Method for the isolation of antibodies with functional activity against cell surface targets

具有针对细胞表面靶标的功能活性的抗体的分离方法

• 批准号: 10478450
• 负责人: Michael P Weiner
• 金额: $ 25.34万
• 依托单位: ABBRATECH, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10478450
• 关键词: Academia; Affinity; Agonist; Animal Model; Antibodies; Bacteriophages; Binding; Binding Sites; Biological; Biological Assay; Biology; Biotechnology; Cell Line; Cell Surface Receptors; Cell surface; Cells; Chemicals; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complement; Complementarity Determining Regions; Complex; Custom; Data; Diagnostic; Enzyme-Linked Immunosorbent Assay; Enzymes; Flow Cytometry; G-Protein-Coupled Receptors; Immunofluorescence Immunologic; Immunoglobulin G; Individual; Interferometry; Ion Channel; Journals; Kinetics; Libraries; Ligand Binding; Ligands; Ligase; Measurement; Methods; Modeling; Monoclonal Antibodies; Peer Review; Peptides; Phage Display; Pharmacologic Substance; Preparation; Proteins; Publishing; Reaction; Reagent; Reporter; Research; Running; Services; Site; Specificity; Structure; Surface; Surface Plasmon Resonance; Testing; Titrations; Triage; Validation; Work; antagonist; antibody libraries; antigen binding; base; commercial application; commercialization; drug development; experimental study; improved; inhibitor; novel; patch clamp; receptor; screening; small molecule; sortase; therapeutic evaluation

“Method for the isolation of antibodies with functional activity against cell surface targets” ABSTRACT We have derived a method for isolating functional mAbs against cell surface receptors that are separately either agonists, antagonists, and partial agonists. We termed the method Directed Ligand Binding (DLB). And in DLB’s first iteration (DLB1) we have successfully used it for peptide- and protein ligands. Using DLB1 these ligands were genetically incorporated into the complementarity determining region (CDR) of an appropriate phage display scFv library. At its essence DLB is a means to direct or bias the initial binding of a subtracted (to remove non-specific- and irrelevant binding) phage displayed Ab library toward a target cell surface receptor binding site by incorporating that receptor’s ligand or inhibitor into the Ab library itself. And then rely on the increased binding ability, via the interaction of both an individual mAb’s CDRs plus the covalently attached ligand to transiently stabilize the Ab::receptor complex to withstand the increasing stringency of the phage biopanning washing cycles used to remove any weak binders. In the DLB1 method, we genetically encoded the ligand into the Ab’s CDR. But that limited us to peptide and protein ligands. We now want to apply the DLB method for the enzymatic conjugation of ligands into an appropriately modified CDR of a complementing library. We refer to this new method as DLB2.0. The significant advantages of the DLB2.0 method are several fold, including: (1) the same library can be used for many screens, and (2) the means for testing mAbs derived from DLB2.0 with and without attached ligand is trivial; just omit the ligand attachment step before any screen. The proposed study uses several GPCR targets as the model and functionality in a cell-based reporter assay as the application. Regarding the commercial application, we are mainly focused on the method. It is true that the targets we have chosen to develop the method, in some cases, do not have exceptional commercial value. When the DLB2.0 generates appropriate IgGs we intend to partner and collaborate with strong academic, and pharmaceutical- and biotech companies by providing them with the IgG clones and/or purified IgG proteins so that they can perform the structural and biological studies needed to both better understand and utilize the mAbs we develop. At Abbratech intend to provide custom services to commercial entities (Pharmaceutical and Biotechnology companies) using the data obtained from this work, including any collaborative results, as a demonstration of the power of DLB2.0.

“具有细胞表面靶标功能的抗体的分离方法” 摘要 我们已经推出了一种分离针对细胞表面受体的功能性单抗的方法 激动剂、拮抗剂或部分激动剂。我们将这种方法称为定向配基结合(DLB)。 在DLB的第一次迭代(DLB1)中，我们成功地将其用于多肽和蛋白质配体。vbl.使用 DLB1这些配体被遗传地整合到阿司匹林的互补决定区 一个合适的噬菌体展示scFv文库。从本质上讲，DLB是一种指导或偏向初始绑定的手段 将消减的(去除非特异性和无关的结合)的噬菌体展示的抗体库 通过将受体的配体或抑制物结合到抗体库本身中来确定细胞表面受体的结合位置。 然后依赖于增强的结合能力，通过两个单抗的CDRs+的相互作用 共价连接的配体暂时稳定抗体：：受体复合体以抵御增加的 用于去除任何弱粘合剂的噬菌体生物扫描洗涤循环的严密性。在DLB1中 方法将该配体基因编码成抗体的CDR。但这限制了我们对肽和蛋白质的研究 配基。我们现在想要将DLB方法应用于配体的酶共轭 适当修改补充性文库的CDR。我们将这种新方法称为DLB2.0。这个 DLB2.0方法的显著优点有几个方面，包括：(1)可以使用相同的库 用于多个屏幕，以及(2)测试从DLB2.0派生的mAb的手段，带和不带附件 配基是微不足道的；只需在任何筛选之前省略配基连接步骤。拟议的研究使用了几个 以GPCR为模型，以功能为基础的细胞报告实验为应用。关于 在商业应用方面，我们主要集中在方法上。诚然，我们的目标是 选择开发的方法，在某些情况下，并不具有特殊的商业价值。当 DLB2.0生成适当的IGG，我们打算与强大的学术合作伙伴合作，并且 通过向制药和生物技术公司提供免疫球蛋白克隆和/或纯化的免疫球蛋白 以便他们能够进行所需的结构和生物学研究，以便更好地理解和利用 我们研发的单抗。在Abbratech打算向商业实体提供定制服务 (制药和生物技术公司)使用从这项工作中获得的数据，包括任何 协作成果，展示了DLB2.0的强大功能。