Projects / Programmes
Development of anti-FREM2 nanobody and its use for targeting glioblastoma cells.
| Code |
Science |
Field |
Subfield |
| 3.03.00 |
Medical sciences |
Neurobiology |
|
| Code |
Science |
Field |
| B000 |
Biomedical sciences |
|
| Code |
Science |
Field |
| 3.01 |
Medical and Health Sciences |
Basic medicine |
Glioblastoma, FREM2, nanobodies, gliomagenesis, cytotoxicity
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
38279 |
PhD Ivana Jovchevska |
Biochemistry and molecular biology |
Head |
2019 - 2022 |
87 |
Organisations (1)
Abstract
Glioblastoma is the most common primary malignancy of the brain which in the majority of the cases presents with fatal consequences. Besides the multimodal and aggressive treatment consisting of maximal surgical resection followed by a combination of temozolomide chemotherapy and radiation the average life expectancy of patients varies between 12 and 18 months after diagnosis. The heterogeneous nature of glioblastomas complicates their clinical management. The two main groups, IDH wild type and IDH mutant, have their own genetic changes. Moreover, genetic genotyping defined three molecular subtypes (classical, proneural and mesenchymal) that present with their own genetic signatures and can be present in one tumor at once. Effort has also been put into identification of bio-markers for glioblastoma; still none of the identified molecules (CD133, EGFR/EGFRvIII, IGHG1, IL-6) has shown enough specificity. Different alternative methods like intranasal delivery, virus therapy and antibody-based trials are currently being explored. A unique unconventional method for identification of molecular changes is the use of llama heavy chain antibody-derived nanobodies. Nanobodies are the smallest antigen binding fragments with exceptional features: small size, high stability to non-physiological conditions, water solubility, and economic production. Due to the specific shape of their antigen-binding fragment, nanobodies can recognize epitopes which are currently invisible to classical antibodies. This makes them suitable for identification of novel targets.
In our previous research we selected FRAS1 related extracellular matrix protein 2 – FREM2 as differentially expressed in glioblastoma cell lines compared to non-malignant astrocytes. In addition, we observed unique surface expression of FREM2 in glioblastoma cells which was not present in the controls. We then confirmed FREM2 presents with increased expression in glioblastoma human tissue samples compared to lower grade gliomas and reference brain samples. Our analysis of TCGA data also implied increased FREM2 expression in gliomas as the malignancy progresses. Moreover, we found a positive correlation between FREM2 gene expression levels and disease recurrence. To confirm our initial findings of FREM2 specificity towards glioblastoma cells, I would like to develop a nanobody targeted against the antigen. For this purpose, I will use an already created phage-displayed nanobody library. After the selection, I will confirm nanobody specificity towards its antigen with combined immunofluorescence using the selected nanobody and a commercially available antibody. To evaluate the effect of anti-FREM2 nanobody on glioblastoma cells, I will perform different tests on cell lines. With live cell imaging I will confirm that the nanobody is able to enter glioblastoma cells. Then, as FREM2 is involved in cell migration, with wound healing assay I will test the ability of the nanobody to inhibit the migratory potential of glioblastoma cells. Next, I will test the effect that anti-FREM2 nanobody has on glioblastoma cell viability. At last, as FREM2 was implicated to play a role in glioblastoma recurrence, I will create a temozolomide-resistant glioblastoma cell line. I will also test the effect of anti-FREM2 nanobody on the temozolomide-resistant cells. At last, I will quantify FREM2 gene expression levels in glioblastoma cells and temozolomide-treated glioblastoma cells.
With this work I expect to obtain a nanobody with high specificity and affinity for the protein of interest – FREM2. Our previous findings suggested involvement of FREM2 in glioblastoma pathology, and in this study I would like to confirm these findings using different functional assays. The proposed study will give new information about the malignant progression of glioblastoma and the role that FREM2 plays in it.
Significance for science
Because of the late diagnosis and aggressive nature which lead to a high mortality rate glioblastoma is a big problem of today’s society. As standard molecular biology methods have not yielded significant improvements in this field, researchers are thinking out of the box to solve the problem.
The antigen FREM2 is an attractive target for development of targeting mechanisms due to its surface expression in glioblastoma cells. After determining the specificity of the nanobody towards its antigen and examining its toxic effect on glioblastoma cells over non-malignant astrocytes, the obtained anti-FREM2 nanobody can be further developed into a biological nano-drug. Developing a nanobody against FREM2 can be useful for targeting glioblastoma cells. Furthermore, the obtained nanobody can also be used as a carrier i.e. to transport a cytotoxic agent to a specific cell type. This research will also open the possibility to explore the mechanism of how nanobodies are transported across the cell membrane.
Because of the small size, nanobodies are expected to pass the blood brain barrier and have greater access to brain tissue. This means that nanobodies can be developed as tools for monitoring and/or diagnosis of brain pathologies in vivo. In laboratories, nanobodies can be used in non-invasive diagnostic techniques such as ELISA. However, for use in clinical practice the pharmacokinetics of nanobodies has to be thoroughly studied. Although their small size is an advantage for diagnosis, it can become a limitation during treatment. Namely, due to the fast excretion, if nanobodies are used in systemic therapy they will require frequent administration. Still, in cases of local drug administration this problem will not be encountered.
Besides the potential limitations, the most important nanobody features for use with human subjects are their natural origin and low immunogenicity. This innovative research will give novel insight into glioblastoma pathology. I expect that the proposed study will show whether FREM2 is a suitable protein to be further evaluated as a glioblastoma bio-marker. Additionally, by examining differences between temozolomide-treated and non-treated glioblastoma cells we can obtain information whether FREM2 is involved in the advancement of the pathology.
The fast progression and high mortality rate of glioblastoma only emphasize the urge for developing novel treatment methods. Despite the growing number of publications on “cancer bio-markers”, there is no single molecule that can be used as a universal marker for the disease. Therapeutic methods should be developed in the direction of targeting a specific subset of cells (for example cancer stem cells) or a cellular characteristic that is crucial for tumor development. For minimizing damage on adjacent tissue, identification of specific glioblastoma bio-markers is necessary for development of targeted therapeutic approaches which will eventually lead to improvement in patient care and life expectancy.
Significance for the country
Because of the late diagnosis and aggressive nature which lead to a high mortality rate glioblastoma is a big problem of today’s society. As standard molecular biology methods have not yielded significant improvements in this field, researchers are thinking out of the box to solve the problem.
The antigen FREM2 is an attractive target for development of targeting mechanisms due to its surface expression in glioblastoma cells. After determining the specificity of the nanobody towards its antigen and examining its toxic effect on glioblastoma cells over non-malignant astrocytes, the obtained anti-FREM2 nanobody can be further developed into a biological nano-drug. Developing a nanobody against FREM2 can be useful for targeting glioblastoma cells. Furthermore, the obtained nanobody can also be used as a carrier i.e. to transport a cytotoxic agent to a specific cell type. This research will also open the possibility to explore the mechanism of how nanobodies are transported across the cell membrane.
Because of the small size, nanobodies are expected to pass the blood brain barrier and have greater access to brain tissue. This means that nanobodies can be developed as tools for monitoring and/or diagnosis of brain pathologies in vivo. In laboratories, nanobodies can be used in non-invasive diagnostic techniques such as ELISA. However, for use in clinical practice the pharmacokinetics of nanobodies has to be thoroughly studied. Although their small size is an advantage for diagnosis, it can become a limitation during treatment. Namely, due to the fast excretion, if nanobodies are used in systemic therapy they will require frequent administration. Still, in cases of local drug administration this problem will not be encountered.
Besides the potential limitations, the most important nanobody features for use with human subjects are their natural origin and low immunogenicity. This innovative research will give novel insight into glioblastoma pathology. I expect that the proposed study will show whether FREM2 is a suitable protein to be further evaluated as a glioblastoma bio-marker. Additionally, by examining differences between temozolomide-treated and non-treated glioblastoma cells we can obtain information whether FREM2 is involved in the advancement of the pathology.
The fast progression and high mortality rate of glioblastoma only emphasize the urge for developing novel treatment methods. Despite the growing number of publications on “cancer bio-markers”, there is no single molecule that can be used as a universal marker for the disease. Therapeutic methods should be developed in the direction of targeting a specific subset of cells (for example cancer stem cells) or a cellular characteristic that is crucial for tumor development. For minimizing damage on adjacent tissue, identification of specific glioblastoma bio-markers is necessary for development of targeted therapeutic approaches which will eventually lead to improvement in patient care and life expectancy.
Most important scientific results
Interim report
Most important socioeconomically and culturally relevant results
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