Dextran modification of a Fab'--beta-lactamase conjugate modulated by variable pretreatment of Fab' with amine-blocking reagents

Bioconjug Chem. 1996 Jan-Feb;7(1):150-8. doi: 10.1021/bc950088j.

Abstract

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.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Antibodies
  • Antigens, Neoplasm / immunology
  • Carcinoembryonic Antigen / immunology
  • Dextrans*
  • Enzyme-Linked Immunosorbent Assay
  • Glycoproteins / immunology
  • Half-Life
  • Humans
  • Imides
  • Immunoglobulin Fab Fragments*
  • Indicators and Reagents
  • Iodine Radioisotopes
  • Mice
  • Mice, Nude
  • Neoplasms / metabolism
  • Radioimmunoassay
  • Structure-Activity Relationship
  • Tissue Distribution
  • Transplantation, Heterologous
  • Tumor Cells, Cultured
  • beta-Lactamases* / metabolism

Substances

  • Antibodies
  • Antigens, Neoplasm
  • Carcinoembryonic Antigen
  • Dextrans
  • Glycoproteins
  • Imides
  • Immunoglobulin Fab Fragments
  • Indicators and Reagents
  • Iodine Radioisotopes
  • tumor-associated antigen 72
  • beta-Lactamases