Hitomi Sudo

Evaluation of 89Zr-Labeled Human Anti-CD147 Monoclonal Antibody as a Positron Emission Tomography Probe in a Mouse Model of Pancreatic Cancer

Introduction Pancreatic cancer is an aggressive cancer and its prognosis remains poor. Therefore, additional effective therapy is required to augment and/or complement current therapy. CD147, high expression in pancreatic cancer, is involved in the metastatic process and is considered a good candidate for targeted therapy. CD147-specfic imaging could be useful for selection of appropriate patients. Therefore, we evaluated the potential of a fully human anti-CD147 monoclonal antibody 059-053 as a new positron emission tomography (PET) probe for pancreatic cancer. Methods CD147 expression was evaluated in four pancreatic cancer cell lines (MIA Paca-2, PANC-1, BxPC-3, and AsPC-1) and a mouse cell line A4 as a negative control. Cell binding, competitive inhibition and internalization assays were conducted with 125I-, 67Ga-, or 89Zr-labeled 059-053. In vivo biodistribution of 125I- or 89Zr-labeled 059-053 was conducted in mice bearing MIA Paca-2 and A4 tumors. PET imaging with [89Zr]059-053 was conducted in subcutaneous and orthotopic tumor mouse models. Results Among four pancreatic cancer cell lines, MIA Paca-2 cells showed the highest expression of CD147, while A4 cells had no expression. Immunohistochemical staining showed that MIA Paca-2 xenografts also highly expressed CD147 in vivo. Radiolabeled 059-053 specifically bound to MIA Paca-2 cells with high affinity, but not to A4. [89Zr]059-053 uptake in MIA Paca-2 tumors increased with time from 11.0±1.3% injected dose per gram (ID/g) at day 1 to 16.9±3.2% ID/g at day 6, while [125I]059-053 uptake was relatively low and decreased with time, suggesting that 059-053 was internalized into tumor cells in vivo and 125I was released from the cells. PET with [89Zr]059-053 clearly visualized subcutaneous and orthotopic tumors. Conclusion [89Zr]059-053 is a promising PET probe for imaging CD147 expression in pancreatic cancer and has the potential to select appropriate patients with CD147-expressing tumors who could gain benefit from anti-CD147 therapy.

ZDHHC8 knockdown enhances radiosensitivity and suppresses tumor growth in a mesothelioma mouse model.

Mesothelioma is an aggressive tumor caused by asbestos exposure, the incidence of which is predicted to increase globally. The prognosis of patients with mesothelioma undergoing conventional therapy is poor. Radiation therapy for mesothelioma is of limited use because of the intrinsic radioresistance of tumor cells compared with surrounding normal tissue. Thus, a novel molecular‐targeted radiosensitizing agent that enhances the radiosensitivity of mesothelioma cells is required to improve the therapeutic efficacy of radiation therapy. ZDHHC8 knockdown reduces cell survival and induces an impaired G2/M checkpoint after X‐irradiation in HEK293 cells. In the present study, we further analyzed the effect of the combination of ZDHHC8 knockdown and X‐irradiation and assessed its therapeutic efficacy in mesothelioma models. SiRNA‐induced ZDHHC8 knockdown in 211H and H2052 mesothelioma cells significantly reduced cell survival after X‐irradiation. In 211H cells treated with ZDHHC8 siRNA and X‐irradiation, the G2/M checkpoint was impaired and there was an increase in the number of cells with micronuclei, as well as apoptotic cells, in vitro. In 211H tumor‐bearing mice, ZDHHC8 siRNA and X‐irradiation significantly suppressed tumor growth, whereas ZDHHC8 siRNA alone did not. Immunohistochemical analysis showed decreased cell proliferation and induction of apoptosis in tumors treated with ZDHHC8 siRNA and X‐irradiation, but not with ZDHHC8 siRNA alone. These results suggest that ZDHHC8 knockdown with X‐irradiation induces chromosomal instability and apoptosis through the impaired G2/M checkpoint. In conclusion, the combination of ZDHHC8 siRNA and X‐irradiation has the potential to improve the therapeutic efficacy of radiation therapy for malignant mesothelioma. (Cancer Sci 2012; 103: 203–209)

Therapeutic Efficacy of C-Kit-Targeted Radioimmunotherapy Using 90Y-Labeled Anti-C-Kit Antibodies in a Mouse Model of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive tumor and prognosis remains poor. Therefore, the development of more effective therapy is needed. We previously reported that high levels of an anti-c-kit antibody (12A8) accumulated in SCLC xenografts. In the present study, we evaluated the efficacy of two antibodies (12A8 and 67A2) for radioimmunotherapy (RIT) of an SCLC mouse model by labeling with the 90Y isotope. Methods 111In- or 125I-labeled antibodies were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays in c-kit-expressing SY cells and in vivo by biodistribution in SY-bearing mice. Therapeutic efficacy of 90Y-labeled antibodies was evaluated in SY-bearing mice upto day 28 and histological analysis was conducted at day 7. Results [111In]12A8 and [111In]67A2 specifically bound to SY cells with high affinity (8.0 and 1.9 nM, respectively). 67A2 was internalized similar to 12A8. High levels of [111In]12A8 and [111In]67A2 accumulated in tumors, but not in major organs. [111In]67A2 uptake by the tumor was 1.7 times higher than for [111In]12A8. [90Y]12A8, but not [90Y]67A2, suppressed tumor growth in a dose-dependent manner. Tumors treated with 3.7 MBq of [90Y]12A8, and 1.85 and 3.7 MBq of [90Y]67A2 (absorbed doses were 21.0, 18.0 and 35.9 Gy, respectively) almost completely disappeared approximately 2 weeks after injection, and regrowth was not observed except for in one mouse treated with 1.85 MBq [90Y]67A2. The area of necrosis and fibrosis increased depending on the RIT effect. Apoptotic cell numbers increased with increased doses of [90Y]12A8, whereas no dose-dependent increase was observed following [90Y]67A2 treatment. Body weight was temporarily reduced but all mice tolerated the RIT experiments well. Conclusion Treatment with [90Y]12A8 and [90Y]67A2 achieved a complete therapeutic response when SY tumors received an absorbed dose greater than 18 Gy and thus are promising RIT agents for metastatic SCLC cells at distant sites.

Development of positron emission tomography probe of 64Cu-labeled anti-C-kit 12A8 Fab to measure protooncogene C-kit expression

INTRODUCTION C-kit is an important diagnostic and therapeutic target molecule for several malignancies, and c-kit-targeted drugs have been used clinically. Because abundant c-kit expression in tumors is a prerequisite for successful c-kit-targeted therapy, imaging of c-kit expression is expected to play a pivotal role in the therapeutic decision for each patient. We evaluated (64)Cu-labeled Fab of anti-c-kit antibody 12A8 as a positron emission tomography (PET) imaging probe. METHODS (111)In- or (125)I-Labeled 12A8 Fab was evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution in mice bearing c-kit-expressing and -non-expressing tumors. Next, Fab fragment was labeled with the positron emitter (64)Cu and evaluated by PET. RESULTS Radiolabeled 12A8 Fab showed specific binding to c-kit-expressing cells with high affinity and internalized into cells after binding to c-kit on cell surface. Although tumor accumulation of [(111)In]Fab was lower than that of [(111)In]IgG, the faster blood clearance of [(111)In]Fab provided higher tumor-to-blood ratio at 6 h postinjection onwards. Blood clearance of (64)Cu-labeled 12A8 Fab was slower than that of [(111)In]Fab, but PET using [(64)Cu]Fab clearly visualized the tumor at 6 h postinjection onwards. CONCLUSION The (64)Cu-labeled 12A8 Fab could be used for c-kit-specific PET imaging and might help in selecting appropriate patients for c-kit-targeted treatments.

18F-FDG PET for Semiquantitative Evaluation of Acute Allograft Rejection and Immunosuppressive Therapy Efficacy in Rat Models of Liver Transplantation

Acute allograft rejection remains a major complication after liver transplantation. We report a semiquantitative imaging method of detecting acute allograft rejection with 18F-FDG PET. Methods: Syngeneic and allogeneic transplanted rats, with or without immunosuppressive treatment, were subjected to serial PET. Autoradiography of the liver was conducted in both the syngeneic and the allogeneic rats. Results: A significant increment of 18F-FDG accumulation in liver allografts was observed by PET on day 2. The 18F-FDG signal was concentrated in the area where inflammatory cells around the vessels were detected by autoradiography. Allotransplanted rats treated with an immunosuppressive agent displayed a marked decrease in hepatic 18F-FDG uptake, compared with allotransplanted rats that were not treated. Conclusion: 18F-FDG PET may be a valid method for facilitating the development of protocols to diagnose graft rejection and to monitor the efficacy of immunosuppressive therapy.

AHNAK is highly expressed and plays a key role in cell migration and invasion in mesothelioma

The worldwide incidence of the highly aggressive tumor mesothelioma is expected to increase. Mesothelioma is classified into three main histological subtypes: epithelioid, sarcomatoid and biphasic. Although the pathological diagnostic markers for epithelioid are established, to date no adequate marker for sarcomatoid mesothelioma has been found. Thus, a reliable diagnostic marker of sarcomatoid mesothelioma is necessary. In this study, to identify an unknown protein with 120 kDa expressed only in the mesothelioma cell line 211H, we conducted proteomic analysis and found five candidate proteins. One such protein, AHNAK, was highly expressed in all seven mesothelioma cell lines (211H, H28, H226, H2052, H2452, MESO1 and MESO4), but not in the mesothelial cell line MeT-5A by RT-PCR and immunofluorescence staining. Furthermore, we confirmed high AHNAK expression not only in xenografts but also in human mesothelioma specimens including sarcomatoid, epithelioid and biphasic mesothelioma using immunohistochemical staining. These findings suggest that AHNAK has the potential to be a new marker for detecting mesothelioma. Since AHNAK is involved in cell migration and invasion in other metastatic tumor cells, we conducted migration and invasion assays in mesothelioma cell lines. The number of migrating cells in six of seven mesothelioma cell lines and the number of invading cells in all seven cell lines were significantly increased compared with those in MeT-5A. Knockdown of AHNAK significantly reduced the cell migration and invasion ability in all seven mesothelioma cell lines. These results support further clinical evaluation of the association of AHNAK and metastasis in mesothelioma.

Evaluation of Efficacy of Radioimmunotherapy with 90Y-Labeled Fully Human Anti-Transferrin Receptor Monoclonal Antibody in Pancreatic Cancer Mouse Models.

Objective Pancreatic cancer is an aggressive tumor and the prognosis remains poor. Therefore, development of more effective therapy is needed. We previously reported that 89Zr-labeled TSP-A01, an antibody against transferrin receptor (TfR), is highly accumulated in a pancreatic cancer xenograft, but not in major normal organs. In the present study, we evaluated the efficacy of radioimmunotherapy (RIT) with 90Y-TSP-A01 in pancreatic cancer mouse models. Methods TfR expression in pancreatic cancer cell lines (AsPC-1, BxPC-3, MIAPaCa-2) was evaluated by immunofluorescence staining. 111In-labeled anti-TfR antibodies (TSP-A01, TSP-A02) were evaluated in vitro by cell binding assay with the three cell lines and by competitive inhibition assay with MIAPaCa-2. In vivo biodistribution was evaluated in mice bearing BxPC-3 and MIAPaCa-2 xenografts. Tumor volumes of BxPC-3 and MIAPaCa-2 were sequentially measured after 90Y-TSP-A01 injection and histological analysis of tumors was conducted. Results MIAPaCa-2 cells showed the highest TfR expression, followed by AsPC-1 and BxPC-3 cells. 111In-TSP-A01 and 111In-TSP-A02 bound specifically to the three cell lines according to TfR expression. The dissociation constants for TSP-A01, DOTA-TSP-A01, TSP-A02, and DOTA-TSP-A02 were 0.22, 0.28, 0.17, and 0.22 nM, respectively. 111In-TSP-A01 was highly accumulated in tumors, especially in MIAPaCa-2, but this was not true of 111In-TSP-A02. The absorbed dose for 90Y-TSP-A01 was estimated to be 8.3 Gy/MBq to BxPC-3 and 12.4 Gy/MBq to MIAPaCa-2. MIAPaCa-2 tumors treated with 3.7 MBq of 90Y-TSP-A01 had almost completely disappeared around 3 weeks after injection and regrowth was not observed. Growth of BxPC-3 tumors was inhibited by 3.7 MBq of 90Y-TSP-A01, but the tumor size was not reduced. Conclusion 90Y-TSP-A01 treatment achieved an almost complete response in MIAPaCa-2 tumors, whereas it merely inhibited the growth of BxPC-3 tumors. 90Y-TSP-A01 is a promising RIT agent for pancreatic cancer, although further investigation is necessary to improve the efficacy for the radioresistant types like BxPC-3.

Preclinical evaluation of 89Zr-labeled human antitransferrin receptor monoclonal antibody as a PET probe using a pancreatic cancer mouse model

ObjectivePancreatic cancer is aggressive and its prognosis remains poor; thus, effective therapy is urgently needed. Transferrin receptor (TfR) is highly expressed in pancreatic cancer and is considered to be a good candidate for molecular-targeted therapy. We radiolabeled and evaluated fully human anti-TfR monoclonal antibodies as a new PET probe for evaluating the biodistribution of the anti-TfR antibody in pancreatic cancer. Materials and methodsTfR expression was evaluated in four human pancreatic cancer (MIAPaCa-2, PANC-1, BxPC-3, and AsPC-1) and murine A4 cell lines. The binding of 125I-labeled anti-TfR antibodies (TSP-A01, TSP-A02, TSP-A03, and TSP-A04) to MIAPaCa-2 cells was compared. 125I-labeled, 67Ga-labeled, and 89Zr-labeled TSP-A01 were evaluated by cell binding, competitive inhibition, and internalization assays. Biodistribution studies of 125I-labeled and 89Zr-labeled TSP-A01 were conducted in mice bearing MIAPaCa-2 and A4 tumors. PET imaging with [89Zr]TSP-A01 was carried out. ResultsMIAPaCa-2 cells showed the highest TfR expression in vitro and in vivo, whereas A4 cells showed no expression. Of the four antibodies, [125I]TSP-A01 showed the highest binding to MIAPaCa-2 cells, but not to A4 cells. The dissociation constant of TSP-A01 was 0.29 nmol/l. Uptake of radiolabeled TSP-A01, especially [89Zr]TSP-A01, was significantly higher in MIAPaCa-2 tumors than in A4 tumors. PET with [89Zr]TSP-A01 clearly visualized MIAPaCa-2 xenografts but not A4 xenografts. Conclusion[89Zr]TSP-A01 is a promising PET probe for evaluating the accumulation of anti-TfR antibody in pancreatic cancer and has the potential to facilitate the selection of appropriate patients who would benefit from anti-TfR antibody therapy.

Comparison of 2-amino-[3-11C]isobutyric acid and 2-deoxy-2-[18F]fluoro-D-glucose in nude mice with xenografted tumors and acute inflammation

Objective2-Deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) accumulates in tumors and also during active inflammation, including therapy-related inflammation. Additional PET tracers that are less avid to inflammation could be useful in differentiating cancer from inflammation and could complement the limitation of [18F]FDG-PET. 2-Amino-[3-11C]isobutyric acid ([3-11C]AIB) is a potential PET tracer for this purpose. We compared [3-11C]AIB and [18F]FDG uptakes in tumors and acute inflammation in a mouse model. MethodsAcute inflammatory lesions were induced in the hind legs of tumor-bearing mice by intramuscular injection of turpentine, and we conducted biodistribution and dynamic PET studies on [3-11C]AIB and [18F]FDG. Results[3-11C]AIB tumor uptake increased with time and was statistically significantly higher than [18F]FDG uptake. In inflamed muscles, [3-11C]AIB uptake was statistically significantly lower than [18F]FDG uptake, and the tumor-to-inflammation ratio for [3-11C]AIB was statistically significantly higher than that for [18F]FDG. Conclusion[3-11C]AIB accumulates more selectively in tumor tissue than does [18F]FDG and thus has the potential of discriminating between tumors and inflammatory lesions better and of complementing the limitation of [18F]FDG.

Immunotargeting of Integrin α6β4 for Single-Photon Emission Computed Tomography and Near-Infrared Fluorescence Imaging in a Pancreatic Cancer Model

To explore suitable imaging probes for early and specific detection of pancreatic cancer, we demonstrated that α6β4 integrin is a good target and employed single-photon emission computed tomography (SPECT) or near-infrared (NIR) imaging for immunotargeting. Expression levels of α6β4 were examined by Western blotting and flow cytometry in certain human pancreatic cancer cell lines. The human cell line BxPC-3 was used for α6β4-positive and a mouse cell line, A4, was used for negative counterpart. We labeled antibody against α6β4 with Indium-111 (111In) or indocyanine green (ICG). After injection of 111In-labeled probe to tumor-bearing mice, biodistribution, SPECT, autoradiography (ARG), and immunohistochemical (IHC) studies were conducted. After administration of ICG-labeled probe, in vivo and ex vivo NIR imaging and fluorescence microscopy of tumors were performed. BxPC-3 tumor showed a higher radioligand binding in SPECT and higher fluorescence intensity as well as a delay in the probe washout in NIR imaging when compared to A4 tumor. The biodistribution profile of 111In-labeled probe, ARG, and IHC confirmed the α6β4 specific binding of the probe. Here, we propose that α6β4 is a desirable target for the diagnosis of pancreatic cancer and that it could be detected by radionuclide imaging and NIR imaging using a radiolabeled or ICG-labeled α6β4 antibody.