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1.
MAbs ; 15(1): 2261509, 2023.
Article in English | MEDLINE | ID: mdl-37823690

ABSTRACT

There are few treatments that slow neurodegeneration in Alzheimer's disease (AD), and while therapeutic antibodies are being investigated in clinical trials for AD treatment, their access to the central nervous system is restricted by the blood-brain barrier. This study investigates a bispecific modular fusion protein composed of gantenerumab, a fully human monoclonal anti- amyloid-beta (Aß) antibody under investigation for AD treatment, with a human transferrin receptor 1-directed Brainshuttle™ module (trontinemab; RG6102, INN trontinemab). In vitro, trontinemab showed a similar binding affinity to fibrillar Aß40 and Aß plaques in human AD brain sections to gantenerumab. A single intravenous administration of trontinemab (10 mg/kg) or gantenerumab (20 mg/kg) to non-human primates (NHPs, Macaca fascicularis), was well tolerated in both groups. Immunohistochemistry indicated increased trontinemab uptake into the brain endothelial cell layer and parenchyma, and more homogeneous distribution, compared with gantenerumab. Brain and plasma pharmacokinetic (PK) parameters for trontinemab were estimated by nonlinear mixed-effects modeling with correction for tissue residual blood, indicating a 4-18-fold increase in brain exposure. A previously developed clinical PK/pharmacodynamic model of gantenerumab was adapted to include a brain compartment as a driver of plaque removal and linked to the allometrically scaled above model from NHP. The new brain exposure-based model was used to predict trontinemab dosing regimens for effective amyloid reduction. Simulations from these models were used to inform dosing of trontinemab in the first-in-human clinical trial.


Subject(s)
Alzheimer Disease , Antibodies, Monoclonal , Animals , Humans , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Amyloid beta-Peptides/immunology , Amyloid beta-Peptides/therapeutic use , Antibodies, Monoclonal/pharmacology , Brain/metabolism , Primates/metabolism
2.
Biotechnol Prog ; 32(3): 776-86, 2016 05.
Article in English | MEDLINE | ID: mdl-27073121

ABSTRACT

Chinese hamster ovary cells (CHO) are widely used in the production of glycosylated therapeutic proteins such as antibodies. During expansion and maintenance, CHO cell lines are prone to production instability, which may be caused by promoter silencing, loss of transgene copies, or post-transcriptional effects. Silencing of recombinant genes may be accompanied by DNA methylation and histone modification. Previously, we demonstrated that cytosine methylation of the human cytomegalovirus major immediate early promoter/enhancer (hCMV-MIE) can be used to predict instability of antibody-producing CHO cell lines. However, the high rate of false prediction motivates the search for further markers of stable promoter activity. To this end, we correlated a variety of histone modifications in the vicinity of hCMV-MIE with production stability over time. Our results suggest that acetylation of histone H3 (H3ac) is a more effective indicator of production stability than DNA methylation. Selecting cell lines with highest CMV promoter H3ac levels enriches stable expressors and improves the average stability of production cell lines. For histone H3 acetylation measurement we employed a method based on chromatin immunoprecipitation (ChIP). In its current form, the method is suitable to evaluate 10-20 cell lines within a few days. We propose to determine H3 acetylation once the number of candidate cell lines has been narrowed based on productivity and product quality. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:776-786, 2016.


Subject(s)
Cytomegalovirus/metabolism , Histones/metabolism , Promoter Regions, Genetic/genetics , Acetylation , Animals , CHO Cells , Cells, Cultured , Cricetulus , Cytomegalovirus/genetics , Gene Expression Profiling
3.
Sci Rep ; 5: 16952, 2015 Nov 19.
Article in English | MEDLINE | ID: mdl-26581326

ABSTRACT

The major immediate-early promoter and enhancer of the human cytomegalovirus (hCMV-MIE) is one of the most potent DNA elements driving recombinant gene expression in mammalian cells. Therefore, it is widely employed not only in research but also in large-scale industrial applications, e.g. for the production of therapeutic antibodies in Chinese hamster ovary cells (CHO). As we have reported previously, multi-site methylation of hCMV-MIE is linked to productivity loss in permanently transfected CHO cells lines. In particular, the cytosine located 179 bp upstream of the transcription start site (C-179) is frequently methylated. Therefore, our objective was to study whether mutation of C-179 and other cytosines within hCMV-MIE might lessen the instability of transgene expression. We discovered that the single mutation of C-179 to G can significantly stabilise the production of recombinant protein under control of hCMV-MIE in permanently transfected CHO cells.


Subject(s)
Cytomegalovirus/genetics , Mutation/genetics , Promoter Regions, Genetic , Recombinant Proteins/biosynthesis , Animals , Base Sequence , CHO Cells , Clone Cells , Cricetinae , Cricetulus , Genes, Immediate-Early , Humans , Molecular Sequence Data
4.
Brain ; 137(Pt 10): 2834-46, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25085375

ABSTRACT

The severity of tau pathology in Alzheimer's disease brain correlates closely with disease progression. Tau immunotherapy has therefore been proposed as a new therapeutic approach to Alzheimer's disease and encouraging results have been obtained by active or passive immunization of tau transgenic mice. This work investigates the mechanism by which immunotherapy can impact tau pathology. We demonstrate the development of Alzheimer's disease-like tau pathology in a triple transgenic mouse model of Alzheimer's disease and show that tau/pS422 is present in membrane microdomains on the neuronal cell surface. Chronic, peripheral administration of anti-tau/pS422 antibody reduces the accumulation of tau pathology. The unequivocal presence of anti-tau/pS422 antibody inside neurons and in lysosomes is demonstrated. We propose that anti-tau/pS422 antibody binds to membrane-associated tau/pS422 and that the antigen-antibody complexes are cleared intracellularly, thereby offering one explanation for how tau immunotherapy can ameliorate neuronal tau pathology.


Subject(s)
Alzheimer Disease/immunology , Alzheimer Disease/pathology , Antibodies/metabolism , tau Proteins/immunology , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Antibodies, Monoclonal/metabolism , Blotting, Western , CA1 Region, Hippocampal/metabolism , CA1 Region, Hippocampal/pathology , Disease Models, Animal , Disease Progression , Fluorescent Antibody Technique , Humans , Image Processing, Computer-Assisted , Lysosomes/metabolism , Lysosomes/pathology , Membrane Microdomains/pathology , Mice , Mice, Transgenic , Phosphorylation , Sarcosine/analogs & derivatives , Sarcosine/chemistry
5.
PLoS One ; 7(7): e40328, 2012.
Article in English | MEDLINE | ID: mdl-22792284

ABSTRACT

Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison of candidate production cell lines as a first stage, followed by an in-depth analysis including identification and quantitation of aberrant sequence variants of selected clones as a second stage. We show that the differential analysis is a suitable tool for sensitive and fast batch to batch comparison of recombinant proteins. The optimized approach allows for detection of not only single amino acid substitutions in unmodified peptides, but also substitutions in posttranslational modified peptides such as glycopeptides, for detection of truncated or elongated sequence variants as well as double amino acid substitutions or substitution with amino acid structural isomers within one peptide. In two case studies we were able to detect sequence variants of different origin down to a sub percentage level. One of the sequence variants (Thr → Asn) could be correlated to a cytosine to adenine substitution at DNA (desoxyribonucleic acid) level. In the second case we were able to correlate the sub percentage substitution (Phe → Tyr) to amino acid limitation in the chemically defined fermentation medium.


Subject(s)
Antibodies, Monoclonal/chemistry , Immunoglobulin G/chemistry , Software , Amino Acid Sequence , Amino Acid Substitution , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/isolation & purification , Base Sequence , CHO Cells , Chromatography, Gel , Cricetinae , High-Throughput Nucleotide Sequencing , Humans , Immunoglobulin G/genetics , Immunoglobulin G/isolation & purification , Molecular Sequence Data , Peptide Fragments/chemistry , Phenylalanine/genetics , Point Mutation , Proteolysis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Reference Standards , Sequence Analysis, DNA , Sequence Analysis, Protein/methods , Tandem Mass Spectrometry/standards , Trypsin/chemistry
6.
Biotechnol Bioeng ; 108(11): 2670-81, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21618470

ABSTRACT

Mammalian cell lines for recombinant protein production need to maintain productivity over extended cultivation times. Long-term stability studies are time and resource intensive, but are widely performed to identify and eliminate unstable candidates during cell line development. Production instability of manufacturing cell lines can be associated with methylation and silencing of the heterologous promoter. We have identified CpG dinucleotides within the human cytomegalovirus major immediate early promoter/enhancer (hCMV-MIE) that are frequently methylated in unstable antibody-producing Chinese hamster ovary (CHO) cell lines. We have established methylation-specific real-time qPCR for the rapid and sensitive measurement of hCMV-MIE methylation in multiple cell lines and provide evidence that hCMV-MIE methylation and transgene copy numbers can be used as early markers to predict production instability of recombinant CHO cell lines. These markers should provide the opportunity to enrich stable producers early in cell line development and allow developers to put more emphasis on other criteria, such as product quality and bioprocess robustness.


Subject(s)
DNA Methylation , Gene Dosage , Gene Expression Regulation , Genomic Instability , Promoter Regions, Genetic , Recombinant Proteins/biosynthesis , Animals , CHO Cells , Cricetinae , Cricetulus , Cytomegalovirus/genetics , Gene Expression Profiling , Real-Time Polymerase Chain Reaction , Recombinant Proteins/genetics , Transgenes
7.
Immunol Lett ; 115(1): 9-15, 2008 Jan 15.
Article in English | MEDLINE | ID: mdl-18079004

ABSTRACT

Soluble forms of CD83, a dendritic cell-specific surface glycoprotein, have been strongly proposed to be of therapeutic utility in inflammatory conditions such as multiple sclerosis and transplantation. We demonstrate here, however, that eukaryotically expressed, recombinant soluble human CD83-Ig molecules fail to achieve efficacy in model systems for those conditions: mouse experimental autoimmune encephalomyelitis models in vivo or in mixed lymphocyte reactions in vitro. These results raise concern as to the viability of a eukaryotically expressed soluble CD83 strategy for clinical therapeutic use.


Subject(s)
Antigens, CD/immunology , Antigens, CD/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Immunoglobulins/immunology , Immunoglobulins/therapeutic use , Lymphocyte Culture Test, Mixed , Membrane Glycoproteins/immunology , Membrane Glycoproteins/therapeutic use , Animals , Antigens, CD/blood , Dendritic Cells/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Humans , Immunoglobulins/blood , Membrane Glycoproteins/blood , Mice , Mice, Inbred C57BL , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/therapeutic use , CD83 Antigen
8.
Immunol Lett ; 2007 Sep 29.
Article in English | MEDLINE | ID: mdl-17935795

ABSTRACT

This article has been withdrawn consistent with Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The Publisher apologizes for any inconvenience this may cause.

9.
Hum Mol Genet ; 12(14): 1767-79, 2003 Jul 15.
Article in English | MEDLINE | ID: mdl-12837699

ABSTRACT

p27(Kip1) regulates cell proliferation by binding to and modulating the activity of cyclin-dependent kinases. The CDK inhibitor is haploinsufficient for tumor suppression and reduced p27 activity is fundamental for the development of many human malignancies. Consistently, reduced p27 protein provides independent prognostic information in various tumors including breast, prostate, colon and gastric carcinomas. In normal cells, p27 protein increases in growth arrest but also oscillates during cell cycle progression. Expression of p27 is regulated through mechanisms including transcription, translation and ubiquitin-mediated degradation. Each of these pathways may contribute to deregulation of p27 in hyperproliferative diseases. p27 translation increases in proliferating cells during G(1) phase and declines as cells enter S phase. To investigate the mechanisms of p27 translational control, we analyzed fragments of the p27 transcript for their contribution to cell cycle regulated translation. We found that an element in the p27 5'-UTR can render reporter translation cell cycle sensitive with maximal translation in G1-arrested cells. This novel element of 114 nt contains a G/C-rich hairpin domain that is predicted to form multiple stable stemloops and also overlaps with a small upstream ORF (uORF). Both structures contribute to cell cycle-regulated translation. The uORF can be translated in vitro and its sequence and position are highly conserved in mice and chickens. Interestingly, the precise sequence or the length of the uORF-encoded peptide are not important for p27 translation, consistent with the idea that ribosomal recruitment to its initiation codon rather than the translation product itself contributes to the regulation.


Subject(s)
5' Untranslated Regions , Cell Cycle Proteins/genetics , Cell Cycle/physiology , Protein Biosynthesis , Tumor Suppressor Proteins/genetics , Cell Cycle Proteins/biosynthesis , Cyclin-Dependent Kinase Inhibitor p27 , HeLa Cells , Humans , Open Reading Frames , Tumor Suppressor Proteins/biosynthesis
10.
Genes Dev ; 16(23): 3087-99, 2002 Dec 01.
Article in English | MEDLINE | ID: mdl-12464637

ABSTRACT

p27Kip1 restrains cell proliferation by binding to and inhibiting cyclin-dependent kinases. To investigate the mechanisms of p27 translational regulation, we isolated a complete p27 cDNA and identified an internal ribosomal entry site (IRES) located in its 5'UTR. The IRES allows for efficient p27 translation under conditions where cap-dependent translation is reduced. Searching for possible regulators of IRES activity we have identified the neuronal ELAV protein HuD as a specific binding factor of the p27 5'UTR. Increased expression of HuD or the ubiquitously expressed HuR protein specifically inhibits p27 translation and p27 IRES activity. Consistent with an inhibitory role of Hu proteins in p27 translation, siRNA mediated knockdown of HuR induced endogenous p27 protein levels as well as IRES-mediated reporter translation and leads to cell cycle arrest in G1.


Subject(s)
5' Untranslated Regions/genetics , Gene Expression Regulation/physiology , Proliferating Cell Nuclear Antigen/genetics , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Base Sequence , ELAV Proteins , Fibroblasts , HeLa Cells , Humans , Molecular Sequence Data , RNA-Binding Proteins/genetics , Ribonucleoproteins/genetics , Sequence Analysis, DNA
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