Periodic Reporting for period 2 - MARVEL (Evolving reversible iMmunocapture by membrane sensing peptides: towARds scalable extracellular VEsicLes isolation)
Reporting period: 2022-03-01 to 2023-04-30
Extracellular vesicles (EV) are submicron membrane vesicles released by most cells with a fundamental role in cell-to-cell communication. Much interest is flourishing towards their exploitation in therapy and diagnostics. However, the fulfilment of the EV promise is hampered by severe limitations in their isolation, characterization and manufacturing. A particularly arduous task is to move the isolation of specific EV subpopulations beyond the analytical scale and towards scalable processes. MARVEL targets a paradigm shift from antibodies to peptides as an alternative class of affinity ligands for EV capturing by introducing membrane-sensing peptides (MSP) as novel ligands for the capturing of small EV, unbiased by differential surface protein expression. MARVEL mission is to combine and implement reversible isolation and MSP technologies, towards the first and best performing ever affinity-based technology for scalable small EV (<200nm) isolation. The modularity in scaling-up of the novel protocols and kits has been demonstrated on medium/large sample volumes in relevant environments for therapeutic and diagnostics use of EVs and specifically: 1) In the lab-scale manufacturing of EVs as a medicinal product for cardiac repair; 2) In urine-based liquid biopsy for bladder cancer prognosis; 3) in a blood-based workflow for cardiac risk assessment.
MARVEL demonstrated (TRL 6):
• The peptide-based workflow is highly effective in isolating and enriching EVs from conditioned medium, urine, plasma and serum with minimal carry over of contaminants
• Peptide conjugated beads enable the release of structurally intact EVs with no decrease in efficacy.
• The peptide-based workflow can be directly applied using various analytic platforms
A business plan based on multiplatform integration of MSP (Ingredient Model) has been developed opening entrepreneurial perspectives
MARVEL demonstrated (TRL 6):
• The peptide-based workflow is highly effective in isolating and enriching EVs from conditioned medium, urine, plasma and serum with minimal carry over of contaminants
• Peptide conjugated beads enable the release of structurally intact EVs with no decrease in efficacy.
• The peptide-based workflow can be directly applied using various analytic platforms
A business plan based on multiplatform integration of MSP (Ingredient Model) has been developed opening entrepreneurial perspectives
MARVEL has the mission to exploit membrane sensing peptides (MSP) and other peptide ligands towards the first affinity-based technology for scalable and reversible small EV (<200nm) isolation. Key figures of merit for peptide ligands are the ease of chemical manipulation, offering virtually unlimited possibilities for system integration, and the limited costs of preparation with well-known possibilities for scale-up of production.
Thanks to its versatile and modular nature, system integration of MARVEL technology will result in a portfolio of products to be applied for EV isolation on different scales: 1) lab-scale and point of need tools for blood and urinary EV enrichment and analysis and 2) integrated TFF-affinity isolation system for EV isolation on a manufacturing scale.
To pursue these goals, MARVEL project successfully implemented 4 Technical Phases:
• Specification phase (WP1): the specifications are laid out that will be used during the system development and in the demonstration phase. Biological samples are collected.
• Component’s phase (WP2): development and optimization of the major components of the project (peptide affinity probes and strategies for EV capture and release)
• Systems phase (WP3): peptide probes are integrated within the analytical and preparative tools at the different scales.
• Demonstration phase (WP4 and WP5): the end-users evaluate in relevant environment the MARVEL technology against the specifications set in WP1.
In WP6 the elaboration of a Business Plan towards multiplatform integration of MSP and other peptide ligands (Ingredient Model) has been perfomed.
Thanks to its versatile and modular nature, system integration of MARVEL technology will result in a portfolio of products to be applied for EV isolation on different scales: 1) lab-scale and point of need tools for blood and urinary EV enrichment and analysis and 2) integrated TFF-affinity isolation system for EV isolation on a manufacturing scale.
To pursue these goals, MARVEL project successfully implemented 4 Technical Phases:
• Specification phase (WP1): the specifications are laid out that will be used during the system development and in the demonstration phase. Biological samples are collected.
• Component’s phase (WP2): development and optimization of the major components of the project (peptide affinity probes and strategies for EV capture and release)
• Systems phase (WP3): peptide probes are integrated within the analytical and preparative tools at the different scales.
• Demonstration phase (WP4 and WP5): the end-users evaluate in relevant environment the MARVEL technology against the specifications set in WP1.
In WP6 the elaboration of a Business Plan towards multiplatform integration of MSP and other peptide ligands (Ingredient Model) has been perfomed.
The aim of the EU-funded MARVEL project is to overcome bottlenecks in EV isolation to scale up the process beyond the analytical. For this purpose reversible isolation has been implemented with membrane-sensing peptides (MSP). This will represent a paradigm shift from antibodies to peptides as an alternative class of affinity ligands characterised by high efficiency of EV capturing. The technology has been applied in the context of lab-scale EV manufacturing for cardiac repair, bladder cancer prognosis in urine samples and cardiac risk assessment by EV associated biomarkers in blood
The main features implemented under MARVEL working program are the following:
1. Taking over of knowledge and protocols from INDEX project.
2. Technical specifications and validation methodology for the demonstration activities.
3. Collection of biological samples for the validation of the urine and blood-based diagnostic workflow.
4. A panel of “second generation” membrane sensing peptide probes (MSPs) with EV capturing capacity improved with respect to the precursor sequence and far superior to tetraspanin antibodies.
5. A panel of specific peptide probes to enrich EV subpopulations based on putative clinically relevant markers with high specificity
6. An innovative strategy for ex novo design of new peptide sequences.
7. A new strategy for rapid, scalable, traceless release of EVs in mild conditions
8. MSP-functionalized magnetic beads demonstrated in EV recovery with negligible pull down of proteins and other non-EV contaminants in cell culture conditioned media, urine , plasma and serum
9. Multiplatform integration of MSP (including TFF membranes) demonstrated in high efficiency capturing of EVs and their complete release in traceless conditions.
10. Market search and IPR analysis report to compare, situate and assess current IPRs and FTO of current and potential MARVEL results.
11. Identification of core value proposition of MARVEL and evaluation of a business model for MARVEL exploitation (Ingredient model)
MARVEL platform will produce direct and heavy impacts on the field of EV by empowering the sustainability of their use in both regenerative medicine (EV-based cell-free therapies) and diagnostics (EV-based liquid biopsy). Such empowerment is expected to increment readiness level of EV technologies and endow them with clinical grade maturity. This will ultimately impact society by providing better clinical outcomes at lower costs through the precision medicine paradigm with implications on life quality and sustainability of public healthcare.
The main features implemented under MARVEL working program are the following:
1. Taking over of knowledge and protocols from INDEX project.
2. Technical specifications and validation methodology for the demonstration activities.
3. Collection of biological samples for the validation of the urine and blood-based diagnostic workflow.
4. A panel of “second generation” membrane sensing peptide probes (MSPs) with EV capturing capacity improved with respect to the precursor sequence and far superior to tetraspanin antibodies.
5. A panel of specific peptide probes to enrich EV subpopulations based on putative clinically relevant markers with high specificity
6. An innovative strategy for ex novo design of new peptide sequences.
7. A new strategy for rapid, scalable, traceless release of EVs in mild conditions
8. MSP-functionalized magnetic beads demonstrated in EV recovery with negligible pull down of proteins and other non-EV contaminants in cell culture conditioned media, urine , plasma and serum
9. Multiplatform integration of MSP (including TFF membranes) demonstrated in high efficiency capturing of EVs and their complete release in traceless conditions.
10. Market search and IPR analysis report to compare, situate and assess current IPRs and FTO of current and potential MARVEL results.
11. Identification of core value proposition of MARVEL and evaluation of a business model for MARVEL exploitation (Ingredient model)
MARVEL platform will produce direct and heavy impacts on the field of EV by empowering the sustainability of their use in both regenerative medicine (EV-based cell-free therapies) and diagnostics (EV-based liquid biopsy). Such empowerment is expected to increment readiness level of EV technologies and endow them with clinical grade maturity. This will ultimately impact society by providing better clinical outcomes at lower costs through the precision medicine paradigm with implications on life quality and sustainability of public healthcare.