Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.Key words: mAb IgG, neonatal Fc receptor (FcRn), pharmacokinetics, subcutaneous bioavailability, animal model, minipig     

Single cell-produced and in vitro-assembled anti-FcRH5/CD3 T-cell dependent bispecific antibodies have similar in vitro and in vivo properties

Bispecific antibody production using single host cells has been a new advancement in the antibody engineering field. We previously showed comparable in vitro biological activity and in vivo mouse pharmacokinetics (PK) for two novel single cell variants (v10 and v11) and one traditional dual cell in vitro-assembled anti-human epidermal growth factor receptor 2/CD3 T-cell dependent bispecific (TDB) antibodies. Here, we extended our previous work to assess single cell-produced bispecific variants of a novel TDB against FcRH5, a B-cell lineage marker expressed on multiple myeloma (MM) tumor cells. An in vitro-assembled anti- FcRH5/CD3 TDB antibody was previously developed as a potential treatment option for MM. Two bispecific antibody variants (designs v10 and v11) for manufacturing anti-FcRH5/CD3 TDB in single cells were compared to in vitro-assembled TDB in a dual-cell process to understand whether differences in antibody design and production led to any major differences in their in vitro biological activity, in vivo mouse PK, and PK/pharmacodynamics (PD) or immunogenicity in cynomolgus monkeys (cynos). The binding, in vitro potencies, in vitro pharmacological activities and in vivo PK in mice and cynos of these single cell TDBs were comparable to those of the in vitro-assembled TDB. In addition, the single cell and in vitro-assembled TDBs exhibited robust PD activity and comparable immunogenicity in cynos. Overall, these studies demonstrate that single cell-produced and in vitro-assembled anti-FcRH5/CD3 T-cell dependent bispecific antibodies have similar in vitro and in vivo properties, and support further development of single-cell production method for anti-FcRH5/CD3 TDBs and other single-cell bispecifics.     

Denitrification in a binary culture and thiocyanate metabolism in Thiohalophilus thiocyanoxidans gen. nov. sp. nov. – a moderately halophilic chemolithoautotrophic sulfur-oxidizing Gammaproteobacterium from hypersaline lakes

Anaerobic enrichment culture with thiocyanate as electron donor and nitrate as electron acceptor at 2 M NaCl inoculated with a mixture of sediments from hypersaline lakes in Kulunda Steppe (Altai, Russia) resulted in a selection of a binary consortium of moderately halophilic, obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) capable of complete denitrification of nitrate with thiosulfate as the electron donor. One consortium member, strain HRhD 3sp, was isolated into pure culture with nitrate and thiosulfate using a density gradient. This strain was responsible for the reduction of nitrate to nitrite in the consortium, while a second strain, HRhD 2, isolated under microoxic conditions with thiosulfate as substrate, was capable of anaerobic growth with nitrite and thiosulfate. Nitrite, either as substrate or as product, was already toxic at very low concentrations for both strains. As a result, optimal growth under anaerobic conditions could only be achieved within the consortium. On the basis of phylogenetic analysis, both organisms were identified as new lineages within the Gammaproteobacteria. As well as thiosulfate, strain HRhD 2 can also use thiocyanate as electron donor, representing a first halophilic SOB capable of growth with thiocyanate at 2–4 M NaCl. Product and enzymatic analysis identified the “carbonyl sulfide (COS) pathway” of primary thiocyanate degradation in this new species. On the basis of phenotypic and genetic analysis, strain HRhD 2 is proposed to be assigned to a new genus and species Thiohalophilus thiocyanoxidans. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microbial sulfide oxidation in the oxic–anoxic transition zone of freshwater sediment: involvement of lithoautotrophic Magnetospirillum strain J10

The oxic–anoxic transition zone (OATZ) of freshwater sediments, where opposing gradients exist of reduced iron and sulfide with oxygen, creates a suitable environment for microorganisms that derive energy from the oxidation of iron or sulfide. Gradient microcosms incubated with freshwater sediment showed rapid microbial turnover of sulfide and oxygen compared with sterile systems. Microcosms with FeS as a substrate also showed growth at the OATZ and subsequent dilution series resulted in the isolation of three novel strains, of which strain J10 grows chemolithoautotrophically with reduced sulfur compounds under microaerobic conditions. All three strains are motile spirilla with bipolar flagella, related to the genera Magnetospirillum and Dechlorospirillum within the Alphaproteobacteria . Strain J10 is closely related to Magnetospirillum gryphiswaldense and is the first strain in this genus found to be capable of autotrophic growth. Thiosulfate was oxidized completely to sulfate, with a yield of 4 g protein mol⁻¹ thiosulfate, and autotrophic growth was evidenced by incorporation of ¹³C derived from bicarbonate into biomass. A putative gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase type II was identified in strain J10, suggesting that the Calvin–Benson–Bassham cycle is used for autotrophic growth. Analogous genes are also present in other magnetospirilla, and in the autotrophically growing alphaproteobacterium magnetic vibrio MV-1.     

Aquatic Habitat Dynamics along a Braided Alpine River Ecosystem (Tagliamento River, Northeast Italy)

Aquatic habitat change caused by flooding was quantified along the Fiume Tagliamento, a morphologically intact gravel-bed river ecosystem in northeast Italy. Five different geomorphic reaches (each around 1.5 km), ranging from near the headwaters at 800 m above sea level (a.s.l.) to near the mouth at 5 m a.s.l., were studied over a 1-year period. All floodplain water bodies in each reach were delineated in August 1999 using a differential global positioning system. Each reach was remapped twice (in December 1999 and August 2000) to investigate the impact of autumn and spring flood seasons on aquatic habitat composition and configuration. A high degree (nearly 62%) of aquatic habitat turnover was documented in a braided headwater floodplain. The degree of aquatic habitat turnover decreased with decreasing elevation to approximately 20% turnover in a meandering reach at 5 m a.s.l. In contrast to turnover, braiding, sinuosity, and aquatic habitat composition changed little in response to flooding in all reaches. Location of aquatic habitats in floodplains changed considerably (turnover), whereas habitat configuration and composition remained relatively stable. These results support the applicability of the shifting mosaic steady-state model to riverine floodplain environments.     

Inundation Dynamics in Braided Floodplains: Tagliamento River, Northeast Italy

The relationships among water level, inundated area, and shoreline dynamics were investigated in a bar-braided and an island-braided floodplain of the Tagliamento River in northeast Italy. Ground-based surveys with a differential global positioning system (aGPS) unit were used to delineate all aquatic-terrestrial interfaces (shorelines) in the active floodplain at different water levels. Despite complex inundation patterns, a highly significant (P < 0.00001) linear relationship between water level and arcsine square root of inundated area was found in both reaches (y = 0.49x + 0.07). A highly significant (P < 0.00009) second-order polynomial relationship occurred between water level and shoreline length (y = 87.83 − 65.85x² + 169.83x). Using these relationships as simple predictive models, we converted several years of water-level data into predictions for degree of inundation and shoreline length. The plot of the simulated degree of inundation strongly resembled the actual hydrograph. Complete inundation of the active floodplains occurred one or two times per year; however, the degree of inundation at lower water levels was highly dynamic during most of the year. Simulated shoreline length averaged 171 m ha⁻¹ (13.6 km km⁻¹), with a maximum of 197 m ha⁻¹ (15.6 km km⁻¹) occurring during periods with intermediate water levels. The corresponding values determined with GPS were somewhat higher, with an average value of 181 m ha⁻¹ (14.4 km km⁻¹) and a maximum of 214 m ha⁻¹ (16.3 km km⁻¹). During major flood events, actual shoreline length decreased to 28 m ha⁻¹ (2.1 km km⁻¹). Braiding index and upstream surface hydrologic connectivity were positively related to water level, whereas total area of isolated water bodies was negatively related to water level. The number of nodes remained high most of the time during the 2-year study period.