Effect of non-repetitive linker on in vitro and in vivo properties of an anti-VEGF scFv.

Single chain antibody fragments (scFvs) are favored in diagnostic and therapeutic fields thanks to their small size and the availability of various engineering approaches. Linker between variable heavy (VH) and light (VL) chains of scFv covalently links these domains and it can affect scFv's bio-physical/chemical properties and in vivo activity. Thus, scFv linker design is important for a successful scFv construction, and flexible linkers are preferred for a proper pairing of VH-VL. The flexibility of the linker is determined by length and sequence content and glycine-serine (GS) linkers are commonly preferred for scFvs based on their highly flexible profiles. Despite the advantage of this provided flexibility, GS linkers carry repeated sequences which can cause problems for PCR-based engineering approaches and immunogenicity. Here, two different linkers, a repetitive GS linker and an alternative non-repetitive linker with similar flexibility but lower immunogenicity are employed to generate anti-Vascular Endothelial Growth Factor scFvs derived from bevacizumab. Our findings highlight a better in vitro profile of the non-repetitive linker such as a higher monomer ratio, higher thermal stability while there was no significant difference in in vivo efficacy in a zebrafish embryonic angiogenesis model. This is the first study to compare in vivo efficacy of scFvs with different linkers in a zebrafish model.

Physicochemical determinants of antibody-protein interactions.

Antibodies are specialized proteins generated by immune system for high specificity and affinity binding to target antigens. Because of their essential roles in immune system, antibodies have been successfully developed and engineered as biopharmaceuticals for treatment of various diseases. Analysis of antibody-protein interactions is always required to get detailed information on effectivity of such antibody-based therapeutics. Although physicochemical rules cannot be generalized for every antibody-protein interaction, there are some features which should be taken into account during antibody development and engineering efforts. In this chapter, physicochemical analysis of antibody paratope-protein epitope interactions will be discussed to highlight important characteristics. First, paratope and non-paratope regions of antibodies will be described and important roles of these regions on binding and biophysical features of antibodies will be discussed. Then, general features of epitope regions of protein antigens will be introduced along with several computational/experimental tools to identify them. Lastly, a rising star of antibody biopharmaceuticals, nanobodies, will be described to show importance of next-generation antibody fragment based biopharmaceuticals in drug development.

Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues.

Phage display is one of the most widely used technology for antibody discovery and engineering. Number of therapeutic antibodies derived from phage display increases rapidly due to its ease of use and ability to control antibody sequence information. Although there are numerous antibody candidates as promising therapeutics, most of them fail at later stages of development due to undesired biophysical properties. Antibody candidates with poor properties should be prevented or improved in early development phases to minimize enormous loss of time and resources. In this study, we showed that phage display derived therapeutic antibodies show higher self-interaction and polyspecificity compared to non-phage display derived ones. To identify molecular determinants behind this, physicochemical properties of CDR regions of 137 therapeutic antibodies were analyzed. We found multiple significant differences in both heavy and light chain CDR regions. Most profoundly, aliphatic content of HCDR3, HCDR2, and LCDR3 regions were enriched in phage display derived antibodies compared to non-phage display derived ones. Physicochemical determinants documented here seem to play important roles in polyspecific and aggregation-prone natures of antibodies which should be avoided in early development phases.

Modified gold surfaces by 6-(ferrocenyl)hexanethiol/dendrimer/gold nanoparticles as a platform for the mediated biosensing applications.

An electrochemical biosensor mediated by using 6-(Ferrocenyl) hexanethiol (FcSH) was fabricated by construction of gold nanoparticles (AuNPs) on the surface of polyamidoamine dendrimer (PAMAM) modified gold electrode. Glucose oxidase (GOx) was used as a model enzyme and was immobilized onto the gold surface forming a self assembled monolayer via FcSH and cysteamine. Cyclic voltammetry and amperometry were used for the characterization of electrochemical response towards glucose substrate. Following the optimization of medium pH, enzyme loading, AuNP and FcSH amount, the linear range for the glucose was studied and found as 1.0 to 5.0mM with the detection limit (LOD) of 0.6mM according to S/N=3. Finally, the proposed Au/AuNP/(FcSH+Cyst)/PAMAM/GOx biosensor was successfully applied for the glucose analysis in beverages, and the results were compared with those obtained by HPLC.

Polysulfone/pyrene membranes: a new microwell assay platform for bioapplications.

The use of PSU-Py prepared by click chemistry as a platform in membrane-bottom microwell plates for oxidase and hydrolase/oxidase-based enzyme assays is studied. For the GOx assay, the postulated fluorescence mechanism is based on the consumption of glucose by dissolved oxygen and GOx in the microwell plates covered with the PSU-Py membrane. For the AG-GOx assay, maltose is used as AG substrate and hydrolyzed to glucose which is then oxidized by the GOx activity. It is shown that the PSU-Py membrane acts as a fluorescence indicator of the enzymatic reactions, and both GOx and AG/GOx enzyme assays are successfully applied for glucose, maltose and acorbose analysis in the range 0.125-2.0 × 10(-3) M glucose, 0.05-0.5 × 10(-3) M maltose, and 0.0125-0.1 mg · mL(-1) acorbose, respectively.