Abstract
BackgroundThe rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene.ResultsThe cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat fut8 gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of fut8 in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 fut8 gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.ConclusionAltogether, these data contribute to a better knowledge of fut8 expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.
Highlights
The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant monoclonal antibody (mAb) due to its lower expression of fut8 gene than other commonly used rodent cell lines
Cell culture The rat cell line YB2/0 was obtained from American Type Culture Collection (Manassas, VA, http://www.atcc. org; catalog n° CRL-1662) and grown in EMSTM medium supplemented with 5% heat inactivated fetal calf serum (FCS) (Invitrogen, Carlsbad, CA, USA) at 37°C under 5% CO2
Isolation and characterization of the rat a1,6 fucosyltransferase cDNA In order to clone the rat fut8 cDNA, the putative rat mRNA sequence of 1728 pb (NM_001002289.1) provided by the NCBI genome annotation division http:// www.ncbi.nlm.nih.gov/sites/entrez was used as a probe for BLAST analysis of rat genomic and expressed sequence tags (EST) divisions of NCBI databases
Summary
The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut gene than other commonly used rodent cell lines. Several independent studies have clearly shown that effector functions of recombinant therapeutic IgG are directly dependent on the glycosylation of the constant region (Fc) [1,2,3]. Cytotoxicity (ADCC) is dependent on appropriate glycosylation of the Fc region of mAbs (for review [7]). Low fucose IgG1 (10-20%) exhibit a higher ADCC activity compared to highly fucosylated IgG (80-90%) either in vitro [8] or in vivo [10]. The most widely used recombinant antibodies are produced by rodent mammalian cell lines with intrinsic fucosyltransferase activity (e.g., Chinese hamster ovary (CHO), mouse myeloma and hybridoma cell lines). Reducing the a1,6-fucose rate of IgG Fc has been a challenge over the last few years to provide maximum efficiency to recombinant mAbs
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