Periodic Reporting for period 1 - OMVCRC (Engineered bacterial Outer Membrane Vesicles (OMVs) for colorectal cancer immunotherapy)
Période du rapport: 2018-01-01 au 2019-06-30
This project originated from recent results obtained in the course of the Advanced ERC Project “OMVAC” (340915), the scope of which was to exploit the unique adjuvanticity properties of bacterial Outer Membrane Vesicles (OMVs) for developing innovative vaccines against infectious diseases and cancer. In particular, Synthetic Biology was applied to engineer OMVs with FAT1, a tumour associated antigen expressed in most primary and metastatic colorectal cancers (CRC). Using cancer models in immunocompetent mice, immunization with FAT1-decorated OMVs inhibited subcutaneous growth of FAT1-positive CT26 cancer cells and protection correlated with an increase in tumour infiltration of CD4+/CD8+ T cells and concomitant decrease of Treg and MDSCs.
The above mentioned promising results prompted the submission of the OMCRC project which had as main objectives: 1) the demonstration that FAT1-OMV immunization can synergize with the protective activity of checkpoint inhibitors and/or other immunotherapeutic strategies, and 2) the development of a scalable FAT1-OMV production and purification process which could allow testing FAT1-OMV/checkpoint inhibitor combination in the clinical setting. In particular, the project was organized in four main work packages (WP): 1) Selection of optimal fusion protein for the production of engineered hFAT1-D8-OMVs, 2) Demonstration, in the mouse model, that FAT1-D8-OMVs immunization can synergize with anti-PD-L1 therapy and/or other immunotherapy strategies, 3) set-up of a laboratory-scale production and purification process for FAT1-D8-OMVs, 4) Set-up of OMV quality assays.
As summarized below, all activities were completed and all objectives successfully achieved.
WP1
Different fusions proteins have been tested for the optimal decoration of OMVs with the FAT1-D8 epitope. One fusion was ultimately selected constituted by a lipoprotein carrying three copies of FAT1-D8 at its C-terminus. The fusion protein accumulated in the OMVs at a concentration of approximately 30% of total OMVs proteins, thus exceeding the 20% threshold reported in the original proposal.
WP2
The synergistic protective activity of FAT1-D8-OMV immunization with other immunotherapeutic strategies was tested using (i) passive administration of anti-PD-L1 antibodies after FAT1-D8-OMV immunization and tumor challenge, and (ii) active immunization with OMVs decorated with five tumor-specific T cell epitopes (neo-epitope-OMVS) after FAT1-D8-OMV immunization and tumor challenge. The data indicate that the combination of FAT1-D8-OMV immunization with anti-PD-L1 antibodies potentiate the inhibition of tumor growth, even if to a non-statistically significant level. Considering that CT26 tumor cells express low level of PD-L1, this is a promising result in light of future clinical applications. Moreover, the combination of FAT1-D8-OMV immunization with neo-epitope-OMV immunization resulted in a substantial improvement of anti-tumor activity (Grandi A. et al. 2018).
WP3
A laboratory scale production and purification process of FAT1-D8-OMVs was successfully set-up, using a 2 liter fermentation unit and tangential flow ultrafiltration. The final yield of purified OMVs was 65.37 (SD +/- 11.30) 60 mg/l, higher than the 50 mg/l threshold reported in the original proposal.
WP4
This last work package was focused on the setting-up of analytical methods necessary to demonstrate quality and lot consistency and OMV preparations. Five analytical methods were established, which allowed the characterization of the chemical-physical properties of OMVs and their adjuvanticity properties. They included: 1) analysis of protein content using two different quantification assays (absorbance at A280 and Lowry), analysis of total protein by SDS-PAGE, 3) analysis of lipid content, 4) analysis of particle size (Q-nano and light scattering), 5) stimulation of IL-6 production by differentiated human monocytes. Importantly, the assays were used to characterize different OMV lots, demonstrating the consistency of the OMV preparation process.
The above mentioned promising results prompted the submission of the OMCRC project which had as main objectives: 1) the demonstration that FAT1-OMV immunization can synergize with the protective activity of checkpoint inhibitors and/or other immunotherapeutic strategies, and 2) the development of a scalable FAT1-OMV production and purification process which could allow testing FAT1-OMV/checkpoint inhibitor combination in the clinical setting. In particular, the project was organized in four main work packages (WP): 1) Selection of optimal fusion protein for the production of engineered hFAT1-D8-OMVs, 2) Demonstration, in the mouse model, that FAT1-D8-OMVs immunization can synergize with anti-PD-L1 therapy and/or other immunotherapy strategies, 3) set-up of a laboratory-scale production and purification process for FAT1-D8-OMVs, 4) Set-up of OMV quality assays.
As summarized below, all activities were completed and all objectives successfully achieved.
WP1
Different fusions proteins have been tested for the optimal decoration of OMVs with the FAT1-D8 epitope. One fusion was ultimately selected constituted by a lipoprotein carrying three copies of FAT1-D8 at its C-terminus. The fusion protein accumulated in the OMVs at a concentration of approximately 30% of total OMVs proteins, thus exceeding the 20% threshold reported in the original proposal.
WP2
The synergistic protective activity of FAT1-D8-OMV immunization with other immunotherapeutic strategies was tested using (i) passive administration of anti-PD-L1 antibodies after FAT1-D8-OMV immunization and tumor challenge, and (ii) active immunization with OMVs decorated with five tumor-specific T cell epitopes (neo-epitope-OMVS) after FAT1-D8-OMV immunization and tumor challenge. The data indicate that the combination of FAT1-D8-OMV immunization with anti-PD-L1 antibodies potentiate the inhibition of tumor growth, even if to a non-statistically significant level. Considering that CT26 tumor cells express low level of PD-L1, this is a promising result in light of future clinical applications. Moreover, the combination of FAT1-D8-OMV immunization with neo-epitope-OMV immunization resulted in a substantial improvement of anti-tumor activity (Grandi A. et al. 2018).
WP3
A laboratory scale production and purification process of FAT1-D8-OMVs was successfully set-up, using a 2 liter fermentation unit and tangential flow ultrafiltration. The final yield of purified OMVs was 65.37 (SD +/- 11.30) 60 mg/l, higher than the 50 mg/l threshold reported in the original proposal.
WP4
This last work package was focused on the setting-up of analytical methods necessary to demonstrate quality and lot consistency and OMV preparations. Five analytical methods were established, which allowed the characterization of the chemical-physical properties of OMVs and their adjuvanticity properties. They included: 1) analysis of protein content using two different quantification assays (absorbance at A280 and Lowry), analysis of total protein by SDS-PAGE, 3) analysis of lipid content, 4) analysis of particle size (Q-nano and light scattering), 5) stimulation of IL-6 production by differentiated human monocytes. Importantly, the assays were used to characterize different OMV lots, demonstrating the consistency of the OMV preparation process.