Although peptide-binding algorithms have greatly enhanced rational peptide design, they are far from perfect. Further, despite their orientation away from the T-cell receptor (TCR), anchor residue substitutions can change pMHC conformation to negatively impact TCR recognition. What is needed then is a bit of magic: a general method for increasing peptide affinity while minimizing changes in TCR specificity. In this issue of the European Journal of Immunology, while seeking to improve the CD8+ T-cell response to the melanocyte differentiation Ag

Gp100, Uchtenhagen et al. [18] appear to achieve the impossible, or at least the improbable. Gp100 expression is greatly enhanced in melanoma, making it an attractive therapeutic vaccine target. Human Hedgehog antagonist Gp10025–33 peptide (KVPRNQDWL (KVP)) presented by the mouse class I Db allomorph elicits self-reactive mouse CD8+ T cells, while the orthologous mouse peptide (EGSRNQDWL (EGS)) does not (Fig. 1) [19, 20]. Both peptides possess canonical p5N and p9L anchor residues for Db (which has a motif of XXXX NXXX[IML], where CCI-779 clinical trial X represents any aa, N

= asparagine, I = isoleucine, M = methionine, L = leucine) [4]. Despite identical anchors, EGS binds Db with 100-fold lower affinity than KVP [15], evincing the contribution of nonanchor residues to Db binding [21, 22]. Systematic crosswise substitution of p1–3 between KVP and EVS revealed greatly enhanced peptide binding [15]) and pMHC stability when simply replacing p3 of EGS with proline (Pro; EGPRNQDWL C1GALT1 (EGP)) [23]. Immunization with EGP elicited higher numbers of EGS-specific CD8+ T cells than EGS itself, and critically, protected against tumor challenge while the homologous peptide did not [23]. Uchtenhagen et al. [18] scrutinized the structural basis for enhanced EGP peptide affinity with surprising and potentially

generally applicable findings. X-ray crystallography of Db complexed with Gp100 peptides KVP, EGS, or EGP revealed a conserved peptide conformation and similar peptide- Db hydrogen bonding in each complex [23]. Thus, the EGP’s increased affinity was not due to large structural alterations in the complex. Notably, in EGP, the pyrrilodine ring of p3P and the hydroxyphenyl group of Db-Y159 formed CH-π interactions, which affords substantial intermolecular-binding energy [24, 25] (and see http://www.tim.hi-ho.ne.jp/dionisio/page/whatis.html). To examine the contribution of CH-π interactions (which occur with aromatic residues) to EGP/Db stability, Uchtenhagen et al. substituted Y159 with either another aromatic (F) or aliphatic residues with a short (A) or long (L) side chain. Intriguingly, the enhanced pMHC stability of EGP versus EGS was abrogated with Db-Y159A or Db-Y159L. An intermediate effect was observed with Y159F, consistent with reduced energetic stabilization of Phe-Pro CH-π interactions compared with that of Tyr-Pro.