In vitroevolution of a T cell receptor with high affinity for peptide/MHC

Abstract

T cell receptors (TCRs) exhibit genetic and structural diversity similar to antibodies, but they have binding affinities that are several orders of magnitude lower. It has been suggested that TCRs undergo selection in vivo to maintain lower affinities. Here, we show that there is not an inherent genetic or structural limitation on higher affinity. Higher-affinity TCR variants were generated in the absence of in vivo selective pressures by using yeast display and selection from a library of V␣ CDR3 mutants. Selected mutants had greater than 100-fold higher affinity (KD Ϸ 9 nM) for the peptide͞MHC ligand while retaining a high degree of peptide specificity. Among the high-affinity TCR mutants, a strong preference was found for CDR3␣ that contained Pro or Gly residues. Finally, unlike the wild-type TCR, a soluble monomeric form of a high-affinity TCR was capable of directly detecting peptide͞MHC complexes on antigen-presenting cells. These findings prove that affinity maturation of TCRs is possible and suggest a strategy for engineering TCRs that can be used in targeting specific peptide͞ MHC complexes for diagnostic and therapeutic purposes.

Transgenic TCR expression: comparison of single chain with full-length receptor constructs for T-cell function

“…In the future, we plan to try an improved tetramer technology 36 to identify the differential affinity between flTCR and scTCR. Holler et al 37 demonstrated that the low affinity of scTCR molecules could be dramatically increased by an in vitro selection system through site-directed mutagenesis. Primary T cells modified with the scTCR-z-28-Lck could lyse low concentrations (10 À10 M) OVA peptide-pulsed EL-4 cells (E:T ¼ 10:1), although at a marginal level.…”

Phase Diagrams of Quasispecies Theory with Recombination and Horizontal Gene Transfer

“…Experiments and theory have emphasized that recombination and gene transfer in various forms increase the rate of laboratory directed protein evolution [4,5,6] (for reviews see [7,8]). Other experiments have amplified this point and have also suggested that, while significant in practice, the advantage of recombination may simply be to speed up the evolutionary process that would naturally occur by mutation alone in the limit of a long enough evolutionary time or a large enough population size [9,10,11].The Eigen [12] and Crow-Kimura [13], or parallel, models of viral quasispecies evolution are among the simplest that capture the basic processes of mutation, selection, and replication that occur in natural evolution. These mathematical models exhibit phase transitions, such that for mutation rates below critical values, an identifiable quasispecies forms.…”

The Case for Absolute Ligand Discrimination: Modeling Information Processing and Decision by Immune T Cells

“…1b): engagement of these receptors (with non-physiological ligands of surface antigens with very large lifetime) has been shown to be necessary and sufficient to activate T cells. In fact, examples of supra-physiological lifetimes for antigen/receptor complexes that lead to T cell activation were derived experimentally by in vitro evolution of the TCR/pMHC complex [30]. In the context of modeling early immune detection, this is relevant as the biophysics of ligand-receptor interaction are very different (with very large binding affinities), yet consistent with the lifetime dogma: antigen/receptor pairs with very strongly-held complexes, and very large lifetimes are indeed very stimulatory.…”