Rapid identification of highly potent human anti-GPCR antagonist monoclonal antibodies.

Complex cellular targets such as G protein-coupled receptors (GPCRs), ion channels, and other multi-transmembrane proteins represent a significant challenge for therapeutic antibody discovery, primarily because of poor stability of the target protein upon extraction from cell membranes. To assess whether a limited set of membrane-bound antigen formats could be exploited to identify functional antibodies directed against such targets, we selected a GPCR of therapeutic relevance (CCR1) and identified target binders using an in vitro yeast-based antibody discovery platform (AdimabTM) to expedite hit identification. Initially, we compared two different biotinylated antigen formats overexpressing human CCR1 in a 'scouting' approach using a subset of the antibody library. Binders were isolated using streptavidin-coated beads, expressed as yeast supernatants, and screened using a high-throughput binding assay and flow cytometry on appropriate cell lines. The most suitable antigen was then selected to isolate target binders using the full library diversity. This approach identified a combined total of 183 mAbs with diverse heavy chain sequences. A subset of clones exhibited high potencies in primary cell chemotaxis assays, with IC50 values in the low nM/high pM range. To assess the feasibility of any further affinity enhancement, full-length hCCR1 protein was purified, complementary-determining region diversified libraries were constructed from a high and lower affinity mAb, and improved binders were isolated by fluorescence-activated cell sorting selections. A significant affinity enhancement was observed for the lower affinity parental mAb, but not the high affinity mAb. These data exemplify a methodology to generate potent human mAbs for challenging targets rapidly using whole cells as antigen and define a route to the identification of affinity-matured variants if required.

An assay for the detection of grapevine leafroll-associated virus 3 using a single-chain fragment variable antibody.

Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevine. A previously described single-chain fragment variable (scFv) antibody (scFvLR3), directed against the coat protein (CP) of GLRaV-3, was expressed in Escherichia coli and used to develop a diagnostic ELISA kit. The antibody was fused to the light chain constant domain of human immunoglobulin to create the bivalent reagent C(L)-LR3, which was purified from the periplasmic fraction, giving a yield of ~5 mg/l. The sensitivity of the reagent against recombinant GLRaV-3 CP was 0.1 ng. The sensitivity, specificity and durability of the reagent was similar to a commercial kit. The C(L)-LR3 showed a weak cross-reaction in immune electron microscopy assays to GLRaV-1 and -7, but not with the phylogenetically more distant GLRaV-2. A fully recombinant kit was developed with the inclusion of a recombinant GLRaV-3 CP expressed in bacteria, thus avoiding problems associated with virus propagation and purification. This system represents a rapid, simple, sensitive and standardized diagnostic protocol for GLRaV-3 detection.

Generation and characterization of a recombinant antibody fragment that binds to the coat protein of grapevine leafroll-associated virus 3.

Pathogen-specific recombinant antibodies have been used to characterize pathogen infections and to engineer resistance in crops. We selected a single-chain antibody fragment (scFvLR3cp-1) specific for the coat protein of grapevine leafroll-associated virus 3 (GLRaV-3), one of the agents of grapevine leafroll (GLR) disease, from a phage display library. The antibody binds specifically to the entire length of GLRaV-3 particles and has a high binding affinity value (K(D)) of 42 nM. The amino acid motif AQEPPRQ located at the N terminus of the GLRaV-3 coat protein was identified as the antibody-binding epitope by PEPSCAN analysis. To evaluate scFv stability in the reducing environment of the plant cell cytosol, transient expression assays were performed using Nicotiana benthamiana as a model plant. Capture ELISA demonstrated that the scFv fragment was produced and retained its antigen-binding capacity in the plant cytosol. Further functional assays showed that scFvLR3cp-1 binds with high specificity to at least four members of the family Closteroviridae. Therefore, the GLRaV-3-specific scFv fragment could be an ideal candidate for mediating broad-spectrum virus resistance if produced in transgenic grapevine plants.

Reduction of the pea ferredoxin-NADP(H) reductase catalytic efficiency by the structuring of a carboxyl-terminal artificial metal binding site.

Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, and adrenodoxin) to redox-based metabolisms in plastids, mitochondria, and bacteria. The FNRs from plants and most eubacteria constitute a unique family, the plant-type ferredoxin-NADP(H) reductases. Plastidic FNRs are quite efficient at sustaining the demands of the photosynthetic process. At variance, FNRs from organisms with heterotrophic metabolisms or anoxygenic photosynthesis display turnover numbers that are 20-100-fold lower than those of their plastidic and cyanobacterial counterparts. To gain insight into the FNR structural features that modulate enzyme catalytic efficiency, we constructed a recombinant FNR in which the carboxyl-terminal amino acid (Tyr308) is followed by an artificial metal binding site of nine amino acids, including four histidine residues. This added structure binds Zn2+ or Co2+ and, as a consequence, significantly reduces the catalytic efficiency of the enzyme by decreasing its kcat. The Km for NADPH and the Kd for NADP+ were increased 2 and 3 times, respectively, by the addition of the amino acid extension in the absence of Zn2+. Nevertheless, the structuring of the metal binding site did not change the Km for NADPH or the Kd for NADP+ of the FNR-tail enzyme. Our results provide experimental evidence which indicates that mobility of the carboxyl-terminal backbone region of the FNR, mainly Tyr308, is essential for obtaining an FNR enzyme with high catalytic efficiency.