Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complementenhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens.Key words: antibody, Fc, complement, CDC, C1q, ADCC, phagocytosis, CD20, CD19, CD40     

Survival of Escherichia coli, Enterococci, and Campylobacter spp. in sheep feces on pastures

The survival of enteric bacteria in 10 freshly collected sheep fecal samples on pastures was measured in each of four seasons. Ten freshly collected feces were placed on pasture, and concentrations of Escherichia coli, enterococci, and Campylobacter spp. were monitored until exhaustion of the fecal samples. In all four seasons, there was an increase in enterococcal concentrations by up to 3 orders of magnitude, with peak concentrations recorded between 11 and 28 days after deposition. E. coli concentrations increased in three out of four seasons by up to 1.5 orders of magnitude, with peak concentrations recorded between 8 and 14 days after deposition. The apparent growth of E. coli and enterococci was strongly influenced by the initial water content of the feces and the moisture gained during periods of rehydration following rainfalls. Conversely, the results suggested that dehydration promoted inactivation. Campylobacter spp. did not grow and were rapidly inactivated at a rate that tended to be faster at higher temperatures. Pulsed-field gel electrophoresis (PFGE) of a selection of Campylobacter spp. suggested that these survival data are applicable to a range of Campylobacter spp., including the most frequently isolated PFGE genotype from sheep in New Zealand, and to genotypes previously observed to cause disease in humans. The results of this study are currently being incorporated into a fecal microbe reservoir model that is designed to assist water managers' abilities to estimate microbial loads on pastures grazed by sheep, including the influence of factors such as rainfall and temperature.     

The roles of the shikimate pathway genes,aroA and aroB,in virulence,growth and UV tolerance of Burkholderia glumae strain 411gr‐6

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B. glumae strains showed pigmentation phenotypes producing at least two (yellow–green and purple) pigment compounds in casein–peptone–glucose agar medium. The B. glumae strains LSUPB114 and LSUPB116 are pigment‐deficient mutant derivatives of the virulent and pigment‐proficient strain 411gr‐6, having mini‐Tn5gus insertions in aroA encoding 3‐phosphoshikimate 1‐carboxyvinyltransferase and aroB encoding 3‐dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B. glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.     

Detrimental Type I Interferon Signaling Dominates Protective AIM2 Inflammasome Responses during Francisella novicida Infection

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Grazing Lawns in Terai Grasslands,Royal Bardia National Park,Nepal1

We compared the community structure, nutritive quality, and aboveground biomass of grazing lawns (patches of shortgrass communities) to neighboring grasslands in the Terai of western Nepal. Grazing lawns differed from the adjacent grasslands in species composition and community structure. Species diversity and species richness were higher on grazing lawns (H = 1.60, S = 20.93) than the grasslands (H = 0.97, S = 8.97). Fencing that excluded grazers for 150 days made areas of grazing lawns indistinguishable from neighboring grasslands in terms of plant height and biomass. Growing shoots of forage from grazing lawns had higher digestibility, crude protein, and sodium than forage from the grasslands. Grazing lawns appear to be maintained by continuous grazing and are enriched by deposition of urine, dung, and by certain plant species not found in the the adjacent grasslands.     

An expeditious synthesis of blood-group antigens,ABO histo-blood group type II antigens and xenoantigen oligosaccharides with amino type spacer−arms

Blood group oligosaccharides are one of the most clinically important antigen families and they may also act as secondary ligands for bacterial toxins from Escherichia coli and Vibrio cholerae. Herein we report the synthesis of spacered (sp = CH₂CH₂CH₂NH₂) glycosides of A antigen {α-D-GalNAc-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-}, B antigen{α-D-Gal-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-}, LewisX{α-D-Gal-(l→4)-[α-L-Fuc-(l→3)]-β-D-GlcNAc-}, A type-II {α-D-GalNAc-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-(1→4)-β-D-GlcNAc-}, B type-II {α-D-Gal-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-(1→4)-β-D-GlcNAc-}, H type-II{α-L-Fuc-(l→2)-β-D-Gal-(1→4)-β-D-GlcNAc-}, xenoantigen {α-D-Gal-(l→3)-β-D-Gal-(1→4)-[α-L-Fuc-(l→2)]-β-D-GlcNAc-} and Linear B Type II {α-D-Gal-(l→3)-β-D-Gal-(1→4)-β-D-GlcNAc-} useful for a range of biochemical investigations. This linker was chosen so as to facilitate the future conjugation of the antigens to proteins or other molecules. We also measured the affinities of some synthesized oligosaccharides against El Tor CTB strain from V. cholera.     

Small-Scale Assays for Studying Dissolution of Pharmaceutical Cocrystals for Oral Administration

The purpose of this study was to better understand the dissolution properties and precipitation behavior of pharmaceutical cocrystals of poorly soluble drugs for the potential for oral administration based on a small-scale dissolution assay. Carbamazepine and indomethacin cocrystals with saccharin and nicotinamide as coformers were prepared with the sonic slurry method. Dissolution of the poorly soluble drugs indomethacin and carbamazepine and their cocrystals was studied with a small-scale dissolution assay installed on a SiriusT3 instrument. Two methodologies were used: (i) surface dissolution of pressed tablet (3 mm) in 20 mL running for fixed times at four pH stages (pH 1.8, pH 3.9, pH 5.4, pH 7.3) and (ii) powder dissolution (2.6 mg) in 2 mL at a constant pH (pH 2). Improved dissolution and useful insights into precipitation kinetics of poorly soluble compounds from the cocrystal form can be revealed by the small-scale dissolution assay. A clear difference in dissolution/precipitation behaviour can be observed based on the characteristics of the coformer used.