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Exploiting Superinfection Exclusion To Express Polyclonal Antibodies In Plants

利用重复感染排除在植物中表达多克隆抗体

基本信息

批准号：
8781667
负责人：
KEITH WYCOFF
金额：
$ 22.5万
依托单位：
PLANET BIOTECHNOLOGY, INC.
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-12 至 2015-09-11
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8781667
关键词：
Affinity Chromatography Agrobacterium Animals Antibodies Antivenins Biological Assay Bontoxilysin Cations Cell Culture Techniques Characteristics Chromatography Codon Nucleotides Disadvantaged Ensure Epitopes Event Exclusion Exhibits Heterogeneity Human IgG1 Immunoblotting Immunoglobulins Individual Infiltration Isoelectric Point Laboratories Light Modeling Monitor Monoclonal Antibodies Mus Nicotiana Plant Leaves Plant Viruses Plantibodies Plants Production Rabies Recombinants Reproducibility Safety Serotyping Serum Snake Bites Specificity System Testing Therapeutic Effect Therapeutic Monoclonal Antibodies Tissues Toxin Transfection Universities Viral Virus Virus Diseases Work base cancer cell cost expression vector in vivo novel polyclonal antibody potency testing prevent public health relevance secondary infection superinfection therapeutic protein

项目摘要

DESCRIPTION (provided by applicant): Cell culture produced monoclonal antibodies (mAbs) have advantages over polyclonal antibodies derived from human or animal sera, such as reproducibility of production and enhanced safety profile. Although targeting multiple epitopes on cancer cells, viruses or toxins with multiple mAbs in combination has demonstrated superior therapeutic effects compared to mAb monotherapy, oligoclonal mAb therapy is not widely practiced partially because of the cost of separately producing and characterizing multiple mAbs for use in combination. In this proposal we will test a novel idea for producing an oligoclonal mixture of mAbs using a plant expression system. Plant viruses exhibit a phenomenon called superinfection exclusion, whereby a preexisting viral infection prevents a secondary infection with the same or a closely related virus. We propose to express multiple mAbs in a single plant simultaneously, exploiting the superinfection exclusion characteristic of plant virus expression vectors to ensure that each heavy chain forms a cognate pair with the appropriate light chain. The heavy and light chain sequences of three mAbs against botulinum neurotoxin serotype A (BoNT/A) will be cloned into our dual viral expression vector, and introduced into Agrobacterum tumefaciens. MAb transient expression will be initiated by infiltration of the Agrobacterium recombinants into benthamiana leaf tissue. Infiltration conditions will be optimized to obtain high-level expression, and to facilitate comparable expression levels of all three mAbs when co-expressed. We will demonstrate that three monoclonal antibodies can be expressed without mixing up heavy and light chains and that the combination can be manufactured reproducibly. The heterogeneity of the individual antibodies and the polyclonality of each batch will be monitored using cation exchange chromatography, taking advantage of the different isoelectric points of the three mAbs. We will then compare the in vivo potency of 1) three anti-BoNT/A mAbs prepared separately in plants and then combined with 2) the same mAbs produced from a single plant infiltration, using the mouse toxin neutralization assay. The percent survival will be determined and the potency (IU/mg) of each plantibody mixture will be calculated.

描述（由申请人提供）：细胞培养产生的单克隆抗体（mAb）具有优于人或动物血清衍生的多克隆抗体的优势，例如生产的重现性和增强的安全性。尽管与mAb单一疗法相比，用组合的多个mAb靶向癌细胞、病毒或毒素上的多个表位已经证明了上级治疗效果，但寡克隆mAb疗法没有被广泛实践，部分原因是单独生产和表征用于组合的多个mAb的成本。在这项提案中，我们将测试一个新的想法，使用植物表达系统生产寡克隆混合物的单克隆抗体。植物病毒表现出一种称为重复感染排斥的现象，即预先存在的病毒感染防止了相同或密切相关的病毒的二次感染。我们建议在单一植物中同时表达多个mAb，利用植物病毒表达载体的超感染排斥特性，以确保每个重链与适当的轻链形成同源对。将三株抗A型肉毒毒素（BoNT/A）的单克隆抗体的重链和轻链序列克隆到我们的双病毒表达载体中，并导入农杆菌中。MAb瞬时表达将通过将农杆菌重组体渗入本塞姆氏菌叶组织中来启动。将优化浸润条件以获得高水平表达，并在共表达时促进所有三种mAb的表达水平相当。我们将证明三种单克隆抗体可以在不混合重链和轻链的情况下表达，并且该组合可以重复生产。利用3种mAb的不同等电点，采用阳离子交换色谱法监测单个抗体的异质性和各批次的多克隆性。然后，我们将使用小鼠毒素中和测定法比较1）在植物中分别制备的三种抗BoNT/A mAb的体内效力，然后与2）由单一植物浸润产生的相同mAb组合。将测定存活百分比并计算每种植物体混合物的效力（IU/mg）。