A novel approach for developing GPCR subtype specific antibodies

开发 GPCR 亚型特异性抗体的新方法

基本信息

批准号：
8330248
负责人：
XIAOMIN FAN
金额：
$ 34.2万
依托单位：
AVANTGEN, INC.
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-08 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8330248
关键词：
3-Dimensional Adverse effects Affect Affinity Agonist Aliquot Animal Model Antibodies Antibody Affinity Antigen Targeting Antigens Binding Binding Sites Biological Assay Biology Brain Cell Line Cell membrane Chinese Hamster Ovary Cell Common Epitope Communities Development Diabetes Mellitus Disease Ensure Environment Enzyme-Linked Immunosorbent Assay Epitope Mapping Epitopes Europium Exhibits Family Family member Functional disorder G Protein-Coupled Receptor Genes G-Protein-Coupled Receptors GALR1 Galanin Receptor Galanin Goals Human Hybridomas In Vitro Individual Label Libraries Ligand Binding Ligands Maps Marketing Mental Health Mental disorders Methods Molecular Molecular Conformation Movement Nature Neuropeptides Neurotransmitters Obesity Outcome Outcome Study Peptides Pharmaceutical Preparations Phase Play Population Preparation Prevalence Production Property Protein Family Proteins Protocols documentation Reagent Research Role Screening procedure Series Signal Pathway Sorting - Cell Movement Specificity Structure Surface Technology Testing Viral Virus Receptors Yeasts analytical tool antigen binding base extracellular galanin receptor hypocretin member novel novel strategies particle psychologic receptor severe mental illness stable cell line tool

项目摘要

DESCRIPTION (provided by applicant): Despite the prevalence of serious mental illness, our current understanding of the pathophysiology of mental disorders is limited. In part, this is due to the complexity of the CNS and difficulty of recapitulating such illnesses in animal models. While G-protein coupled receptor families for various neurotransmitters have been shown to play major roles in normal and dysfunctional mental health, another factor limiting a better understanding of these disorders is that within any one neurotransmitter GPCR family, members share common features, such as the nature of the ligand binding pockets and agonist/antagonist binding sites, necessitating highly specific tools to study their biology. Antibodies can serve as highly specific analytical agents. However, recent studies have called into question the specificity of many commonly available antibodies. Here, we propose to screen for high affinity and highly specific antibodies in vitro by combining two novel platforms: novel yeast antibody display platform and Lipoparticle technology, whereby GPCRs can be successfully assembled at high levels in a native conformation into viral-like particles. This overcomes the problems of low concentration in protein preparations used as immunogens, and poor specificity resulting from linear peptides from discrete domains used as immunogens. Two pairs of related receptors will be used to exemplify this approach; the galanin 1 and 2 (Gal1 and Gal2) receptors and the orexin/hypocretin receptors OX1 and OX2. Each member of a pair binds to the same neuropeptide ligand, but with differing affinities and triggering different downstream signaling pathways. In the case of Gal 1 and 2, the two receptors share limited homology of 40%. In contrast, OX1 and OX2 share 69% identity and 80% similarity and therefore present a greater challenge to isolate antibody clones that can selectively recognize one or the other receptor. After performing a series of positive and subtractive FACS selection steps with each antigen member within a pair to enrich for a population of selective, high affinity antibody clones, we will characterize individual clones with a series of assays on CHO cell-lines expressing each GPCR compared to binding on wild-type CHO cells to determine their specificity and map their respective epitopes on their cognate GPCRs. We will then perform functional assays with these cell lines to determine whether any of the isolated antibodies exhibits agonist or antagonist properties. A positive outcome of these studies will provide a panel of highly specific antibodies against different domains of each of the four GPCRs. Furthermore, this Phase I project will exemplify the power of this novel combined approach to yield highly specific, high affinity antibodies that can distinguish unique epitopes present on GPCRs in their native conformation. This technology can be applied to other GPCRs in Phase II with an overall goal of marketing the antibodies isolated by this research as invaluable research reagents for studying GPCRs.

描述（由申请人提供）：尽管严重精神疾病的率很高，但我们目前对精神疾病的病理生理学的理解仍然有限。在某种程度上，这是由于中枢神经系统的复杂性和在动物模型中概括此类疾病的困难。虽然已显示针对各种神经递质的G蛋白耦合受体家族在正常和功能障碍的心理健康中起主要作用，但另一个因素限制了对这些疾病的更好理解的另一个因素是，在任何一个神经递质GPCR家族中，成员在任何一种神经递质中都具有共同的特征，例如，与辅助袋和agonist tement tooks took interiantintintinitiantinitiantinitiantinitiantinitiant具有粘合剂的依赖性。抗体可以用作高度特定的分析剂。但是，最近的研究质疑许多常用抗体的特异性。在这里，我们建议通过结合两个新型平台来筛选体外高亲和力和高度特异性抗体：新型酵母抗体显示平台和脂肪粒子技术，从而可以将GPCR成功地在天然构型中以高水平的形式成功组装成病毒样颗粒。这克服了用作免疫原子的蛋白质制剂中低浓度的问题，以及由用于免疫原子的离散域的线性肽引起的较差的特异性。将使用两对相关受体来体现这种方法。 Galanin 1和2（GAL1和GAL2）受体以及Orexin/pocretin受体OX1和OX2。一对的每个成员都与同一神经肽配体结合，但具有不同的亲和力并触发了不同的下游信号通路。对于GAL 1和2，两个受体具有40％的有限同源性。相反，OX1和OX2具有69％的身份和80％的相似性，因此对可以选择性地识别一种或另一个受体的抗体克隆提出了更大的挑战。在与一对抗原成员进行一系列积极和减法的选择步骤之后，以丰富选择性，高亲和力抗体克隆的群体，我们将对单个克隆进行特征，并在CHO细胞线上进行一系列测定，表达每个GPCR，与对野生型CHO的结合相比，表达每个GPCR，以确定其特定性并映射他们的特定性并映射其对cogn的表现。然后，我们将使用这些细胞系执行功能测定，以确定任何分离的抗体是否具有激动剂或拮抗剂特性。这些研究的积极结果将为四个GPCR的不同领域提供一系列高度特异性的抗体。此外，这个I阶段项目将体现这种新型组合方法的力量，以产生高度特异性的高亲和力抗体，这些抗体可以区分GPCR上的独特构象中存在的独特表位。该技术可以应用于II阶段的其他GPCR，其总体目标是营销该研究隔离的抗体，作为研究GPCR的宝贵研究试剂。