Monospecific monoclonal antibodies against human protein complexes on an interactome-wide scale.

Project Summary Antibodies can facilitate the study of associations between different proteins in the cell using a technique known as immunoprecipitation IP This approach can reveal differences in protein associations present in healthy vs diseased tissues provide targets for diagnostics and therapeutics and reveal protein protein interfaces that could be druggable targets It is widely recognized that a lack of high quality antibodies against human proteins is negatively impacting biomedical science CDI Laboratories Inc has developed a pipeline that employs the largest content full length human protein array to generate quantifiably monospecific mouse monoclonal antibodies mAbs and has generated a large number of mAbs against human transcription factors TFs The PI of this project has developed methods that enable and optimize the capture of endogenous protein complexes by IP in conjunction with mass spectrometry analysis In Phase I of this STTR project Aim will characterize anti human TF mAbs for their ability to IP endogenous cancer related TF protein complexes This will be accomplished initially using human tissue culture cell lines to establish quantitative functional metrics of the mAbs and then applied to protein complexes isolated from patient derived cancers In Aim cancer related TFs will be used as bait for IP of native complexes and novel mAbs will be generated by immunization with the endogenous TF protein complexes The target specificity of the mAbs will be determined using the human proteome array yielding a catalog of new high quality mAbs with which to study protein protein interactions within cancer cells The long term goal of the project which will be expanded and achieved in Phase II is the production of larger numbers of high quality mAbs against native human proteins that are normally found as constituents of complexes in healthy and cancerous cells Project Narrative This project will combine two defined production pipelines one that isolates intact complexes of human proteins and another that generates highly specific monoclonal antibodies to human protein complexes The resulting combined pipeline will generate information about how intracellular proteins in humans interact with one another in healthy and diseased cancer states and will produce new antibodies that can be used in research settings to experimentally validate and expand on this information