title: Epitope mapping and key amino acid identification of anti-CD22 immunotoxin CAT-8015 using hybrid β-lactamase display.
abstract: Monoclonal antibodies are a commercially successful class of drug molecules and there are now a growing number of antibodies coupled to toxic payloads, which demonstrate clinical efficacy. Determining the precise epitope of therapeutic antibodies is beneficial in understanding the structure-activity relationship of the drug, but in many cases is not done due to the structural complexity of, in particular, conformational protein epitopes. Using the immunotoxin CAT-8015 as a test case, this study demonstrates that a new methodology, hybrid β-lactamase display, can be employed to elucidate a complex epitope on CD22. Following insertion of random CD22 gene fragments into a permissive site within β-lactamase, proteins expressed in Escherichia coli were first screened for correct folding by resistance to ampicillin and then selected by phage display for affinity to CAT-8015. The optimal protein region recognised by CAT-8015 could then be used as a tool for fine epitope mapping, using alanine-scanning analysis, demonstrating that this technology is well suited to the rapid characterisation of antibody epitopes.
authors.affiliation: MedImmune Research, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: D
authors.initials: D
authors.lastname: Bannister
authors.forename: B
authors.initials: B
authors.lastname: Popovic
authors.forename: S
authors.initials: S
authors.lastname: Sridharan
authors.forename: F
authors.initials: F
authors.lastname: Giannotta
authors.forename: P
authors.initials: P
authors.lastname: Filée
authors.forename: N
authors.initials: N
authors.lastname: Yilmaz
authors.forename: R
authors.initials: R
authors.lastname: Minter
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D018360:Crystallography, X-Ray; D018604:Epitope Mapping / Q000379:methods*; D005098:Exotoxins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000235:genetics / Q000276:immunology*; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D001427:Bacterial Toxins; D005098:Exotoxins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2; C541974:immunotoxin HA22; D001618:beta-Lactamases