DEXTRAN MODIFICATION OF A FAB'-BETA-LACTAMASE CONJUGATE MODULATED BY VARIABLE PRETREATMENT OF FAB' WITH AMINE-BLOCKING REAGENTS

The physical and pharmacological properties of proteins can be altered by chemical modification with polymers. Preliminary studies showed th at attachment of oxidized dextran to the bacterial protein, beta-lacta mase (beta L) effectively reduced in vivo immunogenicity in mice with no loss of enzymatic activity. This report describes a general method for differentially dextran modifying the Fab' component of a Fab'-beta -lactamase conjugate by the use of amine-blocking reagents. Methyl ace timidate (MeAcm) and the N-succinimidyl derivative of (methylsulfonyl) ethy1 carbonate (NHS-Msc), reagents which can reversibly block primary amines, were used in model studies to modulate the level of available reactive amines on the F(ab')(2) fragments of both the anti-carcinoem bryonic antigen antibody, ZCE025, and the antitumor-associated glycopr otein-72 antibody, CC49. MeAcm had little or no effect on immunoreacti vity and was maximally effective in modulating dextran attachment, whi le NHS-Msc was much less effective. A comparison of NHS-Msc and MeAcm is described. Treatment of F(ab')(2) with 5-300 mM MeAcm prior to dext ran treatment showed a proportional decline in the level of dextran at tachment as well as intramolecular cross-linking of the protein by the dextran polymers (6 kDa or 33-mer). A conjugate of beta L coupled to MeAcm-treated ZCE025 Fab'[reduced F(ab')(2)] was constructed under sta nd ard conditions using sulfo succinimidyl N-[(4-carboxycyclohexy)meth yl]maleimide. After dextran modification, this conjugate maintained go od immunoreactivity and enzymatic activity. Biodistribution studies in tumor-bearing nude mice of dextranated and nondextranated conjugate s howed comparable overall distribution profiles except that the clearan ce of the dextranated conjugate from both blood and tumor was delayed about 48-72 h.