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.
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.
