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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 21, 2002 as doi:10.1096/fj.01-0994fje. |
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2
* Departments of Neurology and
Immunology, Mayo Medical and Graduate Schools, Mayo Clinic, Rochester, Minnesota, USA
2Correspondence: Department of Immunology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. E-mail: pease.larry{at}mayo.edu
SPECIFIC AIM
This study was designed to provide direct evidence that an antibody present in human serum is capable of inducing remyelination in the central nervous system (CNS) of demyelinated animals. The selected serum was shown in a previous study to induce significant myelin repair after a single 0.5 mg bolus treatment of SJL/J mice chronically infected for 6–10 months with Theiler’s murine encephalomyelitis virus. To test the hypothesis that repair was induced by antibody, a monoclonal human IgM antibody derived from a predominant antibody found in the patient serum was generated by DNA-mediated gene transfer. The monoclonal antibody was compared to serum antibody fractions for its ability to bind myelinated structures in the CNS, induce calcium signals in glial cells in culture, and induce myelin repair in a mouse model of chronic-progressive demyelinating disease.
PRINCIPAL FINDINGS
1. Generation of a human monoclonal IgM antibody by DNA-mediated gene transfer
The DNA sequence of a predominant antibody found in the serum from a patient diagnosed with Waldenstrom’s macroglobulinemia was derived from protein and cDNA sequence analysis. An IgM expression vector was prepared that contained heavy chain, light chain, and dHfR genes under distinct promoters. The heavy chain gene was comprised of a coding sequence for an immunoglobulin-based leader peptide, an exon encoding the complete variable region domain, a truncated intron containing the complete heavy chain enhancer, and a genomic segment of DNA encoding the complete secreted form of the µ constant region. The heavy chain gene was expressed under control of a heavy chain promoter. The light chain gene consisted of sequences coding an artificial leader peptide, fused to a cDNA sequence encoding the complete 
light chain under control of a cmv promoter. The dHfr gene coding sequence was introduced under control of an SV40 promoter.
The assembled IgM expression vector was introduced by electroporation into the immunoglobulin-negative hybridoma cell line F3B6 (ATCC). The cells were selected with 0.2 µM methotrexate. Surviving colonies were selected sequentially with increasing amounts of methotrexate to a final concentration of 200 µM. Immunoglobulin production was monitored at the beginning and end of the selection process. In some cases immunoglobulin production was a substantially increased after selection with methotrexate. In the best case, a colony producing 1.5 µg/mL of antibody was increased to 140 µg/mL. A 5- to 10-fold increase was more typical.
2. Monoclonal rHIgM22 antibody binds oligodendrocytes in brain sections and in culture
The serum-derived antibody sHIgM22 has a characteristic staining pattern on rat cerebellar sections, binding primarily to the white matter but also reacting with Purkinje cells in the granular layer (Fig. 1
A). The recombinant monoclonal antibody rHIgM2B staining pattern was more restricted to the myelin tracts in the white matter (Fig. 1B
). The staining pattern of rHIgM2B overlaps substantially with that of MOG antibody (Fig. 1C, D
), a reagent with known specificity for oligodendrocytes. Oligodendrocytes differentiated in vitro were bound by both the serum derived IgM shIgM22antibodies and the recombinant monoclonal antibody rHIgM22BII (Fig. 1D
) Again, the staining pattern observed with rHIgM22BII and anti-MOG antibody is very similar (Fig. 1E
).
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3. Recombinant rHIgM22 antibody induces calcium signals in glial cells
Treatment of cultured oligodendrocytes with the sHIgM22 antibody preparation mobilized extracellular calcium into the cells (M. P. Soldan, unpublished observations). Two types of calcium influx patterns were observed: an immediate response by some cells and a delayed response by others. Based on morphological comparisons, oligodendrocytes showed a delayed response pattern whereas astroglial cells displayed an immediate response pattern. The recombinant monoclonal rHIgM22BII mobilized calcium influx when added to mixed glial cell cultures in a similar manner (Table 1)
. The induced influx was more of the delayed pattern (specific to oligodendrocytes) than observed with the original sHIgM22. Although both antibody preparations had comparable abilities to induce calcium flux in oligodendrocytes (delayed signal), the recombinant monoclonal antibody had significantly reduced ability (P=0.011) to mobilize calcium in astrocytes (immediate signal). This difference in target specificity in the calcium flux assay is consistent with the observed binding specificity of the rHIgM22BII antibody for myelin tracts (Fig. 1A, B
).
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4. Monoclonal rHIgM22 antibody induces myelin repair in demyelinated lesions
A key question is whether human antibody present in the patient serum is responsible for the observed induction of remyelination. To test this hypothesis, chronically demyelinated SJL mice were treated with a bolus i.p. injection of 0.5 mg of antibody preparation from patient serum (sHIgM2), 0.5 mg of recombinant antibody (rHIgM22BII), or PBS. Five weeks after treatment, the mice were perfused with fixative and their spinal cords were removed for analysis. Spinal cords from individual mice were systematically sampled by serial section. Approximately 10 equally spaced sections encompassing the entire cord were evaluated from each mouse. Individual sections were divided into four quadrants based on the prominent morphological symmetry. Partially intact quadrants whose morphology was disrupted during preparation of slides were systematically excluded. Slides from all animals were blinded before scoring. The number of quadrants containing demyelinated lesions and those containing extensively remyelinated lesions were scored. The amount of remyelination was calculated as the percent repaired lesions, where the total lesions were scored as the sum of the repaired and unrepaired lesions. After treatment of demyelinated mice with the recombinant monoclonal antibody rHIgM22II, the extent of remyelination was equivalent (59.7%) to levels found after treatment with the original sHIgM22 serum antibody preparation (58.1%). In contrast, animals treated with PBS showed spontaneous remyelination at the significantly lower level of 15.8% (P<0.001 vs. either antibody-treated group).
CONCLUSIONS AND SIGNIFICANCE
The key question addressed in this study is whether antibody mediates the regenerative properties characteristic of certain human sera. To answer this question, animals with a persisting infection with Theiler’s virus resulting in chronic-progressive demyelination were treated systemically with recombinant or serum-derived antibodies. Both treatments resulted in significant myelin repair in demyelinated lesions in the CNS compared with the lesions found in spinal cords of PBS-treated animals. This finding provides direct evidence that a single monoclonal antibody present in patient serum is capable of inducing remyelination in chronically disease spinal cords. The monoclonal antibody bound oligodendrocytes in slices of brain tissue and to individual oligodendrocytes in culture.
Our hypothesis is that the antibody binds cell surface structures on oligodendrocytes, promoting intracellular signaling that leads to enhancement of innate repair potential characteristic of these cells (Fig. 2
). The mechanisms underlying antibody-mediated signaling and enhanced remyelination still need delineation. It is not clear whether antibody promotes cell survival, induces differentiation events, or a combination of these possibilities. A direct relationship between the observed calcium flux and specific signaling pathways has not been established. However, the mobilization of calcium in response to antibody treatment strongly suggests that the rHIgM22 antibody directly induces signals by engaging cell surface molecules on oligodendrocytes.
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The ultimate goal of these experiments is to develop a human monoclonal antibody for therapeutic use. In the present study, we have identified a single antibody that can be expressed at high titer in vitro. We have shown previously that the antibody binds to rodent and human oligodendrocytes in vitro and induces calcium mobilization in rodent oligodendrocytes. Most important, the antibody induces remyelination in an animal model of demyelinating disease. We conclude that recombinant antibody rHIgM22BII is a good candidate for further development as a therapeutic tool for treatment of CNS injury involving demyelination.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0994fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.01-0994fje 
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