Over the last decade, mounting preclinical and clinical data have suggested the promising future of 89
Zr-based immunoPET in cancer patient management. The choice of Df as the chelator for 89
Zr is attractive because it has been safely used in the clinic for many years. In the past and ongoing clinical studies, neither adverse reactions nor significant changes in blood and urine values were observed after injection of Df-containing conjugates [2
]. In addition, no antibody responses directed against the Df chelate were observed, indicating that its immunogenicity is very low [34
]. With a new bifunctional chelator (i.e. Df-Bz-NCS) that recently became commercially available, it is expected that 89
Zr-based immunoPET will be the focus of more widespread future investigations.
The currently accepted standard method for quantifying angiogenesis is to assess MVD by performing CD105 immunohistochemistry on tumor tissue, an independent prognostic factor for survival in patients with many types of solid tumors [10
]. 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 such as the VEGFRs (less than 0.2 × 106
copies per cell) [30
]. Non-invasive imaging of CD105 expression has the potential to accelerate anti-angiogenic 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 PET tracer. We have achieved this goal and investigated TRC105 and its conjugates in vitro, in vivo, and ex vivo.
One key challenge in antibody labeling is to minimize the potential interference with its antigen binding affinity/specificity. There is only one lysine residue in each of the complementarity determining region (CDR) of TRC105 [11
], which has a total of ~1400 amino acid residues and ~70 lysines, thus the possibility of Df conjugation at the lysine residue within the CDR is extremely low. Coupling of Df-Bz-NCS to mAbs was very efficient, and it has been reported that a reproducible chelate:mAb ratio of 1.5:1 could be obtained using only a three-fold molar excess of Df-Bz-NCS (which was adopted in this study) [27
]. Such a low chelate:mAb ratio can adequately avoid alteration of the immunoreactivity (and pharmacokinetics) of TRC105, which is confirmed by FACS analysis and microscopy studies ().
The in vivo stability of radiometal-labeled antibodies is always a concern. To confirm that the tumor uptake of 89
Zr-Df-TRC105 visualized by non-invasive PET imaging was indeed CD105 specific, various control experiments (e.g. blocking study and the use of an isotype-matched control) and in vitro/ex vivo experiments (e.g. FACS, microscopy, and histological studies) were performed for validation purposes. Based on the available literature data, the 89
Zr-Df conjugate is very stable in vivo [2
]. Therefore, the key to in vivo stability and antigen binding affinity of an 89
Zr-based tracer lies in the stability of the Df-antibody conjugate (Df-TRC105 in this case). To investigate this aspect, we incubated Df-TRC105 in complete mouse serum at 37 °C for up to 7 days and observed no change in CD105 binding affinity/specificity based on FACS analysis, which confirmed the superb stability of the tracer.
The rationale for choosing the 4T1 murine breast cancer model in this study is that the parent antibody of TRC105 (SN6j, a mAb of murine origin which binds to CD105) has been shown to be an effective anti-angiogenic agent in this model [35
]. Further, this is a rapidly growing tumor. Thus it has highly angiogenic tumor vasculature () which is expected to provide sufficient target density for imaging applications. One 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 [36
]. Thus, the 4T1 tumor model is not optimal for testing TRC105. When compared with other antibody-based PET tracers [31
], tumor uptake in this study is relatively low (~14 %ID/g at the peak). This is largely due to two facts. First, 89
Zr-Df-TRC105 targets the tumor vasculature but not the tumor cells. There are significantly fewer tumor vascular endothelial cells than tumor cells, which are the targets of most antibodies used for cancer imaging. Second, TRC105’s affinity to murine endothelial cells is lower than its affinity to human endothelial cells.
For future investigation, the following strategies may be adopted to better mimic the clinical situation and further improve the tumor uptake/contrast: stably transfect 4T1 cells with human CD105, use transgenic mice with human tumor vasculature, or test an anti-CD105 antibody that binds with high affinity to murine CD105. Follow-up studies are currently underway. In addition, studies have shown that tumor uptake of certain antibody-based PET tracers increased significantly when the specific activity of the tracer gets higher [39
]. Therefore, with further improvement in the specific activity of 89
Zr and optimization of the radiochemistry, tumor uptake of 89
Zr-Df-TRC105 in the currently used 4T1 model may be further improved in future studies. Nonetheless, excellent tumor contrast was achieved in this study, which justifies optimism that this tracer may perform better in cancer patients than in the murine models reported here.
One interesting finding from this study was the clear difference between blood clearance and biodistribution patterns of the two PET tracers, where the uptake of 89
Zr-Df-cetuximabin the liver is much higher and faster than 89
Zr-Df-TRC105 (&). Many factors can affect the circulation half-life of an antibody, and antigen binding is only one of them. The neonatal Fc receptor (FcRn), which binds to the CH2
hinge regions in the constant region (Fc) of IgG antibodies, plays a major role in their serum half-lives [40
]. Although TRC105 and cetuximab are isotype-matched chimeric antibodies, their amino acid sequences in the Fc region may be different [41
]. In addition, variable glycosylation patterns in the Fc regions also add to variability, in addition to variability in amino acid sequences. Thus, the immunocompetent Balb/c may recognize them with very different efficiency, which leads to different blood clearance rate and liver uptake.
In several other studies with 89
Zr-labeled antibodies, the tumor uptake reached a peak at around 24 h and remained stable for up to a week [33
]. Due to the fast growing nature of the 4T1 tumor used in this study, although the total tumor uptake of 89
Zr-Df-TRC105 is quite steady over a period of 4 days, the %ID/g values did drop over time since the tumor volume at 72 h and 96 h was 189 ± 34 % and 221 ± 25 % of its original volume at tracer injection (n = 4). It has been reported that 89
is mostly taken up by the liver and 89
Zr-Df undergoes renal excretion [33
]. The low liver and kidney uptake of radioactivity observed in this study further confirms the superb stability of 89
Zr-Df-TRC105 in vivo. Regarding potential clinical translation, quantitative correlation of PET tracer uptake with CD105 expression level would be highly desirable for treatment monitoring applications, as it would be ideal to non-invasively measure the changes of CD105 expression quantitatively, rather than qualitatively, in each individual patient upon anti-angiogenic therapies.