Immunology Today, voL 7, Nos. 7 & 8, 1986


Multispecific monoclonal antibodies Monoclonal antibodies are frequently shown to participate in unexpected cross reactions involving two apparently dissimilar antigens. This can be attributed either to partial epitope identity or to irrelevant interactions involving additional binding capacity of the antibody. The majority of such interactions appear to fall into the latter category. Such cross reactions are most commonly detected when one of the antigens has a high epitope density and the antibody is multivalent so that a spurious interaction of low intrinsic affinity is amplified by local concentration effects. In this review Souravi Ghosh and Ailsa Campbell discuss the selection of a monoclonal antibody for maximum affinity for the antigens it is designed to study and minimum cross-reactivity. Long before the development of monoclonal antibody technology, Talmage I considered the concept of monoclonal cross reactions, showing considerable forethought in 1959 at a time when both absolute-specificity and lock and key concepts dominated views of antibodyantigen interactions. He reasoned that, if antibodies had the capacity to bind to more than one antigen, although not necessarily simultaneously, then polyclonal sera generated against any epitope would be a mixture of antibodies with mutliple specificities, having only the ability to bind the epitope in common. The overall effect would confer statistically selective specificity to the epitope since each individual antibody in the serum would have the potential to cross react with series of different molecules. Had he written his paper 20 years later, he would doubtless have pointed out that an obvious consequence of this theory is that a monoclonal response would never have the high degree of specificity shown by a polyclonal one. This is, happily, not always the case. However, many of the monoclonal antibodies generated in the last ten years have been produced on the assumption that the concepts of monoclonality and specificity are equally applicable and such optimism is proving to be equally unfounded. While the formal experimental proof of Talmage's theory was supplied by Richards in 19752 who showed that two 'shelf selected' chemicals of totally diverse structure, DNP-lysine and methyl naphthoquinone, could bind significantly, albeit with very low affinity, to a myeloma protein, the essential validity of Talmage's hypothesis has been evident to immunologists for many years. Thus the general definition of 'non specific' or 'low affinity' binding together with its abolition by ill-defined reagents has always been the somewhat arbitrary prerogative of the experimental scientist. Concepts of protein antigen structure have been considerably revised since these seminal cross reaction papers 1'2 were published. In particular, the dynamic nature of protein structure in solution is now becoming apparent 3.4 and consequently the potential interactions between any individual idiotype and a protein antigenic

Department of Biochemistry, Universityof Glasgow, Glasgow G12 BOO, UK ~) 1986, Elsevier Science Publishers B.V, Amsterdam 0167 - 4919/86/$02.00

SouraviGhoshand AilsaM. Campbell epitope may be much wider than early hapten crystallographic papers suggest. The 'lock and key' concept is no longer so rigidly applied and it may be that both the antibody and (flexible) antigen can induce in each other an optimal configuration for binding 5'6 (Fig. I). In addition, now that high affinity interactions of an antibody with an immunizing protein antigen, rather than a random 'shelf chemical' can be analysed7 it is apparent that the extent of interaction between the two molecules is much greater and more wide ranging, involving many more contact points than previously envisaged with earlier artificial systems. A weakly cross reacting antibody molecule able to make even one or two of these contacts with a foreign molecule is therefore likely to be generated, particularly when the flexibility of protein structure is considered. Fig. 2 gives a diagrammatic representation of some possible cross reactions, making the arbitrary assumption that a high affinity contact between antibody and original selecting antigen involves three contact points. These may be hydrogen bonds, hydrophobic interactions, ionic interactions or combinations of the three. A high affinity antibody may make contact with an identical epitope on a cross reacting antigen using all three contact points in which case true identity of the two epitopes is established and such an antigen is

Fig. 1. Apparent immunological identity of two dissimilarproteins due to conformationalalterationsstabilizedby interactionwith antibody. Top:A monodonalantibodyreactingwith a stableconfigurationinvolvingtwo contactpoints. Bottom: The sameantibody reacting with a basicallydissimilarprotein by stabilizingit in an atypicalconformation.


Immunology Today, vol. 7, Nos. 7 & 8, 1986

-reviews Highaffinity Mediumaffinity Lowaffinity Multiplecontacts Reservecapacity Extensivecapacity for non identical for non-identical epitopes epitopes

Antigen structure and presentation One of the most relevant features of antigen structure is epitope density. Thus antigens with frequently repeated epitopes, such as bacterial lipopolysaccharide (LPS), DNA, or carriers heavily substituted with haptens present a single epitope with a limited range of contact GroupII + + + points at high local concentration and may therefore Partial identity. encourage selection of an antibody with a low intrinsic Relevant affinity due to simple kinetic considerations. A similar but /~ + 4less dramatic effect may, of course, operate with (particularly small) protein molecules plated at very high density on say, an FLISA selection assay9. This low ++ ++ intrinsic affinity may leave the antibody with residual capacity for cross reaction with structurally dissimilar Group III antigens. _ Non-identity Another relevant aspect of presentation is the 'block-÷ Irrelevant ing reagent' used to abolish non-specific binding. There are three common reagents. Bovine serum albumin is a _ + ++ 60kDa protein which has a strong negative charge at neutral pH. Serum from non-immunized subjects conFig. 2. The relationship between the affinity of an antibody antigen interaction tains around 70 mg/ml of protein, approximately 50% and the probability of cross reaction with similar (Group II) and dissimilar (Group being albumin and 20% immunoglobulin which is a III) epitopes. comparatively large protein, positive at neutral pH. NonThe high affinity antibody on the left only shows cross reactions with antigens ionic detergents such as Tween 20 are also employed, which have identical (Group I) or partially identical (Group II) epitopes. All cross particularly in immunoblotting and are thought to disreactions of such an antibody are therefore relevant. In contrast, the low affinity rupt primarily hydrophobic interactions. As all three have • antibody on the right has spare capacity for binding to antigens which have no epitope identity (Group III antigens). In addition, its efficiency of detection of quite different effects on protein-protein interactions, it partially identical (Group II) antigens is much reduced. In the intermediate case is quite possible to observe cross reaction with one type shown in the centre, the monodonal antibody crass reacts with most Group II of blocking reagent but not another. In addition, the antigens having partial structural identity but can also cross react with dissimilar concentrations of these reagents employed varies widely Group III antigens. among research groups. Serum from the same species as The majority of monoclonal antibody cross reactions fall into the Group III was used to produce the monoclonal antibody is irrelevant category and do not reflect any structural identity among antigens. obviously the most comprehensive reagent as it contains irrelevant immunoglobulins and should block irrelevant termed a Group I antigen. The high affinity antibody may binding due, for example, to Fc receptors in cellular also make contact with similar structures carrying only assays. While an antibody in tissue-culture supernatant is one or two of these contact points indicating some already in a solution that contains foetal calf serum, this degree of structural similarity between the two antigens. has a very low concentration of immunoglobulin. Antigens of this type, carrying identical contact points are clearly relevant cross reactions and are termed Group Antibody isotype II antigens. In contrast, an antibody with low affinity for It is noticeable that the majority of unexpected cross the original selecting antigen, making only one of the reactions occur with IgM (Table 1) monoclonal antithree contacts has spare capacity to bind totally dissimilar Table 1. Majorparticipantsin unexpectedcrossreactions antigens through these reserve contact points. Such dissimilar antigens are termed Group III antigens. An antibody of intermediate affinity will have the capacity to Molecularstructure Reference make both relevant (Group II) and irrelevant (Group III) Structuralproteins 22, 33, 38-44 interactions. The subject of cross reactions was reviewed by Lane Cellsurfaceproteins(including 13, 21,29-32, 40, 45 and Koprowski in 19828 and, at the time, appeared to immunoglobulins) offer considerable potential for the more detailed analysis of the antigenic similarity and potential structural DNA 28-35 identity among apparently diverse antigens. However, genuine examples of epitope identity or similarity (Group Haptens,phospholipids, 22, 28, 30, 33-35 II, Fig. 2) have only emerged in a very small number of lipopolysaccharidesand cases and the greater number remain unexplained and proteoglycans potentially non-specific Group III interactions. The majority of antigens involved in the cross reactior~s chronicled In all but two casesthe antibodyclassis eitherIgM or not declared.Ref. 13 in the literature of the last three years have as one specificallydescribesIgGcrossreactionsdueto bindingto the Fcreceptorand partner either a cell surface antigen, or an antigen does not implicatethe antibodycombiningsite. It is likelythat a similar present at high local concentration with a repeating phenomenonisoccurringwiththe IgG2ain Ref.21.




GroupI identical




structure (e.g. DNA, actin, myosin, vimentin) and a disproportionate number of the monoclonal antibodies involved have been of the IgM isotype (Table 1). This article attempts to rationalize such findings in terms of current monoclonal immunoassay technology.

+ } Groop,I

immunology Today, vol. 7, Nos. 7 & 8, 1986

bodies which have in general low affinity but high avidity due to their multivalence. The bivalence of IgG antibodies has been shown ~° to be a crucial factor in the analysis of Types I and II cross reactions. The weaker contact with the cross reactive antigen cannot be sustained in a washing-type assay without the cooperative bivalent binding of both available regions of the same antibody (Fig. 3b). Obviously, such an effect may be amplified in an IgM antibody, making unexpected cross reactions more common. It is therefore preferable to generate an IgG antibody if specificity is required. A discussion of the appropriate methodology is covered in several books and articles already published 11'12. In the case of cell surface antigens, cross reaction due to Fc receptor binding may occur with IgG antibodies. Murine antibodies of the Ig G2a and IgG3 subclasses have been reported to be particularly prone to binding to human cells by the Fc region 13.

Antigen-antibody intrinsic affinity From Fig. 2, it is clear that a high contact affinity between an individual antibody variable region and the original selecting antigen is necessary to minimize the chances of detecting an irrelevant (Group III) cross reaction and also to maximize the chances of detecting a relevant (Group II) cross reaction. Several articles detailing methods of production of antibodies with this high intrinsic affinity have been published 11'~2'14 the main requirements being that the immunization schedule is one that matures the immune response to high affinity clones only and that the selection procedure is designed to detect high affinity antibodies only. Presentation and assessment Relevant to the study of cross reactions is the question of how they are determined. The method of assessment frequently controls the definition of whether or. not an antibody is cross reactive. Factors such as pH may alter the cross reacting antigen:desired antigen ratio is. However, the actual type of assay employed is also relevant (Fig. 3). The solid phase ELISA assay is frequently used for hybridoma screening. It involves several but variable washing procedures so that low affinity cross reactions are often partially removed (Fig. 3a), masking the true extent of the cross reaction, whether relevant or irrelevant. In contrast, agglutination, rosetting and complement fixation assays do not involve such washing and lower affinity contacts are more readily detected. tmmunocytochemical assays frequently show cross reactions and it is probably significant that these are frequently reported with major structural proteins but not catalytic ones. The former are present in stoichiometric amounts, with epitopes at high local concentration and are usually highly insoluble. Thus, even where the contact points are weak in comparison to the chosen antigen (Fig. 4b), the cross reaction may be significant. While this type of assay seems superficially similar to an ELISA assay, the latter is frequently conducted with whole unfixed cells so that the antibody has no access to intracellular cross reactive antigens (Fig. 4b) or with purified antigens at much lower local concentrations so that antibody multivalence (Fig. 3b) is less effective. Immunocytochemical assays also suffer from fixative complications since fixation can both destroy and create contact points on the epitope. An extreme case in which


Wasng. b Fig.3. Assay mediated detection of cross reactions. (a) Washing type assaysfail to detect antigenic similarity between the selecting antigen on the left and the cross reactive antigen on the nght. Non-washing assays such as rosetting, agglutination and complement fixation would detect such cross reactions. (b) A common subset of such cases(see Ref. 10). The selecting antigen on the left can bind both whole antibody and F(ab) in a washing assay but the lower affinity cross reactive antigen on the right requires antibody bivalence to stabilize binding by providing a high local concentration of binding sites, and the reaction is abolished by the use of the F(ab) fragment alone. While this illustration shows that potentially relevant (Group II, Fig. 2) cross reactions may not be detected by washing assays, the same principles apply to irrelevant (Group Ill, Fig. 2) antigens. Thesemay display spurious cross reactions on rosetting and agglutination assays which are removed by the use of washing type assays or the use of F(ab) rather than whole antibody, particularly if the antibody is a multivalent IgM.

fixation did both for the same monoclonal antibody has been described 16.

Cross reactions in immunoblotting assays Blotting assays against complex mixtures also tend to display extensive cross reactions, especially with structural proteins or others present in large amounts. Monoclonal antibodies can frequently be shown to react with molecular weight standards which may correspond to Group III epitopes (Fig. 2). In the context of immunoblotting, it is pertinent and possibly alarming to consider the parallel field of DNA-protein blotting, in which it was hoped that highly specific contacts could be readily identified and compared with the multitude of nonspecific contacts which also occurred. With a purified, well characterized DNA binding protein alone on the gel it worked splendidly17'18. With a mixture of chromosomal proteins, it has been a disappointing technique. Lack of success has been attributed to lack of renaturation or to unknown conformational effects caused by the strong and hence presumably potentially deforming binding of the antigen to nitrocellulose paper, together with the concept of a low value for the specific:non-


Immunology Today, vol. 7, Nos. 7 & 8, 1986

-reviews specific binding ratio. These reservations apply equally strongly to immunoblotting. Thus where blotting shows cross reactivity, particularly with major bands on the gel, this could clearly be Group III non-specific binding (Fig. 2). In our laboratory we are able to make a single IgM monoclonal antibody 'identify' different major antigens in a wide variety of protein mixtures, none of which contain the immunizing antigen. However, Lampson and Fisher 19 have made a detailed study of immunoblotting of complex extracts containing HLA antigens using a panel of IgG monoclonal antibodies and defined criteria for obtaining relevant data from mixtures containing HLA antigens. In order to avoid Group III irrelevant cross reactions of the type shown in Fig. 4b, they have suggested that an immunoblot of a total cell extract should be performed before an extensive immunocytochemical study is undertaken.


/ j





Fig. 4. Increasedprobability of cross reactions with antigens at high epitope density(e.g. DNA, LPS,structuralproteins). (a) While the antibodyhas greater intrinsicaffinity for the selectingantigen on the left the epitope densityof the irrelevant crossreactiveantigen on the right favoursantibody binding despitea lower intrinsicaffinity. (b) A common subset of such cases. The antibody is seJected on whole Impermeable cells so that its cross reactive potential is not evident and subsequently found to cross react with structural materialpresent at high epitope density in fixed cells.

Definition of genuine cross reactions In view of all the considerations discussed above, a cross reaction involving epitopes which have significant structural identity, is best defined by an estimate of the precise affinity on the two antigens, and such detailed analyses are rare. The next alternative is to assess the cross reaction in the same assay with the appropriate purified antigen at identical antigen epitope and antibody concentrations. The number of reports of monoclonal cross reactions which can be explained by genuine and structurally relevant interactions as opposed to experimental technique remains rather small. These are detailed below. Cross reactions related to known structural identities Histocompat/b/lily antigens Possibly the best and most detailed studies on multispecificity have been performed with histocompatibility antigens. Such polymorphic antigens may be expected to generate antibodies which identify similar but not identical antigens (Group II, Fig. 2), reacting more weakly but significantly with the antigen with which they can generate fewer contact points. Early IgM monoclonal antibodies were shown to exhibit highly variable assaydependent cross reactivityis and the subsequent elegant work of Parham s'1°'2° has dissected this in more detail with measurement of the appropriate kinetic parameters and differentiation between cross reactions due to identical epitopes and those due to similar ones, the latter being of lower affinity. In addition, by the use of F(ab') fragments in comparison to F(ab')2 fragments he was able to abolish cross reactions where the lower affinity contacts were dependent on bivalent binding (Fig. 3b). However, other groups have shown apparently irrelevant cross reactions involving HLA antigens, the most likely interpretation being Fc receptor binding (Table 1) 13 •21 . However, in this case the additional complication of the relative density of antigen on the surface of two cell types clearly affects the interpretation of cross reactions. Slight cross reaction between two cell types may reflect either the presence of an identical antigen such as Ag 1 at differential density or a Type II or Type III cross reaction. Bacterial lipopolysaccharides (LPS) Bacterial LPS molecules are regularly repeating structures possessing established structural similarities and also distinct serotype specific features and so may be expected to show Group II cross reactions. They are highly antigenic and tend to elicit IgG3 monoclonal antibodies in the mouse and Ig G2b in the rat and also a minority of IgM antibodies in both species. Several cross reactions have been reported and the extent of these cross reactions tends to be assay dependent (Fig. 3a). Agglutination assays with bacteria are historically the main ones used in typing laboratories and a washing type assay such as ELISA is a more recent system generally employed for the initial screening of hybridomas. Thus a monoclonal antibody which was apparently serotype specific on selection, can be shown to cross react when used in agglutination. Additional complexities are thought to be created by the position of the epitope in the LPS chain 23'24. Because of their known structural similarities, cross reactions among serotypes have naturally been interpreted in terms of Group II (Fig. 2) antigens involving LPS core residues common to all gram negative bacteria or

ImmunologyToday,vol. 7, Nos. 7&8, 1986

antigens present on common O-region sugars 25-27. While these may be expected to be Group II relevant cross reactions, no definitive proof has as yet been made available from detailed kinetic analysis. Thus the cross reactions cited may be regarded as potentially of the Group III irrelevant class (Fig. 2). The high epitope density on bacterial LPS molecules is particularly likely to generate low affinity antibodies with potential for Group III (Figs 2 and 4) interactions and unexpected cross reactions between bacterial membrane proteins and LPS molecules 22 will have to be treated with caution until they are further substantiated kinetically and the antibody class is determined. DNA

DNA, like bacterial LPS, has a regular repeating structure with additional subtle variations. However, it differs from bacterial LPS in that it is weakly antigenic so that antibodies to the Watson-Crick DNA structure have only been established from SLE patients or their murine equivalents. Cross reactions among anti-DNA monoclonal antibodies are widely reported (Table 1, Refs 28--35), sometimes with structures where the similarity may be rationalized such as cardiolipin which has two phosphate groupings approximately the same distance apart as those on DNA, but more frequently with proteins, particularly cell surface proteins, and with haptens. No detailed kinetic affinity studies of any of these cross reactions have been published and all the rnonoclonals are of the IgM class. As in the case of LPS, but particularly where IgM antibodies are involved, the high local concentration of both partners in the antibody antigen reaction is likely to favour the selection of very low affinity, and by implication (Fig. 2), low specificity antibodies participating in Group III (Fig. 2) irrelevant cross reactions. Others

The majority of the other unexpected cross reactions reported to date (Table 1) may be significant Group II interactions, but may also be explained by assay dependent phenomena such as epitope density, antibody isotype, Fc receptor binding, or differential assay methodology as discussed above and should be treated with caution until further data is made available. Haptens

One prediction which may be made from analysis of the data cited in this review is that monoclonal antibodies to haptens may be very susceptible to Group III (irrelevant) interactions. There are comparatively few reports of cross reactions with monoclonal antibodies to the classical haptens such as DNP, possibly because these are not customarily tested on immunoblots, with immunocytochemistry or against protein antigens present at high concentration. Nonetheless, cross reactions of anti-DNP antibodies to DNA have been reported 33. Where intrinsic affinity has been determined, it is very low (106) for both IgM and IgG isotype to the same haptens 36. This may be ascribed to several factors. Firstly, the hapten is attached to multiple points on the carrier so that it effectively falls into the class of frequently repeated epitopes similar to bacterial LPS or DNA (Fig. 2, Group III; Fig. 4). Secondly, most small haptens e.g. DNP, phosphotyrosine, acetylcholine, etc.) leave the binding antibody with capacity beyond the hapten structure

alone 36 (Fig. 2). Thus the immunized animal may be presented with many epitopes on the same molecule with the carrier background amino acids being different in each case. Consequently an experimental variant of the Talmage theory is created, and indeed amplified by selection on a molecule of different amino acid sequence and a low affinity monoclonal antibody must be the result. Monoclonal antibodies to haptens are only emerging as reagents generally employed in cellular immunology or biochemistry, but it is possible to predict that the emerging generation of monoclonal antibodies to phosphotyrosine, ADP ribose and other small molecules of considerable interest such as neurotransmitters 37 may have low affinity to the immunizing epitope and consequent considerable cross reactive potential of the Group III irrelevant type (Fig. 2) and will thus be potentially misleading reagents. Such possibilities are seldom investigated in the production on the original antibody and the screening system is not generally designed to investigate irrelevant Group III (Fig. 2) interactions. In the context of haptens, it is interesting to note that antipeptide polyclonal antibodies can be shown to cross react with high density structural proteins 38, presumably because such small molecules can only generate antibodies of limited affinity with a consequent high cross reactive potential. Conclusions

As a consequence of the discussion above, we suggest that the assessment of the significance of a monoclonal antibody cross reaction should take into account the following points. (1) Did the antibody have a high affinity on the original selecting antigen? If not, it may have potential for non specific cross reactions (Fig. 2). (2) Does either of the two antigens involved have a repeated structure (DNA, LPS, dextran, etc.) either naturally or in presentation to the animal (i.e. haptens at a high substitution ratio)? Alternatively, is it present in the assay at high concentrations? Any of these cases may lead to non specific cross reactions (Fig. 4). (3) Is the antibody of the IgM class? If so, then there is an increased likelihood of non specific cross reactions (Table 1). (4) If the antibody is of the IgG class and one of the antigens is a cell surface protein, has possibility of Fc receptor binding been tested? (5) Has the cross reaction been demonstrated under identical experimental conditions employing identical amounts of the two antigens and of pure antibody? (6) Has a kinetic analysis of the intrinsic interactions between antibody isotype and antigenic determinant been performed? Until at least the first four of these questions have been satisfactorily answered, it would seem to be wiser not to interpret a cross reaction between two antigens as evidence of epitope identity. References 1 Talmage, D.W. (1959) Science 129, 1643

2 Richards, F.F,, Konigsberg, W.H., Rosenstern, R.W. eta/. (1975) Science 187, 130 3 Westhof, E., Altschuch, D., Moras, D. etaL (1984) Nature (London) 311,123-127 4 Tainer, J.A., Getzoff, E.D., Alexander, H. et aL (1984) Nature (London) 312, 127 5 Parham, P. (1984)J. ImmunoL 132, 2975-2983 6 Diamond, A.G., Butcher, G.W. and Howard, J.C. (1983) J. ImmunoL 132, 1169-1175


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Multispecific monoclonal antibodies.

Monoclonal antibodies are frequently shown to participate in unexpected cross reactions involving two apparently dissimilar antigens. This can be attr...
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