Hepatitis B virus reactivation in patients treated with monoclonal antibodies

Hepatitis B virus(HBV)reactivation poses a significant clinical challenge,espe-cially in patients undergoing immunosuppressive therapies,including mono-clonal antibody treatments.This manuscript briefly explores the complex rela-tionship between monoclonal antibody therapy and HBV reactivation,drawing upon current literature and clinical case studies.It delves into the mechanisms underlying this phenomenon,highlighting the importance of risk assessment,monitoring,and prophylactic measures for patients at risk.The manuscript aims to enhance the understanding of HBV reactivation in the context of monoclonal antibody therapy,ultimately facilitating informed clinical decision-making and improved patient care.This paper will also briefly review the definition of HBV activation,assess the risks of reactivation,especially in patients treated with monoclonal antibodies,and consider management for patients with regard to screening,prophylaxis,and treatment.A better understanding of patients at risk can help clinicians provide optimum management to ensure successful patient outcomes and prevent morbidity.

Recent progress in the molecular imaging of therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies have become one of the central components of the healthcare system and continuous efforts are made to bring innovative antibody therapeutics to patients in need.It is equally critical to acquire sufficient knowledge of their molecular structure and biological functions to ensure the efficacy and safety by incorporating new detection approaches since new challenges like individual differences and resistance are presented.Conventional techniques for determining antibody disposition including plasma drug concentration measurements using LC-MS or ELISA,and tissue distribution using immunohistochemistry and immunofluorescence are now complemented with molecular imaging modalities like positron emission tomography and near-infrared fluorescence imaging to obtain more dynamic information,while methods for characterization of antibody’s interaction with the target antigen as well as visualization of its cellular and intercellular behavior are still under development.Recent progress in detecting therapeutic antibodies,in particular,the development of methods suitable for illustrating the molecular dynamics,is described here.

Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies

With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.

Production of monoclonal antibodies against AFLM(Ver-1),a middle key protein involved in aflatoxin biosynthesis

Aflatoxins are potent carcinogens,mutagens and teratogens,and are harmful to both humans and animals.As many as 30 genes are involved in aflatoxin biosynthesis.Among them,aflM(ver-1)gene was predicted to encode a 28-kDa NADPH-dependent ketoreductase(AFLM),which catalyzed middle enzymatic steps in aflatoxin biosynthetic pathway.AFLM(Ver-1)was proved to be necessary for conversion of versicolorin A(VERA)to demethylsterigmatocystin(DMST)in aflatoxin B1(AFB1)biosynthesis.For these reasons,aflM gene was cloned and specific monoclonal antibodies for AFLM was developed to better define potential pathways of AFLM involved in AFB1 biosynthesis.Monoclonal antibodies 11B2-1D7 and 3G5-4E7 were successfully screened out by immunizing mouse.Immunoblot analysis revealed that both had high sensitivity and specificity to identify native AFLM protein in A.flavus with detection limit of 11 ng/mL and 8 ng/mL respectively.These results showed that it was suitable for quantitative detection of AFLM in A.flavus isolate.Further investigation revealed that aflatoxin accumulations of various A.flavus were not dependent on AFLM biosynthesis.Overall,this is the first report for development for AFLM monoclonal antibody development and application in A.flavus quantitative detection.

Monoclonal Antibodies Against the Whitefly-Transmitted Tomato Yellow Leaf Curl Virus and Their Application in Virus Detection

Tomato yellow leaf curl virus(TYLCV)is a species of the family Geminiviridae,causing serious yield losses in tomato production.The coat protein(CP)gene of TYLCV isolate SH2 was expressed in Escherichia coli BL21(DE3)using pET-32a as the expression vector.The recombinant protein was purified through Ni+-NTA affinity column and used to immunize BALB/c mice.Three hybridoma cell lines(2B2,2E3 and 3E10)secreting monoclonal antibodies(MAbs)against TYLCV CP were obtained by fusing mouse myeloma cells(Sp 2/0)with spleen cells from the immunized BALB/c mouse.The titers of ascitic fluids of three MAbs ranged from 10-6 to 10-7 in indirect-ELISA.Isotypes and subclasses of all the MAbs belonged to IgG1,κ light chain.Triple antibody sandwich enzyme-linked immunosorbent assay(TAS-ELISA)showed that the MAb 3E10 could react with five begomoviruses infecting tomato,while the other two(2B2 and 2E3)mainly reacted with TYLCV.TAS-ELISA was set up using the MAb 3E10,and the established method could successfully detect virus in plant sap at 1:2 560(w/v,g mL-1).Detection of field samples showed that begomoviruses were common in tomato crops in Zhejiang Province,China.

Generation and application of two monoclonal antibodies targeting conserved linear epitopes in the NP protein of influenza A virus

Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

Availability and Affordability of Therapeutic Monoclonal Antibodies After the New Medical Reform in Hubei Province,China

Objective In 2017,China launched a new round of medical reform(NMR)to address the inaccessibility of high-priced drugs for patients with serious diseases.This study explored the impact of the NMR on the accessibility and affordability of high-priced monoclonal antibodies(mAbs),and the effective promotion policies after the NMR.Methods We used a standard method developed by the World Health Organization to conduct two surveys on the availability of mAbs and their prices before and after the NMR in the public hospitals in Hubei province,China.By interviewing hospital pharmacy experts,we identified the potential value of the current NMR in improving the access to therapeutic mAbs.Results The average availability of 13 mAbs increased by 8.1%in the surveyed hospitals of Hubei province after the NMR.The median unit price of 10 mAbs dropped by 34.3%.The average affordability of a treatment cycle of 10 mAbs dropped from 680 days to 298 days of the disposable daily income for a middle-income resident(56.2%reduction).The drug price negotiation of medical insurance inclusion and the promotion of consistent evaluation of generic and original drugs could effectively promote the accessibility of mAbs.However,the zero markup of drug pricing and the limit on the proportion of drug revenues in public hospitals showed certain negative effects on the availability of mAbs.Conclusion Not all current NMR policies play a positive role in promoting the accessibility of mAbs.To further improve the accessibility of mAbs in the future in China,it is therefore critical to increase the investment in independent research and development of high-quality mAbs,establish localized guidelines for the rational use of mAbs in clinical practice,and have a cost-sharing mechanism for high-priced drugs with multiple stakeholders.

Generation and Characterization of Monoclonal Antibodies Against Red-Spotted Grouper Nervous Necrosis Virus(RGNNV)

Nervous necrosis virus(NNV)can infect more than 120 fish species worldwide and has caused high mortality and sig-nificant economic losses to the aquaculture industry.Among different genotypes of NNV,the red-grouper nervous necrosis virus(RGNNV)is the most widely distributed one with the highest number of susceptible fish species.In this study,the capsid protein(Cp)gene of RGNNV was recombined and expressed in Escherichia coli strain BL21(DE3)and the recombinant Cp(rCp)was used as an immunogen to produce monoclonal antibodies(MAbs)through hybridoma cell fusion technology.Three MAbs were produced and characterized by indirect enzyme-linked immunosorbent assay(ELISA),western blotting,and immunofluorescence assay(IFA).Wes-tern blotting result showed that the MAbs could specifically react with the capsid protein of RGNNV.The result of IFA showed that the MAbs could recognize virions in RGNNV-infected grouper spleen(GS)cells.These results indicate that the MAbs can specifi-cally recognize RGNNV virions and can be used to produce a rapid detection method.This study provides a foundation for further studies on the rapid diagnosis of RGNNV and its infection mechanisms.

^(131)I-labelled anti-hepatoma monoclonal antibodies in targeting therapy of hepatoma xenografts in nude mice

Anti-hepatoma monoclonal antibodies HAb8,HAb18 IgG and HAb18 F（ab’）2were labelled with[131I]according to chloramine T method.The labelling rate andradioactivity were 51% and 9.58×104Bq/mg,respectively.After administration of the[131I]-labelled conjugates through mouse tail vein,hepatoma xenografts were clearly im-aged,Localization index was 2.15,3.09,and 6.99 for HAb8,HAb18 IgG and HAb18F（ab’）2,respectively.A total of 16 hepatoma-bearing nude mice（SMMU-LTNM）were di-vided into 4 groups.When xenografts reached 0.3cm in diameter,the treatment was be-gun by injection of the[131I]-conjugates through mouse tail vein.After 2 weeks of treat-ment,obvious inhibitory or killing effects on the xenografts were found.The tu-mor-bearing mice were killed during the period from 14 to 21d after treatment.Xenografts as well as other organs were sampled and viewed under light microscope ac-cording to routine procedure.The vital organs,such as heart,liver,spleen,lung,kidneyand brain showed no pathological lesions.In hepatoma xenografts,some tumor cellmembranes were found,with obscure cytoplasmic structure and increased eosinophilia,representing necrotic alterations.

Monoclonal Antibodies: An Emerging Class of Therapeutics in Non Small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related deaths in industrialized countries and non small cell lung cancer (NSCLC) accounts for 85% of all lung cancers. Cisplatin doublet based chemotherapy, which is the recommended regimen in first line therapy in advanced or metastatic NSCLC, improves survival but in low proportion. Monoclonal antibodies (mAbs) are a novel promising therapeutic class used with great results in inflammatory diseases such as rheumatoid arthritis. Antibodies are natural proteins with modular structure, specific pharmacodynamics and pharmacokinetics and possibly produced against any antigens, thus giving them several advantages over small drug therapeutics. In solid tumors, therapeutic mAbs improved progression free survival (PFS) and overall survival (OS) of patients with breast and colon cancers and had considerably changed the treatment in clinical practice. In NSCLC, bevacizumab, an anti-VEGF mAb, and cetuximab, an anti-EGFR mAb, are the most studied antibodies. Bevacizumab acts on angiognenesis and improved PFS of non squamous NSCLC but in low proportion as shown in two large phase III trials. It was approved by European Medicines Agency (EMEA) and Food and Drug administration (FDA) as a first line therapy in combination with cisplatin doublet chemotherapy. Cetuximab slightly enhanced OS but did not improve PFS in two large phase III trials. These results added to high adverse effect lead to cetuximab refusal by EMEA and FDA in NSCLC. At first glance, the results of mAbs in NSCLC are somewhat disappointing, in contrast to the benefits obtained with mAb treatments in other solid tumors. However, many other mAbs directed against novel targets, such as IGF1-R or CTLA-4, and new mAbs targeting VEGFR and EGFR pathways with different pharmacodymamical and pharmacokinetic properties are under evaluation and may change our vision of taking care of patients with NSCLC. In conclusion, it seems that mAbs therapy in NSCLC clearly marks the start of a new era in NSCLC treatment, with promises in improving patient survival and quality of life.

Production and characterization of monoclonal antibodies against grass carp CD4-1 and CD4-2

CD4 T helper cells are an important group of cells in the immune system of vertebrates and express CD4 receptor on the cell surface.In mammals,the CD4 receptor is encoded by a single copy gene,whilst in fish,two copies of cd4 genes,namely cd4-1 and cd4-2,are found.In this study,the ectodomains of grass carp(Ctenopharyngodon idella,Ci)CD4-1 and CD4-2 were expressed in the E.coli cells and used to generate monoclonal antibodies in mice.Western blotting,confocal microscopy and flow cytometry were performed to characterize the monoclonal antibodies.It has been shown that the CiCD4-1 and CiCD4-2 monoclonal antibodies had good specificity to react with the recombinant ectodomains of CiCD4-1 and CiCD4-2 expressed in the CHO-S cells and the native CD4 molecules of grass carp.The CiCD4-1 monoclonal antibody did not recognize CiCD4-2 and verse versa.In addition,the CD4-1 and CD4-2 monoclonal antibodies specifically recognized the CD4-1 and CD4-2 receptors expressed in the HEK293 cells and native molecules of fish cells.Further,the percentages of lymphocytes in immune tissues of healthy fish were analyzed by flow cytometry.It was found that 17.6%of lymphocytes were CD4-1^(+)cells and 22.5%were CD4-2^(+)in the head kidney.In the spleen,13.1%of lymphocytes were CD4-1^(+)and 18.6%were CD4-2^(+)while 7.3%of blood lymphocytes were CD4-1^(+)cells and 8.8%were CD4-2^(+)cells.The availability of the CD4-1 and CD4-2 monoclonal antibodies provides antibody-based tools for further elucidation of the functions of CD4 T cells in grass carp.

Hybridoma-derived neutralizing monoclonal antibodies against Beta and Delta variants of SARS-CoV-2 in vivo

Neutralizing monoclonal antibodies(mAb)are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)infection.The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs.In this study,we immunized mice with the receptor-binding domain(RBD)of the SARS-CoV-2 prototypic strain(WIV04)and screened 35 RBDspecific mAbs using hybridoma technology.Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection.The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results.A representative mAb was selected from each category(RD4,RD10,and RD14)to determine the binding kinetics and median inhibitory concentration(IC_(50))of WIV04 and two variants of concern(VOC):B.1.351(Beta)and B.1.617.2(Delta).RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 ng/mL;however,it completely lost neutralizing activity against the B.1.351 variant.RD10 neutralized both variants with an IC50 exceeding 100 ng/mL;whereas RD14 neutralized two variants with a higher IC50(>1 mg/mL).Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections.RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 mg/kg,while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 mg/kg.These results highlight the potential for future modifications of the mAbs for practical use.

Monoclonal antibodies-a proven and rapidly expanding therapeutic modality for human diseases

The study of antibodies has been a focal point in modern biology and medicine since the early 1900s.However,progress in therapeutic antibody development was slow and intermittent until recently.The first antibody therapy,murine-derived murononab OKT3 for acute organ rejection,was approved by the US Food and Drug Administration(FDA)in 1986,more than a decade after César Milstein and Georges Köhler developed methods for the isolation of mouse monoclonal antibodies from hybridoma cells in 1975.As a result of the scientific,technological,and clinical breakthroughs in the 1980s and 1990s,the pace of therapeutic antibody discovery and development accelerated.Antibodies are becoming a major drug modality with more than two dozen therapeutic antibodies in the clinic and hundreds more in development.Despite the progress,need for improvement exists at every level.Antibody therapeutics provides fertile ground for protein scientists to fulfill the dream of personalized medicine through basic scientific discovery and technological innovation.

Both structure and function of human monoclonal antibodies contribute to enhancement of Zika virus infectivity in vitro

Dear Editor,Antibody-dependent enhancement(ADE)has long been recognized for dengue virus(DENV)in vitro and in vivo.It is now clear that antibodies to DENV can also enhance Zika virus(ZIKV)infection in vitro,and vice versa(Dejnirattisai et al.,2016;Stettler et al.,2016).The characteristics of enhancing antibodies,however,remain elusive.Some have suggested that non-neutralizing antibodies or antibodies recognizing specific antigenic sites are

Pharmacogenetics of anticancer monoclonal antibodies

Pharmacogenetics is the study of therapeutic and adverse responses to drugs based on an individual’s genetic background.Monoclonal antibodies(mAbs)are a rapidly evolving field in cancer therapy,however a number of newly developed and highly effective mAbs(e.g.,anti-CTLA-4 and anti-PD-1)possess pharmacogenomic profiles that remain largely undefined.Since the first chemotherapeutic mAb Rituximab was approved in 1997 by the US Food and Drug Administration for cancer treatment,a broad number of other mAbs have been successfully developed and implemented into oncological practice.Nowadays,mAbs are considered as one of the most promising new approaches for cancer treatment.The efficacy of mAb treatment can however be significantly affected by genetic background,where genes responsible for antibody presentation and metabolism,for example,can seriously affect patient outcome.This review will focus on current anticancer mAb treatments,patient genetics that shape their efficacy,and the molecular pathways that bridge the two.

Monoclonal antibodies to the exon 18 encoded moiety of NCAM

Aim: Exon 18 expression of NCAM has been recognized as a biomarker for small cell lung cancer (SCLC). To use this finding for an improved diagnosis of SCLC and personalized treatment of patients, techniques to identify and quantitate E18, the exon 18 encoded protein moiety of NCAM, are needed. We developed three monoclonal antibodies for this purpose. Methods: The his-tagged E18 antigen was expressed in E. coli and, after purification, used to immunize mice. Hybridoma's were isolated by standard procedures and tested for their reaction with E18. Results: Three monoclonal antibodies, MUM-1, MUM-4 and MUM-6 were obtained. They reacted with E18 in western blots, with SCLC cell line NCI-H82, but not with unrelated his-tagged proteins. Only permeabilized NCI-H82 cells stained with the antibodies, confirming the intracellular position of E18. Next an enzyme-linked immunosorbent assay was developed using the earlier isolated monoclonal antibody MUMi-21B2, coated on the surface of microtiter wells as capture antibody and biotinylated MUM-6 as second antibody. Using streptavidin conjugated to horse radish peroxidase a linear dose response curve to his-tagged E18 antigen was obtained between 0 and 5 μg/mL with a sensitivity of at least 0.5 μg/mL or 50 ng/well. Conclusion: Four monoclonal antibodies are available to be used in assays for the identification and quantification of SCLC biomarker E18. This will enable the development of liquid biopsies to follow the tumor load in patients.

Efficacy and safety of monoclonal antibodies in neuromyelitis optica spectrum disorders:A survival meta-analysis of randomized controlled trials

Background Monoclonal antibodies such as rituximab(RTX),eculizumab,inebilizumab,satralizumab,and tocilizumab have been found to be effective therapies for neuromyelitis optica spectrum disease(NMOSD)in several clinical randomized controlled trials.Objective The purpose of this meta-analysis of randomized controlled trials was to assess the efficacy and safety of monoclonal antibodies in the treatment of NMOSD.Methods We searched the following databases for relevant English language literature from the establishment of the database to June 2021:PubMed,Embase,Cohorane Library,the Central Register of Controlled Trials(CENTRAL),and Web of Science.Randomized controlled trials of monoclonal antibodies were the targets of the review.Results We included seven trials containing 775 patients(485 in the monoclonal antibody group and 290 in the control group).Patients in the monoclonal group(HR 0.24,95%CI:0.14 to 0.40,P<0.00001),as well as patients with seropositive AQP4-IgG(HR 0.18,95%CI:0.11 to 0.29,P<0.00001),both had a higher free recurrence rate than that in the control group.In the first year(HR 0.25,95%CI:0.09 to 0.71,P=0.009)and the second year(HR 0.32,95%CI:0.13 to 0.81,P=0.02),no relapses were documented.The average changes of the expanded disability status scale(EDSS)score decreased by 0.29(95%CI:−0.09 to 0.51,P=0.005).Upper respiratory tract infection(OR 1.52,95%CI:0.76 to 3.04,P=0.24),urinary tract infection(OR 0.79,95%CI:0.51 to 1.21,P=0.27),and headache(OR 1.30,95%CI:0.78 to 2.17,P=0.31)were three most frequent adverse reactions.Conclusions Monoclonal antibodies are particularly effective treatments in avoiding recurrence for NMOSD patients,according to this meta-analysis.The associated adverse responses are not significantly different from those seen with traditional immunosuppressants.

Treatment of COVID-19 by monoclonal antibodies and the traditional Chinese medicine

The mortality rate of the recent global pandemic corona virus disease 2019(COVID-19)is currently as high as 7%.The SARS-CoV-2 virus is the culprit behind COVID-19.SARS-CoV-2 is an enveloped single-stranded RNA virus,the genome encodes four types of the structural proteins:S protein,E protein(envelope protein),M protein(matrix protein)and N protein(nucleocapsid protein).In COVID-19,monoclonal antibodies have played a significant role in diagnosis and treatment.This article briefly introduced the development of monoclonal antibodies targeting on S protein and N protein,which represents the main direction of monoclonal antibody drugs used in the diagnosis and treatment of COVID-19.Meanwhile,the traditional Chinese medicine also plays important role in the fight against COVID-19 by regulating human immunity.The article introduced the use of traditional Chinese medicine in fighting against COVID-19.

Preparation and application of a novel monoclonal antibody specific for the heat shock protein 60 of Lawsonia intracellularis

Porcine proliferative enteropathy(PPE),an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis,is commonly associated with diarrhea and reduced weight gain in growing pigs widespread.An accurate method for detecting L.intracellularis is particularly important for preventing and controlling PPE.Heat shock protein 60(Hsp60)is an immunodominant bacterial antigen found in all eukaryotic and prokaryotic organisms.Thus,the purpose of the current investigation was to produce a novel L.intracellularis Hsp60 monoclonal antibody(mAb)useful for immunodiagnostics.Three hybridomas secreted anti-Hsp60 termed 3E5,4E2,and 9G6 were generated,and the titers of ascitic fluids of 3E5,4E2,9G6 were 1:1024000,1:2048000 and 1:2048000,respectively.The Western blotting analysis demonstrated that recombinant Hsp60(rHsp60)was recognized by mAbs 3E5,4E2 and 9G6.Subsequently,analyses of specificity showed all the mAbs were highly specific to L.intracellularis while could not significantly react with other enteric bacteria commonly found in the ileum of pigs,such as Escherichia coli,Salmonella Choleraesuis,Salmonella Typhimurium,and Brachyspira hyodysenteriae.Furthermore,the mAbs were useful for detecting L.intracellularis in the infected monolayer cells and histological sections of the ileum from PPE-affected pigs.Our research will provide a foundation for the development of immunological diagnostic tests.