Periodic Reporting for period 1 - ADC2GBM (Development of brain-permeable masked nanobody-drug conjugates to eliminate glioma stem cells)
Período documentado: 2022-05-01 hasta 2024-04-30
Glioblastoma multiforme (GBM) is the most prevalent and aggressive brain tumour. GBM is the second cause of death from neurological diseases and accounts for 4% of cancer associated deaths. Despite surgical tumour resection followed by chemotherapy and radiotherapy, life expectancy is still limited to 15-20 months due to tumour relapse. Therefore, there is a great need to find more efficient therapies. New treatments need to overcome two main challenges: 1. to reach the tumour margins, where the blood-brain barrier (BBB) is intact, and 2. to eradicate glioma stem cells (GSCs), which are held responsible for tumour relapse after current treatments. Recent advancements at the interface between biotechnology and chemistry provide attractive novel treatment possibilities for GBM. The main objective of the project developed during this MSCA was to engineer a masked antibody derivative with the capacity to overcome the blood-brain barrier (BBB) and selectively kill the remaining glioblastoma stem cells (GSC).
GSCs represent a subpopulation of cells within glioblastoma that are characterized by increased resistance to chemotherapy and radiotherapy. There are several antibody-drug conjugates being developed against GSCs but none has reached clinical approval due to the difficulty in selectively targeting them without affecting other healthy cells expressing the same receptors. A common strategy to direct therapies to GSCs is targeting cell surface makers, such as CD133, a transmembrane glycoprotein. However, this receptor is also expressed in other noncancer cells such as hematopoietic stem cells and epithelial cells with microvilli. Thus, therapies targeting this receptor can result in severe on-target off-site effects. In the last decade, the field of activatable antibodies has emerged, enabling the engagement of antigens that were previously considered undruggable. Therefore, to avoid such deleterious effects, we are developing targeting molecules that are only activated in response to tumour microenvironmental cues, especially enzymatic activity. In this MSCA we have contribute to developing a masked CD133-specific antibody-derivative that is only activated in the tumour environment.
Anti-CD133 masked antibodies may revolutionize the treatment of GBM only if they are capable of reaching all GSCs. Since GSCs are also found in the periphery of the tumour, these biotherapeutics need to cross the BBB. This is mainly formed by brain capillary endothelial cells, which establish tight junctions that hinder paracellular transport. BBB-shuttle peptides are molecules capable of transporting cargoes into the brain parenchyma without disrupting the BBB. Although we and others have developed brain shuttles in the past, there is still a need to discover new and improved shuttles with the capacity to resist protease degradation and transport bigger molecules into the brain. During the course of this action, we have produced a new family of protease-resistant brain shuttles that are able to enhance the transport of antibodies into the brain, which should dramatically increase its efficacy.
The concept of GSC-targeting with activatable antibodies in combination with brain shuttles may change the current paradigm for the treatment of brain tumours. Furthermore, our workflow may be applied to dramatically enhance the efficacy of other biotherapeutics with on-target off-site dose-limiting effects and diseases affecting the brain.
GSCs represent a subpopulation of cells within glioblastoma that are characterized by increased resistance to chemotherapy and radiotherapy. There are several antibody-drug conjugates being developed against GSCs but none has reached clinical approval due to the difficulty in selectively targeting them without affecting other healthy cells expressing the same receptors. A common strategy to direct therapies to GSCs is targeting cell surface makers, such as CD133, a transmembrane glycoprotein. However, this receptor is also expressed in other noncancer cells such as hematopoietic stem cells and epithelial cells with microvilli. Thus, therapies targeting this receptor can result in severe on-target off-site effects. In the last decade, the field of activatable antibodies has emerged, enabling the engagement of antigens that were previously considered undruggable. Therefore, to avoid such deleterious effects, we are developing targeting molecules that are only activated in response to tumour microenvironmental cues, especially enzymatic activity. In this MSCA we have contribute to developing a masked CD133-specific antibody-derivative that is only activated in the tumour environment.
Anti-CD133 masked antibodies may revolutionize the treatment of GBM only if they are capable of reaching all GSCs. Since GSCs are also found in the periphery of the tumour, these biotherapeutics need to cross the BBB. This is mainly formed by brain capillary endothelial cells, which establish tight junctions that hinder paracellular transport. BBB-shuttle peptides are molecules capable of transporting cargoes into the brain parenchyma without disrupting the BBB. Although we and others have developed brain shuttles in the past, there is still a need to discover new and improved shuttles with the capacity to resist protease degradation and transport bigger molecules into the brain. During the course of this action, we have produced a new family of protease-resistant brain shuttles that are able to enhance the transport of antibodies into the brain, which should dramatically increase its efficacy.
The concept of GSC-targeting with activatable antibodies in combination with brain shuttles may change the current paradigm for the treatment of brain tumours. Furthermore, our workflow may be applied to dramatically enhance the efficacy of other biotherapeutics with on-target off-site dose-limiting effects and diseases affecting the brain.
The main objective of the project developed during this MSCA was to engineer a masked antibody-derivative with the capacity to overcome the blood-brain barrier (BBB) and selectively kill the remaining glioblastoma stem cells (GSC).
We have designed and created phage display libraries to generate nanobodies against CD133, a marker of GSC, where the complementary determining regions (CDRs) were randomized. Several phage display libraries have been produced changing the length of the CDRs to allow for more diversity in the selection process. In addition, and to be able to perform the phage display selections, we had to design and express as soluble constructs the extracellular domains of the CD133 receptor. With both the target and the phage display libraries, we are now performing the panning experiments. Preliminary results point to a nanobody sequence with high affinity for CD133.
To selectively kill cancer stem cells, we are developing several masking strategies for antibody-derivatives, facilitating their delivery to their site of action where they will be unmasked and act. We started these studies with an already described single-chain variable fragment antibody (scFv) against the CD133 receptor for which we have developed a steric hindrance mask. With this mask, we can block the interaction with the receptor by 20-folds. We showed that the masking efficiency depends on the conjugation site and the size of the mask. Moreover, the affinity can be fully recovered upon activation by exposure to light at 365 nm or by incubation with matrix metalloproteinases overexpressed in solid tumours (Lucchi et al. New Biotechnology 78, 76-83 (2023)). We have performed biodistribution studies in murine models of carcinoma with this masked anti-CD133 scFv. Preliminary results show some accumulation of the antibodies in the tumour site.
To overcome the BBB, we wanted to discover new brain shuttles with improved characteristics, i.e. higher stability and permeability, which could transport antibodies into the brain. We aimed to develop a methodology that could be applied to a wider variety of peptide shuttles. To this end, we have generated a new family of bicyclic brain shuttles with the capacity to enhance the transport of therapeutic proteins across brain endothelium up to 5-folds. These bicyclic peptides, dubbed BrainBikes, have an increased stability in serum proteases than their linear counterparts and are able to cross the BBB as efficiently. An anti-CD133 scFv was conjugated with the BrainBike peptide and their ability to cross the blood-brain barrier was assessed using a human cell-based BBB model. The results show an increase in the transport capacity when the scFvs are conjugated with the BBB-shuttle than when they are not (Lucana et al RSC Chem Biol 5, 7-11 (2024)).
We have designed and created phage display libraries to generate nanobodies against CD133, a marker of GSC, where the complementary determining regions (CDRs) were randomized. Several phage display libraries have been produced changing the length of the CDRs to allow for more diversity in the selection process. In addition, and to be able to perform the phage display selections, we had to design and express as soluble constructs the extracellular domains of the CD133 receptor. With both the target and the phage display libraries, we are now performing the panning experiments. Preliminary results point to a nanobody sequence with high affinity for CD133.
To selectively kill cancer stem cells, we are developing several masking strategies for antibody-derivatives, facilitating their delivery to their site of action where they will be unmasked and act. We started these studies with an already described single-chain variable fragment antibody (scFv) against the CD133 receptor for which we have developed a steric hindrance mask. With this mask, we can block the interaction with the receptor by 20-folds. We showed that the masking efficiency depends on the conjugation site and the size of the mask. Moreover, the affinity can be fully recovered upon activation by exposure to light at 365 nm or by incubation with matrix metalloproteinases overexpressed in solid tumours (Lucchi et al. New Biotechnology 78, 76-83 (2023)). We have performed biodistribution studies in murine models of carcinoma with this masked anti-CD133 scFv. Preliminary results show some accumulation of the antibodies in the tumour site.
To overcome the BBB, we wanted to discover new brain shuttles with improved characteristics, i.e. higher stability and permeability, which could transport antibodies into the brain. We aimed to develop a methodology that could be applied to a wider variety of peptide shuttles. To this end, we have generated a new family of bicyclic brain shuttles with the capacity to enhance the transport of therapeutic proteins across brain endothelium up to 5-folds. These bicyclic peptides, dubbed BrainBikes, have an increased stability in serum proteases than their linear counterparts and are able to cross the BBB as efficiently. An anti-CD133 scFv was conjugated with the BrainBike peptide and their ability to cross the blood-brain barrier was assessed using a human cell-based BBB model. The results show an increase in the transport capacity when the scFvs are conjugated with the BBB-shuttle than when they are not (Lucana et al RSC Chem Biol 5, 7-11 (2024)).
Health, including the treatment of cancer is one of the main societal challenges defined by the European Commission. Brain tumours have very poor prognosis, thus cancer patients, and especially those suffering from brain cancers, will benefit more directly from the results of the research performed during this MSCA. We have engineered masked antibody derivatives that selectively target cancer stem cells, and a new family of brain shuttle peptides that can overcome the blood-brain barrier. These molecules, together with the ones that we are now developing, can change the paradigm in the treatment of brain cancers.