Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies

Abstract

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gαq protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling.

Accelerating GPCR Drug Discovery With Conformation-Stabilizing VHHs

“…In addition, large panels of VHHs that target extracellular epitopes of native class A (CXCR4, CXCR2, ACKR3, CX3C1, US28, CML1, APJ, OX2R, AGTR2, and OPRM), B (GCGR, GLP1R, PTH1R, and VPAC1), and C (mGluR2, CaSR, mGluR4, and mGluR5) GPCRs have been described ( Jähnichen et al, 2010 ; Huang et al, 2015 ; Peyrassol et al, 2016 ; Peyrassol et al, 2018 ; Van Hout et al, 2018 ; Koehl et al, 2019 ; Cheloha et al, 2020a ; Low et al, 2020 ; Pan et al, 2020 ; Ren et al, 2020 ; De Groof et al, 2021 ; Chen et al, 2021 ; Haubrich et al, 2021 and references in Heukers et al, 2019 ). While most of these VHHs that target extracellular epitopes of GPCRs block receptor signaling or are not demonstrated to interfere with receptor signal transduction, some of these extracellular binders are reported to ortho- or allosterically activate receptor signaling, thus inherently stabilizing active GPCR conformers ( Ma et al, 2020 ; Ren et al, 2020 ; Hong et al, 2021 ; Scholler et al, 2017 ).…”

Therapeutic Potential of Intrabodies for Cancer Immunotherapy: Current Status and Future Directions

“…Interestingly, the function of a new nanobody against a G protein-coupled receptor (GPCR) of the human cytomegalovirus (CMV), US28, which is involved in cancer progression in glioblastoma was demonstrated [ 27 ]. The nanobody (VUN103) inhibits constitutive US28 signaling by G protein displacement and inhibited US28-enhanced spheroid growth in glioblastoma tumor cells.…”

“…They can be selected by phage display or ribosomal display from immune, naïve or synthetic single domain antibody repertoires [ 16 , 17 , 18 , 19 ] and inactivate their targets by altering their conformation or interfere with the binding of the target protein to its corresponding binding partner. sdAbs were isolated against intracellular TAAs [ 19 ], including the recent examples of hypoxia-inducible factor-1 (HIF-1) [ 24 ], serine/threonine protein kinase AKT2 [ 25 ], p53 C-terminal region involved in the interaction with Twist1 [ 26 ] and chemokine receptor US28 [ 27 ]. In addition to targeting intracellular TAAs, intrabodies have been selected against intracellular neoantigens, for example against HRASG12V [ 28 , 29 , 30 ] HRASG12V, KRASG12D, KRASG13D, NRASQ61R, KRASG12V, KRASQ61H [ 31 ] H - and K - Ras G12V [ 32 ], p21Ras [ 33 ] and KRASG12V [ 34 ].…”

Intracellular VHHs to monitor and modulate GPCR signaling

“…By using agonists and antagonists of a given receptor, it is possible to conclude whether a VHH preferentially stabilizes inactive forms of the receptor or if it binds constitutively the active receptor and is displaced by further recruitment of G proteins, or whether it only binds activated conformations and then prevents G protein binding, provided that it has a sufficient affinity for the receptor, as seems to be the case of Nb80 (estimated Kd ≃140 nM) ( 41 ). Several authors have developed a bioluminescence resonance energy transfer (BRET) assay to monitor the interaction of the intra-VHH and a GPCR ( 42 – 44 ). This is the case of the interaction of the kappa-type opioid receptor (OPRK1) with the Nb6 and Nb39 intra-VHHs ( Figure 3 ) ( 42 , 45 ).…”

“…In agreement, the conformation-specific VUN103 intra-VHH competes with Gq and β-arrestin 2 that constitutively interact with US28, and severely impairs inositol phosphate accumulation, nuclear factor of activated T cells (NFAT), signal transducer and activator of transcription 3 (STAT3) and NF-ϰB activation. In contrast, Nb7 does not affect constitutively active US28, but rather stabilizes US28 bound to its fractalkine CX3CL1 ligand, and inhibits the cell signaling it mediates ( 44 ) ( Figure 3 ). Although both Nb7 and VUN103 bind ICL2 and ICL3 ( 46 ), they seem to recognize distinct active conformations of US28.…”