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

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 non-overlapping 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 cross-reactive 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.