Pharmacokinetics of novel Fc-engineered monoclonal and multispecific antibodies in cynomolgus monkeys and humanized FcRn transgenic mouse models.

Monoclonal antibodies (mAbs) are among the fastest growing and most effective therapies for myriad diseases. Multispecific antibodies are an emerging class of novel therapeutics that can target more than one tumor- or immune-associated modulators per molecule. The combination of different binding affinities and target classes, such as soluble or membrane-bound antigens, within multispecific antibodies confers unique pharmacokinetic (PK) properties. Numerous factors affect an antibody's PK, with affinity to the neonatal Fc receptor (FcRn) a key determinant of half-life. Recent work has demonstrated the potential for humanized FcRn transgenic mice to predict the PK of mAbs in humans. However, such work has not been extended to multispecific antibodies. We engineered mAbs and multispecific antibodies with various Fc modifications to enhance antibody performance. PK analyses in humanized FcRn transgenic mouse (homozygous Tg32 and Tg276) and non-human primate (NHP) models showed that FcRn-binding mutations improved the plasma half-lives of the engineered mAbs and multispecific antibodies, while glycan engineering to eliminate effector function did not affect the PK compared with wild-type controls. Furthermore, results suggest that the homozygous Tg32 mouse model can replace NHP models to differentiate PK of variants during lead optimization, not only for wild-type mAbs but also for Fc-engineered mAbs and multispecific antibodies. This Tg32-mouse model would enable prediction of half-life and linear clearance of mAbs and multispecific antibodies in NHPs to guide the design of further pharmacology/safety studies in this species. The allometric exponent for clearance scaling from Tg32 mice to NHPs was estimated to be 0.91 for all antibodies.

Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life.

The neonatal Fc receptor (FcRn) promotes antibody recycling through rescue from normal lysosomal degradation. The binding interaction is pH-dependent with high affinity at low pH, but not under physiological pH conditions. Here, we combined rational design and saturation mutagenesis to generate novel antibody variants with prolonged half-life and acceptable development profiles. First, a panel of saturation point mutations was created at 11 key FcRn-interacting sites on the Fc region of an antibody. Multiple variants with slower FcRn dissociation kinetics than the wildtype (WT) antibody at pH 6.0 were successfully identified. The mutations were further combined and characterized for pH-dependent FcRn binding properties, thermal stability and the FcγRIIIa and rheumatoid factor binding. The most promising variants, YD (M252Y/T256D), DQ (T256D/T307Q) and DW (T256D/T307W), exhibited significantly improved binding to FcRn at pH 6.0 and retained similar binding properties as WT at pH 7.4. The pharmacokinetics in human FcRn transgenic mice and cynomolgus monkeys demonstrated that these properties translated to significantly prolonged plasma elimination half-life compared to the WT control. The novel variants exhibited thermal stability and binding to FcγRIIIa in the range comparable to clinically validated YTE and LS variants, and showed no enhanced binding to rheumatoid factor compared to the WT control. These engineered Fc mutants are promising new variants that are widely applicable to therapeutic antibodies, to extend their circulation half-life with obvious benefits of increased efficacy, and reduced dose and administration frequency.

Feedback from the European Bioanalysis Forum liquid microsampling consortium: capillary liquid microsampling and assessment of homogeneity of the resultant samples.

Following the completion of a detailed experimental protocol into the potential inhomogeneity of capillary liquid microsamples, which was performed at seven European Bioanalysis Forum member companies, the summary and conclusion on the data are reported here. It has been demonstrated that it is possible to generate homogeneous samples using these microsampling techniques; that the resultant microsamples can be accurate and precise and that capillary liquid microsampling data can be consistent with conventional larger volume plasma samples. However, the data contain some variability which is contributed to by the different range of experiences that each investigating site had with these techniques. Therefore, knowledge of the compounds, well-designed experiments and experience with these techniques are essential for the delivery of high quality data.

Feedback from the European Bioanalysis Forum liquid microsampling consortium: microsampling: assessing accuracy and precision of handheld pipettes and capillaries.

Aim: Microsampling in preclinical pharmacokinetics (PK) studies is currently widely adopted across the pharmaceutical industry. Materials & methods: The European Bioanalysis Forum liquid microsampling consortium member companies assessed the accuracy and precision of handheld pipettes and microcapillaries at volumes of less than 10 µl. The following key factors on pipetting performance were also evaluated: Pipette type (positive displacement, air displacement and microcapillary), experience of user and the liquid type. Water was selected as a best-case scenario for accuracy and precision determination and blood plasma as a 'real world' bioanalysis sample type. Conclusion: Accuracy and precision on the pipetted volume decreased at lower volumes and experienced laboratory technicians performed better compared with the infrequent users. With respect to the pipetting devices used, microcapillaries showed better or equivalent accuracy and precision compared with handheld pipettes across the volume range 1-8 µl independent of the matrix used.

In vivo visualization of osteoarthritic hypertrophic lesions.

Osteoarthritis (OA) is one of the most common diseases in the aging population. While disease progress in humans is monitored indirectly by X-ray or MRI, small animal OA lesions detection always requires surgical intervention and histology. Here we introduce bimodal MR/NIR probes based on cartilage-targeting 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid amide (DOTAM) that are directly administered to the joint cavity. We demonstrate applications in healthy and diseased rat joints by MRI in vivo. The same joints are inspected post-mortem by fluorescence microscopy, showing not only the precise location of the reagents but also revealing details such as focal cartilage damage and chondrophyte or osteophyte formation. This allows for determining the distinct pathological state of the disease and the regeneration capability of the animal model and will help to correctly assess the effect of potential disease modifying OA drugs (DMOADs) in the future.