Panning of phage particles
Phages displaying TCRs with low affinity are washed off
FIGURE 7.3 (see color insert following page 178) TCR engineering using (A) phage display and (B) yeast display, adapted from (Boder and Wittrup 1997). A phage display library of TCRs is usually screened by panning phage particles on a surface/matrix coated with purified pMHC complexes, while a yeast display library can be rapidly screened by FACS using fluorophore-labeled (such as phycoerythrin, PE) pMHC complexes.
The highest affinity TCRs yet reported (with picomolar affinities for their specific pMHC ligand) were achieved using phage display and directed evolution (Li et al. 2005) (Figure 7.3A). By using random mutagenesis, human TCR mutants with pMHC affinities up to 26 pM were isolated, representing more than a million-fold improvement over the wild-type TCR. In addition, the half-life of pMHC binding was also improved by ~8000-fold to ~1000 minutes at 25°C. More significantly, these high affinity TCRs showed a high degree of antigen specificity and no cross-reactivity with endogenous pMHC complexes, and they enabled direct visualization of specific pMHC complexes on tumor cells for the first time (Purbhoo et al. 2006). When transfected into human T cells, the mutant TCR with highest pMHC affinity completely lost its antigen specificity, while those expressing TCRs with lower pMHC affinity (with KD values of 450 nM and 4 ^M) responded in an antigen-specific manner (Zhao et al. 2007). This indicates that genetically engineered T cells with midrange pMHC affinity might be useful in immunotherapeutics.
As with MHC proteins, directed evolution and yeast surface display have also been applied to engineer soluble TCRs (Figure 7.3B). By using an E. coli mutator strain XL1-Red, FACS screening, and combining mutations, TCR mutants with increased thermal stability and secretion efficiency were identified (Kieke et al. 1999; Shusta et al. 1999). Selected mutations were combined and resulted in a TCR mutant that was stable for >1 hour at 65°C, had a solubility of over 4 mg/mL, and had a shake-flask expression level of 7.5 mg/L (Shusta et al. 2000). More importantly, although mutations were introduced, the resulting TCRs retained their ligand-binding specificity, making yeast display an attractive engineering platform for engineering TCRs with high affinity to pMHC (Kieke et al. 1999; Shusta et al. 2000). The first example of in vitro affinity maturation of a TCR was reported in 2000 using yeast surface display (Holler et al. 2000). A focused library of MHCI-restricted single-chain TCR was constructed by mutating the CDR3 (complementarity-determining region three) of the a-chain, and mutants with greater than 100-fold higher pMHC binding affinity were identified. Unlike the wild-type TCR, the soluble monomeric form of the high-affinity TCR was capable of directly detecting specific pMHC complexes on APCs. In vivo studies showed that a mouse T cell hybridoma transfected with the high affinity TCR responded to a significantly lower concentration of antigenic peptide (Holler et al. 2001). In several follow-up studies, it was shown that mutations in the CDR1 and CDR2 regions could also contribute to improving the pMHC binding affinity of TCRs (Chlewicki et al. 2005; Weber et al. 2005).
Researchers have used phage display to create TCR-like antibodies, which recognize specific peptides in complex with MHC molecules (Andersen et al. 1996; Denkberg and Reiter 2006). There have been ~100 different TCR-like antibodies generated to date, recognizing antigens involved in several infectious diseases and cancer.
However, the in vivo targeting capability of these TCR-like antibodies has not yet been demonstrated. With further development, this new class of antibodies might lead to novel therapeutic and diagnostic solutions.
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