Tumor Targeting of 211At-Labeled Antibody under Sodium Ascorbate Protection against Radiolysis.

Astatine-211 (211At) is an alpha emitter applicable to radioimmunotherapy (RIT), a cancer treatment that utilizes radioactive antibodies to target tumors. In the preparation of 211At-labeled monoclonal antibodies (211At-mAbs), the possibility of radionuclide-induced antibody denaturation (radiolysis) is of concern. Our previous study showed that this 211At-induced radiochemical reaction disrupts the cellular binding activity of an astatinated mAb, resulting in attenuation of in vivo antitumor effects, whereas sodium ascorbate (SA), a free radical scavenger, prevents antibody denaturation, contributing to the maintenance of binding and antitumor activity. However, the influence of antibody denaturation on the pharmacokinetics of 211At-mAbs relating to tumor accumulation, blood circulation time, and distribution to normal organs remains unclear. In this study, we use a radioactive anti-human epidermal growth factor receptor 2 (anti-HER2) mAb to demonstrate that an 211At-induced radiochemical reaction disrupts active targeting via an antigen-antibody interaction, whereas SA helps to maintain targeting. In contrast, there was no difference in blood circulation time as well as distribution to normal organs between the stabilized and denatured immunoconjugates, indicating that antibody denaturation may not affect tumor accumulation via passive targeting based on the enhanced permeability and retention effect. In a high-HER2-expressing xenograft model treated with 1 MBq of 211At-anti-HER2 mAbs, SA-dependent maintenance of active targeting contributed to a significantly better response. In treatment with 0.5 or 0.2 MBq, the stabilized radioactive mAb significantly reduced tumor growth compared to the denatured immunoconjugate. Additionally, through a comparison between a stabilized 211At-anti-HER2 mAb and radioactive nontargeted control mAb, we demonstrate that active targeting significantly enhances tumor accumulation of radioactivity and in vivo antitumor effect. In RIT with 211At, active targeting contributes to efficient tumor accumulation of radioactivity, resulting in a potent antitumor effect. SA-dependent protection that successfully maintains tumor targeting will facilitate the clinical application of alpha-RIT.

Radioimmunotherapy with an 211 At-labeled anti-tissue factor antibody protected by sodium ascorbate.

Tissue factor (TF), the trigger protein of the extrinsic blood coagulation cascade, is abundantly expressed in various cancers including gastric cancer. Anti-TF monoclonal antibodies (mAbs) capable of targeting cancers have been successfully applied to armed antibodies such as antibody-drug conjugates (ADCs) and molecular imaging probes. We prepared an anti-TF mAb, clone 1084, labeled with astatine-211 (211 At), as a promising alpha emitter for cancer treatment. Alpha particles are characterized by high linear energy transfer and a range of 50-100 µm in tissue. Therefore, selective and efficient tumor accumulation of alpha emitters results in potent antitumor activities against cancer cells with minor effects on normal cells adjacent to the tumor. Although the 211 At-conjugated clone 1084 (211 At-anti-TF mAb) was disrupted by an 211 At-induced radiochemical reaction, we demonstrated that astatinated anti-TF mAbs eluted in 0.6% or 1.2% sodium ascorbate (SA) solution were protected from antibody denaturation, which contributed to the maintenance of cellular binding activities and cytocidal effects of this immunoconjugate. Although body weight loss was observed in mice administered a 1.2% SA solution, the loss was transient and the radioprotectant seemed to be tolerable in vivo. In a high TF-expressing gastric cancer xenograft model, 211 At-anti-TF mAb in 1.2% SA exerted a significantly greater antitumor effect than nonprotected 211 At-anti-TF mAb. Moreover, the antitumor activities of the protected immunoconjugate in gastric cancer xenograft models were dependent on the level of TF in cancer cells. These findings suggest the clinical availability of the radioprotectant and applicability of clone 1084 to 211 At-radioimmunotherapy.

High expression of TMEM180, a novel tumour marker, is associated with poor survival in stage III colorectal cancer.

BACKGROUND: Transmembrane protein 180 (TMEM180) is a newly identified colorectal cancer (CRC)-specific molecule that is expressed very rarely in normal tissue and up-regulated under hypoxic conditions. We developed a monoclonal antibody (mAb) against TMEM180 and decided to examine the medical significance using the mAb. METHODS: A total of 157 patients (86 men and 71 women; median age 63.0 years) with stage III CRC who underwent curative surgery were analyzed for TMEM180 expression as a retrospective cohort design. Immunohistochemistry with anti-TMEM180 mAb was conducted on frozen sections, and the data were evaluated for any correlation with clinicopathological indices or prognosis. SW480 CRC cells were examined to investigate the relationship between the expression of TMEM180 and tumourigenesis of xenografts. RESULTS: In total, 92 cases had low TMEM expression and 65 had high TMEM180 expression. For disease-free survival, hazard ratio in high-TMEM180 cases was 1.449 (95% confidential interval = 0.802-2.619) higher than in low-TMEM180 cases, but the difference was not significant (p = 0.219). For cancer specific survival, hazard ratio in high-TMEM180 cases was 3.302 (95% confidential interval = 1.088-10.020), significantly higher than in low-TMEM180 cases (p = 0.035). In an assay examining in vitro colony-forming activity in soft agar, SW480-WT cells clearly formed colonies, but neither KD1 nor KD2 cells did. The in vivo tumour-initiating activity of SW480 cell lines was positively correlated with the level of TMEM180 expression. CONCLUSION: These results indicate that TMEM180 is a useful marker for clinical prognosis in patients with CRC. We believe that these fundamental data warrant further basic and translational studies of TMEM180, and its mAb, for development of therapeutics against CRC.

Report of the use of patient-derived xenograft models in the development of anticancer drugs in Japan.

Cell line-derived xenograft (CDX) models created by implanting cancer cell lines into immunodeficient mice have contributed largely to the development of cancer drug therapies. However, cell lines often lose their original biological characteristics through many passages and cancer tissues in CDX models have many cancer cells and few cancer stromal cells, therefore CDX models are currently considered not suitable for predicting the results of clinical studies. Conversely, patient-derived xenograft (PDX) models are gaining importance, as human cancer biological characteristics and microenvironments are recreated by implanting tumor tissue into immunodeficient mice. These highly expected, evidently beneficial PDX models have been used in some basic research and are becoming more generalized. However, quality control and quality assurance criteria have not been established for them, and challenges and problems in the utilization of valuable PDX models in drug development have yet to be clarified. In this report, we conducted a questionnaire survey among researchers in Japanese academic institutions and pharmaceutical companies to understand the current status of PDX models in Japan. Based on the questionnaire results, we summarized the situations surrounding respondent's utilization and quality control in the development of anticancer drugs and proposed several measures to facilitate the utilization of PDX models in the development of anticancer drugs.

Evaluation of the antitumor mechanism of antibody-drug conjugates against tissue factor in stroma-rich allograft models.

Tissue factor (TF) is known to be overexpressed in various cancers including pancreatic cancer. The upregulation of TF expression has been observed not only in tumor cells, but also in tumor stromal cells. Because of the potential of TF as a delivery target, several studies investigated the effectiveness of Ab-drug conjugates (ADCs) against TF for cancer therapy. However, it is still unclear whether anti-TF ADC can exert toxicity against both tumor cells and tumor stromal cells. Here, we prepared ADC using a rat anti-mouse TF mAb (clone.1157) and 2 types of in vivo murine pancreatic cancer models, one s.c. and other orthotopic with an abundant tumor stroma. We also compared the feasibility of bis-alkylating conjugation (bisAlk) with that of conventional maleimide-based conjugation (MC). In the s.c. models, anti-TF ADC showed greater antitumor effects than control ADC. The results also indicated that the bisAlk linker might be more suitable than the MC linker for cancer treatments. In the orthotopic model, anti-TF ADC showed greater in vivo efficacy and more extended survival time control ADC. Treatment with anti-TF ADC (20 mg/kg, three times a week) did not affect mouse body weight changes in any in vivo experiment. Furthermore, immunofluorescence staining indicated that anti-TF ADC delivered agents not only to TF-positive tumor cells, but also to TF-positive tumor vascular endothelial cells and other tumor stromal cells. We conclude that anti-TF ADC should be a selective and potent drug for pancreatic cancer therapy.

Utility of epirubicin-incorporating micelles tagged with anti-tissue factor antibody clone with no anticoagulant effect.

Tissue factor (TF), an initiator of the extrinsic blood coagulation cascade, is overexpressed in different types of cancer. Tissue factor overexpression is also known as a poor prognostic factor in pancreatic cancer. We recently developed anti-TF antibody (clone1849)-conjugated epirubicin-incorporating micelles (NC-6300), and reported that this anti-TF1849-NC-6300 showed enhanced antitumor activity against TF-high expressed human pancreatic cancer cells, when compared with NC-6300 alone. However, clone 1849 antibody inhibited TF-associated blood coagulation activity. We studied another anti-TF antibody, clone 1859, which had no effect on blood coagulation and prepared anti-TF1859-NC-6300. In addition, to determine the optimum size of the antibody fragment to conjugate with NC-6300, three forms of the 1859 antibody (whole IgG, F[ab']2 , and Fab') were conjugated to NC-6300. The antitumor effect of each anti-TF1859-NC-6300 was studied in vitro and in vivo, using two human pancreatic cancer cell lines, BxPC3 with high-expressed TF, and SUIT2 with low levels of TF. In vitro, all forms of anti-TF1859-NC-6300 showed higher cytocidal effects than NC-6300 in BxPC3, whereas this enhanced effect was not observed in SUIT2. Likewise, all forms of anti-TF1859-NC-6300 significantly suppressed tumor growth when compared to NC-6300 in the BxPC3, but not in the SUIT2, xenograft model. Among the three forms of conjugates, anti-TF1859-IgG-NC-6300 had a higher antitumor tendency in TF-high expressed cells. Thus, we have confirmed an enhanced antitumor effect of anti-TF1859-NC-6300 in a TF-high expressing tumor; anti-TF1859-IgG-NC-6300 could be used to simplify the manufacturing process of the antibody-micelle conjugation for future clinical studies.

Antitumor effect of antitissue factor antibody-MMAE conjugate in human pancreatic tumor xenografts.

Tissue factor (TF) triggers the extrinsic blood coagulation cascade and is highly expressed in various types of cancer. In this study, we investigated the antitumor effect of an antibody-drug conjugate (ADC) consisting of an anti-TF monoclonal antibody and monomethyl auristatin E (MMAE). MMAE was conjugated to an anti-human TF or anti-mouse TF antibody using a valine-citrulline linker that could be potentially hydrolyzed by cathepsin B in the acidic environment of the lysosome. The cytotoxic and antitumor effects of the ADCs against four pancreatic cancer cell lines were analyzed. Both the ADC with the anti-human TF antibody and that with the anti-mouse TF antibody were stable under physiological conditions. The anti-human ADC was internalized in TF-expressing human tumor cell lines, followed by effective MMAE release. The half maximal inhibitory concentration (IC50 ) of MMAE was approximately 1 nM for all of the cell lines used. Meanwhile, the IC50 of anti-human ADC was 1.15 nM in the cell lines showing high TF expression, while exceeding 100 nM in the cells showing low TF expression levels. Anti-human ADC with passive and active targeting ability exerted significant suppression of tumor growth as compared to that observed in the saline group (p < 0.01). Also significant tumor growth suppressions were seen at the anti-mouse ADC and control ADC groups compared to the saline group (p < 0.01) due to EPR effect. Because various clinical human cancers express highly amount of TF, this new anti-TF ADC may deserve a clinical evaluation.

Enhanced antitumor effect of anti-tissue factor antibody-conjugated epirubicin-incorporating micelles in xenograft models.

For the creation of a successful antibody-drug conjugate (ADC), both scientific and clinical evidence has indicated that highly toxic anticancer agents (ACA) should be conjugated to a monoclonal antibody (mAb) to administer a reasonable amount of ADC to patients without compromising the affinity of the mAb. For ordinary ACA, the conjugation of a mAb to ACA-loaded micellar nanoparticles is clinically applicable. Tissue factor (TF) is often overexpressed in various cancer cells and tumor vascular endothelium. Accordingly, anti-TF-NC-6300, consisting of epirubicin-incorporating micelles (NC-6300) conjugated with the F(ab')2 of anti-TF mAb was developed. The in vitro and in vivo efficacy and pharmacokinetics of anti-TF-NC-6300 were compared to NC-6300 using two human pancreatic cancer cell lines, BxPC3 (high TF expression) and SUIT2 (low TF expression), and a gastric cancer cell line, 44As3 (high TF expression). The intracellular uptake of epirubicin was faster and greater in BxPC3 cells treated with anti-TF-NC-6300, compared with NC-6300. Anti-TF-NC-6300 showed a superior antitumor activity in BxPC3 and 44As3 xenografts, compared with NC-6300, while the activities of both micelles were similar in the SUIT2 xenograft. A higher tumor accumulation of anti-TF-NC-6300 compared to NC-6300 was seen, regardless of the TF expression levels. However, anti-TF-NC-6300 appeared to be localized to the tumor cells with high TF expression. These results indicated that the enhanced antitumor effect of anti-TF-NC6300 may be independent of the tumor accumulation but may depend on the selective intratumor localization and the preferential internalization of anti-TF-NC-6300 into high TF tumor cells.

[Fecal Biomarker for Colorectal Cancer Diagnosis].

The fecal occult blood test (FOBT) is widely used for colorectal cancer (CRC) screening to reduce the mortality rate associated with this cancer. However, several problems exist, as FOBT results can contain some false-negative CRC patients and some-false positive healthy subjects. Thus, to resolve these problems, several fecal biomarkers based on fecal protein, fecal DNA, and fecal RNA have been reported. Fecal calprotectin, which indicates intestinal bleeding or inflammation of the colon mucosa, and fecal tumor M2-PK, which is produced by cancer cells, have been extensively investigated as fecal protein biomarkers. To detect small amounts of CRC-specific proteins, the chemiluminescent enzyme immunoassay (CLEIA), which is a highly sensitive protein detection method using immunomagnetic beads, will be used. DNA mutation of APC, KRAS, and TP53 genes and DNA methylation of VIM, TFPI2, BMP3, NDRG4, and SFRP2 genes were reported as fecal DNA biomarkers. Consequently, a fecal DNA test named Cologuard from Exact Sciences was approved by the FDA in August 2014. Fecal COX2, MMP7, miR-106a, miR-92a, and miR-223 were also reported as fecal RNA biomarkers. This review article summarizes fecal biomarkers using fecal samples for CRC diagnosis.

Effect of combined treatment with the epirubicin-incorporating micelles (NC-6300) and 1,2-diaminocyclohexane platinum (II)-incorporating micelles (NC-4016) on a human gastric cancer model.

Anticancer agent-incorporating polymeric micelles accumulate effectively in tumors via the enhanced permeability and retention effect to exert potent antitumor effects. However, combined use of such micelles has not been elucidated. We compared the effect of combining the epirubicin-incorporating micelle NC-6300 and 1,2-diaminocyclohexane platinum (II) (oxaliplatin parent complex)-incorporating micelle NC-4016 (NCs) with that of epirubicin and oxaliplatin (E/O) in 44As3Luc cells using the combination index method. The in vivo antitumor activities of NCs and E/O were evaluated in mice bearing 44As3Luc xenografts. Pharmacokinetic analysis was performed by high-performance liquid chromatography and mass spectrometry. Cardiotoxicity of NC-6300 and epirubicin was assessed by echocardiography. Neurotoxicity of NC-4016 and oxaliplatin was evaluated by examining the paw withdrawal response to noxious mechanical stimuli. NCs showed a highly synergistic activity equivalent to E/O. In vivo, NCs exhibited higher antitumor activity in the subcutaneous tumor model and longer overall survival in the orthotopic tumor model than E/O (p < 0.001, p = 0.015, respectively). The intratumor concentrations of epirubicin and platinum were significantly higher following NCs than following E/O administration. Moreover, the micelles showed lower cardiotoxicity and neurotoxicity than the corresponding conventional drugs. The combined use of the micelles was associated with remarkable efficacy and favorable toxicities in the human gastric cancer model, and warrants the conduct of clinical trials.

Preparation and characterization of anti-tissue factor single-chain variable fragment antibody for cancer diagnosis.

Tissue factor (TF), which serves as the initiator of the extrinsic blood coagulation cascade, has been found to be overexpressed in various solid tumors, especially brain tumors, pancreatic cancer, and gastric cancer. Overexpression of TF is considered to contribute to the high incidence of thrombotic complications and poor prognosis in patients with such cancers. Therefore, detection or targeting of TF may be a promising approach for the diagnosis and treatment of solid tumors that are known to overexpress the protein. Here, we used the recombinant DNA technology to develop an anti-TF single-chain Fv (scFv) of small size and high affinity for its target. The biochemical characteristics of the anti-TF scFv were evaluated using surface plasmon resonance (SPR) sensing and flow cytometry. The data obtained showed that the affinity of the anti-TF scFv was 2.04 × 10(-8) (KD), and that the protein showed significant binding to the cancer cells. Then, Alexa 647-labeled anti-TF scFv and anti-TF IgG were administered to mice bearing chemically induced spontaneous tumors. The maximum tumor to background ratios of anti-TF scFv and anti-TF IgG were obtained 3 and 24 h after the injections, respectively. This study indicates anti-TF scFv may be suitable as an imaging probe for the diagnosis of solid tumors.

The SCRUM-MONSTAR Cancer-Omics Ecosystem: Striving for a Quantum Leap in Precision Medicine.

The SCRUM-Japan MONSTAR-SCREEN consortium is a nationwide molecular profiling project employing artificial intelligence-driven multi-omics analyses for patients with advanced malignancies, aiming to develop novel therapeutics and diagnostics and deliver effective drugs to patients. Concurrently, studies assessing molecular residual disease-based precision medicine for resectable solid tumors, including CIRCULATE-Japan, are ongoing. The substantial data generated by these platforms are stored within a state-of-the-art supercomputing infrastructure, VAPOR CONE. Since 2015, our project has registered over 24,000 patients as of December 2023. Among 16,144 patients with advanced solid tumors enrolled in MONSTAR-SCREEN projects, 5.0% participated in matched clinical trials, demonstrating a 29.2% objective response rate and 14.8-month median survival (95% confidence interval, 13.4-16.3), for patients treated in the matched clinical trials. Notably, patients who received matched therapy demonstrated significantly prolonged overall survival compared with those who did not (hazard ratio 0.77; 95% confidence interval, 0.71-0.83).

NC-6300, an epirubicin-incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin.

Epirubicin is widely used to treat various human tumors. However, it is difficult to achieve a sufficient antitumor effect because of dosage limitation to prevent cardiotoxicity. We hypothesized that epirubicin-incorporating micelle would reduce cardiotoxicity and improve the antitumor effect. NC-6300 comprises epirubicin covalently bound to PEG polyaspartate block copolymer through an acid-labile hydrazone bond. The conjugate forms a micellar structure of 40-80 nm in diameter in an aqueous milieu. NC-6300 (10, 15 mg/kg) and epirubicin (10 mg/kg) were given i.v. three times to mice bearing s.c. or liver xenograft of human hepatocellular carcinoma Hep3B cells. Cardiotoxicity was evaluated by echocardiography in C57BL/6 mice that were given NC-6300 (10 mg/kg) or epirubicin (10 mg/kg) in nine doses over 12 weeks. NC-6300 showed a significantly potent antitumor effect against Hep3B s.c. tumors compared with epirubicin. Moreover, NC-6300 also produced a significantly longer survival rate than epirubicin against the liver orthotopic tumor of Hep3B. With respect to cardiotoxicity, epirubicin-treated mice showed significant deteriorations in fractional shortening and ejection fraction. In contrast, cardiac functions of NC-6300 treated mice were no less well maintained than in control mice. This study warrants a clinical evaluation of NC-6300 in patients with hepatocellular carcinoma or other cancers.

Application of the fecal microRNA test to the residuum from the fecal occult blood test.

OBJECTIVE: Though the fecal occult blood test is used for colorectal cancer screening worldwide, it does not have a particularly high sensitivity for detecting colorectal cancer. Here we investigated the applicability of the fecal microRNA test to fecal samples that had been used for a previous fecal occult blood test and stored under various conditions. METHODS: Five colorectal cancer patients and five healthy volunteers were enrolled. Fecal samples were stored for 0-5 days at 4°C, room temperature or 37°C. Total RNA was extracted from the fecal occult blood test residuum and microRNA expression was analyzed by real-time reverse transcription polymerase chain reaction. RESULTS: There were no remarkable differences either in colorectal cancer patients or in controls with regard to the concentration of RNA extracted from the fecal occult blood test residuum in any of the storage groups compared with the samples prepared on day 0 (Group 0). Ribosomal RNA stored at room temperature or 37°C degraded rapidly. In contrast, the ribosomal RNA stored at 4°C remained intact for at least 5 days. The microRNAs in samples stored at 4°C and room temperature were conserved; however, the microRNAs stored at 37°C were significantly degraded compared with Group 0 (P < 0.05). In the residuum stored at 4°C up to 5 days, the relative quantification of miR-106a normalized with miR-24 in colorectal cancer patients was significantly higher than those in healthy volunteers (P < 0.05). In contrast, the quantification of normalized miR-106a was remarkably low in samples stored at room temperature and 37°C. CONCLUSIONS: Fecal microRNA of sufficient quality for reverse transcription polymerase chain reaction analysis was extracted from the fecal occult blood test residuum stored at 4°C for up to 5 days.

Special Issue: Evaluation of the Antitumor Mechanism of Armed Antibodies.

This Special Issue focuses on the use of therapeutic antibodies in vitro, in vivo, and in clinical studies [...].

p70S6K/Akt dual inhibitor DIACC3010 is efficacious in preclinical models of gastric cancer alone and in combination with trastuzumab.

The PI3K-Akt-mTOR (PAM) pathway is implicated in tumor progression in many tumor types, including metastatic gastric cancer (GC). The initial promise of PAM inhibitors has been unrealized in the clinic, presumably due, in part, to the up-regulation of Akt signaling that occurs when the pathway is inhibited. Here we present that DIACC3010 (formerly M2698), an inhibitor of two nodes in the PAM pathway, p70S6K and Akt 1/3, blocks the pathway in in vitro and in vivo preclinical models of GC while providing a mechanism that inhibits signaling from subsequent Akt up-regulation. Utilizing GC cell lines and xenograft models, we identified potential markers of DIACC3010-sensitivity in Her2-negative tumors, i.e., PIK3CA mutations, low basal pERK, and a group of differentially expressed genes (DEGs). The combination of DIACC3010 and trastuzumab was evaluated in Her2-positive cell lines and models. Potential biomarkers for the synergistic efficacy of the combination of DIACC3010 + trastuzumab also included DEGs as well as a lack of up-regulation of pERK. Of 27 GC patient-derived xenograft (PDX) models tested in BALB/c nu/nu mice, 59% were sensitive to DIACC3010 + trastuzumab. Of the 21 HER2-negative PDX models, DIACC3010 significantly inhibited the growth of 38%. Altogether, these results provide a path forward to validate the potential biomarkers of DIACC3010 sensitivity in GC and support clinical evaluation of DIACC3010 monotherapy and combination with trastuzumab in patients with HER2- negative and positive advanced GCs, respectively.

Evaluation of Fluorescence Intensity and Antitumor Effect Using Real-Time Imaging in Photoimmunotherapy.

Photoimmunotherapy (PIT) is a promising tumor-selective treatment method that uses light-absorbing dye-conjugated antibodies and light irradiation. It has been reported that IR700 fluorescence changes with light irradiation. The purpose of this study was to investigate the fluorescence intensity and antitumor effect of PIT using real-time fluorescence observation of tumors and predict the required irradiation dose. The near-infrared camera system LIGHTVISION was used to image IR700 during PIT treatment. IR700 showed a sharp decrease in fluorescence intensity in the early stage of treatment and almost reached a plateau at an irradiation dose of 40 J/cm. Cetuximab-PIT for A431 xenografts was performed at multiple doses from 0-100 J/cm. A significant antitumor effect was observed at 40 J/cm compared to no irradiation, and there was no significant difference between 40 J/cm and 100 J/cm. These results suggest that the rate of decay of the tumor fluorescence intensity correlates with the antitumor effect by real-time fluorescence imaging during PIT. In addition, when the fluorescence intensity of the tumor plateaued in real-time fluorescence imaging, it was assumed that the laser dose was necessary for treatment.

Novel virtual cytological analysis for the detection of endometrial cancer cells using autoscan fluoromicroscopy.

The current medical examinations for detecting endometrial cancer can sometimes be stressful and inconvenient for examinees and examiners. Therefore, we attempted to develop an autoscan-virtual cytology system for detecting endometrial cancer without relying on judgment by the human eye. Exfoliated cells from the uterus were retrieved using a tampon inserted for 3 h. More than 100 monoclonal antibodies (mAb) developed by us were screened in three steps of immunohistochemistry to find mAb sets that would enable the cancer and normal endometrium to be perfectly distinguished. The exfoliated cells provided by 30 endometrial cancer patients and a total of 37 samples of 14 non-malignant volunteers including the menstrual cycle were analyzed using imaging cytometry. All samples contained epithelial cells and dysplasia cells, but the pathologist could not definitively diagnose all of them as endometrial cancer cells because most cells had degenerated. Twenty-two of 28 endometrial cancer tissues (79%) were positive with four mAb sets, CRELD1, GRK5, SLC25A27 and STC2, and 22 of 22 normal endometriums (100%) were negative. Our newly developed autoscan-virtual cytology for exfoliated endometrial cells showed overall sensitivity for endometrial cancer patients and overall specificity for volunteers of 50% (15/30) and 95% (35/37), respectively. Our autoscan-virtual cytology combined with cancer-specific mAb and imaging cytometry could be useful for endometrial cancer detection. Autoscan-virtual cytology for endometrial cancer deserves further evaluation for future endometrial cancer screening.

Antitumor effect of humanized anti­tissue factor antibody­drug conjugate in a model of peritoneal disseminated pancreatic cancer.

Tissue factor (TF) is an attractive target for cancer therapy due to its overexpression in multiple types of malignancies. In addition, TF has been reported to play functional roles in both cancer development and metastasis. Several groups have already developed antibody­drug conjugates (ADCs) against TF for use as cancer treatments, and have demonstrated their efficacies in conventional subcutaneous xenograft models and patient­derived xenograft models. However, no previous studies have investigated the effectiveness of anti­TF ADC in an advanced­stage cancer model. The present study developed an original humanized anti­TF monoclonal antibody conjugated with monomethyl auristatin E, and evaluated its in vivo efficacy in a pancreatic cancer xenograft model with peritoneal dissemination. In vitro assays demonstrated that the anti­TF ADC had potent binding affinity and cytotoxic activity against human pancreatic cancer cells that strongly expressed TF antigens. The anti­TF ADC also exhibited greater antitumor effect than that of a control ADC in conventional subcutaneous xenograft models, with efficacy depending on the TF expression in the tumor tissues. Furthermore, the anti­TF ADC significantly inhibited tumor growth in an orthotopic xenograft model, and extended the survival period in a murine peritoneal dissemination model. These results indicated that anti­TF ADC has the potential to be an effective treatment not only for primary tumors, but also for those that are widely disseminated. Therefore, it can be concluded that ADC targeting TF may be a promising agent for advanced pancreatic cancer therapy.

Stabilization of an 211At-Labeled Antibody with Sodium Ascorbate.

211At, an α-particle emitter, has recently attracted attention for radioimmunotherapy of intractable cancers. However, our sodium dodecyl sulfate polyacrylamide gel electrophoresis and flow cytometry analyses revealed that 211At-labeled immunoconjugates are easily disrupted. Luminol assay revealed that reactive oxygen species generated from radiolysis of water caused the disruption of 211At-labeled immunoconjugates. To retain their functions, we explored methods to protect 211At-immunoconjugates from oxidation and enhance their stability. Among several other reducing agents, sodium ascorbate most safely and successfully protected 211At-labeled trastuzumab from oxidative stress and retained the stability of the 211At-labeled antibody and its cytotoxicity against antigen-expressing cells for several days.