The carboxyl terminus of VEGF-A is a potential target for anti-angiogenic therapy

Abstract

Anti-VEGF-A therapy has become a mainstay of treatment for ocular neovascularisation and in cancer; however, their effectiveness is not universal, in some cases only benefiting a minority of patients. Anti-VEGF-A therapies bind and block both pro-angiogenic VEGF-Axxx and the partial agonist VEGF-Axxxb isoforms, but their anti-angiogenic benefit only comes about from targeting the pro-angiogenic isoforms. Therefore, antibodies that exclusively target the pro-angiogenic isoforms may be more effective. To determine whether C-terminal-targeted antibodies could inhibit angiogenesis, we generated a polyclonal antibody to the last nine amino acids of VEGF-A165 and tested it in vitro and in vivo. The exon8a polyclonal antibody (Exon8apab) did not bind VEGF-A165b even at greater than 100-fold excess concentration, and dose dependently inhibited VEGF-A165 induced endothelial migration in vitro at concentrations similar to the VEGF-A antibody fragment ranibizumab. Exon8apab can inhibit tumour growth of LS174t cells implanted in vivo and blood vessel growth in the eye in models of age-related macular degeneration, with equal efficacy to non-selective anti-VEGF-A antibodies. It also showed that it was the VEGF-Axxx levels specifically that were upregulated in plasma from patients with proliferative diabetic retinopathy. These results suggest that VEGF-A165-specific antibodies can be therapeutically useful.

Fig. 1

Exon8apab is specific for VEGF-A₁₆₅a. a An ELISA with Exon8apab as a capture antibody and a biotinylated goat anti-human panVEGF-A detection antibody with increasing concentration of VEGF₁₆₅a (closed circles) or VEGF₁₆₅b (open circles) was carried out. b Western blot using the two antibodies demonstrated specificity of the Exon8apab for 20 ng recombinant human VEGF-A₁₆₅a and a monoclonal antibody to the c terminus of rhVEGF-A₁₆₅b for 20 ng of VEGF-A₁₆₅b. Neither antibody recognised the other protein

Fig. 2

Exon8apab inhibits angiogenesis. a Endothelial cells were seeded onto polycarbonate filters of transwell inserts. Increasing concentrations of Exon8apab and 40 ng/ml rhVEGF-A₁₆₅a was added to the lower well. Cell migration across was measured by counting cells on the lower side of the membrane 24 h after seeding. b The experiment was repeated using either Exon8apab or ranibizumab. IC₅₀ was calculated using a variable slope dose inhibition curve (Prism4.0). c LS174t cells (1 × 10⁹) were implanted into nude mice and tumours allowed to grow to approximately 200 mm³. Animals were then treated with 2 mg/kg Exon8apab antibody at D0 and treatment repeated at D3. Tumour volumes were measured once more at D7. * p < 0.05, one-way ANOVA, Bonferroni test

Fig. 3

Exon8apab inhibits laser-induced CNV in mice. C57/B6 mice underwent retinal lasercoagulation in both eyes (IRIDEX Oculight GLX λ—810 nm, 250 mV, 0.1 s, 75 μm, 4 lesions/eye). Animals were subjected to intraocular injection with either 500 ng (250 ng/µl) of an anti-mouse VEGF neutralising antibody, and control eyes injected with either mouse IgG, or 500 ng (250 ng/µl) of Exon8apab with a control rabbit IgG—controls administered at the same concentration, directly after laser procedure (D0) and on day 7. Staining with isolectin-B4 of the RPE-choroid-sclera complex showed a decrease in lesion size for anti-VEGF and Exon8apab (N, number of eyes; * p < 0.05, one-way ANOVA, Bonferroni post hoc). Merged lesions were excluded from the study. Scale bar 50 µm

Fig. 4

Exon8apab measures VEGF levels in human plasma. VEGF levels were measured in plasma from 32 patients, 5 of whom had proliferative diabetic retinopathy (PDR), 11 non-proliferative diabetic retinopathy (NPDR) and 18 control patients with no diabetes (ND). a VEGF concentrations were measured by ELISA using either the anti-VEGF-A₁₆₅b as a capture antibody, or exon8apab as a capture antibody. b The relative amount of the two isoforms was calculated as a per cent of the total—e.g. 100*VEGF-A_xxxb/(VEGF-A_xxxa + VEGF-A_xxxb). * p < 0.05 compared with VEGF-A_xxxb, Bonferroni