title: Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes.
abstract: 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.
authors.affiliation: Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21287; Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Skora
authors.affiliation: Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Douglass
authors.affiliation: Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
authors.forename: Michael S
authors.initials: MS
authors.lastname: Hwang
authors.affiliation: Oncology Flow Cytometry Core Facility, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
authors.forename: Ada J
authors.initials: AJ
authors.lastname: Tam
authors.affiliation: Oncology Flow Cytometry Core Facility, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
authors.forename: Richard L
authors.initials: RL
authors.lastname: Blosser
authors.affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
authors.forename: Sandra B
authors.initials: SB
authors.lastname: Gabelli
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Jianhong
authors.initials: J
authors.lastname: Cao
authors.affiliation: Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
authors.forename: Luis A
authors.initials: LA
authors.lastname: Diaz
authors.affiliation: Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
authors.forename: Nickolas
authors.initials: N
authors.lastname: Papadopoulos
authors.affiliation: Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Kinzler
authors.affiliation: Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21287; Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21287; bertvog@gmail.com sbzhou@jhmi.edu.
authors.forename: Bert
authors.initials: B
authors.lastname: Vogelstein
authors.affiliation: Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21287; bertvog@gmail.com sbzhou@jhmi.edu.
authors.forename: Shibin
authors.initials: S
authors.lastname: Zhou
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D061505:Cell Surface Display Techniques; D002999:Clone Cells; D000939:Epitopes / Q000235:genetics* / Q000276:immunology*; D006680:HLA Antigens / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010455:Peptides / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000276:immunology*
chemical_list: D000939:Epitopes; D006680:HLA Antigens; D050505:Mutant Proteins; D010455:Peptides; D057127:Single-Chain Antibodies
keywords: antibody engineering; immunotherapy; oncogene; personalized; therapy