Opinion
Generating recombinant antibodies to the complete human proteome

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In vitro antibody generation technologies have now been available for two decades. Research reagents prepared via phage display are becoming available and several recent studies have demonstrated that these technologies are now sufficiently advanced to facilitate generation of a comprehensive renewable resource of antibodies for any protein encoded by the approximately 22,500 human protein-coding genes. Antibody selection in vitro offers properties not available in animal-based antibody generation methods. By adjusting the biochemical milieu during selection, it is possible to control the antigen conformation recognized, the antibody affinity or unwanted cross-reactivity. For larger-scale antibody generation projects, the handling, transport and storage logistics and bacterial production offer cost benefits. Because the DNA sequence encoding the antibody is available, modifications, such as site-specific in vivo biotinylation and multimerization, are only a cloning step away. This opinion article summarizes opportunities for the generation of antibodies for proteome research using in vitro technologies.

Section snippets

Are antibodies needed for every human protein?

The structural and functional individuality of cells as diverse as neurons, myocytes and hepatocytes – all of which share an identical genome – reflect the differential expression, localization and modification of proteins. For the majority of the 22,500 protein-coding genes currently predicted from the human genome (www.ensembl.org), characterization of the expressed protein products and their functions is still lacking. Antibodies are essential reagents for many protein analyses, including

Recombinant antibody fragments versus intact IgG antibodies

Evolution has shaped the antibody molecule as well as the underlying mechanisms that lead to binding site diversity such that a matching structural solution for every protein can always be found, even for antigens that do not occur naturally [7]. Antibody diversity is achieved in precursor B cells by combinatorial DNA rearrangements using a gene fragment lottery to assemble the antigen-binding surface of antibody variable (V) domains. The antibody is completed by combining heavy- and light

Recombinant binders based on non-antibody scaffolds

Non-antibody molecular frameworks have been developed, initially driven primarily by efforts to avoid the restrictions imposed by antibody engineering patents. These frameworks generally carry two or more randomized stretches of amino acids that mimic antibody CDRs. Examples of these alternative scaffolds include Adnectins, Affibodies, Anticalins and DARPins, among others [28]. By combining parts of existing binding sites with randomized repertoires, molecular clamps with high affinity can be

Adopting in vitro methods as the new gold standard for antibody generation

To date, traditional generation of monoclonal antibodies (mAbs) using hybridoma technology has set the performance standard to which emerging recombinant antibody methods and alternative scaffolds are compared. However, with respect to throughput and despite becoming streamlined and automated to a considerable extent [35], hybridoma technology has approached its limits after 35 years of optimization. Hybridoma generation requires immunization of an individual animal, which has several

Systematic recombinant binder generation by phage display

Phage display is an in vitro technique for antibody selection that uses bacteriophage DNA. A very diverse (>109) library of rearranged immunoglobulin genes, typically antigen-binding fragments such as scFv antibody constructs, is obtained either from human B cells or synthetically. The library is cloned into a vector linking the antibody genes in frame to a gene that codes for a phage surface protein such that the antibody fragments are expressed on the outer surface of the phage. This mixture

Concluding remarks and future challenges

It has been proven that phage display can deliver valuable research tools that have started to appear in the antibody catalogues alongside the classical polyclonal and hybridoma antibodies (www.axxora.com). But there is still more to come. In less than 10 years from now, we all might have a desktop robot in our laboratory that is capable of generating a recombinant antibody within a couple of days. Challenges towards this aim include the integration of antigen production and purification into a

Acknowledgements

Research work and resource planning in the authors’ groups are supported by the EU FP6 coordination action ProteomeBinders (contract 026008), and FP7 collaborative projects AffinityProteome (contract 222635) and AFFINOMICS (contract 241481).

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