A novel approach for developing GPCR subtype specific antibodies

描述（由申请人提供）：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理解是有限的。在某种程度上，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精神健康中发挥重要作用，但限制对这些疾病更好理解的另一个因素是，在任何一个神经递质GPCR家族中，成员都具有共同的特征，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它们的生物学。抗体可以作为高度特异性的分析试剂。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，我们提出通过结合两种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Lipoparticle技术，通过这种平台，gpcr可以成功地以高水平的天然构象组装成病毒样颗粒。这克服了用作免疫原的蛋白质制剂中的低浓度问题，以及用作免疫原的离散结构域线性肽导致的低特异性问题。两对相关受体将用于举例说明这种方法；甘丙蛋白1和2 （Gal1和Gal2）受体和食欲素/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通路。在Gal 1和2的情况下，这两个受体有40%的有限同源性。相比之下，OX1和OX2具有69%的同源性和80%的相似性，因此对分离能够选择性识别其中一个受体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进行一系列阳性和负性FACS选择步骤以丰富选择性高亲和抗体克隆群体后，我们将在表达每种GPCR的CHO细胞系上进行一系列测定，并将其与野生型CHO细胞结合，以确定其特异性并将其各自的表位映射到其同源GPCR上。然后，我们将对这些细胞系进行功能测定，以确定是否有任何分离的抗体表现出激动剂或拮抗剂的特性。这些研究的积极结果将提供一组针对四种gpcr不同结构域的高度特异性抗体。此外，这个I期项目将证明这种新型联合方法的力量，以产生高特异性、高亲和力的抗体，这些抗体可以区分gpcr上天然构象的独特表位。该技术可以应用于其他二期gpcr，其总体目标是将本研究分离的抗体作为研究gpcr的宝贵研究试剂推向市场。

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.