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 that attachment of oxidized dextran to the bacterial protein, beta-lactamase (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 acetimidate (MeAcm) and the N-succinimidyl derivative of (methylsulfonyl)ethyl 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-carcinoembryonic antigen antibody, ZCE025, and the antitumor-associated glycoprotein-72 antibody, CC49. MeAcm had little or no effect on immunoreactivity and was maximally effective in modulating dextran attachment, while 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 dextran treatment showed a proportional decline in the level of dextran attachment 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 standard conditions using sulfosuccinimidyl N-[(4-carboxycyclohexyl)methyl]maleimide. After dextran modification, this conjugate maintained good immunoreactivity and enzymatic activity. Biodistribution studies in tumor-bearing nude mice of dextranated and nondextranated conjugate showed comparable overall distribution profiles except that the clearance of the dextranated conjugate from both blood and tumor was delayed about 48-72 h.
Mikolajczyk
S D
SD
Hybritech Inc., San Diego, California 92196-9006, USA.
Meyer
D L
DL
Fagnani
R
R
Hagan
M S
MS
Law
K L
KL
Starling
J J
JJ
D000818:Animals; D000906:Antibodies; D000951:Antigens, Neoplasm / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000276:immunology; D003911:Dextrans; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000276:immunology; D006207:Half-Life; D006801:Humans; D007094:Imides; D007140:Immunoglobulin Fab Fragments; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000378:metabolism; D011863:Radioimmunoassay; D013329:Structure-Activity Relationship; D014018:Tissue Distribution; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D001618:beta-Lactamases / Q000378:metabolism
D000906:Antibodies; D000951:Antigens, Neoplasm; D002272:Carcinoembryonic Antigen; D003911:Dextrans; D006023:Glycoproteins; D007094:Imides; D007140:Immunoglobulin Fab Fragments; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; C047052:tumor-associated antigen 72; D001618:beta-Lactamases