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Identification and optimization of camel single-domain antibodies against diagnostically relevant antigens

Wednesday, 16 November, 2005 - 16:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculty: Science and Bio-engineering Sciences
D
2.01
Dirk Saerens
phd defence

Maintenance and improvement of public health are cornerstones of modern society.
Prognostic and diagnostic measurements on illnesses, cancers or infections can maximize
the chance of remediation and limit infections. But this increases the need for faster, more
sensitive and more specific diagnostic tests. Therefore micro-array, biosensor and biochip
are proposed as the next generation of diagnostic tools due to their advantages in speed
and sensitivity. However, the probes in these novel diagnostic tools remain under a
continuous pressure to increase sensitivity and reusability and to reduce monitoring time,
which puts high demands on size, affinity, stability and immobilization potential of the
probe.

Monoclonal antibodies could serve as good probe candidates. However upon application
their shortcomings became apparent. Even the recombinant antigen-binding fragments
(scFv) have severe limitations due to their lowered stability, and expression/aggregation
problems. The introduction of recombinant antigen-binding fragments of Heavy-chain
antibodies from Camelidae (VHH) might overcome these limitations a lot easier than
antigen-binding fragments of conventional antibodies. This work demonstrates the
potential of these VHHs as probe in diagnostic setups. Two different malignancies are
highlighted in this work, i.e. prostate cancer (PCa) and Trypanosoma parasitic infection.

The identification of 25 VHHs recognizing the human prostate-specific antigen (hPSA), i.e.
the most important serum PCa marker, was successfully performed by panning of two
libraries constructed from different B-cell sources. The VHHs were divided into three nonoverlapping
epitope groups, some of which could inhibit the enzymatic activity of hPSA.
Surprisingly, these VHHs seemed to have allosteric effects or preferential recognition of
isoforms upon hPSA binding. These features could be exploited to study the hPSA
conformational flexibility or to discriminate the stages of PCa.

A biosensor for hPSA was developed based on the most appropriate hPSA-specific VHH,
i.e. cAbPSA-N7. This excellent hPSA-capturing VHH could easily be engineered in different
formats to suit several immobilization schemes. Furthermore, a sandwich detection assay
was optimized for determination of the free to total hPSA ratio, revealing the ability to
detect clinically significant hPSA concentrations for use in PCa diagnostic tests.

Several VHHs were isolated from a library constructed from the B-lymphocytes of a
Trypanosoma-infected dromedary. These VHHs were binding the variant surface
glycoprotein (VSG) from one Trypanosoma strain, but unable to cross-react with other
VSG variants. Modifications on the in vitro selection led to identification of a panel of crossreactive
VHHs, whereby several could specifically label living parasites from many different
strains. Some VHHs recognized a pan-reactive antigen present in de GPI-anchored
membrane fraction of Trypanosoma parasites. Therefore an antibody gene library from an
infected subject can be utilized to isolated highly interesting binding agents for use in
proteomic, diagnostic and therapeutic analysis.

A VHH, the cAbBCII10, was identified to posses a universal framework for grafting
antigen-specificity. This VHH was the best in our collection to combine criteria like high
expression yield, high conformational stability, and no loss of activity upon removal of the
conserved disulfide bond. The loop-grafted chimeras on cAbBCII10 yielded similar
expressions as the parental cAbBCII10. Compared to the loop donor, the affinity of the
chimeras decreased only slightly, but more importantly the conformational stability was in
most cases significantly increased. This universal framework offers the essential properties
for VHH utilization in various biotechnological applications (e.g. use as intrabody for
immunomodulation or in vivo cell staining, or use as probe in biosensors).

By serendipity, two positions in the VHH framework were identified able to accommodate
cysteine residues and to form a disulfide bridge. This extra cystine occurred naturally and
was introduced somatically in one VHH. This bond revealed to be generally introducible in
the VHH framework with only minor effects on the expression yield and equilibrium
dissociation constant. Moreover, this disulfide bond increased the conformational stability
of the VHH, paving the way for a general method to increase stability of a secreted
antigen-binding antibody fragment.