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Project

Characterisation of novel CXCR4-targeting nanobodies with anti-HIV and anti-tumoral activity

Chemokines and their receptors are central players in several diseases and are consequently recognised as interesting therapeutic targets. The chemokine receptor CXCR4 was initially discovered as a coreceptor facilitating HIV entry in human target cells. Additionally, growing evidence supports the crucial role of CXCR4 and its cognate ligand CXCL12 in tumor progression, stem and progenitor cell mobility and inflammatory and autoimmune diseases. Disrupting the crosstalk between CXCL12 and CXCR4 could therefore be an attractive strategy in these disease areas. Multiple CXCR4-targeting ligands of various compound classes  have so far been characterised in in vitro assays and preclinical models. Despite considerable efforts however, only one anti-CXCR4 agent has been approved for clinical use, which emphasizes the demand for improved agents.

In this doctoral project, we first aimed to optimise and validate several in vitro CXCR4-specific assays. For this, we evaluated a panel of 11 commercially available anti-CXCR4 inhibitors (small molecules and peptide-based ligands) that were previously described to inhibit CXCR4 signalling and function. These compounds were tested side-by-side in our cell-based assays to determine their effect on CXCL12 binding, CXCL12-induced calcium mobilization, CXCR4 internalization and cell migration. Our data demonstrated the potential of the CXCL12 competition binding assay as a highly valuable, initial screening assay for the identification of novel CXCR4-interacting ligands. We further developed three additional assays to study the effect of CXCR4-interacting ligands on CXCL12-mediated Akt and ERK activation, and inhibition of cAMP accumulation. Taken together, in the first part of the thesis, several in vitro CXCR4-specific assays that cover many different aspects of CXCR4 activation (i.e. ligand binding, intracellular signalling, cellular response) were developed and validated by existing CXCR4 ligands. These assays formed the methodological basis for the second part of the thesis, in which we aimed to study a relatively novel class of CXCR4-targeting ligands, i.e. nanobodies (Nbs).

A novel panel of CXCR4-targeting Nbs was generated in collaboration with Prof. M.J. Smit (VU Amsterdam, the Netherlands) and Prof. H. de Haard (argenx, Belgium). These newly generated Nbs were characterised by the above-mentioned CXCR4-specific assays. Thereafter, their anti-HIV activity and anti-tumoral efficacy was investigated. Ten unique Nb clones were used in this study, of which two different formats were evaluated: monomeric Nbs (Nbs) (VUN400-VUN409) and their corresponding bivalent Nb-Fc fusion molecules (Nb-Fc) (VUN400-Fc-VUN409-Fc). In an initial characterisation, all Nbs and Nb-Fcs were tested for interaction with CXCR4, inhibition of CXCL12 receptor binding and CXCL12-mediated calcium signalling. All Nbs and Nb-Fcs were active, albeit with varying potencies. Three Nbs and Nb-Fc constructs (VUN400-VUN402 and VUN400-Fc-VUN402-Fc) were studied in more detail to evaluate their inhibitory effect on CXCL12-induced β-arrestin 2 recruitment, inhibition of cAMP accumulation, CXCR4 internalization, chemotaxis and cell morphology changes. While VUN401 and VUN402 were highly potent inhibitors of CXCR4 signalling and cellular response, VUN402 turned out to be a less potent inhibitor. In all different assays, the Nb-Fc constructs were more potent than their monovalent counterparts.

In the next study, we evaluated the potential of CXCR4-targeting Nbs for their inhibition of HIV infection. All Nbs and Nb-Fc constructs inhibited HIV replication. A remarkable finding was made for the monomeric Nbs: their anti-HIV activity did not always correlate well with their potency to inhibit CXCL12-mediated CXCR4 function. When the anti-HIV activity of Nbs VUN400, VUN401 and VUN402 was studied in more detail, it was demonstrated that these Nbs inhibit HIV replication in different relevant cellular systems using both X4 laboratory-adapted HIV strains as well as a X4 clinical isolate. Because the anti-CXCR4 activity of Nb VUN402 was much more moderate, it was postulated that this Nb could serve as an initial starting point for further development of a selective and potent anti-HIV molecule that might have less impact on the normal CXCR4 physiological function.

Finally, given the important role of CXCR4 in cancer progression, we evaluated the anti-tumoral activity of VUN401, a highly potent anti-CXCR4 inhibitor, in an in vivo xenograft mouse model for cancer. The haematological malignancy T-cell acute lymphoblastic leukemia (T-ALL) was used as a study model. After confirming the inhibitory activity of VUN401 on CXCL12-induced MOLT-4/Luc2 cell migration in vitro, we determined its activity in an in vivo model. First, this model was validated using the reference CXCR4 antagonist AMD3100. According to our hypothesis, this small molecule inhibited tumor growth, both as a single agent as well in combination with the chemotherapeutic agent Vincristine. Initial results also indicated that VUN401, whether or not in combination with Vincristine, exerted anti-tumoral activity in our model.

In this PhD project, we characterised a novel panel of CXCR4-targeting Nbs and Nb-Fc constructs. We determined their inhibitory activity on CXCR4 signalling and function. Furthermore, we proposed VUN402 as a candidate molecule for the development of a selective anti-HIV agent. Additionally, our initial results indicated the therapeutic potential of VUN401 for the treatment of T-ALL.

Date:1 Sep 2014 →  10 Oct 2019
Keywords:CXCR4, nanobody, Cancer, HIV
Disciplines:Cancer therapy, Virology
Project type:PhD project