Gram-negative infections continue to cause substantial morbidity and mortality. A major part of the toxicity from these infections is believed to be caused by lipopolysaccharide (LPS) release from dying bacteria, that interacts with host systems leading to secondary inflammation. The availability of an agent with a long half-life that is capable of both killing Gram-negative bacteria and neutralizing its endotoxin would be a major advance which may prove to be therapeutically advantageous. Chemical conjugates of an 18 kD cationic protein present in human granulocytes (CAP18) coupled to human IgG appears to be such an agent. CAP18-IgG fusion proteins are believed to function by binding LPS that is present on or is released from the bacterial cell wall. Bacterial killing is presumed to occur by intercalation and disruption of the outer cell membrane. In preliminary studies CAP18-IgG fusion proteins appear to kill a wide variety of Gram-negative bacteria as well as neutralize bacterial LPS. This study seeks to develop and evaluate the efficacy of fusion proteins of CAP18 conjugated with the human heavy chain IgG in gram negative infections. In order to accomplish this goal the following series of experiments are proposed: 1) Design, construct, express, and purify fusion proteins of CAP18-IgG and CAP18104-135-IgG. 2) Characterize and assess the in vitro ability of these CAP18-IgG fusion proteins to bind and neutralize Gram-negative bacteria and LPS, assess their bactericidal activity, promote opsinophagocytosis of bacteria and bacterial membrane, and determine stability in blood over time. 3) Evaluate these CAP18-IgG fusion proteins in vivo using rabbits and rats to determine their pharmacokinetics, their functional stability in the bloodstream over time and ability to clear radiolabeled LPS and bacteria from the blood. 4) Study the protection by the best CAP18-IgG fusion proteins in two models of Gram-negative sepsis: a) Gram-negative infection in rats made neutropenic with cyclophosphamide, and b) a rat model of peritoneal sepsis.
| Hellman, J; Loiselle, P M; Tehan, M M et al. (2000) Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum. Infect Immun 68:2566-72 |
| Hellman, J; Loiselle, P M; Zanzot, E M et al. (2000) Release of gram-negative outer-membrane proteins into human serum and septic rat blood and their interactions with immunoglobulin in antiserum to Escherichia coli J5. J Infect Dis 181:1034-43 |
| Rao, J; Yan, L; Lahiri, J et al. (1999) Binding of a dimeric derivative of vancomycin to L-Lys-D-Ala-D-lactate in solution and at a surface. Chem Biol 6:353-9 |
