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754 NATURE BIOTECHNOLOGY VOL 18 JULY 2000 http://biotech.nature.com

RESEARCH ARTICLES

The T-cell receptor (TCR) is responsible for specific recognition ofpeptide-major histocompatibility complex (pMHC) antigens incell-mediated immunity. There is significant clinical interest in T-cell-mediated elimination of viruses and cancer cells; additionally,autoimmune diseases such as multiple sclerosis, rheumatoid arthri-tis, and insulin-dependent diabetes mellitus appear to be mediatedat least in part by aberrant T-cell recognition of self pMHC com-plexes. Although the diversity of TCRs is similar to that of antibod-ies, soluble TCRs have not yet been exploited in immunotherapeuti-cal strategies with the potential to provide highly antigen-specificimmunosuppression.

Such applications have not been realized for various reasons.Affinities of TCR/pMHC interactions are quite low (micromolarKd), necessitating infeasibly high concentrations of soluble TCR fortargeting. Many TCRs have low solubility and a high propensity toaggregate1,2, which is improved somewhat by fusion of the TCR vari-able regions to thioredoxin3 or antibody constant regions4–6.Stability of soluble TCRs are low7 compared to antibodies8–10, andrecombinant production yields are low and variable1,11,12. Animproved T-cell receptor scaffold should have the properties of highstability, solubility, and expression level in order to enable furtherengineering of improved antigen-binding affinity.

Antibodies have been improved in these properties through var-ious directed-evolution strategies, but this approach has untilrecently not been successful with TCRs. Yeast surface display hasbeen used to increase the affinity of single-chain antibodies13,14 andto create a single-chain T-cell receptor library15. Antibodies havebeen evolved with relative ease because of their inherent solubilityand stability, whereas the single-chain T-cell receptor from the cyto-toxic T-lymphocyte clone 2C was displayed only after mutation andselection for active mutant scTCRs on the surface of yeast15. These

mutant scTCRs serendipitously exhibited somewhat improved sta-bility and expression characteristics7.

Here we have applied temperature-based screening schemes toimprove scTCR resistance to thermal denaturation and processing atelevated induction temperatures. Combining a total of six selectedmutations yields an extremely stable and soluble mutant scTCR thatbinds specific pMHC ligands (QL9/Ld and SIYR/Kb) and the bacter-ial superantigen SEC3. We also demonstrate the formation of TCRyeast–antigen-presenting cell (APC) conjugates, suggesting the pos-sibility of cell–cell selection strategies using mutated TCR librariesto isolate proteins with increased affinity or altered specificity fortheir pMHC ligands.

ResultsLibrary construction and screening strategy. Based on previouslyobserved correlations among surface display level, soluble secre-tion, and thermal stability7, we hypothesized that yeast surface dis-play efficiency at elevated temperatures could be used as a proxyscreening variable to identify a more robust single-chain scTCRframework. As a starting point, an equimolar mixture of the wild-type 2C scTCR and previously identified mutants mTCR7(αL43P), mTCR15 (βG17E), and mTCR7/15 (αL43P andβG17E)15 was mutagenized by error-prone polymerase chain reac-tion (PCR), and a library of 3 × 105 mutants was displayed on yeastas Aga2p fusions13.

Screening conditions for flow-cytometric sorting were deter-mined using the stability and processing properties of the previ-ously most stable active scTCR mutant, mTCR7/15, as the base-line. Folded scTCR was detected with a conformationally specificantibody, 1B2, that binds only scTCR with properly folded Vαand Vβ domains. 1B2 and the natural pMHC ligand have almost

Directed evolution of a stable scaffold forT-cell receptor engineering

Eric V. Shusta1, Phillp D. Holler2, Michele C. Kieke2, David M. Kranz2*, and K.Dane Wittrup1*

1Department of Chemical Engineering and 2Department of Biochemistry, University of Illinois, Urbana, IL 61801. *Corresponding authors ([email protected]) and ([email protected]).

Received 4 November 1999; accepted 2 May 2000

Here we have constructed a single-chain T-cell receptor (scTCR) scaffold with high stability and solu-ble expression efficiency by directed evolution and yeast surface display. We evolved scTCRs in parallelfor either enhanced resistance to thermal denaturation at 46°C, or improved intracellular processing at37°C, with essentially equivalent results. This indicates that the efficiency of the consecutive kineticprocesses of membrane translocation, protein folding, quality control, and vesicular transport can bewell predicted by the single thermodynamic parameter of thermal stability. Selected mutations wererecombined to create an scTCR scaffold that was stable for over an hour at 65°C, had solubility of over 4mg ml-1, and shake-flask expression levels of 7.5 mg l-1, while retaining specific ligand binding to pep-tide–major histocompatibility complexes (pMHCs) and bacterial superantigen. These properties are com-parable to those for stable single-chain antibodies, but are markedly improved over existing scTCR con-structs. Availability of this scaffold allows engineering of high-affinity soluble scTCRs as antigen-specif-ic antagonists of cell-mediated immunity. Moreover, yeast displaying the scTCR formed specific conju-gates with antigen-presenting cells (APCs), which could allow development of novel cell-to-cell selectionstrategies for evolving scTCRs with improved binding to various pMHC ligands in situ.

Keywords: Yeast display, T-cell receptor, stability maturation, directed evolution

© 2000 Nature America Inc. • http://biotech.nature.com©

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identical epitopes on the TCR16, but 1B2 has a 100-fold higheraffinity, thus serving as a convenient probe for properly foldedscTCR.

The yeast-displayed scTCR library was split and screened in parallelunder in vitro and in vivo stringency conditions. In the in vitro strate-gy, scTCR library surface expression was induced at optimal expressiontemperature (20°C) for 24 h, then shifted to 46°C for 30 min, followingwhich the mTCR7/15 and unscreened library exhibit greatly dimin-ished 1B2 binding (compare Fig. 1B with Fig. 1A). In the in vivo strate-gy, the scTCR library was screened for mutants that were more effi-ciently processed through the yeast secretory apparatus during 24 h ofinduction at an elevated temperature (37°C vs. 20°C used in the “invitro” screen) at which the mTCR7/15 and unscreened library exhibitnegligible surface display (Fig. 1C). The absence of active scTCR on theyeast surface following induction at 37°C is attributable to defectiveintracellular processing during biosynthesis, since folded mTCR7/15 isstable indefinitely on the cell surface at 37°C (ref. 7).

After three consecutive sorting rounds, populations of improvedmutants were identified by both methods (Fig. 1E, I). Mutants iso-lated from the in vitro and in vivo conditions exhibited cross-com-plementarity, as shown in Figure 1F and H. In other words, a statisti-

cally significant sampling of mutant scTCRs with enhanced thermalstability at 46°C were processed and displayed efficiently by yeastinduced at 37°C, and vice versa. This surprising finding indicatesthat screening following induction at 37°C (the “in vivo” method) issufficiently rigorous to identify mutant proteins stable at tempera-tures at least 9°C higher. Furthermore, both enriched mutant popu-lations had an order of magnitude higher level of surface-displayedscTCR upon 20°C induction, compared to the mTCR7/15 (Fig.1D,G). Thus, a screen for improved folding and display at 20°Cwould in principle identify folded proteins that are more thermody-namically stable at a markedly higher temperature, 46°C.

Five clones were sequenced from each enriched population, andfour unique mutants were identified. Each mutant contained thetwo mutations present in mTCR7/15 (αL43P in Vα framework 2and βG17E in Vβ framework 1). Mutant 37.1, enriched by the invivo strategy, contained four mutations in the variable regions ofthe scTCR: βH47Y in Vβ framework 2, βL111V in Vβ framework 4,αY117S in Vα framework 4, and αS67C in Vα HV4. Mutant T4,which was selected in vitro, contained two mutations in Vβ, βA3Gin framework 1 and βL81S in framework 3, in addition to twomutations in the linker (D15E and A3V). Mutant T7 was isolated

Figure 1. Screening and analysis of yeast-displayed scTCR librariesfor enhanced stability or expression at elevated temperatures.Histograms depict immunofluorescent labeling with the 1B2 anti-TCR monoclonal antibody. Induction at 20°C (A,D,G) or stringentlibrary sort conditions of in vitro denaturation (46°C incubation for 31min; B,E,H), or elevated temperature induction conditions (37°Cinduction; C,F,I) are shown. The baseline mTCR7/15 (A–C), the entirepool of mutants isolated by screening for higher stability at 46°C(D–F), all mutants isolated by screening for higher expression at 37°C(G–I), or a negative control yeast (J) are depicted.

Figure 2. Stability and expression properties of selected scTCRmutants. (A) Stability of mutant scTCRs on the surface of yeast.The percentage of 1B2-active scTCR on the surface of yeast after30 min of thermal denaturation at 50°C (dark gray bars) and 55° C(light gray bars) is shown. Duplicate isolates were induced at 20° Cand analyzed in triplicate. (B) Correlation between solubleproduction levels of active scTCR from a low-copy numberplasmid and levels of surface display as measured by flowcytometry. Production levels were determined by 1B2-bindingactivity in an ELISA format. Triplicate cultures from independenttransformants were analyzed for each scTCR clone. Open symbolsreflect data from previously isolated mutants7, and closed symbolsrepresent the mutants described in this communication. (C)Thermal denaturation of soluble scTCR. Yeast supernatantsamples containing scTCR were subjected to the indicatedtemperatures for 1 h. Triplicate samples were analyzed by 1B2-ELISA: TRX-2CTCR (�), mTCR 7/15 (�), LWHI (�). The fractionswere normalized independently to unity by the highest intensityELISA signal having no activity loss. Comparisons are made toTRX-TCR rather than wild-type scTCR because wild-type scTCRwas not detected in yeast supernatants. TRX-TCR and low-temperature mTCR7/15 data are from ref. 7, for comparison toLWHI scTCR.

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independently from both the “in vivo” and “in vitro” selections andcontained four mutations in the variable regions of the scTCR:βG42E in Vβ framework 2, βL81S in Vβ framework 3, αW82R inVα framework 3, and αI118N in Vα framework 4. Mutant T10,which was selected in vitro, had a Vα mutation in CDR3, αL104P.This mutation, located at the interface between the variabledomains, was identified previously but has been shown to be defec-tive in pMHC binding15.

Creation of a stable, well-expressed scTCR scaffold. Severalmutations distant from CDR loops were combined by site-direct-ed mutagenesis in a framework (termed the “L” mutant) consist-ing of mTCRl7/15 and βL81S (observed in two distinct isolatedmutants). The fraction of 1B2-active yeast surface-expressedscTCR remaining after 30 min incubations was determined foreach mutant at 50 and 55°C. Mutations βH47Y (H), αW82R (W),and αI118N (I) were found to contribute additive stabilityimprovements (Fig. 2A). The most stable mutant, LWHI, retainedover 40% of its activity after denaturation on the cell surface for30 min at 55°C.

Soluble mutant scTCRs were expressed and secreted from low-copy yeast plasmids by fusion to a synthetic leader sequence17, and

yeast culture supernatant scTCR levels analyzed by quantitativeELISA. Each of the mutants was expressed at higher levels thanmTCR7/15, and the LWHI mutant produced 20-fold more secretedscTCR (2,000 mg L-1; Fig. 2B). Thermal stability (Fig. 2A) and solu-ble expression (Fig. 2B) correlated over a wide range of expressionlevels. These results expand by an order of magnitude the correla-tion noted previously7.

In order to increase expression levels of LWHI scTCR further, thegene was subcloned into the δ-vector, an integrating vector thatallows for optimal expression level tuning18,19. Secreted LWHI His6-tagged scTCR was purified by immobilized metal-ion affinity chro-matography (IMAC), with a purified, active yield of 7.5 mg L-1 inshake flask culture, a 3.5-fold increase over low-copy plasmidexpression. When the scTCR was produced at these high levels, somepropeptide was incompletely processed by the trans-Golgi Kex2p

Figure 3. Peptide–MHC binding by soluble mutant scTCR. (A) Bindingto cell surface peptide–Ld complexes was monitored in a quantitativeinhibition assay. T2-Ld cells loaded with specific (QL9, QLSPFPFDL)and irrelevant (MCMV, YPHFMPTNL) peptides were incubated in thepresence of 125I-labeled anti-Ld (30-5-7) Fab fragments and variousconcentrations of either soluble LWI scTCR or unlabeled Fabfragments (4.3 µM). Bound and unbound 125I-Fabs were separated bycentrifugation through oil, cell pellets were monitored by γ counting,and the extent of inhibition was calculated. Assays were performed intriplicate. (B) Binding of soluble peptide–MHC complexes to LWHI wasmonitored by ELISA. Purified soluble LWHI scTCR was captured onELISA plates with an anti-His antibody. Binding of pMHC complexeswas examined with soluble multivalent peptide–MHC complexes. SA-HRP, streptavidin–horseradish peroxidase negative control. SIYR/Kb,pMHC biotinylated and complexed with SA-QL9/Ld-IgG followed byanti-mouse IgG-HRP. MCMV/Ld and QL9/Ld, pMHC-IgG fusionsfollowed by anti-mouse IgG-HRP. After the addition of substrate(tetramethylbenzidine and hydrogen peroxide; Kirkegaard and Perry,Gaithersburg, MD), optical density was monitored at 450 nm (A450).

Figure 4. Formation of specific scTCR-yeast–APC complexes. (A)Phase-contrast image of yeast–APC complex. Yeast encircle thelarger APC cell. The sample image is a conjugate between QL2scTCR-displaying yeast and T2-Ld APCs loaded with the cognate QL9peptide. (B) Fluorescence image of yeast–APC complex. Thefluorescently labeled APC is the only cell observed. (C) Analysis ofyeast–APC complexes. The number of yeast per T2-Ld APC loadedwith cognate QL9 (dark gray bars), or irrelevant MCMV (light graybars) peptide are shown for duplicate independent counts of 75 APCs.

Figure 5. Locations of mutations in the LWHI scTCR, shown on thewild-type 2C TCR structure41. All six mutations are in framework(non-CDR) positions. (Note that the Cα and Cβ portions of the TCR,absent in the scTCR construct, are not shown).

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protease. This heterogeneity in scTCR product was effectively elimi-nated by Kex2p overexpression20 (data not shown).

Thermal denaturation of soluble LWHI scTCR (Fig. 2C) illus-trates the striking improvement in stability compared to mTCR7/15and to a soluble thioredoxin–scTCR (TRX-TCR) fusion produced inEscherichia coli3. It should be noted that the experiments representedin Figure 2C reflect the kinetics of denaturation, rather than a directassessment of Tm for reversible denaturation. The LWHI scTCRretained substantial 1B2 activity even after incubation for 1 h at tem-peratures in excess of 65°C. The high stability of this scaffold willprove useful for in vivo applications (i.e., at 37°C) and for furtherbiochemical studies of the TCR.

The yeast production levels of LWHI upon expression level tun-ing are similar in magnitude to those for single-chain antibodies19.As observed with scFv expression19, overexpression of protein-fold-ing chaperones can increase scTCR secretion. Overexpression of theendoplasmic reticulum (ER)-resident chaperone BiP (shown previ-ously to associate with TCRα21), increases expression levels of scTCR(2.0 ± 0.02-fold, data not shown). Another ER-resident chaperone,calnexin, has been shown to associate with each subunit of an αβTCR22, and the production of LWHI scTCR was enhanced by yeastcalnexin overexpression (1.9 ± 0.12-fold, data not shown), possiblydue to the N-linked glycosylation sites present in the Vα (one site)and Vβ (two sites) domains. Although overexpression of DsbA inthe E. coli periplasm increased the yield of a different scTCR11, over-expression of yeast protein disulfide isomerase (PDI) or the yeastPDI relative Eug1p did not affect the LWHI scTCR secretion (datanot shown). Interestingly, overexpression of BiP and PDI togetherdid not yield additive increases as observed previously for single-chain antibody fragments19. It is possible that increased solubility ofthe scTCR limited the necessity for the beneficial effects of BiP andPDI.

Solubly overexpressed LWHI scTCR was found to undergo N-linked glycosylation to a similar extent as the progenitor scTCRmutants described previously7 (data not shown). In order to deter-mine the significance of these N-linked sites for scTCR expressionand stability, each of the three potential N-linked glycosylation sites(N-X-S/T) were changed to Q-X-S/T by site-directed mutagenesis inthe LWI scTCR. Surface expression levels for the nonglycosylatedscTCR were identical to those for the glycosylated form (data notshown). The strong correlation between surface display, solubleexpression, and thermal stability (Fig. 2 and ref. 7) indicate that N-linked glycosylation does not contribute significantly to expressionor stability of these mutant scTCRs.

LWHI scaffold ligand-binding characteristics. The biologicalligand-binding properties of LWHI and LWI scTCR scaffolds weredetermined. The proteins were concentrated to 4 mg ml-1 withoutobservable precipitation, and assayed for ligand binding. SolubleLWI scTCR was shown to compete with a radioiodinated Fab frag-ment for binding to the Ld pMHC on APCs (Fig. 3A). pMHC bind-ing was scTCR-dose-dependent, and required the presence of thecognate QL9 peptide antigen as opposed to the irrelevant MCMVantigen. In an enzyme-linked immunosorbent assay (ELISA)-basedmultivalent pMHC23 binding assay, LWHI scTCR bound specificallyto soluble pMHC QL9/Ld–IgG dimers24,25 but not with an irrelevantpeptide, MCMV/Ld–IgG dimer26 (Fig. 3B). LWHI scTCR bindingwas also detected with another tetrameric pMHC (SIYR/Kb; ref. 27)(Fig. 3B). Finally, binding was detected with the Vβ8-specific super-antigen SEC3 (ref. 28) and SEC3/1A4, a mutant version of the SEC3superantigen that binds the 2C scTCR with higher affinity thanwild-type SEC3 (R.A. Mariuzza, unpublished data). Therefore, theLWHI scTCR retains ligand-binding activity and specificity for allligands tested.

Specific cell–cell complex formation. In cases where solubleforms of the antigen are not readily available, it would be desirable to

screen yeast-displayed polypeptide libraries against cell surface anti-gens in situ. Yeast displaying the LWHI scTCR were capable of bind-ing specifically to QL9/Ld-displaying T2-Ld APCs. LWHI scTCRyeast were incubated with fluorescently labeled APCs loaded withcognate (QL9) or irrelevant (MCMV) peptides, and the mixtureswere examined for conjugates by microscopy (Fig. 4A–C). scTCRyeast surrounded the APCs in a fashion reminiscent of the rosettesformed in classic sheep red blood cell (SRBC) immunoassays.Cell–cell conjugates are observed between QL9-loaded T2-Ld andyeast expressing either LWHI scTCR or a related scTCR, QL2 (amutant of the T7 scTCR that has subsequently been selected forhigher affinity for QL9/Ld; ref. 29.) In contrast, these same scTCRyeast did not form conjugates with APCs loaded with the irrelevantMCMV peptide (Fig. 4C). Formation of cell–cell contacts was sensi-tive to scTCR surface levels, as mTCR7/15 yeast did not form specif-ic complexes (data not shown). Of particular importance for devel-oping screening strategies, cell–cell complex formation was also sen-sitive to scTCR binding affinity, as evidenced by comparison of QL2to LWHI scTCR (Fig. 4C). Yeast displaying an irrelevant anti-lysozyme single-chain antibody do not appreciably form conjugateswith APCs (Fig. 4C). Not all specific APCs were coated with scTCRyeast, perhaps as a consequence of shear forces encountered duringwashing or the heterogeneity in levels of QL9/Ld among the APCs.Nevertheless, control mixtures do not contain any detectablerosettes like that depicted in Figure 4A.

DiscussionA scTCR scaffold with greatly improved stability and secretion char-acteristics has been created using a directed-evolution strategy withyeast surface display. The LWHI scTCR provides a source of solublescTCR for applications including structure/function studies, APClabeling, or antagonism of T-cell activation. Availability of thisrobust scTCR framework will enable engineering and study of T-cellantigen recognition in a manner directly analogous to the prolifera-tion of creative applications of single-chain antibodies. For example,the scTCR scaffold described here has subsequently been matured tonanomolar pMHC binding affinity29.

It is to be expected that the characteristics of other TCR frame-works could be improved by a process similar to that described here.This strategy would not be limited to TCRs for which clonotypicantibodies such as 1B2 are available, since the presence of increasedcell surface scTCR expression can be detected by means of the C-ter-minal c-myc epitope tag13. As we have done previously for 2C,mutants in CDRs could be discarded to reduce the likelihood of lossof functional peptide–MHC binding15. Another approach would beto utilize tetrameric soluble pMHC–streptavidin complexes to detectfunctional scTCR on the yeast surface. The yeast–APC conjugatestrategy (Fig. 4) may be useful to directly select scTCR yeast librariesfor improved binding to pMHC ligands on APCs. Cell–cell complexscreening will also be generally useful for evolving proteins whenthere is difficulty in producing the cognate protein partner as a solu-ble, labeled ligand.

The resistance of LWHI scTCR to thermal denaturation is com-parable to the stability characteristics determined for scFvs anddisulfide-stabilized Fvs8–10. Importantly, the overall success of thescreening strategy was not limited by viability constraints of yeast atthe elevated temperature of 65°C. Recombination of a small numberof mutations identified at screening temperatures of 37°C and 46°Cenabled assembly of the LWHI scTCR with stability well above thosetemperatures. The observation of enhanced surface display of thesemutants at 20°C (Fig. 1D,G) raises the further intriguing possibilityof screening for enhanced display at 20°C in order to identifymutants with stability at much higher temperatures. One could nothave anticipated a priori that screening at 20°C would identifymutants such as the LWHI scTCR that are more stable at 65°C; how-


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ever, the data in Figure 1D,G indicate the feasibility of such anapproach. The rigor of the secretory quality control apparatus isresponsible for this seeming violation (i.e., screen at 20°C for stabil-ity at 65°C) of the axiom of directed evolution that you “get whatyou select for”.

The structural locations of mutations in the LWHI scTCR pro-vide insights to the possible mechanisms that underlie improved sta-bility and expression (Fig. 5). Two of the mutations in LWHI,αI118N and αW82R, are located at what would be the interfacebetween the Vα and Cα domains in the intact TCR. Similar changesin the analogous interface of an anti-fluorescein single-chain anti-body led to a decrease in thermally induced aggregation30. TheLWHI scTCR is soluble at concentrations of at least 4 mg ml-1, whichis 40 times greater than the solubility of E. coli-produced wild-typescTCR for this same 2C clone3. The βL81S and βG17E mutations arelocated in a portion of the Vβ domain shown to interact with the Cβdomain31,32, absent in the scTCR format. Thus, compensation for theexposure of interfaces normally buried in the full TCR complexappears to be a common aspect of the observed stabilizing muta-tions.

We had previously observed that well-displayed mutant scTCRsare more stable7. In the present work, this connection has beenstrengthened by the observation that selection for increased stabilityin turn results in improved display (Fig. 1D,F). These findings sug-gest that in the case of the scTCR, the efficiency of the complexkinetic process of in vivo protein folding can be predicted with a sin-gle thermodynamic parameter, the in vitro stability of the protein(and vice versa). The robustness of this correlation gives confidencethat this approach may be of general utility for obtaining stable scaf-folds in other areas of protein engineering.

Experimental protocolLibrary construction. Wild-type, mTCR7, mTCR15, and mTCR7/15 (ref.15) 2C (ref. 33) scTCRs were subcloned into the yeast surface display vectorpCT-30213 as NheI-XhoI fragments, then used as templates in an error-pronepolymerase chain reaction (PCR). Pooled PCR product was ligated intopCT-302 as a NheI-XhoI fragment, and transformed into yeast EBY100 (refs.13, 34).

Flow cytometry and library sorting. The transformed yeast library wasgrown in synthetic dextrose casamino acids (SD-CAA) to OD600 = 8.0, resus-pended in synthetic galactose casamino acids (SG-CAA) to OD600 = 1.0 for 24h at 20°C (“in vitro” sort) or 37°C (“in vivo” sort). For the “in vitro” sort,yeast were incubated in 100 µl phosphate-buffered saline–bovine serumalbumin (PBS-BSA) at 37°C for 50 min, then 46°C for 31 min, followed by4°C PBS-BSA. Library aliquots from both screening conditions were labeledwith 33 nM biotinylated 1B2 and FITC-antiHA (1:25) (12CA5-FLBoehringer Mannheim, Indianapolis, IN) (4°C, 1 h), then incubated with astreptavidin-phycoerythrin conjugate (1:100) (Pharmingen, San Diego, CA)(4°C, 30 min).

Optimal selection parameters for the “in vitro” sort were determined asdescribed35. mTCR7/15 denaturation rate was determined (0.10 min-1 at46°C), and a denaturation period of 31 min at 46°C was calculated for bestdiscrimination of threefold improvements.

Soluble production of scTCR. Mutant scTCRs L, LH, LI, LW, LWH, andLWHI were ligated into the pRS-GALT7 CEN-based, low-copy expressionplasmid as NheI-XhoI fragments for soluble production19. These plasmidswere transformed into Saccharomyces cerevisiae strain BJ5464, and grown inSD-CAA (+ 20 mg L-1 tryptophan, 48 h, 30°C). The cultures were centrifugedand resuspended in SG-CAA (+ 20 mg L-1 tryptophan, +1 mg ml-1 BSA, 72 h,20°C).

The LWHI reading frame was subcloned into pITY-wt19 as an EagI to SacIfragment (pITY-LWHI). pITY-LWHI was linearized (AhdI), transformedinto BJ5464, and screened on 200, 500, and 1000 µg ml-1 G418 (genetecin)plates18,19.

The Kar2p-overexpressing plasmid pMR-1341 (URA3) results in 10-foldincreased galactose-inducible Kar2p levels19,36. The rat PDI plasmid is pMAL-5.1 (TRP1) and contains the sequence encoding for the yeast ER retentionsignal HDEL37. The Eug1 plasmid, pCT40 (URA3) results in galactose-

inducible overexpression of Eug1p38. The calnexin overexpression plasmid,p90G (URA3), is also under the galactose promoter. Kex2p is overexpressedconstitutively under the TDH promoter using the pAB-KX22 (URA3) plas-mid20.

Purification of scTCR. Strains expressing His6-tagged LWHI scTCR weregrown in 1 L SD-CAA in Tunair flasks (Nalgene, Rochester, NY) (48 h, 30°C),then resuspended in 1 L SG-CAA (+ 1 mg ml-1 BSA + 20 mg L-1 tryptophan +20 mg L-1 uracil (pITY-LWHI), 20°C, 72 h).

Culture supernatants were concentrated to ~50 ml (stirred-cell 10 kDacutoff Amicon concentrator; Millipore, Bedford, MA), then dialyzed againstPBS, pH 8.0. After adding 10 mM imidazole, the solution was loaded onto a5 ml Superflow Ni-NTA (Qiagen, Valencia, CA) column. The column waswashed with 20 volumes 5 mM imidazole, then 3 volumes 20 mM imidazole,then eluted with 250 mM imidazole.

Supernatants and purified protein were quantified by chemiluminescentwestern blot with tetra-His antibody (Qiagen), and the scTCR was deglycosylated (endoglycosidase Hf, New England Biolabs, Beverly, MA).Protein concentration was determined by measuring absorbance at A205 andA280 and using the method of Scopes39.

Antibody and ligand-binding assays. For ELISAs, the wells on anImmunlon plate (Dynex Technologies, Chantilly, VA) were loaded withtetra-His antibody at 10 µg ml-1 overnight at 4°C, then blocked for 2 h inPBS-BT (PBS, pH 7.4 with 1 mg ml-1 BSA, 1 ml L-1 Tween 20), and washedfour times (PBS-BT). scTCR supernatants or thermally denatured scTCRsupernatants (1 h at 37–74°C) were diluted into negative control yeast super-natants, and 50 µl applied to the wells (1 h), washed four times (PBS-BT),and biotinylated 1B2 secondary antibody applied (10 µg ml-1, 30 min). Thiswas followed by 4× PBS-BT washes, then streptavidin–horseradish peroxi-dase (strep-HRP; 1:1,000, 30 min). After an additional 4× PBS-BT washes,then development with peroxidase substrate (Kirkegaard and PerryLaboratories, Gaithersburg, MD) and the optical density at 450 nm deter-mined. Equal signal intensities in the linear range were used for all compar-isons.

For ligand-binding assay, purified LWHI scTCR was captured withtetra-His antibody followed by SIYR/Kb (gift of Steve Jameson and MarkDaniels, University of Minnesota) tetramer conjugated to strep-HRP (~1µg ml-1, 1 h) or biotinylated SEC3 superantigen (1 h, 100 mg ml-1; ToxinTechnology, Sarasota, FL) then strep-HRP (1:1,000, 30 min). For QL9/Ld

binding, the purified scTCR was applied directly to the well at 20 µg ml-1

overnight at 4°C, followed by Ld-IgG dimer (gift of Sean O’Herrin and JeffBluestone, University of Chicago) loaded with either specific QL9 peptideor irrelevant MCMV peptides (3 µg ml-1, 1 h), followed by anti-mouseIgG-HRP (1:1,000, 30 min). Thioredoxin-scTCR was used as a positivecontrol throughout3.

Binding of soluble scTCR to cell surface peptide–Ld complexes was deter-mined by quantitative inhibition assay. Cells with peptide-upregulated (3 hat 37°C) Ld were incubated in the presence of 125I-labeled anti-Ld (30-5-7) Fabfragments in the presence of various concentrations of soluble LWI scTCR orunlabeled Fab fragments (4.3 µM). Bound and unbound 125I-Fabs were sepa-rated by centrifugation through dibutyl phthalate–olive oil, cell pellets weremonitored by γcounting.

Yeast–APC conjugate formation. LWHI scTCR, QL2 scTCR (a mutant ofT7 scTCR), or anti-lysozyme (D1.3) scFv were displayed on the surface ofyeast as described above. Before conjugate formation, the T2-Ld APCs (ref.40) were labeled overnight with 5,7-dimethyl BODIPY-hexadecanoic acid at5 µM, then loaded with QL9 or MCMV at 1 × 10-7 µM peptide for 30 min at37°C. Approximately 1 × 105 peptide-loaded T2-Ld cells were combined with1.5 × 106 yeast cells in a total volume of 120 µl. The cells were pellettedtogether for 30 min at 37°C. A total of 75 APCs, in duplicate, were identifiedfirst by fluorescence, and then bound yeast identified and counted by bright-field microscopy.

AcknowledgmentsThanks to Roy Mariuzza and Peter Andersen for the superantigen mutantSEC3/1A4, Sean O’Herrin and Jeff Bluestone for QL9/Ld dimers, and SteveJameson and Mark Daniels for SIYR/Kb tetramers. This work was funded byNIH GM55757 and NSF BES 95-31407. We would like to acknowledge Dr.Michael Glaser and Jonah Chan for their assistance in performing the fluores-cence microscopy, and Gary Durack of the University of Illinois Flow CytometryFacility for his assistance. Also, special thanks to Dr. Marti Ware for her rosetteassay expertise.


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