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）： 抗体为各种治疗剂提供了出色的靶向能力。几乎具有任何可能的特异性，几种可能的特异性已经大量准备了多种抗体试剂的初始身份，包括SCFV和FAB抗体。但是，铅抗体通常需要进一步优化以最大化其治疗性能：相关细胞中抗体表达和折叠的优化，以及优化抗体对靶抗原的亲和力。承诺针对癌症的靶向抗体的发展经常在这种瓶颈上萎缩。因此，迫切需要促进抗体适应和优化的技术。最好通过随后选择抗体突变体为所需表型而实现抗体优化，因为抗体的有效合理设计目前不可行。多肽显示（例如，噬菌体显示）是一项强大的技术，用于生成和筛选突变多肽的库以用于表型。一种采用有效的真核细胞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。最近，我们证明了逆转录病毒，禽白血病病毒（ALV）的可行性，是一种病毒平台，用于显示包括SCFV在内的各种真核多肽，以及在真核细胞中肽库的有效产生和选择。该R33应用的目的是证明使用ALV显示技术优化SCFV支架的效率，以在真核细胞中有效折叠和表达，并生成一组SCFV，并具有一系列具有优化支架的目标抗原的亲密关系的SCFV。我们将使用ALV显示技术来优化两种具有已知特异性的SCFV，这些SCFV具有肿瘤神经系统的已知特异性：一种抗延纹蛋白SCFV（L36），可抑制多种测定中的血管生成，这可能是由于肿瘤新生儿氏菌形成过程中细胞外基质中层粘连蛋白的暴露；以及识别VEGF的SCFV：受体复合物（LL4）特定于肿瘤新系列中的内皮。非优化的SCFV和优化的SCFV靶向治疗剂的能力将使用肿瘤麻疹病毒评估。具体来说，我们的目标是：1。通过容易发生的PCR创建L36和LL4 SCFV突变体的ALV显示库。 2。筛选SCFV突变体的ALV显示库，以生成一组L36和LL4 SCFV突变体，这些突变体具有一系列已知亲和力（从€M到NM）的目标抗原和优化的SCFV支架。 3。产生重组麻疹病毒，表现出非优化和优化的靶向SCFV，并将其相对于易于生产，SCFV显示效率，颗粒与感染比，复制动力学以及与肿瘤新系列的归属特性进行比较。