Targeted soluble MICA molecules to recruit innate immunity cells to kill specific

描述（由申请人提供）：本提案的总体目标是开发一种平台，该平台广泛地使得能够产生一类新的抗病毒治疗剂，该抗病毒治疗剂募集并激活先天免疫系统以特异性攻击感染有靶向病毒病原体的人细胞。该平台部署了最近发现的可运输多样性生成系统，以创建构建在支架上的大量结合结构域库，该支架天然是云母的组分，MICA是天然免疫系统NK和T细胞上NKG 2D受体的重要配体。云母分子的拟定靶向组分是β结构域，这是一种免疫球蛋白样结构，除了将NKG 2D配体结构域与云母正常附着的细胞表面隔开外，在云母中没有任何已知功能。因此，β结构域是通过专有多样性生成器进行多样化以创建靶向结构域的大规模文库的理想候选者。 分离所需的云母β结构域包括在大肠杆菌表面表达β的多样化文库作为融合蛋白。大肠杆菌，并选择与修饰有预期病毒糖蛋白靶标的荧光纳米脂蛋白颗粒结合的细菌。编码具有所需结合特性的β结构域的基因可直接从分离的E.杆菌 该系统应具有非常广泛的应用，在创造“被动治疗疫苗”的急性治疗病毒感染。我们已经选择追求最初的特点淋巴细胞脉络丛脑膜炎病毒（LCMV）和黄热病病毒（YFV），原型病毒代表高毒力沙粒病毒和黄病毒病原体，分别。 由于它们作为携带NKG 2D的先天免疫效应细胞的直接招募者的显著效力，基于MICA的特异性靶向剂可能最终取代ADCC依赖性治疗性单克隆抗体，包括靶向癌细胞上的肿瘤抗原的单克隆抗体。 公共卫生相关性：对病毒感染的最佳保护是先前被相同的病毒株感染;第二好的是针对感染株的主动疫苗接种。这两种保护机制都依赖于免疫系统的“适应性”手臂，如抗体。适应性免疫需要数周的时间才能对最初的接触或疫苗接种做出反应。 另一方面，先天免疫系统是免疫系统的一个迅速作用的，有效的，虽然是非特异性的手臂，可以抵抗一些病毒和细菌。这项提议涉及到一种名为云母的分子。云母分子经常附着在受感染细胞的外表面，并在病毒或细菌在受感染细胞内复制并传播之前招募先天免疫臂的细胞来破坏受感染的细胞。该项目的目标是将先天免疫转化为一种系统，该系统在特异性和效力方面更接近于适应性免疫，但在药物管理后可以迅速发挥作用。 云母作为“钥匙”（配体）连接到自然杀伤（NK）细胞上的“锁”（受体），NK细胞一旦参与就会杀死云母修饰的病变细胞。然而，许多毒性病毒已经找到了防止其宿主细胞的云母装饰的方法，以便阻碍先天免疫并保护其宿主细胞足够长的时间以供病毒复制和传播。该项目提出将天然人云母转化为可溶性分子，在静脉注射后可以找到并特异性结合受感染的细胞，从而招募天然免疫细胞如NK细胞进行攻击。创建具有不同结合特异性的多种可溶性云母分子的大规模文库的能力将使得能够创建具有广泛应用于特定感染性疾病的急性治疗的平台。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to develop a platform that broadly enables the generation of a new class of anti-viral therapeutics that recruit and activate the innate immunity system to attack specifically human cells infected with targeted viral pathogens. The platform deploys a recently discovered transportable diversity generating system to create massive libraries of binding domains built on a scaffold that is naturally a component of MICA, an important ligand for the NKG2D receptor on NK and T-cells of the innate immunity system. The proposed targeting component of the MICA molecule is the (3 domain, which is an immunoglobin-like structure without any known function in MICA other than spacing the NKG2D ligand domain away from the cell surface to which MICA is normally attached. Thus, the (3 domain is an ideal candidate for diversification by the proprietary diversity generator to create a massive library of targeting domains. The isolation of the desired (3 domain of MICA involves expressing the diversified library of (3 as a fusion protein on the surfaces of E. coli and selecting the bacteria that bind to fluorescent nanolipoprotein particles decorated with the intended viral glycoprotein target. The gene encoding the (3 domain with the desired binding properties can then be directly obtained from the isolated E. coli. The system should have very broad applications in the creation of "passive therapeutic vaccines" for acute treatment of viral infections. We have chosen to pursue initially the well characterized lymphocytic choriomeningitis virus (LCMV) and yellow fever virus (YFV), prototypic viruses representing the highly virulent arenavirus and flavivirus pathogens, respectively. Because of their remarkable potency as direct recruiters of NKG2D-bearing innate immunity effector cells, MICA-based specific targeting agents might eventually replace ADCC-dependent therapeutic monoclonal antibodies, including those targeting tumor antigens on cancer cells. PUBLIC HEALTH RELEVANCE: The best protection from a viral infection is a prior infection by the same viral strain; the second best is active vaccination against the infecting strain. Both of these protective mechanisms depend upon the "adaptive" arm of the immunity system, such as antibodies. Adaptive immunity takes many weeks to develop in response to the initial exposure or vaccination. The innate immunity system, on the other hand, is a promptly acting, potent although rather non-specific arm of the immunity system that can be protective against some viruses and bacteria. This proposal involves engineering a molecule called MICA. MICA molecules frequently appear attached to the outside surface of infected cells and there recruit cells of the innate immunity arm to destroy the infected cells before the virus or bacteria can replicate within the infected cells and spread. The goal of the project is to convert innate immunity into a system that more closely resembles the adaptive immunity in its specificity and potency but yet can act promptly after being administered medically. MICA serves as a "key" (ligand) to the "lock" (receptor) on Natural Killer (NK) cells that once engaged will kill the MICA decorated diseased cell. However, many virulent viruses have found means of preventing the MICA decoration of their host cells so as to thwart the innate immunity and protect their host cells long enough for them, the viruses, to replicate and spread. This project proposes to convert natural human MICA into soluble molecules that after intravenous administration can find and bind specifically to infected cells and thereby recruit the cells of innate immunity such as NK cells to attack. The ability to create a massive library of diverse soluble MICA molecules with different binding specificities will enable the creation of a platform with broad applications for the acute treatment of specific infectious diseases.