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Chemical synthesis of idiotopes. Evidence that antisera to the same JH1 peptide detect multiple binding site-associated idiotopes.

In an attempt to better understand the molecular basis of idiotypy, we have generated several site-specific antisera through immunization of animals with synthetic peptides corresponding to the (JH1) heavy chain joining segment 1 of the mouse heavy chain variable (VH) region. These anti-peptide sera identify several idiotypic determinants present on intact hybridoma and myeloma immunoglobulins. Expression of at least three of these idiotopes is correlated with the antigen specificity of the family of immunoglobulins bearing the determinant. Use of synthetic peptides may prove a powerful technique in the generation of molecularly defined antiidiotypic reagents.

idiotopes on an individual immunoglobulin, called an idiotype, constitutes a unique set of markers highly characteristic of each protein. Indeed, Kunkel (18) used idiotypic analysis to estimate that the variable region repertoire of humans exceeded 107 .
Molecules that share idiotypes could be expected to be structurally and even functionally similar and there are many examples where this is the case (9,17). Antibodies with the same antigen specificity often share idiotypic determinants and this has been shown to reflect common genetic origins and similar structure. In addition, however, molecules that do not share genetic origins, but which have affinity for the same ligand, such as antibodies and T cell receptors specific for the same antige n (19), and antibodies and cell receptors specific for the same hormones (20), have also been shown to share idiotopes. Thus, some idiotopes are functional markers.
In addition to providing useful markers for investigating variable region structure and function, it has become clear that idiotypes may provide an important means of immune regulation. Jerne (2 I) first called attention to the possibility that idiotopes on lymphocyte receptors may be recognized by other lymphocyte receptors with antiidiotope specificity, forming the idiotype network; antibodies, suppressor T cells, and helper T cells have all been shown to recognize idiotopes and to augment or suppress idiotype production (22)(23)(24). Further, antibodies to idiotypic determinants when passively administered result in the elimination of lymphocytes bearing the idiotype (25). If these cells are malignant, antiidiotypic antisera can be an effective therapy (26). Potentially, if the idiotype is shared by a family of autoimmunity-causing clones, antiidiotypic therapy may be curative.
Thus, idiotypic determinants are extremely important from several viewpoints. It is disappointing to realize that the molecular definition of an idiotope has been accomplished in only a few instances (I7). Inability to make molecular assignments of idiotype is due, in part, to the complexity of the process of assignment. Required are both monospecific antiidiotypic antibody and families of idiotypepositive and -negative immunoglobulins that are both completely structurally characterized and sufficiently similar/dissimilar so that the correlation of idiotype with structure can be made by inspection. In the few cases in which idiotype expression can be correlated with certain amino acid residues, it is not clear whether the idiotope is present on these critical residues or, alternatively, on others influenced by the critical residues.
To date, idiotypes have been defined following immunization of animals with intact immunoglobulin. Recently, however, synthetic peptides have been used to generate antibodies that bind both peptide and proteins bearing that sequence (27)(28)(29). We have applied this strategy to immunoglobulins in an effort to generate site-specific antiidiotypic reagents (30, 3 I). Here we show that a peptide corresponding to JH1 of murine Vn regions stimulates a variety of antibodies to immunoglobulin determinants at least some of which are found preferentially on families of antibodies with the same specificity. This indicates that a short, relatively constant part of variable domains actually forms part or all of a multiplicity of determinants. Peptide Synthesis. Peptides were synthesized manually from free amino acids using standard Merrifield techniques (32). Finished peptides were cleaved from the resin with hydrofluoric acid (HF), characterized by thin layer chromatography, and purified by partition chromatography on G-25 Sephadex. Confirmation of the JHI peptide structure was obtained by amino acid sequence analysis. Bombesin, a tetradecapeptide hormone, was obtained commercially (Sigma Chemical Co., St. Louis, MO).
Indirect RIA was used to measure the specificity of binding. Fourfold serial dilutions 5 125 of purified inhibitors were mixed with 4 × 10 cpm of I-myeloma proteins and exposed to microtiter plates coated with a 1:1,000 dilution of rabbit anti-JH~-KLH. After 4 h of incubation, the wells were washed and radioactivity was measured.
Protein Purification. Most proteins used in this study were affinity purified on Sepharose 4B columns conjugated with the appropriate ligand as described elsewhere (38,39). Hybridoma-derived proteins l-B-10, 6A6, and group A carbohydrate (GAC) 123 were used as concentrated serum-free tissue culture supernatants. J606 was purified from ammonium sulfate precipitates of ascites followed by three consecutive euglobulin precipitations.

SDS-PAGE and Western Blot Transfer. M 104 and low molecular weight standards (Bio-
Rad Laboratories, Richmond, CA) were denatured and reduced by brief boiling in 1% sodium dodecyl sulfate (SDS) and 1% 2-mercaptoethanol (2ME). Standards and immunoglobulin heavy and light chains were separated by discontinuous SDS-polyacrylamide gel electrophoresis (PAGE) on a 12.5% gel. Separated proteins were electrically transferred to nitrocellulose (Schleicher & Schuell, Inc., Keene, NH) in a transfer box as described elsewhere (40). Efficiency of separation and transfer was determined by amidoblue black stain of the proteins on the nitrocellulose paper. After transfer, the nitrocellulose was exposed to 1% BSA, 0.05% Tween 20 (Sigma) in PBS. Individual strips were incubated in BSA/Tween/PBS with a 1:2,000 dilution of antisera. After a 1-h incubation with the first antibody, the strips were washed and incubated for 1 h with 2 × 10 n cpm of ~SI-labeled protein A (Sigma). The strips were washed overnight with BSA/Tween/PBS and exposed to x-ray for 24 h at -70°C.
Isoelectric Focusing. Serum antibodies were characterized by IEF in a 0.9% agarose gel (41) (FMC Corp., Rockland, ME) containing 2.5% LKB ampholytes 5-8 and 2.5% LKB ampholyte 8-9.5 (LKB, Gaithersburg, MD). 1-4/A of sera were applied to prepunched wells between a cathodal wick saturated with 100 mM NaOH or imidazole and an anodal wick saturated with 100 mM phosphoric acid or glycylglycine. Proteins were separated with 12 W constant power until the voltage was over 1,000 V. Immunoglobulins were precipitated with 18% sodium sulfate for 20 rain, fixed with 0.01% glutaraldehyde in sodium sulfate for 20 min, and washed extensively. Fixed gels were incubated for 1-2 h with 10 x 108 cpm of l*SI-labeled myeloma protein or normal mouse IgG (42). Gels were then washed for 36 h on a rotary shaker, dried, and exposed to film at -70°C for 1-7 d.

Results
Anti-peptide Sera Bind Both Peptide and Immunoglobulin. Table I shows the sequences and relative positions of the mouse JH segments in the VH domain. The N-terminal region of JH is part of HV3 and intrudes into the binding site. The remainder of Jn lies in the groove separating the VL and VH domains (43). The C-terminus provides the link with the CH~ domain.
A peptide corresponding to JH1 was synthesized (Table I) and coupled to KLH; rats and rabbits were immunized with the conjugate and their sera tested for antibody. Results achieved with a representative serum are shown in Fig. 1. It is clear that JH~-KLH induces antibodies that bind both peptide and immunoglobulins ( Fig. 1). Anti-JHt binds JHl peptide best, but also cross-reacts with the JH2 peptide. Only minimal binding is seen with the 15-amino acid peptide corresponding to the first hypervariable region of MOPC 104 heavy chain (HV 1-M 104; Fig. 1 a). Further, the same antiserum binds substantial amounts of four monoclonal antibodies specific for three separate carbohydrate antigens (Fig. 1 b) known to have different variable region structures (9,17,44). Western blot analysis shows that the anti-immunoglobulin reactivity is heavy chain specific and is capable of detecting denatured heavy chains (Fig. 2, lane 1). The doublet at 70,000 is the glycosylated and deglycosylated M 104 heavy chain as determined by trifluoromethanesulfonic (TFMS) acid deglycosylation experiments (data not shown). The fainter band at ~31,000 is probably a heavy chain breakdown product and has been reported previously (31). The serum shows no activity for light chain (lane 1) or numerous other proteins described in the figure legend (lane 2). A rabbit immunized with KLH-HA (sera 3630) in CFA showed no activity for immunoglobulin heavy or light chain or numerous other proteins (lanes 3 and 4).
Identification of Antiidiotope Antibodies. Direct binding RIA demonstrate that anti-JH] sera bind several immunoglobulins, but cannot determine whether one   or more specificities are expressed within a given serum. Several approaches were used to examine this question. First, anti-peptide sera from several animals were examined, following isoelectric focusing (IEF) in agarose gels, for binding to various labeled immunoglobulin ligands. This technique permits rapid screening of sera for anti-immunoglobulin reactivity, evaluates heterogeneity of antibody activity, permits comparison between different anti-peptide sera, and, most importantly, allows comparison of the heterogeneity and isoelectric point of antibodies in a single serum that binds different ligands. In Fig. 3, a variety of anti-JH1 sera (T and numbered samples) and control sera from animals immune to a variety of irrelevant KLH-coupled peptides (a-d and w-z) were focused on four separate gels, which were subsequently exposed to ~2~I-IgG from normal mouse serum, 125I-J558, an o~ anti-dextran antibody, 125I-Ti5, an o~K anti-PC antibody, and ~25I-GAC34, a ~,sK anti-GAC antibody. Autoradiographs of these gels show that all anti-JH~ sera bind normal mouse IgG (panel A). None of 12 rabbit (data for 4 shown) and 4 rat antisera raised to irrelevant peptides have this specificity. The anti-IgG activity is heterogeneous with a slight suggestion of shared bands between sera. Very different patterns are seen when homogeneous immunoglobulin ligands are used. Many, but not all, sera have reactivity to 1~5I-J558 (panel B); fewer sera are reactive with ~25I-GAC34 (panel C) or I~I-T15 (panel D). In most cases, the heterogeneity of the anti-peptide antibodies reacting with monoclonal immunoglobulins is much more restricted than those to normal IgG. Further, antibodies to any one of the monoclonal Iigands seem unrelated to those reactive with the other monoclonal antibodies. This is best seen in rat serum 11, which binds ~25I-J558 very well, yet shows little or no activity for ~5I-GAC34. On the other hand, rat serum 4 binds ~25I-GAC34 better than ~5I-J558. These results indicate that JH~ induces several separate populations of antibodies that bind different determinants in the J H segment and, secondly, that these determinants have restricted distribution.
T h e second approach to the characterization of anti-peptide antibodies measures the fine specificity of binding to various ligands by competition RIA analysis. In this procedure, the relative capacities of various unlabeled proteins to inhibit the binding of 12~I-labeled monoclonal antibodies to anti-]H1 antibodies measures how closely the unlabeled proteins resemble each other with regard to the antigenic determinant(s) expressed on the labeled proband. Table II summarizes the results of these analyses using a single rabbit anti-JH~ serum (serum T) to bind to three ligands, 1~5I-M104, 1~5I-T15 and ~25I-GAC34. Each protein in FIGURE 3. IEF patterns of rat and rabbit anti-peptide antisera. Rat and rabbit sera were focused as described in Materials and Methods. Gels were overlaid with (A) m25I-mouse IgG, (B) levi-Jr58, (C) 125I-GAC34 or (D) 12SI-T15. Lane T contains rabbit anti-JH1-KLH used in Fig. 2 and Table II. Lanes 1-11 are sera from individual rats immunized withJH1-KLH. Lanes a -d are sera from four rats immunized with bombesin-KLH. Lanes W -Z are sera from four rabbits immunized with HA-KLH. All rat sera demonstrate a nonspecific reactivity with 12~l-T 15 at tow pH.  2  ND  <1  15  ND  <1  ND  100  <1  <1  120  <1  <1  120  <1  3  200  <1  <1  75  <1  <1  200  10  5  6  10  ND  ND  <1  ND  ND  10  ND  100  2  100  2 Numerous proteins are examined for their expression ofJn-Dex, Jri-PC, and JH-GAC. Expression of JH-Dex, JH-PC, and Jn-GAC are reported as a ratio of gg of inhibitor required to generate 50% inhibition as compared to standard inhibitors. Values for 50% inhibition by standards are 40 ng GAC34 for JH-GAC, 270 ng T 15 for Jn-PC, and 36 ng M 104 for JH-DEX. D represents the number of residues assigned to this segment. Table II was tested for the ability to block the binding of each of the three ligands to anti-JH~. The results are clear. All dextran-specific antibodies are capable of blocking 125I-M 104 from binding to anti-JH~; no other protein tested had this ability. Similarly, most of the anti-PC antibodies shared a determinant(s) with T 15 that was absent from all but one other protein, and all six anti-GAC antibodies expressed a determinant(s) absent from nearly all other proteins. Thus, these three distinct determinants, called JH-Dex, Jn-PC, and Jrt-GAC, are highly associated with families of proteins sharing antigen specificity. Surprisingly, at least two of the determinants were not restricted to the JH~-bearing antibodies.
Discussion Our goal at the outset of these studies was to produce antibodies specific for the individual Ju segments. Instead, we found multiple determinants involving JH segments, each highly restricted in its distribution to groups of antibodies with similar antigen-binding specificity. Previously, Andrews and Capra (45), as well as Schiff and co-workers (46), suggested that idiotopes may be located in the J segment, but could not rule out other portions of the variable domains in their assignments. Our surprise comes not from the realization that Jn segments can participate in idiotypic determinants, but from the multiplicity of separate determinants found within a stretch of 16 amino acids. We believe these results require that conformational changes of Jn residues be induced by other segments of the VH domain. To evaluate this hypothesis, it is instructive to consider the available structural information pertaining to VH domains.
Crystallographic studies of the Via domain have demonstrated that this region is composed entirely of/3 pleated sheets (43,47,48). In the intact Fab, this polypeptide chain folds into a nine-stranded beta barrel in which each of the nine strands is stabilized through hydrogen bonds to neighboring strands. The JH segment forms the ninth strand in the beta barrel, is in the immediate vicinity of the antigen binding pocket, and interacts through hydrogen bonding with the first (residues 1-14) and eighth (residues 91-100) strands of the barrel. Conformation of the J segment will be dependent on its own primary sequence, influences from neighboring strands, as well as complex interactions with other regions of the VH, and the CH~ and VL domains. It is conceivable that changes in the length of the D segment, which lies in the hairpin turn of strands eight and nine, may play a crucial role in the conformation of theJ segment. This idea gains support from the fact that Jia-Dex is expressed by proteins with short, tworesidue D segments, while JIa-PC is found on immunoglobulins with 5-7 residue D segments. Unfortunately, crystallographic data examining the effect of insertion or deletion of residues on the secondary and tertiary structure of similar proteins is very limited. High resolution crystallographic data of the Via region are limited to three proteins: Kol, New, and McPC 603 (43,47,48). The New Cm domain has an additional residue relative to McPC 603 Cm (49) that is accompanied by a beta bend with minimal distortion of conformation. On the other hand, the J segments of New and Kol demonstrate differences in conformation; however, interpretation is difficult since only New is sequenced (50). Finally, structural data from the Rei and Rhi Bence-Jones dimers have demonstrated that the molecules show different conformations in sequence-identical regions because of residue changes in neighboring hypervariable regions (51). Therefore, the fragmentary structural data, at the least, indicate that conformational changes can be expected in the J segment that are induced by other parts of the variable region. Indeed, these putative conformational determinants must accommodate significant changes in primary structure of the J segment itself, because, at least in the case of JH-Dex, all fourJ segments express the same putative conformational determinant.
Although it is not possible to decide which parts of the variable domains are important in imparting the conformational changes to Jn, it is interesting that the entire Vn segments of each family of idiotope-bearing molecules are very similar. Each family is encoded by one or several very similar Via segment genes. This is also true for the anti-levan antibody, J606, that expresses the JIa-GAC determinant. It is conceivable that some "VH" or idiotype markers actually reside in the Jia region. The 21A5 idiotype recently described by Morahan and c01-leagues (52) bears some resemblance to our Jn-PC marker. Since their determinant is hapten inhibitable and Cn locus-restricted, the idiotype may be located, at least in part, in the JHI region. We have found that an anti-JH peptide monoclonal antibody reacts exclusively with t~ chains (unpublished results); this suggests that CH regions may influence the conformation of the Jn segments as well.
We believe that anti-peptide antisera will provide an important approach to the localization of variable region markers. With this technique, one can confidently predict in which section of the variable region an idiotope is located. Furthermore, by synthesizing several overlapping peptides it may be possible to map the location of these idiotopes to a key residue or residues. Further, this technique may also be useful in mapping determinants that have already been defined by classical serology. This has recently been demonstrated with antisera generated against peptides corresponding to the third hypervariable region of M104 and J558 (31). Conventional approaches had tentatively localized the private idiotypic determinants of these two proteins to the D segment. Antibodies directed to peptides corresponding to HV3 were shown to distinguish between the proteins in a predictable way, confirming the previous conclusions. More interesting, however, will be the surprises that emerge from use of this new approach.

Summary
In an attempt to better understand the molecular basis of idiotypy, we have generated several site-specific antisera through immunization of animals with synthetic peptides corresponding to the (JHI) heavy chain joining segment 1 of the mouse heavy chain variable (VH) region. These anti-peptide sera identify several idiotypic determinants present on intact hybridoma and myeloma immunoglobulins. Expression of at least three of these idiotopes is correlated with the antigen specificity of the family of immunoglobulins bearing the determinant. Use of synthetic peptides may prove a powerful technique in the generation of molecularly defined antiidiotypic reagents.