Novel Strategies for Selection of Anti-GPCR Antibodies

选择抗 GPCR 抗体的新策略

• 批准号: 7108894
• 负责人: ROBERT F BALINT
• 金额: $ 29.96万
• 依托单位: CYTODESIGN, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7108894
• 关键词: antibody; conformation; human; receptor

DESCRIPTION (provided by applicant): G-protein-coupled receptors (GPCR) are the largest class of signal-transducing receptors on human cells. As a result, GPCRs play essential roles in many human diseases, and today constitute more than 30% of all clinically approved therapeutic targets. It is widely recognized that many GPCRs would make excellent therapeutic targets for antibodies. However, for a variety of reasons, most GPCRs are poor antigens. GPCRs span the plasma membrane 7 times, and thus, most GPCRs cannot be obtained in a usable form for antibody selection in vitro or in vivo. They are also difficult to express at high levels in heterologous hosts, and do not elicit strong responses from animals immunized with GPCR-expressing cells. As a result of these obstacles, no anti-GPCR antibodies have yet been approved for therapeutic use in humans. Peptides corresponding to the extra-cellular structures of GPCRs have been used to raise or select antibodies, but since they lack native conformations, the resulting antibodies have not had adequate affinities or specificities for therapeutic use. We have developed a method for stabilizing extra-cellular structures of receptors in native-like conformations by scaffolding them onto carrier proteins and selecting peptide "chaperones" from random peptide libraries inserted between the extra-cellular domains and the carrier protein, using natural ligands or conformation-sensitive antibodies as the selectors. The carrier of choice is beta-lactamase because it allows for efficient selection for ligand-stabilized receptor conformations from very large chaperone libraries in E. coli by antibiotic resistance. We will use this strategy to stabilize the extra-cellular domains of GPCRs in soluble form, so that high-affinity antibodies can be obtained and developed for therapeutic use. We will focus on chemokine receptors because they play essential roles in many inflammatory diseases, and high-affinity antibodies could have therapeutically useful anti-inflammatory activities with greater specificity and less toxicity than currently available small molecule anti-inflammatorites. In Phase I, we will optimize the extra-cellular domains of 2 key human inflammatory chemokine receptors, CXCR1 and CXCR2, using the neutrophil chemokine IL-8 to select native conformations of these GPCRs on the surface of beta-lactamase. A panel of stable IL-8 binders will then be used to obtain anti-CXCR1 and anti-CXCR2 antibodies. In Phase II, the best antibodies obtained in Phase I will be studied in animal models of inflammatory lung disease. Additional GPCRs will be engineered for antibody selection, and corporate partners will be sought for the development of anti-GPCR antibody products for therapeutic, diagnostic, and research markets.

描述（由申请人提供）：G蛋白偶联受体（GPCR）是人类细胞上最大的一类信号转导受体。因此，GPCR 在许多人类疾病中发挥着重要作用，目前占所有临床批准的治疗靶点的 30% 以上。人们普遍认为许多 GPCR 可以成为抗体的极好治疗靶点。然而，由于多种原因，大多数 GPCR 都是较差的抗原。 GPCR 跨越质膜 7 次，因此，大多数 GPCR 无法以可用于体外或体内抗体选择的形式获得。它们也很难在异源宿主中高水平表达，并且不会引起用 GPCR 表达细胞免疫的动物的强烈反应。由于这些障碍，目前还没有抗 GPCR 抗体被批准用于人类治疗用途。与 GPCR 细胞外结构相对应的肽已被用来产生或选择抗体，但由于它们缺乏天然构象，所得抗体不具有足够的亲和力或特异性用于治疗用途。我们开发了一种将受体的细胞外结构稳定在天然构象的方法，方法是将它们支架到载体蛋白上，并使用天然配体或构象敏感抗体作为选择器，从插入细胞外结构域和载体蛋白之间的随机肽库中选择肽“伴侣”。选择的载体是β-内酰胺酶，因为它允许通过抗生素抗性从大肠杆菌中非常大的伴侣库中有效地选择配体稳定的受体构象。我们将利用这种策略以可溶形式稳定 GPCR 的胞外结构域，从而获得高亲和力的抗体并开发用于治疗用途。我们将重点关注趋化因子受体，因为它们在许多炎症性疾病中发挥重要作用，并且高亲和力抗体可以具有治疗上有用的抗炎活性，比目前可用的小分子抗炎药具有更高的特异性和更低的毒性。在第一阶段，我们将优化 2 个关键人类炎症趋化因子受体 CXCR1 和 CXCR2 的胞外结构域，使用中性粒细胞趋化因子 IL-8 选择这些 GPCR 在 β-内酰胺酶表面的天然构象。然后使用一组稳定的 IL-8 结合剂来获得抗 CXCR1 和抗 CXCR2 抗体。在第二阶段，第一阶段获得的最佳抗体将在炎症性肺病动物模型中进行研究。将设计更多 GPCR 用于抗体选择，并将寻求企业合作伙伴为治疗、诊断和研究市场开发抗 GPCR 抗体产品。