BLT Stages

Abstract Bachelor Project FBT 2018-2019: Study of TNF receptor activation by CD13-AFR and wild-type TNF in endothelial cells

Background: In recent years, major steps have been taken in research into the treatment of cancer. Nevertheless, it remains one of the deadliest diseases. There is a need for new medicines that can completely cure the patient with few side effects. Many cytokines have already shown their therapeutic value in the fight against cancer. The disadvantage of these cytokines is that they bind everywhere and therefore cause many side effects. In addition, only a small portion reaches the final tissue where the treatment is needed. This was solved in the Cytokine Receptor lab by designing AcTaKines. Activaty-on-target cytokines are immunocytokines that have a reduced affinity for the receptor and contain a targeting moiety that recognizes a cell-surface marker on the tumor. In this case, a TNF-based AcTakine, AcTaFactor, was used. After in vitro and in vivo testing, AFR did not stimulate the TNF receptor to the same extent as TNF on endothelial cells.

Aim: The reason behind the difference in activation of the TNF receptor between the CD13-AFR and hTNF is not known and was investigated in this project. We focused on genes and protein involved in signaling towards NF/kB (deubiquitinases, p38/MAPK pathway) and cell death (anti- apoptotic genes, PI3K/Akt pathway). We also measured the expression and secretion of some interleukins as read-out for the NF/kB activity.

Methods: HUVECS wer grown in endothelial growt medium and plated in 6-wells. Subsequently, the cells are stimulated with BCII10-AFR, hTNF, CD13-AFR, BCII10-scTNF and CD13-scTNF in biological triplicates. For qPCR analysis RNA was isolated after the cells were stimulated for 4 hours, 8 hours and 24 hours. RNA was first converted to more stable cDNA before the qPCR analysis with SYBR® Green was done. For the proteins a shorter stimulation period of 10 minutes, 30 minutes and one hour was used. The proteins are then separated using SDS-PAGE and overnight blotted on a PVDF membrane.  The Western Blots are then treated with  antibodies and visualized by ECL imaging.

Results: In accordance with previous results, gene expression after stimulation with CD13-AFR was much lower than wild-type TNF. Interestingly stimulation with the BCII10-scTNF lead to higher gene expression of anti-apoptotic genes, DUBs and interleukines in comparison to CD13- scTNF. Furthermore, after ECL-imaging phosphorylated MK-2 could be detected after stimulation with BCII10-scTNF, hTNF and CD13-AFR, but not CD13-scTNF. The strongest signal was reserved for hTNF and its mutant. For Akt and P38, no significant difference was found.

Conclusion: Our results suggest that the physical coupling of TNF to the CD13 protein on the endothelial cell membrane strongly influences TNF receptor activation. However, further research is needed to confirm this.

Abstract Bachelor Project FBT 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.