Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies.

Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains.

Identification of aggregation inhibitors of the human antibody light chain repertoire by phage display.

Protein aggregation hinders the development of biologics and underpins the molecular basis of many human diseases. Considerable variation of aggregation propensity exists not only between different proteins, but also within a single homologous family, which complicates analyses. A classic example is observed among human antibody light chains, which aggregate in a clonally specific manner, driven by sequence diversity within their variable domains.

Rapid prediction of expression and refolding yields using phage display.

Aggregation limits the recombinant production of many commercially important proteins. We have recently identified mutations that control the aggregation behavior of human antibody variable domains (Dudgeon K., Rouet R.

Generation of human single domain antibody repertoires by Kunkel mutagenesis.

Human antibody single domains are a promising new class of antibody fragments. Here we describe methods for the cloning of human V(H) and V(L) genes into phage and phagemid vectors. Furthermore, we provide detailed protocols for the generation of single domain antibody libraries by Kunkel mutagenesis and the analysis of diversity by DNA sequencing and superantigen binding.

Selection of human VH single domains with improved biophysical properties by phage display.

Human antibody variable heavy (VH) domains tend to display poor biophysical properties when expressed in isolation. Consequently, the domains are often characterized by low expression levels, high levels of aggregation, and increased "stickiness." Here, we describe methods that allow the engineering of human VH domains with improved biophysical properties by phage display.

General strategy for the generation of human antibody variable domains with increased aggregation resistance.

The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (V(H) and V(L)) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requirement to maintain antigen binding.

Expression of high-affinity human antibody fragments in bacteria.

Here we describe protocols for the expression of human antibody fragments in Escherichia coli. Antigen-specific clones are identified by soluble fragment ELISA and concentrated by periplasmic preparation. They are then further purified by affinity chromatography.

Sequence determinants of protein aggregation in human VH domains.

Human antibody variable heavy (VH) domains tend to aggregate upon denaturation, for instance, by heat or acid. We have previously demonstrated that domains resisting protein aggregation can be selected from CDR-only repertoires by phage display. Here we analysed their sequences to identify determinants governing protein aggregation.

KAI1 promoter activity is dependent on p53, junB and AP2: evidence for a possible mechanism underlying loss of KAI1 expression in cancer cells.

A molecular mechanism to explain reduced KAI1 expression in invasive and metastatic tumour cells remains elusive. In this report, we extend an earlier study in bladder cells to confirm that a 76 bp region of the KAI1 promoter (residues -922 to -847), with binding motifs for p53, AP1 and AP2, is required for high level activity of a KAI1 reporter in prostate cancer cell lines. Gel shift and supershift experiments supported binding of p53, junB and heterodimers of AP2alpha/AP2gamma or AP2beta/AP2gamma to this sequence.