Antibody-antigen interactions

Antibody-antigen interactions David M Webster, Andrew H Henry and Anthony R Rees

Univers i t y of Bath, Bath, UK

The structural origins of antibody diversity are well understood as a result of X-ray crystallographic and molecular modelling studies of Fab fragments. A similar understanding of antibody specificity is beginning to emerge from an analysis of structures of hapten, peptide and protein antibody complexes. While the nature of the antibody-antigen interface, and any conformational changes that occur on complex formation, can be described in structural terms, a full explanation of the thermodynamic and mechanistic basis of affinity is less accessible from structure alone. A number of physiochemical studies carried out on wild type and mutant antibodies have raised questions about the natureof theenergetics of the interaction, and the possibility of a key role for water molecules has been discussed. The possibility of 'induced fit' as a common mechanism for antigen-antibody interactions has been raised, and a molecular basis for hapten and protein cross-reactivity proposed. These recent contributions to the field, as well as providing partial solutions to old

problems, have provided exciting new insights.

Current Opinion in Structural Biology 1994, 4:123-129

Introduction

The interaction between an antibody and its antigen has become a paradigm for the study of molecular recognition. Ehrlich and Landsteiner in the early part of the century recognized the importance of antibodies [1]. The manner in which antibodies have been shown to be organized at the genetic level has revealed a potential for molecular diversity unmatched anywhere else in the genome. Translation of this primary code into three-dimensional antibody structures is beginning to unravel the molecular basis of this diversity, and in doing so, reveal unexpected subtleties and new mechanisms for debate.

For example, a relationship between antigen type and the topography of antibody combining sites (ACS) has begun to emerge from X-ray crystallographic studies of antibody complexes (reviewed in [2]). In attempting to uncover the thermodynamics of such interactions by protein engineering and calorimetric studies, pro- posals have emerged to the effect that the affinity of binding is driven by entropic (hydrophobic) effects [3"], by enthalpic (van der Waals, hydrogen-bond, salt bridge) effects [4"], or by entropy-enthalpy compen- sation [5"]. There have also been attempts to explain the long observed phenomenon of 'cross reactivity' whereby an antibody reacts with a number of epi- topes [6", 7",8"]. In one instance, a crystallographic study of Fab-steroid complexes led to the assertion that functionally inert haptens such as steroids may not display sufficient 'molecular character' to induce

high specificity antibodies [6"]. It has also been proposed that the primary mechanism by which antigens bind to antibodies is ' induced fit' [9"].

In reviewing recent work in this field, we will focus on the nature of the antigen interaction and how structural methods, physical chemistry, and molecular biology are together contributing to the debate. In passing, we will address the question: is extrapolation from a few observations to general theorems on the mechanism by which antigens and antibodies interact now possible?

The antibody combining site: variation with antigen type

The current Brookhaven database contains 31 antibody structures, of which 14 are complexes with antigen (see Table 1). Three classes of combining site can be described: cavity (hapten), groove (peptide, DNA, car- bohydrate) and planar (protein), as illustrated in Fig. 1. The amount of Fab surface area buried on complex formation increases in the order cavity < groove < planar (see Table 2), while the percentage of antigen surface area buried in the interface decreases as the size of the antigen increases.

When the antibody-antigen interfaces are inspected, a high degree of shape complementarity is of- ten seen, particularly for protein complexes. In an

Abbreviations ACS~antibody combining sites; CDR--complementarity determining regions.

© Current Biology Ltd ISSN 0959-440X I 23

124 Folding and binding

Table 1. List of antibodies and their fragments whose X-ray coordinates have appeared in the Brookhaven database of protein structures during

the past year,

Brookhaven Resolution Antigen entry Name (I~) type Reference

1 dba, 1 dbb DB3 2.7-3.0 hapten 6 1 ncd NC41 2.9 protein 10 1 igf/2igf B1312 2.8 peptide 20 1 hil/1 hin/1 him 17/9 2.0 peptide 22 lggi, lggb, l g g c 50.1 2.8 peptide 23 ldfb 3D6 2.7 peptide 37 2fbj J539 1.95 CHO 39 ligi 26-10 2.7 hapten 40 7lab NEW 2.0 Myeloma 41 Imam YS*T9.1 2.45 Cl iO 42 I baf ANO2 2.9 hapten 43 8lab HIL 1.8 myeloma a 1 bbd 8F5 2.8 hapten 44 ligm POT 2.3 peptide 45

aFA Saul and RJ Poljak, unpublished results

analysis of the interface of the influenza virus N9 neuraminidase-NC41 Fab complex it was found that the average packing density of atoms at the interface was lower than for a typical protein core [10]. This result is in contrast with the analysis of Walls and Sternberg [11] who found that the packing densi- ties of the three anti-hen egg-white lysozyme antibody interfaces were as high as the protein cores. In contrast, electrostatic complementarity is not always high [12]. For example, in the N9 neuraminidase-NC41 complex, a number of polar residues in the interface are not formally neutralized, though many are in close proxim- ity to shielding residues.

It has also been suggested that water molecules, present at the interface, may mediate antibody-antigen interactions by increasing the packing density and contributing to charge complementarity [10,13]. Wa- ter molecules have been found in the interface of the anti-lysozyme antibody D1.3 [13-15], around the periphery of some anti-protein interfaces [10,15,16], and in the combining site of some anti-peptide antibodies [17]. The proposal that water molecules play a general role in protein recognition may be a little premature in the presence of only a handful of structures [13]. However, it is a reasonable speculation that, where complementarity is imperfect, filling-in by water molecules might occur.

Structural basis of specificity, affinity and cross-reactivity

Analysis of cavity-type antibodies suggests a correlation between buried surface area in combination with

van der Waal contacts, and affinity (Table 2, upper panel). However, it is likely that to generate high affinity and specificity, hydrogen-bonds and/or salt bridges are also required. This is evident when the characteristics of the anti-steroid antibody, DB3, are compared with those of the anti-fluorescein antibody, 4-4-20 (Table 2, upper panel). In the former case, cross- reactivity of the antibody is seen with several steroids which, in the X-ray structures, are seen to bind to alter- native subsites in the combining site [6%18"°], depend- ing on whether the steroid A-ring adopts a syn or anti conformation. The shape complementarity of this ACS to the progesterone immunogen is not totally specific. In comparison, the sheep anti-progesterone antibody, D7B2 has an affinity 200-times higher than DB3, and appears to lack the syn/anti dual accommodation al- lowed by DB3 [191. By contrast, the fluorescein cavity exhibits close complementarity to the hapten and the additionaJ~specificity and affinity observed are likely to result/from extra hydrogen-bonds and a buried salt bridge.

These comparisons draw attention to the fact that our notions of absolute specificity and maximum affinity arise from the systems we have available for inspection. Experience with other systems where small molecule-protein interactions occur, such as biotin-avidin, suggests that the practical upper limit of affinity may be about 1015M -1. This is at least 104 times greater than that of the best murine antibody. While affinities of naturally occurring human antibodies are unknown, sheep antibodies routinely reach affinities of 1011-1014M -1, which suggests that the study of monoclonal antibodies from inbred mouse strains may have placed limits on antigen affinity that are atypical of outbred mammalian species ([19]; P Har- rison, personal communication).

Groove-type binding sites have been seen with anti- peptide antibodies. The first antibody-peptide complex studied by X-ray crystallography involved a 19 residue peptide derived from myohemerythrin [20], of which 10 residues were not visible in the electron density, presumably due to their high mobility. Peptide mobility in the bound state has been studied directly using NMR [21]. While some residues may become very tightly bound on complex formation, others may be as mobile as in the free peptide. Recently, further anti-peptide antibody complexes have been reported [17,22",23"]. The central panel of Table 2 shows that for three complexes there is no obvious trend between surface area, number of contacts and affinity. This may be due to the fact that the flexibility of peptide antigens, and its effect on the entropic com- ponent of the free energy of binding cannot easily be quantified.

Cross-reactivity was also seen [7"] in the binding of a series of peptides of increasing size to the light chain dimer Mcg. In a manner sin~ilar in many respects to the DB3-steroid binding, Mcg was able to bind equiv- alent parts of different peptides to the same region of the combining site, while other parts of the pep-

Antibody-antigen interactions Webster et al. 125

(a)

P

(b) (c)

Fig. 1. Topographic classes of antibody combining site: (a) cavity type, exemplified by anti-fluomscein antibody 4-4-20 [30]; (b) groove type, exemplified by anti-peptide (myohemerythrin) antibody B13 12 [21 ]; (c) planar type, exemplified by influenza virus N9 neuraminidase-NC41 complex [13]. The antigen is shown in dark grey at the top of the figure. The antibody Fv has been pulled apart from the antigen and is the centre image. Residues which lie within 4,~ of any atom in the antigen are coloured dark grey. The bottom image shows the antibody Fv rotated through 90" to highlight the centre of the ACS.

tides adopted different binding modes. These studies draw attention to the so-called 'order-disorder para- dox', observed where anti-peptide antibodies are able to cross-react with the native protein, while anti-protein antibodies do not recognize the peptide [24]. It has been suggested that such cross-reactivity is caused by antibodies that only recognize the denatured protein [25]. However, this position is at odds with recent work in which such cross-reactivity has been clearly observed [26,27"]. This phenomenon still seems to be unresolved.

The planar-type binding site is characteristic of anti- protein antibodies (Fig. 1). Although the great majority of antibodies have been raised against protein antigens, only five high resolution X-ray structures are known for antibody-protein complexes. These are summarized in the lower panel of Table 2. The affinities of the NC41-neuraminidase and D1.3-anti- D1.3 complexes have not been published, but they are likely to be at least 109M -1. What do these data tell us about protein antibody-antigen interactions? First, there is no obvious correlation between the

number or type of interaction seen (van der Waals, hydrogen-bond, salt bridge) and affinity. Second, the buried surface area is not a good indicator of affinity, as might be the case if burial of hydrophobic residues and release of water molecules were the major contributor to t h e free energy of binding. In fact, recent work [4"] provides a contrasting view. During measurements of the enthalpic and entropic contributions to the association of hen egg- white lysozyme with D1.3, using microcalorimetry, it was found that the binding was enthalpically driven, while the entropy decreased (AGo-47.9 kJ mol-1; AH -90.0kJmol-1; AS -42.1 kJmol-1). While this may not always be the case, it is clear that the affinities observed in antibody-antigen complexes have their origins in rather different profiles of interaction.

In a recent study of protein cross-reactivity [8"], the structures of antibody FvD13.11 complexed with pheasant egg lysozyme and a number of uncomplexed cross-reactive avian lysozymes were compared. Of particular interest was the observation of a heteroclitic response (an immune response to an antigen which pro-


Table 2. Some Physico-chemical characteristics of (top panel) antibody-hapten, (centre panel) antibody-peptide and (bottom panel) ant ibody-protein complexes.

Buried surface vdW Salt Antigen area, t~2 contacts H-bonds bridges Ka, M -1 Antibody name

phosphocholine 137 30 2 3 1.7 x 105 McPC603 a aetiocholanolone 227 27 2 0 4.8 x 107 DB3 phenyloxazalone 170 >15* >1 0 1.0 x 108 NQ10/12.5 5,[~-androstane 223 20 2 0 1.3 x 108 DB3 progesterone 11 ,et-hemisuccinate 291 68 2 0 2.8 x 109 DB3 ftuorescein 266 65 5 1 3.4 x 1010 4-4-20

Cholera toxin 503 21 10 1 1.2 x 106 TE33 HIV peptide 475 27 4 0 4.5 x 106 50.1 Influenza haemagglutinin peptide 400 74-81 13-15 1 5.0 x 107 17/9

Hen egg lysozyme 680 75 15 0 1 x 109 D1.3 " " 774 111 14 1 5x109 HyHEL10 " " 750 74 10 3 2 x 1 0 lo HyHEL5

Influenza neuraminidase 879 108 23 1 NA NC41 D1.3 800 99t 9 1 NA anti-D1.3

aData for McPC603, 4-4-20, D1.3, HyHEL10, HyHEL5 and NC41 are taken from [46]; the D1.3-anti-D1.3 data are taken from [47]. Peptide data are as follows: 50.1 [23]; 17/9 [22]; TE33 [17]. The hDB3 structures are from [6]. (t author's estimate, * not yet fully refined).

duces antibodies having a higher affinity to a similar, but not identical antigen) for two of the lysozymes. Residues that differed between the lysozymes were located around the edge of the epitope. Changes in conformational flexibility, coupled with the presence of water molecules around the periphery of the inter- acting surfaces, may have reduced the stereochemical constraints on complex formation.

How far could protein antigen cross-reactivity go? Given the radius of curvature of most proteins and the size of the antibody combining site, multiple paratopes would be forced to overlap. If such redundancy does exist, then proteins rather more different from one another than avian lysozymes may bind to the same antibody, thus increasing the available repertoire far beyond its present size.

Conformational change

The generality of induced fit as a mechanism governing antibody-antigen interactions has frequently been discussed [2,9",13]. In some instances, small changes have been seen in sidechain position o r b a c k b o n e conformation [8",10,18"]; in others, no significant changes to either antigen or antibody are seen on complex formation [6%15]. For situations where the changes are restricted to sidechain reorientation, such that a pre- existing rotamer is preferentially selected in the bound state, the term 'induced fit' may be inappropriate. Per- haps the term 'mutual fit' better describes these types of interactions.

When Fab D1.3 binds to hen egg-white lysozyme [15] no significant changes are seen in the free and bound antigen. No structure for the free D1.3 Fab has yet been determined, but a small difference was seen in the VL-VH pairing of the free and bound forms of FvD1.3 [14]. The shift improves the contacts between the VH domain and the antigen, but is vanishingly small. Without the free Fab, the possibility that the observed change arises from loss of the anchoring effect of CH1 cannot be ruled out. Larger shifts in VL-VH pairing have been reported for the HIV anti- peptide antibody, Fab 50.1 (RL Stanfield et al., unpublished results).

Large movements of complementarity determining regis (CDRs) on complex formation were first seen in an anti-peptide antibody (17/9; central panel Table 2) which showed significant changes between the free and bound forms [22"]. This change was defined as a twisting of the two strands about the long axis of the H3 loop, giving rise to residue movements of around 2-5A. Similar CDR changes have recently been observed in the anti-peptide antibody Fab 50.1 ([23], RL Stanfield et al., unpublished results) and an anti-DNA antibody [28]. For the anti-peptide antibodies, the free conformation of the peptide is unknown but the as- sumption must be that it is disordered. In this situation, the notion of induced fit is entirely appropriate, since large changes in both antigen and antibody appear to be required to attain the complex state. The flexibility exhibited by peptide antigens, however, may impose thermodynamic (particularly entropic) constraints on the maximum affinity attainable.

It has been suggested [29 °] that such flexible ligands may have an upper limit for their association constant of about 108M -1. Thus, for generating antibodies that are required to be cross-reactive with the native protein, peptides may not be the ideal starting point.

Dissection of the antibody interaction and prospects for design

The first attempts to modify antibody affinity in a rational and successful manner were carried out in 1987 using site-directed mutagenesis, in combination with molecular modelling [30]. Despite the arrival of phage display technology [31,32], most systematic studies of the combining site have continued to employ site-specific methods.

'Alanine scanning' mutagenesis was used to identify residues important for antigen binding in the. anti- epidermal growth factor receptor antibody, hu-4D5-5 [3"']. The alanine mutations were chosen to line a shallow depression predicted by a homology model. Only four of the twenty-two mutations carried out resulted in a decrease of more than 10-fold in affinity, as would have been predicted by the 'energetic epitope ' model of Novotny [331, whereby the residues of a sub-region of the physical epi tope contribute the majority of the binding energy. Titration calorimetry of the mutants led to the conclusion that antigen binding was driven by the hydrophobic effect. Of particular interest was the observation that the four critical residues did not exhibit enthalpy-entropy compen- sation during mutagenesis. This is in contrast to the observations of Ito et al. [5"] who, in a study of the D1.3 combining site using a pseudo-rational library (where each residue position is varied using ratio- nally designed subsets of all available amino acids) in which nine putative antigen contact residues were targeted, observed entropy-enthalpy compensat ion in all instances. On the basis of this study these authors conclude, 'It should be very difficult to design mutant antibodies with enhanced affinity based on modelling of antibody-antigen interactions'. Clearly, entropy--enthalpy compensat ion is not a universal phenomenon in antibody recognition and generalisations of this type are therefore not always supportable. Mu- tation of the high affinity (>109 M -1) anti-digoxin antibody, 26-10, has also been carried out using a homology model [34]. The mutants were screened with a panel of digoxin analogues which showed few large differences in specificity or selectivity. This suggests that the affinity was due to a combination of many weak interactions, though it is also possible that the residues with strong interactions were not mutated. Thus, the concept of the energetic epitope, in which only a small number of the interfacial residues contribute to the binding constant, may not be universal [33]. Where good structures or models are known, rational mutations may be more easily designed [29,36"]. A


recent study in which nuclear Overhauser effect data were used to identify residues close to the antigen in an anti-2-phenyloxazol-5-one antibody [36"] supports this notion. Candidate residues were randomly mutated and screened by phage display, allowing identification of a mutant exhibiting a ten-fold increase in binding affinity.

Conclusions

During the past year, evidence has emerged to suggest that general combining site shape may be dictated by particular CDR length combinations. In addition to the three shape classes discussed previously, we have suggested the existence of a fourth, the 'H3 finger' class, characterized by long (>17 residue) H3 loops [29"]. Such loops are frequently seen in anti-viral antibodies, which may bind by a novel mechanism involving an H3 'finger-canyon' (the H3 finger-viral canyon) interaction [38",39].

In attempting to design an antibody combining site a b

initio, t w o premises are implicit. First, that the rules governing the overall coarse topography are known, and second, that fine specificity can be predicted by selection of the most appropriate antigen contact residues. The problem of predicting which antibody residues should form the partners in antibody-antigen pairwise interactions is, at present, a more distant prospect. Furthermore, where mutual, or induced fit processes are at work, the problem will become even more severe.

It is to be hoped that the rapidity with which X-ray structures can now be produced, and the power of phage technology in concert with random mutagenesis, will allow the rules for design to emerge slowly and broaden yet more the available antibody repertoire.

Acknowledgements

We would like to thank SERC, British Biotechnology Ltd, Wellcome Research Ltd and Oxford Molecular Ltd for financial support. All Henry acknowledges SERC for a studentship.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • . of outstanding interest

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DM Webster, AH Henry and AR Rees, School of Biology and Bio- chemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.

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Antibody-antigen interactions