Browsing by Author "Oksanen, Jouni"

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.