Passive immunotherapy using plant-derived broadly HIV-1 neutralizing MAbs to prev

描述（由申请人提供）：虽然许多HIV疫苗方案引发了强大的病毒特异性T细胞反应，但它们无法预防感染。迄今为止，被动免疫疗法与少量广泛中和的单克隆抗体（mab）联合使用，在猴子身上提供了抵抗SHIV攻击的最佳保护。然而，在全球范围内，所需的大剂量将对现有昂贵的微生物发酵或基于细胞的生产系统产生过度需求。植物表达将提供一种廉价、安全、可快速扩展的替代方法。基因工程植物已经成功地用于生产多种复杂蛋白质，如IgG、sIgA、疫苗抗原、酶和生长因子等。这项I期研究的目的是利用农杆菌浸润/注射在植物中产生高表达的单克隆抗体，并评估其在体外广泛中和HIV-1分离株的能力。几种具有良好特征的包膜特异性单克隆抗体将在烟草中产生；2G12, 2F5, 4E10， IgG1b12以及针对cd4诱导的gp120表位的单克隆抗体。为了检验n -糖基化对这些单克隆抗体中和作用的影响，将产生内质网保留的“高甘露糖”和分泌的“复合聚糖”形式，并与cho衍生的蛋白质进行比较。在进行这些研究的同时，将启动较慢的培育稳定转基因烟草植株的进程。这将通过DsRed荧光标记的共表达来识别抗体表达系和追踪转基因后代。在目前的环境下，使用非食品、非饲料作物生产重要的治疗性蛋白质具有许多优点。在第二阶段，这些高水平表达且具有强中和活性的单克隆抗体将由转基因植物或烟草植物的大规模瞬时表达产生，并测试其预防猴感染SHIV分离物的能力。公共卫生相关性：与艾滋病毒疫苗不同，大量单克隆抗体的被动转移可以广泛中和艾滋病毒，在一天后给予SHIV分离物的致命挑战中显示出显著的保护作用。然而，在传统的表达系统中生产这些重组蛋白的成本将是过高的，一个更便宜的系统是当务之急。在这项提议中，这些单克隆抗体将在烟草植物中生产，它们在体外中和HIV和在体内保护猴子的能力将与目前使用的昂贵的cho衍生单克隆抗体进行比较。

DESCRIPTION (provided by applicant): While many HIV vaccine regimens have elicited robust viral-specific T cell responses, they have been unable to prevent infection. To date, passive immunotherapy with a small number of broadly neutralizing monoclonal antibodies (MAbs) given in combination have afforded the best protection against SHIV challenge in monkeys. However, in a global context, the large multiple doses required will create excessive demands on existing expensive microbial fermentation or cell-based production systems. Plant expression will provide an inexpensive, safe, and rapidly scalable alternative. Genetically engineered plants have been successfully used to produce many complex proteins, as diverse as IgG, sIgA, vaccine antigens, enzymes and growth factors. The aim of this Phase I study is to produce highly expressing MAbs in plants using Agrobacterium infiltration/ injection and assess their ability to broadly neutralize HIV-1 isolates in vitro. Several well characterized envelope-specific MAbs will be produced in tobacco; 2G12, 2F5, 4E10, IgG1b12 as well as a MAb against a CD4-inducible gp120 epitope. In order to examine, the effect of N-glycosylation on neutralization by these MAbs, both the ER- retained "high mannose" and the secreted "complex glycan" forms will be produced and compared to CHO-derived proteins. Concomitant with these studies, the slower process of generating stable transgenic tobacco plants will be initiated. This will be aided by coexpression of the DsRed fluorescent marker to identify antibody expressing lines and trace transgenic offspring. In the current environment, the use of a non-food, non-feed crop to produce important therapeutic proteins offers many advantages. In Phase II, those MAbs that are both expressed at high levels and have strong neutralizing activity will be produced by either transgenic plants or by large scale transient expression of tobacco plants and tested for their ability to prevent infection of monkeys challenged with an SHIV isolate. PUBLIC HEALTH RELEVANCE: Unlike HIV vaccines, the passive transfer of large amounts of monoclonal antibodies that broadly neutralize HIV virus has shown dramatic protection against a lethal challenge with a SHIV isolate given one day later. However, the cost of producing these recombinant proteins in traditonal expression systems would be exorbitant and a more inexpensive system is a high priority. In this proposal, these MAbs will be produced in tobacco plants and their ability to neutralize HIV in vitro and to protect monkeys in vivo will be compared to currently used and expensive CHO-derived MAbs.