Engineering the binding properties of the T cell receptor:peptide:MHC ternary complex that governs T cell activity

Abstract

The potency of a T cell is determined in large part by two interactions, binding of a cognate peptide to the MHC, and binding of the T cell receptor (TCR) to this pepMHC. Various studies have attempted to assess the relative importance of these interactions, and to correlate the corresponding binding parameters with the level of T cell activity mediated by the peptide. To further examine the properties that govern optimal T cell activity, here we engineered both the peptide:MHC interaction and the TCR:pepMHC interaction to generate improved T cell activity. Using a system involving the 2C TCR and its allogeneic pepMHC ligand, QL9-L(d), we show that a peptide substitution of QL9 (F5R), increased the affinity and stability of the pep-L(d) complex (e.g. cell surface t(1/2)-values of 13 min for QL9-L(d) versus 87 min for F5R-L(d)). However, activity of peptide F5R for 2C T cells was not enhanced because the 2C TCR bound with very low affinity to F5R-L(d) compared to QL9-L(d) (K(D)=300 microM and K(D)=1.6 microM, respectively). To improve the affinity, yeast display of the 2C TCR was used to engineer two mutant TCRs that exhibited higher affinity for F5R-L(d) (K(D)=1.2 and 6.3 microM). T cells that expressed these higher affinity TCRs were stimulated by F5R-L(d) in the absence of CD8, and the highest affinity TCR exhibited enhanced activity for F5R compared to QL9. The results provide a guide to designing the explicit binding parameters that govern optimal T cell activities.

Fig. 1

Structural features of the 2C:QL9-L^d complex, focusing on QL9 peptide residue Phe 5. (A) End view of the 2C/QL9-L^d complex (Colf 2007) (PDBID:2oi9) from the peptide N-terminus. Peptide QL9 (QLSPFPFDL, green) residues P4, F5, F7, and D8 are located at the interface of the L^d helices and the 2C TCR. Side chains residues Pro 4, Phe 5, Phe 7, and Asp 8 are in contact with both L^d (cyan) and the 2C TCR (CDR3α pink, CDR2α orange, CDR1α purple, CDR3β grey, CDR2β blue, CDR1β red). QL9 residue Phe 5 is positioned closest to the 2C TCR's CDR3α loop (pink). (B) Molecular model of the QL9-F5R-L^d complex. Structure of QL9-L^d (Jones et al., 2008) was modeled with an arginine at QL9 position 5, using Visual Molecular Dynamics software (Humphrey et al., 1996). MHC L^d is represented in grey and peptide QL9 side chains Arg 5 and Asp 8 are represented in cyan.

Fig. 2

Analysis of QL9 and QL9 variant F5R binding to MHC-L^d. (A) Detection of MHC-L^d up-regulation on the surface of T2-L^d with addition of peptides QL9 and F5R. Levels of MHC-L^d were detected with anti-L^d antibody 30.5.7 and flow cytometry. Mean fluorescent units (MFU) above the no-peptide background were plotted versus peptide concentration. Stabilization curves were subjected to non-linear regression to obtain BD₅₀ values, or concentration of peptide required to up-regulate half-maximal L^d. (B) Cell surface lifetimes of QL9/L^d and F5R/L^d complexes at 37°C. Levels of specific peptide/L^d remaining on T2-L^d cells were monitored over time. Data is plotted as % Maximal-peptide L^d, which represents the percentage of peptide/L^d remaining on the cell surface at a specific time. % Maximal-peptide L^d = [(MFU_sample − MFU_{null MCMV}) / (MFU_{max sample} − MFU_{null MCMV})] × 100. Standard deviations were averaged from five independent experiments for QL9/L^d and two independent experiments for F5R/L^d.

Fig. 3

Yeast display and engineering of higher affinity 2C TCR mutants for F5R/L^d. (A) Curves correspond to binding of soluble F5R-L^d-IgG dimer to higher affinity 2C TCR mutants called m1 and m3. Yeast cells displaying m1 and m3 were stained with various concentrations of the dimer followed by detection with PE labeled goat-anti-mouse-IgG and flow cytometry. (B) Specificity of the high-affinity 2C TCR mutant m3 (see panel A) and TCR mutant m6 for various peptide QL9 position 5 variants, in the form of soluble pMHC dimers. Peptide-L^d-IgG dimers at 0.4 μM were incubated with TCR m3 or m6 displayed on the surface of yeast, and detected with PE labeled goat-anti-mouse-IgG followed by flow cytometry. The inset shows the CDR3α amino acid sequences for the wild type 2C and the two higher affinity TCRs m3 and m6.

Fig. 4

Surface plasmon resonance of scTCRs and pMHC complexes QL9-L^d or F5R-L^d. (A) Surface plasmon resonance (SPR) traces for biotinylated QL9-L^d immobilized to a CM5 Biacore 3000 sensor chip, detected with various concentrations of soluble scTCR m3. Concentrations of TCR m3 were: 1000, 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, and 0 nM. (B) SPR traces for soluble 2C TCR immobilized to a CM5 Biacore 3000 sensor chip, detected with soluble F5R-L^d-m31. Concentrations of soluble F5R-L^d-m31 were: 100, 74, 61, 50, 39, 10, and 0 uM. (C–D) SPR traces for biotinylated F5R-L^d immobilized to a CM5 Biacore 3000 sensor chip, detected with higher affinity scTCRs m3 and m6. (C) Concentrations of scTCR m3 were: 50, 25, 12.5, 3.12, 1.56, 0.78, 0.39, 0.19, and 0 μM. (D) Concentrations of TCR m6 were: 20, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, and 0 μM.

Fig. 5

Activation of T cells transfected with wild type TCR 2C and higher affinity TCRs m6 and m3 by QL9-L^d and F5R-L^d. (A) 2C, m3 and m6 T cell transfectants (CD8-negative effector cells) were stimulated in the presence of T2-L^d (targets) and various concentrations of peptide QL9 (QLSPFPFDL). Activation was measured by assaying for levels of IL-2 release in an ELISA. (B) Sensitization doses, SD₅₀, determined from non-linear regression of the activation curves in (A). SD₅₀ values represent the concentration of peptide yielding 50% maximal IL-2 release. Error bars represent standard deviations averaged from three independent experiments. (C) Same as (A) except cells were pulsed with various concentrations of peptide F5R (QLSPRPFDL). (D) Sensitization doses, SD₅₀, determined from non-linear regression of the activation curves in (C). Error bars represent standard deviations averaged from three independent experiments.

Fig. 6

Atomic interactions between peptide QL9 Phe 5 and TCRs 2C, m6, and MHC-L^d. (A) Overlay of structures 2C/QL9-L^d and m6/QL9-L^d (Colf et al., 2007) (PDBID:2oi9 and 2e7l) showing contacts between QL9-Phe5 with the TCRs 2C, m6, and MHC L^d. Van der Waals contacts between peptide QL9-Phe5 (yellow) and the CDR3α loop of 2C (green), or the CDR3α loop of m6 (pink), along with MHC-L^d (cyan) are represented as broken lines. (B) Contact map for peptide QL9 Phe 5 (middle line) with the 2C TCR CDR loops (top line) and m6 TCR CDR loops (bottom line). Dotted lines represent potential Van der Waals contacts, containing a cutoff distance of ≤ 4.5°A, as determined by MacPyMOL software (DeLano Scientific LLC). Interactions between peptide QL9-Phe5 (yellow) and residues of TCRs 2C or m6 are represented in green and pink respectively. The total number of contacts for each pair-wise interaction are listed above or below the highlighted TCR residue.