Nanobody-based microfluidic human Fc assay for preclinical plasma quantification of IgG1/1.1 and IgG1-Fc-conjugates.

BACKGROUND: Therapeutic antibodies and Fc-conjugates are becoming increasingly popular for disease management and accurate and sensitive pharmacokinetic measurements are critical in lead candidate selection in pre-clinical drug discovery. METHODS AND STUDY DESIGN: Human Fc-specific intact monoclonal antibodies, polyclonal antibodies, Fab fragments, aptamers, affibodies and nanobodies were screened for potential as biotinylated capture moieties in a microfluidic assay. Test compounds were Bevacizumab, Rituximab, Infliximab as well as an in-house IgG1.1 and an IgG1-drug conjugate. RESULTS: Capture molecules were tested for specificity in plasma matrices from beagle dog, rat, mouse, pig, rhesus monkey and cynomolgus monkey. We find that the llama nanobody provides the best selectivity across across species. The assay usability were verified in cynomolgus monkey pharmacokinetic studies of in-house IgG1.1 and IgG1-fusion molecules. CONCLUSION: The presented generic nanobody-based assay may find relevance in preclinical testing of future human Fc-containing drug conjugates devoid of Fab fragments and intact monoclonal antibodies.

Duplex and triplex formation of mixed pyrimidine oligonucleotides with stacking of phenyl-triazole moieties in the major groove.

5-(1-Phenyl-1,2,3-triazol-4-yl)-2'-deoxycytidine was synthesized from a modified CuAAC protocol and incorporated into mixed pyrimidine oligonucleotide sequences together with the corresponding 5-(1-phenyl-1,2,3-triazol-4-yl)-2'-deoxyuridine. With consecutive incorporations of the two modified nucleosides, improved duplex formation with a complementary RNA and improved triplex formation with a complementary DNA duplex were observed. The improvement is due to π-π stacking of the phenyl-triazole moieties in the major groove. The strongest stacking and most pronounced positive influence on thermal stability was found in between the uridine analogues or with the cytidine analogue placed in the 3' direction to the uridine analogue. Modeling indicated a different orientation of the phenyl-triazole moieties in the major groove to account for the difference between the two nucleotides. The modified oligonucleotides were all found to be significantly stabilized toward nucleolytic degration.