Project Summary Affinity reagents are useful tools for detecting, monitoring, and quantifying individual proteins in complex mixtures. Modern recombinant biology and protein engineering techniques generate high- affinity, high-specificity reagents that overcome some of the limitations of polyclonal and monoclonal antibodies. However, limited availability of target protein (i.e., antigen) prevents the selection of recombinant affinity reagents, and often traditional antibodies. Many human proteins when overexpressed in bacteria do not fold properly and/or cannot be extracted, and thus are not readily available for selecting affinity reagents that recognize native, folded protein. Even when the proteins are successfully overexpressed and purified, the time, cost, and labor required to produce them is considerable. We propose a solution, Array Based Affinity Selection (ABAS), which combines phage- display with Nucleic Acid Programmable Protein Arrays (NAPPA) to overcome this gap. NAPPA is an innovative protein array platform that circumvents the need of recombinant protein overexpression and purification of target proteins as well as allow multiplexed affinity selection of phage-display libraries. Recombinant DNAs, corresponding to antigen open reading frames fused to HaloTag, are spotted onto an array along with a covalent capture ligand. Once the cDNAs are in vitro transcribed and translated, the HaloTag ligand captures the fusion proteins in situ. To test and validate ABAS, Tango Biosciences and Dr. Joshua LaBaer (Arizona State University) will work together on two specific aims. In Specific Aim 1, we will create a model system with human protein targets, for which we have previously isolated binders by traditional phage-display methods. A number of important variables (i.e., size of spots, incubation time, elution conditions, recovery routes) will be examined to optimize ABAS. In Specific Aim 2, we will generate an array of 10 wild-type and five mutant human GTPases, a family of hydrolase enzymes critical to all cellular functions and are oncogenic drivers of cancer. A recent publication examined 22 commercial antibodies against human GTPases and found that only one is specific in western blots and all failed to work in immunofluorescence and immunohistochemistry experiments. Given that RAS proteins play a major role in cancer, there is a huge unmet need for better affinity reagents to RAS family members and their oncogenic mutations. We will characterize a subset of binders and confirm that they are high-affinity and high-specificity, through probing large arrays of human proteins, mass spectrometry of proteins affinity-purified from cell lysates, and immunofluorescence staining of cell lines. Successful completion of the proposed experiments in the 12-month period will enable Tango Biosciences to enhance its commercial competitiveness by providing access to many more human targets and thus widen its affinity reagent production pipeline. An expanded pipeline will increase Tango's market potential as well as advance academic and pharmaceutical research programs in ways that may ultimately improve diagnosis and treatment of human diseases. Furthermore, validated, affinity reagents that recognize wild-type and mutant RAS members will enable scientists to properly evaluate the role of these proteins in cancer.