U.S. Pat. No. 4,671,958 issued to Rodwell et al. describes a method for site specific covalent attachment of a compound to an antibody molecule by selectively oxidizing a carbohydrate moiety of the antibody, located outside the antigen binding region of the antibody, to form an aldehyde group and then reacting the resultant aldehyde group with an amine group such as a primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, or semicarbazide to form an antibody-compound conjugate which is characterized by substantially the same immunospecificity as the unconjugated antibody.
European Patent Application No. 85401695.3 published March 5, 1986 describes methods for preparing and compositions comprising aqueous soluble antibody-metal ion complexes in which a compatible chelator coordinately bound to a metal ion is covalently attached via an amine group of the chelator to an oxidized carbohydrate moiety of an antibody molecule, located outside the antigen binding region of the antibody. The antibody-metal ion complexes are characterized by (1) substantially the same immunospecificity as the unconjugated antibody molecule; and (2) aqueous solubility such that they are suitable for in vivo administration.
European Patent Application No. 85401776.1 published March 26, 1986 describes methods for preparing and compositions comprising aqueous soluble antibody-therapeutic agent conjugates in which a therapeutic agent is attached, either directly or through a linker, via an amine group to an oxidized carbohydrate moiety of an antibody molecule, located outside the antigen binding region of the antibody. The antibody-therapeutic agent conjugates are characterized by (1) substantially the same immunospecificity as the unconjugated antibody molecule; and (2) aqueous solubility such that they are suitable for in vivo administration.
The methods described in the above references involve site specific attachment of a compound to either a whole antibody molecule or a preformed antibody fragment such as a half antibody molecule (i.e., a single heavy: light chain pair), a Fab, a (Fab').sub.2, or a Fab' fragment to form the antibody-compound conjugates. Thus, according to conventional methods a preformed antibody fragment is coupled to a compound to form an antibody fragment conjugate.
In contrast, the present method involves cleavage of an antibody composition, comprising an antibody attached to a compound, a linker or a linker-compound intermediate, in which the attachment is site specific via an oxidized carbohydrate moiety of the whole antibody. Cleavage of the antibody composition according to the present invention forms an antibody fragment composition. The present invention is based on the surprising discovery that antibody fragment conjugates prepared according to the methods disclosed herein behave significantly different from conventional conjugates when administered in vivo and exhibit in vivo biodistribution which is advantageously used for targeted delivery to in vivo sites.