Bioconjugation of antibodies and affinity proteins for the development of novel diagnostic and therapeutic tools

QC 2024-08-14

Abstract

Antibodies and affinity proteins are essential tools in research, diagnostics and therapy. Antibodies have the ability to bind to a large variety of protein targets with great specificity and selectivity, allowing them to be used for identification, or targeted therapy of devastating diseases such as cancer. Bioconjugation to other molecules enables the extension of antibody function, enhancing its capabilities beyond recognition and binding. By attachment of a cytotoxic molecule such as a cytotoxic drug or a radionuclide to the antibody, a cell-killing agent can be directly delivered to the cancer cells, eliciting localized effect and sparing healthy tissues. Selecting a suitable labelling technique to produce high-quality antibody conjugates is a crucial consideration for many applications. Site-specific labelling is an attractive approach, where the cargo is directed to a predefined location on the antibody, resulting in homogeneous and well-characterized conjugates, without impacting its tumour antigen binding properties. Many different methods have been explored; affinity-based strategies serve as the basis of this thesis. Ligands such as the Protein A- derived Z domain and the FcIII peptide bind to the Fc domain on the antibody framework, a suitable location for delivering a selected payload. 

In this thesis, I have explored affinity ligand-based and glycan engineering-based site-specific antibody labelling methods for the production of antibody conjugates for therapeutic purposes and as research tools. In Papers I-II, we adopted site-specific labelling methods based on the Fc- targeting ligands Z domain and FcIII peptide along with a glycan-modification approach for the preparation of antibody conjugates for radioimmunotherapy (RIT) purposes. Our methods resulted in well-characterized conjugates covalently labelled with a chelator suitable for radiolabelling. The radioimmunoconjugates were evaluated in vivo and showed great potential for future applications in RIT. 

In Paper III, we aimed to improve an earlier developed PNA-based antibody pretargeting system by adopting different labelling strategies for the production of antibody-PNA conjugates. Such systems hold great potential to increase the uptake of radioactivity in the tumour and decrease exposure of healthy tissues to radiation as well as improve imaging contrast for radioimaging. Here, we demonstrated that by carefully reengineering of both the primary and secondary targeting agents, considerable enhancement can be achieved with PNA-based pretargeting. 

Apart from their success as therapeutic and diagnostic agents, antibody conjugates are widely established reagents in basic research. As sequencing-based single-cell protein expression analysis assays are becoming more popular, the production of high-quality antibody-oligonucleotide conjugates gains high importance. In such assays, specific antibody-targeted antigens are identified following the amplification and next-generation sequencing of their corresponding barcodes. In Paper IV, we utilize the Z domain for decorating an antibody panel targeting key immune cell markers with unique barcodes. The barcoded antibodies were used in DBS-Pro, a high-throughput and multiplex single-cell protein analysis method. In this project, DBS-Pro was applied for the identification of major immune cell subpopulations in PBMC samples. i In Paper V, we explored as an alternative to RIT, the potential of using affinity ligands based on endogenous peptides for radionuclide therapy. The short peptide RM26, targeting the cancer antigen gastrin-releasing peptide receptor (GRPR), commonly overexpressed in prostate cancer, is a promising candidate for imaging and therapy. Being a short peptide, RM26 suffers from fast blood clearance, which is a challenge for the therapeutic application of such ligands. Our approach to overcome this issue and to extend the circulatory half-life of RM26 is to conjugate the peptide to an albumin-binding domain (ABD) that can bind to HSA, the most abundant protein in our blood, thus achieving prolonged blood residence time. By designing, producing and evaluating several ABD-RM26 conjugate variants, we were able to investigate the effect of molecular composition on the biodistribution and bioavailability of such molecules, and identifying candidates with the most favourable properties for future development. 

In conclusion, in this thesis I combined recombinant protein techniques with chemical synthesis to produce unique protein conjugates followed by their in vitro and in vivo characterization to evaluate functional and biophysical properties. The experimental work presented in this thesis provides a rationale for the development of novel bioconjugates for a variety of future applications. 

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