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Title:Engineering and characterization of human single-chain T cell receptors
Author(s):Smith, Sheena
Director of Research:Kranz, David M.
Doctoral Committee Chair(s):Kranz, David M.
Doctoral Committee Member(s):van der Donk, Wilfred A.; Gennis, Robert B.; Roy, Edward J.
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):T cell receptors
yeast display
protein engineering
directed evolution
peptide major histocompatibility complex (MHC) antigens
Abstract:All nucleated cells display a sampling of their protein contents in the form of short 9-10 amino acid peptides bound to a product of the major histocompatibility complex (MHC) on the cell surface. This class of MHC-restricted antigens provides the broadest class of potential disease targets, as the peptides displayed are derived from the full antigenic repertoire of the cell, including intracellular, secreted, and cell-surface proteins. These peptides can be from endogenous “self” proteins, or they can result from viral infection or transformation to a cancerous state. Activation of the cell-mediated immune response begins with the binding of “foreign” MHC-restricted antigens by T-cell receptors (TCRs) with an affinity above the threshold for activation by the T cells. Like antibodies, TCRs are generated with large diversity in order to have some TCRs that accomplish this antigen recognition, but unlike antibodies TCRs do not undergo somatic hypermutation and therefore have relatively low affinities (KD = 1-100 μM). The generation of higher-affinity TCR variants provides a useful approach to improve targeting of cancerous cells, and to study the principles of TCR recognition and T cell triggering. TCRs are αβ heterodimers that interact with peptide-MHC (pepMHC) through six complementarity determining region (CDR) loops. The relative positions of these loops is conserved, such that the regions derived from the germline encoded regions of the TCR (i.e. CDR1 and CDR2) are positioned primarily over the MHC helices whereas the diverse regions derived from the junctions of somatically rearranged gene segments (i.e. CDR3) are positioned primarily over the peptide. Over the last 15 years, the Kranz lab has developed a strategy to engineer stable, high affinity TCRs using yeast display of TCRs in a single-chain format (scTCR) that consists of the variable regions of the α and β chains connected by a flexible linker (Vα-linker-Vβ or Vβ-linker-Vα). The focus of this dissertation is the engineering of human high affinity scTCRs for the targeting of cancer antigens, and the use of high affinity scTCRs for understanding the principles MHC-restriction and T cell specificity. In Chapter 2, a human scTCR specific for the melanoma cancer antigen Melan-A/MART-1 was engineered for improved stability and affinity by yeast display. The high affinity scTCR was expressed as a soluble protein in E. coli for MART-1/HLA-A2 binding studies and detection of the specific antigen on the surface of human antigen presenting cells (APCs). This TCR, called T1-S18.45, expresses the stable Vα2 region that is also expressed by two viral-specific TCRs, A6-X15 and 868-Z11, previously engineered by our lab. Using this panel of three high affinity scTCRs, engineered for high-affinity in CDR3 loops, mutational analysis was performed at residues in the CDR1α and CDR2α, testing a prominent hypothesis that holds that there are several evolutionary conserved residues in TCR variable regions that contact MHC. In Chapter 3, a human scTCR specific for Wilms’ Tumor Antigen-1 (WT-1) was engineered for improved stability and affinity by yeast display through a multi-step affinity maturation process. This scTCR was also expressed in E. coli as a soluble scTCR and used to detect WT-1/HLA-A2 on the surface of human antigen presenting cells. The WT-1 antigen is considered a promising therapeutic target for leukemia as well as various solid tumors, and as a result the engineered receptor has been further pursued in adoptive cell therapy models. The conventional approach to engineering high affinity scTCRs to date has required the isolation of specific T cell clones and their clonotypic TCRs prior to in vitro engineering. In Chapter 4 a strategy using in vitro, directed evolution of a single TCR to change its peptide specificity is described. The approach, avoids the need to isolate T cell clones for each MHC-restricted antigen of interest. The human TCR A6, that recognizes the viral peptide Tax in complex with HLA-A2, was converted to TCR variants that recognized the cancer peptide MART-1/HLA-A2 through mutagenesis and selection. Mutational studies and molecular dynamics simulations identified CDR residues that were important in this specificity switch. In addition, TCR variants that exhibited broad cross-reactivity with different peptides were identified, providing opportunities to examine in an unprecedented way the basis of peptide specificity. These finding thus provided a new platform in which in vitro engineering strategies alone could be used to isolate designer TCRs with desired specificities.
Issue Date:2015-01-21
URI:http://hdl.handle.net/2142/72953
Rights Information:Copyright 2014 Sheena Nichole Smith
Date Available in IDEALS:2015-01-21
Date Deposited:2014-12


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