Increased immunogenicity of an anchor-modified tumor-associated antigen is due to the enhanced stability of the peptide/MHC complex: implications for vaccine design

Abstract

The use of "anchor-fixed" altered peptide ligands is of considerable interest in the development of therapeutic vaccines for cancer and infectious diseases, but the mechanism by which successful altered peptide ligands elicit enhanced immunity is unclear. In this study, we have determined the crystallographic structure of a major tumor rejection Ag, gp100(209-217), in complex with the HLA-A*0201 (HLA-A2) molecule, as well as the structure of a modified version of the peptide which substitutes methionine for threonine at position 2 (T2M; gp100(209-2M)). The T2M-modified peptide, which is more immunogenic in vitro and in vivo, binds HLA-A2 with a approximately 9-fold greater affinity and has a approximately 7-fold slower dissociation rate at physiological temperature. Within the limit of the crystallographic data, the T2M substitution does not alter the structure of the peptide/HLA-A2 complex. Consistent with this finding, in peripheral blood from 95 human subjects, we were unable to identify higher frequencies of T cells specific for either the native or modified peptide. These data strongly support the conclusion that the greater immunogenicity of the gp100(209-2M) peptide is due to the enhanced stability of the peptide/MHC complex, validating the anchor-fixing approach for generating therapeutic vaccine candidates. Thermodynamic data suggest that the enhanced stability of the T2M-modified peptide/HLA-A2 complex is attributable to the increased hydrophobicity of the modified peptide, but the gain due to hydrophobicity is offset considerably by the loss of a hydrogen bond made by the native peptide to the HLA-A2 molecule. Our findings have broad implications for the optimization of current vaccine-design strategies.

FIGURE 1

Overview of the gp100₂₀₉/HLA-A2 and gp100_209–2M/HLA-A2 crystal structures. A, Orientation of the two molecules observed in the two asymmetric units. Molecule A is blue and molecule B is yellow. This image shows the T2M-modified structure, but the overall structure with the native peptide is identical. B, 2F_o-F_c electron density maps contoured at 1 σ for the peptides from the A molecules. Native peptide is on top, the T2M variant is on the bottom.

FIGURE 2

Superimposition of the peptides from the gp100₂₀₉/HLA-A2 and the gp100_209–2M/HLA-A2 crystal structures. The native peptide is blue, the T2M-modified peptide is yellow. A and B, Superimposition of the two A molecules (top) and B molecules (bottom). The P2 side chain is indicated as T/M2. Note the repositioning of Q4, F7, and S8 between the A and B molecules (compare top to bottom), but note also how these differences are the same whether one examines the structure with the native or T2M-modified peptide (compare blue to yellow in both images). C, Cross-eyed stereo image of the fit of the Thr² and Met² side chains in the HLA-A2 P2 pocket. Methionine is blue; the carbons of threonine are gray; and the oxygen is red. The surface of HLA-A2 is gray, except for the carboxylate of Glu⁶³ which is red. Glu⁶³ and Lys⁶⁶ are indicated.

FIGURE 3

Interatomic distances between superimposed gp100₂₀₉ and gp100_209–2M peptides. Hatched bars indicate backbone atoms, dark gray indicate side chains. The figure corresponds to the superimpositions in Fig. 2, A and B. Note that the backbone differences are miniscule and without any trends, and that, excluding the site of the substitution, the most significant differences are for atoms in positions 4, 7, and 8, the same positions which show differences between the two molecules in each asymmetric unit. Atoms with distances ≥0.30 Å are labeled.

FIGURE 4

Cavities and hydrogen bonding in and around the P2 pocket in the two peptide/MHC structures. In the structure with the native peptide (A), the hydroxyl group of the Thr² side chain hydrogen bonds with Glu⁶³, a participant in a network of hydrogen bonds strongly conserved in peptide/HLA-A2 structures. B, The hydrogen bond to Glu⁶³ is lost when Thr² is substituted with Met. Numbers indicate hydrogen bond distances. The red sphere is a conserved water whose position is slightly altered in the T2M structure. Two large cavities are present within the P2 pocket when Thr is at position 2 (yellow polygons). The smaller cavity is occupied by the -S-CH₃ moiety of the Met side chain in the modified structure. The larger cavity increases in response to removal of the Thr hydroxyl and repositioning of the γ carbon. This figure was generated using the A molecules, but the conclusions are identical if the B molecules are compared. A conserved hydrogen bond between Glu⁶³ and the amide nitrogen of P2 is not shown for clarity.

FIGURE 5

The frequency of T cells recognizing the native gp100₂₀₉ peptide mirrors the frequency of those recognizing the T2M-modified peptide. The figure shows results from ELISPOT assays conducted on 106 PBMC from 95 individuals, identifying the number of Ag-specific T cells per 10⁵ cells. The line represents a linear regression through the data, characterized by a slope of 1.1 ± 0.1.

FIGURE 6

Dissociation of the gp100₂₀₉ (A) and gp100_209–2M (B) peptides from HLA-A2 as a function of temperature. The decreased dissociation rate of the T2M-modified peptide is evident at all temperatures. Data for lower temperatures were taken for hundreds of hours (>2000 for 4°C), but are not shown for clarity. Lines represent exponential fits to the data as described in the Materials and Methods.

FIGURE 7

Eyring plots for the peptide dissociation data indicate the thermodynamic basis for the enhanced stability of the gp100_209–2M/HLA-A2 complex. Slopes of these lines are proportional to the enthalpic barriers for dissociation, whereas intercepts are proportional to entropic barriers. The steeper slope for the native peptide indicates a more unfavorable enthalpic barrier for dissociation, whereas the greater intercept indicates a more favorable entropic barrier. As the entropic component is larger, the native peptide dissociates faster.