Ryo Tsumura

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.

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.

Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

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.

Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

The present state of therapy for colorectal cancer (CRC) is far from satisfactory, highlighting the need for new targets for this disease. We identified a new colorectal cancer (CRC)-specific molecule, TMEM180, a predicted eleven-pass transmembrane protein that apparently functions as a cation symporter. Our anti-TMEM180 monoclonal antibody (mAb) eradicated SW480 CRC xenografts in mice. The TMEM180 promoter region contains ten hypoxia-responsive element consensus sequences; accordingly SW480 cells upregulated TMEM180 under low-oxygen conditions. TMEM180 expression in SW480 cells was positively correlated with anchorage-independent colony formation and tumourigenesis. TMEM180-positive SW480 cells resided at the tumour–stroma interface manifested by αSMA-positive fibroblasts, also known as the tumour niche. Some clusters of TMEM180-positive cells adjacent to the niche were integrin α6-positive. These data indicate that TMEM180 represents a possible cancer stem cell marker and that a mAb against this protein could be used as antibody-based therapeutic against CRC.

Influence of the dissociation rate constant on the intra-tumor distribution of antibody-drug conjugate against tissue factor

ABSTRACT Antibody‐drug conjugates (ADCs) are currently considered to be promising agents for cancer therapy. However, especially in solid tumors, the uneven distribution of ADCs would decrease their efficacy in clinical studies. We suggest that in addition to optimizing ADC components, such as the linker structure and anticancer agent, it is necessary to consider the distribution of the ADC within tumor tissue. In this study, we established three kinds of anti–tissue factor (TF) ADCs: 1849ADC with a low kd, 444ADC with an intermediate kd, and 1084ADC with a high kd. All three of the anti‐TF ADCs exhibited almost the same in vitro cytotoxicity and pharmacological and biochemical characteristics, although the binding kinetics parameters differed. In vivo, all ADCs exerted equivalent antitumor effects against small BxPC3 tumors. However, on larger BxPC3 tumors, 1084ADC (higher kd) exerted higher antitumor activity than 1849ADC (lower kd). Furthermore, immunofluorescence staining indicated that 1084ADC was distributed throughout the whole tumor, whereas 1849ADC was mainly localized close to tumor vessels. We conclude that the ADC with a higher kd increased the antitumor effect of because it penetrated and distributed evenly throughout the entire solid tumor. These findings highlight the importance of the kd of a mAb in ADC design. Graphical abstract Figure. No caption available.

Abstract 2642: Antibody-drug conjugate for human pancreatic cancer cells using anti-tissue factor monoclonal antibody

Background: The incidence of venous thromboembolism in patients with cancer is reported to be twofold higher than that in patients without cancer. One of the well-characterized procoagulants associated with cancer is tissue factor (TF). TF is expressed not only on the surfaces of the cancer cells, but also in the monocytes and endothelial cells which are stimulated under the cancerous condition. Thus, TF is an appropriate molecule for drug delivery to both cancer cells and cancer stroma. In the present study, the antitumor effect of an antibody drug conjugate (ADC), anti-TF monoclonal antibody (mAb) conjugated with monomethyl auristatin E (MMAE), was investigated. Methods: MMAE was conjugated to anti-human TF (hTF) and -mouse TF (mTF) antibody using a valine-citrulline dipeptide linker. The stability of ADC under the physiological and intracellular conditions were analyzed by HPLC. The bio-distributions of hTF and mTF mAbs were analyzed in vivo. The cytotoxic effects of the ADC in four pancreatic cancer cell lines were analyzed both in vitro and in vivo. Results: The four pancreatic cancer cell lines used in this study were classified into 3 groups; high-TF-expressing cells (BxPC-3), moderate-TF-expressing cells (PSN-1), and low-TF-expressing cells (Capan-1 and Panc-1). Anti-hTF mAb was sufficiently internalized in the cytoplasm of BxPC-3 after 3-h incubation. The ADC was stable under the extracellular condition, and sufficient free MMAE was released into the cytoplasm. hTF mAb permeating from the tumor vessels was distributed at the periphery of the tumors. On the other hand, mTF mAb was distributed in the cancer stroma. The IC50 of MMAE for these pancreatic cancer cell lines was approximately 1 nM, while the IC50 of the MMAE contained in hTF-MMAE for BxPC-3 was 1.15 nM; on the other hand the values for Capan-1 and Panc-1 exceeded 100 nM. BxPC-3 subcutaneous tumor growth was significantly suppressed in the 20 mg/kg hTF-MMAE group as compared with that in the saline group (P Conclusions: ADC measuring 10 nm in size could reach cancer cells through the cancer stroma and could be effectively internalized into cancer cells. Meanwhile, cancer-stroma-targeting mAb (mTF mAb) elicit a partial response as compared to hTF mAb. Our anti-TF mAb conjugated with MMAE warrants clinical evaluation. Citation Format: Yoshikatsu Koga, Ryuta Sato, Ryo Tsumura, Hikaru Machida, Yoshiyuki Yamamoto, Yohei Hisada, Yuki Fujiwara, Masahiro Yasunaga, Shino Manabe, Yasuhiro Matsumura. Antibody-drug conjugate for human pancreatic cancer cells using anti-tissue factor monoclonal antibody. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2642. doi:10.1158/1538-7445.AM2014-2642

Abstract 2648: Enhanced antitumor effect of anti tissue factor (TF) antibody-conjugated epirubicin-incorporating polymeric micelles in human cancer xenografts with high TF expression

Background: Anticancer agent-incorporating polymeric micelles, categorized as passive targeting agents, are expected to accumulate in tumors based on the enhanced permeability and retention effect. Recently, we made epirubicin-incorporating micelles containing acid-sensitive bonds (NC-6300), and reported the enhanced antitumor activity and reduced cardiotoxicity in preclinical studies; a Phase I study of NC-6300 is also under way in Japan. In order to further enhance the accumulation of NC-6300 in tumors using the active targeting strategy, we tried to attach a tumor-specific monoclonal antibody (mAb) to the terminal of polyethylene glycol comprising NC-6300. As the targeting molecule, we selected transmembrane receptor tissue factor (TF), namely, the primary initiator of coagulation. TF, which is known to play an important role in not only blood coagulation, but also in tumor growth and metastasis, is frequently overexpressed in various types of tumor. We have established a mAb against human TF, and succeeded in developing anti TF mAb-conjugated NC-6300 (anti TF-NC-6300). Here, we report the results of in vitro and in vivo studies of this immunoconjugate. Methods: Anti TF-NC-6300 was prepared based on our Antibody/Drug-Conjugated Micelle (ADCM) technology (Japan Patent No.4538666) with slight modification. In the in vitro assay, real-time cell analysis was performed using the xCELLigence system to determine the effects of anti TF-NC-6300 and NC-6300 on pancreatic tumor cell proliferation. Next, image analysis and quantification of the intracellular epirubicin concentration were performed using Array Scan VTI. For evaluating the in vivo antitumor effects, anti TF-NC-6300 (10 mg/kg), NC-6300 (10 mg/kg) and epirubicin (10 mg/kg) were administered intravenously once a week for 3 weeks to mice bearing human pancreatic cancer xenografts or human gastric cancer xenografts with high TF expression (BxPC3 or 44As3). Results: In the real-time cell proliferation assay, anti TF-NC-6300 exerted a greater degree of inhibition of cell growth than NC-6300 in BxPC3. On the other hand, there was no difference in effect between these two drugs in SUIT2, a human pancreatic cancer with low TF expression. In the imaging cytometric analysis, anti TF-NC-6300 was internalized sooner than NC-6300, resulting in higher accumulation of epirubicin in the cytoplasm and nuclei of the BxPC3 cells. On the other hand, no such difference was observed in the case of SUIT2. In the in vivo antitumor assay, as compared to both epirubicin and NC-6300, anti TF-NC-6300 showed significant antitumor activity in xenograft models of BxPC3 and 44As3. Conclusion: These data warrant a clinical evaluation of anti TF mAb-conjugated epirubicin-incorporating polymeric micelles in patients with TF-positive cancer subtypes. Citation Format: Yoshiyuki Yamamoto, Ichinosuke Hyodo, Yoshikatsu Koga, Ryo Tsumura, Ryuta Sato, Toshihumi Obonai, Hirobumi Fuchigami, Masahisa Kudo, Masahiro Yasunaga, Mitsunori Harada, Tatsuyuki Hayashi, Yasuki Kato, Yasuhiro Matsumura. Enhanced antitumor effect of anti tissue factor (TF) antibody-conjugated epirubicin-incorporating polymeric micelles in human cancer xenografts with high TF expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2648. doi:10.1158/1538-7445.AM2014-2648