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The antibody combining site: retrospect and prospect
Immunology Today, vol. 8, No. 2, 1987
The antibodycombiningsite: retrospect and prospect The evolution of our current understanding of how antibodies exert their biological functions has consisted of periods of intense activity punctuated by periods of rather slow progress. Further development of Ehrlich's conceptual advances (which had laid the foundations of molecular immunology) was frustrated by the limitations of the prevailing theoretical framework, the absence of an understanding of antibody heterogeneity and unawareness of the complexity of the antigenic material being used. Forty-five years after Ehrlich dehvered his Croonian lecture to the Royal Society the direct demonstration by Landsteiner 1of apparently limitless numbers of antigenic specificities gave rise to instructive theories of antibody formation. Even after the proposal by Jerne2 and Burnet 3 of the now generally accepted selective theories of antibody formation nearly 60 years later, immunology could still not add much to Ehrlich's simplistic mechanism of antibody-antigen interactions, although it should not be forgotten that the possible contributions of ionic interactions had been anticipated by Arrhenius and Madsen 4. It was not until Porter s and Edelman 6 deduced the multi-chain structure of immunoglobulins that the modern view of the antibody combining site, as a structure consisting of variable plus constant domains, began to emerge. With the protein chemistry problem largely solved, large numbers of sequences of different immunoglobulins appeared and, as a result of the systematic analysis of light chain sequences by Wu and Kabat 7 and of heavy chains by Kehoe and Capra 8, the molecular determinants of complementarity were revealed. The distinctive feature was the presence of hypervariable regions within eacg chain (called complementaritydetermining regions or CDR by Wu and Kabat) bounded by less variable or framework regions. Thus, it became possible to pinpoint particular sequences that, when assembled into an appropriate three-dimensional structure, would form the antigen-binding surface. The sequence information alone, however, was in the end not sufficiently revealing, nor protein structure prediction so well advanced, as to allow the generation of a tertiary fold ab initio. The key to unlocking the structural problem was the highly conserved nature of the framework regions observed when antibocies of different specificity or even species were compared. When the first X-ray crystallographic structure was published by Polja!: et al. ~J the topological origins of this sequence cons( .-. ation became obviou,. Framework regions were seer :0 be organized as 13-strands in.+erconnected by loops, .:orresponding at the N-terminal ends of the strands :0 the CDR. The strar~ds were arranged in a 13-sheet sar~dwich (the 'immunoglobulin fold') the filling of which cogsisted of tightly packed hydrophobic side chains and an invariant disulphide be~d linldng the two sheets together. The constraints on side chain type necessary to pieserve 44
Laboratory of Molecular Biophysics, The Rex Richards Building, Oxford OX13OU, UK
AnthonyR. Rees this 13-fold were thought to be sufficiently stringent to explain the high sequence conservation. The hypervariable loops on the other hand were considered to be free to drift in sequence and conformation without disturbing the framework structure. When structures of other antibody binding site fragments were obtained this was indeed seen to be true. Thus, the antigen binding domain can be viewed as a structurally invariant framework (broadly speaking) on which six hypervariable chains are superimposed. The obvious question of interest is then: can the conformation of the binding site for an antibody of unknown structure be predicted? The earliest attempt at such a prediction was made by Davies and Padlan 1° who constructed a model for the binding site of a rabbit antibody ~pecific for type II polysaccharide using the three-dimensional structure of the mouse anti-phosphorylcholine antibody MOPC603 as a starting point. Their approach, having made the assumption that the framework regions in different antibodies would have essentially the same conformation, was to model the CDR of the rabbit antibody on the conformations of CDR of similar length in other known structures such as MOPC603 (a mouse Fab11), NEW (a human Fab9) and REI (a human light chain dimer~2). The resulting model exhibited a large number of side chains capable of hydrogen bonding, a construction that was particularly persuasive to the authors given the large proportion of -OH groups on the antigen. It is true to say, however, that even with the exhaustive sequence and structural analyses carried out since those early attempts, there was until recently no direct evidence that CDR from one antibody could simply be switched to another without disturbing the framework structure. Our own approach to predicting the hypervariable surface used and extended the methods of Davies and Padlan ~° with the added sophistication contributed by energy minimization 13. The models produced were subjected to test in the computer by attempting to 'dock' the known epitopes to the modelled surfaces and have subsequently been tested by protein engineering ~4,1S. Our analysis of existing structures provided the evidence for the notion that CDR conformation may be more influenced by length than by sequence 1°,13, but it was not until very recently that the 'constant framework: variable CDR' model of antibody combining sites was put to the most critical test of all. Could CDR from one antibody be switched to another's framework and in the process retain the antigen specificity of the CDR donor? Jones et aL 16 have almost answered the question by the elegant use of molecular biology techniques. Their approach was to carry out CDR replacement. A synthetic hybrid gene was constructed in which the heavy chain framework region of a human myeloma, NEWN, was linked with the CDR regions from the mouse anti-NP cap (4-hydroxy-3 nitrophenacetyl caproic acid) antibody, B1-8. The construct was then expressed in a lynnphoid cell ~) 1987. Elsev,erSciencePubhshersB V, Amsterdam 01(:
4919,87l$02 00
Immunology Today, vol. 8, No. 2, 1987
line that secreted only a K-light chain with the parental B1-8 specificity. The hybrid antibody exhibited the binding specificity of B1-8 indicating that, whatever heavy chain conformational determinants were critical for hapten binding, they were not disrupted b' the heterologous framework regions. Of course, it still remains to be formally demonstrated that a similar result would be obtained by light chain 'therapy', but this seems a foregone conclusion. By contrast, when the hybrid antibody was inspected by two different anti-idiotypic antibodies a difference in its reactivity profile was seen. The epitope for one antibody appeared to have been lost completely, while the other exhibited only partial reactivit v. The situation where antigen binding is retained but idiotypy is lost or impaired could lead one to conclude, as did Jones etaL 16 'that the immunoglobulin uses different sites to bind hapten and anti-idiotypic antibodies'. The question of the interrelationship between antigen binding regions and idiotopes has long been of immunological interest. Kieber-Emmons and KOhler 17 have classified idiotopes as: oL-idiotopes - those that lie out of reach of the antigen binding site; 13-idiotopes - those that are close to the antigen binding site so that non-competitive interference is observed; and ~/-idiotopes formed by the binding site itself. The class to which the idiotopes on B1-8 belong, defined by the anti-idiotopic antibodies Ac146 and Ac38, is unclear. The loss of binding may have arisen as a result of small structural perturbations occurring within CDR regions but still sufficiently distant from the binding site as to affect only idiotype interactions. It is well established that such small structural changes can often be accommodated locally 7.1°. Alternatively, the idiotopes may have been associated with residues at or near the framewor~-CDR junctioos where the difference between the native and hybrid antibodies would be most pronounced. Until the mappinq of idiotopes becomes a more precise operation their relationship to antigen binding regions will remain uncertain. When antibodies directed against protein antigens are considered, however, the elimination of idiotype interactions has a rather more obvious origin. Protein epitopes are large by hapten standards 18 and are thus likely to occlude a large proportion of the CDR on formation of antibndy-antigen complex, or so the presumption goes. A description of the structure of a lysozyme-antilysozyme complex at high resolution by Amit et al. 19 has removed any lingering doubt that this was the case. The elegant work of Poljak's laboratory gave us the first Fab structure and, with the new structure, has revealed an antibody-antigen interface of enormous (by hapten standards) proportions. The 20A x 3Oh, surface includes all! SiX CDR, and the degree of complementarity b~ween the two protein surfaces results in a complex sufficiently close-packed as to preclude any possibility of simultaneous anti-idiotype interactions: "... antioj~n binding should sterically hinder the reaction between combining site idiotopes and anti-id0otypic antibodies" 19 Where do we stand today? Although progress in understanding the molecular =sis of antigen recognition and its genetic 3rigins ho~ been ~ramatic, it is still true that we are totally ignorant about how particular combinations of CDR give rise to particular surface topologies. Undoubtedly, understanding will improve as empirical predictive methods are developed that make
use of antibody structure databases. If that were the only approach, however, we might be left waiting for an answer for decades. Rather, it is my view that the resolution will come using an approach that combines prediction with experimental test. Our own version of this exploits the techniques of protein engineering in the testing of computer-derived models of combining sites. More recently, the similar approach of Chothia et al. 2° in which a model of the anti-lysozyme antibody DI3 was produced, was tested directly by comparison with the crystal structure, with a high degree of success. There are still problems to be resolved but in the not too distant future it will surely be possible to construct binding site topologies knowing only the structure of the antigen and the sequences of the matching CDR. When that time arrives, we shall see the realization of Paul Ehrlich's hope "... that an understanding of antibodyantigen interactions will yield rich treasures for biology and therapeutics" 2i. References
1 Landsteiner, K. (1945) The Specificity of Serological Reactions Harvard University Press, Cambridge, MA. Paperback reprint (1962) Dover, New York 2 Jerne, N.K. (1955)Proc. NatlAcad. Sci. USA 41,849 3 Burnet, F.M. (1959) The Clonal Selection Theory of Antibody Formation, Vanderbilt University Press, Nashville 4 Arrhenius, S. (1907)/mmunochemistry, MacMillan Press, New York 5 Porter, R.R. (1959)Biochem. J. 73, 119-126 li Edelman, G.M., Cunningham, B.A., Gall, W.E. etal. (1969) Proc. Natl Acad. Sci. USA 63, 78-85 7 Wu, T.T. and Kabat, E.A. (1970)J. Exp. Med. 132, 21-50 8 Kehoe, J.M. and Capra, J.D. (1971) Proc. NatlAcad. Sci. USA 68, 2019-2021 9 Poljak, R.J.,Amzel, [. M., Avey, H.P. etal. (1973) Proc. Natl Acad. 5ci. USA 70, 3305 10 Davies, D.R. and Padlan, EA. (1976)in Antibodies Human Diagnosis and Therapy (Haber, E. and Krauser, R.M. eds), pp. 119-132, Raven Press, New York 11 Segal, D.M., Padlan, E.A., Cohen, G.H etal. (1974)Proc. NatlAcad. Sci. USA 71, 4298-4302 12 Epp, O., Latham, E., Schiffer, M., Huber, R. and Palm, W. (I 975) Biochemistry 14, 4943-4952 13 de la Paz, P., Sutton, B.J., Darsley, M.J. and Rees, A.R (1986) EMBOJ. 5, 415-425 14 Rees,A.R. and de la Paz, P. (1986) Trends Biochem. Sci. I i, 144-148 15 Roberts, S. and Rees,A.R. (1986) Prot. Eng. I, 59-66 16 Jones, P.T., Dear, P.H., Fnote, J. etal. (~986) ivature (London) 32 !, 522-525 17 Kieber-Emmons, T. and KOhler, H. (1986)Immunol. Rev. 90, 29-48 18 Benjamin, D.C., Berzofsky, J.A., East, l.J. etal. (1984)Ann. Rev. Immunol. 2, 67-101 19 Amit, A.G., Mariuzza, RA., Phillips, S.E.V. and Poljak, R.J. (I 986) Science 233,747-753 20 Chothia, C., Lesk,/-,. M., Levitt, M., Amit, A. G., Mariuzza, R. A., Phillips, S. E. V. ar~cJP~,Ijak,R. J. (I 986) Science 233, 755-758 21 Ehrlich, P. (I 900) in The Collected Papers of Paul Ehrlich (Himmelweit, ;:., ed.), pp. 178-195, Pergamon Press, New York Erratum
The editing of an a~icle o:" clinical,;mrnuno!ogyin Venezuela(Irnrnunol. Today, 1986, 10, 289) introduceo the ~rnpre~sionthat Whittingham and Mackay proposed the structure of a Clinical Imm~no!cgy Department particularly for Venezuela.]his was an error. We apologiseto Dr N Bianco,the article's aumor, for this lapse.
The antibodycombiningsite: retrospect and prospect The evolution of our current understanding of how antibodies exert their biological functions has consisted of periods of intense activity punctuated by periods of rather slow progress. Further development of Ehrlich's conceptual advances (which had laid the foundations of molecular immunology) was frustrated by the limitations of the prevailing theoretical framework, the absence of an understanding of antibody heterogeneity and unawareness of the complexity of the antigenic material being used. Forty-five years after Ehrlich dehvered his Croonian lecture to the Royal Society the direct demonstration by Landsteiner 1of apparently limitless numbers of antigenic specificities gave rise to instructive theories of antibody formation. Even after the proposal by Jerne2 and Burnet 3 of the now generally accepted selective theories of antibody formation nearly 60 years later, immunology could still not add much to Ehrlich's simplistic mechanism of antibody-antigen interactions, although it should not be forgotten that the possible contributions of ionic interactions had been anticipated by Arrhenius and Madsen 4. It was not until Porter s and Edelman 6 deduced the multi-chain structure of immunoglobulins that the modern view of the antibody combining site, as a structure consisting of variable plus constant domains, began to emerge. With the protein chemistry problem largely solved, large numbers of sequences of different immunoglobulins appeared and, as a result of the systematic analysis of light chain sequences by Wu and Kabat 7 and of heavy chains by Kehoe and Capra 8, the molecular determinants of complementarity were revealed. The distinctive feature was the presence of hypervariable regions within eacg chain (called complementaritydetermining regions or CDR by Wu and Kabat) bounded by less variable or framework regions. Thus, it became possible to pinpoint particular sequences that, when assembled into an appropriate three-dimensional structure, would form the antigen-binding surface. The sequence information alone, however, was in the end not sufficiently revealing, nor protein structure prediction so well advanced, as to allow the generation of a tertiary fold ab initio. The key to unlocking the structural problem was the highly conserved nature of the framework regions observed when antibocies of different specificity or even species were compared. When the first X-ray crystallographic structure was published by Polja!: et al. ~J the topological origins of this sequence cons( .-. ation became obviou,. Framework regions were seer :0 be organized as 13-strands in.+erconnected by loops, .:orresponding at the N-terminal ends of the strands :0 the CDR. The strar~ds were arranged in a 13-sheet sar~dwich (the 'immunoglobulin fold') the filling of which cogsisted of tightly packed hydrophobic side chains and an invariant disulphide be~d linldng the two sheets together. The constraints on side chain type necessary to pieserve 44
Laboratory of Molecular Biophysics, The Rex Richards Building, Oxford OX13OU, UK
AnthonyR. Rees this 13-fold were thought to be sufficiently stringent to explain the high sequence conservation. The hypervariable loops on the other hand were considered to be free to drift in sequence and conformation without disturbing the framework structure. When structures of other antibody binding site fragments were obtained this was indeed seen to be true. Thus, the antigen binding domain can be viewed as a structurally invariant framework (broadly speaking) on which six hypervariable chains are superimposed. The obvious question of interest is then: can the conformation of the binding site for an antibody of unknown structure be predicted? The earliest attempt at such a prediction was made by Davies and Padlan 1° who constructed a model for the binding site of a rabbit antibody ~pecific for type II polysaccharide using the three-dimensional structure of the mouse anti-phosphorylcholine antibody MOPC603 as a starting point. Their approach, having made the assumption that the framework regions in different antibodies would have essentially the same conformation, was to model the CDR of the rabbit antibody on the conformations of CDR of similar length in other known structures such as MOPC603 (a mouse Fab11), NEW (a human Fab9) and REI (a human light chain dimer~2). The resulting model exhibited a large number of side chains capable of hydrogen bonding, a construction that was particularly persuasive to the authors given the large proportion of -OH groups on the antigen. It is true to say, however, that even with the exhaustive sequence and structural analyses carried out since those early attempts, there was until recently no direct evidence that CDR from one antibody could simply be switched to another without disturbing the framework structure. Our own approach to predicting the hypervariable surface used and extended the methods of Davies and Padlan ~° with the added sophistication contributed by energy minimization 13. The models produced were subjected to test in the computer by attempting to 'dock' the known epitopes to the modelled surfaces and have subsequently been tested by protein engineering ~4,1S. Our analysis of existing structures provided the evidence for the notion that CDR conformation may be more influenced by length than by sequence 1°,13, but it was not until very recently that the 'constant framework: variable CDR' model of antibody combining sites was put to the most critical test of all. Could CDR from one antibody be switched to another's framework and in the process retain the antigen specificity of the CDR donor? Jones et aL 16 have almost answered the question by the elegant use of molecular biology techniques. Their approach was to carry out CDR replacement. A synthetic hybrid gene was constructed in which the heavy chain framework region of a human myeloma, NEWN, was linked with the CDR regions from the mouse anti-NP cap (4-hydroxy-3 nitrophenacetyl caproic acid) antibody, B1-8. The construct was then expressed in a lynnphoid cell ~) 1987. Elsev,erSciencePubhshersB V, Amsterdam 01(:
4919,87l$02 00
Immunology Today, vol. 8, No. 2, 1987
line that secreted only a K-light chain with the parental B1-8 specificity. The hybrid antibody exhibited the binding specificity of B1-8 indicating that, whatever heavy chain conformational determinants were critical for hapten binding, they were not disrupted b' the heterologous framework regions. Of course, it still remains to be formally demonstrated that a similar result would be obtained by light chain 'therapy', but this seems a foregone conclusion. By contrast, when the hybrid antibody was inspected by two different anti-idiotypic antibodies a difference in its reactivity profile was seen. The epitope for one antibody appeared to have been lost completely, while the other exhibited only partial reactivit v. The situation where antigen binding is retained but idiotypy is lost or impaired could lead one to conclude, as did Jones etaL 16 'that the immunoglobulin uses different sites to bind hapten and anti-idiotypic antibodies'. The question of the interrelationship between antigen binding regions and idiotopes has long been of immunological interest. Kieber-Emmons and KOhler 17 have classified idiotopes as: oL-idiotopes - those that lie out of reach of the antigen binding site; 13-idiotopes - those that are close to the antigen binding site so that non-competitive interference is observed; and ~/-idiotopes formed by the binding site itself. The class to which the idiotopes on B1-8 belong, defined by the anti-idiotopic antibodies Ac146 and Ac38, is unclear. The loss of binding may have arisen as a result of small structural perturbations occurring within CDR regions but still sufficiently distant from the binding site as to affect only idiotype interactions. It is well established that such small structural changes can often be accommodated locally 7.1°. Alternatively, the idiotopes may have been associated with residues at or near the framewor~-CDR junctioos where the difference between the native and hybrid antibodies would be most pronounced. Until the mappinq of idiotopes becomes a more precise operation their relationship to antigen binding regions will remain uncertain. When antibodies directed against protein antigens are considered, however, the elimination of idiotype interactions has a rather more obvious origin. Protein epitopes are large by hapten standards 18 and are thus likely to occlude a large proportion of the CDR on formation of antibndy-antigen complex, or so the presumption goes. A description of the structure of a lysozyme-antilysozyme complex at high resolution by Amit et al. 19 has removed any lingering doubt that this was the case. The elegant work of Poljak's laboratory gave us the first Fab structure and, with the new structure, has revealed an antibody-antigen interface of enormous (by hapten standards) proportions. The 20A x 3Oh, surface includes all! SiX CDR, and the degree of complementarity b~ween the two protein surfaces results in a complex sufficiently close-packed as to preclude any possibility of simultaneous anti-idiotype interactions: "... antioj~n binding should sterically hinder the reaction between combining site idiotopes and anti-id0otypic antibodies" 19 Where do we stand today? Although progress in understanding the molecular =sis of antigen recognition and its genetic 3rigins ho~ been ~ramatic, it is still true that we are totally ignorant about how particular combinations of CDR give rise to particular surface topologies. Undoubtedly, understanding will improve as empirical predictive methods are developed that make
use of antibody structure databases. If that were the only approach, however, we might be left waiting for an answer for decades. Rather, it is my view that the resolution will come using an approach that combines prediction with experimental test. Our own version of this exploits the techniques of protein engineering in the testing of computer-derived models of combining sites. More recently, the similar approach of Chothia et al. 2° in which a model of the anti-lysozyme antibody DI3 was produced, was tested directly by comparison with the crystal structure, with a high degree of success. There are still problems to be resolved but in the not too distant future it will surely be possible to construct binding site topologies knowing only the structure of the antigen and the sequences of the matching CDR. When that time arrives, we shall see the realization of Paul Ehrlich's hope "... that an understanding of antibodyantigen interactions will yield rich treasures for biology and therapeutics" 2i. References
1 Landsteiner, K. (1945) The Specificity of Serological Reactions Harvard University Press, Cambridge, MA. Paperback reprint (1962) Dover, New York 2 Jerne, N.K. (1955)Proc. NatlAcad. Sci. USA 41,849 3 Burnet, F.M. (1959) The Clonal Selection Theory of Antibody Formation, Vanderbilt University Press, Nashville 4 Arrhenius, S. (1907)/mmunochemistry, MacMillan Press, New York 5 Porter, R.R. (1959)Biochem. J. 73, 119-126 li Edelman, G.M., Cunningham, B.A., Gall, W.E. etal. (1969) Proc. Natl Acad. Sci. USA 63, 78-85 7 Wu, T.T. and Kabat, E.A. (1970)J. Exp. Med. 132, 21-50 8 Kehoe, J.M. and Capra, J.D. (1971) Proc. NatlAcad. Sci. USA 68, 2019-2021 9 Poljak, R.J.,Amzel, [. M., Avey, H.P. etal. (1973) Proc. Natl Acad. 5ci. USA 70, 3305 10 Davies, D.R. and Padlan, EA. (1976)in Antibodies Human Diagnosis and Therapy (Haber, E. and Krauser, R.M. eds), pp. 119-132, Raven Press, New York 11 Segal, D.M., Padlan, E.A., Cohen, G.H etal. (1974)Proc. NatlAcad. Sci. USA 71, 4298-4302 12 Epp, O., Latham, E., Schiffer, M., Huber, R. and Palm, W. (I 975) Biochemistry 14, 4943-4952 13 de la Paz, P., Sutton, B.J., Darsley, M.J. and Rees, A.R (1986) EMBOJ. 5, 415-425 14 Rees,A.R. and de la Paz, P. (1986) Trends Biochem. Sci. I i, 144-148 15 Roberts, S. and Rees,A.R. (1986) Prot. Eng. I, 59-66 16 Jones, P.T., Dear, P.H., Fnote, J. etal. (~986) ivature (London) 32 !, 522-525 17 Kieber-Emmons, T. and KOhler, H. (1986)Immunol. Rev. 90, 29-48 18 Benjamin, D.C., Berzofsky, J.A., East, l.J. etal. (1984)Ann. Rev. Immunol. 2, 67-101 19 Amit, A.G., Mariuzza, RA., Phillips, S.E.V. and Poljak, R.J. (I 986) Science 233,747-753 20 Chothia, C., Lesk,/-,. M., Levitt, M., Amit, A. G., Mariuzza, R. A., Phillips, S. E. V. ar~cJP~,Ijak,R. J. (I 986) Science 233, 755-758 21 Ehrlich, P. (I 900) in The Collected Papers of Paul Ehrlich (Himmelweit, ;:., ed.), pp. 178-195, Pergamon Press, New York Erratum
The editing of an a~icle o:" clinical,;mrnuno!ogyin Venezuela(Irnrnunol. Today, 1986, 10, 289) introduceo the ~rnpre~sionthat Whittingham and Mackay proposed the structure of a Clinical Imm~no!cgy Department particularly for Venezuela.]his was an error. We apologiseto Dr N Bianco,the article's aumor, for this lapse.