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Cell surface receptors perform critical functions in the communication<sup></sup>of extracellular information to the inside of the cell. Receptors<sup></sup>for soluble molecules, including hormones, growth factors, or<sup></sup>cytokines, control properties that range from cellular proliferation<sup></sup>to cell migration. Other receptors function in cell-to-cell<sup></sup>communication by interacting with a cognate ligand that is expressed<sup></sup>on the surface of an opposing cell. Frequently, such receptor–ligand<sup></sup>pairs exhibit very low affinities that can make biochemical<sup></sup>and structural analyses difficult (Maenaka <em>et al</em>., 1999<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B41"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). By<sup></sup>engineering the affinities or kinetics of such interactions<sup></sup>it is possible to explore the mechanisms that dictate biological<sup></sup>effects (Rao <em>et al</em>., 2005<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B46"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). Furthermore, engineering high-affinity<sup></sup>soluble receptors or monoclonal antibodies that bind to cell<sup></sup>surface ligands can provide potential therapeutic agents that<sup></sup>antagonize the receptor–ligand interactions or that target<sup></sup>a cell for destruction.<sup></sup><p> To engineer peptides or proteins for improved binding properties,<sup></sup>various methods of directed evolution have been developed. These<sup></sup>methods include systems such as yeast display (Boder and Wittrup,<sup></sup>1997<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B3"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>), phage display (Smith, 1985<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B54"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Bradbury and Marks, 2004<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B8"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>;<sup></sup>Marks and Bradbury, 2004<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B42"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>), <em>Escherichia coli</em> display (Francisco<sup></sup><em>et al</em>., 1993<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B22"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>), baculovirus/insect cell display (Boublik <em>et al</em>.,<sup></sup>1995<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B7"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>) and ribosome display (Hanes <em>et al</em>., 2000<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B28"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). While purified<sup></sup>ligands have been used most often in these approaches, there<sup></sup>has been considerable effort to develop strategies that involve<sup></sup>ligands present in whole cell preparations. For example, in<sup></sup>a process called biopanning, phage display libraries are either<sup></sup>incubated with target cells or introduced <em>in vivo</em> into animals<sup></sup>(Kupsch <em>et al</em>., 1999<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B37"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Giordano <em>et al</em>., 2001<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B26"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Roovers <em>et al</em>.,<sup></sup>2001<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B48"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Trepel <em>et al</em>., 2002<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B58"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). Phage that display a peptide or<sup></sup>antibody against cell- or tissue-specific surface molecules<sup></sup>are then isolated, and the process is repeated for further enrichment.<sup></sup></p> <p> Recently, yeast display has been used as a system to isolate<sup></sup>human scFv fragments that have specificity for various antigens,<sup></sup>with the goal of identifying lead candidates for further directed<sup></sup>evolution (Feldhaus <em>et al</em>., 2003<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B20"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). In several cases, yeast display<sup></sup>has been used to isolate scFv (Boder <em>et al</em>., 2000<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B5"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Rajpal <em>et<sup></sup>al</em>., 2005<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B45"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Razai <em>et al</em>., 2005<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B47"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>) or other cell surface receptors<sup></sup>(Buonpane <em>et al</em>., 2005<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B10"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>) that exhibit picomolar affinity constants<sup></sup>for their ligands. Furthermore, yeast display has been used<sup></sup>to engineer T cell receptors (TCRs) (Holler <em>et al</em>., 2000<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B31"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Kieke<sup></sup><em>et al</em>., 2001<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B36"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Holler <em>et al</em>., 2003<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B32"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Chlewicki <em>et al</em>., 2005<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B12"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>),<sup></sup>natural killer cell receptors (Dam <em>et al</em>., 2003<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B14"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>) and proteins<sup></sup>of the major histocompatibility complex (MHC) (Brophy <em>et al</em>.,<sup></sup>2003<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B9"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Starwalt <em>et al</em>., 2003<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B56"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Esteban and Zhao, 2004<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B18"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>) with improvements<sup></sup>in stability and/or affinity. Each of these cell surface receptors<sup></sup>is involved in cell-to-cell interactions through its specific<sup></sup>binding to cognate ligands on the surface of an opposing cell.<sup></sup>As interactions such as these are typically very low affinity<sup></sup>(Maenaka <em>et al</em>., 1999<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B41"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Davis <em>et al</em>., 2003<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B16"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>), there has been considerable<sup></sup>interest in the development of engineering methods for these<sup></sup>classes of proteins.<sup></sup></p> <p> TCRs are excellent examples of proteins recognizing cell surface<sup></sup>ligands that in many cases are not amenable to isolation and<sup></sup>purification with retention of native conformation. The TCR<sup></sup>recognizes an antigenic peptide, derived from a foreign protein,<sup></sup>presented on the host cell surface bound to a protein of the<sup></sup>MHC (Davis <em>et al</em>., 1998<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B15"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Rudolph and Wilson, 2002<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B49"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). The binding<sup></sup>of a TCR to its cognate peptide-MHC (pMHC) ligand on the target<sup></sup>cell stimulates T cell effector function (e.g. cytokine release,<sup></sup>target cell lysis). Like that of an antibody, the antigen binding<sup></sup>site of a TCR is made up of hypervariable loops called complementarity<sup></sup>determining regions (CDRs) that make contact with ligand (Garcia<sup></sup><em>et al</em>., 1996<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B24"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). While the expression and purification of soluble<sup></sup>pMHC ligands has been an area of intense effort, many pMHC protein<sup></sup>complexes are unstable and/or difficult to manipulate. The variable<sup></sup>success with this class of proteins arises from both the diverse<sup></sup>nature of the antigenic peptides and the extensive polymorphisms<sup></sup>in the MHC. We present here a strategy that allows the isolation<sup></sup>of high-affinity TCRs against different pMHC ligands, without<sup></sup>the need to express and purify soluble forms of pMHC.<sup></sup></p> <p> In the yeast display system, the TCR has been cloned as a V<sub>ß</sub>-linker-V<sub><img src="http://peds.oxfordjournals.org/math/alpha.gif" alt="{alpha}"></sub><sup></sup>single chain (scTCR) fused to the gene for the yeast cell surface<sup></sup>protein AGA2 (Kieke <em>et al</em>., 1999<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B35"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). This fusion construct is<sup></sup>displayed on the surface of yeast, where it is amenable to <em>in<sup></sup>vitro</em> engineering for higher affinity binding to the pMHC ligand.<sup></sup>The 2C TCR, derived from a murine cytotoxic T lymphocyte (CTL)<sup></sup>clone, has been subjected to affinity maturation in this yeast<sup></sup>display system (Holler <em>et al</em>., 2000<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B31"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Holler <em>et al</em>., 2003<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B32"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). This<sup></sup>TCR recognizes a peptide from a mitochondrial protein, QL9 peptide,<sup></sup>presented by the allogeneic MHC molecule L<sup>d</sup> (Udaka <em>et al</em>., 1992<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B59"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>),<sup></sup>as well as the self peptide dEV8 and the foreign peptide SIYR<sup></sup>presented by the syngeneic MHC molecule K<sup>b</sup> (Tallquist and Pease,<sup></sup>1995<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B57"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Udaka <em>et al</em>., 1996<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B60"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). Previously, a library of degenerate<sup></sup>CDR3<sub><img src="http://peds.oxfordjournals.org/math/alpha.gif" alt="{alpha}"></sub> mutants was screened using fluorescent-labeled, soluble<sup></sup>forms of the pMHC ligands and fluorescence-activated cell sorting<sup></sup>(FACS) (Holler <em>et al</em>., 2000<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B31"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Holler <em>et al</em>., 2003<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B32"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). While this<sup></sup>approach was effective for isolating higher affinity TCRs against<sup></sup>the three different pMHC, many T cell systems do not have available<sup></sup>purified and well-characterized soluble pMHC that can be used<sup></sup>for selections by FACS. Furthermore, access to expensive FACS<sup></sup>instrumentation may limit wider application of the approach.<sup></sup></p> Here, we present a strategy to circumvent this requirement by<sup></sup>using intact antigen presenting cells (APCs) as the selecting<sup></sup>platform. Yeast cells that displayed a library of scTCR on their<sup></sup>cell surface were incubated with cells that expressed the selecting<sup></sup>pMHC ligand in its native form on the cell surface. To separate<sup></sup>rare yeast cells that bear the high affinity scTCR mutants from<sup></sup>other yeast cells in the library, the approach takes advantage<sup></sup>of the density differential between lymphoid-derived cells and<sup></sup>yeast cells. Yeast bearing scTCR that bind with high affinity<sup></sup>to the pMHC ligand on the APC can be separated from nonbinding<sup></sup>or low affinity yeast by centrifugation through a discontinuous<sup></sup>density gradient of a commercial media (Ficoll-Paque). In this<sup></sup>single-step selection, yeast that formed stable conjugates with<sup></sup>the pMHC-bearing lymphoid cells were retained at the interface,<sup></sup>whereas unbound yeast sediment to the bottom. The strategy was<sup></sup>validated using yeast that express TCRs with different affinities,<sup></sup>spiked at various frequencies into a population of yeast cells<sup></sup>that express non-binding TCRs. We show that this procedure can<sup></sup>effectively enrich 1000-fold scTCR mutants with affinities in<sup></sup>the nanomolar range. Also, novel high-affinity TCR mutants were<sup></sup>isolated against peptide variants, thereby obviating the need<sup></sup>to purify each peptide variant bound to the MHC ligand. Finally,<sup></sup>the procedure was shown to be effective using TCRs that recognize<sup></sup>either class I or class II MHC ligands, which should prove particularly<sup></sup>useful since class II MHC ligands have been more difficult to<sup></sup>purify (Ferlin <em>et al</em>., 2000<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B21"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Hackett and Sharma, 2002<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B27"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>; Starwalt<sup></sup><em>et al</em>., 2003<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B56"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>). The general procedure should be readily applicable<sup></sup>not only to the isolation of TCRs but also to antibodies that<sup></sup>recognize cell surface tumor antigens now that a human scFv<sup></sup>library in yeast is available (Feldhaus <em>et al</em>., 2003<a href="http://peds.oxfordjournals.org/cgi/content/full/19/6/255#B20"><img src="http://peds.oxfordjournals.org/icons/fig-down.gif" alt="Go"></a>).
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