Head-to-head comparison of three chelates reveals DOTAGA promising for 225 Ac labeling of anti-FZD10 antibody OTSA101.

To select the most suitable chelate for 225 Ac radiolabeling of the anti-FZD10 antibody OTSA101, we directly compared three chelates: S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 2,2',2″-(10-(1-carboxy-4-((4-isothiocyanatobenzyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (p-SCN-Bn-DOTAGA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DO3A-NHS-ester). We evaluated the binding affinity of the chelate-conjugated OTSA101 antibodies, as well as the labeling efficiency and stability in murine serum of 225 Ac-labeled OTSA101 as in vitro properties. The biodistribution, intratumoral distribution, absorbed doses, and therapeutic effects of the chelate-conjugated OTSA101 antibodies were assessed in the synovial sarcoma mouse model SYO-1. Of the three conjugates, DOTAGA conjugation had the smallest impact on the binding affinity (p < 0.01). The labeling efficiencies of DOTAGA-OTSA101 and DO3A-OTSA101 were 1.8-fold higher than that of DOTA-OTSA101 (p < 0.01). The stabilities were similar between 225 Ac-labeled DOTA-OTSA101, DOTAGA-OTSA101, and DO3A-OTSA101in serum at 37 and 4°C. The dosimetric analysis based on the biodistribution revealed significantly higher tumor-absorbed doses by 225 Ac-labeled DOTA-OTSA101 and DOTAGA-OTSA101 compared with 225 Ac-DO3A-OTSA101 (p < 0.05). 225 Ac-DOTAGA-OTSA101 exhibited the highest tumor-to-bone marrow ratio, with bone marrow being the dose-limiting tissue. The therapeutic and adverse effects were not significantly different between the three conjugates. Our findings indicate that among the three evaluated chelates, DOTAGA appears to be the most promising chelate to produce 225 Ac-labeled OTSA101 with high binding affinity and high radiochemical yields while providing high absorbed doses to tumors and limited absorbed doses to bone marrow.

FZD10-targeted α-radioimmunotherapy with 225 Ac-labeled OTSA101 achieves complete remission in a synovial sarcoma model.

Synovial sarcomas are rare tumors arising in adolescents and young adults. The prognosis for advanced disease is poor, with an overall survival of 12-18 months. Frizzled homolog 10 (FZD10) is overexpressed in most synovial sarcomas, making it a promising therapeutic target. The results of a phase 1 trial of ß-radioimmunotherapy (RIT) with the 90 Y-labeled anti-FZD10 antibody OTSA101 revealed a need for improved efficacy. The present study evaluated the potential of α-RIT with OTSA101 labeled with the α-emitter 225 Ac. Competitive inhibition and cell binding assays showed that specific binding of 225 Ac-labeled OTSA101 to SYO-1 synovial sarcoma cells was comparable to that of the imaging agent 111 In-labeled OTSA101. Biodistribution studies showed high uptake in SYO-1 tumors and low uptake in normal organs, except for blood. Dosimetric studies showed that the biologically effective dose (BED) of 225 Ac-labeled OTSA101 for tumors was 7.8 Bd higher than that of 90 Y-labeled OTSA101. 90 Y- and 225 Ac-labeled OTSA101 decreased tumor volume and prolonged survival. 225 Ac-labeled OTSA101 achieved a complete response in 60% of mice, and no recurrence was observed. 225 Ac-labeled OTSA101 induced a larger amount of necrosis and apoptosis than 90 Y-labeled OTSA101, although the cell proliferation decrease was comparable. The BED for normal organs and tissues was tolerable; no treatment-related mortality or obvious toxicity, except for temporary body weight loss, was observed. 225 Ac-labeled OTSA101 provided a high BED for tumors and achieved a 60% complete response in the synovial sarcoma mouse model SYO-1. RIT with 225 Ac-labeled OTSA101 is a promising therapeutic option for synovial sarcoma.

In Vitro Evaluation of No-Carrier-Added Radiolabeled Cisplatin ([189, 191Pt]cisplatin) Emitting Auger Electrons.

Due to their short-range (2-500 nm), Auger electrons (Auger e-) have the potential to induce nano-scale physiochemical damage to biomolecules. Although DNA is the primary target of Auger e-, it remains challenging to maximize the interaction between Auger e- and DNA. To assess the DNA-damaging effect of Auger e- released as close as possible to DNA without chemical damage, we radio-synthesized no-carrier-added (n.c.a.) [189, 191Pt]cisplatin and evaluated both its in vitro properties and DNA-damaging effect. Cellular uptake, intracellular distribution, and DNA binding were investigated, and DNA double-strand breaks (DSBs) were evaluated by immunofluorescence staining of γH2AX and gel electrophoresis of plasmid DNA. Approximately 20% of intracellular radio-Pt was in a nucleus, and about 2% of intra-nucleus radio-Pt bound to DNA, although uptake of n.c.a. radio-cisplatin was low (0.6% incubated dose after 25-h incubation), resulting in the frequency of cells with γH2AX foci was low (1%). Nevertheless, some cells treated with radio-cisplatin had γH2AX aggregates unlike non-radioactive cisplatin. These findings suggest n.c.a. radio-cisplatin binding to DNA causes severe DSBs by the release of Auger e- very close to DNA without chemical damage by carriers. Efficient radio-drug delivery to DNA is necessary for successful clinical application of Auger e-.

Radiolabeled Human Monoclonal Antibody 067-213 has the Potential for Noninvasive Quantification of CD73 Expression.

BACKGROUND: CD73 is an ectonucleotidase regulating extracellular adenosine concentration and plays an important role in adenosine-mediated immunosuppressive pathways. The efficacy of CD73-targeted therapy depends on the expression levels of CD73; therefore, monitoring CD73 status in cancer patients would provide helpful information for selection of patients who would benefit from CD73-targeted therapy. Here, we evaluated the ability of 111In-labeled antibody 067-213, which has high affinity for human CD73, to act as a noninvasive imaging probe. METHODS: Cell binding and competitive inhibition assays for 111In-labeled 067-213 were conducted using MIAPaCa-2 (high CD73 expression) and A431 (low CD73 expression) cells. For in vivo assessments, biodistribution and SPECT/CT studies were conducted in MIAPaCa-2 and A431 tumor-bearing mice. To estimate the absorbed dose in humans, biodistribution and SPECT/CT studies were conducted in healthy rats. RESULTS: 111In-labeled 067-213 bound to MIAPaCa-2 and A431 cells in a CD73-dependent manner and the affinity loss after 111In-labeling was limited. Biodistribution and SPECT/CT studies with 111In-labeled 067-213 in mice showed high uptake in MIAPaCa-2 tumors and lower uptake in A431 tumors. In rats, the probe did not show high uptake in normal organs, including endogenously CD73-expressing organs. The estimated absorbed doses in humans were reasonably low. CONCLUSIONS: 111In-labeled 067-213 showed CD73-expression-dependent tumor uptake and low uptake in normal organs and tissues. Radiolabeled 067-213 holds promise as an imaging probe for noninvasive evaluation of CD73 expression levels in patients. Our data encourage further clinical studies to clarify a role for CD73 monitoring in patients receiving CD73-targeted immune therapy.

Therapeutic efficacy evaluation of radioimmunotherapy with 90 Y-labeled anti-podoplanin antibody NZ-12 for mesothelioma.

Podoplanin is a type I transmembrane sialomucin-like glycoprotein that is highly expressed in malignant mesothelioma. The rat-human chimeric antibody NZ-12 has high affinity for human podoplanin and antibody-dependent cellular cytotoxicity and is applicable for radioimmunotherapy (RIT) to enhance the antitumor effect. In the present study, we evaluated the in vivo and in vitro properties of radiolabeled NZ-12 and the antitumor effect of RIT with 90 Y-labeled NZ-12 in an NCI-H226 (H226) malignant mesothelioma xenograft mouse model. 111 In-labeled NZ-12 bound specifically to H226 cells with high affinity, and accumulation was high in H226 tumors but low in major organs. RIT with 90 Y-labeled NZ-12 significantly suppressed tumor growth and prolonged survival without body weight loss and obvious adverse effects. Higher podoplanin expression levels were observed in human mesothelioma specimens, suggesting higher tumor accumulation of 90 Y-labeled NZ-12 in patients compared with the H226 tumor xenografts. Our findings suggest that 90 Y-labeled NZ-12 is a promising RIT agent as a new therapeutic option for malignant mesothelioma that warrants further clinical studies to evaluate the dosimetry and efficacy in patients.

Antitumor effects of radionuclide treatment using α-emitting meta-211At-astato-benzylguanidine in a PC12 pheochromocytoma model.

PURPOSE: Therapeutic options for patients with malignant pheochromocytoma are currently limited, and therefore new treatment approaches are being sought. Targeted radionuclide therapy provides tumor-specific systemic treatments. The ß-emitting radiopharmaceutical meta-131I-iodo-benzylguanidine (131I-MIBG) provides limited survival benefits and has adverse effects. A new generation of radionuclides for therapy using α-particles including meta-211At-astato-benzylguanidine (211At-MABG) are expected to have strong therapeutic effects with minimal side effects. However, this possibility has not been evaluated in an animal model of pheochromocytoma. We aimed to evaluate the therapeutic effects of the α-emitter 211At-MABG in a pheochromocytoma model. METHODS: We evaluated tumor volume-reducing effects of 211At-MABG using rat pheochromocytoma cell line PC12 tumor-bearing mice. PC12 tumor-bearing mice received intravenous injections of 211At-MABG (0.28, 0.56, 1.11, 1.85, 3.70 and 5.55 MBq; five mice per group). Tumor volumes were evaluated for 8 weeks after 211At-MABG administration. The control group of ten mice received phosphate-buffered saline. RESULTS: The 211At-MABG-treated mice showed significantly lower relative tumor growth during the first 38 days than the control mice. The relative tumor volumes on day 21 were 509.2% ± 169.1% in the control mice and 9.6% ± 5.5% in the mice receiving 0.56 MBq (p < 0.01). In addition, the mice treated with 0.28, 0.56 and 1.11 MBq of 211At-MABG showed only a temporary weight reduction, with recovery in weight by day 10. CONCLUSION: 211At-MABG exhibited a strong tumor volume-reducing effect in a mouse model of pheochromocytoma without weight reduction. Therefore, 211At-MABG might be an effective therapeutic agent for the treatment of malignant pheochromocytoma.

Efficacy Evaluation of Combination Treatment Using Gemcitabine and Radioimmunotherapy with 90Y-Labeled Fully Human Anti-CD147 Monoclonal Antibody 059-053 in a BxPC-3 Xenograft Mouse Model of Refractory Pancreatic Cancer.

The poor prognosis of pancreatic cancer requires the development of more effective therapy. CD147 expresses in pancreatic cancer with high incidence and has a crucial role in invasion and metastasis. We developed a fully human monoclonal antibody (059-053) with high affinity for CD147. Here we evaluated the efficacy of combined treatment using radioimmunotherapy (RIT) with 90Y-labeled 059-053 and gemcitabine in a BxPC-3 xenograft mouse model. Expression of CD147 and matrix metalloproteinase-2 (MMP2) in BxPC-3 tumors was evaluated. In vitro and in vivo properties of 059-053 were evaluated using 111In-labeled 059-053 and a pancreatic cancer model BxPC-3. Tumor volume and body weight were periodically measured in mice receiving gemcitabine, RIT, and both RIT and gemcitabine (one cycle and two cycles). High expression of CD147 and MMP2 was observed in BxPC-3 tumors and suppressed by 059-053 injection. Radiolabeled 059-053 bound specifically to BxPC-3 cells and accumulated highly in BxPC-3 tumors but low in major organs. Combined treatment using RIT with gemcitabine (one cycle) significantly suppressed tumor growth and prolonged survival with tolerable toxicity. The two-cycle regimen had the highest anti-tumor effect, but was not tolerable. Combined treatment with 90Y-labeled 059-053 and gemcitabine is a promising therapeutic option for pancreatic cancer.

Synthesis and evaluation of 11C-labeled coumarin analog as an imaging probe for detecting monocarboxylate transporters expression.

Upregulated monocarboxylate transporters (MCTs) in tumors are considered diagnostic imaging targets. Herein, we synthesized the positron emission tomography probe candidates coumarin analogs 2 and 3, and showed 55 times higher affinity of 2 for MCTs than a representative MCT inhibitor. Whereas [11C]2 showed low tumor accumulation, probably due to adduct formation with plasma proteins, [11C]2 showed high initial brain uptake, suggesting that the scaffold of 2 has properties that are preferable in imaging probes for the astrocyte-neuron lactate shuttle. Although further optimization of 2 is required, our findings can be used to inform the development of MCT-targeted imaging agents.

Immunotargeting of Integrin α6ß4 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 α6ß4integrin is a good target and employed single-photon emission computed tomography (SPECT) or near-infrared (NIR) imaging for immunotargeting. Expression levels of α6ß4were examined by Western blotting and flow cytometry in certain human pancreatic cancer cell lines. The human cell line BxPC-3 was used for α6ß4-positive and a mouse cell line, A4, was used for negative counterpart. We labeled antibody against α6ß4with Indium-111 ((111)In) or indocyanine green (ICG). After injection of(111)In-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(111)In-labeled probe, ARG, and IHC confirmed the α6ß4specific binding of the probe. Here, we propose that α6ß4is 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 α6ß4antibody.

The tyrosine kinase inhibitor nintedanib enhances the efficacy of 90 Y-labeled B5209B radioimmunotherapy targeting ROBO1 without increased toxicity in small-cell lung cancer xenograft mice.

BACKGROUND: Small cell lung cancer (SCLC) has a poor prognosis, and Roundabout homolog 1 (ROBO1) is frequently expressed in SCLC. ROBO1-targeted radioimmunotherapy (RIT) previously showed tumor shrinkage, but regrowth with fibroblast infiltration was observed. The fibroblasts would support tumor survival by secreting growth factors and cytokines. Inhibition of fibroblasts offers a candidate strategy for increasing RIT efficacy. Here, we evaluated the efficacy of combination therapy with 90 Y-labeled anti-ROBO1 antibody B5209B ( 90 Y-B5209B) and the tyrosine kinase inhibitor nintedanib in SCLC xenograft mice. METHODS: Subcutaneous NCI-H69 SCLC xenograft mice were divided into four groups: saline, nintedanib alone, RIT alone, and a combination of RIT with nintedanib (combination). A single dose of 7.4 MBq of 90 Y-B5209B was injected intravenously. Nintedanib was orally administered at a dose of 400 µg five times a week for 4 weeks. Tumor volumes and body weights were measured regularly. Tumor sections were stained with hematoxylin and eosin or Masson trichrome. RESULTS: All six tumors in the combination therapy group disappeared, and four tumors showed no regrowth. Although RIT alone induced similar tumor shrinkage, regrowth was observed. Prolonged survival in the combination therapy group was found compared with the other groups. Temporary body weight loss was observed in RIT and combination therapy. There is no difference in fibroblast infiltration between RIT alone and the combination. CONCLUSION: Nintedanib significantly enhanced the anti-tumor effects of RIT with the 90 Y-B5209B without an increase in toxicity. These findings encourage further research into the potential clinical application of combining RIT with nintedanib.

Wnt1 induces osteoblastic changes in a well-established osteolytic skeletal metastatic model derived from breast cancer.

BACKGROUND: Osteoblastic skeletal metastasis is frequently observed in prostate cancer. An effective therapy has not been developed due to the unclear molecular mechanism. The Wnt family is involved in various biological phenomena including bone metabolism. There is no direct evidence that the family causes osteoblastic skeletal metastasis. AIMS: The present study aims to evaluate whether overexpressed Wnt induces osteoblastic bone metastasis in a well-established osteolytic bone metastatic model. METHODS AND RESULTS: The breast cancer-derived 5a-D-Luc-ZsGreen cells were transfected with Wnt1, Wnt3A, and Wnt5A expression vectors, producing stably highly expressing cells. These cells were intracardially transplanted in nude mice. Bone metastasis development was confirmed by fluorescence imaging. Hind-limb bones including metastasis were dissected and visualized through micro-CT imaging. After imaging, sections were stained with hematoxylin and eosin (H&E), and immunohistochemically stained with an anti-SATB2 antibody. Luminescent imaging confirmed mice with bone metastases in the hind limbs. Micro-CT imaging found an osteoblastic change only in bone metastasis of mice transplanted with Wnt1-expressing cells. This was confirmed on H&E-stained sections. SATB2 immunostaining showed differentiated osteoblasts were at the site of bone metastases in the diaphysis. SATB2 in the Wnt/ß-catenin pathway activated by overexpressed Wnt1 could induce osteoblastic change. CONCLUSION: Our findings provided direct evidence Wnt1 is involved in osteoblastic bone metastasis development. Our model would be a powerful tool for further elucidating molecular mechanisms underlying the disease and developing effective therapies.

Novel Auger-Electron-Emitting 191Pt-Labeled Pyrrole-Imidazole Polyamide Targeting MYCN Increases Cytotoxicity and Cytosolic dsDNA Granules in MYCN-Amplified Neuroblastoma.

Auger electrons can cause nanoscale physiochemical damage to specific DNA sites that play a key role in cancer cell survival. Radio-Pt is a promising Auger-electron source for damaging DNA efficiently because of its ability to bind to DNA. Considering that the cancer genome is maintained under abnormal gene amplification and expression, here, we developed a novel 191Pt-labeled agent based on pyrrole-imidazole polyamide (PIP), targeting the oncogene MYCN amplified in human neuroblastoma, and investigated its targeting ability and damaging effects. A conjugate of MYCN-targeting PIP and Cys-(Arg)3-coumarin was labeled with 191Pt via Cys (191Pt-MYCN-PIP) with a radiochemical purity of >99%. The binding potential of 191Pt-MYCN-PIP was evaluated via the gel electrophoretic mobility shift assay, suggesting that the radioagent bound to the DNA including the target sequence of the MYCN gene. In vitro assays using human neuroblastoma cells showed that 191Pt-MYCN-PIP bound to DNA efficiently and caused DNA damage, decreasing MYCN gene expression and MYCN signals in in situ hybridization analysis, as well as cell viability, especially in MYCN-amplified Kelly cells. 191Pt-MYCN-PIP also induced a substantial increase in cytosolic dsDNA granules and generated proinflammatory cytokines, IFN-α/ß, in Kelly cells. Tumor uptake of intravenously injected 191Pt-MYCN-PIP was low and its delivery to tumors should be improved for therapeutic application. The present results provided a potential strategy, targeting the key oncogenes for cancer survival for Auger electron 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 G(2) /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 G(2) /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 G(2) /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.

The natural sulfoglycolipid derivative SQAP improves the therapeutic efficacy of tissue factor-targeted radioimmunotherapy in the stroma-rich pancreatic cancer model BxPC-3.

α-Sulfoquinovosylacyl-1,3-propanediol (SQAP) is a semi-synthetic derivative of natural sulfoglycolipid that sensitizes tumors to external-beam radiotherapy. How SQAP affects internal radiotherapy, however, is not known. Here, we investigated the effects of SQAP for radioimmunotherapy (RIT) targeting tissue factor (TF) in a stroma-rich refractory pancreatic cancer mouse model, BxPC-3. A low dose of SQAP (2 mg/kg) increased tumor uptake of the 111In-labeled anti-TF antibody 1849, indicating increased tumor perfusion. The addition of SQAP enhanced the growth-inhibitory effect of 90Y-labeled 1849 without leading to severe body weight changes, allowing for the dose of 90Y-labeled 1849 to be reduced to half that when used alone. Histologic analysis revealed few necrotic and apoptotic cells, but Ki-67-positive proliferating cells and increased vascular formation were detected. These results suggest that the addition of a low dose of SQAP may improve the therapeutic efficacy of TF-targeted RIT by increasing tumor perfusion, even for stroma-rich refractory pancreatic cancer.

Precise quantitative evaluation of pharmacokinetics of cisplatin using a radio-platinum tracer in tumor-bearing mice.

OBJECTIVE: The platinum-based antineoplastic drug cisplatin is commonly used for chemotherapy in clinics. This work aims to demonstrate a radio-platinum tracer is useful for precisely quantifying small amounts of platinum in pharmacokinetics studies. METHODS: A cisplatin radiotracer (radio-cisplatin) was synthesized, and a comprehensive evaluation of cisplatin over 7 days after its intravenous injection into nude mice bearing a subcutaneous lung tumor (H460) was conducted. RESULTS: A biphasic retention curve in the whole body and blood was observed [ T1/2 (α) = 1.14 h, T1/2 (ß) = 5.33 days for the whole body, and T1/2 (α) = 23.9 min, T1/2 (ß) = 4.72 days for blood]. The blood concentration decreased within 1 day after injection. Most of the intact cisplatin was excreted via the kidneys in the early time points, and a small part was distributed in tissues including tumors. The plasma protein binding rate of cisplatin increased rapidly after injection, and the protein-bound cisplatin remained in the blood longer than intact cisplatin. The peak uptake in H460 tumors was 4.7% injected dose per gram at 15 min after injection, and the area under the curve (AUC 0-7 days ) was approximately one-half to one-third of the AUC 0-7 days in the kidneys, liver, and bone, where some toxicity is observed in humans. CONCLUSION: The radio-platinum tracer revealed the highly quantitative biodistribution of cisplatin, providing insights into the properties of cisplatin, including its adverse effects. The tracer enables a precise evaluation of pharmacokinetics for platinum-based drugs with high sensitivity.

In vivo validation of the switch antibody concept: SPECT/CT imaging of the anti-CD137 switch antibody Sta-MB shows high uptake in tumors but low uptake in normal organs in human CD137 knock-in mice.

CD137 is an attractive target for cancer immunotherapy, but its expression in normal tissues induces some adverse effects in patients receiving CD137-targeted therapy. To overcome this issue, we developed a switch antibody, STA551, that binds to CD137 only under high ATP concentrations around cells. This study quantified biodistribution of murine switch antibodies in human CD137 knock-in mice to show the viability of the switch antibody concept in vivo. We utilized four antibodies: Sta-MB, Ure-MB, Sta-mIgG1, and KLH-MB. Sta-MB is a switch antibody having the variable region of STA551. The MB is a murine Fc highly binding to murine Fcγ receptor II. Ure-MB has a variable region mimicking the clinically available anti-CD137 agonist antibody urelumab, binding to CD137 regardless of ATP concentration. Sta-mIgG1 has the same variable region as Sta-MB but has the standard murine constant region. KLH-MB binds to keyhole limpet hemocyanin. The four antibodies were radiolabeled with In-111, SPECT/CT imaging was conducted in human CD137 knock-in mice, and the uptake in regions of interest was quantified. 111In-labeled Sta-MB and Sta-mIgG1 showed high uptake in tumors but low uptake in the lymph nodes and spleen in human CD137 knock-in mice. On the other hand, Ure-MB highly accumulated not only in tumors but also in the lymph nodes and spleen. KLH-MB showed low uptake in the tumors, lymph nodes, and spleen. The present study provides evidence that the switch antibody concept works in vivo. Our findings encourage further clinical imaging studies to evaluate the biodistribution of STA551 in patients.

Measurement of biological washout rates depending on tumor vascular status in15O in-beam rat-PET.

Objective.The biological washout of positron emitters should be modeled and corrected in order to achieve quantitative dose range verification in charged particle therapy based on positron emission tomography (PET). This biological washout effect is affected by physiological environmental conditions such as blood perfusion and metabolism, but the correlation to tumour pathology has not been studied yet.Approach.The aim of this study was to investigate the dependence of the biological washout rate on tumour vascular status in rat irradiation. Two types of tumour vascularity conditions, perfused and hypoxic, were modelled with nude rats. The rats were irradiated by a radioactive15O ion beam and time activity curves were acquired by dynamic in-beam PET measurement. Tumour tissue sections were obtained to observe the histology as well. The biological washout rate was derived using a single-compartment model with two decay components (medium decay,k2mand slow decay,k2s).Main results.Allk2mvalues in the vascular perfused tumour tissue were higher than the values of the normal tissue. Allk2mvalues in the hypoxic tumour tissue were much lower than the values of the vascular perfused tumour tissue and slightly lower than the values of the normal tissue.Significance.The dependency of the biological washout on the tumour vasculature conditions was experimentally shown.

Development of Novel 191Pt-Labeled Hoechst33258: 191Pt Is More Suitable than 111In for Targeting DNA.

This study aims to establish new labeling methods for no-carrier-added radio-Pt (191Pt) and to evaluate the in vitro properties of 191Pt-labeled agents compared with those of agents labeled with the common emitter 111In. 191Pt was complexed with the DNA-targeting dye Hoechst33258 via diethylenetriaminepentaacetic acid (DTPA) or the sulfur-containing amino acid cysteine (Cys). The intranuclear fractions of 191Pt- and 111In-labeled Hoechst33258 were comparable, indicating that the labeling for 191Pt via DTPA or Cys and the labeling for 111In via DTPA worked equally well. 191Pt showed a DNA-binding/cellular uptake ratio of more than 1 order of magnitude greater than that of 111In. [191Pt]Pt-Hoechst33258 labeled via Cys showed a higher cellular uptake than that labeled via DTPA, resulting in a very high DNA-binding fraction of [191Pt]Pt-Cys-Hoechst33258 and extensive DNA damage. Our labeling methods of radio-Pt, especially via Cys, promote the development of radio-Pt-based agents for use in Auger electron therapy targeting DNA.

Development of Radiohalogenated Osimertinib Derivatives as Imaging Probes for Companion Diagnostics of Osimertinib.

Osimertinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor approved for treating non-small-cell lung cancer (NSCLC) with EGFR mutations. Genetic testing is required to detect the mutation for selecting patients who can use osimertinib. Here, we report an attempt to develop nuclear imaging probes that detect the EGFR mutations. We designed and synthesized I-osimertinib and Br-osimertinib with a radioactive or nonradioactive halogen atom at an indole ring in osimertinib and evaluated them. In vitro assays suggested that both I-osimertinib and Br-osimertinib exhibit a specifically high activity toward NSCLC with EGFR L858R/T790M mutations. In biodistribution experiments, the accumulation of both [125I]I-osimertinib and [77Br]Br-osimertinib in tumors with mutations was significantly higher than that in blood and muscle. However, these osimertinib derivatives showed a significantly higher accumulation in lungs than in tumors. Therefore, for detecting the mutations in lung cancer, further structural modifications of the probes are required.

Knockdown of COPA, identified by loss-of-function screen, induces apoptosis and suppresses tumor growth in mesothelioma mouse model.

Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes-COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, and RPL18A-would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.