Antiangiogenic Therapy of Established

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[CANCER RESEARCH 61, 7846–7854, November 1, 2001] Antiangiogenic Therapy of Established Tumors in Human Skin/Severe Combined Immunodeficiency Mouse Chimeras by Anti-Endoglin (CD105) Monoclonal Antibodies, and Synergy between Anti-Endoglin Antibody and Cyclophosphamide1

Norihiko Takahashi, Akinao Haba, Fumihiko Matsuno, and Ben K. Seon2 Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263

ABSTRACT Cyclophosphamide was not effective for suppressing human vessels and only weakly suppressive against murine vessels. Combination of SN6j and Endoglin (EDG; CD105) is a proliferation-associated cell membrane cyclophosphamide was effective for completely suppressing human vessels antigen of endothelial cells and is strongly expressed on the tumor- and also effective for partial (i.e., 35%) suppression of murine vessels. The associated angiogenic vascular endothelium. Furthermore, EDG is essen- results show that systemic administration of naked antihuman EDG mAbs tial for angiogenesis and a component of the transforming growth factor can suppress established tumors, and the efficacy is markedly enhanced (TGF)-␤ receptor complex. The present three anti-EDG monoclonal an- by combining a chemotherapeutic drug using an antiangiogenic schedule tibodies (mAbs), SN6f, SN6j, and SN6k, react strongly with proliferating of drug dosing. These mAbs should show stronger antitumor efficacy in human endothelial cells but cross-react very weakly with murine endo- patients whose tumors depend entirely on human blood vessels. thelial cells. Analysis of Scatchard plot of direct binding of these mAbs to proliferating human umbilical vein endothelial cells showed equilibrium constants of 8.3 ؋ 109, 3.1 ؋ 109, and 1.0 ؋ 109 liter/mol, respectively, for INTRODUCTION SN6f, SN6j, and SN6k. These mAbs did not react with MCF-7 human 3 breast cancer cells. To facilitate antiangiogenic tumor therapy by these A homodimer glycoprotein antigen, later termed EDG (CD105), mAbs in animal models, we used human skin/severe combined immuno- was initially identified as a human leukemia-associated cell mem- deficiency (SCID) mouse chimeras bearing tumors of MCF-7. Blood ves- brane antigen (1, 2). Its expression is restricted to immature B-lineage sels in the chimeras were analyzed by immunostaining with species (hu- acute lymphoblastic leukemia cells, myelomonocytic leukemia cells, man or mouse)-specific anti-CD31 and anti-EDG mAbs including an endothelial cells, and a few minor normal cells (1–5). EDG binds antihuman EDG mAb termed SN6h. Blood vessels in the completely TGF-␤1 and TGF-␤3 specifically but fails to bind TGF-␤2 (6). The healed grafted human skins consisted of a mixture of human (43.5%) and role of EDG in the TGF-␤-induced signal transduction is poorly murine (56.5%) vessels, whereas only murine vessels were detected in the understood. EDG is a proliferation-associated antigen on leukemia adjacent murine skins and s.c. tissues. Therefore, murine vessels infiltrate into the human skin grafts from the adjacent murine tissues, whereas the cells (7) and endothelial cells (8–10). Furthermore, EDG is essential growth of human vessels is limited within the boundary of human skins. for angiogenesis (11). Certain anti-EDG mAbs react with tumor- Growth of human MCF-7 tumors in the human skin grafts increased the associated vascular endothelium more strongly than with vascular ratio of human:murine vessels. Analyses of the grafted skins before and endothelium in normal tissues (8, 12–15). Therefore, these selected after tumor transplantation showed that SN6h reacted with tumor- anti-EDG mAbs and their immunoconjugates may be useful for anti- induced angiogenic blood vessels but not with nonangiogenic vessels, angiogenic therapy by targeting EDG on tumor-associated vascular whereas antihuman CD31 mAb reacted with both angiogenic and nonan- endothelium. Recently, we showed that immunoconjugates of selected giogenic vessels. The results show that SN6h is capable of distinguishing anti-EDG mAbs that weakly cross-react with mouse endothelial cells the tumor-induced angiogenic vasculature from the nonangiogenic vascu- were effective for suppressing angiogenesis and tumors in mice (9, 15, lature in the present model. Antiangiogenic therapy of the chimeras 16); these mAbs correspond to externally induced autoantibodies (17). bearing established MCF-7 tumors was carried out by i.v. administration Anti-EDG mAbs were conjugated either with deglycosylated ricin of a mAb(s) via the tail vein of mice. SN6j and SN6k were effective for 125 suppressing the established tumors, whereas tumor suppression was A-chain to obtain immunotoxins (9, 15) or with I to obtain radio- weaker with SN6f. The results indicate an absence of a direct correlation immunoconjugates (16). Although the anti-EDG immunotoxins between antigen-binding avidity and in vivo antitumor efficacy of anti- showed strong antiangiogenic antitumor efficacy at the dose of 24– EDG mAbs and suggest the importance of other factors (e.g., epitopes) in 45% of the LD50, they showed strong toxicity in mice; LD50 of SN6f, antitumor efficacy. No significant toxicity of the mAbs was detected. SN6j, and SN6k immunotoxins ranged between 14.8 and 17.8 ␮g/g of Combination of SN6f and SN6k that define mutually nonoverlapping body weight (9, 15). Therefore, the therapeutic windows of these epitopes showed an additive antitumor effect. Combination of SN6j and immunotoxins are relatively narrow. In contrast to immunotoxins, cyclophosphamide using an antiangiogenic schedule of drug dosing naked anti-EDG mAbs showed no significant toxicity in a dose- showed synergistic antitumor efficacy. The combination therapy induced escalation study in mice (see “Discussion”). Relevant observation was lasting complete regression of the established tumors in two of the eight reported for anti-HER2 immunotoxin and mAbs. An anti-HER2 im- treated chimeras. We examined human and murine blood vessels in large human tumors munotoxin showed a strong hepatotoxicity in patients because hepa- from the chimeras at the end of therapeutic experiment. The test showed tocytes express HER2 antigen (18). Nevertheless, the toxicity of that SN6j therapy resulted in complete suppression of human vessels in anti-HER2 mAb was weak in patients (19, 20) and anti-HER2 mAb the tumors but resulted in only weak suppression of murine vessels. has been widely used in patients, particularly in the combination therapy with a chemotherapeutic drug (e.g., Ref. 21). Received 5/21/01; accepted 9/4/01. In the present study, we investigated antitumor efficacy of naked The costs of publication of this article were defrayed in part by the payment of page (unconjugated) anti-EDG mAbs. A disadvantage of a naked anti-EDG charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by United States Army Medical Research Grant DAMD17-97-1-7197 and 3 The abbreviations used are: EDG, endoglin; SCID, severe combined immunodefi- in part by Roswell Park Cancer Center Support Grant P30 CA16056. ciency; mAb, monoclonal antibody; TGF, transforming growth factor; HUVEC, human 2 To whom requests for reprints should be addressed, at Department of Immunology, umbilical vein endothelial cell; CPA, cyclophosphamide; dgRA, deglycosylated ricin A Roswell Park Cancer Institute, Buffalo, NY 14263. chain; i.d., intradermal/intradermally. 7846

mAb, compared with an immunoconjugate, is weaker antitumor effi- performed as described by others (22–24) with slight modifications. The cacy. To circumvent this problem, we used human skin/SCID mouse procedures are briefly described below. chimeras (22–24) in the present study. Rationale for using the chime- Female SCID mice (7–10 weeks old) were anesthetized, and a section of 2 ras is as follows. The newly formed blood vessels in the transplanted skin (ϳ2cm) was surgically removed. A precut section of fresh, full- human tumors in the immunodeficient animals originate from the thickness, human neonatal foreskin was sutured into place. The grafts were host’s tissues. Our anti-EDG mAbs cross-react very weakly with bandaged securely for 2 weeks to allow healing. EDG-negative MCF-7 cells were inoculated i.d. into the grafted human skins over 7 weeks after the skins mouse endothelial cells, whereas they react much more strongly with were grafted, and the graft healed completely. human endothelial cells (9, 15). The newly formed blood vessels in Immunostaining of Tissues. Serial sections (6 ␮m in thickness) were cut the transplanted human tumors in human skin grafted to SCID mice from the frozen tissues using a cryostat, fixed in cold acetone, and stored at consist of human and mouse vessels (see below). Therefore, our Ϫ70°C until use. Human and mouse blood vessels in the tissue slice were anti-EDG mAbs will be more effective for antiangiogenic tumor determined by immunohistochemical staining using species-specific antiendo- therapy in human skin/SCID mouse chimeras than in SCID mice. Our thelial mAbs, i.e., mouse antihuman EDG mAb (SN6h), mouse antihuman results appear to support this theory. Furthermore, the combination of CD31 mAb, rat antimouse EDG (CD105) mAb, and rat antimouse CD31 mAb. an anti-EDG mAb with CPA showed synergistic antitumor efficacy. In the immunostaining, isotype-matched control murine IgG (MOPC 195 variant; IgG1-␬) and isotype-matched control rat IgG (R35–95; IgG2a-␬) were included as controls. For immunostaining of mouse tissues using mouse mAbs, MATERIALS AND METHODS InnoGenex Mouse-on-Mouse Iso-IHC kit and Vector peroxidase substrate kit DAB were used following the manufacturer’s instructions to reduce back- mAbs and Reagents. Antihuman EDG mAbs SN6f (termed K4–2C10), ground staining. For immunostaining of mouse tissues using rat mAbs, DAKO SN6j, and SN6k that cross-react weakly with mouse endothelial cells were LSAB Kit Peroxidase Universal was used. Counterstaining was performed generated in our laboratory (9, 15). A non-cross-reactive anti-EDG mAb, SN6h with hematoxylin. The ratio of human:mouse vessels in the completely healed (14, 25), and an isotype-matched murine control IgG (MOPC195 variant, human skin grafts (i.e., 8 weeks after the grafting), and in the human skin grafts IgG1-k; Ref. 26) were also generated in our laboratory. Murine antihuman bearing MCF-7 tumors, was determined by counting human and mouse vessels ␬ CD31 mAb JC/70A (IgG1- ) and CPA were obtained from DAKO (Carpin- on four ϫ100 fields (i.e., ϫ10 objective lens and ϫ10 ocular lens) at the area teria, CA) and Sigma Chemical Co. (St. Louis, MO), respectively. Rat anti- of highest vascularization (32) in each tissue section under a microscope. Serial ␬ mouse EDG (CD105) mAb MJ7/18 (rat IgG2a- ), rat antimouse CD31 mAb sections of the human skins were stained with a mixture of SN6h and antihu- ␬ ␬ 390 (rat IgG2a- ) and an isotype-matched rat control IgG (R35–95; IgG2a- ) man CD31 mAb, and a mixture of antimouse CD105 mAb and antimouse were purchased from PharMingen (San Diego, CA). InnoGenex Mouse-on- CD31 mAb. Mouse Iso-IHC kit, peroxidase substrate kit DAB (SK-4100), and LSAB kit In addition, we immunostained mouse and human vessels in the tumors Peroxidase Universal (K568) were purchased from InnoGenex (San Ramon, from the chimera mice that received PBS (control), SN6j, CPA, or SN6j CA), VECTOR (Burlingame, CA), and DAKO, respectively. plus CPA, and were killed at the end of a therapeutic experiment. Large Tissues, Cells, and Mice. Fresh human neonatal foreskins from elective tumors of two different sizes (i.e., 350– 450 mm3 and 100–250 mm3) were circumcisions were obtained from the Cooperative Human Tissue Network obtained from each group of mice for immunostaining and vessel counting. (CHTN) Eastern Division, University of Pennsylvania Medical Center. MCF-7 Sections of the tumors were stained with a mixture of antihuman CD105 human breast cancer cells, KM-3 human leukemia cells, HUVECs, and and CD31 mAbs and a mixture of antimouse CD105 and CD31 mAbs as SVEC4–10 murine endothelial cells (27) were cultured as described previ- described above. Human and mouse vessels were counted separately as ously (9). Female SCID (NCr) mice were obtained from the Frederick Cancer described above. An average of vessel counts in the two tumors was Research and Developmental Center, National Cancer Institute (Frederick, compared between different groups. MD). Mice were maintained in a protected environment in a laminar flow unit Antiangiogenic Therapy of Preformed Human Tumors in Human Skins and given sterilized food and water ad libitum as described previously (28). All Grafted into SCID Mice. MCF-7 cells (8 ϫ 106 cells in 0.1 ml of PBS) were of the handling of SCID mice was performed in a laminar flow hood. transplanted i.d. into human, full-thickness, skins grafted into SCID mice when Antigen-binding Avidity of mAbs. The purified mAbs SN6f, SN6j, and the grafts showed no signs of inflammation, contraction, or rejection. The mice SN6k were individually radiolabeled with 125I using Iodo-Gen as described were left untreated until distinct palpable tumors (3 to 6 mm in diameter in previously (1). The radiolabeled SN6f, SN6j, and SN6k were determined to most cases) appeared. Mice with distinct tumors were divided into groups for contain 1.85, 0.73, and 1.29 iodine atoms per IgG molecule on the average, respectively. Titration experiments which were carried out using a fixed the therapeutic studies. Anti-EDG mAbs and an isotype-matched control IgG ␬ ϫ amount (0.1 ␮g) of each 125I-labeled mAb and 2-fold serial increments of (MOPC 195 variant; IgG1- ) were centrifuged at 100,000 g at 4°Cfor1h, EDG-expressing KM-3 cells showed that 48.6, 50.0, and 55.0%, respectively, and the supernatants were individually filtered through a sterile Millex-GV ␮ of the labeled SN6f, SN6j, and SN6k retained antigen-binding activity. In the filter (0.22 m; Millipore, Bedford, MA) in a laminar flow hood before use. analyses of the binding data for SN6f, SN6j, and SN6k, corrections were made The sterilized solutions were diluted with sterile PBS containing mouse serum ␮ for the above numbers. An analysis of Scatchard plot of binding data was albumin (0.05% final concentration). For the mAb therapy, 200 g/0.2 ml carried out as described previously (29, 30). An equilibrium constant and an mAb or control IgG was administered i.v. via the tail vein of mice. The average maximal number of mAbs bound per cell were estimated by this administration was repeated four times every 2 days. After a break for a week, analysis. the second cycle of the therapy was performed as described for the first cycle. Cellular RIA. An indirect cellular RIA was used to determine the reactiv- For the combination therapy with CPA, 200 ␮g mAb and CPA (80 mg/kg of ities of antihuman EDG mAbs with murine endothelial cells. Details of the body weight) were given i.v. and i.p., respectively. mAbs were given i.v. for assay were described previously (31). Briefly, mAbs and an isotype-matched systemic therapy of tumors. However, CPA was given i.p. because CPA is a control IgG were individually incubated, in triplicate, with target cells in wells small molecule (i.e., Mr 279.1) and would be rapidly eliminated from circu- of 96-well microtiter plates at 4°C for varying periods of time. After the cells lation when mice receive it by bolus i.v. injections. CPA was given following 125 Ј were pelleted and washed three times, I- labeled F(ab )2 of affinity-purified an antiangiogenic schedule (33, 34). Administration of the mAb and CPA was goat antimouse IgG antibodies was added to the cells, and the reaction repeated as indicated in the corresponding figures in “Results.” Three control mixtures were incubated at 4°C for 1 h. After the cells were washed four times, groups received PBS, mAb, and CPA, respectively. the radioactivity in the washed cells was determined in a Cobra Series Auto- Follow-Up of Treatment Efficacy. During the treatment, mice were mon- gamma Counter (Packard Instrument Comp., Meriden, CT). itored daily for tumor and morbidity. Mice were weighed twice a week using Chimeric Human/Mouse Model. Fresh human neonatal foreskins (see an electronic balance (OHAUS Model GT210). Tumor size was measured above) were stored in sterile RPMI 1640 supplemented with 2% fetal bovine twice or three times a week using an electronic caliper (PRO-MAX 6 inch serum, 1% gentamicin, and 2.5 ␮g/ml fungizone. The tissues were used for caliper; Fowler Co., Newton, MA) that was connected to a computer using grafting on the same day as received. The grafting into SCID mice was OptoDemo software (Fowler Co.). The measured tumor diameters were con- 7847

verted to tumor volumes using Excel 97; the tumor volumes were calculated Antibody Avidity and Number of Available Epitopes on EDG- using the following formula (35): expressing Cells. Scatchard plot analyses of direct binding of radiolabeled SN6f, SN6j, and SN6k to EDG-expressing KM-3 leukemia ␲ V ϭ Length ϫ width ϫ height ϫ . cells and subconfluent proliferating HUVECs were carried out (Fig. 6 2). The results show equilibrium constants of 8.02 ϫ 109, 2.85 ϫ 109, ϫ 9 Statistical analysis of the data for the comparison of different groups of mice and 1.01 10 liters/mol, respectively, for SN6f, SN6j, and SN6k to 9 was carried out using Student’s t test. KM-3 cells. The equilibrium constants to HUVECs are 8.32 ϫ 10 , 3.08 ϫ 109, and 1.01 ϫ 109, respectively, for SN6f, SN6j, and SN6k. RESULTS The results show that these mAbs bind to KM-3 cells and HUVECs with very similar avidities. The three mAbs all show good binding Immunostaining of Blood Vessels in Tissues from Human Skin/ avidities, and the rank order of these mAbs for antibody avidity is SCID Mouse Chimeras. Vessels in the human skin grafts and the SN6fϾSN6jϾSN6k. In the same analyses of Scatchard plot, the adjacent mouse tissues were analyzed by immunohistochemical stain- average number of antibody molecules bound per KM-3 cell was ing with species-specific antiendothelial mAbs, i.e., SN6h (an antihu- estimated to be 1.25 ϫ 104, 1.75 ϫ 104, and 1.70 ϫ 104, respectively, man CD105 mAb), antihuman CD31 mAb, antimouse CD105 mAb, at antibody saturation. The number per HUVEC was estimated to be and antimouse CD31 mAb. The ratio of human vessels to mouse 1.56 ϫ 106, 1.13 ϫ 106, and 1.52 ϫ 106, respectively, for SN6f, SN6j, vessels in the completely healed human skin grafts and in the skin and SN6k. Because these mAbs (all IgG1) are bivalent antibodies, the grafts bearing large MCF-7 tumors was determined in serial sections average number of antigens on these cell specimens is likely to be 1- of the tissues by counting human vessels and mouse vessels. Human to 2-fold greater than the antibody number. The present results show vessels were detected by immunostaining the tissue sections with a that EDG is highly expressed on proliferating HUVECs, and they mixture of SN6h and antihuman CD31 mAb, and mouse vessels were express ϳ100-fold as many EDG molecules as do KM-3 cells. detected by immunostaining with a mixture of antimouse CD105 mAb Reactivities of Anti-EDG mAbs with Murine Endothelial Cells. and antimouse CD31 mAb. Blood vessels in the completely healed (8 An indirect cellular RIA was used to determine the reactivities of weeks after grafting) human skin without tumors consisted of 43.5% SN6f, SN6j, and SN6k with proliferating (subconfluent) SVEC4–10 human vessels and 56.5% mouse vessels. Blood vessels in the human murine endothelial cells by incubation for 2 or 24 h. The radioactiv- skin bearing a large established human tumor (250 mm3) consisted of ities (cpm) in the cells treated with SN6f, SN6j, SN6k and an isotype- 59.6% human vessels and 40.4% mouse vessels. Therefore, growth of matched control IgG (IgG1-␬) for 2 h were 414 Ϯ 46 (SD), 381 Ϯ 15, tumors in the human skin grafts depended on both murine and human 394 Ϯ 36, and 290 Ϯ 29, respectively. The radioactivities in the cells vessels. The results also indicate that human tumors stimulate human treated with the mAbs and control IgG for 24 h were 926 Ϯ 32, vessels preferentially, compared with murine vessels. Immunostaining 758 Ϯ 64, 662 Ϯ 30, and 456 Ϯ 60, respectively. In the same assay, of vessels was also performed using individual mAbs. The stained the radioactivities in proliferating HUVECs treated with SN6f and vessels in the human skin grafts and adjacent mouse skins are illus- control IgG for 2 h were 8,227 Ϯ 385 and 196 Ϯ 24, respectively. The trated in Fig. 1, A–D. In addition, vessels in MCF-7 tumors in the results show that the binding of the three anti-EDG mAbs to grafted human skins and the adjacent s.c. murine tissues are illustrated SVEC4–10 murine endothelial cells is weak but significant compared in Fig. 1, E and F). SN6h did not stain any vessels in the grafted with the binding of isotype-matched control IgG. The rank order of human skins or adjacent mouse skins (Fig. 1A). However, SN6h the three mAbs for reactivity with SVEC4–10 cells was stained multiple vessels in tumors in the human skins but not in the SN6fϾSN6j Ϸ SN6k. The reactivities of these mAbs with HUVECs adjacent murine s.c. tissues (Fig. 1E). The results show that SN6h is are much stronger than those with SVEC4–10 murine endothelial capable of distinguishing the tumor-induced angiogenic vessels from cells (see above and Ref. 15). the nonangiogenic vessels. In contrast, antihuman CD31 mAbs Suppression of Established Tumors by Systemic Administra- stained vessels in the tumor-free human skins (Fig. 1B) and also tion of Naked mAbs. MCF-7 human breast cancer cells (8 ϫ 106 vessels in the tumors in the grafted human skins (data not shown). It cells/mouse) were injected i.d. into human skins that had been grafted did not stain any vessels in the adjacent murine skins (Fig. 1B). to SCID mice, and the mice were left untreated until distinct palpable Antimouse CD105 mAbs stained vessels in the human skins as well as tumors appeared. Mice with distinct tumors were divided into groups in the adjacent murine skins (Fig. 1C). It also stained vessels in (n ϭ 6 or 5) and were treated by i.v. administration of PBS, an MCF-7 tumors in the human skins and vessels in the adjacent murine isotype-matched control IgG (MOPC 195 variant; IgG1-␬), SN6f, s.c. tissues (Fig. 1F). Thus, unlike SN6h, the antimouse CD105 mAb SN6j, SN6k, or SN6f plus SN6k (see “Materials and Methods” for was unable to distinguish the nonangiogenic vessels from the tumor- details). SN6f and SN6k define mutually distant epitopes (15). The induced angiogenic vessels. The staining pattern of antimouse CD31 results are presented in Fig. 3. Both SN6j and SN6k showed signifi- mAb is similar to that of antimouse CD105 mAb; the former stained cant antitumor efficacy, whereas SN6f was less effective. The results vessels in the human skins as well as in the adjacent murine skins (Fig. indicate that the antitumor efficacy of anti-EDG mAbs is not directly 1D). The described immunostaining pattern of human skin grafts and proportional to antigen-binding avidities of mAbs because SN6f the adjacent mouse tissues was consistently observed for the tissue shows the strongest antigen-binding avidity among the three mAbs to samples obtained 7, 8, 9, 11, 18, and 20 weeks after grafting of the both HUVECs and murine endothelial cells (see above). The observed human skins. Specificity of the antimouse CD105 and antimouse difference in the antitumor efficacy among SN6f, SN6j, and SN6k is CD31 mAbs for murine vessels was confirmed by immunohistochem- consistent with the difference among the dgRA conjugates of the three ical studies in which these mAbs did not react with any blood vessels mAbs (15). The results suggest the importance of other factors such as in human colon cancer tissues and human neonatal foreskins. The epitopes in the in vivo antitumor efficacy (see “Discussion”). The results show that the growth of human vessels is limited within the combination of SN6f and SN6k showed an additive effect. In addi- boundary of the grafted human skins, but murine vessels infiltrate into tional tests, the effect of the mAb dose on the antitumor efficacy was the grafted human skins from the surrounding murine tissues. There- investigated. An example of such tests is presented in Fig. 4. A 2-fold fore, murine vessels will be more functional for delivering nutrients increase of SN6k from 200 ␮gto400␮g resulted in a small increase and oxygen than human vessels in the human skin grafts. in the antitumor efficacy. Both 200 ␮g and 400 ␮g of SN6k were 7848

Fig. 1. Immunostaining of blood vessels in the human skin/SCID mouse chimeras using species-specific anti- EDG (CD105) and anti-CD31 mAbs. Serial sections were cut from frozen tissues containing the human-mouse skins junction region (A–D; ϫ100). Tissue sections were immunostained with SN6h [an antihuman CD105 mAb (A)], antihuman CD31 mAb (B), antimouse CD105 mAb (C) and antimouse CD31 mAb (D). m, mouse skin; h, human skin. Mouse skin can be distinguished from human skin by the thin epidermis and hair follicles of the mouse skin. SN6h did not react with any blood vessels in either human or mouse skin (A). Antihuman CD31 mAbs stained numerous vessels in the human skin but not in the adjacent murine skin; arrows, a few examples of the stained human vessels (B). Antimouse CD105 mAbs (C) and antimouse CD31 mAbs (D) stained numerous vessels in both human skin and adjacent murine skin; arrowheads, examples of the stained mouse vessels (C and D). In an additional study, serial sections were cut from frozen tissues of the grafted human skins bearing large (250 mm3) MCF-7 human tumors (E and F; ϫ200). SN6h stained multiple vessels in the tumor in the human skin but not in the adjacent murine s.c. tissue (E). Antimouse CD105 mAbs stained multiple vessels in the tumor in the human skin and the murine s.c. tissue (F). Arrows (E), several examples of stained human vessels; arrowheads (F), several examples of stained mouse vessels. Isotype-matched murine control IgG (IgG1-␬) and isotype-matched rat control IgG (IgG2a-␬) showed no significant staining. The staining patterns were similar to that in A.

effective for tumor suppression compared with 400 ␮g of an isotype- Synergistic Antitumor Efficacy by Combination of a mAb with matched control IgG. A similar test was performed with SN6j (Fig. 5). a Chemotherapeutic Drug. To improve antitumor efficacy of the Both 200 ␮g and 400 ␮g of SN6j were effective for tumor suppression naked anti-EDG mAb, SN6j was combined with CPA that was ad- compared with the isotype-matched control IgG. The difference in the ministered i.p. into tumor-bearing chimeras using an antiangiogenic antitumor efficacy between 200 ␮g and 400 ␮g of SN6j was small. schedule of drug dosing (33, 34) or, in another term, a metronomic The results show that naked anti-EDG mAbs can suppress growth of dosing regimen (36). MCF-7 tumors in human skin grafts in SCID established tumors. mice were established as described above. Chimeras with established 7849

average size of tumors was 63.8, 57.5, 54.6, and 97.2 mm3, respectively, for Figs. 3, 4, 5, and 6. Both SN6j and CPA are effective for tumor suppression. Difference in the tumor size between the control PBS group and the SN6j/CPA-treated group is statistically significant (P Ͻ 0.04) after day 45 and until the end of experiment, i.e., day 66. The potential synergy between two antitumor agents was evaluated as described by others (e.g., Ref. 37). Table 1 summarizes relative tumor volume of control and treated groups on five different time points during the therapy. Combination therapy showed more than additive effect on tumor growth suppression. On day 24, 9 days after therapy initiation, there was a slight improvement in antitumor activity in the combination group when compared with the expected additive effect. This improvement became greater during the course of therapy. On days 34 and 45, there were 1.5-fold and 1.6-fold improvements, respectively, in the combination group compared with the expected additive effect (Table 1). This supraadditive effect of the combination therapy persisted until the end of the experiment (i.e., day 66). The results demonstrate that SN6j and CPA exert synergistic antitumor efficacy in the present model. Small decreases in the body weight were observed for the chimeras treated with CPA and CPA plus SN6j; the decreases were within 10% of the control. Therapeutic effect on individual chimeras in each group of Fig. 6 is presented in Fig. 7.

Fig. 2. Binding of 125I-labeled SN6f, SN6j, and SN6k to EDG-expressing KM-3 cells (A) and HUVECs (B). r is the number of antibody molecules bound to one cell at a given dilution, A is the molar concentration of total antibody, x is the molar concentration of bound antibody, so that (A Ϫ x) is the molar concentration of the free antibody. The slope of the binding curve gives the equilibrium constant, K. The intersection point between the binding curve and abscissa gives the maximum number of antibody molecules, n, that can be bound to a single cell. The equilibrium constant for the reaction, K, is expressed in liters per mole.

Fig. 4. Effect of different doses of SN6k on the antitumor efficacy. Established tumors in human skin/SCID mouse chimeras were generated as in Fig. 3. Groups of chimeras (n ϭ 6 in each group) were treated by i.v. administration of an isotype-matched control IgG (400 ␮g), 200 ␮g SN6k, or 400 ␮g SN6k. Injections of the control IgG and SN6k were repeated as in Fig. 3.

Fig. 3. Therapy of established tumors in human skin/SCID mouse chimeras by i.v. administration of naked anti-EDG mAbs. MCF-7 human breast cancer cells were injected into human skin grafts in SCID mice, and therapy was delayed until distinct palpable tumors appeared. Chimeras with distinct tumors were divided into groups [n ϭ 6 except for PBS (n ϭ 5) and MOPC (n ϭ 5) groups] and treated with PBS, an isotype-matched control IgG (MOPC), SN6f, SN6j, SN6k, or SN6f plus SN6k. The therapy was performed in two cycles including 1-week pause between the cycles to relieve the mice from the i.v. injection-associated stress. Each cycle consisted of five injections, at 2-days intervals, of PBS, a control IgG (200 ␮g), a mAb (200 ␮g), or a mixture of SN6f (200 ␮g) and SN6k (200 ␮g) as indicated in the figure. Arrows in the figure, injections of PBS, a control IgG, a mAb, or a mixture of two mAbs. tumors were distributed into four groups (n ϭ 8). Individual groups were treated by systemic administration of PBS, SN6j, CPA, or SN6j plus CPA. The results are presented in Fig. 6. It should be noted that Fig. 5. Therapy of preformed tumors by systemic administration of different doses of SN6j. Three groups of chimeras bearing established tumors (n ϭ 6) were treated by i.v. at the onset of therapy, the average size of tumors in the present set of administration of 400 ␮g of an isotype-matched control IgG, 200 ␮g SN6j, or 400 ␮g experiments is larger than those presented in Figs. 3, 4. and 5; the SN6j. Arrows, injections of control IgG or SN6j. 7850

Growth rate of MCF-7 tumors in the human skin/SCID mouse chi- Table 1 Combination therapy with SN6j and CPA meras was substantially heterogeneous before the onset of the therapy. Fractional tumor volume (FTV)b relative to untreated controls This is in contrast to the more homogeneous growth of the MCF-7 Combination treatment tumors in SCID mice (e.g., Ref. 15). The major reason for this Ratio of expected: heterogeneous growth in the chimeras may be the wider genetic Daya SN6j CPA Expectedc Observed observed FTVd heterogeneity of the human skins compared with SCID mice. To 24 0.824 0.867 0.714 0.651 1.097 34 0.962 0.873 0.840 0.555 1.514 minimize the effect of the heterogeneous tumor sizes on the experi- 45 0.929 0.827 0.768 0.474 1.620 ment, chimeras with tumors of a similar size were distributed nearly 55 0.763 0.702 0.536 0.435 1.232 evenly into different groups. Consequently, the average size of tumors 66 0.743 0.739 0.549 0.426 1.289 in each group became similar among the four groups at the onset of a Day after tumor cell inoculation. Tumor volume was measured twice or three times a week for each mouse (n ϭ 8 for each group of mice). therapy (see Fig. 6). Tumors in 8 chimeras of the control PBS group b FTV, calculated as mean tumor volume experimental/mean tumor volume control. all continued growing, although the growth rate of one tumor was c (Mean FTV of SN6j) ϫ (mean FTV of CPA). substantially slower than others (Fig. 7A). Treatment with SN6j d Obtained by dividing the expected FTV by the observed FTV. A ratio of Ͼ1 indicates a synergistic effect, and a ratio of Ͻ1 indicates a less than additive effect. slowed tumor growth compared with the control group, and a small tumor in one chimera regressed completely (Fig. 7B). CPA therapy using an antiangiogenic schedule slowed the growth of five smaller We immunostained human and murine blood vessels in the large established tumors and induced near regression of one of the tumors. human tumors from the chimera mice after the mice were killed at the However, CPA therapy showed little therapeutic effect on the three end of experiment shown in Fig. 7 (see “Materials and Methods” for large tumors of more than 100 mm3 at the onset of therapy (Fig. 7C). details). Number of murine vessels in the tumors was 52, 47, 47, and Treatment with SN6j and CPA using an antiangiogenic schedule of 34 per 100 ϫ field for groups A, B, C, and D, respectively (see Fig. drug dosing prevented five smaller established tumors from increasing 7 for the groups). Therefore, SN6j (group B) and CPA (group C) in size and induced lasting complete regression of two of the tumors caused only a small decrease (ϳ10%) in the number of murine vessels (Fig. 7D). An additional tumor regressed after day 37 but relapsed in the tumors compared with the control (group A). However, com- soon after the regression. The combination therapy prevented one of bination of SN6j and CPA (group D) caused a larger (i.e., 35%) the three large tumors of more than 100 mm3 from increasing in size decrease in the number of murine vessels. The number of human soon after the initiation of therapy, and the tumor remained stable vessels in the tumors was 33, 0, 36, and 0 per 100 ϫ field for groups during and after the therapy (Fig. 7D). Two of the large tumors A, B, C and D. Therefore, SN6j completely suppressed human vessels continued growing initially but became stable after three injections of in the tumors, whereas CPA was not effective for suppressing human SN6j and CPA; these tumors restarted expanding after cessation of vessels in the tumors. The results show that SN6j is highly effective therapy. The present results show that the combination of SN6j and for suppressing human vessels but only weakly suppressive against CPA is most effective for treating both small and large established murine vessels. CPA was not effective for inhibiting human vessels tumors. The results also indicate that the initial size of the established and only weakly suppressive against murine vessels. However, com- tumors is an important factor for the outcome of the therapy. The bination of SN6j and CPA showed a stronger suppression of murine anti-EDG mAbs, and the combination of an anti-EDG mAb and CPA vessels and was effective in eliminating human vessels in the tumors. should be much more effective for tumor suppression in patients than We would like to add a cautionary note that the tumors used in the in the human skin/SCID mouse chimeras (see “Discussion”). present vessel counting were obtained from chimera mice whose therapy was terminated 3 weeks before the mice were killed and tumors were resected. Therefore, the status of murine and human vessels in these tumors may not be the same as that in the tumors from mice receiving therapy. Nevertheless, the present test reveals different effects of SN6j, CPA, and SN6j plus CPA on tumor vasculature.

DISCUSSION

Antiangiogenic therapy of cancer is highly attractive for several reasons (reviewed in Refs. 38–40). For instance, it can potentially overcome three major problems associated with other anticancer ther- apies, i.e., the problems of drug resistance (41, 42), poor delivery (43, 44), and tumor heterogeneity. One approach to the antiangiogenic therapy is antibody-based targeting of tumor vasculature. We have been targeting EDG on tumor vasculature in vivo (9, 15, 16). Several features of EDG are described above (see “Introduction”). In addition, our anti-EDG mAbs showed little reactivity with normal human bone marrow cells (1). This observation is consistent with our later finding that anti-EDG immunotoxin (i.e., ricin A-chain conjugate of SN6) Fig. 6. Improved antitumor efficacy by the combination of an anti-EDG mAb with a selectively eradicated EDG-expressing cells without severely damag- chemotherapeutic drug and by using an antiangiogenic schedule of drug dosing. Estab- lished tumors of MCF-7 in human skin/SCID mouse chimeras were generated as de- ing normal human hematopoietic progenitors in the colony-forming scribed in Figs. 3, 4, and 5. Groups of chimeras bearing distinct tumors (n ϭ 8 in each unit assays.4 The results suggest that administration of therapeutic group) were treated by administration of PBS, SN6j, CPA, or SN6j plus CPA. CPA (80 doses of an appropriate anti-EDG mAb and immunotoxin may not mg/kg body weight) was injected i.p. every 4 days by using an antiangiogenic schedule (33, 34). SN6j (200 ␮g) was administered i.v. every 3 days for the first five injections and significantly damage hematopoietic progenitors in patients. Neverthe- difference in the tumor size between the less, anti-EDG immunotoxins showed significant toxicity in mice (see ,ء .every 4 days for the remaining five injections PBS group and the SN6j ϩ CPA group is statistically significant (P Ͻ 0.04) after day 45 and until the end of the experiment. Solid arrows, injections of SN6j; dashed arrows, injections of CPA. 4 Y. Haruta, D. Chervinsky, and B. K. Seon. Manuscript in preparation. 7851

Fig. 7. Tumors in individual human skin/SCID mouse chimeras that were treated with PBS, SN6j, CPA, or SN6j plus CPA. Growth rate of MCF-7 tumors in human skin grafts in individual SCID mice varied substantially before initiation of the therapy. Therefore, chimeras with tumors of a similar size were distributed nearly evenly into different groups at the onset of therapy. Consequently, the average size of tumors in each group became similar at the onset of therapy (see Fig. 6). Arrow- heads, injections of PBS; solid arrows, injections of SN6j; dashed arrows, injections of CPA. It should be noted that injections of PBS in A were performed i.v. (upper line) and i.p. (lower line)as controls of SN6j and CPA, respectively. PBS was injected i.p. as a control of CPA in B, and it was injected i.v. as a control of SN6j in C.

“Introduction”). In contrast, no toxicity was detected for naked anticancer, respectively. In view of the observation that the growth of EDG mAb SN6j in a dose-escalation study; the maximum tolerated human vessels was limited to remain within the boundary of human dose was more than 0.344 mg/g body weight of mice.5 skin and that murine vessels infiltrated into human skin from the Previously, Yan et al. (22) developed human skin/SCID mouse surrounding murine tissues, murine vessels will be more functional for chimeras and immunostained blood vessels in a human skin graft and delivering nutrients and oxygen than human vessels in the human skin adjacent murine skin using antimouse PECAM (CD31) mAb, antihu- grafts. man CD31 mAb, and antihuman CD31 polyclonal antibodies. In the Growth rate of MCF-7 tumors in the chimeras is more heteroge- immunostaining, they used human skin that had been transplanted 4 neous than that of MCF-7 tumors in SCID mice (e.g., Ref. 15). The weeks earlier. They detected only human vessels in human skin and heterogeneous growth pattern of human tumors in the chimeras ap- only mouse vessels in the adjacent mouse skin. We immunostained pears to represent tumor growth in patients better than the homoge- blood vessels in several tissue samples from human skin/SCID mouse neously growing tumors in immunodeficient mice. The major reason chimeras whose human skins had been transplanted 7, 8, 9, 11, 18, and for this heterogeneity in the chimeras may be attributable to the 20 weeks earlier. We used antimouse CD105 mAb and SN6h, an genetic heterogeneity of the human skins compared with SCID mice antihuman CD105 mAb, in addition to antimouse CD31 and anti- used for tumor inoculation. Another reason may be that the time human CD31 mAbs in the immunostaining. All of these mAbs are interval between skin grafting and tumor inoculation was variable. species specific (e.g., see “Results”). Both antimouse CD105 mAb This variation occurred because the supply of human skins was scarce and antimouse CD31 mAb consistently stained vessels in human skins and unpredictable. Therefore, a substantial period of time was needed that had been transplanted 7–20 weeks earlier (e.g., see Fig. 1, C and to accumulate enough chimeras for starting an experiment in which D). Antihuman CD31 mAb stained vessels in human skins but not in the grafted human skins were inoculated with tumor cells. adjacent murine skins. These results, showing restricted growth of Despite this problem of heterogeneity, systemic (i.v.) administra- human vessels, are consistent with the observation of Yan et al. (22). tion of naked anti-EDG mAbs SN6j and SN6k consistently showed SN6h stained only tumor-induced angiogenic human vessels (see Fig. significant antitumor efficacy compared with an isotype-matched con- 1). Results of Yan et al. (22) and present studies suggest that the trol IgG and/or PBS. The results showed that naked anti-EDG mAbs infiltrating murine vessels in human skin grafts become immunohis- can suppress established tumors. SN6f that showed the highest anti- tochemically detectable between 4 and 7 weeks after transplantation gen-binding avidity among the three anti-EDG mAbs was less effec- of human skins. Alternatively, the observed difference in the infiltrat- tive for tumor suppression than were SN6j and SN6k. These results ing murine vessels may be attributable to difference in the sensitivity are consistent with our earlier observation that SN6j-dgRA and SN6k- of the immunostaining reagents and/or methods. Despite these ques- tions, the present results demonstrate that human skin grafts contain dgRA were more effective for suppressing established tumors than both human and murine vessels during growth of inoculated tumors. was SN6f-dgRA (15). The results indicate an absence of a direct We inoculated MCF-7 human breast cancer cells into the completely correlation between antigen-binding avidity and in vivo antitumor healed human skin grafts 7 or more weeks after the skin was trans- efficacy for anti-EDG mAbs. Although antigen-binding avidity of a planted. Previously, Juhasz et al. (23) and Brooks et al. (24) success- mAb may still be an important factor for antitumor efficacy, other fully used the chimeras to grow human melanomas and human breast factors such as epitope and specificity may also be important deter- minants involved in antitumor efficacy. SN6f defines an epitope

5 N. Takahashi, A. Haba, M. Tabata, M. Kondo, and B. K. Seon. Manuscript in distinct from those defined by SN6j and SN6k (15). Our recent study preparation. revealed that SN6f epitope resides in peptide 250–305 of EDG 7852

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2001 American Association for Cancer Research. EDG-TARGETED ANTIANGIOGENIC THERAPY molecule, whereas SN6j epitope is in peptide 204–250.6 The molec- creases in the body weight (Ͻ10%) of the chimeras were detected. ular location of SN6k epitope remains to be determined. Concerning The results suggest superiority of the combination therapy over a specificity of the mAbs, SN6j and SN6k showed selective reactivity single-agent therapy, either with CPA or SN6j alone. These results with human tumor vasculature compared with normal human tissue appear to warrant additional studies of the combination therapy. vasculature (15). This selectivity was less distinct for SN6f.7 Effects of TGF-␤ on cancer are complex (reviewed in Refs. 50– Although expression of EDG is highly restricted among different 53). Although TGF-␤ is a potent growth inhibitor in epithelial tissues, tissues and cells in vivo and certain anti-EDG mAbs react with it is both a suppressor and a promoter of tumorigenesis. Recently, tumor-associated vascular endothelium more strongly than with vas- Ananth et al. (54) reported that the neutralizing antibody against cular endothelium in normal tissues (see “Introduction”), EDG is not TGF-␤ inhibited tumorigenesis and, in some cases, regressed estab- a tumor-specific marker and EDG is expressed in varying degrees in lished 786–0 renal cell carcinoma tumors in athymic mice. On the the vasculature of normal tissues. Despite this limitation, we could other hand, Gohongi et al. (55) reported that treatment with neutral- effectively target tumor-associated vasculature using selected anti- izing antibody against TGF-␤ reversed both angiogenesis suppression EDG mAbs and immunoconjugates (9, 15, 16, and this report). We and inhibition of leukocyte rolling induced by gallbladder tumors in believe that this effective targeting of tumor-associated vascular en- SCID mice. Our recent studies suggest that multiple mechanisms are dothelium is attributable to the combined effect of the following: (a) involved in the suppression of tumors by naked anti-EDG mAbs. certain anti-EDG mAbs show a highly restricted reactivity among However, the mechanisms remain to be defined further. different tissues and cells; (b) EDG is a proliferation-associated antigen on endothelial cells and its expression is up-regulated in tumor- ACKNOWLEDGMENTS associated vascular endothelium (8–10, 12–15); and (c) turnover of endothelial cells of normal adult tissue vasculature is very slow (e.g., We thank Hilda Tsai for expert technical assistance, John Parsons and Dr. more than 1,000 days), whereas these endothelial cells undergo rapid William Greco for expert help in establishing an automated system for meas- proliferation during spurts of angiogenesis in tumors (39, 45, 46). uring tumor size, and Dr. Joseph Krasner for help in the preparation of the Therefore, the rapidly dividing endothelial cells of tumor vasculature manuscript. are much more susceptible to killing by anti-EDG mAbs and immunoconjugates than the quiescent vascular endothelium of normal tis- REFERENCES sues. 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Norihiko Takahashi, Akinao Haba, Fumihiko Matsuno, et al.

Cancer Res 2001;61:7846-7854.

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