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VIB, Center for Medical Biotechnology, Vakgroep Prof. Jan Tavernier

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Stageonderwerp 2017-2018: Development of TNF-AcTakines for cancer therapy

Despite huge improvements in therapy, cancer still remains one of the most deadly diseases. There is a huge need of new medicines that can assure complete healing of the patient with almost no side effects. A lot of cytokines, like Tumor Necrosis Factor (TNF), have therapeutic potential but have too much damaging systemic effects. TNF exerts its antitumor effect by activating and damaging tumor vasculature, while its activity on liver, kidney and intestine can cause life-threatening side-effects. In the Cytokine Receptor lab, Activated-by-Targeting Cytokines (AcTakines) are developed for cancer therapy. AcTakines are a novel type of immunocytokines in which mutant cytokines with strongly reduced binding affinity for their receptors are fused to a targeting moiety that recognizes a cell-surface marker of the target cell. In this project, a TNF-AcTakine is fused to a CD13 single chain antibody (VHH). CD13 is a cell-surface marker of tumor neo-vasculature. As such, AcTakines remain inactive through the body and unveil their biological activity only on the tumor vasculature expressing the CD13 protein. TNF-AcTakine with a VHH against BcII10, a bacterial protein, is used as a control. The problem with the current TNF-AcTakines is that they show only minor therapeutic effects in vivo. In vitro experiments show however that TNF-AcTakines can be very effective. The serum half-life of TNF-AcTakines is only 40 minutes. As the protein size is at least 70 kDa, thus the limit for renal clearance is exceeded, the protein is probably filtered out of the circulation by the liver. The aim of this project is to increase the serum half-life by testing two approaches: prevention of N-glycosylation and fusion to mutated antibody Fc regions.

TNF-AcTakine may be glycosylated on a non-physiological way because it is produced in HekF suspension cells. The asialoglycoprotein receptor (ASGPR) in the liver recognizes ‘bad’ glycosylated proteins and removes these proteins out of the circulation. Therefore, an attempt to prevent N-glycosylation of the TNF-AcTakine was implemented in this project. The consensus sequence for N-glycosylation is present three times in the AcTakine. By mutating Asparagine to Glutamine within this motif, N-glycosylation could be prevented. These three mutations were applied separately using the site-directed mutagenesis technique. Subsequently, this mutated DNA-sequence was transfected into HekF-cells. These cells produce the corresponding protein. The mutated protein could be purified out of the supernatans using a nickel column because it has a poly-histidine tag. Protein size and the presence of glycosylation was checked on a protein gel. The mutated protein is injected into mice (by Dr. Huyghe), along with the original AcTakine to compare the serum half-life. The protein can be quantified in the blood samples by performing an anti-mouse TNF Enzyme-Linked Immuno Sorbent Assay (ELISA).

The second method tested in this project, was fusing the TNF-AcTakine to Fc-regions of immunoglobulins. This was done by performing restriction digests on the TNF-Actakine and on a construct containing the Fc-regions. The correct construct can be obtained by ligating the sticky ends of the fragments to each other. As with the first part of the project, the protein is produces in HekF-cells and injected in mice. It can also be quantified using the ELISA.

Mutation of the N-glycosylation motif in the TNF-AcTakine clearly resulted in disappearance of the glycosylation pattern on the protein gel, indicating that the protein was indeed N-glycosylated on this motif. Analysis of the blood samples by ELISA showed however that the serum half-life of the mutated protein was not extended. When the original construct was injected along with an excess of asialofetuin, an ASGPR inhibitor, the serum half-life was significantly longer. This result indicates that the protein is indeed cleared away by the liver. It also suggests that glycosylation is important for its serum half-life but that there are most likely other places in the AcTakine that are glycosylated. Alternatively, proteolytic degradation of the AcTakine in the blood may also shorten its serum half-life. Further research is needed to determine the exact cause of the short half-life of the current TNF-AcTakines.

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Albert Baertsoenkaai 3
9000 Gent
Belgium

Contacts

Leander Huyghe
leander.huyghe@ugent.vib.be
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