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monoclonal antibodies that target the immunogenic proteins expressed in colorectal cancer

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World Journal of Gastrointestinal Oncology 2014年第6期6卷 170-176页

作者： Myron Arlen Philip Arlen Gene Coppa Jim Crawford XuePing Wang Olga Saric Alex Dubeykovskiy Ernesto Molmenti Department of Surgery Division of Surgical Oncology and the Dept. of Pathology North Shore University HospitalManhasset NY 11030 United States Manhasset NY and Precision Biologics Great Neck NY 11021 United States

In an attempt to improve upon the end results obtained in treating colorectal cancer it was apparent that the earlier the diagnosis that could be obtained, the better the chance for obtaining desired results. In the case of more advanced tumors typified by later stage colorectal cancer, surgical debulking is an important part of the treatment strategy. Here the use of additional therapeu-tic modalities including chemotherapy and present day immunotherapy has failed to accomplish the desired im-provements that have been sought after. Adjuvant ther-apy, has offered little to the overall survival. The concept of early detection is now recognized as the initial step in reaching proper end results and can readily be demon-strated from colorectal cancer studies. Here survival has been found to be a reflection of the stage at which the tumor is first identified and treated. When specific mono-clonals targeting colorectal cancer are employed diagnos-tically, we have been able to demonstrate detection of colorectal cancer at its inception as a premalignant lesion, such that genotypic features can be identified before the phenotypic appearance of cancer can be noted.

关键词： Colorectal cancer monoclonal antibodies Immunohistochemistry Antibody dependent cell cytotoxicity Tumor associated antigens

monoclonal antibodies to the exon 18 encoded moiety of NCAM

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Journal of Cancer Metastasis and Treatment 2019年第7期4卷 50-55页

作者： Ann Vander Borght Mieke Duysinx Monique Ummelen Bernard AMvan der Zeijst Department of Genetics and Cell Biology and GROW School for Oncology and Developmental Biology Maastricht UniversityMaastrichtPO Box 6166200 MDThe Netherlands MUbio Products B.V. Maastricht6200 EVThe Netherlands Catherinn B.V. Manuel de Fallapad 1Rotterdam3069 MRThe Netherlands

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.

关键词： Small cell lung cancer tumor biomarker monoclonal antibodies NCAM exon 18

In vivo neutralization assays by monoclonal antibodies against white spot syndrome virus in crayfish (Cambarus proclarkii)

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Acta Oceanologica Sinica 2008年第2期27卷 126-132页

作者： WANG Yinan ZHAN Wenbin XING Jing JIANG Yousheng Laboratory of Pathology and Immunology of Aquatic Animals Laboratory of Maricuhure of Ministry of Education Ocean University of China Qingdao 266003 China

The neutralizing activities of eight monoclonal antibodies （MAbs） against white spot syndrome virus （WSSV）（2D2, 2B2,1D2, 1D5, 1C2, 4A1, 6A4 and 6B4） were analyzed by in vivo experiments. Gills from WSSV-infected shrimp were homogenized and ten-fold serially diluted by PBS, and then incubated with MAbs （hybridoma culture supernatant）, respectively. The mixture of WSSV and MAbs were injected into crayfish （Procambarus clarkii）. After challenge, the death rates of crayfish were counted to determine the neutralizing activities of MAbs. At the same time, the mixture of myeloma culture supernatant and WSSV or PBS was served as positive or negative control, respectively. The results showed that, at each virus dilution, the mean time to death of the crayfish injected with MAb-treated virus was significantly longer than that in the positive control, though they all showed 100% mortality within 25 d, and meanwhile, few crayfish died in the negative control. Among the eight MAbs, 2D2, 2B2, 1D2 and 1D5, especially the former two, delayed the mortality significantly, and 1 C2, 4A1 and 6A4 delayed the mortality as well but not so efficiently, while MAb 6IM was efficient only when the virus concentration increased. The results indicated that the anti-WSSV MAbs can neutralize WSSV in different virus dilutions.

关键词： crayfish WSSV monoclonal antibodies in vivo neutralization

Characterization and application of monoclonal antibodies against Shewanella marisflavi, a novel pathogen of Apostichopus japonicus

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Chinese Journal of Oceanology and Limnology 2011年第5期29卷 973-980页

作者：李强景宏丽李华王轶南徐德海 Key Laboratory of Mariculture Agriculture Ministry of China Dalian Ocean University U.S. Department of Agriculture Agricultural Research Service Aquatic Animal Health Research Laboratory Auburn AL 36832 USA

Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopusjaponicus. In this study, four monoclonal antibodies （MAbs）（3C1, 3D9, 2F2, 2A8） against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no cross reactivity to other gram-negative bacteria, However, 2F2 and 2A8 showed cross reactivity to all tested bacteria. Indirect immunofluorescence, and immunogold electron microscopy, showed the binding antigens of 3C1 and 3D9 were located at the secretion on the surface of strain AP629. The binding antigens of 2F2 and 2A8 were noted on the membrane of the cells. MAbs 3C1 and 3D9 recognized the lipopolysaccharide fraction of strain AP629, and 2F2 and 2A8 recognized in western-blotting protein antigens with molecular weights of 113 and 128 kDa respectively. MAbs 3C1 and 3D9 have the potential for use in pathogen diagnosis, epidemiology and studies on the mechanism of how S. marisflavi infects A. japonicus. Imrnunohistochemistry with 3C1 or 3D9 identified strain AP629 in the body wall of infected A. japonicus. In the adult sea cucumbers that were infected via body wall injection, positive signals were observed at the site of skin ulceration, and at the connective tissue of the non-ulcerated body wall. In addition, some large blue-stained cells aggregated at the connective tissue colonized by large numbers of bacteria. In juveniles infected via immersion infection, positive signals were observed at the cuticle of the body wall only. Our results suggest that 3C1 and 3D9 could be used in various immunological assays to study the invasion mechanism of strain AP629 in A. japonicus, the law of bacterial colonization, proliferation in different tissues of A. japonicus, and correlation between secretion on the surface of strain AP629 and its pathogenesis to A. japonicus.

关键词： Apostichopusjaponicus Shewanella marisflavi monoclonal antibodies

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

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Journal of Pharmaceutical Analysis 2020年第1期10卷 23-34页

作者： Sara Carillo Raquel Peerez-Robles Craig Jakes Meire Ribeiro da Silva Silvia Millan Martín Amy Farrell Natalia Navas Jonathan Bones Characterisation and Comparability Laboratory NIBRT e the National Institute for Bioprocessing Research and TrainingFoster AvenueMount MerrionBlackrockCo.DublinA94 X099Ireland Department of Analytical Chemistry Biohealth Research Institute(ibs.GRANADA)University of GranadaGranadaSpain School of Chemical and Bioprocess Engineering University College DublinBelfieldDublin 4D04 V1W8Ireland

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 *** 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 *** importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality *** N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform *** this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal *** 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.

关键词： N-Glycans Biopharmaceuticals monoclonal antibodies Intact mass analysis Mass spectrometry Native mass spectrometry Glycan analysis Peptide mapping Glycopeptide analysis

Basic regulatory science behind drug substance and drug product specifications of monoclonal antibodies and other protein therapeutics

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Journal of Pharmaceutical Analysis 2024年第6期14卷 785-804页

作者： Patanachai K.Limpikirati Sorrayut Mongkoltipparat Thinnaphat Denchaipradit Nathathai Siwasophonpong Wudthipong Pornnopparat Parawan Ramanandana Phumrapee Pianpaktr Songsak Tongchusak Maoxin Tim Tian Trairak Pisitkun Department of Food and Pharmaceutical Chemistry Faculty of Pharmaceutical SciencesChulalongkorn UniversityPathumwanBangkok10330Thailand Pharmaceutical Sciences and Technology(PST)Graduate Program Faculty of Pharmaceutical SciencesChulalongkorn UniversityPathumwanBangkok10330Thailand Center of Excellence in Systems Biology(CUSB) Faculty of MedicineChulalongkorn UniversityPathumwanBangkok10330Thailand Department of Pharmaceutical Chemistry Faculty of Pharmaceutical SciencesHuachiew Chalermprakiet UniversityBang PhliSamut Prakan10540Thailand Division of Research Affairs Faculty of MedicineChulalongkorn UniversityPathumwanBangkok10330Thailand

In this review,we focus on providing basics and examples for each component of the protein therapeutic specifications to interested pharmacists and biopharmaceutical scientists with a goal to strengthen understanding in regulatory science and *** specifications comprise a list of important quality attributes for testing,references to use for test procedures,and appropriate acceptance criteria for the tests,and they are set up to ensure that when a drug product is administered to a patient,its intended therapeutic benefits and safety can be rendered *** of drug substance or drug product to the specifications is achieved by testing an article according to the listed tests and analytical methods and obtaining test results that meet the acceptance *** attributes are chosen to be tested based on their quality risk,and consideration should be given to the merit of the analytical methods which are associated with the acceptance criteria of the *** criteria are set forth primarily based on efficacy and safety profiles,with an increasing attention noted for patient-centric *** in this work are related guidelines that support the biopharmaceutical specification setting,how to set the acceptance criteria,and examples of the quality attributes and the analytical methods from 60 articles and 23 pharmacopeial *** are also explored on process analytical technologies and other orthogonal tools which are on-trend in biopharmaceutical characterization and quality control.

关键词： Biopharmaceutical analysis Biopharmaceutical quality control Biopharmaceutical specifications monoclonal antibodies Protein therapeutics Regulatory science

Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

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Asian Journal of Andrology 2016年第1期18卷 108-113页

作者： Jana Capkova Alena Kubatova Lukas Ded Olina Tepla Jana Peknicova Laboratory of Reproductive Biology Institute of Biotechnology Academy of Sciences of the Czech Republic v. v. i. Prague Czech Republic Clinic Center IVF ISCARE Prague Czech Republic

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry （FCM） and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies （MoAbs）, which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase （cAMP-dependent protein kinase II, PRKAR2A）, evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.

关键词： asthenozoospermia flow cytometry fluorescence microscopy monoclonal antibodies sperm proteins

Antigenically dominant proteins within the human liver mitochondrial proteome identified by monoclonal antibodies

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Science China(Life Sciences) 2011年第1期54卷 16-24页

作者： JU YanFang YANG JinJu LIU Rong LIU XiaoLan DU XueMei LIU Li CHEN ZhiCheng CHI Jun LIU ShuEr GAO Yuan GAO JianEn JIAO ShunChang HE FuChu SUN QiHong Department of Oncology General Hospital of PLA Beijing 100853 China State Key Laboratory of Proteomics Beijing Proteome Research Center Beijing Institute of Radiation Medicine Beijing 102206 China

Analysis of the mitochondrial proteome would provide valuable insight into the function of this important organelle, which plays key roles in energy metabolism, apoptosis, free radical production, thermogenesis, and calcium signaling. It could also increase our understanding about the mechanisms that promote mitochondrial disease. To identify proteins that are antigenically dominant in human liver mitochondria, we generated >240 hybridoma cell lines from native mitochondrial proteins after cell fusion, screening, and cloning. antibodies that recognized mitochondrial proteins were identified by screening human liver cDNA expression libraries. In this study, we identified 6 major antigens that were recognized by at least 2 different monoclonal antibodies (mAbs). The proteins that were antigenically dominant were: acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase), aldehyde dehydrogenase 1 family member A1, carbamoyl phosphate synthetase 1, dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex), enoyl coenzyme A hydratase 1, and hydroxysteroid (11-beta) dehydrogenase 1. We also determined the subcellular localizations of these enzymes within the mitochondria using immunohistocytochemistry. We believe that these well-characterized antibodies will provide a valuable resource for the Human Liver Proteome Project (HLPP), and will make studies aimed at investigating liver mitochondrial function far easier to perform in future. Our results provide strong evidence that, (i) depletion of dominant proteins from liver mitochondrial samples is possible and, (ii) the approaches adopted in this study can be used to explore or validate protein-protein interactions in this important organelle.

关键词： mitochondria monoclonal antibodies cDNA library screening antigenically dominant proteins liver

Preparation of Anti-cardiac Troponin I monoclonal antibodies and Their Characterization with Surface Plasmon Resonance Biosensor

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Chemical Research in Chinese Universities 2003年第2期19卷 183-189页

作者： WEIJing-yan LIUXia SONGDa-qian BULi-sha HUXing MUYing KeyLaboratoryofMolcularEnzymologyandEngineeringofministryofEducation JilinUniversityChangchun130023P.R.China CollegeofChemistry JilinUniversityChangchun130023P.R.China ThethirdClinicalCollege JilinUniversityChangchun130031P.R.China

Cardiac troponin I(cTnI) was separated and purified from human left ventricular tissue by affinity chromatographic method and used to immunize Balb/c mice by intraperitoneal injection and four hybridoma cell lines, which secreted monoclonal antibody(mAb) against human cTnI, were obtained by cell fusion, identification and cloning twice. Three mAbs(9F5, 2F11, 8C12) were produced from the ascites of Balb/c mice injected intraperitoneally the hybridoma cells and characterized by means of a surface plasmon resonance(SPR) biosensor. An optimal and specific sensing membrane for troponin I was prepared with staphylococcal protein A(SPA) as the intermediate layer and mAb against human cTnI as the capture antibody. On the basis of the sensing membrane, two modes of operation of the SPR biosensor were developed, i.e ., a direct detection of antigen antibody affinity and a sandwich assay. In the sandwich assay detection mode, the mAbs competition was measured by monitoring whether the secondary antibody had been attached to the cTnI already captured by the first antibody on the sensor surface. The SPR biosensor was shown to be able to directly detect the antigen antibody affinity and the order of the affinity was found to be 9F5>2F11>8C12. In the sandwich detection mode, it was found that the different epitopes on the cTnI molecules were recognized by the three mAbs respectively, but the asymmetrical competition was shown between 2F11 and 8C12 and no competition was found between 9F5 and 2F11 or 8c12. Based on these results, a double monoclonal sandwich immunoassay for cTnI was developed by using the optimal antibody pair of 9F5 and 2F11 and the SPR biosensor with SPA substrate membrane, which showed an excellent sensitivity of 0.8 μg/L for both the buffer and the serum samples compared with the direct detection of cTnI for the buffer with the lowest detection limit of 4 μg/L and conventional ELISA with the sensitivity of 1.9 μg/L.

关键词： Troponin I monoclonal antibodies Characterization SPR biosensor