Engineering of camelid VHHs: applications in structural studies, diagnosis and solid tumor therapeutics
Authors:   , Xenaki Aikaterini-Titika 
Published: January 2024

Biotechnological advances allow for the artificial production of antibodies and antibody fragments, in order to exploit their target recognition specificity in basic and translational research, and clinical practice. In this thesis, we describe the engineering of antibody fragments derived from Llamas (VHHs) to be used for the characterization of potential disease-related target proteins and as targeted diagnostics and therapeutics of solid tumors.
We identified VHHs binding tetraspanin CD9, which assisted the structure elucidation of CD9 in complex with its partner protein EWI-F using cryo-electron microscopy. The 3D classifications indicated certain flexibility and structural heterogeneity for the complex of clone 4C8-CD9-EWI-F, while they led to the proposal of a model describing the higher order organisation of tetraspanin-enriched microdomains in cell plasma membranes. Furthermore, dSTORM super-resolution imaging of 4C8-stained CD9-expressing cells revealed that the lipidation status does not affect membrane localization or clustering of CD9. Taking advantage of the function of CD9 as an extracellular vesicle (EV) biomarker, we used clone 4C8 to purify tumor biomarker-containing, cell culture-derived EVs, setting the basis for the development of a straightforward and versatile diagnostic set-up for liquid biopsies.
The therapeutic potential of VHHs against solid tumors was further explored. To improve the pharmacokinetics of the 11A4 HER2-targeted VHH we used an Albumin Binding Domain (ABD) fusion. Such a modification, resulting in increased serum half-life, prolonged 11A4’s accumulation in HER2-expressing xenografts without affecting the homogeneity of their intratumoral distribution. Conjugation of 11A4-ABD to Auristatin F resulted in long-lasting tumor remission in HER2-positive xenograft-bearing mice, without the need for repeated dosing, encouraging the development of VHHs into targeted solid tumor therapeutics, as well.
An increase in molecular size might affect the otherwise homogenous distribution of VHHs. Using an in vitro system of 3D spheroids as a simplified model to recapitulate architectural characteristics of in vivo solid tumors, we sought to understand the effect of several half-life extension strategies on VHH’s distribution. Analysis of the penetration patterns of the different fluorescently labelled constructs suggested that next to molecular size, binding and internalization kinetics are influencing this diffusive transport within spheroids.
Lastly, we aimed at engineering fluorescently labelled VHH-drug conjugates with higher degrees of conjugation or dually modified VHHs that could potentially serve as theranostic agents. We explored a protocol in which both chemical and Sortase A-mediated site-specific conjugation were used, while synthetic peptides amendable to modification with more than one drug molecules using click chemistry were utilized. We were able to produce fluorescent VHH-drug conjugates demonstrating target-selective cell toxicity, while their dual labelling minimally affected the binding affinity of the probes.
Collectively, this thesis describes the use of camelid-derived VHHs in a variety of applications, highlighting the versatility of these naturally-derived antibody fragments. With proper engineering to fit the respective purpose, VHH-based molecules can serve as tools to study protein function, assisting the improvement of currently available diagnostic and treatment options. Ultimately, the development of patient-specific VHH-based drug conjugates can largely improve patients’ quality of life, as personalized treatment options can provide high efficacies with minimal side-toxicities.