Reconciling ice core CO2 and land-use change following New World-Old World contact.

Ice core records of carbon dioxide (CO2) throughout the last 2000 years provide context for the unprecedented anthropogenic rise in atmospheric CO2 and insights into global carbon cycle dynamics. Yet the atmospheric history of CO2 remains uncertain in some time intervals. Here we present measurements of CO2 and methane (CH4) in the Skytrain ice core from 1450 to 1700 CE. Results suggest a sudden decrease in CO2 around 1610 CE in one widely used record may be an artefact of a small number of anomalously low values. Our analysis supports a more gradual decrease in CO2 of 0.5 ppm per decade from 1516 to 1670 CE, with an inferred land carbon sink of 2.6 PgC per decade. This corroborates modelled scenarios of large-scale reorganisation of land use in the Americas following New World-Old World contact, whereas a rapid decrease in CO2 at 1610 CE is incompatible with even the most extreme land-use change scenarios.

Discovery and optimization of a novel anti-GUCY2c x CD3 bispecific antibody for the treatment of solid tumors.

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

Modeling and mitigation of high-concentration antibody viscosity through structure-based computer-aided protein design.

For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported.

Direct Injection Liquid Chromatography High-Resolution Mass Spectrometry for Determination of Primary and Secondary Terrestrial and Marine Biomarkers in Ice Cores.

Many atmospheric organic compounds are long-lived enough to be transported from their sources to polar regions and high mountain environments where they can be trapped in ice archives. While inorganic components in ice archives have been studied extensively to identify past climate changes, organic compounds have rarely been used to assess paleo-environmental changes, mainly due to the lack of suitable analytical methods. This study presents a new method of direct injection high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, without the need of preconcentrating the melted ice, for the determination of a series of novel biomarkers in ice core samples indicative of primary and secondary terrestrial and marine organic aerosol sources. Eliminating a preconcentration step reduces contamination potential and decreases the required sample volume thus allowing a higher time resolution in the archives. The method is characterized by limits of detection (LODs) in the range of 0.01-15 ppb, depending on the analyte, and accuracy evaluated through an interlaboratory comparison. We find that many components in secondary organic aerosols (SOAs) are clearly detectable at concentrations comparable to those previously observed in replicate preconcentrated ice samples from the Belukha glacier, Russian Altai Mountains. Some compounds with low recoveries in the preconcentration steps are now detectable in samples with this new direct injection method significantly increasing the range of environmental processes and sources that become accessible for paleo-climate studies.

A new method for the determination of primary and secondary terrestrial and marine biomarkers in ice cores using liquid chromatography high-resolution mass spectrometry.

The majority of atmospheric compounds measured in ice cores are inorganic, while analysis of their organic counterparts is a less well developed field. In recent years, understanding of formation, transport pathways and preservation of these compounds in ice and snow has improved, showing great potential for their use as biomarkers in ice cores. This study presents an optimised analytical technique for quantification of terrestrial and marine biosphere emissions of secondary organic aerosol (SOA) components and fatty acids in ice using HPLC-MS analysis. Concentrations of organic compounds in snow and ice are extremely low (typically ppb or ppt levels) and thus pre-concentration is required prior to analysis. Stir bar sorptive extraction (SBSE) showed potential for fatty acid compounds, but failed to recover SOA compounds. Solid phase extraction (SPE) recovered compounds across both organic groups but methods improving some recoveries came at the expense of others, and background contamination of fatty acids was high. Rotary evaporation was by far the best performing method across both SOA and fatty acid compounds, with average recoveries of 80%. The optimised preconcentration - HPLC-MS method achieved repeatability of 9% averaged for all compounds. In environmental samples, both concentrations and seasonal trends were observed to be reproducible when analysed in two different laboratories using the same method.

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics.

A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1-5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1-3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics.

High-throughput measurement, correlation analysis, and machine-learning predictions for pH and thermal stabilities of Pfizer-generated antibodies.

Generating stable antibodies is an important goal in the development of antibody-based drugs. Often, thermal stability is assumed predictive of overall stability. To test this, we used different internally created antibodies and first studied changes in antibody structure as a function of pH, using the dye ANS. Comparison of the pH(50) values, the midpoint of the transition from the high-pH to the low-pH conformation, allowed us for the first time to rank antibodies based on their pH stability. Next, thermal stability was probed by heating the protein in the presence of the dye Sypro Orange. A new data analysis method allowed extraction of all three antibody unfolding transitions and showed close correspondence to values obtained by differential scanning calorimetry. T(1%) , the temperature at which 1% of the protein is unfolded, was also determined. Importantly, no correlations could be found between thermal stability and pH(50) , suggesting that to accurately quantify antibody stability, different measures of protein stability are necessary. The experimental data were further analyzed using a machine-learning approach with a trained model that allowed the prediction of biophysical stability using primary sequence alone. The pH stability predictions proved most successful and were accurate to within pH ±0.2.