Preparation and use of immunoconjugates

A soluble immunoconjugate comprising:

• (a) a glycosylated antibody fragment selected from the group consisting of Fab, Fab', F(ab).sub.2, F(ab').sub.2, Fv, and single chain Fv, which comprises a light chain variable region having a carbohydrate moiety attached at about amino acid position 18 of said light chain variable region;
• and
• (b) at least one chelator, wherein said chelator is selected from the group consisting of compounds represented by formula (I) ##STR12## consisting of hydrogen and alkyl, substituted alkyl, aryl or substituted aryl groups;
• Z can be any group capable of reacting with said carbohydrate moiety of said antibody fragment, or Z can be H;
• R.sub.1 is a thiol protecting group which can be removed under conditions which do not significantly diminish the immunoreactivity of said protein:
• R.sub.2 and R.sub.3 can be the same or different, and each represent an acyl group or a substituted acyl group, or hydrogen, alkyl, aryl, substituted alkyl, or substituted aryl, where the substituents on the alkyl or aryl groups are metal-ligating groups selected from the group consisting of sulfhydryl, amine and carboxylic acid or their protected derivatives;
• R.sub.2 and R.sub.3 also can be any group capable of reacting with said carbohydrate moiety of said antibody fragment,
• compounds represented by formula (II) ##STR13## where D is H or CH.sub.2 SR.sub.1 ;
• E can be any group capable of reacting with said carbohydrate moiety of said antibody fragment;
• R.sub.1 is a thiol protecting group which can be removed under conditions which do not significantly diminish the immunoreactivity of said protein;
• and m is 0, 1, 2, or 3, and
• compounds represented by formula (III) ##STR14## where Q can be any group capable of reacting with said carbohydrate moiety of said antibody fragment;
• R.sub.1 is a thiol protecting group which can be removed under conditions which do not significantly diminish the immunoreactivity of said protein;
• and n is 2 or 3, and
• wherein each chelator is covalently bound to said carbohydrate moiety of said antibody fragment,
• and wherein said immunoconjugate retains the immunoreactivity of said antibody fragment.

A method for preparing an immunoconjugate, comprising covalently binding a loaded polyamidoamine dendrimer carrier to the carbohydrate moiety of an antibody fragment,

• wherein said antibody fragment is selected from the group consisting of Fab, Fab', F(ab).sub.2, F(ab').sub.2, Fv, and single chain Fv, wherein said antibody fragment contains a carbohydrate moiety in the light chain of said antibody fragment at about amino acid position 18,
• wherein said loaded polyamidoamine dendrimer carrier has at least one free amine group and a plurality of chelator molecules covalently bound to the polyamidoamine dendrimer carrier,
• wherein said chelator is selected from the group consisting of compounds represented by formula (I) ##STR15## where X is CH, or X and Z taken together can be CO;
• Y is CR.sub.4 R.sub.5, CH.sub.2 CR.sub.4 R.sub.5 or (CH.sub.2).sub.2 CR.sub.4 R.sub.5 where R.sub.4 and R.sub.5 are the same or different and are selected from the group consisting of hydrogen and alkyl, substituted alkyl, aryl or substituted aryl groups;
• Z can be any group capable of reacting with said carbohydrate moiety of said antibody fragment, or Z can be H;
• R.sub.1 is a thiol protecting group which can be removed under conditions which do not significantly diminish the immunoreactivity of said protein;
• R.sub.2 and R.sub.3 can be the same or different, and each represent an acyl group or a substituted acyl group, or hydrogen, alkyl, aryl, substituted alkyl, or substituted aryl, where the substituents on the alkyl or aryl groups are metal-ligating groups selected from the group consisting of sulfhydryl, amine and carboxylic acid or their protected derivatives;
• R.sub.2 and R.sub.3 also can be any group capable of reacting with said carbohydrate moiety of said antibody fragment,
• compounds represented by formula (II) ##STR16## where D is H or CH.sub.2 SR.sub.1 ;
• E can be any group capable of reacting with said carbohydrate moiety of said antibody fragment;
• R.sub.1 is a thiol protecting group which can be removed under conditions which do not significantly diminish the immunoreactivity of said protein;
• and m is 0, 1, 2, or 3, and
• compounds represented by formula (III) ##STR17## where Q can be any group capable of reacting with said carbohydrate moiety of said antibody fragment;
• R.sub.1 is a thiol protecting group which can be removed under conditions which do not significantly diminish the immunoreactivity of said protein;
• and n is 2 or 3
• wherein said loaded polyamidoamine dendrimer carrier is covalently bound through said at least one free amine group of said polyamidoamine dendrimer carrier to said carbohydrate moiety of said antibody fragment,
• and wherein said immunoconjugate retains the immunoreactivity of said antibody fragment.

A method for preparing an immunoconjugate, comprising covalently binding at least one chelator to the carbohydrate moiety of an antibody fragment,

• wherein said antibody fragment is selected from the group consisting of Fab, Fab', F(ab).sub.2, F(ab').sub.2, Fv, and single chain Fv,
• wherein said antibody fragment contains a carbohydrate moiety in the light chain of said antibody fragment at about amino acid position 18,
• wherein said chelator is selected from the group consisting of compounds represented by formula (I) ##STR18## where X is CH, or X and Z taken together can be CO;
• Y is CR.sub.4 R.sub.5, CH.sub.2 CR.sub.4 R.sub.5 or (CH.sub.2).sub.2 CR.sub.4 R.sub.5 where R.sub.4 and R.sub.5 are the same or different and are selected from the group consisting of hydrogen and alkyl, substituted alkyl, aryl or substituted aryl groups;
• Z can be any group capable of reacting with said carbohydrate moiety of said antibody fragment, or Z can be H;
• R.sub.1 is a thiol protecting group which can be removed under conditions which do not significantly diminish the immunoreactivity of said protein;
• R.sub.2 and R.sub.3 can be the same or different, and each represent an acyl group or a substituted acyl group, or hydrogen, alkyl, aryl, substituted alkyl, or substituted aryl, where the substituents on the alkyl or aryl groups are metal-ligating groups selected from the group consisting of sulfhydryl, amine and carboxylic acid or their protected derivatives;
• R.sub.2 and R.sub.3 also can be any group capable of reacting with said carbohydrate moiety of said antibody fragment,
• compounds represented by formula (II) ##STR19## where D is H or CH.sub.2 SR.sub.1 ;
• E can be any group capable of reacting with said carbohydrate moiety of said antibody fragment;
• R.sub.1 is a thiol protecting group which can be removed under conditions which do not significantly diminish the immunoreactivity of said protein;
• and m is 0, 1, 2, or 3, and
• compounds represented by formula (III) ##STR20## where Q can be any group capable of reacting with said carbohydrate moiety of said antibody fragment;
• R.sub.1 is a thiol protecting group which can be removed under conditions which do not significantly diminish the immunoreactivity of said protein;
• and n is 2 or 3,
• and wherein said immunoconjugate retains the immunoreactivity of said antibody fragment.