- Development of a novel strategy for engineering high-affinity proteins by yeast display

Cell surface receptors perform critical functions in the communicationof extracellular information to the inside of the cell. Receptorsfor soluble molecules, including hormones, growth factors, orcytokines, control properties that range from cellular proliferationto cell migration. Other receptors function in cell-to-cellcommunication by interacting with a cognate ligand that is expressedon the surface of an opposing cell. Frequently, such receptor–ligandpairs exhibit very low affinities that can make biochemicaland structural analyses difficult (Maenaka et al., 1999Go). Byengineering the affinities or kinetics of such interactionsit is possible to explore the mechanisms that dictate biologicaleffects (Rao et al., 2005Go). Furthermore, engineering high-affinitysoluble receptors or monoclonal antibodies that bind to cellsurface ligands can provide potential therapeutic agents thatantagonize the receptor–ligand interactions or that targeta cell for destruction.

To engineer peptides or proteins for improved binding properties,various methods of directed evolution have been developed. Thesemethods include systems such as yeast display (Boder and Wittrup,1997Go), phage display (Smith, 1985Go; Bradbury and Marks, 2004Go;Marks and Bradbury, 2004Go), Escherichia coli display (Franciscoet al., 1993Go), baculovirus/insect cell display (Boublik et al.,1995Go) and ribosome display (Hanes et al., 2000Go). While purifiedligands have been used most often in these approaches, therehas been considerable effort to develop strategies that involveligands present in whole cell preparations. For example, ina process called biopanning, phage display libraries are eitherincubated with target cells or introduced in vivo into animals(Kupsch et al., 1999Go; Giordano et al., 2001Go; Roovers et al.,2001Go; Trepel et al., 2002Go). Phage that display a peptide orantibody against cell- or tissue-specific surface moleculesare then isolated, and the process is repeated for further enrichment.

Recently, yeast display has been used as a system to isolatehuman scFv fragments that have specificity for various antigens,with the goal of identifying lead candidates for further directedevolution (Feldhaus et al., 2003Go). In several cases, yeast displayhas been used to isolate scFv (Boder et al., 2000Go; Rajpal etal., 2005Go; Razai et al., 2005Go) or other cell surface receptors(Buonpane et al., 2005Go) that exhibit picomolar affinity constantsfor their ligands. Furthermore, yeast display has been usedto engineer T cell receptors (TCRs) (Holler et al., 2000Go; Kiekeet al., 2001Go; Holler et al., 2003Go; Chlewicki et al., 2005Go),natural killer cell receptors (Dam et al., 2003Go) and proteinsof the major histocompatibility complex (MHC) (Brophy et al.,2003Go; Starwalt et al., 2003Go; Esteban and Zhao, 2004Go) with improvementsin stability and/or affinity. Each of these cell surface receptorsis involved in cell-to-cell interactions through its specificbinding to cognate ligands on the surface of an opposing cell.As interactions such as these are typically very low affinity(Maenaka et al., 1999Go; Davis et al., 2003Go), there has been considerableinterest in the development of engineering methods for theseclasses of proteins.

TCRs are excellent examples of proteins recognizing cell surfaceligands that in many cases are not amenable to isolation andpurification with retention of native conformation. The TCRrecognizes an antigenic peptide, derived from a foreign protein,presented on the host cell surface bound to a protein of theMHC (Davis et al., 1998Go; Rudolph and Wilson, 2002Go). The bindingof a TCR to its cognate peptide-MHC (pMHC) ligand on the targetcell stimulates T cell effector function (e.g. cytokine release,target cell lysis). Like that of an antibody, the antigen bindingsite of a TCR is made up of hypervariable loops called complementaritydetermining regions (CDRs) that make contact with ligand (Garciaet al., 1996Go). While the expression and purification of solublepMHC ligands has been an area of intense effort, many pMHC proteincomplexes are unstable and/or difficult to manipulate. The variablesuccess with this class of proteins arises from both the diversenature of the antigenic peptides and the extensive polymorphismsin the MHC. We present here a strategy that allows the isolationof high-affinity TCRs against different pMHC ligands, withoutthe need to express and purify soluble forms of pMHC.

In the yeast display system, the TCR has been cloned as a Vß-linker-V{alpha}single chain (scTCR) fused to the gene for the yeast cell surfaceprotein AGA2 (Kieke et al., 1999Go). This fusion construct isdisplayed on the surface of yeast, where it is amenable to invitro engineering for higher affinity binding to the pMHC ligand.The 2C TCR, derived from a murine cytotoxic T lymphocyte (CTL)clone, has been subjected to affinity maturation in this yeastdisplay system (Holler et al., 2000Go; Holler et al., 2003Go). ThisTCR recognizes a peptide from a mitochondrial protein, QL9 peptide,presented by the allogeneic MHC molecule Ld (Udaka et al., 1992Go),as well as the self peptide dEV8 and the foreign peptide SIYRpresented by the syngeneic MHC molecule Kb (Tallquist and Pease,1995Go; Udaka et al., 1996Go). Previously, a library of degenerateCDR3{alpha} mutants was screened using fluorescent-labeled, solubleforms of the pMHC ligands and fluorescence-activated cell sorting(FACS) (Holler et al., 2000Go; Holler et al., 2003Go). While thisapproach was effective for isolating higher affinity TCRs againstthe three different pMHC, many T cell systems do not have availablepurified and well-characterized soluble pMHC that can be usedfor selections by FACS. Furthermore, access to expensive FACSinstrumentation may limit wider application of the approach.

Here, we present a strategy to circumvent this requirement byusing intact antigen presenting cells (APCs) as the selectingplatform. Yeast cells that displayed a library of scTCR on theircell surface were incubated with cells that expressed the selectingpMHC ligand in its native form on the cell surface. To separaterare yeast cells that bear the high affinity scTCR mutants fromother yeast cells in the library, the approach takes advantageof the density differential between lymphoid-derived cells andyeast cells. Yeast bearing scTCR that bind with high affinityto the pMHC ligand on the APC can be separated from nonbindingor low affinity yeast by centrifugation through a discontinuousdensity gradient of a commercial media (Ficoll-Paque). In thissingle-step selection, yeast that formed stable conjugates withthe pMHC-bearing lymphoid cells were retained at the interface,whereas unbound yeast sediment to the bottom. The strategy wasvalidated using yeast that express TCRs with different affinities,spiked at various frequencies into a population of yeast cellsthat express non-binding TCRs. We show that this procedure caneffectively enrich 1000-fold scTCR mutants with affinities inthe nanomolar range. Also, novel high-affinity TCR mutants wereisolated against peptide variants, thereby obviating the needto purify each peptide variant bound to the MHC ligand. Finally,the procedure was shown to be effective using TCRs that recognizeeither class I or class II MHC ligands, which should prove particularlyuseful since class II MHC ligands have been more difficult topurify (Ferlin et al., 2000Go; Hackett and Sharma, 2002Go; Starwaltet al., 2003Go). The general procedure should be readily applicablenot only to the isolation of TCRs but also to antibodies thatrecognize cell surface tumor antigens now that a human scFvlibrary in yeast is available (Feldhaus et al., 2003Go).

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