Cecilia Hellström

Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling.

High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions.

Discovery of circulating proteins associated to knee radiographic osteoarthritis

Currently there are no sufficiently sensitive biomarkers able to reflect changes in joint remodelling during osteoarthritis (OA). In this work, we took an affinity proteomic approach to profile serum samples for proteins that could serve as indicators for the diagnosis of radiographic knee OA. Antibody suspension bead arrays were applied to analyze serum samples from patients with OA (n = 273), control subjects (n = 76) and patients with rheumatoid arthritis (RA, n = 244). For verification, a focused bead array was built and applied to an independent set of serum samples from patients with OA (n = 188), control individuals (n = 83) and RA (n = 168) patients. A linear regression analysis adjusting for sex, age and body mass index (BMI) revealed that three proteins were significantly elevated (P < 0.05) in serum from OA patients compared to controls: C3, ITIH1 and S100A6. A panel consisting of these three proteins had an area under the curve of 0.82 for the classification of OA and control samples. Moreover, C3 and ITIH1 levels were also found to be significantly elevated (P < 0.05) in OA patients compared to RA patients. Upon validation in additional study sets, the alterations of these three candidate serum biomarker proteins could support the diagnosis of radiographic knee OA.

Serum autoantibody profiling of patients with paraneoplastic and non-paraneoplastic autoimmune retinopathy

Purpose Although multiple serum antiretinal autoantibodies (ARAs) have been reported in patients with paraneoplastic and non-paraneoplastic autoimmune retinopathy ((n)pAIR), not all retinal antigens involved in (n)pAIR are specified. This study aims to serologically identify patients with presumed (n)pAIR through determination of both known and unknown ARAs by autoantibody profiling. Methods An antigen suspension bead array using 188 different antigens representing 97 ocular proteins was performed to detect ARAs in serum samples of patients with presumed (n)pAIR (n = 24), uveitis (n = 151) and cataract (n = 21). Logistic regressions were used to estimate the associations between ocular antigens and diagnosis. Validation of interphotoreceptor matrix proteoglycan 2 (IMPG2) and recoverin antigens was performed by immunohistochemistry and immunoblot, respectively. Results Samples of patients with presumed (n)pAIR exhibited a broad spectrum of ARAs. We identified retinal antigens that have already been described previously (e.g. recoverin), but also identified novel ARA targets. Most ARAs were not specific for (n)pAIR since their presence was also observed in patients with cataract or uveitis. High titers of autoantibodies directed against photoreceptor-specific nuclear receptor and retinol-binding protein 3 were more common in patients with presumed (n)pAIR compared to uveitis (p = 0.015 and p = 0.018, respectively). The presence of all other ARAs did not significantly differ between groups. In patients with presumed (n)pAIR, anti-recoverin autoantibodies were the most prevalent ARAs. Validation of bead array results by immunohistochemistry (anti-IMPG2) and immunoblot (anti-recoverin) showed concordant results in (n)pAIR patients. Conclusions Patients with (n)pAIR are characterized by the presence of a broad spectrum of ARAs. The diagnosis of (n)pAIR cannot be based on the mere presence of serum ARAs, as these are also commonly present in uveitis as well as in age-related cataract patients.

Untargeted screening for novel autoantibodies with prognostic value in first-episode psychosis

Immunological and inflammatory reactions have been suggested to have a role in the development of schizophrenia, a hypothesis that has recently been supported by genetic data. The aim of our study was to perform an unbiased search for autoantibodies in patients with a first psychotic episode, and to explore the association between any seroreactivity and the development of a Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) disorder characterized by chronic or relapsing psychotic symptoms. We collected plasma samples from 53 patients when they were treated for their first-episode psychosis, and 41 non-psychotic controls, after which the patients were followed for a mean duration of 7 years. Thirty patients were diagnosed with schizophrenia, delusional disorder, schizoaffective disorder, bipolar disorder or a long-term unspecified nonorganic psychosis during follow-up, whereas 23 patients achieved complete remission. At the end of follow-up, plasma samples were analyzed for IgG reactivity to 2304 fragments of human proteins using a multiplexed affinity proteomic technique. Eight patient samples showed autoreactivity to the N-terminal fragment of the PAGE (P antigen) protein family (PAGE2B/PAGE2/PAGE5), whereas no such autoreactivity was seen among the controls. PAGE autoreactivity was associated with a significantly increased risk of being diagnosed with schizophrenia during follow-up (odds ratio 6.7, relative risk 4.6). An immunohistochemistry analysis using antisera raised against the N-terminal fragment stained an unknown extracellular target in human cortical brain tissue. Our findings suggest that autoreactivity to the N-terminal portion of the PAGE protein family is associated with schizophrenia in a subset of patients with first-episode psychosis.

High-Density Serum/Plasma Reverse Phase Protein Arrays

In-depth exploration and characterization of human serum and plasma proteomes is an attractive strategy for the identification of potential prognostic or diagnostic biomarkers. The possibility of analyzing larger numbers of samples in a high-throughput fashion has markedly increased with affinity-based microarrays, thus providing higher statistical power to these biomarker studies. Here, we describe a protocol for high-density serum and plasma reverse phase protein arrays (RPPAs). We demonstrate how a biobank of 12,392 samples was immobilized and analyzed on a single microarray slide, allowing high-quality profiling of abundant target proteins across all samples in one assay.

T20. SEARCHING FOR NOVEL AUTOANTIBODIES WITH CLINICAL RELEVANCE IN PSYCHIATRIC DISORDERS

Abstract Background Immunological reactions may have a role in subgroups of patients suffering from psychiatric disorders. Possible markers for such subgroups may be autoantibodies of currently unknown nature. If identified, they could indicate which patients that would benefit from immunomodulatory treatment in addition to standard interventions. Modern proteomic methods allow analyses of antibody binding to thousands of different human proteins, facilitating the identification of currently undiscovered autoantibodies. Methods We have explored the association between any seroreactivity in plasma samples from first episode psychosis patients against more than 2000 randomly chosen protein fragments derived from human proteins, and the development of disorders characterized by chronic or relapsing psychotic symptoms. Plasma from 53 patients and 41 non-psychotic controls were assessed; the clinical course of the patients were followed for a mean duration of 7 years. The plasma samples were analyzed for IgG reactivity to 2304 fragments (approx 100 a.a. residues in length) of human proteins using a multiplexed affinity proteomic technique, and positive hits validated for binding in two additional assays. Results Thirty patients were diagnosed with schizophrenia, delusional disorder, schizoaffective disorder, bipolar disorder or a long-term unspecified nonorganic psychosis during follow-up, while 23 patients achieved complete remission. Eight patient samples showed autoreactivity to the N-terminal fragment of the PAGE protein family (PAGE2B/PAGE2/PAGE5), whereas no such autoreactivity was seen among the controls. PAGE autoreactivity was associated with a significantly increased risk of being diagnosed with schizophrenia during follow-up (odds ratio 6.7). An antisera raised against the N-terminal fragment stained an unknown extracellular target in human cortical brain tissue (Zandian et al., Transl Psychiatry 7: e1177; doi:10.1038/tp.2017.160). We are currently investigating the identity of this target. In addition, two other putative new autoantibodies found primarily among the patients, and rarely in the controls, will be discussed at the meeting. Discussion Our findings suggest that autoreactivity to the N-terminal portion of the PAGE protein family is associated with schizophrenia in a subset of patients with first-episode psychosis. In addition, we propose that searching for novel autoantibodies in an unbiased way may be feasible using state-of-the-art proteomic methods, and can yield useful biological markers for immune involvement in subgroups of individuals diagnosed with psychiatric disorders.

High-Density Antigen Microarrays for the Assessment of Antibody Selectivity and Off-Target Binding

With the increasing availability of collections of antibodies, their evaluation in terms of binding selectivity becomes an important but challenging task. Planar antigen microarrays are very suitable tools to address this task and provide a powerful proteomics platform for the characterization of the binding selectivity of antibodies toward thousands of antigens in parallel. In this chapter, we describe our in-house developed procedures for the generation of high-density planar antigen microarrays with over 21,000 features. We also provide the details of the assay protocol, which we routinely use for the assessment of binding selectivity of the polyclonal antibodies generated within the Human Protein Atlas.

Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer

OBJECTIVES Cancer-testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies. MATERIALS AND METHODS To comprehensively analyze autoantibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases. RESULTS Altogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analyzed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against cancer-testis antigen family 47; member A (CT47A) genes, P antigen family member 3 (PAGE3), variable charge X-linked (VCX), melanoma antigen family B1 (MAGEB1), lin-28 homolog B (LIN28B) and chromosome 12 open reading frame 54 (C12orf54) were only found in NSCLC patients at a frequency of 1%-4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients. CONCLUSION We identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets.

05.01 Protein profiling in plasma reveals molecular subgroups in systemic lupus erythematosus

Objective Systemic Lupus Erythematosus (SLE) is a heterogeneous systemic autoimmune disease that is currently lacking specific diagnostic biomarkers. The diversity within the patients might obstruct clinical trials and could reflect differences in underlying pathogenesis. Our objective was to identify protein profiles that could be used for diagnosis and to identify molecular subgroups within SLE for patient stratification subjected to different treatment. Method In a cross-sectional study we performed protein profiling of 695 plasma samples from SLE patients and matched controls. This was achieved by utilising an antibody suspension bead array targeting 367 proteins. T-test and ROC analysis was performed to identify differences in the protein profiles between SLE and controls. Unsupervised K-means clustering was performed to identify data-driven SLE subgroups. Results We report that the novel proteins MMP1, SELE, and S100A12 can distinguish between SLE patients and controls with an area under curve of 0.80 in a ROC analysis. In addition, 28 proteins were found to show differences (corrected p-value<0.05) between SLE patients and controls. By unsupervised clustering we identified an IRF5, NOS3 and CLDN8-driven subgroup, an ARID2 and SELE-driven subgroup and one subgroup characterised by low SLC22A2 levels. Conclusion We have identified potential biomarkers of SLE that may be used to improve the diagnosis of SLE patients. Our suggested panel of biomarkers needs to be validated in an additional SLE cohort and also in relation to other systemic autoimmune diseases before it can be used as a diagnostic test. We have also identified subgroups characterised by different molecular patterns, indicating underlying pathogenic differences. These patient groups might benefit from different treatment strategies. Our work adds new information to today’s view of classifying the heterogeneous subgroups within SLE and the importance of personalised medicine.

OP0271 Analysis of the Autoantibody Repertoire in Idiopathic Inflammatory Myopathies Using Antigen Bead Array

Background The Idiopathic Inflammatory Myopathies (IIM), including Polymyositis (PM), Dermatomyositis (DM) and Inclusion Body Myositis (IBM), are a group of rare systemic inflammatory diseases often associated with severe organ manifestations and premature mortality [1]. The identification of new biomarkers is needed to understand underlying biological pathways, improve diagnosis, predict prognosis and tailor treatment to the single patient. Objectives We investigated EDTA-plasma samples of IIM patients and healthy controls (HC) to discover new myositis-associated auto-antigens. Methods Planar antigen microarrays consisting of 5760 human protein fragments were used for the screening of IgG reactivity in 160 samples of patients with Systemic Lupus Erythematosus (SLE) [2]. A set of 355 antigens (Ag) was selected for verification on suspension bead array using 695 SLE samples and 278 IIM samples. The IIM samples were collected from a cohort of 245 IIM patients (91 DM, 125 PM and 29 IBM) regularly followed at the Rheumatology Unit of the Karolinska University Hospital from January 2003 until March 2014. Twenty-eight IIM patients tested positive for anti-hystidyl-tRNA-synthetase antibodies (anti-Jo-1) and 217 were anti Jo-1 negative. Samples from 41 HC were also analysed. Results Reactivity towards 70% of the 355 selected Ag was observed in more than 2% of both IIM and HC samples. Comparing the IIM and the HC groups according to the number of samples which showed reactivity towards each single Ag, reactivity towards 3 Ag was discovered with higher frequencies in the IIM samples (Fisher exact test, p<0.05). We also compared each IIM subset and HC. A higher number of DM and PM samples versus HC showed reactivity towards 4 and 3 Ag, respectively. No statistically significant difference was observed between IBM and HC samples for any of the 355 Ag. Making 2 group comparisons between DM/PM/IBM subsets, a statistically significant different reactivity profile was found for 1 Ag between DM and PM, 3 Ag between DM and IBM and 4 Ag between PM and IBM. The number of reactive samples towards 5 Ag was significantly higher in the anti-Jo-1 positive compared to the anti-Jo-1 negative patients. Conclusions Auto-antigen reactivity was present both in IIM and HC samples. IIM and HC samples showed different frequencies of reactivity towards some of the selected Ag. A validation analysis is ongoing to confirm our preliminary results. References Dalakas MC. Polymyositis, dermatomyositis and inclusion-body myositis. N Engl J Med. 1991;325:1487–98. B. Ayoglu et al., Expert Rev Mol Diagn 11, 219 (Mar, 2011). Disclosure of Interest A. Notarnicola: None declared, C. Mattsson: None declared, H. Idborg: None declared, C. Hellström: None declared, E. Jemseby: None declared, P.-J. Jacobsson Grant/research support from: Astra-Zeneca, P. Nilsson: None declared, I. E. Lundberg Grant/research support from: Novartis, Servier, Astra-Zeneca och Bristol-Myers Squibb