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

Abstract

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

 

S.A. Richman1, S.J. Healan1, K.S. Weber1, D.L. Donermeyer2, M.L. Dossett3,4, P.D. Greenberg3,4, P.M. Allen2 and D.M. Kranz1,5

1 Department of Biochemistry, University of Illinois at Urbana-Champaign 600 S. Mathews Avenue, Urbana, IL 61801, USA 2 Department of Pathology and Immunology, Washington University School of Medicine St Louis, MO 63110, USA 3 Department of Immunology, University of Washington WA 98109, USA 4 The Division of Clinical Research, Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, Seattle, WA 98109, USA

An open access article: Protein Engineering Design and Selection 2006 19(6):255-264; doi:10.1093/protein/gzl00. 

 

Abstract

Yeast display provides a system for engineering high-affinityproteins using a fluorescent-labeled ligand and fluorescence-activatedcell sorting (FACS). In cases where it is difficult to obtainpurified ligands, or to access FACS instrumentation, an alternativeselection strategy would be useful. Here we show that yeastexpressing high-affinity proteins against a mammalian cell surfaceligand could be rapidly selected by density centrifugation.Yeast cell–mammalian cell conjugates were retained atthe density interface, separated from unbound yeast. High-affinityT cell receptors (TCRs) displayed on yeast were isolated usingantigen presenting cells that expressed TCR ligands, peptidesbound to products of the major histocompatibility complex (MHC).The procedure yielded 1000-fold enrichments, in a single centrifugation,of yeast displaying high-affinity TCRs. We defined the affinitylimits of the method and isolated high-affinity TCR mutantsagainst peptide variants that differed by only a single residue.The approach was applied to TCRs specific for class I or classII MHC, an important finding since peptide-class II MHC ligandshave been particularly difficult to purify. As yeast displayhas also been used previously to identify antigen-specific antibodies,the method should be applicable to the selection of antibodies,as well as TCRs, with high-affinity for tumor cell-surface antigens.

 

Keywords: directed evolution/major histocompatibility complex/T cell receptor/yeast display

 


Introduction

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).

Materials and Methods

Peptides

Peptides (SIYR, SIYRYYGL; QL9, QLSPFPFDL) were synthesized bystandard F-moc chemistry at the Macromolecular Core Facilityat Pennsylvania State University. Position 5 variants of theQL9 peptide [Y5 (QLSPYPFDL), R5, H5, and M5] were synthesizedat the Protein Sciences Facility at the University of Illinois.QL9 and SIYR were purified by C-18 reverse-phase HPLC. The Hbpeptide (GKKVITAFNEGLK) was synthesized by standard F-moc chemistryand purified by C-18 reverse-phase HPLC at Washington University.

Cell lines

The endogenous peptide transport-deficient human T-lymphoblastoidcell lines T2-Kb and T2-Ld, which have been transfected withthe Kb or Ld heavy chain, respectively, were provided by P.Cresswell (Alexander et al., 1989Go). The I-Ak, I-Ek –expressingB cell hybridoma, CH27, was used for studies with the classII 3.L2 TCR (Evavold et al., 1992Go). Cells were propagated inRPMI 1640 media /10% fetal bovine serum, supplemented with L-glutamineand 2-mercaptoethanol at 37°C and 5% CO2. G418 (0.5 mg/ml)was added to T2-Kb and T2-Ld cultures.

scTCR yeast display constructs and libraries

Several mutants of the 2C TCR used here were isolated previously.The 2C-T7 scTCR was engineered for increased yeast surface expression(Kieke et al., 1999Go) and it has been shown to have an affinity(KD value) for QL9/Ld of ~3 µM (unpublished data). The2C-m80 TCR mutant was engineered using yeast display and FACSand the full length TCR has been reported previously to havean affinity for SIYR/Kb of 790 nM (Holler et al., 2003Go). Morerecent surface plasmon resonance (SPR) measurements of the E.coli expressed, single-chain form of the 2C-m80 TCR have shownit to have an affinity for SIYR/Kb of 150 nM (unpublished data).As the single-chain form was expressed on yeast in the presentstudies, the KD value of 150 nM will be referred to here asthe affinity of the 2C-m80 scTCR. The 2C-m6 TCR was engineeredusing yeast display and FACS and it has been shown to have anaffinity for QL9/Ld of 6 nM (Holler et al., 2000Go; Holler etal., 2003Go). The 3.L2 mouse TCR recognizes a peptide from anallelic variant of hemoglobin, presented by the I-Ek Class IIMHC molecule (Evavold et al., 1992Go). The mutant 3.L2-M15 wasengineered using yeast display to have high affinity for itsligand, hemoglobin peptide Hb/I-Ek (KD = 25 nM) (Weber et al.,2005Go). Two scTCRs were used as controls that do not bind tothe cognate ligands recognized by the 2C or 3.L2 TCRs. Theseincluded (i) C18-1, a surface-stabilized scTCR mutant (unpublisheddata) of the murine C18 TCR that recognizes a peptide derivedfrom a mutated MAP kinase presented by Kd MHC (Ikeda et al.,1997Go) and (ii) mWT1-B7, a mouse scTCR raised against Wilms tumorantigen-1 and engineered previously for increased yeast surfaceexpression (unpublished data). The 2C CDR3{alpha} library has beendescribed previously (Holler et al., 2000Go). This yeast librarywas cultured in selective SD-CAA liquid media [2% dextrose (w/v),0.67 % yeast nitrogen base (w/v), and 1% casamino acids (w/v)]with kanamycin (50 µg/ml) at 30°C.

Induction of scTCR expression on the yeast cell surface

scTCR expression on the surface of the Saccharomyces cerevisiaestrain EBY100 was induced as described previously (Boder andWittrup, 1997Go). Briefly, the yeast display plasmid pCT302, whichencodes a gene fusion linking the scTCR to the yeast cell surfacegene AGA2, controlled by a galactose-driven promoter, was introducedinto EBY100 yeast cells. Expression of the AGA2:scTCR fusiongene was induced by transferring cells growing in SD-CAA togalactose-containing media and shaking at 20°C for at least24 h.

Flow cytometric analysis of scTCR expression and pMHC binding

Yeast that express 2C scTCR mutants (Vß8.2-positive)were detected with the anti-Vß8.2 antibody F23.2.Yeast that express 3.L2 or C18 scTCR mutants (Vß8.3-positive)were detected with the anti-Vß8.3 antibodies KT-8C1(Cedarlane Laboratories) or 1B3.3-PE (BD Pharmingen). Yeastthat express mWT1-B7 scTCR (Vß11-positive) were detectedwith a PE-conjugated anti-Vß11 antibody, RR3-15-PE(BD Pharmingen). F23.2 or KT-8C1 binding was detected by incubatingwith biotinylated goat-anti-mouse IgG (Rockland Inc.), followedby streptavidin:PE (SA:PE) (BD Pharmingen) or by incubationwith PE-conjugated goat F(ab')2 anti-mouse Ig (Southern Biotech).All antibodies and secondary reagents were diluted in phosphate-bufferedsaline containing 0.5% bovine serum albumin (PBS/BSA). Sampleswere analyzed on a Coulter Epics-XL flow cytometer, gating onyeast cells based on light scattering properties. Flow cytometrywas also used to detect binding of yeast-displayed scTCR tosoluble Ld-Ig fusion protein folded with specific peptide (eitherQL9 or Y5). The QL9/Ld-Ig and Y5/Ld-Ig were produced and purifiedas described previously (Chlewicki et al., 2005Go). To detectpMHC binding by yeast-displayed scTCR, ~400 µg/ml QL9/Ld-Igor ~200 µg/ml Y5/Ld-Ig was incubated with yeast cells onice for 1 h. Cells were washed with PBS/BSA and resuspendedin PE-labeled goat F(ab'2) anti-mouse Ig.

Peptide loading of cells

APCs (T2-Kb, T2-Ld, or CH27) were pelleted by centrifugationand resuspended in fresh media to a concentration of 106 cells/ml.1 ml aliquots were then dispensed into 1.5 ml microfuge tubes.Peptides were incubated with cells at the following final concentrations:1 µM SIYR, 30 µM QL9, and 10 µM Hb. Singleamino acid variants of QL9 at position 5 were incubated at concentrationspreviously determined to be in excess for maximum stabilizationof Ld on the surface of T2-Ld cells (Schlueter et al., 1996Go).Peptide/cell mixtures were rocked at room temperature for 60–90min to allow loading of the peptides.

Density differential centrifugation

Induced yeast cells were counted on a hemacytometer and aliquotedinto tubes that contained the peptide-loaded APCs (~106 yeastcells per tube). Another aliquot of induced yeast served as‘pre-centrifugation’ control. The yeast/APC mixtureswere allowed to rock at room temperature for 1 h. Followingthe incubation, the yeast/APC mixtures were layered onto threemls of Ficoll-Paque PLUS (Pharmacia) in 15 ml conical tubes.The cells were allowed to settle for 10 min at room temperature,and the tubes were then centrifuged at 1500 r.p.m. (~400x g)at 4°C for 30 min. After centrifugation, the visible layerof cells above the Ficoll-Paque (the ‘interface’)was removed from each tube in a 1 ml volume. In some cases,this interface was returned to the rocker for another 30-minincubation, and the procedure was repeated (centrifugation for15 min in this ‘second centrifugation’).

Plasmid rescue and sequencing

Surface display plasmids were rescued from selected yeast clonesusing a Zymoprep I Yeast Plasmid Minipreparation Kit (Zymo Research).Rescued plasmids were introduced into the E. coli strain DH10B(Invitrogen) by electroporation. Plasmids were purified fromE. coli using a QIAprep Spin Mini-Prep Kit (Qiagen) and sequencedat the DNA Core Sequencing Facility at the University of Illinois.

Results

Isolation of yeast:APC conjugates

In a previous study, we showed that it was possible to detectconjugates of a yeast cell bearing a TCR and an APC in whichthe MHC had been loaded with the antigenic peptide (Shusta etal., 2000Go). Using microscopy, these conjugates were shown tobe more prevalent for yeast that expressed a higher affinityTCR. To exploit this observation as a rapid method for selectinghigh-affinity TCRs, a procedure was developed for separatingyeast that are present in conjugates from unbound yeast. Lymphoidcells are known to form a discrete layer above the density mediumFicoll-Paque upon centrifugation, whereas yeast cells sedimentthrough this solution (data not shown). Thus, we reasoned thatyeast cells present as conjugates may be retained at this interface,separating them from unbound yeast.

To determine if yeast that express a high-affinity TCR couldbe isolated using this density centrifugation approach, we tookadvantage of a high-affinity mutant of the 2C-TCR, 2C-m80, whichwas engineered previously using yeast display and binds to thepMHC antigen SIYR/Kb with a KD value of 150 nM (Holler et al.,2003Go). As a control, yeast cells that display the scTCR C18-1,which does not bind to SIYR/Kb, were used. Approximately 106yeast expressing 2C-m80 or C18-1 were incubated with T2-Kb cellsalone (without peptide) or with SIYR-loaded T2-Kb cells, andthe cell mixture was layered onto Ficoll-Paque. Following centrifugation,the discrete interface layer was removed, allowed to incubate,and layered onto another tube of Ficoll-Paque. After this secondcentrifugation, the cells at the interface were again collected.Aliquots from both density selections and the original yeastcell sample were plated on sorbitol medium in order to quantitatethe percentage of 2C-m80 or C18-1 yeast cells that were recoveredfrom the APC interface layer. As shown in Figure 1a, a substantialfraction (16%) of yeast expressing the high affinity 2C-m80TCR remained at the interface following the first centrifugation,and 8% of the yeast remained following the second centrifugation.This retention was SIYR/Kb dependent, as only 0.1% of the high-affinity2C-m80 yeast was recovered when T2-Kb cells were used in theabsence of the SIYR peptide. Furthermore, only 0.1% of yeastexpressing the control C18-1 scTCR were recovered, indicatingthat the high affinity binding of the yeast 2C-m80 TCR to SIYR-Kbon the surface of the APCs was responsible for retaining theseyeast at the APC interface layer.

Quantitative analysis of enrichment and selection potential

Given that yeast bearing the high affinity TCR, but not irrelevantyeast, were retained at the interface, we next addressed whetheror not density differential centrifugation could be used forselection or enrichment methods. A library selection processwas simulated by attempting to isolate high affinity mutantsamong an excess of non-binding yeast. In one experiment, 2C-m80yeast were mixed with non-binding C18-1 yeast at a ratio of1 to 1000, and this mixture was incubated with SIYR-loaded T2-Kbcells and subjected to two sequential rounds of centrifugationthrough Ficoll-Paque (as described above). The ratio of bindingto non-binding yeast, before and after centrifugation, was monitoredby flow cytometry using antibodies specific for the Vßregion of either 2C or C18. As expected, yeast cells that displayedthe 2C-m80 in the pre-centrifugation 1 to 1000 mixture wereundetectable by flow cytometry. Remarkably, almost all yeastrecovered from the interface following centrifugation expressedthe 2C-m80 TCR (Figure 1b). Thus, this procedure yielded almost1000-fold single pass enrichment.

Based on this success, we sought to determine if the procedurewould work with a different high-affinity TCR, and if the procedurewould be capable of selecting high-affinity binders that mightbe even more rare (e.g. 1 in 10 000 or 1 in 100 000). Here,we used yeast that express the high-affinity 2C TCR mutant 2C-m6,that binds to a different ligand, QL9/Ld, with a KD value of6 nM. Yeast cells that express 2C-m6 were mixed at differentratios with excess C18-1 yeast. These yeast cell mixtures wereincubated with QL9-loaded T2-Ld APCs, and the cells were subjectedto two sequential rounds of centrifugation through Ficoll-Paqueas described above. The ratio of 2C-m6 yeast to C18-1 yeastwas monitored as described above, before and after centrifugation,by flow cytometry. Because these ratios are potentially subjectto stochastic variations in the number of ‘binders’(e.g. the starting populations contained only 10 or 100 yeastcells that could bind the APC), the selection procedure wasperformed in triplicate for both the 1:10 000 and 1:100 000experiments. In each case, one of the three replicates yieldeddetectable enrichment following the second centrifugation, asevidenced by the population of yeast that stain positive foran antibody that recognizes 2C but not C18 (Figure 2). Theseresults indicate that it is possible to isolate very rare highaffinity mutants. It was not surprising that enrichment of astarting population consisting of only 10 or 100 desired cellsin 106 is less consistent than when 1000 high affinity cellswere present in the sample. Such variation can be minimizedby over-sampling libraries that might contain rare mutants.

Selection analyzed across a range of TCR affinities In order to explore the affinity ranges that could be selectedusing the density centrifugation procedure, we monitored enrichmentof a high affinity mutant 2C-m6 under circumstances when itsaffinity for different pMHC ligands ranged from >1 µMto 6 nM (including QL9/Ld, discussed in the previous section).The other ligands represented single amino acid variants ofthe QL9 peptide (at position 5 of the QL9 nonamer). Three position5 variants of QL9: M5, H5, and R5, bind to Ld (Schlueter etal., 1996Go), and the affinities of 2C-m6 for these variants inthe context of Ld are ~34, 78 nM, and >1 µM, respectively(Holler and Kranz, 2003Go). The KD value of 2C-m6 for R5/Ld wasjudged to be higher than 1 µM because 2C-m6+ T cells requiredCD8 in order to be stimulated by R5/Ld (Holler and Kranz, 2003Go).T2-Ld cells, loaded with one of each of the three position 5variants, were incubated with 2C-m6 yeast mixed with non-bindingC18-1 yeast at a ratio of 1 to 1000. These cells were then subjectedto centrifugation through Ficoll-Paque, and the ratios of yeastcells that expressed 2C-m6 to C18-1 were monitored before andafter centrifugation by flow cytometry. Not surprisingly, thesingle pass enrichment was correlated with the affinity of theTCR for the pMHC (Figure 3a). Density centrifugation using M5-loadedT2-Ld cells yielded 970-fold enrichment and T2-Ld loaded withH5 (for which 2C-m6 has a ~2-fold lower affinity) yielded 950-foldenrichment. Thus, as observed for selections of the 2C-m80 TCRwith SIYR/Kb, affinities greater than ~100 nM yield almost 1000-foldenrichments in a single centrifugation step.

In an effort to isolate high affinity clones from the 2C CDR3{alpha}library, using a novel peptide that had not yet been used toscreen the library, the procedure described above was performedusing a position 5 variant of the QL9 peptide. T2-Ld cells wereloaded with Y5 peptide (which contains a Tyr substituted forPhe at peptide position 5), incubated with CDR3{alpha} library cells,and subjected to centrifugation through Ficoll-Paque. Five mutantsisolated from this selection, 2C-mY-1 through 2C-mY-5, wereassayed qualitatively for binding to Y5-loaded T2-Ld cells bydensity centrifugation. Three clones, 2C-mY-1, 2C-mY-3 and 2C-mY-4,showed retention levels similar to those of the standard high-affinitypair 2C-m6:QL9/Ld (Figure 4b). One clone, 2C-mY-2, showed aremarkable recovery of ~70%, whereas clone 2C-mY-5 showed reducedrecovery compared to 2C-m6/QL9/Ld but greater than the loweraffinity control (2C-T7/QL9/Ld).

Characterization of TCR clones isolated from a site-directed mutant library

To further characterize the TCR yeast clones isolated by densitycentrifugation with QL9/Ld- or Y5/Ld-bearing APCs, flow cytometrywas performed using anti-TCR antibody F23.2 to evaluate yeastdisplay levels and soluble QL9/Ld or Y5/Ld to evaluate ligandbinding (Figure 5). Yeast cells were stained with F23.2, dimericQL9/Ld (QL9/Ld-Ig), or dimeric Y5/Ld (Y5/Ld-Ig), and bindingwas analyzed using flow cytometry. TCR clones isolated withQL9/Ld or Y5/Ld, the wild-type low affinity TCR 2C-T7 and thepreviously selected mutant 2C-m6 were displayed at similar levels,within 2-fold based on the mean fluorescence units of cellsstained with the anti-TCR antibody. Among the mutants isolatedusing QL9, the mutant that exhibited the highest recovery indensity centrifugation, 2C-mQ-4 (Figure 4a), also showed thehighest level of QL9/Ld-Ig binding (Figure 5a). Similarly, theY5/Ld mutant that showed the highest recovery in density centrifugation,2C-mY-2 (Figure 4b), also showed the highest level of Y5/Ld-Igbinding (Figure 5b). The clone with the lowest recovery by densitycentrifugation, 2C-mQ-5, also showed the lowest level of stainingwith QL9/Ld-Ig. All other QL9/Ld clones were intermediate inboth recovery and staining with QL9/Ld-Ig, while other Y5/Ldclones were very low recovery and staining in the case of Y5/Ld-Ig.As expected, the low affinity 2C TCR 2C-T7 did not show detectablebinding to either QL9/Ld-Ig or Y5/Ld-Ig.

Our previous studies have shown that high-affinity TCR mutantsexhibited distinct sequence motifs in the CDR3{alpha}, which probablycorrelate with the structural requirements for higher affinitybinding. To examine whether or not the ten mutants isolatedby density centrifugation exhibited these same motifs, the plasmidsfrom the yeast clones were sequenced. The amino acid sequencesof the mutated CDR3{alpha}, wild-type CDR3{alpha}, and the CDR3{alpha} of previouslyisolated mutants (2C-m6 and 2C-m12) that contain two sequencemotifs are shown in Figure 6. The five QL9-isolated mutantsdiffered from the wild-type 2C and 2C-m6. Interestingly, allof these mutants contained the Ala101{alpha}Gly substitution that characterizesone of the conserved motifs identified in the CDR3{alpha} of high affinity2C clones, including 2C-m6, isolated during QL9/Ld selectionusing FACS (Holler et al., 2000Go). The five mutants isolatedhere also contained either Ala103{alpha}Arg or Ala103{alpha}Tyr substitutions,both identified as preferential mutations in the original screeningof this library.

Comparison of the CDR3{alpha} sequences of Y5-isolated mutants and2C mutants, isolated previously using QL9/Ld and FACS (e.g.2C-m12), revealed that in this case a second conserved motifwas also present (Figure 6) (Holler et al., 2000Go). This conservedmotif contains three tandem prolines (Pro-Pro-Pro) and it waspresent in 2C-mY-2, the mutant that showed the highest levelof Y5/Ld binding as judged by both density centrifugation andflow cytometry (Figures 4 and 5). A variation of that motif,Pro-Thr-Pro, which was also isolated previously using flow sorting(Holler et al., 2000Go), was identified in mutants 2C-mY-3 and2C-mY-4. Mutant 2C-mY-5 contained the conserved glycine residueat 101{alpha} that was observed in all of the QL9-isolated clones.Finally, mutant 2C-mY-1 did not contain either the proline orthe glycine motif, but its unique sequence (STSWY) was somewhatsimilar to isolates (RWTSG and TWSPF) obtained in previous selections(Holler et al., 2003Go). Thus, despite the fact that these twoligands (QL9 and Y5) differ by only a hydroxyl group, it appearsthat there are significant differences between 2C TCR recognitionof QL9/Ld and Y5/Ld. QL9/Ld may preferentially interact withTCR mutants that contain Gly101{alpha}, while Y5/Ld appears to be capableof using more diverse sequences, and in particular proline-containingmutants.

Quantitation of TCR selection potential in a class II MHC system

Given the effectiveness with which density centrifugation selectedhigh affinity mutants in the 2C TCR/class I MHC system, we extendedour analysis to a TCR/class II MHC-restricted system. The classII restricted TCR 3.L2 binds to a peptide from hemoglobin (Hb)bound to the class II MHC I-Ek (Evavold et al., 1992Go). Previously,a mutant of the 3.L2 TCR, 3.L2-M15 was isolated from sequentialCDR3 libraries using yeast display and FACS (Weber et al., 2005Go).Mutant 3.L2-M15 binds to the Hb/I-Ek ligand with a KD valueof ~25 nM as measured by SPR, an 800-fold increase in affinityover the wild-type 3.L2 TCR. We thus investigated whether yeastcells that express this high affinity mutant 3.L2-M15 couldbe isolated from a 1000-fold excess of non-binding yeast usingdensity centrifugation. The I-Ek-positive mouse cell line CH27was loaded exogenously with 10 µM Hb peptide. Peptide-loadedcells were incubated with yeast that express 3.L2-M15 mixedat a 1 to 1000 ratio with control yeast that express the non-bindingmWT1-B7 TCR. The cell mixture was subjected to one round ofcentrifugation through Ficoll-Paque, and the ratio of 3.L2-M15yeast to mWT1-B7 yeast before and after centrifugation was monitoredby flow cytometry using antibodies specific for either the Vßregion of the 3.L2 TCR or the Vß region of the mWT1TCR (Figure 7a). As expected, prior to centrifugation, onlynon-binding yeast that express mWT1-B7, were detectable by flowcytometry. Following only a single centrifugation, the percentageof cells staining positive for 3.L2-M15 increased from undetectableto 23.6%. Furthermore, the percentage of cells that expressedmWT1-B7 decreased from 70.3 to 39%. This increase in the relativenumber of cells positive for the 3.L2-M15 mutant correlatedwith an enrichment for the high affinity scTCR of ~380-fold (from0.1% of cells to 38% of the cells). The average enrichment forthree experiments with 3.L2-M15 and mWT1-B7 was ~500-fold (Figure 7b).This experiment was repeated using the same high affinity TCR3.L2-m15, but a different non-binding TCR, 2C-T7, and in thiscase an average enrichment of ~200-fold was achieved (Figure 7c).


Discussion

Antibody engineering to facilitate targeting of antigens hasbeen used extensively in the development of antibodies as potentialtherapeutic agents. The most frequent uses of in vitro techniquesseek to optimize antibody binding affinities, typically througha platform such as phage display (Hawkins et al., 1992Go; Markset al., 1992Go). Alternatively, phage display has been used toidentify lead antibodies against targets using either purifiedantigens (Edwards et al., 2003Go) or biopanning procedures employingwhole cells or tissues (Giordano et al., 2001Go; Trepel et al.,2002Go). Like antibodies, TCRs recognize a diverse array of antigens.However, unlike the antigens recognized by antibodies, the antigensrecognized by T cells represent a greater challenge from thebiochemical perspective. These antigens consist of a short peptidethat is non-covalently associated with a cell surface heterodimerencoded by the MHC for class II or the MHC and ß2-microglobulinfor class I. Ternary complexes of peptides and MHC productsexhibit widely diverse stabilities and thus the ability to expressand purify these ligands varies widely. Various laboratories,including the NIH tetramer facility and commercial vendors,provide purified complexes of various well-characterized pMHCligands. However, the variety of MHC alleles (2101 in totalamong human class I and II), and the large number of possiblepeptides makes it difficult to use purified ligands more generally.In order to develop a strategy for engineering TCRs with high-affinityfor a diverse array of antigens, it would be advantageous toavoid the need for purification of the ternary antigen complexes.We show here that it is possible to combine the yeast displaysystem with a rapid density centrifugation method to selecthigh-affinity TCRs. The strategy required only the use of syntheticpeptides that could be loaded exogenously onto an APC that expresseson its surface the proper MHC molecule.

In the examples described here, the density centrifugation methodcan be compared favorably to our previous experience using purifiedpMHC antigens and FACS (Holler et al., 2000Go; Holler et al.,2003Go; Chlewicki et al., 2005Go). In previous studies, three orfour rounds of yeast cell growth, induction and sorting wererequired to identify TCR mutants with high affinity. This processrequired a period of 2–3 weeks, whereas the density centrifugationmethod used here to isolate QL9/Ld or Y5/Ld mutants requiredonly a few hours for selections, through only one or two centrifugations.Thus, this method is rapid, obviates the need for a high-speedflow sorter and does not require purified ligands. While theprecision and ability to perform off-rate based screens remainsan inherent advantage of FACS and yeast display (Boder and Wittrup,1998Go), the present strategy should prove useful for the manysystems that lack purified ligands and for laboratories withlimited access to FACS instrumentation. In a different selectionapproach that also does not require FACS instrumentation, Yeungand Wittrup used biotinylated antigens and streptavidin-coatedmagnetic beads to isolate yeast that express antigen-specificscFvs with an enrichment of ~100-fold (Yeung and Wittrup, 2002Go).While the method described in the present report can be usedonly for ligands that are expressed on cell surfaces, our findingsshow that remarkable enrichments of 1000-fold can be achievedwith only single-pass density centrifugations.

Recent reports have shown that high-affinity TCRs can also beengineered by phage display (Laugel et al., 2005Go; Li et al.,2005Go). It is reasonable to predict that the same type of biopanningthat has been performed with phage-displayed peptides or antibodies(Kupsch et al., 1999Go; Giordano et al., 2001Go; Roovers et al.,2001Go; Trepel et al., 2002Go) could be used for the phage-displayedTCRs, using peptide-loaded APCs. It is possible that this approachmay also find some use in the isolation of ‘lead’pMHC binders from libraries of naïve TCRs, although suchlibraries have not yet been reported. If the density methoddescribed here were to be used with yeast-display librariesof naïve TCRs, clearly the affinities of the TCRs wouldneed to be above a particular threshold (e.g. KD values <1µM). Whether TCRs can be obtained from naive librariesand, if so, whether they retain the typical diagonal dockingorientation on the pMHC ligand (Garcia and Adams, 2005Go) remainsto be determined.

In addition to providing validation that the density centrifugationmethod could be used to isolate high-affinity TCRs from a libraryof mutants, sequence analysis of the panels of high-affinityTCRs against QL9/Ld and Y5/Ld revealed CDR3{alpha} motifs that maycorrelate with recognition differences between these two verysimilar ligands. Accordingly, all mutants isolated against QL9,which contains a Phe at position 5, represented a CDR3{alpha} motifwith a key glycine at residue 101{alpha}. In contrast the additionof a hydroxyl group in the Y5 peptide variant appears to allowthe isolation of CDR3{alpha} mutants with more diversity in their CDR3{alpha}.The molecular explanations for these differences remain to beresolved and will require structural studies of the TCR-peptide/Ldcomplexes. Nevertheless, the data point to the exquisite peptidefine-specificity that is associated with the recognition ofpMHC ligands by TCRs.

As indicated, many pMHC ligands of interest have not yet beensuccessfully expressed and purified in soluble form. This limitationis particularly pronounced for class II MHC ligands (Hackettand Sharma, 2002Go), and, despite significant advances in classII MHC multimer technology, there are still relatively few classII MHC multimers available (Cameron et al., 2002Go). While theexpression of class I MHC molecules in E. coli has been standardizedfor many alleles, it has been more difficult to develop standardizedprotocols for class II MHC production and only a few MHC classII molecules have be produced in E. coli. Some class II MHCmolecules have been successfully isolated following secretionfrom insect cells, often as fusion products with introducedleucine zipper domains to assist in chain association (Scottet al., 1996Go). One fundamental difference between class I andclass II MHC involves the chaperones that facilitate foldingand peptide loading intracellularly (Cresswell and Lanzavecchia,2001Go). Our method of exogenous peptide loading completely eliminatesthe need to develop expression and purification protocols foreach peptide-class II MHC multimer. For example, such an approachmay be useful in the discovery of high-affinity TCRs that bindto class II MHC antigens that are involved in autoimmunity (Lebowitzet al., 1999Go; Masteller et al., 2003Go).

Finally, it is conceivable that this methodology could be extendedto other receptor–ligand interactions, as well as to theengineering of high-affinity antibodies against tumor antigens(Boder and Wittrup, 2000Go; Feldhaus et al., 2003Go; van den Beucken,2003Go; Hoogenboom, 2005Go). In fact, an alternative biopanningstrategy using an antibody–hapten model system was publishedduring the preparation of this manuscript (Wang and Shusta,2005Go). These studies monitored recovery of yeast expressinga high-affinity, fluorescein-specific antibody from a fluorescein-labeled,adherent endothelial cell monolayer. While this approach islimited to adherent cell lines and has yet to be applied toselections from combinatorial libraries, it provides furtherevidence of the potential of yeast display in cell-panning strategies.Furthermore, mammalian proteins other than antibodies or TCRshave been expressed on the cell surface via the yeast displaysystem (Bhatia et al., 2003Go; Schweickhardt et al., 2003Go). Forexample, the cell adhesion molecule E-selectin, which helpsinitiate extravasation of leukocytes by binding sialyl-Lewis-xligand on the leukocyte cell surface, has been expressed asa functional construct on the surface of yeast (Bhatia et al.,2003Go). As the use of yeast display expands as a tool for directedevolution, the density centrifugation strategy for selectionsmay serve to support broadened efforts in library screening,especially when purified soluble selecting agents are not available.

 


References

Alexander J., Payne J.A., Murray R., Frelinger J.A., Cresswell P. (1989) Immunogenetics 29:380–399.[CrossRef][ISI][Medline]

Bhatia S.K., Swers J.S., Camphausen R.T., Wittrup K.D., Hammer D.A. (2003) Biotechnol. Prog. 19:1033–1037.[Medline]

Boder E.T. and Wittrup K.D. (1997) Nat. Biotech. 15:553–557.[CrossRef][ISI][Medline]

Boder E.T. and Wittrup K.D. (1998) Biotechnol. Prog. 14:55–62.[CrossRef][Medline]

Boder E.T. and Wittrup K.D. (2000) Methods Enzymol 328:430–444.[ISI][Medline]

Boder E.T., Midelfort K.S., Wittrup K.D. (2000) Proc. Natl Acad. Sci. USA 97:10701–10705.[Abstract/Free Full Text]

Boublik Y., Di Bonito P., Jones I.M. (1995) Biotechnology (NY) 13:1079–1084.

Bradbury A.R. and Marks J.D. (2004) J. Immunol. Methods 290:29–49.[CrossRef][ISI][Medline]

Brophy S.E., Holler P.D., Kranz D.M. (2003) J. Immunol. Methods 272:235–246.[CrossRef][ISI][Medline]

Buonpane R.A., Moza B., Sundberg E.J., Kranz D.M. (2005) J. Mol. Biol. 353:308–321.[CrossRef][ISI][Medline]

Cameron T.O., Norris P.J., Patel A., Moulon C., Rosenberg E.S., Mellins E.D., Wedderburn L.R., Stern L.J. (2002) J. Immunol. Methods 268:51–69.[CrossRef][Medline]

Chlewicki L.K., Holler P.D., Monti B.C., Clutter M.A., Kranz D.M. (2005) J. Mol. Biol. 346:223–239.[CrossRef][Medline]

Cresswell P. and Lanzavecchia A. (2001) Curr. Opin. Immunol. 13:11–12.[CrossRef][Medline]

Dam J., Guan R., Natarajan K., Dimasi N., Chlewicki L.K., Kranz D.M., Schuck P., Margulies D.H., Mariuzza R.A. (2003) Nat. Immunol. 4:1213–1222.[CrossRef][ISI][Medline]

Davis M.M., Boniface J.J., Reich Z., Lyons D., Hampl J., Arden B., Chien Y. (1998) Annu. Rev. Immunol. 16:523–544.[CrossRef][ISI][Medline]

Davis S.J., Ikemizu S., Evans E.J., Fugger L., Bakker T.R., van der Merwe P.A. (2003) Nat. Immunol. 4:217–224.[CrossRef][ISI][Medline]

Edwards B.M., et al. (2003) J. Mol. Biol. 334:103–118.[CrossRef][Medline]

Esteban O. and Zhao H. (2004) J. Mol. Biol. 340:81–95.[CrossRef][ISI][Medline]

Evavold B.D., Williams S.G., Hsu B.L., Buus S., Allen P.M. (1992) J. Immunol. 148:347.[Abstract]

Feldhaus M., et al. (2003) Nat. Biotechnol. 21:163–170.[CrossRef][ISI][Medline]

Ferlin W., Glaichenhaus N., Mougneau E. (2000) Curr. Opin. Immunol. 12:670–675.[CrossRef][ISI][Medline]

Francisco J.A., Campbell R., Iverson B.L., Georgiou G. (1993) Proc. Natl Acad. Sci. USA 90:10444–10448.[Abstract/Free Full Text]

Garcia K.C. and Adams E.J. (2005) Cell 122:333–336.[CrossRef][ISI][Medline]

Garcia K.C., Degano M., Stanfield R.L., Brunmark A., Jackson M.R., Peterson P.A., Teyton L., Wilson I.A. (1996) Science 274:209–219.[Abstract/Free Full Text]

Garcia K.C., Tallquist M.D., Pease L.R., Brunmark A., Scott C.A., Degano M., Stura E.A., Peterson P.A., Wilson I.A., Teyton L. (1997) Proc. Natl Acad. Sci. USA 94:13838–13843.[Abstract/Free Full Text]

Giordano R.J., Cardo-Vila M., Lahdenranta J., Pasqualini R., Arap W. (2001) Nat. Med. 7:1249–1253.[CrossRef][ISI][Medline]

Hackett C.J. and Sharma O.K. (2002) Nat. Immunol. 3:887–889.[CrossRef][ISI][Medline]

Hanes J., Schaffitzel C., Knappik A., Pluckthun A. (2000) Nat. Biotechnol. 18:1287–1292.[CrossRef][ISI][Medline]

Hawkins R.E., Russell S.J., Winter G. (1992) J. Mol. Biol. 226:889–896.[CrossRef][ISI][Medline]

Holler P.D. and Kranz D.M. (2003) Immunity 18:255–264.[CrossRef][ISI][Medline]

Holler P.D., Holman P.O., Shusta E.V., O'Herrin S., Wittrup K.D., Kranz D.M. (2000) Proc. Natl. Acad. Sci. USA 97:5387–5392.[Abstract/Free Full Text]

Holler P.D., Chlewicki L.K., Kranz D.M. (2003) Nat. Immunol. 4:55–62.[CrossRef][Medline]

Hoogenboom H.R. (2005) Nat. Biotechnol. 23:1105–1116.[CrossRef][ISI][Medline]

Ikeda H., Ohta N., Furukawa K., Miyazaki H., Wang L., Kuribayashi K., Old L.J., Shiku H. (1997) Proc. Natl Acad. Sci. USA 94:6375–6379.[Abstract/Free Full Text]

Kieke M.C., Shusta E.V., Boder E.T., Teyton L., Wittrup K.D., Kranz D.M. (1999) Proc. Natl Acad. Sci. USA 96:5651–5656.[Abstract/Free Full Text]

Kieke M.C., Sundberg E., Shusta E.V., Mariuzza R.A., Wittrup K.D., Kranz D.M. (2001) J. Mol. Biol. 307:1305–1315.[CrossRef][ISI][Medline]

Kupsch J.M., Tidman N.H., Kang N.V., Truman H., Hamilton S., Patel N., Newton Bishop J.A., Leigh I.M., Crowe J.S. (1999) Clin. Cancer Res. 5:925–931.[Abstract/Free Full Text]

Laugel B., et al. (2005) J. Biol. Chem. 280:1882–1892.[Abstract/Free Full Text]

Lebowitz M.S., O'Herrin S.M., Hamad A.R., Fahmy T., Marguet D., Barnes N.C., Pardoll D., Bieler J.G., Schneck J.P. (1999) Cell Immunol. 192:175–184.[CrossRef][ISI][Medline]

Li Y., et al. (2005) Nat. Biotechnol. 23:349–354.[CrossRef][Medline]

Maenaka K., et al. (1999) J. Biol. Chem. 274:28329–28334.[Abstract/Free Full Text]

Marks J.D. and Bradbury A. (2004) Methods Mol. Biol. 248:3161–76.[Medline]

Marks J.D., Hoogenboom H.R., Griffiths A.D., Winter G. (1992) J. Biol. Chem. 267:16007–16010.[Free Full Text]

Masteller E.L., Warner M.R., Ferlin W., Judkowski V., Wilson D., Glaichenhaus N., Bluestone J.A. (2003) J. Immunol. 171:5587–5595.[Abstract/Free Full Text]

Rajpal A., Beyaz N., Haber L., Cappuccilli G., Yee H., Bhatt R.R., Takeuchi T., Lerner R.A., Crea R. (2005) Proc. Natl Acad. Sci. USA 102:8466–8471.[Abstract/Free Full Text]

Rao B.M., Lauffenburger D.A., Wittrup K.D. (2005) Nat. Biotechnol. 23:191–194.[Medline]

Razai A., et al. (2005) J. Mol. Biol. 351:158–169.[CrossRef][ISI][Medline]

Roovers R.C., van der Linden E., de Bruine A.P., Arends J.W., Hoogenboom H.R. (2001) Eur. J. Cancer 37:542–549.[Medline]

Rudolph M.G. and Wilson I.A. (2002) Curr. Opin. Immunol. 14:52–65.[CrossRef][ISI][Medline]

Schlueter C.J., Manning T.C., Schodin B.A., Kranz D.M. (1996) J. Immunol. 157:4478–4485.[Abstract]

Schweickhardt R.L., Jiang X., Garone L.M., Brondyk W.H. (2003) J. Biol. Chem. 278:28961–28967.[Abstract/Free Full Text]

Scott C.A., Garcia K.C., Carbone F.R., Wilson I.A., Teyton L. (1996) J. Exp. Med 183:3.

Shusta E.V., Holler P.D., Kieke M.C., Kranz D.M., Wittrup K.D. (2000) Nat. Biotechnol. 18:754–759.[CrossRef][ISI][Medline]

Smith G.P. (1985) Science 228:1315–1317.[Abstract/Free Full Text]

Speir J.A., Garcia K.C., Brunmark A., Degano M., Peterson P.A., Teyton L., Wilson I.A. (1998) Immunity 8:3553–562.[CrossRef][ISI][Medline]

Starwalt S.E., Masteller E.L., Bluestone J.A., Kranz D.M. (2003) Protein Eng. 16:147–156.[Abstract/Free Full Text]

Tallquist M.D. and Pease L.R. (1995) J. Immunol. 155:2419–2426.[Abstract]

Trepel M., Arap W., Pasqualini R. (2002) Curr. Opin. Chem. Biol. 6:399–404.[CrossRef][ISI][Medline]

Udaka K., Tsomides T.J., Eisen H.N. (1992) Cell 69:989–998.[CrossRef][ISI][Medline]

Udaka K., Wiesmuller K., Kienle S., Jung G., Walden P. (1996) J. Immunol. 157:670–678.[Abstract]

van den Beucken T., Pieters H., Steukers M., van der Vaart M., Ladner R.C., Hoogenboom H.R., Hufton S.E. (2003) FEBS Lett. 546:288–294.[CrossRef][ISI][Medline]

Wang X.X. and Shusta E.V. (2005) J. Immunol. Methods 304:30–42.[Medline]

Weber K.S., Donermeyer D.L., Allen P.M., Kranz D.M. (2005) Proc. Natl Acad. Sci. USA 102:19033–19038.[Abstract/Free Full Text]

Yeung Y.A. and Wittrup K.D. (2002) Biotechnol. Prog. 18:3212–20.[Medline]

 

Received October 30, 2005; revised February 8, 2006; accepted February 9, 2006.

Edited by Hennie Hoogenboom


Figures

mcith_gzl008f1.gif Figure 1 Selective recovery of high affinity yeast after density centrifugation. (A) Percent of yeast recovered from the APC interface layer following centrifugation through Ficoll-Paque. Yeast cells expressing either the high affinity 2C-m80 scTCR or the non-binding C18-1 scTCR were incubated with T2-Kb cells that had been either loaded with exogenous SIYR peptide or used without exogenous peptide loading. The number of yeast present before centrifugation, and after the first and second centrifugation, were determined by plating aliquots of cells at each stage. The percent recovery following the first centrifugation (white bars) and second centrifugation (black bars) was taken as: (# of yeast cells present at interface layer after centrifugation)/(# of yeast cells at interface layer before centrifugation) x 100. (B) Isolation of high affinity 2C-m80 yeast from excess non-binding yeast. A mixture of yeast comprising a 1 to 1000 ratio of 2C-m80 to C18-1 was incubated with SIYR-loaded T2-Kb cells and subjected to two sequential rounds of density differential centrifugation. Aliquots of cells collected before centrifugation and after the second centrifugation were cultured and stained with antibodies specific for the Vß region of either C18-1 (1B3.3, white bars) or 2C-m80 (F23.2, black bars) and analyzed using flow cytometry. Note: The sums of the positive percentages do not equal 100 due to the negative population that is invariably observed in flow cytometric analysis in the yeast display system. See Figure 2.

(Click image to enlarge)

mcith_gzl008f2.gif Figure 2 Enrichment of rare high affinity yeast. Yeast expressing the high affinity 2C-m6 were mixed with non-binding yeast at a ratio of (A) 1 to 10 000 or (B) 1 to 100 000. The mixtures were incubated with QL9-loaded T2-Ld cells and subjected to two sequential rounds of density centrifugation. Aliquots of cells collected before centrifugation, after the first centrifugation and after the second centrifugation were cultured and stained with antibodies specific for the Vß region of either non-binding scTCR C18-1 (1B3.3, grey) or high affinity scTCR 2C-m6 (F23.2, black outline). Note: The negative population in each histogram is invariably observed in the yeast display system and serves as an internal control.

(Click image to enlarge)

mcith_gzl008f3.gif Figure 3 Selection analyzed across a range of affinities. (A) T2-Ld cells were loaded exogenously with the peptide variants R5, H5, and M5. Peptide-loaded APCs were then incubated with a yeast cell mixture comprising a 1 to 1000 ratio of 2C-m6 to C18-1. Cells were then subjected to two sequential rounds of density centrifugation. Aliquots of cells before centrifugation and after the second centrifugation were cultured and stained with antibodies specific for the Vß region of either C18-1 (KT-8C1) or 2C-m80 (F23.2) and analyzed using flow cytometry. The ratios of yeast cells staining positive for F23.2 (binder) to yeast cells staining positive for KT-8C1 (nonbinder) are shown before and after centrifugation (KD value for each 2C-m6/pep/Ld interaction is shown above bars). (B) R5-loaded APCs. Flow cytometry histogram overlays showing C18-1-positive yeast (grey) and 2C-m6-positive yeast (black outline) before and after centrifugation and following a second complete cycle of growth, induction, incubation with R5-loaded T2-Ld and centrifugation.

(Click image to enlarge)

mcith_gzl008f4.gif Figure 4 Analysis of unique TCR mutants by density differential centrifugation. (A) QL9-loaded T2-Ld cells were incubated with yeast expressing mutant scTCRs 2C-T7, 2C-m6 or 2C-mQ-1 through 2C-mQ-5 and (B) Y5-loaded T2-Ld cells were incubated with yeast expressing mutant scTCRs 2C-mY-1 through 2C-mY-5. Cell mixtures were then subjected to one round of density differential centrifugation. The number of yeast present before and after centrifugation was determined by plating aliquots of cells at each stage. The percentages of yeast recovered from the interface, calculated as described for Figure 1, are shown as an average of three trials. The recovery percentages of 2C-m6 (high affinity) and 2C-T7 (wild-type affinity) when incubated with QL9-loaded T2-Ld cells and subjected to density differential centrifugation from (A) are shown in (B) for comparison.

(Click image to enlarge)

mcith_gzl008f5.gif Figure 5 Analysis of unique TCR mutants using flow cytometry. (A) Histograms of yeast cells expressing mutant scTCRs 2C-T7, 2C-m6, and 2C-mQ-1 through 2C-mQ-5 stained with either anti-Vß8.2 (F23.2) (left column) or QL9/Ld-Ig pMHC dimer (right column) and PE-conjugated goat F(ab')2 anti-mouse Ig. (B) Histograms of yeast expressing 2C-T7, 2C-m6, and 2C-mY-1 through 2C-mY-5 stained with either anti-Vß8.2 (F23.2) (left column) or Y5/Ld-Ig pMHC dimer (right column) and PE-conjugated goat F(ab')2 anti-mouse Ig. Mean fluorescence units for each histogram are indicated.

(Click image to enlarge)

mcith_gzl008f6.gif Figure 6 Sequences of TCR mutants. The amino acid sequences of the CDR3{alpha} of mutants 2C-mQ-1 through 2C-mQ-5 and 2C-mY-1 through 2C-mY-5 are shown. The CDR3{alpha} amino acid sequences of previously isolated 2C mutants 2C-m6 and 2C-m12, as well as wild-type 2C CDR3{alpha}, are shown for comparison. 2C-m12* was selected previously using QL9 peptide.

(Click image to enlarge)

mcith_gzl008f7.gif Figure 7 Enrichment of a high affinity MHC class II-restricted scTCR mutant. Yeast expressing the high affinity mutant scTCR 3.L2-M15 were mixed at a 1 to 1000 ratio of (A and B) non-binding mWT1-B7 yeast or (C) non-binding 2C-T7 yeast. The yeast mixtures were incubated with Hb peptide-loaded CH27 APCs. Cells were then subjected to one round of density differential centrifugation. Aliquots of cells that were collected before and after centrifugation were cultured and stained with antibodies specific for the Vß region of either the non-binding scTCRs mWT1-B7 (RR3-15-PE) or 2C-T7 (F23.2) or the high affinity scTCR 3.L2-M15 (1B3.3-PE in A and B, KT-8C1 in C). Stained yeast cells were analyzed using flow cytometry. (A) Histograms of yeast before and after centrifugation stained with antibody specific for Vß region of the high affinity 3.L2-M15 (1B3.3-PE, top row) or mWT1-B7 (RR3-15-PE, bottom row). Percent of cells staining positive in each histogram are shown in the upper right-hand corner. (B and C) Percent of yeast before and after centrifugation staining positive for non-binding scTCR (white bars) or high affinity scTCR (black bars) (average of three trials in B or two trials in C).

(Click image to enlarge)

 


http://www.biology-online.org/articles/development-novel-strategy-engineering-high-affinity.html