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[Cancer Research 66, 970-980, January 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Targeting Cell-Impermeable Prodrug Activation to Tumor Microenvironment Eradicates Multiple Drug-Resistant Neoplasms

Wenyuan Wu1, Yunping Luo1, Chengzao Sun2, Yuan Liu1, Paul Kuo1, Janos Varga3, Rong Xiang1, Ralph Reisfeld1, Kim D. Janda2, Thomas S. Edgington1 and Cheng Liu1

Departments of 1 Immunology and 2 Chemistry, The Scripps Research Institute, La Jolla, California; and 3 California Peptide Research, Inc., Napa, California

Requests for reprints: Cheng Liu, Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, SP258, La Jolla, CA 92037. Phone: 858-785-7734; Fax: 858-785-7756; E-mail: chengliu{at}scripps.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The tumor microenvironment is notably enriched with a broad spectrum of proteases. The proteolytic specificities of peptide substrates provide modular chemical tools for the rational design of cell-impermeable prodrugs that are specifically activated by proteases extracellularly in the tumor microenvironment. Targeting cell-impermeable prodrug activation to tumor microenvironment will significantly reduce drug toxicity to normal tissues. The activated prodrug attacks both tumor and stroma cells through a "bystander effect" without selectively deleting target-producing cells, therefore further minimizing resistance and toxicity. Here, we showed that legumain, the only asparaginyl endopeptidase of the mammalian genome, is highly expressed by neoplastic, stromal, and endothelial cells in solid tumors. Legumain is present extracellularly in the tumor microenvironment, associated with matrix as well as cell surfaces and functional locally in the reduced pH of the tumor microenvironment. A novel legumain-activated, cell-impermeable doxorubicin prodrug LEG-3 was designed to be activated exclusively in the tumor microenvironment. Upon administration, there is a profound increase of the end-product doxorubicin in nuclei of cells in tumors but little in other tissues. This tumor microenvironment–activated prodrug completely arrested growth of a variety of neoplasms, including multidrug-resistant tumor in vivo and significantly extended survival without evidence of myelosuppression or cardiac toxicity. The tumor microenvironment–activated prodrug design can be extended to other proteases and chemotherapeutic compounds and provides new potentials for the rational development of more effective functionally targeted cancer therapeutics. (Cancer Res 2006; 66(2): 970-80)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The local tumor microenvironment differs greatly from that of other tissues. It is enriched in proteolytic activity, is acidic, and is hypoxic. It represents a favorable environment for protease-targeted conversion of inactive compounds to potent cytotoxic agents (1). Targeted activation of prodrug in the tumor extracellular microenvironment can reduce the pressure for selection of tumor cells lacking expression of the target enzyme, and thereby enhance safety and efficacy of tumor destruction. This goal may be achieved by incorporation of cell membrane permeability inhibiting groups that are subject to catalytic removal by a targeted enzyme present and active in the tumor extracellular microenvironment. Such cell-impermeable targeting prodrugs promise to profoundly reduce active drug access to normal tissues, enhance availability to tumors, and therefore significantly reduce toxicity to normal cells and enhance destruction of neoplastic cells.

Legumain is an entirely novel evolutionary offshoot of the C13 family of cysteine proteases (2). It is well conserved from plants to mammals, including humans. First identified in plants as a processing enzyme of storage proteins during seed germination (3, 4), it was subsequently identified in parasites and then in mammals (5). Legumain is a robust acidic cysteine endopeptidase with remarkably restricted specificity absolutely requiring an asparagine at the P1 site of substrate sequence (5). We found legumain to be highly expressed in a majority of tumors, including carcinomas of the breast, colon, and prostate, and in several central nervous system neoplasms (6); on the other hand, expression is not apparent in normal tissues from which the tumors originated. Legumain is present intracellularly in endosome/lysosome systems and is associated with intracellular protein degradation. Importantly, we showed that legumain is also present extracellularly in the tumor microenvironment, associated with matrix as well as cell surfaces and functional locally in the reduced pH of the tumor microenvironment. Although evident in tumors, this endopeptidase is not detectable in the same tumor cell lines in culture that are used to generate the in vivo tumors, inferring an induction of legumain gene expression by the tumor microenvironment (7, 8). In addition to neoplastic cells, we found that legumain is expressed by tumor angiogenic endothelial cells and also here show presence in and on tumor-associated macrophages (9–11), thus presenting multiple local intratumoral cellular targets for prodrug activation (12).

In view of these attractive properties, we designed several legumain-activated prodrugs by covalently linking a cell-impermeable succinyl blocked substrate peptide to the aminoglycoside of doxorubicin. This prototype cell-impermeable targeting tumor microenvironment–activated prodrug is inactive and nontoxic until activated extracellularly by legumain in the acidic tumor microenvironment. Based on in vivo activity, LEG-3 (N-succinyl-ß-alanyl-L-alanyl-L-asparaginyl-L-leucyl-doxorubicin) possessed profoundly reduced toxicity and markedly enhanced efficacy compared with doxorubicin in both murine syngeneic CT26 colon cancer and C1300 neuroblastoma models as well as in the human fibrosarcoma HT1080 and doxorubicin-resistant prostate cancer MDA-PCa-2b xenograft models. Mechanistic and pharmacokinetic evidence support the tumor microenvironment–activated prodrug strategy. The potent in vivo antitumor efficacy and the improved therapeutic index suggest that LEG-3 represents a promising candidate for highly selective chemotherapeutic eradication of tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and cell lines. Rabbit polyclonal antisera against human legumain as well as 293 cells stably expressing human legumain were kindly provided by Dr. D. Roodman (Department of Medicine and Hematology, University of Texas Health Science Center, San Antonio, TX). A legumain substrate peptide was purchased from Bachem, Inc. (King of Prussia, PA). Doxorubicin was purchased from Sigma (St. Louis, MO) and DMEM was from Invitrogen (Carlsbad, CA). The CT26 murine colon carcinoma, C1300 mouse neuroblastoma cell lines, and the human HT1080 fibrosarcoma cells were purchased from American Type Culture Collection (Manassas, VA).

Antibody preparation. Antilegumain antibodies were prepared by immunizing rabbits with keyhole limpet hemocyanin–conjugated peptide CGMKRASSPVPLPP. A cysteine is added to the legumain sequence. The antilegumain antibodies were affinity purified from resultant antisera using peptide antigen coupled to Ultralink Iodoacetyl Gel from Pierce (Rockford, IL). The bound antibodies were eluted by glycine buffer (100 nmol/L, pH 2.7) and neutralized immediately by adding one-tenth volume of 1 mol/L Tris (pH 7.5).

Western blot. Proteins were dissolved in 2x SDS sample buffer for SDS-PAGE analysis using gradient Tris-glycine gels (8-16%). After electrophoresis, the proteins were transferred to nitrocellulose membranes and blocked with nonfat milk. The antilegumain antiserum was used as the first antibody and was incubated with the membranes for 1 hour (1:1,000 dilution). The blot was washed thrice with PBS, incubated with streptavidin-peroxidase for 15 minutes, and developed by the enhanced chemiluminescence method (Sigma).

Flow cytometry analysis. Single-cell suspensions were prepared from organs and tumor tissues as previously reported (6). Rabbit antilegumain antisera diluted 1:5,000 or antigen purified antilegumain antibody at 0.5 µg/mL in PBS are used to detect legumain. This is followed by FITC-conjugated goat anti-rabbit IgG diluted 1:5,000 in PBS (BD PharMingen, La Jolla, CA). For CD14 staining, the phycoerythrin-conjugated anti-mouse CD14 antibody diluted 1:3,000 in PBS was used (BD PharMingen).

Immunohistochemical analysis. Immunohistochemical staining was done on 5-µm-thick frozen sections on poly-L-lysine slides. For endothelial identification, biotinylated rat anti-mouse CD31 monoclonal antibody (MEC 13.3) was used with Texas red–conjugated streptavidin as the secondary reporting reagent. For staining of legumain, rabbit polyclonal anti-legumain antisera was used at 1:500 dilution or antigen-purified antilegumain polyclonal antibody at 0.5 µg/mL and visualized with FITC-conjugated goat anti-rabbit antibody. For the identification of tumor-associated macrophage, rat anti-mouse CD68 antibody was used and followed by an antirat antibody conjugated with Texas red. For identification of collagen I, a biotinylated rabbit anti-mouse collagen I antibody was used at 1:250 dilution and visualized with Texas red–conjugated streptavidin. The slides were analyzed by laser scanning confocal microscope (Bio-Rad, Hercules, CA).

Terminal deoxynucleotidyl transferase–mediated nick end labeling analysis. DNA fragmentation caused by apoptosis was detected by terminal deoxynucleotide transferase–based, in situ cell death detection kit (Roche Applied Science, Mannheim, Germany). The procedure was done according to the instructions of the manufacturer. Briefly, the sections were treated with protein K solution (10 µg/mL in 10 mmol/L Tris/HCl, pH 7.4) for 15 minutes and followed by 15-minute incubation with terminal deoxynucleotidyl transferase (TdT)–mediated nick end labeling (TUNEL) reaction mixture containing TdT and FITC-dUTP. The TUNEL alkaline phosphatase kit (Roche Applied Science) was used for the conversion of fluorescence-based TUNEL detection into a colorimetric labeling. The conversion was achieved by binding of an antifluorescein antibody to FITC-dUTP. The antibody is labeled with alkaline phosphatase. The signals were visualized with Fast Red (Vector Laboratories, Burlingame, CA).

Prodrug synthesis. The synthesis of the succinyl version of the prodrug used the azide method to protect the peptide from racemization. In principle, the N-protected amino acids or peptide esters are converted by hydrazine derivatization to an acid hydrazide. Subsequent reaction with HNO2 or derivatives leads to anacylazide. Thus, the succinyl-Ala-Ala-Asn-Leu-N2H3 peptide was prepared by using liquid phase synthesis. It was directly used to synthesize the target compound. An example of the synthesis is as follows. Solution A: 1,040 mg succinyl-Ala-Ala-Asn-Leu-N2H2+F was dissolved in a small amount of dimethylformamide (DMF) cooled to –10°C and 1.5 mL of 4 N HCl dioxane was added followed by 2.1 mmol/L isoamylnitrite. The mixture was stirred for 30 to 40 minutes at –10°C and then the pH was carefully adjusted to 7.5 with diisopropyl ethylamine. Solution B: 1,210 mg doxorubicin acetate was dissolved in a small amount of DMF at room temperature, the pH was adjusted to 7.5 with DIPEA, and the solution was chilled to –10°C. Solutions A and B were combined and the pH was readjusted to 7.5 and monitored throughout the reaction. The reaction mixture was allowed to warm to 4°C and allowed to stand overnight. High-performance liquid chromatography (HPLC) analysis indicated ~80% completion of the reaction within 24 to 48 hours. The reaction mixture was then diluted 10-fold with 0.1% trifluoroacetic acid (in H2O) and applied directly onto preparative HPLC. A linear acetonitrile gradient was used to elute the target compound. Fractions were analyzed for purity, combined, and lyophilized. HPLC, amino acid analysis, and mass spectrometry were done on the lyophilized powder.

Cytotoxicity assays. The WST-1 (4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disfonate) cell proliferation reagent (Roche Molecular Chemicals, Mannheim, Germany) was used to determine cell proliferation by quantization of cellular metabolic activity. Control 293 cells and legumain+ 293 cells were cultivated in microtiter plates (5 x 103 per well in 100 µL) and were incubated with serial concentrations of LEG or doxorubicin for 48 hours. Subsequently, 10 µL WST-1 solution (1 mg/mL WST-1, 25 µmol/L methyldibenzopyrazine methyl sulfate) was added per well and mixtures were incubated for an additional 4 hours. The tetrazolium salt WST-1 was cleaved by the mitochondrial succinate-tetrazolium-reductase system to formazan in cells in direct correlation with the number of metabolically viable cells in the culture. The amount of formazan salt was quantified in three replicates by absorbance at 450 nm using a microplate reader (Molecular Devices, Palo Alto, CA). All results were derived from replicate experiments with similar results.

Doxorubicin and LEG-3 uptake assay. The legumain+ 293 cells or control 293 cells (2.5 x 105 per well) were seeded in six-well plates. The culture plates were then incubated for 24 hours at 37°C and 5% CO2 and the medium in each well was replaced with 2 mL of serum-free, antibiotic-free medium containing various concentrations of doxorubicin or LEG-type compounds. The cells were incubated 1.5 hours then washed thrice with 2 mL cold PBS. At this point, cell nuclei doxorubicin positivity can be analyzed by fluorescent microscopy. For quantitative assays, the cells were then lysed by adding 0.5 of water and gently rotated on an orbital shaker for 5 minutes at room temperature. The lysed cells were added to 1.5 mL acidified ethanol and incubated at 4°C in the dark for 3 hours. Total doxorubicin and LEG content was measured fluorometrically using a Perkin-Elmer LS-50-B spectrofluorometer (excitation: 470 nm; emission: 590 nm). Fluorescence intensity was translated to drug concentration by use of a standard curve prepared from doxorubicin and LEG solutions in cell lysates that were not previously exposed to the drug. Results are expressed as the mean ± SD of at least three replicates for each experiment.

Determination of marrow toxicity. Groups of healthy BALB/c mice (n = 4) were injected i.p. with a single dose of LEG-3 (49.4 or 4.94 µmol/kg) or doxorubicin (3.4 µmol/kg). On day 7, retro-orbital sinus blood samples were collected into 10 mmol/L EDTA and were counted by hemocytometer after lysis of RBCs with an acidified methyl violet solution.

Determination of maximum tolerable dose. Four six-week-old BALB/c mice were used for each experimental group. The mice were weighed individually and the average weight of the group is used to determine the exact doses. Mice were given i.p. injection daily for 5 days. The maximum tolerable dose (MTD) is defined as the maximum drug dose administered to non–tumor-bearing mice once daily for 5 consecutive days without mortality.

Tissue distribution. LEG compounds or doxorubicin was injected i.p. into mice; 12 hours later, the animals were perfused and the doxorubicin autofluorescence was measured following homogenization in 50% ethanol and then diluted with an equal volume of 50% ethanol containing 0.6 mol/L HCl. Fluorescence measurements were obtained with excitation at 470 nm and emission at 590 nm; concentrations were derived by conversion from a doxorubicin standard curve. Tissues from saline-injected mice provided controls. Blood samples were to 0.75 mL with PBS, centrifuged, the pellets washed with PBS, and doxorubicin was extracted with ethanol and 0.3 mol/L HCl.

Animal models. The CT26 syngeneic murine colon carcinoma model was generated and maintained in The Scripps Research Institute animal facility. This model was produced in BALB/c mice ages 4 to 6 weeks injected with 5 x 105 CT26 tumor cells per s.c. site on the back. The C1300 mouse neuroblastoma model was generated in A/J mice by s.c. injection of 5 x 105 C1300 cells per site on the back. Treatment was initiated when the tumors reached 4 mm in diameter through bolus i.p. (syngeneic tumors) or i.v. (human tumors) injections of the indicated reagents. Treatment was thrice per week for 2 weeks. The human HT1080 fibrosarcoma was xenografted in BALB/c nu/nu mice obtained from The Scripps Research Institute breeding colony. HT1080 cells, 1 x 106 per site, were inoculated s.c. on the back. The MDA-PCa-2b human prostate carcinoma model was generated in WEHI nude mice and these cells (106) were also injected s.c. Tumor growth and signs of physical discomfort were monitored daily including for any gross evidence of tumor necrosis, local tumor ulceration, as well as evidence of toxicity including the mobility of animals, response to stimulus, piloerection, eating, and weight. These procedures have been reviewed and approved by the Institutional Animal Care and Use Committee at The Scripps Research Institute. All the experiments were conducted in The Scripps Research Institute facilities, which are accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. The Scripps Research Institute maintains an assurance with the Public Health Service and is registered with the Department of Agriculture and is in compliance with all regulations relating to animal care and welfare.

Statistical analysis. Statistical significance of data was determined by the two-tailed Student's t test, except for statistical significance of survival curves, which used the log-rank test using GraphPad Prism version 3.00 (GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Legumain is highly expressed by cells in the tumor microenvironment. We have reported that legumain is expressed in vivo by tumor cells and proliferating endothelial cells intracellularly as well as on their cell surfaces (6). In contrast, legumain is not detectable in or on CT26, C1300, HT1080, and MDA-PCa-2b as well as eight other tumor cell lines in culture, the same cells used to generate a set of legumain-expressing tumors in vivo (Fig. 1A). Further, this endopeptidase is not detectable in large-scale survey panels of tumor cell lines in culture, except for THP-1 cells, based on a search of the National Cancer Institute tumor cell expression database.


Figure 1
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  		Figure 1. Legumain expression and cell surface association. A, Western blot analysis of cultured tumor cells and corresponding in vivo tumor-derived cells. B, flow cytometry analysis for cell surface legumain of single-cell suspensions from CT26 tumors with (bottom) and without (top) collagenase treatment. C, flow cytometry analysis of single-cell suspensions prepared from tumor, bone marrow (BM), spleen, and kidney as well as cultured CT26 tumor cells for cell surface legumain. Columns (inset), percentage of cells positive for surface legumain.

 
Using immunohistochemical analysis, legumain is usually present in tissue sections of human colon carcinomas, neuroblastomas, fibrosarcomas, and prostate carcinomas, representative of the types of neoplasms investigated in the present study. Legumain expression was not detectable in the normal tissues of origin for these neoplasms (6). The remarkable local expression of legumain not only by neoplastic cells but also by stromal cells in association with tumor development in vivo infers that this response is in response to novel local aspects of the tumor microenvironment. Legumain is found on the CT26 tumor cell surfaces where it is effectively removed by 30 minutes collagenase digest at 57°C (Fig. 1B). This further establishes the cell surface association of legumain in contrast to the previously identified intracellular localization of legumain, as there is no transmembrane domain or prior evidence of secretion or plasmalemma localization of this lysosomal protease.

To analyze the extracellular localization of legumain in tumors and normal organs, flow cytometry was used to analyze cell surface legumain in single-cell suspensions prepared from tumors, bone marrow, spleen, and kidney, as well as cultured tumor cells. Despite demonstrable intracellular legumain by renal tubular epithelial cells, <2% of isolated viable cells were very weakly positive for cell surface legumain. Spleen cells have considerably less legumain than renal cells; however, ~1% to 2% of spleen cells are weakly positive for cell surface legumain. Furthermore, cell surface legumain is not detectable on cells derived from bone marrow nor is it found on cultured CT26 cells. In contrast, 40% of intact viable cells derived from in vivo CT26 tumors were strongly positive for cell surface legumain (Fig. 1C). A similar pattern was observed for all tumors examined (data not shown), indicating that cell surface and extracellular legumain is uniquely abundant only in tumors. Using confocal microscopy analysis, we have described previously that legumain is expressed by tumor vascular endothelial cells (Fig. 2A; ref. 6). Here, we showed that legumain is expressed by tumor-associated macrophages in tissue sections from CT26 tumors by dual staining with antilegumain and anti-CD68 antibodies (Fig. 2B). Secreted legumain is present in the tumor stroma associating with extracellular matrix proteins, such as collagen I (Fig. 2C). Legumain expression is absent in normal peripheral blood monocytes. Using flow cytometry, legumain is found on the surface of viable endothelial cells and tumor-associated macrophages using both antilegumain antibody and anti-CD31 antibody or anti-CD14 antibody, respectively (Fig. 2D). Interestingly, legumain on endothelial cell and tumor-associated macrophage surfaces is resistant to removal by collagenase, suggesting a mode of cell surface association (Fig. 2E) distinct from that of tumor cells where legumain is removed by collagenase.


Figure 2
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  		Figure 2. Legumain is expressed by stromal cells in tumors. A, double staining of antilegumain antibody (green) and anti-CD31 antibody (red) identifies endothelial cells. Arrows, tumor vascular endothelial cells expressing legumain in tumor (x400). B, double staining of antilegumain antibody (green) and anti-CD68 antibody (red) identifies legumain-expressing, tumor-associated macrophages (arrows; x600). Arrows, tumor-associated macrophage cells expressing legumain. C, double staining of antilegumain (green) with anticollagen I antibody (red; x400). Colocalization of legumain with collagen I in the extracellular matrix (yellow). D, two-dimensional analysis of single-cell suspensions prepared from 4T1 in vivo mouse mammary tumor following mechanical dissociation and of 4T1 tumor cells from tissue culture. The presence of cell surface legumain in tumor vascular endothelial cells and tumor-associated macrophages are shown with the presence of cells that are both legumain+ and CD31+ as well as cells that are both legumain+ and CD14+, respectively. E, two-dimensional flow cytometry of single-cell suspensions prepared from 4T1 tumors using collagenase digestion. Legumain-associated endothelial cells are represented by the group of cells that are legumain+ and CD31+. Legumain-associated, tumor-associated macrophages are represented by legumain+ and CD14+ cells.

 
LEG-3 is activated by tumoral legumain. The ability of the cell-impermeable targeting tumor microenvironment prodrug LEG-3 (Fig. 3A) to kill tumor cells was evaluated first in cell culture (Fig. 3B). With both legumain transfected and control nonproducing cells, we determined the median effective concentration representing the amount of LEG-3 or doxorubicin required for 50% cell death (EC50; Table 1 ). LEG-3 was virtually noncytotoxic to control cells not expressing legumain. However, for cells that were transfected with legumain cDNA and express cell surface legumain, cytotoxicity was significant and EC50 levels were close to those observed for doxorubicin, indicating an efficient conversion of the LEG-3 prodrug. The requirements for activation were shown using an alternate peptide sequence not hydrolyzed by legumain [LEG-4 (N-succinyl-ß-alanyl-L-asparaginyl-L-leucyl-doxorubicin); Fig. 3B] and also by inhibition of LEG-3 activation following inhibition of legumain function (Fig. 3C).


Figure 3
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  		Figure 3. LEG family compounds. A, schematic structure of these LEGs. LEG-2 and LEG-3 are oligopeptidic derivatives of doxorubicin that are cell impermeable and can be hydrolyzed to leucine-doxorubicin by extracellular legumain. LEG-4 is similar but is not subject to legumain hydrolysis. B, cytotoxicity of LEG compounds using legumain+ 293 cells and control wild-type 293 cells. C, legumain-mediated LEG activation and inhibition by cystatin. 1, untreated control; 2, doxorubicin treated; 3, doxorubicin plus cystatin; 4, LEG-3 treated; 5, LEG-3 plus cystatin; 6, LEG-2 treated; 7, LEG-2 plus cystatin. D, cell uptake assay of LEG-3 compared with doxorubicin (Dox). E, localization of doxorubicin in cell nuclei is visualized by autofluorescence (red). In contrast, LEG-3 is not internalized by legumain-negative cells exposed to LEG-3 and they lack nuclear positivity for end product doxorubicin despite presence of extracellular fluorescent signal of the doxorubicin present in the LEG-3.

 

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  		Table 1. In vitro and in vivo toxicity of LEG-3 compared with doxorubicin

 
LEG-3 is activated extracellularly. Tumor cell uptake of LEG-3 was compared with doxorubicin. When added to cell cultures, doxorubicin rapidly entered cells. In contrast, LEG-3 remained extracellular, consistent with its observed lack of cytotoxicity. In contrast, when LEG-3 was added to legumain-expressing cells in culture, the end product doxorubicin was found in cells (Fig. 3D and E).

Legumain is selective and functional in the tumor microenvironment. When doxorubicin was administered as a single i.v. bolus, the plasma concentration very rapidly declined followed by a low concentration and slowly cleared phase, consistent with observations by other investigators (13, 14). The initial decline of infused LEG-3 was much slower, which is attributed to reduced tissue uptake (Fig. 4A). The content of LEG-3 in tumor tissues was determined 12 hours postinjection in mice bearing CT26 tumors. There was significant doxorubicin content in tumors, in contrast to many tissues, including heart, kidney, liver, and brain. The MTD of doxorubicin and a molar equivalent amount of LEG-3 were given i.v. For LEG-3, the amount of drug present in tumors was >10-fold greater than that for doxorubicin administration. LEG-3 was greatly reduced in cardiac tissue (Fig. 4B). Because of the reduced normal tissue uptake and toxicity, larger quantities of LEG-3 could be administered, which resulted in higher drug content in cells within tumors compared with that achieved for doxorubicin administration. Drug accumulation in tissues and tumor was visualized by doxorubicin autofluorescence (Fig. 4C). The data indicate that legumain is selectively found in the tumor microenvironment, and LEG-3 is processed to its cell-permeable Leu-doxorubicin derivative based on the presence of cytoplasmic doxorubicin, which, following processing to the end product doxorubicin, translocated to the nucleus based on intranuclear fluorescence.


Figure 4
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  		Figure 4. In vivo distribution and pharmacokinetics of LEG-3. A, plasma pharmacokinetics of LEG-3 compared with doxorubicin. B, accumulation of LEG-3 and doxorubicin in organs and tumors of mice bearing CT26 colon carcinomas. C, presence of doxorubicin and LEG-3 are visualized with autofluorescence of doxorubicin (red).

 
In vivo toxicity of LEG-3 and doxorubicin. LEG-3 is significantly less toxic than doxorubicin when evaluated in vivo. When given by six repeat i.v. administrations, LEG-3 had a much higher cumulative MTD and reduced LD50 compared with doxorubicin (Table 1). Bone marrow and cardiac toxicity of LEG-3 was examined and compared with doxorubicin. Also, the cytotoxic effect of LEG-3 on peripheral blood leukocyte counts (WBC) was assessed and compared with that of doxorubicin. There were no reductions of total WBC counts in mice treated six times in 12 days with 4.94 µmol/kg LEG-3, and only a slight reduction observed in mice treated with a 10-fold higher dose. In contrast, there was >50% reduction of WBC counts in the group of mice receiving as little as 3.4 µmol/kg doxorubicin by the same schedule (Fig. 5A). LEG-3 produced little evidence of myelosuppression compared with its parent compound doxorubicin. In cardiac tissue, mice similarly treated with 49.4 µmol/kg of LEG-3 exhibited no histologic evidence of cardiac toxicity in contrast to profound cardiac myocyte vacuolization and cell death in mice treated with doxorubicin at 3.4 µmol/kg (Fig. 5B). Cardiac myocytes of mice treated with doxorubicin showed marked apoptosis, which was not observed for LEG-3-treated mice (Fig. 5B).


Figure 5
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  		Figure 5. LEG-3 lacks in vivo toxicity of doxorubicin. A, myelosuppression of LEG-3 and doxorubicin in mice were assessed by determination of peripheral blood leukocyte counts. B, cardiac toxicity was shown by the presence of vacuolar degeneration of myocytes in H&E-stained sections resulting from chronic doxorubicin treatment, which was notably absent in LEG-3-treated mice. TUNEL analysis of cardiac tissue from mice treated with doxorubicin also showed marked apoptosis of myocytes (arrows, red apoptotic nuclei). This was very infrequent in LEG-3-treated mice.

 
In vivo efficacy is dependent on legumain-specific activity. To further characterize the requirements for legumain activation of LEG-3 in vivo, two LEG-type compounds differing in composition of the peptidyl element of the compound were evaluated. LEG-2 (N-succinyl-ß-alanyl-L-threoinyl-L-Asparaginyl-L-Leucyl-doxorubicin) and LEG-4 were analyzed for in vivo efficacy in tumor-bearing mice. The LEG-4 compound is not cleaved by legumain and was devoid of tumoricidal activity (Fig. 6A), whereas LEG-2 and LEG-3, which are converted by legumain, showed in vivo tumoricidal efficacy (Fig. 6B and C).


Figure 6
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  		Figure 6. In vivo specificity and efficacy of LEG-3 in syngeneic mouse tumor models. A, in vivo effects of LEG-4 in mice bearing CT26 tumors (n = 8). B, LEG-2 (n = 8). C, LEG-3 (n = 8), H&E-stained sections of control versus treated tumors. D, in vivo effect of LEG-3 in A/J mice bearing C1300 neuroblastoma (n = 8). H&E-stained sections of control versus treated tumors. E, survival analysis of CT26 tumor-bearing mice treated with LEG-3. F, survival analysis of C1300-bearing mice treated with LEG-3.

 
LEG-3 has effective tumoricidal activity against diverse tumors in vivo. The potential therapeutic efficacy of LEG-3 was evaluated in both syngeneic rodent tumor models and human tumor xenograft models. At a well-tolerated dose (49.4 µmol/L/kg) administered six times in 12 days, LEG-3 effectively arrested growth of murine CT26 colon carcinoma in BALB/c mice and produced complete tumor eradication in some (Fig. 6C). A similar level of efficacy was also observed for the murine C1300 neuroblastoma in A/J mice (Fig. 6D) where LEG-3 treatment induced massive tumor cell death (Fig. 6C and D), tumor eradication, and marked enhancement of survival (Fig. 6E and F).

The in vivo efficacy of LEG-3 on human tumor xenografts in athymic nu/nu mice was assessed and compared with doxorubicin. Legumain protein was not detectable in either HT1080 or MDA-PCa-2b cells in culture. However, robust legumain expression was observed by immunohistochemistry for in vivo tumors propagated from these cells. Indeed, LEG-3 produced potent tumoricidal activity against the HT1080 fibrosarcoma, a fast-growing tumor and a model that is traditionally sensitive to doxorubicin therapy (Fig. 7A). On the other hand, human prostate carcinomas are frequently resistant to doxorubicin therapy. MDA-PCa-2b prostate carcinoma, a known doxorubicin-resistant tumor (15), failed to respond to doxorubicin in vivo. However, administration of LEG-3 led to complete growth arrest (Fig. 7B). LEG-3 was effective and frequently resulted in complete tumor eradication with marked enhancement of survival of the HT1080 as well as the MDA-PCa-2b tumor-bearing mice (Fig. 7C and D). Toxicity of LEG-3 based on weight loss and mortality was negligible (Fig. 7E).


Figure 7
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  		Figure 7. Efficacy of LEG-3 in human xenograft tumor models compared with doxorubicin. A, in vivo effects of LEG-3 (49.4 µmol/kg; n = 8) compared with doxorubicin (1.72 µmol/kg; n = 8) in the HT1080 human fibrosarcoma model and H&E-stained histologic analysis of control HT1080 tumor compared with tumors treated with doxorubicin and LEG-3. B, in vivo effect of LEG-3 (49.4 µmol/kg; n = 8) compared with doxorubicin (1.72 µmol/kg; n = 8) in the MDA-PCa-2b human prostate carcinoma xenograft model and H&E-stained MDA-PCa-2b human prostate carcinoma. C, survival analysis of HT1080 human fibrosarcoma models. D, survival analysis of mice bearing MDA-PCa-2b tumors after treatment. E, gross toxicity of tumor-bearing mice treated with doxorubicin and LEG-3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This cell-impermeable targeting tumor microenvironment–activated prodrug strategy depends on targeting as well as specific catalytic function at a pH <6.8 of a quite unique endopeptidase that is selectively expressed extracellularly in the tumor microenvironment. There, it can convert an effectively designed cell-impermeable prodrug (LEG-3) to a cell-permeable prodrug (Leu-doxorubicin), which, in turn, is converted to the active tumoricidal end product doxorubicin and translocated to cell nuclei for induction of cell death. There are distinct advantages to this strategy in that tissue uptake can occur only in the tumor or comparable pathologic microenvironment. To achieve continuous growth and remodeling, the tumor microenvironment is enriched with a variety of proteases. There are nearly 500 proteases identified thus far in the human genome (16, 17), and many have been associated with the local tumor microenvironment and seem important for tumor invasion and metastasis (18, 19). Drug access to solid tumors is relatively efficient, limited only by diffusion barriers. Compounds, such as LEG-3, converted and activated in the tumor microenvironment can access and be converted by both neoplastic and associated endothelial and stromal cells to produce substantial bystander effects. Whereas most cytotoxic drugs are designed to be cell permeable, here we diminished cell permeability, increased hydrophilic properties, and increased drug solubility to minimize tissue uptake of LEG-3. This correlates with the slower clearance from the blood as well as greatly diminished tissue accumulation.

The design of an effective tumor microenvironment–activated prodrug of this type requires knowledge of the selectivity and expression of the enzyme target, including not only functional characteristics but also in vivo distribution under physiologic and pathologic conditions. In respect to these issues, extracellular accessible legumain represents a promising candidate target because it is the only asparaginyl endopeptidase in the mammalian genome. We discovered that legumain is highly expressed in the majority of solid tumors (6). It is a robust acidic cysteine protease, one that is overexpressed by neoplastic cells as well as intratumoral endothelial cells and macrophages. It requires for function the local acidic tumor microenvironment and is found associated with both the extracellular matrix and cell surfaces in the tumors. In normal tissues, such as kidney, legumain is present in proximal tubular epithelial cells but only as an intracellular lysosomal protein. Although not found, extracellular legumain in normal tissues would be functionally inactive at physiologic pH (5) and such protein that may escape the tumor microenvironment would be inactive for this same reason. Legumain activity in normal tissues could be inhibited by cysteine protease inhibitors, such as cystatin C (20), whereas such inhibitors are commonly down-regulated in tumors (21). In addition to legumain expression, the function of reticuloendothelial system can contribute to the relative higher spleen uptake of prodrug and doxorubicin. However, we did not observe gross spleen enlargement or atrophy in prodrug-treated mice; there is no apparent cellular depletion of lymphoid or myelogenic lineages. Therefore, the legumain-activated prodrugs do not seem to have an enhanced toxicity toward spleen. The cell-impermeable targeting tumor microenvironment–activated prodrug strategy need not be restricted to doxorubicin as the cytotoxic end product. Other compounds are compatible with modification to achieve a similar tumor microenvironment–activated prodrug mode of action. Of particular interest for incorporation into this strategy are a number of highly cytotoxic compounds that have been developed based on their in vitro activity as exemplified by duocarmycin (22, 23).

Tumor-associated proteases have been recognized increasingly as potential targets for selective activation of prodrugs. Thus far, prodrugs were reported that are activated by prostate-specific antigen (24, 25), cathepsin (26–28), plasmin (29), and by undefined tumor-associated proteases (14, 30, 31). Here, we explored the potential of legumain to serve as a tumor-selective target and evaluated the potential of the tumor microenvironment–activated prodrug strategy. The lead candidate compound LEG-3 was analyzed in vitro and in vivo. The aminoglycoside at position C-3' is critical for the ability of doxorubicin to integrate into DNA and to interfere with DNA Topo II and to overcome multiple drug resistance (32, 33). The addition of the legumain substrate peptidyl structure to the C-3'-NH2 abolishes this function. It is critical to our design that charge and the succinyl cap of LEG-3 prevent cell entry. Also, the peptidyl element must not be susceptible to hydrolysis by any other enzyme. In tumor cells overexpressing legumain, LEG-3 was effectively rendered cell permeable, the resultant Leu-doxorubicin prodrug was processed with translocation of end product doxorubicin to nuclei, thereby mediating cytotoxicity. LEG-3 is effective against a variety of multidrug-resistant solid tumors, including not only rapidly progressing syngeneic rodent tumors but also slower growing human tumor xenografts in immunodeficient mice. The specific activation of LEG-3 by legumain in tumors results in higher drug delivery to tumor cells and resultant cytotoxic destruction.

Our data clearly showed that LEG-3 is a tumor microenvironment–activated prodrug that is selectively catalytically converted to end product doxorubicin in the tumor microenvironment. LEG-3 is not found in any significant amount in normal tissues presumably as a result of its cell impermeability. Based on LD50, the toxicity of LEG-3 in the mouse was reduced >10-fold compared with doxorubicin. LEG-3 also exhibited a slower initial reduction in plasma concentration than doxorubicin consistent with the relative tissue impermeability. For cardiac tissue, LEG-3 accumulation was reduced >15-fold. Cardiomyopathy and the development of congestive heart failure is associated clinically with cumulative doxorubicin dosage in excess of 500 to 550 mg/m2, a level readily achieved when required for tumors responsive to the drug, and is the major limitation for therapeutic use of doxorubicin and other anthracyclines (34). This is a notable advantage of compounds like LEG-3 because it is far less cardiotoxic. The toxicity of DNA-intercalating drugs is particularly injurious to tissues with high cell proliferation as exemplified by severe myelosuppression. We found little effect of LEG-3 on cells of myeloid lineage, as mice showed negligible reduction in peripheral blood or marrow myeloid cells at elevated therapeutic doses.

Another advantage of a tumor microenvironment–activated prodrug, such as LEG-3, over doxorubicin is the increased plasma persistence, allowing longer tumor exposure to enhance targeting. Based on the reduced toxicity, larger cumulative dosage of LEG-3 can be administered more rapidly. Consequently, significantly greater tumor inhibition and destruction have been observed for LEG-3 in syngeneic murine colon carcinoma and neuroblastoma models without demonstrable toxicity. LEG-3 was also highly effective against human fibrosarcoma and a doxorubicin-resistant human prostate carcinoma in xenograft models where high levels of intratumoral legumain are present.

Legumain is also produced by endothelial cells and tumor-associated macrophages in the tumor microenvironment. These cells constitute additional local intratumor targets for competent drug activation and therapeutic effects, including tumor microvascular destruction. Evidence that tumor-associated macrophages can be directly tumoricidal and also stimulate tumoricidal activity of T cells is questionable. To the contrary, tumor cells frequently are able to evade the activity of tumor-associated macrophages (11). In some tumors, tumor-associated macrophages account for as many as 50% of cells. Further, evidence has emerged for a symbiotic relationship between tumor cells and tumor-associated macrophages. Tumor cells attract tumor-associated macrophages, which, in turn, provide a considerable array of growth factors and cytokines that can facilitate tumor cell survival. Tumor-associated macrophages have been reported to respond to microenvironmental factors in tumors, such as hypoxia, by producing growth factors including vascular endothelial growth factor (35–37), enzymes such as matrix metalloproteinases (38), cathepsins (39), and now legumain. Through these factors, tumor-associated macrophages stimulate or facilitate tumor angiogenesis, invasion, and growth, similar to the role of macrophages in wound healing. In fact, tumor cells circumvent the need to produce all the factors necessary for their growth and to establish a pseudo-organ, the solid tumor (11, 40), with the aid of tumor-derived molecules that can redirect tumor-associated macrophage activities to promote tumor survival and growth. The potent antitumor activity of LEG-3 in vivo may also be partly attributable to targeting endothelial cells and tumor-associated macrophages in the tumor microenvironment.

We here describe a tumor microenvironment–activated prodrug strategy for cancer therapy. LEG-3, the example, is activated by extracellular legumain in tumors leading to extensive tumor cell death and frequent complete tumor eradication, including drug-resistant tumors. These data support the advance of this strategic class of therapeutics for development as a molecularly targeted cancer therapeutic. Given that this design may be adapted to other cytotoxic compounds, it represents an opportunity to advance cancer therapy.


   Acknowledgments
 
Grant support: Congressionally Directed Medical Research Program grants W81XWH-05-1-0091 and W81XWH-05-1-0318 (C. Liu), NIH program project grant P01 HL016411 (C. Liu and T.S. Edgington), NIH/National Cancer Institute grant CA094193, and the Skaggs Institute of Chemical Biology (K.D. Janda).

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.

We thank Leonard Wong for technical assistance and Barbara Parker for assistance in preparing the manuscript.


   Footnotes
 
Note: W. Wu and Y. Luo contributed equally to this work.

Received 7/22/05. Revised 10/18/05. Accepted 11/11/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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