Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.

000974
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The impact of nanobodies on GPCR structural biology and their potential as

therapeutic agents

David Salom, Arum Wu, Chang C. Liu, and Krzysztof Palczewski

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Primary affiliations:

Gavin Herbert Eye Institute - Center for Translational Vision Research, Department of

Ophthalmology (D.S., A.W., K.P), and Department of Biomedical Engineering (C.C.L),

University of California, Irvine, CA 92697, USA

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Running title: Nanobodies and GPCRs

Corresponding authors:

1- Krzysztof Palczewski, 837 Health Sciences Road, Gillespie Neuroscience Building,

Room 2105, Irvine, CA 92697-4375; phone 949-824-6527, email: [email protected]
2- David Salom, 837 Health Sciences Rd, Gillespie Neuroscience Building, Room 2216,
Irvine, CA 92617; phone 949-824-5154; email: [email protected]
3- Arum Wu, 837 Health Sciences Rd, Gillespie Neuroscience Building, Room 2216,
Irvine, CA 92617; phone 949-824-5154; email: [email protected]
4- Chang C. Liu, ISEB 4064, 419 South Circle View Dr., Irvine, CA 92697; Phone: 949-
824-8534, email: [email protected]

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Manuscript pages: 32
Number of tables: 1
Number of figures: 4
Number of references: 77
Abstract word count: 194
Introduction word count: 255
Discussion word count: no discussion

Abbreviations:
ACKR3: atypical chemokine receptor 3
AR: adrenergic receptor
AT1R: Angiotensin II type I receptor
BRIL: thermostabilized apocytochrome b562
CDR: complementarity-determining region
Cryo-EM: cryogenic electron microscopy
ECD: extracellular domain
GPCR: G protein-coupled receptor
GPCRDB: GPCR database
ICD: intracellular domain
ICL3: intracellular loop 3
Nb: nanobody
OR: opioid receptor
scFv: single-chain variable fragment
T4L: T4 lysozyme
Trx: thioredoxin

Keywords: Nanobodies, GPCRs, single domain antibodies, cryo-EM, crystallography,

therapeutic antibodies, structural biology, ligand, VHHs

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Abstract

The family of human G protein-coupled receptors (GPCRs) is comprised of about 800

different members, with about 35% of current pharmaceutical drugs targeting GPCRs.

However, GPCR structural biology, necessary for structure-guided drug design, has

lagged behind that of other membrane proteins, and it was not until the year 2000 when

the first crystal structure of a GPCR (rhodopsin) was solved. Starting in 2007, the

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determination of additional GPCR structures was facilitated by protein engineering, new

crystallization techniques, complexation with antibody fragments, and other strategies.

More recently, the use of camelid heavy-chain-only antibody fragments (nanobodies) as

crystallographic chaperones has revolutionized the field of GPCR structural biology,

aiding in the determination of more than 340 GPCR structures to date. In most cases,

the GPCR structures solved as complexes with nanobodies (Nbs) have revealed the

binding mode of cognate or non-natural ligands; in a few cases, the same Nb has acted

as an orthosteric or an allosteric modulator of GPCR signaling. In this review we

summarize the multiple ingenious strategies that have been conceived and

implemented in the last decade to capitalize on the discovery of nanobodies to study

GPCRs from a structural perspective.

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Significance Statement:

G protein-coupled receptors (GPCRs) are major pharmacological targets, and the

determination of their structures at high resolution has been essential for structure-

guided drug design and for insights about their functions. Single domain antibodies

(nanobodies) have greatly facilitated the structural determination of GPCRs, by forming

complexes directly with the receptors or indirectly through protein partners.

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Introduction:

The history of nanobodies (Nbs) dates back to 1993, when a serendipitous discovery in

the laboratory of Professor R. Hamers (Vrije Universiteit Brussel) revealed that some

camelid antibodies only have heavy chains (Hamers-Casterman et al., 1993). From this

observation, it was quickly realized that their variable region (VVH) could be engineered

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into a fully functional ~14-kDa antibody fragment with wide potential applications,

including in structural biology (Desmyter et al., 1996; Hamers-Casterman et al., 1993).

Since Nbs have a single domain, they normally cannot bind linear epitopes. Instead,

their three complementarity-determining regions (CDRs) can insert into cavities of the

proteins, making them good at binding difficult-to-access concave epitopes and

stabilizing particular conformations. In the case of GPCRs, several dozen Nbs have

been found to insert one or more CDRs into the GPCR’s ligand-binding pocket, the helix

bundle, or other allosteric sites, stabilizing the active or inactive states.

In the last decade, Nbs have been particularly helpful in structural biology, especially in

obtaining structures of difficult targets such as membrane proteins, in studies

spearheaded by the laboratory of Jan Steyaert in Belgium (Vrije Universiteit Brussel)

(Uchanski et al., 2020). Not surprisingly, the field of GPCR-structural biology has

capitalized on these advances, and the number of structures of GPCRs determined with

the aid of Nbs has been growing since 2011 (Fig. 1). In addition, Nbs are also being

investigated as potential antibody-based therapeutics. Here we review the impact of

Nbs on elucidating structures of GPCRs, either by crystallography or cryo-electron

microscopy (cryo-EM), and the therapeutic potential of Nbs.

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Modes of binding of nanobodies to GPCRs:

The binding mode of a Nb to a GPCR can be classified as direct or indirect, and

intracellular or extracellular. The GPCR database, GPCRDB [GPCRdb.org], lists all the

GPCR structures, including GPCRs bound to Nbs. Unfortunately, GPCRDB has not

been updated since August 2023. In Table S1, we list the structures of GPCR/Nb

complexes that were made public after that date. In Table S2, we list the PDB entries of

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Nb-aided GPCR structures in the GPCR database (except for 214 GPCR structures

with Nb35). Here, we summarize the impact of Nbs in the determination of GPCR

structures, grouped by Nb-binding mode, with an emphasis on Nbs that bind directly to

the extracellular domain of the GPCRs.

1- Direct binding to the intracellular domain: The first-ever Nb-aided GPCR structure

was that of the β2-adrenergic receptor (β2-AR) with Nb80 bound to the intracellular

domain (ICD) (PDB 3P0G) (Rasmussen et al., 2011a). The β2-AR construct used in this

work had the flexible intracellular loop 3 (ICL3) replaced by T4 lysozyme (T4L). Fusions

with T4L and other small, thermostable proteins, such as BRIL (a thermostabilized

apocytochrome b562), thioredoxin (Trx) or rubredoxin are used routinely to increase the

expression and stability of GCPRs for structural studies (Chun et al., 2012). In the case

of the crystal structure of the complex between Nb80 and β2-AR/T4L construct, there

was no interpretable electron density for T4L. However, the CDR3 of Nb80 was clearly

visible binding deep inside the 7-helix bundle, mimicking the binding of G protein and

stabilizing the active conformation of the receptor (Fig. 2A). In most of the GPCR/Nb

structures solved to date, the Nbs bind to the intracellular side of the receptor and are

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specific for the particular receptor (see examples in Fig. 2). Other examples of Nb-

CDRs reaching into the intracellular side of the 7-helix bundle, stabilizing the GPCR

active conformation, include additional structures of Nbs in complex with β2-AR with

different ligands (PDB 6N48 and 4QKX), β1-AR (Fig. 2B) (PDB 6IBL and 7BTS), and

the M2 muscarinic acetylcholine receptor (PDB 4MQS). In most cases, it is the CDR3

loop that penetrates the helix bundle; although in several cases, such as μ-opioid

receptor (μ-OR, PDB 5C1M) (Fig. 2C), and κ-OR (PDB 6B73, 7YIT), the Nbs have a

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different mode of binding, with CDR2 instead being the loop reaching deeper inside the

bundle and stabilizing the active conformation.

In the case of US28, a human cytomegalovirus-encoded chemokine receptor that was

“stolen” from the human genome and evolved by the virus, single-chain fusions of US28

with intracellularly directed nanobodies were screened against yeast cells displaying

CX3CL1 chemokine libraries on their surface (Miles et al., 2018). For this work, the C-

terminus of US28 was fused to the nanobody Nb7 to enable the production of stable

US28 for staining yeast-displayed chemokine libraries. Crystallization of the

CX3CL1.35/US28-Nb7 complex by lipidic cubic phase required an additional nanobody,

raised by alpaca immunization against apo-US28-Nb7 (Fig. 2D). Comparison of the

structure of US28 bound to CX3CL1.35 and the wild type CX3CL1, as well as its

signaling activity bound to different CX3CL1 variants, suggested that signaling of US28

is largely unaffected by even drastic changes to the interactions within the receptor

binding cavity, providing structural basis for US28’s chemokine promiscuity.

Recently, a potentially universal strategy for Nb/GPCR-structural determination was

devised where ICL3 of other GPCRs could be replaced by the ICL3 from κ-OR, which is

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recognized by the negative allosteric modulator Nb6 (Fig. 2E) (Che et al., 2020)

(Robertson et al., 2022). Using this loop-grafting strategy, the structure of several

GPCRs in inactive conformations have been solved in complex with Nb6 by cryo-EM:

histamine receptor 2 (H2R, PDB 7UL3), somatostatin receptor 2 (SSTR2, PDB 7UL5),

neurotensin 1 receptor (NTSR1, PDB 7UL2), α1A-AR (PDB 8HN1, 7YMJ), and

chemokine receptor CXCR3 (PDB 8K2W, 8HNN). In addition, the structure of μ-OR was

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obtained with a megabody (see below) based on Nb6 (PDB 7UL4). Except for SSTR2,

the GPCRs in complex with Nb6 were also bound to antagonists.

2- Direct binding to the extracellular domain: Despite being the most interesting

binding mode because of its therapeutic potential, the structure of only six class-A

GPCRs have been solved to date with a nanobody directly bound to their extracellular

domain; in addition, four class-C GPCRs have been solved with Nbs bound to their

large extracellular domain (ECD) (Fig. 3).

The first structure of a GPCR with a bound extracellular Nb was that of APJ, a target for

the treatment of chronic heart failure (Ma et al., 2020) (Fig. 3A). Thus, Nb-JN241 was

found to make critical contacts with the second extracellular loop of APJ and insert

CDR3 into the ligand-binding pocket. Guided by this crystal structure, the authors

converted antagonist Nb-JN241 into a full agonist Nb-JN241-9 just by inserting a

tyrosine into the CDR3.

Next, the cryo-EM structure of the active orexin receptor 2 (OX2R) in complex with its G

protein and a small-molecule agonist was solved by stabilizing the complex with a

bound Nb (Hong et al., 2021) (Fig. 3B). OX2R is a potential target for the treatment of

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narcolepsy; however, in this work, no modulation of activity of the receptor by the Nb

was reported.

μ-Opioid receptor (μ-OR) is the molecular target of opioid analgesics such as morphine

and fentanyl. The cryo-EM structure of μ-OR bound to the antagonist-nanobody NbE

was solved recently (Fig. 3C) (Yu et al., 2023). NbE displays a unique ligand-binding

mode and achieves selectivity for µ-OR through interactions with extracellular receptor

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loops and deep penetration into the orthosteric pocket. Based on the structure of the

CDR3 loop of NbE, the authors designed short-peptide analogs that retain antagonism

toward µ-OR. In addition to NbE, the μ-OR complex also included an anti-Nb Fab and

an anti-Fab Nb. The work of Yu et al. illustrates the potential of Nbs to uniquely engage

with GPCRs, and it describes novel μ-OR ligands that can serve as the basis for

therapeutic development.

Rhodopsin is the prototypical, class-A GPCR, and multiple mutations in rhodopsin have

been identified to cause retinal degeneration. In the case of rhodopsin, the covalently

bound antagonist (11-cis-retinal) photo-isomerizes into the full agonist all-trans-retinal;

then, through a series of short-lived conformations, rhodopsin transforms into the fully

activated conformation Meta-II. Our lab recently developed a battery of Nbs that bind to

the extracellular domain of rhodopsin and act as allosteric modulators, preventing

rhodopsin to transition from the ground state to the Meta-II state upon photoactivation

(Wu et al., 2023). Furthermore, photoactivation of rhodopsin in isolation followed by

incubation with the Nb, shifts the equilibrium from active meta II-rhodopsin toward the

inactive Meta-I conformation, despite the binding pocket being occupied by the full

agonist all-trans-retinal. This Nb-induced conformational shift is also observed with apo-

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rhodopsin (Fig. 3D), whose conformation in isolation is otherwise similar to that of Meta-

II. The crystal structures of Nb2 in complex with ground-state rhodopsin, photo-activated

rhodopsin and apo-rhodopsin revealed extensive interactions of the three CDRs of Nb2

with the N-terminus, ELC2 and, surprisingly, the two native N-glycans of rhodopsin.

These observations serve as a reminder that post-translational modifications of native

GPCRs like glycosylation, which often are overlooked, need to be taken into

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consideration in the development of biologics. Additionally, we found out that the non-

variable framework region 2 (FR2) of Nb2 also was involved in extensive interactions

with the native glycans of rhodopsin. There was also another unexpected finding in our

work. Notably, unlike the other available structures of extracellular Nb/class-A GPCR

complexes where the Nb-CDR3 loop binds deep inside the orthosteric pocket of the

GPCR, the antagonistic activity of Nb2 toward rhodopsin is achieved by preventing

small conformational changes in the extracellular domain of rhodopsin that are

concomitant with the transition from ground-state to activated state. This effect is in

contrast with the mode of action of intracellular Nb modulators, since their activity is

facilitated by the major opening of the helix bundle upon GPCR activation.

Angiotensin II type I receptor (AT1R) modulates renal and cardiovascular function in

response to the eight-amino-acid peptide-hormone angiotensin II. Recently, the

structure of two fusion proteins Nb-AT1R/BRIL bound to anti-BRIL Fab have been

solved by cryo-EM (PDBs 8TH3, 8TH4) (Skiba et al., 2024). In both cases, the CDR3s

of the fused Nbs (AT118-H and AT118-L) were found to occupy the orthosteric binding

pocket and act as antagonists by competing with peptide ligands for binding to AT1R;

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however, the two Nbs exhibited different levels of interference with small-molecule

antagonists.

The α1A-AR responds to adrenaline and noradrenaline and is involved in smooth muscle

contraction and cognitive function. Two structures of Nb29/α1A-AR/miniGsq have been

solved (Fig. 3E), in which an agonist (noradrenaline or oxymetazoline) is bound in the

orthosteric binding pocket, while the Nb29-CDR3 loop occupies the extracellular

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vestibule of this binding pocket (PDB 7YMH and 7YM8). Nb29 covers the extracellular

surface of α1A-AR, analogous to the anti-APJ nanobody JN241 and anti-PAR2 Fab;

whereas the CDR3 loop of Nb29 binds to a site similar to that of LY2119620 (a weak

positive allosteric modulator) binding to M2R (Toyoda et al., 2023).

The Nb-aided structures of dimeric class-C GPCRs, which have large extracellular

domains (ECD), represent a special case. In recent work (Kumar et al., 2023), the cryo-

EM structures of mGlu5 by itself (8T7H) or bound to Nb43 through the ECD (PDB 8T6J,

8T7H, 8T8M, 8TAO) enabled the authors to propose a model for a sequential, multistep

activation mechanism for mGlu5 (Fig. 3F). Other structures of class-C GPCRs with Nb

bound to the extracellular domain are mGlu1 in an intermediate state (PBD 7DGE),

active mGlu2 (PDB 7EPB) and inactive CaSR (PDB 7E6U).

3- Indirect binding to the intracellular domain: The majority (~80%) of the Nb-

facilitated structures of GPCRs consist of complexes with a bound G protein which, in

turn, is bound to Nb35 (Fig. 4A). This nanobody stabilizes the active GPCR/G protein

complex by interacting with the Gαs and Gβ subunits, preventing dissociation of the

nucleotide-free complex by the nonhydrolyzable GTP analog GTPγS (Rasmussen et al.,

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2011b). All GPCR/Gs/Nb35 structures have been determined by cryo-EM, except for

the D1A dopamine receptor (PDB 7JOZ) and β2-AR (PDB 3SN6), which were solved by

X-ray crystallography. In a few cases, a single-chain variable fragment (scFv) also

formed part of the complex.

Another mode of indirect binding in GPCR/Nb complexes is that of an anti-BRIL Fab

fragment bound to a GPCR/BRIL fusion construct, as shown for inactive Frizzled

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receptor FZD3 (Fig. 4B). Finally, to obtain the cryo-EM structure of the inactive

glucagon receptor/Arr2 complex, without or with glucagon bound (PDB 8JRU and 8JRV,

respectively), multiple modifications were performed on the proteins to stabilize the

complex: the C-terminus of the receptor was replaced by the C-terminus of the

vasopressin V2 receptor to achieve optimal binding to a Cys-free Arr2; Arr2 was fused

to a scFv; and a Nb that binds to active Arr2 was added to the complex (Fig. 4C).

4- Indirect binding to the extracellular domain:

The only Nb-facilitated GPCR structure where the Nb is indirectly bound to the

extracellular domain of the GPCR is of that of atypical chemokine receptor 3 (ACKR3)

bound to chemokine CXCL12 (also called stromal cell-derived factor 1) (PDB 7SK7). In

that case, a Fab fragment that directly contacts the GPCR/ligand complex serves as an

intermediate binding partner between the Nb and the GPCR (Yen et al., 2022). In the

same article, the authors also solved another structure of the ACKR3/CXCL12 complex

with a Fab fragment plus a Nb, but in this second structure the Nb and Fab were bound

to the intracellular domain of the GPCR. These two structures, along with five additional

structures of ACKR3 without Nb but with different ligands and mutations, together with

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functional studies, have provided insights about the ligand-binding promiscuity of

ACKR3, why it fails to couple to G proteins, and why it is biased toward regulation by

arrestin-2.

Use of nanobodies for therapeutic intervention and clinical imaging

Since the discovery of nanobodies, the interest in developing therapeutic nanobodies

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has been growing rapidly, leading to the introduction of two FDA-approved nanobody

drugs into the clinical market (Morrison, 2019; Natrajan et al., 2024). More than three

dozen nanobodies have been entered into the clinical realm for various human-disease

indications, including solid tumors, chronic inflammatory disease, autoimmune and

infectious disorders, and brain neuroimaging (Table S3). Half of the clinical-stage

nanobody-drug candidates have been targeted to cancer-expressed antigens whose

high expression is often associated with tumor progression and immune response.

Over 370 GPCRs are potential pharmacological targets for drug discovery, as they are

implicated in many diseases and therapeutic strategies. Yet the vast majority of GPCR-

targeting nanobodies are still in the discovery stages of research and development, they

are currently being developed for a broad spectrum of disease indications, including

asthma, cancer, cardiovascular diseases, HIV infection, hyperparathyroidism,

hyperthyroidism, immune dysfunctions, inflammatory diseases, kidney dysfunction,

metabolic syndromes, migraine, neurological and neurodegenerative diseases,

osteoporosis, pain, psychiatric disorders, and vision disorders (Table 1). The selectivity

and regiospecificity of the binding of nanobodies enable diverse pharmacological effects

on the activities of the GPCRs by regulating various interactions, including occupation of

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orthosteric binding pockets or allosteric sites, ligand binding affinity, stabilization of

specific conformations based on the Nb-epitope binding sites, and penetration of Nb-

CDRs into the receptor cores.

A unique feature of nanobodies from a therapeutic perspective is their use as molecular

chaperones for misfolded proteins associated with degenerative diseases, such as

amyloidosis and rhodopsin-associated retinal degeneration. Potentially therapeutic

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nanobodies have been developed against amyloidogenic targets such as rhodopsin

(Wu et al., 2023), lysozyme (Dumoulin et al., 2003), Aβ-peptides (Habicht et al., 2007;

Lafaye et al., 2009), α-synuclein (Lafaye et al., 2009), β2-microglobulin (Domanska et

al., 2011), and gelsolin, which are involved, respectively, in autosomal dominant retinitis

pigmentosa, systemic amyloid disease, Alzheimer’s Disease, Parkinson’s disease, renal

failure, and gelsolin amyloidosis (Van Overbeke et al., 2014). One of the underlying

mechanisms of anti-amyloidogenic nanobodies is to inhibit amyloid fibril formation by

stabilizing oligomers and trapping them in intermediate fibrils. A recent publication

reported that rhodopsin-targeting Nb2 acted as a molecular chaperone to restore the

proteostasis of the misfolded P23H-mutant rhodopsin protein by stabilizing it in an

intermediate conformation (Wu et al., 2023).

Clinical applications of radiolabeled or fluorescent-labeled nanobodies have emerged

for in vivo molecular imaging methods, such as optical and positron-emission

tomography (PET)/computer tomography (CT) nuclear imaging, particularly in cancer

and brain. High specificity, affinity for the target antigen, and rapid renal clearance of

nanobody tracers are major advantages for in vivo imaging. Due to their small size,

nanobodies are expected to achieve a uniform distribution and deep tissue penetration,

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an important rate-limiting factor in diagnostic imaging (Debie et al., 2020; Zheng et al.,

2022).

Current challenges, knowledge gaps and perspective:

Nbs are commonly generated by immunization of llamas or alpacas with the target

protein. However, large animal immunization has several downsides including cost and

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accessibility, animal welfare challenges, and limitations on addressable targets since

immunological tolerance of self-antigens makes it difficult to raise nanobodies that bind

conserved epitopes. Over the past few years, several technological developments in

synthetic Nb generation have taken off. For example, in vitro discovery of Nbs using

phage display or yeast surface display (McMahon et al., 2018; Moutel et al., 2016;

Uchanski et al., 2019) have led to the discovery of countless Nbs, including ones

targeting GPCRs such as AT1R on both the intracellular and extracellular interface

(McMahon et al., 2020; Wingler et al., 2019). For such approaches to yield potent Nbs,

large synthetic Nb libraries must be constructed from which target-specific binders can

be enriched. Often, the enriched binders have weak affinity and need to undergo affinity

maturation through repeated rounds of error-prone PCR and selection. More recently, in

vitro Nb discovery was upgraded through the implementation of autonomously

diversifying yeast-displayed Nbs (Wellner et al., 2021) that encode Nbs or Nb libraries

on a rapidly hypermutating plasmid system (Ravikumar et al., 2018; Rix et al., 2023).

These systems allow for Nbs to continuously diversify, and therefore rapidly affinity

mature, as they are subjected to simple rounds of sorting for target binding and growth.

Another way to produce heavy-chain only single-domain antibodies is to generate

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transgenic mouse lines with either fully humanized or hybrid llama/human variable

domains genes (Eden et al., 2024; Janssens et al., 2006; Teng et al., 2020; Xu et al.,

2021). These methods still rely on animal immunization, but of mice instead of alpacas

or llamas, providing obvious advantages. A remarkable recent advancement is the

possibility of de novo computational design of Nbs in cases where the structure of the

epitope is known (Bennett et al., 2024). Here, diffusion-based generative models for

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protein design nominate epitope-specific synthetic Nb designs that can be further

screened and improved by yeast display, with the eventual goal of direct design of Nbs

against any epitope without needing animal immunization nor in vitro engineering

approaches.

A particular gap in our knowledge is how Nbs can modulate the activity of GPCRs from

the extracellular side. The presence of PTMs such as glycosylation could be a major

obstacle for translating Nbs from in vitro proof-of-principle status to in vivo therapeutic

application, since most GPCRs are N- and/or O-glycosylated at the N-terminus and

extracellular loops. The discovery and characterization of antibodies targeting GPCRs is

usually done using recombinant GPCRs, in many cases with mutations, deletions, or

deglycosylase treatments to remove N-glycans. The case of a Nb targeting bRho that

does not recognize deglycosylated or hyperglycosylated bRho (Wu et al., 2023)

illustrates how the presence of native glycans in the GPCR is necessary for relevant

development and characterization of Nbs as potential therapeutics.

The future of Nb-facilitated GPCR structural biology seems to be tied to advances in

strategies to increase the molecular weight (MW) of the complexes required for

structural determination by cryo-EM. Once a GPCR-targeted Nb becomes available, it is

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possible to increase its MW by engineering a megabody (Mb), which are generated by

circular permutation of a Nb with a stable, mid-size unrelated protein (Uchanski et al.,

2021). μ-OR/Mb6 was the first GPCR/Mb complex solved, although most of the Mb

structure was not solved (PDB 7UL4) (Robertson et al., 2022). Other similar strategies

involve adding to the protein/Nb complex an anti-Nb Fab fragment plus an MBP/Protein-

A construct (Legobodies) (Wu and Rapoport, 2021). Finally, it is possible to increase the

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MW of a GPCR/Nb complex by addition of a Fab fragment that recognizes the Nb; and,

then, an anti-Fab Nb (for example, Fig. 3C in (Bloch et al., 2021)).

Another interesting approach is the use of intrabodies in GPCR research (Manglik et al.,

2017), where nanobodies are expressed de novo within cells for multiple applications,

such as to visualize or modulate antigens in the living cell (Wagner and Rothbauer,

2020); or to study protein conformational states (Uchanski et al., 2020). Among the

reported applications of anti-GPCR intrabodies are inhibition of G protein activation,

GRK-mediated receptor phosphorylation, β-arrestin recruitment, and receptor

internalization (Manglik et al., 2017).

18
 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
This article has not been copyedited and formatted. The final version may differ from this version.

Acknowledgements

We thank Dr. Jeffrey L. Benovic, Montserrat Salom and members of the Center

for Translational Vision Research and the Gavin Herbert Eye Institute for their

comments and insights on this manuscript.

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 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
This article has not been copyedited and formatted. The final version may differ from this version.

Data Availability Statement

The authors declare that all the data supporting the findings of this study are available

within the paper and its Supplemental Data

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20
 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
This article has not been copyedited and formatted. The final version may differ from this version.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Salom D., Wu, A., Liu C., and

Palczewski K.

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 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
This article has not been copyedited and formatted. The final version may differ from this version.

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and Casellas R (2021) Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants.
Nature 595(7866): 278-282.
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Tesmer JJG and Handel TM (2022) Structures of atypical chemokine receptor 3 reveal the basis
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 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
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Footnotes

Financial support: This work was supported in part by grants from the National

Institutes of Health [grants R01EY009339, R01EY0034519, P30EY034070] (K.P., K.P.

and core grant to Department of Ophthalmology, Gavin Herbert Eye Institute at the

University of California, Irvine). The authors acknowledge support to the Department of

Ophthalmology Gavin Herbert Eye Institute at the University of California, Irvine from an

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unrestricted Research to Prevent Blindness award. The authors also acknowledge the

Institute for Rapid Antibody Engineering and Evolution, part of the Engineering+Health

Initiative of the UCI Samueli School of Engineering, for additional support.

Conflict of interest:

CCL is a co-founder of K2 Biotechnologies, Inc., which uses OrthoRep for protein engineering.

K.P. is a consultant for Polgenix Inc. and serves on the Scientific Advisory Board at Hyperion

Eye Ltd.

Reprint requests: Krzysztof Palczewski, 837 Health Sciences Road, Gillespie

Neuroscience Building, Room 2105, Irvine, CA 92697-4375, email: [email protected]

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 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
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Figure Legends

Figure 1: Number of PDB entries of nanobody-facilitated GPCR structures, solved

either via X-ray crystallography or cryo-EM. The GPCR database

(gpcrdb.org/structure) was used to compile the structures released up to 8-16-2023, and

the Protein Data Base (rcsb.org) for structures released after that date. Note that some

GPCRs are represented by multiple PDB entries.

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* The value of PDB entries for 2024 is estimated based on the number of PDBs

released at the time of submission of this review

Figure 2: Five representative crystal structures of nanobodies in direct contact with the

intracellular domain of a GPCR. A) β2-adrenergic receptor (PDB 3P0G); B) T4L/β1-

adrenergic receptor fusion protein (PDB 7BTS); C) µ-opioid receptor (PDB 5C1M); D)

US28/Nb7 fusion protein (PDB 5WB2); E) κ-opioid receptor (PDB 6VI4).

Figure 3: Six (crystal or cryo-EM) structures of GPCRs with nanobodies directly bound

to their extracellular domains. A) APJ/rubredoxin fusion protein (PDB 6KNM); B) orexin

receptor 2/Gs protein complex (PDB 7L1V); C) µ-opioid receptor (PDB 8QOT); D) rod

opsin (PDB 8FD0); E) α1-adrenergic receptor in complex with an engineered minimal

Gsq protein (PDB 7YM8); F) metabotropic glutamate receptor mGlu5 (PDB 8TAO).

Figure 4: Three examples of cryo-EM structures of GPCRs in complex with different

proteins, facilitated by an Nb not in direct contact with the GPCR. A) Adenosine-A2A

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 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
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receptor in complex with a mini-Gs protein (PDB 6GDG); B) Frizzled receptor

FZD3/BRIL fusion protein bound to anti-BRIL Fab fragment (PDB 8JHC); C) glucagon

receptor in complex with arrestin-2 fused to a scFv (PDB 8JRV).

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 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
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Table 1. GPCR-targeting nanobodies (as of June 2024)

Target Nb clones Epitope Pharmacology References

Rho Nb2 ECD Antagonist (Wu et al., 2023)
CXCR2 2B2, 127D1, 54B12, 97A9, ECD Antagonist, inverse agonist (Bradley et al., 2015)
163E3, 163D2,
bivalent 2B2-35GS-2B2,
163E3-35GS-163E3
and 127D1-35GS-163E3
CXCR4 238D2, 238D4, bivalent ECD Antagonist, inverse agonist (Jahnichen et al., 2010)
L3-L8
CXCR4 10A10, bivalent 10A10 ECD Antagonist, inverse agonist (de Wit et al., 2017)
CXCR4 VUN401-VUN409 ECD Antagonist (Van Hout et al., 2018)
CXCR4 VUN400-Fc, VUN401-Fc, ECD Antagonist (Bobkov et al., 2018)

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VUN402-Fc
CXCR4 Bispecific Nb PX4 ECD Antagonist (Hao et al., 2022)
(PD-V1 and CXCR4)
ACKR3 NB1-5, Nb38 ECD Antagonist, inverse agonist (Maussang et al., 2013)
(CXCR7)
ACKR3 VUN701 ECD Antagonist (Kleist et al., 2022)
(CXCR7)
CX3CR1 54A12, 54D05, 66B02 ECD Antagonist (Low et al., 2020)
66G01, BI655088, BI655089
US28 Nb7 ICD Active (Burg et al., 2015)
US28 VUN103 ICD Antagonist (De Groof et al., 2021)
US28 US28-Nb, bivalent US28-Nb ECD Antagonist, inverse agonist (Heukers et al., 2018)
ChemR23 CA4910, CA5183, ECD Antagonist (Peyrassol et al., 2016)
bivalent CA4910
mGlu2 DN1, DN10, DN13 ECD Partial, full agonist PAM (Scholler et al., 2017)
mGlu2/3
mGlu2/4
mGlu4 DN42 ECD Nanobody-based TR-FRET sensors (Meng et al., 2022)
mGlu2/4
mGlu4 DN45 ECD Agonist, PAM (Haubrich et al., 2021)
mGlu2/4
mGlu5 Nb43 ECD PAM (Koehl et al., 2019)
mGlu5 Nb5A ICD PAM (Eshak et al., 2024)
M2R Nb9-1, Nb9-8, Nb9-20 ICD Active (Kruse et al., 2013)
μ-OR NbE ECD Antagonist (Yu et al., 2023)
μ-OR Nb39 ICD Active (Huang et al., 2015)
κ-OR Nb6/7, Nb39 ICD Active (Che et al., 2018)
β1-AR Nb80, Nb6B9 ICD Active (Warne et al., 2019)
β2-AR Nb80 ICD Active (Rasmussen et al., 2011a)
β2-AR Nb6B9 ICD Active (Ring et al., 2013)
β2-AR Nb60, Nb61, Nb64, Nb65, ICD Active or Inactive (Staus et al., 2014)
Nb71
β2-AR Nb.b201, Nb.c200 ICD Active (McMahon et al., 2018)
Nb.c203
α1A-AR Nb29 ECD Weak PAM or neutral (Toyoda et al., 2023)

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 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
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AT1R Nb.AT110, Nb.AT110i1 ICD Active (Wingler et al., 2019)

AT1R AT118, AT118i4 ECD Antagonist, inverse agonist (McMahon et al., 2020)
AT1R AT118-H, AT118-L ECD Antagonist (Skiba et al., 2024)
A2AR Nb.AD101, Nb.AD102 ICD Active (McMahon et al., 2018)
APJ JN241, JN241-9 ECD Antagonist, full agonist (Ma et al., 2020)
OX2R Sb51 ECD Active (Hong et al., 2021)
PTHR1 VHH∙PTH ECD Antibody ligand conjugation (Cheloha et al., 2020)
SMO NbSmo8 ICD Active (Deshpande et al., 2019)
CaSR Nb32 ECD NAM (Cui et al., 2024)
CaSR Nb-2D11, Nb88 ECD Inactive (Chen et al., 2021)
SUCNR1 Nanobody6 ICD NAM (Haffke et al., 2019)
5-HT2AR VGS-Nb2 ICD PAM (English et al., 2019)

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GPR68 VGS-Nb6 ICD PAM (English et al., 2019)
GPR75 NbH3 ICD Active (Lv et al., 2022)

The list of nanobodies were compiled through a literature search, including two review articles
(Heukers et al., 2019; Wingler and Feld, 2022). This table only includes nanobodies that bind directly
to GPCR proteins, and it cites the original publications that first identified the nanobodies.
Abbreviations: α1A-AR, α1A adrenergic receptor; A2AR, A2A adenosine receptor; ACKR, atypical
chemokine receptor; APJ, apelin receptor; AT1R, angiotensin II type 1 receptor; β1-AR, β1-
adrenergic receptor; β2-AR, β2-adrenergic receptor; CaSR, Calcium Sensing Receptor; ChemR23,
Chemerin receptor 23; CXCR, C-X-C motif chemokine receptor; GPR75, G protein-coupled receptor
75; GRP68, G protein-coupled receptor 68; 5-HT2AR, serotonin 2A receptor; κ-OR, kappa opioid
receptor; M2R, M2 muscarinic acetylcholine receptor; mGlu, metabotropic glutamate receptor; μ-OR,
μ-opioid receptor; NAM, negative allosteric modulator; OX2R, orexin receptor type 2; PAM, positive
allosteric modulator; PTHR1, parathyroid hormone receptor 1; Rho, rhodopsin; SMO,
transmembrane transducer smoothened.

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Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
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Figure 1
Number of PDB entries with Nbs

100

80

60 Cryo-EM
X-ray
40

20

0
2010 2015 2020 2025
Year of PDB release*
 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
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)LJXUH

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A B C D E
&;&/
T4L ȕ$5
fusion

ȕ$5 ȝ25 ț25

86Nb7
fusion
Nb6B9
1E

Nb39 Nb6

1E%
 Molecular Pharmacology Fast Forward. Published on August 6, 2024 as DOI: 10.1124/molpharm.124.000974
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Figure 3

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A B C D E F
Nb43
anti-Fab Nb

1
Nb JN241 anti-Nb Fab
Nb2 Nb29
Sb
Nb Sb51
NbE

OX2R ȝ25 rod Į$5

opsin
svFv16

mGlu5

APJ/Rubredoxin Gs
fusion heterotr
heterotrimer miniGsq
Figure 4

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A B C glucagon

anti-Fab Nb
glucagon
AA2AR receptor

FZD3/BRIL
miniGs
fusion
heterotrimer

Arr2/scFv30
anti-BRIL fusion
Fab

Nb35 Nb32