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Antimicrobial Agents and Chemotherapy, March 1998, p. 583-588, Vol. 42, No. 3
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Covalent Polymyxin B Conjugate with Human Immunoglobulin G as an Antiendotoxin Reagent

Joseph J. Drabick,1,* Apurba K. Bhattacharjee,1 David L. Hoover,1 George E. Siber,2 Vivian E. Morales,1 Lynnette D. Young,1 Scott L. Brown,3 and Alan S. Cross4

Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20307-51001; Wyeth-Lederle Vaccines and Pediatrics, Pearl River, New York 109652; Microbiology Section, Department of Pathology and Clinical Laboratory Services, Walter Reed Army Medical Center, Washington, D.C. 20307-50003; and Division of Infectious Diseases and Program in Oncology, Department of Medicine, School of Medicine, University of Maryland, Baltimore, Maryland 21201-17344

Received 1 May 1997/Returned for modification 11 August 1997/Accepted 18 December 1997

Polymyxin B (PMB) is a cyclic decapeptide antibiotic which also binds and neutralizes endotoxin. Unfortunately, PMB can be considerably nephrotoxic at clinically utilized doses, thereby limiting its utility as a therapeutic antiendotoxin reagent. We sought to change the pharmacokinetics and toxicity profile of PMB by covalently linking it to a human immunoglobulin G (IgG) carrier. Conjugates of PMB with IgG were prepared by EDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]-mediated amide formation. Analysis by dot enzyme-linked immunosorbent assay with an anti-PMB monoclonal antibody showed that the purified conjugate contained bound PMB. The IgG-PMB conjugate reacted with lipid A and J5 lipopolysaccharide in Western blot assays in a manner comparable to that of whole antiserum with anti-lipid A reactivity; unconjugated IgG had no reactivity. The PMB bound in the conjugate retained its endotoxin-neutralizing activity compared to that of unbound PMB as evidenced by its dose-dependent inhibition of tumor necrosis factor release by endotoxin-stimulated human monocytes in vitro; unconjugated IgG had no activity. By this assay, the PMB-IgG conjugate was determined to have approximately 3.0 µg of bound functional PMB per 100 µg of total protein of conjugate (five molecules of PMB per IgG molecule). The PMB-IgG conjugate was also bactericidal against clinical strains of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae relative to unconjugated IgG with MBCs of <4 µg of conjugate per ml for each of the tested strains. The conjugate appeared to be nontoxic at the highest doses deliverable and provided statistically significant protection from death to galactosamine-sensitized, lipopolysaccharide-challenged mice in a dose-dependent fashion when administered prophylactically 2 h before challenge. However, neither free PMB nor the PMB-IgG conjugate could protect mice challenged with endotoxin 2 h after administration. This suggests that these reagents can play a role in prophylaxis but not in therapy of sepsis. These experiments demonstrated that the PMB-IgG conjugate retains bound yet functional PMB as evidenced by its endotoxin-neutralizing activity both in vitro and in vivo. Further work is required to define the role that this or related conjugate compounds may play in the prophylaxis of endotoxin-mediated disease.

* Corresponding author. Mailing address: Hematology/Oncology Service, Walter Reed Army Medical Center, Washington, DC 20307-5100. Phone: (202) 782-5749. Fax: (202) 782-3256. E-mail: jdrabino{at}aol.com.

Antimicrobial Agents and Chemotherapy, March 1998, p. 583-588, Vol. 42, No. 3
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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