Binding Of Monoclonal Antibodies Research Articles

P-107 MICROFLUIDIC SPERM SEXING: An XLD (X-linked Disorder) model for preventing genetic disorders in newborns

Abstract Study question Can sperm sexing based on unique surface membrane markers – enable genotypic sperm sorting by allosomal content to prevent certain X-linked disorders in newborns? Summary answer In an X-Linked-Disorder prevention model, we utilized a microfluidic device functionalized with H-Y monoclonal antibodies attached to magnetic beads to enrich Y-chromosome bearing sperm sorts. What is known already About 4% of newborns are at risk of being born with an X-linked disease such as Fragile X Syndrome. The current clinically approved mitigation plan for preventing such genetic disorders is to consider Preimplantation Genetic Testing for Monogenetic diseases (PGT-M) which involves an invasive microsurgical procedure on fragile embryos. To minimize this invasiveness, we proposed a less invasive microsurgical exclusion approach to prevent such genetic disorders by genotypically sorting sperm cells that can be used for IUI/IVF/ICSI – with microfluidics – using their allosomal characteristics. Study design, size, duration Semen samples purchased from FairFax Cryobank from anonymous donors were used in immuno-magnetic microfluidic sorting protocols with the use of phycoerythrin (PE) binding Tetrameric Antibody Complexes (TAC) coupled with magnetic beads to tag H-Y monoclonal antibody (mab) binding activities on the sperm surface membrane. Participants/materials, setting, methods Immuno-magnetic sorting was conducted with specially fabricated microfluidic-sperm-sorting (MSS) chips. Sort yield and purities of positively selected sperm populations were assessed by flow-cytometry, immunofluorescence, fluorescent-in-situ-hybridization (FISH) and by quantitative-PCR (qPCR – Cq & Tm). Quantitative experiments were run in triplicates and the results were expressed as absolute means for comparison between paired variables. Statistical analyses were performed using Student’s t-test and a p-value of less than 0.05 was considered to be statistically significant. Main results and the role of chance For the assessment of H-Y sperm sorts, our preliminary data showed that flow cytometry was able to confirm the positive sperm selection (which were mostly Y-chromosome bearing sperm cells) in a sperm genotyping protocol – initially utilizing immuno-magnetic sort tubes prior to flowing through microfluidic chips. Both immuno-magnetic sort tubes and the microfluidic chips were able to optimally confirm from qPCR-Cq means – the negative selection – being the X-chromosome bearing sperm that may have been largely unselected by the H-Y-mab. Values for Cq means compared under H-Y-qPCR data sets were statistically significant with a p value of 0.000023 (p values is significant at < 0.05). Our study was able to elucidate a potential less invasive clinical application of genotypically sorting sperm cells with mab-functionalized microfluidic chips for an XLD-model. Molecular down stream applications like flow cytometry may therefore be utilized to experimentally confirm sort purities of positively selected sperm populations (with H-Y markers) – based on the utility of the SMCX and SMCY gene in the X and Y sperm chromosome bearing sperm cells respectively. Limitations, reasons for caution Physiological effect of magnetic fields of varying tesla or gauss strengths on sperm selected with immuno-magnetic beads may warrant further safety studies prior to actual clinical applications. Optimal selection results may be achieved with the use of freshly collected normospermic rather than frozen-thawed samples. Wider implications of the findings Although we had de-emphasized the clinical application of the microfluidic sperm sexing protocol for legitimate use in sex selection and gender balancing, this study design was solely focused on adapting a less invasive method of preventing X-linked genetic disorders with the potential exclusion of microsurgical procedures leading to PGT-M. Trial registration number NA

Preventing skin toxicities induced by EGFR inhibitors by topically blocking drug-receptor interactions.

Epidermal growth factor receptor (EGFR) inhibitors are used to treat many advanced-stage epithelial cancers but induce severe skin toxicities in most treated patients. These side effects lead to a deterioration in the quality of life of the patients and compromise the anticancer treatment. Current treatment strategies for these skin toxicities focus on symptom reduction rather than preventing the initial trigger that causes the toxicity. In this study, we developed a compound and method for treating "on-target" skin toxicity by blocking the drug at the site of toxicity without reducing the systemic dose reaching the tumor. We first screened for small molecules that effectively blocked the binding of anti-EGFR monoclonal antibodies to EGFR and identified a potential candidate, SDT-011. In silico docking predicted that SDT-011 interacted with the same residues on EGFR found to be important for the binding of EGFR inhibitors cetuximab and panitumumab. Binding of SDT-011 to EGFR reduced the binding affinity of cetuximab to EGFR and could reactivate EGFR signaling in keratinocyte cell lines, ex vivo cetuximab-treated whole human skin, and A431-injected mice. Specific small molecules were topically applied and were delivered via a slow-release system derived from biodegradable nanoparticles that penetrate the hair follicles and sebaceous glands, within which EGFR is highly expressed. Our approach has the potential to reduce skin toxicity caused by EGFR inhibitors.

Genetic or therapeutic neutralization of ALK1 reduces LDL transcytosis and atherosclerosis in mice

Low-density lipoprotein (LDL) accumulation in the arterial wall contributes to atherosclerosis initiation and progression1. Activin A receptor-like type 1 (ACVRL1, called activin-like kinase receptor (ALK1)) is a recently identified receptor that mediates LDL entry and transcytosis in endothelial cells (ECs)2,3. However, the role of this pathway in vivo is not yet known. In the present study, we show that genetic deletion of ALK1 in arterial ECs of mice substantially limits LDL accumulation, macrophage infiltration and atherosclerosis without affecting cholesterol or triglyceride levels. Moreover, a selective monoclonal antibody binding ALK1 efficiently blocked LDL transcytosis, but not bone morphogenetic protein-9 (BMP9) signaling, dramatically reducing plaque formation in LDL receptor knockout mice fed a high-fat diet. Thus, our results demonstrate that blocking LDL transcytosis into the endothelium may be a promising therapeutic strategy that targets the initiating event of atherosclerotic cardiovascular disease.

Mechanisms of inhibition of human monoclonal antibodies in immune thrombotic thrombocytopenic purpura

AbstractAntibody binding to a plasma metalloprotease, a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13), is necessary for the development of immune thrombotic thrombocytopenic purpura (iTTP). Inhibition of ADAMTS13-mediated von Willebrand factor (VWF) cleavage by such antibodies clearly plays a role in the pathophysiology of the disease, although the mechanisms by which they inhibit ADAMTS13 enzymatic function are not fully understood. At least some immunoglobulin G–type antibodies appear to affect the conformational accessibility of ADAMTS13 domains involved in both substrate recognition and inhibitory antibody binding. We used single-chain fragments of the variable region previously identified via phage display from patients with iTTP to explore the mechanisms of action of inhibitory human monoclonal antibodies. Using recombinant full-length ADAMTS13, truncated ADAMTS13 variants, and native ADAMTS13 in normal human plasma, we found that, regardless of the conditions tested, all 3 inhibitory monoclonal antibodies tested affected enzyme turnover rate much more than substrate recognition of VWF. Hydrogen-to-deuterium exchange plus mass spectrometry experiments with each of these inhibitory antibodies demonstrated that residues in the active site of the catalytic domain of ADAMTS13 are differentially exposed to solvent in the presence and absence of monoclonal antibody binding. These results support the hypothesis that inhibition of ADAMTS13 in iTTP may not necessarily occur because the antibodies directly prevent VWF binding, but instead because of allosteric effects that impair VWF cleavage, likely by affecting the conformation of the catalytic center in the protease domain of ADAMTS13. Our findings provide novel insight into the mechanism of autoantibody-mediated inhibition of ADAMTS13 and pathogenesis of iTTP.

Abstract 5556: Lemur tyrosine kinase 3 serves as a predictor of patient outcome and a target for the treatment of ovarian cancer

Abstract The objective of these in vitro and in vivo studies was to validate Lemur Tyrosine Kinase 3 (LMTK3) as a specific target and predictor of clinical outcome in ovarian cancer. LMTK3 belongs to a family of regulated tyrosine kinases with three structurally related isoforms, LMTK1, LMTK2, and LMTK3. Both nuclear and cytoplasmic LMTK3 expressions correlated with tumor grade and patient survival in cancers such as breast and colorectal cancer. We tested the clinical significance of the LMTK3 gene by immunohistochemistry (IHC) using LMTK3 monoclonal antibody on formalin-fixed paraffin-embedded sections (FFPE) collected from 204 early-stage (stage I-II) ovarian cancer patients. Results from this IHC LMTK3 study revealed a higher cytoplasmic to nuclear localization of LMTK3 correlated with worse overall survival (P<0.01). Further investigation of LMTK3’s prognostic value by screening LMTK3 signaling in 270 stage III-IV ovarian cancer patients is ongoing. We utilized the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for testing the killing efficacy of targeting LMTK3 by a monoclonal antibody, siRNA, and specific LMTK3 binding peptides (LMTK3BP) in ovarian cancer cell lines SKOV3, MDAH-2774, A2780, and TOV-21G. Treatment with LMTK3 specific monoclonal antibody, siRNA, and LMTK3BP significantly induced killing of both chemosensitive and chemoresistant ovarian cancer cells without affecting normal cells in vitro. Moreover, we observed this killing as synergistic with both Cisplatin and Taxotere treatment in vitro. Lastly, we used an A2780 cell line derived orthotopic xenograft mouse model of ovarian cancer to test the efficacy of specific LMTK3BP in vivo. Strikingly, LMTK3BP 20 mg/kg IV dose given three times a week for three weeks showed a 35% tumor reduction. Furthermore, in vivo safety studies showed no signs of toxicity of the LMTK3BP, even at a very high dose of 40 mg/kg. Thus, with dose optimization in ongoing experiments, we could expect a higher efficacy. In conclusion, this study highlighted the importance of LMTK3 as a predictor of patient clinical outcome and potential target for treatment in ovarian cancer. Citation Format: Thea K. Kirsch-Mangu, Axel S. Tullberg, Anna Portela, Khalil Helou, Ghassan M. Saed. Lemur tyrosine kinase 3 serves as a predictor of patient outcome and a target for the treatment of ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5556.

NMDA-receptor-Fc-fusion constructs neutralize anti-NMDA receptor antibodies.

N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most common subtype of autoimmune encephalitis characterized by a complex neuropsychiatric syndrome usually including memory impairment. Patients develop an intrathecal immune response against NMDARs with antibodies that presumably bind to the amino-terminal domain of the GluN1 subunit. The therapeutic response to immunotherapy is often delayed. Therefore, new therapeutic approaches for fast neutralization of NMDAR antibodies are needed. Here, we developed fusion constructs consisting of the Fc part of immunoglobulin G and the amino-terminal domains of either GluN1 or combinations of GluN1 with GluN2A or GluN2B. Surprisingly, both GluN1 and GluN2 subunits were required to generate high-affinity epitopes. The construct with both subunits efficiently prevented NMDAR binding of patient-derived monoclonal antibodies and of patient CSF containing high-titre NMDAR antibodies. Furthermore, it inhibited the internalization of NMDARs in rodent dissociated neurons and human induced pluripotent stem cell-derived neurons. Finally, the construct stabilized NMDAR currents recorded in rodent neurons and rescued memory defects in passive-transfer mouse models using intrahippocampal injections. Our results demonstrate that both GluN1 and GluN2B subunits contribute to the main immunogenic region of the NMDAR and provide a promising strategy for fast and specific treatment of NMDAR encephalitis, which could complement immunotherapy.

Immunoliposomes: A Targeted Drug Delivery System for Cancer Therapeutics and Vaccination.

Cancer has become one of the world's most lethal and life-threatening disorders, resulting in many deaths. Drug targeting and managing drug delivery are concepts that are implemented to increase a drug's therapeutic index by enhancing its specificity to particular cells, tissues, or organs and reducing its action and harmful side effects. Liposomes have proven to be one of the most innovative drug delivery systems in medicine. Immunoliposomes, also known as antibody-coupled liposomes, have gained a lot of attention as a homing device for targeted therapies. Monoclonal antibodies or antibody fragments that combine with liposomes to create immunoliposomes have been considered a leading technique for targeted delivery. Various functionalization strategies are adopted for the non-covalent and covalent binding of monoclonal antibodies and their components to liposomal surfaces, such as thiolation, amide bonds, hydrazone bonds, and electrostatic interactions, hydrophobic interactions, hydrogen bonding, etc. for cancer-specific targeting. This provides an overview of various stimulus-responsive immunoliposomes capable of regulating drug release in response to an exogenous magnetic field, changes in temperature or pH, enzyme concentration, endogenous stimuli, and applications of immunoliposomes in vaccination and cancer therapeutics and endogenous immune response stimulation.

Ultrasensitive immunosensor for monitoring of CA 19-9 pancreatic cancer marker using electrolyte-gated TiS3 nanoribbons field-effect transistor

Measuring CA 19-9 antigen level is critical for early diagnosis of pancreatic cancer, monitoring the treatment process, and predicting disease recurrence. The purpose of this research is to assess the application of novel few-layered TiS3 nanoribbons material as a channel material in electrolyte-gated field-effect transistor immunosensor for rapid detection of CA 19-9 antigen as a cancer marker. Accordingly, TiS3 nanoribbons were produced through liquid-phase exfoliation of as-synthesized TiS3 whiskers in N, N-dimethylformamide. Then, dispersed TiS3 nanoribbons were drop cast onto the FET surface to form an active channel material between source and drain electrodes. Subsequently, the channel surface was modified by utilizing 1-naphthylamine (NA) and glutaraldehyde (GA) to strengthen the binding of monoclonal antibody 19-9 to TiS3 nanoribbons. Spectroscopic and microscopic methods were utilized for comprehensive characterizations. Electrical characterization of electrolyte-gated TiS3 nanoribbons field-effect transistor represented a depletion-mode n-type behavior with field-effect mobility of 0.059 cm2/Vs, current on/off ratio of 10.88 and subthreshold swing (SS) of 450.9 mV/decade. With increasing in CA 19-9 antigen concentration from 1.0 × 10−12 U/mL to 1.0 × 10−5 U/mL, a decrease in the drain current occurred with high sensitivity of 0.04 μA/decade and a detection limit of 1.3 × 10−13 U/mL. Additionally, the proposed TiS3 nanoribbons FET immunosensor exhibited outstanding selectivity, and its good performance was compared with an enzyme-linked immunosorbent assay (ELISA) for spiked real human serum samples. The good and satisfactory obtained results of the proposed immunosensor suggest that the developed platform can be a superb candidate for cancer diagnosis and therapeutic monitoring.

Netrin-1 Monoclonal Antibody-Functionalized Nanoparticle Loaded with Metformin Prevents the Progression of Abdominal Aortic Aneurysms.

Abdominal aortic aneurysms (AAAs) are a global health and economic burden. Therapeutic strategies to inhibit the progression of AAAs are currently lacking. Recently, the therapeutic effect of metformin on aneurysms has attracted considerable interest. However, the unfavorable pharmacokinetic properties of metformin limit its feasibility for AAA treatment. We constructed a metformin-loaded netrin-1-responsive AAA-targeted nanoparticle (Tgt-NP-Met) for AAA management. Evaluation of the therapeutic effect of Tgt-NP-Met was performed by in vitro and in vivo experiments. Our results showed that the binding of netrin-1 monoclonal antibodies enhanced the AAA-targeting capability of nanoparticles (NPs). Moreover, Tgt-NP-Met administration prevented AAA development and reduced the aneurysm diameter in apolipoprotein E (ApoE)-deficient (ApoE-/-) mice that received continuous infusion of angiotensin II. Furthermore, metformin prevented AAA progression by inhibiting the transformation of vascular smooth muscle cells (VSMCs) from a contractile phenotype to a synthetic phenotype, which is mediated by macrophage infiltration and activation. Our findings identify metformin as a functional suppressor for macrophage-mediated phenotypic transformation of VSMCs and Tgt-NP-Met as an efficient therapeutic strategy for AAA management.

Safety, Tolerability, and Pharmacokinetics of Benralizumab: A Phase 1, Randomized, Single-Blind Study of Healthy Chinese Participants.

Biological therapies targeting eosinophils have been shown to be effective in treating patients with severe eosinophilic asthma. Benralizumab (Fasenra®, AstraZeneca) is a humanized monoclonal antibody binding to the alpha subunit of the interleukin-5 receptor, which rapidly depletes eosinophils via antibody-dependent cellular cytotoxicity. The aim of this Phase 1 study was to assess the safety, tolerability, and pharmacokinetics of benralizumab in healthy Chinese individuals. In this randomized, single-blind study (NCT03928262), healthy Chinese adult participants aged 18 to 45 years, weighing 50 to 100 kg, were randomized 1:1:1 to receive a single subcutaneous (SC) injection of benralizumab 10 mg, 30 mg, or 100 mg in the upper arms on Day 1. Safety was monitored throughout the study (up to Day 85), and blood samples were taken to determine serum benralizumab concentrations and for detection of anti-drug antibody. A non-compartmental analysis was conducted to estimate the pharmacokinetic parameters. Thirty-six healthy participants were enrolled, 12 in each dose group (mean [SD] age 26 [6] years). Following a single SC injection of benralizumab, 13 adverse events were reported by 10 participants (28%), with one mild injection-site reaction assessed as related. The mean serum benralizumab concentrations increased in a dose proportional manner, followed by exponential decreases. The mean terminal half-lives were 15.1 days for the 10 mg dose, 14.4 days for the 30 mg dose, and 15.4 days for the 100 mg dose. All doses resulted in near-complete depletion of eosinophils on Day 2, which was maintained throughout the study to Day 85. A single SC injection of benralizumab was well tolerated by healthy Chinese participants, with no new or unexpected safety findings. The pharmacokinetics of benralizumab in Chinese participants was dose-proportional and consistent with those of non-Chinese participants observed in previous studies. NCT03928262 (https://clinicaltrials.gov/ct2/show/NCT03928262).

Ligand Dilution Analysis Facilitates Aptamer Binding Characterization at the Single-Molecule Level.

Cell-specific aptamers offer a powerful tool to study membrane receptors at the single-molecule level. Most target receptors of aptamers are highly expressed on the cell surface, but difficult to analyze in situ because of dense distribution and fast velocity. Therefore, we herein propose a random sampling-based analysis strategy termed ligand dilution analysis (LDA) for easily implemented aptamer-based receptor study. Receptor density on the cell surface can be calculated based on a regression model. By using a synergistic ligand dilution design, colocalization and differentiation of aptamer and monoclonal antibody (mAb) binding on a single receptor can be realized. Once this is accomplished, precise binding site and detailed aptamer-receptor binding mode can be further determined using molecular docking and molecular dynamics simulation. The ligand dilution strategy also sets the stage for an aptamer-based dynamics analysis of two- and three-dimensional motion and fluctuation of highly expressed receptors on the live cell membrane.

Ligand Dilution Analysis Facilitates Aptamer Binding Characterization at the Single‐Molecule Level

AbstractCell‐specific aptamers offer a powerful tool to study membrane receptors at the single‐molecule level. Most target receptors of aptamers are highly expressed on the cell surface, but difficult to analyze in situ because of dense distribution and fast velocity. Therefore, we herein propose a random sampling‐based analysis strategy termed ligand dilution analysis (LDA) for easily implemented aptamer‐based receptor study. Receptor density on the cell surface can be calculated based on a regression model. By using a synergistic ligand dilution design, colocalization and differentiation of aptamer and monoclonal antibody (mAb) binding on a single receptor can be realized. Once this is accomplished, precise binding site and detailed aptamer‐receptor binding mode can be further determined using molecular docking and molecular dynamics simulation. The ligand dilution strategy also sets the stage for an aptamer‐based dynamics analysis of two‐ and three‐dimensional motion and fluctuation of highly expressed receptors on the live cell membrane.

Antibodies targeting the neuraminidase active site inhibit influenza H3N2 viruses with an S245N glycosylation site

Contemporary influenza A H3N2 viruses circulating since 2016 have acquired a glycosylation site in the neuraminidase in close proximity to the enzymatic active site. Here, we investigate if this S245N glycosylation site, as a result of antigenic evolution, can impact binding and function of human monoclonal antibodies that target the conserved active site. While we find that a reduction in the inhibitory ability of neuraminidase active site binders is measurable, this class of broadly reactive monoclonal antibodies maintains protective efficacy in vivo.

Characterization of FcγRIa (CD64) as a Ligand Molecule for Site-Specific IgG1 Capture: A Side-By-Side Comparison with Protein A.

Fc γ receptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin-biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (ka 50-80 × 105 M-1 s-1) increased in comparison to His capture method (1.9-2.4 × 105 M-1 s-1). In addition, the dissociation rate (kd 10-5 s-1) seemed slower over the His capture method (10-4 s-1) and provided stability on the chip surface during the dissociation phase. The KD values for FcγRIa were found in the picomolar range (2.1-10.33 pM from steady-state affinity analysis and 37.5-46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification.

Quantitative systems pharmacology model of the amyloid pathway in Alzheimer's disease: Insights into the therapeutic mechanisms of clinical candidates.

Despite considerable investment into potential therapeutic approaches for Alzheimer's disease (AD), currently approved treatment options are limited. Predictive modeling using quantitative systems pharmacology (QSP) can be used to guide the design of clinical trials in AD. This study developed a QSP model representing amyloid beta (Aβ) pathophysiology in AD. The model included mechanisms of Aβ monomer production and aggregation to form insoluble fibrils and plaques; the transport of soluble species between the compartments of brain, cerebrospinal fluid (CSF), and plasma; and the pharmacokinetics, transport, and binding of monoclonal antibodies to targets in the three compartments. Ordinary differential equations were used to describe these processes quantitatively. The model components were calibrated to data from the literature and internal studies, including quantitative data supporting the underlying AD biology and clinical data from clinical trials for anti-Aβ monoclonal antibodies (mAbs) aducanumab, crenezumab, gantenerumab, and solanezumab. The model was developed for an apolipoprotein E (APOE) ɛ4 allele carrier and tested for an APOE ɛ4 noncarrier. Results indicate that the model is consistent with data on clinical Aβ accumulation in untreated individuals and those treated with monoclonal antibodies, capturing increases in Aβ load accurately. This model may be used to investigate additional AD mechanisms and their impact on biomarkers, as well as predict Aβ load at different dose levels for mAbs with known targets and binding affinities. This model may facilitate the design of scientifically enriched and efficient clinical trials by enabling a priori prediction of biomarker dynamics in the brain and CSF.

Complement component C3 and C5b-9 deposition on hypoxia reperfused endothelial cells by non-HLA antibodies against RhoGDI2: A player involved in graft failure?

Antibodies against Rho GDP-dissociation inhibitor 2 (RhoGDI2) are associated with inferior graft survival in transplant patients receiving a kidney from deceased donors. Although this suggests that these antibodies contribute to graft injury because of ischemia, it remains unknown whether they are also pathogenically involved in the process of graft loss. To study this, we firstly analyzed the IgG subclass profile of anti-RhoGDI2 antibodies in kidney transplant recipients, and whether antibody titers change over time or because of acute rejection. Next, we investigated the expression of RhoGDI2 on primary kidney and lung endothelial cells (ECs) upon hypoxia reperfusion. In addition, the complement-fixing properties of anti-RhoGDI2 antibodies were studied using imaging flow cytometry. Anti-RhoGDI2 antibodies in patients are mainly IgG1, and titers remained stable and seemed not be changed because of rejection. Antibodies against RhoGDI2, which surface expression seemed to increase upon hypoxia reperfusion, co-localized with C3 on ECs. Binding of human IgG1 monoclonal anti-RhoGDI2 antibodies as well as patient derived antibodies, resulted in complement activation, suggesting that these antibodies are complement fixing. This study suggested a potential pathogenic role of anti-RhoGDI2 antibodies in kidney graft loss. During ischemia reperfusion, the ability of these antibodies to fix complement could be one of the mechanisms resulting in tissue injury.

Clonal IgA and IgG autoantibodies from individuals at risk for rheumatoid arthritis identify an arthritogenic strain of Subdoligranulum.

The mucosal origins hypothesis of rheumatoid arthritis (RA) proposes a central role for mucosal immune responses in the initiation or perpetuation of the systemic autoimmunity that occurs with disease. However, the connection between the mucosa and systemic autoimmunity in RA remains unclear. Using dual immunoglobulin A (IgA) and IgG family plasmablast-derived monoclonal autoantibodies obtained from peripheral blood of individuals at risk for RA, we identified cross-reactivity between RA-relevant autoantigens and bacterial taxa in the closely related families Lachnospiraceae and Ruminococcaceae. After generating bacterial isolates within the Lachnospiraceae/Ruminococcaceae genus Subdoligranulum from the feces of an individual, we confirmed monoclonal antibody binding and CD4+ T cell activation in individuals with RA compared to control individuals. In addition, when Subdoligranulum isolate 7 but not isolate 1 colonized germ-free mice, it stimulated TH17 cell expansion, serum RA-relevant IgG autoantibodies, and joint swelling reminiscent of early RA, with histopathology characterized by antibody deposition and complement activation. Systemic immune responses were likely due to mucosal invasion along with the generation of colon-isolated lymphoid follicles driving increased fecal and serum IgA by isolate 7, because B and CD4+ T cell depletion not only halted intestinal immune responses but also eliminated detectable clinical disease. In aggregate, these findings demonstrate a mechanism of RA pathogenesis through which a specific intestinal strain of bacteria can drive systemic autoantibody generation and joint-centered antibody deposition and immune activation.

Efficacy and safety of monoclonal antibody against calcitonin gene-related peptide or its receptor for migraine patients with prior preventive treatment failure: a network meta-analysis

ObjectiveThe relative effects of monoclonal antibody against calcitonin gene-related peptide (CGRP) or its receptor for adult migraine patients with prior treatment failure remains uncertain. Therefore, this study systematically assessed the comparative effectiveness of different CGRP binding monoclonal antibodies (mAbs) for these patients.MethodsSeveral online databases including Ovid MEDILNE, Ovid EMBASE, Cochrane Library, and ClinicalTrials.gov were systematically searched from inception to June 15, 2022. We included randomized clinical trials (RCT) of adult migraine patients with previous treatment failure that assessed any CGRP monoclonal antibody. The primary efficacy outcome was change in monthly migraine days (MMDs), and the primary safety outcome was treatment-emergent adverse events (TEAEs).ResultsOverall, seven studies totaling 3, 052 patients were included. Three-node analysis showed that CGRP mAbs was superior to CGRP receptor mAbs in reducing MMDs (MD: -1.55, 95% CrI: − 2.43 to − 0.44) and improving at least 50% response rates (RR: 1.52, 95% CrI: 1.04 to 2.21). Nine-node analysis showed galcanezumab 240 mg ranked first in reducing MMDs (MD -4.40, 95% CrI − 7.60 to − 1.19) and improving 50% response rates (RR: 4.18, 95% CrI: 2.63 to 6.67). Moreover, treatment with fremanezumab or eptinezumab 300 mg provides a significant advantage over erenumab 140 mg regarding an improved response rate of at least 50%. The analysis did not show difference in incidences of TEAEs and serious adverse events in any of the comparisons.ConclusionsIt appears that CGRP mAbs, especially galcanezumab 240 mg, monthly fremanezumab, and eptinezumab 300 mg, seem to be the best choice for the treatment of migraine patients with previous treatment failures. This finding also calls for future research that examine the associations between these medications in migraine therapy among the same patient group to testify the present findings.

Multicolor fluorescence activated cell sorting to generate humanized monoclonal antibody binding seven subtypes of BoNT/F.

Generating specific monoclonal antibodies (mAbs) that neutralize multiple antigen variants is challenging. Here, we present a strategy to generate mAbs that bind seven subtypes of botulinum neurotoxin serotype F (BoNT/F) that differ from each other in amino acid sequence by up to 36%. Previously, we identified 28H4, a mouse mAb with poor cross-reactivity to BoNT/F1, F3, F4, and F6 and with no detectable binding to BoNT/F2, F5, or F7. Using multicolor labeling of the different BoNT/F subtypes and fluorescence-activated cell sorting (FACS) of yeast displayed single-chain Fv (scFv) mutant libraries, 28H4 was evolved to a humanized mAb hu6F15.4 that bound each of seven BoNT/F subtypes with high affinity (KD 5.81 pM to 659.78 pM). In contrast, using single antigen FACS sorting, affinity was increased to the subtype used for sorting but with a decrease in affinity for other subtypes. None of the mAb variants showed any binding to other BoNT serotypes or to HEK293 or CHO cell lysates by flow cytometry, thus demonstrating stringent BoNT/F specificity. Multicolor FACS-mediated antibody library screening is thus proposed as a general method to generate multi-specific antibodies to protein subtypes such as toxins or species variants.

A platform technology for generating subunit vaccines against diverse viral pathogens.

The COVID-19 pandemic response has shown how vaccine platform technologies can be used to rapidly and effectively counteract a novel emerging infectious disease. The speed of development for mRNA and vector-based vaccines outpaced those of subunit vaccines, however, subunit vaccines can offer advantages in terms of safety and stability. Here we describe a subunit vaccine platform technology, the molecular clamp, in application to four viruses from divergent taxonomic families: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), Lassa virus (LASV) and Nipah virus (NiV). The clamp streamlines subunit antigen production by both stabilising the immunologically important prefusion epitopes of trimeric viral fusion proteins while enabling purification without target-specific reagents by acting as an affinity tag. Conformations for each viral antigen were confirmed by monoclonal antibody binding, size exclusion chromatography and electron microscopy. Notably, all four antigens tested remained stable over four weeks of incubation at 40°C. Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge.