Binding Of Monoclonal Antibodies Research Articles

Emerging variants develop total escape from potent monoclonal antibodies induced by BA.4/5 infection

The rapid evolution of SARS-CoV-2 is driven in part by a need to evade the antibody response in the face of high levels of immunity. Here, we isolate spike (S) binding monoclonal antibodies (mAbs) from vaccinees who suffered vaccine break-through infections with Omicron sub lineages BA.4 or BA.5. Twenty eight potent antibodies are isolated and characterised functionally, and in some cases structurally. Since the emergence of BA.4/5, SARS-CoV-2 has continued to accrue mutations in the S protein, to understand this we characterize neutralization of a large panel of variants and demonstrate a steady attrition of neutralization by the panel of BA.4/5 mAbs culminating in total loss of function with recent XBB.1.5.70 variants containing the so-called ‘FLip’ mutations at positions 455 and 456. Interestingly, activity of some mAbs is regained on the recently reported variant BA.2.86.

Real-world clinical remission of severe asthma with benralizumab in Spanish adults with severe asthma

Objective Patients with severe eosinophilic asthma experience high risk of exacerbations and reduced quality of life. Benralizumab, a monoclonal antibody binding to IL-5 receptor α subunit, is an approved drug for its treatment. The objective was to describe clinical remission after benralizumab prescription in routine clinical practice. Methods Retrospective multicenter study with data from four hospitals in Valencian Community (Spain) with asthma units between 2019 and 2020. Data was gathered at baseline and after 12 months. We considered clinical remission after 1 year if the patient remained without exacerbations and use of systemic corticosteroids and with good clinical control and normal lung function. Results Data from 139 patients was gathered. At the 12-month follow-up, 44.1% were in clinical remission, since 84.0%, 77.5%, 51.0% and 95.5% of patients did not experience exacerbations, had total asthma control test score of ≥20, prebronchodilator FEV1 of ≥80% and did not use systemic corticosteroids. A significant reduction of long-acting muscarinic antagonists (p = 0.0001), leukotriene receptor antagonists (p = 0.0326), oral corticosteroids (p < 0.0001) and short-acting beta agonists (p = 0.0499) was observed. Baseline factors with greatest individual influence on clinical remission were employment situation, tobacco use, comorbidity number, eosinophil value, number of exacerbations, FEV1, emergency visit number, and ACT, MiniAQLQ and TAI scores. Final analysis of multiple logistic regression indicated that having baseline FEV1 value below 80% increases remission chance 9.7 times a year compared to FEV1 >80%. Conclusion Clinical remission after treatment with benralizumab is achievable in a high percentage of patients with severe asthma eosinophilia not controlled in real life.

Potent Apoptosis Induction by a Novel Trispecific B7-H3xCD16xTIGIT 2+1 Common Light Chain Natural Killer Cell Engager.

Valued for their ability to rapidly kill multiple tumor cells in succession as well as their favorable safety profile, NK cells are of increasing interest in the field of immunotherapy. As their cytotoxic activity is controlled by a complex network of activating and inhibiting receptors, they offer a wide range of possible antigens to modulate their function by antibodies. In this work, we utilized our established common light chain (cLC)-based yeast surface display (YSD) screening procedure to isolate novel B7-H3 and TIGIT binding monoclonal antibodies. The chicken-derived antibodies showed single- to low-double-digit nanomolar affinities and were combined with a previously published CD16-binding Fab in a 2+1 format to generate a potent NK engaging molecule. In a straightforward, easily adjustable apoptosis assay, the construct B7-H3xCD16xTIGIT showed potent apoptosis induction in cancer cells. These results showcase the potential of the TIGIT NK checkpoint in combination with activating receptors to achieve increased cytotoxic activity.

Evidence to Date on the Therapeutic Potential of Zolbetuximab in Advanced Gastroesophageal Adenocarcinoma.

Gastric adenocarcinoma (GAC) continues to be a prevalent worldwide malignancy and a leading cause of cancer death, and it is frequently cited as incurable. Targeted therapy in GAC has lagged behind other solid tumors. The human epidermal growth factor receptor-2 (HER-2) represented the single target in GACs for many years, seen in approximately 20% of patients with advanced GAC. Recent advances in management now include the addition of immunotherapy checkpoint inhibition to select front-line advanced GACs. Unfortunately, outcomes remain poor for most patients. We anticipate finding a key to future discoveries in GACs in next-generation sequencing and more targeted approaches. Claudin 18.2 (CLDN18.2) has emerged as a therapeutic target in GACs. CLDN18.2 is reportedly expressed in 14-87% of GACs, and CLDN18.2 is available for monoclonal antibody (mAb) binding as it is expressed on the outer cell membrane. Here, we review the exploration of CLDN18.2 as a target in GACs via the use of zolbetuximab (IMAB362). Zolbetuximab is now under priority FDA review for GACs, and we eagerly await the review outcome.

Design of an Ara h 2 hypoallergen from conformational epitopes.

Adverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapysuggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA). X-ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum. A ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site-specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant. These studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.

Zolbetuximab for Claudin18.2-positive gastric or gastroesophageal junction cancer.

Claudins (CLDNs) are a family of major membrane proteins that form components of tight junctions. In normal tissues, CLDNs seal the intercellular space in the epithelial sheets to regulate tissue permeability, paracellular transport, and signal transduction. Claudin18.2 (CLDN18.2), a member of the CLDN family, is expressed specifically in gastric mucosal cells in normal tissue, and its expression is often retained in gastric cancer cells. CLDN18.2 is ectopically expressed in many cancers other than gastric cancer such as esophageal cancer, pancreatic cancer, biliary tract cancer, non-small-cell lung cancer, and ovarian cancer. Structurally, CLDN18.2 is localized on the apical side of the cell membrane and has extracellular loops capable of binding monoclonal antibodies. Upon malignant transformation, CLDN18.2 is exposed to the cell surface of the whole membrane, which enables the binding of monoclonal antibodies. Based on these characteristics, CLDN18.2 was considered to be optimal for target therapy, and zolbetuximab was developed which is a first-in-class chimeric immunoglobulin G1 monoclonal antibody highly specific for CLDN18.2. It binds to CLDN18.2 on the tumor cell surface and stimulates cellular and soluble immune effectors that activate antibody-dependent cytotoxicity and complement-dependent cytotoxicity. Recently, zolbetuximab combined with chemotherapy demonstrated a survival benefit in patients with CLDN18.2-positive and HER-2-negative gastric or gastroesophageal junction cancers in the global phase III SPOTLIGHT and GLOW trials. From these clinically meaningful results, CLDN18.2-targeting therapy including zolbetuximab has attracted a lot of attention. In this review, we summarize the clinical implications of CLDN18.2-positive gastric or GEJ cancer, and CLDN18.2-targeting therapy, mainly for zolbetuximab.

Engagement of CD300c by a novel monoclonal antibody induces the differentiation of monocytes to M1 macrophages

Human CD300c is expressed on various immune or cancer cells and is a novel B7 family member, functioning as an activity modulator on immune cells. To elucidate the function of CD300c, we developed CL7, a human CD300c-specific monoclonal antibody, and assessed its biological activity. The specific binding of CL7 monoclonal antibody against recombinant CD300c antigen was confirmed using enzyme-linked immunosorbent assay and surface plasmon resonance analysis. The binding affinity of CL7 was strong at the sub-nanomolar level. Furthermore, CL7 effectively bound to exogenously expressed CD300c on 293T cells. CL7 antibody differentiated monocytes to M1 macrophages, as evidenced by the upregulated expression of M1-specific cell surface markers and increased secretion of M1-specific cytokines in vitro in THP-1 cells and primary macrophages, as well as the increased population size of M1 macrophages in tumors grafted into mice. Additionally, CL7 treatment upregulated PD-L1 expression on THP-1 cells. We confirmed that the mechanism of M1 macrophage differentiation was through the mitogen-activated protein kinase and NF-κB signaling pathways. CD300c expression on various immune and cancer cells was similar to that of the well-known immune checkpoint PD-L1, suggesting the possibility of CD300c as a novel tumor biomarker. We also confirmed that the tumor size was substantially reduced by CL7 antibody treatment in the CT26 mouse model. Our study supports that CD300c is a potential therapeutic target in immuno-oncology. Overall, the CD300c-specific monoclonal antibody, CL7, is a promising immunotherapeutic agent, and it induces enhanced differentiation of M1 macrophages and/or their infiltration into the tumor microenvironment.

Quantitative Rapid Magnetic Immunoassay for Sensitive Toxin Detection in Food: Non-Covalent Functionalization of Nanolabels vs. Covalent Immobilization.

In this study, we present a novel and ultrasensitive magnetic lateral flow immunoassay (LFIA) tailored for the precise detection of zearalenone, a mycotoxin with significant implications for human and animal health. A versatile and straightforward method for creating non-covalent magnetic labels is proposed and comprehensively compared with a covalent immobilization strategy. We employ the magnetic particle quantification (MPQ) technique for precise detection of the labels and characterization of their functionality, including measuring the antibody sorption density on the particle surface. Through kinetic studies using the label-free spectral phase interferometry, the rate and equilibrium constants for the binding of monoclonal antibodies with free (not bound with carrier protein) zearalenone were determined to be kon = 3.42 × 105 M-1s-1, koff = 7.05 × 10-4 s-1, and KD = 2.06 × 10-9 M. The proposed MPQ-LFIA method exhibits detection limits of 2.3 pg/mL and 7.6 pg/mL when employing magnetic labels based on covalent immobilization and non-covalent sorption, with dynamic ranges of 5.5 and 5 orders, correspondingly. We have successfully demonstrated the effective determination of zearalenone in barley flour samples contaminated with Fusarium graminearum. The ease of use and effectiveness of developed test systems further enhances their value as practical tools for addressing mycotoxin contamination challenges.

High-Throughput Screening of Surface Engineered Cyanine Nanodots for Active Transport of Therapeutic Antibodies into Solid Tumor.

The successful delivery of therapeutic biomacromolecules into solid tumorholds great challenge due to their high resistance to penetrate through the complex tumor microenvironments. Here, active-transporting nanoparticles are harnessed to efficiently deliver biomacromolecular drugs into solid tumors through cell transcytosis. A series of molecularly precise cyanine 5-cored polylysine G5 dendrimers (Cy5 nanodots) with different peripheral amino acids (G5-AA) is prepared. The capability of these positively charged nanodots to induce cell endocytosis, exocytosis, and transcytosis is evaluated via fluorescence-based high-throughput screen. The optimized nanodots (G5-R) are conjugated with αPD-L1 (a therapeutic monoclonal antibody binding to programmed-death ligand 1) (αPD-L1-G5-R) to demonstrate the nanoparticle-mediated tumor active transport. The αPD-L1-G5-R can greatly enhance the tumor-penetration capability through adsorption-mediated transcytosis (AMT). The effectiveness of αPD-L1-G5-R is tested in treating mice bearing partially resected CT26 tumors, mimicking the local immunotherapy of residual tumors post-surgery in clinic. The αPD-L1-G5-R embedded in fibrin gel can efficiently mediate tumor cell transcytosis, and deliver αPD-L1 throughout the tumor, thereby enhancing immune checkpoint blockade, reducing tumor recurrence, and significantly prolonging the survival time. The active-transporting nanodots are promising platforms for efficient tumor delivery of therapeutic biomacromolecules.

Lecanemab Clarity AD: Quality-of-Life Results from a Randomized, Double-Blind Phase 3 Trial in Early Alzheimer’s Disease

BackgroundLecanemab is a humanized IgG1 monoclonal antibody binding with high affinity to amyloid-beta protein protofibrils. In phase 3 development, lecanemab has been shown to reduce markers of amyloid in early Alzheimer’s disease and reduce decline on clinical endpoints of cognition and function at 18 months.ObjectivesTo describe the health-related quality-of-life (HRQoL) results from Clarity AD which were exploratory outcomes in this trial.DesignClarity AD was an 18-month, multi-center, doubleblind, phase 3 trial.SettingEarly Alzheimer’s disease.ParticipantsIndividuals 50–90 years of age with a diagnosis of mild cognitive impairment or mild dementia due to Alzheimer’s disease and positron emission tomography or cerebrospinal fluid evidence of cerebral amyloid accumulation.InterventionPlacebo or lecanemab 10-mg/kg IV biweekly.MeasurementsHRQoL was measured at baseline and every 6 months using the European Quality of Life–5 Dimensions (EQ-5D-5L; by subject) and Quality of Life in AD (QOL-AD; by subject and proxy). Study partner burden was measured using the Zarit Burden Interview (ZBI).ResultsA total of 1795 participants were enrolled (lecanemab:898; placebo:897). At month 18, adjusted mean change from baseline in EQ-5D-5L and QOL-AD by subject showed 49% and 56% less decline, respectively. QOL-AD rated by study partner as proxy resulted in 23% less decline. ZBI adjusted mean change from baseline at 18 months resulted in 38% less increase of care partner burden. Individual HRQoL test items and dimensions also showed lecanemab benefit.ConclusionsLecanemab was associated with a relative preservation of HRQoL and less increase in caregiver burden, with consistent benefits seen across different quality of life scales and within scale subdomains. These benefits provide valuable patient reported outcomes which, together with previously reported benefits of lecanemab across multiple measures of cognition, function, disease progression, and biomarkers, demonstrate that lecanemab treatment may offer meaningful benefits to patients, care partners, and society.

Riding the Wave: Unveiling the Conformational Waves from RBD of SARS-CoV-2 Spike Protein to ACE2.

The binding affinity between angiotensin-converting enzyme 2 (ACE2) and the receptor-binding domain (RBD) plays a crucial role in the transmission and reinfection of SARS-CoV2. Here, microsecond molecular dynamics simulations revealed that point mutations in the RBD domain induced conformational transitions that determined the binding affinity between ACE2 and RBD. These structural changes propagated through the RBD domain, altering the orientation of both ACE2 and RBD residues at the binding site. ACE2 receptor shows significant structural heterogeneity, whereas its binding to the RBD domain indicates a much greater degree of structural homogeneity. The receptor was more flexible in its unbound state with the binding of RBD domains inducing structural transitions. The structural heterogeneity observed in the ACE2 unbound form plays a role in the promiscuity of viral entry, as it may allow the receptor to interact with various related and unrelated ligands. Furthermore, rigidity may be important for stabilizing the complex and ensuring the proper orientation of the RBD-binding interface with ACE2. The greater structural homogeneity observed in the ACE2-RBD complex revealed the effectiveness of neutralizing antibodies and vaccines that are primarily directed toward the RBD-binding interface. The binding of the B38 monoclonal antibody revealed restricted conformational transitions in the RBD and ACE2 receptors, attributed to its potent binding interaction.

Epitope editing enables targeted immunotherapy of acute myeloid leukaemia.

Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia1,2, the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens3-5. Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning.

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 &amp; 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 &amp;lt; 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&amp;lt;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.