115025 Pathobody: A proteogenomic pipeline for capture and sequencing and production of pathogen-specific antibodies

AbSano have changed the patient criteria for inclusion in our study and we now only select patients that had an infection with Covid 19 and that also got vaccinated against Covid 19. This yields the best number of B-cells in the FACS selection. AbSano has generated and tested the TSN (concentration) for the B-cell selection and also has set up the labeling conditions for the cellist the FACS. Absano has tested this and is now using the conditions for the B-cell cultures. We have ELISA assays running for each individual B-cell culture in which we test for: antibody production, antibody specificity, and neutralisation capacity. GenXPro has been working on the development of sequencing protocols for the antigen-binding fragments of antibodies. They developed and tested NGS library preparation protocols for this in a two step way. First they experimentally designed the development, and second they test sequence analysed this protocol using control B-cells provided by AbSano. Erasmus MC has received ~2000 FACS selected B-cells for 10x Genomics sequencing. The sequencing is still ongoing. Erasmus MC is also working on the validation of the affinity enrichment of supernatants of B-cell samples. They performed a succesful affinity enrichment and could identify immunoglobulin related peptides in a proteomics analysis of the affinity enriched product. They could show that proteomics data could be matched to available NGS data of the reference culture, thereby demonstrating that the workflow is sound. Pubgene has done a literature review to analyse current antibodies, their status on clinical trials, development status, latest scientific findings and patent situation. PubGene have developed a method that allows constant monitoring of the scientific progress in this field. Furthermore, Pubgene has initiated the creation of a database and framework allowing the storage and mapping of the data elements from the different analyses, and by this creating a foundation for data handling, storage and analysis.

The development of active vaccines is time-consuming and normally takes 5-10 years. If a rapid response to a pathogen is required, recombinant mAbs provide a faster path towards the protection of sensitive groups. Moreover, mAbs may be important in post-exposure therapies, where active immunization can no longer raise a protective response in time to limit an infectious process. This project aims to develop a fast platform for the recovery of pathogen-specific antibody molecules from exposed patients. High-value monoclonal antibody leads derived from Covid-19 patients will be the first output. The novel combination of NGS, phage display, and proteomics pioneered in our project will be the basis of an offer to industrial parties that wish to develop antibodies against other pathogens and to have a response capability in future pandemics. The PathoBody technology, integrating LC-MS/MS, NGS, phage display and bioinformatics tools, is a breakthrough development that represents a significant improvement over current state-of-the-art methods. Our new approach combines protein and RNA sequence analysis. The protein sequences provided by LC-MS/MS combined NGS of the whole complete immunoglobulin repertoire creates a reference cDNA database, which can be used to make a phage display library. This approach allows us to generate the full relevant Ab sequence to be used for cloning and production. This approach allows fast selection of target-specific mAbs, rather than laborious cell isolation, expansion and clone detection methods. Marketable outputs of this project are 1) safe, efficient patient-derived therapeutic Covid-19 monoclonals 2) an efficiency-demonstrated, novel platform-technology to produce such antibodies and 3) IP covering the different steps of the process. Potential customers of these products are large- and medium-sized pharma companies able to carry drugs through all steps of approval.