Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry.

Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.

Immune complexome analysis of a rich variety of serum immune complexes identifies disease-characteristic immune complex antigens in systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular endothelial dysfunction and skin fibrosis. Recently, the presence and pathogenic role of immune complexes (ICs) of SSc patients were reported. However, the identities of antigens in these ICs are unknown. Therefore, we examined ICs in the serum of SSc patients to elucidate SSc pathogenesis. In this study, IC concentrations in serum samples from SSc and systemic lupus erythematosus (SLE) patients were measured by C1q enzyme-linked immunosorbent assays; immune complex analysis was used for comprehensive identification and comparison of antigens incorporated into ICs (IC-antigens). The expression patterns of SSc-specific IC-antigens in skin sections were investigated by immunohistochemistry. Compared with SLE patients who developed disease because of IC deposition, SSc patients had a greater number of IC-antigens and a smaller difference in IC concentrations, suggesting that SSc pathogenesis is affected by the proteins present in ICs. In contrast, the IC concentration and number of IC-antigens did not significantly differ according to the clinical phenotype of SSc. We identified 478 IC-antigens in SSc patients, including multiple RNAP II-associated proteins that were targeted by antibodies previously associated with SSc pathogenesis. The most frequently detected RNAP II-associated protein, RNA polymerase II transcription subunit 30 (MED30), was strongly expressed at lesion sites and reportedly regulates endothelial differentiation. Therefore, increased expression of MED30 in lesions may have an antigenic effect, and MED30 function may be impaired or inhibited by IC formation. RNAP II-associated proteins may SSc pathogenesis through mechanisms such as the MED30 pathway.

Immune complexome analysis of serum samples from non-small-cell lung cancer patients identifies predictive biomarkers for nivolumab therapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have achieved important outcomes in cancer treatment. However, current clinical biomarker tests are not suitable for some patients because they require tumor tissues and have poor predictive value for treatment responses. Therefore, the identification of biomarkers that enable screening tests in all patients is necessary. METHODS: We performed an immune complexome analysis of non-small cell lung cancer patients treated with nivolumab to comprehensively identify and compare antigens incorporated into immune complexes (IC-antigens) in serum samples from the responders (n = 15) and non-responders (n = 20). Additionally, combinations of IC-antigens characteristic to the responder group were evaluated by logistic regression analysis and receiver operating characteristics curves to examine their predictiveness for ICI treatment responses. RESULTS: The combination of predictive biomarkers detected before treatment was profilin-1, purine nucleoside phosphorylase, alpha-enolase, and nucleoside diphosphate kinase A [p = 0.0043, odds ratio = 2.26, 95% confidence interval (CI) = 1.19-4.28, area under the curve = 0.76]. The combination of predictive biomarkers detected after treatment was peptidyl-prolyl cis-trans isomerase A, ubiquitin-like modifier-activating enzyme 1, complement component C8 beta chain, and apolipoprotein L1 (p = 0.0039, odds ratio = 2.56, 95% CI = 1.25-5.23, area under the curve = 0.77). CONCLUSION: Combinations of serum IC-antigens may predict the therapeutic effect of nivolumab in non-small cell lung cancer patients.

Coronin1C Is a GDP-Specific Rab44 Effector That Controls Osteoclast Formation by Regulating Cell Motility in Macrophages.

Osteoclasts are multinucleated bone-resorbing cells that are formed by the fusion of macrophages. Recently, we identified Rab44, a large Rab GTPase, as an upregulated gene during osteoclast differentiation that negatively regulates osteoclast differentiation. However, the molecular mechanisms by which Rab44 negatively regulates osteoclast differentiation remain unknown. Here, we found that the GDP form of Rab44 interacted with the actin-binding protein, Coronin1C, in murine macrophages. Immunoprecipitation experiments revealed that the interaction of Rab44 and Coronin1C occurred in wild-type and a dominant-negative (DN) mutant of Rab44, but not in a constitutively active (CA) mutant of Rab44. Consistent with these findings, the expression of the CA mutant inhibited osteoclast differentiation, whereas that of the DN mutant enhanced this differentiation. Using a phase-contrast microscope, Coronin1C-knockdown osteoclasts apparently impaired multinuclear formation. Moreover, Coronin1C knockdown impaired the migration and chemotaxis of RAW-D macrophages. An in vivo experimental system demonstrated that Coronin1C knockdown suppresses osteoclastogenesis. Therefore, the decreased cell formation and fusion of Coronin1C-depleted osteoclasts might be due to the decreased migration of Coronin1C-knockdown macrophages. These results indicate that Coronin1C is a GDP-specific Rab44 effector that controls osteoclast formation by regulating cell motility in macrophages.

Ceruloplasmin Levels in Cancer Tissues and Urine Are Significant Biomarkers of Pathological Features and Outcome in Bladder Cancer.

BACKGROUND/AIM: A previous report showed that immune complex-ceruloplasmin (CP) in urine is associated with carcinogenesis and malignant behavior in bladder cancer (BC). We investigated the pathological significance and prognostic roles of urine and tissue levels of CP protein in BC patients. MATERIALS AND METHODS: Urine CP levels were measured using an enzyme-linked immunosorbent assay in 97 patients. CP expression in BC tissues was evaluated by immunohistochemical analysis in 176 patient samples. RESULTS: Urine CP levels were positively associated with tumor grade and pT stage in non-muscle invasive BC (NMIBC). CP expression in BC tissues was positively associated with tumor growth and progression. Multivariate analysis demonstrated that high urine CP levels was an independent predictor of recurrence in the urinary tract in NMIBC (hazard ratio=2.87, p=0.016). CONCLUSION: CP-related markers, especially urine CP levels, are useful biomarkers of malignant potential and prognosis in NMIBC.

Detection of Novel Urine Markers Using Immune Complexome Analysis in Bladder Cancer Patients: A Preliminary Study.

BACKGROUND/AIM: Little is known on urine biomarkers that are associated with malignant behavior in patients with bladder cancer (BC). Our aim was to identify BC-related factors in urine samples using our original method "immune complexome analysis", based on detecting the immune complex (IC). PATIENTS AND METHODS: Immune complexome analysis was performed using urine samples from 97 BC patients, including 67 with non-muscle invasive BC (NMIBC). RESULTS: Eight IC-antigens were recognized as candidates for BC-related factors from 20,165 proteins. IC-serum albumin, -fibrinogen γ chain, -hemoglobin subunit α, -hemoglobin subunit ß, -ceruloplasmin, and fibrinogen ß chain were significantly associated with either pathological features and/or outcome. IC-ceruloplasmin was most widely associated with pathological features in all BC patients and lamina propria invasion and urinary tract recurrence in NMIBC. CONCLUSION: Based on detection of IC-antigens it was demonstrated that six IC-antigens, especially IC-ceruloplasmin, are potential urine biomarkers in BC.

Optimization of pH Elution Conditions in Immune Complexome Analysis for Comprehensive Identification of Immune Complex Antigens.

The identification of antigens incorporated into immune complexes (IC-antigens) is important for studying the pathophysiology of immunological diseases. Immune complexome analysis identifies IC-antigens by analyzing ICs collected from biological fluids by IC-capturing beads. In this study, we optimized the method to improve its comprehensiveness while maintaining selectivity for IC-antigens by comparing the number of identified peptides (model IC experiment) or proteins (human pooled serum) eluted from Protein G beads using different pH solutions (pH 2.0 - 11.0).

Revisiting immune complexes: Key to understanding immune-related diseases.

Immune complexes (ICs) formed by foreign or self-antigens and antibodies in biological fluids affect various tissues and are thought to cause several diseases. Biological and physical properties of IC, abnormal IC amounts, IC deposition and their relationships with disease pathogenesis had been studied. However, the relationship between ICs and each disease is not well understood and little is known of what determined ICs deposition in particular organ and why different organs are affected in different diseases. Recent technological advance enables identification of ICs in particular its antigens in tissues and body fluids, which may provide a key to discover an important trigger for immunological abnormality occurrence. Further identification of their epitopes, that are the exact origin of antigenicity, is developing and may be useful for diagnosis, elucidation of pathogenesis and treatment against IC-induced diseases. Here, we first make an overview of clearance of ICs, IC-induced pathogenesis and biological properties of ICs. Then, we introduce various methods developed to recover ICs from biological fluids or to identify antigens incorporated into ICs. Furthermore, several methods that can be used in epitope mapping for IC antigens are also documented.

Identification of Antigens in Immune Complexes.

Comprehensive identification and profiling of antigens in immune complexes (IC-antigens) is useful to provide insights into pathophysiology and could form the basis for novel diagnostic and treatment strategies for many immune-related diseases. Immune complexome analysis is the method for comprehensively identifying and profiling IC-antigens in biological fluids (such as serum and cerebrospinal fluid). Here, we describe an IC-antigen detection method; specifically, ICs in biological fluids are captured by using protein G- or protein A-coated beads, are subjected to papain-digestion, elution, and tryptic digestion, and are analyzed by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS).

Selective and Sensitive Mass Spectrometric Identification of Immune Complex Antigens in Cerebrospinal Fluid.

Comprehensive identification of immune complex antigens (IC-antigens) in cerebrospinal fluid (CSF) is useful to provide insights into pathophysiology and could form the basis for novel diagnostic and treatment strategies for central nervous system autoimmune diseases and other neurological disorders. Immune complexome analysis is the method for comprehensively identifying IC-antigens in biological fluids (such as serum and CSF). Here, we describe IC-antigens detection method; specifically, ICs in CSF are captured and are subjected to papain-digestion elution and tryptic digestion, and are analyzed by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS).

Comprehensive immune complexome analysis detects disease-specific immune complex antigens in seminal plasma and follicular fluids derived from infertile men and women.

BACKGROUND: Autoimmune reactions and subsequent inflammation may underlie spermatogenic dysfunction and endometriosis-related infertility. The aim of this study is to identify disease-specific antigens in immune complexes (ICs) in seminal plasma (SP) and in follicular fluid (FF). METHODS: Immune complexome analysis, in which nano-liquid chromatography-tandem mass spectrometry is employed to comprehensively identify antigens incorporated into ICs in biological fluids, was performed for specimens collected from infertile couples undergoing assisted reproduction. Forty-two male patients consisting of subjects with oligozoospermia (n = 6), asthenozoospermia (n = 8), and normal semen analysis (n = 28). Fifty-eight female patients consisting of subjects with ovarian endometriosis (n = 10) and control women without disease (n = 48). RESULTS: Four disease-specific antigens were identified in subjects with oligozoospermia, while five disease-specific antigens were detected in subjects with asthenozoospermia, some of which are involved in sprematogenesis. Eight antigens were detected only in subjects with endometriosis. CONCLUSION: Functional characteristics of disease-specific antigens were found to correspond to the pathogenesis of male and female infertility. The formation of ICs may contribute to spermatogenic dysfunction and endometriosis-related infertility via loss of function of the related proteins. Immune complexome analysis is expected to be a valuable tool for the investigation of novel diagnostic methods and treatment strategies for infertility.

Selective, sensitive and comprehensive detection of immune complex antigens by immune complexome analysis with papain-digestion and elution.

Comprehensive identification and profiling of antigens in immune complexes (ICs) in biological fluids, such as serum and cerebrospinal fluid, is useful for developing early diagnostic markers and specific treatments for many diseases. We have developed a method, designated "immune complexome analysis", to comprehensively identify the antigens in ICs. In this method, we first purify ICs from biological fluid by using Protein G- or Protein A-coated beads, then these ICs are subjected to tryptic digestion on the beads and subsequent analysis using nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). We previously used this method to find specific antigens in circulating ICs (CIC-antigens) in serum for autoimmune diseases, infectious disease and cancers. However, this method detects not only CIC-antigens but also antibodies and proteins bound non-specifically to the beads, which restricts the detection of minor peptides released by the digestion of CIC-antigens whose amounts are generally much less than antibodies and the proteins. To selectively detect CIC-antigens with enhanced sensitivity, in this study we compared three methods (Method A, direct tryptic digestion on the beads; Method B, low-pH elution and tryptic digestion; Method C, papain-digestion, elution, and tryptic digestion) and examined which method selectively elutes CIC-antigens from CICs bound to the beads and selectively detects CIC-antigens using nano-LC-MS/MS. We also compared three types of CIC-capturing beads (Protein G-coated magnetic beads, Protein A-coated magnetic beads and Proceptor™-sepharose beads) to examine if parallel use of these beads aids the comprehensive detection of CIC-antigens in immune complexome analysis. Comparison showed that Method C provided the most selective and sensitive detection of CIC-antigens, without interference by antibodies and proteins non-specifically bound to the beads. In addition, using three types of beads allowed the examination of a wide range of CIC-antigens in immune complexome analysis. Therefore, combining Method C with three types of beads should allow the selective and sensitive identification of IC-antigens present in biological fluids from patients with a variety of diseases. The identification of IC-antigens may lead to the development of diagnostic methods and protocols for specific treatments for these diseases.

Proteomic approach to profiling immune complex antigens in cerebrospinal fluid samples from patients with central nervous system autoimmune diseases.

BACKGROUND: Immune complexes (ICs) may clearly reflect immunological abnormalities caused by disease, especially for autoimmune diseases. Although ICs have been detected in cerebrospinal fluid (CSF) from patients with CNS autoimmune diseases, identities of antigens in such ICs have not been comprehensively determined. METHODS: We used immune complexome analysis, in which nano-liquid chromatography-tandem mass spectrometry is employed to comprehensively identify antigens incorporated into ICs in biological fluids, to characterize ICs in CSF samples from patients with CNS autoimmune diseases, and to find disease-specific IC antigen to a certain CNS autoimmune disease. Also, we compared the IC antigens we identified with the reported CSF proteome or with the published plasma proteome to examine if the method is distinguished from the conventional CSF proteome analysis. RESULTS: We identified 176 antigens in 78 CSF samples. We then assessed the overlaps among these antigens, the CSF proteome, and the plasma proteome; 140 of the 176 antigens were found to be exclusively detected by our method. Notably, IC-associated suprabasin in CSF was 100% specific to neuropsychiatric systemic lupus erythematosus (NPSLE). CONCLUSIONS: This report is the first to comprehensively identify the antigens incorporated into ICs in CSF. There was limited overlap between the antigens we identified and the CSF proteome or the plasma proteome; therefore, our method can be distinguished from the conventional CSF proteome analysis. Although the sensitivity of disease-specific IC-antigens detected in immune complexome analysis screening, the sensitivity may be improved by developing an ELISA method specifically for detecting the ICs. Immune complexome analysis of CSF may be a new and promising path to biomarker discovery for diagnosis and study for CNS autoimmune diseases.

Immune complexome analysis of antigens in circulating immune complexes from patients with acute cellular rejection after living donor liver transplantation.

Liver transplantation is a life-saving procedure for many end-stage liver diseases; however, rejection after transplantation is still occurs in some recipients. The most common form of rejection is T cell-related acute cellular rejection (ACR). To understand the mechanism of rejection, it is necessary to identify immune targets. Since the development of B cell immunity depends upon concordant T cell immunity, we hypothesized that rejection-specific antigens in circulating immune complexes (CICs) may be present in the sera of recipients experiencing rejection, and as such, may be useful as diagnostic biomarkers for ACR. The purpose of this study was to investigate rejection-specific antigens in CICs (CIC-antigens) in serum of ACR patients. We applied immune complexome analysis, in which CICs are separated from whole serum and then subjected to direct tryptic digestion and identification of CIC-antigens by nano-liquid chromatography-tandem mass spectrometry, to sera of 32 living donor liver transplant recipients (10 recipients experienced ACR and the others did not experience). CIC-antigens were compared between rejection and non-rejection groups to elucidate those that were only detected in the rejection group. We identified 11 CIC-antigens that were only detected in patients who experienced rejection, 4 of which (thrombospondin-1, apolipoprotein E, apolipoprotein C-III, and complement factor H) were only detected during ACR.

Immune complexome analysis reveals the specific and frequent presence of immune complex antigens in lung cancer patients: A pilot study.

Cancer immunotherapies such as antibodies targeting T cell checkpoints, or adaptive tumor-infiltrating lymphocyte (TIL) transfer, have been developed to boost the endogenous immune response against human malignancies. However, activation of T cells by such antibodies can lead to the risk of autoimmune diseases. Also, the selection of tumor-reactive T cells for TIL relies on information regarding mutated antigens in tumors and does not reflect other factors involved in protein antigenicity. It is therefore essential to engineer therapeutic interventions by which T cell reactivity against tumor cells is selectively enhanced (i.e., "focused cancer immunotherapy") based on tumor antigens that are specifically expressed in the tumor of a certain cancer and in many patients with this cancer. Immune complexes (ICs) are the direct and stable products of immunological recognition by humoral immunity. Here, we searched for tumor-specific IC antigens in each of five cancers (lung (n = 28), colon (n = 20), bladder (n = 20), renal cell (n = 15) and malignant lymphoma (n = 9)), by using immune complexome analysis that comprehensively identifies and profiles the constituent antigens in ICs. This analysis indicated that gelsolin and inter-alpha-trypsin inhibitor heavy chains were specifically and frequently detected (at a frequency higher than 80%), and that phosphoproteins (VENTX, VCIP135) were also specifically present in the ICs of lung cancer patients. Immune complexome analysis successfully identified several tumor-specific IC antigens with high detection frequency in lung cancer patients. These specific antigens are required to validate the clinical benefit by further analysis using a large number of patients.

Proteomic profiling of antigens in circulating immune complexes associated with each of seven autoimmune diseases.

OBJECTIVE: Immune complexes (ICs) trigger humoral immune responses. Therefore, the identification of constituent antigens within ICs would have very different clinical significance than identification of free antigens. DESIGN AND METHODS: Here, we applied immune complexome analysis of serum to the study of seven major autoimmune diseases-anti-neutrophil cytoplasmic antibody-associated vasculitis, Takayasu's arteritis, mixed connective tissue disease, dermatomyositis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus-and healthy donors to comprehensively identify antigens incorporated into circulating ICs and to find disease-specific antigens. RESULTS: We identified 468 distinct IC-associated antigens using this method. Importantly, 62 of those antigens were disease-specific antigens, and there were at least three disease-specific antigens for each of the seven autoimmune diseases. Of the disease-specific antigens identified, coiled-coil domain-containing protein 158 and spectrin were identified as potential autoantigens important to SSc and SS pathogenesis, respectively; notable titin and spectrin autoantibodies are reportedly found in SSc and SS patients, respectively. CONCLUSION: Immune complexome analysis may be generally applicable to the study of the relationship between ICs and autoimmune diseases in animals and humans.