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. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30-33, 35 in V H and 24, 49-53, 56 in V L ). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (V H 3 and V κ 1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant V H and V L domains revealed a surprising degree of structural conservation, indicating compatibility with V H ∕V L pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.biotechnology | monoclonal antibodies | protein aggregation | protein engineering | antibody therapeutics P rotein aggregation represents a key bottleneck in the generation of antibody-based reagents and hinders the development and production of human therapeutics (1). It is generally believed that the aggregation propensity of larger antibody reagents (such as immunoglobulin G and Fab) is mostly determined by their variable domain components (V H and V L ), although there is currently little understanding of the mechanisms involved (1, 2). Indeed, significant differences of aggregation rates have been reported for antibodies that differ exclusively in their variable domains (1, 2). Aggregation propensity is even more pronounced for smaller antibody reagents, which lack the interdomain stabilization of their larger counterparts (3, 4). This is a major problem in biotechnology due to an increasing trend toward smaller antibody formats for imaging and tumor targeting applications (4). Common formats include human single chain fragments (scFv) and human single domain antibodies, both of which frequently display poor biophysical properties (1, 3).The aggregation propensity of human variable domains is in marked contrast to the variable heavy domains of camels and llamas, which are generally nonaggregating and soluble ("V HH domains") (5-7). More favorable properties have also been described for "camelized" and other engineered human V H model domains (8-12) but not for human V L domains. Their p...
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 À922 to À847), 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 AP2a/AP2c or AP2b/AP2c to this sequence. Introduction of mutations into specific motifs demonstrated an essential requirement for p53 and junB to reporter activity, and that functional synergy between these two factors enhanced activity. A further elevation of reporter activity required AP2. Roles of individual p53, junB and AP2 proteins, as well as functional synergy between p53 and junB, were confirmed in transfection experiments. Western blotting analysis showed that an absence of wild-type p53, and/or a loss of junB and AP2 protein expression, correlated with downregulation of KAI1 mRNA levels in a series of prostate cancer cell lines. A loss of p53 function and/or expression of junB, combined with reduced expression of specific AP2 proteins may underly downregulated KAI1 expression in tumour cells.
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. We found that, while many different CDR sequences conferred aggregation-resistance, certain physico-chemical properties were strongly selected for. Thus, hydrophobicity and beta-sheet propensity were significantly lower among the selected domains, whereas net negative charge was increased. Our results provide guidelines for the design of human VH repertoires with reduced levels of protein aggregation.
Background: Camelid antibody domains are naturally stable and capable of cleft binding. Results: Protein engineering can endow human antibody domains with such properties. Conclusion: Our strategy does not require undesirable antibody framework changes. Significance: Robust building blocks for human therapeutic applications.
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. This article provides an overview of expression and purification strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.
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., Kokmeijer I., Schofield P., Stolp J., Langley D., Stock D. and Christ D. (2012) Proc Natl Acad Sci USA, 109, 10879-10884. This has allowed the generation of a panel of human antibody variable heavy domains with a defined range of aggregation propensities. Here we utilize this unique resource to validate a previously reported heat-denaturation method on phage (Jespers L., Schon O., Famm K. and Winter G. (2004) Nat Biotechnol, 22, 1161-1165. Our experiments revealed that the method is not only robust in respect to denaturation conditions on phage, but also highly indicative of solution behavior. In particular, it is an excellent predictor of expression and refolding yields.
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.
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