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

We used the software and and db developed under this project to compare the uniqueness and overlap of our antibody CDRH3 domain with the Cov-AbDab database. We found 56 mappings between the two data sets of which 8 Pathobody sequences were unique. These sequences bind 17 different SARS-CoV2 variants. This suggests that we can identify new antibodies with potential therapeutic effects that would not infringe on already existing commercial COVID antibodies. Based on the phagemid constructs that were transferred to Erasmus, bacterias were expanded to produce phage particles. An aliquot of these phage particles was used as the input of the first selection round. Complexes of covid-specific phages and the spike protein were collected using recombinant biotinylated spike protein (Delta variant) and streptavidin coated magnetic particles. Remaining phages were used to infect bacteria for the next expansion step. GenXPro completed the antigen-binding fragment (Fab) libraries for Next-Generation Sequencing (NGS). The Fab libraries were sequenced on a MiSeq using paired-end sequencing with 300 base pair reads. The methodology has been further optimized for user-friendly processing and the development of a commercial kit for Fab NGS analysis has begun in test series. PubGene finalized the first version of the database and pipeline for the data analysis form the project. The pipeline includes routines for importing libraries of interest, import/loading and analysis of the Pathobody CDR3-IMGT sequences, loading/import of the comparative sequence database, including algorithms for matching and analyzing the sequence data. We have also added algorithms and routines for calculating the likelihood for which SARS-CoV2 variants our sequences might bind, might not and might and might not neutralize. Finally, the pipeline includes tools for matching the Pathobody CDR3-IMGT sequences to PDB 3D structures.

Through this project we have confirmed that we are able to identify new antibodies with potential therapeutic effects that would not infringe on already existing commercial COVID antibodies. Further more we have together with our partners developed a pipeline with necessary protocols and software that could be used in response to future emerging threats. Regarding the commercialisation and out licensing of the pipeline, this is expected to take a bit more time than originally anticipated. The reason for this is that the envisioned/primary commercialisation partner in the project, AbSano had to withdraw from the project due to a very unfortunate event within the corporate management. We, the remaining partners are currently discussing how to proceed with the commercialisation of the project results.

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.