity of a biosimilar and its reference is demonstrated by physico-chemical and structural as well as in vitro functional comparability studies. Non-clinical in vivo studies are rarely needed. The extent of clinical studies depends on the possibilities to demonstrate high similarity by analytical tests. When the high analytical similarity has been established and the comparable pharmacokinetics and -dynamics, safety and efficacy have been demonstrated in one therapeutic indication, extrapolation safety and efficacy to other indications is expected. Several national regulatory agencies endorse physician-guided switches between biosimilars and reference products without additional clinical trials.

Clinical development of a novel drug has traditionally been seen as a series of four phases, each having its own objectives in establishing the efficacy and safety of the drug. Increasingly individualized medicine and the changing mechanisms of drug action are also changing the designs of clinical drug testing. The borders of development phases become blurred and the traditional large, controlled multicenter studies may in part be replaced by individual and risk-based approaches. The indications for drugs are more precisely targeted from biological starting points, and a target-oriented development may guide the designs of clinical testing at all stages of development. Utilization of data from registries along with modeling will become more common in clinical drug testing.

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid ligand binding assay (LBA)/LCMS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for biotherapeutics, biomarkers and immunogenicity assays using hybrid LBA/LCMS and regulatory agencies' inputs. Part 1 (LCMS for small molecules, peptides and small molecule biomarkers) and Part 3 (LBA: immunogenicity, biomarkers and pharmacokinetic assays) are published in Volume 9 of Bioanalysis, issues 22 and 24 (2017), respectively.

The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3–7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event – a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC–MS, hybrid ligand-binding assay (LBA)/LC–MS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC–MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC–MS for biotherapeutics and regulatory agencies’ inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.

Despite the establishment of a specific approval pathway, the issuance of detailed scientific guidelines for the development of similar biological medicinal products (so-called “biosimilars”) and the approval of several biosimilars in the European Union, acceptance of biosimilars in the medical community continues to be low. This is especially true in therapeutic indications for which no specific clinical trials with the biosimilar have been performed and that have been licensed based on extrapolation of efficacy and safety data from other indications. This article addresses the concerns frequently raised in the medical community about the use of biosimilars in such extrapolated indications and explains the underlying scientific and regulatory decision making including some real-life examples from recently licensed biosimilars.

In the EU, the EMA has been working with biosimilars since 1998. This experience is crystallized in the extensive set of guidelines, which range from basic principles to details of clinical trials. While the guidance may appear complicated, it has enabled the development of biosimilars, of which 21 have managed to get marketing authorization. Currently marketed biosimilars in the EU have a good track record in safety and traceability. No biosimilars have been withdrawn from the market because of safety concerns. The most controversial issues with biosimilars are immunogenicity and extrapolation of therapeutic indications. The available data for these topics do not raise concerns among EU regulators. Interchangeability and substitution are regulated by individual EU member states.

The risks of biotechnology-derived products and other biologicals are different from the risks of small chemical entities and, thus, their risk management will have special features. For many biological products, the safety is mainly dependent on the starting materials and the manufacturing process. Therefore, their risk management must be focused accordingly. In certain issues, such as immunogenicity of recombinant DNA products, a proper risk management plan can only be built during the pre-licensing studies. Application of special epidemiological methods for investigation of very rare safety signals is crucial for some biologicals, e.g. vaccines. Genetically modified viruses used as vaccines and vectors for gene transfer, as well as xenogeneic cells, carry major public health risks. Therefore, the use of such products is possible only with extensive risk management programmes. Some elements of these programmes may be difficult to enforce because of legal, ethical and practical obstacles. The main special risk of biologicals is the transmission of infectious agents. The mitigation of this risk is complicated by the ever-changing epidemiological situation and the long latency of some infections. The impact of any measure for risk reduction of essential products, such as vaccines and plasma-derived medicinal products, must be considered carefully from the overall benefit/risk point of view. The risk must be reduced to the lowest level that is reasonably achievable without unduly putting the availability of essential products at risk. Adequate expertise and consultations with special experts are important in the risk management of the new advanced therapies in order to ensure the full understanding of the risks and to avoid creating unnecessary barriers for innovation. Due to the sensitive issues linked with biotechnology-derived products and other biologicals, risk communication is of critical importance. From the regulatory point of view, the risk management of biologicals is far from optimal, especially in the EU where legislation lags behind the progress in the field, and where the concerted actions of the national regulatory agencies need improvement. From the industry point of view, the risk management plans for biologicals should be designed in multidisciplinary teams.

The possibilities for rational combination therapy improved with the introduction of new non-cytotoxic drugs with a defined mode of action. The practical difficulties in demonstration of efficacy of combination therapy for autoimmune diseases are qualitatively the same as those for solo therapy [4]. The additional hurdle is due to the study designs. Combinations of new, often biotechnological agents, and old medicinal products have been successfully applied by using the add-on or even the “combo” vs single components design [5]. However, it must be realised that scientifically ideal study designs are not always feasible for ethical and logistic reasons. In the field of autoimmune diseases, as for other severe and chronic diseases, compromises have to be made in order not to block the progress in patient care. Experience gained in other areas, such as cancer, acute transplant rejection, and HIV infection, may be helpful. These areas may offer good (and also bad!) examples for the development of combination treatments in difficult circumstances. The present guidelines for autoimmune diseases are not very helpful for the development of combination therapies. However, regulatory authorities are pleased to offer scientific and regulatory advice for developers of combination therapies on a case by case basis.

We evaluated the sensitivity and prognostic value of an enzyme-linked immunosorbent assay (ELISA) for the measurement of antifilaggrin antibodies (AFA), using filaggrin purified from human skin as an antigen. The AFA test was applied to a series of 306 patients with various recent-onset inflammatory joint diseases. The results were compared to those of the conventional immunofluorescence tests for antikeratin antibody (AKA) and antiperinuclear factor (APF) and of the rheumatoid factor (RF) tests from a previous study. There was a very good agreement between the results of the tests for APF and AFA (kappa-value 0.79 in patients with peripheral poly/oligoarthritis). The agreement between the tests for AKA and AFA was significant but less pronounced (kappa-value 0.50). The AFA test detected 10/22 of the RF-negative erosive cases, particularly those with a large number of erosive joints. Thus, the test for AFA supplements RF in the prediction of erosiveness.

Objective: To identify patterns of maternal antibodies associated with anincreased risk of having a child with congenital heart block (CHB) and to provide a basis for counseling women with a previously affected child. Methods: This retrospective clinical study of the obstetric histories of 46Finnish women with a CHB child compared the strength and specificity of the immune response to SS-A/Ro and SS-B/La, as determined by immunoblot and ELISA, in 44 affected women with 85 women with systemic lupus erythematosus (SLE) and 32 women with primary Sjogren's syndrome (SS) with healthy children. Results: High levels of anti-SS-A/Ro and anti-SS-B/La by practically all assays were associated with a significantly increased risk of having a CHB child. The best single test to identify high-risk mothers was anti-52kd SS-A/Ro by immunoblot (OR 18.9), and it was the only assay to detect mothers at increased risk of CHB as compared with controls with primary SS. Low risk of CHB was indicated by undetectable or low levels of antibodies in the ELISA assays and no reactivity on immunoblot. Mothers with a previous child with CHB had a history of fetal loss (mostly spontaneous abortions) or a history of recurrent fetal losses (⪖3) slightly more often than controls. Late-trimester obstetric complications in non-CHB pregnancies were insignificant. The relative risk for a female child compared with a male child to have CHB was 1.9 (1.2-2.9, P = .009), and the risk of the mother having another child with CHB was 12% (4 of 34). Conclusion: Although there is no unique antibody profile specific for CHB, mothers with a high or low risk of having a child with CHB can be identified. Female children appear to have an increased risk of CHB, but the risk of the mother having another child with CHB is low.

Dear Sir, Danese and Gomollon issued an ECCO position statement on the use of similar biological medicinal products (biosimilars) in inflammatory bowel disease (IBD)1, arguing that the use of biosimilars in patients with IBD will require clinical trials in this particular patient population, with comparison to the innovator product. The authors state that subtle differences in structure may lead to profound differences in immunogenicity and efficacy, even with the same biological medicine. Indeed, when changes are introduced in the manufacturing process of any biological product, its new version is not identical, and therefore, manufacturers must demonstrate …

Objective. To study the long-term outcome of mothers of children with isolated congenital heart block (CHB) and to characterize the maternal autoantibody response to SS-A/Ro and SS-B/La. Methods. A retrospective clinical study of 33 mothers a mean of 11.2 years (SD 9.2 years, range 0–32 years) after the delivery of their first child with CHB. A clinical and immunologic study of 31 of these mothers, compared with 89 healthy mothers, 45 mothers with systemic lupus erythematosus (SLE), and 19 mothers with primary Sjogren's syndrome (SS), all of whom had healthy children. The specificity of the autoantibody responses to SS-A/Ro and SS-B/La was studied by enzyme-linked immunosorbent assays using purified human recombinant antigens and affinity-purified antigens. Results. By the time of the analysis, 2 (6%) of the 33 mothers of CHB children had died and 6 (18%) had met the criteria for SLE. As a group, mothers of CHB children had clinical and immunologic characteristics more closely related to primary SS than to SLE or any other connective tissue disease. The predominant autoantibody response was to the SS-A/Ro antigens, notably to the 52-kd SS-A/Ro protein (prevalence 97%). Compared with controls with SLE, mothers of CHB children had higher titers of antibodies to recombinant 52-kd and 60-kd SS-A/Ro proteins and to the affinity-purified SS-A/Ro antigen (P < 0.05, P < 0.01, and P < 0.001, respectively). Compared with controls with primary SS, the autoantibody responses were similar. Conclusion. The predominant autoimmune disorder in mothers of children with CHB is subclinical primary SS. Antibodies to SS-A/Ro appear to be a prerequisite for the development of CHB.

Objective. To study the relationship between antibodies that recognize human retroviral proteins and the presence of clinical features and ribonucleoprotein antibodies in patients with autoimmune connective tissue diseases (CTDs). Methods. Antibodies against native human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia/lymphoma virus type I, recombinant HIV-1 Nef protein, and ribonucleoprotein antigens were determined by immunoblot of sera from 65 prospectively studied patients with definite or suspected CTDs of autoimmune type. Results. Antibodies to retroviral proteins (ARP), most frequently to HIV Gag proteins p55 and p24, were found in 64% of 22 patients with systemic lupus erythematosus (SLE), in 63% of 8 patients with discoid LE (DLE), in 75% of 8 patients with mixed connective tissue disease (MCTD), and in 26% of 19 individuals with chronic biologically false-positive (CBFP) seroreactions, but not in 8 patients with subacute cutaneous lupus erythematosus. No clear correlation of ARP with antibodies to any specific small nuclear RNP antigen was observed. The most striking finding was that recurrent infections, both in LE patients and in those with CBFP reactions and widespread, acral discoid skin lesions, occurred significantly more often in ARP-positive patients. Conclusion. The occurrence of antibodies reacting with human retroviral proteins is associated with severe skin lesions and recurrent infections in SLE, DLE, and MCTD patients, and with a disposition toward developing systemic disease in CBFP reactors.

Objective. We sought to determine whether circulating antikeratin antibodies (AKA) precede the onset of rheumatoid arthritis (RA). Methods. By matching the registers of 2 previous population studies with the registry of patients receiving antirheumatic drugs several years later, pre-illness serum specimens could be obtained from 39 individuals who subsequently developed RA. AKA were assayed with the standard indirect immunofluorescence technique. Results. Ten of 39 serum specimens from individuals who subsequently developed seropositive RA, and 1 of 15 sera from individuals who developed seronegative RA, were positive for IgG-class AKA by immunofluorescence assay. The AKA-positive sera were also positive for rheumatoid factors. Conclusion. The findings focus attention on the role of pre-illness immunologic events in the pathogenesis of RA.

Increased serum levels of hyaluronate (HA) have been found in patients with rheumatoid arthritis (RA). This probably reflects increased leakage of HA from the inflamed joints into the circulation. In a prospective study of 40 patients with early RA, we evaluated the relationship of serum HA to clinical, laboratory, and radiologic parameters of disease activity. The patients were followed for 12 months; all had active disease at study entry. We confirmed the previous finding of higher serum HA concentrations in RA patients compared with healthy controls. At study entry, the patients' serum HA levels correlated positively with clinical and laboratory parameters of acute inflammation. Despite marked clinical improvement during therapy with second-line drugs, the serum HA levels increased during the followup period. At the end of 1 year, these levels correlated with the radiologic progression of joint lesions, whereas they showed a less pronounced correlation with clinical or laboratory parameters of inflammation. We conclude that, in early RA, serum HA levels may reflect ongoing joint destruction and may even predict subsequent joint damage.

The Summary of Product Characteristics (SPC) approved by the European Medicines Agency (EMEA) and the Package Insert (PI) approved by the Food and Drug Administration (FDA) were examined for 32 biopharmaceutical products. The aim was to identify differences in the product information since such information may have an impact on the planning of global clinical development programmes. The EU SPC contained more detailed instructions to the prescriber, including the positioning of the product with regard to the stage of the disease and to other therapies. The approach to safety information, notably to contraindications and warnings was more conservative in the EU SPC. The conservative approach in the EU may reflect the central position of the SPC in risk management of new pharmaceuticals. A typical feature of the US PI was the detailed description of the efficacy and safety result of the pivotal clinical trials.

Objective—To evaluate the clinical significance of antifilaggrin antibodies (AFA) measured by an enzyme linked immunosorbent assay (ELISA) in serial specimens from patients with recent onset rheumatoid arthritis (RA). Methods—Filaggrin was purified from human skin and used as an antigen in ELISA. The AFA test was applied to five serial specimens from 78 patients with recent onset RA followed up for three years. Rheumatoid factor (RF) had been measured earlier from the same samples by quantitative immunoturbidimetry. Results—The mean AFA level was highest at entry (54% positive), followed by a statistically significant decline at six months and a slight increase at three years. AFA were persistently positive in 23 patients and persistently negative in 28 patients. Eleven of the latter patients were persistently negative for RF. At study entry AFA levels correlated to some degree with RF levels. In general, raised AFA levels at entry were associated with an active and treatment resistant disease, but they did not predict radiological progression. Conclusions—The test for AFA has potential for an additional immunological test for RA. (Ann Rheum Dis 2001;60:32‐35)

Background: The so-called antikeratin antibody (AKA) and the antiperinuclear factor (APF) that recognize proteins related to human epidermal filaggrin belong to the most specific serological markers of rheumatoid arthritis (RA). However, assays for the detection of AKA and APF are currently based on immunofluorescence, a method that is subject to arbitrary interpretation and inadequate standardization of the substrates. Methods: Proteins extracted from human epidermis were separated by reversed-phase high-performance liquid chromatography (HPLC). Filaggrin-containing fractions, identified in immunoblotting by monoclonal antifilaggrin antibodies, were then subjected to gel filtration HPLC and, finally, to a second reversed-phase HPLC step. Tryptic digestion, amino acid sequencing and mass spectrometry were used to cornfirm the identity of the purified protein. Filaggrin was used as antigen in enzyme-linked immunosorbent assay (ELISA) to measure IgG class antifilaggrin antibodies. Results: The filaggrin preparation obtained gave a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, binding monoclonal antifilaggrin antibody in immunoblotting. Amino acid sequences of all 10 tryptic peptides analyzed were shown to originate from human filaggrin. Antifilaggrin antibody levels exceeded the 99th percentile level of 100 middle-aged blood donors in 26/55 (47%) RA sera. At a similar cutoff level 28/55 (51%) of the RA sera were positive in the AKA test. Of the 26 antifilaggrin-positive sera, 21 were also AKA-positive. Conclusion: Human filaggrin can be purified by standard biochemical techniques, despite the heterogeneity of the protein, and used in ELISA for testing autoantibodies to filaggrin. The sensitivity of the assay equals that of the AKA test.

OBJECTIVE—Rheumatoid arthritis (RA) is associated with several autoantibodies that can precede the clinical disease. The immunoglobulin concentrations in serum samples before illness were studied to learn more about the immunological process before RA. METHODS—A case-control study was nested within a Finnish cohort of 19 072 adults who had neither arthritis nor a history of it at the baseline examination during 1973-1977. By late 1989, 124 had developed RA, of which 89 were positive for rheumatoid factor (RF). Three controls per each incident case were individually matched for sex, age, and municipality. The concentrations of IgG, IgA, and IgM were measured from stored serum samples. RESULTS—Serum IgG before illness was found to be directly proportional to the risk of RF positive RA, and a non-linear association was present between serum IgA and the risk of RF positive RA. These associations were constant between men and women and other subgroups of the study population and not confounded by serum orosomucoid concentration, level of education, smoking, alcohol intake or body mass index. As adjusted for these factors, the odds ratios (95% confidence intervals) of RF positive RA in the lowest, mid, and highest tertiles of IgG distribution were 1.00, 1.55 (0.81, 2.97), and 2.22 (1.16, 4.26), and in the tertiles of IgA 1.00, 2.23 (1.14, 4.36), and 1.78 (0.89, 3.57), respectively. The associations persisted throughout the entire observation period but were most distinct when the period to the onset of clinical RA was ≥ 10 years. IgM carried no predictive significance. None of the serum immunoglobulins predicted the development of RF negative RA. CONCLUSIONS—Increased IgG levels may reflect some, at present unknown process in the early events leading to the development of RA, typically occurring ≥ 10 years before the onset of clinical disease.

The purpose of this study was to examine the relationship between circulating antibodies to stratum corneum (AKA) and antiperinuclear factors (APF) on one hand, and the x-ray progression of joint damage in chronic poly/oligoarthritis on the other hand. The analysis involved 133 patients with either rheumatoid or nonspecific arthritis derived from a cohort of 442 patients with recent onset arthritis. The patients were followed up for eight years with regular clinical, laboratory, and radiological evaluations. Radiographic evidence of joint destruction was quantitated by a radiographic index based on the Larsen grading. AKA and APF were detected, either at entry or at follow-up, in 26 and 54 patients, respectively. Seventy-six of the 133 patients had developed erosions. All AKA-positive patients had a rheumatoid factor-positive erosive poly-arthritis. The presence of APF was also associated with a progressive arthritis although four APF-positive patients had a non-erosive disease. Neither AKA nor APF were able to distinguish a particularly severe form of progressive RA.

Publisher Summary This chapter discusses the antibodies to keratin and associated intermediate filaments in rheumatoid arthritis and other connective tissue diseases. The cytoskeleton of practically every type of cell is constituted by three types of filament microfilaments, microtubules, and intermediate filaments. The latter are to a certain extent cell-type specific. The intermediate filament expression and distribution patterns in cells and tissues reflect their differentiation or functional specialization state, their histogenesis, and any malignant or viral transformation. The concept that autoantibodies to specific components of the cytoskeleton may play a role in the initiation or perpetuation of autoimmune diseases is an intriguing possibility. This chapter focuses on the antibodies to the cytoskeleton of epithelial cells. The most prominent cytoskeletal structure in epithelial cells is the keratin filament network. Keratin filaments show extensive heterogeneity between different epithelial cells and between the same cells at different stages of differentiation.

This study focuses on the consequences of T-lymphocyte activation in chronic polyarthritis in terms of expression of cell surface receptors interacting with extracellular matrix (ECM). The expression of the VLA group of integrins was studied on in vitro-stimulated peripheral-blood T cells, and on peripheral-blood and synovial-fluid mononuclear cells (MNC) of patients with polyarthritis. The VLA expression was measured by flow cytometry using monoclonal antibodies (MoAbs) against alpha-subunits of the VLA family. VLA-alpha 4 and VLA-alpha 5, but not VLA-alpha 1, were expressed on a major fraction of unstimulated peripheral-blood T cells both in the patients with polyarthritis and in healthy individuals. Two distinct populations, VLA-alpha 5-high and VLA-alpha 5-low, were found in resting peripheral-blood T lymphocytes. Two days after stimulation by phorbol 12-myristate 13-acetate (PMA) and concanavalin A, most T cells became VLA-alpha 5-high. In patients with chronic polyarthritis, the expression of VLA-alpha 1 and VLA-alpha 5 was always higher on synovial-fluid T cells than on peripheral-blood T cells. These results give further support to the hypothesis that upon activation the induction of the VLA adhesion-molecule expression may be a factor contributing to the accumulation of T cells in the inflamed synovium.

A case of HLA identical twins with one affected by congenital heart block is reported. Both twins, as their mother, had more than 12-fold higher anti-Ro antibody titers compared to healthy controls, but no differences were observed between the affected and the healthy baby. It is possible that there is a third factor causing the manifestation of this disease.

Mouse monoclonal antibodies against ED sequence-containing cellular fibronectin (cFn) were used to show that Fn in the inflamed synovium is distinct from the major form of plasma Fn (pFn). An accumulation of cFn was seen at sites of hyperplasia of the rheumatoid synovial membrane and in the walls of small vessels in the synovium by immunofluorescence microscopy. cFn was also found in rheumatoid synovial fluid by immunoblotting. Approximately one-fifth of the T lymphocytes from rheumatoid synovial fluid bound to Fn. The binding of synovial fluid T cells was always higher than that from peripheral blood. These results have two implications. On the one hand, the cellular type of Fn may be an indicator of synovial inflammation. On the other hand, the deposition of Fn may be a factor contributing to the infiltration of mononuclear cells into the synovium.

The diagnostic and prognostic significance of the different types of serum smooth muscle antibodies (SMA) were investigated in sera of 24 patients with chronic active hepatitis (CAH) and 15 patients with primary biliary cirrhosis (PBC). SMA of IgG class were found in 92% of sera from patients with CAH but in only 20% of sera from PBC patients, whereas the incidence of IgM-SMA was higher in PBC (67%) than in CAH (38%). All six patients with the atypical cholestatic form of CAH has SMA of IgM class, whereas other CAH patients had SMA of mainly IgG class. SMA reacting with rabbit liver (bile canaliculus antibodies, BCA) and with rat glomeruli (glomerulus antibodies) were of anti-actin specificity and were more common in CAH than in PBC. Organ specific BCA or glomerulus antibodies were not found. Anti-actin antibodies were detected in the majority of the investigated sera by an immunoenzymatic anti-actin assay. The results suggest that the determination of SMA titres with heavy chain specific antisera may help in the assessment of diagnosis and prognosis of chronic hepatitis.

Results consistent with a general humoral immune stimulation were found when 127 sera from 89 patients with Yersinia enterocolitica infection were studied. Significantly increased gammaglobulin concentrations and elevated isohemagglutinin titers were seen in these sera as compared to sera from normal blood donors and patients with streptococcal infection. Antinuclear and anti-smooth muscle antibodies were demonstrated in both yersinia and streptococcal infection. The prevalence of non-organ specific antiepithelial antibodies reacting with gastrointestinal and thyroid epithelial cells was significantly increased in yersinia infection.

Previous studies have suggested that cytoskeletal elements in an altered form in vivo (alcoholic hyalin , AH) are targets for an autoimmune reaction in alcoholic liver disease (ALD). In this study we assayed autoantibodies to cytoskeletal cytoskeletal cytokeratin filaments, intermediate filaments of epithelial cells, by indirect immunofluorescence technique (IIF) using cultured human amnion epithelial cells and PtK 2 cells as targets. Sera from 20 patients with ALD, 38 patients with urogenital cancer, 22 patients with renal diseases, and from 18 healthy controls were studied. Patients with ALD had a significantly increased prevalence of cytokeratin filament autoantibodies using both PtK 2 cells (69%) and human amnion epithelial cells (69%) as substrate, as compared to other groups. The antibodies were mainly of IgA and IgM class and could be demonstrated in all forms of ALD. Interestingly, cytoskeleton antibodies have previously been observed in diseases of viral and "autoimmune" origin.

This case report describes a patient who initially had a pleuritis and arthalgias. During the follow-up he developed first a membranous glomerulonephritis with nephrotic syndrome and subsequently a crescentic, rapidly progressive glomerulonephritis with glomerular basement membrane antibodies (anti-GBM). An analysis of the serum samples obtained during the follow-up revealed no infections at the onset of renal failure. However, anti-GBM could be demonstrated in the serum samples obtained 2 months before the deterioration of the renal function. The anti-GBM did not react with alveolar BM and the patient had no signs of pulmonary hemorrhage. The etiology and the sequence of the pathological events of rapidly progressive glomerulonephritis is discussed in the light of these observations.

A solid phase enzyme-linked-immunosorbent assay (ELISA) for the determination of antibodies against denatured, single-stranded (ss-) DNA is described. Polystyrene cuvettes coated with ss-DNA were incubated with serum samples and the anti-ss-DNA antibodies bound were detected by means of an anti-IgG-alkaline phosphatase conjugate. The binding of anti-ss-DNA antibodies in individual sera was expressed as units calculated as % of the absorbance in relation to the absorbance value obtained with a reference pool. Absorption experiments showed that the assay is specific for antibodies against denatured DNA. By using immunologically purified anti-ss-DNA antibodies the assay was shown to detect specific antibodies in concentrations down to 1 ng/ml. Antibodies against DNA could be detected in 94% of sera with antinuclear antibodies.

The role of autoantibodies against cardiac tissue in the pathogenesis of acute myopericarditis is poorly understood – partly because the specificity of the antibodies is unknown. We assayed antibodies against desmin, a cytoskeletal muscle protein, in 18 patients with acute infectious myopericarditis (AIM), in 24 patients with acute uncomplicated infections, in ten patients with acute myocardial infarction, and in 68 blood donors by an immunoenzymatic assay using purified desmin from Purkinje fibers of cow heart. In addition, anti-heart antibodies were assayed by indirect immunofluorescence. Anti-heart antibodies were detected in all groups by indirect immunofluorescence. On the other hand, anti-desmin antibody levels exceeding the mean + 2 SD of the blood donors could be demonstrated in seven (39%) AIM patients but in only one patient in the other groups. The analysis of serial samples indicated that the antibody level was highest in the acute phase of AIM and declined during the recovery.

Human smooth muscle antibodies (SMA) reacting with cytoplasmic intermediate filaments were characterized by the indirect immunofluorescence technique. These antibodies were noted to comprise a major type of SMA. When cultured human embrynic fibroblasts treated with a microtubulus-disrupting drug, vinblastine, were used, the SMA reacting with intermediate filaments (IMF-SMA), could be readily distinguished from other types of SMA by the immunofluorescence staining pattern. Absorption studies with intermediate filaments prepared from cultured human embryonic fibroblasts and from bovine smooth muscle tissue indicated that there are more than one autoantigen in the intermediate filaments of smooth muscle. The major subunit protein of intermediate filaments having a molecular weight of 55,000 was enriched in the antigenically active preparations of intermediate filaments. Five of 26 patients positive for IMF-SMA had signs of liver diseases and only two had chronic hepatitis.