PREPARATION AND PRECLINICAL EVALUATION OF HUMANIZED A33 IMMUNOCONJUGATES FOR RADIOIMMUNOTHERAPY

A humanised IgG1/k version of A33 (hA33) has been constructed and expr essed with yields up to 700 mg l(-1) in mouse myeloma NSD cells in sus pension culture. The equilibrium dissociation constant of hA33 (K-D = 1.3 nM) was shown to be equivalent to that of the murine antibody in a cell-binding assay. hA33 labelled with yttrium-90 using the macrocycl ic chelator 12N4 (DOTA) was shown to localise very effectively to huma n colon tumour xenografts in nude mice, with tumour levels increasing as blood concentration fell up to 144 h. A Fab' variant of hA33 with a single hinge thiol group to facilitate chemical cross-linking has als o been constructed and expressed with yields of 500 mg l(-1). Trimalei mide cross-linkers have been used to produce a trivalent Fab fragment (hA33 TFM) that binds antigen on tumour cells with greater avidity tha n hA33 IgG. Cross-linkers incorporating 12N4 or 9N3 macrocycles have b een used to produce hA33 TFM labelled stably and site specifically wit h yttrium-90 or indium-111 respectively. These molecules have been use d to demonstrate that hA33 TFM is cleared more rapidly than hA33 IgG f rom the circulation of animals but does not lead to accumulation of th ese metallic radionuclides in the kidney. Y-90-labelled hA33 TFM there fore appears to be the optimal form of the antibody for radioimmunothe rapy of colorectal carcinoma.