Immunoglobulin variable region sequences of humanmonoclonal anti-DNA, antibodies

Seminars in

Arthritis and Rheumatism VOL 28, NO 3

DECEMBER 1998

I m m u n o g l o b u l i n Variable Region Sequences of H u m a n M o n o c l o n a l Anti-DNA Antibodies Anisur Rahman, David S. Latchman, and David A. Isenberg Objective: Anti-DNA antibodies are believed to be important in the pathogenesis of systemic lupus erythematosus (SLE). Antibodies that bind specifically and with high affinity to dsDNA are most closely involved in tissue damage. Analysis of the sequences of the variable regions of human monoclonal anti-DNA antibodies is useful in defining the structural features that give rise to these binding properties. This article systematically reviews the evidence derived from such sequences. Method: Previous reviews of this subject have been hampered by incomplete knowledge of the human immunoglobulin variable region repertoire. In this article, the original sequence data from reports of over 50 human monoclonal antibodies (mAb) are reinterpreted by alignment to the most similar alleles of the most similar germline genes. This allows accurate estimation of the site and nature of somatic mutations. Results: Human IgG monoclonal anti-DNA antibodies generally carry more mutations than IgM. In many cases these have been selected by an antigen-driven process. In many of the more specific, higher affinity dsDNA binders, there is an accumulation of basic residues in the complementarity determining regions. However, many exceptions to this rule exist, particularly among IgM mAb. Conclusions: Unlike murine anti-DNA antibodies, these human mAb show little evidence for preferential use of particular VH, V~ and V~ genes or families to encode antibodies of this specificity. Semin Arthritis Rheum 28:141-154. Copyright © 1998 by W.B. Saunders Company

INDEX WORDS: Monoclonal antibody; immunogiobulin variable region; DNA sequence. NTIBODIES TO DNA were first described in the blood of patients with systemic lupus erythematosus (SLE) in 1957 (1, 2). A number of studies have shown that the levels of antibodies to double-stranded DNA (anti-dsDNA), but not always of antibodies to single-stranded DNA (antissDNA) are related to the rise and fall of disease activity in patients with SLE (3, 4). This is particularly true in cases in which the disease activity takes the form of glomerulonephritis (5). More direct evidence that anti-dsDNA antibodies are implicated in causing glomerular damage in SLE

A

From Bloomsbury Rheumatology Unit~Centrefor Rheumatology, Department of Medicine and Department of Molecular Pathology, University College, London, UK. Anisur Rahman, PhD, MRCP: Wellcome Trust Clinical Research Training Fellow; David S. Latchman, PhD, DSc: Professor of Molecular Pathology; David A. Isenberg, MD, FRCP: Arthritis Research Campaign Diamoml Jubilee Professor of Rlwumatolog~: Dt: Rahman is supported by Wellcome Trust Research Training Fellowship 040 366/Z/94/Z. Address reprint requests to Anisur Rahman, PhD, MRCP, Bloomsbury Rheumatology Unit, Arthur Stanley House, 40-50 Tottenham St, London W1P 9PG, United Kingdom. Copyright © 1998 by W.B. Saunders Company 0049-0172/98/280.3-000158.00/0

Seminars in Arthritis and Rheumatism, Vol 28, No 3 (December), 1998: pp 141-154

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comes from reports that these antibodies are often found in renal biopsies from patients with lupus nephritis but not from those with other types of nephritis (6, 7). Some murine monoclonal antiDNA antibodies have been shown to deposit in mouse (8) or rat (9) glomeruli with associated proteinuria. More recently, some human monoclonal IgG anti-dsDNA antibodies have been introduced into severe combined immunodeficiency (SCID) mice with resulting glomerular deposition and proteinuria (10, 11) suggesting that such antibodies may play a direct pathogenic role in lupus nephritis. IDENTIFICATION OF A PATHOGENIC SUBSET OF ANTI-dsDNA

Not all patients with high blood anti-dsDNA levels have highly active disease (4). Similarly, not all the monoclonal anti-DNA antibodies tested in the mouse and rat models previously mentioned could form glomerular deposits (8-11). These findings suggest that the ability to bind dsDNA is not sufficient to render an antibody molecule capable of causing tissue damage in SLE. A subset of anti-dsDNA antibodies is particularly closely linked to the development of such damage. This subset consists primarily of positively charged IgG antibodies that bind specifically to dsDNA with high affinity. Okamura et al (12) showed that, in 40 untreated Japanese patients with lupus nephritis, the degree of histological damage seen in renal biopsy specimens was closely correlated with the level of IgG but not IgM anti-dsDNA in the patient's serum. Suzuki et al (13) found that patients with active lupus nephritis were more likely to have high titers of positively charged anti-dsDNA antibodies in their serum than those with inactive disease. While recognizing that not all patients with active SLE have high titers of high affinity antidsDNA antibodies (14), it is important to investigate the particular structural features that differentiate these molecules from other immunogtobulins. By building up a database of sequence information from monoclonal anti-DNA antibodies derived from both humans and mice, it has been possible to deduce that certain sequence features appear to occur commonly in antibodies of this specificity, particularly in high affinity IgG antibodies. This helps both in the study of how such antibodies come to develop in some individuals but not in

others, and in constructing an image of the antibodyDNA binding site. By comparing the sequence data with the ability of these antibodies to cause tissue damage in experimental models, it may be possible to define sequence characteristics that are related to pathogenicity. The design of drugs that interfere with binding of antibodies to DNA may represent an avenue for the treatment of SLE in the future. INTERPRETATION OF ANTIBODY SEQUENCES

An immunoglobulin molecule consists of two identical heavy chains and two identical light chains as shown in Fig 1. There are two types of light chain, designated kappa and lambda. The antigen binding site, however, is encoded by just the amino terminal domains of these chains. These domains vary considerably in sequence between different antibody molecules and are therefore known as the light chain and heavy chain variable regions (VL and VH, respectively). The carboxyl terminal parts of the chains have almost the same sequence in all antibodies of a given isotype and are known as the constant regions. In comparing the sequence features of a number of different monoclonal antibodies (mAb), it is therefore only necessary to consider the variable regions. Within these regions, variability of sequence is not uniform. Instead, three areas of very high variability, known as the complementarity determining regions (CDRs) are separated by longer stretches of sequence which are much more similar in different antibodies (see Fig i). These are called the framework regions (FRs) because evidence from crystallographic studies suggests that they form a scaffold from which loops encoded by the CDRs extend to make contact with the antigen (15). Thus the sequences of the CDRs of the VH and VL regions are particularly important in determining the antigen binding properties of an antibody. Both the VH and VL domains are produced by fusion of gene segments that are separate in germline DNA but are brought together by a tissue specific recombination process in B cells. The VL domain is encoded by two segments (the VL and JL genes) and the VH domain by three segments (the VH, DH, and JH genes). The mechanism of recombination has been reviewed extensively elsewhere (16, 17). The overall effect of this process is to provide each B cell with a large potential repertoire of variable domain sequences. This large repertoire arises partly because there is a relatively large

SEQUENCES OF HUMAN ANTI-DNA ANTIBODIES

143

VH

VH

VL

VL CHI

CHI

CL

Fig 1, Schematic diagram of the basic structure of an immunoglobulin molecule. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain also contains a single variable domain (VH} but the heavy chain constant region consists of three constant do-

Flexible hinge region s--TT'-

CH2

CH2

CH3

CH3

mains (OH1, CH2, and CH3) and a flexible hinge region. Within VH and VL variability of sequence is highest within the complementarity determining regions (CDRs), whereas the intervening framework regions (FRs) are more similar in sequence in different antibody molecules. S-S, disulphide bond between chains.

In each V region CDRs and FRs are distinguished as shown below.

CDR

CDR FR

number of VH, DH, JH, VL, and JL gene segments from which those expressed can be chosen. In addition, the junctions between the segments are somewhat flexible. Nucleotides can be lost from one or both genes at a junction, or new nucleotides can be added by the enzyme terminal deoxynucleotidyltransferase. Finally, B cell variable (but not constant) regions are subject to a localized hypermutation mechanism (18) that increases the repertoire of possible sequences still further. It has been argued that somatic mutations (ie, mutations that develop only in a particular clone of cells and are not passed on through the germline) are particularly important in the development of

FR

CDR FR

IgG antibodies that bind to antigens with high affinity. When a number of mAb to the same antigen are derived from a single mouse, the IgG mAb almost invariably carry more mutations than the IgM mAb (19, 20). Furthermore, mutations in these high-affinity antibodies tend not to be distributed randomly. Replacement mutations, which alter the amino acid sequence of the antibody, are concentrated in the CDRs. Silent mutations, which do not alter the amino acid sequence, are not concentrated in this way. The explanation is that, in the presence of antigen, those antibody-secreting clones that develop mutations enhancing the affinity of antibody for antigen are stimulated to divide

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faster than other clones (19) and therefore undergo selective expansion. Clearly, the mutations most likely to alter binding affinity in this way will be replacement mutations in the CDRs. Shlomchik et al (21) proposed that the finding of a high ratio of replacement to silent (R:S) mutations in the CDRs but not the FRs of the sequence of a mAb should be taken as evidence that this sequence arose as the result of such an antigen-driven process. The aims of studying anti-DNA mAb sequences are to deduce the extent to which particular variable region gene segments are used preferentially to encode antibodies of this specificity and the extent to which high-affinity binding is dependent on the presence of somatic mutations in these genes. However, these deductions can only be accurate if it is possible to be sure which gene segments have been used to encode each antibody. This has not been possible for human mAb until very recently, because the complete repertoire of human VH, V~, and Vx genes was not known until the publication of comprehensive maps of these loci during the last few years (22-25). THE REPERTOIRE OF HUMAN VH, VK AND Vx GENE SEGMENTS

Functional VH, V~, and Vx genes are found on human chromosomes 14, 2, and 22, respectively. Although some VH and VK genes are found on other chromosomes, there is no evidence that these outliers can be expressed in the form of antibodies. In all three of these loci, there are a large number of pseudogenes (genes that cannot give a functional product), as well as functional genes. The genes can be classified into families on the basis of sequence homology. Two VH genes belong to the same family if they share the same nucleotide sequence at more than 80% of positions. Similar rules can be applied to the classification of VK and Vx genes. There are seven VH, seven V,, and 10 Vx families. The families are not equal in size. For example, the vast majority of VH genes belong to just three families: VH 1, 3, and 4. Similarly, four V~ families (V~ I-IV) and three Vx families (Vx 1-3) are larger than the others. Although it is possible that a few undiscovered VH, V~, or Vx genes exist in some haplotypes, almost all expressed sequences can be accounted for by derivation from genes which have been mapped (22-25). The potential repertoire of functional variable region gene segments available to a human anti-

body secreting cell comprises approximately 50 VH (22, 23), 40 VK (24), and 30 Vx (25) genes. However, not all of these genes are equally likely to be expressed. It has long been recognized that in the fetal immune system, a subset of the available VH genes are rearranged preferentially in the expression of antibodies (26). In adult B lymphocytes, studies using in situ hybridization (27), production of cDNA libraries (28), and single cell polymerase chain reaction (PCR) (29) have also shown that certain VH genes are more likely to be productively rearranged than the others. Brezinschek et al (29) estimated that a group of just 8 genes from three families (VH 1, 3, and 4) accounted for over 50% of all productive rearrangements in IgM secreting peripheral B cells. Similarly, Cox et al (30) have reported that just 11 of the 40 potentially functional V~ genes account for 90% of expressed V~ sequences. It is important to note that this preferential rearrangement of certain VH and VL gene segments is independent of antigen specificity. It appears to be a bias intrinsic to the rearrangement process, and it is important to allow for this when attempting to decide whether particular genes or families are used preferentially to encode anti-DNA antibodies. Because much of the evidence differentiating the potential from the expressed repertoire is so new, it has been difficult to make this allowance in previous reviews of the subject (31, 32). THE IMPORTANCE OF ALLELIC POLYMORPHISM Allelic polymorphism leads to small differences in sequence between alleles of the same gene in different haplotypes. This observation is important in the interpretation of sequences of human mAb because it can lead to uncertainty in determining the site and nature of somatic mutations. In many reports of human monoclonal anti-DNA antibodies, mAb cDNA sequences were compared with the most similar published sequences of germline VH, VK, or Vx genes rather than with the experimentally determined sequences of those genes in the individuals from whom the mAb were derived. Under these circumstances, it is difficult to be sure whether differences between the expressed and germline sequences are true somatic mutations or arise because the individual from whom the mAb was derived possesses a different allele from the individual whose germline sequence was published.

SEQUENCESOF HUMAN ANTI-DNAANTIBODIES

This difficulty is reduced where the germline gene in question is known to display little or no polymorphism (ie, it is likely to have practically the same sequence in everyone). For most human VH, VK, and Vx genes, polymorphism does indeed appear to be very limited. Review of all published alleles of VH genes (33) shows that the majority of alleles differ by only 1 to 2 nucleotides. It seems reasonable to assume, therefore, that where the VH sequence of a mAb differs at many positions from all published alleles of the most similar germline gene, these differences result from mutation rather than polymorphism. Similar conclusions can be reached from review of published alleles of human VK (33) and Vx (25) genes. It is important, however, in interpreting mAb sequences, to compare the mAb cDNA sequence with the most similar allele of the most similar gene. Although it would be premature to conclude that all alleles of all human VH, VK, and Vx genes are now known, it does seem likely that the vast majority- of them have been recognized. This allows much more certainty in the alignment of mAb sequences to the germline genes from which they are most likely to have been derived than has previously been possible. Therefore, it is timely to undertake a systematic review of the sequences of monoclonal human anti-DNA antibodies in comparison with these germline genes. Similar reviews of the more numerous published sequences of murine monoclonal anti-DNA antibodies have allowed the derivation of some general principles, as described in the next section.

EVIDENCE FROM STUDIES OF MURINE MONOCLONAL ANTI-DNA ANTIBODIES

Monoclonal anti-DNA antibodies from a number of different mouse models of SLE have been produced and sequenced (20, 34-36). Review of these sequences enables a number of general conclusions to be drawn (37). Certain mouse VH and VK genes and families appear to be used preferentially to encode antiDNA antibodies. The strongest evidence for this is the finding that the same genes are often used to encode anti-DNA antibodies derived from different models in different laboratories. Radic mad Weigert (37) found that members of a group of 13 murine VH genes were used very frequently, encoding the

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heavy chains of almost one third of over 300 anti-DNA mAb reviewed. High-affinity binding and specificity for dsDNA rather than ssDNA are commonly associated with IgG isotype (as in human SLE) and with the accumulation of replacement mutations in the CDRs (20, 37). This is particularly evident in the heavy chains. The result of such mutations is often an increase in the number of basic residues in the CDRs including arginine (R), asparagine (N), and lysine (K). The role of R residues has been emphasized, particularly in CDR3 of the heavy chain, where they may arise from junctional diversity and use of unusual reading frames as well as by somatic mutation. It is believed that the presence of R, N, and K residues in the CDRs enhances the affinity of antibodies for the negatively charged DNA molecule both by charge interactions and by the formation of hydrogen bonds (37). SEQUENCES OF HUMAN MONOCLONAL ANTI-DNA ANTIBODIES

Earlier reviews (31, 32, 38) of the sequences of human monoclonal anti-DNA antibodies suggested that somatic mutations and basic residues were important in conferring high affinity to DNA, as in the mouse. However, no definite conclusion as to preferential use of genes or families could be made, and these reviews were hampered by alignment of mAb sequences to V region genes that were not, in fact, the closest germline counterparts, because of incomplete knowledge of the human V gene repertoire at the time of the original reports. Such alignments would tend to overestimate the number of somatic mutations. Since 1982, sequences of over 50 human mAb that bind ssDNA and/or dsDNA have been reported. These antibodies were produced by a number of different methods and their binding properties defined by different assays. This must be kept in mind when makdng comparisons between them. With this caveat, the sequence features of all these mAb are summarized in Tables 1, 2, and 3. In interpreting these sequences, it is important to ask four questions: 1. Is there preferential use of particuIar genes or families to encode anti-DNA mAb? In the current analysis, each sequence has been aligned to the closest allele of the most similar gene from the repertoire recognized today. Gene names in the table are those

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designated in the currently accepted maps of the human VH (22, 23), VK (24), and Vx (25) loci. 2. Are the antibodies somatically mutated? This can be estimated from the degree of homology to the germline gene. Lower homologies suggest more extensive mutation. For example, a homology figure of 92% implies that there are approximately 20 somatic mutations within the sequence. 3. Is there evidence for antigen-driven clonal expansion? Perhaps the best evidence for clonal expansion is the isolation of two or more clonally related mAb from a single person. Clonally related mAb can be recognized by the fact that they share the same VHCDR3 sequence including the same VD and D J junctions. Because of junctional diversity and flexibility in the use of D genes, variability of VHCDR3 is so great that the chance of the same sequence being produced in cells from different clones is vanishingly small. Because the frequency of hybridoma formation is low, the isolation of two or more clonally related mAb implies that the clone from which these mAb were derived must have many members. The clearest evidence that clonal expansion has been driven by antigen is the finding of high R:S ratios in CDRs but not FRs. This approach has limitations, because some antibody sequences show many replacement mutations in both CDRs and FRs. 4. Are concentrations of basic residues important? To some extent the definition of such a sequence feature is arbitrary. For the purpose of the tables, a high concentration of basic residues is taken to mean accumulation of three or more basic residues in VHCDR3 or of three to four such residues within a five amino acid stretch in any other CDR. These limits were chosen because, in germline genes, VHCDR2 and VLCDR1 are often 15 to 20 amino acids long and may well contain three basic residues, but rarely in such close proximity. HUMAN IgM MONOCLONAL ANTI-DNA ANTIBODIES

Shoenfeld et al (39) were the first group to produce human monoclonal anti-DNA antibodies.

All the mAb produced were of IgM isotype and were polyreactive (40). Most monoclonal antiDNA antibodies produced over the next 10 years were also polyreactive IgM. A wide variety of antigens other than DNA was reported to be bound by these mAb. In using polyreactivity as a distinguishing mark for this group of mAb, however, it is important to recognize that the antigen-binding assays performed in each case were not directly comparable. Some groups tested mAb against a variety of different antigens, whereas others merely reported binding to DNA and one other antigen. Different groups used different assay methods, so that specificities and affinities cannot be compared in any quantitative way. The sequence features of these polyreactive IgM antibodies are shown in Table 1. In many of the earlier reports, only the heavy chain was sequenced. The majority of the antibodies sequenced show homologies of 98% to 100% to germline VH and VL genes indicating very little somatic mutation. However, 5 of the 25 mAb in the table have high R:S ratios in the CDRs. This suggests that antigen-driven accumulation of mutations is not necessarily accompanied by the development of specificity for DNA and can occur in IgM antibodies. In the majority of these antibodies, there was no link between antigen-driven somatic mutation and accumulation of basic residues in the CDRs. In the Vn sequence of POP (45), somatic mutations actually remove K and N residues in FR3, whereas inAb47 (46), Cl19 (52), andA5 (53), the mutations neither increase nor decrease the number of basic residues in the sequence. However, in A431 (44), mutations created an extra N residue in VHCDR2. Accumulations of basic residues in CDRs occurred in six polyreactive IgM antibodies. These did not arise by somatic mutation. In WRI176 (49), RT 79 (50), and RT 84 (51), these basic residues were present in VHCDR3 and arose from a number of different D genes. In 21/28 (41), 8El0 (41), and B122 (52), they occurred in the unmutated CDR2 sequences of genes from the VH1 family. There are good reasons for considering these polyreactive mAb to be representatives of the class of natural autoantibodies (54) because, in many cases, they were produced from tissues of healthy people rather than those with SLE, and they typically bound a number of antigens with relatively low affinity. They may therefore not be

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SEQUENCES OF HUMAN ANTI-DNA ANTIBODIES

Table 1: Sequence Characteristics of Human Polyreactive IgM That Bind DNA

mAb

18/2 1/17 21/28 8E10 C6B2 Kim 4.6 A10 A431 L16 ML1 POP Ab47 BEG 2 B19.7 WRI176 RT79 RT72 RT84 9500 9604 C119 C471 B122 86204 A5

Homology

High R:S in CDR

Basic Residues in CDR

Ref

NP NP NP NP NP

NP NP NP NP NP

No No No No No

No No Yes Yes No

41 41 4I 41 42

lb NP NP NP NP L6 NP NP NP L16 A3/A19 L12 NP L8 3m L6 L2 L12 A27 3p

100% NP NP NP NP 99% NP NP NP 99% 99% 98% NP 98% 98% 100% 100% 98% 99.7% 97%

No No Yes No No Yes Yes No No No No No No No No Yes No No No Yes

No No No No No No No No No Yes Yes No Yes No No No No Yes No No

43 44 44 44 44 45 46 47 48 49 50 51 51 52 52 52 52 52 52 53

VH

VH

VL

VL

VL

Origin

Gone

Homology

Family

Gene

SLE PBL SLE PBL SLE PBL Leprosy Sickle cell spleen Healthy tonsil Healthy PBL Healthy PBL Fetal liver Fetal spleen CLL Healthy PBL Fetal liver SLE PBL SLE spleen SLE spleen SLE spleen SLE spleen SLE PBL SLE PBL SLE PBL SLE PBL Healthy PBL Healthy PBL Healthy PBL

3-23 3-23 1-03 1-03 4-61

100% 100% 100% 100% 97%

NP NP NP NP NP

3-30 6-01 6-01 6-01 6-01 3-23 7-4.1 4-61 3-23 3-33 4-34 4-b 4-34 3-33 3-21 3-23 3-64 1-18 3-23 3-23

100% 99% 98% 100% 100% 96% 98% 99% 100% 99.6% 100% 100% 100% 100% 96% 96% 99.6% 97% 97% 100%

Vxl NP NP NP NP VKIII NP NP NP VKIII VKII VKI NP VJ Vx3 VKIII VKIII VKI V,III Vx3

Abbreviations:SLE, systemic lupus erythematosus; PBL, peripheral blood lymphocytes; R:S, ratio of replacementto silent mutation~ mAb, monoclonal antibodies; CLL, chronic lymphocytic leukemia; NP, not published; CDR, complementarity determining regions.

representative of the specific high-affinity antidsDNA antibodies believed to be most important in the pathogenesis of SLE. Table 2 shows the characteristics of monoclonal human IgM believed to be more pathologically relevant, because they are specific for DNA and/or carry idiotypes characteristically found on antibodies deposited in tissues damaged by SLE. Manheimer-Lory et al (55) studied several anti-dsDNA antibodies that carried the 3I idiotype believed to be important in lupus nephritis (56). Four of these antibodies, III-3R, III-2R, II-1, and IC-4, were IgM. Similarly, Hirabayashi et al (57) studied mAb NE1 and NE13, which were clonally related, bound only ssDNA and dsDNA, and carried the NE1 idiotype. NE1 is also detectable on anti-DNA in kidneys damaged by lupus nephritis. Kim 11.4 (58), B8807, and B8815 (52) do not carry idiotypes

relevant to lupus, but are of interest because they are monospecific binders to DNA, showing no binding to several other antigens tested. These nine mAb do not carry more mutations in either VH or VL than the potyreactive mAb in Table 1. NE1, NEt3, B8815, and Kim 11.4 have accumulations of basic residues in VnCDR3, VHCDR2, and VLCDR2, respectively, but this is not a feature of the other five mAb. Berek and Milstein (19) suggested that the earliest phase of the antibody response to a particular antigen was dominated by clones whose sequence permitted binding to that antigen with minimal mutation. Only later would highly mutated IgG antibodies take over because of antigen-driven clonal expansion. Perhaps the nine specific antiDNA mAb in Table 2 represent this class of minimally mutated, nonantigen-driven IgM that

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RAHMAN, LATCHMAN, AND ISENBERG

Table 2: Sequence Characteristics of More Specific Human IgM Antibodies to DNA

mAb

Origin

VH Gene

111-2R 111-3R IC-4 11-1 NE1 NE13 Kim 11.4 B8807 B8815

SLE spleen SLE spleen SLE spleen SLE PBL SLE PBL SLE PBL Healthy tonsil RA PBL RA PBL

1-69 3-07 4-59 5-51 4-34 4-34 4-39 3-23 1-03

VH Homology

VL Family

VL Gene

96% 99% 97% 98% 100% 100% 98% 100% 100%

VKI VKI VKI VKIII VKI VKI V~I VKI VKIII

A20 O18 O18 L16 L5 L5 lc A30 L6

VL Homology 100% 99% 96% 98% 99.7% 100% 98% 99.6% 99.3%

High R:S in CDR

Basic Residues in CDR

Ref

No No No No No No Yes No No

No No No No Yes Yes Yes No Yes

55 55 55 55 57 57 58 52 52

Abbreviations: mAb, monoclonal antibodies; R:S, ratio of replacement to silent mutations; CDR, complementarity determinin regions; SLE, systemic lupus erythematosus; PBL, peripheral blood lymphocytes,

may nevertheless be important in mounting a specific challenge to a given antigen. The idea that minimally mutated IgM can have intrinsic properties that enhance binding to a particular antigen is supported by consideration of human IgM monoclonal antiphospholipid antibodies (APL). APL in mice and humans often contain basic amino acids in the CDRs, particularly those antibodies that show specificity for negatively charged phospholipids. Like the anti-DNA antibodies in Table 2, the five monospecific human IgM APL that have been reported contain very few somatic mutations (55). All of them, however, have concentrated areas of basic residues in the CDRs, usually in VHCDR3. VDJ rearrangements that create a highly positive VHCDR3 could, in some cases, produce an intrinsically high affinity for negatively charged phospholipids (PL) or DNA, even in the absence of isotype switching or further somatic mutation. Taking the IgM mAb in Tables 1 and 2 as a whole, it seems clear that there is no evidence for preferential use of VH or VL families. All VH families are represented with the exception of the small and rarely expressed VH2. Similarly, the four major VK and two of the three major Vx families all encode mAb in these tables. The largest families encode the most mAb. Thus VH 1, 3, and 4 collectively account for all but six of these IgM. In fact, VH6 is probably overrepresented because the four mAb A10, A431, L16, and ML1 (44) were derived from an experiment that set out specifically to find clones which rearranged this gene. V3-23 is the most commonly used VH gene, occurring in 8 of the 34 IgM anti-DNA antibodies

listed (24%). This figure is higher than the 13% of all productive VH rearrangements estimated to include this gene by Brezinschek et al (29). V3-23 may therefore be used preferentially in anti-DNA antibodies. About half (18 of 34) of the IgM are encoded by VH genes of the group of eight genes shown to encode 56% of productive IgM rearrangements (29). Thus, apart from V3-23, there is little to suggest that the pattern of gene usage in these IgM is different from that intrinsic to the recombination process. Almost all the VK genes used in these antibodies belong to the group of 11 estimated by Cox et al (30) to encode 90% of the expressed repertoire. The exceptions are A30 and L5. A30 is interesting because it has also been reported to encode cationic anti-DNA antibodies extracted from blood of patients with lupus nephritis (60). There is no evidence for preferential usage of anY JH or JL gene in either IgM or IgG anti-DNA antibodies. Use of DH genes is difficult to analyze because most of the mAb have complicated VHCDR3 regions to which more than one DH segment contributes. However, the DH gene DXP' 1 contributes the sequence YYGS to a number of IgM anti-DNA including 18/2, 1/17 and 21/28 (41), Kim 4.6 (43), and B122 (52). YYGS is also found in CDRs of some pathogenic mouse monoclonal anti-DNA antibodies (61). It has been suggested that this tyrosine-rich motif could contribute to the DNA-binding site, perhaps by stacking interactions between the planar tings of tyrosines and those of purine and pyrimidine bases (62).

SEQUENCES OF HUMAN ANTI-DNA ANTIBODIES

HUMAN IgG MONOCLONAL ANTI-DNA ANTIBODIES The sequence features of 18 IgG mAb are shown in Table 3. All these mAb are specific for dsDNA with the exceptions of 33.F12 (63), which also binds cardiolipin (CL), D5 (50), and RH-14 (11), which also bind ssDNA, 9702 (52), which binds ssDNA and platelets but not dsDNA and R149 (69), which binds ssDNA and CL but not dsDNA. There are no unmutated VH or VL among these 18 mAb. These IgG are much less homologous to their germline genes than the IgM previously discussed. The degree of mutation is greater in VH than VL in almost every case. In 13 of the mAb, high R:S ratios in the CDRs suggest antigen-driven clonal expansion, although no investigator has yet produced two clonally related IgG anti-DNA antibodies from the same individual. In three of the remaining five mAb, 33.C9 (63), SD6 (68), and D5 (50) R mutations are present in the CDRs, but because there are also R mutations in FRs and S mutations in CDRs, it is not possible to be certain that an antigen-driven process has occurred. Accumulations of basic residues also occur in CDRs of most of these mAb. They are not confined to VHCDR3 and often arise by mutation. WinNer et al (63) showed that among the six mAb 32.B9, 33.Hll, 33.F12, 33.C9, 35.21, and 19.E7, mutations to basic residues were common and occurred in all six CDRs. Ehrenstein et al (66) described the mAb B3, which has no basic residues in VHCDR3 but two successive R residues resulting from somatic mutation in VLCDR1. In contrast, an important role for VHCDR3 was suggested by consideration of antibodies encoded by the gene V4-34. This gene encodes all human cold agglutinins and heavy chains derived from V4-34 carry the idiotope 9G4, which is commonly present in patients with SLE (70). Comparison of the 9G4 positive IgG anti-DNA antibodies T14 (64) and D5 (50) with cold agglutinins shows that the main difference is the presence of basic residues in VHCDR3 of the anti-DNA antibodies but not the cold agglutinins. The VHCDR3 region of T14 is also rich in tyrosine residues, a feature that has also been noted in other IgG (68) and IgM (43) antiDNA antibodies. There is no strong indication that particular V~{ or VK families are used preferentially in these IgG antibodies. The 18 mAb use VH genes from the largest families VH 1, 3 and 4, and all four major V,

149

families are represented. Gene usage is similar in the IgG and IgM anti-DNA antibodies with the following exceptions. A single Vx gene, 2a2, encodes five of the six lambda chains shown to occur in IgG anti-DNA antibodies. Conversely, neither 2a2 nor any other Va2 family gene is used in any of the IgM anti-DNA antibodies reported thus far. This difference between IgG and IgM is striking, although it would be premature to draw any conclusions as to preferential usage of Vx 2 genes from such a small number of antibodies. The VH 3 family gene V3-11 also is not used in IgM anti-DNA, but encodes three IgG anti-DNA. In two of these three, 33.F12 (63) and H2F (55) V3-11 is practically unmutated. Perhaps V3-11 can encode anti-dsDNA but not anti-ssDNA. Whereas other antibody secreting clones might start as anti-ssDNA secretors and develop anti-dsDNA activity through sequential somatic mutation, clones that rearrange V3-11 might start as high affinity anti-dsDNA secretors. V3-11 differs from all other germline VH genes in that its CDR1 contains the sequence YYMS, which is very similar to the YYGS motif believed to be important in VHCDR3 of some other anti-DNA antibodies (41, 43, 62). HUMAN IgG ANTI-DNA Fab DERIVED FROM REPERTOIRE CLONING

A major limitation in this field is the difficulty in producing human monoclonal anti-DNA antibodies by the immortalization of B lymphocytes. Whereas splenocytes are generally used for the production of mouse mAb, large numbers of human splenocytes are rarely available. Many groups have used peripheral blood lymphocytes (PBL) instead, but the numbers of antibody secreting hybridomas produced are typically low. Even when splenocytes are used to produce large numbers of such clones, the vast majority of these secrete IgM antibodies (71). It has proved particularly difficult to produce human IgG monoclonal anti-DNA antibodies, although the reasons for this are not entirely clear (72). In view of this, it is important to develop new methods of producing such mAb. Silverman's group have reported the production of nine human IgG Fab fragments that bind to dsDNA. These were produced by a method that was not dependent on the immortalization of B cells (73, 74). This technique involved the in vittv expression of unselected VH and VL cDNA se-

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Table 3: Sequence Characteristics of Human IgG Anti-DNA Antibodies

mAb

Origin

VH Gene

32.B9 33.H 11 33.F12 33.C9 35.21 19.E7 T14 2A4 I-2a H2F D5 B3 KS3 SD6 9702 B8801 R149 RH-14

SLE PBL SLE PBL SLE PBL SLE PBL SLE PBL SLE PBL SLE PBL Myeloma SLE spleen SLE PBL SLE PBL SLE PBL SLE PBL Healthy PBL SLE PBL RA PBL SLE PBL SLE PBL

3-23 3-07 3-11 4-39 3-74 3-30 4-34 4-61 3-30 3-11 4-34 3-23 4-34 3-30 1-46 3-11 1-69 3-07

VH Homology

VL Family

VL Gene

VL Homology

High R:S in CDR

97% 95% 98% 94% 95% 99% 96% 92% 94% 99% 94% 94% 94% 97% 98% 96% 97% 96%

Vx8 Vx2 VKIII VKI NP VKIII VKIII VKI VKI V,IV VKIII Vx2 Vx2 Vx2 VJII VKI VKII Vx2

8a 2a2 A27 L12 NP L6 A27 02 L8 B3 A27 2a2 2a2 2a2 A27 A30 A3 2a2

98% 99.4% 99.7% 98% NP 99.7% 99% 94% 95% 99% 96% 93% 96% 96% 99% 97% 99.4% 99%

Yes Yes Yes No Yes No Yes Yes Yes No No Yes Yes No Yes Yes Yes Yes

Basic Residues in CDR Yes Yes Yes No Yes No Yes Yes No Yes Yes Yes Yes Yes No No Yes No

Ref 63 63 63 63 63 63 64 55, 65 55 55 50 66 67, 68 67, 68 52 52 69 11

Abbreviations: mAb, monoclonal antibodies; R:S, ratio of replacement to silent mutations; CDR, complementarity determininc

regions; SLE, systemic lupus erythematosus; PBL, peripheral blood lymphocytes; RA, rheumatoid arthritis; NP, not published.

quences derived from PBL of healthy people or patients with SLE. These sequences were cloned into a phagemid expression vector. The products of expression were Fab molecules that were covalently bound to the surface of the phage particle. This allowed particles carrying high-affinity antidsDNA Fab to be selected by binding to a surface that had been coated with DNA. This method permitted the screening of a large repertoire of human VH/VL combinations to find those that were most favorable for binding to DNA. This approach has been termed repertoire cloning. The Fab derived from repertoire cloning have not been included in Table 3 because they differ in one fundamental respect from the monoclonal IgG in that table. The mAb are derived by immortalization of single cells, which implies that in the donor, there must be at least one clone of cells that expresses exactly the same VH/VL combination seen in the mAb. In the Fab derived from repertoire cloning, however, there is no guarantee that the VH and VL sequences expressed by the phages coexist in any cells of the original donor. In other words, it is not certain that these combinations would ever be produced by the processes of recombination, mutation, and clonal expansion occurring in vivo. Evi-

dence that these Fab may represent clones occurring in the donor came from the finding that heavy chain and light chain idiotypes from a single phage-expressed Fab derived from a patient with SLE were also found to coexist on a proportion of anti-dsDNA antibodies in the blood of the same patient (74). Consideration of the VH and VL sequences of these phage-expressed Fab shows that genes from a number of different families are used. As in the monoclonal antibodies of both isotypes previously discussed, VH3 genes were used most commonly. Of the nine Fab reported, six used VH3 genes (three V3-23, three V3-30), one VH1 (V1-02), one VH5 (V5-51), and one VH6 (V6-01). Two Fab used the A27 gene from the VKIII family, three used the Vx 1 gene le, two used Vx3 genes (3I and 3r), one the Vx2 gene 2a2, and one the Vx7 gene 7a. Reference to Tables 1, 2 and 3 shows that most of these genes have previously been shown to encode monoclonal anti-DNA antibodies. Because the production of mAb is a method of sampling the output of the antibody secreting cells that develop in vivo, this suggests that the immune system is good at selecting from the available repertoire all those VH and

SEQUENCES OF HUMAN ANTI-DNA ANTIBODIES

VL regions that have the potential to form DNAbinding sites. Unlike IgG mAb, however, the IgG Fab derived from repertoire cloning did not show a correlation between extensive mutation and high affinity for dsDNA (73, 74). Homology to the germline genes varied between 91.8% and 99.6%, but the highest affinity binders tended to have the least mutations with no evidence for high R:S ratios in the CDRs (73). However, all the Fab that showed high affinity for dsDNA did have accumulations of basic residues in CDRs, particularly VHCDR3. FUTURE DIRECTIONS

Although the method used in this article to identify accumulations of basic residues shows a clear difference between IgG and IgM mAb, it is too crude to identify the exact positions of residues that may be involved in antibody-DNA interactions (as opposed to the regions in which these residues may be found). By comparing the sequences of much larger numbers of murine monoclonal antiDNA antibodies, Radic and Weigert identified positions at which R, N, and K were particularly likely to occur in such antibodies (37). This analysis suggested, for example, a tendency for antiDNA antibodies to possess an R residue at position 96 of the light chain and/or basic residues at positions 50, 53, and 54 of VLCDR2. The number of human monoclonal anti-DNA mAb for which sequence information is available is at present too small for a similar analysis to provide useful data, particularly because so many of the IgM antibodies described are practically unmutated. A further difficulty arises from the fact that the VH and VL sequences of these human mAb arise from a wide variety of different genes and families, so that direct comparisons between them may be difficult. Even where it has been possible to compare the sequences of antibodies derived from the same VH or VL gene, this has not yet led to the identification of particular positions at which basic residues are especially likely to result from somatic mutation in human anti-DNA antibodies. For example, the three IgG antibodies D5 (50), T14 (64), and KS3 (67, 68) all use the VH gene V4-34. The V H sequences of these antibodies contain 11, 7, and 11 replacement mutations, respectively when compared with V4-34. However, only two of these mutations are found in both D5 and T14, a different mutation is common to KS3 and T14, and KS3 and

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D5 have no replacements in common at all. None of the shared mutations leads to any change in the number of basic residues. The production of many more human monoclonal anti-DNA antibodies in the future may make it easier to locate potentially important positions within the sequence by the type of comparison previously described. An alternative approach would be to compare the three-dimensional structures of antibodies rather than their sequences. Although no human monoclonal anti-DNA antibodies have been crystallized, it is possible to use computer programs to predict the three-dimensional structure of an antibody from its V~ and VL sequences (75). Each CDR loop of an antibody can adopt one of a limited number of possible conformations (called canonical forms). The conformation adopted by a particular loop is usually predictable from the presence of certain key residues in its sequence. By modelling several anti-DNA antibodies, it may therefore be possible to discover whether they tend to use particular canonical forms or to have basic residues at particular positions in the CDR loops. This approach already has been applied to murine anti-DNA antibodies, suggesting that residues at the tips of VHCDR1 and VHCDR2 might interact with the major groove of the DNA double helix, whereas VHCDR3 could straddle one of the phosphate backbones of the molecule (37). Too few human monoclonal anti-DNA antibodies have been modelled to allow a similar analysis. However, Kalsi et al (76) have produced computer models of the IgM mAb WRI176 (49) and the IgG mAb B3 (66). B3 appears to bind dsDNA in a cleft. The interaction is stabilized by interactions with R residues derived from VLCDR1, VLCDR2, and VHCDR2. WRI176 does not have a cleft, but a tryptophan residue in VHCDR2 may participate in stacking interactions with nucleotides in DNA. CONCLUSIONS

Advances in knowledge of the extent and use of the human immunoglobulin variable region repertoire have made it important to reappraise sequence data from the human monoclonal anti-DNA antibodies reported over the last decade. This reappraisal provides some answers to the four questions posed earlier in this article. The importance of antigendriven clonal expansion in the production of highaffinity anti-DNA antibodies, particularly those of the IgG isotype, is confirmed. However, some

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highly mutated antibodies can be polyreactive and some unmutated antibodies can bind dsDNA specifically and with high affinity. The presence of basic residues in the CDRs is a common, important, but not invariable feature of anti-dsDNA antibodies. With the possible exceptions of V3-23, V3-11, and 2a2, there is currently no good evidence that any human variable region genes or families are used preferentially to encode anti-DNA antibodies. The next steps in analysis of these data are likely to involve comparison of the primary sequences

and the three-dimensional structures of larger numbers of human monoclonal anti-DNA antibodies to more precisely locate the positions at which somatic mutations and basic residues can be influential in binding to DNA. It is to be hoped that the analysis undertaken in this article will facilitate such studies in the future. ACKNOWLEDGEMENT Dr. Rahman is supported by Wellcome Trust Research Training Fellowship 040 366/Z/94/Z.

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