The glycosylated adhesion domain of human CD2: glycan structure and significance for the interaction with CD58

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Abstracts

[7] Stanfield, R.L., Fieser, T.M., Leruer, R.A. and Wilson, I.A. (1990) Structure of an anti-peptide antibody B1312 and its complex with a peptide antigen. Science 248, 637644. [8] Haynes, M.R., Stura, E.A. Hilvert, D. and Wilson, I.A. (1994) Routes to catalysis: The structure of a catalytic antibody and comparison with its natural counterpart. Science 263, 646-652. Oiigosaecharide epitopes and antibody binding sites of Fab and single chain Fv molecules. D.R. Bundl@, J.-R. Brisson~, M. Cygler b, C.R. MacKenzie~, S.A. Narang¢, N.M. Young¢, aDepartment of Chemistry, Uni-

versity of Alberta, Edmonton, Alberta, bBiotechnology Research Institute, Montreal, Clnstitute for Biological Sciences, Ottawa, National Research Council of Canada, Canada. Crystal structures have shown that the combining sites of three distinct antibodies, are complimentary to oligosaccharide epitopes that are relatively small, i.e. 2-4 hexoses residues, and may be either of a groove or cavity type. The monoclonal antibody S.e.-155.4 has been studied in most detail: crystal structures of native Fab, six oligosaccharide-Fab complexes [1,2] (including two mutant Fab-oligosaccharide complexes) and one trisaccharide-single chain Fv complex [3]. Aromatic amino acids located in 5 hypervariable loops are the major contact residues. Three hexose residues fill the site and weak to medium strength hydrogen bonds involve neutral donor-acceptor pairs. Two distinct bound ligand conformations correlate with solution thermodynamics and NMR measurements [2]. Bound water molecules amplify the number of hydrogen bonds between protein and carbohydrate and, in two structures, a water molecule modifies the conformation of an exposed saccharide residue, Structural modification of the antigen by functional group replacement has identified the crucial structural elements of the epitope [4] and thermodynamic data point of the importance of hydrophobic interactions [5]. Mutagenesis has been used to investigate the opportunities for redesign of carbohydrate binding sites [6]. Fully functional Fab was expressed in E. coli using chemically-synthesized genes and these genes were rearranged to provide active singlechain Fv constructs [7]. Screening of phage display libraries for tighter binding mutants demonstrated a bias for selection of single chain binding site mutants that formed multivalent oligomers with avidity up to 1000-fold that of the wild-type univalent Fv protein [8]. Binding site affinity was not increased by rational design and only weakly by screening phage display libraries. [1] Cygler, M., Rose, D.R. and Bundle, D.R. (1991) Science 253, 442-446. [2] Bundle, D.R., Baumann, H., Brisson, J.R., Gagne, S.M., Zdanov, A. and Cygler, M. (1994) Biochemistry 33, 51835192. [3] Zdanov, A., Li, Y., Bundle, D.R., Deng, S.-J., MacKenzie, C.R., Narang, S.A., Young, N.M. and Cygler, M. (1994) Proc. Natl. Acad. Sci. USA 91, 6423-6427.

[4] Bundle, D.R., Eichler, E., Gidney, M.A.J., Meldal, M., Ragauskas, A., Sigurskjold, B.W., Sinnott, B., Watson, D.C., Yaguchi, M. and Young, N.M. (1994) Biochemistry 33, 5172-5182. [5] Sigurskjold, B. and Bundle, D.R. (1992) J. Biol. Chem. 267(12), 8371-8376. [6] Brummell, D.A., Sharma, V.P., Anand, N.N., Bilous, D., Dubuc, G., Michniewicz, J., MacKenzie, C.R., Sadowska, J., Sigurskjold, B.W., Sinnott, B., Young, N.M., Bundle, D.R. and Narang, S.A. (1993) Biochemistry 32, 11801187. [7] Deng, S.-J., MacKenzie, C.R., Sadowska, J., Michniewicz, J., Young, N.M., Bundle, D.R. and Narang S.A. (1994) J. Biol. Chem. 269, 9533-9538. [8] Deng, S.-J., MacKenzie, C.R., Hirama, T., Brosseau, R., Lowary, T., Young, N.M., Bundle, D.R. and Narang S.A. (1995) Proc. Natl. Acad. Sci. USA 92, 4992-4996. The glycosylated adhesion domain of human CD2: glycan structure and significance for the interaction with CD58. Gerhard Wagnera, Johnathan S. Cholia, Jing Lia, Alex Smolyarb, Ellis Reinherzb, Maria H. Knopper~, Kevin J. Willisc, Antonio R.N. Arulanandam~, Daniel F. Wyssc, aHarvard

Medical School, Boston, MA, 02115, bDana Father Cancer Institute, Boston, MA 02115, cProcept Inc, Cambridge, MA 02139, USA. The human T-cell surface glycoprotein receptor CD2 is important for T lymphocytes to mediate their regulatory and effector function. The amino-terminal domain of human CD2 is responsible for cell adhesion by binding to the counter receptor CD58 on antigen - - presenting cells or target cells. The adhesion domain of human CD2 contains a single highmannose N-glycan which is, at least in part, crucial for the adhesion function. Here, a refined NMR structure of the adhesion domain of hCD2 is presented. The binding site of the counter receptor CD58 was identified from mutations of surface exposed side chains, as revealed by the solution structure. The single high mannose glycan is linked to the side chain of Asn65. The chemical structure and the heterogeneity of the glycan has been characterized with electrospray mass spectroscopy and the composition of the different forms has further been elucidated by NMR, in particular by betero-nuclear 1H-~3C experiments. The conformation of the carbohydrate was determined based on numerous NOEs between the glycan and the protein and within the glycan. Three of the four branches of the carbohydrate show a well defined conformation. Presence of the single high-mannose glycan attached to Asn65 is crucial for adhesion function of hCD2. Complete removal of the glycan by PNGaseF or by mutation of Asn65 or Thr67 leads to loss of adhesion function as measured in cell-based assays, hCD2 treated with endo H which removes all of the glycan but the first GlcNAc retains adhesion function. As found by circular dichroism spectroscopy, the loss of hCD2 function upon glycan elimination is due to destablilization and unfolding of the adhesion domain. By mutational screening of protein residues near the carbohydrate, we found that the K61E mutation eliminates the need of a carbohydrate

Abstracts for hCD2 function. K61 is located for the center of a cluster of five lysines. This cluster of positive charges des~b'dizcs the protein, an effect that is counter balanced by the stabilizing effect of the nearby giycan. The K61E mutation partially neutralizes the excess of positive charge and eliminates the need for a stabilizing glycan. Molecular basis of antigen mimicry by an anti-idiotopic antibody. Fernando A. GoldbaumL Barry A. Fields b, Carlos A. Fossatia, William Dall'Acqua b, Roberto J. Poljak b, Xavier Ysern% Roy A. Mariuzza b, ~Instituto de Estudios de la Immunidad Humoral, Buenos Aires, Argentina, bCARB, University of Maryland Biotechnology Institute, Rockville, AID, cCenter for Drug Evaluation and Research F.D.A., Rockville, AID, USA. Functional mimicry of biological macromolecules by antiidiotypic antibodies has been described in several systems and has encouraged the use of anti-idiotypic antibodies as surrogate antigens. To explore the molecular basis of the functional antigen mimicry by anti-idiotypic antibodies, we have determined the crystal structure of an idiotope-anti-idiotope complex between the protein engineered, bacterially expressed Fv fragments of the anti-lysozyme antibody D1.3 (BALB/c; IgG1, •) and the anti-D1.3 antibody E5.2 (BALB/c; IgGl, x), to 1.9 A resolution. The six CDRs of each Fv participate in the idiotope-anti-idiotope contacts; the VHCDR3 of E5.2 accounts for 77% of the total contacts to D1.3. Except to V L Tyr49 of both antibodies, no framework residues are involved in the contacts. Of the 18 Dl.3 residues contacting E5.2 and the 17 that contact HEL, 13 contact both E5.2 and HEL. Thus, E5.2 mimics HEL in its binding to Dl.3. Conservation of hydrogen bonding is an important aspect of this mimicry: six of the l l interface hydrogen bonds in the Dl.3-E5.2 interface are structurally equivalent to those in the DI.3-HEL complex. The atoms by which the anti-idiotopic E5.2 contacts D1.3 are close in space to those of lysozyme that contact D1.3. Thus, the observed structural mimicking is functional, providing a substitute binding structure rather then an exact molecular replica. It does not require sequence homology between antigen and anti-idiotopic antibody. To verify these structural observations, E5.2 was used as an immunogen in BALB/c and C57BL mice, and was capable of inducing an anti-lysozyme response in some of the immunized animals. In further experiments to clarify the structural basis of mimicking and the idiotypic network in this system, we have used the Abl, D1.3, to immunize rabbits. The xenogeneic, polyclonal anti-idiotypic antibodies thus obtained were in turn used as immunogens in BALB/c mice. This approach gave a strong anti-anti-idiotypic response in which an anti-E5.2 and an anti-HEL activity could be substantiated. An anti-anti-idiotypic monoclonal antibody, AF14 (IgG1, x) was obtained that reacts with E5.2 and with HEL. These reactions are inhibited by Fab D1.3. Amino acid sequence determination and crystallization attempts will be performed using Fab and Fv fragments from AFI4, free and complexed with antigen and with the anti-idiotope E5.2, to further investigate the molecular basis of this idiotypic network. In parallel with this approach, we have obtained a

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eDNA library of V n and V L genes which will be expressed in a phage display system to search for a single chain Fv fragment reactive against HEL and E5.2. Human antibody diversity. Ian M. Tomlinson a, Gerald Waltera'b, Peter T. Jones ax, Paul H. Deaff, Erik L.L. Sonnhammerd, Greg Winter~l'c, ~MRC Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, U.K., bCambridge Antibody Technology Ltd., The Science Park, Melbourn, Cambridgeshire SG8 6JJ, U.K., CMRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K., dSanger Centre, Hinxton Hall, Cambridge CB10

IRQ, u.K. Our knowledge of the human germline V gene repertoire is complete. There are surprisingly few V, D and J segments and they encode a small repertoire of main-chain conformations, or canonical structures. Sequence diversity encoded by these germline segments in the primary repertoire is concentrated at the centre of the antigen binding site. In contrast, somatic mutation spreads diversity to regions at the periphery of the binding site that are highly conserved in the primary repertoire, whilst conserving the canonical structures of the antigen binding loops. The two patterns of sequence diversity are therefore complementary, antibodies in turn using two intermeshing regions of the antigen binding site to make and/or facilitate binding contacts. We propose that evolution has favoured this complementarity as an efficient strategy for searching sequence space. Some sites have undergone germline diversification, whilst others have been conserved for alteration by somatic hypermutation. In this way, somatic hypermutation has left an evolutionary imprint on the sequences of the human V gene segments. An understanding of how natural antibody repertoires are produced in vivo should facilitate the design and construction of synthetic antibody repertoires which mimic these processes in vitro.

Engineering of anti-digoxin anybody combining sites. Michael N. Margolies a, Mary K. Short a, Christian C. Titlow a, Philip D. Jeffreyb, Steven Sheriff:, aMassachusetts General Hospital and Harvard Medical School, Boston, MA 02114, bMemorial Sloan-Kettering Cancer Center, New York, "BristolMyers Squibb Pharmaceutical Research Institute, Princeton, USA. Murine monodonal digoxin-specific antibodies display exceptional affinity for their site-filling, relatively rigid hydrophobic ligand, for which there are numerous analogues of known stereochemistry. Anti-digoxin antibodies utilize diverse V region genes and exhibit varying specificity for these cardiac glycoside analogues. Studies of structure-function relationships and antibody combining site engineering are made feasible by the X-ray crystallographic structures of two different antidigoxin Fabs, utilizing entirely different variable region sequences, in complex with digoxin (Fab 26-10) and with ouabain (Fab 40-50). DNA encoding these anti-digoxin Fabs were doned into the bacteriophage expression vector pComb3. Mutant Fabs with unique specificity obtained from phage-displayed libraries produced by saturation mutagenesis of com-