In solid tumors, angiogenesis occurs in the setting of a defective vasculature and impaired lymphatic drainage that is associated with increased vascular permeability and enhanced tumor permeability. These universal aspects of the tumor microenvironment can have a marked influence on intratumoral drug delivery that may often be underappreciated. In this study, we investigated the effect of blood vessel normalization in tumors by the antiangiogenic drug bevacizumab on antibody uptake by tumors. In mouse xenograft models of human ovarian and esophageal cancer (SKOV-3 and OE19), we evaluated antibody uptake in tumors by positron emission tomographic imaging 24 and 144 hours after injection of Zr-IgG (SKOV-3) before or after treatment with bevacizumab. Intratumor distribution was assessed by fluorescence microscopy along with mean vessel density (MVD) and vessel normalization. Notably, bevacizumab treatment decreased tumor uptake and intratumoral accumulation compared with baseline in the tumor models relative to controls. Bevacizumab treatment also reduced MVD in tumors and increased vessel pericyte coverage. These findings are clinically important, suggesting caution in designing combinatorial trials with therapeutic antibodies due to a possible reduction in tumoral accumulation that may be caused by bevacizumab cotreatment. Cancer Res; 73(11); 3347-55. Ó2013 AACR.

Vascular endothelial growth factor (VEGF) pathway targeting agents have been combined with other anticancer drugs, leading to improved efficacy in carcinoma of the cervix, stomach, lung, colon and rectum, ovary, and breast. Vessel normalization induced by VEGF pathway targeting agents influences tumor drug uptake. Following bevacizumab treatment, preclinical and clinical studies have shown a decrease in tumor delivery of radiolabeled antibodies and two chemotherapeutic drugs. The decrease in vessel pore size during vessel normalization might explain the decrease in tumor drug uptake. Moreover, the addition of bevacizumab to cetuximab, or panitumumab in colorectal cancer patients or to trastuzumab in breast cancer patients, did not improve efficacy. However, combining bevacizumab with chemotherapy did increase efficacy in some cancer types. Novel biomarkers to select patients who may benefit from combination therapies, such as the effect of an angiogenesis inhibitor on tumor perfusion, requires innovative trial designs and large clinical trials. Small imaging studies with radiolabeled drugs could be used in the interphase to gain further insight into the interplay between VEGF targeted therapy, vessel normalization and tumor drug delivery.

Transforming growth factor-b (TGF-b) promotes cancer invasion and metastasis and is therefore a potential drug target for cancer treatment. Fresolimumab, which neutralizes all mammalian active isoforms of TGF-b, was radiolabeled with 89 Zr for PET to analyze TGF-b expression, antibody tumor uptake, and organ distribution. Methods: 89 Zr was conjugated to fresolimumab using the chelator N-succinyldesferrioxamine-B-tetrafluorphenol. 89 Zr-fresolimumab was analyzed for conjugation ratio, aggregation, radiochemical purity, stability, and immunoreactivity. 89 Zr-fresolimumab tumor uptake and organ distribution were assessed using 3 protein doses (10, 50, and 100 mg) and compared with 111 In-IgG in a human TGF-b-transfected Chinese hamster ovary xenograft model, human breast cancer MDA-MB-231 xenograft, and metastatic model. Latent and active TGF-b1 expression was analyzed in tissue homogenates with enzyme-linked immunosorbent assay. Results: 89 Zr was labeled to fresolimumab with high specific activity (.1 GBq/ mg), high yield, and high purity. In vitro validation of 89 Zr-fresolimumab showed a fully preserved immunoreactivity and long (.1 wk) stability in solution and in human serum. In vivo validation showed an 89 Zr-fresolimumab distribution similar to IgG in most organs, except for a higher uptake in the liver in all mice and higher kidney uptake in the 10-mg group. 89 Zr-fresolimumab induced no toxicity in mice; it accumulated in primary tumors and metastases in a manner similar to IgG. Both latent and active TGF-b was detected in tumor homogenates, whereas only latent TGF-b could be detected in liver homogenates. Remarkably high 89 Zr-fresolimumab uptake was seen in sites of tumor ulceration and in scar tissue, processes in which TGF-b is known to be highly active. Conclusion: Fresolimumab tumor uptake and organ distribution can be visualized and quantified with 89 Zr-fresolimumab PET. This technique will be used to guide further clinical development of fresolimumab and could possibly identify patients most likely to benefit.

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