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

单抗IgGs是生物技术领域最成功的发现之一，被用作生物化学、诊断和治疗人类疾病的试剂。尽管取得了成功，但由于免疫球蛋白分子是大的(~150 kDa)四聚体结构，因此具有实际的局限性。它们的大小和结构复杂，使得它们难以制造，而且由于需要冷藏，分发成本高昂。免疫球蛋白的局限性刺激了对其他新近发现的免疫蛋白的研究，如鲨鱼VNAR(IgNAR的可变区，或新的抗原受体)。IgNARs是一类独特的蛋白质，已在软骨鱼的血清中发现。VNAR可分离为12-15 kDa的单体结合域，其较小的尺寸使其具有潜在的穿透致密组织的潜力，使其成为免疫球蛋白的替代品。此外，VNAR已被确定为可能的生物疗法，基于它们的健壮性和溶解性，结合到抗原裂隙和阻断酶的活性部位的倾向，以及对一系列抗原的高结合亲和力。因此，辉瑞公司开发了基于VNAR结构域的设计师合成库。虽然像骆驼VHH这样的技术近亲已经很好地描述了VHH的特征，并正在进行一些应用的临床试验，但VNAR在结构和生物物理上还不太清楚。VNAR结构域与T细胞受体Va和Ig G VK链具有相同的结构特征，但序列同源性较低(~35%)。VNAR包含一个相对较短的CDR1环(CDR=互补决定区)和一个较长的CDR3环，这两个环创建了结构域的主要结合表面。辉瑞公司开发了包含这些关键结合环中合成多样性的大型VNAR文库，并使用这些文库衍生出识别和拮抗各种药物靶点的结构域，包括高级糖基化终产物受体(RAGE)。RAGE是免疫球蛋白超家族中的一个多配体成员，与败血症反应有关。该项目将研究已知的靶向RAGE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，而不是自然D节段编码的多样性，它们的结构很可能是新的，无法先验预测。事实上，即使对于天然的IgNARs，CDR也显示出诱导的共晶结构匹配的证据。因此，该项目将侧重于通过溶液核磁共振得出一些特定于RAGE的合成VNAR的结构。每个克隆的结构都将在自由和抗原复合状态下获得。项目计划：1.通过噬菌体展示鉴定合成的靶向RAGE的VNAR。因此，它们在细菌中很好地表达，并将被克隆，以便在大肠杆菌中过度表达。2.RAGE胞外结构域和一些序列结构域缺失将被表达和纯化，用于与从(1)中纯化的VNAR的结合分析。这些分析将确定VNAR和随后选择的RAGE亚域之间的区域特异性，以用同位素标记表达(根据Dixon实验室建立的方案)。3.从(1)和(2)中表达的蛋白质将用于衍生溶液核磁共振结构，研究CDR环的结构、动力学和抗原识别模式。4.将根据(3)中的数据选择主要的VNAR克隆。这个克隆人将经历为期6个月的基于展示的亲和力成熟过程(学生在辉瑞都柏林实验室的安置期)。5.利用获得CDR区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机制。

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.