Potent and Efficacious Inhibition of CXCR2 Signaling by Biparatopic Nanobodies Combining Two Distinct Modes of Action

Abstract

Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacologic characterization of nanobodies selectively blocking CXCR2, the most promiscuous of all chemokine receptors. Two classes of selective monovalent nanobodies were identified, and detailed epitope mapping showed that these bind to distinct, nonoverlapping epitopes on the CXCR2 receptor. The N-terminal-binding or class 1 monovalent nanobodies possessed potencies in the single-digit nanomolar range but lacked complete efficacy at high agonist concentrations. In contrast, the extracellular loop-binding or class 2 monovalent nanobodies were of lower potency but were more efficacious and competitively inhibited the CXCR2-mediated functional response in both recombinant and neutrophil in vitro assays. In addition to blocking CXCR2 signaling mediated by CXCL1 (growth-related oncogene a) and CXCL8 (interleukin-8), both classes of nanobodies displayed inverse agonist behavior. Bivalent and biparatopic nanobodies were generated, respectively combining nanobodies from the same or different classes via glycine/serine linkers. Interestingly, receptor mutation and competition studies demonstrated that the biparatopic nanobodies were able to avidly bind epitopes within one or across two CXCR2 receptor molecules. Most importantly, the biparatopic nanobodies were superior over their monovalent and bivalent counterparts in terms of potency and efficacy.

Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers

“…PEs were produced as described [53]. Briefly, E.coli WK6 cells were transformed with Nb-coding pHEN4 plasmids and cultured in 10 mL Terrific broth (TB) containing 100 μg/mL Ampicillin (Fermentas) for 6h at 37°C.…”

Nanobodies to Study G Protein–Coupled Receptor Structure and Function

“…Additionally, these antibodies induce stem cell mobilization in vivo, perhaps providing utility as alternatives for currently approved small-molecule drugs such as plerixafor. Antagonist nanobodies that bind other chemokine receptors, including CXCR2 or CXCR7, have also been described (127, 128). As nanobodies are only approximately 15 kDa, they may provide better penetration into tumors, the central nervous system, or other bodily compartments difficult to access by conventional antibodies.…”

Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding

“…The following nanobody sequences were obtained and ordered as G-blocks from IDT: -Nb-BF10 (Srivastava et al, 2013); -Nb-CA52 (Smolarek et al, 2010); -Nb-2B2, Nb-127D1, and Nb-54B12 (Bradley et al, 2015); -Nb-P2 (Fatima et al, 2014); -Nb-gp41 (Lutje . All peptide array sequences were ordered from Genewiz.…”