Human IgE Chimeras to Map the FcϵR Binding Site of IgE

METHODS: A Companion to Methods in Enzymology 8, 124–132 (1995)

The Use of Mouse/Human IgE Chimeras to Map the FceR Binding Site of IgE Ahuva Nissim,1 Shelley Schwarzbaum, and Zelig Eshhar2 Department of Chemical Immunology of the Weizmann Institute of Science, Rehovot, Israel 76100

The binding of IgE antibodies to their specific receptors on mast cells is a crucial step in the allergic response and can serve as a paradigm for the study of receptor–ligand interactions. Intense efforts have been made to identify amino acid sequences and structural motifs on the IgE molecule that may be involved in the binding to the Fce receptor. Studies using short IgE peptides or fragments are restricted by the conformation of these polypeptides, as a sequence that corresponds to the receptor-binding epitope on the IgE molecule may adopt a different, nonbinding conformation in solution. To avoid this difficulty, we have used exon shuffling between mouse and human IgE to identify the domain involved in binding to the mouse and human low- and high-affinity Fce receptors. The results obtained using such human–mouse IgE chimeras strongly suggest that the Ce3 domain is sufficient for species-specific binding to both the low- and the high-affinity Fce receptors. Binding is observed even upon deletion of the Ce2 domain. Within the Ce3 domain, conformational determinants composed of residues from throughout the domain are needed to form the binding site for the FceRI. For the low-affinity receptor, the binding site appears to reside in the C-terminal part of Ce3. q 1995 Academic Press, Inc.

The interaction between immunoglobulin E (IgE)3 and its high-affinity receptor (FceRI) on mast cells and basophils is a key step in the allergic response. Bridging of receptor-bound IgE by a specific antigen triggers mast cell degranulation and release of substances mediating type I immediate hypersensitivity (1, 2). In ad1 Current address: Center for Protein Engineering, MRC Research Centre, Hills Road, Cambridge, CB2-2QH England. 2 To whom correspondence should be addressed. Fax: 972-8344141. 3 Abbreviations used: IgE, immunoglobulin E; hIgE, human IgE; mIgE, murine IgE; FceRI, high-affinity Fc receptor for immunoglobulin E; FceRII, low-affinity Fc receptor for immunoglobulin E; Ce1,2,3,4, first, second, third, and fourth heavy chain constant region domains of IgE; MAb, monoclonal antibody.

dition to the high-affinity Fce receptor found on mast cells, certain B cells express a low affinity receptor for IgE. This receptor, known as the FceRII, or CD23, is thought to be involved in the down-regulation of IgE production (3). Because of the central role of IgE and its receptors in the allergic response, identification of the precise IgE/receptor interaction site(s) is an important first step toward the development of agents able to inhibit this interaction and thereby to modulate allergic disease. Early studies, which attempted to identify the Fc receptor-binding site on IgE, relied on the use of protease digestion to produce large constant region fragments that maintained receptor binding (4, 5). Such studies implicated the entire Fce region in the binding site. Somewhat more specific mapping was performed using protease protection studies (6), which demonstrated that a region in the cleft between the second and the third domain of rodent IgE was protected from proteolysis when bound to the FceRI. Inasmuch as crystallographic data on the Fc region exist only for IgG, attempts have been made to construct a model of the Fc region of the IgE molecule in search of sites that may be involved in the interaction with the FceR (7, 8). Such models were able to identify peptides and specific amino acid residues that were likely to be exposed on the surface of the IgE molecule. Nevertheless, these models did not identify any particular structural feature that indicated the location of the FceR-binding site(s). Studies using short peptides corresponding to various IgE sequences, or monoclonal antibodies (MAb) against such peptides, were also unable to identify a discrete interaction site. IgE peptides bound to the FceRI either weakly (9) or irreproducibly (10, 11), probably because these peptides did not maintain their native conformation. Antibiody blocking studies (12, 13) usually do not have sufficient resolution to distinguish between a true receptor-binding site and stearic inhibition by the large blocking MAb. A more useful approach to elucidate the precise site

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1046-2023/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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on the IgE molecule that interacts with the FceRI utilizes recombinant DNA technology. Cloned Ce gene segments of both human (14, 15) and mouse (16, 17) IgE were expressed in bacteria (18–20), yielding functional molecules. A 76-amino-acid monomeric recombinant peptide (rE2*-3*), spanning the Ce2–Ce3 junction of human IgE, was found to bind to the human FceRI with an affinity similar to that of native IgE (21). Nevertheless, the use of recombinant IgE fragments tends to overestimate the size of the minimal binding sequence. Nonbinding residues or domains must often be included to help maintain the conformation of the actual binding region. A smaller octapeptide, containing sequences included in the Ce3 part of the rE2*-3* fragment, was reported to specifically inhibit histamine release by human peripheral basophils (22). However, no direct measurements of the binding of this peptide to mast cells or the affinity of binding to the FceRI were performed. Only limited information is available on the region of IgE that interacts with the low-affinity FceRII. Studies with anti-IgE mAbs and studies using IgE fragments produced in bacteria suggest that the Ce3 domain also contains the epitopes that interact with the FceRII (23, 24). Using bacterially produced human IgE fragments, it was demonstrated that the FceRII can recognize a motif in the Ce3 domain that is formed upon dimerization of one or both of the flanking (Ce2 and Ce4) domains (25). One of the aims in elucidating the receptor binding site is to design an IgE analogue that will enable effective blocking of allergic responses. Thus, we have focused our efforts on murine IgE, which allows the testing of the feasibility of such an approach using in vivo model systems. In earlier studies, we constructed and expressed recombinant murine IgE, and by deletions, truncations, and site-directed mutagenesis compiled evidence suggesting Ce3 as the principal domain responsible for the receptor binding of murine IgE (26). However, because some of the effects observed in such studies could, in fact, result from mutations distal to the actual binding site, which induced gross conformational changes in the molecule, we have developed a complementary approach designed to minimize such changes. In these studies, rather than destroy the murine receptor-binding site through mutagenesis, we attempted to introduce the binding domains or epitopes into a molecule that allows the conservation of their overall conformation. To achieve this, we took advantage of the fact that despite the large degree of sequence homology between mouse and human IgE (17, 27), human IgE does not bind to the rodent FceRI, while both murine and human IgE bind with similar affinity to the human FceRI (28). To study the role played by the different Ce domains

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or epitopes of murine IgE in the binding to the FceRI, various domains or defined regions in the human molecule were replaced with their murine counterparts. Thus, human IgE provided an appropriate framework, able to conserve the native conformation of mouse IgE fragments. In addition, the strict species specificity of the low-affinity FceRII allowed us to use the same set of human–mouse IgE chimeras to map the FceRII binding site. We describe here some of our results, which indicate that the residues that participate in the binding to both the high- and the low-affinity receptors for IgE are contained within the Ce3 domain. We also summarize similar results reported by other groups, using the analogous approach of IgE–IgG chimeras.

DESCRIPTION OF METHODS Generation of IgE Chimeras Two principal expression vectors were used, MupSVCe and Hu-pSVCe. The Mu-pSVCe vector, which was constructed in our laboratory (26), contains a murine anti-NP VH segment and the entire mouse Ce region gene. It also contains the gpt selectable marker and Ig enhancer approximately 1 kb upstream from the leader exon (E, Fig. 1). The Hu-pSVCe vector is similar to the one described previously (29) and contains a murine anti-NP VH segment and the entire human Ce region gene. To construct it, the murine Ce of the Mu-pSVCe was replaced with the gene encoding human Ce (14). Upon transfection into the l light chain-expressing J558L myeloma, relatively large amounts (ca. 3–10 mg/ml) of chimeric human IgE with anti-NIP specificity could be obtained from the supernatants of stable transfectants; even higher levels were obtained in ascites fluid of mice injected with the transfectomas. A schematic diagram of the different human–mouse chimeric IgE mutants generated by the exon-shuffling approach is depicted in Fig. 1. CHM2, CHM3, and CHM2M3 are chimeric human Ce molecules containing the murine Ce2, Ce3, and Ce2 / Ce3 domains, respectively. In addition, to study the involvement of the Ce2 in the FceRI binding of IgE, we constructed two deletion mutants, one in which the entire Ce2 is missing and another in which only 16 amino acids from the carboxy terminus of the murine Ce2 are present. Thus, the PCDD mutant contains human Ce1, murine Ce3, and human Ce4. The CSPD mutant contains, in addition, 16 amino acids (from Val314 to Pro329) of the murine Ce2. Because of the localization of the two interheavy chain disulfide bonds within the Ce2 (Cys241 and Cys328), we preserved Cys328 in both PCDD and CSPD deletion mutants to ensure proper dimerization.

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FIG. 1. Structure of expression vectors containing mouse and human IgE heavy chain sequences (top) and of the mouse–human chimeric IgE molecules (bottom). The letters at the left indicate our designation for each chimera. Human sequences are shown as white boxes, and mouse sequences are depicted by shaded regions.

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To construct the human–mouse Ce3 hybrid chimeras, the same principal expression vector (Hu-pSVCe) was used. In view of the studies implicating the interface between the Ce2 and the Ce3 domain as the site of interaction between the IgE and its FceRI (6, 21), 16 or 26 amino acids from the amino-terminal portion of human Ce3 were swapped with their murine analogues (amino acids 330–346 for C3BX and 330–356 for C3HD). To study the contribution of Ce2 to the interaction, additional variants were made in which the human Ce2 domains of these chimeric molecules were replaced with murine Ce2 (C2C3BX and C2C3HD, respectively). The involvement of the carboxy end of the Ce3 domain in the interaction with the FceRI and FceRII was analyzed using the C3NT mutant, in which two separate stretches from both ends of Ce3 (amino acids 330–356 and 400–447) were replaced with their murine counterparts. The molecular genetic manipulations that we have used are described in detail in our previous publications (26, 29, 30). Binding Studies The ability of the various chimeric and mutated IgE molecules to bind to the FceRI and FceRII was evaluated by several independent assays. The RBL-2H3 rat mast cell line (31), expressing high levels of surface receptors, served as the source for the rodent FceRI. The human CHO-3D10 cell line (32), made by transfecting CHO cells with a chimeric human FceRI a chain containing an IL2 receptor b-chain anchor, was used for human FceRIbinding studies. As a source of FceRII, we used the 01.2A3 mouse B-cell hybridoma (33) and the human EBVtransfected B-cell line, RPMI-8866 (34). To measure binding to the high-affinity receptor, the ability of our various IgE chimeras to inhibit the binding of radiolabeled IgE to RBL and CHO-3D10 cells was tested. In addition, some of the chimeric antibodies were tested for their ability to form rosettes between FceR-bearing target cells and NIP-coupled sheep red blood cells. Finally, binding to RBL cells was also evaluated by testing the ability of the chimeric antibodies, in the presence of antigen, to induce degranulation of the RBL cells. Detailed experimental methods have been described previously (26, 29, 35).

RESULTS Binding to the High-Affinity FceRI We first performed competitive inhibition experiments to compare the ability of the various purified chimeric IgE molecules to block the binding of radiolabeled mouse or human IgE to their respective FceR.

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Table 1 summarizes our data from a series of such experiments. The recombinant murine IgE could effectively block the binding of native mouse IgE to the rodent receptor. On the other hand, recombinant IgE containing the entire human Ce could not bind to the rodent receptor even at very high concentrations. Exchanging the second constant domain of the human IgE with its murine counterpart (CHM2) did not endow the human IgE with the ability to bind to the rodent receptor. However, exchanging the third domain (CHM3), or both Ce2 and Ce3 domains (CHM2M3), conferred on the human IgE full reactivity toward the rodent FceRI. Thus, the Ce3 domain determines the species specificity of binding. In addition, as an independent test of FceRI binding, we found (29) that the chimeric human–mouse IgE molecules that contained the murine Ce3 domain retained the effector function of IgE, i.e., the ability to mediate mast cell degranulation. Because of sequence homology between the human and the mouse Ce2 (36%) (16), and because several studies implicated both the Ce2 and the Ce3 domains in the IgE–FceRI interaction (36, 37), the studies described above do not exclude the possibility that amino acids from a homologous portion of the human Ce2 play a part in the binding of the CHM3 chimera to the rodent FceRI. To test this possibility, the biological activity of the Ce2 deletion mutants was studied. We have shown by both blocking (Table 1) and degranulation experiments (29) that both the partially deleted CSPD and the fully deleted PCDD mutants maintained binding to RBL cells. The results described above show that the third constant domain of IgE (Ce3) is the sole domain involved in the binding to the rodent FceRI. Because of several reports implicating the Ce2–Ce3 interface as the major site responsible for IgE binding to the human FceRI (20, 21), we further determined whether the Ce2 domain is necessary for the binding of IgE to the human receptor. To this end, the ability of the Ce2-deleted mutants to bind to the CHO-3D10 cells was tested. Both CSPD and PCDD mutants inhibited the binding of human and mouse IgE to the human FceRI expressed on CHO-3D10 cells (Table 1). Thus, the Ce2 domain is not necessary for binding to either the rodent or the human FceRI. Having assigned the determinants that interact with the human and mouse high-affinity Fce receptors to Ce3, we next attempted to identify the regions within the Ce3 domain that are involved in receptor binding. Replacing of 16 or 26 amino acids from the amino terminal of Ce3 (C3BX and C3HD mutants) was not sufficient to endow the human IgE with the ability to bind to the rodent receptor. Likewise, addition of the mouse Ce2 domain to these chimeras (C2C3BX and C2C3HD) was equally ineffective. Nevertheless, these mutants

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maintained their ability to bind to human FceRI. In addition, we tested the binding of the mouse–human Ce3 chimeras to anti-human and anti-mouse Ce3 MAbs to ensure that all of these Ce3 hybrids, except the C3NT mutant, maintained the conformational epitopes recognized by the antibodies (30). Thus, it appears that the N-terminal residues of Ce3 are not involved in determining the species specificity of the IgE–FceRI interaction. Exchange of longer stretches from the carboxy and amino ends of human Ce3 with their murine analogues (C3NT) resulted in an IgE chimera that was unable to bind to either the rodent or the human receptors (30). Binding to the Low-Affinity FceRII The various chimeric and mutated IgE molecules were used to study the site on the IgE molecule that interacts with the murine and human B-cell FceRII. The ability of the various chimeric IgEs to bind to the receptor was determined by the rosette assay (30) and by a competitive inhibition assay (Table 1). Both human and mouse IgE demonstrate species-specific binding to the FceRII. Human chimeric IgE molecules that contain the murine Ce3 (CHM3) bound to the rodent FceRII and lost their ability to bind to the human FceRII. Thus, the species specificity observed is contained within the third domain. The binding of the CHM3 to 01.2A3 cells is, however, of lower affinity than that of the nonmutated mouse IgE. Exchanging both the Ce2 and the Ce3 domains (CHM2M3) conferred on human IgE full reactivity toward the mouse FceRII. As with the high-affinity receptor, the Ce2 domain does

not appear to take part in the actual interaction with the FceRII, as the CSPD mutant, lacking almost the entire Ce2 domain, retains the ability to bind to the murine low-affinity receptor, although with somewhat reduced affinity. Swapping of shorter fragments of the amino-terminal portion of human Ce3 with their murine analogues (C3BX and C3HD) was not sufficient to endow the human IgE with the ability to bind to the mouse FceRII, even after the introduction of the murine second domain (C2C3BX and C2C3HD). This suggests that additional mouse sequences at the carboxy end of Ce3 might also contribute to the recognition sequence. However, as in the case of the high-affinity receptor, the C3NT mutant failed to bind to the Mu-FceRII, presumably due to conformational changes in Ce3. The human chimeric molecules that contain the murine Ce3 (CHM3 and CHM2M3) lost their ability to bind to the human FceRII (Table 1). However, CHM2, in which the second human domain is replaced by its murine homolog, binds with the same affinity as the unmutated Hu-rIgE. Furthermore, the data obtained using the set of chimeras containing human–mouse hybrid Ce3 domains indicate that binding to the human receptor is reduced as longer mouse sequences are introduced into the human Ce3 backbone. The C3BX chimera has a fivefold lower affinity for the human FCeRII than that of the native human IgE, measured by the competition assay. However, the binding affinity of C3HD and C2C3HD is further reduced; they formed rosettes but barely inhibited the binding of I125-labeled Hu-rlgE. Again, the C3NT mutant, which has mouse sequences at both the amino and the carboxy ends of

TABLE 1 Binding of IgE Chimeras to the Low- and High-Affinity Fce Receptors FCeRI Chimera

Ce1

C e2

C e3

FceRII

Ce4

Human

Rodent

Human

Murine

Murine IgE Human IgE

M H

M H

M H

M H

//(/) ///

/// 0

0 ///

/// 0

CHM3 CHM2 CHM2M3 CSPD PCDD

H H H H H

H M M 16 aa 1 aa

M H M M M

H H H H H

//(/) /// //(/) // //

/// 0 /// // //

0 /// 0 0 0

// 0 /// // ND

C3BX C2C3BX C3HD C2C3HD C3NT

H H H H H

H M H M H

M/H M/H M/H M/H M/H/M

H H H H H

/// /// /// /// 0

0 0 0 0 0

// // / / 0

0 0 0 0 0

Note. Binding of chimeric IgE molecules to the rodent and human FceRI and -II was determined by measuring the ability of the chimeric IgE to compete the binding of radiolabeled murine or human IgE to cell lines bearing the appropriate FceR. M, murine; H, human.

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Ce3, lost its ability to bind to the human FceRII. These results suggest that the binding site for the human FceRII can be localized to the carboxy-terminal end of Ce3; nevertheless, conformational changes in any part of the domain can interfere with binding.

DISCUSSION The high degree of structural and sequence homology between immunoglobulins of various classes and from various species enables the exchange or grafting of equivalent regions from within these molecules. Such chimeras should maintain the gross conformation and function of the various domains of the parental molecules. In this review we have summarized our studies using hybrid mouse–human IgE molecules to demonstrate that the FceRI and FceRII binding site of IgE can be assigned to the third constant region domain (Ce3). In addition, we have generated intra-Ce3 chimeras in an attempt to map the precise interaction site(s) within this domain. Binding to the High-Affinity Receptor We chose this strategy to overcome the ambiguity resulting from the mutational approach, in which a loss of activity following a change in a site has been used to indicate the involvement of this site in the binding interaction. Our alternative, more direct approach involves the construction of the binding site in an analogous but nonbinding molecule. The system makes use of the well-defined species specificity of IgE and the fact that human IgE does not bind to the rodent receptor. Because of the substantial homology in sequence (Ce1, 40%; Ce2, 36%; Ce3, 47%; Ce4, 51%) (16, 17) and overall similarity in tertiary and quarternary structure between the mouse and the human IgE molecules, mouse–human IgE chimeras were expected to be an excellent system to study the contribution of different murine Ce fragments to the FceRI-binding site. Thus, by exchanging murine domains with their corresponding human homologs, it was possible to maintain the overall conformation of the molecule and to pinpoint more precisely the region responsible for the binding of mouse and human IgEs to their FceRI. The use of chimeras has enabled us to begin to delineate the minimal sequences that, when held in their native conformation, are necessary for receptor binding. In our system, the Ce3 domain is sufficient to account for FceRI binding. However, studies analyzing IgE fragments produced in bacteria or mammalian expression systems have suggested that parts of the Ce2 domain (21) or, alternatively, the Ce4 domain (38) are needed in addition to Ce3 for binding to the FceRI. The

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necessity of Ce2 or Ce4 sequences for fragment binding is probably an artifact caused by the use of isolated IgE sequences that do not otherwise adopt their native configuration. One study that tested the binding of IgE fragments used IgE polypeptides expressed in COS cells (38). In addition to an Fce fragment (235–547) that spans the entire Fc domain, only two other fragments, Fce(315– 547) and Fce(329–547), bound to FceRI. Fce(315–547) is similar to our CSPD mutant, which lacks most of Ce2 except 16 amino acids at its carboxy end, including Cys 328. Fce(329–547) is similar to our PCDD mutant except that it does not contain Cys 328. In a competition assay with native IgE, Fce(315–547) and intact IgE were indistinguishable in their activity, whereas Fce(329–547) was approximately fivefold less active. Fragments containing only the Ce2–Ce3 interface and the Ce3 domain did not bind, in contrast to similar fragments produced in bacteria, which were studied by Helm (20, 21). The fragments produced in COS cells do not address the role of Ce4. While Ce4 must be included in these polypeptides to obtain FceRI binding, it is likely that this domain helps stabilize the conformation of the binding epitopes and does not contain the actual recognition sequences. The advantage of the chimera approach is that it allows the differentiation between domains with an actual role in binding and domains that only provide a framework for the proper molecular conformation and subunit association. Other studies attempting to identify the FceRI-binding site made use of chimeras between mouse or human IgE and human IgG1, based on modeling that indicated that the Ce3 domain of IgE is structurally similar to the Fcg2 domain of IgG1. Studies by Weetall et al. (36, 37) demonstrated that Ce4 does not take part in the binding to the rodent FceRI. In their studies, chimeric mIgE in which the Ce4 was replaced by Cg3 bound similarly to intact IgE. However, chimeric IgG1 molecules into which either Ce3 alone or Ce3 plus Ce4 were added, and modified IgE molecules in which the Ce3 was replaced by Cg2, failed to bind to the FceRI. Based on these data, the authors suggested that both Ce2 and Ce3 are needed to bind the high-affinity receptor on mast cells. We believe that the g/Ce3 chimeric molecule is unable to bind to the FceRI because the Ce3 domain, when inserted into the IgG molecule, may assume a spatial orientation that differs from its native conformation. The failure to bind may be due, as well, to differences in flexibility between the two immunoglobulin molecules. Segmental flexibility in Ig is considered to be an important control element in mediating the effector function of antibodies (39). Comparative studies of segmental motion in Ig indicate that IgE may be less flexible than IgG (40). In fact, in the same studies cited above (36, 37), it was shown that a chimeric

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e/Cg2 molecule, in which the Ce3 domain was replaced by Cg2, failed to bind to the FcgRI, although it has been well established that Cg2 is the crucial domain responsible for binding to the FcgRI (41). Taken together, these results support our view that failure to bind does not necessarily indicate that the domain in question is not capable of interacting with its receptor when placed in the proper conformation or framework. To further elucidate the exact site within the Ce3 domain that is responsible for the binding to the highaffinity receptor, we replaced various human Ce3 subdomains with their murine homologues, testing their ability to confer upon human IgE the ability to bind to the rodent FceRI. Because previous studies implicated the Ce2–Ce3 interface as the site of the IgE–FceRI interaction (8, 21, 36, 44), attention was first focused on the amino-terminal end of Ce3. Our results show that swapping of 16 or 26 amino acids from the aminoterminal end of Ce3 (mutants C3BX and C3HD) was insufficient to enable the human IgE to bind to the rodent receptor. It therefore appears very likely that the contact residues on the IgE molecule that interact with the FceRI are contained in several regions of the Ce3 and that the maintenance of the native conformation of the entire domain is necessary for binding. The chimera approach has been further refined in recently published work by Presta et al. (42), who attempted to identify sequences within Ce3 that mediate FceR binding. In this study, human IgG chimeras were made containing IgE sequences from Ce3 loops that were thought, based on modeling and mutagenesis studies, to comprise the FceRI contact sites. A chimeric molecule containing these three loops together with the IgE hinge region (contained in the Ce2–Ce3 interface) was able to bind to the FceRI. The binding was of reduced affinity, apparently because of conformational differences between the IgE and the IgG molecular frame. These studies also addressed the controversy as to the role of the hinge region (Fce2–Fce3 junction) in the binding site. Site-specific mutations in this interface did not have an appreciable effect on binding (42), indicating that the Fce2–Fce3 junction is not directly involved in the IgE–FceRI interaction. In another study (43), a point mutation at residue 333 reduced binding to the FceRI by about fivefold. The authors of this study interpret this in support of an important role for the Ce2–Ce3 junction (hinge region) in the IgE–FceRI interaction. Interestingly, this mutation did not affect binding to the FceRII. A site-specific point mutation at residue 404 (near the C-terminal end) of murine Ce3 (26) reduced the binding to the rodent FceRI by approximately 50%. While either or both of these mutations could have altered residues directly involved in the binding site, it is more likely

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that they induced conformational changes, which indirectly affected receptor binding. Binding to the Low-Affinity Receptor Unlike the FceRI, binding to the FceRII is completely species specific. Thus, we were able to use the same mouse–human IgE chimeras to show that a site(s) on the Ce3 domain is also required for the binding of mouse and human IgE to the appropriate FceRII. Studies with the human–mouse Ce3 hybrids suggest that both mouse and human FceRII recognize determinants at the carboxy end of Ce3. Our results, demonstrating the importance of Ce3 in IgE–FceRII binding, are consistent with those reported by other groups (23–25). Using recombinant chimeric mIgE–human IgG1 molecules, Keegan et al. (24) showed the importance of Ce3 in the binding to mouse FceRII. However, because a e/Cg3 chimera in which Ce4 was exchanged by Cg3 was less active than the native murine IgE, the Ce4 domain was also suggested to contribute to the interaction site. Nevertheless, the fact that our CHM2M3 chimeric IgE, which bears a human Ce4, retains its full reactivity toward the mouse low-affinity receptor argues that the role of Ce4 in the actual binding is minor. Furthermore, a g/Ce3 chimera (24), in which murine Ce3 was introduced into the human IgG backbone, did not bind to the murine FceRII, suggesting that the IgG backbone does not provide the proper conformational environment to allow binding of IgE domains to the FceRII. Results reported with recombinant human e-chain fragments expressed in bacteria indicate that all three Fce domains appear necessary to obtain full reactivity (25). Ce4 and Ce2 are thought to have a role in stabilizing dimerization of the bacterially expressed fragments. In fact, our data demonstrate that Ce3 alone determines the species specificity and that the Ce2 domain does not have any direct role in the binding to the human FceRII. The current set of human–mouse chimeric IgE molecules does not test involvement of Hu-Ce4 on the binding to the Hu-FceRII. Attempts were also made to map the fine specificity of the low-affinity receptor binding site using peptides and anti-peptide Abs. Vercelli et al. (25) demonstrated that a recombinant e-chain fragment spanning the Ce2–Ce3 junction (Gln301 –Arg376) failed to bind to the human FceRII. Ghaderi and Stanworth (45) identified two peptides representing two b-sheets within the Ce3 domain, which exhibited binding to the low-affinity receptor. A peptide corresponding to a sequence near the N-terminal end of Ce3 failed to bind in this study. These studies agree with our results, obtained using the intra-Ce3 chimeras, and together suggest that the binding site for the low-affinity receptor is composed of epitopes at the C-terminal end of the Ce3 domain.

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Nevertheless, since the peptide studies rely on the lack of binding by small IgE fragments or peptides, alterations in the native conformation cannot be discounted.

CONCLUSIONS While we remain far from a complete understanding of the spatial arrangement in IgE–FceR binding, our studies using mouse–human antibody hybrids have elucidated certain aspects of the structural requirements of the IgE-binding site. We have shown that the Ce3 domain is both necessary and sufficient for speciesspecific binding of IgE to the high- and low-affinity receptors. While the Ce2 domain might have an important role in the stabilization of the IgE molecule, sequences from Ce2 do not participate in and are not necessary for direct binding to either the high- or the low-affinity IgE-specific receptors. Within the Ce3 domain, binding is not contributed by a distinct sequence, but rather most likely by a conformational determinant generated by several stretches of this domain. Neither the Ce2–Ce3 junction nor the external loop at the cleft formed between the Ce3 and the Ce4 domains (amino acids 346–356) are sufficient to determine the FceRI binding. The low-affinity receptor binding site(s) is localized to the carboxy end of the Ce3, and it is likely that the internal loops of this domain are involved in binding to the FceRII. The complementary approaches of binding studies using IgE polypeptides, IgE point mutants, and exonshuffling chimeras have yielded much information about the location of the receptor-binding site. For allergy therapy, it is unfortunate that the determinant comprising the FceRI-binding site is conformation dependent and is not composed of a short, contiguous amino acid sequence. In the absence of an actual structural solution for Fce, the conformational nature of the binding determinant and the slow rate of dissociation of the IgE–FceRI complex does not suggest any simple approach to the treatment of allergy through receptor blockade. It is hoped that the elucidation of the X-ray crystal structure of the IgE molecule and its receptors will permit the design of powerful FceR antagonists.

ACKNOWLEDGMENTS The work described here was supported in part by a grant from the Leo and Julia Forchheimer Center for Molecular Genetics at the Weizmann Institute of Science.

REFERENCES 1. Ishizaka, K., Ishizaka, T., and Hornbrook, M. M. (1966) J. Immunol. 97, 840–853.

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