Iwao Ohizumi

Identification of Soluble NH2-Terminal Fragment of Glypican-3 as a Serological Marker for Early-Stage Hepatocellular Carcinoma

For detection of hepatocellular carcinoma (HCC) in patients with liver cirrhosis, serum α-fetoprotein has been widely used, but its sensitivity has not been satisfactory, especially in small, well-differentiated HCC, and complementary serum marker has been clinically required. Glypican-3 (GPC3), a heparan sulfate proteoglycan anchored to the plasma membrane, is a good candidate marker of HCC because it is an oncofetal protein overexpressed in HCC at both the mRNA and protein levels. In this study, we demonstrated that its NH2-terminal portion [soluble GPC3 (sGPC3)] is cleaved between Arg358 and Ser359 of GPC3 and that sGPC3 can be specifically detected in the sera of patients with HCC. Serum levels of sGPC3 were 4.84 ± 8.91 ng/ml in HCC, significantly higher than the levels seen in liver cirrhosis (1.09 ± 0.74 ng/ml; P < 0.01) and healthy controls (0.65 ± 0.32 ng/ml; P < 0.001). In well- or moderately-differentiated HCC, sGPC3 was superior to α-fetoprotein in sensitivity, and a combination measurement of both markers improved overall sensitivity from 50% to 72%. These results indicate that sGPC3 is a novel serological marker essential for the early detection of HCC.

Anti–Glypican 3 Antibody as a Potential Antitumor Agent for Human Liver Cancer

Human glypican 3 (GPC3) is preferentially expressed in the tumor tissues of liver cancer patients. In this study, we obtained a monoclonal antibody (mAb) against the COOH-terminal part of GPC3, which induced antibody-dependent cellular cytotoxicity (ADCC). The mAb, designated GC33, exhibited marked tumor growth inhibition of s.c. transplanted Hep G2 and HuH-7 xenografts that expressed GPC3 but did not inhibit growth of the SK-HEP-1 that was negative for GPC3. GC33 was efficacious even in an orthotopic model; it markedly reduced the blood alpha-fetoprotein levels of mice intrahepatically transplanted with Hep G2 cells. Humanized GC33 (hGC33) was as efficacious as GC33 against the Hep G2 xenograft, but hGC33 lacking carbohydrate moieties caused neither ADCC nor tumor growth inhibition. Depletion of CD56+ cells from human peripheral blood mononuclear cells markedly abrogated the ADCC caused by hGC33. The results show that the antitumor activity of hGC33 is mainly attributable to ADCC, and in human, natural killer cell-mediated ADCC is one possible mechanism of the antitumor effects by GC33. hGC33 will provide a novel treatment option for liver cancer patients with GPC3-positive tumors.

Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells

Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.

Association of CD44 with OTS-8 in tumor vascular endothelial cells

Endothelial cells in solid tumors play an important role in tumor growth, invasion and metastasis through angiogenesis. We have recently cloned two tumor vascular antigens from isolated rat tumor vascular endothelial cells (TEC). One is CD44, a family of cell surface proteins implicated in adhesion interactions and tumor metastasis. The other is OTS-8, a marker for osteoblast into osteocyte transition and type I alveolar epithelial cells termed as E11 antigen and RTI40, respectively. To test for a possible interaction between the two antigens on endothelial cells in tumor angiogenesis, we examined in vivo association of CD44 with OTS-8 using lysates of isolated rat TEC and COS-7 cells cotransfected with CD44 and OTS-8 expression plasmids. The association was detected by direct co-immunoprecipitation of the two types of cells lysed with digitonin, whereas the detection was lost when lysed with Nonidet P-40. To confirm this association, intact COS-7 cells cotransfected were reacted with homobifunctional N-hydroxysuccinimide ester crosslinking reagents. Immunoblot analysis showed a crosslinked CD44/OTS-8 protein complex of 120 kDa, suggesting the proximity of the two proteins. These findings provide evidence of a weak physical association between CD44 and OTS-8 in TEC, and suggest that OTS-8 may alter the mode of endothelial cell growth and/or migration induced by CD44 in tumor angiogenesis.

A new disulfide-linked dimer of a single-chain antibody fragment against human CD47 induces apoptosis in lymphoid malignant cells via the hypoxia inducible factor-1α pathway

CD47 belongs to the immunoglobulin superfamily and is associated with β‐integrins. Recently it was reported that CD47 ligation rapidly induces apoptosis in B‐chronic lymphocytic leukemia (CLL) cells. Chronic lymphocytic leukemia is still an incurable hematological malignancy even with the novel therapeutic agents; therefore, new and effective agents for the treatment of CLL in clinical settings are urgently needed. We generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Subsequently, we created a disulfide‐stabilized dimer of a single‐chain antibody fragment of MABL (S‐S diabody) to get rid of the adverse effect of MABL such as hemagglutination. In this study, we analyzed the effects of this new antibody on cellular proliferation, and the molecular mechanism of CD47‐mediated apoptosis in human lymphoid malignant cells. Treatment with S‐S diabody alone induced apoptosis of CD47‐positive primary B‐CLL and leukemic cells (MOLT‐4 and JOK‐1). In addition, administration of S‐S diabody significantly prolonged the survival of severe combined immunodeficiency (SCID) mice inoculated with JOK‐1 cells. In gene expression profiling of the S‐S diabody‐treated MOLT‐4 cells, hypoxia inducible factor (HIF)‐1α downstream genes (RTP801 and BNIP3) were upregulated, and the mRNA expression levels of HIF‐1α, RTP801 and BNIP3 were increased. Knockdown of HIF‐1α by siRNA repressed S‐S diabody‐induced apoptosis in MOLT4 cells. In conclusion, CD47 will be a molecular target for the treatment of lymphoid malignancies, and S‐S diabody might have potential as a novel therapeutic agent for B‐CLL. (Cancer Sci 2011; 102: 1208–1215)

Effective Cancer Targeting Using an Anti‐tumor Tissue Vascular Endotheliumspecific Monoclonal Antibody (TES‐23)

Immunoconjugate targeting of solid tumors has not been routinely successful because the endothelial cells of blood vessels act as a physical barrier against the transport of macromolecules, such as antibodies. In the present study, we attempted to achieve tumor vascular targeting with an antitumor tissue endothelium‐specific monoclonal antibody (TES‐23). TES‐23, an IgG1 monoclonal antibody raised against rat KMT‐17 fibrosarcoma‐derived endothelial cells, was covalently conjugated with neocarzinostatin (NCS) in a previous study. The TES‐23‐NCS conjugate induced tumor hemorrhagic necrosis, and showed marked anti‐tumor effects against rat KMT‐17 fibrosarcoma. This result prompted us to investigate whether this approach would be applicable to various other types of solid tumors. One hour after injection of 125I‐labeled TES‐23 into BALB/c mice bearing Meth‐A fibrosarcoma and Colon 26 adenocarcinoma, the tumor accumulation of TES‐23 was greater than that of the control IgG. In the present study, we report the anti‐tumor effects of this monoclonal antibody in mice bearing Meth‐A fibrosarcoma. Mice treated with the immunoconjugate showed improved survival with no side effects. This result indicates that common antigens may be found in different kinds of tumor endothelial cells, and that TES‐23 might recognize these antigens.

Therapy of rat tracheal carcinoma IC-12 in SCID mice: vascular targeting with [213Bi]-MAb TES-23.

In previous work, we have demonstrated that vascular targeting of [213Bi], an alpha-emitter, to lung blood vessels could efficiently destroy tumour colonies growing in the lung. In order to expand this approach to treatment of tumours growing in other sites, we employed the monoclonal antibody (MAb) TES-23, which reacts with CD44H, preferentially expressed on new blood vessels in tumours. Biodistribution studies of N-succinimidyl [125I] 3-iodobenzoate (SIB)-radiolabelled MAb TES-23 in ICR-severe combined immunodeficient (SCID) mice bearing subcutaneous (s.c.) and intramuscular (i.m.) IC-12 tumours, demonstrated efficient tumour uptake. At 24 h, accumulation in small tumours was 45%ID/g for s.c. tumours, and 58%ID/g for i.m. tumours and in large tumours it was 25%ID/g for s.c. tumours and 17%ID/g for i.m. tumours. Micro-autoradiography data confirmed that radiolabel accumulated in or near tumour blood vessels. Normal tissues had very low levels of radioactivity. Treatment of mice bearing small IC-12 tumours with [213Bi] MAb TES-23 retarded tumour growth relative to animals treated with cold MAb TES-23. Biodistribution and therapy experiments were also performed in BALB/c mice bearing both s.c. and i.m. syngeneic, lung carcinoma (line 498) tumours. [I(125)] SIB MAb TES-23 accumulated efficiently in both s.c. and i.m. tumours (14%ID/g and 15%ID/g, respectively, at 4 h); however, no therapeutic effect of [213Bi] MAb TES-23 treatment could be demonstrated in this model system. The data demonstrate that the timing of vascularisation of the tumours and the delivery kinetics of MAb relative to the half-life of the therapeutic radionuclide are critical for effective therapy.

IDENTIFICATION OF TUMOR VASCULAR ANTIGENS BY MONOCLONAL ANTIBODIES PREPARED FROM RAT-TUMOR-DERIVED ENDOTHELIAL CELLS

We have reported the isolation and specific in vitro properties of tumor‐derived endothelial cells (TEC) from rat KMT‐17 fibrosarcomas transplanted into rats. To develop antibody‐based tumor vascular targeting therapy for solid tumors, we have generated monoclonal antibodies (MAbs) using passive immunization of outside‐out membrane vesicles of rat epididymal‐fat‐pad‐derived capillary endothelial cells (FCEC) followed by active immunization of those of rat TEC. The MAbs produced were screened against TEC and FCEC. Of all cultured hybridomas, 75 (3.3%) of the secreted MAbs preferentially recognized TEC. We selected a total of 7 MAbs which detected antigens highly abundant in TEC, although 5 of the 7 MAbs were weakly positive for FCEC in cell‐ELISA and FACS analyses. The antigens recognized by these MAbs, with the exception of MAb TES‐7, were present on endothelial cells of tumor blood vessels in KMT‐17 fibrosarcoma tissues, as shown by immunohistochemical analysis. Antigens of 40‐ and 80‐kDa were recognized by MAbs TES‐1, 7, 17, 21 and 26 and by MAbs TES‐23 and 27 respectively. Although the function of these antigens, which are preferentially expressed on rat tumor‐derived endothelial cells, is still unknown, we believe that future studies of such antigens will help elucidate the role of endothelial cells in tumor vasculature. Our results indicate that MAbs may provide a novel tool for the development of antibody‐based therapy targeting tumor vasculature. Int. J. Cancer 77:561–566, 1998.

Molecular cloning and characterization of antigens expressed on rat tumor vascular endothelial cells.

We have previously prepared monoclonal antibodies (MAbs) against tumor vasculature using cultured rat tumor endothelial cells (TECs) isolated from solid KMT‐17 tumors and identified 40 and 80 kDa antigens recognized by TES‐17 and TES‐23 MAbs, respectively. To clarify the nature of antigens on tumor vasculature, molecular cloning was conducted by screening the rat TEC cDNA library. Two antigens were detected: a 40 kDa OTS‐8 antigen, which had been defined as a differentiation marker for osteoblastic lineage, and an 80 kDa CD44H antigen. Northern blots showed that OTS‐8 mRNA was expressed exclusively in the lung, in addition to TECs, while CD44H mRNA was detected in the lung, intestine, spleen, thymus and peripheral blood cells, in addition to TECs. Immuno‐histochemistry of KMT‐17 tumors revealed that OTS‐8 and CD44 were expressed on sprouting TECs. In addition, TES‐23 MAb stained TECs of tubular vessels as well as sprouting TECs, but anti‐rat CD44 MAbs stained only sprouting TECs. PCR showed that CD44 cDNA with a splice in exon 6 (CD44ex6) was present in rat TECs at low levels. Our results indicate that OTS‐8 and CD44 are expressed on rat sprouting TECs and that TES‐23 MAb recognizes CD44H on sprouting TECs as well as an unknown epitope on TECs of tubular vessels that could not be recognized by anti‐rat CD44 MAbs. Int. J. Cancer 86:799–805, 2000.

Suppression of solid tumor growth by a monoclonal antibody against tumor vasculature in rats : Involvement of intravascular thrombosis and fibrinogenesis

We have reported that immunization of rat tumor‐derived endothelial cells (TEC) isolated from KMT‐17 solid tumors results in the generation of several monoclonal antibodies (MAbs). TES‐23, one of these MAbs, recognizes a naturally occurring 80‐kDa antigen expressed on endothelial cells of tumor blood vessels. To determine whether such MAbs can suppress solid tumor growth in vivo by impairment of endothelial cells in tumors following direct binding, we tested the biodistribution of 125I‐labeled TES‐23 in rats bearing KMT‐17 solid tumors. We also examined the effect of treatment using unconjugated TES‐23 on tumor growth and histo‐pathological changes in tumor tissues. Biodistribution studies showed localization of TES‐23 into tumor tissues 60 min after intravenous injection. TES‐23 suppressed significantly the growth of KMT‐17 solid tumors following administration for 5 days. Histo‐pathological examination showed that TES‐23 caused degeneration, apoptosis and/or necrosis and denudation of endothelial cells in viable tumor areas following local aggregation and adhesion of lymphocytes, with subsequent intravascular thrombus formation by platelets and fibrin. Our results indicate that TES‐23, which recognizes TEC, can target endothelial cells of solid tumor vasculature directly, resulting in growth suppression in vivo by reduction of blood flow due to intravascular thrombosis. Our results also suggest that targeting tumor vasculature is a potentially attractive approach for the treatment of solid tumors. Int. J. Cancer 82:853–859, 1999.