ANTIGENS, ANTIBODIES AND THEIR USES

Abstract: A purified Candidosis antigen, having a molecular weight of 45 to 55 kD, can be obtained and used in a vaccine. Antibodies, e.g. a human monoclonal antibody, can be obtained and used in therapy, e.g. together with an anti-fungal agent.

Description: AXON -1- ANTIGENS, ANTIBODIES AND THEIR USES FIELD OF THE INVENTION This invention relates to antigens, their isolation from subjects suffering from the associated malady, to antibodies against the antigens, and to their respective uses in therapy and diagnosis. BACKGROUND OF THE INVENTION Candida is a fungus which is pathogenic in humans who are i muno-suppressed. Systemic candidosis is a well-known complaint. It is associated with dissemination of Candida species throughout the blood stream and invading deep tissues. This occurs in patients with an underlying lympho-proliferative disorder, or after surgery. The diagnostic criteria are (1) cultural and histological evidence from a deep organ biopsy at necropsy or during life, and (2) positive blood cultures. EP-A-0141616 and O-A- (PCT/GB85/00476) disclose monoclonal antibodies against antigens of the genus Candida. A description of the various species within the genus is given in the latter, the relevant contents of which are incorporated herein by reference. SUMMARY OF THE INVENTION A first aspect of the present invention lies in candidosis antigens, for therapeutic or diagnostic use. A second aspect of the present invention lies in antibodies, more preferably human antibodies and most preferably human monoclonal antibodies, against a candidosis antigen, e.g. an antigen as defined above, for therapeutic or diagnostic use, and in such human monoclonal antibodies per se. DETAILED DESCRIPTION OF THE INVENTION An antigen of the invention may be obtained by purification of a blood sample obtained from a subject suffering from systemic candidosis, followed by purification. An antigen of the invention may be provided in suitable form for therapeutic or diagnostic use, e.g. in substantially pure form and/or as part of the kit containing one or more other reagents. The antigen is preferably provided for use in the form of a vaccine, e.g. for intravenous or percutaneous administration to a human subject. An antigen of the present invention may be characterised by a molecular weight o± from 45 to 55 kD, and more specifically about 47 kD. An antigen of the invention may be used to raise serum in an animal, e.g. in rabbits. The rabbit anti-serum may then be used to detect candidal antigens in human sera. An antigen of the invention may be of general use as a diagnostic reagent, as the basis of antigen tests for systemic candidosis. Antigens of the invention, refractionated if necessary, may be used in the invention .described in O-A-8600927. Antibodies of the invention may be provided for use in the form of a pharmaceutical composition. They may be used in the treatment of systemic candidosis. They may be given directly or coupled to certain drugs, e.g. anti-candidosis drugs such as amphotericin B or 5-fluorocytosine. The following Examples 1, 2, 3 and 5 illustrate the invention. The following abbreviations are used: PBS = phosphate-buffered saline; BSA = bovine serum albumin; BSA-TRIS = 3% BSA in TRIS-buffered saline, pH 7.4. Percentages are by weight, or by volume with respect to liquids. ("Millipore" , "Millex" , "Sepharose" and "Tween" may be registered Trade Marks) . Example 1 This Example illustrates how an antigen of the invention may be obtained from yeast. Yeasts (Candida albicans species) were grown overnight in Sabouraud's agar or in a defined minimal mediu supplemented with amino-acids. They were harvested in distilled water, and crushed using a press; a "pressate" was prepared by fragmentation in an "X press" (LKB, Bromnia, Sweden^ at a pressure of 200 mPa. Any suitable form of mechanical destruction may be used instead of a press, e.g. glass balls may be used. The pressate was frozen at -70 C overnight and then loaded with yeast. This product was then placed within plastic bags in a 70% methanol bath containing cardice, for 30 minutes. The plastic bags prevented methanol from penetrating into the pressate containing the yeast. The pressate was taken from the bath, the plastic bags were removed, and a pressure of 200 mPa was exerted. The process was repeated three times, to obtain 90% or more disruption of cells. The distintegrated cells were removed from the press and centrifuged at 38,000 g for 1.5 hours at 4 C. The supernatant was filtered through a 0.22 μm Millipore-membrane (Millex) , to ensure sterility. Example 2 This Example illustrates the preparation of an antigen from human serum. Serum was obtained from patients with systemic candidosis, and desalted. This desalted serum was used as an antigen preparation in an immunoblotting system. It was run on a SDS Polyacrylamide electrophoresis gel to separate it from the other components of serum. A Transblotting set was used to transfer it from the gel to nitrocellulose paper. It was detected by a modified enzyme-linked immunosorbent assay, using a rabbit anti-serum raised against a yeast "pressate" (see above) . Transfer was performed at room temperature, using a current of 350 mA for 1.5 hours, in a Bio-Rad Trans-Blot cell. Free protein sites were saturated by incubation in 3% BSA in buffered saline (0.9% NaCl - 10 mM TRIS, pH 7.4) at 4 C overnight. The nitrocellulose was then incubated at 25 C for 2 hours with the anti-serum raised against the yeast "pressate", diluted 1:50 in 3% BSA - 0.05% Tween 20 in buffered saline. After washing five times over 30 minutes in 0.9% saline - 0.05% Tween 20, it was incubated for 1 hour at 25 C with alkaline phosphatase-conjugated goat anti-rabbit IgG. The conjugate was diluted 1:1000, immediately before use in 3% BSA in buffered saline. After washing, the nitrocellulose was incubated for 15 minutes at 25 C in a freshly prepared and filtered mixture of equal volumes of naphthol ASMX phosphate and Fast Red TR salt. Example 3 This Example illustrates the further purification of an antigen obtained, as in Example 2, from human serum. Serum was obtained from a patient with candidosis, and was shown, by enzyme immunoassay against antigen prepared as in Examples 1 and 2, to contain antibody against antigens of Candida albicans, including the antigen of 45 to 47 kD described above. Immunoglobulin G (IgG) from the serum was purified by adsorption on to protein A (which has a specific affinity for IgG) and then elution. This yielded 110 mg IgG which was shown, by polyacrylamide gel electrophoresis, to be pure. The IgG was coupled to 20 ml Sepharose CL 6B using cyanogen bromide (a standard method) . The IgG-Sepharose complex was packed into a column and washed, firstly with 3 column volumes of glycine-HCl buffer, pH 2.8, and secondly with 5 column volumes of PBS. 2-3 ml desalted serum (as from Example 2) were passed through the column at a rate of 0.5 l/min, and the column was then washed with PBS until the optical densi ' ty of the filtrate, measured at 280 nm, was zero. The column was then washed through with glycine-HCl buffer, pH 2.8, to elute the Candida antigen specifically bound to the IgG-Sepharose. The eluate was collected in 5 vessels containing solid TRIS , to raise the pH to 8.0. The column was then washed again with PBS in readiness for the next sample of desalted serum. Each sample of eluted antigen was placed in a dialysis sac, and covered with solid sucrose to 10 concentrate the antigen. The antigen was then dialysed against distilled water. Example 4 This Example illustrates an assay for antigen obtained by any of the methods described in Examples 1, 2 15 and 3. A sodium dodecyl polyacrylamide gel was prepared by . a standard method. Aliquots of the antigen (0.025 ml) in distilled water were applied into sample slots of the gel, and a current of 50 mA at 150 volts -was applied 20 across the gel for 6 hours. During this time, the components of the sample migrated in the gel at a rate roughly proportional to their molecular size. The current was switched off. The gel was removed from its glass supporting plates and mounted, together 25 with a sheet of nitrocellulose paper, in a Bio-Rad Transblot cell. A current of 350 mA was applied for 1.5 hours, so that antigen was transferred from the gel to the paper. The paper was then removed, and soaked in BSA-TRIS at 4 C overnight, to saturate free binding sites 30 on the paper. _% • The nitrocellulose paper was then incubated with anti-serum prepared as in Example 3 (or from patients with, or convalescing from, candidosis) diluted 1:50 in BSA-TRIS with 0.05% Tween 20 for 2 hours at 25 C. The 35 paper was then washed five times in normal saline with 0.05% Tween 20, and then incubated at 25 C for 1 hour with a goat antibody against rabbit or human (as appropriate) immunoglobulin conjugated with alkaline phosphatase, diluted 1:1000 in BSA-TRIS. The paper was washed as before, and then incubated for 15 min at 25 C in a freshly prepared and filtered mixture of equal volumes of naphthol ASMX phosphate and Fast Red TR salt. The paper was then washed again as before, and dried in air, when characteristic bands of stained immune complexes were visible on the paper. Instead of anti-serum, monoclonal antibody may be used, prepared by the procedure described in either WO-A-8600927 or O-A-8600928 , the contents of which are incorporated herein by reference. The antibody may be diluted 1:7 in the buffered BSA. Example 5 The antigenic supernatant obtained by the procedure of Example 1 is used to raise hyperimmune serum in New ' Zealand white rabbits. The rabbit anti-serum is then used to detect candidal antigens in human sera as described in Example 4. The rabbits are immunised either by direct intravenous injection or else by combining antigen with Full Freund's adjuvant and immunising subcutaneously.

Claims: CLAIMS 1. A candidosis antigen, for therapeutic or diagnostic use. 2. An essentially pure candidosis antigen. 3. An antigen according to claim 1 or claim 2, which has a molecular weight of from 45 to 55 kD. 4. An antigen according to claim 1 or claim 2, which has a molecular weight of about 47 kD. 5. A human antibody against a candidosis antigen, for therapeutic or diagnostic use. 6. A human monoclonal antibody against a candidosis antigen. 7. A human monoclonal antibody against a candidosis antigen, for therapeutic or diagnostic use. 8. An antibody according to any of claims 5 to 7, in which the antigen is as defined in claim 3 or claim 4. 9. A vaccine which comprises an antigen according to any of claims 1 to 4. 10. A -pharmaceutical composition which comprises an antibody according to any of claims 5 to 8. 11. A composition according to claim 10, which additionally comprises an anti-candidosis drug. 12. A composition according to claim 11, in which the drug is amphotericin B or 5-fluorocytosine. 13. A method of treating a human or other animal subject prone to or suffering from candidosis, which comprises administering to the subject an anti-candidosis effective amount of an antigen according to any of claims 1 to 4, an antibody according to any of claims 5 to 8, a vaccine according to claim 9 or a composition according to any of claims 10 to 12.

Inventors: STARKIE SELBY JOHN, WILLIAMS GWYNFOR REES, STARKIE, SELBY, JOHN, WILLIAMS, GWYNFOR, REES
Applicants: AXON HEALTHCARE LTD, AXON HEALTHCARE LTD.
Application year: 1986
Main classification: A61K39
IPC codes: A61K38, A61K39, C07K1, C07K14, C07K16, G01N33