Periodic Reporting for period 5 - ONCOINTRABODY (Targeting common oncogenes with intracellular monobodies)
Berichtszeitraum: 2022-12-01 bis 2023-12-31
Cancer is a leading cause of death in Europe. The process of cancer development is driven by the activation of specific cancer-causing genes, so-called oncogenes. This results in the reprogramming of normal cells and lets them acquire common functional hallmark of cancer. Most cancers are still treated by surgically removing the tumour, killing tumour cells by irradiation or chemotherapy. In many cases, tumour growth can initially be kept under control, but disseminated tumor cells may become insensitive to the treatments and seed metastasis, which are hard to control and commonly lead to the death of the patient. Over the past 20 years, so-called targeted anti-cancer drugs entered clinical practice. These targeted drugs act specifically on the gene products of the cancer-causing oncogenes – the oncoproteins. In a few cases these new treatments resulted in a therapeutic breakthrough. In contrast, the majority of targeted drugs suffer from short-lived responses due to drug resistance. In addition, the majority of oncoproteins remain untargeted. Therefore, novel approaches are needed to broaden the variety of targeted oncoproteins. This is expected to result in therapies with less side-effects and a better survival of cancer patients.
My lab has pioneered studies to target oncoproteins with monobody proteins. Monobodies are small engineered binding proteins that can be regarded as mini-antibodies. This makes them much easier to handle and to produce. Monobodies can be developed in the laboratory and do not require the immunization of animals. On the other hand, monobodies display most pharmacologic features of antibodies, which include a highly specific interaction and the tight binding to its targets. We demonstrated that monobodies can be developed to several oncoproteins for which no specific drugs exist. However, major questions regarding the possible use of monobodies as precision cancer therapeutics remain to be answered. In particular, it is unclear, if monobodies can be be engineered to enter cancer cells in sufficient quantities. In addition, the pharmacokinetics and immunogenicity of monobodies is completely unexplored.
In this ERC project, my lab developed monobodies to oncoproteins for which no chemical inhibitors or drugs exist. We developed new technologies to deliver monobody proteins into cancer cells. 'Mirror-image' monobodies were for the first time developed and we showed their improved stability and better pharmacological behaviour. Finally, we determined and improved the plasma stability and biodistribution of monobodies. The overall objectives of the project were achieved, and we made great progress towards our goal to establish monobodies as a novel class of intracellular protein-based therapeutics.
My lab has pioneered studies to target oncoproteins with monobody proteins. Monobodies are small engineered binding proteins that can be regarded as mini-antibodies. This makes them much easier to handle and to produce. Monobodies can be developed in the laboratory and do not require the immunization of animals. On the other hand, monobodies display most pharmacologic features of antibodies, which include a highly specific interaction and the tight binding to its targets. We demonstrated that monobodies can be developed to several oncoproteins for which no specific drugs exist. However, major questions regarding the possible use of monobodies as precision cancer therapeutics remain to be answered. In particular, it is unclear, if monobodies can be be engineered to enter cancer cells in sufficient quantities. In addition, the pharmacokinetics and immunogenicity of monobodies is completely unexplored.
In this ERC project, my lab developed monobodies to oncoproteins for which no chemical inhibitors or drugs exist. We developed new technologies to deliver monobody proteins into cancer cells. 'Mirror-image' monobodies were for the first time developed and we showed their improved stability and better pharmacological behaviour. Finally, we determined and improved the plasma stability and biodistribution of monobodies. The overall objectives of the project were achieved, and we made great progress towards our goal to establish monobodies as a novel class of intracellular protein-based therapeutics.
In this ERC project, we developed monobodies to several proteins that are critically involved in cancer development. We have engineered monobody binders to various oncoproteins for which no drugs exist yet. These monobodies bound the target oncoproteins efficiently and with a high selectivity. The project team then continued to determine how the monobodies act outside and inside of cancer cells and how they can inhibit signalling pathways that are critical for the growth of tumour cells.
We made also excellent progress in developing new technologies to bring monobody proteins into cancer cells.:
1. Monobody delivery through chimeric bacterial toxin subunits: We hijacked how bacteria that secrete toxin proteins bring these proteins into their host cells. These toxins have a modular composition, and we only took those parts that are required for recognizing the host cells and transferring proteins into the host cells. These parts were then fused with monobodies, which we could thereby delivered into cancer cells. Our work provided a first unequivocal example of the delivery of a stoichiometric protein inhibitor to cancer cells.
2. As a novel delivery approach, we have changed the backside of monobodies to carry a high density of positive charges (‘super-charged monobody’). Supercharging is also used in nature by bacteria and viruses to bring specific peptides and proteins to their host cells. The super-charged monobodies enabled delivery of functional monobodies to the cytoplasm of cells in high enough concentrations to elicit a specific inhibition of the target proteins.
3. We have also reprogramed the type III secretion system (T3SS) injectisome of non-pathogenic bacteria to translocate monobody proteins into tumour cells. Direct injection into the cytoplasm and high accumulation of monobody proteins in different tumour cell lines was achieved. This resulted in signaling inhibition and specific induction of cell death.
Another important objective was to construct mirror-image D-monobodies. Protein therapeutics, such as antibodies, constantly encounter enzymes that could degrade them. Mirror-image proteins have some advantages including protection from degradation, and they cannot evoke an unwanted immune response. Unfortunately, mirror-image proteins are technically very challenging to engineer and produce. We have now managed to make the first mirror-image monobodies that bind and inhibit the central leukemia oncoprotein Bcr-Abl. We also demonstrated the high stability of these proteins in blood plasma.
To enable a possible application of our findings in vivo, we studied the pharmacokinetics, biodistribution and plasma stability of monobodies. We subsequently engineered monobody fusions with an albumin binding domain, which enabled the binding of monobodies to the major blood protein albumin. We were able to show that the half-life of monbodies was dramatically increased in mice.
Our results were published (or are on its way to be published) in international peer-reviewed scientific journals. All team members presented and discussed results of this project at various local, national and international conferences, workshops and summer schools for PhD students/postdocs. In addition, the PI presented results of the project in different academic institutions, at seminar series of professional scientific societies, at seminars at pharma/biotech companies and in different virtual conference during the pandemic.
We made also excellent progress in developing new technologies to bring monobody proteins into cancer cells.:
1. Monobody delivery through chimeric bacterial toxin subunits: We hijacked how bacteria that secrete toxin proteins bring these proteins into their host cells. These toxins have a modular composition, and we only took those parts that are required for recognizing the host cells and transferring proteins into the host cells. These parts were then fused with monobodies, which we could thereby delivered into cancer cells. Our work provided a first unequivocal example of the delivery of a stoichiometric protein inhibitor to cancer cells.
2. As a novel delivery approach, we have changed the backside of monobodies to carry a high density of positive charges (‘super-charged monobody’). Supercharging is also used in nature by bacteria and viruses to bring specific peptides and proteins to their host cells. The super-charged monobodies enabled delivery of functional monobodies to the cytoplasm of cells in high enough concentrations to elicit a specific inhibition of the target proteins.
3. We have also reprogramed the type III secretion system (T3SS) injectisome of non-pathogenic bacteria to translocate monobody proteins into tumour cells. Direct injection into the cytoplasm and high accumulation of monobody proteins in different tumour cell lines was achieved. This resulted in signaling inhibition and specific induction of cell death.
Another important objective was to construct mirror-image D-monobodies. Protein therapeutics, such as antibodies, constantly encounter enzymes that could degrade them. Mirror-image proteins have some advantages including protection from degradation, and they cannot evoke an unwanted immune response. Unfortunately, mirror-image proteins are technically very challenging to engineer and produce. We have now managed to make the first mirror-image monobodies that bind and inhibit the central leukemia oncoprotein Bcr-Abl. We also demonstrated the high stability of these proteins in blood plasma.
To enable a possible application of our findings in vivo, we studied the pharmacokinetics, biodistribution and plasma stability of monobodies. We subsequently engineered monobody fusions with an albumin binding domain, which enabled the binding of monobodies to the major blood protein albumin. We were able to show that the half-life of monbodies was dramatically increased in mice.
Our results were published (or are on its way to be published) in international peer-reviewed scientific journals. All team members presented and discussed results of this project at various local, national and international conferences, workshops and summer schools for PhD students/postdocs. In addition, the PI presented results of the project in different academic institutions, at seminar series of professional scientific societies, at seminars at pharma/biotech companies and in different virtual conference during the pandemic.
This ERC project focused on an entirely novel approach to expand the spectrum of targetable oncoproteins. Our work in this ERC project has enabled the development of monobody inhibitors of several notoriously difficult to target oncoproteins. In addition, two key unresolved obstacles for protein-based intracellular therapeutics were addressed: We have developed efficient protein delivery approaches to enable cell entry of monobodies and inhibition of targets in the cytoplasm of cancer cells. Secondly, we have developed mirror-image D-monobodies that limit monobody immunogenicity and increase its stability. We have employed a variety of state-of-the-art chemical biology and protein engineering methods and developed new assays to study the pharmacology of monobodies. This innovative project addressed a central problem in cancer medicine and we provided strong support that monobodies can be further developed to more effectively and more specifically target cancer.