title: Selection of human single domain antibodies recognizing the CMYC protein using enhanced intracellular antibody capture.
abstract: Protein functions that are mediated by interaction with other proteins (protein-protein interactions, PPI) are important for normal cell biology and also in disease. Molecules that can interfere with PPI are required as laboratory tools to dissect function, as lead drug surrogates for target validation and as templates for drug discovery. We describe enhanced developments to Intracellular Antibody Capture (IAC) technology that can select antibody fragments able to interact with targets in cells. This is illustrated by the isolation of single heavy chain variable region domains binding to the basic-helix-loop-helix and leucine zipper region of the CMYC oncogenic protein. The enhanced IAC (eIAC) methodology deploys screening in yeast cells of a single diverse library initially with randomization only of CDR3. Further sequential randomization of CDR2 and CDR1 of three independently selected anti-CMYC clones illustrates an in vivo affinity maturation process. This concise eIAC approach facilitates the rapid selection of antibody fragments to explore the proteome interaction spectrum of mammalian cells and disease targeting.
authors: Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
authors: J
authors: J
authors: Zeng
authors: Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
authors: H C
authors: HC
authors: Li
authors: Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
authors: T
authors: T
authors: Tanaka
authors: Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
authors: T H
authors: TH
authors: Rabbitts
mesh_terms: D000595:Amino Acid Sequence; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007158:Immunologic Techniques; D016350:Leucine Zippers / Q000235:genetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology*; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; C489427:MYC protein, human; D019151:Peptide Library; D016271:Proto-Oncogene Proteins c-myc; D061905:Single-Domain Antibodies
keywords: Antibody; Cancer; Chromosomal translocations; Intrabody; Intracellular antibody; Leukemia