Research Report 2003 - Max-Planck-Institut für molekulare Genetik

Department of Vertebrate Genomics
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recombinant and natural proteins, peptides and small chemical compounds (Rhyner et al., 2003;
Konthur et al., submitted). Interestingly, we found, that panning on the same target in folded and
denatured form yields partially different antibodies, some recognising conformational epitopes
only. These antibody fragments are of particular interest for in vivo diagnostic and therapeutic
applications.
Automating the selection process increases the general selection throughput, but also shifts the
bottleneck of the selection pipeline further towards the isolation and evaluation of monospecific
binders. Therefore, we are developing and testing different techniques for downstream characterization
of selected antibody fragments. In collaboration with the protein group (J. Kreutzberger),
we are investigating the use of protein microarrays as a tool for the elucidation of monospecificity
of individual scFv’s (Konthur et al., unpubl.) and have established a multiplex assay format
on protein microarrays called MIST (multiple spotting technique; Angenedt et al., 2003), which
will allow the parallel testing of thousands of single scFv-expressing clones at a time. Also, in
collaboration with J. Gobom, we are in the process of establishing a mass spectrometry-based
assay format for the elucidation of antibody complexity in an enriched, target specific oligoclonal
pool of antibody fragments by peptide mass fingerprinting.
The successful implementation of these novel evaluation tools will greatly facilitate the scale-up
of artificial binder selection to high-throughput in the future. The ultimate goal is to obtain large
sets of artificial binders against given sets of proteins, such as all proteins encoded on the human
chromosome 21, specific proteins of the human brain, oncoproteins and transcription factors (in
collaboration with M.L. Yaspo, K. Büssow & B. Korn (RZPD), respectively). The artificial
binders shall be used to generate binder arrays applicable in protein expression profiling.
(2) Selection of natural binding partners
We have established protocols for the identification of natural binding partners utilising M13
and T7 phage displayed tissue and species specific cDNA expression product libraries. By
panning these libraries on immunoglobulins (Ig) from sera of individuals with allergies and/or
autoimmune diseases, we were able to identify novel allergens and autoantigens, which can be
used for clinical diagnosis in future (Crameri et al., 2001). Since selections on complex mixtures,
such as immunoglobulins, yield a large number of binding proteins, we have devised a
DNA-array based method for the elucidation of gene complexity of the enriched library by
consecutive rounds of hybridisation. For example, in collaboration with Prof. R. Crameri (Swiss
Institute of Asthma and Allergy Research), we have enriched an Aspergillus fumigatus cDNA
expression product library on patient IgE, picked ~ 13,000 single clones and identified additional
60 novel allergens next to the 14 known allergens by less than 15 DNA-hybridisations
(Kodzius et al., 2003). For the future, we envisage that monitoring the presence or absence of
IgE recognising a set of allergens can be used for the fine-diagnosis of different clinical manifestations
of Aspergillus fumigatus allergies.
In addition to allergy research, we have applied cDNA expression product phage display screening
to autoimmunity research in collaboration with Prof. G. Burmester and Dr. K. Skriner (Charité
- Humboldt University, Berlin). By screening a human foetal brain cDNA library displayed on
T7 phage, we have identified numerous novel autoantigens, which are now under further investigation.
Ultimately, we would like to use this technology also in functional genomic applications
for the identification of other than antibody-antigen interactions, such as protein-protein,
protein-DNA and protein-chemical compound interactions.
Future development and perspectives
In future, we would like to extend our portfolio of in vitro ligand screening technologies by
establishing other methods in the group, such as DNA aptamer technology (SELEX) and ribosome
display. These technologies will complement limitations observed with phage display.
The group is participating in numerous NGFN-2, EU FP6, and BMBF proposals.