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Clinical Cancer Research Vol. 5, 371-382, February 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics

Induction of Lasting Complete Regression of Preformed Distinct Solid Tumors by Targeting the Tumor Vasculature Using Two New Anti-Endoglin Monoclonal Antibodies1

Fumihiko Matsuno, Yuro Haruta, Masao Kondo, Hilda Tsai, Maurice Barcos and Ben K. Seon2

Departments of Immunology [F. M., Y. H., M. K., H. T., B. K. S.] and Pathology [M. B.], Roswell Park Cancer Institute, Buffalo, New York 14263

ABSTRACT

Endoglin (EDG, CD105) is a proliferation-associated antigen on endothelial cells. In this study, two new anti-EDG monoclonal antibodies (mAbs) Y4-2F1 (or termed SN6j) and P3-2G8 (SN6k) were generated and used for treating distinct preformed tumors. These mAbs, both IgG1-{kappa} antibodies, cross-reacted weakly with mouse endothelial cells but defined epitopes different from the epitope defined by a previously reported anti-EDG mAb K4-2C10 (B. K. Seon et al., Clin. Cancer Res., 3: 1031–1044, 1997). SN6j and SN6k reacted strongly with human endothelial cells and vascular endothelium of malignant human tissues but showed no significant reactivity with tumor cells per se. The deglycosylated ricin A chain (dgRA) conjugates of the two mAbs showed a weak but specific cytotoxic activity against murine endothelial cells in vitro.

In the therapeutic studies, severe combined immunodeficient mice were inoculated s.c. with MCF-7 human breast cancer cells and left untreated until palpable tumors of distinct size (4–6 mm in diameter) appeared. Mice with the distinct tumors were treated by i.v. administration of individual anti-EDG conjugates, unconjugated mAbs, or a control conjugate. Long-lasting complete regression of the tumors was induced in the majority of tumor-bearing mice (n = 8 for each conjugate) when 40 µg of the individual conjugates were administered three times via the tail vein. It is remarkable that the tumors remained regressed without further therapy for as long as the mice were followed (i.e., 100 days). Control conjugate did not induce regression of the tumors in any of the treated mice, although weak nonspecific effects were observed in some of the mice (n = 8). The effects of unconjugated mAbs were small with the dose used, i.e., 34 µg three times. The anti-EDG conjugates showed antiangiogenic activity in the dorsal air sac assay in mice. The results suggest good potential of these conjugates for the clinical application.

INTRODUCTION

The progressive growth of solid tumors beyond clinically occult sizes (a few mm3) requires the continuous formation of new blood vessels, a process known as tumor angiogenesis (1) . Tumor growth and metastasis are angiogenesis dependent. Therefore, either prevention of tumor angiogenesis or selective destruction of the tumors’ existing blood vessels may present a potentially effective strategy for the prevention and treatment of solid tumors (2, 3, 4, 5, 6, 7) . The primary objectives of the present studies are therapy of preformed tumors by targeting the tumor vasculature.

To effectively target tumor vasculature, it is imperative to develop appropriate reagents that selectively destroy tumor-associated vasculature without severely damaging normal tissues. Endothelium in the normal adults is considered quiescent because the turnover of these cells is very low (e.g., thousands of days; Refs. 1 , 3 , 8 , and 9 ). However, the same endothelial cells can undergo rapid proliferation during spurts of angiogenesis. Therefore, an appropriate proliferation-associated antigen on endothelial cells could be a candidate target in antiangiogenic therapy. EDG3 is such a proliferation-associated marker, and its tissue distribution is highly restricted (see below).

In 1986, we reported a novel human leukemia-associated glycoprotein homodimer that was identified by a mAb termed SN6 (10) . The antigen was a homodimer of Mr 160,000 and termed GP160 (10) . SN6 showed a highly restricted reactivity to immature B-lineage ALL cells and myeloid/monocytic leukemia cells but not with various normal human peripheral blood cells. Approximately 1% of normal bone marrow cells reacted with SN6 (10) . Studies of CFU assays showed that the majority of CFU-GEMM pluripotent progenitor cells and CFU-GM myeloid progenitor cells in the normal human bone marrow were intact after incubation with SN6 IT under conditions where >99% of the colony formed by EDG-expressing NALM-6 leukemia cells were suppressed.4 In addition, we reported that the expression of GP160 on leukemia cells was strongly up-regulated by transformation with 12-O-tetradecanoylphorbol-13-acetate (11) ; therefore, we concluded that GP160 is a transformation/proliferation-associated antigen. In 1988, Gougos and Letarte (12) reported a leukemia-associated glycoprotein homodimer of Mr 170,000 that showed an identical cell distribution to GP160. They found that the Mr 170,000 homodimer was present on endothelial cells as well as leukemia cells and determined the nucleotide sequence of cDNA encoding the Mr 170,000 homodimer, termed EDG (13) . Recently, EDG was shown to specifically bind TGF-ß, and its deduced amino acid sequence possessed a strong homology to that of betaglycan, a TGF-ß receptor type III (14) . The role of EDG in the TGF-ß-induced signal transduction is poorly understood. The existence of two forms of EDG was reported recently, i.e., the smaller form (Mr 160,000, termed S-EDG) and the larger form (Mr 170,000, L-EDG); a small difference between the two EDGs was due to the different size of the cytoplasmic portions of the proteins (15) . At the recent 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens (November 10–14, 1996 in Kobe, Japan), SN6 was determined to be an anti-EDG mAb (16) . EDG is a proliferation-associated marker on endothelial cells (17 , 18) as well as on leukemia cells (11) . Some anti-EDG mAbs were reported to react strongly with vascular endothelium of tumor tissues but less so with the vascular endothelium of normal tissues (17 , 19 , 20) .

Although EDG on tumor-associated blood vessels in patients appears to be an attractive target for antiangiogenic therapy, this potential has not been explored until recently (18) . The major reason is that the newly formed blood vessels in the transplanted human tumors in the immunodeficient animals originate from the host’s tissues, and conventional anti-EDG mAbs do not react with endothelial cells of animals such as mice and rats (17) . Recently, we reported an anti-EDG mAb K4-2C10 (or termed SN6f) that cross-reacted with mouse endothelial cells, presumably by cross-reacting with mouse endoglin (18) . Such mAbs would correspond to a case of externally induced autoantibodies (21) . The dgRA conjugate of K4-2C10 was highly effective for antiangiogenic therapy when therapy was initiated before distinct tumors were detected (18) . In a preliminary study, however, K4-2C10-dgRA was not strongly effective for inducing the regression of preformed distinct solid tumors. In the present studies, we generated two new anti-EDG mAbs that cross-react weakly with mouse endothelial cells but define epitopes different from the epitope defined by K4-2C10. Comparative studies indicate that dgRA conjugates of the new mAbs are more effective than K4-2C10 in vivo. Therefore, these new mAbs and their immunoconjugates were used to treat distinct palpable tumors, the results for which are presented in this report.

MATERIALS AND METHODS

Cells, Tissues, and Animals.
The SVEC4-10 murine endothelial cell line (22) , HUVECs, and MCF-7 human breast cancer cell line (23) were cultured as described previously (18) . Various human hematological cell lines were cultured in RPMI 1640 supplemented with 4–10% fetal bovine serum, 100 units/ml penicillin, and 50 µg/ml streptomycin as described previously (10) . A buffy coat specimen from a patient with ALL was provided by Roswell Park Cancer Institute. Various human tissues were obtained from the Tissue Procurement Facility of Roswell Park Cancer Institute. Normal female BALB/c mice and female SCID (C.B.-17/ICr) mice were obtained from Taconic (Germantown, NY). SCID mice were maintained in a protected environment in a laminar flow unit and given autoclaved food and water ad libitum as described previously (24) . SCID mice, 6–7 weeks of age, were used in the present experiments. All handling of SCID mice was performed in a laminar flow hood.

mAb and Reagents.
Anti-GP160 (EDG) mAb SN6 and an isotype-matched murine control IgG (MOPC 195 variant, IgG1-{kappa}) were generated in our laboratory (10) . dgRA and SMPT were purchased from Inland Labs (Austin, TX) and Pierce (Rockford, IL), respectively. Mouse serum albumin (Fraction V Powder) and FITC isomer 1 were purchased from Sigma Chemical Co. (St. Louis, MO) and Calbiochem (San Diego, CA), respectively. Immunohistochemical reagents were obtained from DAKO (Carpinteria, CA). Diffusion chamber rings and cellulose membrane filters (0.45 µm pore size; 13-mm diameter) were purchased from Millipore (Bedford, MA).

Isolation of EDG.
EDG was isolated from ALL cells as described previously (18) . The purity of the isolated antigen was examined by SDS-PAGE analysis and silver staining of the gels (18) .

Generation of mAbs.
Two female BALB/c mice were immunized with the isolated EDG preparation as described previously (18) . Cell fusion, hybridoma screening, cloning, and mAb class determination were carried out as described previously (10 , 25) . A hybridoma producing mAb Y4-2F1 (SN6j) and another hybridoma producing mAb P3-2G8 (SN6k) were derived from mouse 1 and mouse 2, respectively.

Radioimmunoprecipitation and SDS-PAGE Analysis.
The EDG preparation was radiolabeled with 125I using Iodo-Gen and immunoprecipitated using mAb and an isotype-matched control IgG coupled to AminoLink gel (Pierce Chemical; Ref. 18 ). The immunoprecipitated antigen was analyzed by SDS-PAGE, and an autoradiograph was prepared as described previously (18) .

Cellular RIA.
Cellular RIA was used to determine the reactivity of mAb with various cells. Details of the assay were described previously (25) .

Epitope Comparison.
A competitive binding assay was performed to compare the epitopes defined by K4-2C10, SN6j, and SN6k. The assay was carried out as described previously (26) .

Flow Cytometry.
The purified mAbs SN6j and SN6k and an isotype-matched control IgG were individually conjugated to FITC as described previously (18) . Binding of the FITC-labeled mAb and control IgG to target cells was analyzed using Becton Dickinson FACScan. In the analysis of SVEC4-10 mouse endothelial cells from confluent cultures, the cultures were kept confluent for 3 days before the cells were used for the analysis as described previously (18) .

Immunohistochemical Staining of Tissues.
Tissues were frozen, air-dried, and fixed with acetone (18) . Fixed tissues were cut on a cryostat and stained using DAKO LSAB + Kits following the supplier’s recommended procedure. Counterstaining was performed with hematoxylin.

Preparation of IT and in Vitro Cytotoxicity Test.
mAbs and an isotype-matched control IgG were individually conjugated with dgRA using SMPT, which generates an in vivo stable disulfide linker (27) , as described previously (18 , 24) . The in vitro cytotoxic activity of IT and control conjugate was evaluated as described previously (18) . The results were expressed as a percentage of the [3H]thymidine incorporated by cells treated with the medium only.

Determination of the Maximum Tolerated Dose of IT in Mice.
The maximum tolerated dose of an IT was determined as described previously (18) but with modifications. Groups of four normal female BALB/c mice (7 weeks of age) were injected i.v. (via the tail vein) with an IT at doses of 0.025, 0.05, 0.1, 0.2, and 0.4 mg; an IT was injected in three equal portions on 3 consecutive days. In the case of SN6k-dgRA, SCID mice as well as BALB/c mice were used. Mice were weighed prior to injection and daily thereafter and were observed for morbidity and mortality for 2 weeks. The LD50 was determined by plotting the percentage of mortality versus injected dose to determine the dose resulting in 50% mortality.

Transplantation of MCF-7 into SCID Mice.
Transplantability of MCF-7 in SCID mice was investigated by s.c. inoculation of varying doses of MCF-7 into the left flank of the nonconditioned (e.g., not irradiated) SCID mice. At the doses over 8 x 106 cells (in 0.1 ml PBS), 100% of the inoculated mice developed tumors.

Comparison of in Vivo Antitumor Efficacy.
The dgRA conjugates of K4-2C10, SN6j, and SN6k were compared for their antitumor efficacy under the conditions where these conjugates were only partially effective for tumor suppression. Individual ITs were sterilized and mixed with a sterilized mouse serum albumin solution in PBS (final concentration, 0.1%). Twenty µg of individual ITs were given into mice of different groups (n = 7 for each group) via the tail vein on day 7 after tumor inoculation; palpable tumors were barely detected on day 7. Control group mice (n = 4) were untreated. Administration of the IT was repeated once on day 9. Mice were monitored daily as described below.

Therapy of Tumor-inoculated Mice.
IT and mAb were individually sterilized by filtering through Millex-GV filter (0.22 µm; Millipore) in a laminar flow hood. The sterilized solutions were diluted with sterile PBS containing mouse serum albumin (final concentration, 0.1%). SCID mice inoculated with 8 x 106 MCF-7 cells were left untreated until palpable distinct tumors appeared. Mice with distinct tumors (40–90 mm3 tumor volume) were divided into groups for the therapeutic studies. Groups of the SCID mice (n = 8 or 7) were untreated (control) or treated by i.v. administration of 34 µg/0.2 ml unconjugated mAb (SN6j or SN6k) or 40 µg/0.2 ml mAb-dgRA (SN6j-dgRA or SN6k-dgRA) via the tail vein. The treatment was repeated twice on days 2 and 4 after the onset of the therapy. In an additional control test, SCID mice bearing distinct MCF-7 tumors were divided into two groups (n = 8 for each group); one group was untreated, whereas the other group was treated by i.v. administration of 40 µg of dgRA conjugate of an isotype-matched control IgG. The treatment was repeated twice as described above. The total dose (i.e., 120 µg) of the IT corresponded to 42.8 and 43.0% of the LD50 of SN6j-dgRA and SN6k-dgRA, respectively.

Follow-Up of Treatment Efficacy.
During the treatment, mice were monitored daily for the tumor size and morbidity. Mice were weighed twice a week using an electronic balance (OHAUS Model GT210). Tumor volumes were estimated as described previously (18) . Statistical analysis of the data for the comparison of different groups of mice was carried out using Student’s t test.

Assay of Tumor Angiogenesis in Mice.
Tumor angiogenesis and antiangiogenic activity of IT were evaluated by using the dorsal air sac method as described previously (18) , but with modifications. Briefly, 1.6 x 107 MCF-7 cells in 0.2 ml PBS were placed into a Millipore chamber (14 mm in diameter), which was sealed at each chamber end with a cellulose membrane filter (0.45 µm pore size). A chamber, so prepared, was implanted into a dorsal air sac of each of several female BALB/c mice; the dorsal air sac was generated by injecting 7 ml of air. Three groups of three mice each implanted with a chamber containing MCF-7 were untreated (angiogenesis-positive control) or treated with SN6j-dgRA or SN6k-dgRA. Another group, the fourth group, of three mice was implanted with a chamber containing PBS (angiogenesis-negative control). The IT (30 µg/0.2 ml) was administered via the tail vein 5 h before implantation of the chamber and repeated twice 24 and 48 h after the chamber implantation. The mice were sacrificed on day 3, and blood vessels in the excised skin were examined under a stereoscopic microscope and photographed. The experiments were repeated twice.

RESULTS

Generation and Characterization of mAbs.
Anti-EDG mAbs that were generated by immunizing mice with an isolated EDG preparation were initially identified by testing against a variety of hematopoietic cell lines in a cellular RIA and by immunoprecipitating EDG as described previously (10 , 18) . In addition, the generated mAbs were tested against various malignant tissues by immunohistochemical staining. In this report, we present data of anti-EDG mAbs Y4-2F1 (or termed SN6j) and P3-2G8 (SN6k) that cross-react with mouse endothelial cells; both SN6j and SN6k were determined to be IgG1-{kappa} antibodies. The prototype anti-EDG mAb SN6 (10 , 20) was included in the various tests of SN6j and SN6k as a reference. Both SN6j and SN6k as well as SN6 (IgG1-{kappa} antibody) reacted with all of the four immature B-lineage leukemia cell lines tested (KM-3, REH, NALM-1, and NALM-6) and all of the three myelomonocytic leukemia cell lines tested (ML-2, HL-60, and U937). However, they did not react with any of the three mature B-lineage leukemia-lymphoma cell lines (BALL-1, BALM-2, and Daudi), any of the seven T leukemia cell lines (MOLT-4, JM, CCRF-CEM, CCRF-HSB2, Ichikawa, HPB-MLT, and HUT-78), or any of the three EBV-transformed B cell lines (CCRF-SB, RPMI 1788, and RPMI 8057). This pattern of reactivity is typical for anti-EDG mAbs (10 , 12 , 18 , 28) . In addition, SN6j and SN6k reacted strongly with HUVECs (see below). From the 125I-labeled antigen preparation, both SN6j and SN6k immunoprecipitated a single major component of Mr 170,000 under unreduced conditions (Fig. 1)Citation . Under reduced conditions, a single major component of Mr 90,000 was detected for both SN6j and SN6k immunoprecipitates (data not shown). SN6 showed the same pattern of immunoprecipitation under both reduced and unreduced conditions (18) . The results indicate that SN6j and SN6k define EDG. In an additional test, reactivity of SN6j and SN6k against several malignant tissues was investigated by immunohistochemical staining; these included malignant tissues of breast, colon, rectum, kidney, and lung. Both mAbs reacted strongly with vascular endothelium of all of the tested malignant tissues. An isotype-matched control IgG did not show any significant staining in any tested tissues. The reactivity of these anti-EDG mAbs with the tissues was restricted to vascular endothelium, and the mAbs did not react with tumor cells per se. Some of the test results obtained using SN6j and SN6k are illustrated in Figs. 2Citation and 3Citation . In the results shown in Fig. 2Citation , SN6j (A) and SN6k (B) react strongly with vascular endothelium of rectal carcinoma tissues. In Fig. 3Citation , malignant and normal breast tissues are compared for the vascular reactivity of SN6j and SN6k. At appropriate dilutions of the hybridoma ascites (e.g., 10,000-fold and 5,000-fold dilution), numerous blood vessels of the malignant breast tissues were stained with SN6j (Fig. 3A)Citation and SN6k (Fig. 3C)Citation , but much weaker or minimal reactivity of these ascites samples was detected with a few blood vessels of the normal breast tissues (Fig. 3, B and D)Citation . An isotype-matched control IgG (IgG1-{kappa}) did not stain either malignant or normal breast tissues (Fig. 3, E and F)Citation . SN6j and SN6k showed no significant reactivity with MCF-7 human breast cancer cells in a cellular RIA. This observation is consistent with our recent finding that another cross-reactive anti-EDG mAb K4-2C10 did not react with MCF-7 in the cellular RIA (18) .



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  		Fig. 1. SDS-PAGE of immunoprecipitates from the 125I-labeled EDG preparation. In the immunoprecipitation, we used P3-2G8 (or termed SN6k; A), Y4-2F1 (SN6j; B), and an isotype-matched control IgG (MOPC 195 variant; C). Samples were analyzed on 6.5% gels. Bio-Rad kaleidoscope prestained marker proteins were used as references. The single major immunoprecipitated component in each of Lanes A and B is indicated by an arrow.

 


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  		Fig. 2. Reactivity of SN6j and SN6k with vascular endothelium of rectal carcinoma tissues. Frozen and acetone-fixed human tissue samples were allowed to react with a 10,000-fold dilution of SN6j ascites (A), SN6k ascites (B) or an isotype-matched control IgG ascites (C) and stained with DAKO staining kits. Counterstaining was performed with hematoxylin (x50). An example of the stained blood vessels is indicated by an arrow in A and B. The reactivity of SN6j and SN6k is restricted to the vascular endothelium of the tissues, and these mAbs did not react with tumor cells per se. Control IgG did not show any staining.

 


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  		Fig. 3. Stronger reactivity of SN6j and SN6k with malignant tissues than with normal tissues. Frozen/acetone-fixed samples of malignant and normal human breast tissues were allowed to react with a 10,000-fold dilution of SN6j ascites (A and B), a 5,000-fold dilution of SN6k ascites (C and D), and a 5,000-fold dilution of control IgG ascites (MOPC 195 var; E and F), and then stained and counterstained (x50) as described above. Numerous blood vessels of the malignant tissues were stained with SN6j (A) and SN6k (C), whereas much weaker or minimal reactivity of SN6j and SN6k was detected with a few blood vessels of the normal breast tissues (B and D). Control IgG did not react with either malignant or normal breast tissue (E and F). The reactivity of SN6j and SN6k was localized to the blood vessels of the tissues. An example of the stained blood vessels is indicated by an arrow in A and C. A, C, and E, breast carcinoma; B, D, and F, normal breast.

 
Epitope Comparison.
The epitopes defined by K4-2C10, SN6j, and SN6k were compared by a competitive binding assay. An isotype-matched control IgG (MOPC 195 variant, IgG1-{kappa}) was included in the assay; K4-2C10, SN6j, and SN6k are all IgG1-{kappa} antibodies. The results are shown in Fig. 4Citation . Preincubation of EDG-expressing KM-3 cells with K4-2C10 blocked the subsequent binding of 125I-labeled K4-2C10 completely at the maximum, whereas preincubation of KM-3 cells with an isotype-matched control IgG showed no significant blocking. The results show that the blocking is specific. SN6j blocked the subsequent binding of 125I-labeled K4-2C10 only up to 80% at the maximum, whereas SN6k blocked binding of 125I-labeled K4-2C10 up to 20%. The results show that the epitope defined by SN6k (SN6k epitope) is in a relatively distant position from both K4-2C10 epitope and SN6j epitope. SN6j epitope is in closer proximity to K4-2C10 epitope but not close enough to block 100%. Therefore, the three epitopes defined by K4-2C10, SN6j, and SN6k, respectively, are individually different.



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  		Fig. 4. Competitive binding between cross-reactive anti-EDG mAbs as measured by a cellular RIA. EDG-expressing KM-3 leukemia cells (2 x 105 cells) were incubated with serial dilutions of purified K4-2C10, SN6j, SN6k, and an isotype-matched control IgG (IgG1-{kappa}; MOPC) for 1 h at 4°C in individual wells of 96-well microtiter plates. 125I-labeled K4-2C10 (4 x 104 cpm) was then added, and the incubation was continued for an additional 1 h. Cells were washed three times, and the radioactivity in the washed cell pellet was determined in a {gamma}-ray spectrometer.

 
Reactivity of SN6j and SN6k with Mouse and Human Endothelial Cells.
Reactivity of mAbs SN6j and SN6k with SVEC4-10 mouse endothelial cells, HUVECs, and EDG-negative BALL-1 control cells was analyzed by flow cytometry. In the case of SVEC4-10 cells, we compared proliferating cells from subconfluent cultures and quiescent cells from confluent cultures for their reactivity with SN6j and SN6k. The results of SN6k are illustrated in Fig. 5Citation . SN6j and SN6k showed significant binding to SVEC4-10 cells from subconfluent cultures (Fig. 5A)Citation , whereas they bound less to SVEC4-10 cells from confluent cultures (Fig. 5B)Citation . MFI of SVEC4-10 cells from subconfluent cultures was 17.97, 16.96, and 4.58, respectively, for SN6j, SN6k, and an isotype-matched control IgG, whereas MFI of the cells from confluent cultures was 12.19, 10.36, and 3.90, respectively, for SN6j, SN6k, and the control IgG. Sixty-two and 63% of the SVEC4-10 cells from subconfluent cultures reacted with SN6j and SN6k, respectively, whereas only 21 and 19% of the cells from confluent cultures reacted with SN6j and SN6k, respectively. Therefore, SN6j and SN6k react with the proliferating SVEC4-10 cells more strongly than with the quiescent SVEC4-10 cells. No significant reaction of the mAbs was observed with EDG-negative BALL-1 control cells (Fig. 5C)Citation . The mAbs reacted strongly with proliferating HUVECs (Fig. 5D)Citation . MIF of HUVECs was 209.0, 211.2, and 4.95, respectively, for SN6j, SN6k, and the control IgG. More than 99% of HUVECs reacted with both SN6j and SN6k.



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  		Fig. 5. Flow cytometry analysis of binding of SN6k to SVEC4-10 mouse endothelial cells (A and B), EDG-negative BALL-1 human leukemia cells (C), and HUVECs (D). Binding of FITC-labeled SN6k and control IgG of the same isotype (MOPC 195 variant, IgG1-{kappa}) to target cells was measured using Becton Dickinson FACScan. Proliferating cells from subconfluent cultures (A) and quiescent cells from confluent cultures (B) were compared for SVEC4-10 cells. HUVECs tested were proliferating cells from subconfluent cultures. Reactivities of SN6j with SVEC4-10, BALL-1, and HUVECs were very similar to those of SN6k.

 
Reactivity of SN6j and SN6k with Tumor Vasculature in the Mouse as Determined by Immunohistochemical Staining.
In a previous report (18) , using an immunohistochemical staining assay we showed that a cross-reactive anti-EDG mAb K4-2C10 specifically reacted with endothelium of tumor-associated vasculature in the s.c. tissues of SCID mice bearing human tumors. In the present immunohistochemical staining, SN6j and SN6k specifically reacted with tumor-associated vasculature of s.c. tissues of SCID mice bearing s.c. tumors of BALL-1a human tumors (24) .

Cytotoxic Activity of dgRA Conjugates of SN6j and SN6k in Vitro.
dgRA conjugates of SN6j, SN6k, and an isotype-matched control IgG were tested against SVEC4-10 cells after incubation for 48 h at 37°C. The results are shown in Fig. 6Citation . Both SN6j-dgRA and SN6k-dgRA show a weak but significant cytotoxicity against mouse endothelial cells; the IC50s of these conjugates were 16 and 26 nM, respectively. It should be noted that the IC50 of K4-2C10-dgRA was 29 nM(18) . Control dgRA conjugate shows a weak cytotoxic effect at concentrations higher than 30 nM (3 x 10-8M). Previously, such nonspecific cytotoxic effects of control RA and dgRA conjugates against various target cells in vitro were often observed at concentrations higher than 30 nM(17 , 29) .



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  		Fig. 6. Cytotoxic activity of dgRA conjugates of SN6j, SN6k, and an isotype-matched control IgG (MOPC) against SVEC4-10 murine endothelial cells. Both SN6j-dgRA and SN6k-dgRA showed weak but significant cytotoxic activity. The IC50s of SN6j-RA and SN6k-dgRA are 16 and 26 nM, respectively.

 
Comparison of the in Vivo Antitumor Efficacy of Three Cross-Reactive Anti-EDG ITs.
Antitumor efficacy of K4-2C10-dgRA, SN6j-dgRA, and SN6k-dgRA was compared using a low dose of individual ITs under the conditions where these ITs were only partially effective for tumor suppression (see "Materials and Methods" for details). The average tumor volume ± SD was 65.1 ± 46.6, 58.4 ± 38.8, and 43.2 ± 23.2 mm3 for the three groups of mice (n = 7 for each group) treated with K4-2C10-dgRA, SN6j-dgRA, and SN6k-dgRA, respectively, on day 58 after tumor inoculation. The value of the untreated control group (n = 4) was 183.1 ± 98.2 mm3. The results indicate that SN6j-dgRA and SN6k-dgRA are more effective than K4-2C10-dgRA or at least as effective as K4-2C10-dgRA for in vivo tumor therapy.

Maximum Tolerated Dose of IT.
The LD50 of SN6j-dgRA in normal BALB/c mice was 16.6 µg/g body weight. The LD50 of SN6k-dgRA in normal BALB/c and SCID mice was 16.5 and 17.5 µg/g body weight, respectively.

Therapy of SCID Mice Bearing Distinct Tumors of Human Breast Cancer.
SCID mice were inoculated s.c. with 8 x 106 MCF-7 cells/mouse into the left flank of the mice and left untreated until palpable tumors of distinct size (4–6 mm in diameter) appeared in the mice. It took 10–14 days in most cases until the distinct tumors appeared. Mice with distinct tumors were divided into groups, and then the mice were treated by i.v. administration of IT (SN6j-dgRA or SN6k-dgRA) or unconjugated mAb (SN6j or SN6k; see "Materials and Methods" for details). The control group was untreated. The results are shown in Figs. 7Citation and 8Citation . Both SN6j-dgRA and SN6k-dgRA showed remarkable antitumor efficacy after only three injections. Tumors in all of the control group mice (n = 8) continued growing (Figs. 7ACitation and 8ACitation ). SN6j-dgRA induced lasting complete regression in five of the eight treated mice (Fig. 7B)Citation . The effect of unconjugated mAb SN6j was marginal (Fig. 7C)Citation . SN6k-dgRA induced lasting regression of tumors in five of the eight treated mice (Fig. 8B)Citation , whereas the effect of unconjugated mAb SN6k was small (Fig. 8C)Citation . The antitumor efficacy of SN6j-dgRA and SN6k-dgRA was statistically significant (P < 0.0001 for both conjugates). It is remarkable that the tumors remained regressed without further therapy for as long as the mice were followed. In additional tests, effects of dgRA conjugate of an isotype-matched control IgG (MOPC 195 variant, IgG1-{kappa}) on the distinct tumors were investigated, and the results are presented in Fig. 9Citation . The control IT did not induce regression of the tumors in any of the treated mice (n = 8), although some effects on the tumors were observed in the majority of the mice (Fig. 9B)Citation . These effects are probably nonspecific ones because the same control IT showed weak nonspecific cytotoxic effects against murine endothelial cells in an in vitro assay (see Fig. 6Citation ). All of the treated mice tolerated well the IT therapy, although a transient weight loss was observed in the treated mice.



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  		Fig. 7. Induction of lasting complete regression of the preformed human solid tumors in SCID mice by systemic administration of SN6j-dgRA. Mice were inoculated s.c. with MCF-7 human breast cancer cells and left untreated until palpable tumors of distinct size (4–6 mm in diameter) appeared in the majority of the inoculated mice. Mice with distinct tumors were selected and divided into three groups: untreated (control, A), treated by administration of SN6j-dgRA (B) or unconjugated SN6j (C) via the tail vein. The treatment was repeated twice on days 2 and 4 after the onset of the therapy against distinct palpable tumors (see arrows at the bottom of the figure).

 


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  		Fig. 8. Strong antitumor efficacy of SN6k-dgRA in vascular targeting therapy of distinct tumors. SCID mice with distinct palpable tumors (4–6 mm in diameter) of MCF-7 were untreated (control, A), or treated with SN6k-dgRA (B) and unconjugated SN6k (C) in the same manner as described for Fig. 7Citation .

 


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  		Fig. 9. Effect of the control dgRA conjugate on the tumor growth in SCID mice. Mice with distinct MCF-7 tumors (4–6 mm in diameter) were untreated (A) or treated with the dgRA conjugate of an isotype-matched control IgG (MOPC-dgRA) (B) under the same conditions as described for Figs. 7Citation and 8Citation .

 
Suppression of Angiogenesis by SN6j-dgRA and SN6k-dgRA.
Tumor-associated angiogenesis was induced in mice by the dorsal air sac method (see "Materials and Methods" for details), and the antiangiogenic activity of SN6j-dgRA and SN6k-dgRA was evaluated by observing short-term (3 days) effects of the individual ITs on the angiogenesis. Representative examples of the results are illustrated in Fig. 10Citation . MCF-7 tumor cells induced weak but significant angiogenesis in the dorsal skin (A) compared with the control dorsal skin (D). Previously, HT1080 tumor cells induced stronger angiogenesis in the same assay (18) . Nevertheless, MCF-7 was used in the present assay because we have been using MCF-7 tumors as the target in the antitumor therapy (Figs. 7Citation 8Citation 9)Citation . Numerous neomicrovessels are observed in the angiogenesis-induced dorsal skin (Fig. 10A)Citation . In addition, distinct tortuous neovessels were generated in the skin, as indicated by arrows in the figure (Fig. 10A)Citation . The neomicrovessels and tortuous neovessels were significantly inhibited by SN6j-dgRA (B) and SN6k-dgRA (C). These findings are consistent with the observed long-term effects of the ITs on the distinct tumors, i.e., induction of long-lasting regression of the tumors.



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  		Fig. 10. Induction of angiogenesis by MCF-7 in a dorsal air sac in mice and inhibition of the angiogenesis by SN6j-dgRA and SN6k-dgRA. MCF-7 tumor cells in an implanted Millipore chamber induced angiogenesis in the murine dorsal skin. Numerous neomicrovessels and distinct tortuous neovessels were generated in the dorsal skin (A); two examples of the tortuous neovessels are indicated by arrows. SN6j-dgRA (B) and SN6k-dgRA (C) inhibited significantly the formation of the tortuous neovessels and neomicrovessels. The control murine dorsal skin (D) was obtained by implanting a Millipore chamber containing cell culture medium without MCF-7 cells in a dorsal air sac.

 
DISCUSSION

Antiangiogenic therapy has several advantages over conventional tumor cell targeting in the therapy of solid tumors: (a) this approach may allow us to circumvent the problem of acquired drug resistance (30 , 31) . The rationale is that drug-resistant mutants are easily generated from tumor cells because of the genetic instability of tumor cells. However, genetically stable normal cells, such as vascular endothelial cells, would be far less adept at generating such mutants. Indeed, drug resistance has not been a significant problem in antiangiogenic therapy of patients (4) ; (b) antiangiogenic therapy may be able to overcome the problem of tumor heterogeneity from the reason described above; tumor heterogeneity is a major problem with the tumor cell targeting therapy; (c) physiological barriers for the high molecular weight drugs (such as antibodies and immunoconjugates) to penetrate into solid tumors (32 , 33) will be circumvented by targeting a tumor’s vasculature rather than tumor cells. The reason is that unlike tumor cells in the solid tumors, the vascular endothelial cells are directly accessible to circulating high molecular weight drugs; (d) many thousands of dependent tumor cells will die of nutrient and oxygen deprivation if a capillary or a sector of the capillary bed fails (6 , 7) . Therefore, killing of only a minority of vascular endothelial cells of tumors may be sufficient to eradicate most malignant cells in tumors; and (e) a single agent developed for antiangiogenic therapy could be applied to most types of solid tumors and angiogenesis-associated diseases.

Several angiogenesis inhibitors are being evaluated for their antitumor efficacy in patients (reviewed in Refs. 4 , 34, 35, 36 ). They showed varying degrees of promising antitumor efficacy, although none of them showed curative antitumor efficacy. A major problem with these agents is that they need to be administered continuously because tumors regrow when the treatment is discontinued. Other interesting angiogenesis inhibitors that may be potentially useful for clinical application include antivascular endothelial growth factor mAb (37) , angiostatin (38) , interleukin 12 (39) , integrin {alpha}{nu}ß3 antagonists (40) , endostatin (31) , vascular endothelial growth factor-toxin conjugate (41) , and drug conjugates of {alpha}{nu} integrin-binding peptides (42) .

mAb-based antiangiogenic therapy of cancer is highly attractive conceptually because one of the most important features of a mAb is its fine specificity. However, few good immuno-target markers on vasculature have been known (7) . Previously, Thorpe and co-workers (43 , 44) used artificially induced murine MHC II antigens as the marker for vascular targeting. Brooks et al. (40) reported that a mAb or cyclic peptide antagonist of integrin {alpha}{nu}ß3 disrupted ongoing angiogenesis on the chick chorioallantoic membrane. In addition, anti-{alpha}{nu}ß3 mAb LM609 prevented tumor growth in a study of a SCID mouse/human chimeric model with transplanted human skin containing human tumor cells (45) . Recently, Brekken et al. (46) reported that the vascular endothelial growth factor: receptor (Flk-1) complex may be a good target marker of tumor endothelium.

The endothelium in normal adults is considered quiescent because the turnover of the endothelial cells is very low (1 , 3 , 8 , 9) . The endothelium of normal adult tissues is the most quiescent of all tissues in the body (3) . However, the same endothelial cells can undergo rapid proliferation during spurts of angiogenesis. Therefore, a proliferation-associated antigen on endothelial cells may be a good candidate for antiangiogenic therapy. EDG is a proliferation-associated antigen on endothelial cells (17 , 18) as well as on leukemia cells (11) . Furthermore, anti-EDG mAbs show a highly restricted reactivity to proliferating endothelial cells, immature ALL cells, myeloid/monocytic leukemia cells, and a few minor normal cells (10 , 12 , 28) . In our recent study, dgRA conjugate of a novel anti-EDG mAb K4-2C10 that cross-reacted with mouse endothelial cells showed a strong antiangiogenic activity and inhibited completely human breast tumors in SCID mice when therapy was initiated before palpable tumors appeared (18) . In a preliminary study, however, K4-2C10-dgRA was not strongly effective for inducing regression of preformed distinct tumors. In the present study, two new anti-EDG mAbs SN6j and SN6k that cross-react with mouse endothelial cells but define epitopes different from the K4-2C10 epitope were generated and compared with K4-2C10. Comparative studies indicate that SN6j-dgRA and SN6k-dgA are more potent for treating preformed tumors than K4-2C10-dgRA. Therefore, the new mAbs and their dgRA conjugates were used for antiangiogenic therapy of distinct preformed tumors. i.v. administration of the dgRA conjugates of SN6j and SN6k induced long-lasting complete regression of distinct s.c. tumors in the majority of the treated mice (see Figs. 7Citation and 8Citation ). SN6j-dgRA and SN6k-dgRA were prepared using SMPT that generates an in vivo stable linker (see "Materials and Methods"). The mechanisms by which ricin A-chain and dgRA ITs kill target cells are well characterized (reviewed in Ref. 47 ). SN6j-dgRA and SN6k-dgRA are internalized into endothelial cells after the mAb part of individual conjugates binds to EDG on endothelial cells. In the cells, dgRA is delivered to polyribosome, where dgRA inactivates the ribosomes by cleaving the N-glycosidic bond of an adenine residue of 28S rRNA (48) . Consequently, dgRA inhibits the protein synthesis and kills the cells. Histological examination of various tissues (e.g., spleen, kidney, and heart) of SN6j-dgRA- and SN6k-dgRA-treated mice showed no vascular damage. These results are consistent with the selective targeting of tumor vasculature by these ITs in the therapeutic studies. In the present in vitro studies, we tested the SVEC4-10 mouse endothelial cell line and HUVECs. SVEC4-10 was generated by SV40 virus infection and grows in media without addition of any defined growth factors (22) . On the other hand, we need to grow HUVECs in media containing growth factors to obtain sufficient number of cells for experiments. Furthermore, cell culture perturbs endothelial cells from their quiescent in vivo state (0.1% replications per day) to an activated state (1–10% replications per day) with a loss of specialized functions associated with diverse vessels and organ systems (49) . In view of this limitation of cultured HUVECs for obtaining quiescent endothelial cells, we tested the endothelium of normal human tissues in comparison with malignant tissues. Reactivity of SN6j and SN6k with normal tissue endothelium was much weaker than that with malignant tissue endothelium. The reactivity with malignant tissues was localized to the tumor vasculature, and no reactivity was detected with tumor cells per se.

The highly effective in vivo antitumor efficacy of SN6j-dgRA and SN6k-dgRA is remarkable in view of the fact that these mAbs react only weakly with mouse endothelial cells, and their dgRA conjugates are only weakly cytotoxic to mouse endothelial cells in vitro. Furthermore, the present ITs appear to have distinct advantages over many other reported antiangiogenic agents in that these ITs were able to exert long-lasting antitumor effects with only three i.v. injections. Antitumor effects of most antiangiogenic agents are brief, and these agents need to be administered continuously. However, the described apparent advantages of the present anti-EDG ITs need to be tested in patients.

Results of the present and previous studies (18) indicate that immunoconjugates of anti-EDG mAbs defining the cross-reactive epitopes between human and mouse endothelial cells are very useful for animal model studies of human solid tumors. In addition, such immunoconjugates appear to have good potential for clinical therapy of solid tumors and other angiogenesis-associated diseases (4 , 35) in view of: (a) the long-lasting antitumor efficacy of the immunoconjugates; (b) much stronger reactivity of the anti-EDG mAbs with HUVECs than with mouse endothelial cells; (c) restricted primary reactivity of anti-EDG mAbs with certain types of human leukemia cells and proliferating endothelial cells; and (d) absence of damaging effects of anti-EDG IT on the human hematopoietic progenitors.

ACKNOWLEDGMENTS

We are grateful to Dr. K. A. O’Connell for SVEC4-10 cell line and Dr. H. Slocum for human tissues. We thank Dr. M. Tabata for help in the radioimmunoprecipitation experiments and C. Zuber and C. Steger for help in the preparation of the manuscript.

FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by USPHS Grant R01 CA19304, United States Army Medical Research Grant DAMD17-97-1-7197, American Cancer Society Grant IM-741/RPG-91-005-05-IM and Roswell Park Alliance grant, and supported in part by Roswell Park Cancer Center Support Grant P30 CA16056. Roswell Park Cancer Institute is a comprehensive cancer center-National Cancer Institute designated. Back

2 To whom requests for reprints should be addressed, at Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263. Phone: (716) 845-3141; Fax: (716) 845-8906. Back

3 The abbreviations used are: EDG, endoglin; mAb, monoclonal antibody; CFU, colony-forming unit; TGF, transforming growth factor; ALL, acute lymphoblastic leukemia; dgRA, deglycosylated ricin A chain; SCID, severe combined immunodeficient; IT, immunotoxin; SMPT, 4-succinimidyloxycarbonyl-{alpha}-methyl-{alpha}-(2-pyridyldithio)toluene; HUVEC, human umbilical vein endothelial cell; IC50, 50% inhibitory concentration; LD50, 50% lethal dose; MFI, mean fluorescence intensity. Back

4 Y. Haruta, D. Chervinsky, and B. K. Seon, manuscript in preparation. Back

Received 6/29/98; revised 9/24/98; accepted 11/ 6/98.

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