OBJECTIVES:Autoantibodies targeting melanoma differentiation associated protein 5 (MDA5) are strongly associated with dermatomyositis (DM) and may contribute to its pathogenesis. Here we aimed to investigate MDA5+ B cells, their phenotype and generate MDA5 monoclonal antibodies to assess their epitope specificity. METHODS:MDA5-reactive B cells were captured from peripheral blood of patients with anti-MDA5+ DM (n = 3) using an MDA5-fluorescent probe. B cell receptor (BCR) sequences were analysed from single-sorted B cells (n = 240). Selected clones were re-expressed as IgG1 monoclonal antibodies (mAbs, n = 23). Reactivity was assessed using recombinant MDA5 protein constructs, peptide epitope mapping, ELISA, western blot and a commercial line blot assay. RESULTS:Of 240 anti-MDA5+ sorted B cells, 23 BCRs were re-expressed as mAbs, two of which showed high reactivity and specificity for MDA5. These antibody sequences originated from one CD19+IgD-CD27-CD38+ and one CD19+IgD-CD27+CD38+ IgG+ B cell with low somatic hypermutation (SHM). Both mAbs had nanomolar apparent affinity and bound to sites within the helicase domains of the MDA5 protein but with distinct epitope recognition. Serology screening confirmed targeting of a linear epitope identified in the mAb studies. CONCLUSION:Our results show that anti-MDA5+ B cells recognize the helicase domains, which are the enzymatically active domains of the protein. These results have implications for understanding the etiopathology of anti-MDA5+ DM and development of new antigen-specific therapies.

Genetic studies are revolutionizing our understanding of neurodegenerative diseases by identifying hundreds of new genetic risk factors, yet the ability to link these variants to phenotype is constrained by the lack of knowledge on the encoded proteins. Moreover, for any given disease, elucidating the functional connections among the genetic risk factors has been challenging. Using 33 risk genes for amyotrophic lateral sclerosis (ALS) as a model, we set out to explore connections among the encoded proteins using antibodies identified within a systematic framework exploiting knockout-based antibody characterization. We characterized 296 antibodies against 33 genetically linked to ALS—ACSL5, ALS2, ANG, ANXA11, ATXN2, C9orf72, CAV1, CCNF, CHCHD10, CHMP2B, FIG4, FUS, HNRNPA1, HNRNPA2B1, KIF5A, LGALS1, MATR3, NEK1, OPTN, PFN1, SETX, SIGMAR1, SOD1, SPG11, SQSTM1, TAF15, TARDBP, TBK1, TIA1, TUBA4A, UBQLN2, VAPB, and VCP—and identified high-quality reagents for most proteins. Using these reagents, as the first step in characterizing the ALS genetic risk proteome, we profiled protein expression across human induced pluripotent stem cell–derived and primary neurological cells. Protein-level analyses uncovered diverse and unexpected expression signatures, discordant with RNA datasets. Notably, 22 proteins were enriched in glial populations, with ten dynamically regulated by inflammatory cues in microglia, underscoring immune-responsive pathways as key contributors to ALS pathogenesis. This work provides both a reference resource for ALS biology and a generalizable framework for translating genetic discoveries into mechanistic protein insights in neurodegeneration. ### Competing Interest Statement The authors have declared no competing interest. Motor Neurone Disease Association ALS Association ALS Society of Canada Genome Canada, OGI-210 Génome Québec, OGI-210 Ontario Genomics, https://ror.org/00c68jz96, OGI-210 Québec Consortium for Drug Discovery Canadian Institutes of Health Research, https://ror.org/01gavpb45, PJT-195804 ALS FindingACure

OBJECTIVES:To determine prevalence and clinical associations of anti-Four-and-a-half-LIM-domain 1 (FHL1) autoantibodies in patients with idiopathic inflammatory myopathies (IIM) and to evaluate autoantibody levels over time. METHODS:Sera at the time of diagnosis from patients with IIM (n = 449), autoimmune disease controls (DC, n = 130), neuromuscular diseases (NMDs, n = 16) and healthy controls (HC, n = 100) were analysed for anti-FHL1 autoantibodies by enzyme-linked immunosorbent assay (ELISA). Patients with IIM FHL1+ and FHL1- were included in a longitudinal analysis. Serum levels were correlated to disease activity. RESULTS:Autoantibodies to FHL1 were more frequent in patients with IIM (122/449, 27%) compared with DC (autoimmune DC and NMD, 13/146, 9%, P < 0.001) and HC (3/100.3%, P < 0.001). Anti-FHL1 levels were higher in IIM [median (IQR)=0.62 (0.15-1.04)] in comparison with DC [0.22 (0.08-0.58)], HC [0.35 (0.23-0.47)] and NMD [0.48 (0.36-0.80)] P < 0.001. Anti-FHL1+ patients with IIM were younger at the time of diagnosis compared with the anti-FHL1- group (P = 0.05) and were seronegative for other autoantibodies in 25%.In the first follow-up, anti-FHL1+ sample 20/33 (60%) positive at baseline had turned negative for anti-FHL1 autoantibodies. Anti-FHL1 autoantibodies rarely appeared after initiating treatment. Anti-FHL1 autoantibody levels correlated with CK (r = 0.62, P= 0.01), disease activity measured using the Myositis Disease Activity Assessment Tool (MYOACT) (n = 14, P = 0.004) and inversely with Manual Muscle Test-8 (r = -0.59, P = 0.02) at baseline. CONCLUSION:Anti-FHL1 autoantibodies were present in 27% of patients with IIM; of these, 25% were negative for other autoantibodies. Other autoimmune diseases had lower frequencies and levels. Anti-FHL1 levels often decreased with immunosuppressive treatment, correlated with disease activity measures at diagnosis and rarely appeared after start of treatment.

Abstract A substantial fraction of amyotrophic lateral sclerosis (ALS)-associated proteins remain poorly characterized, in part because of the limited availability of validated research antibodies. We established knockout (KO)-based antibody characterization workflows and demonstrated that widely used antibodies against the major ALS-associated protein C9orf72 lacked specificity (Laflamme et al., 2019). We subsequently scaled this framework to systematically benchmark research antibodies, revealing that up to 61% fail to perform as recommended by manufacturers (Ayoubi et al., 2023). Here, we extend this approach by establishing the ALS-Reproducible Antibody Platform (ALS-RAP), a comprehensive effort to generate a publicly available dataset of KO-validated antibodies targeting proteins encoded by ALS risk genes. In total, we characterized 303 antibodies against 33 ALS-associated proteins to identify high-quality reagents for use in western blot, immunoprecipitation, and immunofluorescence. Using these antibodies, we profiled protein levels across human induced pluripotent stem cell (iPSC)-derived and primary neurological cell types. These analyses revealed diverse cellular distributions and higher levels of several ALS-associated proteins in glial populations, consistent with emerging evidence for immune contributions to ALS. Together, ALS-RAP provides a validated antibody toolbox and protein expression resource to support the study of ALS-associated proteins.

The oleaginous microalga Microchloropsis gaditana (formerly Nannochloropsis gaditana ) has gained large interest due to its potential to produce lipids for a wide range of biotechnological applications. To optimize M. gaditana growth conditions and develop new strains to enhance lipid synthesis and accumulation, a broad understanding of the organism metabolism is essential. Computational models such as genome-scale metabolic models constitute powerful tools for unravelling microorganism metabolism. In this work we present iMgadit23, a new genome-scale metabolic model for M. gaditana . Model covers 2330 reactions involving 1977 metabolites and associated with 889 genes. Pathways involved in membrane and storage glycerolipid biosynthesis and degradation have undergone thorough manual curation and have been comprehensively described based on current knowledge of M. gaditana lipid metabolism. Additionally, we developed a detailed 2D-pathway map of model content to provide a systems-level visualization of M. gaditana metabolism. We demonstrated the predictive capabilities of iMgadit23, validating its ability to qualitatively and quantitatively capture in vivo growth phenotypes under diverse environmental and genetic conditions. Model was also able to capture the role of the Bubblegum acyl-CoA synthetase in remodeling M. gaditana lipid metabolism. iMgadit23 and its 2D map constitute valuable tools to increase understanding of M. gaditana metabolism and deciphering mutant phenotypes, specifically in the context of lipid metabolism. The model holds significant promise in predicting M. gaditana metabolic capabilities, facilitating strain engineering, and optimizing cultivation processes for a broad range of industrial applications. ### Competing Interest Statement The authors have declared no competing interest.

OBJECTIVES Clinical observations in patients with dermatomyositis (DM) and autoantibodies against the melanoma differentiation-associated protein 5 (MDA5) suggest that the autoantibodies contribute to the pathogenesis of MDA5(+) DM. To gain insight into the role of the anti-MDA5 autoantibodies, we aimed to identify their binding sites on the different domains of the MDA5 protein. METHODS We developed an in-house ELISA to assess the reactivity against the MDA5 domains (conformational epitopes) in plasma (n = 8) and serum (n = 24) samples from MDA5(+) patients with varying clinical manifestations and disease outcomes. The reactivities were also assessed using Western Blot (linearized epitopes). An ELISA-based depletion assay was developed to assess cross-reactivity among the different MDA5 domains. RESULTS All eight plasma samples consistently showed reactivity towards conformational and linearized epitopes on the helicase domains of the MDA5 protein. The ELISA-based depletion assay suggests that anti-MDA5 autoantibodies specifically target each of the three helicase domains. Twenty-two of the 24 serum samples showed reactivity in the in-house ELISA and all 22 displayed reactivity towards the helicase domains of the MDA5 protein. CONCLUSIONS Our data revealed that the main immunogenic targets of anti-MDA5 autoantibodies from MDA5(+) patients are the helicase domains. Considering that the helicase domains are responsible for the enzymatic activity and subsequent triggering of an inflammatory response, our findings suggest that binding of anti-MDA5 autoantibodies could alter the canonical activity of the MDA5 protein and potentially affect the downstream induction of a pro-inflammatory cascade.

Background Anti-synthetase syndrome (ASSD), a sub-group of idiopathic inflammatory myopathies (IIM) is characterized by the presence of autoantibodies targeting aminoacyl tRNA synthetases (aaRS) and specific clinical manifestations such as myositis and interstitial lung disease (ILD) [1]. Some of the most common anti-aaRS autoantibodies in ASSD are anti-Jo1, -PL7, -PL12 and-EJ. In addition, many anti-aaRS positive patients are also positive for anti-Ro52. Having the combination of anti-Jo1 and anti-Ro52 increases the risk of developing ILD [2]. The presence of autoantibodies is an important part of the classification of ASSD, however only autoantibodies of IgG isotype are usually analyzed in the clinical setting. In rheumatoid arthritis there is evidence that anti-citrullinated protein/peptide antibodies (ACPA) can be found as IgG, IgA and IgM, and importantly, specific isotypes might correlate with disease activity [3, 4]. Objectives To verify if other autoantibody isotypes, besides IgG, might be present in sera of patients with IIM/ASSD and to compare with the corresponding frequencies in population controls (PC). Methods Stored sera collected from consecutive 366 IIM patients and 156 age/gender matched PC at Karolinska University Hospital were retrospectively selected. The serum samples were screened for the presence of autoantibodies of isotypes IgG, IgA and IgM, against a panel of 20 antigens representing Jo1 (HisRS), PL7 (ThrRS), PL12 (AlaRS), EJ (GlyRS), and Ro52 (TRIM21) using a multiplex bead array assay. Results We identified IIM patients with autoantibodies of different isotypes, and a low frequency in PC (Figure 1). For anti-Jo1 autoantibodies we could detect IIM patients with only IgG (n=13), only IgM (n=8) and only IgA (n=4), but the majority had a combination of two (n=32) or three isotypes (n=16). For the other anti-aaRS autoantibodies the distribution was more equal to each of the three isotypes with anti-PL12 and anti-PL7 being represented by a slightly higher frequency of IgG and only a few patients had antibodies of more than one isotype targeting PL12, PL7 or EJ. The majority of anti-Ro52 positive IIM patients (n=52) only harbored IgG isotype. The combination of anti-Ro52 and anti-aaRS autoantibodies was identified in 28 patients (anti-Jo1 (n=19), -PL12 (n=2), -PL7 (n=3), and -EJ (n=4)). Most patients with such combination had anti-Ro52 IgG together with anti-aaRS IgG or IgG in combination with IgA and/or IgM. The exception was observed for three anti-Jo1 positive patients who had the combination anti-Ro52 IgG with only anti-Jo1 IgM and one anti-PL7 positive patient who had anti-Ro52 IgA together with anti-PL7 IgA and IgG. Figure 1. Venn diagrams showing reactivity in idiopathic inflammatory myopathies (IIM) (top) and population controls (PC) (bottom) for the three autoantibody isotypes IgG, IgA and IgM against five myositis antigens: Jo1 (HisRS), PL12 (AlaRS), ThrRS (PL7), EJ (GlyRS) and Ro52 (TRIM21). Conclusion The frequency of the different autoantibody isotypes seems to be autoantigen dependent. Our results suggest that for anti-aaRS autoantibodies it could be important to investigate additional autoantibody isotypes, as some patients only harbor autoantibodies of IgM or IgA isotypes but not IgG. The clinical relevance of the different antibody isotypes still needs to be determined. References [1]Mahler, M., et al., Rev, 2014. 13 (4-5): p. 367-71. [2]Huang, H.L., et al., J Clin Neurosci, 2020. [3]Arlestig, L., et al., Ann Rheum Dis, 2012. 71 (6): p. 825-9. [4]Roos Ljungberg, K., et al., Arthritis Res Ther, 2020. 22 (1): p. 274. Table 1. Total number of individuals and percentage (n (%)) in each group for each of the isotypes and antigens. anti-Jo1 anti-PL12 anti-PL7 anti-EJ anti-Ro52 IIM PC IIM PC IIM PC IIM PC IIM PC IgG 61 (16.7) 1 (0.6) 7 (1.9) 0 (0.0) 7 (1.9) 0 (0.0) 3 (0.8) 0 (0.0) 54 (14.8) 5 (3.2) IgA 20 (5.5) 0 (0.0) 2 (1.2) 1 (0.6) 3 (0.8) 2 (1.3) 1 (0.3) 1 (0.6) 3 (0.8) 1 (0.6) IgM 56 (15.3) 1 (0.6) 1 (0.3) 2 (1.3) 7 (1.9) 0 (0.0) 1 (0.3) 0 (0.0) 3 (0.8) 2 (1.3) Acknowledgements SciLifeLab facilities Autoimmunity and Serology Profiling and Human Antibody Therapeutics (Drug Discovery and Development). IMI project EUbOPEN, This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 875510. The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA and Ontario Institute for Cancer Research, Royal Institution for the Advancement of Learning McGill University, Kungliga Tekniska Hoegskolan, Diamond Light Source Limited. Disclosure of Interests Charlotta Preger Grant/research support from: IMI project EUbOPEN, Grant no 875510, Antonella Notarnicola: None declared, Cecilia Hellström: None declared, Edvard Wigren Grant/research support from: IMI project EUbOPEN, Grant no 875510, Ingrid E. Lundberg Shareholder of: Roche and Novartis, Consultant of: Corbus Pharmaceuticals Inc, Astra Zeneca, Bristol Myer´s Squibb, Corbus Pharmaceutical, EMD Serono Research & Development Institute, Argenx, Octapharma, Kezaar, Orphazyme, and Janssen, Grant/research support from: Astra Zeneca, Per-Johan Jakobsson Shareholder of: Gesynta Pharma, Consultant of: UCB, Grant/research support from: Gesynta Pharma, Helena Persson Employee of: Affibody AB, Susanne Gräslund Grant/research support from: IMI project EUbOPEN, Grant no 875510

Objectives To determine the prevalence and associations of autoantibodies targeting a muscle-specific autoantigen, four-and-a-half-LIM-domain 1 (FHL1), in South Australian patients with histologically-confirmed idiopathic inflammatory myopathies (IIM) and in patients with SSc. Material and methods Sera from patients with IIM (n = 267) from the South Australian Myositis Database (SAMD), SSc (n = 174) from the Australian Scleroderma Cohort Study (ASCS) and healthy controls (HC, n = 100) were analysed for anti-FHL1 autoantibodies by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results Autoantibodies to FHL1 were more frequent in patients with IIM (37/267, 13.8%) compared with SSc (12/174, 7%) (P < 0.02) and HC (2/100, 2%) (P < 0.001). The most common IIM subtypes among FHL1(+) IIM patients were (32%) and IBM (2/37, 32%). No statistically significant differences in muscular or extra-muscular manifestations of IIM were found when comparing patients who were anti-FHL1(+) with their anti-FHL1(-) counterparts. In 29/37 (78%) anti-FHL1(+) patients, no myositis-specific autoantibodies (MSA) were present. In FHL1(+) muscle biopsies, there was less frequent infiltration by CD45(+) cells (P = 0.04). There was a trend for HLA alleles DRB1*07 and DRB1*15 to be more frequent in anti-FHL1(+) compared with anti-FHL1(-) patients (9/25 vs 19/113, P = 0.09 and 8/25 vs 15/114, P = 0.09, respectively). Conclusions We report a substantial prevalence (13.8%) of anti-FHL1 autoantibodies in a large cohort of patients with histologically confirmed IIM; 75% of these cases did not have a detectable myositis-specific autoantibody. Anti-FHL1 autoantibodies were also detected in a subgroup of patients with SSc (7%), indicating that anti-FHL1 autoantibodies may not be myositis-specific. The trend towards an HLA-DR association might indicate a specific immune response to the FHL1 protein.

Background The association between anti-melanoma differentiation association protein 5 autoantibodies (aMDA5) and rapidly progressive interstitial lung disease (RP-ILD) in clinically amyopathic dermatomyositis is well established in Asian population cohorts. In western cohorts, ILD has been strongly associated with aMDA5 but data regarding RP-ILD have been more conflicting. It is also suggested that western cohorts have more pronounced myopathic features than Asian. Objectives To characterise the disease manifestations of a Swedish aMDA5 positive idiopathic inflammatory myositis (IIM) cohort and to explore antigen reactivity of the MDA5 protein. Methods First available serum samples collected from 28 consecutive patients with IIM and positive aMDA5 ever tested by ELISA, Line Blot (LB) or Immunoprecipitation, attending Karolinska University Hospital between 1999 and 2021, were included. Clinical data including presence of anti-SSA autoantibodies by ELISA or LB was retrieved retrospectively. An in-house ELISA was used to screen serum samples for reactivity against a recombinant MDA5 protein (rMDA5, aa A110-D1025, UniProt ID Q9BYX4) and seven MDA5-derived constructs containing different domains. Correlations between aMDA5 reactivity levels and clinical data were explored. Results Nine patients showed no reactivity to any of the rMDA5 constructs by ELISA and were excluded from further analysis. Reactivity against rMDA5 was confirmed by ELISA in 19 patients (median 184.7 µg/mL (interquartile range (IQR) 277.07). The cohort included 13 male and 6 female patients, 94% Caucasian, with mean age at diagnosis of 41.05 years (standard deviation (SD) 10.5). Median disease duration at time of sampling was 0 months (IQR 1). All patients except one had signs of muscle involvement (muscle weakness, elevated muscle enzymes, muscle oedema or muscle biopsy consistent with myositis). At diagnosis 63.2% of patients reported muscle weakness (21.1 % had a manual muscle test 8 score <75). Dermatological findings were observed in 17/19 (89.7 %). During disease course nine patients (47.4%) had confirmed arthritis. ILD was diagnosed in 16/19 patients (84.2%), four of these (25%) developed a RP-ILD. One patient passed away due to RP-ILD and one required a lung transplant. Patients with ILD had a statistically significant higher mean age at diagnosis than those without (42.8.5 (SD 10.3) vs 31.3 (SD 4.7) years, p=0.02). Patients developing RP-ILD were not significantly older than patients with chronic ILD. Respiratory symptoms were reported by 75% of patients with ILD at time of diagnosis. The mean total lung capacity (TLC) of the ILD cohort was 68% (SD 17), mean diffusion capacity of carbon monoxide (DLCO) was 59% (SD 15) and mean forced vital capacity (FVC) was 62% (SD 19). There was a higher proportion of patients with CRP ≥ 3 times the reference range at diagnosis amongst patients with FVC <70 % than patients with FVC >70 % (88.9 % vs 16.7 %, p= 0.01). Ten patients (52.6%) had anti-SSA autoantibodies, all had ILD. Anti-SSA positive patients had a statistically significant lower TLC than those without (62% vs 79% respectively, p=0.04) and a lower FVC (57% vs 76% respectively, p=0.05). We found a weak non-statistically significant negative correlation between titres of aMDA5 and TLC, DLCO and FVC (Pearson coefficients -0.187, -0.289, -0.130 respectively). Frequency of ILD was higher in patients with aMDA5 titres >100 µg/mL than those with titers <100, but not statistically significant (81.3% vs 18.8%, respectively). Conclusion In this Caucasian cohort of aMDA5 positive IIM patients, ILD was present in over 80% of patients, of these, one quarter had RP-ILD. Older patients were more likely to present with ILD. Anti-SSA positivity and higher CRP levels were associated with worse lung function. We found a weak negative correlation between aMDA5 titres and lung function tests, as well as a trend of higher frequency of ILD in patients with higher aMDA5 titres. Muscle and skin involvement were found in a high proportion of patients. Acknowledgements D. Demirdal & E. Van Gompel contributed equally to this abstract. Disclosure of Interests Deniz Demirdal: None declared, Eveline Van Gompel: None declared, Edvard Wigren: None declared, Maryam Dastmalchi: None declared, Begum Horuluoglu: None declared, Angeles Shunashy Galindo-Feria: None declared, Susanne Gräslund: None declared, Karine Chemin: None declared, Ingrid E. Lundberg Shareholder of: Roche and Novartis., Consultant of: Consulting fees from Corbus Pharmaceuticals Inc, Astra Zeneca, Bristol Myer´s Squibb, Corbus Pharmaceutical, EMD Serono Research & Development Institute, Argenx, Octapharma, Kezaar, Orphazyme, and Janssen, Grant/research support from: Research grants from Astra Zeneca, Antonella Notarnicola Speakers bureau: compensation for lecture at conference sponsored by Boehringer Ingelheim.

Background To address the reactivity and affinity against histidyl-transfer RNA synthetase (HisRS) autoantigen of anti-Jo1 autoantibodies from serum and bronchoalveolar lavage fluid (BALF) in patients with idiopathic inflammatory myopathies/anti-synthetase syndrome (IIM/ASSD). To investigate the associations between the reactivity profile and clinical data over time. Methods Samples and clinical data were obtained from (i) 25 anti-Jo1(+) patients (19 sera with 16 longitudinal samples and 6 BALF/matching sera at diagnosis), (ii) 29 anti-Jo1(-) patients (25 sera and 4 BALF/matching sera at diagnosis), and (iii) 27 age/gender-matched healthy controls (24 sera and 3 BALF/matching sera). Reactivity towards HisRS full-length (HisRS-FL), three HisRS domains (WHEP, antigen binding domain (ABD), and catalytic domain (CD)), and the HisRS splice variant (SV) was tested. Anti-Jo1 IgG reactivity was evaluated by ELISA and western blot using IgG purified from serum by affinity chromatography. In paired serum-BALF, anti-Jo1 IgG and IgA reactivity was analyzed by ELISA. Autoantibody affinity was measured by surface plasmon resonance using IgG purified from sera. Correlations between autoantibody reactivity and clinical data were evaluated at diagnosis and longitudinally. Results Anti-Jo1 IgG from serum and BALF bound HisRS-FL, WHEP, and SV with high reactivity at the time of diagnosis and recognized both conformation-dependent and conformation-independent HisRS epitopes. Anti-HisRS-FL IgG displayed high affinity early in the disease. At the time of IIM/ASSD diagnosis, the highest autoantibody levels against HisRS-FL were found in patients ever developing interstitial lung disease (ILD) and arthritis, but with less skin involvement. Moreover, the reactivity of anti-WHEP IgG in BALF correlated with poor pulmonary function. Levels of autoantibodies against HisRS-FL, HisRS domains, and HisRS splice variant generally decreased over time. With some exceptions, longitudinal anti-HisRS-FL antibody levels changed in line with ILD activity. Conclusion High levels and high-affinity anti-Jo1 autoantibodies towards HisRS-FL were found early in disease in sera and BALF. In combination with the correlation of anti-HisRS-FL antibody levels with ILD and ILD activity in longitudinal samples as well as of anti-WHEP IgG in BALF with poor pulmonary function, this supports the previously raised hypothesis that the lung might have a role in the immune reaction in anti-Jo1-positive patients.

Mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer's disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signaling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterized. Here, we present phage display generated antibody single-chain variable fragments (scFvs) to human TREM2 immunoglobulin-like domain. Co-crystal structures revealed the binding of two scFvs to an epitope on the TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species, which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterization of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.

ABSTRACT Objectives Autoantibodies are thought to play a key role in the pathogenesis of idiopathic inflammatory myopathies (IIM). However, up to 40% of IIM patients, even those with clinical manifestations of anti-synthetase syndrome (ASSD), test seronegative to all known myositis-specific autoantibodies (MSAs). We hypothesized the existence of new potential autoantigens among human cytoplasmic aminoacyl tRNA synthetases (aaRS) in patients with IIM. Methods Plasma samples and clinical data from 217 patients with, 50 patients with ASSD, 165 without, and two with unknown ASSD status were included retrospectively, as well as serum from 156 age/sex-matched population controls. Samples were screened using a multiplex bead array assay for presence of autoantibodies against a panel of 118 recombinant protein variants, representing 33 myositis-related proteins, including all 19 cytoplasmic aaRS. Results We identified reactivity towards 16 aaRS in 72 of the 217 patients. Twelve patients displayed reactivity against nine novel aaRS. The novel autoantibody specificities were detected in four patients previously seronegative for MSAs and in eight with previously detected MSAs. We also confirmed reactivity to four of the most common aaRS (Jo1, PL12, PL7, and EJ (n=45)) and identified patients positive for anti-Zo, -KS, and -HA (n=10) that were not previously tested. A low frequency of anti-aaRS autoantibodies was detected in controls. Conclusion Our results suggest that most, if not all, cytoplasmic aaRS may become autoantigenic. Autoantibodies against new aaRS may be found in plasma of patients previously classified as seronegative with potential high clinical relevance.

The presence of myositis specific anti-melanoma differentiation associated protein 5 (MDA5) autoantibodies is associated with mucocutaneous ulcerations, rapidly progressing interstitial lung disease (RPILD), arthritis and mild muscle involvement in patients. RPILD is the major cause of mortality. At present it is unknown which domain of the MDA5 protein is the main elicitor of an immunogenic response.The aim of this study is to delineate the domains in the MDA5 protein that are the target of autoantibodies.Anti-MDA5 IgG were isolated from MDA5(+) patient plasma (7 UPMC, 1 KI and 1 KULeuven) by affinity chromatography using an in-house affinity column as described earlier in Ossipova et al, 2014(1). 8 constructs covering different regions of the MDA5 protein were recombinantly produced in E.coli (Uniprot ID Q9BYX4, Figure 1). An in-house ELISA was developed to identify the domains with the main epitope(s) by measuring the reactivity of the plasma samples and purified autoantibodies against these MDA5 protein constructs, similar to what was reported by Fernandes-Cerqueira et al, 2018(2). The biotinylated MDA5 proteins were immobilized on streptavidin coated plates and subsequently incubated with primary antibodies (purified autoantibodies(2) or original plasma) and a HRP-conjugated secondary antibody. The ELISA was developed by the addition of TMB substrate and the optical density (OD) was measured at 450 nm.Figure 1.Graphical presentation of the constructs representing different (combinations of) domains of the MDA5 protein.The preliminary data suggest the main reactivity of the plasma samples and the corresponding purified autoantibodies is directed towards the helicase domains and that there is variability between the patients in the reactivity towards domains located at the end of the protein.The study aims to resolve the main immunogenic domain of the MDA5 protein, which will lead to more insight in the disease mechanisms. The preliminary results suggest this domain is in the center of the MDA5 protein, but further experiments are necessary. We will use this set up to study differences in reactivity between patients (from different cohorts) and assess if differences in antibody reactivity could be linked to clinical features such as RPILD. Such correlations might be beneficial to predict the disease progression and to apply personal treatment approaches.[1]Ossipova E, Cerqueira CF, Reed E, Kharlamova N, Israelsson L, et al. Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint. Arthritis Res Ther. 2014;16(4):R167.[2]Fernandes-Cerqueira C, Renard N, Notarnicola A, Wigren E, Gräslund S, et al. Patients with anti-Jo1 antibodies display a characteristic IgG Fc-glycan profile which is further enhanced in anti-Jo1 autoantibodies. Scientific reports. 2018;8(1):17958.Eveline Van Gompel: None declared, Catia Cerqueira: None declared, Edvard Wigren: None declared, Susanne Gräslund: None declared, Karine Chemin: None declared, Begum Horuluoglu: None declared, Ellen De Langhe: None declared, Olivier Benveniste: None declared, Ingrid E. Lundberg Consultant of: Consulting fees from Corbus Pharmaceuticals, Inc, Grant/research support from: Research grants from Bristol Myers Squibb and AstraZeneca.