IE76312B1 - Neoglycoproteins the preparation and use thereof - Google Patents

Abstract

The synthesis and use of neoglycoproteins is described. Monosialolactose and disialolactose are isolated from cow colostrum and coupled using a coupling reagent to human serum albumin (HSA) as carrier protein. These neoglycoproteins are suitable as vaccines and as ligands for the localisation and therapy of lectin-expressing tumour tissues.

Description

The invention relates to the synthesis and use of neoglycoproteins. Monosialolactose and disialolactose are isolated from bovine colostrum and coupled by means of a coupling reagent to human serum albumin (HSA) as carrier protein. The neoglycoproteins are suitable as vaccines and, moreover, as ligands for the localization and therapy of lectin-expressing tumor tissues.

Gangliosides, i.e. glycosphingolipids which contain sialic acid, are plasma membrane lipids composed of a hydrophobic ceramide portion and a hydrophilic carbohydrate portion. Glycosphingolipids are anchored with the ceramide portion, which is composed of sphingosine and a long-chain fatty acid, in the outer plasma membrane in such a way that the oligosaccharide chains project into the extracellular space. Because of the fact that they form on the cell . surface cell-differentiating and species-specific patterns, it is thought that they have a biologically important function in cellular events.

Glycosphingolipids are typical of all mammalian cells but usually occur in only small amounts. Glycosphingolipids which contain sialic afcid, i.e. gangliosides, are particularly concentrated on neuronal plasma membranes. Gangliosides are also strongly expressed on the cell surfaces of tumors of neuroectodermal origin (such as melanoma, astrocytoma, neuroblastoma etc.). Some of these gangliosides are ones which occur on normal mammalian cells, but also in the brain and on nerve fibers, only in relatively small amounts. This is why the relevant gangliosides GM2, GM3, GD2 and GD3 have become interesting as tumor-associated antigens. The possibilities of a tumor immunotherapy of tumors of neuroectodermal origin with these gangliosides as target antigen are also being investigated by various research groups. Two different approaches have been followed to date with some success: 1. The use of of monoclonal antibodies directed against the appropriate gangliosides and - 2 2. active immunization with the appropriate gangliosides.

Investigations have shown that antibodies against purified GM2 ganglioside cannot be raised in melanoma patients. Not until adjuvants such as Bacillus CalmetteGuerin (BCG) or Salmonella minnesota were used, and the patients were pretreated with cyclophosphamide, were IgM antibodies induced against ganglioside GM2 (Livingston et al. (1987), Proc. Natl. Acad. Sci. 84, 2911-2915).

Moreover, a whole-cell vaccine composed of human or murine melanoma or astrocytoma cell lines having a high GM2 ganglioside content have been injected into melanoma patients and resulted in induction of anti-GM2 antibodies of the IgM isotope (Livingston et al. loc. cit.).

Studies to date on melanoma patients have shown that an elevated titer of antibodies against ganglioside GM2 correlates with an increased survival time.

The central problems in the design of a ganglioside vaccine are the poor immunogenicity of the purified gangliosides and the difficulty of isolating them.

Such difficulties also exist in general in the investigation of sugar-binding proteins. This is the reason, for example, for the development of neoglycoproteins containing a simple sugar such as, for example, galactose and a carrier protein such as, for example, bovine serum albumin (BSA) in order to analyze the binding of lectins better [J. Histochem. and Cytochem. (1984), 32 (10), pp. 1091-1098].

One approach to increasing the immunogenic ity of the ganglioside GM2 is to be found in U.S. Patent No. 4,557,931- This discloses a neoglycoprotein whose sugar portion derives from GM2 and whose protein portion is HSA. This oligosaccharide-HSA conjugate is used as vaccine for inducing antibodies which react with GM2, and as diagnostic agent.

It was also known that the sugar portions of gangliosides GM3 and GD3, monosialolactose and disialolactose respectively, can easily be obtained from bovine colostrum. We have found, surprisingly, that monosialolactose and disialolactose, coupled to HSA by means of a coupling reagent, react with monoclonal antibodies which have been raised against gangliosides GM3 and GD3 respectively. Conversely, the abovementioned neoglycoproteins are able to raise antibodies which react with gangliosides GM3 and GD3 and are therefore suitable as vaccines for specific tumor immunotherapy. Furthermore, they are also suitable, for example, as ligands for the localization and therapy of lectin-expressing tumor tissues.

Accordingly, the invention relates to a) neoglycoproteins composed of monosialo- or disialolactose, with carrier proteins, including enzymes, preferably human serum albumin, b) a process for the preparation thereof, the preferred starting material being bovine colostrum as source of sugar, c) and the use thereof as vaccines or as diagnostic aids, for example for detecting gangliosides in competitive radioimmunoassays as part of tumor diagnosis as well as therapeutic or diagnostic agents for lectin-expressing tumors.

The invention is further contained in the examples and the patent claims. •'4 Examples 1. Isolation of monosialolactose and disialolactose from bovine colostrum Bovine colostrum was worked up by the method of v. Nicolai, with minor modifications (v. Nicolai, Thesis, Bonn University, 1971).

Bovine colostrum (10 liters) was centrifuged to remove fats, and proteins were precipitated with 50 % acetone in the cold. After renewed centrifugation the supernatant was concentrated in a rotary evaporator to 1/10 of the original volume.

The crude fraction was passed through a Sephadex G-15 column to remove salts, and the sugar-positive and chloride-negative fractions were combined and concentrated. The individual sugars were separated by ion exchange on a DEAE-Sephadex A-25 column in 0.1 M Tris/HCl buffer, pH 7.5, using a sodium chloride gradient. The appropriate sugar fractions were combined, concentrated and passed through a Sephadex G-15 column to remove salts.

Sugars were detected in the individual fractions from the column working-up steps by the Ehrlich and anthrone tests or by fractionation by thin-layer chromatography on silica gel plates using the solvent mixture pyridine/ ethyl acetate/glacial acetic acid/water (6:3:1:3) and spraying the plate with resorcinol. 2. Coupling of monosialolactose and disialolactose to human serum albumin (HSA) by means of the coupling reagent SPDP There are several possibilities in principle for the coupling of the sugars to protein. The sugars can, for example, be coupled directly to the free epsilon-aminolysyl groups of the protein, or else via homo- or hetero5 bifunctional coupling reagents.

The coupling reactions using N-succinimidyl 3- (2-pyridyldithio)propionate (SPDP) as coupling reagent are described hereinafter. The reaction steps substantially correspond to the published method (J. Carlsson et al. (1978), Biochem. J. 173, 723-737). a) Reductive amination of monosialolactose and disialolactose The method of Wiegandt was used for the redutive amina10 tion at the Cx atom of the glucose in monosialolactose and disialolactose in methanolic ammonium acetate solution with the addition of sodium cyanoborohydride (H. Wiegandt, W. Ziegler, (1974) Hoppe-Seyler's Z.

Physiol. Chem. 355, 11-18).

The reductively aminated sugar derivative was purified on a Biogel P2 column.

Rx and R2 hereinafter are defined as follows: r1 = .GaiBl· NeuAc ' NeuAc eL NeuAc cC Abbreviations : Gal: NeuAc galactose N-acetylneuraminic acid CH->0H sodium cyanoborohydride Ri .CH-j-NH-R, OH L_7 b) Reaction of reductively aminated sugar with SPDP I The derivatization was carried out in 0.1 M sodium phosphate buffer + 0.1 M sodium chloride, pH 7.5, with a 3-5-fold molar excess of SPDP. The reaction time at room temperature was 2-12 hours.

The sugar-NH2-SPDP derivative was separated from N-hydroxysuccinimide and unreacted SPDP on a Biogel P2 column. If the derivative was contaminated with SPDP an additional purification step was carried out on a small silica gel column which was eluted with solvent mixtures of increasing polarity. c). Reaction of HSA with SPDP The protein derivatization was carried out under the same conditions as the sugar derivatization in 0.1 M sodium phosphate buffer, pH 7.5, with a 3-5-fold molar excess, based on free epsilon-aminolysyl groups, of SPDP.

Protein -NH2 II Protein-N-C-CH9-CH9 H The protein-SPDP derivative was separated off on a Sephadex G-25 column which was eluted with the buffer for the subsequent reactions (0.1 M sodium phosphate buffer, pH 6, 5 mM EDTA). d) Reduction of the HSA-SPDP derivative The disulfide bridges newly introduced by the derivatization in the HSA-SPDP derivative were reduced, with the elimination of 2-thiopyridone, in 0.1 M sodium phosphate buffer, pH 6, 5 mM EDTA with the addition of 25 mM dithiothreitol. The native disulfide bridges of the protein are not reduced under these reaction conditions. The reaction was carried out at room temperature, and the reaction time was 1-2 hours.

II Proiein-N~C~CH~CH~'-S~5 H 22 Dithiothreitol Protein-'M- CPU-SH H 2 2 The reduced HSA-SPDP derivative was separated off on a Sephadex G-25 column using 0.1 M sodium phosphate buffer, pH 6, 5 mM EDTA as eluting buffer. e) Coupling of the sugar and of the protein derivative Reduced HSA-SPDP derivative was immediately reacted with the sugar-NH2-SPDP derivative: the sugar-NH2-SPDP derivative was used in a 2-5-fold molar excess based on epsilon-aminolysyl groups in the protein, and the reaction time was 24-48 hours at room temperature.

Rj ΜΗ The coupling product or neoglycoprotein was separated from the other reaction products by column chromatography on Sephadex G-25.

In the procedure described, reductively aminated sugars 5 are coupled via SPDP to free epsilon-aminolysyl groups of the protein. An alternative possibility is to reduce the disulfide bridges of the protein and to couple the reductively aminated sugars via SPDP to the sulfhydryl groups, which are now free, of the cysteine residues. 3. Detection methods for the identification of the intermediate products The following detection methods were used by choice for the identification of the intermediate products: A) for monosialolactose and disialolactose and the corresponding derivatives a) a change, after reductive amination and SPDPderivafcization, in the migration behavior on thin-layer chromatography on silica gel G-60 plates in the following solvents: pyridine/ethyl acetate/glacial acetic acid/water (6:3:1:3) chloroform/methanol/O.2% aqueous calcium chlo5 ride (60:35:8) and stainability of the bands with iodine vapor, ninhydrin and resorcinol. b) Quantification of SPDP-derivatization by a slight modification of the determination of neuraminic acid with 1,2-diamino-4,5-dimethoxybenzene (S.

Hara et al. (1986) J. Chromatography 377, lll119) and by elimination of 2-thiopyridone with dithiothreitol. The amount of 2-thiopyridone liberated, which corresponds to the amount of SPDP-derivatized sugar, can be measured by measuring the increase in extinction at the wavelength of lambda = 343 nm in a photometer, and the concentration can be calculated using the Lambert-Beer law. The molar extinction coeffi20 cient for 2-thiopyridone at lambda = 343 nm is 7.06 χ 103 χ Μ*1 x cm-1 (D.R. Grassetti & J.F. Murray, (1967) Arch. Biochem. Biophys. 119, 41-49). c) GC Analysis of the individual sugar derivatives after reductive amination and SPDP-derivatization by the method of J. Lechner et al. (1985) J. Biol. Chem. 260, 860-866.

B) For HSA a) Protein determination was carried out with the -30 Bio-Rad protein assay. b) the SPDP-derivatization was quantified by protein determination with the Bio-Rad protein assay and by elimination of 2-thiopyridone with dithio11 threitol. The liberated 2-thiopyridone, which corresponds to the amount of bound SPDP, was measured by the increase in extinction at the wavelength lambda = 343 nm in a photometer, and the concentration was calculated using the Lambert-Beer law. The molar extinction coefficient for 2-thiopyridone at lambda = 343 nm is 7.06 x 103 x M1 x cm'1 (Grassetti & Murray, loc. cit.). c) HSA and HSA-SPDP derivatives show different migration behaviors on SDS gel electrophoresis, stained with Coomassie blue. 4. Synthesis of disialolactose-HSA conjugate In a characteristic experimental mixture, 10 mg (10.75 /xmol) of disialolactose in 2 ml of a methanolic ammonium acetate solution were reductively aminated with the addition of 20 mg of sodium cyanoborohydride. The reductively aminated sugar was purified on a Biogel P2 column and then, in 13 ml of 0.1 M sodium phosphate buffer, pH 7.5, reacted with 13 mg of SPDP dissolved in 1050 μΐ of ethanol, and the disialolactose-NH2-SPDP derivative was purified on a Biogel P2 column and a silica gel column. The yield, measured by determination of neuraminic acid with 1,2-diamino-4,5-dimethoxybenzene and by elimination of 2-thiopyridone with dithiothreitol, was about 50 % (4.8-5.2 μιηοΐ) of the amount of disialolactose used.

For the derivatization of HSA, 5.6 mg of SPDP were dissolved in 450 μΐ of ethanol and added to 6 mg of HSA in 5550 μΐ of 0.1 M sodium phosphate buffer, pH 7.5. Purification on a Sephadex G-25 column resulted in about 4.8 mg of HSA-SPDP derivative with a derivatization of 45-53 SPDPmolecules per HSA molecule.

The disulfide bridges newly introduced in the HSA-SPDP derivative were cleaved with 25 mM dithiothreitol in 0.1 M sodium phosphate buffer + 5 mM EDTA, pH 6. The reaction time was 1 hour at room temperature. After purification on a Sephadex G-25 column, the reduced HSA-SPDP derivative was immediately reacted with the disialolactose-NH2-SPDP derivative in 0.1 M sodium phosphate buffer + 5 mM EDTA, pH 6. It was possible to follow the coupling reaction in a photometer by an increase in the extinction at the wavelength lambda = 343 nm due to the elimination of 2-thiopyridone. There was no further increase in extinction after 24-48 hours, the coupling reaction was complete.

Purification of the coupling product on a Sephadex G-25 column resulted in 2.1-2.5 mg of disialolactose-HSA conjugate which was derivatized with 30-33 disialolactose molecules per HSA molecule.

The degree of derivatization of the coupling product was determined by 1.) protein determination with the Bio-Rad protein assay and 2.a) by determination of neuraminic acid in the coupling product with 1,2-diamino-4,5-dimethoxybenzene, b) by -’calculating the concentration of liberated 2-thiopyridone (in the final coupling step) by measuring the extinction at the wavelength lambda = 343 nm and inserting the appropriate parameters into the Lambert-Beer equation and c) by determining the concentration of recovered unreacted disialolactose-NH2-SPDP derivative using the methods mentioned under a) and b).

. Immunological reactions of the disialolactose-HSA conjugate The coupling product (disialolactose-HSA conjugate) was subsequently tested in a Bio-Dot assay with monoclonal anti-GD3 antibodies: both- mab 704/16 and mab 624/253 (both directed against the GD3 ganglioside) reacted with the neoglycoprotein.

It was also possible after SDS gel, electrophoresis under non-reducing conditions and subsequent Western blotting to show the reaction of the monoclonal antibodies mab 704/16 and mab 625/253 with the disialolactose-HSA conjugate.

Immunization experiments on mice have shown that the disialolactose-HSA conjugate is considerably more immunogenic than the GD3 ganglioside, i.e. elicits a stronger humoral immune response.

Claims (15)

1. Patent claims

1. A neoglycoprotein consisting of a carrier protein and the sugar mono- or disialolactose. 5

2. A neoglycoprotein as claimed in claim 1, wherein the carrier protein is human serum albumin (HSA).

3. A process for the preparation of a neoglycoprotein as claimed in claim 1, which comprises coupling the sugar to the carrier protein. 1.0'

4. The process for the preparation of a neoglycoprotein as claimed in claim 1, which comprises covalently coupling the sugar to the carrier protein.

5. The process for the preparation of a neoglycoprotein as claimed in claim 4, wherein N-succinimidyl 3-(215 pyridyldithio)propionate (SPDP) is used as coupling reagent.

6. The process for the preparation of a neoglycoprotein as claimed in claim 3, wherein bovine colostrum is used as source of sugar. 20

7. The use of a neoglycoprotein as claimed in claim 1 or obtainable as claimed in claim 3 for the production of a vaccine.

8. The use of a neoglycoprotein as claimed in claim 1 or obtainable as claimed in claim 3 for the produc25 tion of a diagnostic agent.

9. The use of a neoglycoprotein as claimed in claim 1 in a competitive radioimmunoassay for detecting gangliosides.

10. A vaccine which contains a neoglycoprotein as claimed in claim 1.

11. A neoglycoprotein as claimed in claim 1, substantially as hereinbefore described and exemplified.

12. A process for the preparation of a neoglycoprotein as claimed in claim 1, substantially as hereinbefore 5 described and exemplified.

13. A neoglycoprotein as claimed in claim 1, whenever prepared by a process claimed in a preceding claim.

14. Use as claimed in claim 8 or 9, substantially as hereinbefore described.

15. A vaccine as claimed in claim 10, substantially as hereinbefore described.