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Proc Natl Acad Sci U S A. 2015 Aug 11;112(32):9967-72. doi: 10.1073/pnas.1511996112. Epub 2015 Jul 27.

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

Author information

1
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;
2
Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
3
Oncology Flow Cytometry Core Facility, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
4
Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21287;
5
Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
6
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.
7
Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21287; bertvog@gmail.com sbzhou@jhmi.edu.

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.

KEYWORDS:

antibody engineering; immunotherapy; oncogene; personalized; therapy

PMID:
26216968
PMCID:
PMC4538619
DOI:
10.1073/pnas.1511996112
[Indexed for MEDLINE]
Free PMC Article

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