660 NATURE BIOTECHNOLOGY VOL 17 JULY 1999 http://biotech.nature.com RESEARCH Aberrant glycosylation is fundamental to tumor progression, since cells acquire competence for metastasis and a faster clonal growth through newly synthesized carbohydrate structures 1 . Tumor- expressed carbohydrate antigens are therefore considered important targets for active specific and passive immunotherapy, and carbohy- drate-based cancer vaccines are under investigation to modulate immune responses to carbohydrate antigens both qualitatively and quantitatively 2 . Carbohydrates, however, are typically poorly immunogenic, difficult to purify in large quantities, difficult to syn- thesize, and usually result in mostly short-lived IgM-type antibodies in a vaccinated host without long-lasting immunity. Additionally, most carbohydrate antigens are T-cell independent, reflecting their inability to stimulate MHC class II–dependent T-cell help 3 . Consequently, carbohydrates by themselves do not elicit a sufficient anamnestic or secondary immune response leading to the develop- ment of polysaccharide–protein conjugates 2,4 . Molecular mimicry of carbohydrate antigens as a means to con- vert the T-cell–independent response to a T-cell–dependent one is an attractive alternative. The idea of using surrogate antigens as immunogens requires that antigenic mimicry, accomplished using amino acids in place of sugars, elicits an immune specificity pattern for the nominal antigen. Anti-idiotypic antibodies 5 and peptides that mimic tumor-associated carbohydrate (TAC) antigens 6–8 or their glycosphingolipid constituents 9 have been described. Here, we demonstrate that designed peptides that consist of carbohydrate- mimicking motifs presented as multiple-antigen peptides (MAPs) are effective at eliciting (primarily) IgM antibody responses in mice that react with neolactoseries-expressing human and murine tumor cells. The anti-peptide serum mediates complement-dependent- cytotoxicity (CDC) of human breast tumor cells that express the neolactoseries antigen Lewis Y (LeY) in the presence of human com- plement as effectively as or more effectively than serum provoked by synthetic LeY. The effectiveness of peptide mimotopes as priming agents is an anticipated requirement for longer-lasting immune responses. Although there is no adequate in vivo tumor model for LeY since this antigen is not expressed on murine tumor cells, a sia- lylated homolog Lewis X (sLeX) is expressed on Meth A fibrosarco- ma tumor on a Balb/c background. Vaccination with peptide mimo- topes diminishes tumor growth in vivo in graded Meth A tumor challenge studies. Results Immunogenic mimicry of peptide motifs. Epitope mapping tech- niques have repeatedly demonstrated that antibodies bind to epi- topes of three to five amino acids within a sequence. The fine speci- ficity of some carbohydrate-binding proteins maps to peptides con- taining central W/YXY residues. Peptides with the generalized aro- matic motif W/YXY are defined to mimic several carbohydrate sub- units. These include YPY as a mimic of mannose 10,11 , WRY found to mimic a(1–4) glucose 12 , WLY to mimic LeY 6 , and YRY as a mimic of the major C polysaccharide a(2–9) sialic acid (MCP) of Neisseria meningitidis 13 . This generalized-motif feature is associated with pep- tides that are defined as mimics of glycosphingolipid constituents 9 . The neolactoseries structures Lewis X (LeX), sLeX, Lewis a (Lea), sialyl-Lea (sLea) and Lewis b (Leb) all share a common epitope topography 14 with constituents that are structurally similar to carbo- hydrates that are mimicked by the W/YXY motif 15 . Consequently, Vaccination with carbohydrate peptide mimotopes promotes anti-tumor responses Thomas Kieber-Emmons 1* , Ping Luo 1 , Jianping Qiu 1 , Tylis Y. Chang 1 , Insug O 2 , Magdalena Blaszczyk-Thurin 2 , and Zenon Steplewski 3 1 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia PA. 19104. 2 The Wistar Institute, Philadelphia PA 19104. 3 Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia PA 19104. *Corresponding author (tom@xray.med.upenn.edu) Received 2 November 1998; accepted 14 April 1999 Tumor-associated carbohydrate (TAC) antigens are important targets in cancer vaccine efforts. Carbohydrates are, however, frequently poor immunogens, in that they are T-cell–independent antigens. Molecular mimicry of TAC by peptides is an alternative approach to generating anti-carbohydrate immune responses. Here we demonstrate that peptide mimotopes can elicit antibody responses that cross-react with representative human TAC antigens. Primary immunization with such a multiple antigenic peptide, along with QS-21 as adjuvant, elicits cytotoxic antibodies reactive with naturally occurring forms of TAC expressed on tumor cells, and vaccination of mice with peptide mimotopes reduced tumor growth and prolonged host survival in a murine tumor model. Keywords: vaccine, cancer, mimotype, mimic, carbohydrate, Lewis Y, Meth A Table 1. Peptides used in these studies. Peptide name Sequence a Source 105 GGIYYPY DIYYPY DIYYPY D Designed synthetic 106 GGIYWR Y DIYWR Y DIYWR Y D Designed synthetic 107 GGIYYR Y DIYYR Y DIYYR Y D Designed synthetic 104 IMILLIFSLLWFGGA Isolated from peptide library with BR55-2 C1 GDTRYIPALQHGDKK Irrelevant control a The respective aromatic-X-aromatic motifs are underlined. © 1999 Nature America Inc. • http://biotech.nature.com © 1999 Nature America Inc. • http://biotech.nature.com