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K1, mAb

David Reinhart, Lukas Damjanovic, Christian Kaisermayer, Wolfgang Sommeregger, Andreas Gili, Bernhard Gasselhuber, Andreas Castan, Patrick Mayrhofer, Clemens Grünwald-Gruber, Renate Kunert
Chinese hamster ovary (CHO) cells comprise a variety of lineages including CHO-DXB11, CHO-K1, CHO-DG44 and CHO-S. Despite all CHO cell lines sharing a common ancestor, extensive mutagenesis and clonal selection has resulted in substantial genetic heterogeneity among them. Data from sequencing shows that different genes are missing in individual CHO cell lines and each cell line harbors a unique set of mutations with relevance to the bioprocess. However, not much literature is available about the influence of genetic differences of CHO on the performance of bioprocess operations...
April 27, 2018: Biotechnology Journal
Sujeewa D Wijesuriya, Elizabeth Pongo, Milan Tomic, Fangjiu Zhang, Consuelo Garcia-Rodriquez, Fraser Conrad, Shauna Farr-Jones, James D Marks, Arnold H Horwitz
Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies...
April 12, 2018: Protein Expression and Purification
Mahmood Rahimi-Zarchi, Seyed Abbas Shojaosadati, Mohammad Mehdi Amiri, Mahmood Jeddi-Tehrani, Fazel Shokri
The effects of all-trans retinoic acid (RA) and sodium butyrate (NaBu) on growth, viability and antibody production of two types of transfected Chinese hamster ovary cell lines (CHO-K1 and CHO-S) were investigated using a batch mode cell culture. By adding 0.5 mM NaBu in the CHO-K1 cell culture, the cell specific productivity (Qp ) and antibody concentration increased by five- and threefold, respectively. The optimal concentration of RA was 100 nM which resulted in twofold increase in antibody production...
April 4, 2018: Bioprocess and Biosystems Engineering
Shinji Yamada, Shunsuke Itai, Takuro Nakamura, Miyuki Yanaka, Yao-Wen Chang, Hiroyoshi Suzuki, Mika K Kaneko, Yukinari Kato
Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L1 Mab-13 (IgG1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses...
April 2, 2018: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Soo Min Noh, Seunghyeon Shin, Gyun Min Lee
To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia...
March 29, 2018: Scientific Reports
C Augustsson, A Svensson, B Kjaer, T-Y Chao, X Wenjuan, B O Krogh, J Breinholt, J T Clausen, I Hilden, H H Petersen, L C Petersen
Essentials Activated FVII (FVIIa) and FX (FXa) are inhibited by tissue factor pathway inhibitor (TFPI). A monoclonal antibody, mAb2F22, was raised against the N-terminal fragment of TFPI (1-79). mAb2F22 bound exclusively to the K1 domain of TFPI (KD ∼1 nm) and not to the K2 domain. mAb2F22 interfered with inhibition of both FVIIa and FXa activities and restored clot formation. SUMMARY: Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI)...
May 2018: Journal of Thrombosis and Haemostasis: JTH
Oliver Popp, Samuel Moser, Jörg Zielonka, Petra Rüger, Silke Hansen, Oliver Plöttner
Novel biotherapeutic glycoproteins, like recombinant monoclonal antibodies (mAbs) are widely used for the treatment of numerous diseases. The N-glycans attached to the constant region of an antibody have been demonstrated to be crucial for the biological efficacy. Even minor modifications of the N-glycan structure can dictate the potency of IgG effector functions such as the antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Here, we present the development of a glycoengineered CHO-K1 host cell line (HCL), stably expressing β1,4-N-Acetylglucoseaminyltransferase III (GnT-III) and α-mannosidase II (Man-II), for the expression of a-fucosylated antibodies with enhanced Fc-mediated effector function...
February 2018: MAbs
Jessna H M Yeo, Steven C L Ho, Mariati Mariati, Esther Koh, Shi Jie Tay, Susanto Woen, Peiqing Zhang, Yuansheng Yang
There are several selection markers which are suitable for generating stably transfected Chinese hamster ovary (CHO) cell lines. Due to their different modes of action, each selection marker has its own optimal selection stringency in different host cells for obtaining high productivity. Using an internal ribosome entry site (IRES)-mediated tricistronic vector and a set of IRES variants with different strengths, the expression of five antibiotics resistance genes (ARGs) in CHO-K1 cells and dihydrofolate reductase (DHFR) in CHO DG44 cells is optimized to enhance the stringency of selection for high producing cells...
December 2017: Biotechnology Journal
Tanja Diana, Christian Wüster, Paul D Olivo, Angelica Unterrainer, Jochem König, Michael Kanitz, Artur Bossowski, Brigitte Decallonne, George J Kahaly
BACKGROUND: The measurement of TSH receptor (TSHR) antibodies is warranted for diagnosis of Graves' disease (GD). OBJECTIVE: The performance, detection sensitivity, and specificity of 6 TSHR immunoassays were compared. METHODS: Two bioassays and 4 binding assays (Kronus, Immulite, Kryptor, Dynex) were compared in a dilution study performed in patients with autoimmune thyroid disease. Both bioassays were compared to 2 binding assays using stimulatory (M22) and blocking (K1-70) monoclonal antibody (MAb) mixtures...
September 2017: European Thyroid Journal
Shun Xin Wang-Lin, Ruth Olson, Janet M Beanan, Ulrike MacDonald, Joseph P Balthasar, Thomas A Russo
Acinetobacter baumannii has become an important concern for human health due to rapid development and wide spread of antimicrobial-resistant strains and high mortality associated with the infection. Passive immunizations with antisera targeting outer membrane proteins (OMPs) have shown encouraging results in protecting mice from A. baumannii infection, but monoclonal anti-OMP antibodies have not been developed, and their potential therapeutic properties have not been explored. The goal of this report is to evaluate the antibacterial activity of monoclonal antibodies (MAbs) targeting outer membrane protein A (OmpA) of A...
December 2017: Infection and Immunity
Shinji Yamada, Shunsuke Itai, Takuro Nakamura, Miyuki Yanaka, Noriko Saidoh, Yao-Wen Chang, Saori Handa, Hiroyuki Harada, Yumiko Kagawa, Osamu Ichii, Satoru Konnai, Mika K Kaneko, Yukinari Kato
Podoplanin (PDPN) is expressed in several normal tissues, such as lymphatic endothelial cells, podocytes of renal glomerulus, and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. Although monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, dog PDPN, and bovine PDPN have been established, anticat PDPN (cPDPN) mAbs have not been developed. In this study, we immunized mice with Chinese hamster ovary (CHO)-K1 cell lines expressing cPDPN, and developed anti-cPDPN mAbs...
October 2017: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Mikhail S Karbyshev, Evgeniya S Grigoryeva, Viktor V Volkomorov, Elisabeth Kremmer, Alexander Huber, Irina V Mitrofanova, Evgeniya V Kaigorodova, Marina V Zavyalova, Julia G Kzhyshkowska, Nadezda V Cherdyntseva, Evgeny L Choynzonov
Transmembrane prostate androgen-induced protein 1 (TMEPAI) is a single-span membrane protein, functionally involved in transforming growth factor beta signaling pathway. The particular protein presented in cells in three isoforms, which differs in the length of the soluble N-terminal extracellular domain, making it challenging for the immunochemical recognition. By using quantitative real-time polymerase chain reaction, we identified significant upregulation of PMEPA1 gene expression in malignant tissues of patients with gastric adenocarcinoma...
April 2018: Pathology Oncology Research: POR
Elizabeth Diago-Navarro, Isabel Calatayud-Baselga, Donglei Sun, Camille Khairallah, Inderjit Mann, Amaia Ulacia-Hernando, Brian Sheridan, Meiqing Shi, Bettina C Fries
Hypervirulent Klebsiella pneumoniae (hvKp) strains are predicted to become a major threat in Asia if antibiotic resistance continues to spread. Anticapsular antibodies (Abs) were developed because disseminated infections caused by hvKp are associated with significant morbidity and mortality, even with antibiotic-sensitive strains. K1-serotype polysaccharide capsules (K1-CPS) are expressed by the majority of hvKp strains. In this study, K1-CPS-specific IgG Abs were generated by conjugation of K1-CPS to immunogenic anthrax protective antigen (PA) protein...
January 2017: Clinical and Vaccine Immunology: CVI
Soo Min Noh, Jin Hyoung Park, Myung Sin Lim, Jong Won Kim, Gyun Min Lee
Chinese hamster ovary (CHO) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. To reduce ammonia production, the glutamine synthetase (GS) system was used to develop therapeutic monoclonal antibody (mAb)-producing CHO cells (SM-0.025). Additionally, the lactate dehydrogenase-A (LDH-A) was downregulated with shRNA to reduce lactate production in SM-0.025. The resulting mAb-producing cell lines (#2, #46, and #52) produced less ammonia than the host cell line during the exponential phase due to GS protein overexpression...
February 2017: Applied Microbiology and Biotechnology
Sae-Hae Kim, Yu Na Kim, Thang Thua Truong, Nguyen Thi Thu Thuy, Le Quynh Mai, Yong-Suk Jang
Dengue virus (DENV) is a mosquito-borne pathogen that annually infects more than 390 million people in 100 different countries. Symptoms of the viral infection include a relatively weak dengue fever to severe dengue hemorrhagic fever/dengue shock syndrome, which are mortal infectious diseases. As of yet, there is no commercially available vaccine or therapeutic for DENV. Currently, passive immunotherapy using DENV-specific antibody (Ab) is a considered strategy to treat DENV infection. Here, we developed a monoclonal Ab (mAb), EDIIImAb-61, specific to the DENV domain III of the envelope glycoprotein (EDIII) with broad-spectrum detection ability to all four DENV serotypes (DENV-1∼4) to use as a therapeutic Ab...
May 13, 2016: Biochemical and Biophysical Research Communications
J Furmaniak, J Sanders, R Núñez Miguel, B Rees Smith
The availability of human monoclonal antibodies (MAbs) to the TSHR has enabled major advances in our understanding of how TSHR autoantibodies interact with the receptor. These advances include determination of the crystal structures of the TSHR LRD in complex with a stimulating autoantibody (M22) and with a blocking type autoantibody (K1-70). The high affinity of MAbs for the TSHR makes them particularly suitable for use as ligands in assays for patient serum TSHR autoantibodies. Also, M22 and K1-70 are effective at low concentrations in vivo as TSHR agonists and antagonists respectively...
September 2015: Hormone and Metabolic Research, Hormon- und Stoffwechselforschung, Hormones et Métabolisme
Jadwiga Furmaniak, Jane Sanders, Bernard Rees Smith
TSH receptor (TSHR) autoantibodies (TRAbs) play a key role in the pathogenesis of Graves' disease. In the majority of patients, TRAbs stimulate thyroid hormone synthesis via activation of the TSHR (stimulating TRAbs, TSHR agonists). In some patients, TRAbs bind to the receptor but do not cause activation (blocking TRAbs, TSHR antagonists). Isolation of human TSHR monoclonal antibodies (MAbs) with either stimulating (M22 and K1-18) or blocking activities (5C9 and K1-70) has been a major advance in studies on the TSHR...
April 2013: Auto- Immunity Highlights
Kentaro Shoji, Tadanobu Takahashi, Kohta Kurohane, Koki Iwata, Takeshi Matsuoka, Shogo Tsuruta, Takatomo Sugino, Masaki Miyake, Takashi Suzuki, Yasuyuki Imai
Secretory immunoglobulin (Ig) A (SIgA), comprised of dimeric IgA and secretory component (SC), is believed to provide a defense mechanism on the mucosal surface. Influenza A virus (IAV) hemagglutinin (HA)-specific SIgA is thought to play an important role in the prevention of IAV infection. However, the topical application of preformed IAV-specific SIgA has not been shown to prevent IAV infection. This is due to the difficulty in the production of antigen-specific IgA monoclonal antibodies (mAbs) and monoclonal SIgA...
April 2015: Viral Immunology
Stephanie Kapelski, Alexander Boes, Holger Spiegel, Melanie de Almeida, Torsten Klockenbring, Andreas Reimann, Rainer Fischer, Stefan Barth, Rolf Fendel
BACKGROUND: Monoclonal antibodies (mAbs) are essential tools in biological research, diagnosis and therapy, and are conventionally produced in murine hybridoma cell lines. Professional applications of mAbs depend on the steady supply of material. Because hybridoma cultures can stop producing the antibody or even die, preservation of the unique epitope specificity of mAbs by rescue of the sequences encoding the antibody variable domains (V regions) is important. The availability of these sequences enables not only the recombinant expression of the original antibody for further applications, but opens the road for antibody engineering towards innovative diagnostic or therapeutic applications...
2015: Malaria Journal
Maiken L Henriksen, Kirstine L Madsen, Karsten Skjoedt, Soren Hansen
Immunoaffinity chromatography is a powerful fractionation technique that has become indispensable for protein purification and characterization. However, it is difficult to retrieve bound proteins without using harsh or denaturing elution conditions, and the purification of scarce antigens to homogeneity may be impossible due to contamination with abundant antigens. In this study, we purified the scarce, complement-associated plasma protein complex, collectin LK (CL-LK, complex of collectin liver 1 and kidney 1), by immunoaffinity chromatography using a calcium-sensitive anti-collectin-kidney-1 mAb...
November 2014: Journal of Immunological Methods
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