Development of a pre-glycoengineered CHO-K1 host cell line for the expression of antibodies with enhanced Fc mediated effector function

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. Glycoengineered HCLs were generated in a two-step strategy, starting with generating parental HCLs by stable transfection of CHO-K1 cells with GnT-III and Man-II. In a second step, parental HCLs were stably transfected a second time with these two transgenes to increase their copy number in the genetic background. Generated glycoengineered CHO-K1 cell lines expressing two different mAbs deliver antibody products with a content of more than 60% a-fucosylated glycans. In-depth analysis of the N-glycan structure revealed that the majority of the Fc-attached glycans of the obtained mAbs were of complex bisected type. Furthermore, we showed the efficient use of FcγRIIIa affinity chromatography as a novel method for the fast assessment of the mAbs a-fucosylation level. By testing different cultivation conditions for the pre-glycoengineered recombinant CHO-K1 clones, we identified key components essential for the production of a-fucosylated mAbs. The prevalent effect could be attributed to the trace element manganese, which leads to a strong increase of a-fucosylated complex- and hybrid-type glycans. In conclusion, the novel pre-glycoengineered CHO-K1 HCL can be used for the production of antibodies with high ratios of a-fucosylated Fc-attached N-glycans. Application of our newly developed FcγRIIIa affinity chromatography method during cell line development and use of optimized cultivation conditions can ultimately support the efficient development of a-fucosylated mAbs.     

Changes in microvascular fluid filtration capacity during 120 days of 6 degrees head-down tilt.

We used venous congestion strain gauge plethysmography (VCP) to measure the changes in fluid filtration capacity (K(f)), isovolumetric venous pressure (Pv(i)), and blood flow in six volunteers before, on the 118th day (D118) of head-down tilt (HDT), and 2 days after remobilization (Post). We hypothesized that 120 days of HDT cause significant micro- and macrovascular changes. We observed a significant increase in K(f) from 3.6 +/- 0.4 x 10(-3) to 5.7 +/- 0.9 x 10(-3) ml. min(-1). 100 ml(-1). mmHg(-1) (+51.4%; P < 0.003), which returned to pretilt values (4.0 + 0.4 x 10(-3) ml. min(-1). 100 ml(-1). mmHg(-1)) after remobilization. Similarly, Pv(i) increased from 13.4 +/- 2.1 mmHg to 28.9 +/- 2.8 mmHg (+105.8%; P < 0.001) at D118 and was not significantly different at Post (12.4 +/- 2.6 mmHg). Blood flow decreased significantly from 2.3 +/- 0.3 to 1.3 +/- 0.2 ml. min(-1). 100 ml tissue(-1) at D118 and was found elevated to 3.4 +/- 0.7 ml. min(-1). 100 ml tissue(-1) at Post. We believe that the increased K(f) is caused by a higher microvascular water permeability. Because this may result in edema formation, it could contribute to the alterations in fluid homeostasis after exposure to microgravity.     

Zeitverlauf der Änderungen an energiereichen Phosphorverbindungen im Meerschweinchenherzen nach Asphyxie und Wiederbeatmung

Zusammenfassung 1. Nach verschieden langer Dauer von Asphyxie und anschließender Wiederbeatmung wurde im Meerschweinchenherzen ATP, ADP, AMP, Kreatinphosphat und anorganisches Phosphat bestimmt.2. Bereits 1 Minute nach Asphyxie kam es zu einem deutlichen Abfall von Kreatinphosphat und Anstieg von anorganischem Phosphat; 5 Minuten nach Asphyxie war Kreatinphosphat nicht mehr nachweisbar.3. ATP fiel 2 Minuten nach Asphyxie ab und zeigte einen weiteren Abfall bis 15 Minuten nach Asphyxie, während AMP zuerst einen Anstieg, später einen Abfall zeigte.4. Bei Asphyxiedauer von 1 Minute regenerierten sich die energiereichen Phosphorverbindungen im Meerschweinchenherzen nach Widerbeatmung von 1/2 Minute und bei 5 Minuten Asphyxiedauer nach 9 Minuten Wiederbeatmung.5. Dauert die Asphyxie 7 Minuten, so ist keine vollständige Regeneration der energiereichen Phosphorverbindungen mehr erreichbar.6. War bei der Wiederbeatmung eine 50- bis 60prozentige Regeneration des Kreatinphosphatanteiles eingetreten, so verschwanden im EKG regelmäßig die Rhythmusstörungen.

---

Time course of changes in energy-rich phosphate compounds in the heart of the guinea pig following asphyxia and renimation

In the myocardium of guinea pigs the levels of ATP, ADP, AMP, creatine phosphate and inorganic phosphate, respectively, were determined following different periods of asphyxia and subsequent reanimation. Depending upon the duration of asphyxia, all energy-rich phosphate compounds decreased, creatine phosphate demonstrating this effect most markedly. Following reanimation, the energy-rich phosphate compounds underwent partial or complete regeneration, unless asphyxia lasted longer than 5 to 7 minutes.

     

Presumptive monosomy 21 with neuronal migration disorder re-diagnosed as de novo unbalanced translocation t(18p;21q) by fluorescence in situ hybridisation

We present clinical and cytogenetic data of a one year old boy with partial monosomy for both 21q and 18p, resulting from a de novo unbalanced translocation. The initial diagnosis of a seemingly full monosomy 21 was revised after fluorescence in situ hybridisation (FISH) with whole chromosome painting probes and a locus-specific chromosome 21 probe. The karyotype was reinterpreted as 45,XY,der(18)t(18;21)(p11.2;q22.1),-21. This karyotype, to our knowledge, has not been previously described. The boy presented with a spectrum of clinical features previously described for (partial) monosomy 18p only, for monosomy 21q only, or for both of these aneusomies. The radiological finding of a neuronal migration disorder with localised polymicrogyria (cortical dysplasia) has not been described for either monosomy before.     

Functional diversity along the transverse axis of hippocampal area CA1

Decades of neuroscience research have shed light on the hippocampus as a key structure for the formation of episodic memory. The hippocampus is divided into distinct subfields – CA1, CA2 and CA3. While accumulating evidence points to cellular and synaptic heterogeneity within each subfield, this heterogeneity has not received much attention in computational and behavioural studies and subfields have until recently been considered functionally uniform. However, a couple of recent studies have demonstrated prominent functional differences along the proximodistal axis of the CA1 subfield. Here, we review anatomical and physiological differences that might give rise to heterogeneity along the proximodistal axis of CA1 as well as the functional implications of such heterogeneity. We suggest that such heterogeneity in CA1 operates dynamically in the sense that the CA1 network alternates, on a subsecond scale, between a state where the network is primarily responsive to functionally segregated direct inputs from entorhinal cortex and a state where cells predominantly are controlled by more integrated inputs from CA3.