Antibodiescanfacilitatethestudyofassociationsbetweendifferentproteinsinthecellusinga 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,andrevealprotein--proteininterfacesthatcouldbedruggabletargets.Itiswidely recognizedthatalackofhighqualityantibodiesagainsthumanproteinsisnegativelyimpacting 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 1 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 Aim2, cancer--relatedTFswillbeusedasbaitforIPofnativecomplexes,andnovel 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 cancercells.Thelongtermgoaloftheproject,whichwillbeexpandedandachievedinPhaseII, istheproductionoflargernumbersofhighqualitymAbsagainstnativehumanproteinsthatare normallyfoundasconstituentsofcomplexesinhealthyandcancerouscells.
Thisprojectwillcombinetwodefinedproductionpipelines,onethatisolates 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 settingstoexperimentallyvalidateandexpandonthisinformation.
