ANTIBODIES AGAINST SPECIFIC POSITIONS OF HLA CLASS II MOLECULES, A PROCESS AND A REAGENT FOR DETECTING GENE PRODUCTS FOR IDENTIFYING HUMANS HAVING A GREAT TENDENCY TO DEVELOP IDDM, AS WELL AS A PROCESS FOR PRODUCING SUCH A REAGENT

Abstract: A reagent for detecting gene products characteristic of humans tending to develop insulin-dependent diabetes mellitus (IDDM) consists of or contains antibodies directed specifically against one of the positions 13, 26, 45 or 57 in the HLA-DQw7 or HLA-DQw8 allele.

Description: Antibodies against specific positions of HLA class II mole¬ cules, a process and a reagent for detecting gene products for identifying humans having a great tendency to develop IDDM, as well as a process for producing such a reagent. The present invention concerns HLA antibodies against HLA class II molecules and a reagent for detecting gene products for identifying humans having a great tendency to develop IDDM, as well as a process for producing such a reagent. Insulin-dependent Diabetes mellitus, also called IDDM, is a disease which is often developed in an early age. IDDM is thus the most common chronic metabolic disease in children. The etiology has not been fully determined as yet, but it is assumed that the disease can be released by actuation of a still unknown virus or by other external effects. The pathogenesis comprises phenomena of an autoimmune nature, including the presence of insulitis islet cell autoantibodies or organospecific autoantibodies or diseases of an autoimmune nature, cf. Gepts, W. "Pathologic anatomy and the pancreas in juvenile diabetes mellitus". Diabetes 14:619-633 (1965). Foulis A. K. and J. A. Stewart. "The pancreas in recent-onset Type 1 (insulin-dependent) diabetes mellitus: insulin content of islets, insulitis and associated changes in exocrine acinar tissue". Diabetologia. 26:456-461 (1984). Humans having certain hereditary characterisitcs have a particularly great tendency to develop IDDM. Although the hereditary factor has not been fully explained, it has been found that 95% of IDDM patients have the tissue type antigenes HLA-DR 3 and/or 4; Platz. P., B. K. Jakobsen, M. Morling, L. P. Ryder, A. Svejgaard, M. Thomsen, M. Christy, H. Kromann, J. Benn, J. Nerup and A. Green, "A genetic analysis of insulin-dependent diabetes mellitus, Diabetologia 21, 108-115 (1982), and Wolf, E., K. M. Spencer and A. G. Cudworth "The genetic susceptibility to type I (insulin-dependent) diabetes. "Analysis of the HLA-DR association", Diabetologia 24, 224-230 (1983). HLA-DR 3/4 heterozygous humans thus have the greatest risk of developing IDDM. Though almost all IDDM patients have HLA-DR 3 and/or 4 antigenes, determination of these gene products is not suitable for identification of humans having a great tendency to develop IDDM, since the frequency of humans having such specific hereditary characteristics is about 60% of the entire population, and just a small fraction actually develops IDDM. Even in families with two or more children having IDDM, the risk of an HLA-DR identical sister or brother is only 15-30%, cf. the above-mentioned literature. It is known to use an HLA-DQ β-chain cDNA probe, Bδhme, J., B. Carlsson, J. Wallin, E. Mδller, B. Persson, P. A. Peterson and L. Rask. "Only one DQ β restriction fragment pattern of each DR specificity is associated with insulin- ependent diabetes", J. Immunol., 1986, p. 941- 947, for detection of gene structures which are characteristic of humans having a great tendency to develop IDDM. Such a probe can be used for tissue type determination of humans having a great tendency to develop IDDM since the probe hybridizes to HLA-DQ β-chain genes. Such a probe is produced from genomic DNA from lymphocytes by cleavage with BamHI restriction enzyme and isolation of a 3.7 kb fragment. It is also known that the HLA-DQ β-chain probe is non¬ specific. J. Clin. Invest., vol. 79, April 1987, p. 1144-1152 discloses an agent for detection of the gene structure in humans having a tendency to develop insulin-dependent diabetes mellitus (IDDM). This agent consists of a DNA sequence from the HLA-DQ β-chain gene with increased specificity and is therefore suitable as a probe in tisue type determinations for identifying humans having a great risk of developing IDDM. According to the present invention antibodies have been found with a particularly high specificity against positions in polypeptides expressed from HLA-DR or the HLA-DQ β-chain gene from humans having a great risk of developing IDDM, and such heretofore unknown antibodies are suitable for detection of the characteristic gene products. Thus, the invention concerns heretofore unknown HLA antibodies directed specifically against specific positions of HLA class 2 molecules. The positions involved are in particular 13, 26, 45 and 57 in the HLA- DQw7 or HLA-DQw8 allele. It has been found that the HLA-DQ β-locus is more closely associated with susceptibility to diabetes than HLA-DR. DR4 is associated with at least 2 different DQ alleles. DQw7 and DQw8, of which in particular the DQw8 allele is increased in frequency in insulin-dependent diabetes (6, 7). The precise specificity of an immuno reagent at amino acid level is known only in rare cases. One way of circumventing this is by generating antibodies against synthetic peptides which can represent sequences of a native protein (19). This method has been used for producing antibodies of predetermined specificity for a plurality of purposes, such as identification of gene products (20) and analysis of the orientation of plasma membrane proteins (21). Position-specific antibodies can react with the native protein from which the sequence has been obtained and can distinguish highly specifically between proteins which are only different in a single amino acid (22). Owing to the allelic polymorphism of the HLA antigenes, frequently based on multiple amino acid substitutions, it has previously been attempted to generate position specific antibodies for these molecules (23-31). Several works have focused on generation of peptide antibodies for HLA class I antigenes (23, 26, 28, 29, 31). Thus, it has been shown that a polymorphic determinant, divided from HLA-B17, subtype Bw57, Bw58 and HLA-A2, is present in the amino acid positions 56-69 of the first domaine (29). Rabbit antibodies raised against a peptide corresponding to these amino acids of the HLA- A2 protein reacted specifically with both HLA-A2 and HLA- B17 proteins (29). In another case, rabbit antisera were raised against a peptide representing position 63-84 of the hypervariable region of the HLA-B27.1 subtype (31). These antibodies were found to be HLA-B27.1 specific by means of compliment mediated cell kill analysis on lymphoblastoid cell lines. An antibody recognized the B27.1 subtype specifically and did not react with the other 3 B27 subtypes, one of these only different from HLA-B27.1 by a single amino acid substitution (31). In a third case, polyclonal antisera were produced for the N- terminal parts of HLA-DR and -DQ β-chains. These antisera were found to be locus specific and reacted with the native proteins both by immuno blotting technique and by cell surfaceimmuno fluorescence analysis (27) . The 2 HLA-DR4 associated DQ alleles, DQw7 and -8, can be distinguished at protein level, i.a. by the monoclonal antibody TA10, which is believed to recognize an epitope in the first domaine (34, 35). As mentioned, the two alleles differ by four amino acids in the first domaine, but it has not been shown conclusively which precise amino acids condition the TA10 specificity. It has recently been proposed through the use of an oligo nucleotide probe corresponding to the amino acids in position 24-30 in the first domaine of the HLA-DQw7 β- chain that the TA10 specificity is attached to these amino acids (35). The purpose of the present invention is to produce position-specific antibodies which can distinguish closely related HLA class II allelic gene products which only vary in the individual amino acids. It has thus been found possible to generate polyclonal peptide antisera which can with high affinity distinguish HLA-DQ β-chain alleles, different in one amino acid in a predetermined position. It has been found possible to produce considerable amounts of antisera by immunizing rabbits. An unlimited supply of antibody can be obtained by establishing monoclonal antibodies with the same characteristics as for the polyclonal ones. Thus, by the process of the invention it will be possible to obtain HLA typing reagents with a specificity which was possible in the past only by nucleotide sequencing of genes or cDNA. Such antibodies are useful for diagnostication of susceptibility to develop certain autoimmune diseases. It is remarkable that it is possible to raise specific polyclonal or monoclonal antibodies which can distinguish between HLA-DQw7 and -8 β-chain proteins on the basis on one amino acid substitution, e.g. aspartic acid and alanine in position 57. The amino acid sequence in the various DQw alleles appears from the following table: DQβ ALLELES βCHAIN AMINO ACID RESIDUES 40-70 40 50 60 70 DQw7 (7) FDSDVEVYRAVTPLGPPDAEYWNSQKEVLER DQw8 (13) DQw3.3 (8) DQwl.19 (8) DQwl.l (36) G Q-R-V G DQwl.2 (8) GDQwl.AZH (8) G Q-R-S G DQwl.12 (8) G Q-R-D DI DQw2 (18) GEF L—L-A DI DQ- (8) G RLD DI--E The invention will be illustrated below by means of some examples with reference to the drawing, in which figs. 1- 3 show immunoblot analysis on sera diluted 1:100. EXAMPLE 1 Peptides having 12-13 amino acid residues, representing the following sequences of the HLA-DQw7 and w8 β-chain: (47-59) and (49-60) were synthesized on an automatic solid face peptide synthesis machine. These peptides were coupled to a carrier protein, in this case keyhole limpet hemocyanine through an additional attached amino or carboxy cysteine amino acid. Rabbits were immunized according to a standard regime with synthetic peptides mixed in Freund's adjuvant. Lymphoblastoid cells, formed by Epstein-Barr virus transformation of peripheral blood lymphocytes, were used for characterizing the antibodies. The tissue types of these cells were established by means of standard typing techniques. The reactivity of the antibodies with HLA-DQ β-chains were studied by standard technique by means of electrophoresis and Western blotting. Results It is shown by immuno blotting analysis that 3 antisera produced against DQw8 peptides (HLA-DQw8 position 49-60) exhibit intense binding reaction to a 29 kD band from DQw8 cells (see fig. 1). This band was identical in size with a band recognized by monoclonal antibody directed against HLA-DR3DQ β-chains (see fig. 1). The 3 antisera only exhibited weak reaction to a 29 kD band from DQw7 cells (see fig. 2). It was found by dilution tests that the 3 HLA-DQw8 specific antibodies in a dilution 1:33 recognized the 29 dK band from DQw7 cells, while there was no reaction to the 29 kD band from DQw7 cells at greater dilutions (see fig. 2). Antisera against DQw8 clearly recognized the 29 kD band from DQwδ cells even at a dilution of 1:300. The selective binding of antisera against DQw8 was confirmed in an inhibition experiment with free peptides. Binding of DQw8 antisera to DQw8 cells was completely blocked after addition of free DQw8 peptide, but only partially after addition of a DQw7 peptide (see fig. 3). 2 antisera formed against DQw7 peptide (HLA-DQβw7 position 47-59 and 49-69, respectively) reacted primarily with DQw7 cells. These sera showed strong binding to a 29 kD band from DQw7 cells, but reacted only slightly or not at all with DQwδ cells. It was found in dilution experiments that a DQw7 specific serum did not react with DQw8 cells even at a dilution of 1:33, whereas the other DQw7 specific serum bound the 29 kD band from DQw8 cells with low intensity at this dilution. Both antisera recognized the 29 kD band from DQw7 cells even at a 1:300 dilution. The selective binding of these antibodies was confirmed by inhibition experiments with free peptides. Free DQw7 peptide completely blocked the binding of the DQw7 specific antibody to DQw7 cells, whereas DQw8 peptide only blocked the binding partially. EXAMPLE 2 Example 1 is repeated with the modification that peptides of another length with 5 amino acids from numbers 53-57 inclusive than in example 1 are used, but so that at least 5 connected amino acids are used, and that the amino acid in position 57 is incorporated in the sequence. EXAMPLE 3 Example 1 is repeated with the modification that peptides coupled to a carrier protein by glutaraldehyde coupling are used for immunization. EXAMPLE 4 Example 1 is repeated, but the resulting antibodies are analysed by cell surface fluorescence analysis, so-called FACS analysis. EXAMPLE 5 Example 1 is repeated, but the resulting antibodies are analysed by complement mediated cell kill analysis. EXAMPLE 6 Examples 1-5 are repeated, producing monoclonal antibodies with the same characteristics as described in example 1. Prior art (Kδhler, G. and Milstein, C, Nature, 1975, 256:495) is used for the production of monoclonal antibodies, such as immunization of a test animal, such as a mouse, a rat or a rabbit, with the synthetic peptide, isolation of spleen cells from the animal, fusing with myeloma cells and cultivation, incubation and isolation of produced monoclonal antibody. E.g. , a mouse is immunized with a synthetic peptide representing the β-chain of HLA-DQw8, amino acids (47-59) coupled to a carrier protein and mixed in Freund's adjuvant according to the following scheme: Day 1: Injection of 25 μg of peptide coupled to a carrier protein mixed with complete Freund's adjuvant. Day 14: Injection of 25 ng of peptide coupled to a carrier protein mixed with incomplete Freund's adjuvant. Day 21: Injection of 25 u.g of peptide coupled to a carrier protein mixed with incomplete Freund's adjuvant. Day 28: The spleen is removed, and a cell suspension of the organ is prepared. These cells are mixed with myeloma cells (e.g. of the type SP2/0) in the ratio of 5 spleen cells to 1 myeloma cell, and the cells are fused under the action of 50% polyethylene glycol for 2 min. The fused cells are distributed in small chambers (960) in culture medium, and after 7-14 days the medium is examined for presence of reactive antibodies. The cells are removed from chambers in which antibodies are present, said cells being cultivated through dilution cloning until homogeneity. These cells are now cultivated in larger volumes from which antibodies can be isolated, or the cells are injected into the abdominal cavity of mice to produce ascites with a high concentration of antibodies, from which this can be isolated. EXAMPLE 7 Example 6 is repeated, but antibodies are produced which can specifically recognize each of the two other amino acids described in HLA-DQ β-chains, position 57, namely serine and valine, respectively. EXAMPLE 8 Example 6 is repeated, but antibodies are produced which can specifically recognize each of the two amino acids by which HLA-DQw7 and -DQw8 β-chains differ from each other in position 45, namely glutamic acid and glycine, respectively. EXAMPLE 9 Example 6 is repeated, but antibodies are produced which can specifically recognize each of the two amino acids by which HLA-DQw7 and -DQwδ β-chains differ from each other in position 26, namely tyrosine and leucine, respectively. EXAMPLE 10 Example 6 is repeated, but antibodies are produced which can specifically recognize each of the two amino acids by which HLA-DQw7 and -DQw8 β-chains differ from each other in position 13, namely alanine and glycine, respectively. REFERENCE LIST 1Z 1. Lernmar , A. (1984) Diabetologia 28, 195-203. 2. Platz, P., Jakobsen, B.K., Morl ng, M., Rydec, L.P., Svejgaard, A., Thomsen, M. , Christy, M. , Kromann, H., Benn, J., Nerup, J. ~ Green, A. (1982) Diabetolooia 21, 108-115. 3. Owerbach, D., Lernmark, A., Platz, p., Ryder, L.P., Rask, L. , Peterson, P.A. £ Ludvigsson, J. (1983) Mature (Lond.) 303, 815-817. 4. Cohen-Haguenauer, 0., Robbins,, E., Massart, C, Busson, M. , Deschamps, I., Hors, J., Lalouel, J.M., Dausset, J. - Cohen, D. 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Claims: P a t e n t C l a i m s 1. An HLA antibody, c h a r a c t e r i z e d in that it is directed specifically against specific positions of HLA class 2 molecules. 2. An antibody according to claim 1, c h a r a c - t e r i z e d in that it is directed specifically aginst peptides which are synthetized from coding sequences of HLA-DR or HLA-DQ genes. 3. An antibody according to claim 1 or 2, c h a r a c - t e r i z e d in that it is polyclonal and directed specifically against position 57 in HLA-DQ molecules. 4. An antibody according to claim 1 or 2, c h a r a c ¬ t e r i z e d in that it is monoclonal and directed specifically against position 57 in HLA-DQ molecules. 5. A reagent for detecting gene products in humans having a great tendency to develop IDDM, c h a r a c ¬ t e r i z e d by containing an antibody according to claims 1-4. 6. A process for producing the reagent stated in claim 5, c h a r a c t e r i z e d in that a synthetic polypeptide fragment with an amino acid sequence identical with or corresponding to a region around position 57 in a polypeptide, expressed by the HLA-DQ β- chain gene in chromosome No. 6, is coupled to a carrier protein used for immunizing an animal in a manner known per se, following which the developed antibody is extracted and processed into a reagent. 7. A process for diagnosing humans having a great tendency to develop IDDM, c h a r a c t e r i z e d in that lymphocytes or other cells from a human in question is incubated with the reagent defined in claim 5, following which bound antibody is detected in a manner known per se.

Inventors: DYRBERG THOMAS, DYRBERG, THOMAS
Applicants: NOVONORDISK AS, NOVO-NORDISK A/S
Application year: 1989
Main classification: G01N33
IPC codes: G01N33