Effective cancer targeting using an anti-tumor tissue vascular endothelium-specific monoclonal antibody (TES-23).

Immunoconjugate targeting of solid tumors has not been routinely successful because the endo-thelial 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 anti-tumor 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 (125)I-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.

Gonadotropins stimulate growth of MCF-7 human breast cancer cells by promoting intracellular conversion of adrenal androgens to estrogens.

Estrogen receptor (ER)-positive breast cancers initially respond well to estrogen ablation treatment but finally acquire refractoriness, the phenomenon that is a major clinical problem. Because some breast cancers synthesize estradiol (E(2)) and E(2) synthesis is regulated by gonadotropins in normal ovaries, and because circulating gonadotropins are elevated in postmenopausal women and during estrogen ablation treatment, we hypothesized that gonadotropins might modulate estrogen synthesis/metabolism in breast cancer tissue as well. To test this possibility, MCF-7 cells were treated with dehydroepiandrosterone (DHEA) or human chorionic gonadotropin (hCG; approximately LH), each alone or in combination. Cell growth (3-day treatment) was assayed by the MTT method and estrogen synthesis (24-hour treatment) was measured using the ERE-luciferase reporter system. First, MCF-7 cell growth was stimulated by DHEA in a concentration-dependent manner with a maximal effect at 10(-4) M. Although hCG alone did not have a significant proliferative effect, hCG significantly and dose dependently stimulated MCF-7 cell growth in the presence of a submaximal concentration of DHEA (10(-7 )M). This stimulatory effect of DHEA and hCG was blocked by a pure antiestrogen ICI182,780 and an aromatase inhibitor, arimidex. Using MCF-7 cells transfected with the ERE-luciferase reporter system, hCG treatment was shown to increase ERE-mediated transcription. These results indicate that MCF-7 cells intrinsically converted DHEA into E(2) upon hCG stimulation, then grew their own cells DHEA- and hCG-dependently. We conclude that gonadotropins can act on breast cancer cells and accelerate conversion of DHEA into estrogens, thereby stimulating growth of estrogen-dependent tumor cells. This phenomenon, at least in part, could explain: (1) an increased tissue concentration of E(2) in postmenopausal breast cancer; (2) acquisition of hormone refractoriness during estrogen ablation treatment, and (3) the effectiveness of GnRH antagonist/superagonist in some postmenopausal breast cancer patients.

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.

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.

Recognition of human activated CD44 by tumor vasculature-targeted antibody.

TES-23 monoclonal antibody (MAb), which targets rat CD44H on tumor vascular endothelial cells (TEC), dominantly reacted to human activated CD44 rather than human inactive CD44. TES-23 MAb reacted to HT-1080 fibrosarcoma cells almost comparably to anti-human CD44 MAb and moderately to HUVEC; however, it hardly reacted to PBMC. The binding of soluble hyaluronate to HT-1080 cells and HUVEC was clearly noted, but not to PBMC. In addition, stimulation with phorbol 12-myristate 13-acetate induced soluble hyaluronate binding of MOLT-4 human T lymphoma cells and relatively increased the reactivity of TES-23 MAb. Our results suggest that TES-23 MAb can potentially recognize human activated CD44 and hence might be potentially useful for the treatment of human solid tumors containing TEC that express activated CD44.

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 (125)I-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.

Antibody-based therapy targeting tumor vascular endothelial cells suppresses solid tumor growth in rats.

We have developed a new approach to antibody-based therapy of solid tumors by targeting tumor vascular endothelial cells (EC) which are essential for the growth of solid tumors. We investigated the effect of an antibody against tumor-derived endothelial cells (TEC) on the growth of solid tumors in rats. Intravenous administration of TES-23, a monoclonal antibody generated by TEC isolated from rat KMT-17 solid tumors, at 1 mg/rat/day for 5 days resulted in significant suppression of KMT-17 tumor growth. Histopathological analysis of tumors administered with TES-23 showed that adhesion of lymphocytes to EC followed by denudation of EC in the viable tumor area. In contrast, little obvious toxicity was observed in most of the rat organs examined. These findings suggest that the concept of an antibody-based therapy with targeting tumor vascular EC would be promising in treatment of solid tumors.

Flat revertant inducers, styryl diphenylamine derivatives, inhibit growth and metastasis of murine tumor cells in vivo.

Previously, we reported that a novel styryl diphenylamine derivative, RX-465, reverts the transformed phenotype of human fibrosarcoma HT1080 cells in vitro. To determine whether agents inducing such phenotypic reversion show antitumor and/or antimetastatic effects in vivo, we assessed tumor growth and metastasis in mice inoculated with either murine B16-, B16BL6 melanoma or M5076 reticulosarcoma. These mice received orally our new derivatives of RX-465, namely RX-512 and RX-549. We found that these-derivatives inhibited B16 melanoma and M5076 reticulosarcoma growth by greater than 80% in vivo. RX-512 also inhibited the metastatic dissemination of B16BL6 melanoma into the lung and that of M5076 reticulosarcoma into the liver by 98% and 100% at doses of 60 and 40 mg/kg/day, respectively. These treatments did not cause significant decreases in body weight. Our data strongly demonstrate that styryl diphenylamine derivatives that revert transformed phenotype in vitro potentially inhibit tumor growth and metastasis in mice with negligible toxicity at effective doses.

Therapeutic potential of humanized anti-interleukin-6 receptor antibody: antitumor activity in xenograft model of multiple myeloma.

A xenograft model of human multiple myeloma (MM) was established in athymic nude mice using S6B45 cells whose growth is dependent on IL-6 in an autocrine fashion. S6B45 cells were inoculated s.c. into mice pretreated with 500 cGy X-ray and anti-asialo GM1 antibody. In more than 90% of the mice, a palpable tumor emerged within 30 days at the inoculation site. Histological observation of the tumor section revealed that the tumor mass was composed of two different phenotypes of myeloma cells, corresponding to plasmablasts and mature plasma cells. I.v. injection of more than 0.125 mg of mouse monoclonal antibody (PM1) against human IL-6R (hIL-6R) on days 1, 3 and 5 markedly delayed the time of tumor incidence. One mg of anti-hIL-6 antibody (MH166) also strongly inhibited the growth of S6B45, whereas control antibody (MOPC31C) and anti-hIL-6R antibody without neutralizing activity (AUK181-6) produced no significant effects. To reduce the antigenicity of PM1 in human, mouse-human chimeric PM1 (chPM1) with human IgG1 constant region and humanized PM1 (hPM1), human IgG1 with mouse complimentarity determining regions, were constructed and evaluated for their in vivo antitumor activity in our model. The in vivo efficacy of these recombinant antibodies (chPM1 and hPM1) was shown to be equivalent to that of the original PM1. These results indicate that the antitumor activity of PM1 is completely recreated in hPM1, and that blocking of the IL-6 signal by this humanized antibody could be a potent therapy for MM.