Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-07-29
2001-04-10
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007500, C435S070210, C436S548000, C530S388150
Reexamination Certificate
active
06214568
ABSTRACT:
The invention concerns human monoclonal antibodies which react with islet cells of the pancreas, their use as a standard in the determination of antibodies against an islet cell antigen, as well as a method for the determination of antibodies against an islet cell antigen of the pancreas.
One often finds antibodies in the sera of prediabetics or of newly diagnosed diabetics which react with the endocrine cells of the pancreas, namely the islet cells. These antibodies are usually denoted islet cell antibodies (ICA). The typical ICAs are of the IgG isotype (G. Botazzo et al., Lancet 2 (1974) 1279-1283). They react selectively with the endocrine cells of the pancreas but not with the exocrine cells, the excretory ducts or the connective tissue. In contrast the autoreactive antibodies of the IgM isotype which can also be detected do not show a preferential staining of the islet cells, but also react in the same manner with other tissues such as the pituitary gland, thyroid gland, T-lymphocytes and adrenal gland (E. Garzelli et al., J. Clin. Invest. 77, 1986, 1627-1631; S. Srikanta et al., Mol. Biol. Med. 3, 1986, 113-127). These are probably types of natural autoantibodies of low specificity and low affinity which are also present in the blood of healthy persons (Seigneurin et al., Blood 71 (1988), 581). In contrast the presence of ICAs of the IgG isotype in serum is a predictive marker for the so-called insulin-dependent diabetes (synonymous with juvenile diabetes or type I diabetes). These autoantibodies can be detected in the serum up to nine years before the onset of diabetes.
The production of human monoclonal antibodies which are reactive against islet cells has already been attempted several times. In this connection G. Eisenbarth et al. (Nature 300, 1982, 264-267) obtained a monoclonal antibody which, however, is of the IgM isotype. E. Garzelli et al. (J. Clin. Invest. 77, 1986, 1627-1631) obtained antibodies of the IgM isotype which in addition to reacting with pancreatic tissue also reacted with the thyroid gland, peripheral nerves, the oesophagus and T-lymphocytes. S. Spitalnik et al. (International Research Symposium “The Immunology of Diabetes”, American Diabetes Association Woods Hole, MA, Oct. 27-30, 1987, Abstr. 113) also obtained a polyreactive antibody of the IgM isotype. W. Richter et al. (Horm. Metabol. Res. 21, 1989, 686-688) succeeded in immortalizing the lymphocytes which produce IgG against islet cells but did not succeed in cloning these B-lymphocytes on a single cell basis. E. Houssaint et al. (Clin. exp. Immunol. 82 (1990), 44-51) obtained a human monoclonal IgG antibody which, however, only showed a positive reaction towards rat and hamster islet cell lines.
S. Srikanta and G. Eisenbarth (Mol. Biol. Med. 3 (1986), 113-127) described antigens of islet cells which were, however, not specific for islet cells. The antigens or epitopes of these antigens can also be detected in other endocrine tissues such as e.g. the pituitary gland, thyroid gland, parathyroid gland, thymus, adrenal gland and in melanocytes.
S. Baekkeskov et al. (Nature 347 (1990), 151-156) identified a further 64 kD islet cell antigen as glutamate decarboxylase (GAD). Apart from the islet cells of the pancreas, this antigen is also expressed at a high rate by the neurones of the CNS which secrete &ggr;-aminobutyric acid. Patients with a rare neurological disease, the so-called “stiff-man-syndrome”, develop autoantibodies against GAD. Almost all patients with the “stiff-man-syndrome” in addition have autoantibodies against islet cells of the pancreas so that the “stiffman-syndrome” is often accompanied by a type I diabetes mellitus. The functional relevance of GAD as an autoantigen in the “stiff-man-syndrome” as well as in type I diabetes mellitus was deduced from these observations.
It was therefore the object of the present invention to provide human monoclonal antibodies of the IgG isotype which react with an human islet cell antigen and to provide a method for the determination of antibodies against an islet cell antigen.
This object is achieved by human monoclonal antibodies of the IgG isotype against human pancreatic islet cells which can be obtained by immortalizing human lymphocytes of prediabetics or diabetics, treating the culture supernatant of the immortalized cells with a conjugate of antibodies against human Fc &ggr; and a label, subsequently treating with human immunoglobulin, incubating with immobilized human pancreatic islet cells or immobilized GAD, identifying an immortalized human cell culture which produces an antibody against pancreatic islet cells via determination of the label bound to the immobilized islet cells or to the immobilized GAD, isolating a human immortalized cell which produces this antibody, propagating this immortalized cell and isolating the monoclonal antibody produced by these cells.
It surprisingly turned out that the monoclonal antibodies which can be obtained according to the present invention react specifically with purified GAD. After incubation with different tissue sections, the antibodies according to the present invention react with islet cells of the pancreas and with the cerebellum but not with several other tissues such as e.g. from the adrenal gland, stomach, intestine, pituitary gland, cerebral cortex, lung, liver or thyroid gland.
In this connection it surprisingly turned out that binding of the antibodies according to the present invention to pancreatic sections can be weakened or completely prevented by preincubation with a diabetic serum. This shows that the antibodies according to the present invention are not only found in the blood of the particular donor from whose blood they were obtained, but are found generally in diabetic sera and that these antibodies have a general relevance for type I diabetes mellitus.
The immortalization of human lymphocytes from prediabetics or diabetics is preferably carried out by transformation with Epstein-Barr virus (EBV). In addition other methods familiar to one skilled in the art (e.g. hybridoma technique) can also be used.
In order to detect immortalized cells which produce the desired antibody, a conjugate of antibodies against human Fc &ggr; and a label is used. In this case a conjugate of Fab fragments binding human Fc &ggr; and a label is preferred such as that which can be obtained according to the method of Ishikawa et al., J. Immunoassay 4 (1983), page 209.
Monovalent Fab or F(ab′) fragments as well as divalent F(ab′)
2
fragments can be used as the Fab fragments which bind Fc &ggr;.
It is usual to use an enzyme, a fluorescent or chemiluminescent dye or a radioactive isotope as the label.
The conjugate of antibodies against human Fc &ggr; and a label is first incubated with the culture supernatant of the immortalized cells, then treated with human immunoglobulin and finally incubated with immobilized human pancreatic islet cells or immobilized GAD. In this case pancreatic tissue sections are preferably used as the immobilized islet cells.
The immortalized cell line producing an antibody against pancreatic islet cells is then identified via determination of the label bound to the immobilized islet cells or to the immobilized GAD. Depending on the type of label, this determination is carried out by means of an appropriate enzymatic reaction or by measuring the fluorescence or chemiluminescence.
For the cloning, the cells whose culture supernatant shows a positive reaction are isolated. This is preferably carried out by means of a fluorescence-activated cell sorter. The cell clones isolated in this way are propagated and those cell clones which produce the antibodies against pancreatic islet cells are identified as described above. The antibody is then isolated from the culture supernatant of these cell clones according to methods familiar to one skilled in the art.
Those monoclonal antibodies which belong to the subclass IgG1 or IgG3 are preferred.
A preferred embodiment of the invention are monoclonal antibodies which are capable of binding to human pancreatic islet cells or
Albert Winfried
Brandt Michael
Eiermann Thomas
Endl Josef
Jungfer Herbert
Fulbright & Jaworski LLP
Roche Diagnostics GmbH
Wortman Donna C.
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