@phdthesis{Hauptstein2023,
  author    = {Hauptstein, Niklas},
  title     = {Site directed molecular design and performances of Interferon-α2a and Interleukin-4 bioconjugates with PEG alternative polymers},
  doi       = {10.25972/OPUS-29691},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-296911},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Serum half-life elongation as well as the immobilization of small proteins like cytokines is still one of the key challenges for biologics. This accounts also for cytokines, which often have a molecular weight between 5 and 40 kDa and are therefore prone to elimination by renal filtration and sinusoidal lining cells. To solve this problem biologics are often conjugated to poly(ethylene glycol) (PEG), which is the gold standard for the so called PEGylation. PEG is a synthetic, non-biodegradable polymer for increasing the hydrodynamic radius of the conjugated protein to modulate their pharmacokinetic performance and prolong their therapeutic outcome. Though the benefits of PEGylation are significant, they also come with a prize, which is a loss in bioactivity due to steric hindrance and most often the usage of heterogeneous bioconjugation chemistries. While PEG is a safe excipient in most cases, an increasing number of PEG related side-effects, such as immunological responses like hypersensitivity and accelerated blood clearance upon repetitive exposure occur, which highlights the need for PEG alternative polymers, that can replace PEG in such cases. Another promising method to significantly prolong the residence time of biologics is to immobilize them at a desired location. To achieve this, the transglutaminase (TG) Factor XIIIa (FXIIIa), which is an important human enzyme during blood coagulation can be used. FXIIIa can recognize specific peptide sequences that contain a lysine as substrates and link them covalently to another peptide sequence, that contains a glutamine, forming an isopeptide bond. This mechanism can be used to link modified proteins, which have a N- or C-terminal incorporated signal peptide by mutation, to the extracellular matrix (ECM) of tissues. Additionally, both above-described methods can be combined. By artificially introducing a TG recognition sequence, it is possible to attach an azide group containing peptide site-specifically to the TG, recognition sequence. This allows the creation of a site-selective reactive site at the proteins N- or C-terminus, which can then be targeted by cyclooctyne functionalized polymers, just like amber codon functionalized proteins. This thesis has focused on the two cytokines human Interferon-α2a (IFN-α2a) and human, as well as murine Interleukin-4 (IL-4) as model proteins to investigate the above-described challenges. IFN-α2a has been chosen as a model protein because it is an approved drug since 1986 in systemic applications against some viral infections, as well as several types of cancer. Furthermore, IFN-α2 is also approved in three PEGylated forms, which have different molecular weights and use different conjugation techniques for polymer attachment. This turns it into an ideal candidate to compare new polymers against the gold standard PEG. Interleukin-4 (IL-4) has been chosen as the second model protein due to its similar size and biopotency. This allows to compare found trends from IFN-α2a with another bioconjugate platform and distinguish between IFN-α2a specific, or general trends. Furthermore, IL-4 is a promising candidate for clinical applications as it is a potent anti-inflammatory protein, which polarizes macrophages from the pro-inflammatory M1 state into the anti-inflammatory M2 state.},
  subject      = {Cytokine},
  language  = {en}
}