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

ABSTRACT: 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.

SUBMITTER: Smith SN

PROVIDER: S-EPMC4225554 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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

Smith Sheena N SN Wang Yuhang Y Baylon Javier L JL Singh Nishant K NK Baker Brian M BM Tajkhorshid Emad E Kranz David M DM

Nature communications 20141107

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 singl ...[more]

PMID: 25376839

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Project description:UnlabelledThe actions of many bacterial toxins depend on their ability to bind to one or more cell-surface receptors. Anthrax toxin acts by a sequence of events that begins when the protective-antigen (PA) moiety of the toxin binds to either one of two cell-surface proteins, ANTXR1 and ANTXR2, and is proteolytically activated. The activated PA self-associates to form oligomeric pore precursors, which, in turn, bind the enzymatic moieties of the toxin and transport them to the cytosol. We introduced a double mutation into domain 4 of PA to ablate its native receptor-binding function and fused epidermal growth factor (EGF) to the C terminus of the mutated protein. The resulting fusion protein transported enzymatic effector proteins into a cell line that expressed the EGF receptor (A431 cells), but not into a line lacking this receptor (CHO-K1 cells). Addition of excess free EGF blocked transport of effector proteins into A431 cells via the fusion protein, but not via native PA. We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor. PA fusion proteins with altered receptor specificity may be useful in biological research and could have practical applications, including ablation or perturbation of selected populations of cells in vivo.ImportanceBacterial toxins that act within mammalian cells have receptor-dependent mechanisms to transport their enzymatic components to the cytoplasmic compartment. By inactivating or otherwise modifying their respective intracellular targets, these intracellular effectors disrupt metabolic pathways and in some cases cause death of the cell. Our results show that the receptor specificity of the transport protein of anthrax toxin may be readily changed, raising the possibility that receptor-redirected forms of protective antigen (PA) and PA homologs may be useful for research and medical applications requiring modification or ablation of designated populations of cells.

Dataset Information

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

Publications

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

Similar Datasets

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