Synthetic nanobodies as angiotensin receptor blockers Conor McMahona,1, Dean P. Stausb,c,1, Laura M. Winglerb,c,1,2, Jialu Wangc, Meredith A. Skibaa, Matthias Elgetid,e, Wayne L. Hubbelld,e, Howard A. Rockmanc,f, Andrew C. Krusea,3, and Robert J. Lefkowitzb,c,g,3 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; bHoward Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710; cDepartment of Medicine, Duke University Medical Center, Durham, NC 27710; dJules Stein Eye Institute, University of California, Los Angeles, CA 90095; eDepartment of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; fDepartment of Cell Biology, Duke University Medical Center, Durham, NC 27710; and gDepartment of Biochemistry, Duke University Medical Center, Durham, NC 27710 Edited by K. Christopher Garcia, Stanford University, Stanford, CA, and approved July 13, 2020 (received for review May 6, 2020) There is considerable interest in developing antibodies as functional a need for more broadly applicable methodologies to discover modulators of G protein-coupled receptor (GPCR) signaling for both antibody fragments explicitly directed to the membrane- therapeutic and research applications. However, there are few an- embedded domains with limited surface exposure. tibody ligands targeting GPCRs outside of the chemokine receptor The angiotensin II type 1 receptor (AT1R) is a GPCR that group. GPCRs are challenging targets for conventional antibody dis- exemplifies the opportunities and the challenges surrounding an- covery methods, as many are highly conserved across species, are tibody drug development. Both the endogenous peptide agonist of biochemically unstable upon purification, and possess deeply buried the AT1R (angiotensin II) and small-molecule inhibitors (angio- ligand-binding sites. Here, we describe a selection methodology to tensin receptor blockers, ARBs) bind deep within the 7TM bundle enrich for functionally modulatory antibodies using a yeast- – displayed library of synthetic camelid antibody fragments called (14 17). Since this receptor serves as one of the principal regu- “nanobodies.” Using this platform, we discovered multiple nano- lators of blood pressure and renal function, ARBs are frontline bodies that act as antagonists of the angiotensin II type 1 receptor treatments for hypertension and kidney disease. While ARBs are (AT1R). Following angiotensin II infusion in mice, we found that an safe and effective in most patients, they readily cross the placenta affinity matured nanobody antagonist has comparable antihyper- and exhibit severe on-target fetal toxicity that prevents their use tensive activity to the angiotensin receptor blocker (ARB) losartan. during pregnancy (18). This toxicity restricts pharmacological The unique pharmacology and restricted biodistribution of nano- treatments for hypertensive disorders during pregnancy, including body antagonists may provide a path for treating hypertensive dis- preeclampsia, a leading cause of maternal and fetal death (19). orders when small-molecule drugs targeting the AT1R are The development of biologic-based AT1R antagonists may pro- contraindicated, for example, in pregnancy. vide an alternative therapeutic approach for treating hypertensive disorders during pregnancy, as small proteins cannot cross the nanobodies | angiotensin | ligand | GPCR | hypertension placental barrier (20). protein-coupled receptors (GPCRs) are the largest family of Significance Gtransmembrane proteins in humans and constitute the sin- gle largest class of small-molecule drug targets (1). Still, many The G protein-coupled receptor (GPCR) family is responsible for GPCR-targeted drugs lack selectivity for a single receptor sub- regulating much of human physiology, and GPCRs have be- type, and some therapeutically relevant GPCRs have not been come the most successful target class for drug development. successfully targeted by small-molecule drugs. Antibody-based Although many biologics have gained clinical approval, GPCR- therapeutics could provide a path to target otherwise undrug- targeted drugs remain almost exclusively small molecules, and gable GPCRs, as well as achieve high receptor subtype speci- developing GPCR-targeted antibodies remains challenging and ficity, by recognizing extended epitopes outside the conserved often unsuccessful. We describe a methodology to isolate an- ligand-binding pocket of GPCRs. Furthermore, antibodies are tibody fragment antagonists of a peptide-binding GPCR, and inherently endowed with potentially advantageous pharmaco- we show that one of these antibody fragments can be used to logical properties distinct from small molecules, such as long modulate blood pressure in vivo in mice. Antibody-based ap- half-lives, restricted distribution in vivo, and the ability to induce proaches may be useful in treating conditions where small- targeted cytotoxic effects by triggering immune responses via molecule drugs cannot be developed or where the unique their Fc regions. Despite this potential, few functionally modu- pharmacological properties of antibodies are desirable. latory GPCR antibodies have been reported, and no broadly applicable approaches for GPCR antibody discovery have been Author contributions: C.M., D.P.S., L.M.W., J.W., M.A.S., M.E., W.L.H., H.A.R., A.C.K., and established, although new screening methods are emerging (2). R.J.L. designed research; C.M., D.P.S., L.M.W., J.W., M.A.S., and M.E. performed research; To date, most examples of GPCR-targeted antibodies act on C.M. contributed new reagents/analytic tools; C.M., D.P.S., L.M.W., J.W., M.A.S., M.E., members of the chemokine receptor family, which bind small W.L.H., H.A.R., A.C.K., and R.J.L. analyzed data; and C.M., D.P.S., L.M.W., M.A.S., A.C.K., protein ligands (3–8). These antibodies likely function by oc- and R.J.L. wrote the paper. cluding ligand recognition motifs on the receptors’ amino ter- Competing interest statement: C.M., D.P.S., L.M.W., M.A.S., A.C.K., and R.J.L. are coinven- tors on a patent application for AT1R blocking nanobodies. A.C.K. is a cofounder and mini rather than by directly altering receptor signaling or consultant for Tectonic Therapeutic Inc. and for the Institute for Protein Innovation, a competing for ligand binding to the seven-pass transmembrane nonprofit research institute. (7TM) domain (7, 8). Similarly, the known antibody fragments This article is a PNAS Direct Submission. targeting family B GPCRs such as the glucagon receptor and Published under the PNAS license. GLP-1 receptor (9, 10), parathyroid hormone receptor (11), and 1C.M., D.P.S., and L.M.W. contributed equally to this work. calcitonin gene-related peptide receptor (12) function by 2Present address: Department of Pharmacology and Cancer Biology, Duke University blocking receptor ectodomains rather than interacting with the Medical Center, Durham, NC 27710. 7TM core. However, the vast majority of GPCRs lack extracel- 3To whom correspondence may be addressed. Email: [email protected] or lular ligand recognition regions and instead bind small-molecule [email protected]. and peptide ligands entirely within their 7TM domain. Although This article contains supporting information online at https://www.pnas.org/lookup/suppl/ functional antibody fragments were recently obtained via im- doi:10.1073/pnas.2009029117/-/DCSupplemental. munization for the peptide binding apelin receptor (13), there is First published August 4, 2020. 20284–20291 | PNAS | August 18, 2020 | vol. 117 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.2009029117 Downloaded by guest on September 28, 2021 We recently developed a fully in vitro camelid antibody frag- antagonist (Fig. 1C). Sequences of selected nanobodies are sum- ment (“nanobody”) discovery platform (21), which allowed iso- marized in SI Appendix, Table S1. lation of conformation-stabilizing nanobodies for the AT1R that bind the intracellular face of the receptor (14). We reasoned that Affinity Maturation of Nanobody AT118. Both nanobody ligands similar approaches might equally apply to targeting the extra- showed a reduced ability to compete with radioligands in cellular side of the receptor and yield antagonists and agonists. membranes (SI Appendix,TableS2). However, we reasoned We designed a yeast selection scheme to enrich orthosteric li- that the reduced competition could be overcome by increasing gands, resulting in multiple nanobodies targeting the extracel- the nanobody’s affinity for AT1R through affinity maturation. lular side of AT1R. These nanobodies effectively bind the Using error-prone PCR to mutagenize AT118, we generated a 7 receptor in vitro, and a lead clone antagonizes AT1R signaling new yeast-displayed library of 8.4 × 10 mutant AT118 clones. through both the G protein and β-arrestin pathways. Moreover, FACS selections were performed to enrich for higher-affinity in mice this nanobody clone shows antihypertensive activity and AT118 mutants. Three mutations that were highly enriched blocks the pressor activity of angiotensin II infusion comparably after two sequential FACS selections were combined into a to the small-molecule ARB losartan. These results demonstrate consensus clone called AT118i4 (Fig. 2A). that synthetic yeast display library selections can effectively de- This consensus nanobody clone exhibited nearly 70-fold en- liver receptor-modulating antibody fragments with activity both hanced binding affinity when compared to the parent clone