We describe a novel cancer therapy of unusual potency and selectivity and elucidate its mechanism. Collectively, our data demonstrate the safety and effectiveness of specific delivery of short-ranged alpha particles to endothelial cells in inhibiting endothelial cell proliferation and tumor new vessel formation, resulting in suppression of tumor growth. Additionally, the treatment resulted in a relatively mature remaining tumor vasculature and an enhanced overall anti-tumor response when combined with subsequently administered chemotherapy.
Most anti-angiogenic therapies that are being developed or have been approved target cytokines, growth factors or their receptors. However, tumors may circumvent therapies aimed at a single signaling pathway via up-regulation of alternate pathways and therefore, targeting of multiple angiogenic pathways has been suggested 
. Selective killing of endothelial cells in the tumor neovasculature or their progenitors with a cytotoxic agent is an attractive alternative approach to overcome the acquired resistance. We exploited the exclusive binding specificity of the antibody E4G10 for the endothelial cells of the neovasculature as well as VE Cadherin positive EPCs in the bone marrow and blood via its proposed targeting of an epitope exposed only on the monomeric, unengaged form of VE cadherin, which gets masked on the formation of adherens junctions between adjacent endothelial cells (Fig. S1
). Therefore, based on the proposed mechanism, the antibody should not target established vasculature. Our in vivo imaging and post-mortem biodistribution data confirm the proposed selectivity of the antibody for neovasculature and EPCs. The uptake of radioactivity seen in the lungs and the heart was due to the presence of radiolabeled antibody in the blood pool in these organs and it declined in proportion to the blood clearance of radiolabeled antibody. As a consequence of selective targeting of a minor subpopulation of cells, in conjunction with the short range of the alpha particles, no discernible toxicity was seen in therapy studies with 225
Ac-E4G10. Additionally, administration of supra-therapeutic doses of 225
Ac-E4G10 in mice did not result in any histopathologic abnormality, vascular leak or hemorrhage in normal organs as has been seen with other VE-cadherin-binding antibodies that disrupt adherens junctions in established vessels 
. This result further validated the pharmacokinetic data that E4G10 did not specifically accumulate in normal tissues with established blood vessels as the target VE-cadherin epitope for EG410 is masked in those vessels.
Ac was considered as a suitable cytotoxic agent for coupling to E4G10 because of its four alpha particle emissions per decay of a 225
Ac atom, which contributes to the enhanced the potency of 225
Ac labeled constructs. 225
Ac labeled antibodies have been shown to be safe and potent anti-tumor agents in mouse models of solid prostatic carcinoma, disseminated lymphoma, intra-peritoneal ovarian cancer and in a rat model of meningeal neuroblastoma 
. The high energy (5–8 MeV) and short path-length (50–80 µm) makes alpha particles the most appropriate form of radiation for targeting of individual endothelial cells 
Even though EG410 did not bind to the LNCaP cells, treatment with 225
Ac labeled E4G10 resulted in an inhibition of tumor growth, lower serum PSA and enhanced survival in prostate cancer xenograft-bearing mice, accompanied by a decrease in tumor blood vessel density (as evidenced by vWF immunostaining). Although 225
Ac-E4G10 inhibited tumor growth, it did not eradicate tumors when used as a single agent. The result is consistent with the vascular-targeting mechanism of action of 225
Ac-E4G10. Since the tumor cells are not targeted, the residual tumor cells (as seen in ) can initiate a second phase of angiogenesis which results in a resurgence in tumor growth. Specific depletion of VE-cadherin positive endothelial cells of the tumor neovasculature with 225
Ac labeled E4G10 is one explanation for the observed decrease in tumor blood vessel density. Recently, it has been shown that hypoxic stress can enhance the release of endothelial progenitors from the bone marrow and their recruitment and incorporation into tumor vasculature 
. Selective alpha particle-mediated killing of the VE-cadherin positive late endothelial progenitors in the bone marrow or circulating endothelial progenitor cells in the blood stream (which are readily accessible to the radiolabeled antibody) is another mechanism via which 225
Ac-E4G10 may have inhibited tumor angiogenesis. Our related manuscript (Nolan et al,
submitted) describes, in a Lewis lung cancer model, the mobilization of VE cadherin positive EPCs from the bone-marrow into the peripheral circulation and their incorporation into tumor neovessels. Treatment with 225
Ac-E4G10 resulted in a significant decrease in the bone-marrow derived endothelial cell progenitors in the tumor and a lower tumor vessel density. Another plausible contribution to the pronounced inhibition of tumor growth, besides direct cytotoxicity to tumor neovascular endothelial cells or their progenitors, can be from the local release of α-particle emitting daughters of 225
Ac (francium-221, astatine-217 and bismuth-213
in the tumor microenvironment as a result of 225
Ac decay following binding of 225
Ac-E4G10 to VE cadherin positive endothelial cells in nascent tumor vasculature.
Histopathologic examination of control tumors revealed a network of dilated, anastomosing vascular spaces that formed between tumor cell nests and were filled with extravasated RBCs. Although these tumors displayed significantly greater staining for vWF as compared to 225
Ac-E4G10 treated ones, most of these vascular channels were not lined by endothelial cells and therefore, did not stain for vWF. Moreover, most vWF positive structures in the control tumors did not possess a lumen and may represent endothelial sprouts growing into the tumor 
. These findings are consistent with previous data on this tumor model 
. A transient breach in vessel wall integrity secondary to growth factor-driven active endothelial cell proliferation and sprouting may have resulted in the extravasation of RBCs and the resultant intra-tumoral hemorrhage 
Blood vessels in tumors are abnormal in structure (dilated and torturous with abnormal basement membrane and inadequate pericyte coverage) and function (hyperpermeable; 
. Tumor vessel leakiness correlates closely with histologic tumor grade 
. A functionally compromised vasculature also precludes efficient delivery of oxygen and chemotherapeutics to the tumors. Furthermore, tumor hypoxia makes cancer cells resistant to radiation damage 
. Previous studies have shown that inhibition of VEGF signaling can “normalize” the blood vessels and therefore, overcome these pharmacokinetic barriers to drug and oxygen delivery 
. An interesting finding in our study was that treatment with 225
Ac-E4G10, in addition to reducing tumor blood vessel density, also resulted in a structurally mature residual tumor vasculature wherein a greater proportion of vascular endothelial cells had pericyte coverage as compared to control tumors. This could be attributed to pruning of immature tumor vessels via killing of excess endothelial cells or EPCs by treatment with 225
Ac-E4G10. Therefore, inhibition of abnormal endothelial cell proliferation and the resultant vessel leak may possibly be the reason for the relatively normal residual tumor vasculature seen in our studies. The role of pericyte coverage in inhibiting metastasis in a murine pancreatic cancer model has been shown recently 
. An inverse correlation between pericyte coverage and hematogenous spread has also been observed in colorectal cancer patients 
. The effects of 225
Ac-E4G10 treatment on tumor metastasis and invasiveness are currently being investigated.
Combination therapy wherein 225
Ac-E4G10 treatment was followed by a course of paclitaxel resulted in an enhancement of the overall anti-tumor response. One plausible explanation for that effect is that the structural normalization of tumor vasculature by 225
Ac-E4G10 treatment resulted in increased efficiency of the vessels in delivering the chemotherapeutic to the tumor cells, thereby leading to synergy. Alternatively, the two treatments may also have an additive effect by killing two distinct populations of cells (endothelial and tumor cells). Paclitaxel has also been shown to possess anti-angiogenic properties 
but recent data by Kerbel et al
suggests that the cremophor-based paclitaxel formulation (which was used in our experiments) does not have a significant impact on the tumor vasculature or viability of circulating endothelial progenitors 
. Nonetheless, killing of tumor cells by a cytotoxic agent can possibly decompress blood vessels in a tumor and therefore, increase blood flow. The exact mechanism of the enhanced overall response is currently being investigated.
Our results allude to the development of an integrative approach to cancer therapy wherein 225Ac-E4G10 therapy is precisely timed with chemotherapy or radiation to maximize the delivery of the chemotherapeutic and to improve radiation sensitivity of tumors. Delivering the two treatment modalities in a carefully planned temporal fashion can potentially result in a synergistic effect on tumor-cell killing. Importantly, our data suggest that targeting the tumor cells or their microenvironment may not be necessary to slow cancer growth if the angiogenic progenitors, a relatively small but possibly sensitive cell population, can be selectively depleted.