Monovalent Nanobodies Research Articles

CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells

The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.

Downregulation of EGFR by a novel multivalent nanobody-liposome platform

The epidermal growth factor receptor (EGFR) is a recognized target for tumor therapy and monoclonal antibodies (mAbs, e.g. cetuximab) have been developed to inhibit receptor activation. Besides blocking ligand (e.g. EGF) binding to the receptor, reports have shown that mAbs promote slow receptor internalization and degradation in lysosomes, i.e. downregulation. The efficacy of receptor downregulation was recently shown to depend on the size of receptor clusters formed at the cell surface. In this study, a multivalent platform is presented, consisting of nanobodies recognizing the ectodomain of EGFR (EGa1) coupled to PEG-liposomes, and the in vitro and in vivo effects of this system on EGFR internalization and downregulation were investigated. Nanobodies are the smallest functional antigen-binding immunoglobulin fragments and the EGa1 nanobody has been described as an EGFR-antagonist. EGa1-liposomes (EGa1-L) induced a more than 90% removal of EGFR from the cell surface, as a result of receptor internalization. Furthermore, this massive sequestration of EGFR mediated by EGa1-L lead to receptor degradation, while no degradation was detected with the monovalent nanobody. The downregulatory capacity here reported was found to be independent of the epitope on EGFR recognized by the grafted nanobody, and exclusive to the nanobody-liposomes, as anti-EGFR single chain variable fragments (scFv) coupled to liposomes were unable to induce this effect. Importantly, EGa1-L induced a significant inhibition of tumor cell proliferation, in vitro, an effect likely mediated by the combination of receptor downregulation and receptor antagonism. Also in vivo, EGFR downregulation was observed in tumors of mice intravenously injected with EGa1-L, indicating that this multivalent platform blocks ligand binding to the receptor and simultaneously induces the downregulation of EGFR.

Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis

Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal disorder. Systemic treatment of IBD patients with anti-tumor necrosis factor (TNF)-α antibodies has proven to be a highly promising approach, but several drawbacks remain, including side effects related to systemic administration and high cost of treatment. Lactococcus lactis was engineered to secrete monovalent and bivalent murine (m)TNF-neutralizing Nanobodies as therapeutic proteins. These therapeutic proteins are derived from fragments of heavy-chain camelid antibodies and are more stable than conventional antibodies. L. lactis-secreted anti-mTNF Nanobodies neutralized mTNF in vitro. Daily oral administration of Nanobody-secreting L. lactis resulted in local delivery of anti-mTNF Nanobodies at the colon and significantly reduced inflammation in mice with dextran sulfate sodium (DSS)-induced chronic colitis. In addition, this approach was also successful in improving established enterocolitis in interleukin 10 (IL10)–/– mice. Finally, L. lactis-secreted anti-mTNF Nanobodies did not interfere with systemic Salmonella infection in colitic IL10–/– mice.In conclusion, this report details a new therapeutic approach for treatment of chronic colitis, involving in situ secretion of anti-mTNF Nanobodies by orally administered L. lactis bacteria. Therapeutic application of these engineered bacteria could eventually lead to more effective and safer management of IBD in humans.