A novel approach for developing GPCR subtype specific antibodies

描述(申请人提供)：尽管严重的精神疾病很普遍，但我们目前对精神障碍的病理生理学的了解是有限的。在一定程度上，这是由于中枢神经系统的复杂性，以及在动物模型中重述此类疾病的难度。虽然各种神经递质的G蛋白偶联受体家族在正常和功能障碍的精神健康中发挥着重要作用，但另一个限制人们更好地理解这些疾病的因素是，在任何一个神经递质GPCR家族中，成员都有共同的特征，如配体结合口袋和激动剂/拮抗剂结合部位的性质，这就需要高度特异的工具来研究它们的生物学。抗体可以作为高度特异的分析试剂。然而，最近的研究对许多常见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的平台来筛选高亲和力和高特异性的抗体：新型酵母抗体展示平台和脂肪制品技术，借此GPCRs可以成功地以天然构象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的问题，以及用作免疫原的离散结构域线性多肽导致的特异性差的问题。两对相关的受体将被用来举例说明这一方法：甘丙素1和2(Gal1和Gal2)受体以及增食欲素/下克隆素受体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通路。在Gal1和Gal2的情况下，这两个受体的同源性有限，仅为40%。相比之下，OX1和OX2有69%的同源性和80%的相似性，因此对分离能够选择性识别一个或另一个受体的抗体克隆提出了更大的挑战。在对一对中的每个抗原成员执行一系列正向和消减FAC选择步骤以丰富选择性、高亲和力抗体克隆群后，我们将在表达每个GPCR的CHO细胞系上进行一系列分析来表征单个克隆，并将其与野生型CHO细胞结合进行比较，以确定它们的特异性，并将它们各自的表位映射到它们的同源GPCRs上。然后，我们将对这些细胞系进行功能分析，以确定是否有任何分离的抗体表现出激动剂或拮抗剂特性。这些研究的阳性结果将提供一组针对四个GPCR的不同区域的高度特异性抗体。此外，这个第一阶段的项目将展示这种新的组合方法的力量，以生产高特异性、高亲和力的抗体，能够区分GPCR上存在的天然构象中的独特表位。这项技术可以应用于第二阶段的其他GPCRs，总体目标是将本研究分离的抗体作为研究GPCRs的宝贵研究试剂进行营销。 公共卫生相关性：心理健康障碍影响了3.5%的美国人口，并将在他们的一生中困扰15%的人口。尽管开发了许多新的治疗方法，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。这些药物针对的是大脑中关键神经递质(或大脑信使)的受体，这些受体有许多共同的特征，但在选择性作用模式上有明显的不同。关于这些受体的三维结构(构象)，以及这些蛋白质家族相关成员之间存在的确切差异，信息不足。目前的治疗方法针对这些家族中的许多不同受体，因此是非特异性的，导致了积极的药物效果和不希望看到的副作用。该项目的目标是使用一种新的方法来开发针对G蛋白偶联受体亚型的抗体，并研究这些受体的天然形式。

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.