Over the last decade, molecular imaging of angiogenesis has gained tremendous interest since angiogenesis is a fundamental process in both normal physiology and many disease processes such as tumor development and metastasis [1
]. Two of the most intensively studied angiogenesis-related targets are integrin αv
and vascular endothelial growth factor receptors (VEGFRs). Several tracers targeting these two receptors are already in clinical investigation [35
]. Besides these two widely explored targets, CD105 is another very important tumor angiogenesis-related target. In clinical practice, the currently accepted standard method for quantifying tumor angiogenesis is to assess microvessel density (MVD) by performing CD105 immunohistochemistry on tumor tissue, which is an independent prognostic factor for survival in patients with many types of solid tumors [11
]. CD105 has the advantage of being selectively expressed on proliferating endothelial cells at significantly higher levels (up to 3 × 106
copies per cell) than other angiogenic targets like the VEGFRs (less than 0.2 × 106
copies per cell) [31
]. Surprisingly, molecular imaging of CD105 has not been well-studied in the literature [13
Non-invasive imaging of CD105 expression has the potential to accelerate drug development by providing a reliable measure of angiogenesis in the entire body as an intact system, thereby facilitating individualized treatment monitoring and dose optimization in animal models, clinical trials, and ultimately in the day-to-day management of cancer patients. Therefore, the goal of this study was to develop a CD105-specific NIRF agent for non-invasive imaging of tumor angiogenesis. We have achieved this goal and investigated 800CW-TRC105 in vitro, in vivo, and ex vivo. The experimental setup in this study represents the “best-case scenario” for optical imaging in that: 1) the emission maximum of 800CW is 806 nm, which is in the optically clear NIR window; 2) the tumors were subcutaneously inoculated, thus tissue penetration is not a major issue; 3) the use of laser excitation gives stronger signal than other excitation sources; and 4) the mouse hair was removed before NIRF imaging and the skin color of the mouse is very light. In clinical settings, NIRF imaging can be used for imaging tissues close to the surface of the skin (e.g. breast imaging), tissues accessible by endoscopy (such as the esophagus and colon), and intraoperative visualization (typically image-guided surgery).
The rationale for choosing the 4T1 tumor model for this study is that the parent antibody of TRC105 (SN6j, a monoclonal antibody of murine origin which binds to CD105) has been shown to be effective as an anti-angiogenic agent in this model [27
]. Further, 4T1 tumor has highly angiogenic tumor vasculature () which is expected to provide sufficient target density for imaging applications. We realized that one major limitation of this model is that the tumor vasculature is of murine origin. TRC105 has significantly higher affinity to human CD105 than its murine homolog [40
]. Thus, the 4T1 tumor model is not optimal for testing TRC105. For future investigation, the following strategies may be evaluated to better mimic the clinical situation: using transgenic mice with human tumor vasculature or testing an anti-CD105 antibody that binds with high affinity to murine CD105. Follow-up studies are currently underway. Nonetheless, excellent tumor contrast was achieved in this study, even at early time points. The higher avidity of this agent to human CD105 is expected to exhibit even better tumor targeting capability in human patients (e.g. image-guided tumor resection) than we see here in mouse models of cancer.
One interesting finding from this study was the difference between the pharmacokinetic and biodistribution patterns of 800CW-TRC105 and 800CW-Cetuximab. The tumor uptake of 800CW-TRC105 (due to specific TRC105-CD105 interaction) increased quite rapidly and plateaued while tumor uptake of 800CW-Cetuximab (due to passive targeting only) increased much more slowly. The circulation half-lives were very different for these two isotype-matched antibodies. In recent human studies, a surprisingly short circulation half-life for TRC105 was also observed in cancer patients [28
]. At doses of 20 mg or less (0.3 mg/kg), TRC105 is only detectable in the blood for 4 h or less. The half-life at a dose of 700 mg (10 mg/kg) is less than 24 h (unpublished data). The half-life of TRC105 will be longer at higher doses once CD105 in the tumor vasculature is saturated and after the tumor bulk is reduced.
The working hypothesis for such findings is that CD105 is an intravascular target that is immediately available to TRC105 upon injection (unlike tumor targets that require an antibody to diffuse out of the circulation and into a tumor before it can bind to its target). Tumor endothelial cells express CD105 at high copy numbers, which serves as an intravascular CD105 sink that rapidly sequesters the TRC105. Since TRC105 inhibits angiogenesis, the tumor becomes more hypoxic thereby inducing more CD105 expression on previously quiescent endothelial cells, thus functioning as a nearly endless TRC105 sink. Another explanation for such an obvious difference between isotype-matched antibodies (both TRC105 and Cetuximab are human/murine chimeric IgG1 type) is the lack of cross-reactivity with murine tissues for Cetuximab, which leads to longer circulation half-life and slower clearance/sequestration than TRC105 (which cross-reacts with murine CD105 although with lower affinity than human CD105).