Faculty of Pharmacy

Oncolytic adenoviruses are a new cancer treatment platform which aims to eliminate cancer through direct lysis of cancer cells by viral replication and the activation of the immune system by the release of tumor antigens upon oncolysis. In the PeptiCRAd technology, the activation of an anti-cancer immune response is enhanced by the addition of poly-lysine modified cancer peptides, where the antigen presentation to the immune system is improved in comparison to plain oncolytic viruses. PeptiCRAd complexes have been assumed to form solely by electrostatic interactions, but the thermodynamic profiles and mechanisms involved in the complexation have not been previously addressed. Thus, by adding isothermal titration calorimetry as part of the analysis repertoire provides valuable information of the characteristics of PeptiCRAd complexes. In this study, the applicability of isothermal titration calorimetry in PeptiCRAd complexation analyses was evaluated based on initial peptide-to-virus and virus-to-peptide titrations, and a method of analysis was created for the thermodynamics of the interactions of the complex. Optimization of the experimental method (i.e., titration protocol) and the data analysis (i.e., calculation models) remains inconclusive for quantitative analysis as data obtained from the measurements was mainly of bad quality, thus requiring further optimization to obtain reliable data. However, using surface plasmon resonance as an already established method for poly-lysine peptide-virus interaction studies gave robust data and can be used as a base or guideline to further develop isothermal titration calorimetry analyses for characterizing PeptiCRAd complexes. Although isothermal titration calorimetry measurements were unsuccessful for quantification purposes, it was possible to qualitate the mechanisms of PeptiCRAd complexation for four different peptides with fair confidence. The peptides showed low heats of binding, and positive and negative cooperative binding in ionic and non-ionic solutions, respectively. Based on this, the binding of peptides in PeptiCRAd complexes was determined to be driven by hydrophobic inter-peptide interactions on the virus surface, although an electrostatic attraction is indeed present at the virus-peptide interface, initiating the binding event. Also, improvements to the titration protocol for PeptiCRAd analyses with isothermal titration calorimetry are suggested for further optimizations in the future to conclusively determine the applicability of the isothermal titration calorimetry technique for characterizing peptide-virus interactions of PeptiCRAd complexes.

Vascular endothelial growth factor C (VEGF-C) is the most studied of the growth factors that control the growth of lymphatic vessels (lymphangiogenesis) and belongs to the same VEGF family as VEGF-A, which controls the growth of blood vessels. The growth of blood vessels and lymphatic vessels is centrally related to the pathophysiology of several cancers that form solid tumours and wet macular degeneration. Unlike VEGF-A, VEGF-C is not currently (2023) a target molecule of any approved drugs, but in clinical trials in the indications mentioned above, combining a VEGF C inhibitor with VEGF-A inhibitors has provided better results than VEGF-A inhibitor monotherapy. The study's objective was converting a phage display library containing single-chain antibody variable fragments (scFvs) screened against VEGF-C into full IgG class antibodies. The scFvs had shown a binding affinity towards the human, mouse, or both VEGF-C variants. The DNA sequences of the best binders of the library had previously been cloned into pLK06H plasmids. The scFvs comprise the variable region of the light and heavy chain (VL and VH) but do not contain the constant regions of the antibody (CL and CHx). Using single-chain antibody fragments as drugs is limited because, in most indications, better stability of whole antibodies, lower immunogenicity, and a longer half-life enabling less frequent dosing is desirable. In addition, the Fc part of whole antibodies often mediates the drug effect, such as complement activation, and whole antibodies are also used as research tools. Secondly, the study aimed to investigate how changing the antibody format affects the binding affinity. To produce whole antibodies, original DNA sequences of pVitro-trastuzumab-IgGk1 plasmid encoding VH and VL regions were replaced with new VH and VL sequences from the phage display library. Several recombinant DNA technology methods were utilised, but the most crucial method was the commercially available NEBuilder HiFi DNA Assembly, which enabled the seamless joining of several DNA fragments into a recombinant DNA molecule in a single-tube reaction. The cloning workflow proved uncertain, as only one constructed antibody production plasmid was sufficiently amplified and expressed in bacterial and mammalian cell cultures. Suboptimal overlapping of DNA fragments and insufficient competence of the bacterial strain used in the transformation were probable bottlenecks. Therefore, as such, the method is not suitable for use on a large scale to convert single-chain antibody fragments into whole antibodies. Also, binding tests were not performed. However, the work done and the antibody production plasmid built is a good basis for further optimisation of the method. In the optimisation, attention should be paid, especially to the quality of the DNA primers and the competence of the bacterial cell line. Also, alternative cloning methods, such as restriction enzymes and ligases, could be used instead of the NEBuilder HiFi DNA Assembly.

Prolyl oligopeptidase (PREP) is endopeptidase which cleaves short proline containing peptides. Abnormalities in brain PREP activity has been connected to neurodegenerative diseases. Recently it has been detected that besides its proteolytic activity PREP interacts directly with other proteins which might contribute to generation of neurodegenerative diseases. Further it has been discovered that certain small molecular PREP inhibitors are able to modify these protein-protein interactions (PPIs) and thus have a potential to alleviate the progression of neurodegenerative diseases. This has led to the development of novel second generation PREP ligands which lack the strong inhibitory activity but are potent compounds on modifying the PPIs. Thiazole structure containing PREP modulators has provided most promising class of compounds. It has been detected that these compounds mediate their effects via novel binding site on the enzyme and these effects are not connected to the inhibition of the enzymatic activity. The synthesis of these thiazole containing PREP modulators has proven to be demanding since it have involved a usage of laborious synthesis route and provided low yields. The aim of this research was to examine the synthesis of 2-(2-benzimidazol-1-yl)ethyl)- 4-methyl thiazole containing PREP modulators via previously reported synthesis route. Another aim was to design and develop a synthesis route for 2-(2-(benzimidazol-1- yl)ethyl)-5-bromo-4-methylthiazole, a molecule which serves as valuable intermediate for the lead optimization and generation of second-generation PREP modulators. A synthetic route for 2-(2-(benzimidazol-1-yl)ethyl)-5-bromo-4-methylthiazole was successfully developed. Despite that the total yield of the route remained low. When searching the reasons for the low obtained yield the chemistry behind a thiazole creating cycloaddition reaction and an aromatic halogenation was examined. This led to the discovery of a rare cationic compound which was found to be synthesized from previously undescribed starting materials.