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Abstract. ING-1(heMAb), a human-engineered monoclonal anti- body (MAb) that specifically targets the epithelial cell adhesion molecule (Ep-CAM), kills ...
RESEARCH ARTICLE

Neoplasia . Vol. 5, No. 6, November/December 2003, pp. 489 – 494

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ING-1(heMAb), a Monoclonal Antibody to Epithelial Cell Adhesion Molecule, Inhibits Tumor Metastases in a Murine Cancer Model Harry H. Ruan *, Kristen R. Scott *, Eddie Bautista y and W. Steve Ammons * *Department of Pharmacology and y Department of Molecular Immunology, XOMA (US) LLC, Berkeley, CA 94710, USA Abstract ING-1(heMAb), a human-engineered monoclonal antibody (MAb) that specifically targets the epithelial cell adhesion molecule (Ep-CAM), kills adenocarcinoma cells in vitro and inhibits tumor growth in vivo. In the current study, we evaluated the efficacy of ING-1(heMAb) in a murine model of cancer metastases. Mice received intravenous dosing of 1 mg/kg ING-1(heMAb), twice a week, starting on day 2 or day 5. A negative control group received 1 mg/kg human immunoglobulin G with the same dose frequency starting on day 2. A positive control group received weekly 100 mg/kg 5-flurouracil/leucovorin starting on day 2. ING-1(heMAb)/day 2 treatment significantly reduced both the number of visible tumor nodules in body cavities ( P < .01) and the number of metastases on lung surfaces ( P < .005). The treatment also resulted in a 91% reduction of micrometastases in lung tissues ( P < .0001). Delaying ING-1(heMAb) treatment until day 5 caused 54% reduction in micrometastases ( P < .005). Our results indicate that a number of parameters, including treatment starting day, dose level, and dose frequency, are critical in achieving the optimal efficacy of ING-1(heMAb). We conclude that ING-1(heMAb) effectively reduced tumor metastases in a murine cancer model. Immunotherapy with ING-1(heMAb) may be beneficial in treating human metastatic diseases. Neoplasia (2003) 5, 489 – 494 Keywords: Metastases, Ep-CAM, micrometastases, cancer model, ING-1(heMAb).

Introduction Epithelial cell adhesion molecule (Ep-CAM) is a 40-kDa glycoprotein, which is overexpressed on the membranes of most adenocarcinoma cells, some squamous cell carcinomas, and renal cell carcinoma cells [1 – 3]. Ep-CAM functions as a homophilic adhesion molecule that promotes weak, flexible, intercellular interactions and is thought to play a physiological role in morphogenesis and embryonic development [4]. Although the exact function of Ep-CAM during tumorigenesis, tumor invasion, and metastasis is still unknown, increased expression of Ep-CAM is associated with active proliferation of epithelial cells and has been reported to correlate with malignant progression of tumors [5,6].

Overexpression of Ep-CAM is detected in a wide variety of adenocarcinomas, including the colorectum, prostate, pancreas, lungs, and breasts [1]. Clinically, Ep-CAM has long been recognized as a valid tumor target for monoclonal antibody (MAb) – based cancer immunotherapy. For example, a murine anti – Ep-CAM antibody called edrecolomab was among the first MAbs to enter clinical trials for treatment of Duke’s stage C colon cancer [7]. Overall, previous studies have demonstrated that Ep-CAM is a validated target for passive immunotherapy. MAbs that recognize tumor-associated antigens are increasingly used for treatment of cancer and metastasis. The therapeutic MAb used in the present study, ING-1(heMAb), is a human-engineered immunoglobulin G (IgG)-1 MAb that binds the Ep-CAM antigen with high affinity [8]. Human engineering technology uses a unique residue-by-residue analysis to convert a nonhuman antibody variable region to human and permits direct construction of a fully active human chimeric antibody [8]. As a result of the human engineering process, ING-1(heMAb) has minimal immunogenicity in humans [9] and still retains the antigen-binding specificity of its murine counterpart, Br-1 [10,11]. Br-1 is a mouse antitumor antibody that was originally generated against breast carcinoma tissues [12]. In addition, ING-1(heMAb) has a half-life of 10 to 20 days in animal studies, comparable to that of a native IgG [13]. In addition, ING-1(heMAb) half-life in humans was between 16 and to 35 hours, as determined in a Phase I dose-escalating study [9]. These properties make ING-1(heMAb) an excellent candidate for clinical development as a cancer therapeutic. We have previously demonstrated that ING-1(heMAb) can promote killing of human tumor cells in the presence of immune effector cells [13]. The main mechanism underlying the antitumor effect of ING-1(heMAb) has been suggested as antibody-dependent cell-mediated cytotoxicity (ADCC) [14]. In addition, ING-1(heMAb) significantly inhibited the establishment of tumor xenografts [13]. However, MAbs targeting EpCAM in general lack the efficacy to eradicate large tumor masses. We therefore postulated that ING-1(heMAb) would

Abbreviations: 5-FU, 5-fluorouracil; ADCC, antibody-dependent cell-mediated cytotoxicity; Ep-CAM, epithelial cell adhesion molecule; IgG, immunoglobulin G; i.v., intravenous; LV, leucovorin; MAb, monoclonal antibody Address all correspondence to: Harry H. Ruan, Department of Pharmacology, XOMA (US) LLC, 2910 7th Street, Berkeley, CA 94710, USA. E-mail: [email protected] Received 29 July 2003; Revised 1 October 2003; Accepted 8 October 2003. Copyright D 2003 Neoplasia Press, Inc. All rights reserved 1522-8002/03/$25.00

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be most effective when used to eliminate metastases and residual tumor cells. In the current study, we examined the efficacy of ING-1(heMAb) in treating tumor metastases in a murine cancer model. In this model, human colon cancer cells (HT-29) were injected intravenously into nude mice, resulting in establishment of visible tumor nodules in body cavities and formation of metastases on lung surfaces and micrometastases within lung tissues. ING-1(heMAb) treatment was found to be effective in inhibiting the growth of small tumor nodules, metastases, and micrometastases. The efficacy of ING-1(heMAb) was especially pronounced against formation of micrometastases. In most types of solid cancers, the disseminated metastatic disease, rather than the primary lesion, is the cause of death of the majority of patients. Therefore, we believe the potential application of ING-1(heMAb) in treating cancer metastases offers promise for therapeutic outcome for cancer patients.

Materials and Methods Cell Line and Mice The human colon adenocarcinoma cell line, HT-29, was purchased from the American Type Culture Collection (Manassas, VA). Cells were maintained as monolayers in Eagle’s defined minimal essential medium (DMEM) supplemented with 10% fetal bovine serum, sodium pyruvate, and nonessential amino acids, and incubated at 37jC with 5% CO2. Female NCr nu/nu mice (Simonsen Laboratory, Gilroy, CA), aged 6 to 7 weeks and weighing 20 to 22 grams, were used. All animals were maintained in a sterile environment. Cages, bedding, food, and water were all autoclaved, and animals were handled in a laminar flow hood. They were housed in an animal facility approved by the American Association for Accreditation of Laboratory Animal Care. The animal room was maintained on daily 12-hour light/12hour dark cycle. All animal studies were conducted under the guidelines of the Institutional Animal Care and Use Committee (IACUC). All animal study protocols have been approved by the IACUC. Reagents and Drugs ING-1(heMAb) was manufactured at XOMA (US) LLC (Berkeley, CA). Human IgG (in lyophilized form), 5-flurouracil (5-FU), leucovorin (LV), and Bouin’s solution were purchased from Sigma (St. Louis, MO). ING-1(heMAb) was supplied as 5 mg/ml in a formulation buffer (20 mM sodium phosphate, 0.15 M sodium chloride, 0.005% polysorbate 80, pH 7.2). Prior to study initiation, human IgG was reconstituted to appropriate concentrations in the ING-1(heMAb) formulation buffer, and 5-FU and LV were diluted in phosphate-buffered saline (PBS) solution. All reagents and drugs were kept at 2jC to 8jC during the study period. Experimental Metastasis Model in IL-1 – Pretreated Athymic Nude Mice Female athymic mice were injected through the lateral tail vein with PBS containing 0.5 mg of recombinant human IL-1b

(purity 97%, ED50 5 – 10 pg/ml in D10 assay; R&D Systems, Minneapolis, MN) in order to increase the efficiency of tumor metastasis in lung tissues [15,16]. Two to 3 hours later, single-cell suspensions of HT-29 cells (1.5  107 cells/ml in DMEM) were injected through the intravenous route (0.2 ml per mouse). On day 2, mice were randomly divided into various groups (10 per group). Mice received ING-1(heMAb) once or twice weekly, intravenously, for 3 weeks starting on either day 2 or day 5. A negative control group received 1 mg/kg human IgG twice weekly for 3 weeks, starting on day 2. In the positive control group, 100 mg/kg 5-FU/LV was injected intraperitoneally once weekly for 4 weeks, starting on day 2. At the end of the study (8 weeks), all mice were sacrificed and subjected to necropsy and tissue collection. Visible tumor nodules (> 3 mm in diameter) throughout animal body cavities were examined and counted during necropsy. Lung tissues were dissected and fixed in a neutralbuffered formalin/Bouin’s fixative solution (4:1 vol/vol) for 18 to 24 hours followed by changing into 70% ethanol. The number of tumor nodules on lung surfaces was counted under a dissecting microscope. Histology Lung tissue samples were sent to IDEXX Laboratories (West Sacramento, CA) for histological analysis. Trimmed tissues were processed by dehydration, paraffin embedding, and sectioning. From each lung tissue, five step sections (7 mm thick each), separated by ~250 mm, were stained with hematoxylin and eosin. Sections were examined by a pathologist and the number of micrometastasis foci was counted. Statistical Analysis The ANOVA post hoc Bonferroni/Dunn test was used to compare the results on visible tumor nodules, tumor metastases, and micrometastases. The analyses were performed between the IgG group and various study groups. A P value < .05 was considered to be statistically significant. Unless otherwise noted, all data are presented as mean ± SE.

Results ING-1(heMAb) Eliminated Visible Tumor Nodules in Body Cavities of Some Animals At the end of the study, all animals were humanely sacrificed and necropsy was performed. To determine whether ING-1(heMAb) was effective in reducing the size of large, well-established metastases, visible tumor nodules (diameter z 3 mm) inside the cavities of animal bodies were examined and counted. Due to difficulties in counting some individual nodules, values for each animal were determined based on a scoring system, as defined in Table 1. As shown in Table 1A, 90% of animals in the IgG group developed tumor nodules, with 50% of them scoring more than 20 nodules. ING-1(heMAb), 1 mg/kg, starting on day 2 resulted in significant reduction of visible tumor nodules in the body cavities; 70% of the mice were tumor-free at the study

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termination (at 8 weeks, P < .01, versus IgG group). 5-FU/LV, 100 mg/kg, starting on day 2 prevented 50% of animals from developing visible tumors ( P < .01, versus IgG group). ING-1(heMAb) treatment, starting on day 5, did not significantly reduce the incidence or the number of tumor nodules. In a second study, similar results were obtained with ING-1(heMAb) at 1 mg/kg (data not shown). Furthermore, as shown in Table 1B, ING-1(heMAb), starting on day 2, remained effective at 0.3 mg/kg, preventing 50% of the mice from developing visible tumors ( P < .01, versus IgG group). However, the efficacy of ING-1(heMAb) at 0.3 mg/kg was diminished when the dose frequency was reduced from twice a week to once a week (Table 1B).

Reduction of Tumor Metastases on Lung Surfaces in Mice Treated with ING-1(heMAb) After necropsy, organs including the lungs, liver, spleen, and kidneys were collected and fixed in diluted Bouin’s solution. To evaluate the efficacy of ING-1(heMAb) against metastases on organ surfaces, tissues were examined and metastases were counted under a dissecting microscope. No metastases were detected on the liver, spleen, or kidneys in any group (data not shown). In contrast, a

Table 1. Visible Tumor Nodules During Necropsy. A IgG

ING-1/d2y

ING-1/d5

5-FU/LVy

1 mg/kg

1 mg/kg

1 mg/kg

100 mg/kg

+++ +++ +++ +++ +++ ++ ++ + + 0

++ ++ + 0 0 0 0 0 0 0

+++ +++ +++ +++ +++ ++ ++ ++ + 0

+++ ++ ++ + + 0 0 0 0 0

Animal

IgG

ING-1/2  wy

ING-1/1  w

1 mg/kg

0.3 mg/kg

0.3 mg/kg

1 2 3 4 5 6 7 8 9 10

+++ +++ +++ ++ ++ ++ ++ ++ ++ +

+ + + + + 0 0 0 0 0

+++ ++ ++ ++ ++ ++ + + + +

Animal*

1 2 3 4 5 6 7 8 9 10 B

In each study group, animals were listed based on the order of the number of visible tumor nodules, from the highest to the lowest. In both panels, 0: no tumor; (+) 1 to 10 tumor nodules; (++) 11 to 20 tumor nodules; (+++) > 20 tumor nodules. IgG: IgG starting on day 2. In panel (A), ING-1/d2: ING-1(heMAb) starting on day 2; ING-1/d5: starting on day 5; 5-FU/LV: 5FU/LV starting on day 2. In panel (B), ING-1/2  w: ING-1(heMAb)/day 2, twice weekly; ING-1/1  w: ING-(heMAb)/day 2, once weekly. *Mice received 3  106 HT-29 cells intravenously on study day 1. y P < .01, versus IgG group.

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significant number of metastases were observed on lung surfaces in the IgG control group (Figure 1). As shown in Figure 1A, 1 mg/kg ING-1(heMAb)/day 2 treatment resulted in 86% reduction of the average number of metastases when compared with the IgG group (from 12.1 to 1.7) ( P < .005, versus IgG group). However, when ING-1(heMAb) treatment was delayed until day 5, no significant reduction of metastasis nodules was observed (Figure 1A). 5-FU/LV, 100 mg/kg, starting on day 2 also resulted in 79% of metastases reduction (from 12.1 to 2.5) ( P < .005, versus IgG group). In a second study, similar results were obtained with ING-1(heMAb) at 1 mg/kg (data not shown). As shown in Figure 1B, ING-1(heMAb)/day 2 treatment at 0.3 mg/kg still inhibited tumor metastases by 89% (from 21.3 to 2.3) ( P < .005, versus IgG group). When the dose frequency was reduced to once weekly, 0.3 mg/kg ING-1(heMAb) inhibited metastases by 46%; however, the inhibition failed to reach statistical significance (Figure 1B). Inhibition of Micrometastases Formation in Lung Tissues by ING-1(heMAb) After being examined under a dissecting microscope, lung tissues from all animals were processed into slides and stained with hematoxylin and eosin for cell morphology. The slides were examined by a pathologist in a blind fashion to determine the total number of micrometastasis foci from each lung section. Examination of lung sections for animals treated with ING-1(heMAb) demonstrated that micrometastases were reduced in all three treatment groups compared to the IgG group. As shown in Figure 2A, compared with the IgG group, 1 mg/kg ING-1(heMAb)/day 2 treatment reduced the average number of micrometastasis foci by 91% (from 17.3 to 1.6) ( P < .0001, versus IgG group). 5-FU/LV, 100 mg/kg, resulted in a 76% decrease of the number of foci (from 17.3 to 4.1) ( P < .0001, versus IgG group). ING1(heMAb) treatment starting on day 5 also resulted in an inhibitory effect against micrometastases, reducing the foci number by 54% (17.3 to 8.0) ( P < .005, versus IgG group). In a second study, similar results were observed with ING1(heMAb) at 1 mg/kg (data not shown). In contrast to the results obtained on visible tumor nodules and tumor metastases, ING-1(heMAb)/day 2 treatment at 0.3 mg/kg, either twice a week or once a week, was effective in inhibiting micrometastases. As shown in Figure 2B, twice weekly dosing led to 84% reduction of micrometastases ( P < .0001, versus IgG group), whereas once weekly dosing resulted in a 73% reduction ( P < .0001, versus IgG group).

Discussion The dissemination of cancer metastases remains the principal cause of death in cancer patients. Despite the lethal nature of cancer, to date, no effective treatment is available to counter its progression, and prognosis for patients with metastatic diseases remains very poor [17]. Innovative therapeutic strategies are needed in order to effectively treat metastases and to prolong patients’ lives. Therapeutic MAbs, either alone or conjugated with cytotoxic agents, have

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Figure 1. ING-1(heMAb) treatment on day 2 reduces number of tumor metastases on lung surfaces. Upon necropsy, lung tissues from all mice were dissected and fixed in diluted Bouin’s solution. Each lung sample was examined under a dissecting microscope and tumor metastases were identified and counted. The column represents the average number of tumor metastases based on 10 mice per group. The error bar is standard error. In both panels, IgG: IgG starting on day 2. In panel (A), ING-1/d2: ING-1(heMAb) starting on day 2; ING-1/d5: ING-1(heMAb) starting on day 5; 5-FU/LV: 5-FU/LV starting on day 2. In panel (B): ING-1/2W: ING-1(heMAb) at 0.3 mg/kg treated twice weekly; ING-1/1W: ING-1(heMAb) at 0.3 mg/kg treated once weekly. **P < .005 (ANOVA post hoc Bonferroni/Dunn test).

shown promise in inhibiting tumor invasion and metastasis [18]. Here we have investigated the use of the humanengineered MAb, ING-1(heMAb), as a means of specifically targeting immune effector cells to Ep-CAM expressing tumor metastases in vivo.

Previous studies on MAbs targeting human Ep-CAM have demonstrated significant antitumor activities, both in vitro and in vivo [19,20]. The mechanism by which anti – Ep-CAM antibodies, including ING-1(heMAb), exert their therapeutic efficacy appears to be the activation of ADCC through

Figure 2. ING-1(heMAb) treatment at all dose schedules significantly inhibited tumor micrometastases in lung tissues. The lung tissues were sent to IDEXX Laboratories for slide processing and hematoxylin and eosin staining. The column represents the average number of micrometastases per slide and the error bar is the standard error. In both panels, IgG: IgG starting on day 2. In panel (A), ING-1/d2: ING-1(heMAb) starting on day 2; ING-1/d5: ING-1(heMAb) starting on day 5; 5-FU/LV: 5-FU/LV starting on day 2. In panel (B): ING-1/2W: ING-1(heMAb) at 0.3 mg/kg treated twice weekly; ING-1/1W: ING-1(heMAb) at 0.3 mg/kg treated once weekly. **P < .005, ***P < .0001 (ANOVA post hoc Bonferroni/Dunn test).

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interaction with host immune effector cells. We have used nude mice in all previous and present in vivo studies. Although lacking functional T-cell – mediated immunity, these mice retain the effector cells required for mounting ADCC responses; those cells include macrophages, natural killer cells, and neutrophils. We have previously reported that ING-1(heMAb) can significantly suppress the growth of human colon tumor HT-29 and prostate tumor PC-3 in murine xenograft models [13]. The degree of growth suppression is dose-dependent, with 1 mg/kg providing the greatest benefit. Although it is highly effective in preventing and/or inhibiting tumor establishment in xenograft models, other preclinical studies evaluating the in vivo efficacy of ING-1(heMAb) have not yet demonstrated its ability to eradicate large, wellestablished, solid tumors in xenograft models (Ruan et al., unpublished data). Similar findings were associated with other anti – Ep-CAM antibodies [20]. A number of factors— including antibody localization into the tumor, tumor vascularization, and immune effector cells—that may limit the effectiveness of some MAbs have been identified [21,22]. ING-1(heMAb) may be particularly useful as an adjuvant therapy after resection of primary tumors to prevent tumor recurrence and to eliminate tumor metastases. In this study, we investigated the efficacy of ING-1(heMAb) in a cancer metastasis model, in which metastases were formed by injecting tumor cells directly into the blood vessels. We selected this model over the traditional flank tumor model because this model provides a simple, reproducible approach to studying metastases. In contrast, traditional flank models rarely generate the necessary metastasis characteristics to show the optimal efficacy of ING-1(heMAb). In this experimental cancer model, the efficacy of ING-1(heMAb) was clearly demonstrated in inhibiting tumor metastases and micrometastases. In this study, we have systematically analyzed metastasis data at visual, microscopic, and cellular levels. Three different endpoints were chosen for determining ING-1(heMAb) efficacy: visible tumor nodules, tumor metastases on lung surfaces, and micrometastases in lung tissues. We found that ING-1(heMAb) at 1 mg/kg, starting on day 2, was highly effective in inhibiting all three endpoints. First, ING-1(heMAb) significantly reduced and, in some animals, completely eliminated visible tumor nodules as determined during necropsy ( P < .01, versus IgG group). Second, a significant reduction of metastases on lung surfaces was observed in the ING-1(heMAb)/day 2 – treated group ( P < .005, versus IgG group). Lastly, ING-1(heMAb) treatment, starting on day 2, resulted in 91% reduction of micrometastases in lung tissues ( P < .005, versus IgG group). Among the three endpoints, the whole body nodule data suggest that ING-1(heMAb) is the most effective against nodules in body cavities because 50% of animals had no nodules. We postulate that tumor nodules in body cavities are most accessible for immune effector cells; therefore, ING-1(heMAb) elicits the most potent ADCC activity against these nodules. Delaying ING-1(heMAb) treatment until day 5 resulted in loss of

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efficacy on the first two endpoints. However, day 5 treatment still led to 54% reduction in micrometastases ( P < .005, versus IgG group). These results indicate that treatment started early (day 2) was more effective than delayed (day 5) treatment. The difference in results obtained with early and delayed treatment may be due to a difference in time available for growth of disseminated tumor cells. Thus, on day 2, tumor cells were still in early stages of micrometastasis formation; however, by day 5, metastases likely have started active growth. This explanation is consistent with the previously proposed mechanism of action for ING-1(heMAb), which predicts that the size of metastasis nodules limits the access of the MAb and the immune effector cells to the target cells and, consequently, affects its activity in vivo [20]. These data suggest that ING-1(heMAb) should be most active in the adjuvant setting, where the targets are circulating tumor cells and metastases. This proposed clinical use is consistent with the clinical findings that many MAb treatment trials in patients with measurable tumor burdens have not been successful. Our studies also identified experimental parameters that contribute to the optimal efficacy of ING-1(heMAb) against metastases. In addition to the starting day of MAb treatment as discussed above, we have observed that the dose level and dose frequency are interdependent in determining ING-1(heMAb) efficacy. When its dose frequency was twice weekly, ING-1(heMAb) remained active at 0.3 mg/kg as determined by the three endpoints. When the dose frequency was reduced to once weekly, ING-1(heMAb) at 0.3 mg/kg was less active: activity against visible nodules and metastases was not evident, but activity against micrometastases was retained. These results indicate that in order to achieve its full antimetastasis potential, ING-1(heMAb) needs to be administered at a sufficient dose frequency to maintain its steady-state level in the blood. From our current study, we conclude that, at a minimum, twice weekly dosing is required for ING-1(heMAb) to be effective against metastases in this model. In addition, among the three endpoints, micrometastases appear to be most susceptible to ING-1(heMAb), again suggesting that the earlier the stage of metastatic disease is, the more effective is the ING-1(heMAb) therapy. In summary, the following conclusions can be derived from these studies. First, our results support that a MAb designed to elicit ADCC activity such as ING-1(heMAb) can be a useful means of inhibiting metastasis. Previous examples of successful development of MAbs that are dependent, at least partially, on ADCC activity include herceptin and rituximab from Genentech, Inc. (South San Francisco, CA) [23]. Moreover, our studies provided proof-of-principle that ING-1(heMAb) treatment can lead to significant reduction of metastases and micrometastases, and this protection is at least equal to that provided by the standard 5-FU/LV chemotherapy. Lastly, ING-1(heMAb) treatment was more effective when it was administered at earlier stages of metastases development. ING-1(heMAb) was particularly effective when administered early in the study (on day 2), whereas day 5 treatment was also effective in reducing

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micrometastases. Taken together, ING-1(heMAb) effectively reduced tumor metastases in a murine model of human cancer and these results support the use of ING-1(heMAb) as a clinical therapy against tumor metastases. We believe that ING-1(heMAb) may show potential as a targeted therapy in treating minimal residual diseases or early metastases. In addition, to obtain further enhancement of antimetastasis activity, the combined modality using ING-1(heMAb) with other anticancer treatments should also be considered.

[10]

[11]

[12]

Acknowledgements The authors acknowledge Patrick Scannon, Robert Gundel, Nathalie Dubois-Stringfellow, Trudy Vanhove, Anne Dollard, and Janet McNicholas for their helpful suggestions in the writing of this manuscript.

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