Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and 3

Abstract

Vascular endothelial growth factor C (VEGF-C) is a key mediator of lymphangiogenesis, acting via its receptors VEGF-R2 and VEGF-R3. High expression of VEGF-C in tumors correlates with increased lymphatic vessel density, lymphatic vessel invasion, sentinel lymph node metastasis and poor prognosis. Recently, we found that in a chemically induced skin carcinoma model, increased VEGF-C drainage from the tumor enhanced lymphangiogenesis in the sentinel lymph node and facilitated metastatic spread of cancer cells via the lymphatics. Hence, interference with the VEGF-C/VEGF-R3 axis holds promise to block metastatic spread, as recently shown by use of a neutralizing anti-VEGF-R3 antibody and a soluble VEGF-R3 (VEGF-C/D trap). By antibody phage-display, we have developed a human monoclonal antibody fragment (single-chain Fragment variable, scFv) that binds with high specificity and affinity to the fully processed mature form of human VEGF-C. The scFv binds to an epitope on VEGF-C that is important for receptor binding, since binding of the scFv to VEGF-C dose-dependently inhibits the binding of VEGF-C to VEGF-R2 and VEGF-R3 as shown by BIAcore and ELISA analyses. Interestingly, the variable heavy domain (VH) of the anti-VEGF-C scFv, which contains a mutation typical for camelid heavy chain-only antibodies, is sufficient for binding VEGF-C. This reduced the size of the potentially VEGF-C-blocking antibody fragment to only 14.6 kDa. Anti-VEGF-C VH-based immunoproteins hold promise to block the lymphangiogenic activity of VEGF-C, which would present a significant advance in inhibiting lymphatic-based metastatic spread of certain cancer types.

Biological Basis of Therapeutic Lymphangiogenesis

“…[167][168][169][170][171][172] To block tumor lymphangiogenesis and metastasis, antilymphangiogenic strategies using neutralizing antibodies against VEGFR-3, VEGF-C, and VEGF-D, as well as soluble VEGFR-3 fusion proteins (VEGF-C/D trap) and VEGF-C siRNA molecules, have been used. [173][174][175][176][177][178][179] The efficacy of neutralizing antibodies against VEGFR-3 was shown to be increased through the use of a combination of 2 distinct classes of antibodies directed toward functionally different regions of the receptor that are involved in ligand binding and receptor dimerization. 180 Some inhibition of tumor-induced lymphangiogenesis is also provided by antibodies against the neuropilin-2 coreceptor, which, however, is also expressed in a variety of neuronal tissues.…”

Long noncoding RNA BLACAT2 promotes bladder cancer–associated lymphangiogenesis and lymphatic metastasis

“…Accumulating evidence has shown that upregulated VEGF-C, a lymphangiogenic growth factor, positively correlates with regional LN metastasis and poor survival in multiple human malignancies, including bladder cancer (9,(11)(12)(13)(14)(15). Several groups have reported that when VEGF-C signaling is blocked via either small interfering RNAs or a neutralizing antibody to VEGF-C or VEGF-R3, the lymphatic-based metastatic spread of human malignancies can be inhibited (9,(16)(17)(18). Importantly, the VEGF-C monoclonal antibody (VGX-100) has been tested in a phase I clinical trial for advanced or metastatic solid tumors that are refractory to standard treatments (ClinicalTrials.gov NCT01514123).…”

Inhibition of lymphangiogenic factor VEGF-C expression and production by the histone deacetylase inhibitor suberoylanilide hydroxamic acid in breast cancer cells

“…The targeting of VEGF-C has not received significant attention in the past decade. By using antibody phage-display, Rinderknecht et al identified a VEGF-C-blocking antibody which could effectively inhibit the interaction between VEGF-C and VEGFR3 and suppress its downstream signaling (30). The underlying mechanism mediating the upregulation of VEGF-C in cancer cells is largely unclear.…”