Periodic Reporting for period 4 - SUPRAVACC (Supramolecular engineering of glycan-decorated peptides as synthetic vaccines)
Okres sprawozdawczy: 2023-10-01 do 2025-03-31
The main and most important feature of vaccines is the induction of an immunological memory response, which is key to providing long-term protection against pathogens. The current strategies for potent antibacterial and antiviral vaccines employ conjugation of pathogen specific entities onto carrier proteins, and are limited to formulations that suffer from low stability and short shelf-lives, and are thus not viable in developing countries. Strategies for the development of new vaccinations against endogenous diseases like cancer further remain an unmet challenge, since current methodologies suffer from a lack of a modular and tailored vaccine-specific functionalisation. SupraVacc therefore proposes a new design approach for the development of fully synthetic molecular vaccines, using carbohydrate and glycopeptide appended epitopes that are grafted onto supramolecular building blocks. These units can be individually designed to attach disease specific antigens and immunostimulants. Due to their self-assembling properties into nanoscaled pathogen mimetic particles, they serve as a supramolecular subunit vaccine toolbox. By developing a universal supramolecular polymer platform, we will construct multipotent vaccines from glycan-decorated peptides, that combine the activity of protein conjugates with the facile handling, precise composition and increased stability of traditional small molecule pharmaceutical compounds. SUPRAVACC will pioneer the design of minimalistic and broadly applicable vaccines, and will evaluate the supramolecular engineering approach for immunisations against antibacterial diseases, as well as for applications as antitumour vaccine candidates.
Multicomponent supramolecular copolymerization of functionalized peptide amphiphiles yields pathogen mimetic particles with precise composition of bioactive components that are co-presented on the surface of the fibers. For further development the understanding of the mechanism, the dynamics and the stability of the assembly, and the internalization and localization in livings cells is of particular interest in order to develop a flexible fully synthetic vaccine platform. For the study of the dynamics of the system the successful synthesis of the peptide amphiphiles was performed using functionalized dyes that are suitable for the fluorescence spectroscopic and microscopic investigations. By supramolecular co-polymerization of unfunctionalized peptide amphiphiles and FRET pair labels the timescale of the exchange of the supramolecular building blocks within the assembly of nanofibers was studied. The FRET efficiency was measured spectroscopically and showed an increase of energy transfer in the timescale of hours to days, depending on the molecular structure. The slow dynamics of the system is advantageous and has enabled the microscopic investigation of the nanofibers in physiological buffers using laser scanning confocal microscopy (LSCM). Imaging of the nanostructures show micrometer long fibers. Incubation of the fibers with bone marrow derived murine macrophages (BMDM) or RAW 309 murine macrophages (RAW 309) show an efficient internalization into cells. More advanced techniques like fluorescence lifetime imaging microscopy (FLIM) will further be used to gather information on the stability of the fibers inside immune cells and in vivo. This successful synthetic route will also enable the introduction of receptor agonists for receptor mediated internalization into living cells, address different cell populations and finally modulate the immunoresponse of supramolecular vaccines. With respect to the development of synthetic vaccines, we have aim for bacterial (I) and anti-tumour vaccines(II):
(I) For example the bacterial pathogen streptococcus pneumoniae can be classified into different serotypes due to the molecular structure of the capsular polysaccharide. In our fully synthetic approach, oligosaccharide antigens are currently being synthesized and subsequently conjugated to supramolecular monomers that facilitate the modular construction of a multivalent vaccine. Precursor molecules for the syntheses of antigens, derived from the capsular polysaccharides of S. Pneumoniae serotype 3 and 14 have been successfully synthesized in the first 24 months of the project.
(II) Breast cancer is the most common cancer worldwide. A promising target structure for therapy of breast cancer is tumor-associated mucin 1 (TA-MUC1) which is detected in more than 90% of all breast cancers. Due to its characteristic aberrant glycosylation, MUC1 is a promising tumor-specific antigen. Basis of a vaccine are its specific antigenic structures as well as stimulating additives. In this respect, we successfully accomplished the organic synthesis of three tumor-associated carbohydrate antigens (TACA): the Thomsen-nouveau-antigen (TN), a sialylated Thomsen-nouveau-antigen (STN) and a sialylated Thomsen-Friedenreich-antigen (2,3-ST) – which were subsequently incorporated into a MUC1 derived peptide backbone resulting in the TA-MUC1 sequences PAHGVT11(TN)SAPDTRPAPGST18(2,3-ST)APPA and PAHGVTSAPDTRPAPGS17(STN)TAPPA. Additionally, T cell epitope oligopeptide sequences derived from tetanus toxoid, p2 and p30, as well as a pan DR-binding epitope (PADRE) were produced via solid phase peptide synthesis. The synthesis of an immunostimulant on the basis of an imidazoquinoline structural motif was accomplished and optimized to provide a high affinity Toll-like receptor 7/8 (TLR7/8) agonist, which is able to link the innate immune response with adaptive immunity. Immunological evaluation of these successfully prepared building blocks has been started, and optimization studies using multicomponent vaccines are currently ongoing.
(I) For example the bacterial pathogen streptococcus pneumoniae can be classified into different serotypes due to the molecular structure of the capsular polysaccharide. In our fully synthetic approach, oligosaccharide antigens are currently being synthesized and subsequently conjugated to supramolecular monomers that facilitate the modular construction of a multivalent vaccine. Precursor molecules for the syntheses of antigens, derived from the capsular polysaccharides of S. Pneumoniae serotype 3 and 14 have been successfully synthesized in the first 24 months of the project.
(II) Breast cancer is the most common cancer worldwide. A promising target structure for therapy of breast cancer is tumor-associated mucin 1 (TA-MUC1) which is detected in more than 90% of all breast cancers. Due to its characteristic aberrant glycosylation, MUC1 is a promising tumor-specific antigen. Basis of a vaccine are its specific antigenic structures as well as stimulating additives. In this respect, we successfully accomplished the organic synthesis of three tumor-associated carbohydrate antigens (TACA): the Thomsen-nouveau-antigen (TN), a sialylated Thomsen-nouveau-antigen (STN) and a sialylated Thomsen-Friedenreich-antigen (2,3-ST) – which were subsequently incorporated into a MUC1 derived peptide backbone resulting in the TA-MUC1 sequences PAHGVT11(TN)SAPDTRPAPGST18(2,3-ST)APPA and PAHGVTSAPDTRPAPGS17(STN)TAPPA. Additionally, T cell epitope oligopeptide sequences derived from tetanus toxoid, p2 and p30, as well as a pan DR-binding epitope (PADRE) were produced via solid phase peptide synthesis. The synthesis of an immunostimulant on the basis of an imidazoquinoline structural motif was accomplished and optimized to provide a high affinity Toll-like receptor 7/8 (TLR7/8) agonist, which is able to link the innate immune response with adaptive immunity. Immunological evaluation of these successfully prepared building blocks has been started, and optimization studies using multicomponent vaccines are currently ongoing.
The induction of a robust immune response and an immunological memory necessitates the co-presentation of the specific antigens (B cell epitope) together with immunogenic T cell epitopes as well as immunomodulating adjuvant structures. This is traditionally accomplished in pharmaceutical industry or academic research labs by conjugating B cell epitopes to a highly immunogenic carrier protein (e.g. tetanus toxoid) or by post-functionalization of polymers. As these techniques lack flexibility, reproducibility, as well as stability and are hard to characterize, this research program has aimed for the supramolecular engineering of glycopeptides into synthetic anti-tumor vaccines. Our ongoing vaccination studies have shown that tumor-associated mucins based vaccines can be applied via a supramolecular scaffold and results in a potent immune response in mice, depending on the molecular composition of the multicomponent copolymers. This is a significant breakthrough in the interdisciplinary chemical immunology and immunotherapy field.