The 18-kD cationic protein CAP18 is an antimicrobial protein first isolated from rabbit granulocytes that binds lipopolysaccharide and inhibits many of its biological activities (Larrick et al., 1995). The most active approximately 30 amino acid fragment of CAP18 has potent antimicrobial activity versus both gram negative and gram-positive bacteria. The overall goal of this project is to create a novel anti-LPS/anti-microbial therapy for severe infections by genetically engineering a CAP18 peptide-immunoglobulin fusion protein. To increase the effective half-life of the peptide, to reduce the probability of renal toxicity and to augment the clearance of LPS and bacteria, we will replace the VH region of human IgG1 with our most potent human CAP18 peptide. The IgG1 constant region of this construct will contain selected mutations in its CH2 and CH3 domains. These mutations are known to augment FcRIII binding affinity for enhanced bacterial/LPS clearance and to improve FcRN binding to increase serum half-life. In Phase I we will a) prepare cDNA constructs of CAP18 peptide fused to novel Ig heavy chain; b) express and purify CAP18-Ig using protein G; c) demonstrate the capacity of CAP18-Ig to neutralize LPS in whole blood. The PHASE II goals will include scale-up production of CAP18-Ig for animal efficacy studies and toxicity studies in support of an IND. This work will produce a novel antimicrobial broad-spectrum agent suitable for prophylaxis and/or therapy.
