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Differences in heavy chain amino acid sequences affecting the specificity of antibodies for variants of cytochrome c
0161-5890/93$6.00 + 0.00
Molecular Immunology, Vol. 30, No. 12, pp. 1107-I 114, 1993 Printed in Great Britain.
0 1993 Pergamon Press Ltd
DIFFERENCES IN HEAVY CHAIN AMINO ACID SEQUENCES AFFECTING THE SPECIFICITY OF ANTIBODIES FOR VARIANTS OF CYTOCHROME c* RONALD JEMMERSON,~CAROLYN MUELLER and DEBORAH FLAA Department
of Microbiology,
University of Minnesota MN 55455, U.S.A.
Medical School, Minneapolis,
(First received 1 December 1992; accepted in revised form 9 March 1993) Abstract-In a previous study [Goshorn ef al. (1991) 1. biof. Chem. 266, 2134-21421, several mAb specific for the same region on different cytochromes c were shown to have similar H or L chains. To determine the effect of differences in individual chains on antigenic variant s~ificity in the present study, chimeric mAb were prepared by recombining the H and L chains of mAb having the same or a different cytochrome c specificity. The H and L chains of two mAb to the region around residue 60 on horse cytochrome c (lF5.Dl and 2ES.GlO) were functionally interchangeable even though the H chain differed by 11 amino acid residues in the complementarity-determining regions (CDR) and 15 amino acids overall in the variable regions. The L chains only differed by four amino acid residues in the CDR (five residues overaIl). Neither the H nor L chain of a mAb binding the same region of rat cytochrome c (6H2.B4) was functionally interchangeable with the chains of the two horse cytochrome c-specific mAb. The L chain of this mAb is very different from the other L chains which were derived from a different V, family, but the H chain is nearly as similar to the horse cytochrome c-specific H chains as they are to each other. Most of the differences occur in CDR3 and result from the use of a distinct D, segment. The results indicate that, in some cases, the specificity of a mAb for a particular variant of a protein Ag, at least in regard to the H chain, is determined by only a few amino acid differences. The differences in the sequences of the H chains of the three mAb in this study and in the structures of their specific Ag provide insight into a possible molecular basis for the specificity of these mAb.
et al., 1991) or DNA (Schlomchik
structures of H$ and L chains of mAb to a variety of Ag have been determined (Kaartinen et al., 1983; Darsley and Rees, 1985; Wysocki ef al., 1986; Caton et al., 1986; Stenzf-Poore and Rittenberg, 1989; Schmitter et al., 1990; Wang et al., 1990; Schlomchik er al., 1990). The Ig structures can be relatively restricted, as in the antibody response to certain haptens (Kaartinen et al., 1983; Wysocki et al., 1986) or carbohydrates (Wang et al., 1990), and among mAb of shared idiotype (Bedzyk et al., 1990), or can be quite diverse as in the response to more complex Ag such as proteins (Caton The primary
et al., 1990). This diversity extends even to mAb which bind the same region of a protein Ag (Clarke et al., 1990). It is not known to what extent the diversity of epitopes in an antigenic region contributes to the diversity in Ig structures. When mAb to an antigenic region are grouped on the basis of fine specificities, often greater structural similarities among the mAb begin to emerge (Schmitter et al., 1990; Kavaler et al., 1990). Indeed, mAb to identical or similar sites on protein Ag can be very similar in the sequences of their H and L chains (Schmitter et ai., 1990; Kavaler et al., 1990; Goshorn et ai., 1991), although this is not necessarily always the case. With this premise in mind, we sought to determine: (1) whether mAb to the same region of different variants of a protein Ag were similar in primary structure, and (2) if so, whether the number of amino acid differences between mAb specific for different variants would be small enough to begin to understand how the specificity of mAb for a particular antigenic variant is encoded in the primary structure. We previously reported the amino acid sequences of the H and L chains of several mAb to two antigenic regions on horse, cow, and rat cytochromes c (Goshorn et al., 1991). Examples were observed where either the H or L chains of mAb specific for different cytochromes c were similar, but none where both chains were similar. To examine the effects of differences in only the H or L chain on mAb specificity,
*This work was supported by a grant from the National Science Foundation (DMB-901918 1). The nucleotide sequences reported in this paper have been submitted to the GenBank~m/EMBL Data Bank with accession numbers M57989-M57991 (H chains), M57987 and LO2500-LO2501 (L chains). TAuthor to whom correspondence should be addressed. &4bbreviations: Ag, antigen; cDNA, complementary deoxy ribonucleic acid; CDR, complementarity-dete~ining region; D, diversity segment; ELISA, enzyme-linked immunosorbent assay; H chain, immunoglobulin heavy chain; mQ,concn of Ag that inhibits the binding of an antibody in ELISA by 50%; Ig, immunoglobulin; J, joining segment; L chain, Ig light chain; mAb, monoclonal antibody; PCR, polymerase chain reaction; V, variable. 1107
1108
R. JEMMERSON
in the present study chimeric mAb were prepared by recombining the H and L chains of mAb having the same or different specificities. The H chains of two mAb with the same specificity for horse cytochrome c and differing at 15 amino acid positions in the V region could be substituted for one another in combination with the same L chain. The L chains differed by five amino acid residues and were also functionally interchangeable. In contrast, the H chains of an mAb specific for the same region but on rat cytochrome c, and, overall, nearly as similar in amino acid sequence to the other two H chains as they are to each other, could not substitute for the other two H chains. It did differ at several residues within the CDR that are shared by the other two chains, in particular CDR3. The L chain of this mAb could not functionally substitute for the horse cytochrome cspecific L chains and was very different from them in amino acid sequence. In this particular case, only a few amino acid residue changes in an mAb, at least in regard to the H chain, are necessary to impart specificity for one variant of a protein Ag over another. Furthermore, the results observed for the two horse cytochrome c-specific mAb support the view that mAb can differ markedly in amino acid sequence within the CDR yet bind the same epitope on a protein Ag if they share contact residues necessary for Ag binding. EXPERIMENTAL
PROCEDURES
mAb and Ag Horse and rat cytochromes c and BALB/c mouse B cell hybridomas secreting antibodies specific for one or the other of these Ag were obtained as previously described (Goshorn et al., 1991). Two of the mAb employed in this study were IgGl, K (2ES.GlO and 6H2.B4) and one was IgG2a, K (lF5.Dl). The mAb were purified from ascites fluid in two steps. First, the Ig fractions were precipitated in 50% saturated ammonium sulfate, centrifuged, and washed twice. The pellets were resuspended in phosphate-buffered saline, pH 7.2 and dialyzed against 1 1 of the same buffer with several changes. The mAb were adsorbed to protein G-agarose (GammaBind G, Genex Corp., Gaithersburg, MD) and eluted in 0.5 M acetic acid. The eluates were neutralized by titration with 1 M Tris and dialyzed against 1 1 of phosphate-buffered saline with several buffer changes. Nucleotide and protein sequences Using polydT-purified mRNA as template, L chain cDNA was synthesized, amplified by the PCR employing previously described primers, cloned into M 13mp 18 and M13mp19, and sequenced in both directions (Goshorn et al., 1991). The H chain nucleotide sequences, previously reported as partial sequences, were confirmed from clones of PCR products expressed in the TA cloning system, version 1.3 (Invitrogen, San Diego, CA) and extended in this study to include the 5’ ends. To eliminate sequence ambiguities resulting from the use of degenerate 5’ primers, the amino-terminal sequences of
et al.
purified polypeptide chains were determined employing a gas-phase automated protein sequenator (model 477A, Applied Biosystems, Inc., Foster City, CA).
Separation of H and L chains The disulfide bonds between H and L chains were reduced by treating 1 mg mAb in l-2 ml phosphatebuffered saline with 1 mg dithiothreitol for 60 min under an atmosphere of N,. Iodoacetamide (6 mg) was then added for 30 min (Hamel et al., 1984). The solution was dialyzed against 4.5 M urea, 0.75 M propionic acid for 4 hr and was then chromatographed on a I .O x 50 cm column of Sephadex G-100 equilibrated in 0.9 M urea, 0.75 M propionic acid (Olander and Little, 1975). The separated chains were stored at 4°C in the same solution for up to 2 weeks.
Recombination of H and L chains Equimolar amounts of H and L chains [calculated assuming a molar extinction coefficient ratio at 280 nm of 3: 1 for H: L; see Fasman (1976)] were dialyzed together for 18 hr against 2 I of distilled water, then 4 hr against 20mM acetate, pH 7.0, and finally, overnight against phosphate-buffered saline. To determine that the H and L chains had recombined, portions of the products were chromatographed using a 1.5 x 100 cm column of Sephadex G-200 (Pharmacia, Piscataway, NY). To increase the sensitivity of detection of the small amounts of eluting material, 50 ~1 aliquots of the fractions collected were directly adsorbed to wells of a microtiter plate and the presence of intact mAb, free H. or free L chain was determined by ELISA using goat anti-mouse IgG (whole molecule) conjugated to horseradish peroxidase (Sigma Chemical Co., St Louis, MO).
ELISA Binding to cytochrome c of native mAb, free H and L chains (dialyzed using the same conditions as in the recombination experiments), or recombined antibodies was determined by ELISA. Either horse or rat cytochrome c was adsorbed to wells of a microtiter plate (Nunc-Immuno Maxisorp plates, Gibbco, Coon Rapids. MN), and then the antibodies or free chains at varying dilution were incubated on the plate. The concns of Ig proteins were determined from the absorbance at 280 nm assuming an extinction coefficient of 1.5 for a 1 mg/ml solution (Fasman, 1976). Binding of the mAb to cytochrome c was detected as described above. In some experiments, the relative affinities of the native mAb and chimeric antibodies were determined by a competitive ELISA. The antibodies were incubated on Ag-coated microtiter plates, at concns of antibody which half-saturated the binding sites on the plate, in the presence of increasing amounts of soluble Ag. The amount of Ag resulting in 50% inhibition of binding, denoted [I],,, is an estimate of the affinity of the antibodies (Silvestri and Taniuchi, 1988).
Sequence variation in mAb specific for different cytochromes c
1109
H chain of 2E5.GlO as is the H chain of lFS.Dl, both of which are specific for horse cytochrome c. The procedure used was that described by Maniatis The amino acid sequences of the two L chains of the et al. (1982). DNA was purified from 5 x lo’-10’ hybrihorse cytochrome c-specific mAb had not been deterdoma cells. After cell lysis in 0.5% sodium dodecylsulmined previously and it was not known whether they fate and treatment with proteinase K, DNA was were similar to each other or to the L chain of 6H2.B4. extracted in phenol and precipitated in ethanol. The We now report that the L chain of 6H2.B4 (determined DNA (15 pg) was digested with 150 U BamHI or EcoRI previously) [see Goshorn et al. (1991)] is very different restriction endonuclease (Bethesda Research Laborafrom the L chains of the two horse cytochrome c-specific tories, Gaithersburg, MD) for 18 hr at 37°C. The mAb and that these are, in fact, similar to each other digested DNA was precipitated in ethanol, elec(Fig. 1). The Vu of 6H2.B4 is a member of the V,14 trophoresed overnight in a 0.7% agarose gel, and the family and the J segment is J,5. The V, of both lF5.Dl gel was blotted onto a nitrocellulose membrane. Ig and 2ES.GlO are members of the V, 12/13 family and the DNA was detected using as the radiolabeled probe a J, segments are J,2 (Potter et al., 1982; Sakano et al., 2 kb segment of cDNA, pJ11, representing the 1979). The two horse cytochrome c-specific L chains BamHI-EcoRI fragment which extends from the only differ at five amino acid positions in the V region, J,2-J,3 intron to 3’ of the enhancer (Marcu et al., four of these within the CDR. 1980). pJ11 was labeled with deoxycytidine 5’-[~r-~‘P] Since the nucleotide sequences of the H and L chain triphosphate (Amersham, Arlington Heights, IL) by V regions were determined from PCR products, it is nick translation. The blot was exposed to Kodak Xpossible that some nucleotide base changes may have OMAT AR film for 8 days at -80°C. occurred in vitro. The infidelity of base pairing in the PCR is well known and the frequency of error can approach one nucleotide in 300 (Eckert and Kunkel, RESULTS 1991). We examined more than one M 13 clone and Primary structure of the mAb sometimes several for each V region examined and the Three mAb were employed in this study. Two of the only variations observed were at the 5’ end in the region mAb, lF5.Dl and 2E5.Gl0, bind the surface of horse ternplated in the PCR by the degenerate 5’ primers. cytochrome c opposite that of the exposed heme edge Ambiguities in the nucleotide sequence were resolved by around residues 60 and 62. The other mAb, 6H2.B4, sequencing the mAb H and L chains directly. The amino binds the same surface on rat cytochrome c (Jemmerson acid sequences were then used to infer the nucleotide and Johnson, 1991). The horse cytochrome c-specific sequences in the regions where the 5’ primers overlapped mAb weakly cross-react with the rat protein in solution, within the coding sequences. but the rat cytochrome c-specific mAb does not bind horse cytochrome c. These mAb were chosen for this Separation of H and L chains study because their H chains are similar in sequence Following reduction of the disulfide bonds, the free (Table 1) [see Goshorn et al. (1991)]. The two mAb specific for horse cytochrome c differ at 15 amino acid chains of the mAb were readily isolated using Sephadex residue positions in the V region of the H chain, 11 of G- 100 in a solution of propionic acid and urea. A typical elution profile is presented in Fig. 2. The fractions were them within the CDR. The mAb specific for rat cytochrome c differs from these at 14 residues relative to pooled as shown. Silver staining of gels from 2E5.GlO and 18 residues relative to lF5.D1, 11 of 14 and SDSpolyacrylamide gel electrophoresis of these frac13 of 18 differences, respectively, occurring within the tions indicated that the H chain fractions contained CDR. On the whole, the H chain of 6H2.B4 which is mostly monomer, variable amounts of dimer, and at specific for rat cytochrome c is nearly as similar to the most a small amount (approximately less than 5%) of Southern blot analysis
Table 1. Amino acid sequence comparison of H chain V regions from cytochrome c-specific mAb” Residue position
“The amino acid sequences were deduced from the nucleotide sequences of PCR amplified cDNA (obtained using mRNA as template) except for the N-terminal residues which were determined by protein sequencing. Except as indicated at position 4, 5’ nucleotide sequences are identical to that of the germ-line gene HlO (Goshorn, et aI., 1991). The polypeptides are identical at all positions not given. A dash indicates identity with the sequence of the lFS.Dl H chain.
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Fig. 1. Nudeotide and deduced amino acid sequences of the L chains of two anti-horse cytochrome c-specific mAb (lFS.Dl and 2E5.GlO) and of a rat cytochrome c-specific mAb (6H2.B4). A dash indicates identity of the chains with the lF5.Dl L chain. Several of the codons at the 5’ end were modified to conform to the amino acid sequences determined from the purified proteins. The nucleotide ambiguities arose from the use of degenerate 5’ primers in the PCR. See text for details.
intact Ig (results not shown). The L chains appeared to be pure, but some contamination with H chains is possible. Recombination of H and L chains The H and L chains of each mAb were recombined with each other or with the complementary chain from a different mAb to form chimeras. To demonstrate that the combined chains formed intact Ig, they were chromatographed on Sephadex G-200. The presence of Ig, H or L chains in the eluting fractions was detected by ELISA. As shown in Fig. 3, the recombined products of heterologous chains (6H2H : 2E5L and 1F5H : 2E5L) in the examples presented eluted at a position similar to that of native Ig (2E5 :GlO). Similar results were observed for the other chimeric mAb. Functional reconstitution of the mAb was shown by their ability to bind cytochrome c in ELISA. In all three cases, the autologous recombinations resulted in functional reconstitution as exemplified in Fig. 4 for mAb 6H2.B4. Binding of the recombined product to rat cytochrome c adsorbed to microtiter plates approached that of the native mAb, although approximately threefold more of the recombined product was required for
a similar level of binding. This difference was observed for all three autologous recombinations in this study and is comparable to what has been observed previously for in vitro recombination of H and L chains of other mAb (Hamel et al., 1987; Smith-Gill et al., 1987). The basis for this difference is not clear. It does not appear to be due to a change in affinity. In competition ELISA, all three autologously reconstituted Ig showed [&, values comparable to those of the native mAb (results not shown). It is possible that upon recombining only a fraction of the H and L molecules recombined to the native state. This would not be surprising since the mAb were reduced and denatured in acid/urea to separate the chains. The isolated H and L chains did appear to bind the
Froctton
Fractton
Fig. 2. Separation of H and L chains by gel filtration chromatography. mAb 6H2.B4 was reduced and alkylated, dialyzed against 4.5 M urea, 0.75 M propionic acid, and applied to a 1 x SOcm column of Sephadex G-100 equilibrated in 0.9 M urea, 0.75 M propionic acid.
Fig. 3. Get filtration chromatography of chimeric and native antibodies. The chime& antibodies IFSH :2ESL (---) and 6H2H :2ESL (- - -), and native mAb 2E5.GlO (. .) were applied to a 1.5 x 100 cm column of Sephadex G-200 equilibrated in phosphate-buffered saline. The presence of Ig proteins in the eluting fractions was demonstrated by ELISA. The centers of the eluting fractions for the free H and L chains used in separate runs to standardize the column are indicated by the arrows.
in mAb specific for different cytochromes c
Sequence variation
E CYi 3
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-8 [Ab]
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Fig. 4. Binding of native mAb, recombined antibody, and H and L chains to cytochrome c. Rat cytochrome c was adsorbed to microtiter plates and the binding of native 6H2.B4 (O), 6H2H : 6H2L (@), 6H2L (c]), and 6H2H (H) to the platebound Ag was examined by ELISA. Each point represents the average of three trials with the standard deviation indicated by the vertical bars.
Ag in the solid-phase assay employed but much more weakly than the intact Ig. While binding of V, domains to Ag has been observed (Ward et al., 1989), it cannot be ruled out in this study that the binding attributed to the free H chain may be due to contaminating intact Ig present in the H chain fraction. The Sephadex gel used for separating H and L chains did not allow isolation of the H chains from the intact Ig. However, the binding attributed to the L chain appears to be due to the L chain. After rechromatography of the L chain fractions on the same column used for its initial isolation, comparable binding to the antigen was observed (results not shown). This binding is likely due to L chain dimers since after chromatography on Sephadex G-200 most of the Ag binding was found in the fractions eluting just prior to the free L chain. Reconstitution of Ag binding with heterologous II and L chains A compilation of the data from Ag-binding assays of recombined Ig from two independent experiments is presented in Table 2. Relative binding was calculated as the amount (mol. per liter) of chimeric Ig necessary to occupy half the binding sites on the ELISA plates divided by the corresponding amount of the free H or L chain listed for comparison. Binding to both horse and rat cytochromes c was examined in all cases; only the results for binding to the Ag for which the H or L chains are specific is given in Table 2 since the specificities observed were consistent with the original specificities of the native mAb. For example, from the first set of data from experiment 2 in Table 2, in which binding of chimeric antibodies to horse cytochrome c is compared with the background binding of 1FSL alone, the chimera 6H2H : 1F5L bound slightly poorer in ELISA to horse cytochrome c than did the L chain of lFS.Dl, yielding a relative binding ratio of 0.7. Both chimeras 2E5H : 1F5L and 1F5H : 1FSL bound signi~cantly better
1111
with ratios of 56 and 79, respectively, but not at the level of native 1F5.D1, with a relative binding ratio of 280. As mentioned above we have consistently observed that recombined H and L chains do not have the same ability to bind Ag as the native mAb. Although the relative binding ratios varied between experiments the relative magnitudes of the ratios were comparable. Throughout Table 2 it can be seen that the complementary chains of both 2E5.GlO and 1FS.Dl can be substituted for one another. The substitutions gave reasonably comparable results whether the chains were derived from 2E5.GlO or lF5.Dl. However, the autologous partners were generally slightly more effective than the heterologous partners. For example the chimera lF5H: lF5L was a more effective antibody than 2ESH : lF5L. Similarly, the 2E5H : 2E5L chimera was slightly more effective than 1F5H : 2E5L. The L chain of 6H2.B4 when paired with the H chain of either 2E5.GlO or lF5.Dl did not regain Ag reactivity. Similarly, the H chain of 6H2.B4 when paired with the L chains of lF5.Dl or 2E5.GlO did not bind the Ag.
Table 2. Binding of recombined mAbs to cytochrome c Relative binding” mAb 6H2H : 1FSL 2ESH: lF5L lF5H: IFSL Native lF5 6H2H : 2E5L 2ESH : 2E5L IFSH : 2E5L Native 2E5 6HZH : 6H2L 2E5H : 6H2L 1F5H : 6H2L Native 6H2 lF5H:6H2L lFSH:2E5L lF5H:lF5L Native IF5 2ESH : 6H2L 2E5H : 2ESL 2E5H:lF5L Native 2E5 6H2H : 6H2L 6H2H : 2E5L 6H2H : 1F5L Native 6H2
Compared to LFSL lF5L lF5L lF5L 2E5L 2E5L 2E5L 2E5L 6H2L 6H2L 6H2L 6H2L lF5H lF5H IFSH lF5H 2E5H 2E5H 2E5H 2E5H 6H2H 6H2H 6H2H 6H2H
Experiment 1 0.3 7 28 89 0.7 63 20 447 112 CO.1 CO.1 282 0.1 45 56 141 0.2 100 11 708 316 0.8 1.0 794
Experiment 2 0.7 56 79 280 3.2 28 25 160 44 0.3 0.5 125 1.3 450 250 710 1.3 180 110 790 I78 2.2 1.3 500
“Calculated as the molarity of whole mAb resulting in halfsaturation of cytochrome c binding sites in ELISA divided by the molar&y of free H or L chain in the comparison resulting in the same extent of binding. Horse cytochrome c was the Ag in comparisons with lFS.Dl and 2ES.GlO free H and L chains and rat cytochrome c was the Ag in comparisons with the 6H2B4 H and L chains. Data listed under experiments 1 and 2 were carried out using preparations of H and L chains obtained at different times. The data within each group were tabulated from several smaller experiments.
R. JEMMERSONet al.
1112
DISCUSSION In vitro recombination of H and L chains to examine antibody specificity has been employed in several studies (Smith-Gill et al., 1987; Radic et al., 1991; Bedzyk and Voss, 1991). As others have reported, we did not observe complete reconstitution of binding activity when homologous pairs of H and L chains were recombined. This is likely due to reassociation of the chains in a non-native manner since size-fractionation of the recombined products indicated that reassociation of the chains had occurred to a significant extent. Although reconstituted binding activity did not reach that of the native mAb, it is clear that enhanced binding over that of the individual chains did occur with certain H and L chain combinations and, for every combination of H and L chain showing enhancement, this enhanced binding was reproducible in two independent experiments. This strongly supports the validity of this approach. The results show that the H and L chains of the two horse cytochrome c-specific mAb, lF5.Dl and 2ES.Gl0, are functionally interchangeable, although there appears to be some preference for pairing with the original chain partner. However, neither the H nor the L chain of the rat cytochrome c-specific mAb, 6H2.B4, is interchangeable with the chains of the other two mAb. The three mAb bind the same region of their respective cytochrome c Ag based on immunochemical comparisons of their reactivities with variant cytochromes c (Jemmerson and Johnson, 1991). From the information available it is not yet possible to determine whether these mAb contact the same residues of the Ag and, hence, bind the same epitope. Since the H and L chains of the two horse cytochrome c-specific mAb, lFS.Dl and 2E5.Gl0, are functionally interchangeable to a considerable extent it seems likely that these two mAb do bind the same epitope. However, resolution of this question awaits the results of X-ray diffraction analysis. Crystals of the F(ab) fragments of both lF5.Dl and 2E5.GlO complexed to horse cytochrome c have been obtained and diffraction data have been collected.* The H and L chains of these two mAb are similar to a third mAb, E8, specific for the same region of horse cytochrome c which has also been crystallized as a F(ab) complexed to the antigen (Mylvaganam et al., 1991). From the recombination of the H and L chains between the rat cytochrome c-specific mAb, 6H2.B4, and the two horse cytochrome c-specific mAb, it is clear that both H and L chains contribute to the specificity of the mAb. The failure of the L chain of 6H2.B4 to functionally substitute for the other two L chains was expected since it is derived from a different V, family and is very different in amino acid sequence. The H chain of the 6H2.B4 is, however, similar to the H chains of the
*Jemmerson R., Buron S., Sanishvili R., Margoliash
E., Westbrook E. and Westbrook M. (1993) Crystallization of two monoclonal Fab fragments of similar amino acid sequence bound to the same area of horse cytochrome c and interacting by potentially distinct mechanisms. Submitted.
two horse cytochrome c-specific mAb. It differs from the H chain of 2E5.GlO in the V region at 14 amino acid residues and from the H chain of IF5.Dl at 18 amino acid residues, the majority of these differences occurring within the CDR. Thus, the V region of the H chain of 6H2.B4 is not much more different from the horse cytochrome c-specific H chains than they are from each other. The failure of the rat cytochrome c-specific H chain to functionally substitute for the horse cytochrome c-specific H chains, and vice versa, indicates that the differences in antigenic variant specificity between them is due to their few amino acid residue differences. Most of these occur in CDR3 and result from the utilization of a distinct D, segment. A single amino acid residue change in CDR2 could also be important in H chain interaction with the antigen. The H chain of 6H2.B4 has aspartic acid at position 56 while the other two H chains have asparagine. The other differences among these H chains where 6H2.B4 is unique are conservative in nature and would not necessarily be expected to significantly alter antigen recognition. It has been suggested from a study of a few mAb to different variants of influenza virus hemagglutinin that subtle changes in a protein epitope can result in dramatically different structures in the mAb elicited against a site (Meek et al., 1989). However, the boundaries of the epitopes or each variant were not determined so it is not known how similar the different epitopes were. In another study of numerous mAb to two variants of influenza virus hemagglutinin that also differed at only one amino acid residue, examples were observed of H or L chains of mAb with different specificity which were similar in amino acid sequence (Stark and Caton, 1991). However, distinct H and L chain combinations were elicited in response to the two variants. These findings are not incompatible with those in the present study. While we have examined fewer mAb than in the hemagglutinin study, we also observed the use of similar H or L chains in response to two different cytochromes c but not the same combination of H and L chains. The L chain of 6H2.B4, while distinct from those of the two horse cytochrome c-specific mAb in this study, is similar to the L chains of two other horse cytochrome c-specific mAb, 5F8 and 3A3 (Goshorn et al., 1991). We are currently recombining the H and L chains of this group of mAb to examine the effect of differences in the L chains on antigenic variant specificity. The finding that the H and L chains of at least some of the mAb apparently binding the same epitope are similar in sequence is not surprising and is consistent with observations in other antigen systems (Schmitter et al., 1991; Kavaler et al., 1990). A relatively large number of sequence changes is allowable within the CDR, of the H chain in particular, while maintaining Ag specificity. The H chains of 2E5.GlO and lF5.Dl differ at 15 amino acid residue positions, 11 of them within the CDR. Only a few residues from each CDR have been shown to be involved in antigen contact in the X-ray crystallographic studies completed to date (Davies et al..
Sequence variation in mAb specific for different cytochromes c
1988). Presumably, the Ag contact residues are conserved in the two H chains and the differences between them do not significantly affect the conformations of the CDR. It is also possible that different residues of the two H chains could contact the same residue(s) on cytochrome c (see below), thus allowing for the same specificity to be encoded by slightly different amino acid sequences. The amino acid differences between 2E5.GlO and lF5.Dl are not due to somatic mutation alone. Based on nucleotide sequencing, the H chains are derived from distinct J, segments, indicating that these two mAb, while obtained from the same mouse, are not clonally related. We attempted Southern analyses to determine if these two mAb were derived from the same V, gene. However, the interpretation was difficult because the mAb utilize different J, genes which would result in distinct bands even if they were derived from the same VH gene, and both chromosomes of the B cells rearranged, making assignment of the V, being examined to a particular band impossible. This latter problem also prevented comparison of the V, genes of 6H2.B4 and lF5.Dl even though they utilize the same Jr, segment. The number of nucleotide differences between these three V, segments is, however, within the range of the number of substitutions that can occur by somatic mutation (French et al., 1989). Comparison of the H chain amino acid sequences in Table 1 and the sequences of horse and rat cytochromes c on the antigenic surface bound by these mAb (Margoliash et al., 1961; Carlson et al., 1977; Bushnell et al., 1990) reveals a possible strategy for H chain-cytochrome c contact and for Ag specificity. For horse cytochrome c, lysine 60 is an immunodominant residue which could form a salt bridge with an acid group of the mAb (Jemmerson and Johnson, 1991). The horse cytochrome c-specific H chains have aspartic acid at either position 100 or 101 which could serve in such an electrostatic interaction. In contrast, the H chain of the rat cytochrome c-specific H chain not only lacks an aspartic acid residue at both positions but has an arginine at position 100 which could cause steric repulsion when contacting lysine 60 of horse cytochrome c. Rat cytochrome c has glycine at position 60 (Carlson et al., 1977) and its binding to this mAb would not be adversely affected by the arginine at position 100 in the H chain. Alternatively, it is also possible that the aspartic acid residues in CDRl that are unique to the H chains of IF5.Dl and 2ES.GlO play a role in forming a salt linkage. CDR2 of the 6H2.B4H chain has an aspartic acid at position 56, unique to this H chain, that could cause charge interference if CDR2 of the other two H chains contacts residue 92 of horse cytochrome c, a glutamic acid residue which may be part of the epitope(s) for the anti-horse cytochrome c mAb. In rat cytochrome c the residue at position 92 is alanine. To our knowledge the only other protein Ag examined for binding of chimeric antibodies made by the recombination of H and L chains is hen egg-white lysozyme (Smith-Gill et al., 1987). The H and L chains of two
1113
different mAb having similar fine specificities, HyHEL-8 and HyHEL-10, were shown to be functionally interchangeable. Within the CDR, the L chains differed by only one amino acid residue. The H chains were identical in CDRl, differed by only one amino acid residue in CDR3, and differed by four conservative replacements in CDR2. Thus, they were more similar than the H chains in the present study. The HyHEL-8 and HyHEL-10 H chains could not be substituted in the recognition of lysozyme by an H chain from a DNP-specific mAb that differed at only eight amino acid positions in the CDR, five of them occurring in CDR3. This suggests that the specificity for lysozyme over DNP is encoded in one or more of these eight amino acid residues. Interestingly, the L chain of the DNP-specific mAb that differed from the L chain of HyHEL-10 at only two amino acid residues in the CDR was functionally interchangeable with it. The results of this cited study and the present study show how useful H and L chain recombination can be in attempting to understand the molecular basis for antibody recognition of protein Ag; in particular, the relationship between amino acid sequence of the H and L chains of mAb and Ag specificity. These types of studies raise interesting questions that are not as easily provoked through X-ray diffraction analysis of Ag-F(ab) crystals but that will require X-ray crystallography for their resolution. Acknowledgements-The authors thank Drs Kathy Staskus and Brian Van Ness, University of Minnesota, for their guidance in Southern analysis, and Dr Chris Pennell, University of Minnesota for advice regarding cDNA cloning, for providing the pJl1 probe and for critically reading the manuscript. The secretarial assistance of Jan Smith and artwork of Tim Leonard are appreciated.
REFERENCES
Bedzyk W. D., Herron J. N., Edmundson A. B. and Voss E. W., Jr (1990) Active site structure and antigen binding properties of idiotypically cross-reactive anti-fluorescein monoclonal antibodies. J. biol. Chem. 265, 133-138. Bedzyk W. D. and Voss E. W., Jr (1991) Active site contributions to fluorescein binding determined by in vitro heavy and light chain reassociations. Molec. Immun. 28, 21-34. Bushnell G. W., Louie G. V. and Brayer G. D. (1990) High resolution three-dimensional structure of horse heart cytochrome c. J. molec. Biol. 214, 585-595. Carlson S. S., Mross G. A., Wilson A. C., Mead R. T., Wolin L. D., Bowers S. F., Foley N. T., Muijsers A. 0. and Margoliash E. (1977) Primary structure of mouse, rat, and guinea pig cytochrome c. Biochemistry 16, 1437-1442. Caton A. J., Brownlee G. G., Staudt L. M. and Gerhard W. (1986) Structural and functional implications of a restricted antibody response to a defined antigenic region on the influenza virus hemagglutinin. EMBO J. 5, 1577-1587. Caton A. J., Stark S. E., Kavaler J., Staudt L. M., Schwartz D. and Gerhard W. (1991) Many variable region genes are utilized in the antibody response of BALB/c mice to the influenza virus A/PR/8/34 hemagglutinin. J. Immun. 147, 1675-1686.
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Clarke S. H., Staudt L. M., Kavaler J., Schwartz D., Gerhard W. U. and Weigert M. G. (1990) V region gene usage and somatic mutation in the primary and secondary responses to influenza virus hemagglutinin. J. immun. 144, 279552801. Darsley M. J. and Rees A. R. (1985) Nucleotide sequences of five anti-lysozyme monoclonal antibodies. EMBO J. 4, 393-398.
Davies D. R., Sheriff S. and Padlan E. A. (1988) Antibody-antigen complexes. J. biol. Chem. 263, 10541-10544. Eckert K. A. and Kunkel T. A. (1991) DNA polymerase fidelity and the poiymerase chain reaction. PCR Meth. Appiic. 1, 17-24. Fasman G. D. (Ed.) (1976) Handbook of Biochemistry and Molecular Biology, 3rd Edn., pp. 452-457. Chemical Rubber Co., Cleveland, OH. French D. L., Laskov R. and Scharff M. D. (1989) The role of somatic hype~utation in the generation of antibody diversity. Science 244, 1152-I 157. Goshorn S. C., Retzel E. and Jemmerson R. (1991) Common structural features among monoclonal antibodies binding the same antigenic region of cytochrome c. J. biol. Chem. 266, 2134-2142.
Hamel P. A., Isenman D. E., Klein M. H., Luedtke R. and Dorrington K. J. (1984) Structural basis for the preferential association of autologous immunoglobulin subunits: role of the J region of the light chain. Molec. Immun. 21, 277-283. Hamel P. A., Klein M. H., Smith-Gill S. J. and Dorrington K. J. (1987) Relative noncovalent association constant between immunoglobulin H and L chains is unrelated to their expression or antigen-binding activity. .I. Immun. 139, 3012-3020.
Jemmerson R. and Johnson J. G. (1991) Different functional boundaries for the major antigenic region of two cytochromes c. Proc. natn. Acad. Sci. U.S.A. 88, 44284432. Kaartinen M., Griffiths G. M., Markham A. F. and Milstein C. (1983) mRNA sequences define an unusually restricted IgG response to 2-phenyloxazolone and its early diversification. Nature 304, 320-324. Kavaler J., Caton A. J., Staudt L. M., Schwartz D. and Gerhard W. (1990) A set of closely related antibodies dominates the primary antibody response to the antigenic site CB of the A/PR~8/34 influenza virus hemagglutinin. J. Immun. 145, 2312-2321.
Maniatis T., Fritsch E. F. and Sambrook J. (1982) Molecuiur Cloning-a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Marcu K. B., Banerji B., Penncavage N. A., Lang R. and Arnheim N. (1980) 5’ flanking region of immunoglobulin heavy chain constant region genes displays length leterogeneity in germlines of inbred mouse strains. Cell 22, 187-196. Margoliash E., Smith E. L., Kreil G. and Tuppy H. (1961) Amino acid sequence of horse heart cytochrome c. The complete amino acid sequence. Nature 192, 1125-I 127. Meek K., Johansson B., Schulman J., Bona C. and Capra J. D. (1989) Nucleotide changes in sequential variants of influenza virus hemagglutinin genes and molecular structures of corresponding monoclonal antibodies specific for each variant. Proc. natn. Acad. Sci. U.S.A. 86, 4664-4668.
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Mylvaganam S. E., Paterson Y., Kaiser K., Bowdish K. and Getzoff E. D. (1991) Biochemical implications from the variable gene sequences of an anti-cytochrome c antibody and crystallographic characterization of its antigen-binding fragment in free and antigen-complexed forms. J. molec. Bio!. 221, 4551162.
Olander J. and Little J. R. (1975) Preferential homologous recombination of H and L chains from mouse myeloma proteins which bind DNP ligands. Immunochemistry 12, 383-387.
Potter M., Newell J. B., Rudikoff S. and Haber E. (1982) Classification of mouse V, groups based on the partial amino acid sequence to the first invariant tryptophan: impact of 14 new sequences from IgG myeloma proteins. Molec. Immun. 19, 1619-1630. Radic M. Z., Mascelli M. A., Erikson J., Shan H. and Weigert M. (1991) Ig H and L chain contributions to autoimmune specificities. J. Immun. 146, 176182. Sakano H., Hiippi K., Heinrich G. and Tonegawa S. (1979) Sequences at the somatic recombination sites of immunoglobulin light-chain genes. Nature 280, 288-294. Schlomchik M., Mascelli M., Shan H., Radic M. Z., Pisetsky D., Marshak-Rothstein A. and Weigert M. (1990) Anti-DNA antibodies from auto~mmune mice arise by clonal expansion and somatic mutation. J. exp. Med. 171, 264297. Schmitter D., Poch O., Zeder G., Heinrich G. F., Kocher H.-P., Quesniaux V. F. J. and van Rogenmortel M. H. V. (1990) Analysis of the structural diversity of monoclonal antibodies to cyclosporine. Molec. Immun. 27, 1029-1038.
Silvestri I. and Taniuchi H. (1988) A study of fine specificity of monoclonal antibodies to yeast iso-l -cytochrome c. J. biol. Chem. 263, 18702-18715. Smith-Gill S. J., Hamel P. A., Lavoie T. 3. and Dorrington K. J. (1987) Contributions of immunoglobulin heavy and light chains to antibody specificity for lysozyme and two haptens. J. Immun. 139, 41354144. Stark S. E. and Caton A. J. (1991) Antibodies that are specific for a single amino acid interchange in a protein epitope use structurally distinct variable regions. J. exp. Med. 174, 613-624.
Stenzl-Poore M. P. and Rittenberg M. B. (1989) Clonal diversity, somatic mutation, and immune memory to phosphocholine-keyhole limpet hemocyanin. J. Immun. 143, 41234133.
Wang D., Chen H.-T., Liao J., Akolkar P. N., Sikder S. K., Gruezo F. and Kabat E. A. (1990) Two families of monoclonal antibodies to s((1 -+ 6)dextran, V, 19.1.2 and Vn9.14.7, show distinct patterns of Jk and J, minigene usage and amino acid substitutions in CDR3. J. Immun. 145, 3002-3010.
Ward E. S., G&sow D., Griffiths A. D., Jones P. T. and Winter G. (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Esfherichia coli. Nature 341, 544-546. Wysocki L., Manser T. and Gefter M. L. (1986) Somatic evolution of variable region structures during an immune response. Proc. natn. Acad. Sci. U.S.A. 83, 1847-1851.
Molecular Immunology, Vol. 30, No. 12, pp. 1107-I 114, 1993 Printed in Great Britain.
0 1993 Pergamon Press Ltd
DIFFERENCES IN HEAVY CHAIN AMINO ACID SEQUENCES AFFECTING THE SPECIFICITY OF ANTIBODIES FOR VARIANTS OF CYTOCHROME c* RONALD JEMMERSON,~CAROLYN MUELLER and DEBORAH FLAA Department
of Microbiology,
University of Minnesota MN 55455, U.S.A.
Medical School, Minneapolis,
(First received 1 December 1992; accepted in revised form 9 March 1993) Abstract-In a previous study [Goshorn ef al. (1991) 1. biof. Chem. 266, 2134-21421, several mAb specific for the same region on different cytochromes c were shown to have similar H or L chains. To determine the effect of differences in individual chains on antigenic variant s~ificity in the present study, chimeric mAb were prepared by recombining the H and L chains of mAb having the same or a different cytochrome c specificity. The H and L chains of two mAb to the region around residue 60 on horse cytochrome c (lF5.Dl and 2ES.GlO) were functionally interchangeable even though the H chain differed by 11 amino acid residues in the complementarity-determining regions (CDR) and 15 amino acids overall in the variable regions. The L chains only differed by four amino acid residues in the CDR (five residues overaIl). Neither the H nor L chain of a mAb binding the same region of rat cytochrome c (6H2.B4) was functionally interchangeable with the chains of the two horse cytochrome c-specific mAb. The L chain of this mAb is very different from the other L chains which were derived from a different V, family, but the H chain is nearly as similar to the horse cytochrome c-specific H chains as they are to each other. Most of the differences occur in CDR3 and result from the use of a distinct D, segment. The results indicate that, in some cases, the specificity of a mAb for a particular variant of a protein Ag, at least in regard to the H chain, is determined by only a few amino acid differences. The differences in the sequences of the H chains of the three mAb in this study and in the structures of their specific Ag provide insight into a possible molecular basis for the specificity of these mAb.
et al., 1991) or DNA (Schlomchik
structures of H$ and L chains of mAb to a variety of Ag have been determined (Kaartinen et al., 1983; Darsley and Rees, 1985; Wysocki ef al., 1986; Caton et al., 1986; Stenzf-Poore and Rittenberg, 1989; Schmitter et al., 1990; Wang et al., 1990; Schlomchik er al., 1990). The Ig structures can be relatively restricted, as in the antibody response to certain haptens (Kaartinen et al., 1983; Wysocki et al., 1986) or carbohydrates (Wang et al., 1990), and among mAb of shared idiotype (Bedzyk et al., 1990), or can be quite diverse as in the response to more complex Ag such as proteins (Caton The primary
et al., 1990). This diversity extends even to mAb which bind the same region of a protein Ag (Clarke et al., 1990). It is not known to what extent the diversity of epitopes in an antigenic region contributes to the diversity in Ig structures. When mAb to an antigenic region are grouped on the basis of fine specificities, often greater structural similarities among the mAb begin to emerge (Schmitter et al., 1990; Kavaler et al., 1990). Indeed, mAb to identical or similar sites on protein Ag can be very similar in the sequences of their H and L chains (Schmitter et ai., 1990; Kavaler et al., 1990; Goshorn et ai., 1991), although this is not necessarily always the case. With this premise in mind, we sought to determine: (1) whether mAb to the same region of different variants of a protein Ag were similar in primary structure, and (2) if so, whether the number of amino acid differences between mAb specific for different variants would be small enough to begin to understand how the specificity of mAb for a particular antigenic variant is encoded in the primary structure. We previously reported the amino acid sequences of the H and L chains of several mAb to two antigenic regions on horse, cow, and rat cytochromes c (Goshorn et al., 1991). Examples were observed where either the H or L chains of mAb specific for different cytochromes c were similar, but none where both chains were similar. To examine the effects of differences in only the H or L chain on mAb specificity,
*This work was supported by a grant from the National Science Foundation (DMB-901918 1). The nucleotide sequences reported in this paper have been submitted to the GenBank~m/EMBL Data Bank with accession numbers M57989-M57991 (H chains), M57987 and LO2500-LO2501 (L chains). TAuthor to whom correspondence should be addressed. &4bbreviations: Ag, antigen; cDNA, complementary deoxy ribonucleic acid; CDR, complementarity-dete~ining region; D, diversity segment; ELISA, enzyme-linked immunosorbent assay; H chain, immunoglobulin heavy chain; mQ,concn of Ag that inhibits the binding of an antibody in ELISA by 50%; Ig, immunoglobulin; J, joining segment; L chain, Ig light chain; mAb, monoclonal antibody; PCR, polymerase chain reaction; V, variable. 1107
1108
R. JEMMERSON
in the present study chimeric mAb were prepared by recombining the H and L chains of mAb having the same or different specificities. The H chains of two mAb with the same specificity for horse cytochrome c and differing at 15 amino acid positions in the V region could be substituted for one another in combination with the same L chain. The L chains differed by five amino acid residues and were also functionally interchangeable. In contrast, the H chains of an mAb specific for the same region but on rat cytochrome c, and, overall, nearly as similar in amino acid sequence to the other two H chains as they are to each other, could not substitute for the other two H chains. It did differ at several residues within the CDR that are shared by the other two chains, in particular CDR3. The L chain of this mAb could not functionally substitute for the horse cytochrome cspecific L chains and was very different from them in amino acid sequence. In this particular case, only a few amino acid residue changes in an mAb, at least in regard to the H chain, are necessary to impart specificity for one variant of a protein Ag over another. Furthermore, the results observed for the two horse cytochrome c-specific mAb support the view that mAb can differ markedly in amino acid sequence within the CDR yet bind the same epitope on a protein Ag if they share contact residues necessary for Ag binding. EXPERIMENTAL
PROCEDURES
mAb and Ag Horse and rat cytochromes c and BALB/c mouse B cell hybridomas secreting antibodies specific for one or the other of these Ag were obtained as previously described (Goshorn et al., 1991). Two of the mAb employed in this study were IgGl, K (2ES.GlO and 6H2.B4) and one was IgG2a, K (lF5.Dl). The mAb were purified from ascites fluid in two steps. First, the Ig fractions were precipitated in 50% saturated ammonium sulfate, centrifuged, and washed twice. The pellets were resuspended in phosphate-buffered saline, pH 7.2 and dialyzed against 1 1 of the same buffer with several changes. The mAb were adsorbed to protein G-agarose (GammaBind G, Genex Corp., Gaithersburg, MD) and eluted in 0.5 M acetic acid. The eluates were neutralized by titration with 1 M Tris and dialyzed against 1 1 of phosphate-buffered saline with several buffer changes. Nucleotide and protein sequences Using polydT-purified mRNA as template, L chain cDNA was synthesized, amplified by the PCR employing previously described primers, cloned into M 13mp 18 and M13mp19, and sequenced in both directions (Goshorn et al., 1991). The H chain nucleotide sequences, previously reported as partial sequences, were confirmed from clones of PCR products expressed in the TA cloning system, version 1.3 (Invitrogen, San Diego, CA) and extended in this study to include the 5’ ends. To eliminate sequence ambiguities resulting from the use of degenerate 5’ primers, the amino-terminal sequences of
et al.
purified polypeptide chains were determined employing a gas-phase automated protein sequenator (model 477A, Applied Biosystems, Inc., Foster City, CA).
Separation of H and L chains The disulfide bonds between H and L chains were reduced by treating 1 mg mAb in l-2 ml phosphatebuffered saline with 1 mg dithiothreitol for 60 min under an atmosphere of N,. Iodoacetamide (6 mg) was then added for 30 min (Hamel et al., 1984). The solution was dialyzed against 4.5 M urea, 0.75 M propionic acid for 4 hr and was then chromatographed on a I .O x 50 cm column of Sephadex G-100 equilibrated in 0.9 M urea, 0.75 M propionic acid (Olander and Little, 1975). The separated chains were stored at 4°C in the same solution for up to 2 weeks.
Recombination of H and L chains Equimolar amounts of H and L chains [calculated assuming a molar extinction coefficient ratio at 280 nm of 3: 1 for H: L; see Fasman (1976)] were dialyzed together for 18 hr against 2 I of distilled water, then 4 hr against 20mM acetate, pH 7.0, and finally, overnight against phosphate-buffered saline. To determine that the H and L chains had recombined, portions of the products were chromatographed using a 1.5 x 100 cm column of Sephadex G-200 (Pharmacia, Piscataway, NY). To increase the sensitivity of detection of the small amounts of eluting material, 50 ~1 aliquots of the fractions collected were directly adsorbed to wells of a microtiter plate and the presence of intact mAb, free H. or free L chain was determined by ELISA using goat anti-mouse IgG (whole molecule) conjugated to horseradish peroxidase (Sigma Chemical Co., St Louis, MO).
ELISA Binding to cytochrome c of native mAb, free H and L chains (dialyzed using the same conditions as in the recombination experiments), or recombined antibodies was determined by ELISA. Either horse or rat cytochrome c was adsorbed to wells of a microtiter plate (Nunc-Immuno Maxisorp plates, Gibbco, Coon Rapids. MN), and then the antibodies or free chains at varying dilution were incubated on the plate. The concns of Ig proteins were determined from the absorbance at 280 nm assuming an extinction coefficient of 1.5 for a 1 mg/ml solution (Fasman, 1976). Binding of the mAb to cytochrome c was detected as described above. In some experiments, the relative affinities of the native mAb and chimeric antibodies were determined by a competitive ELISA. The antibodies were incubated on Ag-coated microtiter plates, at concns of antibody which half-saturated the binding sites on the plate, in the presence of increasing amounts of soluble Ag. The amount of Ag resulting in 50% inhibition of binding, denoted [I],,, is an estimate of the affinity of the antibodies (Silvestri and Taniuchi, 1988).
Sequence variation in mAb specific for different cytochromes c
1109
H chain of 2E5.GlO as is the H chain of lFS.Dl, both of which are specific for horse cytochrome c. The procedure used was that described by Maniatis The amino acid sequences of the two L chains of the et al. (1982). DNA was purified from 5 x lo’-10’ hybrihorse cytochrome c-specific mAb had not been deterdoma cells. After cell lysis in 0.5% sodium dodecylsulmined previously and it was not known whether they fate and treatment with proteinase K, DNA was were similar to each other or to the L chain of 6H2.B4. extracted in phenol and precipitated in ethanol. The We now report that the L chain of 6H2.B4 (determined DNA (15 pg) was digested with 150 U BamHI or EcoRI previously) [see Goshorn et al. (1991)] is very different restriction endonuclease (Bethesda Research Laborafrom the L chains of the two horse cytochrome c-specific tories, Gaithersburg, MD) for 18 hr at 37°C. The mAb and that these are, in fact, similar to each other digested DNA was precipitated in ethanol, elec(Fig. 1). The Vu of 6H2.B4 is a member of the V,14 trophoresed overnight in a 0.7% agarose gel, and the family and the J segment is J,5. The V, of both lF5.Dl gel was blotted onto a nitrocellulose membrane. Ig and 2ES.GlO are members of the V, 12/13 family and the DNA was detected using as the radiolabeled probe a J, segments are J,2 (Potter et al., 1982; Sakano et al., 2 kb segment of cDNA, pJ11, representing the 1979). The two horse cytochrome c-specific L chains BamHI-EcoRI fragment which extends from the only differ at five amino acid positions in the V region, J,2-J,3 intron to 3’ of the enhancer (Marcu et al., four of these within the CDR. 1980). pJ11 was labeled with deoxycytidine 5’-[~r-~‘P] Since the nucleotide sequences of the H and L chain triphosphate (Amersham, Arlington Heights, IL) by V regions were determined from PCR products, it is nick translation. The blot was exposed to Kodak Xpossible that some nucleotide base changes may have OMAT AR film for 8 days at -80°C. occurred in vitro. The infidelity of base pairing in the PCR is well known and the frequency of error can approach one nucleotide in 300 (Eckert and Kunkel, RESULTS 1991). We examined more than one M 13 clone and Primary structure of the mAb sometimes several for each V region examined and the Three mAb were employed in this study. Two of the only variations observed were at the 5’ end in the region mAb, lF5.Dl and 2E5.Gl0, bind the surface of horse ternplated in the PCR by the degenerate 5’ primers. cytochrome c opposite that of the exposed heme edge Ambiguities in the nucleotide sequence were resolved by around residues 60 and 62. The other mAb, 6H2.B4, sequencing the mAb H and L chains directly. The amino binds the same surface on rat cytochrome c (Jemmerson acid sequences were then used to infer the nucleotide and Johnson, 1991). The horse cytochrome c-specific sequences in the regions where the 5’ primers overlapped mAb weakly cross-react with the rat protein in solution, within the coding sequences. but the rat cytochrome c-specific mAb does not bind horse cytochrome c. These mAb were chosen for this Separation of H and L chains study because their H chains are similar in sequence Following reduction of the disulfide bonds, the free (Table 1) [see Goshorn et al. (1991)]. The two mAb specific for horse cytochrome c differ at 15 amino acid chains of the mAb were readily isolated using Sephadex residue positions in the V region of the H chain, 11 of G- 100 in a solution of propionic acid and urea. A typical elution profile is presented in Fig. 2. The fractions were them within the CDR. The mAb specific for rat cytochrome c differs from these at 14 residues relative to pooled as shown. Silver staining of gels from 2E5.GlO and 18 residues relative to lF5.D1, 11 of 14 and SDSpolyacrylamide gel electrophoresis of these frac13 of 18 differences, respectively, occurring within the tions indicated that the H chain fractions contained CDR. On the whole, the H chain of 6H2.B4 which is mostly monomer, variable amounts of dimer, and at specific for rat cytochrome c is nearly as similar to the most a small amount (approximately less than 5%) of Southern blot analysis
Table 1. Amino acid sequence comparison of H chain V regions from cytochrome c-specific mAb” Residue position
“The amino acid sequences were deduced from the nucleotide sequences of PCR amplified cDNA (obtained using mRNA as template) except for the N-terminal residues which were determined by protein sequencing. Except as indicated at position 4, 5’ nucleotide sequences are identical to that of the germ-line gene HlO (Goshorn, et aI., 1991). The polypeptides are identical at all positions not given. A dash indicates identity with the sequence of the lFS.Dl H chain.
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R
Fig. 1. Nudeotide and deduced amino acid sequences of the L chains of two anti-horse cytochrome c-specific mAb (lFS.Dl and 2E5.GlO) and of a rat cytochrome c-specific mAb (6H2.B4). A dash indicates identity of the chains with the lF5.Dl L chain. Several of the codons at the 5’ end were modified to conform to the amino acid sequences determined from the purified proteins. The nucleotide ambiguities arose from the use of degenerate 5’ primers in the PCR. See text for details.
intact Ig (results not shown). The L chains appeared to be pure, but some contamination with H chains is possible. Recombination of H and L chains The H and L chains of each mAb were recombined with each other or with the complementary chain from a different mAb to form chimeras. To demonstrate that the combined chains formed intact Ig, they were chromatographed on Sephadex G-200. The presence of Ig, H or L chains in the eluting fractions was detected by ELISA. As shown in Fig. 3, the recombined products of heterologous chains (6H2H : 2E5L and 1F5H : 2E5L) in the examples presented eluted at a position similar to that of native Ig (2E5 :GlO). Similar results were observed for the other chimeric mAb. Functional reconstitution of the mAb was shown by their ability to bind cytochrome c in ELISA. In all three cases, the autologous recombinations resulted in functional reconstitution as exemplified in Fig. 4 for mAb 6H2.B4. Binding of the recombined product to rat cytochrome c adsorbed to microtiter plates approached that of the native mAb, although approximately threefold more of the recombined product was required for
a similar level of binding. This difference was observed for all three autologous recombinations in this study and is comparable to what has been observed previously for in vitro recombination of H and L chains of other mAb (Hamel et al., 1987; Smith-Gill et al., 1987). The basis for this difference is not clear. It does not appear to be due to a change in affinity. In competition ELISA, all three autologously reconstituted Ig showed [&, values comparable to those of the native mAb (results not shown). It is possible that upon recombining only a fraction of the H and L molecules recombined to the native state. This would not be surprising since the mAb were reduced and denatured in acid/urea to separate the chains. The isolated H and L chains did appear to bind the
Froctton
Fractton
Fig. 2. Separation of H and L chains by gel filtration chromatography. mAb 6H2.B4 was reduced and alkylated, dialyzed against 4.5 M urea, 0.75 M propionic acid, and applied to a 1 x SOcm column of Sephadex G-100 equilibrated in 0.9 M urea, 0.75 M propionic acid.
Fig. 3. Get filtration chromatography of chimeric and native antibodies. The chime& antibodies IFSH :2ESL (---) and 6H2H :2ESL (- - -), and native mAb 2E5.GlO (. .) were applied to a 1.5 x 100 cm column of Sephadex G-200 equilibrated in phosphate-buffered saline. The presence of Ig proteins in the eluting fractions was demonstrated by ELISA. The centers of the eluting fractions for the free H and L chains used in separate runs to standardize the column are indicated by the arrows.
in mAb specific for different cytochromes c
Sequence variation
E CYi 3
I.0
“0 a
-8 [Ab]
-7
-6
fW
Fig. 4. Binding of native mAb, recombined antibody, and H and L chains to cytochrome c. Rat cytochrome c was adsorbed to microtiter plates and the binding of native 6H2.B4 (O), 6H2H : 6H2L (@), 6H2L (c]), and 6H2H (H) to the platebound Ag was examined by ELISA. Each point represents the average of three trials with the standard deviation indicated by the vertical bars.
Ag in the solid-phase assay employed but much more weakly than the intact Ig. While binding of V, domains to Ag has been observed (Ward et al., 1989), it cannot be ruled out in this study that the binding attributed to the free H chain may be due to contaminating intact Ig present in the H chain fraction. The Sephadex gel used for separating H and L chains did not allow isolation of the H chains from the intact Ig. However, the binding attributed to the L chain appears to be due to the L chain. After rechromatography of the L chain fractions on the same column used for its initial isolation, comparable binding to the antigen was observed (results not shown). This binding is likely due to L chain dimers since after chromatography on Sephadex G-200 most of the Ag binding was found in the fractions eluting just prior to the free L chain. Reconstitution of Ag binding with heterologous II and L chains A compilation of the data from Ag-binding assays of recombined Ig from two independent experiments is presented in Table 2. Relative binding was calculated as the amount (mol. per liter) of chimeric Ig necessary to occupy half the binding sites on the ELISA plates divided by the corresponding amount of the free H or L chain listed for comparison. Binding to both horse and rat cytochromes c was examined in all cases; only the results for binding to the Ag for which the H or L chains are specific is given in Table 2 since the specificities observed were consistent with the original specificities of the native mAb. For example, from the first set of data from experiment 2 in Table 2, in which binding of chimeric antibodies to horse cytochrome c is compared with the background binding of 1FSL alone, the chimera 6H2H : 1F5L bound slightly poorer in ELISA to horse cytochrome c than did the L chain of lFS.Dl, yielding a relative binding ratio of 0.7. Both chimeras 2E5H : 1F5L and 1F5H : 1FSL bound signi~cantly better
1111
with ratios of 56 and 79, respectively, but not at the level of native 1F5.D1, with a relative binding ratio of 280. As mentioned above we have consistently observed that recombined H and L chains do not have the same ability to bind Ag as the native mAb. Although the relative binding ratios varied between experiments the relative magnitudes of the ratios were comparable. Throughout Table 2 it can be seen that the complementary chains of both 2E5.GlO and 1FS.Dl can be substituted for one another. The substitutions gave reasonably comparable results whether the chains were derived from 2E5.GlO or lF5.Dl. However, the autologous partners were generally slightly more effective than the heterologous partners. For example the chimera lF5H: lF5L was a more effective antibody than 2ESH : lF5L. Similarly, the 2E5H : 2E5L chimera was slightly more effective than 1F5H : 2E5L. The L chain of 6H2.B4 when paired with the H chain of either 2E5.GlO or lF5.Dl did not regain Ag reactivity. Similarly, the H chain of 6H2.B4 when paired with the L chains of lF5.Dl or 2E5.GlO did not bind the Ag.
Table 2. Binding of recombined mAbs to cytochrome c Relative binding” mAb 6H2H : 1FSL 2ESH: lF5L lF5H: IFSL Native lF5 6H2H : 2E5L 2ESH : 2E5L IFSH : 2E5L Native 2E5 6HZH : 6H2L 2E5H : 6H2L 1F5H : 6H2L Native 6H2 lF5H:6H2L lFSH:2E5L lF5H:lF5L Native IF5 2ESH : 6H2L 2E5H : 2ESL 2E5H:lF5L Native 2E5 6H2H : 6H2L 6H2H : 2E5L 6H2H : 1F5L Native 6H2
Compared to LFSL lF5L lF5L lF5L 2E5L 2E5L 2E5L 2E5L 6H2L 6H2L 6H2L 6H2L lF5H lF5H IFSH lF5H 2E5H 2E5H 2E5H 2E5H 6H2H 6H2H 6H2H 6H2H
Experiment 1 0.3 7 28 89 0.7 63 20 447 112 CO.1 CO.1 282 0.1 45 56 141 0.2 100 11 708 316 0.8 1.0 794
Experiment 2 0.7 56 79 280 3.2 28 25 160 44 0.3 0.5 125 1.3 450 250 710 1.3 180 110 790 I78 2.2 1.3 500
“Calculated as the molarity of whole mAb resulting in halfsaturation of cytochrome c binding sites in ELISA divided by the molar&y of free H or L chain in the comparison resulting in the same extent of binding. Horse cytochrome c was the Ag in comparisons with lFS.Dl and 2ES.GlO free H and L chains and rat cytochrome c was the Ag in comparisons with the 6H2B4 H and L chains. Data listed under experiments 1 and 2 were carried out using preparations of H and L chains obtained at different times. The data within each group were tabulated from several smaller experiments.
R. JEMMERSONet al.
1112
DISCUSSION In vitro recombination of H and L chains to examine antibody specificity has been employed in several studies (Smith-Gill et al., 1987; Radic et al., 1991; Bedzyk and Voss, 1991). As others have reported, we did not observe complete reconstitution of binding activity when homologous pairs of H and L chains were recombined. This is likely due to reassociation of the chains in a non-native manner since size-fractionation of the recombined products indicated that reassociation of the chains had occurred to a significant extent. Although reconstituted binding activity did not reach that of the native mAb, it is clear that enhanced binding over that of the individual chains did occur with certain H and L chain combinations and, for every combination of H and L chain showing enhancement, this enhanced binding was reproducible in two independent experiments. This strongly supports the validity of this approach. The results show that the H and L chains of the two horse cytochrome c-specific mAb, lF5.Dl and 2ES.Gl0, are functionally interchangeable, although there appears to be some preference for pairing with the original chain partner. However, neither the H nor the L chain of the rat cytochrome c-specific mAb, 6H2.B4, is interchangeable with the chains of the other two mAb. The three mAb bind the same region of their respective cytochrome c Ag based on immunochemical comparisons of their reactivities with variant cytochromes c (Jemmerson and Johnson, 1991). From the information available it is not yet possible to determine whether these mAb contact the same residues of the Ag and, hence, bind the same epitope. Since the H and L chains of the two horse cytochrome c-specific mAb, lFS.Dl and 2E5.Gl0, are functionally interchangeable to a considerable extent it seems likely that these two mAb do bind the same epitope. However, resolution of this question awaits the results of X-ray diffraction analysis. Crystals of the F(ab) fragments of both lF5.Dl and 2E5.GlO complexed to horse cytochrome c have been obtained and diffraction data have been collected.* The H and L chains of these two mAb are similar to a third mAb, E8, specific for the same region of horse cytochrome c which has also been crystallized as a F(ab) complexed to the antigen (Mylvaganam et al., 1991). From the recombination of the H and L chains between the rat cytochrome c-specific mAb, 6H2.B4, and the two horse cytochrome c-specific mAb, it is clear that both H and L chains contribute to the specificity of the mAb. The failure of the L chain of 6H2.B4 to functionally substitute for the other two L chains was expected since it is derived from a different V, family and is very different in amino acid sequence. The H chain of the 6H2.B4 is, however, similar to the H chains of the
*Jemmerson R., Buron S., Sanishvili R., Margoliash
E., Westbrook E. and Westbrook M. (1993) Crystallization of two monoclonal Fab fragments of similar amino acid sequence bound to the same area of horse cytochrome c and interacting by potentially distinct mechanisms. Submitted.
two horse cytochrome c-specific mAb. It differs from the H chain of 2E5.GlO in the V region at 14 amino acid residues and from the H chain of IF5.Dl at 18 amino acid residues, the majority of these differences occurring within the CDR. Thus, the V region of the H chain of 6H2.B4 is not much more different from the horse cytochrome c-specific H chains than they are from each other. The failure of the rat cytochrome c-specific H chain to functionally substitute for the horse cytochrome c-specific H chains, and vice versa, indicates that the differences in antigenic variant specificity between them is due to their few amino acid residue differences. Most of these occur in CDR3 and result from the utilization of a distinct D, segment. A single amino acid residue change in CDR2 could also be important in H chain interaction with the antigen. The H chain of 6H2.B4 has aspartic acid at position 56 while the other two H chains have asparagine. The other differences among these H chains where 6H2.B4 is unique are conservative in nature and would not necessarily be expected to significantly alter antigen recognition. It has been suggested from a study of a few mAb to different variants of influenza virus hemagglutinin that subtle changes in a protein epitope can result in dramatically different structures in the mAb elicited against a site (Meek et al., 1989). However, the boundaries of the epitopes or each variant were not determined so it is not known how similar the different epitopes were. In another study of numerous mAb to two variants of influenza virus hemagglutinin that also differed at only one amino acid residue, examples were observed of H or L chains of mAb with different specificity which were similar in amino acid sequence (Stark and Caton, 1991). However, distinct H and L chain combinations were elicited in response to the two variants. These findings are not incompatible with those in the present study. While we have examined fewer mAb than in the hemagglutinin study, we also observed the use of similar H or L chains in response to two different cytochromes c but not the same combination of H and L chains. The L chain of 6H2.B4, while distinct from those of the two horse cytochrome c-specific mAb in this study, is similar to the L chains of two other horse cytochrome c-specific mAb, 5F8 and 3A3 (Goshorn et al., 1991). We are currently recombining the H and L chains of this group of mAb to examine the effect of differences in the L chains on antigenic variant specificity. The finding that the H and L chains of at least some of the mAb apparently binding the same epitope are similar in sequence is not surprising and is consistent with observations in other antigen systems (Schmitter et al., 1991; Kavaler et al., 1990). A relatively large number of sequence changes is allowable within the CDR, of the H chain in particular, while maintaining Ag specificity. The H chains of 2E5.GlO and lF5.Dl differ at 15 amino acid residue positions, 11 of them within the CDR. Only a few residues from each CDR have been shown to be involved in antigen contact in the X-ray crystallographic studies completed to date (Davies et al..
Sequence variation in mAb specific for different cytochromes c
1988). Presumably, the Ag contact residues are conserved in the two H chains and the differences between them do not significantly affect the conformations of the CDR. It is also possible that different residues of the two H chains could contact the same residue(s) on cytochrome c (see below), thus allowing for the same specificity to be encoded by slightly different amino acid sequences. The amino acid differences between 2E5.GlO and lF5.Dl are not due to somatic mutation alone. Based on nucleotide sequencing, the H chains are derived from distinct J, segments, indicating that these two mAb, while obtained from the same mouse, are not clonally related. We attempted Southern analyses to determine if these two mAb were derived from the same V, gene. However, the interpretation was difficult because the mAb utilize different J, genes which would result in distinct bands even if they were derived from the same VH gene, and both chromosomes of the B cells rearranged, making assignment of the V, being examined to a particular band impossible. This latter problem also prevented comparison of the V, genes of 6H2.B4 and lF5.Dl even though they utilize the same Jr, segment. The number of nucleotide differences between these three V, segments is, however, within the range of the number of substitutions that can occur by somatic mutation (French et al., 1989). Comparison of the H chain amino acid sequences in Table 1 and the sequences of horse and rat cytochromes c on the antigenic surface bound by these mAb (Margoliash et al., 1961; Carlson et al., 1977; Bushnell et al., 1990) reveals a possible strategy for H chain-cytochrome c contact and for Ag specificity. For horse cytochrome c, lysine 60 is an immunodominant residue which could form a salt bridge with an acid group of the mAb (Jemmerson and Johnson, 1991). The horse cytochrome c-specific H chains have aspartic acid at either position 100 or 101 which could serve in such an electrostatic interaction. In contrast, the H chain of the rat cytochrome c-specific H chain not only lacks an aspartic acid residue at both positions but has an arginine at position 100 which could cause steric repulsion when contacting lysine 60 of horse cytochrome c. Rat cytochrome c has glycine at position 60 (Carlson et al., 1977) and its binding to this mAb would not be adversely affected by the arginine at position 100 in the H chain. Alternatively, it is also possible that the aspartic acid residues in CDRl that are unique to the H chains of IF5.Dl and 2ES.GlO play a role in forming a salt linkage. CDR2 of the 6H2.B4H chain has an aspartic acid at position 56, unique to this H chain, that could cause charge interference if CDR2 of the other two H chains contacts residue 92 of horse cytochrome c, a glutamic acid residue which may be part of the epitope(s) for the anti-horse cytochrome c mAb. In rat cytochrome c the residue at position 92 is alanine. To our knowledge the only other protein Ag examined for binding of chimeric antibodies made by the recombination of H and L chains is hen egg-white lysozyme (Smith-Gill et al., 1987). The H and L chains of two
1113
different mAb having similar fine specificities, HyHEL-8 and HyHEL-10, were shown to be functionally interchangeable. Within the CDR, the L chains differed by only one amino acid residue. The H chains were identical in CDRl, differed by only one amino acid residue in CDR3, and differed by four conservative replacements in CDR2. Thus, they were more similar than the H chains in the present study. The HyHEL-8 and HyHEL-10 H chains could not be substituted in the recognition of lysozyme by an H chain from a DNP-specific mAb that differed at only eight amino acid positions in the CDR, five of them occurring in CDR3. This suggests that the specificity for lysozyme over DNP is encoded in one or more of these eight amino acid residues. Interestingly, the L chain of the DNP-specific mAb that differed from the L chain of HyHEL-10 at only two amino acid residues in the CDR was functionally interchangeable with it. The results of this cited study and the present study show how useful H and L chain recombination can be in attempting to understand the molecular basis for antibody recognition of protein Ag; in particular, the relationship between amino acid sequence of the H and L chains of mAb and Ag specificity. These types of studies raise interesting questions that are not as easily provoked through X-ray diffraction analysis of Ag-F(ab) crystals but that will require X-ray crystallography for their resolution. Acknowledgements-The authors thank Drs Kathy Staskus and Brian Van Ness, University of Minnesota, for their guidance in Southern analysis, and Dr Chris Pennell, University of Minnesota for advice regarding cDNA cloning, for providing the pJl1 probe and for critically reading the manuscript. The secretarial assistance of Jan Smith and artwork of Tim Leonard are appreciated.
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