Biomarkers and clinical characteristics of autoimmune chronic spontaneous urticaria: Results of the PURIST Study.

BACKGROUND: Autoimmune chronic spontaneous urticaria (aiCSU) is an important subtype of chronic spontaneous urticaria (CSU) in which functional IgG autoantibodies to IgE or its high-affinity receptor (FcεRI) induces mast cell degranulation and subsequent symptom development. However, it has not been tightly characterized. This study aimed to better define the clinical and immunological features and to explore potential biomarkers of aiCSU. METHODS: This was a multinational, multicenter study of 182 CSU patients. The clinical features studied included: urticaria activity and impact (UAS7 and quality of life); autologous serum skin test (ASST); IgG anti-FcεRI and IgG anti-IgE; IgG-anti-thyroperoxidase (IgG anti-TPO); total serum IgE; and basophil reactivity (BASO) using the basophil activation test (BAT) and basophil histamine release assay (BHRA). RESULTS: Of the 182 patients, 107 (59%) were ASST+, 46 (25%) were BASO+, and 105 (58%) were IgG anti-FcεRI+/IgE+. Fifteen patients (8%) fulfilled all three criteria of aiCSU. aiCSU patients appeared more severe (UAS7 21 vs 9 P < 0.016) but showed no other clinical or demographic differences from non-aiCSU patients. aiCSU patients also had markedly lower total IgE levels (P < 0.0001) and higher IgG anti-TPO levels (P < 0.001). Of biomarkers, positive BAT and BHRA tests were 69% and 88% predictive of aiCSU, respectively. CONCLUSIONS: aiCSU is a relatively small but immunologically distinct subtype of CSU that cannot be identified by routine clinical parameters. Inclusion of BHRA or BAT in the diagnostic workup of CSU patients may aid identification of aiCSU patients, who may have a different prognosis and benefit from specific management.

B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera.

BACKGROUND: Autoimmune diseases like rheumatoid arthritis and inflammatory bowel disease are treated with TNF-alpha-blocking antibodies such as infliximab and adalimumab. A common side effect of therapeutic antibodies is the induction of anti-drug antibodies, which may reduce therapeutic efficacy. METHODS: In order to reveal immunogenic epitopes on infliximab which are responsible for the adverse effects, sera from patients treated with infliximab were screened by ELISA for anti-infliximab antibodies. Sera containing high levels of anti-drug-antibodies (>1.25 µg/ml) were analyzed in an oligopeptide microarray system containing immobilized 15-meric oligopeptides from the infliximab amino acid sequence. Immunogenic infliximab IgG-epitopes were identified by infrared fluorescence scanning and comparison of infliximab-treated patients versus untreated controls. RESULTS: Six relevant epitopes on infliximab were recognized by the majority of all patient sera: 4 in the variable and 2 in the constant region. Three of the epitopes in the variable region are located in the TNF-alpha binding region of infliximab. The fourth epitope of the variable part of infliximab is located close to the TNF-alpha binding region and contains an N-glycosylation sequon. The sera positive for anti-infliximab antibodies do not contain antibodies against adalimumab as determined by ELISA. Thus, there is no infliximab-adalimumab cross-reactivity as determined by these systems. CONCLUSIONS: Our data shall contribute to a knowledge-based recommendation for a potentially necessary therapy switch from infliximab to another type of TNF-alpha-blocker. The characterization of immunogenic epitopes on therapeutic monoclonal antibodies using unprocessed patient sera shall lead to direct translational aspects for the development of less immunogenic therapeutic antibodies. Patients benefit from less adverse events and longer lasting drug effects.

The European Bioanalysis Forum recommendation on establishing appropriate drug tolerance levels in antidrug antibody assays.

The European Bioanalysis Forum, in collaboration with several key industry stakeholders, has recently led discussions that address international immunogenicity guidance documents, specifically the three tier approach for immunogenicity testing strategies, after more than 20 years of experience with biotherapeutics. As part of this, the strategy and methods used to assess drug tolerance across all immunogenicity assays are challenged, emphasizing that bioanalytical scientists need to consider the context-of-use of each assay. Here, recommendations for drug tolerance assessments, driven by strong scientific rationale and subject to reevaluation as needed, are provided. This includes carefully considering the drug and positive control concentrations considered to be appropriate and which tiers are most relevant for performing drug tolerance assessments.

Proceedings of the 14th European immunogenicity platform open symposium on immunogenicity of biopharmaceuticals.

Biologics have revolutionized disease management in many therapeutic areas by addressing unmet medical needs and overcoming resistance to standard-of-care treatment in numerous patients. However, the development of unwanted immune responses directed against these drugs, humoral and/or cellular, can hinder their efficacy and have safety consequences with various degrees of severity. Health authorities ask that a thorough immunogenicity risk assessment be conducted during drug development to incorporate an appropriate monitoring and mitigation plan in clinical studies. With the rapid diversification and complexification of biologics, which today include modalities such as multi-domain antibodies, cell-based products, AAV delivery vectors, and nucleic acids, developers are faced with the challenge of establishing a risk assessment strategy sometimes in the absence of specific regulatory guidelines. The European Immunogenicity Platform (EIP) Open Symposium on Immunogenicity of Biopharmaceuticals and its one-day training course gives experts and newcomers across academia, industry, and regulatory agencies an opportunity to share experience and knowledge to overcome these challenges. Here, we report the discussions that took place at the EIP's 14th Symposium, held in April 2023. The topics covered included immunogenicity monitoring and clinical relevance, non-clinical immunogenicity risk assessment, regulatory aspects of immunogenicity assessment and reporting, and the challenges associated with new modalities, which were discussed in a dedicated session.

Endocannabinoids limit excessive mast cell maturation and activation in human skin.

BACKGROUND: Mast cells (MCs) crucially contribute to many inflammatory diseases. However, the physiological controls preventing excessive activities of MCs in human skin are incompletely understood. OBJECTIVE: Since endocannabinoids are important neuroendocrine MC modifiers, we investigated how stimulation/inhibition of cannabinoid 1 (CB1) receptors affect the biology of human skin MCs in situ. METHODS: This was investigated in the MC-rich connective tissue sheath of organ-cultured human scalp hair follicles by quantitative (immuno)histomorphometry, ultrastructural, and quantitative PCR techniques with the use of CB1 agonists or antagonists, CB1 knockdown, or CB1 knockout mice. RESULTS: Kit+ MCs within the connective tissue sheath of human hair follicles express functional CB1 receptors, whose pharmacological blockade or gene silencing significantly stimulated both the degranulation and the maturation of MCs from resident progenitor cells in situ (ie, enhanced the number of tryptase+, FcεRIα, or chymase+ connective tissue sheath-MCs). This was, at least in part, stem cell factor-dependent. CB1 agonists counteracted the MC-activating effects of classical MC secretagogues. Similar phenomena were observed in CB1 knockout mice, attesting to the in vivo relevance of this novel MC-inhibitory mechanism. CONCLUSION: By using human hair follicle organ culture as an unconventional, but clinically relevant model system for studying the biology of MCs in situ, we show that normal skin MCs are tightly controlled by the endocannabinoid system. This limits excessive activation and maturation of MCs from resident progenitors via "tonic" CB1 stimulation by locally synthesized endocannabinoids. The excessive numbers and activation of MCs in allergic and other chronic inflammatory skin diseases may partially arise from resident intracutaneous MC progenitors, for example, because of insufficient CB1 stimulation. Therefore, CB1 stimulation is a promising strategy for the future management of allergy and MC-dependent skin diseases.

Pharmacokinetic and pharmacodynamic similarity of biosimilar natalizumab (PB006) to its reference medicine: a randomized controlled trial.

BACKGROUND: PB006 (Polpharma Biologics S.A; marketed as Tyruko®, Sandoz) is an approved biosimilar to natalizumab (Tysabri®; Biogen [ref-NTZ]). This multicenter, double-blind, randomized, single-dose study was conducted to demonstrate pharmacokinetic/pharmacodynamic (PK/PD) similarity between PB006 and ref-NTZ. RESEARCH DESIGN AND METHODS: Healthy participants (N = 453) were randomized to receive 3 mg/kg infusion of PB006, US-licensed, or EU-approved ref-NTZ before an 85-day follow-up. Primary PK endpoint was total natalizumab serum concentration over time; secondary PK endpoints explored concentration changes. Primary PD endpoints compared CD19+ cell counts and percentage α4-integrin receptor saturation, per natalizumab's mechanism of action. Secondary PD endpoints explored serum changes in sVCAM-1 and sMAdCAM-1, CD34+, and CD19+ cells. Safety, tolerability, and immunogenicity were assessed. RESULTS: The primary PK endpoint was met, with 90% confidence intervals (CIs) of the geometric mean for serum test/reference ratios contained within a prespecified margin (0.8-1.25). All primary PD endpoints were met, with 90% and 95% CIs within this similarity margin for baseline-adjusted CD19+ cell counts and percentage α4-integrin receptor saturation. All secondary endpoints were similarly contained, except sVCAM. No notable differences in safety, tolerability, or immunogenicity were observed. CONCLUSION: Similarity was confirmed, with PB006 demonstrating PK/PD behavior consistent with that of ref-NTZ. CLINICAL TRIAL REGISTRATION: EudraCT number 2019-003874-15.

PB006 (developed by Polpharma Biologics S.A; and marketed as Tyruko® by Sandoz) is an approved biosimilar to the reference medicine, natalizumab (Tysabri®, Biogen [ref-NTZ]) used to treat relapsing forms of multiple sclerosis. Approved biosimilar medicines have been shown to be as safe and effective as their reference medicines via different types of comparisons to the reference medicine, confirming that physicians and patients can expect the same clinical outcome.This study was conducted to confirm that PB006 acts the same way in the body as ref-NTZ. Healthy participants received one dose of either PB006, ref-NTZ from the US or ref-NTZ from Europe. During the study, blood samples were tested to confirm how much of each medicine was present in participants' blood, as well as to assess changes in immune cells or proteins related to how natalizumab works. The study also measured whether any treatment caused unwanted side effects or caused any changes in the immune system that may stop the medicine working.The results showed that PB006 behaved in the same way as ref-NTZ in the blood. All reported side effects were similar between groups and were as expected for this medicine, and neither PB006 nor ref-NTZ caused any important or unexpected changes to the immune system. This study showed that biosimilar natalizumab, PB006, behaves in the same way as ref-NTZ, and the same treatment outcomes can be expected.

When to Extend Monitoring of Anti-drug Antibodies for High-risk Biotherapeutics in Clinical Trials: an Opinion from the European Immunogenicity Platform.

The determination of a tailored anti-drug antibody (ADA) testing strategy is based on the immunogenicity risk assessment to allow a correlation of ADAs with changes to pharmacokinetics, efficacy, and safety. The clinical impact of ADA formation refines the immunogenicity risk assessment and defines appropriate risk mitigation strategies. Health agencies request for high-risk biotherapeutics to extend ADA monitoring for patients that developed an ADA response to the drug until ADAs return to baseline levels. However, there is no common understanding in which cases an extension of ADA follow-up sampling beyond the end of study (EOS) defined in the clinical study protocol is required. Here, the Immunogenicity Strategy Working Group of the European Immunogenicity Platform (EIP) provides recommendations on requirements for an extension of ADA follow-up sampling in clinical studies where there is a high risk of serious consequences from ADAs. The importance of ADA evaluation during a treatment-free period is recognized but the decision whether to extend ADA monitoring at a predefined EOS should be based on evaluation of ADA data in the context of corresponding clinical signals. If the clinical data set shows that safety consequences are minor, mitigated, or resolved, further ADA monitoring may not be required despite potentially detectable ADAs above baseline. Extended ADA monitoring should be centered on individual patient benefit.

Thyrotropin releasing hormone (TRH): a new player in human hair-growth control.

Thyrotropin-releasing hormone (TRH) is the most proximal component of the hypothalamic-pituitary-thyroid axis that regulates thyroid hormone synthesis. Since transcripts for members of this axis were detected in cultured normal human skin cells and since human hair follicles (HFs) respond to stimulation with thyrotropin, we now have studied whether human HF functions are also modulated by TRH. Here we report that the epithelium of normal human scalp HFs expresses not only TRH receptors (TRH-R) but also TRH itself at the gene and protein level. Stimulation of microdissected, organ-cultured HFs with TRH promotes hair-shaft elongation, prolongs the hair cycle growth phase (anagen), and antagonizes its termination by TGF-beta2. It also increases proliferation and inhibits apoptosis of hair matrix keratinocytes. These TRH effects may be mediated in part by reducing the ATM/Atr-dependent phosphorylation of p53. By microarray analysis, several differentially up- or down-regulated TRH-target genes were detected (e.g., selected keratins). Thus, human scalp HFs are both a source and a target of TRH, which operates as a potent hair-growth stimulator. Human HFs provide an excellent discovery tool for identifying and dissecting nonclassical functions of TRH and TRH-mediated signaling in situ, which emerge as novel players in human epithelial biology.

Erythropoietin and amyotrophic lateral sclerosis: Plasma level determination.

In amyotrophic lateral sclerosis (ALS), respiratory muscle weakness causes ventilatory insufficiency and tissue hypoxia, which induces a number of metabolic pathways, and in particular increases erythropoietin (EPO) synthesis. EPO is a glycoprotein with neuroprotective properties that stimulates erythropoiesis. Here, EPO plasma level in a large population of ALS patients, with and without respiratory failure, was measured. Plasma EPO level of patients with ALS (n = 98), controls with other neuromuscular diseases (n = 58) and healthy controls (n = 20) has been quantified by ELISA. No significant difference was found between ALS patients and the two control groups. EPO level was not different between bulbar- and spinal-onset patients and was not correlated with disease duration or functional impairment. However, in the ALS group EPO level was higher in females (p = 0.0006) and correlated positively with age (p = 0.006). The subgroup of ALS patients with respiratory failure had higher plasma levels of EPO compared with ALS patients with preserved respiratory function (p = 0.016), but short-term non-invasive ventilation did not change EPO level. In conclusion, EPO levels were found to be significantly higher in ALS patients with respiratory impairment representing preservation of this homeostatic mechanism.

Monitoring of nuclear factor of activated T-cell-regulated gene expression in de novo and long-term liver transplant recipients treated with cyclosporine a.

Pharmacodynamic drug monitoring might allow an improved use of immunosuppressive medication in transplant recipients. We assessed whether drug concentrations reflect the effect of cyclosporine (CsA) on expression of nuclear factor of activated T-cells-regulated cytokines. CsA drug concentrations and expression of interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor in stimulated blood lymphocytes were determined predose (C0) and 2 hours after (C2) CsA intake in 20 de novo (less than 3 months) and 20 long-term (3 months to 10 years) liver transplant patients. The residual cytokine expression at C2 relative to C0 was calculated. Mean CsA C0 and C2 concentrations were 236 and 776 µg/L in de novo and 100 and 573 µg/L in long-term liver transplant patients, respectively. Two hours after CsA intake, the residual cytokine expression for all cytokines was comparable in both groups (de novo patients mean 16%; long-term patients mean 17%). CsA C2 concentrations showed a significant (P < 0.01) correlation with the residual cytokine expression of interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor in both de novo and long-term patients, whereas CsA C0 concentrations did not. The data suggest that CsA C2 concentrations, but not C0 concentrations, reflect the effect of CsA on downregulation of cytokine expression in both de novo and long-term liver transplant patients.

Erythropoietin and the skin: a role for epidermal oxygen sensing?

Erythropoietin (EPO), long appreciated as the chief endocrine regulator of red blood cell formation, is now recognized to exert many additional functions outside the bone marrow. Thus, the quest is on to define the full range of EPO functions in the physiology and pathology of non-hematopoietic tissues. Two recent studies in man and mice have highlighted the importance of the mammalian skin as one peripheral tissue with a previously unsuspected role in EPO biology; both, as a target and as a source of EPO. In addition, the skin has been proposed to function as an oxygen sensor. The present hypothesis essay critically reviews the currently available evidence for this and provides a unifying theoretical scenario for intracutaneous EPO functions and for a potential role of the skin in the control of EPO production. Mainly, we propose that the skin itself directly contributes to the up-regulation of EPO plasma levels in response to hypoxia.

Autoimmune Diseases Are Linked to Type IIb Autoimmune Chronic Spontaneous Urticaria.

PURPOSE: Patients with chronic spontaneous urticaria (CSU) have an increased risk for comorbid autoimmune diseases. In this retrospective multicenter study of CSU patients, we evaluated clinical and laboratory features of CSU associated with a higher risk of comorbid autoimmune diseases. METHODS: We analyzed records of CSU patients (n = 1,199) for a history or presence of autoimmune diseases. Patients were diagnosed with type IIb autoimmune CSU (aiCSU) if all 3 tests were positive: autologous serum skin test (ASST), basophil histamine release assay (BHRA) and/or basophil activation test (BAT), and IgG autoantibodies against FcεRIα/IgE detected by immunoassay. RESULTS: Twenty-eight percent of CSU patients had at least 1 autoimmune disease. The most prevalent autoimmune diseases were Hashimoto's thyroiditis (HT) (≥ 21%) and vitiligo (2%). Two percent of CSU patients had ≥ 2 autoimmune diseases, most frequently HT plus vitiligo. Comorbid autoimmune diseases, in patients with CSU, were associated with female sex, a family history of autoimmune diseases, and higher rates of hypothyroidism and hyperthyroidism (P < 0.001). Presence of autoimmune diseases was linked to aiCSU (P = 0.02). The risks of having autoimmune diseases were 1.7, 2.9 and 3.3 times higher for CSU patients with a positive ASST, BHRA and BAT, respectively. In CSU patients, markers for autoimmune diseases, antinuclear antibodies and/or IgG anti-thyroid antibodies were associated with non-response to omalizumab treatment (P = 0.013). CONCLUSIONS: In CSU, autoimmune diseases are common and linked to type IIb autoimmune CSU. Our results suggest that physicians assess and monitor all adult patients with CSU for signs and symptoms of common autoimmune diseases, especially HT and vitiligo.

2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation (Part 3 - Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global 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 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.

Does erythropoietin modulate human hair follicle melanocyte activities in situ?

Erythropoietin (EPO) is now appreciated for not only drive erythopoiesis, but also to exert additional functions. Since we had previously shown that human hair follicles (HFs) are both an extra-renal source and an extra-medullary target of EPO, we have now studied whether one such function is the regulation of HF pigmentation. Human anagen VI HFs were treated with EPO (100 IU/ml) in serum-free organ culture. Unexpectedly, we noticed greatly divergent pigmentary effects of EPO, since both up- and down-regulation of HF melanin content and tyrosinase activity in situ was seen in HF derived from different individuals. These divergent effects could not be attributed to differences in skin regions, the total HF melanocyte number or specific traits of individual HF donors. Our pilot study provides first evidence suggesting that EPO may modulate normal human melanocyte functions under physiologically relevant conditions in situ.

Are autoantibodies against the beta1-adrenergic receptor markers for dilated cardiomyopathy?

BACKGROUND: Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure in the western world but there is still no specific and early diagnosis available. Besides a genetic predisposition and viral infections, autoimmune reactions play an important role in the pathogenesis of DCM. The beta1-adrenergic receptor (beta1-AR) has been described as the major target structure in autoimmune DCM. METHODS: In this study a recombinant GST-beta1-AR fusion protein comprising the second extracellular loop was generated as a target for the analysis of autoantibodies in sera from 115 patients with different heart failure diseases (41 DCM, 30 non-ischemic secondary cardiomyopathy [NISCM], 44 coronary artery disease [CAD]). Sera were collected from a non-selected population of heart failure patients in consecutive order. RESULTS: Autoantibodies against the beta1-AR were detected in 37% of DCM, 30% of NISCM, and 36% of CAD patients but none of the controls were positive. Furthermore, our data show that cardiomyopathy patients with anti-beta1-AR antibodies are younger (54 years vs. 61 years [DCM], 53 years vs. 56 years [NISCM], 61 years vs. 61 years [CAD]. Regardless of diagnosis antibody-positive patients had lower EF levels (29% vs. 32%, p = 0.0001 [DCM]; 23% vs. 25%, p < 0.0001 [NISCM]; 23% vs. 25%, p < 0.0001 [CAD]) than the antibody-negative counterparts but, nevertheless, also lower NT-proBNP levels compared to antibody negative patients (567 pg/mL vs. 1296 pg/mL, p = 0.0005 [DCM]; 224 pg/mL vs. 1135 pg/mL, p = 0.0002 [NISCM]; 605 pg/mL vs. 940 pg/mL, p = 0.0005 [CAD]). CONCLUSIONS: We conclude that DCM patients should be further characterized and differentiated by the detection of autoantibodies against beta1-AR. Autoimmune DCM patients are younger compared with their non-autoimmune counterparts, possibly due to the autoimmune trigger of the disease or reflecting an early stage of the disease. Surprisingly, the autoimmune patients have worse clinical manifestations but show less excessive NT-proBNP levels. It is not clear yet, though, whether these autoantibodies have a direct impact on the NT-proBNP levels. Whether or not these data are a consequence of pathogenic antibodies has to be elucidated in further studies.

Current Considerations on Characterization of Immune Response to Multi-Domain Biotherapeutics.

Compounds containing two or more structural domains with a distinct mode of action relevant to functionality have been defined as multi-domain biotherapeutics (MDBs). Several modalities, including endogenous protein fusions with an antibody Fc fragment or another polypeptide, bispecific antibodies, antibody-drug conjugates, as well as polyethylene glycol conjugates have been viewed as examples of MDBs. Similar to other biotherapeutics, MDBs have the potential to induce a host immune response, commonly detected in the form of anti-drug antibodies (ADAs). The need to characterize ADA specificity to a particular domain of the MDB has been identified as a potential regulatory requirement based on the compound nature of the drug and associated immunogenicity risk factors. MDB-related immunogenicity risk factors are discussed herein. The relative risk level of each of the immunogenicity factors was analyzed based on publicly available information. It is proposed that MDB-related immunogenicity risk factors can be divided into major and minor categories. Major risk category factors include (a) presence of immunogenic structural or linear epitopes of either non-human or human sequence origin and (b) significant homology of an MDB domain to an endogenous protein with a specific and unique function. Proposed minor risk category factors include (a) epitope spread, (b) repetitive antigenic structure of MDB, and (c) hapten-like effect due to chemical conjugation or fusion with a larger protein. Detailed modality-based information on several examples of MDBs is presented to support this proposal.

European Immunogenicity Platform 11th Open Scientific Symposium on immunogenicity of biopharmaceuticals.

Given the expanding number of complex therapeutic protein drugs and advanced therapy medicinal products that are being developed, improving our ability to assess the potential immunogenicity of biologics is critical to ensuring treatment efficacy and patient safety. In this context, the European Immunogenicity Platform annual meeting provides opportunities for experts from industry and academia, regulators and clinicians to convene and discuss immunogenicity assessment methods and tools. This report summarizes the key messages on immunogenicity testing, prediction, clinical relevance and advanced therapy medicinal products discussed at the 11th Open Scientific European Immunogenicity Platform Symposium on Immunogenicity of Biopharmaceuticals, Lisbon, Portugal, 17-19 February 2020.

Immunogenicity of rituximab in patients with severe pemphigus.

Pemphigus is a life-threatening autoimmune bullous disease associated with autoantibodies to desmoglein 1 and/or 3. The anti-CD20 chimeric mouse monoclonal antibody rituximab has previously been successfully applied in more than 130 reported pemphigus patients with severe and/or refractory disease. Since antibodies against other therapeutics such as IFNalpha and beta, erythropoietin, and TNFalpha antagonists had led to decreased efficacy of these drugs, we determined anti-rituximab antibodies in 11 patients with pemphigus before rituximab administration as well as 3, 9, and 15 months thereafter. For this purpose, a novel, affinity capture elution assay was established using rabbit IgG against the F(ab)2 fragment of rituximab. In addition, serum levels of rituximab were determined by a competition ELISA. In 2 of 11 pemphigus patients, antibodies to rituximab were detected. In both patients, only a partial remission was observed under treatment. In addition, when followed over a longer period of time, the occurrence of anti-rituximab antibodies paralleled an increase in disease activity. Of the 9 patients without development of antiantibodies to rituximab, in 5, all lesions healed and in 4, partial remissions were seen. These observations show that antibodies to rituximab are generated in some patients during rituximab treatment and may be associated with a less favourable response to treatment.

Thyroid hormones directly alter human hair follicle functions: anagen prolongation and stimulation of both hair matrix keratinocyte proliferation and hair pigmentation.

CONTEXT: Both insufficient and excess levels of thyroid hormones (T3 and T4) can result in altered hair/skin structure and function (e.g. effluvium). However, it is still unclear whether T3 and T4 exert any direct effects on human hair follicles (HFs), and if so, how exactly human HFs respond to T3/T4 stimulation. OBJECTIVE: Our objective was to asses the impact of T3/T4 on human HF in vitro. METHODS: Human anagen HFs were isolated from skin obtained from females undergoing facelift surgery. HFs from euthyroid females between 40 and 69 yr (average, 56 yr) were cultured and treated with T3/T4. RESULTS: Studying microdissected, organ-cultured normal human scalp HFs, we show here that T4 up-regulates the proliferation of hair matrix keratinocytes, whereas their apoptosis is down-regulated by T3 and T4. T4 also prolongs the duration of the hair growth phase (anagen) in vitro, possibly due to the down-regulation of TGF-beta2, the key anagen-inhibitory growth factor. Because we show here that human HFs transcribe deiodinase genes (D2 and D3), they may be capable of converting T4 to T3. Intrafollicular immunoreactivity for the recognized thyroid hormone-responsive keratins cytokeratin (CK) 6 and CK14 is significantly modulated by T3 and T4 (CK6 is enhanced, CK14 down-regulated). Both T3 and T4 also significantly stimulate intrafollicular melanin synthesis. CONCLUSIONS: Thus, we present the first evidence that human HFs are direct targets of thyroid hormones and demonstrate that T3 and/or T4 modulate multiple hair biology parameters, ranging from HF cycling to pigmentation.