Technology to Optimize scFvs for Targeting Therapeutics

描述(由申请人提供)： 抗体为各种治疗剂提供了优越的靶向能力。几种技术极大地促进了各种抗体试剂的初步鉴定，包括单链抗体和Fab抗体，几乎具有任何可能的特异性。然而，铅抗体往往需要进一步的优化才能最大限度地发挥其治疗作用：优化抗体在相关细胞中的表达和折叠，以及优化抗体与靶抗原的亲和力。有希望的抗癌靶向抗体的发展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针对所需表型选择抗体突变体来实现，因为目前还不可行有效、合理的抗体设计。多肽展示(如噬菌体展示)是一种用于产生和筛选表型突变多肽文库的强大技术。利用真核细胞高效的蛋白质合成和质量控制系统的真核展示技术将最大限度地优化靶向抗体的治疗参数。我们最近证明了一种逆转录病毒，即禽白血病病毒(ALV)，作为展示包括单链抗体在内的各种真核多肽的病毒平台的可行性，以及在真核细胞中高效地产生和选择多肽库的可行性。本R33应用程序的目的是展示使用ALV展示技术优化scFv支架的效率，以便在真核细胞中高效折叠和表达，并利用优化的支架产生一组与其靶抗原具有一定亲和力的scFv。我们将使用ALV展示技术来优化两个已知具有肿瘤新生血管特异性的scFv：抗层粘连蛋白scFv(L36)，它在各种检测中抑制血管生成，可能是由于在肿瘤新血管形成过程中细胞外基质中暴露出层粘连蛋白；以及一个scFv，它识别肿瘤新血管内皮细胞特异性的血管内皮生长因子受体复合体(LL4)。未优化和优化的单链抗体靶向治疗肿瘤新生物的能力将使用溶瘤麻疹病毒进行评估。具体来说，我们的目标是：1.通过易错聚合酶链式反应建立L36和LL4单链抗体突变体的ALV展示文库。2.筛选单链抗体突变体的ALV展示文库，以产生一组L36和LL4单链抗体突变体，这些突变体与其靶抗原具有一系列已知的亲和力(从M到NM)，并具有优化的scFv支架。3.制备具有非优化和优化靶向单链抗体的重组麻疹病毒，并比较它们的生产简便性、单链抗体展示效率、颗粒与感染性比、复制动力学和对肿瘤新血管的归巢特性。

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.