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The sequences of over 300 murine monoclonal anti-DNA antibodies have been reported. These antibodies were derived from many different murine models of SLE of

which there are two main types. The first are known as autoimmune strains. These strains develop autoantibodies and clinical features similar to those o f SLE. Fi hybrids of New Zealand Black (NZB) mice and New Zealand white (NZW) mice ([NZB/NZW]Fi) spontaneously develop autoimmune disease closely paralleling the symptoms o f human SLE. At two to four months of age, these mice spontaneously develop a range of autoantibodies (including antibodies against nuclear proteins and DNA, glomerulonephritis caused by the deposition of immune complexes in the kidney and die by 18 months. Interestingly, as in human SLE, the incidence o f autoimmunity in the (NZB/NZW) Fi hybrids is much greater in females (Howie et a l, 1968).

In the strain MRL Ipr/lpr which is characterised by the homozygous presence of a mutation termed Ipr (for lymphoproliferation) the mice develop massive lymphoid organ enlargement, a range o f autoantibodies (including anti-dsDNA and anti-Sm) and accelerated severe lupus-like disease in early life (50% die from glomerulonephritis within six months) (Andrews et a l, 1978). The Ipr mutation is in fact a defective fas gene. The protein encoded by the fa s gene is a cell surface protein that interacts with its ligand to produce a signal that leads to apoptotic death of those cells carrying the fas- protein on their surface. Fas-induced cell death is involved in the clonal deletion of self­ reactive lymphocytes. Consequently a defect in the fas gene may lead to the persistence of autoreactive lymphocytes as found in SLE or lupus-like syndromes in mice.

The second main type o f lupus-prone murine models are those in which autoimmune dysfunctions similar to that of human SLE have been experimentally induced in normal mice through immunisation with proteins/DNA complexes or antibodies some of which carry public idiotypes associated with autoantibodies. For example, normal, healthy mice immunised with monoclonal (Mendlovic et a l, 1988) or polyclonal (Tincani et a l,

1993) antibodies that bear the public idiotype 16/6 have been seen to produce autoantibodies and develop glomerulonephritis. However this model is controversial, as it has been difficult to reproduce in other laboratories (Isenberg et a l, 1991).

However despite the different origins of these murine monoclonal anti-DNA antibodies, sequence analysis has led to the identification of recurrent characteristics amongst them (Radie et a l, 1994). Firstly, anti-DNA antibodies from various different models tend to use the same Vh and Vk genes. In an extensive review of information from the sequence analysis of over 300 monoclonal anti-DNA antibodies derived from various murine

Sequence Analysis of Monoclonal Anti-DNA Antibodies

models. Radie et al. (1994) found that 65% o f all the Vh sequences were derived from just one Vh family, J558. A further 22% were derived from a second Vh family (known as 7183) with the remainder (13%) derived from various other families. Use of V^ families was much less restricted. Although J558 is the largest murine Vh family, it is unlikely that this fully explains the apparent preferential usage of this family to encode anti-DNA antibodies since it was found that there is also preferential usage o f individual genes within the Vh families encoding anti-DNA antibodies. For example, ten J558 genes and three 7183 genes were found to encode a third of the published Vh sequences. Moreover just eleven Vk genes were found to encode just under a third o f published Vk sequences. Given the large number o f V genes in the mouse genome, it is unlikely that the preferential usage of certain genes to encode anti-DNA antibodies occurs by chance (Kofler et a l, 1992).

Secondly, sequence analysis of anti-DNA antibodies from various murine models has highlighted the importance of clonal expansion and antigen driven accumulation of somatic mutations in determining the ability o f these antibodies to bind dsDNA. Marion and colleagues analysed the sequences o f 117 monoclonal anti-DNA antibodies from eleven different (NZB x NZW)Fi mice (Marion et a l, 1992). In some cases, IgG produced by a single mouse were derived from the same expanded B cell clone whereas the majority of IgM were not. Clonally related IgM and IgG hybridomas were obtained from two individual mice. Within a single clone, more mutations were found in the IgG than the IgM, particularly in the CDRs. This increase in mutations was associated with increased ability to bind dsDNA (Marion et a l, 1992).

Similar results were seen when Shlomchik et a l (1987) produced four monoclonal anti- DNA antibodies from an MRL Ipr/lpr mouse. All four mAb (3H9, lA l, 2F2 and 4H8) were derived from the same expanded B cell clone, but were not identical due to the presence of somatic mutations. All four o f the antibodies had a high percentage of replacement mutations in their CDRs compared to the FRs, thus providing substantial evidence that these anti-DNA antibodies developed in these MRL Ipr/lpr mice due to antigen driven clonal expansion.

Thirdly, sequence analysis of these murine anti-DNA antibodies has led to the speculation that certain amino acids are present in the CDRs o f anti-DNA antibodies at a higher frequency than in antibodies with other antigen specificities (Radie et a l,

1994). In the 117 anti-DNA mAh analysed by Marion et al. (1992) it was found that as the immune response matured from IgM to IgG, more mutations accumulated leading to an increased ability to bind dsDNA rather than just ssDNA. The mutations that led to high avidity binding to dsDNA were those that increased the number o f arginine residues in the CDRs, especially VhCDRS.

Shlomchik et al. (1987) found that one of the mAb derived from the MRL Ipr/lpr, 3H9 only differs from the other clone members by several different somatic mutations and by its unique ability to bind dsDNA. Since clonally related B cells inherit the same V region genes, the binding difference must be due to the somatic mutations that occurred during clonal expansion. In particular one of these somatic mutations involved the replacement of a glycine with an arginine residue.

Radie et al. (1994) also noted that in many murine anti-dsDNA IgG antibodies, antigen- driven accumulation of somatic mutations has led to higher frequencies of certain amino acids, including arginine, asparagine and lysine being present at various positions in the CDRs. It has been suggested that the structures of these amino acids allow them to form electrostatic interactions and/or hydrogen bonds with the negatively charged DNA phosphodiester backbone. Arginine and lysine are positively charged and therefore could form charge interactions directly with this backbone. Asparagine is uncharged. However both asparagine and arginine have the ability to function as either a donor or recipient of hydrogen bonds with nucleotides within the helix (Radie et a l, 1994).

In conclusion, sequence analysis of murine monoclonal anti-DNA antibodies has shown that there is preferential use of Vh and Vk genes to encode these antibodies and that there is antigen-driven accumulation of somatic mutations in the CDRs leading to the prevalence of certain amino acid residues, in particular arginine, in the CDRs.