Proline substitution independently enhances H‐2D^b complex stabilization and TCR recognition of melanoma‐associated peptides

Abstract

Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe identification of major histocompatibility complex (MHC) restricted tumor-associated antigens (TAAs) as targets for CD8+ cytotoxic T-cell lymphocytes (CTLs) has opened new possibilities for immunotherapy and led to intense efforts to harness and increase immune responses against tumors [1]. T-cell responsesCorrespondence: Dr. Adnane Achour e-mail: adnane.achour@ki.se against MHC/TAA complexes are constrained by immune tolerance and most of the identified TAAs are poorly immunogenic [2]. In general, cancer-associated epitopes display weak MHCbinding affinity and low complex stabilization capacity, and the avidity of TAA-reactive CTLs is moderate at best [2]. Therefore, the use of altered peptide ligands (APLs) with enhanced stabilization capacity and immunogenicity may allow overcoming tolerance and the intrinsic low immunogenicity of WT TAAs. Nevertheless, the appropriate design and possible clinical application of APLs has proven difficult and still represents one of the major hurdles in the design of cancer vaccines. Eur. J. Immunol. 2013. 43: 3051-3060 The most commonly used approach to improve the immunogenicity of TAAs is to modify peptide anchor residues in order to optimize the interactions of the targeted epitope with the MHC peptide binding groove [3][4][5]. However, it has been demonstrated that even subtle anchor-residue modifications may compromise molecular mimicry, therefore limiting the capacity of these APLs to elicit effective CTL responses against native TAAs [6][7][8][9]. Therefore, successful design of structurally conservative peptide modifications yielding enhanced MHC stabilization capacity and immunogenicity would represent a key advance in T-cell epitope immunogen design.Expression of the differentiation antigen gp100, normally restricted to melanocytes, is significantly increased in malignant melanoma [10], rendering it an attractive vaccine candidate [11,12]. Although the mouse gp100-derived epitope EGS (gp100 25-33 EGSRNQDWL) contains the theoretically optimal motif required for binding to H-2D b (D b ), with an asparagine at position 5 (p5N) and a leucine at the C-terminus (p9L), it binds with very weak affinity to D b and is poorly immunogenic [11,12].Peptide position 3 has been described as an auxiliary anchor position for D b -restricted epitopes [13][14][15][16]. Nevertheless, the dramatically increased MHC-I affinity, binding stability, and immunogenicity resulting from the substitution of serine at position 3 of gp100 to a proline at peptide position 3 (p3P) in the optimized epitope EGP (EGPRNQDWL) was unexpected [11]. Vaccination of C57BL/6 mice with EGP elicited high frequencies of endogenous CTLs that cross-reacted with D b /EGS complexes on the surface of melanoma cells, leading to tumor cell lysis and impairing tumor progression [11,17]. Crystal structure analysis revealed that the pyrrolidine ring of p3P closely interacts with the side chain ...

Altered Peptide Ligands Revisited: Vaccine Design through Chemically Modified HLA-A2–Restricted T Cell Epitopes

“…This result can be explained by more hydrophobic contact between the large alkyl side chains and HLA-A*0201 residues forming the pocket. In addition, CH-p interactions between the alkyl chains and Tyr7 could contribute to enhanced affinity, similar to those reported for Pro at P 3 and Tyr159 within H-2D b :: peptide structures (58). However, we cannot exclude that extension of the P 2 side chain beyond that of NVA also affects the overall conformation of the peptide or TCR interaction, as has been shown for other APLs harboring P 2 or P 3 substitutions (see Supplemental Fig.…”

“…The NVA residue on P 2 protrudes into the hydrophobic anchor pocket and shows two different conformations in one of the peptides of the asymmetric unit, suggesting some degree of flexibility upon interaction with HLA-A*0201. The NVA residue could stabilize the interaction between peptide and HLA-A*0201 via van der Waals contact, but also via CH-p interactions with Tyr7, similar to the CH-p interactions reported between proline at P 3 and Tyr159 within the H-2D b ::peptide complex (57,58). The side chain of the PRG residue on P C fits well into the hydrophobic F-pocket, whereas the main chain atoms form hydrogen bonds with Asp77, Thr143, and a water molecule (Fig.…”

“…2C). The contribution of aromatic-p interactions to the binding of peptide and MHC H chain has been described previously, in particular the effect of an APL harboring a proline substitution at P 3 in which CH-p interactions between this proline and Tyr159 are noteworthy to mention, as these interactions have been shown to increase the binding affinity significantly between H-2D b and peptide (57) and to enhance the stability of the H-2D b ::peptide complex (58). The NVA residue on P 2 protrudes into the hydrophobic anchor pocket and shows two different conformations in one of the peptides of the asymmetric unit, suggesting some degree of flexibility upon interaction with HLA-A*0201.…”

“…Three mice were immunized with each peptide. set out to improve the affinity of two well-known viral epitopes: Influenza Matrix 1 (58)(59)(60)(61)(62)(63)(64)(65)(66) epitope GILGFVFTL (50), as a stringent model epitope already having high affinity for HLA-A*0201, and CMV pp65 (495-503) peptide NLVPMVATV (51) as an intermediate model peptide having a moderate affinity for HLA-A*0201 (52). To explore the general scope of substitutions with proteogenic amino acids, we systematically introduced all 20 proteogenic residues on all nine positions in the peptides and screened these peptides for binding capacity to HLA-A*0201 using an MHC exchange fluorescence polarization FP assay (Supplemental Table I) (38).…”

Dipeptides promote folding and peptide binding of MHC class I molecules

“…Depending on the class I allotype, high-affinity peptides must be 8 to 10 aa long (such that the termini can form hydrogen bonding networks) and have defined side chains at particular positions that bind into specificity pockets at the bottom of the binding groove (3)(4)(5). This knowledge has been used to predict the binding affinity of any peptide sequence to a given class I allotype (6), and, together with theoretical simulations of the conformational movements of class I-peptide complexes, it has allowed the design of altered peptide ligands with higher binding affinities (7). The high-affinity peptides thus identified can be used to fold many bacterially expressed denatured class I molecules into their native state (8).…”