Chemically induced dimerization modules as a platform for plant biosensor engineering

Protein biosensors for small molecules have important applications in agriculture, medicine, and security, but it remains difficult to rapidly produce a high-affinity sensor for a given ligand. This is partly due to two major challenges. First, most small molecule ligands have only a small number of residues with which a protein can make energetically favorable contacts, making it difficult to engineer high-affinity binding. Second, even if a high-affinity binding protein is engineered, it is difficult to transduce the binding event into an output. The majority of plant hormone perception occurs by chemically induced dimerization, where binding of the hormone to a soluble receptor causes a conformational change that allows the receptor to form a heterodimer with an interaction partner. These CID modules make an ideal platform for engineering small molecule biosensors because they naturally address the two primary challenges above: their unique architecture allows sensitive biosensors to be constructed from low-affinity receptors and protein dimerization provides a natural method of ligand binding transduction. The ability to engineer CID modules would lead directly to in planta biosensors and would also have broader applications to biosensor design in other biological systems. Here we describe the development of a general biosensor engineering platform using the abscisic acid receptor PYR1 of Arabidopsis thaliana, which was previously engineered to sense the agrochemical mandipropamid.1 We combine comprehensive mutagenesis2,3, high-throughput screening, deep sequencing, and machine learning to rapidly construct a model of the fitness landscape for binding of PYR1 to a specific ligand. We then use this model to design a targeted library to screen for higher affinity sensors. For high-throughput screening, we use both an established yeast two-hybrid (Y2H) screen and a novel yeast surface display (YSD) system. These techniques offer complementary advantages: Y2H is straightforward to implement and requires no purified protein, while YSD offers higher throughput and more stringent quantification of protein-protein interactions. Finally, we describe early development of two additional CID modules from the gibberellin and strigolactone sensing networks of A. thaliana. (1) Park, S.-Y.; Peterson, F. C.; Mosquna, A.; Yao, J.; Volkman, B. F.; Cutler, S. R. Agrochemical Control of Plant Water Use Using Engineered Abscisic Acid Receptors. Nature 2015, 520 (7548), 545–548. https://doi.org/10.1038/nature14123. (2) Wrenbeck, E. E.; Klesmith, J. R.; Stapleton, J. A.; Adeniran, A.; Tyo, K. E. J.; Whitehead, T. A. Plasmid-Based One-Pot Saturation Mutagenesis. Nat. Methods 2016, 13 (11), 928–930. https://doi.org/10.1038/nmeth.4029. (3) Medina-Cucurella, A. V.; Steiner, P. J.; Faber, M. S.; Beltrán, J.; Borelli, A. N.; Kirby, M. B.; Cutler, S. R.; Whitehead, T. A. User-Defined Single Pot Mutagenesis Using Unpurified Oligo Pools. Re

Enhanced T cell receptor specificity through framework engineering

Development of T cell receptors (TCRs) as immunotherapeutics is hindered by inherent TCR cross-reactivity. Engineering more specific TCRs has proven challenging, as unlike antibodies, improving TCR affinity does not usually improve specificity. Although various protein design approaches have been explored to surmount this, mutations in TCR binding interfaces risk broadening specificity or introducing new reactivities. Here we explored if TCR specificity could alternatively be tuned through framework mutations distant from the interface. Studying the 868 TCR specific for the HIV SL9 epitope presented by HLA-A2, we used deep mutational scanning to identify a framework mutation above the mobile CDR3β loop. This glycine to proline mutation had no discernable impact on binding affinity or functional avidity towards the SL9 epitope but weakened recognition of SL9 escape variants and led to fewer responses in a SL9-derived positional scanning library. In contrast, an interfacial mutation near the tip of CDR3α that also did not impact affinity or functional avidity towards SL9 weakened specificity. Simulations indicated that the specificity-enhancing mutation functions by reducing the range of loop motions, limiting the ability of the TCR to adjust to different ligands. Although our results are likely to be TCR dependent, using framework engineering to control TCR loop motions may be a viable strategy for improving the specificity of TCR-based immunotherapies

Preferential Identification of Agonistic OX40 Antibodies by Using Cell Lysate to Pan Natively Paired, Humanized Mouse-Derived Yeast Surface Display Libraries

To discover therapeutically relevant antibody candidates, many groups use mouse immunization followed by hybridoma generation or B cell screening. One modern approach is to screen B cells by generating natively paired single chain variable fragment (scFv) display libraries in yeast. Such methods typically rely on soluble antigens for scFv library screening. However, many therapeutically relevant cell-surface targets are difficult to express in a soluble protein format, complicating discovery. In this study, we developed methods to screen humanized mouse-derived yeast scFv libraries using recombinant OX40 protein in cell lysate. We used deep sequencing to compare screening with cell lysate to screening with soluble OX40 protein, in the context of mouse immunizations using either soluble OX40 or OX40-expressing cells and OX40-encoding DNA vector. We found that all tested methods produce a unique diversity of scFv binders. However, when we reformatted forty-one of these scFv as full-length monoclonal antibodies (mAbs), we observed that mAbs identified using soluble antigen immunization with cell lysate sorting always bound cell surface OX40, whereas other methods had significant false positive rates. Antibodies identified using soluble antigen immunization and cell lysate sorting were also significantly more likely to activate OX40 in a cellular assay. Our data suggest that sorting with OX40 protein in cell lysate is more likely than other methods to retain the epitopes required for antibody-mediated OX40 agonism

Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds

A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense-response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense-response applications