Platform for high-throughput antibody selection using synthetically-designed antibody libraries.

Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>10(20)), the practical diversity that can be achieved by transformation of Escherichia coli is limited to about 10(10). To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target.

AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins.

Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes.

Phage ESCape: an emulsion-based approach for the selection of recombinant phage display antibodies.

Antibody phage display technology is a well established method for selecting specific antibodies against desired targets. Although phage display is the most widely used method of generating synthetic antibodies, it is laborious to perform multiple selections with different antigens simultaneously using conventional manual methods. We have developed a novel approach to the identification and isolation of cells secreting phage encoding desirable antibodies that utilizes compartmentalization and Fluorescence Activated Cell Sorting (FACS). This method, termed Phage Emulsion, Secretion, and Capture (ESCape), allows us to individually query each phage against the antigen. Here, we demonstrate the ability of Phage ESCape to identify novel scFvs against a phosphopeptide epitope of the Her2 kinase from a phage display library containing approximately 10(8) synthetically diversified antibodies. Clones were analyzed by monoclonal phage ELISA against the Her2 phosphopeptide, and positive binders were identified as those showing a signal greater than 3-fold higher than the background signal against an irrelevant antigen. We isolated antibodies recognizing the phosphopeptide in a single round of selection by Phage ESCape, but the strength and specificity of the hits was substantially improved when the library was pre-enriched by a single round of biopanning. By minimizing the selection rounds required for phage display and using a FACS machine as a 'colony picker' equivalent, Phage ESCape has the potential to dramatically increase the throughput of in vitro screening methods.

1-Benzylbenzimidazoles: the discovery of a novel series of bradykinin B(1) receptor antagonists.

The design, synthesis, and structure-activity studies of a novel series of BK B(1) receptor antagonists based on a 1-benzylbenzimidazole chemotype are described. A number of compounds, for example, 38g, with excellent affinity for the cynomolgus macaque and rat bradykinin B(1) receptor were discovered.

A high-throughput HERG potassium channel function assay: an old assay with a new look.

In this paper, we describe an assay using radioactive rubidium (86Rb) efflux to screen functional human ether-a go-go-related gene (HERG) K+ channels in a high-throughput screening (HTS) format. This assay offers an alternative way to examine junctional interactions between chemical compounds and HERG K+ channels. Follow-up experiments and discussions were carried out to address a variety of factors that affect potency evaluation within the Rb efflux assay. Factors that can affect the assay results, such as assay time, efflux rate, and compound blocking kinetics, are discussed in detail. Our results provide some explanations for the variances of the assay results and offer some guidelines for using the Rb efflux assay to evaluate compound interactions with HERG K+ channels in the pharmaceutical industry.