Murine Monoclonal Antibody Research Articles

Phase 1 study of intraventricular 131I-omburtamab targeting B7H3 (CD276)-expressing CNS malignancies

BackgroundThe prognosis for metastatic and recurrent tumors of the central nervous system (CNS) remains dismal, and the need for newer therapeutic targets and modalities is critical. The cell surface glycoprotein B7H3 is expressed on a range of solid tumors with a restricted expression on normal tissues. We hypothesized that compartmental radioimmunotherapy (cRIT) with the anti-B7H3 murine monoclonal antibody omburtamab injected intraventricularly could safely target CNS malignancies.Patients and methodsWe conducted a phase I trial of intraventricular 131I-omburtamab using a standard 3 + 3 design. Eligibility criteria included adequate cerebrospinal fluid (CSF) flow, no major organ toxicity, and for patients > dose level 6, availability of autologous stem cells. Patients initially received 74 MBq radioiodinated omburtamab to evaluate dosimetry and biodistribution followed by therapeutic 131I-omburtamab dose-escalated from 370 to 2960 MBq. Patients were monitored clinically and biochemically for toxicity graded using CTCAEv 3.0. Dosimetry was evaluated using serial CSF and blood sampling, and serial PET or gamma-camera scans. Patients could receive a second cycle in the absence of grade 3/4 non-hematologic toxicity or progressive disease.ResultsThirty-eight patients received 100 radioiodinated omburtamab injections. Diagnoses included metastatic neuroblastoma (n = 16) and other B7H3-expressing solid tumors (n = 22). Thirty-five patients received at least 1 cycle of treatment with both dosimetry and therapy doses. Acute toxicities included < grade 4 self-limited headache, vomiting or fever, and biochemical abnormalities. Grade 3/4 thrombocytopenia was the most common hematologic toxicity. Recommended phase 2 dose was 1850 MBq/injection. The median radiation dose to the CSF and blood by sampling was 1.01 and 0.04 mGy/MBq, respectively, showing a consistently high therapeutic advantage for CSF. Major organ exposure was well below maximum tolerated levels. In patients developing antidrug antibodies, blood clearance, and therefore therapeutic index, was significantly increased. In patients receiving cRIT for neuroblastoma, survival was markedly increased (median PFS 7.5 years) compared to historical data.ConclusionscRIT with 131I-omburtamab is safe, has favorable dosimetry and may have a therapeutic benefit as adjuvant therapy for B7-H3-expressing leptomeningeal metastases.Trial registration: clinicaltrials.gov NCT00089245, August 5, 2004.

Production and characterization of a murine anti-dal monoclonal antibody for blood typing in dogs

Considering the strong immunogenicity of the Dal antigen, and that > 98% of dogs, including blood donors, are Dal-positive, finding compatible blood for a previously transfused Dal-negative patient may be challenging. This is exacerbated by limited access to typing reagents, which currently rely on polyclonal antibodies (PAb) produced following sensitization of dogs. Therefore, the objective of this study was to produce and characterize an anti-Dal murine monoclonal antibody (MAb). Conventional hybridoma technology was used to produce MAb directed against canine red blood cells (cRBC). Briefly, female BALB/c mice were immunized via repeated intraperitoneal injections of washed Dal-positive cRBC (DEA 1,3,7 negative; DEA 4,5 positive) until serologic titers were sufficient (>1:1000). Following fusion with myeloma cells, 573 hybridoma cell culture supernatants were obtained and screened for MAb of interest using a gel column agglutination technique and known Dal-negative and Dal-positive cRBC. Fifteen supernatants led to cRBC agglutination, but only one had the desired pattern (i.e. anti-Dal). To assess its specificity and sensitivity, Dal blood typing of 62 canine EDTA-blood samples was performed using the anti-Dal MAb and two canine PAb: 45 Dal-positive and 17 Dal-negative were identified with 100% agreement between reagents (kappa =1). The anti-Dal MAb produced was further determined to be an IgG1. Conventional hybridoma technology, aided by a gel column technique, has enabled the production of a murine MAb specific against the canine Dal antigen. This will ensure long-term perennity of Dal blood typing, facilitate clinical management and research, as well as avoid resorting to repeat dog sensitization.

Molecular mimicry of the receptor-binding domain of the SARS-CoV-2 spike protein: from the interaction of spike-specific antibodies with transferrin and lactoferrin to the antiviral effects of human recombinant lactoferrin.

The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves dysregulations of iron metabolism, and although the mechanism of this pathology is not yet fully understood, correction of iron metabolism pathways seems a promising pharmacological target. The previously observed effect of inhibiting SARS-CoV-2 infection by ferristatin II, an inducer of transferrin receptor 1 (TfR1) degradation, prompted the study of competition between Spike protein and TfR1 ligands, especially lactoferrin (Lf) and transferrin (Tf). We hypothesized molecular mimicry of Spike protein as cross-reactivity of Spike-specific antibodies with Tf and Lf. Thus, strong positive correlations (R2 > 0.95) were found between the level of Spike-specific IgG antibodies present in serum samples of COVID-19-recovered and Sputnik V-vaccinated individuals and their Tf-binding activity assayed with peroxidase-labeled anti-Tf. In addition, we observed cross-reactivity of Lf-specific murine monoclonal antibody (mAb) towards the SARS-CoV-2 Spike protein. On the other hand, the interaction of mAbs produced to the receptor-binding domain (RBD) of the Spike protein with recombinant RBD protein was disrupted by Tf, Lf, soluble TfR1, anti-TfR1 aptamer, as well as by peptides RGD and GHAIYPRH. Furthermore, direct interaction of RBD protein with Lf, but not Tf, was observed, with affinity of binding estimated by KD to be 23nM and 16nM for apo-Lf and holo-Lf, respectively. Treatment of Vero E6 cells with apo-Lf and holo-Lf (1-4mg/mL) significantly inhibited SARS-CoV-2 replication of both Wuhan and Delta lineages. Protective effects of Lf on different arms of SARS-CoV-2-induced pathogenesis and possible consequences of cross-reactivity of Spike-specific antibodies are discussed.

Advancing humanized monoclonal antibody for counteracting fentanyl toxicity towards clinical development

ABSTRACT Innovative therapies to complement current treatments are needed to curb the growing incidence of fatal overdoses related to synthetic opioids. Murine and chimeric monoclonal antibodies (mAb) specific for fentanyl and its analogs have demonstrated pre-clinical efficacy in preventing and reversing drug-induced toxicity in rodent models. However, mAb-based therapeutics require extensive engineering as well as in vitro and in vivo characterization to advance to first-in-human clinical trials. Here, novel murine anti-fentanyl mAbs were selected for development based on affinity for fentanyl, and efficacy in counteracting the pharmacological effects of fentanyl in mice. Humanization and evaluation of mutations designed to eliminate predicted post-translational modifications resulted in two humanized mAbs that were effective at preventing fentanyl-induced pharmacological effects in rats. These humanized mAbs showed favorable biophysical properties with respect to aggregation and hydrophobicity by chromatography-based assays, and thermostability by dynamic scanning fluorimetry. These results collectively support that the humanized anti-fentanyl mAbs developed herein warrant further clinical development for treatment of fentanyl toxicity.

Treatment with activin type ii receptor antibody improves cardiac function in mice after myocardial infarction

Abstract Background Activin type ii receptor (ActRII) binding to its natural ligands including myostatin, activin, and growth development factor 11 negatively regulates muscle growth. Blockade of the ligands/ActRII signaling with human ActRII monoclonal antibody has been shown to increase body lean mass and reduce fat mass in patients with type 2 diabetes. However, the blockade effect on the ligands/ActRII signaling in heart failure with or without cardiac cachexia remains controversial. We hypothesized that the blockade effect on the ligands/ActRII signaling with a murine ActRII monoclonal antibody would improve cardiac function in a mouse model of heart failure. Methods Under anesthesia with isoflurane in oxygen, C57BL/6 mice were subjected to sham surgery or myocardial infarction (MI) by ligation of left anterior descending coronary artery to induce heart failure. Mice were randomized into treatment groups based on ejection fraction measured by Vevo 3100 Imaging System two weeks after MI. MI mice were treated weekly with vehicle (n=11), 10 mg/kg (n=12), or 20 mg/kg (n=12) ActRII antibody by subcutaneous injection for 8 weeks. Cardiac function was determined by Vevo 3100 Imaging system at 4, 8 weeks post-treatment, respectively. Body lean and fat mass were determined by Bruker's Whole Body Composition Analyzer. Myocardial infarct size was determined by Masson's trichrome staining on heart sections. Results Myocardial infarction markedly impaired cardiac function in mice. Ejection fraction reduced to 21.3±1.9% in MI mice compared to 56.3±1.2% in sham-operated mice (p&amp;lt;0.0001). Weekly treatment with 10 mg/kg or 20 mg/kg antibody significantly improved ejection fraction to 32.1±2.8%, and 35.9±2.9%, respectively (p&amp;lt;0.01 and p&amp;lt;0.001). Antibody treatment in MI mice also reduced MI-induced lung congestion by 5% and 8% (p&amp;lt;0.01), respectively. However, heart weight and myocardial infarct size in antibody treated mice were comparable to those in vehicle-treated mice. Moreover, antibody treatment dose-dependently increased body weight, which is further confirmed by increases in muscle mass and body lean mass measured by gravimetric analysis and body composition analyzer. Conclusion ActRII antibody dose-dependently improved cardiac function independent of myocardial infarct size post-MI. Although antibody significantly increased body weight and lean mass, it did not significantly increase heart weight, indicating that antibody treatment did not cause adverse remodeling post-MI while it remarkably improved cardiac function. Our data suggest that ActRII antibody could be a novel treatment for heart failure with reduced ejection fraction and may be particularly beneficial in patients with cachexia. Funding Acknowledgement Type of funding sources: None.

Molecular imaging of experimental atherosclerosis using anti-malondialdehyde-modified low-density lipoprotein humanised antibody fragment targeted nanoparticles

Abstract Introduction Oxidative modification of low-density lipoprotein (LDL), for example by malondialdehyde (MDA) adduction with subsequent uptake by macrophages to form foam cells and later the plaque necrotic core, is a key initiating event in atherogenesis. Accordingly, a larger lipid necrotic core is a key plaque vulnerability factor, predisposing plaques to rupture and subsequent thrombosis and development of an acute coronary syndrome. Thus, MDA-LDL is an attractive focus for the molecular targeting of atherosclerotic plaques. Purpose To develop antibody fragment-targeted nanoparticles that can be utilised for both the molecular imaging and therapeutics of vulnerable atherosclerotic plaques. Methods LO1 is an IgG3k natural monoclonal murine antibody that reacts with MDA-LDL. Humanised LO1Fab fragments have been engineered to reduce immunogenicity and improve lesion penetration. These humanised LO1Fab fragments were used to functionalise fluorescent poly(lactic-co-glycolic acid) (PLGA) - polyethylene glycol (PEG) nanoparticles. Nanoparticle in vitro function was assessed, prior to fluorescence molecular tomography (FMT) co-registered with micro-CT, four-hours after iv injection in atherosclerotic LDL-receptor−/− mice fed a high-fat diet for 40-weeks. Results Humanised LO1Fab fragment conjugated fluorescent PLGA-PEG nanoparticles were formulated with 210nm size and polydispersity index (variability of nanoparticle size around the average) of &amp;lt;0.2. Antibody conjugation efficiency was 30%. In vitro function was confirmed on ELISA versus the blank untargeted nanoparticles with MDA-LDL on solid phase, detecting nanoparticle presence via the conjugated LO1Fab, PEG corona or fluorescence. Fluorescence microscopy on stained aortic root cryosections from atherosclerotic mice confirmed binding to fatty lesions. Construct in vivo in half-life was 90-minutes for both the targeted and untargeted nanoparticles in a two-phase model in LDL-receptor−/− mice, based on fluorescence analysis of serial tail vein blood samples. There was greater uptake in the region-of-interest (heart and aortic arch vessels) in mice injected with LO1Fab-conjugated nanoparticles versus untargeted nanoparticles (mean ± standard deviation) (64.7±22.9 versus 25.2±26.5pmol of Cy5; n=7; p=0.02). Ex vivo analysis fluorescence reflectance imaging and quantitative FMT of the extracted aortae confirmed these findings (1.0±0.3 versus 0.5±0.2pmol of Cy5; n=7; p=0.002; Figure 1). Conclusions Humanised antibody Fab fragment fluorescent nanoparticles have been developed that successfully target MDA-LDL and localise to atherosclerotic plaques in murine experimental atherosclerosis. These targeted nanoparticles have the potential to amplify fluorescent signal for imaging and carry a therapeutic cargo for targeted drug delivery direct to atherosclerotic plaques. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Wellcome Trust Clinical Research Fellowship

Role of antibody engineering in generation of derivatives starting from MOv19 MAb: 40years of biological/therapeutic tools against folate receptor alfa.

In the 1980s, we developed and characterized numerous murine monoclonal antibodies (MAbs) directed against human tumor-associated antigens. This mini review is focused on the generation of derivatives of an anti-folate receptor α (FRα) MAbs, named MOv19, exploiting the antibody-engineering progresses in the last 40 years. The FRα location on the luminal surface of proliferating epithelial cells, inaccessible to circulation, versus its over-expression in the entire surface of numerous carcinomas suggested a role for anti-FRα MAbs in the diagnosis and/or treatment of solid tumors. Presently, two MOv19 derivatives are in clinical trials: a chimeric resurfaced version in an antibody-drug conjugate format (SORAYA trial, 2022) and the murine scFv in a second generation chimeric antigen receptor, CAR-T (Phase Ia, 2021). MOv19 and its derivatives could be considered a relevant example that well-characterized anti-tumor murine Mabs and antibody engineering could be combined to generate useful therapeutic tools.

Tetraplex Fluorescent Microbead-Based Immunoassay for the Serodiagnosis of Newcastle Disease Virus and Avian Influenza Viruses in Poultry Sera.

Virulent Newcastle disease virus (NDV) as well as highly pathogenic avian influenza (HPAIV) subtypes H5 and H7 induce contagious and lethal systemic disease in poultry. In contrast, low pathogenic AIV H5 and H7 may circulate clinically unnoticed in poultry but eventually generate HPAIV. Low pathogenic NDV strains are widely used as live-attenuated vaccines against ND. Serological tools are essential to conduct active surveillance for infections with notifiable AIV-H5, -H7 and to control vaccination against NDV and HPAIV in poultry populations. Here, recombinant nucleocapsid proteins (NP) of AIV and NDV, and haemagglutinin protein fragment-1 (HA1) of AIV subtypes H5 and H7 were expressed in E. coli. Purification and refolding were required before coating fluorescent microspheres via streptavidin-biotin linkage. The tetraplexed inhibition fluorescent microsphere immunoassay (iFMIA) was then assembled for analysis on a Luminex®-like platform (Bioplex®) using murine monoclonal antibodies specific for each of the four targets. The assay was evaluated by testing galliform poultry sera derived from experimental infections (n = 257) and from farms (n = 250), respectively. The tetraplex iFMIA compared favorably with commercially available ELISAs and the “gold standard” hemagglutination inhibition assay. Tetraplexed iFMIA provided a specific and sensitive tool to detect and discriminate AIV- and NDV-specific antibodies in the sera of galliform poultry.

IgG against the Membrane-Proximal Portion of the Desmoglein 3 Ectodomain Induces Loss of Keratinocyte Adhesion, a Hallmark in Pemphigus Vulgaris

Pemphigus vulgaris is a severe autoimmune blistering disease characterized by IgG autoantibodies (auto-abs) against the desmosomal adhesion molecules desmoglein (DSG) 3 and DSG1. Underlying mechanisms leading to blister formation upon binding of DSG-specific IgG auto-abs are not fully understood. Numerous studies showed the pathogenicity of IgG auto-ab binding to the aminoterminal region 1 (EC1) of the DSG3 ectodomain. However, auto-abs in pemphigus vulgaris are polyclonal, including IgG against both aminoterminal- and membrane-proximal epitopes of the DSG3 ectodomain. In this study, the pathogenicity of a previously uncharacterized murine monoclonal IgG antibody, 2G4, directed against the membrane-proximal region (EC5) of the DSG3 ectodomain was characterized and tested in various specificity and functionality assays. The results clearly show that 2G4 is capable of inhibiting intercellular keratinocyte adhesion and of inducing cellular DSG3 redistribution by activation of the p38MAPK signal transduction pathway. In this study, we provide evidence that an IgG auto-abs directed against the membrane-proximal region EC5 of DSG3 induces acantholysis, the hallmark in pemphigus vulgaris. These findings challenge the current concept that IgG auto-abs targeting the NH2-terminal portion of the DSG3 ectodomain are pathogenic only. Our study provides further aspects for a deeper understanding of desmosomal keratinocyte adhesion and improves our insight into the complex auto-ab‒induced blister formation in pemphigus vulgaris.

Identification of Novel Retinal Pericyte-Targeting rAAV Vectors Through Directed Evolution

PurposeRetinal pericytes play a vital role in maintaining retinal homeostasis, and their dysfunction underlies pathogenesis in such vascular eye diseases as diabetic retinopathy and wet age-related macular degeneration. Consequently, retinal pericytes are attractive therapeutic targets for gene therapy, but effectively targeting pericytes with recombinant adeno-associated virus (rAAV) vectors remains a challenge.MethodsWe introduced genetic modifications into the surface-exposed variable regions of the rAAV2/2 capsid to generate a complex library (>1 × 107) of capsid mutants that were then screened for preferential tropism toward retinal pericytes. Using the Tg(Cspg4-DsRed.T1)1Akik/J reporter mouse model, which has red fluorescent pericytes that can be isolated via flow cytometry in order to recover vector genomes, we performed three rounds of screening and identified seven putative mutants capable of transducing retinal pericytes.ResultsFollowing intravitreal administration of mutant vectors packaging ubiquitously expressing green fluorescent protein reporters and postmortem flow cytometry of enzymatically digested retinae, two mutants in particular, Peri-E and Peri-G, demonstrated significantly greater transduction of retinal pericytes than unmodified rAAV2/2 (1.4-fold and 2.8-fold, respectively).ConclusionsAlthough difficult to characterize the effect of each point mutation in the context of multiple amino acid variations from the wild-type AAV2 sequence, we identified several point mutations that may play critical roles in limiting HSPG binding, evading neutralization by murine A20 monoclonal antibodies, modulating antigenicity, and evading ubiquitination to ultimately improve transduction efficiency of retinal pericytes.Translational RelevanceIdentification of novel retinal pericyte targeting rAAV vectors enables the development of new, long-lasting gene therapies for retinal diseases such as diabetic retinopathy and wet age-related macular degeneration.

A Comparison of Murine PD-1 and PD-L1 Monoclonal Antibodies.

Blockade of the PD-L1/PD-1 pathway has proven to be a broadly effective cancer immunotherapy. FDA-approved therapeutic monoclonal antibodies (mAbs) targeting the pathway have high affinity, blocking capacity, and low antibody effector activity. A number of rat antimouse mAbs have been used to model cancer immunotherapy in mouse models. We set forth the amino acid sequences of mAbs specific for mouse PD-1 (29F.1A12) and PD-L1 (10F.9G2) and compare their avidities, blocking capacities, biological activities, and epitope recognition with other commonly used mAbs. Further manipulation of these sequences should facilitate better modeling of immunotherapy in mouse models and the generation of novel agents.

Development of an efficient reproducible cell-cell transmission assay for rapid quantification of SARS-CoV-2 spike interaction with hACE2

SummaryEfficient quantitative assays for measurement of viral replication and infectivity are indispensable for future endeavors to develop prophylactic or therapeutic antiviral drugs or vaccines against SARS-CoV-2. We developed a SARS-CoV-2 cell-cell transmission assay that provides a rapid and quantitative readout to assess SARS-CoV-2 spike hACE2 interaction in the absence of pseudotyped particles or live virus. We established two well-behaved stable cell lines, which demonstrated a remarkable correlation with standard cell-free viral pseudotyping for inhibition by convalescent sera, small-molecule drugs, and murine anti-spike monoclonal antibodies. The assay is rapid, reliable, and highly reproducible, without a requirement for any specialized research reagents or laboratory equipment and should be easy to adapt for use in most investigative and clinical settings. It can be effectively used or modified for high-throughput screening for compounds and biologics that interfere with virus-cell binding and entry to complement other neutralization assays currently in use.

Abstract 2858: Development of a first-in-class humanized antibody targeting CXCL1 in bladder cancer

Abstract Introduction and Objectives: Chemokine (C-X-C motif) ligand 1 (CXCL1) plays a crucial role in tumor initiation, promotion, and progression in bladder cancer (BCa) development. Previously, we reported on a monoclonal murine antibody (HL2401) towards CXCL1 which was capable of inhibiting growth in both in vitro and in vivo models. Herein, we have developed a first-in-class humanized monoclonal antibody neutralizing CXCL1 (NTC-001). This study is final step in pre-clinical stage. In this study, we aimed to evaluate efficacy and safety of the antibody to move into phase 1 clinical trials in near future. Methods: We humanized mouse anti-human CXCL1 antibody resulting in NTC-001, and then tested for the following: molecular weight and pH, specificity, binding affinity to ligand and to albumin, ability to inhibit in vitro angiogenesis, ability to inhibit cellular proliferation alone or with chemotherapy, and pharmacokinetics and pharmacodynamics. Results: NTC-001 had a molecular weight of 150kD, and was an acidic antibody (pH5.1). NTC-001 specifically bound human CXCL1 in both WB and ELISA. NTC-001 was bound to albumin and had minimal hepatic metabolism. NTC-001 significantly inhibited tube formation in HUVEC, and more efficiently with the formulation. NTC-001 significantly inhibited T24 cell proliferation (p&amp;lt;0.001). Inhibitory effect of NTC-001 on cell proliferation was correlated with the levels of CXCL1 expression in BCa cell lines. Compared to CXCL1 expression-low group (RT112 and UMUC-14), CXCL1 expression-high group (T24, 5637, 253J, 253J-BV) tended to show low IC50. Synergistic combination efficacy was observed in NTC-001 in combination with gemcitabine (combination index&amp;lt;1) though not with cisplatin. PK study demonstrated a half-life of 324 hours and 18 hours in CD-1 and NSG-SGM3, respectively. Conclusions: NTC-001 is a first in class humanized neutralizing monoclonal antibody towards CXCL1, and could be a promising therapeutic agent in BCa. We are going to proceed to complete our investigational new drug enabling studies in both small and large animals. Citation Format: Kaoru Murakami, Yuka Sasaki, Hideki Furuya, Charles Rosser. Development of a first-in-class humanized antibody targeting CXCL1 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2858.

Role of heat shock protein 60 in primed and naïve states of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) exist in at least two distinct states in mammals: naïve pluripotency that represents several molecular characteristics in pre-implantation epiblast and primed pluripotency that corresponds to cells poised for differentiation in post-implantation epiblast. To identify and characterize the surface molecules that are necessary for the maintenance of naïve hPSCs, we generated a panel of murine monoclonal antibodies (MAbs) specific to the naïve state of hPSCs. Flow cytometry showed that N1-A4, one of the MAbs, bound to naïve hPSCs but not to primed hPSCs. Cell surface biotinylation and immunoprecipitation analysis identified that N1-A4 recognized heat shock protein 60 (HSP60) expressed on the surface of naïve hPSCs. Quantitative polymerase chain reaction (qPCR) analysis showed that HSP60 expression was rapidly downregulated during the embryoid body (EB) differentiation of primed hPSCs. HSP60 knockdown led to a decrease in the expression of pluripotency genes in primed hPSCs. HSP60 depletion also led to a decrease in the expression of pluripotency genes and representative naïve-state-specific genes in naïve hPSCs. Taken together, the results suggest that HSP60 is downregulated during differentiation of hPSCs and is required for the maintenance of pluripotency genes in both primed and naïve hPSCs, suggesting that HSP60 is a regulator of hPSC pluripotency and differentiation.

VEGF expression in cetuximab resistance development in patients with squamous cell carcinoma of the tongue and oral mucosa.

e18072 Background: Clinical course and aggressiveness of a tumor, as well as the subtle mechanisms of its pathogenesis are related to vascularization, which in turn is determined by the ability of transformed cells to synthesize a wide range of biologically active substances. Among modern approaches to cancer treatment, special attention is paid to anti-EGFR agents reducing tumor vascularization. The purpose of this study was to analyze VEGF expression in tumor vessels in patients with squamous cell carcinoma of the tongue and oral mucosa developing cetuximab resistance. Methods: The study included 45 patients with squamous cell carcinoma of the tongue and oral mucosa T3-4N0-1M0. The patients were divided into 3 groups: group 1 – all patients before treatment; group 2 – patients after standard chemotherapy with cisplatin and fluorouracil; group 3 – patients after chemotherapy in combination with targeted therapy with cetuximab. Immunohistochemical study was performed on sections from paraffin blocks of tumors using monoclonal murine anti-VEGF antibodies (clone VG1, Thermo Scientific) diluted 1:150. The Reveal Polyvalent HRP-DAB Detection System was used. The significance of differences was assessed using the Mann Whitney and Wilcoxon tests calculated using the STATISTICA 10.0 program (StatSoftInc., USA). Results: Changes in the density of vessels stained with the VEGF marker were studied in tumor cells of patients in all groups. Patients in group 1 had from 3 to 10 vessels in one field of view; in group 2 – from 2 to 6 vessels in 70% of cases (21) and up to 15 vessels in 30% of cases (9); in group 3 – only solitary vessels. The average numbers of microvessels stained with the VEGF marker per field of view were 6.5±1.52; 4.5±1.71; 1.0±0.28, respectively. Quantification of intratumoral microvascular density showed that the minimum number of microvasculature vessels in the field of view was observed in patients who received chemotherapy and targeted therapy with cetuximab. The value was 5 times lower compared to initial values, and 4 times lower than in patients who received only standard chemotherapy (the Mann Whitney test, p &lt; 0.05). The statistical analysis according to the Wilcoxon test on the effect of targeted therapy (comparison between groups 2 and 3) revealed a statistically significant effect of cetuximab for this marker (p = 0.028). Conclusions: Immunohistochemical analysis of the VEGF neoangiogenesis factor in squamous cell carcinoma of the tongue and oral mucosa confirmed the effectiveness of targeted therapy with cetuximab.

FLORA-5/GOG3035: Frontline chemo-immunotherapy (paclitaxel-carboplatin-oregovomab [PCO] versus chemotherapy (paclitaxel-carboplatin-placebo [PCP]) in patients with advanced epithelial ovarian cancer (EOC)—Phase III, double-blind, placebo-controlled, global, multinational study.

TPS5619 Background: Oregovomab, a murine IgGκ1 monoclonal antibody, has high affinity binding to tumor associated antigen CA125, thus, rendering the target antigen CA125 more immunogenic or “neoantigen-like” through altered and enhanced antigen processing and presentation to specific T cells. This phenomenon is hypothesized to bypass tumor-associated immune suppression when oregovomab is combined with chemotherapy. In a randomized phase II study, oregovomab in combination with paclitaxel and carboplatin (PC) induced tumor immunity and demonstrated significant improvement in median PFS (41.8 months(m) PCO vs 12.2 m PC, HR 0.46, p=0.0027) and median OS (N.E. PCO vs 43.2 m PC, HR O.35, p=0.043) in patients with previously untreated EOC. Oregovomab combined with PC had a favorable toxicity profile. FLORA-5/GOG3035, the definitive confirmatory global registration trial, is currently recruiting patients in the front-line setting. Methods: The study is a phase 3, multicenter, double-blind, placebo-controlled trial. Optimally debulked patients with FIGO III/IV EOC and serum CA125 ≥ 50 U/ml receiving adjuvant (Cohort 1) or patients receiving neoadjuvant chemotherapy post-interval cytoreductive surgery (Cohort 2) will be randomized to PC + oregovomab or placebo (PCO vs. PCP). Patients with germline BRCA1/2 mutations are excluded. Chemotherapy will be administered every 3 weeks in both cohorts. Oregovomab/placebo is administered simultaneously at cycles 1, 3, and 5 of chemotherapy with an additional dose at 12 weeks following cycle 5 in Cohort 1. Neoadjuvant patients will be administered oregovomab/placebo after debulking surgery at cycles 4 and 6 with two additional doses at 6- and 18-weeks following cycle 6 in Cohort 2. No other front-line maintenance therapy is permitted. The primary objective is PFS determined by RECIST 1.1. Cohort 1 will recruit 372 patients with a 90% power to detect a difference with an alpha of 0.025 and a hazard ratio of 0.65 when 252 PFS events have been observed. Cohort 2 will be analyzed separately recruiting 232 patients with a 90% power to detect a difference with an alpha of 0.025 and a hazard ratio of 0.60 when 165 PFS events have been observed. An interim futility analysis will be performed. Secondary objectives include OS, frequency and severity of AEs, and QoL. Exploratory objectives include iRECIST, TFST, TSST, PFS2, and evaluation of correlative biomarkers. The study is actively enrolling in the US, Canada, Belgium, Italy, Spain, Czech Republic, Hungary, Poland, Korea, Taiwan, Mexico, Argentina, and Chile. 179 patients were enrolled at time of submission. Clinical trial information: NCT04498117.

Cellular Prion Protein Expression in the Brain Tissue from Brucella ceti-Infected Striped Dolphins (Stenella coeruleoalba).

Simple SummaryBrucella ceti, a zoonotic bacterial pathogen, is known to exhibit a strong neurotropism and neuropathogenicity for striped dolphins (Stenella coeruleoalba), often leading to their stranding and death. Given the lack of information on B. ceti infection’s neuropathogenesis, we investigated, for the first time, cellular prion protein (PrPc) expression in the brain tissue from B. ceti-infected, neurobrucellosis-affected striped dolphins. Our study was inspired by previous work, reporting PrPc as the host cell receptor for B. abortus on the surface of murine macrophages. Immunohistochemistry (IHC) and Western blot (WB) analyses were carried out on brain tissues from 12 striped dolphins found stranded along the coasts of Italy (11 specimens) and the Canary Islands (one individual), five of which served as negative controls. While PrPc IHC yielded inconclusive results, WB analyses showed a clear-cut PrPc expression, albeit of different intensity, in the brain tissue of all the herein investigated, B. ceti-infected and neurobrucellosis-affected individuals. In this respect, the aforementioned PrPc expression patterns could be influenced by a number of intrinsic host-related factors, as well as by several extrinsic factors including simultaneously occurring neuropathies and/or coinfections by other neurotropic pathogens. Additionally, an upregulation of PrPc mRNA in the brain tissue of striped dolphins could be also hypothesized during the different stages of B. ceti infection, in a similar fashion to what is already shown in murine bone marrow cells challenged with Escherichia coli. In conclusion, much more work is needed in order to properly assess the role of PrPc, if any, as a host cell receptor for B. ceti in striped dolphins. Brucella ceti, a zoonotic pathogen of major concern to cetacean health and conservation, is responsible for severe meningo-encephalitic/myelitic lesions in striped dolphins (Stenella coeruleoalba), often leading to their stranding and death. This study investigated, for the first time, the cellular prion protein (PrPc) expression in the brain tissue from B. ceti-infected, neurobrucellosis-affected striped dolphins. Seven B. ceti-infected, neurobrucellosis-affected striped dolphins, found stranded along the Italian coastline (6) and in the Canary Islands (1), were investigated, along with five B. ceti-uninfected striped dolphins from the coast of Italy, carrying no brain lesions, which served as negative controls. Western Blot (WB) and immunohistochemistry (IHC) with an anti-PrP murine monoclonal antibody were carried out on the brain parenchyma of these dolphins. While PrPc IHC yielded inconclusive results, a clear-cut PrPc expression of different intensity was found by means of WB analyses in the brain tissue of all the seven herein investigated, B. ceti-infected and neurobrucellosis-affected cetacean specimens, with two dolphins stranded along the Italian coastline and one dolphin beached in Canary Islands also exhibiting a statistically significant increase in cerebral PrPc expression as compared to the five Brucella spp.-negative control specimens. The significantly increased PrPc expression found in three out of seven B. ceti-infected, neurobrucellosis-affected striped dolphins does not allow us to draw any firm conclusion(s) about the putative role of PrPc as a host cell receptor for B. ceti. Should this be the case, an upregulation of PrPc mRNA in the brain tissue of neurobrucellosis-affected striped dolphins could be hypothesized during the different stages of B. ceti infection, as previously shown in murine bone marrow cells challenged with Escherichia coli. Noteworthy, the inflammatory infiltrates seen in the brain and in the cervico-thoracic spinal cord segments from the herein investigated, B. ceti-infected and neurobrucellosis-affected striped dolphins were densely populated by macrophage/histiocyte cells, often harboring Brucella spp. antigen in their cytoplasm, similarly to what was reported in macrophages from mice experimentally challenged with B. abortus. Notwithstanding the above, much more work is needed in order to properly assess the role of PrPc, if any, as a host cell receptor for B. ceti in striped dolphins.

Generation and characterization of monoclonal antibodies against the hemagglutinin of H3N2 influenza A viruses

Seasonal influenza viruses are highly contagious, leading to 290,000–650,000 mortalities every year globally. Among the influenza viruses, influenza A virus (H3N2) has attracted much attention due to its high frequency of antigenic variations, resulting in poor protection by vaccination. We generated a panel of murine neutralizing monoclonal antibodies (mAbs) against A/Texas/50/2012 (H3N2) and identified the relevant epitopes that potentially influence the antigenicity by selecting mAb-resistant mutants. The epitopes were mainly in antigenic site A (1/9, 11.1%), B (6/9, 66.7%), and C (1/9, 11.1%), which is consistent with recent reports on the immunodominance of antigenic site B. The amino acid substitutions at positions 156, 157, 159, 160, and 189 at antigenic site B resulted in decreased mAb capability for blocking receptor binding. In addition, the neutralizing spectra of three mAbs (1F8, 1G9 and 1H5) were different, suggesting that their epitopes may be different but partially overlapping, and it required further study. Further, the mAb 3F9 selected a new substitution, D53G/N, at antigenic site C and showed in vitro neutralizing activity against A/Victoria/361/2011 (H3N2), A/Texas/50/2012 (H3N2), and A/Hong Kong/2671/2019 (H3N2), suggesting a potential epitope on H3 hemagglutinin for inducing broad neutralizing antibody responses. Continuous research and regular monitoring of novel epitopes are of great importance for improving vaccine strain selection.

A Reciprocal Cross-Reactivity between Monoclonal Antibodies to SARS-CoV-2 Spike Glycoprotein S1 and Human CXCR2—An Implication of a Viral Mimic of Human CXCR2

Some viruses contain mimics of host chemokine receptors that influence host immunity; however, such viral mimics have not yet been reported for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, I focused on C-X-C motif chemokine receptor 2 (CXCR2) as a candidate chemokine receptor exploited by SARS-CoV-2. Similarities between the extracellular domain (ECD) of human CXCR2 and the SARS-CoV-2 spike glycoprotein S1 (CoV2S1) were investigated. Flow cytometric analysis of healthy donor-derived peripheral leukocytes was performed to examine the cross-reactivity between specific monoclonal antibodies against these two proteins. The results showed that CR3022, a monoclonal antibody to the receptor binding domain of CoV2S1, recognized the CXCR2 ECD, and a murine monoclonal antibody to human CXCR2 recognized recombinant CoV2S1. This reciprocal cross-reactivity suggests that CoV2S1 harbors a mimic of the CXCR2 ECD.

Antibodies to Cartilage Oligomeric Matrix Protein Are Pathogenic in Mice and May Be Clinically Relevant in Rheumatoid Arthritis

ObjectiveCartilage oligomeric matrix protein (COMP) is an autoantigen in rheumatoid arthritis (RA) and experimental models of arthritis. This study was undertaken to investigate the structure, function, and relevance of anti‐COMP antibodies.MethodsWe investigated the pathogenicity of monoclonal anti‐COMP antibodies in mice using passive transfer experiments, and we explored the interaction of anti‐COMP antibodies with cartilage using immunohistochemical staining. The interaction of the monoclonal antibody 15A11 in complex with its specific COMP epitope P6 was determined by x‐ray crystallography. An enzyme‐linked immunosorbent assay and a surface plasma resonance technique were used to study the modulation of calcium ion binding to 15A11. The clinical relevance and value of serum IgG specific to the COMP P6 epitope and its citrullinated variants were evaluated in a large Swedish cohort of RA patients.ResultsThe murine monoclonal anti‐COMP antibody 15A11 induced arthritis in naive mice. The crystal structure of the 15A11–P6 complex explained how the antibody could bind to COMP, which can be modulated by calcium ions. Moreover, serum IgG specific to the COMP P6 peptide and its citrullinated variants was detectable at significantly higher levels in RA patients compared to healthy controls and correlated with a higher disease activity score.ConclusionOur findings provide the structural basis for binding a pathogenic anti‐COMP antibody to cartilage. The recognized epitope can be citrullinated, and levels of antibodies to this epitope are elevated in RA patients and correlate with higher disease activity, implicating a pathogenic role of anti‐COMP antibodies in a subset of RA patients.