Targeting Viral Envelope Glycoproteins with Synthetic Antibodies

描述（由申请方提供）：针对1型人类免疫缺陷病毒（HIV-1）、流感病毒和埃博拉病毒等膜病毒包膜糖蛋白的单克隆抗体是解读病毒进入机制和鉴定免疫治疗或疫苗开发表位的必要试剂。最近在抗体工程方面的工作已经证明，特异性的高亲和力抗体可以从简单的噬菌体展示文库中分离，其中抗体互补决定区（CDR）的多样性由设计的、合成衍生的寡核苷酸（“合成抗体”）编码。因此，合成抗体方法规避了传统抗体分离方法的许多限制，从而扩大了抗原可被靶向的范围和特异性。该提案的总体目标是开发使能用于病毒膜融合研究的合成抗体技术。在目标1中，我们提出开发基于混杂生殖系片段VH 1 -69的针对病毒抗原的合成抗体文库。从该祖细胞产生的许多病毒抗体表现出与其病毒抗原的类似的相互作用模式，表明VH 1 -69可以用作开发病毒特异性合成抗体文库的支架。我们将生产基于VH 1 -69的合成抗体文库，并针对病毒靶标对其进行筛选，以评估这一假设。在目标2中，我们提出鉴定靶向HIV-1 gp 41的近膜外部区域（MPER）的构象特异性抗体。MPER是几种广泛中和抗体（NAb）的靶点，因此是疫苗开发的重点研究对象。我们将鉴定针对结构受限的MPER肽的合成抗体，然后表征这些抗体的中和效力，以深入了解MPER表位构象对膜融合抑制的作用。在目标3中，我们提出分离针对埃博拉病毒GP 1和GP 2的推定融合中间体的合成抗体。与HIV-1和流感相比，已经分离出相对较少的针对GP 1和GP 2的抗体，这极大地阻碍了对该病毒的膜融合的机制的理解。我们的方法将克服未能产生GP 1或GP 2特异性抗体的其他抗体分离方法的局限性，并产生用于融合中间体和潜在免疫治疗剂的解剖的新试剂。

DESCRIPTION (provided by applicant): Monoclonal antibodies directed against the envelope glycoproteins of membrane viruses such as human immunodeficiency virus type 1 (HIV-1), influenza, and ebolavirus are essential reagents for deciphering mechanisms of viral entry, and identifying epitopes for immunotherapy or vaccine development. Recent work in antibody engineering has demonstrated that specific, high-affinity antibodies can be isolated from simple phage display libraries in which diversity at the antibody complementarity determining regions (CDRs) is encoded by designed, synthetically-derived oligonucleotides ('synthetic antibodies'). Therefore, the synthetic antibody approach circumvents many limitations of traditional antibody isolation methods thereby expanding the scope and specificity with which antigens may be targeted. The overall goal of this proposal is to develop enabling synthetic antibody technologies for applications in the study of viral membrane fusion. In Aim 1, we propose to develop synthetic antibody libraries focused toward viral antigens based on the promiscuous germline segment VH1-69. Many viral antibodies borne from this progenitor exhibit similar modes of interaction with their viral antigens, suggesting that VH1-69 could serve as a scaffold for development of virus- specific synthetic antibody libraries. We will produce VH1-69-based synthetic antibody libraries and screen them against viral targets to evaluate this hypothesis. In Aim 2, we propose to identify conformation-specific antibodies that target the membrane-proximal external region (MPER) of HIV-1 gp41. The MPER is the target of several broadly neutralizing antibodies (NAbs) and therefore the subject of intense investigation for vaccine development. We will identify synthetic antibodies against structurally-constrained MPER peptides, and then characterize neutralization potency of these antibodies to gain insight into the role of MPER epitope conformation on inhibition of membrane fusion. In Aim 3, we propose to isolate synthetic antibodies against the putative fusion intermediates of ebolavirus GP1 and GP2. In comparison to HIV-1 and influenza, relatively few antibodies against GP1 and GP2 have been isolated which has greatly impeded mechanistic understanding of membrane fusion for this virus. Our approach will overcome limitations of other antibody isolation methods that have failed to produce GP1- or GP2-specific antibodies, and yield novel reagents for dissection of fusion intermediates and potential immunotherapeutics.