Human cell lines for biopharmaceutical manufacturing: history,status, and future perspectives

Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.     

Mutation of Threonine 34 in Mouse Podoplanin-Fc Reduces CLEC-2 Binding and Toxicity in Vivo While Retaining Anti-lymphangiogenic Activity

The lymphatic system plays an important role in cancer metastasis and inhibition of lymphangiogenesis could be valuable in fighting cancer dissemination. Podoplanin (Pdpn) is a small, transmembrane glycoprotein expressed on the surface of lymphatic endothelial cells (LEC). During mouse development, binding of Pdpn to the C-type lectin-like receptor 2 (CLEC-2) on platelets is critical for the separation of the lymphatic and blood vascular systems. Competitive inhibition of Pdpn functions with a soluble form of the protein, Pdpn-Fc, leads to reduced lymphangiogenesis in vitro and in vivo. However, the transgenic overexpression of human Pdpn-Fc in mouse skin causes disseminated intravascular coagulation due to platelet activation via CLEC-2. In the present study, we produced and characterized a mutant form of mouse Pdpn-Fc, in which threonine 34, which is considered essential for CLEC-2 binding, was mutated to alanine (PdpnT34A-Fc). Indeed, PdpnT34A-Fc displayed a 30-fold reduced binding affinity for CLEC-2 compared with Pdpn-Fc. This also translated into fewer side effects due to platelet activation in vivo. Mice showed less prolonged bleeding time and fewer embolized vessels in the liver, when PdpnT34A-Fc was injected intravenously. However, PdpnT34A-Fc was still as active as wild-type Pdpn-Fc in inhibiting lymphangiogenesis in vitro and also inhibited lymphangiogenesis in vivo. These data suggest that the function of Pdpn in lymphangiogenesis does not depend on threonine 34 in the CLEC-2 binding domain and that PdpnT34A-Fc might be an improved inhibitor of lymphangiogenesis with fewer toxic side effects.     

Experience Does Not Equal Expertise in Recognizing Infrequent Incoming Gunfire: Neural Markers for Experience and Task Expertise at Peak Behavioral Performance

For a soldier, decisions to use force can happen rapidly and sometimes lead to undesired consequences. In many of these situations, there is a rapid assessment by the shooter that recognizes a threat and responds to it with return fire. But the neural processes underlying these rapid decisions are largely unknown, especially amongst those with extensive weapons experience and expertise. In this paper, we investigate differences in weapons experts and non-experts during an incoming gunfire detection task. Specifically, we analyzed the electroencephalography (EEG) of eleven expert marksmen/soldiers and eleven non-experts while they listened to an audio scene consisting of a sequence of incoming and non-incoming gunfire events. Subjects were tasked with identifying each event as quickly as possible and committing their choice via a motor response. Contrary to our hypothesis, experts did not have significantly better behavioral performance or faster response time than novices. Rather, novices indicated trends of better behavioral performance than experts. These group differences were more dramatic in the EEG correlates of incoming gunfire detection. Using machine learning, we found condition-discriminating EEG activity among novices showing greater magnitude and covering longer periods than those found in experts. We also compared group-level source reconstruction on the maximum discriminating neural correlates and found that each group uses different neural structures to perform the task. From condition-discriminating EEG and source localization, we found that experts perceive more categorical overlap between incoming and non-incoming gunfire. Consequently, the experts did not perform as well behaviorally as the novices. We explain these unexpected group differences as a consequence of experience with gunfire not being equivalent to expertise in recognizing incoming gunfire.     

Testing the Role of Climate Change in Species Decline: Is the Eastern Quoll a Victim of a Change in the Weather?

To conserve a declining species we first need to diagnose the causes of decline. This is one of the most challenging tasks faced by conservation practitioners. In this study, we used temporally explicit species distribution models (SDMs) to test whether shifting weather can explain the recent decline of a marsupial carnivore, the eastern quoll (Dasyurus viverrinus). We developed an SDM using weather variables matched to occurrence records of the eastern quoll over the last 60 years, and used the model to reconstruct variation through time in the distribution of climatically suitable range for the species. The weather model produced a meaningful prediction of the known distribution of the species. Abundance of quolls, indexed by transect counts, was positively related to the modelled area of suitable habitat between 1990 and 2004. In particular, a sharp decline in abundance from 2001 to 2003 coincided with a sustained period of unsuitable weather over much of the species’ distribution. Since 2004, abundance has not recovered despite a return to suitable weather conditions, and abundance and area of suitable habitat have been uncorrelated. We suggest that fluctuations in weather account for the species’ recent decline, but other unrelated factors have suppressed recovery.