Recombinant Humanized Antibody Research Articles

Successful treatment of pulmonary hypertension and right heart failure by antibody-based functional blocking of the extra-domain A of fibronectin in a preclinical animal model

Abstract Background Pulmonary Hypertension (PH) as heterogeneous disease group is pathogenetically accompanied by pulmonary vascular and right ventricular tissue remodelling showing similarities between the clinical groups 1 (Pulmonary Arterial Hypertension), 2 (PH associated with left heart disease) and 3 (PH associated with lung disease). Since the remodelling process entails the abundant re-expression of extra-domain A containing fibronectin (ED-A+ Fn), which is virtually absent in healthy adult organs, a potential functional role in PH development and progression has been suggested and recently proven by our group. The human recombinant antibody F8 (IgG) specifically recognises ED-A+ Fn and is capable to block its function at least in vitro. Thus, F8 might serve as a novel therapeutic agent for the treatment or even prevention of PH. Purpose Against that background, the current study was aimed to test the therapeutic efficacy of F8 after systemic administration in a well-established preclinical model of PH in comparison to a non-specific control antibody and a dual endothelin receptor antagonist as standard of care. Methods Induction of PH was realized by subcutaneous injection of Monocrotaline (MCT) in mice. In total, 41 animals were included in the study approach and were divided into the following experimental groups: sham-treated controls (controls; n=7), PH mice without a specific treatment (PH_untreated; n=12), PH mice treated with the dual endothelin receptor antagonist Macitentan (PH_MAC; n=6), PH mice treated with the F8 antibody (PH_F8; n=8) and PH mice treated with an antibody of irrelevant antigen specificity in the murine system (PH_KSF; n=8). Treatment effects were evaluated on the haemodynamic, echocardiographic and microscopic level. Results In comparison to controls, untreated PH mice showed significantly higher right ventricular systolic pressure values (RVPsys, p<0.05) as well as impaired echocardiographic parameters of right ventricular morphology and function, i.e., enlarged basal right ventricular (RVbasal, p=0.016) diameters and reduced tricuspid annular plane systolic excursion values (TAPSE, p=0.008). In the PH_F8 group, haemodynamic and echocardiographic parameters were significantly improved compared to the PH_untreated or the PH_KSF group (p<0.05 for the majority of parameters). Histological evaluation revealed significant lung tissue damage after in the PH_untreated group compared to controls (p=0.008), which were significantly attenuated in the PH_F8 (p=0.04) but not the PH_KSF group (p=n.s.). Conclusion The therapeutic efficacy of the human recombinant F8 antibody specific to ED-A+ Fn has been proven for the first time in vivo in a well-established preclinical model of PH. F8 acts as a disease-modifier by attenuating PH associated lung tissue remodelling resulting in remarkable improvements of pulmonary hemodynamics and echocardiographic parameters of right ventricular load.

Impaired functional connectivity of the hippocampus in translational murine models of NMDA-receptor antibody associated neuropsychiatric pathology

Decreased hippocampal connectivity and disruption of functional networks are established resting-state functional MRI (rs-fMRI) features that are associated with neuropsychiatric symptom severity in human anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. However, the underlying pathophysiology of NMDAR encephalitis remains poorly understood. Application of patient-derived monoclonal antibodies against the NR1 (GluN1) subunit of the NMDAR now allows for the translational investigation of functional connectivity in experimental murine NMDAR antibody disease models with neurodevelopmental disorders. Using rs-fMRI, we studied functional connectivity alterations in (1) adult C57BL/6 J mice that were intrathecally injected with a recombinant human NR1 antibody over 14 days (n = 10) and in (2) a newly established mouse model with in utero exposure to a human recombinant NR1 antibody (NR1-offspring) at the age of (2a) 8 weeks (n = 15) and (2b) 10 months (n = 14). Adult NR1-antibody injected mice showed impaired functional connectivity within the left hippocampus compared to controls, resembling impaired connectivity patterns observed in human NMDAR encephalitis patients. Similarly, NR1-offspring showed significantly reduced functional connectivity in the hippocampus after 8 weeks, and impaired connectivity in the hippocampus was likewise observed in NR1-offspring at the age of 10 months. We successfully reproduced functional connectivity changes within the hippocampus in different experimental murine systems that were previously observed in human NMDAR encephalitis patients. Translational application of this method within a combined imaging and histopathological framework will allow future experimental studies to identify the underlying biological mechanisms and may eventually facilitate non-invasive monitoring of disease activity and treatment responses in autoimmune encephalitis.

A novel bispecific antibody dual-targeting approach for enhanced neutralization against fast-evolving SARS-CoV-2 variants.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has caused unprecedented health and socioeconomic crises, necessitating the immediate development of highly effective neutralizing antibodies. Despite recent advancements in anti-SARS-CoV-2 receptor-binding domain (RBD)-specific monoclonal antibodies (mAbs) derived from convalescent patient samples, their efficacy against emerging variants has been limited. In this study, we present a novel dual-targeting strategy using bispecific antibodies (bsAbs) that specifically recognize both the SARS-CoV-2 RBD and fusion peptide (FP), crucial domains for viral attachment to the host cell membrane and fusion in SARS-CoV-2 infection. Using phage display technology, we rapidly isolated FP-specific mAbs from an established human recombinant antibody library, identifying K107.1 with a nanomolar affinity for SARS-CoV-2 FP. Furthermore, we generated K203.A, a new bsAb built in immunoglobulin G4-(single-chain variable fragment)2 forms and demonstrating a high manufacturing yield and nanomolar affinity to both the RBD and FP, by fusing K102.1, our previously reported RBD-specific mAb, with K107.1. Our comprehensive in vitro functional analyses revealed that the K203.A bsAb significantly outperformed the parental RBD-specific mAb in terms of neutralization efficacy against SARS-CoV-2 variants. Furthermore, intravenous monotherapy with K203.A demonstrated potent in vivo neutralizing activity without significant in vivo toxicity in a mouse model infected with a SARS-CoV-2 variant. These findings present a novel bsAb dual-targeting strategy, directed at SARS-CoV-2 RBD and FP, as an effective approach for rapid development and management against continuously evolving SARS-CoV-2 variants.

Neuronal binding by antibodies can be influenced by low pH stress during the isolation procedure

Low pH stress and its influence on antibody binding is a common consideration among chemists, but is only recently emerging as a consideration in Immunological studies. Antibody characterizations in Multiple Sclerosis (MS), an autoimmune disease of the Central Nervous System (CNS) has revealed that antibodies in the cerebrospinal fluid (CSF) of patients with Multiple Sclerosis bind to myelin-related and non-myelin antigen targets. Many laboratories have used molecular biology techniques to generate recombinant human antibodies (rhAbs) expressed by individual B cells from healthy donors and patients with systemic autoimmune disease to identify antigen targets. This approach has been adapted within the Neuroimmunology research community to investigate antigen targets of individual B cells in the CSF of MS patients. Our laboratory determines which antibodies to clone based on their immunogenetics and this method enriches for cloning of rhAbs that bind to neurons. However, newer technologies to assist in purification of these rhAbs from culture supernatants use an acidic elution buffer which may enhance low pH stress on the antibody structure. Our laboratory routinely uses a basic elution buffer to purify rhAbs from culture supernatants to avoid low pH stress to the antibody structure. Our goal was to investigate whether acidic elution of our rhAbs using Next Generation Chromatography would impact the rhAbs' ability to bind neurons. The limited data presented here for two neuron-binding rhAbs tested indicated that acidic elution buffers used during rhAb purification impacted the ability of rhAbs with low CDR3 charge to maintain binding to neuronal targets. Reproducibility in a larger panel of rhAbs and factors underlying these observations remain untested.

Efficacy and safety of KN046, a novel bispecific antibody against PD-L1 and CTLA-4, in patients with non-small cell lung cancer who failed platinum-based chemotherapy: a phase II study

BackgroundThis study aimed to evaluate the efficacy and safety of KN046, a novel recombinant humanised antibody targeting PD-L1 and CTLA-4 in advanced non-small cell lung cancer (NSCLC) patients after failure or intolerance to platinum-based chemotherapy. MethodsIn this multi-centre, open-label phase II clinical trial, patients were enroled after failure or intolerance to platinum-based chemotherapy. KN046 at 3 mg/kg or 5 mg/kg was administered intravenously every 2weeks. The primary end-point was objective response rate (ORR) evaluated by a blinded independent review committee (BIRC). ResultsA total of 30 and 34 patients were included in the 3 mg/kg (cohort A) and 5 mg/kg (cohort B) cohorts. On 31st August 2021, the median follow-up duration was 24.08 months (interquartile [IQR], 22.28, 24.84) and 19.35months (IQR, 17.25, 20.90) in the 3 mg/kg and 5 mg/kg cohorts, respectively. BIRC-assessed ORRs were 13.3% and 14.7% in the 3 mg/kg and 5 mg/kg cohorts, respectively. Median progression-free survival was 3.68 (95% confidence interval [CI] 3.22–7.29) and 3.68 (95%CI 1.81–7.39) months, while overall survival was 19.70 (95.5%CI 15.44-not estimated [NE]) and 13.04 (95.5%CI 9.86-NE) months, respectively. The most common treatment-related adverse events (TRAEs) were anaemia (28.1%), hyperglycaemia (26.7%), and infusion-related reactions (26.7%). The incidence rates of grade ≥ 3 TRAEs and TRAEs leading to treatment discontinuation were 42.2% and 14.1%, respectively. ConclusionsBoth 3 mg/kg and 5 mg/kg KN046 showed promising efficacy and favourable safety profile for advanced NSCLC after failure or intolerance to previous platinum-based chemotherapy. Trial registration numberNCT03838848

Phase 1 Trial of Antibody NI006 for Depletion of Cardiac Transthyretin Amyloid

BackgroundTransthyretin amyloid (ATTR) cardiomyopathy is a progressive and fatal disease caused by misfolded transthyretin. Despite advances in slowing disease progression, there is no available treatment that depletes ATTR from the heart for the amelioration of cardiac dysfunction. NI006 is a recombinant human anti-ATTR antibody that was developed for the removal of ATTR by phagocytic immune cells.MethodsIn this phase 1, double-blind trial, we randomly assigned (in a 2:1 ratio) 40 patients with wild-type or variant ATTR cardiomyopathy and chronic heart failure to receive intravenous infusions of either NI006 or placebo every 4 weeks for 4 months. Patients were sequentially enrolled in six cohorts that received ascending doses (ranging from 0.3 to 60 mg per kilogram of body weight). After four infusions, patients were enrolled in an open-label extension phase in which they received eight infusions of NI006 with stepwise increases in the dose. The safety and pharmacokinetic profiles of NI006 were assessed, and cardiac imaging studies were performed.ResultsThe use of NI006 was associated with no apparent drug-related serious adverse events. The pharmacokinetic profile of NI006 was consistent with that of an IgG antibody, and no antidrug antibodies were detected. At doses of at least 10 mg per kilogram, cardiac tracer uptake on scintigraphy and extracellular volume on cardiac magnetic resonance imaging, both of which are imaging-based surrogate markers of cardiac amyloid load, appeared to be reduced over a period of 12 months. The median N-terminal pro–B-type natriuretic peptide and troponin T levels also seemed to be reduced.ConclusionsIn this phase 1 trial of the recombinant human antibody NI006 for the treatment of patients with ATTR cardiomyopathy and heart failure, the use of NI006 was associated with no apparent drug-related serious adverse events. (Funded by Neurimmune; NI006-101 ClinicalTrials.gov number, NCT04360434.)

Synthesis of Human Antibodies Against HBsAg in Newly Established Chinese Hamster Lung (CHL-YN) Cell Line

Hepatitis B immunoglobulin (HBIG) is an effective treatment for hepatitis B, including postexposure prophylaxis of HBV infection, prevention of HBV reinfection in liver transplant patients, and reducing sexual transmission. This study investigated the synthesis of human IgG antibodies that specifically target HBsAg subtype adr in CHL-YN cells, a newly established cell line that grows faster than CHO-K1. To achieve the synthesis of human IgG antibodies, a plasmid vector encoding DNA sequences for human IgG antibodies against HBsAg was constructed and then transiently transfected into CHL-YN cells. The expression and antigen-binding capacity of the recombinant human IgG antibodies were analyzed using western blot and ELISA. The results showed successful expression and secretion of human IgG antibodies that recognize HBsAg subtype adr in CHL-YN cells. The ELISA test confirmed the specificity of the human IgG antibodies towards HBsAg subtype adr. Thus, this study concluded that human IgG antibodies that target HBsAg subtype adr were transiently expressed in CHL-YN cells.

Synthesis of Human Antibodies Against HBsAg in Newly Established Chinese Hamster Lung (CHL-YN) Cell Line

Hepatitis B immunoglobulin (HBIG) is an effective treatment for hepatitis B, including postexposure prophylaxis of HBV infection, prevention of HBV reinfection in liver transplant patients, and reducing sexual transmission. This study investigated the synthesis of human IgG antibodies that specifically target HBsAg subtype adr in CHL-YN cells, a newly established cell line that grows faster than CHO-K1. To achieve the synthesis of human IgG antibodies, a plasmid vector encoding DNA sequences for human IgG antibodies against HBsAg was constructed and then transiently transfected into CHL-YN cells. The expression and antigen-binding capacity of the recombinant human IgG antibodies were analyzed using western blot and ELISA. The results showed successful expression and secretion of human IgG antibodies that recognize HBsAg subtype adr in CHL-YN cells. The ELISA test confirmed the specificity of the human IgG antibodies towards HBsAg subtype adr. Thus, this study concluded that human IgG antibodies that target HBsAg subtype adr were transiently expressed in CHL-YN cells.

Generation of Rat Monoclonal Antibodies Against Human Epidermal Growth Factor Receptor 2

Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor without any known ligands and a member of the epidermal growth factor receptor (EGFR) family. It is a proto-oncogenic protein that, through signaling cascades, promotes cell proliferation and inhibits apoptosis in cancer cells through homo- and heterodimerization with other EGFR family receptors. Since several cancers, including breast cancer, overexpress HER2, it is a target of tumor therapy. Both trastuzumab and pertuzumab are recombinant humanized monoclonal antibodies (mAbs) used in clinical trials that target the extracellular domain (ECD) of HER2. Therefore, it is important to generate antibodies against various ECDs of HER2. In this study, we describe rat mAbs, which were generated against the ECD of human HER2. The human breast cancer cell line SK-BR-3 was subjected to immunofluorescence staining as it expresses HER2, and mAbs can detect both intact and endogenous HER2 within the cell line.

Cross-reactivity of human monoclonal antibodies with canine peripheral blood mononuclear cells

In drug development, the dog is often used as a model for non-rodent preclinical safety studies. In particular, immunophenotyping in dogs can be important to characterize the toxicological profile of a test item. A wide range of antibodies specific to surface antigens is needed, however, commercially available antibodies to dog are scarce. To date, numerous studies have reported the cross-reactivity of human monoclonal antibodies with canine peripheral blood mononuclear cells (PBMC). In this study, we aimed to increase the number of canine-specific antibodies and took a rather novel approach to further determine cross-reactivity of 378 human recombinant antibodies lacking Fc regions to surface antigens on canine PBMC. The screening resulted in 30 human monoclonal antibodies well reactive to canine PBMC. Sequence homology of the targeted human and canine antigens was analyzed with Basic Local Alignment Search Tool. Thirteen human cross-reactive antibodies of interest were analyzed with cells from canine whole blood in combination with lineage markers. Finally, ten antibodies were identified as useful markers for the application in dog. Except for CD27, the remaining nine antibodies are already commercially available human cross-reactive antibodies. This study provides a new source for all ten antibodies described here.

Novel bispecific human antibody platform specifically targeting a fully open spike conformation potently neutralizes multiple SARS-CoV-2 variants

Rapid emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted an urgent need for the development of broadly applicable and potently neutralizing antibody platform against the SARS-CoV-2, which can be used for combatting the coronavirus disease 2019 (COVID-19). In this study, based on a noncompeting pair of phage display-derived human monoclonal antibodies (mAbs) specific to the receptor-binding domain (RBD) of SARS-CoV-2 isolated from human synthetic antibody library, we generated K202.B, a novel engineered bispecific antibody with an immunoglobulin G4-single-chain variable fragment design, with sub- or low nanomolar antigen-binding avidity. Compared with the parental mAbs or mAb cocktail, the K202.B antibody showed superior neutralizing potential against a variety of SARS-CoV-2 variants in vitro. Furthermore, structural analysis of bispecific antibody-antigen complexes using cryo-electron microscopy revealed the mode of action of K202.B complexed with a fully open three-RBD-up conformation of SARS-CoV-2 trimeric spike proteins by simultaneously interconnecting two independent epitopes of the SARS-CoV-2 RBD via inter-protomer interactions. Intravenous monotherapy using K202.B exhibited potent neutralizing activity in SARS-CoV-2 wild-type- and B.1.617.2 variant-infected mouse models, without significant toxicity in vivo. The results indicate that this novel approach of development of immunoglobulin G4-based bispecific antibody from an established human recombinant antibody library is likely to be an effective strategy for the rapid development of bispecific antibodies, and timely management against fast-evolving SARS-CoV-2 variants.

Optimization of recombinant antibody production based on the vector design and the level of metabolites for generation of Ig- producing stable cell lines

BackgroundThe biopharmaceutical industry is significantly growing worldwide, and the Chinese hamster ovary (CHO) cells are used as a main expression host for the production of recombinant monoclonal antibodies. Various metabolic engineering approaches have been investigated to generate cell lines with improved metabolic characteristics for increasing longevity and mAb production. A novel cell culture method based on the 2-stage selection makes it possible to develop a stable cell line with high-quality mAb production. ResultsWe have constructed several design options of mammalian expression vectors for the high production of recombinant human IgG antibodies. Versions for bipromoter and bicistronic expression plasmids different in promoter orientation and cistron arrangements were generated.The aim of the work presented here was to assess a high-throughput mAb production system that integrates the advantages of high-efficiency cloning and stable cell clones to stage strategy selection reducing the time and effort required to express therapeutic monoclonal mAbs.Development of a stable cell line using bicistronic construct with EMCV IRES-long link gave an advantage in high mAb expression and long-term stability. Two-stage selection strategies allowed the elimination of low-producer clones by using metabolic level intensity to estimate the IgG production in the early steps of selection. The practical application of the new method allows to reduce time and costs during stable cell line development.

Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain α-neurotoxins from snakes

Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, broadly-neutralizing human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and high neutralization capacity. Here, we report the establishment of a pipeline based on phage display technology for the discovery and optimization of high affinity broadly-neutralizing human monoclonal antibodies. This approach yielded a recombinant human antibody with superior broadly-neutralizing capacities in vitro and in vivo against different long-chain α-neurotoxins from elapid snakes. This antibody prevents lethality induced by Naja kaouthia whole venom at an unprecedented low molar ratio of one antibody per toxin and prolongs the survival of mice injected with Dendroaspis polylepis or Ophiophagus hannah whole venoms.

Obtaining and investigating immunochemical properties of monoclonal antibodies against rCTLA-4 protein

Monoclonal antibodies are used to block control points of the tumor development of many oncological pathologies. One of the critical control points of tumor development of several oncological pathologies is the receptor for cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Monoclonal antibodies against the CTLA-4 receptor are laboratory-derived humanized antibodies. An essential step in the humanization of antibodies is the production of murine hybrid cells producing monoclonal antibodies. This article describes studies of mice monoclonal antibodies against a recombinant human CTLA-4 receptor (rCTLA-4) expressed in Escherichia coli. To obtain strains of hybrid cells producing monoclonal antibodies were used methods of hybridoma technology. As a result, hybrid cells producing monoclonal antibodies to CTLA-4 were obtained. Strains of hybrid cells have high productive activity in vitro and in vivo. Monoclonal antibodies react with rCTLA-4 protein, belong to the class of IgG1, and have a high binding constant. They efficiently bind to the rCTLA-4 receptor and block the interaction of rCTLA-4 with the commercial recombinant human B7-1 Fc and rhesus monkey PD-1 hFc proteins. These monoclonal antibodies to rCTLA-4 can be used to obtain recombinant humanized monoclonal antibodies to the human CTLA-4 receptor.

A phase Ia dose-escalation trial of Ametumumab (a fully human monoclonal antibody against epidermal growth factor receptor) in patients with advanced solid malignancies.

Epidermal growth factor receptor (EGFR) is a well-known target for cancer treatment. However, the authorized anti-EGFR monoclonal antibodies generally cause several toxic effects, especially severe cutaneous toxicities as well as infusion reactions, and the clinical indications are limited. Here we developed Ametumumab, a fully human recombinant anti-EGFR monoclonal antibody. To assess the safety, tolerability, pharmacokinetics (PK), and immunogenicity of Ametumumab. A first-in-human phase Ia dose escalation study of Ametumumab in patients with advanced solid malignancies. An open-label, first-in-human dose escalation study was done in 22 patients with advanced malignancies who received six ascending dosages ranging from 75 to 750 mg/m2. Following a single dosage and a 28-day dose-limiting toxicity (DLT) monitoring period, patients were given repeated doses weekly. Blood samples were taken to determine the PK parameters of Ametumumab and anti-drug antibody concentrations. Every 8 weeks, radiographic tumor evaluations were conducted. In this trial, no DLT was observed, and the maximum tolerated dose was not reached at doses up to 750 mg/m2. There were no severe adverse events but mild and moderate adverse effects, such as headache, proteinuria, and rash. Single-dose PK results demonstrated a straightforward linear relationship with dosage escalation. The medication concentrations accumulated and attained steady-state after four rounds of injections. It was calculated that 10 patients with disease control would be observed in the 22 evaluable patients. The disease control rate was 45.5%. The Ametumumab was well tolerated and safe in patients with advanced solid malignancies, exhibiting minimal immunogenicity, a long half-life, high levels of drug exposure in the blood, and preliminary effectiveness. The trial was registered with CTR20170343 on 10 April 2017, The China Center for Drug Evaluation.

Construction of Human Immune and Naive scFv Phage Display Libraries.

Antibody phage display is a widely used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. In order to successfully generate antibodies using phage display, the basis is the construction of high-quality antibody gene libraries. Here, we describe detailed methods for the construction of such high-quality immune and naive scFv gene libraries of human origin. These protocols were used to develop human naive (e.g., HAL9/10) and immune libraries, which resulted in thousands of specific antibodies for all kinds of applications.

Технологии создания вируснейтрализующих антител человека на примере SARS-CoV-2

Monoclonal antibodies (mAbs) are the most promising and most intensively replenished type of bioactive pharmaceuticals. Currently, there are over 100 different mAbs approved by the FDA and other regulating agencies for treatment of oncological, infectious, systemic, autoimmune and other diseases. Design of antibodies neutralizing pathogens of socially significant infections, such as HIV, hepatitis viruses, SARS-CoV-2, is a separate direction. The SARS-CoV-2 pandemic has shown how urgent it is to have a technological platform enabling production of fully human antibodies. The development of recombinant DNA technology and antibody phage display enabled compilation of libraries of antigen-binding fragments and screening with target antigens. This review discusses the advantages and disadvantages of phage display, including use of single-domain antibody technology based on the heavy chain variable domain. We describe the state-of-the-art (and practical results of its application) technology enabling production of human antibodies by sorting and sequencing the genome of individual memory B cells, using monoclonal virus-neutralizing antibodies against SARS-CoV-2 as an example. The prospects of further development of the recombinant human antibody production technology are discussed; in particular, we consider creation of sequences of variable fragments of antibodies with the help of artificial intelligence.

Recombinant Humanized IgG1 Antibody Promotes Reverse Cholesterol Transport through FcRn-ERK1/2-PPARα Pathway in Hepatocytes

Hyperlipidemia-associated lipid disorders are considered the cause of atherosclerotic cardiovascular disease. Reverse cholesterol transport (RCT) is a mechanism by which excess peripheral cholesterol is transported to the liver and further converted into bile acid for excretion from the body in feces, which contributes to reducing hyperlipidemia as well as cardiovascular disease. We previously found that the recombinant humanized IgG1 antibody promotes macrophages to engulf lipids and increases cholesterol efflux to high-density lipoprotein (HDL) through ATP-binding cassette sub-family A1 (ABCA1), one of the key proteins related to RCT. In the present study, we explored other RCT related proteins expression on hepatocytes, including scavenger receptor class B type I (SR-BI), apolipoprotein A-I (ApoA-I), and apolipoprotein A-II (ApoA-II), and its modulation mechanism involved. We confirmed that the recombinant humanized IgG1 antibody selectively activated ERK1/2 to upregulate SR-BI, ApoA-I, and ApoA-II expression in mice liver and human hepatocellular carcinoma cell lines HepG2 cells. The rate-limiting enzymes of bile acid synthesis, including cholesterol 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1), exhibited a significant increase when treated with the recombinant humanized IgG1 antibody, as well as increased excretion of bile acids in feces. Besides, abolishment or mutation of peroxisome proliferator-activated receptor α (PPARα)/RXR binding site on SR-BI promoter eliminated SR-BI reporter gene luciferase activity even in the presence of the recombinant humanized IgG1 antibody. Knock down the neonatal Fc receptor (FcRn) on hepatocytes impaired the effect of recombinant humanized IgG1 antibody on activation of ERK1/2, as well as upregulation of SR-BI, ApoA-I, and ApoA-II expression. In conclusion, one of the mechanisms on the recombinant humanized IgG1 antibody attenuates hyperlipidemia in ApoE-/- mice model fed with high-fat-diet might be through reinforcement of liver RCT function in an FcRn-ERK1/2-PPARα dependent manner.

Trial-in-Progress: A Phase 2 Study of Mitoxantrone, Etoposide and Gemtuzumab Ozogamicin (MEGO) for Acute Myeloid Leukemia That Persisted after the 1st Induction Regimen

Background Acute myeloid leukemia (AML) is a disease characterized by a clonal proliferation of myeloid precursors with reduced capacity to differentiate into more mature cellular elements. The standard treatment for newly diagnosed AML has not changed appreciably over the last few decades and consists primarily of an anthracycline combined with a nucleoside analogue. Complete remission (CR) rates of 65-80% can be expected in younger patients but decrease with older age. There is no agreed standard of care for AML patients that do not respond to initial first line induction therapy. Although remission after second-line therapy may occur, the CR rate is lower. A few chemotherapy regimens have been used in patients with resistant disease with overall CR rates of 20-40%. Gemtuzumab ozogamicin (GO) is an antibody drug conjugate consisting of a recombinant humanized antibody to CD33 which is linked to calicheamicin. Although GO is currently approved for induction therapy in combination with "7+3", its benefit is mostly evident in favorable risk disease (Hills et al Lancet Onc 2014) and thus its use is mostly limited in this setting. Pre-clinical data suggest that GO is most effective when it saturates CD33 which happens in low-burden disease (Van der Velden et al Leukemia 2004). Thus, we decided to proceed with a trial that incorporates gemtuzumab ozogamicin on the 2nd induction regimen for patients that did not achieve complete remission with the first. Methods This is a phase II, singe-center clinical trial of GO in combination with mitoxantrone and etoposide in patients with AML that persisted after their 1st induction regimen. The patients are eligible if they have at least 10% of blasts in their day 14 bone marrow biopsy and CD33 expression in at least 30% of leukemic blasts in the bone marrow. Patients with repeat bone marrows after day 14 showing progression of AML (>5% blasts) were also eligible. Patients also need to have ECOG performance status 0-2, left ventricular ejection fraction (LVEF) >50%, GFR > 30 ml/min, AST/ALT < 2.5 x upper normal limits and total bilirubin <2 x upper normal limit. Patients that received CPX-531 for induction are excluded due to concerns for prolonged thrombocytopenia. The standard of care for our center for this patient population is 10mg/m2 of mitoxantrone on days 1-5 and 100mg/m2 of etoposide on days 1-5. Trial patients receive the same regimen and a single dose of GO 3mg/m2 (capped at 4.5mg) on day 6. The primary endpoint of the trial is complete remission rate (CR). The hypothesis is that addition of GO to mitoxantrone, and etoposide will increase our historical CR rate of 35% to 50%. The trial has a Simon's two-stage design, with an interim analysis planned after 16 evaluable patients have been enrolled. The trial plans to accrue 44 evaluable patients. This design has an alpha of 0.15 and 80% power. Secondary objectives include progression-free survival, overall survival, and treatment-related mortality. To-date we have enrolled 15 patients, 13 of them evaluable. 2 patients died from sepsis before the administration of GO. We have not recorded any episodes of VOD so far. Recruitment is continuing and this trial is registered on clinicaltrials.gov; NCT03839446.

Rabies Virus Neutralizing Activity, Safety, and Immunogenicity of Recombinant Human Rabies Antibody Compared with Human Rabies Immunoglobulin in Healthy Adults

ObjectivePreliminary assessment of rabies virus neutralizing activity, safety and immunogenicity of a recombinant human rabies antibody (NM57) compared with human rabies immunoglobulin (HRIG) in Chinese healthy adults. MethodsSubjects were randomly (1:1:1) allocated to Groups A (20 IU/kg NM57), B (40 IU/kg NM57), or C (20 IU/kg HRIG). One injection was given on the day of enrollment. Blood samples were collected on days –7 to 0 (pre-injection), 3, 7, 14, 28, and 42. Adverse events (AEs) and serious AEs (SAEs) were recorded over a period of 42 days after injection. ResultsAll 60 subjects developed detectable rabies virus neutralizing antibodies (RVNAs) (> 0.05 IU /mL) on days 3, 7, 14, 28, and 42. The RVNA levels peaked on day 3 in all three groups, with a geometric mean concentration (GMC) of 0.2139 IU/mL in Group A, 0.3660 IU/mL in Group B, and 0.1994 IU/mL in Group C. At each follow-up point, the GMC in Group B was significantly higher than that in Groups A and C. The areas under the antibody concentration curve over 0–14 days and 0–42 days in Group B were significantly larger than those in Groups A and C. Fifteen AEs were reported. Except for one grade 2 myalgia in Group C, the other 14 were all grade 1. No SAEs were observed. ConclusionThe rabies virus neutralizing activity of 40 IU/kg NM57 was superior to that of 20 IU/kg NM57 and 20 IU/kg HRIG, and the rabies virus neutralizing activity of 20 IU/kg NM57 and 20 IU/kg HRIG were similar. Safety was comparable between NM57 and HRIG.