Generation and characterization of heavy chain antibodies derived from Camelids

Antibodies and antibody fragments are essential tools in basic
research, diagnostics and therapy. Conventional antibodies consist
of two heavy and two light chains with both chains contributing to
the antigen-binding site. In addition to these conventional
antibodies, camelids (llamas, alpacas, dromedaries and camels)
possess so-called heavy chain antibodies (hcAbs) that lack the
light chains. The antigen binding site of these unusual antibodies
is formed by one single domain only, the so called VHH domain. The
VHH domain represents the smallest intact antigen binding fragment
(~ 15 kDa) and is characterized by very high stability, solubility
and specificity. These unique features render VHHs a promising
alternative to conventional antibodies and antibody fragments with
a multitude of possible applications. In the course of this thesis,
we aimed to generate and characterize VHHs suitable for different
applications ranging from biochemical studies to immunofluorescence
assays and live cell imaging. In order to meet the diverse
requirements of the intended downstream applications, a new
selection method differing from traditional phage display, called
native panning, was developed and established. In combination with
the protein-protein interaction fluorescent two-hybrid (F2H) assay,
a new process to identify antigen specific VHHs functional inside
living cells was developed. We used the immune system of alpacas to
generate VHH libraries against various antigens, ranging from small
peptides to large proteins. The libraries were screened by phage
display and antigen specific VHHs were identified by phage ELISA.
We were able to identify antigen specific VHHs against different
antigens which were characterized and their functionality was
tested in various applications. Furthermore, we could demonstrate
that the selection method influences which VHHs are identified and
therefore needs to be chosen very carefully with regard to the
intended biochemical and cell biological application. In summary,
we developed and established an efficient and versatile process to
screen and identify antigen specific VHHs suitable for different
downstream applications.