Cathelijne Frielink

Comparison of a monomeric and dimeric radiolabeled RGD-peptide for tumor targeting.

The alpha v beta 3 integrin, a transmembrane heterodimeric protein expressed on sprouting endothelial cells, binds to the arginine-glycine-aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin. Growing malignant tumors continuously require angiogenesis. As a result, alpha v beta 3 is preferentially expressed in growing tumors and is a potential target for radiolabeled RGD-peptides. In this study we compared the tumor targeting characteristics of a monomeric radiolabeled RGD-peptide with those of a dimeric analogue. Both peptides were radiolabeled with 99mTc via the hydrazinoni-cotinamid (= HYNIC) moiety to form 99mTc-HYNIC-c(RGDfK) and 99mTc-HYNIC-E-[c(RGDfK)]2. In vitro, the IC50 showed a 10-fold higher affinity of the dimer for the alpha v beta 3 integrin as compared to the monomer (0.1 vs. 1.0 nM). In athymic female BALB/c mice with subcutaneously growing OVCAR-3 ovarian carcinoma xenografts, tumor uptake peaked at 5.8 +/- 0.7% ID/g and 5.2 +/- 0.6% ID/g for the dimer and the monomer, respectively. At 1, 2, and 4 h postinjection (p.i.) uptake of the dimer in the tumor was significantly higher than that of the monomeric analogue. Tumor-to-blood ratios were highest at 24 h p.i. at a value of 63 for both compounds. At all timepoints kidney retention of the dimer was significantly higher as compared to kidney retention of the monomer. In conclusion, in this mouse model the dimeric RGD-peptide showed better retention in the tumor than the monomeric analogue, most likely due to the bivalent interaction with the target cell. Furthermore, kidney retention of the dimeric peptide was higher than that of the monomeric peptide.

αvβ3 integrin-targeting of intraperitoneally growing tumors with a radiolabeled RGD peptide

Ovarian cancer is the fourth most common cause of cancer deaths among females in the western world after cancer of the breast, colon and lung. The inability to control the disease within the peritoneal cavity is the major cause of treatment failure in patients with ovarian cancer. The majority of ovarian carcinomas express the αvβ3 integrin. Here we studied the tumor targeting potential of an 111In‐labeled cyclic RGD peptide in athymic BALB/c mice with intraperitoneally (i.p.) growing NIH:OVCAR‐3 human ovarian carcinoma tumors. The cyclic RGD peptide, c(RGDfK)E, was synthesized, conjugated with DOTA and radiolabeled with 111In. The targeting potential of 111In‐DOTA‐E‐c(RGDfK) was studied in athymic mice with i.p. growing NIH:OVCAR‐3 xenografts and the optimal dose of this compound was determined (0.01 μg up to 10 μg). The biodistribution at optimal peptide dose was determined at various time points (0.5 up to 72 hr). Furthermore, the therapeutic potential of 177Lu‐DOTA‐E‐c(RGDfK) was studied in this model. Two hours after i.p. administration, 111In‐DOTA‐E‐c(RGDfK) showed high and specific uptake in the i.p. growing tumors. Optimal uptake in the i.p. growing tumors was observed at a 0.03–0.1 μg dose range. Tumor uptake of 111In‐DOTA‐E‐c(RGDfK) peaked 4 hr p.i. [(38.8 ± 2.7)% ID/g], gradually decreasing at later time points [(24.0 ± 4.1)% ID/g at 48 hr p.i.]. Intraperitoneal growth of OVCAR‐3 could be significantly delayed by injecting 37 MBq 177Lu‐labeled peptide i.p. Radiolabeled DOTA‐E‐c(RGDfK) is suitable for targeting of i.p. growing tumors and potentially can be used for peptide receptor radionuclide therapy of these tumors.

Biodistribution of 131I-, 186Re-, 177Lu-, and 88Y-Labeled hLL2 (Epratuzumab) in Nude Mice with CD22-Positive Lymphoma

UNLABELLED Radioimmunotherapy (RIT) is a new and effective treatment modality in patients with non-Hodgkins lymphoma. The monoclonal antibody (mAb) hLL2 (epratuzumab), a humanized mAb directed against the CD22 antigen, and which internalizes, can be labeled with various radionuclides. The biodistribution of hLL2 labeled with (131)I, (186)Re, (177)Lu, and (88)Y was studied in nude mice with subcutaneous human lymphoma xenografts in order to determine the most suitable of these four radionuclides for RIT with hLL2. METHODS Human Ramos lymphoma xenografts were transplanted in cyclophosphamide-pretreated athymic BALB/c mice. Four groups of mice were injected intravenously with (131)I-, (186)Re-, (88)Y-, or (177)Lu-labeled hLL2, respectively. To determine the nonspecific tumor uptake, two groups of mice received (88)Y-labeled or (131)I-labeled control antibody, cG250. The biodistribution of the radiolabel was determined 1, 3, and 7 days postinjection (p.i.). RESULTS Radiolabeled hLL2 had a higher tumor uptake than the nonspecific mAb at all time-points, irrespective of the radiolabel used. Tumor accretion of (88)Y- and (177)Lu-hLL2 was higher than tumor uptake of (131)I- and (186)Re-hLL2. Activity in the bone, represented by the femur without bone marrow, was higher for (177)Lu- and (88)Y-hLL2 than for (131)I- and (186)Re-hLL2 on day 7 p.i. CONCLUSION The use of the residualizing radiolabels (88)Y and (177)Lu in combination with a mAb directed against an internalizing antigen resulted in higher uptake and better retention of the radiolabel in the tumor.

Diannexin protects against renal ischemia reperfusion injury and targets phosphatidylserines in ischemic tissue

Renal ischemia/reperfusion injury (IRI) frequently complicates shock, renal transplantation and cardiac and aortic surgery, and has prognostic significance. The translocation of phosphatidylserines to cell surfaces is an important pro-inflammatory signal for cell-stress after IRI. We hypothesized that shielding of exposed phosphatidylserines by the annexin A5 (ANXA5) homodimer Diannexin protects against renal IRI. Protective effects of Diannexin on the kidney were studied in a mouse model of mild renal IRI. Diannexin treatment before renal IRI decreased proximal tubule damage and leukocyte influx, decreased transcription and expression of renal injury markers Neutrophil Gelatinase Associated Lipocalin and Kidney Injury Molecule-1 and improved renal function. A mouse model of ischemic hind limb exercise was used to assess Diannexin biodistribution and targeting. When comparing its biodistribution and elimination to ANXA5, Diannexin was found to have a distinct distribution pattern and longer blood half-life. Diannexin targeted specifically to the ischemic muscle and its affinity exceeded that of ANXA5. Targeting of both proteins was inhibited by pre-treatment with unlabeled ANXA5, suggesting that Diannexin targets specifically to ischemic tissues via phosphatidylserine-binding. This study emphasizes the importance of phosphatidylserine translocation in the pathophysiology of IRI. We show for the first time that Diannexin protects against renal IRI, making it a promising therapeutic tool to prevent IRI in a clinical setting. Our results indicate that Diannexin is a potential new imaging agent for the study of phosphatidylserine-exposing organs in vivo.

Imaging of Prostate Cancer with Immuno-PET and Immuno-SPECT Using a Radiolabeled Anti-EGP-1 Monoclonal Antibody

hRS7 is a humanized IgG1 monoclonal antibody directed against the epithelial glycoprotein-1 (EGP-1; also known as TROP2). This antigen is found in many epithelial cancers, including prostate cancer, and therefore this antibody could be suitable for targeting this cancer. In this study, the characteristics of hRS7 for targeting prostate cancer were examined. The potential for immuno-PET with 89Zr-hRS7 and immuno-SPECT with 111In-hRS7 was assessed using nude mice with human prostate cancer xenografts. Methods: EGP-1 expression was assessed by immunohistology in human primary and metastatic prostate cancer samples and in PC3 xenografts. The optimal antibody protein dose for prostate cancer targeting was examined in nude mice with subcutaneous PC3 xenografts, and then the biodistribution of 111In-, 125I-, and 89Zr-labeled hRS7 was determined in subcutaneous PC3 xenografts at 1, 3, and 7 d after injection. Immuno-PET and immuno-SPECT were performed with 89Zr-hRS7 and 111In-hRS7 in mice with subcutaneous and intraprostatic PC3 xenografts, respectively. Results: Immunohistochemical analysis showed abundant EGP-1 expression in human primary and metastatic prostate cancers and in PC3 xenografts. 111In-hRS7 and 89Zr-hRS7 preferentially and specifically accumulated in PC3 xenografts, with tumor uptake as high as 60% injected dose per gram at a protein dose of 0.1 μg per mouse. PC3 tumors in nude mice were clearly visualized with both tracers with immuno-PET and immuno-SPECT. Conclusion: hRS7 shows excellent in vivo tumor targeting in human prostate cancer xenografts. Therefore, hRS7 is a potential vehicle for targeting prostate cancer.

A New Tri-Fab Bispecific Antibody for Pretargeting Trop-2–Expressing Epithelial Cancers

RS7 is an internalizing anti-Trop-2 pancarcinoma antibody capable of targeting most epithelial cancers. Because pretargeting strategies could improve the tumor localization of radionuclides, a new anti-Trop-2 × antihapten bispecific antibody for pretargeting, based on humanized RS7, was prepared and evaluated with a radiolabeled hapten-peptide in vitro and in vivo to determine whether its internalization properties would interfere with pretargeting. Methods: The anti-Trop-2 × antihapten bispecific antibody, TF12, was prepared using the modular dock-and-lock method. TF12 and humanized RS7 binding was assessed by cell binding assays and fluorescence-activated cell sorting analysis in a variety of human carcinoma cell lines. The internalization of TF12 was evaluated in vitro using a fluorescent TF12 conjugate or hapten-peptide and 111In-labeled TF12 and RS7. The biodistribution of TF12 and its use as a pretargeting agent with an 111In-labeled hapten-peptide were assessed in several human epithelial cancer xenografts. Dose optimization was examined in 2 tumor models. Results: TF12 internalizes, but a substantial fraction remained accessible on the tumor surface. Fluorescence-activated cell sorting analysis showed only a minor change in fluorescent signal when the tumor was probed with a fluorescent hapten-peptide over 4 h, and microscopy showed substantial membrane staining when reassessed at 24 h after TF12 exposure. Only 40.1% of 111In-TF12 was internalized after 24 h. In vivo, excellent tumor localization of the 111In-labeled peptide was observed in several tumor models. Conclusion: TF12 was retained sufficiently on the cell surface in several epithelial cancers, thereby making it suitable for pretargeted imaging and therapy of various Trop-2–expressing carcinomas.

Interferons can upregulate the expression of the tumor associated antigen G250-MN/CA IX, a potential target for (radio)immunotherapy of renal cell carcinoma.

BACKGROUND Interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) can induce therapeutic responses in a minority (5-25%) of patients with metastatic renal cell carcinoma (RCC). G250-MN/CA IX, a tumor-associated antigen expressed on the majority of clear cell RCCs, is a potential (radio)immunotherapeutic target for G250-antibody based (radio)immunotherapy. We investigated the effect of the biological response modifiers (BRMs) IL-2, IFN-alpha, and IFN-gamma on the expression of the G250 antigen on RCC cells. METHODS In vitro, the expression of the G250 antigen was measured by flow cytometry (FCM) after culturing RCC cells in the presence of various concentrations of the BRMs. Additionally, the number of G250 epitopes per cell was determined quantitatively by Scatchard analysis. RESULTS Upregulation of G250 expression was observed on RCC cells cultured in the presence of IFN-alpha or IFN-gamma, whereas the addition of IL-2 had no effect. For both IFNs a clear dose-response relation between G250 antigen expression and IFN dose was observed, with IFN-gamma being the more potent agent. G250 expression could be upregulated four-fold. Interestingly, the effect of combining IFN-alpha and IFN-gamma revealed a more pronounced upregulation of G250 expression than either one of the IFNs alone. CONCLUSIONS On the basis of in vitro experiments, G250 expression can be upregulated by IFN-alpha and IFN-gamma. In vivo studies are warranted to investigate whether due to IFN treatment increased G250 expression occurs, and whether increased G250 expression can enhance the therapeutic efficacy of G250-antibody based (radio)immunotherapy.

111In-exendin uptake in the pancreas correlates with the beta cell mass and not with the alpha cell mass

Targeting of the GLP-1 receptor with 111In-labeled exendin is an attractive approach to determine the β-cell mass (BCM). Preclinical studies as well as a proof-of-concept study in type 1 diabetic patients and healthy subjects showed a direct correlation between BCM and radiotracer uptake. Despite these promising initial results, the influence of α-cells on the uptake of the radiotracer remains a matter of debate. In this study, we determined the correlation between pancreatic tracer uptake and β- and α-cell mass in a rat model for β-cell loss. The uptake of 111In-exendin (% ID/g) showed a strong positive linear correlation with the BCM (Pearson r = 0.82). The fraction of glucagon-positive cells in the total endocrine mass was increased after alloxan treatment (26% ± 4%, 43% ± 8%, and 69% ± 21% for 0, 45, and 60 mg/kg alloxan, respectively). The uptake of 111In-exendin showed a negative linear correlation with the α-cell fraction (Pearson r = −0.76). These data clearly indicate toward specificity of 111In-exendin for β-cells and that the influence of the α-cells on 111In-exendin uptake is negligible.

Pretargeting of carcinoembryonic antigen-expressing tumors with a biologically produced bispecific anticarcinoembryonic antigen x anti-indium-labeled diethylenetriaminepentaacetic acid antibody.

Purpose: The aim of these studies was to develop a pretargeting strategy for CEA-expressing cancers using biologically produced bispecific monoclonal antibodies (bsMAb). The bsMAbs used in this system have affinity for the carcinoembryonic antigen on the one hand, and for indium-labeled diethylenetriaminepentaacetic acid (DTPA), on the other. Experimental Design: Stable quadroma clones producing bsMAb MN-14xDTIn-1 were isolated. LS174T tumor–bearing mice were injected with 1 to 100 μg of bsMAb followed by 1 to 60 ng of an 111In-labeled bivalent peptide [Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH2]. Mice were killed at 24 hours postinjection and the biodistribution of the radiolabel was determined. The biodistribution of diDTPA labeled with four different radionuclides (111In, 99mTc, nonresidualizing 125I, and residualizing 125I) was determined at various time points postinjection following pretargeting of LS174T tumors with bsMAb MN-14xDTIn-1. Results: Optimal tumor targeting was observed when tumors were pretargeted with 10 μg of bsMAb MN-14xDTIn-1 and when 6 ng of a radiolabeled peptide was given 72 hours later. The uptake of the four radiolabels in LS174T tumors at 4 hours postinjection was similar. However, at later time points, the 111In-label and residualizing 125I-label were better retained in the tumor than the nonresidualizing 125I label. Although the absolute uptake in the tumor (in terms of percentage of injected dose per gram of tissue) was 5-fold lower than the uptake obtained with directly labeled MN-14, the pretargeting strategy revealed much higher tumor-to-blood ratios due to the rapid clearance of the radiolabel from the circulation as compared with 111In-MN-14 (445 ± 90 and 5.3 ± 1.1, respectively, at 72 hours postinjection). Conclusions: Effective targeting of carcinoembryonic antigen-expressing tumors was achieved with a newly produced bispecific antibody. The 111In-labeled l-amino acid peptide and 125I-d-amino acid peptide were better retained in the tumor than the 99mTc- and 125I-l-amino acid peptide. Very high tumor-to-blood ratios were obtained due to rapid background clearance.

Imaging Integrin αvβ3 on Blood Vessels with 111In-RGD2 in Head and Neck Tumor Xenografts

Arginine-glycine-aspartic acid (RGD)–based imaging tracers allow specific imaging of integrin αvβ3, a protein overexpressed during angiogenesis, leading to the possibility that it might serve as a tool to stratify patients for antiangiogenic treatment. However, these tracers have generally been characterized in xenograft models in which integrin αvβ3 was constitutively expressed by the tumor cells themselves. In the studies presented here, the use of 111In-RGD2 as a tracer to image only integrin αvβ3 expression on blood vessels in the tumor was determined using tumor xenografts in which tumor cells were integrin αvβ3-negative. Methods: DOTA-E-[c(RGDfK)]2 was radiolabeled with 111In (111In-RGD2), and biodistribution studies were performed in squamous cell carcinoma of the head and neck (HNSCC) xenograft mouse models to determine the optimal peptide dose to image angiogenesis. Next, biodistribution and imaging studies were performed at the optimal peptide dose in 3 HNSCC mouse models, FaDu, SCCNij3, and SCCNij202. Immunohistochemical analysis of tumor vascular and cell surface expression of integrin αvβ3 and correlation analysis of vascular integrin αvβ3 and autoradiography were completed. Results: All 3 HNSCC xenografts expressed integrin αvβ3 on the vessels only. The optimal peptide dose of 111In-RGD2 was 1 μg or less for specific integrin αvβ3–mediated uptake of the tracer. SPECT/CT imaging showed clear uptake of the tracer in the periphery of the tumors, corresponding with well-vascularized areas of the tumor. Within the tumor, 111In-RGD2 autoradiography coincided with vascular integrin αvβ3 expression, as determined immunohistochemically. Integrin αvβ3–mediated uptake was also detected in nontumor tissues, which, through immunohistochemical analysis, proved positive for integrin αvβ3. Conclusion: 111In-RGD2 allows the visualization of integrin αvβ3 in xenograft models in which integrin αvβ3 is expressed only on the neovasculature, such as in the HNSCC tumors. Thus, 111In-RGD2 allows specific visualization of angiogenesis in tumor models lacking constitutive tumoral integrin αvβ3 expression but may be less useful for this purpose in many tumors in which tumor cells express integrin αvβ3.