Technology to Optimize scFvs for Targeting Therapeutics

描述（由申请人提供）： 抗体为多种治疗剂提供上级靶向能力。 几种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化其治疗性能：优化相关细胞中的抗体表达和折叠，以及优化抗体对靶抗原的亲和力。 有希望的针对癌症的靶向抗体的开发往往在这个瓶颈上停滞不前。 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抗体突变体用于所需表型来实现，因为抗体的有效合理设计目前不可行。 多肽展示（例如，噬菌体展示）是产生和筛选表型突变多肽文库的有力技术。 采用真核细胞的有效蛋白质合成和质量控制系统的真核展示技术将最佳地优化靶向抗体的治疗参数。 我们最近已经证明了逆转录病毒，禽白血病病毒（ALV），作为一个病毒平台的各种真核多肽，包括scFv的展示，并在真核细胞中的肽库的有效生成和选择的可行性。 该R33申请的目的是证明使用ALV展示技术优化scFv支架以在真核细胞中有效折叠和表达的效率，以及使用优化的支架产生一组对其靶抗原具有一系列亲和力的scFv的效率。 我们将使用ALV展示技术来优化两种对肿瘤新生血管具有已知特异性的scFv：抗层粘连蛋白scFv（L36），其在多种测定中抑制血管生成，推测是由于在肿瘤新生血管形成期间层粘连蛋白暴露于细胞外基质中;以及识别对肿瘤新生血管中的内皮具有特异性的VEGF：受体复合物（LL 4）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。 具体而言，我们的目标是：1。通过易错PCR创建L36和LL 4 scFv突变体的ALV展示文库。 2. 筛选scFv突变体的ALV展示文库以产生一组L36和LL 4 scFv突变体，其对其靶抗原具有一系列已知亲和力（从μ M至nM）并且具有优化的scFv支架。 3. 产生展示非优化和优化的靶向scFv的重组麻疹病毒，并比较它们在生产的容易性、scFv展示的效率、颗粒与感染性比率、复制动力学和对肿瘤新血管的归巢特性方面。

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.