Immunologicat Reviews 1992, No. 130 Published by Munksgaard, Copenhagen, Denmark . . , ^ u _/ x No part may be reproduced by any process without written permission from the author(s)

Genetically-Engineered Antibodies: Tools for the Study of Diverse Properties ofthe Antibody Molecule SEtJNG-UoN

SHIN*, ANN WRIGHT*, VINCENT

BoNAGURAt &

SHERIE L. MORRISON*

INTRODUCTION The capacity to produce genetically-engineered antibodies has altered our ability to investigate antibody function and to produce antibodies that are useful for both in vitro and in vivo applications. Among the getietically-engineered antibodies are chimeric antibodies in which segments from immunoglobulins from diverse species are joined together. Mouse/human chimeric antibodies in particular are potentially useful for immunotherapy for they should exhibit the same specificity but reduced immunogenicity compared to their murine counterparts. Chimeric antibody production has progressed through several generations producing ever more imaginative combinations of antigen specificity and effector functions. Chimeric antibodies have proven to be invaluable tools for the study of immunoglobulins. The availability of cloned constant region genes has made it possible to produce rare antibodies, such as IgD and IgE, in quantities sufficient for study. The technology allows the systematic study of structure-function relationships in antibodies and the determination ofthe structural differences which lead to variation in biological activities such as complement activation and Fc receptor binding. Gene transfection and expression has also been a valuable approach to the study of post-translational modifications such as glycosylation. Genetically-engineered antibodies can also be used as antigens to determine the specificity of immune recognition. This review will focus primarily on experiments from our laboratory which have addressed these issues. * Department of Microbiology and Molecular Genetics and the Molecular Biology Institute, UCLA, Los Angeles, CA 90024, t Schneider Children's Hospital, Department of Pediatrics, Long Island Jewish Medical Center, New Hyde Park, NY 11042, USA. Correspondence: Sherie L. Morrison, Ph.D., UCLA, Molecular Biology Institute, Los Angeles, CA 90024, USA.

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The organization of the antibody genes with the domain structure of antibody reflected in the exon structure of the genes makes them particularly amenable to such manipulations as domain exchange {"exon shuffling") (Fig. 1). Domain shuffling has also made it possible to identify the region of the antibody which is critical to the function of interest and to produce fusion proteins with domains joined together in novel combinations or expressed with non-immunoglobulin proteins. As the sequences of immunoglobulin genes have become available for comparison, it has become possible to predict residues critical for certain effector functions and evaluate their role by site-directed mutagenesis. Although it is possible to use transient transfection to produce sufficient quantities of antibodies for limited studies, we have primarily utilized stable transfectants in our studies. Stable transfectants provide a continuous, well-characterized source of antibodies. Electroporation, protoplast fusion (Wright & Shin 1991) and lipofection (Feigner et al. 1987) are all effective means of obtaining stable antibody-producing cell lines. Suitable cellular recipients include non-producing murine myeloma cell lines such as SP2/0 and P3X63Ag8.653 and Chinese hamster ovary cells. INVESTIGATION OF FUNCTIONAL PROPERTIES OF ANTIBODIES /. Complement activation By modifying antibody structure using genetic engineering we can gain insights into antibody function and produce antibodies with improved functional properties. Complement is an important soluble system triggered by interaction with antibody. Antibodies of most species have the ability to activate complement; however, by focusing attention on the four human IgG isotypes, which are very similar in amino acid sequence but differ in the ability to activate complement, it is possible to gain insights into the structural requirements for complement activation. The complement cascade is complex with many difTerent proteins interacting and being modified during the activation process. Cl, the first component ofthe cascade, is comprised of one molecule of Clq, two of Clr, and two of CIs. This first component of complement binds tightly to immune complexes, with binding followed by the cleavage of Clr and CIs both of which are proteases in the activated state. Clq interacts only weakly in solution with a single IgG (Shumaker et al. 1976) but the afiinity of interaction increases when IgG becomes multimeric in an immune complex (Tschopp et al. 1980). Clustering of several antibody molecules on a multivalent determinant appears to be sufficient to provide the activation signal (Metzger 1978). Activation is regulated by Cl inhibitor which binds the complex, blocking its enzymatic activity. Activated CIs is highly specific for its substrates, C2 and CA. C4 is cleaved into C4a and C4b. C2 binds at a site on C4b and is cleaved by nearby CIs. Amplification of the complement cascade occurs at both of these steps with the

PROPERTIES OF RECOMBINANT ANTIBODIES VH

CHI

H

CH2

CH3

CH3

VH

CHI

H

CH2

CH3

CIHIHHZXZl—

Figure I. Schematic diagrams of domain shuffling in antibodies. The mouse-human heavy chain genes are shown al the top, and the resulting antibodies at the bottom. The dark crossed region represents a murine anti-dansyl (DNS) variable region and the open and shaded regions represent the constant regions of human lgG2 and lgG3, respectively. In the resulting antibodies, the CH2 domain of anti-DNS chimeric IgG3 has been replaced with thai of IgG2.

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i

isotypes apparently differing in their amplification ability (Bruggeman et al. 1987, Bindon et al. 1988, 1990). The C4b,2a complex forms the enzyme for the cleavage of C3 and, in association with C3b,C5. The C5 cleavage product, C5b, assembles with C6-C9 to generate the membrane attack complex, which causes lysis. Antibodies can differ in their ability to interact with Clq, to amplify the cascade, and to avoid inactivation by complement inhibitors. In the human, the IgGl and IgG3 isotypes are the most effective in complement activation. IgG2 is much less effective and functions under only some assay conditions; IgG4 is unable to bind Clq and activate complement (Dangl et al. 1988). It remains to be ascertained which residues determine the isotype-specific differences. The CH2 domain plays an important role in complement activation. Facb (IgG depleted of CH3) and CH2 fragments bind Cl and activate complement, while Fab and CH3 fragments do not show any activity (Utsumi et al. 1985, Yasmeen et al. 1976, Colomb & Porter 1975). Disruption of CH2 by removing the carbohydrate added at Asn297 profoundly decreased the ability of the molecule to activate complement (Tao & Morrison 1989, Gillies & Wesolowski 1990); molecules produced in yeast with the yeast high mannose carbohydrate and chimeric antibodies lacking CH2 are unable to carry out complement-mediated cytolysis (Horwitz et al. 1988, Gillies & Wesolowski 1990). While these studies have demonstrated the importance of CH2, it remains unclear which residues in CH2 constitute the Clq binding site and contribute to complement activation. Site-directed mutagenesis has demonstrated the importance of GIu318, Lys320 and Lys322 along an exposed j9-strand in C,.,2 for complement activation (Fig. 2) (Duncan & Winter 1988). However, these residues are not polymorphic among the human isotypes and so cannot explain the observed isotype-specific differences. Exon shuffling has confirmed the importance ofthe CH2 domain in complement activation and intra-exon shuffling has located the structures primarily responsible for the differential ability of human IgGl and IgG4 to activate complement to the COOH-terminal part (from residue 292 to 340) of the CH2 domain (Tao et al. 1991). Within this region, IgGl differs from IgG4 at only four residues: 296 (Tyr vs Phe), 327 (Ala vs Gly), 330 (Ala vs Ser) and 331 (Pro vs Ser). The side chain of residue 327 is mostly buried inside the molecule and is probably not directly involved in Cl binding; however, the greater flexibility afforded by the Gly residue may influence the conformation of the nearby complement binding site and thereby influence complement activation. Residue 296 from the opposing domain is accessible and may also contribute to the Clq binding site. Both IgGI and IgG3, the most effective isotypes in complement activation, have Tyr 296 while IgG2 and IgG4 have Phe296. Residues 330 and 331 fold into proximity with the previously identified 318-320-322 residues (Duncan & Winter 1988). Therefore these residues together may provide the binding site for Clq (Fig. 2).

PROPERTIES OF RECOMBINA^JT ANTIBODIES

-331

Figure 2. Space-filling model ofthe Fe region of human IgG. Residues implicated as being important for either complement activation or FcyRl binding are indicated. The crystal structure is from Diesenhofer (1981). The graphic was made using Maclmdad.

Indeed, site-directed mutagenesis has shown that alteration of Ser331 to Pro in the 1.1.1/4.4 molecule improves its ability to activate complement. Similarly, substitution of Ser for Pro331 in IgG3 decreased its ability to activate complement (Tao, Smith, Morrison, in preparation). The hinge regions serve as spacers separating the antibody's Fab arms from the Fc and, in contrast to the C region domains, are structurally diverse. They vary in amino acid sequence as well as length. IgGl, IgG2, and IgG4 have hinge regions of from 12-15 amino acids while IgG3 has an extended hinge region of 62 amino acids including 21 prolines and 11 cysteines. The functional hinge as deduced from crystallographic studies is more extensive than the genetic hinge and can be divided into three regions: the upper hinge, the core and the lower hinge (Burton 1985, Feinstein et al. 1986). The upper hinge includes the amino acids from the end of CHI to the first residue in the hinge that restricts motion, generally the first cysteine that forms an inter-heavy chain disulfide bond. The length of the upper hinge correlates with the segmental flexibility of the antibody (Dangl et al. 1988). Tlie core hinge contains the inter-H chain disulfide bridges and the lower hinge connects to the CH2 domain and includes amino acids in CH2.

Even though the binding site for Clq is located in CH2, the structure of the hinge region influences the ability of IgG to activate complement. Molecules lacking a hinge region are unable to activate complement, probably because access to the region involved in Clq binding is restricted (Tan et al. 1990). While studies have indicated that hinge length and segmental flexibility correlate with

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the ability ofthe IgG to consume complement (Dangl et al. 1988, Oi et al. 1984), this correlation is not absolute since IgG3 molecules with altered hinge regions as rigid as IgG4 activate complement effectively (Tan et al. 1990). Similarly, the inability of IgG4 to activate complement is not the result of its rigid hinge, since IgG4 with a flexible hinge was still unable to activate complement (Tan et al. 1990, Sandlie et al. 1989). When the influence of variations in antigen concentration, epitope patchiness, antibody binding affinity and complement concentration (Michaelsen et al. 1991) were studied the IgG3 subclass was generally most effective in inducing cytolysis, particularly at high patchiness, which might be expected from its greater flexibility. Only at high antigen concentration did the IgGl subclass mediate more efficient cytolysis than IgG3. A major focus has been the identification of the sequences within the normal Ig molecule which contribute to complement activation. However, non-Ig sequences can also influence the ability of antibody molecules to activate complement. In our studies we have made the surprising observation that antibody molecules in which insulin-like growth factor 2 (IGF2) was fused to the CH3 domain of mouse/human chimeric IgG3 showed an approximately 100-fold increase in the ability to effect complement-mediated cell lysis, compared to the chimeric lgG3 (Fig. 3) (Shin et al. unpublished data). The initial demonstration of this was made using antigen-coated SRBC. The lysis has been demonstrated to be antigen-specific and not just a non-specific effect ofthe IGF2 on the SRBC. The mechanism by which IGF2 effects this potentiation remains unclear at the present time.

Cone, of Fusion Protein (|iM) Figure 3. Complement-dependent cell lysis by fusion proteins. Anti-DNS chimeric IgG (square) and anti-DNS chimeric IgG3-lGF2 fusion proteins (circles) were incubated at varying concentrations with DNS-coupled (filled symbols) or non-coupled (open symbols) sheep red blood cells (SRBC) and SRBC lysis monitored.

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2. Specificity ofthe interaction with FcyRI Important for antibody function is the triggering of host cellular effector systems through the interaction of cellular receptors with antibodies or immune complexes. The antibody molecule serves as a bridge, with one polypeptide chain able to simultaneously bind the foreign antigen and immune effector cells. IgG interacts through its Fc region with a family of receptor molecules. The human Fc receptor family can be divided into three groups based on IgG subclass specificity and recognition by monoclonal antibodies: FcyRI, II, and III. FcyRI (CD64) is distinctive in being the only receptor capable of binding monomeric IgG with high affinity (Unkeless 1989) while FcyRII and FcyRIII require the presentation of ligand as either soluble IgG complexes or IgG-coated particles. Receptor interaction consists both of binding and cell triggering and the two events may have different structural requirements. Human IgG can be subdivided into three categories based on its recognition by FcyRI; IgGl and IgG3 bind with high affinity (A, 10^-10' M"'), IgG4 binds with approximately a 10-fold reduction in affmity, while IgG2 does not show any significant binding. CH2 plays a central role in determining binding by FcyRI. Monoclonal antibodies against CH2 are able to block interaction with the receptor (Partridge et al. 1986), and a CH2 domain-deleted IgGl myeloma protein failed to bind FcyRI (Woofet al. 1984). Exon exchange experiments have also supported a dominant role for CH2 (Shopes et al. 1990) and, in the context ofa complete IgG molecule, substitution of CH2 of IgG2 into either IgGl (Chappel et al. 1991) or IgG3 (Canfield & Morrison 1991) was sufficient to abolish interaction with FcyRI. Antibodies lacking the conserved carbohydrate present in the CH2 domain of IgG molecules fail to bind to the human FcyRI, indicating that the carbohydrate interposed between CH2 domain of human IgG is necessary to maintain the appropriate structure ofthe CH2 domain (Tao & Morrison 1989). Aglycosylation changes the environment of His268 (Lund et al. 1990) and also alters the recognition of the antibody by some members of a panel of monoclonal antibodies (Tao 1990). Sequences folding proximal to the hinge must be accessible for FcyRI to interact and antibodies lacking a hinge region fail to bind to FcyRI, probably as a consequence of decreased accessibility resulting from the close proximity of the Fab (Woofet al. 1984. Canfield & Morrison 1991, Tan et al. 1990). Deletion of the CHI domain from IgGl also results in a greater than 10-foId decrease in affmity for the receptor; when CHI is deleted, the light chain constant region may impede access (Canfield & Morrison 1991). An interesting question is what changes in IgG2 lead to its failure to bind FcyRI. Sequence comparison of IgG2 with IgGI and IgG3 shows only 7 positions within CH2 at which there are sequence differences with the differences concen-

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trated in the hinge proximal loop of residues 233-236. The sequence of this region for IgGl and IgG3 is Glu-Leu-Leu-Gly; the comparable sequence in IgG2 is ProVal-Ala. Therefore, IgG2 shows 3 amino acid substitutions and an amino acid deletion. Of central importance in determining non-binding is Glu235; in the context of human IgG, alteration of Leu235 to Glu was sufficient to destroy receptor binding (Canfield & Morrison 1991). However, the other amino acid changes in this region also infiuence binding, since alteration of any ofthe amino acids at 234-236 of IgGl to the comparable residues of IgG2 decreased or destroyed its ability to bind FcyRI (Chappel et al. 1991). This region of the molecule is not resolved in the solved crystal structure (Deisenhofer 1981). Sequence differences in the hinge proximal region have been shown to contribute to the decreased ability of IgG4 to bind FcyRI. IgG4 has a Phe at 234 while both IgGl and IgG3 have Leu at this position. Substitution of Phe into IgG3 at this position decreased its ability to bind FcyRI; the reciprocal substitution of Leu into IgG4 increased the affinity for FcyRI. In one case the substitution fully restored the ability of IgG4 to bind FcyRI to that of IgGl and IgG3 (Chappel et al. 1991); using different antibodies there was an increase in affmity but it was still less than that of IgGl and IgG3 (Canfield & Morrison 1991). Htunan IgG4 and IgGl and IgG3 also differ in sequence in the loop of amino acids, 327-331, which folds in proximity to the hinge. Indeed, substitution of Pro331 by Ser within IgG3 resulted in a decreased affmity for FcyRI (Canfield & Morrison 1991). No studies have addressed the impact ofthe Gln-His polymorphism at residue 268 on FcyRI binding. The context of the CH2 domain can also influence the interaction with FcyRI. Insertion of the CH2 domain of IgG3 into IgG2 resulted in a molecule with some reduction in affmity compared to IgG3 and either shortening of lengthening the hinge of IgG3 somewhat impaired its receptor binding. Substituting the hinge of IgG4 into IgGl had negligible impact on binding affmity (Canfield & Morrison 1991).

RECOMBINANT ANTIBODIES AS ANTIGENS: SPECIFICITY OF RECOGNITION BY RHEUMATOID FACTORS A primary focus of attention has been the functional properties of geneticallyengineered antibody molecules. However, they can also be used as antigens to determine the specificity of recognition by antibodies directed to human immunoglobulins and have been useful in determining the specificity of recognition of a panel of murine antibodies specific for different isotypes (Hamilton 1990, Hamilton & Morrison submitted). One particular question which we have attempted to address using genetically-engineered antibodies is the binding specificity of rheumatoid factors (RFs).

PROPERTIES OF RECOMBINANT ANTIBODIES

95

RFs are autoantibodies that bind to the constant region of IgG. IgM and IgG RFs are abundant in pannus tissue obtained from the joints of RF-positive rheumatoid arthritis (RA) and are thought to contribute to the synovial inflammation (Mellors et al. 1960, Pernis et al. 1984). However, the presence of RFs is not specific for rheumatoid arthritis (Estes & Christian 1971, Carson et al. 1978, Bunim et a). 1964, Dresner & Trombly 1959). Monoclonal IgM RFs are present in the sera of some patients with Waldenstrom's macroglobulinemia (WMac), a lymphoproliferative disease without articular symptoms. RFs also occur in apparently health individuals (Dresner & Trombly 1959, Fong et al. 1985) and are elicited by antigen-antibody complexes as part ofthe normal immune response (Levine & Axelrod 1985). An intriguing question is why rheumatoid disease is found in some individuals with RFs but not in others. We have attempted to determine if the RFs present in individuals with rheumatoid disease had different binding specificities from those found in individuals lacking disease and if this difference in specificity correlates with pathogenesis. Previous studies evaluated RF-binding specificities by measuring RF binding to myeloma IgG. Proteolytic digestion was used to produce antibody fragments which were then used to identify the regions on IgG recognized by the RF (Sasso et al. 1988, Nardella et al. 1981). However, using this approach it is difficult to purify myeloma proteins completely free of contamination by other normal IgG isotypes and proteolytic fragments may exhibit an altered conformation which would have an impact on their recognition by the RF. To precisely localize the RF binding sites on IgG we have instead utilized a system in which chimeric mouse-human IgG genes manipulated in vitro and expressed as mAb after transfection into lymphoid cells are used as antigens. A combination of site-directed mutagenesis and domain shuffling can then be used to precisely map the epitopes on IgG recognized by the RFs. This approach has the advantage of greater flexibility because any potential epitope can be mutagenzied to test its importance. In addition, all changes are made in the context of an intact IgG molecule so potential problems of steric changes caused by fragmentation are avoided. To obtain definitive immunochemical information, it is necessary that both the antigen (the genetically-engineered chimeric antibody) and the antibody be monoclonal. Our initial studies focused on RFs from WMac which provide a ready source of monoclonal proteins. More recently we have shifted our attention to RFs produced by continuous lines derived from individuals with RA. In our studies of 18 monoclonal RFs from patients with WMac we found that the majority (14) bound IgG 1, 2 and 4, but did not react with IgG3. This reactivity pattern corresponded to the GA specificity previously reported (Allen & Kunkel 1966, Normansell & Young 1975). Only 3 of the 18 monoclonal WMac RFs bound lgG3 well. By shuffling the C region domains between IgG3 and IgG4, it was possible to demonstrate that sequence variation in the CH3 domain is responsible for the differential binding to IgG3 and IgG4. For these RFs, His435 in

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CH3 is a critical residue for binding, and replacing it with Arg, the residue present in IgG3, destroys or reduces RF binding (Artandi et al. 1991). However, additional residues are important in constituting the epitope recognized. Among the important residues are amino acids 252-254 (Met-Ile-Ser) and 309-311 (LeuHis-Gln) located within CH2. These residues are conserved among IgG isotypes and comprise two loops of amino acids on the surface of the domains (Fig. 4). The residues in these two loops were replaced with either glycines, which removed the existing amino acid side chains, or with prolines. Substitution of positions 252-254 of IgG4 with either three Gly or Pro residues eliminated binding by 16 of the 17 WMac RFs reactive with IgG4. The loop containing residues 309-311 is farther from the CH2-CH3 interface than the loop containing residues 252-254 (See Fig. 4). Replacing the amino acids in this more distal loop with Gly eliminated binding by 14 of the 17 RFs. However, substitution of Pro residues at these positions resulted in loss of binding by only 6. Eight RFs unable to bind the mutant containing the Gly substitutions bound the mutant containing Pro; all of these RFs express the Wa cross-reactive idiotype (Artandi et al. 1992). Only one Wa cross-reactive idiotype-positive RF tested did not exhibit this reactivity pattern. Staphylococcal Protein A (SPA) did not bind to any of the IgG4 antibodies with substitutions in the CH2 loops, indicating that the binding specificities of these monoclonal WMac RFs and SPA are not identical. These studies have therefore demonstrated that three regions of exposed loops make a major contribution to the epitopes recognized by the WMac RFs we have

309-311

252-254

Figure 4. Residues implicated as playing a role in forming the epitope recognized by many rheumatoid factors. The 252-254, 309-311 loops and His435 are dark and labeled. Two different views with the antibody turned 90° are shown. The crystal structure is from Diesenhofer (1981) and the graphic was made using Maclmdad.

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examined: residues 252-254 and 309-311 in C^l and residue 435 in CH3 (Fig. 4). These three regions, noncontiguous in linear sequence, fold into close proximity to form an epitope similar to other protein epitopes recognized by monoclonal antibodies. Although this epitope contains many ofthe same residues as the SPA binding site on IgG, the binding specificities of SPA and monoclonal WMac are not identical. Our more recent studies have investigated the binding specificities of RFs produced by virus-transformed cells lines from the synovium or blood of RA patients. It was reasoned that the specificity of these RFs would be more likely to represent specificities involved in the pathogenesis of RA. The studies have indeed demonstrated that the RFs from this source have binding specificities distinct from what we observed for WMac RFs. To date we have examined 18 RFs produced by cell lines derived from patients with RA (Bonagura et al. submitted). Half of these bound IgGl, 2, and 4, but not IgG3, the GA specificity observed with RFs from WMac. However, six bound all four IgG subclasses, a pattern observed only in the one RF derived from a patient who had WMac and RA. The remaining three RFs had subclass specificities unlike any observed with WMac-derived RFs. The influence ofthe C^l loops on binding was investigated for the RA-derived RFs. For 13 ofthe 15 RA-derived RFs that bound IgG4 strongly, substitution of the proximal CH2 loop (252-254) with either Gly or Pro eliminated binding. For five of the nine RFs with the GA reactivity pattern, substitution of Ile253 with Ala was sufficient to destroy reactivity. However, the remaining four RFs and SPA bound IgG4 containing AIa253; the reactivity of SPA was surprising because Ile253 is a contact residue for SPA (Diesenhofer 1981). Unlike the WMacderived RFs, several RA-derived RFs bound IgG outside of the discontinuous epitope comprised of the residues from the two loops of CH2 and the histidine of CH3. One bound IgG in CH2, another in CH3, and a third at an undetermined site outside of the CH2-CH3 interface. Unlike WMac-derived RFs, several RAderived RFs are not inhibited in binding by SPA. Thus we have demonstrated that the repertoire of RFs expressed by RA patients shows unique binding specificities for IgG epitopes not found among the WMac-derived RFs we examined. It remains to be demonstrated if this difference in specificity has any influence on the pathogenesis of the disease.

PRODUCTION OF ANTIBODIES USUALLY AVAILABLE IN LIMITED QUANTITIES One elear advantage of gene transfection is the ability to produce large quantities of proteins which would otherwise be available only in limited quantities. For example, it has been difficult to obtain information on the role of circulating IgD

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because there are few sources of antigen-specific secretory IgD. To address this problem, we have produced a mouse/human chimeric IgD molecule in which the variable region from the murine anti-dansyl myeloma 27-44 was joined to the human IgD constant region (Shin et al. 1992). When expressed along with a chimeric murine anti-dansyl/human kappa light chain, a fully assembled HjL; molecule of IgD was produced which retains specificity for the hapten dansyl and is conformationally and functionally indistinguishable from human IgD in its ability to upregulate IgD receptors on human T cells. Human IgD contains three sites of N-linked glycosylation; Asn297 in CH2 and Asn386 and Asn437 in CH3 (Mellis & Baer^iger 1983a); five to seven sites of O-linked glycosylation are located in the first hinge (Takayasu et al. 1982, Mellis & Baenziger 1983a). Both the cytoplasmic and secreted chimeric IgD heavy chains produced by the transfectoma appear heterogeneous in size because of differential glycosylation. Pulse-chase experiments indicate that one N-linked carbohydrate residue is added cotranslationally with the remaining two added post-translationally. Endo H treatment ofthe chimeric IgD molecule demonstrated that at least one carbohydrate moiety on the secreted HjLj was of the high mannose type (Shin et al. 1992). In naturally occurring IgD the carbohydrate at residue 297 in the 0^2 domains is high mannose form while the two carbohydrate residues in C^3 are complex. The availability of stable lines producing IgD made it possible to study the assembly of IgD. The pathway of chimeric IgD assembly was H -)- L -* HL -» H;L;; neither H2 nor H2L intermediates were observed. Interfering with the N-linked glycosylation of chimeric IgD by Tunicamycin (TM) treatment prevented the assembly of complete H2L2 molecules; instead, TM treatment stopped IgD assembly at the H L half-molecule stage and completely inhibited secretion (Shin et al. 1992). Secretion of murine IgD and htiman and murine IgG is largely unaffected by lack of carbohydrate (Hickman & Komfeld 1978, Vasilov & Ploegh 1982, Tao & Morrison 1989) while secretion of murine IgA, IgM and IgE is inhibited 65-80%, 80% and 100% respectively by TM treatment (Hickman et al. 1977, Hickman & Komfeld 1978). A common feature of murine IgA, IgM and IgE and of human IgD is that a single disulfide bond at the end ofthe hinge links the heavy chains. In contrast, murine and human IgG have several inter-heavy chain disulfide bonds located more amino terminal in the hinge. It seems likely that the perturbation resulting from the lack of carbohydrate extends into the hinge to the region participating in the inter-heavy chain disulfide and prevents its formation. Therefore, glycosylation of CS2 in human IgD may play an important role in stabilizing the CH2 domain and carboxy-terminus of the hinge, enabling a proper conformation to be achieved to form the inter-heavy chain disulfide bond. POST-TRANSLATIONAL MODIFICATION Antibody molecules are glycosylated in their Fc regions at characteristic positions according to their isotype (Sutton & Phillips 1983). All IgG antibodies are

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glycosylated in CH2 at Asn297; the conservation of this structure among species suggests that it plays an important part in maintaining the structure and functions of IgG. Early studies in which carbohydrate-depleted mouse and rabbit monoclonal antibodies were produced by glycosidase or TM treatment indicated that the presence of carbohydrate is essential for some effector functions (Nose & Wigzell 1983, Leatherbarrow et al. 1985). Chimeric antibodies provide a useful tool for the study of the effect of glycosylation on antibody function. Genetically removing the carbohydrate addition sequence leads to molecules completely deficient in carbohydrate at a defmed position. Furthermore, comparative studies of human isotypes and IgG subclasses are readily attainable. Site-directed mutagenesis was used to produce human IgGI and IgG3 antibodies in which Asn297, the carbohydrate attachment residue in CH2, was altered (Tao & Morrison 1989). The aglycosylated antibodies were assembled and secreted at levels similar to wild-type transfectomas. The aglycosylated antibodies showed normal levels of antigen binding as well as binding to SPA. Compared to the corresponding wild-type IgGs, the aglycosylated IgGl and lgG3 were more sensitive to most proteases, especially pepsin and chymotrypsin, which cleave the molecule in CH2. IgG3 is generally more sensitive to protease attack, possibly due to its extended hinge. As described above, aglycosylated IgGl and IgG3 were both unable to activate complement and bind human FcyRI receptors on monocytic cells (Tao & Morrison, \ 989, Walker et al. 1989). Carbohydrate-deficient IgG I was unable to bind Clq even at high concentrations, and is thus deficient in the first step of complement activation. IgG3 showed only partial loss of Clq binding but was nonetheless unable to activate complement; this is consistent with the observation that Clq binding is a necessary but not the sole requirement for complement activation (Bindon 1988). Apparently the Clq binding site in CH2 is altered by the absence of carbohydrate. The constant region of IgG contributes to the exceptionally long serum half-life ofthe protein. When measured in mice, the serum half-life of human IgGl was not affected by the loss of CH2-associated carbohydrate, but that of human IgG3 was reduced by approximately one-third. Similar results were obtained in other studies with murine IgG2b (Wawrzynczak et al. 1992). These results suggest that, while carbohydrate may play a role in IgG catabolism, it is not the sole determinant. It is widely recognized that all antibodies contain carbohydrate in their constant regions, which contributes to their biologic properties. In addition, a significant proportion of antibodies also possesses carbohydrate addition sites in the V region (ICabat et al. 1987). These carbohydrate attachment sequences vary in position but among anti-carbohydrate antibodies a site is frequently seen at Asn58 in CDR2; however, the presence of a carbohydrate addition sequence does not guarantee the presence of carbohydrate and the presence of V^-associated carbohydrate has been demonstrated in only a few cases.

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Anti-dextran antibodies with potential N-linked glycosylation sites in position Asn58 in CDR2 have higher affmity for a(l -*6) dextran than those otherwise identical in sequence but lacking such sites. Experiments demonstrated that it was the presence of carbohydrate in VH that increased the affinity ofthe antibody for antigen more than 10-fold (Wallick et al. 1988). These dextran-specific antibodies provide a useful system for studying the role of V-region glycosylation on antigen binding. Single amino acid changes have been used to place carbohydrate addition sequences at novel positions in CDR2 of nonglycosylated anti-dextran V regions and the resulting antibodies have been evaluated for the effect of glycosylation on antigen binding (Wright et al. 1991). Carbohydrate addition sites were placed in CDR2 at position 54 (Ser54 -• Asn54) and at position 60 (Lys62 ->• Thr62). Both sites were glycosylated and, as was seen for carbohydrate added at Asn58, the resulting carbohydrate was accessible for binding by lectin. The efTect of carbohydrate on antigen binding varied depending on its position. Carbohydrate at Asn60 increased affinity for antigen three-fold, compared to the greater than 10-foid increase in affmity conferred by carbohydrate at Asn58. In contrast, glycosylation at Asn54 abolished antigen binding (Table I). Surprisingly, a high mannose, rather than complex, carbohydrate was attached at Asn60 indicating that carbohydrate attached at two nearby residues can be processed differentially as the protein is directed through thecell. Thus, slight changes in the position ofthe N-linked carbohydrate in CDR2 ofthe antidextran result in substantially different effects on antigen binding. The model of

TABLE I Apparent binding constants for dextran B5I2 Carbohydrate attachment site ASN-58 ASN-54 ASN-60 None

TM treatment' + + + -

aKa ( + S.D.)

LlOx 10* (0.153) L17x 10'(0.441) . M>CLCNACfii

ipiGLCNAC

\t

Figure 6 (A). Complement consumption by chimeric IgG molecules produced in murine myeloma cells and the Chinese hamster ovary glycosylation mutant Led. 8 /g of chimeric antibody was incubated with decreasing amounts of dansyl-BSA and 2CHso units of guinea pig complement at 37'C for 45 min. Hemoiysin-sensitized ''Cr-sensitized sheep red blood cells were added and incubated further for 45 min. The amount of "Cr released was quantitated and complement consumption was calculated as {l-(cpm Ag-i-Ab-i-C/cpm Ab+-C)| X t(K)%. (B)The general carbohydrate structure associated with the CH2 of human IgG; the structure may vary with the presence of the underlined residues. The truncated structure produced by the Lecl mutant is indicated.

ACKNOWLEDGMENTS Research in the laboratory is supported by grants CA 16858 and AI29470 from the National Institutes of Health, by grants IM-550 and IM-603 from the American Cancer Society, and by fellowship support from Centocor.

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