Methods for engineering and characterization of advanced therapeutics

QC 2024-09-05

Abstract

Protein therapeutics are used worldwide to treat a multitude of diseases. Most therapeutic proteins on the market today are antibodies, and one of the characteristics that has made antibodies so useful for therapeutic use is their propensity to specifically and selectively bind other proteins with high affinity. While antibodies themselves can be very effective for treatment of different diseases, the field has also started to progress towards more advanced formats and entities. Monoclonal antibodies are generally monospecific. In many cases, however, binding to two or more therapeutically relevant proteins simultaneously can be beneficial. To overcome this limitation, different bispecific antibody formats have been engineered. The AffiMab, for example, is a bispecific antibody format, where a monoclonal antibody is fused to a much smaller affinity protein called an affibody molecule, allowing two independent affinity proteins to be combined in a modular fashion. To achieve this, the individual affinity proteins first needs to be developed, and this can be achieved using protein engineering methods, such as phage display. While phage display enables selection of affinity proteins binding new target proteins, simply binding may not be enough, and methods for discovering more diverse affinity proteins and characterizing their binding are therefore essential.

Bispecific antibodies constitute only one example where advanced therapeutic entities have emerged. Another example of advanced therapeutics are gene therapies, where adeno-associated viruses (AAV) have transpired as one of the most successful platforms. AAV-based gene therapy has, in several recent approvals, proved its ability to be used to cure inherited genetic disorders, such as spinal muscle atrophy and haemophilia, with a single treatment. The lack of precise targeting of AAV vectors, however, has prompted researchers to develop engineered AAVs with improved targeting. While these engineered variants are yet to reach the clinic, they hold promise of gene therapies with less side-effects and higher effective doses in the desired tissues. Compared to protein therapeutics, AAVs add new dimensions to characterization of quality of the produced therapeutic. As AAVs are a combination of proteins and DNA, both these components need to be verified, and as such, methods used for characterization of proteins only cover a part of it. Analysis and characterization of the DNA inside the AAV capsids is crucial, both for drug discovery, to ensure the correct gene is being delivered, and for understanding the observed effects of treatment. Standardized methods for sequencing and analysis of recombinant AAV genomes are yet to be established but this, combined with novel AAV variants, could help leverage a new generation of AAV-based therapies. 

All of these topics are explored in this thesis. In Study I, AffiMabs were developed for treatment of HER2 resistant gastric carcinoma, with improved cytotoxic effect observed in vitro compared to the parental proteins trastuzumab and Z_EGFR. In Study II, a method for rapid, residue level, eptiope mapping was developed, and the eptiopes of three new antibodies targeting the SARS-CoV-2 spike protein were determined. In Study III, phage display was combined with deep sequencing to enable discovery of affibody molecules with different binding kinetics and epitopes. In Study IV, affibody molecules were fused to AAV capsids to achieve receptor specific targeting. The capsids were shown to be both functional, superior to the parental serotype for receptor-specific transduction, and modular, as different affibody molecules could be incorporated. Finally, in Study V, a method for deep sequencing and analysis of AAV genomes was established, allowing identification of a wide array of different DNA encapsidated in AAVs.

In summary, the studies presented in this thesis explores a broad selection of new and improved methods for both engineering and characterization of advanced therapeutics. 

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