Enhanced-affinity murine T-cell receptors for tumor/self-antigens can be safe in gene therapy despite surpassing the threshold for thymic selection

Schmitt, Thomas M.; Aggen, David H.; Stromnes, Ingunn M.; Dossett, Michelle L.; Richman, Sarah A.; Kranz, David M.; Greenberg, Philip D.

doi:10.1182/blood-2013-01-478164

Cited by 59 publications

(55 citation statements)

References 45 publications

(54 reference statements)

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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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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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“…Dextramers of class II pMHC molecules improve detection of antigen-specific CD4 + T cells (16), which are emerging as important components of anti-tumor immunity (17,18) and are generally lower-affinity than CD8 + T cells(19). Moreover, TCRs derived from tumor-reactive CD8 + T cells are constrained by thymic selection to a low-affinity range (1,4) and are generally assisted in pMHC recognition by CD8:MHC I interaction (20). We transduced PBMCs with TCRs of varying affinity for A2/MART1 to determine the extent to which dextran doping improves pMHC multimer staining of low- and high-affinity TCRs expressed on CD8 + or CD8 - T cells.…”

Section: Resultsmentioning

confidence: 99%

“…However, T cells bearing low-affinity TCRs (>80 M) are poorly detected using pMHC tetramers (3). This is particularly problematic for detection of T cells mediating autoimmunity and anti-tumor immunity, as thymic selection eliminates T cells with high affinity for non-mutated self-antigens (1,4). …”

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Preparation of Peptide–MHC and T-cell Receptor Dextramers by Biotinylated Dextran Doping

Bethune

Comı́n-Anduix

et al. 2017

BioTechniques

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Peptide-major histocompatibility complex (pMHC) multimers enable the detection, characterization, and isolation of antigen-specific T-cell subsets at the single-cell level via flow cytometry and fluorescence microscopy. These labeling reagents exploit a multivalent scaffold to increase the avidity of individually weak T-cell receptor (TCR)-pMHC interactions. Dextramers are an improvement over the original streptavidin-based tetramer technology because they are more multivalent, improving sensitivity for rare, low-avidity T cells, including self/tumor-reactive clones. However, commercial pMHC dextramers are expensive, and in-house production is very involved for a typical biology research laboratory. Here, we present a simple, inexpensive protocol for preparing pMHC dextramers by doping in biotinylated dextran during conventional tetramer preparation. We use these pMHC dextramers to identify patient-derived, tumor-reactive T cells. We apply the same dextran doping technique to prepare TCR dextramers and use these novel reagents to yield new insight into MHC I-mediated antigen presentation.

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“…5 Similarly, intracellular proteins such as Wilms tumor antigen-1 and minor histocompatibility antigens have been validated in preclinical experiments as attractive targets for the treatment of hematologic malignancies. [6][7][8][9] TCR gene therapy trials targeting these antigens are currently open for recruitment of leukemia patients. The TCR recognition is focused on short linear peptide epitopes presented by HLA class I molecules (9-10 amino acid peptides) and HLA class II molecules (15-18 amino acids).…”

Section: Tcrs or Cars To Target Malignant Cellsmentioning

confidence: 99%

Optimizing T-cell receptor gene therapy for hematologic malignancies

Morris

Stauss

2016

Blood

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Recent advances in genetic engineering have enabled the delivery of clinical trials using patient T cells redirected to recognize tumor-associated antigens. The most dramatic results have been seen with T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19, a differentiation antigen expressed in B cells and B lineage malignancies. We propose that antigen expression in nonmalignant cells may contribute to the efficacy of T-cell therapy by maintaining effector function and promoting memory. Although CAR recognition is limited to cell surface structures, T-cell receptors (TCRs) can recognize intracellular proteins. This not only expands the range of tumor-associated self-antigens that are amenable for T-cell therapy, but also allows TCR targeting of the cancer mutagenome. We will highlight biological bottlenecks that potentially limit mutation-specific T-cell therapy and may require high-avidity TCRs that are capable of activating effector function when the concentrations of mutant peptides are low. Unexpectedly, modified TCRs with artificially high affinities function poorly in response to low concentration of cognate peptide but pose an increased safety risk as they may respond optimally to cross-reactive peptides. Recent gene-editing tools, such as transcription activator–like effector nucleases and clustered regularly interspaced short palindromic repeats, provide a platform to delete endogenous TCR and HLA genes, which removes alloreactivity and decreases immunogenicity of third-party T cells. This represents an important step toward generic off-the-shelf T-cell products that may be used in the future for the treatment of large numbers of patients.

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Enhanced-affinity murine T-cell receptors for tumor/self-antigens can be safe in gene therapy despite surpassing the threshold for thymic selection

Abstract: Key Points High-affinity tumor/self antigen-specific TCRs that surpass the threshold for normal thymic selection can be safe for TCR gene therapy. T cells that express endogenous TCRs that are self-reactive can survive in the periphery with diminished TCR expression levels.

Cited by 59 publications

References 45 publications

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

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

Preparation of Peptide–MHC and T-cell Receptor Dextramers by Biotinylated Dextran Doping

Optimizing T-cell receptor gene therapy for hematologic malignancies

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