Facilitation of endoglin-targeting cancer therapy by development/utilization of a novel genetically engineered mouse model expressing humanized endoglin (CD105).

Endoglin (ENG) is a TGF-ß coreceptor and essential for vascular development and angiogenesis. A chimeric antihuman ENG (hENG) monoclonal antibody (mAb) c-SN6j (also known as TRC105) shows promising safety and clinical efficacy features in multiple clinical trials of patients with various advanced solid tumors. Here we developed a novel genetically engineered mouse model to optimize the ENG-targeting clinical trials. We designed a new targeting vector that contains exons 4-8 of hENG gene to generate novel genetically engineered mice (GEMs) expressing functional human/mouse chimeric (humanized) ENG with desired epitopes. Genotyping of the generated mice confirmed that we generated the desired GEMs. Immunohistochemical analysis demonstrated that humanized ENG protein of the GEMs expresses epitopes defined by 7 of our 8 anti-hENG mAbs tested. Surprisingly the homozygous GEMs develop normally and are healthy. Established breast and colon tumors as well as metastasis and tumor microvessels in the GEMs were effectively suppressed by systemic administration of anti-hENG mAbs. Additionally, test result indicates that synergistic potentiation of antitumor efficacy can be induced by simultaneous targeting of two distinct epitopes by anti-hENG mAbs. Sorafenib and capecitabine also showed antitumor efficacy in the GEMs. The presented novel GEMs are the first GEMs that express the targetable humanized ENG. Test results indicate utility of the GEMs for the clinically relevant studies. Additionally, we generated GEMs expressing a different humanized ENG containing exons 5-6 of hENG gene, and the homozygous GEMs develop normally and are healthy.

[Splenomegaly Associated with FOLFOX Therapy].

BACKGROUND: Oxaliplatin is one of the key drugs for the treatment of colorectal cancer, although it is known to cause hepatic sinusoidal injury. METHOD: Thirty-one patients underwent modified FOLFOX6 therapy from April 2006 and December 2009 at our hospital. Four patients were excluded from this study because they had too many intervals in their therapeutic courses. The size of the spleen and the blood platelet count were analyzed before and after 6 months of FOLFOX therapy. The spleen was measured using CT scan, and the splenic index(SI=length×width×height)was evaluated. RESULTS: After 6 months of treatment, the mean SI was increased from 229 cm3 to 323 cm3(p<0. 01). At the same time, the mean platelet count was decreased from 26. 9 ×10 / / 4 mm3 to 17. 1×104 mm3(p<0. 01). A negative correlation was found between the ratio of SI and the platelet count after 6 months of treatment to baseline(r=-0. 42, p=0. 030). SI increased by>50% in 12 patients (44. 4%). The platelet count decreased more severely in patients whose SI increased by>50%(p=0. 028). CONCLUSIONS: Splenomegaly and thrombocytopenia were observed in patients who underwent FOLFOX therapy. These are candidate parameters for evaluating hepatic sinusoidal injury.

Anti-endoglin monoclonal antibodies are effective for suppressing metastasis and the primary tumors by targeting tumor vasculature.

Anti-metastatic activity of an antitumor agent is exceedingly important because metastasis is the primary cause of death for most solid cancer patients. In this report, we show that 3 anti-endoglin (ENG) monoclonal antibodies (mAbs) SN6a, SN6j and SN6k which define individually distinct epitopes of ENG of tumor vasculature are capable of suppressing tumor metastases in the multiple metastasis models. The metastasis models were generated by i.v., s.c. (into flank) or mammary gland fat pad injection of 4T1 murine mammary carcinoma cells and splenic injection of two types of colon26 murine colorectal carcinoma cells. Individual mAbs were injected i.v. via the tail vein of mice. SN6a and SN6j effectively suppressed the formation of metastatic colonies of 4T1 in the lung in all of the three 4T1 metastatic models. In addition, these mAbs were effective for suppressing the primary tumors of 4T1 in the skin and mammary fat pad. These mAbs effectively suppressed microvessel density and angiogenesis in tumors as measured by the Matrigel plug assay in mice. No significant side effects of the administered mAbs were detected. Furthermore, SN6a and SN6j extended survival of the tumor-bearing mice. SN6j, SN6k and their immunoconjugates with deglycosylated ricin A-chain were all effective for suppressing hepatic metastasis of colon26. The findings in the present study are clinically relevant in view of the ongoing clinical trial of a humanized (chimerized) form of SN6j.

Anti-tumor activity of an anti-endoglin monoclonal antibody is enhanced in immunocompetent mice.

In the present study, we investigated the mechanisms by which anti-endoglin (EDG; CD105) monoclonal antibodies (mAbs) suppress angiogenesis and tumor growth. Antihuman EDG mAb SN6j specifically bound to murine endothelial cells and was internalized into the cells in vitro. SN6j effectively suppressed angiogenesis in mice in the Matrigel plug assay. We found that SN6j is more effective for tumor suppression in immunocompetent mice than in SCID mice. We hypothesized that T cell immunity is important for effective antitumor efficacy of SN6j in vivo. To test this hypothesis, we investigated effects of CpG oligodeoxynucleotides (ODN) and depletion of CD4(+) T cells and/or CD8(+) T cells on antitumor efficacy of SN6j in mice. Systemic (i.v.) administration of a relatively small dose (0.6 mug/g body weight/dose) of SN6j suppressed growth of established s.c. tumors of colon-26 in BALB/c mice and improved survival of the tumor-bearing mice. Addition of CpG ODN to SN6j synergistically enhanced antitumor efficacy of SN6j. In contrast, such enhancing effects of CpG ODN were not detected in SCID mice. Antitumor efficacy of SN6j in BALB/c mice was abrogated when CD4(+) T cells and/or CD8(+) T cells were depleted; effect of CD8(+) T cell depletion was stronger. Interestingly, CD4-depletion decreased tumor growth while CD8-depletion enhanced tumor growth in the absence of SN6j. SN6j induced apoptosis in human umbilical vein endothelial cells in a dose-dependent manner which indicates an additional mechanism of antiangiogenesis by SN6j. (c) 2008 Wiley-Liss, Inc.

Preoperative evaluation of hepatic functional reserve by converted ICGR15 calculated from Tc-GSA scintigraphy.

BACKGROUND AND AIM: Conversion of data from technetium 99 m diethylenetriaminepentaacetic acid galactosyl human serum albumin (99mTc-GSA) scintigraphy to ICGR15 (indocyanin green retention at 15 min) is an easy and convenient method for obtaining parameters to determine the appropriate and safe extent of liver resection. We investigated a conversion method which also accounts for LHL15 (receptor index: uptake ratio of the liver to the liver plus heart at 15 min) and HH15 (blood clearance index: uptake ratio of the heart at 15 min to that at 3 min) characteristics. METHODS: Cases included 282 patients undergoing hepatic resection following 99mTc-GSA scintigraphy and an ICG tolerance test. Degree of liver dysfunction was classified as A, B, or C according to criteria of the Liver Cancer Study Group of Japan. RESULTS: HH15 demonstrated a larger distribution in patients with liver damage A, while LHL15 demonstrated a larger distribution in patients with liver damage B. In liver damage A, the conversion formula ICGR15 = 87.0-79.6 x LHL15 was obtained, and in liver damage B, the conversion formula ICGR15 = -23.3 + 72.4 x HH15 was obtained, and correlation with ICGR15 was higher (r = 0.61, P < 0.0001) than when the data were not segregated by liver damage severity. Furthermore, postoperative hyperbilirubinemia significantly occurred in cases where both ICGR15 and converted ICGR15 were high. CONCLUSIONS: Conversion models based on data segregated by severity of liver damage were more closely correlated with ICGR15 than conversion models not based on segregated data. By using this converted ICGR15, preoperative estimation of hepatic functional reserve can become more reliable.

She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast, Saccharomyces cerevisiae.

She4p/Dim1p, a member of the UNC-45/CRO1/She4p (UCS) domain-containing protein family, is required for endocytosis, polarization of actin cytoskeleton, and polarization of ASH1 mRNA in Saccharomyces cerevisiae. We show herein that She4p/Dim1p is involved in endocytosis and actin polarization through interactions with the type I myosins Myo3p and Myo5p. Two-hybrid and biochemical experiments showed that She4p/Dim1p interacts with the motor domain of Myo3/5p through its UCS domain. She4p/Dim1p was required for Myo5p localization to cortical patch-like structures. Using random mutagenesis of the motor region of MYO5, we identified four independent dominant point mutations that suppress the temperature-sensitive growth phenotype of the she4/dim1 null mutant. All of the amino acid substitutions caused by these mutations, V164I, N168I, N209S, and K377M, could suppress the defects of endocytosis and actin polarization of the she4/dim1 mutant as well. She4p/Dim1p also showed two-hybrid interactions with the motor domain of a type II myosin Myo1p and type V myosins Myo2p and Myo4p, and was required for proper localization of Myo4p, which regulates polarization of ASH1 mRNA. Our results suggest that She4p/Dim1p is required for structural integrity or regulation of the motor domain of unconventional myosins.

Percutaneous transhepatic gallbladder drainage followed by elective laparoscopic cholecystectomy in patients with moderate acute cholecystitis under antithrombotic therapy.

BACKGROUND: Standard treatment for acute cholecystitis (AC) in patients receiving antithrombotic drugs has not been established. We evaluated the safety of percutaneous transhepatic gallbladder drainage (PTGBD) followed by elective laparoscopic cholecystectomy (LC) in patients with moderate AC who were receiving antithrombotics. METHODS: Seventy-five patients received PTGBD from January 2006 to March 2013 followed by elective LC for moderate AC. Patients were divided into Group A, which consisted of patients receiving antithrombotic therapy (n = 23), and Group B, which included the remaining patients (n = 52). We analyzed clinical outcomes and perioperative complications between groups. RESULTS: No hemorrhagic events occurred during PTGBD insertion regardless of antithrombotic treatment. The open conversion rate was not significantly different between the two groups. Postoperative complications were found in 10 patients (13.3%). The rate of postoperative complications in Group A was slightly higher than that in Group B, but the difference was not significant (21.7% vs. 9.6%; P = 0.15). Complications associated with PTGBD occurred in six patients (8%). There were no significant differences in the incidence of these complications, operation time, intraoperative blood loss, or length of postoperative hospital stay. CONCLUSIONS: Percutaneous transhepatic gallbladder drainage followed by elective LC may be an effective therapeutic strategy for moderate AC in patients receiving antithrombotic therapy.

Hepatic sinusoidal obstruction associated with S-1 plus cisplatin chemotherapy for highly advanced gastric cancer with paraaortic lymph node metastases: report of a case.

A 56-year-old man who was diagnosed with gastric cancer with multiple paraaortic lymph node metastases was treated with S-1 plus cisplatin. The spleen gradually enlarged during the therapeutic courses. After the 6th course of therapy, the primary gastric lesion and paraaortic lymphadenopathies disappeared. He underwent a curative resection, including a distal gastrectomy with regional and paraaortic lymph node dissections. Irregularly distributed congestion of the liver was noted during the surgery. Histological examinations revealed residual cancer cells in 3 regional lymph nodes and no cancer cells in the primary site and paraaortic lymph nodes. Hepatic sinusoidal obstruction syndrome (SOS) was also confirmed histologically. This is the first report of a case with SOS after S-1 plus cisplatin therapy. S-1 plus cisplatin therapy can cause SOS, although it is a promising preoperative chemotherapy for highly advanced gastric cancer.

Endoglin-targeted cancer therapy.

Vascular-targeting antiangiogenic therapy (VTAT) of cancer can be advantageous over conventional tumor cell targeted cancer therapy if an appropriate target is found. Our hypothesis is that endoglin (ENG; CD105) is an excellent target in VTAT. ENG is selectively expressed on vascular and lymphatic endothelium in tumors. This allows us to target both tumor-associated vasculature and lymphatic vessels to suppress tumor growth and metastasis. ENG is essential for angiogenesis/vascular development and a co-receptor of TGF-ß. Our studies of selected anti-ENG monoclonal antibodies (mAbs) in several animal models and in vitro studies support our hypothesis. These mAbs and/or their immunoconjugates (immunotoxins and radioimmunoconjugates) induced regression of preformed tumors as well as inhibited formation of new tumors. In addition, they suppressed metastasis. Several mechanisms were involved in the suppressive activity of the naked (unconjugated) anti-ENG mAbs. These include direct growth suppression of proliferating endothelial cells, induction of apoptosis, ADCC (antibody-dependent cell-mediated cytotoxicity) and induction of T cell immunity. To facilitate clinical application, we generated a human/mouse chimeric anti-ENG mAb termed c-SN6j and performed studies of pharmacokinetics, toxicology and immunogenicity of c-SN6j in nonhuman primates. No significant toxicity was detected by several criteria and minimal immune response to the murine part of c-SN6j was detected after multiple i.v. injections. The results support our hypothesis that c-SN6j can be safely administered in cancer patients. This hypothesis is supported by the ongoing phase 1 clinical trial of c-SN6j (also known as TRC105) in patients with advanced or metastatic solid cancer in collaboration with Tracon Pharma and several oncologists (NCT00582985).

The upstream regulator, Rsr1p, and downstream effectors, Gic1p and Gic2p, of the Cdc42p small GTPase coordinately regulate initiation of budding in Saccharomyces cerevisiae.

BACKGROUND: Cdc42p, a Rho family small GTPase, is essential for budding initiation in the yeast Saccharomyces cerevisiae. The homologous proteins Gic1p and Gic2p (Gic1/2p) are effectors of Cdc42p, but their precise functions remain unknown. Rsr1p/Bud1p is a Ras family small GTPase that controls the selection of the budding site. Previous observations suggested that Rsr1p-GTP recruits Cdc24p, a GDP/GTP exchange factor for Cdc42p, at the incipient bud site. However, this model only addresses how Rsr1p determines the budding site, because the rsr1 mutant normally initiates budding. RESULTS: Here we show that a rsr1 gic1 gic2 mutant fails to initiate budding, resulting in unbudded, large, and multinucleated cells. Expression of a dominant active or dominant negative mutant of RSR1 also inhibited the growth of the gic1 gic2 mutant, suggesting that cycling of Rsr1p between the GTP- and GDP-bound forms is required for budding initiation in the gic1 gic2 mutant. Among the mutations in effectors of CDC42, only the gic1 gic2 mutation demonstrated a synthetic lethal interaction with rsr1. Increased gene dosage of CDC42 suppressed defects in budding initiation of rsr1 gic1 gic2 mutants containing additional mutations in other effectors of CDC42, including BNI1, CLA4 or STE20. The polarized localization of Bni1p-GFP (green fluorescent protein) and Cla4p-GFP was lost after depletion of Gic1p in the rsr1 gic2 mutant. CONCLUSION: We propose that Gic1/2p may stabilize or maintain a complex consisting of Cdc42p-GTP and its effectors at the budding site, which are assembled by the action of the Rsr1p-Cdc24p system.