Synthetic Nanobodies As Angiotensin Receptor Blockers

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 single 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 AT118 in vitro and in vivo, providing a path to drugging GPCRs. in a radioligand binding assay using membranes from HEK 293 cells overexpressing AT1R (Fig. 2B and SI Appendix,TableS2). Results Notably, the affinity of AT118i4 for AT1R on membranes was Discovery of Nanobody Ligands to the Angiotensin Receptor. To comparable to that of losartan, a commonly prescribed ARB. discover nanobodies targeting the human AT1R, we employed a fully synthetic library of 5 × 108 unique nanobodies displayed on Functional Characterization of Nanobody AT118i4. Competitive the surface of Saccharomyces cerevisiae yeast (21). Cells were binding with orthosteric ligands suggested AT118 and AT118i4 incubated with purified AT1R protein in detergent, and likely bind the extracellular face of the receptor. However, this receptor-binding nanobody clones were enriched using magnetic- result could, in principle, also arise from an intracellular binder activated cell sorting (Fig. 1A). The resulting population was used as a starting point to discover AT1R ligands. We reasoned that nanobodies that are orthosteric ligands would be able to Fluorophore

AB PHARMACOLOGY displace low-affinity ligands but would be less effective at com- Library High Affinity peting for binding with high-affinity ligands. Therefore, we αFLAG-FITC 1.5 μM AT1R AT1R Ligand Micelle antibody stained the yeast simultaneously with two different populations AT1R of AT1R labeled with distinct fluorophores, one bound to a high- Round 1 αFLAG-647 affinity ligand (olmesartan, population 1) and another bound to 1.5 μM AT1R a low-affinity ligand (TRV055, population 2). The two ligands antibody Nanobody Low Affinity are mutually competitive with one another, both binding to the Round 2 MACS Ligand AT1R’s canonical orthosteric binding site (22). Additionally, the 500 nM AT1R 500 nM AT1R staining mixture contained 2.5 μM of free TRV055 (Fig. 1B)as + olmesartan + TRV055 Yeast cell AT1R an additional selective pressure to favor enrichment of high- Round 3 FACS affinity nanobodies capable of effectively competing with the low-affinity ligand. While ligand exchange is possible under these C = conditions, the slow off rate of olmesartan (T1/2 72 min) (23) 100 AT118 (K = 100 nM) minimizes mixing of the populations during the timescale of the i nding experiment. We performed fluorescence-activated cell sorting i AT120 (Ki = 385 nM)

(FACS) to isolate yeast cells whose nanobodies preferentially nb Losartan (Ki = 187 nM) ta r 50 bound to the TRV055 population. a After FACS, single clones were isolated and screened in on- s yeast binding assays using flow cytometry. First, 48 clones were lme (% of maximum) -O

screened for preferential binding to TRV055-bound AT1R com- H] 3 pared to AngII-bound AT1R. Nanobody ligands that compete [ 0 with AngII and TRV055 would be expected to preferentially -11 -10 -9 -8 -7 -6 -5 displace TRV055 due to its much lower AT1R-binding affinity [Ligand] (log M) than AngII. Two candidate clones from this screen were further evaluated for binding to olmesartan-bound AT1R. Both of these Fig. 1. Discovery of synthetic nanobody ligands for the angiotensin recep- clones, AT117 and AT118, had weak binding to AT1R in the tor. (A) Flowchart of the selection method. Two rounds of MACS were presence of high-affinity ligands (AngII and Olm) and stronger performed on a yeast library displaying synthetic nanobodies to enrich for AT1R binders, as previously described (14). For the third round of selection, binding in the presence of a low-affinity ligand (TRV055). After yeasts were simultaneously stained with two receptor populations, isolating these two clones, we searched for additional antagonist olmesartan-bound AT1R and TRV055-bound AT1R, and FACS was used to nanobodies using a similar but less stringent approach by screen- enrich for yeast displaying nanobodies that preferentially interact with the ing 48 clones for binding to either unliganded AT1R or TRV055-bound AT1R population. (B) Cartoon representing FACS selection olmesartan-bound AT1R. One candidate nanobody was chosen for yeast displaying nanobody ligands to AT1R. Yeasts were stained with two (AT120) for further evaluation from this screen. Clone AT117 receptor populations, AT1R bound to a high-affinity (olmesartan, pink) or showed very low receptor binding affinity, so we performed de- low-affinity (TRV055, yellow) ligand. Each of these receptor populations was tailed characterization of only clones AT118 and AT120. Radio- labeled by a different fluorophore through dye-conjugated anti-FLAG antibody. Yeasts that preferentially interact with the low-affinity ligand-AT1R ligand competition binding using purified AT1R in detergent 3 3 population were collected. (C) Radioligand competition binding of [ H]- showed that both nanobodies competed with the ARB [ H]- olmesartan and nanobodies AT118 or AT120 to AT1R. AT118 (red), AT120 olmesartan with mid- to low-nanomolar affinities, confirming (green), and positive control losartan (black) compete with antagonist they bind to the receptor at the same site as a conventional AT1R olmesartan for binding to detergent-solubilized AT1R.

McMahon et al. PNAS | August 18, 2020 | vol. 117 | no. 33 | 20285 Downloaded by guest on September 28, 2021 A CDR1 CDR2 AT120 QVQLQES--GLVQAGGSLRLSCAASGNIFGGVGMGWYRQAPGKEREFVAGINNGGTTNY 57 AT118 QVQLQESGGGLVQAGGSLRLSCAASGYIFRKYRMGWYRQAPGKEREFVAGINGGSSTNY 59 AT118i4 QVQLQESGGGLVQAGGSLRLSCAASGYIYRRYRMGWYRQAPGKEREFVAGINGGSSTNY 59

CDR3 AT120 ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVVRPSFDYHVYWGQGTQVTVSS 116 AT118 ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYRIVWDLLVYWGQGTQVTVSS 118 AT118i4 ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYRIVWDLRVYWGQGTQVTVSS 118

B 100

75 AT120 AT118 50 AT118i4 25 Losartan (% ofmum) maxi H]-Olmesartan binding 3 [ 0

-11 -10 -9 -8 -7 -6 -5 [Ligand] (log M)

Fig. 2. Affinity maturation of nanobody ligand AT118. (A) Sequence of affinity-matured AT118. The sequences of AT118, affinity-matured AT118i4, and AT120 were aligned. Residues mutated to generate AT118i4 are highlighted in red. (B) Radioligand competition binding of AT118i4 and [3H]-olmesartan to AT1R in cell membranes. AT118i4 (blue) competes with olmesartan for binding to AT1R in HEK cell membranes, demonstrating higher affinity than AT118 (red).

that has a high degree of negative cooperativity with orthosteric used double electron–electron resonance (DEER) spectroscopy to ligands. In order to definitively determine if AT118i4 binds to monitor the effects of AT118i4 binding on the intracellular regions the extracellular side of the AT1R we stained nonpermeabilized of the AT1R responsible for transducer activation. DEER mea- HEK suspension cells overexpressing the AT1R with AT118i4. sures the distribution of distances between two nitroxide spin la- Strong binding was observed by flow cytometry in the absence of bels, which are added to engineered cysteine residues on the AT1R ligands, but this was attenuated in the presence of AngII, AT1R. We previously validated pairs of AT1R sites that report on confirming that AT118i4 binds on the extracellular side of the key conformational changes that occur upon binding to small- AT1R (Fig. 3A). One potential benefit of antibody-based ther- molecule inverse agonists, AngII, and agonists that are biased apeutics that target GPCRs is the possibility of achieving very toward the activation of either Gq- or β-arrestin-mediated path- high receptor subtype specificity, which may reduce side effects ways. Analysis of AT118i4’s effect on several of these pairs shows in clinical applications. Indeed, when we stained cells expressing that the nanobody stabilizes conformations sampled by the unli- either AT1R or angiotensin II type 2 receptor (AT2R), we found ganded receptor and has a profile distinct from both ARBs and that there was little or no binding of AT118i4 to AT2R agonists (Fig. 4). In the TM1-TM6 pair, which reports on the ∼10 expressing cells (Fig. 3B), while AT118i4 showed strong, satu- Å outward movement of TM6 that typically occurs upon GPCR rable binding to AT1R-expressing cells (half-maximal effective activation (42.5 Å, AngII-stabilized peak in the AT1R), two concentration [EC50] = 31 nM). conformations are present in the absence of ligand (31.5 and 34 Å Next, we sought to investigate the functional properties of peaks). Small-molecule ARBs shift the distribution toward the AT118i4. In order to determine whether AT118i4 effectively shorter 31.5 Å distance, but AT118i4 slightly stabilizes the 34 Å antagonizes AT1R signaling, we treated HEK suspension cells distance, although much more weakly than the β-arrestin-biased stably overexpressing AT1R with AngII in the presence of ligand TRV026. Like TRV026, AT118i4 depletes the population AT118i4 (Fig. 3C and SI Appendix, Table S3). Gq signaling, of distances at >25 Å in the ICL2-TM5 pair, which ARBs like assessed by inositol monophosphate accumulation, was signifi- candesartan strongly stabilize. However, like ARBs, AT118i4 fails cantly reduced in cells treated with AT118i4 (EC50 = 89 nM) to induce conformational changes in helix 8 (ICL2-H8 pair) that = when compared with vehicle (EC50 3.5 nM). We also observed are characteristic of either TRV026 (25.5 Å) or AngII (32 Å). a modest but reproducible decrease in basal activation of Gq signaling (Fig. 3C), indicating that AT118i4 acts as an inverse Activity of AT118i4 In Vivo. Given its potent inhibition of angio- agonist. AT118i4 also reduced AT1R internalization (Fig. 3D tensin II signaling in cells that is comparable to clinically used and SI Appendix, Table S3), indicating that signaling through ARBs, we reasoned that AT118i4 could serve as an antihyper- β-arrestin is suppressed. In addition, ERK phosphorylation, tensive in vivo. To examine whether AT118i4 could be functional which is activated by both Gq and β-arrestin signaling, was in a mouse model, we performed a radioligand competition profoundly reduced in cells treated with AT118i4 (SI Appendix, binding assay with [3H]-olmesartan and AT118 using human Fig. S1). AT1R or murine angiotensin II receptor type 1a (AT1Ra). Reduced basal activation of the AT1R suggested that AT118i4 Compared to human AT1R, murine AT1aR has substantially is likely stabilizing an inactive conformation of the receptor (i.e., it reduced (19-fold) affinity for AT118i4 but not [3H]-olmesartan is an inverse agonist rather than a simple neutral antagonist). We (SI Appendix, Fig. S2). Since murine AT1Ra and human AT1R

20286 | www.pnas.org/cgi/doi/10.1073/pnas.2009029117 McMahon et al. Downloaded by guest on September 28, 2021 AT1R + AT118i4 + AngII A B AT1R AT1R + AT118i4 AT2R No AT1R + AT118i4

ity 5 800 5x10 ens 4x105

3x105 400 2x105 Cell number 1x105 Mean fluorescence int

(AU; background subtracted) 0 1 2 3 4 0 10 10 10 10 0 200 400 600 Binding (fluorescence) [AT118i4] (nM)

C D

100 No nanobody 100 No nanobody

ion AT118i4 AT118i4 ivat ct a

q 50 50 -arrestin internalization β lative G (% of maximum) (% of maximum)

Re 0 0 Relative -12 -10 -8 -6 -4 -13 -11 -9 -7 -5 [Angiotensin II] (log M) [Angiotensin II] (log M)

Fig. 3. Functional characterization of AT118i4. (A) Binding of AT118i4 to cells expressing AT1R demonstrated by flow cytometry. HEK suspension cells PHARMACOLOGY transiently expressing AT1R were treated with AT118i4-His (blue) or both AT118i4-His and AngII (red). Cells not expressing AT1R (transiently transfected with empty vector) were also treated with AT118i4-His (gray). Cells were stained with anti-His-488 before flow cytometry analysis. Data in A are representative of three independent experiments. (B) Subtype specificity of AT118i4 assessed by flow cytometry. HEK suspension cells transiently transfected with AT1R (blue) and AT2R (orange) were treated with AT118i4-V5-His and anti-V5 488, then analyzed by flow cytometry. Data are representative of three independent experiments. Error bars represent SEM from replicates. (C) Inhibition of Gq-mediated inositol monophosphate accumulation by AT118i4 in response to AngII. HEK cells were treated with 5 μM AT118i4 (blue) or no antibody (black). (D) AT118i4 treatment reduces translocation of β-arrestin2 to endosomes upon AngII stimulation.

are very highly conserved and differ at only nine positions in data demonstrate that AT118i4 and other nanobodies derived their extracellular regions, we introduced these differences as from our yeast display library are readily amenable to optimization mutations into human AT1R to determine if they influence for therapeutic applications. AT118 binding. Extracellular mutations (I164V/F170Y and Q187R) significantly decreased AT118i4 affinity, while other Discussion mutations were tolerated. Drugs targeting GPCRs represent roughly 35% of marketed We then tested whether AT118i4 could suppress AngII acti- therapeutics (1), illustrating the profound physiological impor- vation of the AT1R in vivo. Anesthetized mice were pretreated tance of this class of receptors. There has been growing interest with an intravenous (i.v.) bolus of the ARB losartan (10 mg/kg), in targeting GPCRs with antibody-based therapeutics, which AT118i4 (7 to 10 mg/kg), or the control nanobody AT110S offer advantages over small-molecule drugs for certain applica- (10 mg/kg, which binds the intracellular surface of the AT1R), tions. Importantly, antibodies have the potential for exquisite followed by a graded dose of AngII (Fig. 5A and SI Appendix, subtype specificity by virtue of their large and extensive inter- Fig. S3). Continuous monitoring of aortic blood pressure with a action surfaces with target antigens. The ability to target specific high-fidelity micromanometer catheter showed that, like losartan GPCRs within closely related families could reduce side effects (Fig. 5B), AT118i4 significantly attenuates the AngII-induced and enable therapeutic development for previously inaccessible rise in blood pressure (Fig. 5C). In contrast, a control nano- GPCRs. Likewise, antibodies that recognize naturally occurring body that binds the intracellular side of the AT1R did not block GPCR variants could be useful for treating rare diseases (24). the physiological effects of AngII (Fig. 5C). Notably, AT118i4 Notably, as genetically encoded entities, additional attributes can was even more efficacious in blocking the effects of AngII in be engineered readily into antibody ligands. For example, tissue- female mice than in male mice (SI Appendix, Table S3). or cell-type-specific activity can be achieved through engineering Another critical step in adapting antibody fragments for ther- bispecific antibodies, and Fc-dependent immune effector func- apeutic applications is humanization to reduce immunoreactivity. tions can be used to achieve antibody-dependent cell-mediated Encouraged by the in vivo activity of AT118i4, we tested whether cytotoxicity. its camelid framework could be humanized without adversely af- Despite the therapeutic potential of GPCR-targeted antibody fecting its functional properties. We created a panel of nanobody ligands, there are a number of challenges associated with their variants with varying numbers of humanizing mutations to better discovery. Many GPCRs are highly conserved across species, resemble the heavy chain of a human antibody. AT118i4h32, with creating barriers to generating antibody therapeutics from animal- 11 total humanizing amino acid changes, was the most aggressively derived libraries or immunization approaches. Moreover, most mutagenized clone with unaltered affinity for the AT1R (SI Ap- GPCRs are biochemically unstable upon purification, resulting in pendix,Fig.S4). AT118i4h32 further displays enhanced protein rapid denaturation following injection into an animal. Our in vitro expression, solubility, and stability compared to AT118i4. These selection approach addresses both challenges. Since our library is

McMahon et al. PNAS | August 18, 2020 | vol. 117 | no. 33 | 20287 Downloaded by guest on September 28, 2021 A AT1R active AT1R inactive

TM6 H8

TM5 TM1 90°

ICL2

B TM1-TM6 ICL2-TM5 ICL2-H8

31.5 Å 34.0 Å 42.5 Å 24.0 Å 27.0 Å 25.5 Å 32.0 Å 40.5 Å

AngII (endogenous) TRV026 (biased) AT118i4 No ligand Candesartan (antagonist) 25 30 35 40 45 50 15 20 25 30 35 40 20 25 30 35 40 45 Distance (Å) Distance (Å) Distance (Å)

Fig. 4. Conformations sampled by AT1R. (A) Labeling sites in the intracellular regions of the AT1R. Spheres indicate positions of Cα of labeled residues in the receptor in inactive (gray, Protein Data Bank [PDB] ID code 4YAY) and active (orange, PDB ID code 6DO1) conformations. (B) DEER distance probability distributions in the presence of AT118i4 and various classes of ligands (candesartan, ARB; TRV026, β-arrestin-biased ligand; AngII, endogenous agonist) for AT1R pairs monitoring the conformations of the cytoplasmic ends of the indicated transmembrane helices, intracellular loop 2, and helix 8.

fully synthetic, it has not been negatively selected for tolerance of the AT2R receptor subtype. Despite reduced affinity for murine self. In addition, selections can be performed on short timescales AT1R, AT118i4 significantly reduced the rise in blood pressure in under optimized conditions (e.g., low temperature, defined buffer mice in response to infusion of the endogenous agonist angiotensin composition, no endogenous proteases or receptor ligands), en- II. In both in vitro and in vivo experiments, we observed a reduc- suring that receptors remain folded during the selection process. tion in basal AT1R signaling, suggesting that AT118i4 acts as an In addition, counterselections can be carried out simultaneously, inverse agonist. DEER spectroscopy indicates that AT118i4 sta- increasing the probability of finding a candidate molecule pos- bilizes an inactive state of the AT1R that is distinct from small- sessing the desired properties. molecule AT1R antagonists. The small convex paratope and long CDR3 loops of nanobodies AT1R-targeted nanobody ligands may have therapeutic potential may make these antibody fragments particularly suited to target- for treating hypertensive and kidney diseases during pregnancy, as ing the buried pockets typically responsible for ligand binding. ARBs and ACE inhibitors both exhibit severe fetal toxicity, making Indeed, nanobodies are frequently used in place of prototypical pharmacologic modulation of the renin–angiotensin–aldosterone antibodies to access the cytosolic transducer binding pocket of system largely impossible with existing therapeutics. Unlike tradi- GPCRs and to stabilize active conformations as “G protein mi- tional small-molecule drugs, a protein would not pass through the metics” for crystallography. Recently, a camelid-derived nanobody placental barrier without the capacity to bind to the FcRn receptor was reported to antagonize the human apelin receptor (APJ) (27, 28). Our approach for discovering an orthosteric nanobody may through extensive interactions between the nanobody and extra- serve as a blueprint for future efforts aimed at generating other cellular loops of APJ and insertion of CDR3 into the activating GPCR-targeted antibody ligands. peptide binding site (13). The APJ nanobody antagonist was ra- tionally engineered into an APJ agonist, demonstrating the po- Materials and Methods tential for rapid modification of antibody ligand pharmacology Nanobody Discovery. A yeast-displayed library of nanobody AT1R binders was through protein engineering. Nanobody attachments to GPCR generated using magnetic-activated cell sorting (MACS), which has been ligands have also been developed in order to impart additional previously described (14). In summary, 1 × 1010 cells displaying a synthetic functionality to the ligands (25, 26). library of nanobodies were stained for 40 min at 4 °C with anti-fluorescein We discovered two nanobody antagonists to the AT1R from a isothiocyanate (FITC) microbeads (Miltenyi) and FITC-labeled anti-FLAG an- synthetic yeast-displayed nanobody library. Our selection method- tibody. After centrifugation and resuspension in buffer (20 mM Hepes [pH ology took advantage of the range of affinities of small-molecule 7.5], 150 mM NaCl, 2.8 mM CaCl2, 0.05% maltose neopentyl glycol [MNG], 0.005% cholesterol hemisuccinate [CHS], 0.1% bovine serum albumin [BSA], AT1R ligands, which facilitates a selection methodology that en- 0.2% maltose), cells were passed through an LD column (Miltenyi) to remove riches for nanobodies that are competitive with orthosteric ligands. yeast displaying nanobodies that bound to FLAG antibody or microbeads. The high-affinity AT1R nanobody antagonist, AT118i4, antago- Collected yeast was then stained again, with 1.5 μM of FLAG-tagged AT1R nizes AT1R signaling comparably to clinically used antagonists and and 1 μM of anti-FLAG FITC antibody for 1 h at 4 °C, followed by staining exhibits exceptional subtype specificity, with negligible binding to with anti-FITC microbeads for 15 min at 4 °C, and then passed into an LS

20288 | www.pnas.org/cgi/doi/10.1073/pnas.2009029117 McMahon et al. Downloaded by guest on September 28, 2021 A Angiotensin II (ng) V5-epitope (GKPIPNPLLGLDST) was inserted prior to the His-tag for flow Vehicle/Drug 0.1 1 5 10 20 50 cytometry experiments. All constructs were verified by Sanger sequencing. Escherichia coli strain BL21(DE3) was transformed with nanobody 2 min 10 min 3 min 3 min 3 min 3 min 3 min 3 min encoding plasmids. Transformed cells were grown in Terrific Broth media at 37 °C with 50 μgmL−1 kanamycin to an optical density at 600 nm = 0.7 to 1.5, cooled to 17 °C, and induced with 200 μM isopropyl β-D-1-thiogalactopyr- BCVehicle (n=6) anoside overnight. Cell pellets were resuspended in 50 mL sucrose, ethyl- 150 Ctrl nanobody (n=6) Vehicle (n=6) e 150 enediaminetetraacetic acid (EDTA), Tris buffer (20 mM Tris [pH 8], 500 mM AT118i4 (n=8) Losartan (n=6) sucrose, 500 μM EDTA) per liter of culture; 100 mL cold H2O and 100 U of 100 100

pressur benzonase nuclease per liter of culture supplemented with 5 mM MgCl2 g) g)

**** c **** # **** H were added to resuspended pellets and stirred for 1 h at room temperature. olic pressure 50 # t

toli 50 **** # ** # Resuspended cells were cleared by centrifugation (14,000 × g, 30 min). Su- sys (mm H sys (mm 0 * 0 † pernatant was supplemented with 100 mM NaCl and stirred at room temperature for 15 min. Supernatant was passed through a glass microfiber ΔAortic -50 ΔAortic -50 filter and applied to protein A agarose resin (GoldBio) or a HiTrap FF rPro- l sal 0.1 1550 10 20 a 0.1 1550 10 20 /Los tein A column (GE Healthcare). The resin was washed with 10 column vol- Ba as e/Nb Angiotensin II (ng) B Angiotensin II (ng) umes of wash buffer (10 mM NaH2PO4 [pH 7.5], 100 mM NaCl). Protein was Vehicle Vehicl eluted with 100 mM NaH2PO4 (pH 2.5) and 100 mM NaCl directly into 2 M Fig. 5. AT118i4 blocks hypertension caused by angiotensin II infusion in Hepes (pH 8; 0.2 mL/mL elution). Purified nanobody was buffer exchanged mice. (A) Treatment protocol for measuring changes in mouse blood pres- into 20 mM Hepes (pH 7.5), 150 mM NaCl, and 10% glycerol through size × sure upon exposure to AngII. (B) Measurements of the effect of losartan on exclusion chromatography (Superdex S75 10 300 mm) or dialysis. aortic systolic pressure in mice. The effects of the antihypertensive or ARB To remove endotoxin for animal experiments, AT118i4 was immobilized losartan (green) on systolic pressure in mice exposed to increasing concen- on Nickel NTA resin (GoldBio) and washed with 50 column volumes of sterile trations of AngII were compared to untreated mice (black) in a positive endotoxin-free phosphate-buffered saline (PBS) and 0.1% Triton X-114 at a control experiment. Statistical analysis was performed using two-way flow rate of 1 mL/min at 4 °C. Triton X-114 was removed by washing the ANOVA with Sidak multiple comparison posttests; *P < 0.05, **P < 0.01, resin with five column volumes of sterile PBS four times at a flow rate of ****P < 0.0001, compared with the vehicle-treated mice. (C) Measurements 1 mL/min at 4 °C. AT118i4 was eluted in sterile PBS containing 200 mM of the effect of nanobody AT118i4 on aortic systolic pressure in mice. Systolic imidazole. Imidazole was subsequently removed using a PD-10 column, and pressure in mice exposed to increasing concentrations of AngII was mea- AT118i4 was concentrated to 1 mg/mL, filter sterilized (0.2 μm), snap frozen sured after pretreatment with nanobody AT118i4 (blue), negative control in liquid nitrogen, and stored at −80 °C. Endotoxin levels were measured nanobody (red), or vehicle PBS (black). Statistical analysis was performed using the limulus amebocyte assay (Thermo Fisher), and all AT118i4 prepa-

† PHARMACOLOGY using two-way ANOVA with Sidak multiple comparison posttests; P < 0.01, rations contained less than five endotoxin units per milligram of protein. #P < 0.0001, compared with the vehicle-treated mice. Radioligand Binding. In competition radioligand binding experiments with detergent-solubilized Flag-AT1R wild type,∼60 ng of purified receptor were 3 column (Miltenyi). Bound yeast was eluted and grown for a second round of added to serial dilutions of nanobodies and 2.5 nM [ H]-olmesartan MACS. The second round was performed similarly to the first, but with 4 × (American Radiolabeled Chemicals) in 20 mM Hepes (pH 7.4), 100 mM μ 108 cells and anti-FLAG antibody labeled with Alexa Fluor 647 and anti-647 NaCl, 0.01% maltose lauryl neopentyl glycol, and 0.1% BSA in 200 L reac- microbeads. Yeast enriched for binding to AT1R after these two rounds of tions. After 90 min at room temperature, reactions were harvested on GF/B MACS was simultaneously stained with 500 nM AT1R prebound to the high- filters using a 96-well Brandel harvester and quickly washed three times with cold 20 mM Hepes (pH 7.4) and 100 mM NaCl. Crude cell membranes for affinity ligand olmesartan and 500 nM AT1R bound to a lower-affinity li- radioligand binding experiments were prepared as previously described (22) gand, TRV055, and the receptors were fluorescently labeled using 150 nM of from Expi293F Inducible cells (Thermo Fisher) transfected with the indicated M1 anti-FLAG 647 antibody and M1 anti-FLAG 488, respectively, before AT1R construct cloned in empty pcDNA-Zeo-tetO (29) according to the staining. The staining buffer was composed of 20 mM Hepes (pH 7.5), manufacturer’s instructions. Membranes were incubated with serially di- 150 mM NaCl, 3 mM CaCl ,2.5μM TRV055, 0.1% MNG, 0.01% CHS, 0.1% 2 luted nanobodies and 2.5 nM [3H]-olmesartan in 50 mM Tris (pH 7.4), BSA, 0.2% maltose. FACS was performed on the stained yeast, and yeast cells 150 mM NaCl, 12.5 mM MgCl , 0.2% BSA, leupeptin, and benzamidine. After displaying nanobodies that preferentially bound to the AT1R-TRV stained 2 90 min at room temperature, reactions were harvested on GF/B filters using population were collected and grown. From this FACS library, AT118 was a 96-well Brandel harvester and quickly washed three times with cold 50 mM isolated by screening with a BD Accuri C6 Plus flow cytometer for yeast- Tris (pH 7.4). Data points were fit to a one-site competition binding model in displayed nanobody clones that interact more robustly with TRV055- GraphPad Prism. To determine inhibitory constant (K ) values, radioligand bound AT1R versus olmesartan- or angiotensin II–bound AT1R, and AT120 i affinity was determined by saturation binding using a serial dilution of [3H]- was isolated by screening for clones that interact more strongly with unli- olmesartan in the presence or absence of 10 μM candesartan. Data were fit ganded AT1R versus olmesartan-bound AT1R. to a one-site saturation-binding model in GraphPad Prism. Data shown represent the mean and SE of three independent experiments. Affinity Maturation. Error-prone PCR was performed on AT118 using the GeneMorph II Random Mutagenesis Kit (Agilent). The error-prone PCR Flow Cytometry Assays. Expi293F Inducible cells were transiently transfected product was then amplified with Q5 High-Fidelity DNA Polymerase (New with empty pcDNA-Zeo-tetO (29), pcDNA-Zeo-tetO-FLAG-AT1R wild type England Biolabs) and transformed into BJ5465 yeast using an ECM 830 (human), or pcDNA-Zeo-tetO-FLAG-AT2R wild type (human) according to Electroporator (BTX-Harvard Apparatus). The resulting yeast-displayed li- the manufacturer’s protocol and induced with doxycycline (4 μg/mL) and × 7 brary comprised 8.4 10 transformed clones with an average mutation rate sodium butyrate (5 mM) 2 d later. Three days posttransfection, cells were of 1.8 amino acid changes per clone. The nanobody expression in the error- harvested and resuspended in cold assay buffer (Hank’s Balanced Salt So- prone yeast library was induced, and cells were stained with 250 nM of AT1R lution, 10 mM Hepes [pH 7.4], 3 mM CaCl , 0.1% BSA) at a density of 8 × 106 μ 2 and then subsequently with 2 g/mL anti-HA-488 (Cell Signaling Technolo- live cells/mL and kept at 4 °C for the duration of the experiment. Cells were gies) and 400 nM of M1 anti-FLAG 647 antibody. Thirty-nine million cells incubated with or without 250 μM AngII for 30 min before the addition of were sorted, and 17,000 cells were collected using a gate drawn for cells His-tagged AT118i4 (160 nM). Cells were incubated with the nanobody for displaying higher-affinity nanobodies. A second FACS was performed using a 30 min, washed twice with assay buffer, and then incubated with a 1:250 lower concentration of AT1R to further enrich for cells displaying higher- dilution of anti-6X His Dylight 488 (abcam) and 15 nM Dylight 650–labeled affinity nanobodies. To generate high-affinity clones, 50 colonies were M1 anti-FLAG for 30 min. Cells were washed twice and then resuspended in Sanger sequenced, and enriched mutations were identified and added to a assay buffer for flow cytometry (BioRad S3e cell sorter). Data were analyzed consensus clone, AT118i4. with FlowJo 10 software. AT1R- and AT2R-transfected cells were gated for M1-positive singlets. For saturation-binding experiments cells were har- Protein Expression and Purification. DNA encoding AT118 and AT120 was vested, washed, and resuspended in flow cytometry buffer (20 mM Hepes cloned into pET26b, which contains an N-terminal pelB signal sequence for [pH 7.5], 150 mM NaCl, 0.1% BSA) 3 d posttransfection; 1 × 105 cells were periplasmic expression and C-terminal His-tag for purification. A C-terminal stained with AT118i4 containing a C-terminal V5 epitope tag for 1 h at 4 °C.

McMahon et al. PNAS | August 18, 2020 | vol. 117 | no. 33 | 20289 Downloaded by guest on September 28, 2021 Cells were then washed twice with flow cytometry buffer, supplemented receptor and incubated at ambient temperature for 3 h. Spin-labeled receptor

with 1 mM CaCl2, and incubated with a 1:500 dilution of Alexa Fluor was purified and buffer exchanged (40 mM Hepes [pH 7.4], 100 mM NaCl, 657-labeled anti-V5 antibody (Thermo Fisher) and 100 nM Alexa Fluor 0.01% MNG, 0.001% CHS, D2O) by size exclusion chromatography. After 488-labeled M1 anti-FLAG for 20 min. Cells were washed, resuspended in monomeric fractions were concentrated, ligands (10-fold excess) or AT118i4

flow cytometry buffer with 1 mM CaCl2, and analyzed with a BD Accuri C6 (twofold molar excess) were added to the receptor, along with dimethyl flow cytometer. AT1R- and AT2R-transfected cells were gated for M1- sulfoxide and d8-glycerol to final concentrations of 2% and 20%, respectively, positive singlets. Data were analyzed with BD Accuri C6 Plus software. so that all samples had the same buffer composition. Samples were incubated at room temperature for 1 h and then flash frozen in 1.4/1.7 mm or (inner Cellular Assays. diameter/outer diameter) borosilicate capillaries (VitroCom). Gq activation. Expi293F cells stably expressing wild-type FLAG-AT1R (human) Q-band four-pulse DEER measurements were conducted on an Elexsys were diluted to 2 × 106 cells/mL the day before the experiment. On the day E580 pulsed electron paramagnetic resonance spectrometer equipped with of the experiment, 2 × 104 cells/well were plated in a low-volume 96-well an ER5106QT-2 cavity resonator (Bruker). During experiments a recirculating plate, pretreated ±5 μM AT118i4, and then stimulated with the indicated helium cryocooling system (ColdEdge Technologies) was used to maintain concentration of AngII for 1 h at 37 °C. IP1 was quantified using the IP-One sample temperature at 50 K. Observed pulse lengths were 18 to 22 ns (π/2) Gq Kit (Cisbio) on a CLARIOstar plate reader (BMG Labtech). and 36 to 44 ns (π), as determined by an echo nutation experiment. A linear β-arrestin2 internalization. U2OS cells stably expressing β-arrestin2 with an frequency-swept (chirp) pulse of 50 MHz half-width, generated by an arbi- enzyme acceptor tag and endosome-localized ProLink were transiently trary waveform generator (Bruker) and applied 70 MHz below the observer transfected with wild-type FLAG-AT1R (human). One day posttransfection, frequency, served as the pump pulse. All pulses were further amplified via an cells were plated at a density of 35,000 cells per well. Two days post- external 150 W TWT amplifier (Applied Engineering Systems). LongDistances transfection, cells were treated with ±5 μM AT118i4 for 30 min and then (version 590 or newer, available at http://www.biochemistry.ucla.edu/Faculty/ with the indicated concentration of AngII for 3 h at 37 °C. Chem- Hubbell/software.html) was used for background correction and model-free iluminescence resulting from the complementation of the enzyme acceptor/ analysis of dipolar evolution data. The L-curve criterion was used to select ProLink β-galactosidase fragments due to β-arrestin endocytosis was de- a single smoothness parameter for each spin pair that was used for all ligand tected with a PathHunter kit (Eurofins, DiscoverX) using a CLARIOstar plate conditions. reader (BMG Labtech). PhosphoERK stimulation. Expi293F cells stably expressing wild-type FLAG-AT1R Blood Pressure Measurements in Mice. For this study 10- to 14-wk-old C57BL/6J × 6 (human) were diluted to 2 10 cells/mL the day before the experiment. On wild-type mice of both sexes were used. Animal experiments carried out for μ ± μ the day of the experiment, 500 L of cells were pretreated with 5 M this study were handled according to approved protocols and animal wel- AT118i4 and then with 10 nM AngII for the indicated time at 37 °C. Cells fare regulations mandated by the Institutional Animal Care and Use Com- × were solubilized by the addition of 4 Laemmli sample buffer and sonicated. mittee of Duke University Medical Center. Total and phosphorylated ERK were detected by Western blotting using Mice were anesthetized with ketamine (100 mg/kg) and xylazine (2.5 mg/kg). anti-MAPK 1/2 (1:10,000; EMD Millipore) and anti-p44/42 MAPK (1:4,000; Cell After bilateral vagotomy, a 1.4 French (0.46 mm) high-fidelity micromanometer Signaling) and quantified by densitometry (ImageJ, NIH). Densitometry catheter (ADInstruments) connected to a pressure transducer (ADInstruments) values for pERK were normalized to their respective total ERK values, and was inserted into the right carotid artery just above the aortic valve to monitor relative pERK values in each experiment were normalized to the 5 min time aortic blood pressure. Drugs were administered by i.v. injection through a point in the absence of a nanobody. Data shown represent the mean and SE jugular vein. Recording of basal blood pressure was performed at steady state of three independent experiments. after the catheter insertion (2 to 3 min after insertion). Animals then received vehicle (100 μL PBS), the AT1R nanobody AT118i4 (7 to 10 mg/kg, in 100 μL DEER Spectroscopy. AT1R constructs were purified and spin labeled with bis- PBS), the control nanobody AT110s (10 mg/kg, in 100 μl PBS), or the angio- (2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-yl)disulfide (Enzo) to install the V1 tensin II receptor blocker, losartan (10 mg/kg, in 100 μL PBS). Ten minutes after spin label as previously described (30). Briefly, minimal cysteine versions of mice received nanobody or vehicle solutions blood pressure was recorded, and N-terminal FLAG-tagged human AT1R with engineered cysteines (F55C D236C, then mice were injected with a graded dose of AngII (0.1 to 50 ng, each in TM1-TM6; R139C K220C, ICL2-TM5; R139C R311C, ICL2-H8) in pACMV-tetO 100 μL water) at 3 min intervals. The blood pressure was continuously moni- were transiently transfected into Expi293F cells stably expressing the tetracy- tored and recorded as described. Data analysis was performed using LabChart cline repressor (29) using an ExpiFectamine transfection kit (Thermo Fisher) 8 software (ADInstruments). according to the manufacturer’s instructions. Two days after transfection, Data are expressed as mean ± SEM. Statistical significance was determined doxycycline (4 μg/mL), sodium butyrate (5 mM), and losartan (5 μM) were with two-way repeated-measures ANOVA with Sidak correction for multiple added to the cultures, and cells were harvested and flash frozen with liquid comparison in GraphPad Prism. Differences were considered statistically nitrogen ∼30 h thereafter. Following hypotonic lysis (10 mM Tris [pH 7.4], significant at P < 0.05. 2 mM EDTA, 10 mM MgCl2,1μM losartan, 2.5 U/mL benzonase [Sigma], protease inhibitors benzamidine and leupeptin), cell pellets were solubilized with lauryl MNG (0.5%, 0.05% CHS, 20 mM Hepes [pH 7.4], 500 mM NaCl, Data Availability. All study data are included in the article and SI Appendix.

10 mM MgCl2,1μM losartan, 2.5 U/mL benzonase, benzamidine, leupeptin) for 2h at 4 °C. After insoluble material was cleared by centrifugation (18,000 × ACKNOWLEDGMENTS. Funding to support this research was provided by NIH g for 30 min at 4 °C), 2 mM CaCl was added to the solubilized material, which Grants R01HL056687 (H.A.R.), R01HL075443 (H.A.R.), DP5OD021345 (A.C.K.), 2 R21HD101596 (A.C.K.), and R01HL16037 (R.J.L.); the Vallee Foundation was loaded onto M1 anti-FLAG resin at 4 °C. After washing with the same (A.C.K.); the Smith Family Foundation (A.C.K.); the Helen Hay Whitney buffer containing a 50-fold lower concentration of MNG and CHS and Hepes Foundation (M.A.S.); and the Mandel Center for Hypertension and Athero- at pH 6.8, the receptor was eluted with 5 mM EDTA and 0.2 mg/mL FLAG sclerosis at Duke (R.J.L.). We thank the Duke Cardiovascular Research Center peptide (in wash buffer without CaCl2). A 10-fold molar excess of bis- Small Animal Physiology Core for performing the in vivo mouse hemodynamic (2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-yl)disulfide was added to the experiments.

1. R. Santos et al., A comprehensive map of molecular drug targets. Nat. Rev. Drug 7. R. S. Boshuizen et al., A combination of in vitro techniques for efficient discovery of Discov. 16,19–34 (2017). functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2). 2. H. Ren et al., Function-based high-throughput screening for antibody antago- mAbs 6, 1415–1424 (2014). nists and agonists against G protein-coupled receptors. Commun. Biol. 3, 146 8. L. Peng, M. M. Damschroder, K. E. Cook, H. Wu, W. F. Dall’Acqua, Molecular basis for (2020). the antagonistic activity of an anti-CXCR4 antibody. mAbs 8, 163–175 (2016). 3. M. Jo, S. T. Jung, Engineering therapeutic antibodies targeting G-protein-coupled 9. C. M. Koth et al., Molecular basis for negative regulation of the glucagon receptor. receptors. Exp. Mol. Med. 48, e207 (2016). Proc. Natl. Acad. Sci. U.S.A. 109, 14393–14398 (2012). 4. C. J. Hutchings, M. Koglin, F. H. Marshall, Therapeutic antibodies directed at G 10. S. Hennen et al., Structural insight into antibody-mediated antagonism of the protein-coupled receptors. mAbs 2, 594–606 (2010). glucagon-like peptide-1 receptor. Sci. Rep. 6, 26236 (2016). 5. D. Maussang et al., Llama-derived single variable domains (nanobodies) directed 11. K. Sarkar et al., Modulation of PTH1R signaling by an ECD binding antibody results in against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo. inhibition of β-arrestin 2 coupling. Sci. Rep. 9, 14432 (2019). J. Biol. Chem. 288, 29562–29572 (2013). 12. F. Garces et al., Molecular insight into recognition of the CGRPR complex by migraine 6. S. Jähnichen et al., CXCR4 nanobodies (VHH-based single variable domains) potently prevention therapy aimovig (Erenumab). Cell Rep. 30, 1714–1723.e6 (2020). inhibit chemotaxis and HIV-1 replication and mobilize stem cells. Proc. Natl. Acad. Sci. 13. Y. Ma et al., Structure-guided discovery of a single-domain antibody agonist against U.S.A. 107, 20565–20570 (2010). human apelin receptor. Sci. Adv. 6, eaax7379 (2020).

20290 | www.pnas.org/cgi/doi/10.1073/pnas.2009029117 McMahon et al. Downloaded by guest on September 28, 2021 14. L. M. Wingler, C. McMahon, D. P. Staus, R. J. Lefkowitz, A. C. Kruse, Distinctive acti- 23. M. T. Le, M. K. Pugsley, G. Vauquelin, I. Van Liefde, Molecular characterisation of the vation mechanism for angiotensin receptor revealed by a synthetic nanobody. Cell interactions between olmesartan and telmisartan and the human angiotensin II AT1 176, 479–490.e12 (2019). receptor. Br. J. Pharmacol. 151, 952–962 (2007). 15. L. M. Wingler et al., Angiotensin and biased analogs induce structurally distinct active 24. A. S. Hauser et al., Pharmacogenomics of GPCR drug targets. Cell 172,41–54.e19 – conformations within a GPCR. Science 367, 888 892 (2020). (2018). 16. H. Zhang et al., Structural basis for ligand recognition and functional selectivity at 25. R. W. Cheloha et al., Improved GPCR ligands from nanobody tethering. Nat. Commun. – angiotensin receptor. J. Biol. Chem. 290, 29127 29139 (2015). 11, 2087 (2020). 17. H. Zhang et al., Structure of the Angiotensin receptor revealed by serial femtosecond 26. H. Farrants et al., SNAP-tagged nanobodies enable reversible optical control of a G crystallography. Cell 161, 833–844 (2015). protein-coupled receptor via a remotely tethered photoswitchable ligand. ACS Chem. 18. M. Bullo, S. Tschumi, B. S. Bucher, M. G. Bianchetti, G. D. Simonetti, Pregnancy out- Biol. 13, 2682–2688 (2018). come following exposure to angiotensin-converting enzyme inhibitors or angiotensin 27. N. E. Simister, Placental transport of immunoglobulin G. Vaccine 21, 3365–3369 receptor antagonists: A systematic review. Hypertension 60, 444–450 (2012). 19. B. W. J. Mol et al., Pre-eclampsia. Lancet 387, 999–1011 (2016). (2003). 20. M. G. Buse, W. J. Roberts, J. Buse, The role of the human placenta in the transfer and 28. S. Roy et al., M281, an anti-FcRn antibody, inhibits IgG transfer in a human ex vivo metabolism of insulin. J. Clin. Invest. 41,29–41 (1962). placental perfusion model. Am. J. Obstet. Gynecol. 220, 498.e1-498.e9 (2019). 21. C. McMahon et al., Yeast surface display platform for rapid discovery of conforma- 29. D. P. Staus et al., Sortase ligation enables homogeneous GPCR phosphorylation to tionally selective nanobodies. Nat. Struct. Mol. Biol. 25, 289–296 (2018). reveal diversity in β-arrestin coupling. Proc. Natl. Acad. Sci. U.S.A. 115, 3834–3839 22. R. T. Strachan et al., Divergent transducer-specific molecular efficacies generate bi- (2018). ased agonism at a G protein-coupled receptor (GPCR). J. Biol. Chem. 289, 14211–14224 30. L. M. Wingler et al., Angiotensin analogs with divergent bias stabilize distinct re- (2014). ceptor conformations. Cell 176, 468–478.e11 (2019). PHARMACOLOGY

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