Strong in Vivo Maturation Compensates for Structurally Restricted H3 Loops in Antibody Repertoires

A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites. Much of the diversity of this repertoire is provided by varying one antigen binding loop, created by inserting randomly a D (diversity) gene out of a small pool between the V and J genes. It is therefore assumed that any particular D-encoded region surrounded by different V and J regions adopts a different conformation. We have solved the structure of two lysozyme-specific variable domains of heavy-chain antibodies isolated from two strictly unrelated dromedaries. These antibodies recombined identical D gene sequences to different V and J precursors with significant variance in their V(D)J junctions. Despite these large differences, the D-encoded loop segments adopt remarkably identical architectures, thus directing the antibodies toward identical epitopes. Furthermore, a striking convergent maturation process occurred in the V region, adapting both binders for their sub-nanomolar affinity association with lysozyme. Hence, on a structural level, humoral immunity may rely more on well developed maturation and selection systems than on the acquisition of large primary repertoires. A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites. Much of the diversity of this repertoire is provided by varying one antigen binding loop, created by inserting randomly a D (diversity) gene out of a small pool between the V and J genes. It is therefore assumed that any particular D-encoded region surrounded by different V and J regions adopts a different conformation. We have solved the structure of two lysozyme-specific variable domains of heavy-chain antibodies isolated from two strictly unrelated dromedaries. These antibodies recombined identical D gene sequences to different V and J precursors with significant variance in their V(D)J junctions. Despite these large differences, the D-encoded loop segments adopt remarkably identical architectures, thus directing the antibodies toward identical epitopes. Furthermore, a striking convergent maturation process occurred in the V region, adapting both binders for their sub-nanomolar affinity association with lysozyme. Hence, on a structural level, humoral immunity may rely more on well developed maturation and selection systems than on the acquisition of large primary repertoires. The interaction of conventional antibodies with antigens is mediated by up to six dedicated hypervariable loops, three (H1-H3) 1The abbreviations used are: H1, H2, H3, the first, second, and third hypervariable loops of the variable domain of the heavy chain of an antibody, respectively; L1, L2, L3, the first, second, and third hypervariable loops of the variable domain of the light chain of an antibody, respectively; VH, variable domain of the heavy chain of an immunoglobulin; VL, variable domain of the light chain of an immunoglobulin; VHH, variable domain of a heavy-chain antibody; cAb, camel single domain antibody; V, variable gene; D, diversity gene; J, joining gene; VH, variable gene of the variable domain of the heavy chain; VL, variable gene of the variable domain of the light chain; VHH, variable gene of a camelid heavy-chain antibody; JH, joining gene of the variable domain of the heavy chain; HEWL, hen egg white lysozyme; ΔASA, change of accessible surface area. in the variable domain of the heavy chain (VH) and three (L1-L3) in the variable domain of the light chain (VL) (1.Padlan E.A. Adv. Protein Chem. 1996; 49: 57-133Crossref PubMed Google Scholar). Within the antigen-combining site, the H3 loop is the major determinant of antibody diversity and the major contributor for overall antigen affinity and specificity (1.Padlan E.A. Adv. Protein Chem. 1996; 49: 57-133Crossref PubMed Google Scholar, 2.Milstein C. Neuberger M.S. Adv. Protein Chem. 1996; 49: 451-485Crossref PubMed Google Scholar, 3.Xu J.L. Davis M.M. Immunity. 2000; 13: 37-41Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). This loop is generated after recombining one V, one D, and one J gene out of a pool. Imprecision in the V(D)J recombination event, with concomitant nucleotide deletions and N or P nucleotide additions (4.Tonegawa S. Nature. 1983; 302: 575-581Crossref PubMed Scopus (3177) Google Scholar, 5.Weill J.C. Reynaud C.A. Immunol. Today. 1996; 17: 92-97Abstract Full Text PDF PubMed Scopus (83) Google Scholar), makes it possible for a particular D gene to occur in different locations and reading frames within the H3-encoded region (4.Tonegawa S. Nature. 1983; 302: 575-581Crossref PubMed Scopus (3177) Google Scholar, 5.Weill J.C. Reynaud C.A. Immunol. Today. 1996; 17: 92-97Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Hence, the sequence of the H3 loop becomes the most diverse of all antigen binding loops. The crystal structure determination of different antibodies revealed the presence of canonical structures for all antigen binding loops (L1-L3, H1, and H2) except for the H3 loop (6.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. Colman P.M. Spinelli S. Alzari P.M. Poljak R.J. Nature. 1989; 342: 877-883Crossref PubMed Scopus (1084) Google Scholar, 7.Al-Lazikani B. Lesk A.M. Chothia C. J. Mol. Biol. 1997; 273: 927-948Crossref PubMed Scopus (591) Google Scholar). The folding of a particular loop into a canonical structure is dictated by a small number of conserved key residues within the hypervariable sequences. The immense sequence diversity of the H3 loop with the D-encoded part, which can be located anywhere within the loop, obviously precludes an easy assignment of such key residues. Moreover, because the number of possible V(D)J recombinations exceeds by far the number of crystal structures that are available, it is as yet impossible to investigate whether the D-encoded part of the loop adopts different or similar backbone architectures when present in a different V-J surrounding. Likewise, it remains an open question whether the same epitope on a large antigen will be recognized by antibodies originating from different B cell lineages that employed the same D gene in their V(D)J recombination. This might be expected if the structure of H3 loop is largely determined by the D gene-encoded segment and if this part of the paratope dominates the antigen specificity. However, this would also mean that the structural repertoire of the H3 loop is less diverse than predicted by the immense sequence diversity. These fundamental questions might be easier to assess with antibodies from camelids because a considerable fraction of their serum antibodies lack light chains (8.Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Nature. 1993; 363: 446-448Crossref PubMed Scopus (2205) Google Scholar, 9.Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-295Crossref PubMed Scopus (133) Google Scholar). Up to three loops (H1-H3) of the variable domain of these heavy-chain antibodies (VHH) constitute the antigen-binding site. The VHH, like VH, is generated after V(D)J gene rearrangements (10.Nguyen V.K. Hamers R. Wyns L. Muyldermans S. EMBO J. 2000; 19: 921-930Crossref PubMed Scopus (216) Google Scholar). Despite the absence of the light chain, the heavy-chain antibodies recognize, via their VHHs, a wide range of antigens with affinity constants that are comparable with those found for conventional antibodies (11.Muyldermans S. Cambillau C. Wyns L. Trends Biochem. Sci. 2001; 26: 230-235Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). The H3 loop of VHHs is, on average, longer than in human or mouse VHs (9.Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-295Crossref PubMed Scopus (133) Google Scholar). It was therefore postulated that the extended H3 loop compensates for the loss of VH-VL combinatorial diversity, making its contribution to the diversity of the repertoire even more significant than in conventional antibodies. Furthermore, the H3 loop of a VHH is even more crucial for specific antigen recognition than the H3 loop of a VH-VL paratope. This is illustrated by the crystal structures of VHH-antigen complexes where up to 100% of the contacts with the antigen can be provided by the H3 loop (12.Decanniere K. Desmyter A. Lauwereys M. Ghahroudi M.A. Muyldermans S. Wyns L. Structure. 1999; 7: 361-370Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 13.Desmyter A. Decanniere K. Muyldermans S. Wyns L. J. Biol. Chem. 2001; 276: 26285-26290Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Here we report the structure of two VHHs, cAb-Lys2 and D2-L19, generated from different V and J genes but sharing identical D gene sequences. These binders had matured in two different dromedaries immunized with hen egg white lysozyme (HEWL) to the same epitope and were helped by striking convergent hypermutations within the H2 loop region. The structure solution of these VHHs in complex with their antigen demonstrates that the in vivo recombination of an identical D gene segment can lead to congruent H3 loop architectures even when occurring in a different V-J context and masked by different somatic mutations. Isolation and Purification of the VHHs—The dromedary immunization in Morocco, library generation, and pannings to retrieve the lysozyme binder cAb-Lys2 have been described elsewhere (14.Ghahroudi M.A. Desmyter A. Wyns L. Hamers R. Muyldermans S. FEBS Lett. 1997; 414: 521-526Crossref PubMed Scopus (590) Google Scholar). Other dromedaries kept in Dubai (United Arab Emirates) were immunized over a 2-month period with lysozyme, and phage-displayed libraries of the VHHs were made during the work described by Lauwereys et al. (15.Lauwereys M. Ghahroudi M.A. Desmyter A. Kinne J. Holzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (398) Google Scholar). Panning of these libraries with immobilized antigen yielded D2-L19 and six additional lysozyme-specific VHHs. D2-L19 and cAb-Lys2 were recloned in an expression vector and tagged with a His6 tail. The recombinant VHHs were produced in Escherichia coli and purified to homogeneity by IMAC and gel filtration according to Conrath et al. (16.Conrath K.E. Lauwereys M. Galleni M. Matagne A. Frere J.M. Kinne J. Wyns L. Muyldermans S. Antimicrob. Agents Chemother. 2001; 45: 2807-2812Crossref PubMed Scopus (279) Google Scholar). Kinetic and Affinity Measurements of the VHH-HEWL Interaction— The kinetic constants were determined by surface plasmon resonance on a Biacore 3000® (BIAcore, Uppsala, Sweden). HEWL was immobilized on a CM5 chip by amine coupling using N-ethyl-N′-(dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), and ethanolamine according to the manufacturer's recommendations. 100 resonance units of protein were immobilized in flow cell 2 of the chip. The surface in flow cell 1 was used as a reference and treated only with EDC, NHS, and ethanolamine. Sensorgrams of five concentrations (500-30 nm (D2-L19) and 50-3 nm (cAb-Lys2)) plus a zero concentration (injection of running buffer) were collected. The curves were fitted to a 1:1 Langmuir binding model with the BIAevaluation software (version 3.2) (BIAcore) after subtraction of the reference and zero concentration data. Identical ka and kd values were obtained for measurements with antibodies immobilized (100 resonance units) to the matrix surface and HEWL in the mobile phase. Crystallization of the VHH-HEWL Complexes—The VHH-HEWL complexes were obtained following existing protocols (17.Desmyter A. Transue T.R. Ghahroudi M.A. Thi M.H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (417) Google Scholar). 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PubMed Scopus Google Scholar). of D2-L19 and VHHs, cAb-Lys2 from a dromedary immunized in in and D2-L19 from a dromedary immunized in in were isolated by (14.Ghahroudi M.A. Desmyter A. Wyns L. Hamers R. Muyldermans S. FEBS Lett. 1997; 414: 521-526Crossref PubMed Scopus (590) Google Scholar, M. Ghahroudi M.A. Desmyter A. Kinne J. Holzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (398) Google Scholar). these VHHs employed the same D gene that was to different V and J the are to it that the D gene the E. T. K. and C. of of S. D. Scholar), a 100 and in cAb-Lys2 for The of these sequences in the VHH genes revealed five nucleotide to 2 The most J K. and were used for the recombination of cAb-Lys2 and D2-L19, A in the VHH (10.Nguyen V.K. Hamers R. Wyns L. Muyldermans S. EMBO J. 2000; 19: 921-930Crossref PubMed Scopus (216) Google revealed that the V region of cAb-Lys2 most from the gene sequence which nucleotide and the V region of D2-L19 most from the gene sequence sequence of D2-L19 and that the had been and to and were These two genes to the VHH that is most used to heavy-chain antibodies in dromedary (10.Nguyen V.K. Hamers R. Wyns L. Muyldermans S. EMBO J. 2000; 19: 921-930Crossref PubMed Scopus (216) Google Scholar). The number of and the that are in these binders are for antibodies obtained after in vivo affinity maturation C. Neuberger M.S. Adv. Protein Chem. 1996; 49: 451-485Crossref PubMed Google Scholar, J. C. Nature. PubMed Scopus Google Scholar, J. Sci. S. A. 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Scopus Google Scholar). the VHH residues from the H2 and H3 loops in antigen However, the of contacts are mediated by the H3 loop for D2-L19 and for of which the part by the D region and the two J crucial contacts with the The and of H3 for to the paratope of of the between the VHH and HEWL are found The loop segment of cAb-Lys2 for to the of its paratope with of the of H3 and of the H2 remarkably congruent is by the and by the D gene and an that to the number of possible in the Furthermore, the part of the H3 loop in VHH over and with the 2 region of the as been (9.Nguyen V.K. Desmyter A. Muyldermans S. Adv. Immunol. 2001; 79: 261-295Crossref PubMed Scopus (133) Google Scholar). However, the different hypermutations in the D-encoded during in and its (D2-L19) a different of and residues Hence, the of the D and J gene-encoded of H3 is by residues. with the conserved of the and the by the of both HEWL binders adopt different the backbone of the loop and the of the H2 loop are different in the two VHHs The paratope residues in H2 of D2-L19 and cAb-Lys2 somatic of which a striking convergent in cAb-Lys2 and in D2-L19 all these convergent the occurring in both VHHs in particular a for HEWL interaction because it a between the H2 and H3 loops. The of HEWL into this and is surrounded by residues from this that is an of the VHH-HEWL interaction L. Chothia C. J. J. Mol. Biol. 1999; PubMed Scopus Google Scholar, D. S. S. Sci. S. A. PubMed Scopus Google Scholar). in both the of all epitope for and and making two with cAb-Lys2 and three with The that and which were from and in cAb-Lys2 and and in D2-L19 The convergent maturation of the in an between this and the chain of A and The chain of the matured with the a between the chain of and located the of the VHH 2 region. This the of the H3 The also contacts via its chain, a between the of and the on the chain of in the complex or a in the The in of the chain in the two VHH-HEWL complexes might have might be from different of the or complexes within the the might have been in these VHHs for than structural VHHs, cAb-Lys2 and D2-L19, were obtained after from two different immunized dromedaries. revealed that both VHHs from the recombination of a D gene with different V and J to different and junctions. Despite significant in these and the D-encoded adopt an identical conformation. the hypermutations in the D-encoded part of the H3 loop segments change the loop but a different in the residues and The D-encoded are located in the VHH-HEWL and key with the Furthermore, a striking convergent maturation process occurred in the H2 region, adapting both binders for their high affinity association with an identical epitope on It is that the two VHHs described in this report are from because from two different The of the D gene sequences in these matured binders for a of the D-encoded to with a particular This is from an isolated as it is by the of the sequences of VHHs with antigen specificity or The revealed that an D gene and convergent maturation via hypermutations toward the same loop sequences are Moreover, revealed that the binders sharing identical D segments also to on their De K. S. and L. V, D, and J gene recombination been for conventional antibody particular C. Neuberger M.S. Adv. Protein Chem. 1996; 49: 451-485Crossref PubMed Google Scholar, J. C. Nature. PubMed Scopus Google Scholar, T. Mol. Immunol. PubMed Scopus Google Scholar), it yet been for large protein The of D genes in antibodies the same antigen also from a structural repertoire of this loop within the primary antigen-combining sites. The crystal structures of D2-L19 and cAb-Lys2 with a identical for the part of the H3 loop by a D gene this We whether the of this part of the loop would also occur when by D genes. of all VHH crystal structures revealed that the by the of is in three VHHs. These VHHs employed different D genes and are unrelated the the of the in E. R. J. T. G. Muyldermans S. Wyns L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), A (12.Decanniere K. Desmyter A. Lauwereys M. Ghahroudi M.A. Muyldermans S. Wyns L. Structure. 1999; 7: 361-370Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and human lysozyme M. A.M. Desmyter A. Decanniere K. D. G. A. J. Muyldermans S. Wyns L. C. Matagne A. Robinson Nature. PubMed Scopus Google the part of the H3 of these makes with the The identical backbone of these is dictated one by a or the between two in 2 and The following residues a of a the the is to the conserved the of the an by the J gene and conserved in all camelid genes in D2-L19 or in toward the VHH and an with and and The chains are to different loop sequences. therefore that the recombination of identical D genes to different VHH and J precursors would lead to identical H3 loop The of a structural within part of the H3 loop two the structural of this and of its key residues have a for VHHs. it for a of the structural repertoire of the antigen-combining in the primary This that a longer H3 loop in camelid antibodies with a possible sequence repertoire the structural of H3 loops (11.Muyldermans S. Cambillau C. Wyns L. Trends Biochem. Sci. 2001; 26: 230-235Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). A striking convergent maturation occurred in the H2 loops of cAb-Lys2 and This maturation is by significant structural The hypermutations a in the paratope that the chain of of the is to and to the of the VHH-HEWL The convergent the and the for a of the interaction with the it that the somatic to a convergent maturation during the selection process is an to high affinity Moreover, this process the VHHs that are from the same D sequence in their maturation process toward the same considerable on the of somatic in the antigen maturation process Nat. Immunol. PubMed Scopus Google Scholar). an maturation been for in the of affinity somatic for antigens been affinity antibodies have been in during their with a A. Sci. S. A. PubMed Google or a protein antigen A. C.A. M.M. Poljak R.J. J. Immunol. 1999; Google Scholar). This can be by the number of between an antibody and a as to protein which more than of interaction surface L. Chothia C. J. J. Mol. Biol. 1999; PubMed Scopus Google Scholar, Adv. Protein Chem. Scopus Google Scholar). that somatic also a significant in the of the antigen-combining toward a large This maturation for the selection of a of antigen specificity of the that from a selection process from VHHs with a structural a an large structural diversity within the VHH primary this it is that VHHs the same D gene can adopt a wide of H3 structures when present in a different V and J However, the antigen in the selection of only a VHH that by the D-encoded part in a similar conformation. it can be that the particular antigen (HEWL) used in this can only with a of paratope that all VHHs with H3 loop were from the it can postulated that the camelid heavy-chain antibody maturation and VHHs constitute an that be to conventional antibodies because lack the combinatorial diversity provided by the association and the interaction might more H3 loop these be the successful immunization of only one or a number of and genes J.L. 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