ANTIGENS AGAINST AUTOIMMUNE DISEASES

Abstract: An antigenic determinant against a species-related autoantibody, in purified form can be used in the treatment of autoimmune diseases. A hybrid cell comprising a heteromyeloma cell fused to a species-related somatic cell, which is capable of secreting complete cell products of the somatic cell can be used, inter alia, to produce the novel pure determinants.

Description: ANTIGENS AGAINST AUTOIMMUNE DISEASES Field of the Invention This invention relates to antigenic determinants for use in the prevention and treatment of autoimmune diseases, and also to hybrid cells which code for, express and secrete biological cell and membrane products, their production and use. Background of the Invention Autoantibodies which are produced by B lymphocytes are involved in the pathogenesis of a number of autoimmune diseases, such as thyroiditis or diabetes, which involve organs such as the thyroid, pancreatic islets or adrenal glands. Some autoantibodies bind cell membrane receptors -(or antigens) and trigger an immune response which leads to destruction of the cell or antigen. What triggers the autoimmune response is not known. Autoantibodies can also bind antigens, causing stimulation or blockage of biological processes. The circulating levels of autoantibodies in the body may be low, but such autoantibodies are highly potent and have to be neutralised to prevent damage to organs and cells. Current evidence suggests that Type 1 diabetes mellitus is a chronic autoimmune disease involving two types of islet cell antibodies: (i) islet cell cytoplasmic antibodies (ICCA) ; (ii) islet cell surface antibodies (ICSA) . The ICSA are believed to be central to the initial destruction of the beta cells, whereas ICCA is secondary to the damage. ICSA have been detected in 40-67% of recent-onset diabetics and are present in susceptible individuals long before clinical onset. As such, they may be used as an early predictor of diabetes and as a marker of the silent, ongoing beta cell damage in prediabetic individuals. Various approaches have been adopted to neutralise autoantibodies, including the use of immunosuppressants , e.g. cyclosporin, and of mouse monoclonal anti-idiotypic antibody neutralisation techniques, but neither has been very successful. Immunosuppressants are too non-specific and will attack all B and T cells, while the use of mouse monoclonal anti-idiotypic antibodies often results in the production of human anti-mouse antibodies in patients, considerably reducing the effectiveness of treatment. Desired biological products can be produced by growing bacterial or yeast cells whose DNA has been modified, by recombinant technology, to express the products. Current genetic transfer/fusion techniques provide cells which are capable of secreting desired polypeptides. However, a desired product containing an amino-acid sequence and an additional moiety such as a carbohydrate, lipid or glycolipid cannot be secreted. The additional moiety must be added to the amino-acid sequence of the product at a later stage in product synthesis. Examples of biological products having such additional moieties are glycoproteins, lipoproteins and glycolipids. Moieties such as lipids or carbohydrates are important in the synthesis of biological products; their presence may be crucial in determining the stability and the biological/immunological activity of the final product. It is known to produce immortal hybrid cells by transfection/fusion of oncogenes or myeloma lines with somatic cells, usually of the same species. The use of such hybrid cells to produce a desired product carries high risk that cancer products will also be produced. Contaminating cancer products could induce cancer growth in individuals that are treated with the product. An object behind the present invention is to produce biological products comprising amino-acid sequences combined with an additional moiety, preferably in the absence of oncogenic products. Summary of the Invention The present invention provides antigenic determinants, i.e. antigens or antigenic fragments, which are specific for autoantibodies and which can be used to neutralise circulating antibodies and to target the B lymphocytes producing the autoantibodies. The present invention provides also a hybrid cell line which is the product of cell fusion between a heteromyeloma cell and a species-related somatic cell. The hybrid cells are capable of secreting complete cell and membrane products of the somatic cell. The heteromyeloma cells have undergone membrane alteration so that they are species-related to the somatic cells, but have been derived from myeloma cells which are not species-related to .the somatic cells. Detailed Description of the Invention Antigenic determinants containing the antigenic sequences specific for autoantibody binding sites can be identified by standard gene-sequencing techniques. Once the amino-acid sequence is known, the peptide fragments for the antigen sequence may be synthetically produced. Synthetic production, e.g. by recombinant technology, may be the preferred route for antigenic determinants that are composed of amino-acids only. However, for antigenic determinants which are glycoproteins, lipoproteins or glycolipidε, synthesis is less easy. The preferred route to produce such antigenic determinants is from hybrid cell lines of the invention; they are described in mere detail below. Enzyme digestion or chemical treatment of the antigens can be carried out to produce individual antigenic fragments which are able to bind specifically to the autoantibodies, so causing a neutralising effect. An antigenic fragment inevitably has lower molecular weight than the corresponding antigen and may therefore be less immunogenic than whole antigens when administered to patients _in vivo. An antigenic determinant of the invention may have as its sole function the blocking of an autoantibody. The determinant may also have other functions, and any such other function which is undesirable can be blocked by using a complementary antigen binder. Antigenic determinants of the invention are essentially free of naturally-associated material. They may be purified by standard techniques. Examples of such techniques are affinity purification, HPLC and electrophoresis. A determinant of the invention may be incorporated into a composition which can be used to treat patients having autoimmune diseases. Long-term treatment of autoimmune diseases may involve not only the neutralisation but also the destruction of the B lymphocytes which secrete autoantibodies. Antigenic determinants of the invention can be tagged with radioactive tracers such as iodine or cytotoxic drugs which can then be used to target and destroy autoantibcdy-secretinσ B lymphocytes. Tagging may be of particular value in the prevention or prophylaxis of diabetes, where it is believed that antoantibodies can be detected 5 to 7 years before the patient becomes insulin-dependent; there may therefore be adequate time for individuals to be treated with neutralising agents such as antigenic determinants specific for the autoantibody, up to the stage when 50% (or more) of the pancreas has been destroyed. Antigenic determinants may also be used to screen biological samples from individuals before an autoimmune disease is manifested. For screening, the antigenic determinants may be incorporated into diagnostic kits. Hybrid cells of the present invention can be used to produce desired biological products that are expressed by the somatic cell. It is an advantage of the invention that, if the myeloma cells are of, say, animal origin, no human oncogenic products are secreted in addition to the desired product when the hybrid cell products are used in humans. It is preferred that the hybrid cells are the product of cell . fusion between human heteromyeloma cells and human somatic cells. Standard gene cloning or fusion technologies can be used to produce a human or other heteromyeloma cell line which is derived from, say, another animal myeloma cell line. Standard techniques can be used to fuse cells from the heteromyeloma cell line with cells from a line of selected human somatic cells such as thyroid cells, to produce a hybrid cell line. All the genes of the thyroid cell are incorporated into the hybrid and expressed, and cell products ' are secreted. A hybrid human heteromyeloma/ hyroid cell line has been deposited on 18th February 1988 at the European Collection of Animal Cell Cultures, Porton Down, England. The accession number is ECACC 88021801. The deposited hybrid cell line secretes human thyroid cell and membrane products such as TSH (thyroid-stimulating hormone) receptor, thyroglobulin and thyroid peroxidase enzyme. There is minimal secretion of human oncogenic products. Similar techniques to those outlined above can be used to produce hybrid cell lines derived from human somatic cells which are, for example, pituitary cells, pancreatic endocrine and exocrine cells, adrenal medulla and cortex cells, blood cells, liver cells, lung cells, brain ceils, bone marrow cells, gut cells, placental cells, ovary cells and testicular cells. The hybrid cells can be cultured to express and secrete products expressed by the somatic cell; provided that the hybrid cells retain the appropriate genes, the products will continue to be secreted. Insulin and other pancreatic hormones and enzymes can be produced from hybrid-containing pancreatic cells, while LH (luteinising hormone) , FSH (follicle-stimulating hormone) , GH (growth hormone) , prolactin and TSH can be " roduced from hybrid-containing pituitary cells . LH and FSH are recognised to enhance the success rate of in vitro fertilisation procedures. Other products of interest are erythropoietin and TPA (tissue plasminogen activator) . By the procedure of the following illustrative Example, islet cell membrane antigens (ICMA) have been purified from cell lines that express the antigen and have demonstrated binding of the ICMA with human islet cell autoantibodies (ICA) . The ICMA can be digested by a variety of enzymes or chemical treatment, and purified fragments tested for antigenic bonding with ICA. ("Guildhay" refers to Guildhay Antisera * Ltd. , 6 Riverside Business Centre, Walnut Tree Close, Guildford, Surrey, England) . Example An anti-anti-idiotypic mouse monoclonal antibody (Moyle et al, 1988 Diabetes 3_7_, 206; Moyle et al, 1988 Biochem. Soc. Trans., in press) was used for the purification of the ICMA. 5 mg purified monoclonal antibodies were coupled on to 1 g activated beads (Guildhay) . The column was washed 3 times with phosphate-buffered saline (PBS) , and a solubiliεed islet cell membrane preparation, passed through a 0.2 μm filter (Millipore) , was applied to the column. The column was then washed with PBS until there was no protein in the eluate. Bound ICMA was eluted at 4°C, and fractions containing ICMA were pooled and dialysed against PBS. The purified ICMA was characterised in the following ways : (a) Nitrocellulose blotting: Purified ICMA was spotted on to nitrocellulose membranes, incubated with ICSA-positive diabetic sera and anti-anti-idiotypic MAb. Staining was visualised by coomasie blue. (b) Enzyme-linked immunosorbent assay (ELISA) : Purified ICMA was coated on to sensitised microtitre • wells, incubated with ICSA-positive diabetic sera and anti-anti-idiotypic MAb. Binding was visualised by adding either anti-human-HRP or anti-Mouse-HRP conjugate (Tan et al, Diabetes 1988, 37:204) . (c) Displacement ELISA: The purified ICMA will displace binding of ICSA-positive diabetic sera in the ICSA ELISA kit available from Guildhay. (d) SDS-gel Electrophoresis: By classical SDS polyacrylamide gel electrophoresis, the ICMA appeared as a single band with a molecular weight of 60,000-65,000. (e) Nature: The ICMA is apparently a glycoprotein with sialic acid terminal groups. Neuraminidase treatment, however, did not affect the binding of the ICMA to ICSA-positive diabetic sera. Because it is believed that circulating ICSA initiates beta cell damage when bound to ICMA., it is possible to use complementary peptides or chemicals to bind the ICMA and thus prevent ICSA binding. In this way, the beta cells could effectively be prevented from damage. The design of these "protective" peptides or chemical compounds follows from knowledge of the amino-acid sequence of the antigenic site of ICMA.

Claims: CLAIMS 1. An antigenic determinant against a species-related autoantibody, in purified form. 2. A determinant according to claim 1, in which the species is human. 3. A determinant according to claim 1 or claim 2, which is a lipoprotein, glycoprotein or glycolipid. 4. A determinant according to any preceding claim, which is a pancreatic islet cell membrane antigen. 5. A determinant according to any preceding claim, which is labelled. 6. A determinant according to any preceding claim, which is bound to a cytotoxic drug. 7. A determinant according to any preceding claim, for use in the treatment of an autoimmune disease, in which the determinant is species-related to the autoantibodies which cause the autoimmune disease. 8. A vaccine composition comprising a determinant according to any of claims 1 to 7. 9. A method of treating an autoimmune disease in a subject, which comprises administering to the subject a determinant according to any of claims 1 to 7. 10. A hybrid cell comprising a heteromyeloma cell fused to a species-related somatic cell, which is capable of secreting complete cell products of the somatic cell. 11. A hybrid cell according to claim 10, in which the somatic cell is a pituitary cell. 12. A hybrid cell according to claim 10, in which the somatic cell is an adrenal medulla or cortex cell. 13. A hybrid cell according to claim 10, in which the somatic cell is a pancreatic cell. 14. A hybrid cell according to any of claims 10 to 13, in which the heteromyeloma cell and somatic cell are human cells. _ Q _ 15. A hybrid cell according to claim 14, in which the heteromyeloma cell contains genes from an animal cell. 16. A hybrid cell according to claim 10, having the deposit accession number ECACC 88021801. 17. A hybrid cell according to any of claims 10 to 16, which is capable of secreting a lipoprotein, glycoprotein or glycolipid. 18. A method of producing a biological cell and/or membrane product, which comprises culturing a hybrid cell according to any of claims 10 to 17. 19. A method according to claim 19, in which the biological product is a lipoprotein, a glycoprotein or a glycolipid. 20. A method according to claim 19 or claim 20, in which the biological product is a determinant according to any of claims 1 to 6, and which comprises the further step of purifying the determinant. 21. A method according to claim ' 20, which comprises the additional step of enzymatically digesting or chemically treating the antigenic determinant to produce antigenic fragments.

Inventors: TAN KIM SZE, TAN, KIM, SZE
Applicants: TAN KIM SZE, TAN, KIM, SZE
Application year: 1989
Main classification: C07G17
IPC codes: A61K39, A61K47, C07K14, C12N5, C12N15, C12R1