Periodic Reporting for period 4 - ONCOINTRABODY (Targeting common oncogenes with intracellular monobodies)
Periodo di rendicontazione: 2021-10-01 al 2022-11-30
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. Those include the uncontrolled division of cells and the prevention of cell death. 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 metastatis, 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 technologies and approaches are urgently needed to broaden the variety of targeted oncoproteins. This is expected to result in an increase in efficacy and a better tolerability of therapies with less side-effects.
To address these challenges, my lab has pioneered studies to target oncoproteins with monobody proteins. Monobodies are small engineered binding proteins that can be regarded as mini-antibodies. Monobodies are more than 10-times smaller than monobodies and are composed of a single protein chain. This makes them much easier to handle and to produce. In contrast to antibodies, monobodies can be developed in the laboratory and do not required the immunization of animals. On the other hand, monobodies display most of the favourable pharmacologic features as antibodies, which included a highly specific interaction and the tight binding to its targets.
My laboratory has demonstrated that monobodies can be developed to several oncoproteins for which no specific drugs exist. In particular, monobodies are particularly suited to interfere with the interactions of oncoproteins with other proteins. However, major questions regarding the possible use of monobodies as precision protein-based cancer therapeutics remain to be answered. In particular, it is unclear, if monobodies can be developed to target a broad spectrum of oncoproteins from different protein families. A largely unsolved problem is the inability of proteins, such as monobodies, to enter cells on their own. While some techniques were developed to facilitate uptake of proteins into cancer cells, it is unknown if any such technique will enable the delivery of monobody proteins in sufficient quantities to cancer cells. Finally, the pharmacokinetics and immunogenicity of monobodies is completely unexplored.
In this ERC-project, my lab aims at developing monobody binders to key oncoproteins for which no chemical inhibitors exist. We will develop methods to deliver monobody proteins into cancer cells using available and newly developed technologies. 'Mirror-image' monobodies, composed of particular amino acids, will be developed and tested and promise to further improve the pharmacological behaviour of monobodies. Finally, we plan to test the developed monobodies and delivery systems in relevant models for cancer patients. Our goal is to establish monobodies as a novel class of intracellular protein-based therapeutics. We hope to kick off their use beyond basic research tools towards possible applications in cancer patients.
To address these challenges, my lab has pioneered studies to target oncoproteins with monobody proteins. Monobodies are small engineered binding proteins that can be regarded as mini-antibodies. Monobodies are more than 10-times smaller than monobodies and are composed of a single protein chain. This makes them much easier to handle and to produce. In contrast to antibodies, monobodies can be developed in the laboratory and do not required the immunization of animals. On the other hand, monobodies display most of the favourable pharmacologic features as antibodies, which included a highly specific interaction and the tight binding to its targets.
My laboratory has demonstrated that monobodies can be developed to several oncoproteins for which no specific drugs exist. In particular, monobodies are particularly suited to interfere with the interactions of oncoproteins with other proteins. However, major questions regarding the possible use of monobodies as precision protein-based cancer therapeutics remain to be answered. In particular, it is unclear, if monobodies can be developed to target a broad spectrum of oncoproteins from different protein families. A largely unsolved problem is the inability of proteins, such as monobodies, to enter cells on their own. While some techniques were developed to facilitate uptake of proteins into cancer cells, it is unknown if any such technique will enable the delivery of monobody proteins in sufficient quantities to cancer cells. Finally, the pharmacokinetics and immunogenicity of monobodies is completely unexplored.
In this ERC-project, my lab aims at developing monobody binders to key oncoproteins for which no chemical inhibitors exist. We will develop methods to deliver monobody proteins into cancer cells using available and newly developed technologies. 'Mirror-image' monobodies, composed of particular amino acids, will be developed and tested and promise to further improve the pharmacological behaviour of monobodies. Finally, we plan to test the developed monobodies and delivery systems in relevant models for cancer patients. Our goal is to establish monobodies as a novel class of intracellular protein-based therapeutics. We hope to kick off their use beyond basic research tools towards possible applications in cancer patients.
In this ERC project, we made good progress in developing 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. In all cases these monobodies bound the target oncoproteins efficiently and with an extremely high selectivity, which limits possible side-effects. The project team then continued to determine how the monobodies act outside and inside of cells.
In particular for two targets, two publications in renowned international journal could be finished:
The Src kinases are important oncoproteins and drug targets, but also play important roles in normal development and signaling. Genetic, cell biological and biochemical studies have established Src kinases as important drug targets, but it has been difficult to selectively target Src kinases with various approaches. We have successfully developed eleven monobody clones that act as potent inhibitors of different Src kinases and recognize their targets with an unprecedented level of selectivity. We also determined the three-dimensional structure of three different monobodies, which showed us how they can be so selectivity and enabled experiments in which we could switch their binding properties. Finally, we showed that the developed monobodies selectively perturb signaling of specific Src kinases in cancer cells.
In a second manuscript, we provide the first evidence that a class of proteins for which no drugs exist on the market, can be inhibited by monobody proteins. These are the transcription factors, which are proteins that bind to DNA and regulate the expression of genes in cells. In cancer cells, transcription factors can be altered and often activate genes that are normally not activated in healthy cells. We have focused on the transcription factor STAT3, which is activated in a large number of tumor types and is even mutated in certain blood cancers. In our work, we demonstrate a novel way to target STAT3 activity with monobodies and acting through an unsuspected mechanism-of-action. Furthermore, novel insight on STAT3 structural biology and alternative STAT3 signaling is provided. We foresee that targeting of STAT3 in different cancers, but also in chronic inflammatory diseases could be a new therapeutic approach.
In particular for two targets, two publications in renowned international journal could be finished:
The Src kinases are important oncoproteins and drug targets, but also play important roles in normal development and signaling. Genetic, cell biological and biochemical studies have established Src kinases as important drug targets, but it has been difficult to selectively target Src kinases with various approaches. We have successfully developed eleven monobody clones that act as potent inhibitors of different Src kinases and recognize their targets with an unprecedented level of selectivity. We also determined the three-dimensional structure of three different monobodies, which showed us how they can be so selectivity and enabled experiments in which we could switch their binding properties. Finally, we showed that the developed monobodies selectively perturb signaling of specific Src kinases in cancer cells.
In a second manuscript, we provide the first evidence that a class of proteins for which no drugs exist on the market, can be inhibited by monobody proteins. These are the transcription factors, which are proteins that bind to DNA and regulate the expression of genes in cells. In cancer cells, transcription factors can be altered and often activate genes that are normally not activated in healthy cells. We have focused on the transcription factor STAT3, which is activated in a large number of tumor types and is even mutated in certain blood cancers. In our work, we demonstrate a novel way to target STAT3 activity with monobodies and acting through an unsuspected mechanism-of-action. Furthermore, novel insight on STAT3 structural biology and alternative STAT3 signaling is provided. We foresee that targeting of STAT3 in different cancers, but also in chronic inflammatory diseases could be a new therapeutic approach.
Our knowledge on the biological mechanisms and targeting of cancer has expanded enormously, but overall survival has not improved for a majority of cancer types over the past decades. In this project, we work on an entirely novel approach to expand the spectrum of targetable oncoproteins by advancing monobodies to novel protein-based intracellular therapeutics. Our previous work that focused on a limited number of targets, established monobodies as efficient tools to inhibit oncogenic signalling. Their small size and quick evolvability make them optimal candidates, but their potential to act as protein-based therapeutics is completely unexplored. We have selected notoriously difficult to target oncoproteins. Besides targeting highly relevant oncoproteins, two key unresolved obstacles for protein-based intracellular therapeutics are addressed: Enabling efficient protein delivery to cancer cells and limiting monobody immunogenicity. We will systematically evaluate protein delivery methods and employ new chemical biology methods. This innovative endeavour uses state-of-the-art protein engineering techniques to address a central problem in cancer medicine and may provide a ground-breaking new approach to more effectively and more specifically target cancer.