Neovascularization is a particularly important hallmark of breast tumor growth and metastatic spread [46–50
]. The growth factor VEGF-A and the receptor KDR have both been implicated in highly metastatic breast cancers [51–53
]. We have previously demonstrated that the VEGF121
/rGel growth factor fusion toxin specifically targets Flk-1/KDR-expressing tumor vascular endothelial cells and inhibits the growth of subcutaneously implanted human tumor xenografts [38
]. The current study was designed to evaluate its effect on the development of breast cancer metastases in lungs following intravenous injection of MDA-MB-231 cells.
The salient finding of our study of the VEGF121
/rGel construct is that: this fusion toxin is specifically cytotoxic to cells overexpressing the KDR receptor for VEGF. However, the human breast MDA-MB-231 cells employed for these studies do not express this receptor and, therefore, were not directly affected by this agent (). Although the antitumor effects of VEGF121
/rGel observed from our in vivo
studies appear to be solely the result of targeting the Flk-1-expressing tumor vasculature and not the tumor cells themselves, one cannot rule out a direct effect on tumor cells or a combination of targeting both the tumor and the vasculature. Administration of the VEGF121
/rGel construct to mice previously injected (i.v.) with tumor cells dramatically reduced the number of tumor colonies found in the lung, their size, and their vascularity. In addition, the number of cycling breast tumor cells within lung metastatic foci was found to be reduced by an average of 60%. This reduction compares favorably to the effect of DT-VEGF on the growth of pancreatic cancer [54
] and to other vascular targeting agents such as Avastin, which had an overall clinical response rate of 9.3% in a Phase I/II dose escalation trial in previously treated metastatic breast cancers [55
]. In addition to the reduced number of blood vessels present in lung metastases of treated mice, we also found that the few vessels present had a greatly reduced expression of VEGFR-2. Therefore, this construct demonstrated an impressive long-term impact on the growth and development of breast tumor metastatic foci found in the lungs.
Targeting tumor vasculature with a variety of technologies has been shown to inhibit the growth and development of primary tumors as well as metastases. Recently, Shaheen et al. [56
] demonstrated that small-molecule tyrosine kinase inhibitors active against the receptors for VEGF, fibroblast growth factor, and platelet-derived growth factors were also capable of inhibiting microvessel formation and metastases in tumor model systems. Previously, Seon et al. [57
] demonstrated long-term antitumor effects of an antiendoglin antibody conjugated with ricin-A chain (RTA) in a human breast tumor xenograft model.
Surprisingly, one finding from our study was that administration of VEGF121
/rGel resulted in a three-fold decrease in the number of Ki-67-labeled (cycling) cells in the metastatic foci present in the lung (). Clinical studies have suggested that tumor cell cycling may be an important prognostic marker for disease-free survival in metastatic breast cancer, but that Ki-67 labeling index, tumor MVD, and neovascularization appear to be independently regulated processes [58,59
]. To our knowledge, this is the first report of a significant reduction in tumor labeling index produced by a vascular targeting agent.
Another critical finding from our studies is the observation that the vascular-ablative effects of the VEGF121
/rGel fusion construct alone were unable to completely eradicate lung metastases. Although the growth of larger pulmonary metastases was completely inhibited by this therapeutic approach, the development of small, avascular, metastatic foci within lung tissues was observed. Our findings indicate that vasculature in the small and mid-sized metastatic lesions (diameter < 500 µm) was much more susceptible to the action of VEGF121
/rGel than that in colonies with diameters larger than 500 µm. Several explanations might account for this observation. First, the number of VEGF receptors on endothelial cells within the small, exponentially expanding colonies might be higher than that in the well-established lesions. This would lead to an increase in binding sites for VEGF121
/rGel and, hence, an increased toxicity toward vessels specifically in small colonies. Second, vascular endothelial cells in small colonies might have a reduced capacity to survive after drug assault compared to vessels in established lesions. This could be due to insufficient recruitment of supporting cells (pericytes/smooth muscle cells) to the newly formed vessels, and/or derangements in the production of and interaction with components of basement membrane. Currently, the precise mechanism of the differential anti-vascular toxicity on different size colonies is not completely understood. However, these data strongly suggest that the combination of vascular targeting agents with chemotherapeutic agents or with radiotherapeutic agents, which directly damage tumor cells themselves, may provide for greater therapeutic effect. Studies of several vascular targeting agents in combination with chemotherapeutic agents have already demonstrated a distinct in vivo
antitumor advantage of this combination modality against experimental tumors in mice [60
]. Studies by Pedley et al. [61
] have also suggested that the combination of vascular targeting and radioimmunotherapy may also present a potent antitumor combination. Finally, studies combining hyperthermia and radiotherapy with vascular targeting agents have demonstrated an enhanced activity against mammary carcinoma tumors in mice [62
]. Studies in our laboratory combining VEGF121
/rGel and various chemotherapeutic agents, biologic agents, or therapeutic agents targeting tumor cells are currently ongoing.
The rGel toxin is a single-chain N
-glycosidase that is similar in its action to ricin-A chain [39
]. However, unlike ricin-A chain, the use of rGel does not appear to result in VLS [44
]. Side effects have been observed with clinical administration of RTA-based, diphtheria toxin-based, and Pseudomonas
exotoxin-based fusion proteins. These side effects include liver toxicity, development of neutralizing antibodies, and development of VLS. The development of neutralizing antibodies was found in 69% of patients treated with RTA immunotoxins [63
], 37% of patients treated with PE-based constructs [64
], and 92% of patients treated with DT constructs [65
]. In contrast, our ongoing clinical trial with an rGel-based conjugate currently demonstrates a relatively low antigenicity of the rGel component, with only 2 of 22 patients developing antibodies to the rGel portion of the drug [44
]. In addition, the development of hepatoxicity and VLS is commonly observed, with toxin molecules thus far not having been observed for rGel-based agents. These findings support continuing development in a clinical setting of targeted therapy using rGel. In addition, our laboratory continues to develop designer toxins with reduced antigenicity and size [66
The presented findings demonstrate that VEGF121/rGel can clearly and specifically target Flk-1/KDR-expressing tumor vasculature both in vitro and in vivo and that this agent can have an impressive inhibitory effect on tumor metastases. Studies are continuing in our laboratory to examine the activity of this agent alone and in combination against a variety of orthotopic and metastatic tumor models.