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Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled …

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

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).

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