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：
8198932
负责人：
XIAOMIN FAN
金额：
$ 34.97万
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 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8198932
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. PUBLIC HEALTH RELEVANCE: Mental health disorders affect 3.5% of the U.S. population and will afflict 15% of the population over their lifetime. Despite the development of many new therapies, they are all limited by serious side effects, including extrapyramidal effects (uncontrolled movements), obesity and diabetes. These agents target receptors of key neurotransmitters (or brain messengers) in the brain that share many common features yet differ markedly in their selective modes of action. There is insufficient information about the 3-D structure (conformation) of these receptors, and the precise differences that exist between related members of these protein families. Current therapies target many different receptors within these families and are therefore non-specific, leading to positive drug effects as well as undesired side effects. The goal of this project is to use a novel approach to develop antibodies that are specific for subtypes of G-protein couple receptors and to study these receptors in their native form.

描述（由申请人提供）：尽管严重的精神疾病普遍存在，但我们目前对精神障碍的病理生理学的了解仍然有限。部分原因是中枢神经系统的复杂性以及在动物模型中重现此类疾病的难度。虽然各种神经递质的 G 蛋白偶联受体家族已被证明在正常和功能失调的心理健康中发挥着重要作用，但限制更好地了解这些疾病的另一个因素是，在任何一个神经递质 GPCR 家族中，成员都有共同的特征，例如配体结合袋和激动剂/拮抗剂结合位点的性质，需要高度特异性的工具来研究其生物学。抗体可以用作高度特异性的分析剂。然而，最近的研究对许多常用抗体的特异性提出了质疑。在这里，我们建议通过结合两个新颖的平台来体外筛选高亲和力和高特异性的抗体：新颖的酵母抗体展示平台和脂质颗粒技术，从而GPCR可以以天然构象成功地以高水平组装成病毒样颗粒。这克服了用作免疫原的蛋白质制剂浓度低以及用作免疫原的来自离散结构域的线性肽导致的特异性差的问题。将使用两对相关受体来举例说明这种方法；甘丙肽 1 和 2（Gal1 和 Gal2）受体以及食欲素/下丘脑分泌素受体 OX1 和 OX2。一对中的每个成员都与相同的神经肽配体结合，但具有不同的亲和力并触发不同的下游信号传导途径。对于 Gal 1 和 2，这两种受体的同源性有限，仅为 40%。相比之下，OX1 和 OX2 具有 69% 的同一性和 80% 的相似性，因此对分离能够选择性识别一种或另一种受体的抗体克隆提出了更大的挑战。对一对中的每个抗原成员进行一系列正向和消减 FACS 选择步骤以富集选择性、高亲和力抗体克隆群体后，我们将通过对表达每个 GPCR 的 CHO 细胞系进行一系列分析来表征各个克隆结合野生型 CHO 细胞以确定其特异性并将其各自的表位绘制在其同源 GPCR 上。然后，我们将对这些细胞系进行功能测定，以确定任何分离的抗体是否表现出激动剂或拮抗剂特性。这些研究的积极成果将提供一组针对四种 GPCR 的不同结构域的高度特异性抗体。此外，这一第一阶段项目将体现这种新颖的组合方法的力量，以产生高度特异性、高亲和力的抗体，这些抗体可以区分 GPCR 上天然构象中存在的独特表位。该技术可应用于第二阶段的其他 GPCR，总体目标是将本研究分离的抗体作为研究 GPCR 的宝贵研究试剂进行销售。公共卫生相关性：精神健康障碍影响着 3.5% 的美国人口，并将影响 15% 的人口一生。尽管开发了许多新疗法，但它们都受到严重副作用的限制，包括锥体外系效应（不受控制的运动）、肥胖和糖尿病。这些药物以大脑中关键神经递质（或大脑信使）的受体为目标，这些受体具有许多共同特征，但其选择性作用模式却存在显着差异。关于这些受体的 3-D 结构（构象）以及这些蛋白质家族相关成员之间存在的精确差异的信息不足。目前的疗法针对这些家族中的许多不同受体，因此是非特异性的，导致积极的药物作用以及不希望的副作用。该项目的目标是使用一种新方法来开发针对 G 蛋白偶联受体亚型的特异性抗体，并研究这些受体的天然形式。