Semi-synthetic nanobody-ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor-1

Antibodies have proven highly valuable for therapeutic development; however, they are typically poor candidates for applications that require activation of G protein-coupled receptors (GPCRs), the largest collection of targets for clinically approved drugs. Nanobodies (Nbs), the smallest antibody fragments retaining full antigen-binding capacity, have emerged as promising tools for pharmacologic applications, including GPCR modulation. Past work has shown that conjugation of Nbs with ligands can provide GPCR agonists that exhibit improved activity and selectivity compared to their parent ligands. The neurokinin-1 receptor (NK1R), a GPCR targeted for the treatment of pain, is activated by peptide agonists such as Substance P (SP) and neurokinin A (NKA), which induce signaling through multiple pathways (Gs, Gq and β-arrestin). In this study, we investigated whether conjugating NK1R ligands with Nbs that bind to a separate location on the receptor would provide chimeric compounds with distinctive signaling properties. We employed sortase A-mediated ligation to generate several conjugates consisting of Nbs linked to NK1R ligands. Many of these conjugates exhibited divergent and unexpected signaling properties and transcriptional outputs. For example, some Nb-NKA conjugates showed enhanced receptor binding capacity, high potency partial agonism, prolonged cAMP production, and an increase in transcriptional output associated with Gs signaling; whereas other conjugates were virtually inactive. Nanobody conjugation caused only minor alterations in ligand-induced upstream Gq signaling with unexpected enhancements in transcriptional (downstream) responses. Our findings underscore the potential of nanobody conjugation for providing compounds with advantageous properties such as biased agonism, prolonged duration of action, and enhanced transcriptional responses. These compounds hold promise not only for facilitating fundamental research on GPCR signal transduction mechanisms but also for the development of more potent and enduring therapeutics.

Excess/unbound ligand was removed, fresh media with 1 µM spantide I was added, and cAMP concentration was measured for an additional 30 min (washout) (n = 3).Washout assay data is summarized as the area under the curve (AUC) recorded for a 100 nM of peptide or conjugate.Data represent mean ± SEM from n=3 independent experiments.Representative concentration-response curves or cAMP production kinetics (mean ± SD) for washout assays.

Figure S2 .
Figure S2.Comparison of the activity NKA and SP6-11 to their triglycine analogues for inducing Gs, β-arrestin2 and Gq responses.Representative concentration-response curves (mean ± SD from technical replicates).EC50 was calculated from the fitting of a sigmoidal concentration-response model to data from three or more independent experiments using GraphPad Prism.

Figure S3 .
Figure S3.MS of the nanobodies before and after conjugation to G3NKA or G3SP6-11.Mass spectra and deconvoluted masses were acquired according to Methods.

Figure S4 .
Figure S4.Replicate experiments of substance P (SP) activation of epitope-tagged NK1R via Glosensor assay.Data points correspond to mean ± SD from technical replicates.

Figure S5 .
Figure S5.Independent replicates for flow cytometry analysis of biotin-labeled nanobodies binding to epitope-tagged NK1R cells.Staining and analysis was performed according to Methods.

Figure S6 .
Figure S6.Effects of peptides and conjugates on cAMP production in NK1R wild-type cells.HEK293 cells stably transfected with Glosensor cAMP reporter (Promega Corp.) 23 and wild-type NK1R were treated with varying concentrations (1 pM -10 mM) of the indicated peptides or conjugates.Activation was assessed by cAMP production after 6 min (n = 3) as described in Methods.(A, B) Representative concentration-response curves (mean ± SD).Insets of concentration-responses without G3-peptides included are shown (under table) to allow better visualization of the activity of the weaker compounds.Curves result from the fitting of a sigmoidal concentration-response model to data.*Maxactivity values were calculated by normalizing the response at 1 mM for conjugates to that of G3NKA or G3SP6-

Figure S8 .
Figure S8.Binding assay for measurement of Nb-ligand conjugate competition with labeled SP. (A) Chemical structure and high-resolution MS analysis of SP-AF647.Binding assay was performed using flow cytometry to assess the ability of (B) NKA and Nb-NKA or (C) SP6-11 and Nb-SP6-11 to outcompete fluorescently labeled SP for binding to epitope-tagged NK1R.Values correspond to mean ± SEM from 3 biological replicates.Differences were evaluated for statistical significance with a one-way ANOVA followed by Dunnett correction.*p < 0.03, **p < 0.002, ***p < 0.0002, **** p < 0.0001

Figure S10 .
Figure S10.Impact of spantide I on the dissipation of cAMP signaling (washout) for Nb6e-NKA and G3NKA.

Figure S11 .
Figure S11.Evaluation of G3SP6-11 peptide and conjugate performance on Gq transcription.Transcriptional responses were evaluated through transfection of cells expressing epitope tagged NK1R with a luciferase reporter plasmid reporting on Gq signaling.Cells were incubated with ~35 nM of the indicated peptides or conjugates for 17 hours and transcription was measured as described in Methods.Data represent mean ± SEM from n=3 independent experiments.