Systematic amino acid substitutions improved efficiency of GD 2 -peptide mimotope vaccination against neuroblastoma Matthias Bleeke a,e , Stefan Fest a,b,c,e , Nicole Huebener a , Christiane Landgraf d , Burkhart Schraven c , Gerhard Gaedicke d , Rudolf Volkmer d, * , Holger N. Lode a, * a Charite ´ – Universita ¨tsmedizin Berlin, Department of Pediatrics, Experimental Oncology, Berlin, Germany b Otto-von-Guericke University of Magdeburg, Faculty of Medicine Magdeburg, Department of Pediatrics, Magdeburg, Germany c Otto-von-Guericke University of Magdeburg, Faculty of Medicine Magdeburg, Institute of Molecular and Clinical Immunology, Magdeburg, Germany d Charite ´ – Universita ¨tsmedizin Berlin, Department of Medical Immunology, Berlin, Germany ARTICLE INFO Article history: Received 18 March 2009 Received in revised form 14 July 2009 Accepted 22 July 2009 Keywords: Immunotherapy Neuroblastoma Ganglioside Mimotope SPOT ABSTRACT The likelihood of identifying peptides of sufficient quality for the development of effective cancer vaccines by screening of phage display libraries is low. Here, we introduce the sequential application of systematic amino acid substitution by SPOT synthesis. After the substitution of two amino acids within the sequence of a phage display-derived mimo- tope of disialoganglioside GD 2 (mimotope MA), the novel mimotope C3 showed improved GD 2 mimicry in vitro. Peptide vaccination with the C3 mimotope induced an 18-fold increased anti-GD 2 serum response associated with reduction of primary tumour growth and spontaneous metastasis in contrast to MA mimotope controls in a syngeneic neuro- blastoma model. In summary, SPOT provides an ideal optimisation tool for the develop- ment of phage display-derived cancer vaccines. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction The translation of carbohydrates or glycolipids into peptide surrogates by application of phage display libraries is an established tool to overcome the weak antigenicity of the nominal antigens. The generated peptides are mimotopes of the nominal antigen and their protective efficacy as vaccines against infective agents, including bacteria and viruses, as well as against cancer was demonstrated. 1–3 However, the application of phage display libraries is restricted by the tech- nically limited number of actually expressed and screened peptide antigens and may therefore fail to identify the most efficient peptide mimotope. 4–6 We addressed the question if optimisation of a phage display-derived mimotope can be car- ried out by systematic alteration of the amino acid sequence resulting in improved antibody binding and vaccine efficacy. Here, we establish proof of concept that such an optimisation procedure is effective for a peptide mimotope of disialogan- glioside GD 2 (mimotope MA) used as a vaccine against neuroblastoma. Ganglioside GD 2 is an established target for immunother- apy against neuroblastoma, the most common extracranial solid tumour during early childhood. Infusions of monoclonal anti-GD 2 antibodies (mAbs), namely chimeric mAb ch14.18 0959-8049/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejca.2009.07.026 * Corresponding authors: Address: Forum 4, R 2.0407, Augustenburger Platz 1, 13353 Berlin, Germany. Tel.: +49 (0) 30 450 666233; fax: +49 (0) 30 450 559917 (H.N. Lode). Address: Institut fu ¨ r Medizinische Immunologie, Abt. Molekulare Bibliotheken, Hessische Str. 3-4, 10115 Berlin, Germany. Tel.: +49 (0) 30 450 524267; fax: +49 (0) 30 450 524942 (R. Volkmer). E-mail addresses: rve@charite.de (R. Volkmer), holger.lode@charite.de (H.N. Lode). e Equally contributed to this paper. EUROPEAN JOURNAL OF CANCER xxx (2009) xxx – xxx available at www.sciencedirect.com journal homepage: www.ejconline.com Please cite this article in press as: Bleeke M et al., Systematic amino acid substitutions improved efficiency of GD 2 -peptide mimotope vaccination against neuroblastoma, Eur J Cancer (2009), doi:10.1016/j.ejca.2009.07.026 ARTICLE IN PRESS