Solid-Phase Epitope Recovery: A High Throughput Method for Antigen Identification and Epitope Optimization

Abstract

Self tolerance to MHC class I-restricted nonmutated self Ags is a significant hurdle to effective cancer immunotherapy. Compelling evidence is emerging that altered peptide ligands can be far more immunogenic than their corresponding native epitopes; however, there is no way to reliably predict which modifications will lead to enhanced native epitope-specific immune responses. We reasoned that this limitation could be overcome by devising an empirical screen in which the nearly complete combinatorial spectrum of peptides of optimal length can be rapidly assayed for reactivity with a MHC class I-restricted cytotoxic T cell clone. This method, solid-phase epitope recovery, quantitatively ranks all reactive peptides in the library and allows selection of altered peptide ligands having desirable immunogenic properties of interest. In contrast to rationally designed MHC anchor-modified peptides, peptides identified by the present method are highly substituted in predicted TCR contact residues and can reliably activate and expand effector cell populations in vitro which lyse target cells presenting the wild-type epitope. We demonstrate that solid-phase epitope recovery peptides corresponding to a poorly immunogenic epitope of the melanoma Ag, gp100, can reliably induce wild-type peptide-specific CTL using normal donor T cells in vitro. Furthermore, these peptides can complement one another to induce these responses in an overwhelming majority of normal individuals in vitro. These data provide a rationale for the design of superior vaccines comprising a mixture of structurally diverse yet functionally convergent peptides.

Design of Peptide-Based Vaccines for Cancer

“…Furthermore, in mouse models, immunization with H-2K b -binding mEphA2 682-689 , and I-A b -binding mEphA2 [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] loaded DC in C57BL/6 mice induced specific CTL lysis in vitro. Mice immunized with peptides, EphA2 671-679 , EphA2 682-679 , or EphA2 [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] peptides showed protection against a EphA2 negative B16 tumor challenge and induced significant suppression of lung metastases after challenge with B16-BL6 tumor cells [183]. Mechanism of EphA2 protection was due to inhibition of vascular endothelial growth factor (VEGF) induced angiogenesis.…”

“…It is important to be cautious in designing modifications to peptides for cancer immunotherapy to improve them of higher affinity, since this can effect T cell reactivity. To overcome these problems, solid-phase epitope recovery method has been used to determine reactive peptides with immunogenic properties of interest [30]. APLs have also been used (antagonists) for autoimmune diseases [31,32] and for infectious diseases.…”

“…1) improved MHC binding affinity and exhibited potent anti-tumor immunity with no autoimmune responses in mice [19]. Enhanced CTL activation has been achieved with 'anchor' residue modification of the melanoma antigen MART-1 [27][28][29][30][31][32][33][34][35] (AAGIGILTV) to LAGIGILTV [20]. Making anchor motif modifications to include high affinity anchors, does not necessarily indicate that the peptide will bind with high affinity to the MHC as one must consider proximal/distal amino acids [21].…”

“…In clinical trials immunization with CAP-1-6D, induced enhanced CTL responses in patients compared to CAP-1 peptide [26]. In addition, a single amino acid substitution of MART1 [27][28][29][30][31][32][33][34][35] (AAGIGILTV) (substitution of Leu in position 1) enhances MART1 [27][28][29][30][31][32][33][34][35] T cell activity, by inducing IFN-γ and IL-2 in vitro and these T cells are insensitive to inhibitory effects of MART1 [27][28][29][30][31][32][33][34][35] antagonist peptides [20]. Mutated gp100 209-217 peptides with preferred HLA-A2 anchor residues bound with higher affinity to HLA-A2 as compared to the native gp100 peptide [27].…”

Capturing viral diversity for in-vitro test reagents and HIV vaccine immunogen design

“…The use of single sequence-based test sets or even the combination of a number of few such sequences will probably not fully accomplish this goal, and approaches that mediate large sequence diversity coverage are thus urgently needed. For instance, the use of combinatorial peptide libraries has revealed sequence variants with potentially increased in-vivo immunogenicity and the ability to induce cross-reactive responses; however, cross-reactivity of de-novo generated responses has generally not been assessed, and generic combinatorial peptide libraries would probably not be suitable for selective detection of HIV-specific responses only [28,29].…”