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Molecular & Cellular Analysis Technologies
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Antibodies provide superior targeting capabilities to a variety of therapeutic agents. Several technologies have greatly facilitated the initial identification of a variety of antibody reagents, including scFv and Fab antibodies, with virtually any possible specificity. However, lead antibodies often require further optimization to maximize their therapeutic performance: optimization of antibody expression and folding in relevant cells, and optimization of the affinity of the antibody for the target antigen. The development of promising targeting antibodies against cancer often languishes at this bottleneck. Therefore, technologies to facilitate antibody adaptation and optimization are urgently needed. Antibody optimization is best achieved by the randomization and subsequent selection of antibody mutants for the desired phenotypes since efficient rational design of antibodies is currently not feasible. Polypeptide display (e.g., phage display) is a powerful technology for the generation and screening of libraries of mutant polypeptides for a phenotype. A eukaryotic display technology that employs the efficient protein synthesis and quality control system of eukaryotic cells would best optimize the therapeutic parameters of targeting antibodies. We have recently demonstrated the feasibility of a retrovirus, avian leukosis virus (ALV), as a viral platform for the display of a variety of eukaryotic polypeptides including scFvs, and the efficient generation and selection of a peptide library in eukaryotic cells. The goal of this R33 application is to demonstrate the efficiency of using the ALV display technology for the optimization of the scFv scaffold for efficient folding and expression in eukaryotic cells and for generating a panel of scFvs with a range of affinities for their target antigen with an optimized scaffold. We will use the ALV display technology to optimize two scFvs with known specificity for tumor neovasculature: an anti-laminin scFv (L36) that inhibits angiogenesis in a variety of assays, presumably due to the exposure of laminin in the extracellular matrix during tumor neovessel formation; and a scFv that recognizes a VEGF:receptor complex (LL4) specific to endothelium in tumor neovessels. The ability of the nonoptimized and the optimized scFvs to target a therapeutic agent to tumor neovessels will be assessed using oncolytic measles viruses. Specifically, we aim to: 1. Create ALV display libraries of L36 and LL4 scFv mutants by error-prone PCR. 2. Screen the ALV display libraries of scFv mutants to generate a panel of L36 and LL4 scFv mutants with a range of known affinities (from ¨M to nM) for their target antigen and with an optimized scFv scaffold. 3. Generate recombinant measles viruses displaying nonoptimized and optimized targeting scFvs and compare them with respect to ease of production, efficiency of scFv display, particle to infectivity ratios, replication kinetics, and homing properties to tumor neovessels.