Versatile strategy for controlling the specificity and activity of engineered T cells.

The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR-T--cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell. Here, we describe the design and synthesis of structurally defined semisynthetic adaptors we refer to as "switch" molecules, in which anti-CD19 and anti-CD22 antibody fragments are site-specifically modified with FITC using genetically encoded noncanonical amino acids. This approach allows the precise control over the geometry and stoichiometry of complex formation between CD19- or CD22-expressing cancer cells and a "universal" anti-FITC-directed CAR-T cell. Optimization of this CAR-switch combination results in potent, dose-dependent in vivo antitumor activity in xenograft models. The advantage of being able to titrate CAR-T-cell in vivo activity was further evidenced by reduced in vivo toxicity and the elimination of persistent B-cell aplasia in immune-competent mice. The ability to control CAR-T cell and cancer cell interactions using intermediate switch molecules may expand the scope of engineered T-cell therapy to solid tumors, as well as indications beyond cancer therapy.

Therapeutic applications of an expanded genetic code.

To date, over 100 noncanonical amino acids (ncAAs) have been genetically encoded in living cells in order to expand the functional repertoire of the canonical 20 amino acids. More recently, this technology has been expanded to the field of protein therapeutics, where traditional chemical methods typically result in heterogeneous mixtures of proteins. The site-specific incorporation of ncAAs with orthogonal chemical groups allows unprecedented control over the site of conjugation and the stoichiometry, thus facilitating the rational optimization of the biological functions and/or pharmacokinetics of biologics. Herein, we discuss the recent contribution of ncAA technology in enhancing the pharmacological properties of current protein therapeutics as well as developing novel therapeutic modalities.

A genetically encoded aza-Michael acceptor for covalent cross-linking of protein-receptor complexes.

Selective covalent bond formation at a protein-protein interface potentially can be achieved by genetically introducing into a protein an appropriately "tuned" electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs that genetically encode three aza-Michael acceptor amino acids, N(ε)-acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a specific surface lysine ε-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 in vitro or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein in vitro or in living cells.

Mutational analysis of 48G7 reveals that somatic hypermutation affects both antibody stability and binding affinity.

The monoclonal antibody 48G7 differs from its germline precursor by 10 somatic mutations, a number of which appear to be functionally silent. We analyzed the effects of individual somatic mutations and combinations thereof on both antibody binding affinity and thermal stability. Individual somatic mutations that enhance binding affinity to hapten decrease the stability of the germline antibody; combining these binding mutations produced a mutant with high affinity for hapten but exceptionally low stability. Adding back each of the remaining somatic mutations restored thermal stability. These results, in conjunction with recently published studies, suggest an expanded role for somatic hypermutation in which both binding affinity and stability are optimized during clonal selection.

A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli.

To site-specifically incorporate an unnatural amino acid (UAA) into target proteins in Escherichia coli, we use a suppressor plasmid that provides an engineered suppressor tRNA and an aminoacyl-tRNA synthetase (aaRS) specific for the UAA of interest. The continuous drive to further improve UAA incorporation efficiency in E. coli has resulted in several generations of suppressor plasmids. Here we describe a new, highly efficient suppressor plasmid, pUltra, harboring a single copy each of the tRNA and aaRS expression cassettes that exhibits higher suppression activity than its predecessors. This system is able to efficiently incorporate up to three UAAs within the same protein at levels up to 30% of the level of wild-type protein expression. Its unique origin of replication (CloDF13) and antibiotic resistance marker (spectinomycin) allow pUltra to be used in conjunction with the previously reported pEVOL suppressor plasmid, each encoding a distinct tRNA/aaRS pair, to simultaneously insert two different UAAs into the same protein. We demonstrate the utility of this system by efficiently incorporating two bio-orthogonal UAAs containing keto and azido side chains into ketosteroid isomerase and subsequently derivatizing these amino acid residues with two distinct fluorophores, capable of Förster resonance energy transfer interaction. Finally, because of its minimal composition, two different tRNA/aaRS pairs were encoded in pUltra, allowing the generation of a single plasmid capable of dual suppression. The high suppression efficiency and the ability to harbor multiple tRNA/aaRS pairs make pUltra a useful system for conducting single- and multiple-UAA mutagenesis in E. coli.