Daniel I. R. Spencer 1 Lynda Robson 1 Des Purdy 2 Nick R. Whitelegg 3 N. Paul Michael 2 Jeetendra Bhatia 1 Surinder K. Sharma 1 Anthony R. Rees 3 Nigel P. Minton 2 Richard H. J. Begent 1 Kerry A. Chester 1 1 CRC Targeting and Imaging Group, Department of Oncology, Royal Free and University College Medical School, UCL, London, UK 2 Department of Molecular Microbiology, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts., UK 3 Centre for Protein Analysis and Design, University of Bath, Bath, UK A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics Antibodies are highly specific recognition molecules which are increasingly being applied to target therapy in patients. One type of developmental antibody-based ther- apy 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 carboxy- peptidase 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. Keywords: Surface enhanced laser desorption/ionization / Antibody directed enzyme prodrug therapy / Phage display / Single chain antibody variable fragment / Mass spectrometry PRO 0172 1 Introduction Protein and peptide therapeutics are of growing impor- tance in the treatment of disease but frequently induce an antibody response. This occurs with both foreign pro- teins [1–4] and recombinant human proteins [5–7]. The antibodies induce adverse reactions and reduce potency of the therapeutics making them unusable. This problem is of major clinical and economic importance. Knowledge of the amino acid sequence of a protein allows identifi- cation of linear epitopes by reaction of overlapping pep- tides with antibody [8]. Mutations based on this strategy have been used successfully to give clinically valuable reduction in immunogenicity [1]. However, reaction with linear peptides is not suited to identification of conforma- tional epitopes recognized in antibody responses to highly structured proteins; in these cases a means of identifying the epitope within the folded antigen is required. The human immune response to an antigen is typically polyclonal, but to identify individual epitopes on the anti- gen it is necessary to use antibodies of a single specificity. Phage display antibody libraries are a source of diverse antibodies for this purpose [9, 10]. In the system we have developed, a combinatorial phage library of single chain Fv antibodies (sFvs), derived from an immunized mouse, is used for selection of antigen-reactive sFvs. Surface enhanced laser desorption/ionization affinity mass spec- trometry (SELDI-AMS) is then used to map where the indi- vidual sFvs bind to the antigen. The sFv is attached to the SELDI chip and reacted with native antigen. The parts of the antigen not interacting with sFv are then removed by proteolysis [11, 12]. Remaining (sFv-bound) peptides are characterized directly from the chip by MS. Bioinformatic algorithms, using data on proteolytic sites, peptide mass and a structural model, are then applied to define the sFv- binding epitope of the antigen, guiding the redesign pro- cess to reduce or eliminate immunoreactivity. Correspondence: Dr. Kerry A. Chester, Department of Oncology, Royal Free and University College Medical School, UCL, Royal Free Campus, Rowland Hill Street, London, NW3 2PF, UK E-mail: k.chester@ucl.ac.uk Fax: +44-20-7794-3341 Abbreviations: ADEPT , antibody directed enzyme prodrug ther- apy; AMS, affinity mass spectrometry; CEA, carcinoembryonic antigen; CPG2, carboxypeptidase enzyme G2; CPG2, fusion protein; DSSP , define secondary structure of proteins; MFE-23, anti-CEA sFv; MFE-CP , anti-CEA sFv; SELDI, surface enhanced laser desorption/ionization; sFv , single chain antibody variable fragment Proteomics 2002, 2, 271–279 271 ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 1615-9853/02/0303–271 $17.50+.50/0