Sandra Heskamp

ImmunoSPECT and immunoPET of IGF-1R expression with the radiolabeled antibody R1507 in a triple-negative breast cancer model.

The insulinlike growth factor 1 receptor (IGF-1R) is a new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti–IGF-1R antibodies. Therefore, the aim of the present study was to develop a noninvasive, in vivo imaging method, using radiolabeled antibodies, to visualize IGF-1R expression. Methods: R1507 is a monoclonal antibody directed against the IGF-1R. In vitro, the affinity and internalization kinetics of 111In-R1507 were determined using the IGF-1R–expressing triple-negative breast cancer cell line SUM149. In vivo, the pharmacodynamics of 111In-R1507 and 125I-R1507 were determined in mice with subcutaneous SUM149 tumors. 111In-R1507 SPECT and 89Zr-R1507 PET images of mice with subcutaneous SUM149 tumors were acquired at 1, 3, and 7 d after injection. Results: 111In-R1507 (concentration required to inhibit binding by 50%, 0.1 nM) was slowly internalized by SUM149 cells. 111In-R1507 specifically and efficiently accumulated in the SUM149 xenografts: the tumor uptake was 20 percentage injected dose per gram (%ID/g), 33 %ID/g, and 31 %ID/g at 1, 3, and 7 d after injection, respectively. 125I-R1507 accumulated in the tumor less efficiently. Small-animal SPECT and small-animal PET of mice clearly visualized the subcutaneous SUM149 xenograft, with increasing contrast at later time points. Conclusion: 111In-R1507 and 89Zr-R1507 are new tracers to noninvasively determine IGF-1R expression in vivo in breast cancer xenografts using SPECT and PET. In the future, these techniques may enable patient selection for IGF-1R–targeted therapy.

PSMA Ligands for Radionuclide Imaging and Therapy of Prostate Cancer: Clinical Status

Prostate cancer (PCa) is the most common malignancy in men worldwide, leading to substantial morbidity and mortality. At present, imaging of PCa has become increasingly important for staging, restaging, and treatment selection. Until recently, choline-based positron emission tomography/computed tomography (PET/CT) represented the state-of-the-art radionuclide imaging technique for these purposes. However, its application is limited to patients with high PSA levels and Gleason scores. Prostate-specific membrane antigen (PSMA) is a promising new target for specific imaging of PCa, because it is upregulated in the majority of PCa. Moreover, PSMA can serve as a target for therapeutic applications. Currently, several small-molecule PSMA ligands with excellent in vivo tumor targeting characteristics are being investigated for their potential in theranostic applications in PCa. Here, a review of the recent developments in PSMA-based diagnostic imaging and therapy in patients with PCa with radiolabeled PSMA ligands is provided.

Noninvasive Imaging of Tumor PD-L1 Expression Using Radiolabeled Anti–PD-L1 Antibodies

Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive antitumor activity. Patients with tumors expressing PD-L1 are most likely to respond to this treatment. The aim of our study was to develop a noninvasive imaging technique to determine tumor PD-L1 expression in vivo. This could allow selection of patients that are most likely to benefit from anti-PD-1/PD-L1 treatment and to monitor PD-L1 expression during therapy. The monoclonal antibody PD-L1.3.1 was radiolabeled with Indium-111 ((111)In) and characterized using PD-L1-expressing MDA-MB-231 cells. Subsequently, the optimal antibody dose and time point for imaging was determined in mice with MDA-MB-231 xenografts. Finally, SPECT/CT imaging was performed in xenograft models with different PD-L1 expression levels and tumor sections were analyzed for PD-L1 expression using IHC. The optimal antibody dose of (111)In-PD-L1.3.1 (Kd = 1 nmol/L) for SPECT/CT imaging was ≤1 μg. Highest tumor-to-normal tissue contrast was obtained at days 3 and 7 after injection. (111)In-PD-L1.3.1 SPECT/CT showed efficient accumulation in high PD-L1-expressing tumors (MDA-MB-231 and SK-Br-3), whereas no specific uptake was observed in tumors with low or no detectable levels of PD-L1 (SUM149, BT474, and MCF-7). SPECT/CT and autoradiography showed a very heterogeneous distribution of (111)In-PD-L1.3.1 within the tumor. In conclusion, this is the first study showing the feasibility of noninvasive in vivo imaging of PD-L1 expression in tumors. (111)In-PD-L1.3.1 showed efficient and specific uptake in PD-L1 expressing xenografts. This technique may enable patient selection for PD-1 and PD-L1-targeted therapy.

Imaging of Human Epidermal Growth Factor Receptor Type 2 Expression with 18F-Labeled Affibody Molecule ZHER2:2395 in a Mouse Model for Ovarian Cancer

Affibody molecules are small (7 kDa) proteins with subnanomolar targeting affinity. Previous SPECT studies in xenografts have shown that the Affibody molecule 111In-DOTA-ZHER2:2395 can discriminate between high and low human epidermal growth factor receptor type 2 (HER2)–expressing tumors, indicating that radiolabeled Affibody molecules have potential for patient selection for HER2-targeted therapy. Compared with SPECT, PET with positron-emitting radionuclides, such as 18F, may improve imaging of HER2 expression because of higher sensitivity and improved quantification of PET. The aim of the present study was to determine whether the 18F-labeled NOTA-conjugated Affibody molecule ZHER2:2395 is a suitable agent for imaging of HER2 expression. The tumor-targeting properties of 18F-labeled ZHER2:2395 were compared with 111In- and 68Ga-labeled ZHER2:2395 in mice with HER2-expressing SK-OV-3 xenografts. Methods: ZHER2:2395 was conjugated with NOTA and radiolabeled with 18F, 68Ga, and 111In. Radiolabeling with 18F was based on the complexation of Al18F by NOTA. The 50% inhibitory concentration values for NOTA-ZHER2:2395 labeled with 19F, 69Ga, and 115In were determined in a competitive cell-binding assay using SK-OV-3 cells. Mice bearing subcutaneous SK-OV-3 xenografts were injected intravenously with radiolabeled NOTA-ZHER2:2395. One and 4 h after injection, PET/CT or SPECT/CT images were acquired, and the biodistribution was determined by ex vivo measurement. Results: The 50% inhibitory concentration values for 19F-, 69Ga-, and 115In-NOTA-ZHER2:2395 were 5.0, 6.3, and 5.3 nM, respectively. One hour after injection, tumor uptake was 4.4 ± 0.8 percentage injected dose per gram (%ID/g), 5.6 ± 1.6 %ID/g, and 7.1 ± 1.4 %ID/g for 18F-, 68Ga-, and 111In-NOTA-ZHER2:2395, respectively, and the respective tumor-to-blood ratios were 7.4 ± 1.8, 8.0 ± 1.3, and 4.8 ± 1.3. Tumor uptake was specific, because uptake could be blocked efficiently by coinjection of an excess of unlabeled ZHER2:2395. PET/CT and SPECT/CT images clearly visualized HER2-expressing SK-OV-3 xenografts. Conclusion: This study showed that 18F-NOTA-ZHER2:2395 is a promising new imaging agent for HER2 expression in tumors. Affibody molecules were successfully labeled with 18F within 30 min, based on the complexation of Al18F by NOTA. Further research is needed to determine whether this technique can be used for patient selection for HER2-targeted therapy.

Bevacizumab reduces tumor targeting of antiepidermal growth factor and anti-insulin-like growth factor 1 receptor antibodies.

Bevacizumab (antivascular endothelial growth factor [anti‐VEGF]) and cetuximab (antiepidermal growth factor receptor [anti‐EGFR]) are approved antibodies for treatment of cancer. However, in advanced colorectal cancer, the combination fails to improve survival. As the reason for the lack of activity is unknown, our study aims to determine the effect of bevacizumab on targeting of anti‐EGFR and insulin‐like growth factor 1 receptor (IGF‐1R) antibodies in tumors with single‐photon emission computed tomography (SPECT)/CT imaging. Mice with subcutaneous EGFR and IGF‐1R‐expressing SUM149 xenografts received a single dose of bevacizumab (10 mg/kg) or saline. After 4 days, mice were injected with radiolabeled cetuximab or R1507, an anti‐IGF‐1R antibody. A control group received a radiolabeled irrelevant IgG (hLL2). Three days later, SPECT/CT images were acquired and mice were dissected to determine the concentration of antibodies in the tissues. Tumors were analyzed immunohistochemically to determine vascular density (CD34), VEGF, EGFR and IGF‐1R expression. SPECT/CT imaging revealed that bevacizumab treatment significantly reduced tumor targeting of radiolabeled cetuximab by 40% from 33.1 ± 1.1 %ID/g to 19.8 ± 5.7 %ID/g (p = 0.009) for untreated and bevacizumab‐treated tumors, respectively. A similar effect was found for 111In‐R1507: tumor targeting of R1507 decreased by 35%. No significant differences in tumor uptake were observed in mice that received an irrelevant IgG. Uptake in normal organs was not altered by bevacizumab. Immunohistochemical analysis showed that vascular density decreased with 43%, whereas EGFR and IGF‐1R expression was unaltered. In conclusion, bevacizumab treatment significantly reduces tumor targeting of anti‐EGFR and anti‐IGF‐1R antibodies. This emphasizes the importance of timing and sequencing of bevacizumab in combination with other antibodies.

Predicting IGF-1R therapy response in bone sarcomas: immuno-SPECT imaging with radiolabeled R1507

Purpose: To investigate whether indium-111–labeled R1507 (111In-R1507) immuno-SPECT (single—photon emission computed tomography), a novel noninvasive, in vivo screening method to visualize membranous insulin-like growth factor 1 receptor (IGF-1R) expression and accessibility, can be used to predict IGF-1R treatment (R1507) response in bone sarcomas. Experimental Design: BALB/c nude mice were subcutaneously implanted with IGF-1R–expressing human bone sarcoma xenografts (OS-1, EW-5, and EW-8) which showed high, modest, or no response, respectively, to R1507, a monoclonal antibody targeting the extracellular domain of IGF-1R. An IGF-1R–negative tumor (OS-33), unresponsive to IGF-1R inhibitors, was examined as well. Mice were injected with 111In-R1507. Biodistribution and immuno-SPECT/computed tomography imaging studies were carried out 1, 3, and 7 days p.i. in mice with OS-1 and EW-5 xenografts and 3 days p.i. in mice with EW-8 and OS-33 xenografts. Results: Biodistribution studies showed specific accumulation of 111In-R1507 in OS-1 and EW-5 xenografts (27.5 ± 6.5%ID/g and 14.0 ± 2.8%ID/g, 3 days p.i., respectively). Most importantly, 111In-R1507 uptake in IGF-1R positive, but unresponsive, EW-8 xenografts (6.5 ± 1.5%ID/g, 3 days p.i.) was similar to that of the IGF-1R–negative OS-33 tumor (5.5 ± 0.6%ID/g, 3 days p.i.). Uptake in normal tissues was low and nonspecific. Corresponding immuno-SPECT images clearly discriminated between high, modest, and nonresponding tumors by showing a homogeneous (OS-1), heterogeneous (EW-5), or nonspecific (EW-8 and OS-33) tumor uptake of 111In-R1507. Conclusions: 111In-R1507 immuno-SPECT is an excellent method to visualize membranous IGF-1R expression and target accessibility in vivo in human bone sarcoma xenografts and may serve as an independent marker to predict IGF-1R therapy (R1507) response in bone sarcoma patients. Clin Cancer Res; 17(24); 7693–703. ©2011 AACR.

Better survival in patients with metastasised kidney cancer after nephrectomy: a population-based study in the Netherlands.

AIM Cytoreductive nephrectomy is considered beneficial in patients with metastasised kidney cancer but only a minority of these patients undergo cytoreductive surgery. Factors associated with nephrectomy and the independent effect of nephrectomy on survival were evaluated in this study. METHODS Patients were selected from the population-based cancer registry and detailed data were retrieved from clinical files. Factors associated with nephrectomy were evaluated by logistic regression analyses. Cox proportional hazard regression analysis was performed to evaluate factors associated with survival; a propensity score reflecting the probability of being treated surgically was included in order to adjust for confounding by indication. RESULTS 37.5% of 328 patients diagnosed with metastatic kidney cancer between 1999 and 2005 underwent nephrectomy. Patients with a low performance score, high age, ≥2 comorbid conditions, ≥2 metastases, low or high BMI, weight loss, elevated lactate dehydrogenase, elevated alkaline phosphatase, female gender and liver or bone metastases were less likely to be treated surgically. Three year survival was 25% and 4% for patients with and without nephrectomy, respectively (p<0.001). After adjustment for other prognostic factors including the propensity score, nephrectomy remained significantly associated with better survival (Hazard ratio: 0.52, 95% Confidence interval: 0.37-0.73). CONCLUSIONS Even after accounting for prognostic profile, patients still benefit from a nephrectomy; an approximately 50% reduction in mortality was observed. It is, therefore, recommended that patients with metastasised disease receive cytoreductive surgery when there is no contraindication. Trial results on cytoreductive surgery combined with targeted molecular therapeutics are awaited for.

Theranostic applications of antibodies in oncology

Targeted therapies, including antibodies, are becoming increasingly important in cancer therapy. Important limitations, however, are that not every patient benefits from a specific antibody therapy and that responses could be short‐lived due to acquired resistance. In addition, targeted therapies are quite expensive and are not completely devoid of side‐effects. This urges the need for accurate patient selection and response monitoring.

Towards personalized treatment of prostate cancer: PSMA I&T, a promising prostate-specific membrane antigen-targeted theranostic agent

Prostate-specific membrane antigen (PSMA) is a well-established target for nuclear imaging and therapy of prostate cancer (PCa). Radiolabeled small-molecule PSMA inhibitors are excellent candidates for PCa theranostics—they rapidly and efficiently localize in tumor lesions. However, high tracer uptake in kidneys and salivary glands are major concerns for therapeutic applications. Here, we present the preclinical application of PSMA I&T, a DOTAGA-chelated urea-based PSMA inhibitor, for SPECT/CT imaging and radionuclide therapy of PCa. 111In-PSMA I&T showed dose-dependent uptake in PSMA-expressing tumors, kidneys, spleen, adrenals, lungs and salivary glands. Coadministration of 2-(phosphonomethyl)pentane-1,5-dioic acid (2-PMPA) efficiently reduced PSMA-mediated renal uptake of 111In-PSMA I&T, with the highest tumor/kidney radioactivity ratios being obtained using a dose of 50 nmol 2-PMPA. SPECT/CT clearly visualized subcutaneous tumors and sub-millimeter intraperitoneal metastases; however, high renal and spleen uptake in control mice (no 2-PMPA) interfered with visualization of metastases in the vicinity of those organs. Coadministration of 2-PMPA increased the tumor-to-kidney absorbed dose ratio during 177Lu-PSMA I&T radionuclide therapy. Hence, at equivalent absorbed dose to the tumor (36 Gy), coinjection of 2-PMPA decreased absorbed dose to the kidneys from 30 Gy to 12 Gy. Mice injected with 177Lu-PSMA I&T only, showed signs of nephrotoxicity at 3 months after therapy, whereas mice injected with 177Lu-PSMA I&T + 2-PMPA did not. These data indicate that PSMA I&T is a promising theranostic tool for PCa. PSMA-specific uptake in kidneys can be successfully tackled using blocking agents such as 2-PMPA.

89Zr-Immuno-Positron Emission Tomography in Oncology: State-of-the-Art 89Zr Radiochemistry

Immuno-positron emission tomography (immunoPET) with 89Zr-labeled antibodies has shown great potential in cancer imaging. It can provide important information about the pharmacokinetics and tumor-targeting properties of monoclonal antibodies and may help in anticipating on toxicity. Furthermore, it allows accurate dose planning for individualized radioimmunotherapy and may aid in patient selection and early-response monitoring for targeted therapies. The most commonly used chelator for 89Zr is desferrioxamine (DFO). Preclinical studies have shown that DFO is not an ideal chelator because the 89Zr–DFO complex is partly unstable in vivo, which results in the release of 89Zr from the chelator and the subsequent accumulation of 89Zr in bone. This bone accumulation interferes with accurate interpretation and quantification of bone uptake on PET images. Therefore, there is a need for novel chelators that allow more stable complexation of 89Zr. In this Review, we will describe the most recent developments in 89Zr radiochemistry, including novel chelators and site-specific conjugation methods.