Technology to Optimize scFvs for Targeting Therapeutics

优化 scFv 用于靶向治疗的技术

• 批准号: 6962134
• 负责人: Mark J Federspiel
• 金额: $ 26.82万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-19 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6962134
• 关键词: SCID mouse; angiogenesis inhibitors; antibody; antigen antibody reaction; avian leukosis virus; biotechnology; gene expression; gene mutation; genetic screening; growth factor receptors; human genetic material tag; immunoglobulin structure; immunologic substance development /preparation; laminin; measles virus; mutant; neoplasm /cancer blood supply; neoplasm /cancer immunotherapy; peptide library; phage display; polymerase chain reaction; protein folding; recombinant virus; technology /technique development; vascular endothelial growth factors; vascular endothelium

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）： 抗体为多种治疗剂提供卓越的靶向能力。 多项技术极大地促进了各种抗体试剂的初步鉴定，包括 scFv 和 Fab 抗体，几乎具有任何可能的特异性。 然而，先导抗体通常需要进一步优化，以最大限度地提高其治疗性能：优化抗体在相关细胞中的表达和折叠，以及优化抗体对靶抗原的亲和力。 有前景的抗癌靶向抗体的开发常常陷入这个瓶颈。 因此，迫切需要促进抗体适应和优化的技术。 抗体优化最好通过随机化和随后选择所需表型的抗体突变体来实现，因为有效合理的抗体设计目前还不可行。 多肽展示（例如，噬菌体展示）是用于生成和筛选表型突变多肽文库的强大技术。 采用真核细胞高效蛋白质合成和质量控制系统的真核展示技术将最好地优化靶向抗体的治疗参数。 我们最近证明了逆转录病毒、禽白血病病毒（ALV）作为病毒平台的可行性，用于展示包括 scFv 在内的多种真核多肽，以及在真核细胞中有效生成和选择肽库。 该 R33 应用的目标是证明使用 ALV 展示技术优化 scFv 支架的效率，以便在真核细胞中有效折叠和表达，并通过优化的支架生成一组对其靶抗原具有一系列亲和力的 scFv。 我们将使用 ALV 显示技术来优化两种已知对肿瘤新生血管具有特异性的 scFv：一种抗层粘连蛋白 scFv (L36)，它在各种测定中抑制血管生成，可能是由于肿瘤新生血管形成过程中层粘连蛋白暴露在细胞外基质中；以及识别肿瘤新生血管内皮特异的 VEGF：受体复合物 (LL4) 的 scFv。 将使用溶瘤麻疹病毒评估未优化和优化的 scFv 将治疗剂靶向肿瘤新血管的能力。 具体来说，我们的目标是： 1. 通过易错 PCR 创建 L36 和 LL4 scFv 突变体的 ALV 展示库。 2. 筛选 scFv 突变体的 ALV 展示文库，生成一组 L36 和 LL4 scFv 突变体，其靶抗原具有一系列已知的亲和力（从 ¿M 到 nM），并且具有优化的 scFv 支架。 3. 生成展示未优化和优化的靶向 scFv 的重组麻疹病毒，并比较它们的生产难易程度、scFv 展示效率、颗粒与感染性比率、复制动力学和肿瘤新血管的归巢特性。