Related Experiment Video
Updated: Apr 6, 2026
Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes
Andrew D Skora1, Jacqueline Douglass2, Michael S Hwang2
1Ludwig 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;
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.
Insights
Researchers developed a new method to create MANAbodies, which are full-length antibodies that target cancer neoantigens on cell surfaces. This approach successfully identified antibodies for KRAS and EGFR mutations, offering a promising new avenue for cancer therapy.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Cancer neoantigens are typically intracellular and inaccessible to conventional antibodies.
- Targeting cell surface-presented mutant peptides requires novel antibody strategies.
Purpose of the Study:
- To develop a method for identifying single-chain variable fragments (scFvs) specific for mutant peptides bound to Human Leukocyte Antigen (HLA) molecules.
- To convert these scFvs into full-length antibodies, termed MANAbodies, for targeting mutation-associated neoantigens.
Main Methods:
- Construction of a diverse phage display library of scFv sequences.
- Utilizing a competitive selection protocol to identify scFvs specific for mutant peptides presented by specific HLA types.
- Conversion of a selected scFv into a full-length MANAbody.
Main Results:
- Identified two scFvs specific for KRAS and epidermal growth factor receptor (EGFR) mutant peptides, respectively, when complexed with HLA-A2 and HLA-A3.
- Demonstrated that the MANAbody specifically recognizes mutant peptide-HLA complexes, distinguishing them from wild-type forms even with a single amino acid difference.
Conclusions:
- The developed approach enables the identification and generation of antibodies targeting cell surface-presented cancer neoantigens.
- MANAbodies represent a novel class of antibodies with potential for precise cancer therapy by targeting specific mutant peptide-HLA complexes.
Related Concept Videos
01:28Diversity of Antigen Receptors
Before encountering any antigen, lymphocytes express these receptors. On B cells, the antigen receptor is a membrane-bound antibody molecule called BCR; on T cells, it is a T cell receptor or TCR. B and T cell receptors are composed of two...
01:26Antigens Involved in Adaptive Immunity
Complete Antigens
Complete antigens possess both immunogenicity and...
01:31Hybridoma Technology
Hybridoma Selection
Commonly used fusion techniques — electroporation,...
01:31Antigen Processing Pathways
MHC Class I: Presenting Endogenous...
01:22T Cell Activation and Clonal Selection
Naive T cells that have not yet encountered an antigen express two primary CD...
01:24B Cell Activation and Differentiation
When naive B cells encounter a specific antigen that can bind to the B cell receptor (BCR) on their surface, they undergo sensitization to respond to the antigen's presence. Sensitization begins with...