Periodic Reporting for period 1 - Lipobodies (Targeting intracellular proteins and protein-protein interactions with site-specific lipobodies: a new approach for intracellular antibodies)
Reporting period: 2021-10-01 to 2023-09-30
The main scientific problem addressed during this project was the lack of effective delivery strategies for antibodies and antibody fragments. It proposed that the development of a practical, general, and modular strategy for the efficient delivery and cell uptake of antibodies and antibody fragments could help further optimize these therapeutics, which have already proven high value in the treatment of cancer and many other diseases. As the outcomes of this project could positively impact the development of next-generation therapeutics, they could be of high interest not only to the scientific community but to the general society.
The main strategy proposed by this project was to study the covalent lipidation of antibodies or antibody fragments as a tool to efficiently boost their cell internalization. To assemble different lipo-antibodies bearing a variable number of lipid chains as well as labels for intracellular monitoring and suitable handles for bioconjugation, a unique combination between multicomponent reactions (MCRs) and site-selective conjugation chemistries was projected (Figure 1).
During this period, I developed and optimized the conditions for both the assembly of modular systems for the modification of the antibody fragments and their final conjugation. The utilization of MCRs allowed me to generate a library of constructs, including fluorescently labeled molecules for cell internalization and trafficking monitoring. Lipidation of therapeutics such as the engineered antibody THIOMAB V205C, different anti-HER2 affibodies, and anti-mesothelin nanobodies, proved efficient at increasing their cell permeability properties, without strongly affecting their target binding capacity. As a follow-up to this work, I also aim to study lipidation of therapeutics as a strategy to foster half-life extension, which has been described as one of the main drawbacks of antibody fragments.
In addition to this work, I designed and developed a new multicomponent process based on the combination of the isonitrile-tetrazine (4+1) cycloaddition and the Ugi four-component reaction (Ugi-4CR) which allowed me to access a new chemical space of pyrazole amide derivatives and has been already well received by the scientific community.
The main strategy proposed by this project was to study the covalent lipidation of antibodies or antibody fragments as a tool to efficiently boost their cell internalization. To assemble different lipo-antibodies bearing a variable number of lipid chains as well as labels for intracellular monitoring and suitable handles for bioconjugation, a unique combination between multicomponent reactions (MCRs) and site-selective conjugation chemistries was projected (Figure 1).
During this period, I developed and optimized the conditions for both the assembly of modular systems for the modification of the antibody fragments and their final conjugation. The utilization of MCRs allowed me to generate a library of constructs, including fluorescently labeled molecules for cell internalization and trafficking monitoring. Lipidation of therapeutics such as the engineered antibody THIOMAB V205C, different anti-HER2 affibodies, and anti-mesothelin nanobodies, proved efficient at increasing their cell permeability properties, without strongly affecting their target binding capacity. As a follow-up to this work, I also aim to study lipidation of therapeutics as a strategy to foster half-life extension, which has been described as one of the main drawbacks of antibody fragments.
In addition to this work, I designed and developed a new multicomponent process based on the combination of the isonitrile-tetrazine (4+1) cycloaddition and the Ugi four-component reaction (Ugi-4CR) which allowed me to access a new chemical space of pyrazole amide derivatives and has been already well received by the scientific community.
The assembly of modular constructs for lipidation (lipo-TAGs), that is scaffolds incorporating one or two lipid chains, fluorescent labels for intracellular localization monitoring, and proper spacer/linker systems for bioconjugation, was the first task planned and accomplished during this project. The utilization of the Ugi-4CR, allowed me to build diverse lipo-TAGs in a highly efficient and modular way. These lipo-TAGs contain two lipid chains, either two palmitic or one palmitic and one myristic fatty-acid derived chains, spacers with different lengths and hydrophobic character, and eventually carboxyfluorescein, which was chosen as a fluorophore for internalization monitoring. Different linkers were either directly or post-Ugi-4CR introduced, i.e. containing the azide and dibenzocyclooctyne (DBCO) groups, allowing protein conjugation via a strain-promoted azide-alkyne cycloaddition (SPAAC), and maleimide, allowing site-specific cysteine modification via Michael addition.
As part of my endeavors using the Ugi-4CR for the assembly of the lipo-TAGs, I conceived the idea of developing a new multicomponent reaction based on the combination of the isonitrile-tetrazine (4+1) cycloaddition and the Ugi-4CR. This new multicomponent reaction allowed the synthesis of novel pyrazole amide derivatives and proved highly efficient for both the assembly of small molecules and the modification of peptides, including peptide stapling (Figure 2). The results of this work were recently published in a high-impact journal and presented at the 8th International Conference on Multicomponent Reactions and Related Chemistry, Burgos, Spain. The main significance of this part of the research lies in the growing demand for high diversity- and complexity-oriented processes, not only allowing the efficient assembly of modular systems such as the lipo-TAGs but also structurally complex and diverse libraries of molecules meeting different medicinal chemistry purposes.
The next task consisted of the synthesis of the lipobodies, that is the expression, purification, and modification of the different antibody formats. The main targets proposed in this research project were different oncogenic RAS proteins, but unfortunately, the expression of the intracellular domain antibodies targeting RAS was extremely challenging. Anticipating this type of risk, I decided to move forward with the proof of concept and selected other oncogenic targets such as the human epidermal growth factor receptor 2 (HER2) and mesothelin. As anti-HER2 formats, I chose two anti-HER2 affibodies with different cysteine mutation sites and the engineered antibody THIOMAB V205C. After efficiently achieving conjugation of these formats to the lipo-TAGs, the analytical and biophysical characterization of the obtained lipobodies allowed us to asses that neither the affibodies' structure nor their binding to the HER2 receptor was significantly affected in comparison with the parent affibodies. Thereafter, I modified these formats with fluorescently labeled lipo-TAGs, which allowed me to study their cell-internalization properties in comparison with the non-lipidated constructs.
The results of the current project were presented in internal group seminars as well as in the Halle-Glacier workshop “From Immunology to bioactive compounds". The most recent communication of the results of this project was at the Bayer Boundary Breaking Science Lecture on “Multicomponent reactions: exploring a wider chemical space toward medicinal and biomedical applications” which took place online on the 19th of October, 2023. Other dissemination forms include different interviews from the Bayer Science Foundation and the Women in Research #LINO23 blog.
As part of my endeavors using the Ugi-4CR for the assembly of the lipo-TAGs, I conceived the idea of developing a new multicomponent reaction based on the combination of the isonitrile-tetrazine (4+1) cycloaddition and the Ugi-4CR. This new multicomponent reaction allowed the synthesis of novel pyrazole amide derivatives and proved highly efficient for both the assembly of small molecules and the modification of peptides, including peptide stapling (Figure 2). The results of this work were recently published in a high-impact journal and presented at the 8th International Conference on Multicomponent Reactions and Related Chemistry, Burgos, Spain. The main significance of this part of the research lies in the growing demand for high diversity- and complexity-oriented processes, not only allowing the efficient assembly of modular systems such as the lipo-TAGs but also structurally complex and diverse libraries of molecules meeting different medicinal chemistry purposes.
The next task consisted of the synthesis of the lipobodies, that is the expression, purification, and modification of the different antibody formats. The main targets proposed in this research project were different oncogenic RAS proteins, but unfortunately, the expression of the intracellular domain antibodies targeting RAS was extremely challenging. Anticipating this type of risk, I decided to move forward with the proof of concept and selected other oncogenic targets such as the human epidermal growth factor receptor 2 (HER2) and mesothelin. As anti-HER2 formats, I chose two anti-HER2 affibodies with different cysteine mutation sites and the engineered antibody THIOMAB V205C. After efficiently achieving conjugation of these formats to the lipo-TAGs, the analytical and biophysical characterization of the obtained lipobodies allowed us to asses that neither the affibodies' structure nor their binding to the HER2 receptor was significantly affected in comparison with the parent affibodies. Thereafter, I modified these formats with fluorescently labeled lipo-TAGs, which allowed me to study their cell-internalization properties in comparison with the non-lipidated constructs.
The results of the current project were presented in internal group seminars as well as in the Halle-Glacier workshop “From Immunology to bioactive compounds". The most recent communication of the results of this project was at the Bayer Boundary Breaking Science Lecture on “Multicomponent reactions: exploring a wider chemical space toward medicinal and biomedical applications” which took place online on the 19th of October, 2023. Other dissemination forms include different interviews from the Bayer Science Foundation and the Women in Research #LINO23 blog.
The lipidation of the different antibody formats proved efficient at enhancing the cell internalization properties of these constructs. These results open an avenue of opportunities for the development of further projects, including the design of lipo-antibody drug conjugates and bispecific formats targeting both extra- and intracellular receptors. Another property of the lipo-TAGs that our research group is interested in investigating is their capability to increase the half-life of antibody fragments, which has been extensively described as one of the major drawbacks of these constructs. These would signify increasing the likelihood of these therapeutics, which have already proven to be less immunogenic when compared to full antibodies, to reach clinical trials and be introduced to the market. As the project results related to the development of the lipo-TAGs and lipobodies are of highly innovative and translational character, we are planning to patent and license them in the near future.
On the other hand, the successful design and development of a new multicomponent process, although parallel to the main project, not only makes it possible to meet the demand for high diversity- and complexity-oriented processes, but also to access a novel chemical space that can be of high interest when designing combinatorial libraries of compounds with medicinal applications.
On the other hand, the successful design and development of a new multicomponent process, although parallel to the main project, not only makes it possible to meet the demand for high diversity- and complexity-oriented processes, but also to access a novel chemical space that can be of high interest when designing combinatorial libraries of compounds with medicinal applications.