**1. Introduction**

G-protein-coupled receptors (GPCRs), which make up the largest superfamily of human membrane proteins, play pivotal roles in mediating intracellular signaling and inducing cell proliferation, cell growth, and cell motility through the association and subsequent dissociation of G-proteins in response to external stimuli (Figure 1) [1,2]. Many clinical studies have revealed that abnormal functions of GPCRs are highly related to a variety of human diseases and affect the patient survival rate [3–5]. Therefore, GPCRs are crucial drug targets to treat patients with various diseases, and their targeting drugs represent more than 30 percent of all US Food and Drug Administration (FDA)-approved drugs [6–8]. Annual sales of these drugs have increased to about USD 180 billion in 2018 [9].

**Figure 1.** Schematic diagram of G-protein-coupled receptor (GPCR) signaling pathways mediated by Gα protein subunits. Downstream signaling triggered by binding of G proteins changes the concentrations of phospholipase C-beta (PLCβ), phosphoinositide 3-kinases-gamma (PI3Kγ), diacylglycerol (DAG), inositol trisphosphate (IP3), and cyclic adenosine monophosphate (cAMP) and regulates various cellular functions such as cell motility, cell growth, cell proliferation, and cancer progression and metastasis.

Compared to small-molecule chemical drugs and small peptides, therapeutic antibodies have many advantages in terms of higher target specificity, fewer side effects, and superior serum circulating half-life [10]. However, despite clinical and marketing successes of monoclonal antibody products to treat numerous diseases, only two anti-GPCR therapeutic antibody drugs, Amgen's erenumab (trade name: Aimovig), targeting calcitonin gene-related receptor (CGRPR) to treat migraine (Figure 2a) [11] and Kyowa Kirin's mogamulizumab (trade name: Poteligeo), targeting chemokine receptor 4 (CCR4) to treat refractory mycosis fungoides and Sézary syndrome (Figure 2b), have been approved to date [12].

Generally, amenable techniques to isolate therapeutic human antibodies include (1) humanization of candidate antibodies followed by the selection of hybridoma cells derived from immunized animals; (2) screening of the human naïve antibody library displayed on the surface of bacteriophages, bacteria, or yeast, which take advantage of a physical linkage between genotype and phenotype; and (3) hybridoma selection after immunizing an antigen into humanized transgenic animals, referred to as XenoMouseTM, which contains the genes for variable regions of the heavy (VH) and light (VL) chains of the human antibody repertoire [13].

**Figure 2.** US FDA-approved anti-GPCR antibodies erenumab and mogamulizumab. (**a**) Erenumab is an antagonistic monoclonal antibody against calcitonin gene-related peptide receptor (CGRPR) consisting of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) for treatment of chronic migraine. (**b**) Mogamulizumab is an antibody against chemokine receptor 4 (CCR4) for treatment of T-cell leukemia by inactivating the GPCR and clearance of target cells by enhanced antibody-dependent cell-mediated cytotoxicity (ADCC).

Regardless of the antibody isolation technique, preparing pure GPCR antigens with the native conformation of the human in vivo condition is essential for successful isolation of therapeutic functional human anti-GPCR antibodies. In particular, GPCRs containing seven transmembrane α-helices are usually expressed at very low levels in heterologous expression systems; therefore, it is very hard to purify the antigen with a native conformation as a soluble form. Furthermore, the limited surface area of the extracellular region of GPCRs in the whole GPCR structure makes it very difficult to prepare the GPCR antigen as a target for therapeutic anti-GPCR antibodies. Even though antigen preparation is one of the most difficult steps in development of therapeutic anti-GPCR antibodies, two anti-GPCR antibodies have overcome the challenges and have been recently approved. In addition, dozens of anti-GPCR antibodies are under clinical development or are waiting for clinical evaluations of therapeutic efficacy and toxicity. In this review, we focus on therapeutic anti-GPCR antibodies that have been recently approved or those that are subject to clinical trials (Table 1) and how the various types of GPCR antigens are prepared to isolate the highly challenging therapeutic anti-GPCR antibodies that have entered the clinical development phase.

**Table 1.** Anti-GPCR antibodies approved by the US FDA or subject to clinical trial.

