Molecular Immunology 42 (2005) 1321–1330 Subtle sequence differences in a tumour-associated peptide epitope translate into major changes in antigenicity Jonas Persson a , Johan Lantto a, 1 , Torbj¨ orn Drakenberg b , Mats Ohlin a, ∗ a Department of Immunotechnology, Lund University, P.O. Box 7031, S-22007 Lund, Sweden b Biophysical Chemistry, Lund University, Box 124, SE-22100 Lund, Sweden Received 18 November 2004; accepted 17 December 2004 Available online 16 February 2005 Abstract Antigenicity, the ability to bind to members of repertoire of diverse immune receptors, is a concept that is poorly characterised with respect to its defining parameters. To learn more about its makeup, we have investigated the ability of two peptides with highly related sequences, derived from the tumour-associated antigen mucin-1, to recruit in vitro members from a large na¨ ıve repertoire of synthetic human antibody fragments. One of the peptides represents the epitope that is immunodominant in mice. We now demonstrate that the other peptide, which differs from the first only by a very conservative aspartate-threonine to glutamate-serine change, is much less antigenic than the first peptide. This is so despite the fact that there is no observable difference in the tendency of the two peptides to adopt a structure in solution. Furthermore, the peptides differ in their immunodominant parts and the less antigenic peptide selects for antibody fragments targeting residues outside of the epitope considered to be immunodominant in mice. We conclude that subtle sequence changes greatly, affect antigenicity and immunodominance of epitopes in this important tumour-associated antigen. © 2005 Elsevier Ltd. All rights reserved. Keywords: CD227; Antigenicity; Epitope; Phage display; Peptide 1. Introduction Antigenicity is the property of being recognisable by a specific T- or B-cell receptor available in the immune sys- tem. This property is to some extent coupled to the even more complex phenomenon termed immunogenicity, i.e. the capacity not only to be recognised by but also to induce a spe- cific immune response when presented to the immune sys- tem. The importance of the immune system in the protection against disease and our ability to induce protective immu- Abbreviations: CDR, complementarity determining region; NMR, nu- clear magnetic resonance; NOE, nuclear overhauser effect; NOESY, nuclear overhauser effect spectroscopy; scFv, single-chain antibody fragment(s); TOCSY, total correlated spectroscopy; VNTR, variable number of tandem repeats ∗ Corresponding author. Tel.: +46 46 2224322; fax: +46 46 2224200. E-mail address: mats.ohlin@immun.lth.se (M. Ohlin). 1 Symphogen A/S, Elektrovej, Building 375, DK-2800 Lyngby, Denmark. nity through vaccination makes it important to understand the principles underlying these processes. Despite much ef- fort (summarised by Pellequer et al., 1994), it is still difficult to predict the actual antigenicity/immunogenicity of differ- ent structures within a more complex molecule. It depends not only on the context in which the molecule is presented but also on the characteristics of the immune repertoire that is to recognise it (Van Regenmortel, 2001). The first fac- tor, the nature of the antigen itself, is a critical component in this respect. For example, a few residues in the amino- terminus of hen egg lysozyme were shown to be particularly well recognised upon immunisation, and their removal di- minished the immunogenicity of the entire protein (Wicker et al., 1984). Similarly, the immune response in many anti- gens is focused to one or a few immunodominant structure(s) while leaving the rest of the molecule largely unable to mount a response. Specific mutations of an antigen may; however, reduce the immunogenicity or shift it to other regions of 0161-5890/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2004.12.010