Sequences of monoclonal antiphospholipid antibodies: variations on an anti-DNA antibody theme

Sequences of Monoclonal Antiphosphol ipid Antibodies: Variations on an Anti-DNA Antibody Theme

Anisur Rahman, Sanjeev Menon, David S. Latchman, and David A. Isenberg

Antiphospholipid antibodies (aPL) are recognized increasingly as a probable cause of clinical features such as thrombosis and recurrent miscarriages, particularly in a subset of patients with systemic lupus erythematosus (SLE) and those with the antiphospholipid antibody syndrome (APS). A powerful method of studying the origin of these antibodies and delineating their binding sites is to sequence monoclonal aPL. The few reports of mouse aPL sequences suggest that gene families J558 and VK23 may be used preferentially but without extensive mutation of complementarity determining regions (CDR). Polyreac- tive human aPL, which bind DNA as well as phospholipids, generally use germline genes with few mutations. Specific immunoglobulin (Ig) M aPL also tend to use relatively unmutated genes but often have high concentrations of positive residues in CDR, which may enhance binding to anionic phospholipids. IgG aPL show many more antigen-selected mutations, particularly in heavy chain CDR. This difference between isotypes is similar to that seen in anti-DNA antibodies, but the role of positively charged residues in aPL is less evident, and additional motifs are likely to be important in antigen binding. Semin Arthritis Rheum 26:515-525. Copyright © 1996 by W.B. Saunders Com- pany

INDEX WORDS: Antiphospholipid; sequence; monoclonal.

I N THE PAST DECADE, there has been increasing interest in the occurrence and

clinical effects of antiphospholipid antibodies (aPL). They occur in 1.5% to 5% of the popula- tion, 1 but are found more frequently in some patients with infections or autoimmune diseases, particularly systemic lupus erythematosus (SLE). It has been estimated that aPL are present in 25% to 40% of SLE patients. 2 These antibodies bind primarily to anionic phospholipids, such as cardiolipin, and the term anticardiolipin antibody (aCL) is often used synonymously with aPL. In some cases the presence of aPL is associated with clinical features including recurrent arterial and venous thrombosis, cerebral infarcts, and recurrent miscarriages? ,4 These features are believed to arise from the effects of aPL on platelets and the blood-clotting mechanism. In patients with autoimmune diseases, but not infection, aPL may bind in association with a serum cofactor, [32-glycoprotein I ([32GPI), which is able to form complexes with negatively charged but not neutral phospholipids. This cofactor-dependent binding to anionic phospholipids correlates with the occurrence of thrombosis. 5,6 When aPL and these typical clinical lea-

tures are found in the absence of any other autoimmune disease, the patient is said to have the primary antiphospholipid antibody syndrome (PAPS). 4

Measurements of aPL levels in patients with these conditions show that particular clinical features of the syndrome are related more closely to the levels of immunoglobulin (Ig) G

From the Bloomsbury Rheumatology Unit~Division of Rheu- matology, Department of Medicine; and the Department of Molecular Pathology, University College, London.

Anisur Rahman, MA, MRCP: Research Fellow, Blooms- bury Rheumatology Unit~Division of Rheumatology, Depart- ment of Medicine; Sanjeev Menon, MA, MRCP: Research Fellow, Bloomsbury Rheumatology Unit~Division of Rheuma- tology, Department of Medicine; David S. Latchman, DSc: Professor of Molecular Pathology, Department of Molecular Pathology; David A. Isenberg, MD, FRCP: Professor of Rheumatology, Bloomsbury Rheumatology Unit~Division of Rheumatology, Department of Medicine.

Dr Rahman is supported by Wellcome Trust Research Fellowship no. 040 366/Z/94/Z.

Address reprint requests to Anisur Rahman, M_A, MRCP, Bloomsbury Rheumatology Unit, Arthur Stanley House, 40-50 Tottenham St, London W1P 9PG, UK,

Copyright © 1996 by W.B. Saunders Company" 0049-0172/96/2602-000555.00/0

Seminars in Arthritis and Rheumatism, Vo126, No 2 (October), 1996: pp 515-525 515

516 RAHMAN ET AL

aPL than IgM aPL. This has been shown for both venous thromboembolic disease 7 and fetal loss. 7,8 This distinction is reminiscent of that seen with anti-double-stranded DNA antibodies (anti-ds DNA) in SLE. Thus there is consid- erable evidence 9,1° to show that levels of IgG anti-ds DNA correlate more closely with clinical features of SLE, particularly nephritis, than do those of the IgM isotype. The IgG antibodies are generally more specific for ds DNA and bind with higher affinity, properties which may be relevant to their clinical consequences.

The similarities between anti-ds DNA antibodies and aPL are therefore clear. Both occur in SLE, both bind primarily to negatively charged antigens, and in each case there is a distinction in properties between IgG and IgM isotypes. These properties of anti-ds DNA antibodies have been investigated by studying the sequences of monoclonal antibodies derived from mouse models and from patients. Monoclonal aPL have been produced comparatively recently, and information on their sequences is now becoming available. In this review, we can thus begin to answer the question of whether similarities in aPL and anti-ds DNA arise from similarities in their genetic origin.

THE GENETIC ORIGIN OF ANTIGEN SPECIFICITY: GENE FAMILIES AND SOMATIC

MUTATION

The antigen-binding site of an Ig molecule is formed primarily by the complementarity determining regions (CDR) of its heavy and light chains. There are three CDRs in each variable domain of the molecule. These CDRs are encoded by linear stretches of highly variable nucleotide sequence within the Vn, V~, and Vx genes, with the exception of heavy chain CDR 3, which arises from the fused D and Jn gene segments. Not all CDR play an equal role in determining the antigen specificity of an antibody. For example, a number of authors have suggested that heavy chain CDR 3 is particularly crucial in the binding of ds DNA. 11a2

There are two main sources for differences between CDR sequences in different antibodies. Firstly, there is a large repertoire of variable region genes from which the antibody-producing cell can choose when expressing an antibody molecule. As increasing numbers of germline

VH genes have been sequenced, it has become clear that they can be divided into families on the basis of sequence similarity. This applies equally to V~ and Vx genes and is true in both humans and mice. The entire human VH locus has now been mapped and consists of approxi- mately 120 genes, which can be divided into seven families of varying size. 13qs The largest families are VH 1, 3, and 4. If selection of a VH gene for the production of an antibody of a particular specificity were purely random, one would expect to find such antibodies using Vu genes from each family. Although this is true for IgM anti-ds DNA derived from both healthy individuals and those with SLE, most of the IgG anti-ds DNA so far sequenced use genes from families VH3 and VH4. 9 This may have arisen by chance, simply because so few IgG monoclonals have been produced and sequenced, but it is also possible that genes from these families carry sequence motifs within their CDRs that give them an advantage in binding to DNA with high affinity. This idea is consistent with the fact that certain genes within these families (notably V3-23 and V4-34) are also preferred. Murine monoclonal IgG anti-ds DNA antibodies may also use certain VH and V~ genes preferentially, aa This raises the question of whether similar family and gene preferences would be seen in IgG aPE

The second source of variability in CDR sequence is somatic mutation of variable region genes in the antibody-producing cell. A replacement (R) mutation causes a change in the amino acid sequence, whereas a silent (S) mutation does not, because of the degeneracy of the genetic code. For a particular monoclonal antibody, the extent of somatic mutation can be assessed by comparing the sequence of variable- region complementary DNA (cDNA) with that of the closest germline gene. Where this has been done for human and murine anti-ds DNA antibodies, there appears to be a much higher degree of somatic mutation in IgG than IgM molecules, particularly in VH .9'16 Because germline human V genes of most families are known to exhibit a degree of allelic polymorphism, 17 it is difficult to be sure how many somatic mutations are present unless comparison is made with the sequence of the germline gene in the individual from whom the antibody was derived.

SEQUENCES OF ANTIPHOSPHOLIPID ANTIBODIES 517

Because germline sequences are not available for aI1 such individuals, a degree of uncertainty is introduced into the interpretation of the number and distribution of such mutations. Bearing this in mind, it still appears evident that R (but not S) mutations are not distributed randomly but tend to be concentrated in the CDRs, exactly where they might best contribute to the increased binding affinity of IgG antibodies. This is usually expressed as a high ratio of R to S mutations (R:S) in CDRs. This finding supports the idea that the development of somatic mutation in these antibodies has been antigen driven. In the presence of antigen, a clone of antibody-producing cells divides, and some of the daughters accumulate mutations in the CDRs. Those cells in which the mutations increase antigen-binding affinity are stimulated to divide faster and come to dominate the clone. Over time this leads to a preponderance of antibodies in which CDR are hypermutated compared with framework regions (FR). I8 The best evidence for the presence of such expanded clones; comes from the production of clonally related antigen-specific hybridomas from individual patients or mice. 18a9 Descent from the same ancestor clone is deduced from the presence in such antibodies of almost identical VH and VL sequences, particularly at the recombination points where the germline V, D, and J segments have been brought together. Some of these clonally related antibodies show very few mutations, indicating that where a germline sequence is sufficient to confer high antigen- binding affinity, clonal expansion does not de- pend on the accumulation of mutations. Thus, the presence of expanded clones secreting autoantibodies is persuasive evidence for an antigen- driven process, even where high R:S ratios in the CDRs do not occur.

As :noted, CDR 3 of the heavy chain is encoded by the fused D and Ju segments. Because there are a large number of D segments and they can be linked together or inverted, this CDR region shows particularly high variability. It therefore appears significant that sequences of a number of anti-DNA antibodies show a common feature in VH CDR 3. This is the presence of a number of positively charged amino acids, particularly arginine. 9a1,~2,2° It has been argued that these enhance binding

to negatively charged DNA. Other authors, however, have shown that positively charged residues can also accumulate in different CDR of the heavy or light chains, sometimes arising by somatic mutation.11,16,21 Because the phospholipids to which aPL bind are also negatively charged, it is reasonable to speculate that similar concentrations of positive charge might occur in their CDRs.

Thus, in considering the evidence on sequences of monoclonal aPL, it is important to consider the following questions:

1. Are particular genes and families used preferentially?

2. Are the antibodies somatically mutated? Is there a difference between IgG and IgM?

3. Is there evidence of an antigen-driven process?

4. Are concentrations of positive charge important?

MONOCLONAL MURINE APL

In contrast to the wealth of evidence from murine anti-DNA antibodies, there has been relatively little published on monoclonal murine aPL. This is partially because of the lack of a well-characterized mouse model of the syndrome. A new model of experimental antiphospholipid syndrome (APS) produced by immuniz- ing mice with human aPL has been described and may lead to the development of more mouse aPL for sequencing. 22

Some information, however, can be derived from consideration of existing mouse models, which are characterized by the production of autoantibodies, but do not have all the clinical features of APS. The MRL-lpr/lpr mouse is an established model of autoimmune disease. Ani- mals of this strain develop clinical features such as arthritis and nephritis, and their serum con- tains a variety of autoantibodies, including aPL. Kita et a123 have produced 14 monoclonal aCL, 13 of which were IgG, by fusing the splenocytes of MRL-lpr/lpr mice with mouse myeloma cells. Sequence analysis showed that different antibodies from a single mouse tended to be clonally related, as would be predicted from the clonal expansion model outlined in the previous section. 18 Allowing for this, the 14 antibodies could be shown to derive from nine different clones.

518 RAHMAN ET AL

Six of these clones used VH genes of the J558 family, but mutations in VI~ were almost all in FR. Although some of the antibodies had VH CDR 3 regions that were rich in arginine, there was no apparent relationship between arginine content and affinity of binding to cardiolipin. The apparent preference for the J558 family must not be overinterpreted, because almost 50% of all spontaneously activated B cells from MRL-lpr/lpr mice use VH genes from this family. 24 However, 43% of the aCL in this study used light chain genes of the VK23 family as opposed to less than 5% of anti-DNA antibodies and rheumatoid factors from this strain. The pathological relevance of these antibodies is uncertain, because the mice do not develop characteristic features of APS, and the antibodies were not specific phospholipid binders. Twelve of 14 also bound single-stranded DNA (ssDNA).

More recently, the same group have studied six monoclonal aCL derived from a different murine model, the (NZW x BXSB) F1 male mousey These mice do develop some features characteristic of human APS, such as thrombo- cytopenia and vascular disease. They produce serum aCL, some of which are of IgG isotype and show binding to cardiolipin, which is enhanced by [32GPI. These properties were used to distinguish two of six monoclonal aCL produced as "pathogenic." These two antibodies also induced thrombosis when injected into mice. Sequence analysis showed that both used J558 VH genes and one used a VK23 gene. The other four "nonpathogenic" antibodies were less likely to use these families. Because the number of antibodies studied was so small, it is impossible to draw any firm conclusion about family preference in murine aPE Although one of the pathogenic antibodies showed a number of differences in CDR 1 and 2 of the heavy chain, compared with the nearest known germline gene, the overall homology was poor enough to make it unclear whether the antibody does in fact derive from this gene. Hence the degree of somatic mutation is uncertain. As with MRL-lpr/ lpr-derived aCL, there was no relationship between the CDR arginine content and binding to cardiolipin. Indeed, neither pathogenic aCL had any positive residues in VH CDR 3.

MONOCLONAL HUMAN APL

The characteristics of the VH regions of these antibodies are shown in Table 1 and those of the V~ regions in Table 2. Antibodies considered in this section have been derived from a wide variety of subjects, some with clinical features of APS, some with SLE but without APS, and others who were healthy. This makes it difficult to make direct comparisons between these antibodies, particularly because the assays used to define binding characteristics were not the same in each report. Bearing these limitations in mind, monoclonal human aPL can be considered in three groups:

1. Polyreactive IgM antibodies 2. More specific IgM aPL 3. IgG antibodies

Polyreactive IgM aPL

Historically, it has proved easier to produce polyreactive monoclonal IgM antibodies than more specific ones, particularly those of IgG isotype. 9 One consequence of this was that a subset of the early IgM anti-DNA antibodies sequenced also showed significant binding to phospholipids. We can therefore consider these as low-affinity monoclonal aPL and reassess their sequences in comparison with specific aPL of more recent provenance.

In 1987, Hoch and Schwaber 26 produced a hybridoma C6B2 from splenocytes of a child with sickle cell disease. This clone secreted an IgMK antibody, which bound ssDNA, dsDNA, and cardiolipin. Only the heavy chain was sequenced, and no germline assignment could be made at the time. Comparison with recognized VH germline genes is difficult, because the sequence of C6B2 appears to contain two oppos- ing frameshifts near the beginning of CDR 1, causing four successive amino acid replacements. Excluding this region, the closest germline gene is V4-61 with 15 nucleotide differences, most of which are silent. There is no concentration of R mutations in the CDR and no accumulation of positive residues.

Dersimonian et a119 studied three IgM monoclonals produced previously in the same laboratory from peripheral blood lymphocytes (PBL) of a patient with SLE. 19 Two of these, 18-2 and 1-17, showed identical V, D, and J sequences and recombination points and were thus clonally

SEQUENCES OF ANTIPHOSPHOLIPID ANTIBODIES

T a b l e 1: VH C h a r a c t e r i s t i c s o f Human Monoclonal aPL

519

Positive Antigens Germline* High R:S Residues

Antibody Origin Isotype Bound Gene &Nt in CDR in CDR JH Reference

C6B2 Sickle cell M ssDNA 4-61 15 No No 3 26

splenocytes dsDNA

CL

18-2 SLE PBL M ssDNA, CL 3-23:~ 2 No No 5 19

platelets

1-17 SLE PBL M ssDNA, CL 3-23:[: 2 No No 5 19

platelets

Kim 4.6 Healthy child M ssDNA 3-30:1: 0 No No 6 27

tonsil lym- dsDNA

phocytes RNA, CL

A10 Healthy adult M ssDNA 6-1:1: 3 Yes No 3 30

PBL dsDNA, CL

A431 Healthy adult M ssDNA 6-1:1: 6 Yes No 4 30

PBL dsDNA, CL

L16 Fetal l iver M ssDNA 6-1:i: 0 No No 3 30

lymphocytes CL

C11£ SLE PBL M ssDNA 3-23:1: 11 Yes No 3 31

PL

platelets

C471 SLE PBL M ssDNA 3-64 1 No No 4 31

platelets

PL

B122 Healthy adult M ssDNA 1-18 8 No No 4 31 PBL platelets

PL

B6204 Healthy adult M ssDNA 3-23:1: 7 No No 4 31

PBL platelets

PL, RF

Kim 13.1 Healthy child M CL 1-69:i: 1 No Yes 5 32

tonsil lym-

phocytes

REN CLL cells M CL 4-61 11 No? Yes 3 33

ssDNA

BH 1 PAPS PBL M PL 3-33:~ 0 No Yes 6 35

STO 103 Healthy child M PL 4-61 0 No Yes 4 36 tonsil lyrn- piatelets

phocytes

RSP 4. SLE spleen M CL 3-30:i: 10 No Yes 4 37

R149 SLE PBL G1 CL 1-69:~ 10 Yes Yes 6 38 AH 2 SLE PBL G1 PL 5-51:~ 16 Yes No 6 39

Histones

DA 3 SLE PBL G3 PL 5-515 15 Yes No 6 39

UK 4 SLE PBL G1 PL 3-23:1: 21 Yes Yes 4 39

Abbreviations: aPL, antiphospholipid antibodies; CDR, complementarity determining region; SLE, systemic lupus erythematosus; PBL, peripheral blood lymphocytes; ss, single-stranded; ds, double-stranded; CL, cardiolipin; PL, phospholipid; CLL, chronic lymphocytic leukemia; RF, rheumatoid factor; PAPS, primary antiphospholipid syndrome. *Germline genes are named according to the new nomenclature of Matsuda et al. 13 Both the old and new names are given in the text, tAN = Number of nucleotide differences compared with the germline gene. :[:Denotes genes that are members of the fetally expressed repertoire.

520 RAHMAN ET AL

Table 2: VL Characteristics of Human Monoclonal aPL

Positive Germline High R:S Residues

Antibody Isotype V L Family Gene &N in CDR in CDR JL Reference

Kim 4.6 Lambda M Humlv 117 0 No Yes Jh3 27

C119 Kappa KIIla Vg 0 No No JK4 31

C471 Kappa KIIla kv328h5 0 No No JK2 31

B122 Kappa KI L12a 3 No No JK2 31

B6204 Kappa KIIla A27 1 No No JK1 31

Kim 13.1 Kappa KIIla Vg 0 No No J•4 32 REN Lambda X8 VX8 1 No No J)t3 33

BH 1 Lambda kill VMII.1 3 No No JXl 35

STO 103 Kappa K4 Humk 18 1 No Yes JK2 36 RSP 4 Lambda Xl IGLV1S2 5 No Yes JXl 37

R149 Kappa K2 A3 2 No No JK2 38

LJ 1 Lambda XlII IGLV3S2 6 No Yes? Jk2 39

AH 2 Lambda hi IGLV1S2 4 No Yes? Jk2 39

DA 3 Lambda Xl IGLV1S2 6 No Yes? JX2 39

UK 4 Lambda Xll DPL 11 15 Yes Yes JX2 39

NOTE. The origins and binding characteristics of all antibodies are shown in Table 1, with the exception of LJ 1, which was derived from systemic lupus erythematosus peripheral blood lymphocytes and binds phospholipid and histones. Abbreviations: aPL, antiphospholipid antibody; CDR, complementarity determining region.

related. Both bound cardiolipin as well as DNA and showed only two nucleotide differences from germline gene VH26 (now V3-23). Only one of these differences gave rise to an amino acid substitution. Siminovitch et a127 produced the polyreactive antibody Kim 4.6 from healthy tonsillar tissue. Both chains were sequenced and found to be identical to VH 1.9III (V3-30) and Vx humlv117, respectively. There were no somatic mutations, but a run of four successive positively charged residues was present in VL CDR 2.

Germline genes with minimal or no mutation may thus encode both heavy and light chains of polyreactive aPL. Not all germline genes are equally likely to be used by such antibodies. Members of the fetally expressed repertoire, in particular, seem to be overrepresented. This repertoire comprises the subset of V genes shown to be expressed in fetal liver cells. 28,29 It includes the genes V3-23 and V3-30 men- tioned. 13 Another member is V6-1, the sole gene in the VH6 family. In a study to investigate the properties of antibodies derived from this family, Logtenberg et al 3° screened a large number of Epstein-Barr virus (EBV)-trans- formed PBL and fetal liver cells with a probe to

detect production of VH6-derived RNA. 3° The PBL were derived from healthy adult volunteers without either SLE or APS. Of four positive clones, three produced IgM antibodies that bound both ssDNA and cardiolipin. One (L16) derived from fetal liver had the same sequence as germline V6-1, whereas A10 and A431 derived from adult PBL had three and six somatic mutations, respectively. The R:S ratios were higher in CDR than FR, suggesting an antigen- driven process.

In a more r e c e n t s tudy, 31 Rioux et al produced four polyreactive IgMK aPL from PBL, two (Cl19 and C471) from patients with SLE, and two (B122 and B6204) from healthy individuals. Each used a different VK gene with few somatic mutations. In contrast, three of four VH genes showed between 7 and 11 nucleotide differences from the closest germline sequence. Only in Cl19 was there a significantly high R:S ratio in CDR. However, this antibody, like the others, showed binding to a range of antigens. Thus, extensive mutation, even if apparently antigen driven, is not always associated with specific binding. Both Cl19 and B6204 used germline gene V3-23, also found in several other aPL (Table 1). Both had long stretches of


serine residues in CDR 2 of the heavy chain, but there was no accumulation of charged residues in any CDR.

Because the antibodies described in this section were not derived from patients known to have APS or serum aPL, their clinical relevance is questionable. Though information derived from their sequences is useful in determining the role of germline gene preference and mutation in conferring the ability to bind phospholipids, it tells us nothing about pathogenicity. Therefore, in recent years attempts have been made to study monoclonals, which can be considered more specific aPL, either because they are derived from patients with APS, because they bind solely to phospholipids, or because they exhibit [32GPI cofactor dependence.

More Specific Monoclonal IgM aPL

Again using tonsillar cells from a healthy donor,, Siminovitch et aP 2 produced and sequenced antibody Kim 13.1. In contrast to their earlier monoclonal Kim 4.6, this was specific for cardiolipin. Despite this more specific binding, there was no evidence of extensive mutation. V~j was encoded by the gene 51P1 (1-69) with a single nucleotide substitution, and VK by the gene Vg with no substitutions. Both germline genes are expressed in the fetus. The D region of this antibody could not be aligned to any published sequence but contained one asparagine and two arginine residues with positive charge.

Mariette et a133 studied a 58-year-old man with chronic lymphocytic leukemia (CLL). This patient: had high-titer serum aCL and clinical features including Raynaud's phenomenon and thrombophlebitis. An IgMk monoclonal REN was produced by fusing the CLL cells with a mouse myeloma line. Although this antibody bound ssDNA as well as cardiolipin, affinity for the latter was enhanced by [32GPI. The heavy chain was encoded by VH4 71-2 (V4-61). There were 11 nucleotide differences compared with the published germline sequence of this gene, with a particularly high concentration of 4R and no S mutations in CDR 2. One of these replacements creates an extra arginine residue. How- ever, the authors pointed out that 71-2 is a polymorphic gene and that each of the nucleo-

tide differences they detected had been seen previously in an allelic variant or closely related gene. They therefore concluded that the sequence of REN VR was derived by allelic polymorphism rather than somatic mutation. This interpretation is questionable because no other sequence had been reported to have all 11 of these differences in concert, and at least some of them might represent mutational "hotspots. ''34 The only way to resolve this would be to sequence the germline gene from the same patient. VH CDR 3 contained two arginines and one asparagine within a stretch of four amino acids close to the D-J junction. The light chain was encoded by the Vx8 gene with a single R mutation in CDR 1.

The antibody BH 1 was produced from PBL of a patient with PAPS. 35 BH 1 binds negatively charged phospholipids but not neutral ones or DNA. It does not bind phospholipid in the absence of serum. The V~j chain of this antibody may derive from a variant of 301969 (V3-33) and is identical to the germline gene in the same patient. 3019b9 is a member of the fetal repertoire. BH 1 Vx is homologous to germline gene VxIII. 1 with two amino acid replacements creat- ing new isoleucine residues in FR. There were four positively charged residues in VH CDR 2 and a run of three asparagines at the beginning of VH CDR 3. These may contribute to binding of negatively charged phospholipids. The clinical relevance of BH 1 in this patient is supported by the fact that idiotypes expressed on this monoclonal are also present in the patient's serum. However, the binding properties of the monoclonal are more like those of serum IgG than IgM, suggesting that it may not be a typical representative of serum aPL in this individual.

Denomme et aP 6 reported similar results from analysis of an IgMK antibody STO 103, which binds platelets and negatively charged phospholipids only. STO 103 uses the unmutated 71-2 (V4-61) gene and the Humk18 VK gene with a single silent mutation. CDR 1 of the light chain contained a run of four successive positively charged residues.

The IgMh antibody RSP 4 was produced from splenocytes of a patient with SLE who had serum antieardiolipin antibodies. 37 RSP 4 binds specifically to cardiolipin, and its VH and VL

522 RAHMAN ET AL

sequences show 97% and 98% homology to the germline genes V3-30 and IGLV1S2, respectively. There is no convincing evidence that the few R mutations seen are clustered in the CDRs, but there is a run of four successive positively charged residues in VLCDR2 and one of three such residues at the beginning of VHCDR3.

Thus, like IgM anti-DNA antibodies, IgM aPL are mainly characterized by germline VH and VL sequences with little mutation. How- ever, the more specific and better defined aPL differ from the polyreactive ones in their content of positively charged amino acids in the CDR. It is somewhat surprising that these antibodies do not bind DNA, showing that attraction of opposite charges alone cannot be responsible for their aPL activity. The precise three-dimensional arrangement of charge is likely to be important. The repeating double- helical structure of DNA could present a regu- lar array of negatively charged groups to the antigen-binding site of an antibody, whereas this is not true of phospholipids. The role of antigen in the development of these antibodies is intriguing, especially because two of them (REN and BH 1) may have pathological relevance. Perhaps germline CDR sequence in these molecules is so favorable to antigen binding that most mutations would tend to reduce affinity. Under these circumstances, the continuing presence of antigen would lead to selection of the unmutated sequence.

IgG Monoclonal aPt.

Van Es et aP 8 have produced an IgG1 antibody, R149, by EBV transformation of PBL from an SLE patient. This antibody binds both cardiolipin and ssDNA. Although the patient had serum aCL, she did not have any features of APS, so the clinical relevance of R149 is questionable despite its isotype. The VH region was encoded by 51P1 (1-69) with 10 nucleotide differences compared with the germline sequence in the same patient. The VL region was encoded by A3, with only two nucleotide changes. All of the R mutations were in CDR, and all S mutations were in FR. This pattern suggests strongly that the mutations have been selected by antigen. A striking feature was the

presence of five arginine residues in CDR 3 of the heavy chain. This is reminiscent of both IgG anti-ds DNA antibodies and the specific IgM aPL previously described.

More recently, Menon et aP 9 have produced four IgG aPL from PBL of three patients with SLE, all of whom had clinical features of APS. All four monoclonals bind negatively charged phospholipids but not neutral ones or DNA. None of the antibodies, however, showed 132GPI-dependent binding. Two of these aPL, AH 2 and DA 3, were derived from the same patient, and sequence analysis showed them to be clonally related. Their VH regions were encoded by VH251 (V5-51), a fetally expressed member of the small VH5 family. UK 4 VH was encoded by V3-23.

In each of these heavy chains there were many somatic mutations with high R:S in the CDR. Statistical analysis by the method of Chang and Casali 4° confirmed that this pattern was likely to have arisen by an antigen-driven process in each case. None of the CDR 3 regions contained any positive residues, but mutations resulted in production of a new arginine and asparagine in CDR 2 of UK 4 VH.

Amongst the light chains only UK 4 VL showed a high degree of mutation away from its germline counterpart DPL 11. Again, this resulted from selection by antigen. Two mutations in CDR 3 lead to adjacent arginine and asparagine residues. The other three lambda chains used Vx genes (IGLVIS2 and IGLV3S2) from two different families with few mutations. In each case, however, mutation in at least one CDR led to an increase in the number of positively charged amino acids.

UK 4 uses the same VH and Vx genes as B3, an IgG anti-DNA antibody produced in the same laboratory from PBL of a different SLE patient. Comparison of the sequences of these two antibodies with their germline genes (3-23 and DPL 11) showed that both differed from the germline sequences at a number of sites, but that the positions of these differences were not the same in UK 4 as in B3. Because the germline DNA of the patients involved was not sequenced, it is uncertain whether all of these differences are actually caused by mutation. If they are, one might postulate that these germ-


line genes can give rise to either anti-ds DNA antibodies or aPL, depending on the particular sets of mutations they accumulate. In the individual SLE patient, a similar mechanism might enable a single potentially autoreactive clone to give rise to descendants, which secrete autoanti- bodie,~ of widely different specificity.

The limited information available on sequences of human IgG aPL shows that, like IgG anti-DNA antibodies, they tend to carry more somatic mutations than their IgM counterparts, primarily in the heavy chains. If there is any VH family restriction, it is not the same as that in anti-DNA, because VH5-derived IgG aPL have already been demonstrated. There may, however, be a preference for genes of the fetal repertoire as seen in the more numerous monoclonal IgM aPE Although each of the IgG aPL sequences described has some accumulation of positive charge in CDR, this is not as clearcut as in marly anti-ds DNA antibodies and not generally biased toward VHCDR3.

FUTURE DIRECTIONS

Although a modest number of human monoclonal aPL have now been sequenced, all may be criticized concerning their relevance to the clinical syndrome. The potential pathogenicity of any particular antibody is difficult to quantify because current knowledge does not allow us to define essential properties for pathogenicity of aPL. The ideal antibodies to study would prob- ably be IgG monoclonals derived from patients with APS, which should show specific high- affinity binding to phospholipids enhanced by the recognized cofactor [32GPI. Idiotypes expressed on each such antibody should be present in the patient's serum, and this expression should vary with disease activity. Though a number of groups have approached this ideal, none has yet attained it, and production of such antibodies is an important goal for the future.

Comparison of the sequences of aPL and anti-DNA antibodies shows that gene prefer-

ence and antigen-driven somatic mutation are common features in the development of both. The accumulation of positively charged residues in CDR is seen frequently, but is clearly not sufficient for binding to either antigen. Thus, some aPL have highly positive CDR but do not bind DNA. The same is true for some anti-DNA antibodies, which do not bind phospholipids. This implies that other, less obvious, sequence motifs are likely to be important and that the different spatial arrangements of the positively charged residues in antibodies of different specificity must be considered. Understanding of such arrangements can be enhanced by using amino acid sequences to generate computer models of the three-dimensional structure of antibodies or by crystallizing the antibodies themselves. To shed light on these areas, we need to sequence and compare many more aPL. It clearly will be easier to derive large numbers of such antibodies from mice than from humans, and the development of new murine models either in inbred strains or by immuniz- ing healthy animals will be important. When human monoclonal aPL do become available, it will be important to gain a precise picture of the distribution of mutations within their variable regions. This will be aided by the continuing efforts to clone and sequence all the alletic forms of human germline V gene segments.

Sequence analysis will help to explain the processes involved in the development of pathogenic antibodies. We also may be able to build up a picture of the antigen-binding site of aPL. The more accurate this picture becomes, the better will be our chances of designing molecules to interfere with the antigen-antibody interaction and thus, possibly, to influence the course of this important clinical syndrome.

ACKNOWLEDGMENT

Dr Rahman is supported by Wellcome Trust Research Fellowship no. 040 366/Z/94/Z.

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Sequences of monoclonal antiphospholipid antibodies: variations on an anti-DNA antibody theme

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