Structural Investigation of Novel Single Domain Synthetic Biotherapeutics Targeting a Receptor Ectodomain

针对受体胞外域的新型单域合成生物治疗药物的结构研究

• 批准号: BB/I015965/1
• 金额: $ 11.71万
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI015965%2F1
• 关键词: Structural; Investigation; Novel; Single; Domain

Monoclonal IgGs have been one of the most successful discoveries in biotechnology and are used as reagents in biochemistry, diagnostics and treatment of human disease. Despite their successes, IgG molecules have practical limitations as they are large (~150 kDa) tetrameric structures. Their size and structural complexity renders them problematic to manufacture and expensive to distribute due to the need for cold storage. The limitations of the IgG have stimulated the investigation of other, recently discovered, immune proteins such as the shark VNAR (variable domain of the IgNAR, or Novel Antigen Receptor). IgNARs are a unique class of protein that have been identified in the serum of cartilaginous fish. The VNAR can be isolated as a monomeric binding domain of 12-15 kDa in size, and their smaller size makes them an attractive alternative to IgG as they have the potential to penetrate dense tissues that may be inaccessible to IgG. In addition, VNARs have been identified as possible biotherapeutics based on their robustness and solubility, propensity to bind to antigen clefts and block active sites of enzymes, and high binding affinities for a range of antigens. Pfizer has therefore developed designer synthetic libraries based on the VNAR domain. While technological relatives like the camelid VHH have been well characterized and are in clinical trials for a number of applications, the VNAR is much less well understood structurally and biophysically. The VNAR domain shares structural features with the T-cell receptor Va and the IgG Vk-chain, but sequence homology with these domains is low (~35%). The VNAR contains a relatively short CDR1 loop (CDR = Complementarity Determining Region) and a longer CDR3 loop, which create the main binding surface of the domain. Pfizer has developed large libraries of VNAR containing synthetic diversity in these key binding loops and has used these libraries to derive domains which recognise and antagonise a wide variety of drug targets, including the Receptor for Advanced Glycation End-products (RAGE). RAGE is a multi-ligand member of the immunoglobulin super-family that is implicated in the septic response. This project will investigate synthetic VNARs known to target the RAGE protein and establish a structural understanding of synthetic VNAR CDR loops and their mechanism of antigen binding. As the CDR loops have come from random amino acid diversity, rather than natural D-segment encoded diversity, their structures are likely to be novel and cannot be predicted a priori. Indeed, even for natural IgNARs, the CDRs have shown evidence for induced fit in co-crystal structures. The project will therefore focus on deriving the structures of a number of RAGE-specific synthetic VNARs via solution NMR. The structure of each clone will be derived in both the free and antigen-complexed states. Project plan: 1.Synthetic VNAR targeting RAGE were identified previously via phage display. They therefore express well in bacteria and will be cloned for overexpression in E. coli. 2.The RAGE ectodomain and a number of sequential domain deletions will be expressed and purified for use in binding assays with the purified VNARs from (1). These assays will identify domain-specificity among the VNARs and the subsequent choice of RAGE sub domains to be expressed with isotopic labels (according to established protocols in Dixon lab). 3.The expressed proteins from (1) and (2) will be used to derive solution NMR structures, investigating the structure, dynamics and antigen recognition modality of the CDR loops. 4.A lead VNAR clone will be chosen based on data from (3). This clone will undergo a 6 month process of display-based affinity maturation (placement period for the student, in Pfizer laboratories Dublin). 5.Repeat (1-3) using the affinity matured synthetic VNARs that have acquired mutations in the CDR regions, to investigate dynamics of CDR structure and mechanism of antigen recognition.

单克隆IgG是生物技术中最成功的发现之一，并用作生物化学、诊断和治疗人类疾病的试剂。尽管它们取得了成功，但IgG分子具有实际限制，因为它们是大的（~150 kDa）四聚体结构。它们的尺寸和结构复杂性使得它们的制造成问题，并且由于需要冷藏而使得分配昂贵。IgG的局限性刺激了对其他最近发现的免疫蛋白的研究，如鲨鱼VNAR（IgNAR的可变结构域，或新抗原受体）。IgNAR是一类独特的蛋白质，已在软骨鱼的血清中鉴定。VNAR可以分离为大小为12-15 kDa的单体结合结构域，并且它们的较小大小使它们成为IgG的有吸引力的替代物，因为它们具有穿透IgG可能无法接近的致密组织的潜力。此外，VNAR已被鉴定为可能的生物治疗剂，这是基于其稳健性和溶解性、结合抗原裂缝和阻断酶活性位点的倾向以及对一系列抗原的高结合亲和力。因此，Pfizer开发了基于VNAR结构域的设计师合成文库。虽然像骆驼科VHH这样的技术亲属已经得到了很好的表征，并且正在进行许多应用的临床试验，但VNAR在结构和生物药理学上的理解要少得多。VNAR结构域与T细胞受体Va和IgG Vk链共享结构特征，但与这些结构域的序列同源性较低（~35%）。VNAR包含相对短的CDR 1环（CDR =互补决定区）和较长的CDR 3环，其产生结构域的主要结合表面。辉瑞公司已经开发了在这些关键结合环中含有合成多样性的VNAR的大型文库，并且已经使用这些文库来衍生识别和拮抗各种药物靶标的结构域，包括晚期糖基化终产物受体（Receptor for Advanced Glycation End-products，RGP）。免疫球蛋白是免疫球蛋白超家族的多配体成员，与脓毒症反应有关。该项目将研究已知靶向VNAR蛋白的合成VNAR，并建立对合成VNAR CDR环及其抗原结合机制的结构理解。由于CDR环来自随机氨基酸多样性，而不是天然D-区段编码的多样性，因此它们的结构可能是新颖的，并且不能先验预测。事实上，即使对于天然IgNAR，CDR也显示出共晶体结构中诱导拟合的证据。因此，该项目将侧重于通过溶液NMR推导出一些RAGE特异性合成VNAR的结构。每个克隆的结构将在游离和抗原复合状态下推导。项目计划：1.合成的VNAR靶向噬菌体展示技术已被鉴定。因此，它们在细菌中表达良好，并将被克隆用于在E.杆菌2.将表达并纯化胞外域和许多连续的结构域缺失，以用于与来自（1）的纯化的VNAR的结合测定。这些测定将鉴定VNAR之间的结构域特异性和随后选择的待用同位素标记表达的VNAR亚结构域（根据狄克逊实验室中建立的方案）。3.来自（1）和（2）的表达蛋白将用于推导溶液NMR结构，研究CDR环的结构、动力学和抗原识别模式。4.将根据（3）中的数据选择领先的VNAR克隆。该克隆将经历6个月的基于展示的亲和力成熟过程（学生的安置期，在Pfizer实验室都柏林）。5.使用在CDR区中获得突变的亲和力成熟的合成VNAR重复（1-3），以研究CDR结构的动力学和抗原识别的机制。