RATIONAL DESIGN TO MODULATE ANTIBODY AFFINITY

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 作为治疗剂的单抗因其特异性和激发免疫反应的能力，拮抗信号通路，以及作为在疾病部位运送细胞毒性化合物的载体而被公认。临床上的许多单抗通常识别疾病细胞表面人源性抗原的过度表达，最突出的是针对Erb家族的西妥昔单抗(Erbitux)和曲妥珠单抗(Herceptin)(例如，EGFR和Her2)。这些受体经常在实体肿瘤中过度表达，包括转移性结直肠癌、头颈部和乳腺癌，但通常也在上皮细胞中表达。在治疗剂量下，正常组织中的受体也被激活，导致副作用(例如，心脏毒性和PML)。这些副作用降低了疗效，缩小了治疗窗口，并限制了单抗治疗的持续时间。为了解决这些严重的并发症，我们最近开发了一种方法来调节治疗性单抗的抗原亲和力，并使用肿瘤相关蛋白酶在疾病部位激活单抗。我们发现，‘前抗体’的裂解可以恢复单抗的抗原亲和力。我们现在对偶联小分子以微调抗体亲和力的调节感兴趣。为此，我们将使用溴化小分子文库来浸泡治疗性Fab片段的晶体，并使用同步辐射通过SAD/MAD相变来识别结合片段。这些信息将使我们能够将这些小分子偶联到多肽上，并产生新型的掩蔽剂。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Monoclonal antibodies (mAbs) as therapeutic agents are recognized for their specificity, and ability to elicit an immune response, antagonize signaling pathways, and as vehicles to deliver cytotoxic compounds at the disease site. Many of the mAbs in the clinic typically recognize the overexpression of human-derived antigens on the surface of diseased cells, the most prominent being cetuximab (Erbitux) and trastuzumab (Herceptin) which target the Erb family (e.g., EGFR and Her2). These receptors are frequently overexpressed in solid tumors including metastatic colorectal, head and neck and breast cancers, but are also normally expressed in epithelial cells. At therapeutic doses, the receptors in normal tissues are also engaged, leading to side effects (e.g., cardiotoxicity and PML). These side effects reduce the efficacy, narrow the therapeutic window, and limit the length of the administration of mAb treatment. To address these serious complications, we have recently developed a method to modulate the antigen affinity of therapeutic mAbs and use a tumor-associated protease to active the mAb at the disease site. We show that cleavage of the 'pro-antibody' restores the mAb antigen affinity. We are now interested in coupling small molecules to fine tune the modulation of antibody affinity. To do so, we will use a brominated, small molecule library to soak crystals of the therapeutic Fab fragment, and use synchrotron radiation to identify bound fragments through SAD/MAD phasing. This information will allow us to couple these small molecules to peptides and generate novel masking agents.