University of Groningen

Molecular aspects of antibody-antigen interactionsSchellekens, Gerardus Antonius

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Chapter 1

General Introduction

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1.1 The antibody molecule

All vertebrates produce antibodies as a response to an invasion of micro-organismsor foreign substances. Antibody or immunoglobulin (Ig) molecules are responsiblefor two major biological functions: (1) a receptor function for the recognition offoreign antigens such as toxins, viruses, and exposed molecules on the surface ofpathogenic organisms; and (2) an effector function which results in the eliminationor inactivation of the foreign antigen or the cell marked by the presence of thatantigen [Roitt et al., 1993]. This ability to differentiate between antigens has madeantibodies indispensable tools in diagnostic medicine and in the biomolecularsciences. The discovery of Köhler and Milstein [1975] that antibodies of a singlepredetermined specificity can be produced in vitro as homogeneous proteinmolecules known as monoclonal antibodies (mAbs), has extended the possibility tostudy the nature of the antibody-antigen interaction at the structural and functionallevel.

Figure 1. Schematic representation of the antibody (IgG) molecule showing itsdomains and other features.

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The antibody molecule consists of four polypeptide chains. Two identical lightchains with a mass of around 25 kD and two identical heavy chains with a mass ofaround 50 kD. The polypeptides are linked by disulfide bridges connecting eachlight chain to a heavy chain and linking the two heavy chains to one another. The N-terminal parts of both heavy and light chain contain a variable domain (VH and VL)that together form the antigen binding region, of which two are present in themolecule (IgG serves as an archetype in this description; see figure 1). Comparisonof the amino acid sequences of VH and VL domains revealed that in each chain threeregions of high variability can be distinguished. These hypervariable regions, oftenreferred to as ‘complementarity determining regions’ (CDRs) determine antibodyspecificity as postulated by Wu and Kabat [1970]. The ability of the immune systemto produce specific antibodies with high affinity against a countless pool of antigensimplies that the diversity in the genes encoding the variable regions must be veryhigh.

1.2 The specificity of antibodies

Two processes determine the diversity of the antibody repertoire that is necessaryfor a specific antibody response. In the first, that occurs during the differentiation ofpre-B cell to mature B cell, gene segments rearrange to create a large diverse pool ofVH and VL germline sequences. In the second process, ‘somatic hypermutation’, thatoccurs during the differentiation of B cell to plasma cell, the affinity and specificityof the antibody response is fine-tuned by the introduction of mutations within thegermline sequences of VH and VL.

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Figure 2. The formation of the functional V L and VH domains by V-J and V-D-Jjoining.

The V domains of both light and heavy chain are the product of a series of generearrangements. For example, the VL domain is formed by the joining of two geneticelements, Vκ (or Vλ) and Jκ (or Jλ). The Vκ elements, of which around 50 functionalelements are present, encode the first three framework regions (FR1, 2 & 3), the firsttwo CDRs entirely, and the amino terminal portion of CDR3. The Jκ element, ofwhich five are present, encodes the remaining portion of CDR3 and the completeFR4 (figure 2).

Because each Vκ gene segment has the potential to rearrange to any of the five Jκelements, 250 distinct V domains can be encoded. Additional diversity is generatedat the site at which gene segments connect due to the fact that the termini of eachgene segment can undergo loss of 1-5 nucleotides during the recombination process(‘junctional diversity’). It is evident that at this V-J junction the majority of theinitial diversity in the light chain can be found.

The rearrangements that occur in the formation of the V domain of the heavy chainis more complex. The domain is formed by the joining of three genetic elements: theVH gene segment, of which more than 100 are present, is connected to one of 30 Dgene elements, that itself can be linked to one of 6 JH gene segments. The VH

element encodes FR1, 2 & 3, CDR1 & 2, and the JH element encodes FR4 entirely.

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All three VH, D and JH segments contribute to the diversity of the heavy chain CDR3(figure 2). Combinatorial rearrangements of VH, D and JH can theoretically generate1.8 x 104 different variable domains. During the splicing process nucleotides can belost from the genetic elements, or added in a random (‘N’ nucleotides) or templated(‘P’ junction) fashion. In total, potentially more than 109 different V-D-J junctionscan be generated. Random combination of H and L chain partners yields a potentialpre-immune antibody repertoire larger than 1013 different immunoglobulins.

Upon exposure to antigen and T-cell help, the variable domain genes undergosomatic mutation. This mechanism, unique to B cells, allows the affinity maturationof the antibody response to repeated immunization. The rate of somatic mutation canbe as high as 10-3 per base per cell cycle during the transition of B cell to plasma cell[Wysocki et al., 1986].

1.3 Antibody-antigen interactions

Studies on how the molecular diversity of antibodies, due to the organization at thegenetic level, is translated into antigen specificity at the molecular level can bedivided into structural studies and functional studies. Structural studies areperformed by determining the three-dimensional structures of antibody fragmentsand antibody-antigen complexes using X-ray crystallography. Functional studiesfocus on the kinetics and thermodynamics of the antibody-antigen interaction oftencombined with mutagenesis. Knowledge concerning the relation between structureand function is indispensable in efforts to rationally adapt antibodies to specificneeds. However, some aspects regarding the nature of the antibody-antigeninteraction are still subject of debate.

1.3.1 Structural studies

The number of structures of antibody-antigen complexes determined at a resolutionthat is sufficient for a detailed inspection of the antibody-antigen interface islimited. Five medium-resolution crystal structures (2.5-3.0 Å) of complexes betweenFab fragments and protein antigens are studied in detail [Webster et al., 1994].Three antibodies are directed against lysozyme (D1.3, HyHEL10 and HyHEL5)[Amit et al., 1986; Sheriff et al., 1987; Padlan et al., 1989], one against influenzavirus neuraminidase (NC41) [Colman et al., 1987] and an antiidiotype antibodyagainst D1.3 [Bentley et al., 1990]. More refined structures have been reported forthe Fab-antigen complex of D1.3 (2.5 Å) [Fischmann et al., 1991] and NC41 (2.9 Å)

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[Tulip et al., 1992]. The more recently reported high-resolution structure (1.8 Å) ofthe D1.3 Fv fragment, free and complexed to lysozyme [Mariuzza & Poljak, 1993],has led to adjustments of some aspects deduced from the earlier structures i.e. thenumber of water molecules either buried or very near the interface was much largerthan reported in the medium-resolution studies. The general features of theantibody-protein complexes can be summarized as follows: contacts betweenantibody and antigen are spread over a relatively large buried surface area (680-879Å2). There is a high degree of size complementarity, but electrostaticcomplementarity is not always high [Novotny & Sharp, 1992]. Antibody residues inclose contact with the antigen are mainly found in the CDRs. Conformationalchanges of the Fab upon antigen binding are small (1-2 Å for backbone, around 2 Åfor side chains) [Bhat et al., 1990]. Evidence for conformational changes can only bepresented in those cases where the structure of the free Fab is determined.

Three medium-resolution structures of Fabs complexed to peptide antigens werereported. These include Fab 17/9 complexed to a peptide from influenzahaemagglutinin [Rini et al., 1992], Fab TE33 complexed to cholera toxin [Shoham,1993], and Fab 50.1 complexed to the V3 loop peptide of gp120 of humanimmunodeficiency virus [Rini et al., 1993]. The buried surface area is smaller thanfor protein antigens (400-500 Å) and the combining site can be described better as agroove rather than the planar combining site found in Fab-protein complexes. Alarge conformational change occurs upon peptide binding in Fab 17/9 and Fab 50.1.A twist occurs about the long axis of the CDR3 loop that gives rise to residuemovement of up to 5 Å [Webster et al., 1994]. In the bound state the V3 loop andcholera toxin peptides adopt a regular secondary structure when bound to theantibody in the form of a β-turn. It is however not certain if these structuralconstraints are already present in solution or if these secondary structure elementsare the same as in the original protein [Rini et al., 1992]. The binding mechanismhere can be described as an ‘induced fit’ because large conformational changes inthe antibody and possibly in the antigen are needed for complex formation.

By examination of the known Fab structures, Chothia et al. [1989] found that five ofthe six hypervariable loops (L1, L2, L3, H1 & H2) are limited to a few main chainconformations depending on length and the occurrence of specific residues atspecific sites in the CDR or close to the CDR. This means that the occurrence ofcertain residues and the length of the CDR can predict the conformation of these

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loops. No such prediction can be made for the H3 CDR that is the most variable bothin sequence as well as in length [Davies & Chacko, 1993].

Structural studies on antibody complexes can give information about the nature ofthe antibody-antigen interface and any conformational changes that occur oncomplex formation. It can, however, not fully explain the mechanisms behind theaffinity and specificity of the interactions. There is, for example, no clear correlationbetween the affinity of the antibody for its antigen and the number of contactingresidues or the size of the contact surface area [Webster et al., 1994].

1.3.2 Functional studies

Comparison of X-ray data of antibody-antigen complexes with immunological datamay give paradoxes. The number of residues that define an epitope is typicallyaround four, yet the number of residues involved in contact, as seen in X-ray data, isabout ten to fifteen on each side [Novotny et al., 1989]. Nuss et al. [1993] identifiedfive residues of influenza virus neuraminidase with a large contribution to bindingto mAb NC41. The total number of residues in contact with the antibody, as seen inthe complex structure, is 19. Kelley & O’Connell [1993] used micro-calorimetry andsurface plasmon resonance combined with mutagenesis of the CDRs of antibodyhu4D5-5 to identify four residues that make a large contribution to the free energyof binding and the affinity of the antibody for the antigen. Estimates made of thebinding strength using structural data, concerning all contact residues, exceed by farthose derived from direct measurements [Novotny et al., 1989]. From theexperimental data it can be concluded that only a fraction of the residues in closecontact in the complex contribute to binding.

There is still much debate concerning the actual force that drives the affinity ofbinding. Experimental data was presented that suggests that hydrogen bonds, saltbridges and van der Waals interactions determine affinity (enthalpy driven) [Tello etal., 1993]. Other experimental data was presented which suggests a large role for theso-called ‘hydrophobic effect’; the loss of the ordered structure of water moleculespresent at the surfaces that are buried by the interacting molecules upon binding.This implies that the interaction is entropy driven [Kelley & O’Connell, 1993].Experimental data [Ito et al., 1993; Hibbits et al., 1994], confirming thethermodynamic model of enthalpy-entropy compensation presented by Novotny etal. [1989], suggests that complex formation is entropy driven at low temperatures,but enthalpy driven at higher temperatures when favorable and unfavorable entropic

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effects balance each other. In the cases where entropic forces seem to play a key rolein the interaction, the most important structural feature of complex formation issurface complementarity; an exact ‘fit’ of the antibody and antigen surfacesinvolved in the interaction. This would have implications for efforts to modulate thebinding characteristics of antibody molecules by ‘protein engineering’. Subtlechanges in the shape of the antigen binding-site of antibody molecules by aminoacid replacement could have a large and unpredictable effect on the bindingcharacteristics, even if these residues are not within a CDR or even in contact withthe antigen. Such effects on binding by replacement of non-contact frameworkresidues have been reported [Foote & Winter, 1992].

It is likely that antibody molecules show, as they do in their amino acid sequence, alarge amount of variability in the way they bind the antigen in respect to kinetics andthermodynamics of the interaction. Therefore it is difficult to formulate general rulesthat govern binding, especially if there are only a few examples of complexes thatare studied in detail at both the structural and functional level. Multi-dimensionalnuclear magnetic resonance (NMR) spectroscopy of antibody Fv fragments [Freundet al., 1994] and antibody-antigen complexes [Anglister et al., 1993] could not onlybe a faster way to obtain structural information, but could also allow the study of thedynamic processes occurring during complex formation. However, the NMRtechnique puts restrictions on the size, solubility and stability of the proteins underinvestigation [Gronenborn & Clore, 1994]. Therefore small, soluble, stable andmonomeric antibody fragments that should also retain antigen-binding capability areneeded.

1.4 Engineering antibodies

Antibodies are used widely in medicine and science as indicator molecules. Thespecific binding properties are used in countless clinical diagnostic tests, and for theidentification and quantification of antigens under study in the laboratory, wheretechniques as immunoblotting, immunoprecipitation, enzyme-linked immunosorbentassay (ELISA) and radioimmuno- assay (RIA) are indispensable. Antibodies are alsoincreasingly used for other applications such as purification of biomolecules(immunoaffinity chromatography), for both diagnostic (imaging) and therapeuticapplications in vivo, and even for catalysis of chemical reactions [Plückthun, 1990;Li et al., 1994]. There are three main topics in modification of antibodies for specificapplications: size reduction of antibodies, humanization of antibodies and binding-

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modulation by mutation including ways to select and produce antibody fragments byusing E. coli as a host. The latter two topics will be discussed briefly.

Human monoclonal antibodies can not be made by hybridoma technology.Humanization of antibodies of rodent origin tries to circumvent the problem of animmune response against these antibodies in in vivo applications. The strategies thatcan be used range from the construction of chimeric molecules, CDR grafting, andthe selection and production of totally human antibody fragments in E. coli [Lefranc& Lefranc, 1990; Winter & Milstein, 1992; George, 1994].

For binding modulation the method developed by Winter and coworkers to expresslibraries of antibody-fragments coding genes on the surface of a filamentousbacteriophage has received considerable attention. Antibody fragments can beselected on binding in vitro and can be subsequently expressed in the periplasm ofE. coli. The antibodies can be of naive source [Hoogenboom & Winter, 1992] orderived after immunization [Clackson et al., 1991] or even semi-synthetic byrandomizing one or more CDRs [Griffiths et al., 1994]. Antibody fragments fromrodent as well as human source can be selected and produced in this way. This workhas stimulated interest in the production and modifications of recombinantantibodies of which only a few examples can be mentioned: fusion of recombinantantibody fragments to toxins [Buchner et al., 1992] or reporter enzymes [Kohl et al.,1991], the production of bivalent or bispecific recombinant antibody fragments[Pack et al., 1993; Holliger et al., 1993], the production of antigenized antibodies[Zaghouani et al., 1993] and recently the production of ‘chelating recombinantantibodies’ (CRAbs). CRAbs are bispecific dimeric scFv fragments constructed torecognize two independent epitopes on the same antigen that bind the antigen withvery high affinity [Neri et al., 1995]. One of the advantages of selecting antibodiesfrom phage libraries is that antigens can be used that give difficulties inimmunization e.g. instable antigens or autoantigens. Obviously the production ofantibody fragments in E coli offers other advantages e.g. the possibility ofsimplifying mutagenesis studies and the possibility of labeling the antibodyfragment for structural studies using multidimensional NMR spectroscopy [Skerra,1993].

Size reduction has several advantages in different fields. As can be seen in figure 1,only a small part of the antibody molecule is involved in antigen binding. Theantigen binding domain makes up one sixth of the molecule. Reduction of the size ofthe antibody molecule to the size of the Fv, or even smaller, would be beneficial in

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immunoaffinity and biosensor applications because of the gain in stability of theligand [Welling et al., 1990-91]. Smaller molecules are also shown to have a bettertissue penetration and faster blood clearance in in vivo applications [George, 1994;Gruber et al., 1994]. Smaller molecules would also come within the size limits ofNMR investigation, as already discussed in the previous section.

A classical method used in the size reduction of antibody molecules is the digestionof immunoglobulins with pepsin, which results in the bivalent F(ab')2 fragment, orpapain that yields the monovalent Fab fragment [Roitt et al., 1993]. Occasions atwhich proteolytic cleavage yields an Fv (‘fragment variable’) are very rare [Givol,1991]. These Fv molecules consist of the VH and VL fragments held together, in anunstable manner, by non-covalent interactions. The recombinant version of the Fvfragments can be stabilized by linking the VH and VL domain with a flexible peptidelinker. Such a molecule is referred to as a single chain Fv (scFv) [Bird et al., 1988].Fvs can also be stabilized by chemical cross-linking of VH and VL usingglutaraldehyde or introducing disulfide bridges by substitution of cysteine residuesat appropriate sites in the VH and VL domain [Glockshuber et al., 1990]. Antigenbinding of isolated VH domains has been reported [Ward et al., 1989] but theirusefulness is limited due to their low solubility. This is probably caused by thepresence of hydrophobic regions, normally buried by interaction with VL.

1.5 Antigen binding peptides

An even more rigorous way of size-reduction of the antibody would be the use ofsynthetic peptides based on the sequence of one or two CDRs. The advantages of theuse of synthetic peptides would be their small size and stability and the ability toproduce peptides in large quantities through solid phase synthesis. Syntheticpeptides, based on the sequence of a single CDR, with affinity towards the antigenhave been reported [Taub et al., 1989-92; Williams et al., 1989-90; Welling et al.,1990-91; Levi et al., 1993]. In the work of Taub and Williams, which is reviewed byTaub & Green [1992], CDR peptides, derived from anti-receptor antibodies, sharedbiological activities with the original ligand. Also similar sequence patterns sharedby the CDR peptide and the receptor-binding part of the ligand were identified.Welling and coworkers [1990] showed that synthetic peptides derived from anti-lysozyme antibodies retarded lysozyme in affinity-chromatography. Levi andcoworkers [1993] reported a CDR peptide derived from an antibody raised against

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the V3 loop of gp120 with affinity towards a V3 loop peptide and gp120. Thesereports will be discussed in more detail in section 2.4.3.

1.6 Random peptide libraries and antibody-antigen interactions

The idea of constructing large libraries of peptides by inserting random stretches ofDNA in the gene coding for a coat protein of filamentous bacteriophage [Smith,1985] and the development of experimental procedures for the selection of phagebinding to an antibody [Parmley & Smith, 1988] opened up the possibility to screenlarge peptide pools for antibody binding [Scott & Smith, 1990; Cwirla et al., 1990;Barret et al., 1992; Stephen & Lane, 1992; Dower, 1992; Christian et al., 1992;Böttger & Lane, 1994; Böttger et al., 1995; Stephen et al., 1995]. In most cases theconformation-independent epitope could be identified. In the reports whereantibodies were used that recognize conformation-dependent epitopes [Felici et al.,1993; Hoess et al., 1994] binding peptides were identified that had no sequencesimilarity with the antigen. It was suggested that these peptides mimic the epitopesshape and they are referred to as ‘mimotopes’. Phage libraries have also been usedfor the selection of peptides binding to non-antibody targets e.g. streptavidin[Devlin et al., 1990], tumor necrosis factor α [Oldenburg et al., 1992], concanavalinA [Scott et al., 1992], fibrinogen receptor GPIIb/IIIa [O’Neil et al., 1992], molecularchaperone BiP [Blond-Elguindi et al., 1993]. Recently it was investigated whetherphage-displayed peptide libraries can be used to identify disease-specific epitopesby screening with polyclonal sera from rheumatoid arthritis patients [Dybwad, et al.,1993] and from individuals that were immunized with a hepatitis B virus vaccine[Folgori et al., 1994]. Synthetic peptide libraries derived by the split-synthesisstrategy have also been useful in identifying peptide ligands [Houghten et al., 1991;Lam et al., 1991]. The phage display technology has not been restricted to librariesof peptides or antibody-fragments. A growing number of partly randomized proteindomains was succesfully displayed on phage [Hoess, 1993; Wu et al., 1995].

1.7 Aim of this study

The aim of this study is to identify the minimal parts of both antibody and antigeninvolved in antigen binding. For this purpose sub-fragments of the VH and VL

domain, either cloned and expressed in E. coli or synthetic peptides based on CDRs,were produced and binding was evaluated. Monoclonal antibody A16, directedagainst herpes simplex virus, was used as a model system (chapter 2). In chapter 3

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the use of random peptide libraries combined with peptide synthesis for highresolution epitope mapping are explored. Also the more general use of such librariesfor the identification of peptides interacting with a non-antibody target wasevaluated. In chapter 4 a method is presented to identify binding clones from aphage display library using biosensor technology.

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1.8 References

Amit, A.G., Mariuzza, R.A., Phillips, S.E.V. & Poljak, R.J. (1986) Three-dimensionalstructure of an antigen-antibody complex at 2.8 Å resolution. Science 233, 747-758.

Anglister, J., Scherf, T., Zilber, B., Levy, R. Zvi, A., Hiller, R. & Feigelson, D. (1993)Two-dimensional NMR investigations of the interaction of antibodies with peptideantigens. FASEB Journal 7, 1154-1162.

Barrett, R.W., Cwirla, S.E., Ackerman, M.S., Olsen, A.M., Peters, E.A. & Dower, W.J.(1992) Selective enrichment and characterization of high affinity ligands fromcollections of random peptides on filamentous phage. Analytical Biochemistry 204,357-364.

Bentley, G.A., Boulot, G., Riottot, M.M. & Poljak, R.J. (1990) 3-dimensionalstructure of an idiotype-anti-idiotype complex. Nature 348, 254-257.

Bhat, T.N., Bentley, G.A., Fischmann, T.O., Boulot, G., & Poljak, R.J. (1990) Smallrearrangements in structures of Fv and Fab fragments of antibody D1.3 on antigenbinding. Nature 347, 483-485.

Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lec, S.M.,Lee, T. & Popo, S.H. (1988) Single chain antigen binding fragment. Science 242,423-426.

Blond-Elguindi, S., Cwirla, S.E., Dower, W.J., Lipshutz, R.J., Sprang, S.R., Sambrook,J.F. & Gething, M.H. (1993) Affinity panning of a library of peptides displayed onbacteriophages reveals the binding specificity of BiP. Cell 75, 717-728.

Böttger, V. & Lane, E.B. (1994) A monoclonal antibody epitope on keratin 8identified using a phage peptide library. Journal of Molecular Biology 235, 61-67.

Böttger, V., Böttger, A., Lane, E.B. & Spruce, B.A. (1995) Comprehensive epitopeanalysis of monoclonal anti-proenkephalin antibodies using phage display librariesand synthetic peptides: revelation of antibody fine specificities caused by somaticmutations in the variable region genes. Journal of Molecular Biology 247, 932-946.

Buchner, J., Pastan, I. & Brinkmann, U. (1992) A method for increasing the yield ofproperly folded recombinant fusion proteins: single-chain immunotoxin fromrenaturation of bacterial inclusion bodies. Analytical Biochemistry 205, 263-270.

Chothia, C., Lesk, A.M., Tramontano, A., Levitt, M., Smith-Gill, S.J., Air, G., Sheriff,S., Padlan, E.A., Davies, D. & Tulip, W.R. (1989) The conformations ofimmunoglobulin hypervariable regions. Nature 342, 877-883.

Clackson, T., Hoogenboom, H.R., Griffiths, A.D. & Winter, G. (1991) Makingantibody fragments using phage display libraries. Nature 352, 624-628.

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Colman, P.M., Laver, W.G., Varghese, J.N., Baker, A.T., Tulloch, P.A., Air, P.A. & Webster, R.G. (1987) Three-dimensional structure of a complex of antibody withinfluenza virus neuraminidase. Nature 326, 358-363.

Christian, R.B., Zuckerman, R.N., Kerr, J.M., Wang, L. & Malcolm, B.A. (1992)Simplified method for construction, assessment and rapid screening of peptidelibraries in bacteriophage. Journal of Molecular Biology 227, 711-718.

Cwirla, S.E., Peters, E.A., Barret, R.W. & Dower, W.J. (1990) Peptides on phage: avast library of peptides for identifying ligands. Proceedings of the NationalAcademy of Science USA 87, 6378-6382.

Davies, D.R. & Chacko, S. (1993) Antibody structure. Accounts of ChemicalResearch 26, 421-427.

Devlin, J.J., Panganiban, L.C. & Devlin, P.E. (1990) Random peptide libraries: asource of specific protein binding molecules. Science 249, 404-406.

Dower, W.J. (1992) Phage power. Current Biology 2, 251-253.

Dybwad, A., Øystein, F., Kjeldsen-Kragh, J., Natvig, J.B. & Sioud, M. (1993)Identification of new B cell epitopes in the sera of rheumatoid arthritis patients usinga random nanopeptide phage library. European Journal of Immunology 23, 3189-3193.

Felici, F., Luzzago, A., Folgori, A. & Cortese, R. (1993) Mimicking of discontinuousepitopes by phage-displayed peptides, II. Selection of clones recognized by aprotective monoclonal antibody against the Bordetella pertussis toxin from phagepeptide libraries. Gene 128, 21-27.

Fischman, T.O., Bentley, G.A., Bhat, T.N., Boulot, G., Mariuzza, R.A., Phillips, S.E.V.,Tello, D. & Poljak, R.J. (1991) Crystallographic refinement of the three-dimensionalstructure of the FabD1.3-lysozyme complex at 2.5-Å resolution. Journal ofBiological Chemistry 266, 12915-12920.

Folgori, A., Tafi, R., Meola, A., Felici, F., Galfré, G., Cortese, R., Monaci, P. &Nicosa, A. (1994) A general strategy to identify mimotopes of pathological antigensusing only random peptide libraries and human sera. EMBO Journal 13, 2236-2243.

Foote, J. & Winter, G. (1992) Antibody framework residues affecting theconformation of the hypervariable loops. Journal of Molecular Biology 224, 486-499.

Freund, C., Ross, A., Plückthun, A. & Holak, T.A. (1994) Structural and dynamicproperties of the Fv fragment and the single-chain Fv fragment of an antibody insolution investigated by heteronuclear three-dimensional NMR spectroscopy.Biochemistry 33, 3296-3303.

George, A.J.T. (1994) Antibody engineering. Endeavour 18, 27-31.

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Givol, D. (1991) The minimal antigen-binding fragment of antibodies: Fv fragments.Molecular Immunology 28, 1379-1386.

Glockshuber, R., Malia, M., Pfitzinger, I. & Plückthun, A. (1990) A comparison ofstrategies to stabilize Immunoglobulin Fv-Fragments. Biochemistry 29, 1362-1367.

Griffiths, A.D., Williams, S.C., Hartley, O., Tomlinson, I.M., Waterhouse, P., Crosby,W.L., Konterman, R.E., Jones, P.T., Low, M., Allison, T.J., et al. (1994) Isolation ofhigh affinity human antibodies directly from large synthetic repertoires. EMBOJournal 13, 3245-3260.

Gronenborn, A.M. & Clore (1994) Where is NMR taking us? Proteins: Structure,Function, and Genetics 19, 273-276.

Gruber, M., Schodin, B.A., Wilson, E.R. & Kranz, D.M. (1994) Efficient tumor lysismediated by bispecific single chain antibody expressed in Escherichia coli. Journalof Immunology 152, 5368-5374.

Hibbits, K.A., Gill, D.S. & Wilson, R.C. (1994) Isothermal titration calorimetric studyof the association of hen egg lysozyme and the anti-lysozyme antibody HyHEL-5.Biochemistry 33, 3584-3590.

Hoess, R.H. (1993) Phage display of peptides and protein domains. Current Opinionin Structural Biology 3, 572-579.

Hoess, R.H., Mack, A.J., Walton, H. & Reilly, T.M. (1994) Identification of astructural epitope by using a peptide library displayed on filamentousbacteriophage. Journal of Immunology 153, 724-729.

Holliger, P., Prospero, T. & Winter, G. (1993) “Diabodies”: Small bivalent andbispecific antibody fragments. Proceedings of the National Academy of Science USA90, 6444-6448.

Hoogenboom, H.R. & Winter, G. (1992) By-passing immunisation. Humanantibodies from synthetic repertoires of germline VH gene segments rearranged invitro. Journal of Molecular Biology 227, 381-388.

Houghten, R.A., Pinella, C., Blondelle, S.E., Appel, J.R., Dooley, C.T. & Cuervo, J.H.(1991) Generation and use of synthetic peptide combinatorial libraries for basicresearch and drug discovery. Nature 354, 84-86.

Ito, W., Iba, Y. & Kurosawa, Y. (1993) Effects of substitution of closely relatedamino acids at the contact surface in an antibody-antigen complex onthermodynamic parameters. Journal of Biological Chemistry 268, 16639-16647.

Kelley, R.F. & O’Connell, M.P. (1993) Thermodynamic analysis of an antibodyfunctional epitope. Biochemistry 32, 6828-6835.

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Kohl, J., Ruker, F., Himmler, G., Razazzi, E. & Katinger, H. (1991) Cloning andexpression of an HIV-1 specific single-chain Fv region fused to Escherichia colialkaline phosphatase. Annals of the New York Academy of Science 646, 106-114.

Köhler, G. & Milstein, C. (1975) Continuous cultures of fused cells secretingantibodies of predefined specificity Nature 256, 495-497.

Lam, K.S., Salmon, S.E., Hersh, E.M., Hruby, V.J., Kazmierski, W.M. & Knapp, R.J.(1991) A new type of synthetic peptide library for identifying ligand-bindingactivity. Nature 354, 82-84.

Lefranc, G. & Lefranc, M.P. (1990) Antibody engineering and perspectives intherapy. Biochimie 72, 639-651.

Levi, M., Sällberg, M., Rudén, U., Herlyn, D., Maruyama, H., Wigzell, H., Marks, J.and Wahren, B. (1993) A complementarity-determining region synthetic peptide actsas a miniantibody and neutralizes human immunodeficiency virus type 1 in vitro.Proceedings of the National Academy of Science USA 90, 4374-4378.

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