A novel approach for developing GPCR subtype specific antibodies

描述（由申请人提供）：尽管严重精神疾病的流行，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和在动物模型中重现此类疾病的困难。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，但限制更好地理解这些疾病的另一个因素是，在任何一种神经递质GPCR家族中，成员具有共同的特征，例如配体结合口袋和激动剂/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：新的酵母抗体展示平台和Lipoparticle技术，从而可以成功地将GPCR以高水平以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，以及由用作免疫原的来自离散结构域的线性肽导致的低特异性问题。两对相关受体将用于验证这种方法;甘丙肽1和2（Gal 1和Gal 2）受体以及食欲素/下丘脑泌素受体OX 1和OX 2。一对中的每个成员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。在Gal 1和2的情况下，两种受体共享40%的有限同源性。相比之下，OX 1和OX 2共有69%的同一性和80%的相似性，因此对分离可以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。在对一对中的每种抗原成员进行一系列阳性和消减FACS选择步骤以富集选择性高亲和力抗体克隆群体后，我们将通过一系列测定对表达每种GPCR的CHO细胞系进行表征，与野生型CHO细胞上的结合进行比较，以确定其特异性并将其各自的表位定位在其同源GPCR上。然后，我们将用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。这些研究的积极结果将提供一组针对四种GPCR中每一种的不同结构域的高度特异性抗体。此外，该I期项目将增强这种新型组合方法的能力，以产生高度特异性、高亲和力的抗体，这些抗体可以区分天然构象中GPCR上存在的独特表位。该技术可应用于II期的其他GPCR，总体目标是将本研究分离的抗体作为研究GPCR的宝贵研究试剂上市。

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.