Application of platform process development approaches to the manufacturing of Mabcalin™ bispecifics.

Bispecific biotherapeutics offer potent and highly specific treatment options in oncology and immuno-oncology. However, many bispecific formats are prone to high levels of aggregation and instability, leading to prolonged development timelines, inefficient manufacturing, and high costs. The novel class of Mabcalin™ molecules consist of Anticalin® proteins fused to an IgG and are currently being evaluated in pre-clinical and clinical studies. Here, we describe a robust high-yield manufacturing platform for these therapeutic fusion proteins providing data up to commercially relevant scales. A platform upstream process was established for one of the Mabcalin bispecifics and then applied to other clinically relevant drug candidates with different IgG target specificities. Process performance was compared in 3 L bioreactors and production was scaled-up to up to 1000 L for confirmation. The Mabcalin proteins' structural and biophysical similarities enabled a downstream platform approach consisting of initial protein A capture, viral inactivation, mixed-mode anion exchange polishing, second polishing by cation exchange or hydrophobic interaction chromatography, viral filtration, buffer exchange and concentration by ultrafiltration/diafiltration. All three processes met their target specifications and achieved comparable clearance of impurities and product yields across scales. The described platform approach provides a fast and economic path to process confirmation and is well comparable to classical monoclonal antibody approaches in terms of costs and time to clinic.

Hyperactivity of the Arabidopsis cryptochrome (cry1) L407F mutant is caused by a structural alteration close to the cry1 ATP-binding site.

Plant cryptochromes (cry) act as UV-A/blue light receptors. The prototype, Arabidopsis thaliana cry1, regulates several light responses during the life cycle, including de-etiolation, and is also involved in regulating flowering time. The cry1 photocycle is initiated by light absorption by its FAD chromophore, which is most likely fully oxidized (FADox) in the dark state and photoreduced to the neutral flavin semiquinone (FADH°) in its lit state. Cryptochromes lack the DNA-repair activity of the closely related DNA photolyases, but they retain the ability to bind nucleotides such as ATP. The previously characterized L407F mutant allele of Arabidopsis cry1 is biologically hyperactive and seems to mimic the ATP-bound state of cry1, but the reason for this phenotypic change is unclear. Here, we show that cry1L407F can still bind ATP, has less pronounced photoreduction and formation of FADH° than wild-type cry1, and has a dark reversion rate 1.7 times lower than that of the wild type. The hyperactivity of cry1L407F is not related to a higher FADH° occupancy of the photoreceptor but is caused by a structural alteration close to the ATP-binding site. Moreover, we show that ATP binds to cry1 in both the dark and the lit states. This binding was not affected by cry1's C-terminal extension, which is important for signal transduction. Finally, we show that a recently discovered chemical inhibitor of cry1, 3-bromo-7-nitroindazole, competes for ATP binding and thereby diminishes FADH° formation, which demonstrates that both processes are important for cry1 function.

Lifetimes of Arabidopsis cryptochrome signaling states in vivo.

One crucial component in light signaling is the quantity of photoreceptor present in the active signaling state. The lifetime of the signaling state of a photoreceptor is limited because of thermal or otherwise back reversion of the chromophore to the ground state, and/or degradation of the photoreceptor in the light-activated state. It was previously shown that the lit state of plant cryptochromes contains flavin-neutral semiquinone, and that the half-lives of the lit state were in the range of 3-4 min in vitro. However, it was unknown how long-lived the signaling states of plant cryptochromes are in situ. Based on the loss of degradation of cry2 after prolonged dark incubation and loss of reversibility of photoactivated cry1 by a pulse of green light, we estimate the in vivo half-lives of the signaling states of cry1 and cry2 to be in the range of 5 and 16 min, respectively. Based on electron paramagnetic resonance measurements, the lifetime of the Arabidopsis cry1 lit state in insect cells was found to be ~6 min, and thus very similar to the lifetime of the signaling state in planta. Thus, the signaling state lifetimes of plant cryptochromes are not, or are only moderately, stabilized in planta.

Degradation of Arabidopsis CRY2 is regulated by SPA proteins and phytochrome A.

The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.

Photoreduction of the folate cofactor in members of the photolyase family.

Cryptochromes and DNA photolyases are related flavoproteins with flavin adenine dinucleotide as the common cofactor. Whereas photolyases repair DNA lesions caused by UV radiation, cryptochromes generally lack repair activity but act as UV-A/blue light photoreceptors. Two distinct electron transfer (ET) pathways have been identified in DNA photolyases. One pathway uses within its catalytic cycle, light-driven electron transfer from FADH(-)* to the DNA lesion and electron back-transfer to semireduced FADH(o) after photoproduct cleavage. This cyclic ET pathway seems to be unique for the photolyase subfamily. The second ET pathway mediates photoreduction of semireduced or fully oxidized FAD via a triad of aromatic residues that is conserved in photolyases and cryptochromes. The 5,10-methenyltetrahydrofolate (5,10-methenylTHF) antenna cofactor in members of the photolyase family is bleached upon light excitation. This process has been described as photodecomposition of 5,10-methenylTHF. We show that photobleaching of 5,10-methenylTHF in Arabidopsis cry3, a member of the cryptochrome DASH family, with repair activity for cyclobutane pyrimidine dimer lesions in single-stranded DNA and in Escherichia coli photolyase results from reduction of 5,10-methenylTHF to 5,10-methyleneTHF that requires the intact tryptophan triad. Thus, a third ET pathway exists in members of the photolyase family that remained undiscovered so far.

Assessment of postischemic neurogenesis in rats with cerebral ischemia and propofol anesthesia.

BACKGROUND: Postischemic endogenous neurogenesis can be dose-dependently modulated by volatile anesthetics. The intravenous anesthetic propofol is used during operations with a risk of cerebral ischemia, such as neurosurgery, cardiac surgery, and vascular surgery. The effects of propofol on neurogenesis are unknown and, therefore, the object of this study. METHODS: Eighty male Sprague-Dawley rats were randomly assigned to treatment groups with propofol administration for 3 h: 36 mg x kg(-1) x h(-1) propofol with or without cerebral ischemia and 72 mg x kg(-1) x h(-1) propofol with or without cerebral ischemia. In addition, 7 rats with propofol administration for 6 h and 14 treatment-naive rats were investigated. Forebrain ischemia was induced by bilateral carotid artery occlusion and hemorrhagic hypotension. Animals received 5-bromo-2-deoxyuridine for 7 days. 5-Bromo-2-deoxyuridine-positive neurons were counted in the dentate gyrus after 9 and 28 days. Spatial learning in the Barnes maze and histopathologic damage of the hippocampus were analyzed. RESULTS: Propofol revealed no impact on basal neurogenesis. Cerebral ischemia increased the amount of new neurons. After 28 days, neurogenesis significantly increased in animals with low-dose propofol administered during cerebral ischemia compared with naive animals, whereas no significant difference was observed in animals with high-dose propofol during ischemia. Neuronal damage in the CA3 region was increased at 28 days with high-dose propofol. Postischemic deficits in spatial learning were not affected by propofol. CONCLUSIONS: Independent effects of propofol are difficult to ascertain. Peri-ischemic propofol administration may exert secondary effects on neurogenesis by modulating the severity of histopathologic injury and thereby regenerative capacity of the hippocampus.