PROCEDURE FOR THE PRODUCTION OF NEW PROTEIN DERIVATIVES
Deactivated protein derivatives Technisches area Immunotoxine are connections, which e.g. consist generally of an immunologically effective part, like an antibody, preferably a monoclonal antibody, and a Toxin. Immunotoxine are used for selective killing of neoplastischen cells in the framework of the cancer therapy. in case of of Immunotoxinen, which consist of an antibody and a nonspecific Toxin, the Toxin can become effective opposite cells by the fact that a) the Immunotoxin over the Toxinanteil is bound and internalized nonspecific at cells or b) the Immunotoxin of an enzymatic Lyse is subject, whereby the connection between antibodies and nonspecific Toxin is solved and the Toxin is set free; the Toxin becomes nonspecific effective also in this case. Both cases the death of cells enters by the Toxinanteil of the assigned Immunotoxins, without a selective controlling of the Toxins on goal cells is possible. Ziel to the invention Die available invention aims off to create new deactivated protein derivatives it at a certain group of actually nonspecific celltoxically effective Toxinen, i.e. with Lectinen and Bakterientoxinen, make possible, to prevent a connection of these Toxine at the cell surface. The invention aims further at the creation of connections suitable for the deactivation of proteins and procedure for the production of these protein derivatives and connections as well as at the use of the deactivated protein derivatives with the production from Immunotoxinen. Ausführliche description of the invention Unter Lectinen is to be understood in the context of the invention a group from proteins to, which exhibit at least two connection places for groups of coal hydrates. Bakterientoxine are of pathogenen bacteria produced proteins, which consist of two components, of which the one component toxically work and which other component, which is even not toxic, makes possible the connection of the Toxins to the cell membrane over connection places for coal hydrates as well as its penetration into the cell. Den both types of the aforementioned proteins, i.e. the Lectinen and the Bakterientoxinen, are common that they are cytotoxic substances, which exhibit two different sections in their structures. With a structural section they can be bound over coal hydrate connection places to the cell surface, whereby a Internalisation is made possible into the cell. The other structural section can determined, for the cell vitally necessary enzyme systems block and thus in a monomolecular reaction cell death to cause. By the available invention succeeds to block and by to the Toxin the ability taking coal hydrate connection places in proteins of the type of the Lectine and Bakterientoxine to the cell surface to be bound and into the cell penetrate. Die new, deactivated protein derivatives in accordance with the available invention have the general formula EMI2.1 where n a number from 1 to 3 are, A for the remainder of a protein of the type of the Lectine or Bakterientoxine stands, for g 1 a remainder of a coal hydrate from the mono, Di, the trichloroethylene and Tetrasaccharide and their derivatives comprehensive group meant and R2 is a hydrogen atom, a C1-C10-Alkylrest or the Nitrogruppe. In the general formula (I) can A for the remainder of any protein of the type of the Lectine or Bakterientoxine stand. If preferential remainders of the type of the Lectine are the Ricin, the Abrin and the Viscum album Lectin remainder, preferential remainders of the type of the Bakterientoxine are Diphtherietoxin, E.colitoxin, protein A and Pseudomonastoxin remainder. Die new protein derivatives of of the general formula (I) contain proteins, whose coal hydrate connection places are blocked by the remainder of a Aminobenzoesäurederivats, which contains a remainder of g 1. Preferential remainders of g 1 are Galactose, lactose, Mannose, Galactosamin, N-Acetylgalactosamin, Glucosamin or N-Acetylglucosamin-remainders. Preferential alkyl residues R2 have 1 to 5 carbon atoms, in particular 1, 2 or 3 carbon atoms, like methyl, ethyl and Propyl. In the deactivated protein derivatives according to invention (I) is intact structural portion of the protein remainder of The affecting the cell system, cannot however due to being present the group of blockings with the remainder of g 1 not into the cell penetrate and from there there not become effective. Die new protein derivatives of the general formula (I) are valuable for the use with the production of Immunotoxinen. Um the Toxine of the deactivated protein derivatives according to invention of the general formula (I), which a remainder of A of a protein of the type of the Lectine or Bakterientoxine contained to let become celleffective needs this blocked Toxine a specific carrier, that makes the penetration possible into the cell. The absence of such a carrier excludes a nonspecific, toxic effect of the Toxinanteils of the new deactivated protein derivatives. Examples of such carrier, the one cell-specific, toxic effect of the deactivated protein derivatives according to invention of the general formula (I) release, are monoclonal antibodies. Zur production of the new deactivated protein derivatives of the general formula (I) sets one a Azidobenzoesäurederivat of the general formula EMI4.1 where g 1 and has R2 the meanings indicated above, with a protein of the type of the Lectine or Bakterientoxine to a protein derivative of the general formula (I) over. The proteins (Toxine), used with this manufacturing process, in addition, are to be acquired generally available, can by extraction from plants and/or their seeds as well as from bacteria be won. The cleaning is made appropriately affinitätschromatographisch, preferably by a column, which is filled with appropriate immobilized coal hydrate, and by means of ion exchange chromatography, preferably using Cm-Sepharose. Die Azidobenzoesäurederivate of the general formula (II) used with the above manufacturing process is again and is also the subject of the available invention. They can be manufactured, by converting Azidobenzoesäuren of the formula EMI4.2 or their reactive derivatives, where R2 has the meaning indicated above, with coal hydrates from the mono, Di, the trichloroethylene and Tetrasaccharide and their derivatives comprehensive group. Preferred reactive derivatives of Azidobenzoesäuren of the formula (III) are N-Hydroxysuccinimidester of the appropriate Azidobenzoesäure. Nach a special execution form of the procedure according to invention for the production of the Azidobenzoesäurederivate of the general formula (II) shifting of the N-Hydroxysuccinimidester with Aminozuckern takes place in H2O/DMF-Lösungsmittelgemi. Die cleaning of the connections of the general formula (II) takes place appropriately via Säulenchromatographie on silicagel derivatives with a mixture from water and acetonitrile as Eluens. Die conversion of the Azidobenzoesäurederivate of of the general formula (II) with the one remainder of A containing proteins (Toxinen) can be accelerated by influence of light. Appropriately one works when using Azidobenzoesäurederivaten of the general formula (II), where R2 means a hydrogen atom or a C1-C10-Alkylrest, under irradiation with UV light in homogeneous, aqueous solution. When using Azidobenzoesäurederivaten of the general formula (II), where R2 means the Nitrogruppe, working under irradiation with visible light is favourable in homogeneous, aqueous solution. The duration of the irradiation for the photo activation of the Azidobenzoesäurederivate of the general formula (II) several hours can amount to with 4 DEG C. Die separation and cleaning of the new deactivated protein derivatives of the general formula (I) been made appropriately via affinity chromatography, best by a column filled with appropriate immobilized coal hydrate. Beispiele 1) production of N-4-Azidobenzoylgalactosamin 100 mg Galactosaminhydrochlorid in 5 ml H2O are dest. solved and 150 mg N-Hydroxisuccinimidester of the 4-Azidobenzoesäure as well as 100 mg Dicyclohexylcarbodiimid are solved in 30 ml dimethylformamide. The two solutions are united in a suitable container and the pH value of this solution by means of NaHCO3 is placed to 7 - 7.5. Diese solution is < in the locked container at ambient temperature at least 12; h> agitated. After the end of the reaction the solvents in the water jet vacuum are removed and the arrears in 50 ml H2O are dest. taken up. Nach separating the insoluble components in such a way won solution on 10 x 1 cm column, filled with LiChroprep TM RP-18 (grain size 0.04 - 0, o63 mm) and with H2O dest. equillibriert, given up and after reaching a sturdy baseline by means of acetonitrile gradients (0 100% acetonitrile linear over 500 ml) eluiert. In such a way won pure substance is lyophilisiert and kept with 4 DEG C (yield 114 mg, d.i. 84% D. Th.). N-4-Azidobenzoylgalactosamin was characterized by IR, NMR and UV spectroscopy as well as by an CHN analysis: IR spectrum: Azide gang with 2120 cm< - > < 1> UV spectrum: Maximum with 270 Nm NMR spectrum: Protons parasubstituierten aromatics with 7,05-8,14 ppm, relationship of the integrals of the aromatic protons to those of the coal hydrate portion of 4 to 7th EMI6.1 2) production of N-5-Azido-2-nitrobenzoylgalactosamin 100 mg Galactosaminhydrochlorid in 5 ml H2O are dest. solved and 175 mg n-Hydroxisuccinimidester of the 5-Azido-2-nitrobenzoesäure as well as 100 mg Dicyclohexylcarbodiimid are solved in 30 ml Dioxan. The two solutions are united in a suitable container and the pH value of the solution by means of NaHCO3 is placed to 7 - 7.5. Diese solution is < in the locked container at ambient temperature at least 12; h> agitated. After the end of the reaction the solvents in the water jet vacuum are removed and the arrears in 50 ml H2O are dest. taken up. Nach separating the insoluble components in such a way won solution on 10 x 1 cm column, filled with LiChroprep TM RP-18 (grain size 0.04 - 0.063 mm) and with H2O dest. equillibriert, given up and after reaching a sturdy baseline by means of acetonitrile gradients (0 100% acetonitrile linear over 1000 ml) eluiert. In such a way won, pure substance is lyophilisiert and is characterized with 4 DEG C kept (yield 122 mg, d.i. 78% d.Th.) N-5-Azido-2-nitrobenzoylgalactosamin by IR, NMR and UV spectroscopy as well as by an CHN analysis: IR spectrum: Azide gang with 2120 cm< - > < 1> as well as NO2-Bande at 1340 cm< - > < 1> UV spectrum: Maximum with 315 Nm NMR spectrum: Protons meta para disubstituierten aromatics with 6,98-7,88 ppm, relationship of the integrals of the aromatic protons to those of the coal hydrate portion of 3 to 7th EMI7.1 3) production of at the Galactosebindungsstelle blocked Ricin 50 mg Ricin, solved in 1 ml 10 mm of trichloroethylene/HCl buffer pH 7.7, in a suitable container with 3 mg N-4-Azidobenzoylgalactosamin are shifted and with a 40-W Germizidleuchtstoffröhre254 without filter cover from 5 cm distance at least 30 min. exposed. Nach end of the reaction is removed surplus destroyed N-4-Azidobenzoylgalactosamin by Entsalzen over for 10 x 2 cm column filled with bio gel P6DG and the developed product by means of affinity chromatography over 5 x 3 cm column is cleaned filled with lactose Sepharose. Das so won product is kept in a concentration of 1 mg/ml in 50 mm sodium phosphate buffer pH 7.2 under additive of 0,02% NaN3 with 4 DEG C. Yield: 3.7 mg d.i. 7.4% d.Th The coal hydrate connection place blocked Ricin its decreased toxicity on Zellinien became (human Zellinie HL 60) by its activity in the cell-free protein synthesis system (Reticulozytenlysat from rabbits), and its molecular weight in SDS electrophoresis as well as by its to bind with the cleaning used, inability appropriate coal hydrates characterized. EMI8.1 * of one of the two Ricin subunits does not give it to two, only forms different in the molecular weight, a double gang with 60 and 62 kD is from there typically for Ricin and under any circumstances as pollution to be regarded. 4) production of at the Galactosebindungsstelle blocked Ricin by means of N-5-Azido-2-nitrobenzoylgalactosamin 50 mg Ricin in 1 ml 10 mm of trichloroethylene/HCl buffers pH 7.7 are solved, shifted with 3,5 mg N-5-Azido-2-nitrobenzoylgalactosamin and exposed in a suitable container from 20 cm distance with 500 W a mercury vapor lamp with emission maximum with 320 Nm using a focusing lens at least 20 min. Nach end of the reaction is removed surplus destroyed N-5-Azido-2-Nitrobenzoylgalactosamin by Entsalzen over for 10 x 2 cm column filled with bio gel P6DG and the developed product by means of affinity chromatography over 5 x 3 cm column is cleaned filled with lactose Sepharose. Das so won product is kept in a concentration of 1 mg/ml in 50 mm sodium phosphate buffer pH 7.2 under additive of 0,02% NaN3 with 4 DEG C. Yield: 2.9 mg, d.i. 5.8% d.Th The coal hydrate connection place blocked Ricin its decreased toxicity on Zellinien became (human Zellinie HL 60) by its activity in the cell-free protein synthesis system (Reticulozytenlysat from rabbits), and its molecular weight in SDS electrophoresis as well as by its to bind with the cleaning used, inability appropriate coal hydrates characterized. EMI9.1 * of one of the two Ricin subunits gives it to two, only forms different in the molecular weight, a double gang with 60 and 62 kD is typical from there for Ricin and no-falls as pollution to regard. 5) production of Abrin by means of N-5-Azido-2-nitrobenzoylgalactosamin, blocked in the coal hydrate connection place, 20 mg Abrin in 1 ml 20 mm sodium phosphate buffers (pH 6.5) are suspended and shifted in an appropriate container with 2 mg N-5-Azido-2-nitrobenzoylgalactosamin. Die photo activation with 500 W Quecksilberhochdrucklampe from approximately 20 cm distance under intensive cooling on 10 DEG C. Das surplus reagent becomes effected after complete photolysis via dialysis against the o. A. Buffer removes. The blocked is separated from the still intact Abrin over a 1 x 5 cm column, filled also to epoxi activated Sepharose of coupled lactose, whereby this procedure is three times repeated. Dieses product is kept in 10 mm of sodium phosphate buffers under additive of 0,8% NaCl and 0.02% NaN3 with 4 DEG C. Das in the coal hydrate connection place blocked Abrin was characterized by the following parameters: - Molecular weight in SDS electrophoresis - activity in the cell-free protein synthesis system - decreased toxicity on the Zellinie HL 60 - inability to bind lactose. EMI10.1 6) production of Viscum album Lectin blocked in the coal hydrate connection place 20 mg Viscum album Lectin are suspended in 1 ml 20 mm of sodium phosphate buffers (pH 6.5) and shifted in an appropriate container with 2 mg N-5-Azido-2-Nitrobenzoylgalactosamin. Die photo activation with 500 W mercury high pressure vapor lamp from approximately 20 cm Enfernung under intensive cooling on 10 DEG C. Das surplus reagent becomes effected after complete photolysis via dialysis against the o. A. Buffer removes. The blocked Viscum album Lectin is separated from still intact Viscum album Lectin over a 1 x 5 cm column, whereby this procedure is three times repeated. Dieses product is kept in 10 mm of sodium phosphate buffers under additive of 0,8% NaCl and 0.02% NaN3 with 4 DEG C. Yield: 1.4 mg, that are 7% D. Th. Das to the Galactosebindungsstelle blocked Viscum album Lectin was characterized by the following parameters: - Molecular weight in SDS electrophoresis - activity in the cell-free protein synthesis system - decreased toxicity on the Zellinie HL 60 - inability to bind lactose. EMI11.1 The protein derivatives have the general formula <IMAGE> in which n is a number from 1 to 3, A is the residue of a protein of the lectin or bacteriotoxin type, R1 is a residue of a carbohydrate from the group comprising mono-, di-, tri- and tetrasaccharides and derivatives thereof, and R2 is a hydrogen atom, a C1-C10-alkyl radical or the nitro group, and suppress binding of lectins or bacteriotoxins to the cell surface; compounds suitable for inactivating proteins and processes for preparing these protein derivatives and compounds, and the use of the inactive protein derivatives for preparing immunotoxins are described. 1. Protein derivatives of the general formula EMI12.1 where n a number from 1 to 3 is, A for the remainder of a protein of the type of the Lectine or Bakterientoxine stands, for g 1 a remainder of a coal hydrate from the mono, Di, the trichloroethylene and Tetrasaccharide and their derivatives comprehensive group meant and R2 is a hydrogen atom, a C1-C10-Alkylrest or the Nitrogruppe. 2. Protein derivatives according to requirement 1, where n is a number from 1 to 3, A for the Ricin or Abrinrest and g 1 and R2 the meanings indicated above stand have. 3. Protein derivatives according to requirement 1, where n is a number from 1 to 3, A for the Diphtherietoxin, E.colitoxin, protein A or Pseudomonastoxin remainder and g 1 and R2 the meanings indicated above stand have. 4. Protein derivatives after one of the requirements 1 to 3, by it characterized that g 1 means a Galactose, a lactose, a Mannose, a Galactosamin, a N-Acetylgalactosamin, a Glucosamin or a N-Acetylglucosamin-remainder, n a number from 1 to 3 and A and R2 the meanings indicated above have. 5. Azidobenzoesäurederivate of the general formula EMI12.2 where g 1 a remainder of a coal hydrate from the mono, Di, the trichloroethylene and Tetrasaccharide and their derivatives comprehensive Grup `PE meant and R2 is a hydrogen atom, a C1-C10-Alkylrest or the Nitrogruppe. 6. Azidobenzoesäurederivate according to requirement 5, where g 1 a Galactose, a lactose, a Mannose, a Galactosamin, a N-Acetylgalactosamin, a Glucosamin or a N-Acetylglucosamin-remainder meant and R2 has the meaning indicated above. 7. Procedure for the production of new protein derivatives of the general formula EMI13.1 where n a number from 1 to 3 is, A for the remainder of a protein of the type of the Lectine or Bakterientoxine stands, for g 1 a remainder of a coal hydrate from the mono, Di, the trichloroethylene and Tetrasaccharide and their derivatives comprehensive group meant and R2 a hydrogen atom, a C1-C10-Alkylrest or the Nitrogruppe is, thereby characterized that one a Azidobenzoesäurederivat of the general formula EMI13.2 where g 1 and R2 the meanings indicated above have with a protein of the type of the Lectine or Bakterientoxine to a protein derivative of the general formula (I) converts. 8. Procedure according to requirement 7, by the fact characterized that one begins as protein of the type of the Lectine Ricin, Abrin or Viscum album Lectin. 9. Procedure according to requirement 7, by the fact characterized that one begins as protein of the type of the Bakterientoxine Diphtherietoxin, E.colitoxin, Pseudomonastoxin or protein A. 10. Procedure after one of the requirements 7 to 9, by the fact characterized that one as Azidobenzoesäurederivat of the general formula (II) such begins, where R2 a hydrogen atom or a C1-C10-Alkylrest meant and g 1 has the meaning indicated above, and the conversion under irradiation with UV light in homogeneous, aqueous solution accomplishes. 11. Procedure after one of the requirements 7 to 9, by the fact characterized that one as Azidobenzoesäurederivat of the general formula (II) such begins, where R2 the Nitrogruppe meant and g 1 has the meaning indicated above, and the conversion under irradiation with visible light in homogeneous, aqueous solution accomplishes. 12. Procedure for production of Azidobenzoesäurederivaten general formula EMI14.1 where g 1 remainder carbohydrate from mono, Di, trichloroethylene and Tetrasaccharide and their derivatives comprehensive group meant and R2 hydrogen atom, C1-C10-Alkylrest or Nitrogruppe is, thereby characterized that one converts Azidobenzoesäuren of the formula EMI14.2 or their reactive derivatives, where R2 means a hydrogen atom, a C1-C10-Alkylrest or the Nitrogruppe with coal hydrates from the mono, Di, the trichloroethylene and Tetrasaccharide and their derivatives comprehensive group. 13. Procedure according to requirement 12, by the fact characterized that one as reactive derivatives of Azidobenzoesäuren of the formula (III) N-Hydroxysuccinimidester of the appropriate Azidobenzoesäure begins. 14. Procedure according to requirement 12 or 13, by the fact characterized that one uses comprehensive group of Galactose, lactose, Mannose, Galactosamin, N-Acetylgalactosamin, Glucosamin or N-Acetylglucosamin or their derivatives as coal hydrates from the mono, Di, the trichloroethylene and Tetrasaccharide and their derivatives. 15. Use of protein derivatives of the general formula EMI15.1 where n a number from 1 to 3 is, A for the remainder of a protein of the type of the Lectine or Bakterientoxine stands, for g 1 a remainder of a coal hydrate from the mono, Di, the trichloroethylene and Tetrasaccharide and their derivatives comprehensive group meant and R2 a hydrogen atom, a C1-C10-Alkylrest or the Nitrogruppe is, with the production of Immunotoxinen.