Antibody engineering to facilitate targeting of antigens has
been used extensively in the development of antibodies as potential
therapeutic agents. The most frequent uses of in vitro
seek to optimize antibody binding affinities, typically through
a platform such as phage display (Hawkins et al
; Markset al
). Alternatively, phage display has been used to
identify lead antibodies against targets using either purified
antigens (Edwards et al
) or biopanning procedures employing
whole cells or tissues (Giordano et al
; Trepel et al
). Like antibodies, TCRs recognize a diverse array of antigens.
However, unlike the antigens recognized by antibodies, the antigens
recognized by T cells represent a greater challenge from the
biochemical perspective. These antigens consist of a short peptide
that is non-covalently associated with a cell surface heterodimer
encoded by the MHC for class II or the MHC and ß2-microglobulin
for class I. Ternary complexes of peptides and MHC products
exhibit widely diverse stabilities and thus the ability to express
and purify these ligands varies widely. Various laboratories,
including the NIH tetramer facility and commercial vendors,
provide purified complexes of various well-characterized pMHC
ligands. However, the variety of MHC alleles (2101 in total
among human class I and II), and the large number of possible
peptides makes it difficult to use purified ligands more generally.
In order to develop a strategy for engineering TCRs with high-affinity
for a diverse array of antigens, it would be advantageous to
avoid the need for purification of the ternary antigen complexes.
We show here that it is possible to combine the yeast display
system with a rapid density centrifugation method to select
high-affinity TCRs. The strategy required only the use of synthetic
peptides that could be loaded exogenously onto an APC that expresses
on 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., 2000; Holler et al.,2003; Chlewicki et al., 2005). 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,1998), 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, 2002).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., 2005; Li et al.,2005). It is reasonable to predict that the same type of biopanningthat has been performed with phage-displayed peptides or antibodies(Kupsch et al., 1999; Giordano et al., 2001; Roovers et al.,2001; Trepel et al., 2002) 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, 2005) 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 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 motifwith a key glycine at residue 101. In contrast the additionof a hydroxyl group in the Y5 peptide variant appears to allowthe isolation of CDR3 mutants with more diversity in their CDR3.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, 2002), and, despite significant advances in classII MHC multimer technology, there are still relatively few classII MHC multimers available (Cameron et al., 2002). 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., 1996). One fundamental difference between class I andclass II MHC involves the chaperones that facilitate foldingand peptide loading intracellularly (Cresswell and Lanzavecchia,2001). 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., 1999; Masteller et al., 2003).
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, 2000; Feldhaus et al., 2003; van den Beucken,2003; Hoogenboom, 2005). In fact, an alternative biopanningstrategy using an antibody–hapten model system was publishedduring the preparation of this manuscript (Wang and Shusta,2005). 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., 2003; Schweickhardt et al., 2003). 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.,2003). 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.