Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 32; pp. 9967 - 9972
• Main Authors: Skora, Andrew D., Douglass, Jacqueline, Hwang, Michael S., Tam, Ada J., Blosser, Richard L., Gabelli, Sandra B., Cao, Jianhong, Diaz, Luis A., Papadopoulos, Nickolas, Kinzler, Kenneth W., Vogelstein, Bert, Zhou, Shibin
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 11.08.2015; National Acad Sciences
• Subjects: Amino acids; Biological Sciences; Cell Membrane - metabolism; Cell Surface Display Techniques; Cells; Clone Cells; Epidermal growth factor; Epitopes - genetics; Epitopes - immunology; Genes; HLA Antigens - genetics; HLA Antigens - immunology; Humans; Molecules; Mutant Proteins - metabolism; Mutants; Mutation; Mutation - genetics; Peptides; Peptides - metabolism; Single-Chain Antibodies - immunology; antibody engineering; oncogene; therapy; personalized; immunotherapy
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1511996112

Mutant epitopes encoded by cancer genes are virtually always located in the interior of cells, making them invisible to conventional antibodies. We here describe an approach to identify single-chain variable fragments (scFvs) specific for mutant peptides presented on the cell surface by HLA molecules. We demonstrate that these scFvs can be successfully converted to full-length antibodies, termed MANAbodies, targeting “Mutation-Associated Neo-Antigens” bound to HLA. A phage display library representing a highly diverse array of single-chain variable fragment sequences was first designed and constructed. A competitive selection protocol was then used to identify clones specific for mutant peptides bound to predefined HLA types. In this way, we obtained two scFvs, one specific for a peptide encoded by a common KRAS mutant and the other by a common epidermal growth factor receptor (EGFR) mutant. The scFvs bound to these peptides only when the peptides were complexed with HLA-A2 (KRAS peptide) or HLA-A3 (EGFR peptide). We converted one scFv to a full-length antibody (MANAbody) and demonstrate that the MANAbody specifically reacts with mutant peptide–HLA complex even when the peptide differs by only one amino acid from the normal, WT form.