Identification and engineering of human variable regions that allow expression of stable single-chain T cell receptors

Aggen, David H.; Chervin, Adam S.; Insaidoo, Francis K.; Piepenbrink, Kurt H.; Baker, Brian M.; Kranz, David M.

doi:10.1093/protein/gzq113

Cited by 39 publications

(66 citation statements)

References 55 publications

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“…We chose the human TCR A6 as an initial scaffold based on the wealth of information available and previous findings that it is amenable to yeast display and directed evolution 34 . The A6 TCR recognizes at least three distinct HLA-A2-restricted ligands, Tax (LLFGYPVYV), derived from HTLV-1, and two structural mimics of Tax called Tel1p (MLWGYLQYV) and HuD (LGYGFVNYI) 6,15,49 .…”

Section: Discussionmentioning

confidence: 99%

Changing the peptide specificity of a human T-cell receptor by directed evolution

et al. 2014

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Binding of a T cell receptor (TCR) to a peptide/major histocompatibility complex is the key interaction involved in antigen specificity of T cells. The recognition involves up to six complementarity determining regions (CDR) of the TCR. Efforts to examine the structural basis of these interactions and to exploit them in adoptive T cell therapies has required the isolation of specific T cell clones and their clonotypic TCRs. Here we describe a strategy using in vitro, directed evolution of a single TCR to change its peptide specificity, thereby avoiding the need to isolate T cell clones. The human TCR A6, which recognizes the viral peptide Tax/HLA-A2, was converted to TCR variants that recognized the cancer peptide MART1/HLA-A2. Mutational studies and molecular dynamics simulations identified CDR residues that were predicted to be important in the specificity switch. Thus, in vitro engineering strategies alone can be used to discover TCRs with desired specificities.

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Section: Discussionmentioning

confidence: 99%

Changing the peptide specificity of a human T-cell receptor by directed evolution

et al. 2014

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“…Single-codon libraries of the A6-c134 and RD1-MART HIGH TCRs were cloned as single chains (Vβ-linker-Vα) for yeast display (Aggen et al, 2011). Primers were synthesized by Integrated DNA technologies and PCR products containing degenerate NNK codons were generated via overlap extension PCR.…”

Section: Methodsmentioning

confidence: 99%

An Engineered Switch in T Cell Receptor Specificity Leads to an Unusual but Functional Binding Geometry

et al. 2016

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Summary Utilizing a diverse binding site, T cell receptors (TCRs) specifically recognize a composite ligand comprised of a foreign peptide and a major histocompatibility complex protein (MHC). To help understand the determinants of TCR specificity, we studied a parental and engineered receptor whose peptide specificity had been switched via molecular evolution. Altered specificity was associated with a significant change in TCR binding geometry, but this did not impact the ability of the TCR to signal in an antigen-specific manner. The determinants of binding and specificity were distributed among contact and non-contact residues in germline and hypervariable loops, and included disruption of key TCR-MHC interactions that bias αβ TCRs towards particular binding modes. Sequence/fitness landscapes identified additional mutations that further enhanced specificity. Our results demonstrate that TCR specificity arises from the distributed action of numerous sites throughout the interface, with significant implications for engineering therapeutic TCRs with novel and functional recognition properties.

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“…In addition, because of their naturally low affinities, they represent protein engineering targets for both stability and affinity. Previously, we reported successful affinity engineering of TCRs by directed evolution using yeast display (2,(17)(18)(19)(20)(21) and mammalian cell display (7,22). We also described structure-guided design strategies that estimated the binding energies of both favorable and unfavorable mutations and led to the design of additional high affinity TCRs (23,24).…”

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confidence: 99%

Deep Mutational Scans as a Guide to Engineering High Affinity T Cell Receptor Interactions with Peptide-bound Major Histocompatibility Complex

Harris

Wang

Riley

et al. 2016

Journal of Biological Chemistry

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Edited by Peter CresswellProteins are often engineered to have higher affinity for their ligands to achieve therapeutic benefit. For example, many studies have used phage or yeast display libraries of mutants within complementarity-determining regions to affinity mature antibodies and T cell receptors (TCRs). However, these approaches do not allow rapid assessment or evolution across the entire interface. By combining directed evolution with deep sequencing, it is now possible to generate sequence fitness landscapes that survey the impact of every amino acid substitution across the entire protein-protein interface. Here we used the results of deep mutational scans of a TCR-peptide-MHC interaction to guide mutational strategies. The approach yielded stable TCRs with affinity increases of >200-fold. The substitutions with the greatest enrichments based on the deep sequencing were validated to have higher affinity and could be combined to yield additional improvements. We also conducted in silico binding analyses for every substitution to compare them with the fitness landscape. Computational modeling did not effectively predict the impacts of mutations distal to the interface and did not account for yeast display results that depended on combinations of affinity and protein stability. However, computation accurately predicted affinity changes for mutations within or near the interface, highlighting the complementary strengths of computational modeling and yeast surface display coupled with deep mutational scanning for engineering high affinity TCRs.The process of increasing the affinity of a protein occurs naturally with antibodies, where somatic mutation within the variable region genes is followed by antigen-driven selection of B cells that express membrane-bound antibodies. In contrast, T cell receptors (TCRs) 3 do not undergo somatic mutations and bind to their antigen, a peptide-MHC (pepMHC), with low (micromolar) affinities. However, improvements in TCR affinity to the same levels of antibodies can be achieved by in vitro approaches involving the generation of mutant TCR libraries followed by antigen selection (1-3).For therapeutic purposes, the affinity of a variety of proteinprotein interactions, and especially antibody-antigen interactions, has been enhanced using in vitro directed evolution approaches, including phage, yeast, ribosomal, and mammalian display (e.g. see Refs. 4 -7). These methods rely on the generation of large libraries of mutants at residues within the proteinprotein interface, followed by several rounds of selection for desired parameters (such as affinity, stability, and expression levels) (8, 9).Although directed evolution using larger degenerate libraries has been successful, the most recent techniques involving deep sequencing of single-codon libraries have the potential both to provide mechanistic structural information about a binding site and at the same time to provide leads for affinity improvements. Sequence fitness landscapes have successfully been utilized to map protein-DNA...

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Identification and engineering of human variable regions that allow expression of stable single-chain T cell receptors

Cited by 39 publications

References 55 publications

Changing the peptide specificity of a human T-cell receptor by directed evolution

Changing the peptide specificity of a human T-cell receptor by directed evolution

An Engineered Switch in T Cell Receptor Specificity Leads to an Unusual but Functional Binding Geometry

Deep Mutational Scans as a Guide to Engineering High Affinity T Cell Receptor Interactions with Peptide-bound Major Histocompatibility Complex

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