Class II-restricted T cell receptor engineeredin vitrofor higher affinity retains peptide specificity and function

Abstract

The T cell receptor (TCR) ␣␤ heterodimer determines the peptide and MHC specificity of a T cell. It has been proposed that in vivo selection processes maintain low TCR affinities because T cells with higher-affinity TCRs would (i) have reduced functional capacity or (ii) cross-react with self-peptides resulting in clonal deletion. We used the class II-restricted T cell clone 3.L2, specific for murine hemoglobin (Hb͞I-E k ), to explore these possibilities by engineering higher-affinity TCR mutants. A 3.L2 single-chain TCR (V␤-linker-V␣) was mutagenized and selected for thermal stability and surface expression in a yeast display system. Stabilized mutants were used to generate a library with CDR3 mutations that were selected with Hb͞I-E k to isolate a panel of affinity mutants with KD values as low as 25 nM. Kinetic analysis of soluble single-chain TCRs showed that increased affinities were the result of both faster on-rates and slower off-rates. T cells transfected with the mutant TCRs and wild-type TCR responded to similar concentrations of peptide, indicating that the increased affinity was not detrimental to T cell activation. T cell transfectants maintained exquisite hemoglobin peptide specificity, but an altered peptide ligand that acted as an antagonist for the wild-type TCR was converted to a strong agonist with higher-affinity TCRs. These results show that T cells with high-affinity class II reactive TCRs are functional, but there is an affinity threshold above which an increase in affinity does not result in significant enhancement of T cell activation.antigen specificity ͉ major histocompatibility complex ͉ T cell activity

Deep Mutational Scans as a Guide to Engineering High Affinity T Cell Receptor Interactions with Peptide-bound Major Histocompatibility Complex

“…The TCRs are in single-chain form (V␤-linker-V␣) as Aga2 fusions for yeast display, whereas in T cells, the V␣ and V␤ each contain transmembrane-spanning constant regions. Despite these differences, we have invariably observed with other TCRs that the mutations yielding higher affinity in the yeast display system also yielded higher affinity when used as full-length TCRs transferred into T cells (18,37,50,51). Thus, although not formally tested, we have reason to believe that the mutations of RD1-MART HIGH TCRs identified in the present work would exhibit binding with enhanced affinities when transferred to the full-length TCR context.…”

“…In addition, because of their naturally low affinities, they represent protein engineering targets for both stability and affinity. Previously, we reported successful affinity engineering of TCRs by directed evolution using yeast display (2,(17)(18)(19)(20)(21) and mammalian cell display (7,22). We also described structure-guided design strategies that estimated the binding energies of both favorable and unfavorable mutations and led to the design of additional high affinity TCRs (23,24).…”

Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions

“…Transduced CD8 ϩ T cells expressing a high-affinity 2C TCR variant but not the wild-type (WT) 2C TCR also recognized normal non-peptide-pulsed H-2K bϩ murine target cell lines, indicating that this variant confers upon CD8 ϩ T cells the ability to cross-recognize endogenously processed self ligands that presumably fail to trigger with the WT 2C TCR due to the lower avidity of their cellular interactions (16). Using a similar approach, AAS were also identified in the CDR1 and CDR3 regions of a murine class II-restricted TCR that resulted in a Ͼ100-fold increase in the affinity of the TCR for the cognate peptide/MHC complex that did not appear to alter the cognate Ag complex specificity of T cells transfected with these TCRs (17).…”

Engineering High-Affinity T Cell Receptor/ Cytokine Fusions for Therapeutic Targeting

“…Our goal to target the immunosuppressive effects of IL-10 locally to particular class II MHC epitopes may be modeled by targeting the murine class II MHC allele I-E k presenting a peptide derived from the beta chain of the minor d allele of hemoglobin (Hbβ d [64][65][66][67][68][69][70][71][72][73][74][75][76]). This Hb/I-E k complex is recognized by a wild-type TCR called 3.L2 with an affinity of 20 µM for the peptide-MHC (Weber et al 2005). Engineering efforts to improve this interaction are detailed below.…”

“…Engineered receptors with 1000-fold or more increased affinity compared to their original, wild-type TCRs have been isolated, and the soluble forms of these TCRs bind to the targeted peptide-MHC with a high level of specificity (Chervin et al 2008;Holler et al 2003;Holler et al 2000;Li et al 2005b;Weber et al 2005). The higher affinities achieved for TCR binding to peptide-MHC (nanomolar and picomolar) are in the range of affinity-matured antibodies, and are thus sufficient to evaluate for their ability to serve as soluble therapeutics.…”