Martine Duonor-Cérutti

Molecular cloning and expression of a human hST8Sia VI (α2,8-sialyltransferase) responsible for the synthesis of the diSia motif on O-glycosylproteins

Based on BLAST analysis of the human and mouse genome databases using the human CMP sialic acid; alpha2,8-sialyltransferase cDNA (hST8Sia I; EC 2.4.99.8), a putative sialyltransferase gene, was identified on human chromosome 10. The genomic organization was found to be similar to that of hST8Sia I and hST8Sia V. Transcriptional expression analysis showed that the newly identified gene was constitutively expressed at low levels in various human tissues and cell lines. We have isolated a full-length cDNA clone from the breast cancer cell line MCF-7 that encoded a type II membrane protein of 398 amino acid residues with the conserved motifs of sialyltransferases. We have established a mammary cell line (MDA-MB-231) stably transfected with the full-length hST8Sia VI and the analysis of sialylated carbohydrate structures expressed at the cell surface clearly indicated the disappearance of Neu5Acalpha2-3-sialylated structures. The transient expression of a truncated soluble form of the enzyme in either COS-7 cells or insect Sf-9 cells led to the production of an active enzyme in which substrate specificity was determined. Detailed substrate specificity analysis of the hST8Sia VI recombinant enzyme in vitro, revealed that this enzyme required the trisaccharide Neu5Acalpha2-3Galbeta1-3GalNAc (where Neu5Ac is N-acetylneuraminic acid and GalNAc is N-acetylgalactosamine) to generate diSia (disialic acid) motifs specifically on O-glycans.

ADAPTATION OF AN INSECT CELL LINE OF SPODOPTERA FRUGIPERDA TO GROW AT 37° C: CHARACTERIZATION OF AN ENDODIPLOID CLONE

SummarySf21 and Sf9 cell lines established from the lepidoptera Spodoptera frugiperda do not display major induction of heat shock proteins when exposed to a temperature of 37°C. After some months of adaptation at 37°C we obtained two new cell lines, Sf21-HT and SF9-HT, which have now been established for several years in our laboratory. The Sf9-HT line displays a slightly shorter doubling time at 37°C than the wild type at 28°C, but cell lethality gives rise to an earlier growth arrest. The composition of total lipid extract from heat-adapted cells reveals a higher sphingomyelin to phosphatidylcholine ratio and a higher percentage of saturated fatty acids, which are expected for the lower membrane fluidity, required for thermotolerance. The cell volume of Sf9-HT is doubled, and by flow cytometry we showed that the DNA content is twice that in the parental cell line. Karyotypic examination of metaphasic cells achieved under epifluorescence microscopy revealed a doubled chromosome number in Sf9-HT.

Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies

We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VH-CH1-hinge domains of mAb2 are fused through a peptidic linker to the N terminus of mAb1 H chain, and paired mutations at the CH1-CL interface mAb1 are introduced that force the correct pairing of the two different free L chains. Two different sets of these CH1-CL interface mutations, called CR3 and MUT4, were designed and tested, and prototypic bispecific Abs directed against CD5 and HLA-DR were produced (CD5xDR). Two different hinge sequences between mAb1 and mAb2 were also tested in the CD5xDR-CR3 or -MUT4 background, leading to bispecific Ab (BsAbs) with a more rigid or flexible structure. All four Abs produced bound with good specificity and affinity to CD5 and HLA-DR present either on the same target or on different cells. Indeed, the BsAbs were able to efficiently redirect killing of HLA-DR+ leukemic cells by human CD5+ cytokine-induced killer T cells. Finally, all BsAbs had a functional Fc, as shown by their capacity to activate human complement and NK cells and to mediate phagocytosis. CD5xDR-CR3 was chosen as the best format because it had overall the highest functional activity and was very stable in vitro in both neutral buffer and in serum. In vivo, CD5xDR-CR3 was shown to have significant therapeutic activity in a xenograft model of human leukemia.

The α1,6-Fucosyltransferase Gene (fut8) from the Sf9 Lepidopteran Insect Cell Line: Insights into fut8 Evolution

The core alpha1,6-fucosyltransferase (FUT8) catalyzes the transfer of a fucosyl moiety from GDP-fucose to the innermost asparagine-linked N-acetylglucosamine residue of glycoproteins. In mammals, this glycosylation has an important function in many fundamental biological processes and although no essential role has been demonstrated yet in all animals, FUT8 amino acid (aa) sequence and FUT8 activity are very well conserved throughout the animal kingdom. We have cloned the cDNA and the complete gene encoding the FUT8 in the Sf9 (Spodoptera frugiperda) lepidopteran cell line. As in most animal genomes, fut8 is a single-copy gene organized in different exons. The open reading frame contains 12 exons, a characteristic that seems to be shared by all lepidopteran fut8 genes. We chose to study the gene structure as a way to characterize the evolutionary relationships of the fut8 genes in metazoans. Analysis of the intron-exon organization in 56 fut8 orthologs allowed us to propose a model for fut8 evolution in metazoans. The presence of a highly variable number of exons in metazoan fut8 genes suggests a complex evolutionary history with many intron gain and loss events, particularly in arthropods, but not in chordata. Moreover, despite the high conservation of lepidoptera FUT8 sequences also in vertebrates and hymenoptera, the exon-intron organization of hymenoptera fut8 genes is order-specific with no shared exons. This feature suggests that the observed intron losses and gains may be linked to evolutionary innovations, such as the appearance of new orders.

Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging

PURPOSE For early atherosclerosis imaging, magnetic oil-in-water nanoemulsion (NE) decorated with atheroma specific monoclonal antibody was designed for Magnetic Particle Imaging (MPI) and Magnetic Resonance Imaging (MRI). MPI is an emerging technique based on direct mapping of superparamagnetic nanoparticles which may advantageously complement MRI. METHODS NE oily droplets were loaded with superparamagnetic iron oxide nanoparticles of 7, 11 and 18nm and biofunctionalized with atheroma specific scFv-Fc TEG4-2C antibody. RESULTS Inclusion of nanoparticles inside NE did not change the hydrodynamic diameter of the oil droplets, close to 180nm, nor the polydispersity. The droplets were negatively charged (ζ=-30mV). In vitro MPI signal was assessed by Magnetic Particle Spectroscopy (MPS). NE displayed MRI and MPS signals confirming its potential as new contrast agent. NE MPS signal increase with NPs size close to the gold standard (Resovist). In MRI, NE displayed R2* transversal relaxivity of 45.45, 96.04 and 218.81mM-1s-1 for 7, 11 and 18nm respectively. NE selectively bind atheroma plaque both in vitro and ex vivo in animal models of atherosclerosis. CONCLUSION Magnetic NE showed reasonable MRI/MPS signals and a significant labelling of the atheroma plaque. These preliminary results support that NE platform could selectively image atherosclerosis.

Data on atherosclerosis specific antibody conjugation to nanoemulsions

This article present data related to the publication entitled “Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging” (Prévot et al., 2017) [1]. Herein we describe the engineering in the baculovirus-insect cell system and purification processes of the human scFv-Fc TEG4-2C antibody, specific of platelets within the atheroma plaque. For molecular targeting purpose, atheroma specific antibody was conjugated to nanoemulsions (NEs) using a heterobifunctional linker (DSPE-PEG-maleimide). Atheroma labelling was assayed by immunochemistry on arterial sections from rabbits.