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

描述(由申请人提供)：这项提案的总体目标是开发一个平台，广泛地使一类新的抗病毒疗法的产生能够招募和激活先天免疫系统，以特别攻击感染目标病毒病原体的人类细胞。该平台部署了最近发现的可运输多样性产生系统来创建大量结合结构域的文库，该结合结构域建立在支架上，该支架自然是MICA的组成部分，MICA是天然免疫系统NK和T细胞上NKG2D受体的重要配体。MICA分子的靶向成分是(3)结构域，这是一种免疫球蛋白样结构，在MICA中除了将NKG2D配体结构域与MICA通常连接的细胞表面隔开外，没有任何已知功能。因此，(3)域是专有多样性生成器进行多样化的理想候选者，以创建一个庞大的目标域库。 MICA所需的(3)结构域的分离包括在大肠杆菌表面以融合蛋白的形式表达(3)的多样化文库，并选择与带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。然后，可以从分离的大肠杆菌中直接获得编码具有所需结合特性的(3)结构域的基因。 该系统在创造用于急性治疗病毒感染的“被动治疗性疫苗”方面应该有非常广泛的应用。我们最初选择了特征良好的淋巴细胞性脉络膜脑膜炎病毒(LCMV)和黄热病病毒(YFV)，这两种病毒分别代表强毒和黄病毒的病原体。 由于其作为携带NKG2D的天然免疫效应细胞的直接招募者的显著效力，基于MICA的特异性靶向试剂最终可能取代依赖于ADCC的治疗性单抗，包括那些针对癌细胞上的肿瘤抗原的单抗。 公共卫生相关性：对病毒感染的最好保护是先前感染相同的病毒株；其次是对感染株进行主动疫苗接种。这两种保护机制都依赖于免疫系统的“适应性”臂，如抗体。适应性免疫需要数周时间才能形成，以应对最初的暴露或疫苗接种。另一方面，先天免疫系统是免疫系统的一个迅速起作用的、强大的、尽管相当非特异性的手臂，可以保护自己免受某些病毒和细菌的伤害。这项提议涉及设计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，在病毒或细菌可以在受感染细胞内复制和传播之前，它们招募先天免疫臂的细胞来摧毁受感染细胞。该项目的目标是将先天免疫转化为一种在特异性和效力上与获得性免疫更相似的系统，但在医学上给予后仍能迅速发挥作用。云母是自然杀伤(NK)细胞上“锁定”(受体)的“钥匙”(配体)，一旦参与，将杀死云母装饰的疾病细胞。然而，许多强毒病毒已经找到了防止其宿主细胞装饰MICA的方法，从而挫败了天然免疫，并保护它们的宿主细胞足够长的时间，使它们--病毒--能够复制和传播。该项目提出将天然的人MICA转化为可溶性分子，静脉给药后可以发现并与感染细胞特异性结合，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性的不同可溶性MICA分子的大规模文库的能力将使创建一个具有广泛应用的平台，用于特定传染病的急性治疗。

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.