In this study, we successfully synthesized and evaluated 64Cu-NOTA-bevacizumab for imaging of VEGF levels in RCC tumor xenografts. This compound was stable, specifically accumulated in tumors, and could be blocked by the addition of unlabeled bevacizumab. Additionally, a RAD001-mediated decrease in VEGF expression was clearly visualized by PET imaging using 64Cu-NOTA-bevacizumab. Tracer uptake diminution was concomitant with static effect of RAD001 on tumor growth. Thus, 64Cu-NOTA-bevacizumab may be a novel surrogate biomarker for disease stabilization mediated by rapalog or mTOR kinase inhibitor therapies.
Traditionally solid tumor therapeutic response is scored based on tumor dimensional reduction, either measured physically, radiographically, or by CT and MRI. The Response Evaluation Criteria in Solid Tumors (RECIST) and its more recent RECIST1.1 version provides a standardized methodology for efficacy comparisons across drugs and studies. However, the emergence of molecularly targeted therapies challenges RECIST as an efficacy benchmark 
. When molecularly targeted therapies are effective, they principally produce stable disease (SD) or partial response (PR) with minimal changes in tumor size. Limitations of RECIST for molecular therapy efficacy determination motivates development of CT, MRI, and ultrasound imaging algorithms and techniques tailored in particular for tumor vascular targeting drugs. These algorithms are designed to detect changes in overall tumor vascular density, angiogenic factor mediated vascular leakage, and tumor vascular flow 
Another approach to assess therapy efficacy is PET. Tumors are characterized by enhanced glucose and thymidine uptake in response to genetic dysregulation of metabolism and proliferation 
. The radiotracers 18
F-fluorodeoxyglucose (FDG) and 18
F-fluorothymidine (FLT) 
, image tumor glucose uptake and DNA synthesis detecting primary tumor and metastasis and monitoring therapeutic response. Both FDG and FLT PET can reveal therapeutic efficacy prior to tumor volume changes. These two PET tracers are challenged by the fact that some cancers such as RCC (and prostate) are predominantly FDG insensitive 
, and FLT underestimates proliferation in tumors with activated pyrimidine de-novo synthesis or salvage pathway upregulation 
. However, in the minority of FDG positive RCCs, PET detects tumor response earlier than size reduction and predicts better progression free survival (PFS) 
Despite limitations in cancers such as RCC, a new dimension in PET has become apparent in the last few years. “ImmunoPET” tracers are antibody-radionuclide conjugates binding to proteins either attached to the cell surface, or secreted into the tumor microenvironment 
. Using bifunctional chelators such as the NCS (isothiocyanate) or NHS (N-Hydroxysuccinimide) forms of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-N,N’N”-triacetic acid (NOTA), radionuclide metal ions can be complexed in the chelator cores while covalent bonds are formed with free lysine groups on antibodies or peptides 
. The power of this approach is that DOTA/NOTA chelated PET tracers can be conjugated to antibodies specific for a repertoire of angiogenic factors. As VEGF is the principal angiogenic factor produced by RCC (and most other cancers as well), this angiogenic factor has been explored as an imaging target. Prior studies have evaluated the use of radiolabeled bevacizumab for PET imaging of VEGF expression. For example, bevacizumab has been radiolabeled with 89
for preclinical imaging of ovarian xenograft models. Despite the ability of these agents to image VEGF, the high energy and abundance of the gamma decay from these radionuclides are an exposure concern compared to 64
Cu which has a much lower high energy gamma decay abundance. Paudyal et al.
Cu-DOTA- bevacizumab for preclinical VEGF expression imaging in colorectal xenografts 
. Liver uptake was relatively high,17.2% ID/g, in contrast to our 64
Cu-NOTA-bevacizumab tracer where it was 4.8% ID/g. An explanation for the differential liver uptake could be the higher 64
Cu binding affinity of NOTA compared to DOTA. Nagengast et al
. demonstrated the ability of 89
Zr-radiolabeled ranibizumab to measure dynamic changes in VEGF expression following sunitinib treatment, a multi-tyrosine kinase inhibitor also used in metastatic renal cancer 
. However, low tumor (~5–6% ID/g) and high kidney uptake 
may limit the use of radiolabeled ranibizumab in intra-abdominal tumors. In contrast, we found relatively high 64
Cu-NOTA-bevacizumab accumulation (~30% ID/g) in untreated renal cancer xenografts tumors in comparison to normal organs such as liver, kidney, GI tract, and bladder, similar to other groups using this tracer 
. Our increased VEGF signal-to-noise facilitated tumor imaging enhancement and its rapalog-mediated decrease.
A potential limitation of our study is that bevacizumab binds only to human and not to mouse VEGF. As such, host-derived VEGF sources such as stromal fibroblasts, myeloid cells, and vascular supporting cells were undetectable 
. Therefore, in humans, the potential exists for increased tumor to background ratio of 64
Cu-NOTA-bevacizumab uptake due to VEGF production from multiple sites in the tumor microenvironment. Moreover, inhibition of VEGF secretion from tumor associated myeloid cells could be of near equal therapeutic importance as malignant cellular VEGF production. In addition, the ability of humanized bevacizumab to detect VEGF in the heart, lung, kidney, and brain could be important predictors of VEGF signaling inhibitor toxicities such as proteinuria, pulmonary hemorrhage, and cardiotoxicity 
. Signal to noise and utility of VEGF content imaging in normal organs, for therapy toxicity monitoring will be interesting venues for investigation in patient studies.
Although rapid tumor growth prevented assessment of the predictive ability, of 64
Cu-NOTA-bevacizumab PET, the concordance of imaging and tracer biodistribution with our preclinical disease stabilization suggests a translational role for immunoPET during patient selection for molecularly targeted therapies. mTORC1 regulation of “weak” mRNA translation encompasses secreted growth factors many of which are angiogenic factors 
. Therefore, VEGF, or an expanded repertoire of immunoPET tracers, cognate for other secreted molecules that are mTOCR1 targets, could detect patients that may be inhibitor sensitive. This potential ability for rapid patient selection based on functional PET is particularly important as substantial percentages of solid tumors either possess signaling pathways bypassing the mTORC1 translational requirement, or upregulate or amplify genes encoding downstream mTORC1 translational effectors 
. In addition, breakthrough from mTORC1-mediated stable disease to progressive disease could also be detected prior to measurable tumor expansion. Conversely, 64
Cu-NOTA-bevacizumab PET could also be used to track elevations of tumor VEGF during VEGFR inhibitor therapies. Drug dosage could then be increased in those patients to match the increase in tumor production. In addition, libraries of immunoPET tracers could potentially pinpoint which compensatory angiogenic factor(s) are induced during mTORC1 or VEGFR inhibitor therapies and an appropriate alternative pathway-specific inhibitor deployed. As such, the concept of immunoPET reporting on tumor angiogenic factor, chemokine, or cytokine production offers great potential for therapy tailored personalized medicine. However, in order to supplant or even augment criteria such as RECIST that was established from data acquired for large patient populations, PET studies will similarly need to be tested in large patient cohorts preferably encompassing several different tumor histotypes.