title: A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics.
abstract: Antibodies are highly specific recognition molecules which are increasingly being applied to target therapy in patients. One type of developmental antibody-based therapy is antibody directed enzyme prodrug therapy (ADEPT) for the treatment of cancer. In ADEPT, an antibody specific to a tumor marker protein delivers a drug-activating enzyme to the cancer. Subsequent intravenous administration of an inactive prodrug results in drug activation and cytotoxicity only within the locale of the tumor. Pilot clinical trials with chemical conjugates of the prodrug activating enzyme carboxypeptidase G2 (CPG2) chemically conjugated with an antibody to and carcinoembryonic antigen (CEA), have shown that CPG2-mediated ADEPT is effective but limited by formation of human antibodies to CPG2 (HACA). We have developed a recombinant fusion protein (termed MFE-CP) of CPG2 with an anti-CEA single chain Fv antibody fragment and we have developed methods to address the immunogenicity of this therapeutic. A HACA-reactive discontinuous epitope on MFE-CP was identified using the crystal structure of CPG2, filamentous phage technology and surface enhanced laser desorption/ionization affinity mass spectrometry. This information was used to create a functional mutant of MFE-CP with a significant reduction (range 19.2 to 62.5%, median 38.5%) in reactivity with the sera of 11 patients with post-therapy HACA. The techniques described here are valuable tools for identifying and adapting undesirable immunogenic sites on protein therapeutics.
authors.affiliation: CRC Targeting and Imaging Group, Department of Oncology, Royal Free and University College Medical School, UCL, London, UK.
authors.forename: Daniel I R
authors.initials: DI
authors.lastname: Spencer
authors.forename: Lynda
authors.initials: L
authors.lastname: Robson
authors.forename: Des
authors.initials: D
authors.lastname: Purdy
authors.forename: Nick R
authors.initials: NR
authors.lastname: Whitelegg
authors.forename: N Paul
authors.initials: NP
authors.lastname: Michael
authors.forename: Jeetendra
authors.initials: J
authors.lastname: Bhatia
authors.forename: Surinder K
authors.initials: SK
authors.lastname: Sharma
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Rees
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Minton
authors.forename: Richard H J
authors.initials: RH
authors.lastname: Begent
authors.forename: Kerry A
authors.initials: KA
authors.lastname: Chester
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D014408:Biomarkers, Tumor; D017321:Clinical Trials, Phase I as Topic; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011623:gamma-Glutamyl Hydrolase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000906:Antibodies; D014408:Biomarkers, Tumor; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D011623:gamma-Glutamyl Hydrolase