Periodic Reporting for period 1 - Chemical Mutagenesis (Chemical mutagenesis: a powerful tool for the creation of a library of nanobodies)
Okres sprawozdawczy: 2022-03-01 do 2024-02-29
Therapies based on monoclonal antibodies (mAb) have revolutionized different medical fields, particularly, oncology.
By recognizing and targeting some checkpoints overexpressed on the surface of cancer cells, mAb can trigger an immune response that may result in cell apoptosis. Namely, Nivolumab (OPDIVO®) and Pembroluzimab (Keytruda®) disrupt the interaction between T cells (immune cells) and cancer cells by blocking a transmembrane protein called PD-L1, a checkpoint commonly overexpressed in tumors. As a result, T cells are no longer capable of recognizing these malignant cells.
mAb affinity towards certain receptors can also be harnessed to deliver cytotoxic payloads specifically to the tumor environment, thereby decreasing the off-target effect of the drug. As an example, Adcetris®, used in the treatment of certain lymphomas, consists of a mAb (brentuximab) chemically modified to incorporate a very potent antimitotic agent (Monomethyl auristatin E), which is delivered mainly in the tumor.
These mAbs are called Antibody Drug Conjugates (ADCs) and, so far there are 14 already approved by the FDA and around 100 are presently being tested.
However, some mAbs are currently failing in clinical trials due to its low tissue penetration. Their size (molecular weight over 100KDa) drastically reduces the diffusion within the tumor, which limits their efficacy as they only reach the surface of the tumor, while the inside is still growing.
In this sense, smaller proteins might be a solution as their diffusion is higher. Some constructions as nanobodies have been deeply studied to overcome mAb limitations. Nevertheless, these systems are fast cleared from the body, which also impact on their efficacy, as this fast removal may decrease the levels of drug reaching the tumor.
For all these reasons, finding a proper macromolecule that balances size and clearance remains a challenge and could be the key to achieve more effective treatment for cancer.
In this project, we envisioned the possibility of increasing the half-life of small proteins by stablishing a covalent bond with their targets. This covalent interaction would increase the tissue retention as these proteins, once bound to their receptors, would not be cleared that fast.
With this aim, we propose the chemical modification of small proteins to incorporate a relatively reactive motif able to interact only with receptor PD-L1.
By recognizing and targeting some checkpoints overexpressed on the surface of cancer cells, mAb can trigger an immune response that may result in cell apoptosis. Namely, Nivolumab (OPDIVO®) and Pembroluzimab (Keytruda®) disrupt the interaction between T cells (immune cells) and cancer cells by blocking a transmembrane protein called PD-L1, a checkpoint commonly overexpressed in tumors. As a result, T cells are no longer capable of recognizing these malignant cells.
mAb affinity towards certain receptors can also be harnessed to deliver cytotoxic payloads specifically to the tumor environment, thereby decreasing the off-target effect of the drug. As an example, Adcetris®, used in the treatment of certain lymphomas, consists of a mAb (brentuximab) chemically modified to incorporate a very potent antimitotic agent (Monomethyl auristatin E), which is delivered mainly in the tumor.
These mAbs are called Antibody Drug Conjugates (ADCs) and, so far there are 14 already approved by the FDA and around 100 are presently being tested.
However, some mAbs are currently failing in clinical trials due to its low tissue penetration. Their size (molecular weight over 100KDa) drastically reduces the diffusion within the tumor, which limits their efficacy as they only reach the surface of the tumor, while the inside is still growing.
In this sense, smaller proteins might be a solution as their diffusion is higher. Some constructions as nanobodies have been deeply studied to overcome mAb limitations. Nevertheless, these systems are fast cleared from the body, which also impact on their efficacy, as this fast removal may decrease the levels of drug reaching the tumor.
For all these reasons, finding a proper macromolecule that balances size and clearance remains a challenge and could be the key to achieve more effective treatment for cancer.
In this project, we envisioned the possibility of increasing the half-life of small proteins by stablishing a covalent bond with their targets. This covalent interaction would increase the tissue retention as these proteins, once bound to their receptors, would not be cleared that fast.
With this aim, we propose the chemical modification of small proteins to incorporate a relatively reactive motif able to interact only with receptor PD-L1.
Collaborators from the Institute for Protein Design (Seattle, USA) designed and expressed several mutants of a small protein (minibinder) with high affinity to PD-L1. Those mutants contained free cysteines at different positions facing the surface of PD-L1. On our side, we studied different methodologies to incorporate new functionalities onto the surface of those minibinders via site selective cysteine modification.
Aiming to improve the affinity of these minibinders, we successfully introduce some relatively stable functionalities known as latent electrophiles. These warheads are able to react with some functional groups (present on a protein) only in proximity.
That way, we created a library of 40 new minibinders with different affinities to PD-L1, and we selected those able to stablish a covalent interaction with that receptor. From those, we selected the fastest one to study it in vitro. Preliminary results obtained in collaboration with the Instituto de Medicina Molecular (IMM) from Lisbon, suggest that this new minibinder not only establishes a covalent interaction with PD-L1 on cell surface, but also induces its degradation.
These results have been disseminated at the EFMCE-ISCB International Symposium on Chemical Biology celebrated at Basel in November 2023, and once the undergoing experiments have been accomplished, the main outputs of the project will be published in a high-impact journal.
Aiming to improve the affinity of these minibinders, we successfully introduce some relatively stable functionalities known as latent electrophiles. These warheads are able to react with some functional groups (present on a protein) only in proximity.
That way, we created a library of 40 new minibinders with different affinities to PD-L1, and we selected those able to stablish a covalent interaction with that receptor. From those, we selected the fastest one to study it in vitro. Preliminary results obtained in collaboration with the Instituto de Medicina Molecular (IMM) from Lisbon, suggest that this new minibinder not only establishes a covalent interaction with PD-L1 on cell surface, but also induces its degradation.
These results have been disseminated at the EFMCE-ISCB International Symposium on Chemical Biology celebrated at Basel in November 2023, and once the undergoing experiments have been accomplished, the main outputs of the project will be published in a high-impact journal.
PD-L1 interaction with its counterpart PD-1 leads to a dampened antitumor response. In this project we managed to covalently block and degrade this checkpoint which may allow T cell recognition and increase the immune response to cancer.
However, the degradation mechanism and the in vivo cytotoxicity and tumor selectivity remain unknown. Some more experiments are currently undergoing to provide some insight into these matters. As soon as these questions have been answered, this platform could be patented to be exploited in a company/institution able to perform clinical trials and possibly develop a new improved treatment against PD-L1+ cancers.
However, the degradation mechanism and the in vivo cytotoxicity and tumor selectivity remain unknown. Some more experiments are currently undergoing to provide some insight into these matters. As soon as these questions have been answered, this platform could be patented to be exploited in a company/institution able to perform clinical trials and possibly develop a new improved treatment against PD-L1+ cancers.