Identification of Highly Cross-Reactive Mimotopes for a Public T Cell Response in Murine Melanoma.

While immune checkpoint blockade results in durable responses for some patients, many others have not experienced such benefits. These treatments rely upon reinvigorating specific T cell-antigen interactions. However, it is often unknown what antigens are being recognized by T cells or how to potently induce antigen-specific responses in a broadly applicable manner. Here, we characterized the CD8+ T cell response to a murine model of melanoma following combination immunotherapy to determine the basis of tumor recognition. Sequencing of tumor-infiltrating T cells revealed a repertoire of highly homologous TCR sequences that were particularly expanded in treated mice and which recognized an antigen from an endogenous retrovirus. While vaccination against this peptide failed to raise a protective T cell response in vivo, engineered antigen mimotopes induced a significant expansion of CD8+ T cells cross-reactive to the original antigen. Vaccination with mimotopes resulted in killing of antigen-loaded cells in vivo yet showed modest survival benefit in a prophylactic vaccine paradigm. Together, this work demonstrates the identification of a dominant tumor-associated antigen and generation of mimotopes which can induce robust functional T cell responses that are cross-reactive to the endogenous antigen across multiple individuals.

Screening for CD19-specific chimaeric antigen receptors with enhanced signalling via a barcoded library of intracellular domains.

The immunostimulatory intracellular domains (ICDs) of chimaeric antigen receptors (CARs) are essential for converting antigen recognition into antitumoural function. Although there are many possible combinations of ICDs, almost all current CARs rely on combinations of CD3𝛇, CD28 and 4-1BB. Here we show that a barcoded library of 700,000 unique CD19-specific CARs with diverse ICDs cloned into lentiviral vectors and transduced into Jurkat T cells can be screened at high throughput via cell sorting and next-generation sequencing to optimize CAR signalling for antitumoural functions. By using this screening approach, we identified CARs with new ICD combinations that, compared with clinically available CARs, endowed human primary T cells with comparable tumour control in mice and with improved proliferation, persistence, exhaustion and cytotoxicity after tumour rechallenge in vitro. The screening strategy can be adapted to other disease models, cell types and selection conditions, and could be used to improve adoptive cell therapies and to expand their utility to new disease indications.

Yeast Display for the Identification of Peptide-MHC Ligands of Immune Receptors.

T cells detect peptide antigens presented by major histocompatibility complex (MHC) proteins via their T cell receptor (TCR). The sequence diversity of possible antigens, with trillions of potential peptide-MHC targets, makes it challenging to study, characterize, and manipulate the peptide repertoire of a given TCR. Yeast display has been utilized to study the interactions between peptide-MHCs and T cell receptors to facilitate high-throughput screening of peptide-MHC libraries. Here we present insights on designing and validating a peptide-MHC yeast display construct, designing and constructing peptide libraries, conducting selections, and preparing, processing, and analyzing peptide library sequencing data. Applications for this approach are broad, including characterizing peptide-MHC recognition profiles for a TCR, screening for high-affinity mimotopes of known TCR-binding peptides, and identifying natural ligands of TCRs from expanded T cells.

A Gradient of Sitewise Diversity Promotes Evolutionary Fitness for Binder Discovery in a Three-Helix Bundle Protein Scaffold.

Engineered proteins provide clinically and industrially impactful molecules and utility within fundamental research, yet inefficiencies in discovering lead variants with new desired functionality, while maintaining stability, hinder progress. Improved function, which can result from a few strategic mutations, is fundamentally separate from discovering novel function, which often requires large leaps in sequence space. While a highly diverse combinatorial library covering immense sequence space would empower protein discovery, the ability to sample only a minor subset of sequence space and the typical destabilization of random mutations preclude this strategy. A balance must be reached. At library scale, compounding several destabilizing mutations renders many variants unable to properly fold and devoid of function. Broadly searching sequence space while reducing the level of destabilization may enhance evolution. We exemplify this balance with affibody, a three-helix bundle protein scaffold. Using natural ligand data sets, stability and structural computations, and deep sequencing of thousands of binding variants, a protein library was designed on a sitewise basis with a gradient of mutational levels across 29% of the protein. In direct competition of biased and uniform libraries, both with 1 × 109 variants, for discovery of 6 × 104 ligands (5 × 103 clusters) toward seven targets, biased amino acid frequency increased ligand discovery 13 ± 3-fold. Evolutionarily favorable amino acids, both globally and site-specifically, are further elucidated. The sitewise amino acid bias aids evolutionary discovery by reducing the level of mutant destabilization as evidenced by a midpoint of denaturation (62 ± 4 °C) 15 °C higher than that of unbiased mutants (47 ± 11 °C; p < 0.001). Sitewise diversification, identified by high-throughput evolution and rational library design, improves discovery efficiency.

Toward Bioremediation of Methylmercury Using Silica Encapsulated Escherichia coli Harboring the mer Operon.

Mercury is a highly toxic heavy metal and the ability of the neurotoxin methylmercury to biomagnify in the food chain is a serious concern for both public and environmental health globally. Because thousands of tons of mercury are released into the environment each year, remediation strategies are urgently needed and prompted this study. To facilitate remediation of both organic and inorganic forms of mercury, Escherichia coli was engineered to harbor a subset of genes (merRTPAB) from the mercury resistance operon. Protein products of the mer operon enable transport of mercury into the cell, cleavage of organic C-Hg bonds, and subsequent reduction of ionic mercury to the less toxic elemental form, Hg(0). E. coli containing merRTPAB was then encapsulated in silica beads resulting in a biological-based filtration material. Performing encapsulation in aerated mineral oil yielded silica beads that were smooth, spherical, and similar in diameter. Following encapsulation, E. coli containing merRTPAB retained the ability to degrade methylmercury and performed similarly to non-encapsulated cells. Due to the versatility of both the engineered mercury resistant strain and silica bead technology, this study provides a strong foundation for use of the resulting biological-based filtration material for methylmercury remediation.