Patent EP-0269188-B1 (EP)
IPC: A61K47/00
Filed: 1985-06-18
Granted: 1992-03-04
Abstract: 1. Claims for the Contracting States BE, CH, DE, FR, GB, IT, LI, LU, NL, SE An immunotoxin formed by the coupling of an antibody P with the sub-unit A of ricin, having the following statistical formula : P'-W'-A' in which P' represents the radical of a protein P which is an antibody or a fragment of an antibody, as such or appropriately chemically modified, from which one or more of its own functions have been removed and in which other functional groups are optionally blocked, A' represents the radical of a protein which is the sub-unit A of ricin, as such or appropriately chemically modified, from which one or more of its own functions have been removed and in which other funtional groups are optionally blocked, and W' represents a divalent covalent structure selected from : (a) a group of the formula : -Z"-Y-E-S-S-(E'-Y'-Z')n - or (b) a group of the formula : -(NH-Y'-E')n -S-S-E-Y-Z'-, in which : - Z' and Z' represents the functions belonging to the proteins A and P, chosen from the oxygen atom originating from the hydroxyl of one of the tyrosine residues, the carbonyl group originating from one the terminal carboxyls or the free carboxyls of the aspartic and/or glutamic acids of A and P ; Z" also represents the group originating from the dialdehyde structure obtained after oxidation of one of the carbohydrate structures of P with periodic acid ; Z' also represents the -NH- group originating from one of the terminal amines of A or from one of the amines in the epsilon position of one of the lysine residues ; - Y and Y' represent functional groups capable of bonding covalently with any one of the functions Z' and Z" of the proteins A and P ; - E and E' represent inert spacing structures ; and - n represents zero or 1. 1. Claims for contracting state AT A process for the preparation of an immunotoxin having the following statistical formula : P'-W'-A' in which P' represents the radical of a protein P which is an antibody or a fragment of an antibody, as such or appropriately chemically modified, from which one or more of its own functions have been removed and in which the other functional groups are optionally blocked, A' represents the radical of a protein A which is the sub-unit A of ricin, as such or appropriately chemically modified, from which one or more of its own functions have been removed and in which the other functional groups are optionally blocked, and W' represents a divalent structure chosen from : (a) a group of the formula : -Z"-Y-E-S-S-(E'-Y'-Z')n or (b) a group of the formula : -(NH-Y'-E')n -S-S-E-Y-Z'-, in which : - Z' and Z" represent the groups belonging to the proteins A and P, chosen from the oxygen atom originating from the hydroxyl of one of the tyrosine residues, the carbonyl group originating from one of the terminal carboxyls or from one of the free carboxyls of the aspartic and/or glutamic acids of A and P ; Z" represents also the group originating from the dialdehyde structure obtained after oxidation of one of the carbohydrate structure of P with periodic acid ; Z' represents also the -NH- group originating from one of the terminal amines of A or from one of the amines in the epsilon position of one of the A lysine residues ; - Y and Y' represent functional groups capable of bonding covalently with any one of the functions Z' and Z" of the proteins A and P ; - E and E' represent inert spacing structures ; and - n represents zero or 1, characterized in that a protein of formula : P1'-(Z1 -Y'-E')n -SH is reacted, in aqueous solution and at ambient temperature, with a protein of the formula : P'2 -Z1 -Y-E-G in which P'1 and P'2 represent the radicals of proteins P1 and P2 which are indifferently the proteins A and P as defined in claim 1, from which one or more of its own functions are removed or one of the radicals of the proteins P1 or P2 originating from the opening of the carbohydrate nuclei of antibodies or antibody fragments by reaction with periodic acid, Y, Y', E and E' are as defined above Z1 represents Z' or NH and Z2 represents Z' or Z" and G represents a group -S-S-X, in which X is an activating group, it being understood that, when P'1 is the sub-unit A of ricin, n is 1 or alternatively n can be zero, but, in this case, Z2 is other than -NH-.