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Large Molecule Therapeutics

Antitumor Synergism and Enhanced Survival with a Tumor Vasculature–Targeted Enzyme Prodrug System, Rapamycin, and Cyclophosphamide

John J. Krais, Needa Virani, Patrick H. McKernan, Quang Nguyen, Kar-Ming Fung, Vassilios I. Sikavitsas, Carla Kurkjian and Roger G. Harrison
DOI: 10.1158/1535-7163.MCT-16-0263 Published September 2017

Abstract

Mutant cystathionine gamma-lyase was targeted to phosphatidylserine exposed on tumor vasculature through fusion with Annexin A1 or Annexin A5. Cystathionine gamma-lyase E58N, R118L, and E338N mutations impart nonnative methionine gamma-lyase activity, resulting in tumor-localized generation of highly toxic methylselenol upon systemic administration of nontoxic selenomethionine. The described therapeutic system circumvents systemic toxicity issues using a novel drug delivery/generation approach and avoids the administration of nonnative proteins and/or DNA required with other enzyme prodrug systems. The enzyme fusion exhibits strong and stable in vitro binding with dissociation constants in the nanomolar range for both human and mouse breast cancer cells and in a cell model of tumor vascular endothelium. Daily administration of the therapy suppressed growth of highly aggressive triple-negative murine 4T1 mammary tumors in immunocompetent BALB/cJ mice and MDA-MB-231 tumors in SCID mice. Treatment did not result in the occurrence of negative side effects or the elicitation of neutralizing antibodies. On the basis of the vasculature-targeted nature of the therapy, combinations with rapamycin and cyclophosphamide were evaluated. Rapamycin, an mTOR inhibitor, reduces the prosurvival signaling of cells in a hypoxic environment potentially exacerbated by a vasculature-targeted therapy. IHC revealed, unsurprisingly, a significant hypoxic response (increase in hypoxia-inducible factor 1 α subunit, HIF1A) in the enzyme prodrug–treated tumors and a dramatic reduction of HIF1A upon rapamycin treatment. Cyclophosphamide, an immunomodulator at low doses, was combined with the enzyme prodrug therapy and rapamycin; this combination synergistically reduced tumor volumes, inhibited metastatic progression, and enhanced survival. Mol Cancer Ther; 16(9); 1855–65. ©2017 AACR.

This article is featured in Highlights of This Issue, p. 1727

Introduction

As research continues to unravel the molecular circuitry behind cancers, targeted therapies will likely enhance the duration and quality of life of cancer patients through creative approaches, including toxic payload delivery, signaling attenuation, immunostimulation, and combination strategies (1–3). One targeted adaptation of a toxic payload delivery involves the local activation of chemotherapeutic prodrugs within the tumor using enzyme prodrug therapies (4, 5). Enzyme prodrug strategies localize an enzyme to the tumor microenvironment either through gene delivery (6, 7), antibody targeting (8, 9), local administrations, or cellular delivery systems (10). Once the enzyme is localized to the tumor environment, a nontoxic prodrug is administered that is then enzymatically converted to a toxic drug in the tumor, minimizing systemic exposure to the cytotoxic agent.

Enzyme prodrug therapies attempt to achieve a high therapeutic index and selectivity through the generation of toxic levels of drug from an inert prodrug at the tumor. The methionine gamma-lyase (MGL) enzyme prodrug system converts the prodrug selenomethionine to methylselenol, enhancing cytotoxicity of the compound by several orders of magnitude (11, 12). The methylselenol created in the tumor leads to a rapid antitumor effect due to oxidative stress as a result of the generation of toxic reactive oxygen species, which activate the caspase cascade and apoptosis (11, 13). Alone, selenomethionine exhibits minimal toxicity because mammalian cells do not express the enzymes necessary to convert it to a toxic selenol compound (14, 15), allowing for controlled activation of the prodrug through the targeting of the MGL system.

A primary criterion for enzyme prodrug system success, site-specific prodrug activation, requires the use of foreign enzymes to avoid off-target cytotoxic activity, and in the case of MGL, the Pseudomonas putida enzyme is often utilized. Administration of large foreign proteins presents complications even for immunocompetent preclinical models. Cystathionine gamma-lyase (CTH) acts in a mammalian cystathionine metabolism pathway in a manner similar to bacterial methionine salvage with MGL. Wild-type mammalian CTH displays no activity toward methionine or selenomethionine; however, three amino acid substitutions impart MGL activity to CTH (16), providing a viable alternative to the administration of a foreign protein in the MGL enzyme prodrug system.

Current enzyme prodrug systems face limitations regarding enzyme localization, including gene-directed enzyme prodrug therapy and its derivative virus-directed enzyme prodrug therapy. Most significantly, gene delivery strategies suffer from poor gene expression in vivo; however, selective delivery, insertional mutagenesis, and immunogenicity are persistent concerns as well (17–19). Delivery of active enzyme, as with antibody-directed enzyme prodrug therapies, circumvents expression issues; however, antibody–enzyme conjugate accessibility to the tumor-specific targets can limit this approach (17, 20).

A potential target for delivery of therapies is phosphatidylserine, which is expressed externally on cancer cells and the tumor vasculature but remains on the cytoplasmic side of the membrane of healthy cells and normal vasculature (21–25). Targeting of an enzyme to phosphatidylserine can be achieved through the fusion of the enzyme to a member of the Annexin protein family, which binds to anionic phospholipids, such as phosphatidylserine, with high affinity (26–30). Phosphatidylserine acts as a unique target for the delivery of an enzyme prodrug system because adhesion to the endothelial wall of tumor vessels allows for tumor killing through the destruction of vasculature and permeation of the small-molecule cytotoxic drug into the tumor, creating a bystander killing effect (31, 32). The Annexin-assisted delivery of mutant mammalian CTH (mCTH) to the tumor vasculature and the subsequent occlusion of vessels following conversion of selenomethionine to methylselenol present a unique opportunity for anticancer combination therapies to address the tumor response to the resultant intensification of the hypoxic tumor environment.

Hypoxia-inducible factors (HIF) activate mechanisms to improve oxygenation as well as reprogram metabolic processes to enhance cell survival under oxygen deprivation, such as in the case of a vasculature-directed cytotoxic delivery system. HIF1 and HIF2 regulate over 1,000 genes involved in cancer biology, including the proangiogenic VEGF and enzymes responsible for matrix remodeling and cellular migration (33–35). The hypoxic tumor microenvironment activates this cellular survival mechanism, which cascades and ultimately plays a role in the progression of a number of the hallmark traits of cancer, including angiogenesis, metastasis and invasion, altered metabolism, and apoptotic resistance. Rapamycin, an inhibitor of mTOR, acts as an upstream antagonist of HIF1 α subunit (HIF1A). mTOR regulates a signaling cascade through control of phosphorylation of proteins S6K1 and 4E-BP-1, important for translation of mRNAs involved in cell growth and proliferation processes, including HIF (10, 36). Effective reduction of HIF1A levels with rapamycin could significantly reduce the prosurvival signaling of the hypoxic response and inhibit tumor progression. Combination therapy with rapamycin and vasculature-targeted enzyme prodrug therapies may be especially complementary as targeted enzyme prodrug therapy has been shown to result in necrotic tumor cores and reduced blood flow (27).

Induction of tumor cell apoptosis with methylselenol and reduction of prosurvival signaling with rapamycin can be further exploited through augmentation of antitumor immunity. Tumor cell apoptosis increases antigen presentation and stimulation of tumor-specific effector T cells (37), an effect that could be amplified through the dose-dependent immunomodulatory properties of cyclophosphamide. In particular, low-dose cyclophosphamide has been shown to selectively deplete regulatory T cells (Treg), potentially tipping the balance from immune tolerance and evasion to an active anticancer immunity. Cyclophosphamide acts by alkylating DNA, creating interstrand and intrastrand cross-links that ultimately result in cell death (38, 39). Increased apoptosis and decreased immunosuppressive activity of Tregs upon low-dose cyclophosphamide treatment result from a hypersensitivity possibly related to a decreased capacity for DNA damage repair (39). Combined with other antitumor therapies, low-dose cyclophosphamide can enhance the antitumor immune response, leading to tumor rejection and improved survival (40–42).

The work described aims to address the efficacy of a three-pronged anticancer strategy: targeted delivery of a cytotoxic compound with an enzyme prodrug system, modulation of the prosurvival hypoxic response within the tumor microenvironment, and promotion of antitumor immunity. Together, this strategy aims to address many of the hallmark traits of cancer while minimizing the impact on healthy tissue. We validate the efficacy of the targeted enzyme prodrug system using human MDA-MB-231 and murine 4T1 triple-negative breast cancer models. Our findings using the highly metastatic 4T1 mammary tumor in immunocompetent mice demonstrate a synergistic effect on tumor volumes when combining rapamycin with the enzyme prodrug therapy and a significant antimetastatic effect when including cyclophosphamide.

Materials and Methods

Fusion protein construction, expression, and purification

The methionine gamma-lyase and Annexin A5 (ANXA5) fusion protein (MGL-ANXA5) was produced and purified as described previously (26). Wild-type human CTH displays no activity toward l-methionine or l-selenomethionine; however, three mutations impart MGL activity to human CTH (16). Equivalent mutations were applied to mouse CTH to impart MGL activity (E58N, R118L, E338N). Mutant mouse CTH (mCTH), ANXA5, and Annexin A1 (ANXA1) gene fragments were codon optimized for Escherichia coli (E. coli) protein production using DNAWorks (Helix Systems; NIH, Bethesda, MD) and synthesized (Life Technologies) with each fusion, mCTH-ANXA5 and mCTH-ANXA1, consisting of three fragments. Fragment sizes ranged from 371 to 1,000 amino acids with 40-bp overlapping regions on each end of the fragment. Gene fragments were directly assembled into pET-30Ek/LIC (EMD Chemicals) using the Gibson Assembly method (43). Gibson assembly Master Mix (New England Biolabs) was held at 50°C for 60 minutes in a thermocycler with the gene fragments and vector. The assembled product was transformed into NEB 5-alpha competent E. coli, expanded, and cultured on kanamycin plates. Colonies were sequenced (Oklahoma Medical Research Foundation) and transformed into BL21(DE3) competent cells for expression or into T7 Express lysY competent cells (New England Biolabs) for enhanced protein yields.

BL21(DE3) E. coli were grown in Luria broth medium at 37°C and 200 rpm to an OD600 nm of 0.6. T7 Express lysY E. coli were grown in TB medium at 37°C and 200 rpm to an OD600 nm of 1.2. Growth occurred in two steps: initially at 10 mL and then expanded to 1 L. Isopropyl β-D-thiogalactopyranoside (IPTG) was added to a concentration of 0.4 mmol/L for 6 hours at 30°C at 180 rpm for mCTH-ANXA1 or 1.0 mmol/L for 19 hours at 25°C and 200 rpm for mCTH-ANXA5.

Recombinant fusion proteins were purified using immobilized metal affinity chromatography with immobilized Ni2+ to isolate the fusion protein as described previously (26, 28, 29). An endotoxin removal step using a 1% Triton X-114 wash was included, and the 6× His Tag was removed with HRV-3C protease (Thermo Scientific Pierce).

Endotoxin levels were assessed through a chromogenic Limulus Amebocyte Lysate assay (Lonza) according to the manufacturer's instructions. Purity was assessed through a densitometric analysis with ImageJ software (FIJI build; NIH) from SDS-PAGE gels with Coomassie brilliant blue staining of protein samples. Enzyme activity with l-methionine and l-selenomethionine was assessed according to the methioninase assay described previously (26).

Cell lines and culture conditions

Human MDA-MB-231 and murine 4T1 breast cancer cell lines were purchased from ATCC in 2010 and 2011, respectively. Human endothelial HAAE-1 cells were purchased from Coriell Cell Repositories in 2012. All cells were cultured according to conditions recommended by the suppliers and described previously (29, 44). ATCC verifies cell identity with isoenzymology methods and short tandem repeat analysis. Coriell Cell Repositories perform cell identification with nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase isoenzyme electrophoresis and chromosome analysis. No further authentication was performed; however, no cells were maintained in culture for more than 6 months. Experiments were performed within six passages, except in the generation of TdTomato-expressing 4T1 cells and GFP-expressing MDA-MB-231 cells.

In vitro binding and cytotoxicity analysis

The specific, calcium-dependent binding of the Annexin fusion proteins was quantified as described previously (26, 28, 29). Live cell imaging with GFP-expressing MDA-MB-231 cells and Dylight 680–conjugated fusion protein confirmed binding to the cell membrane (see Supplementary Fig. S1). The cytotoxicity assays were performed over 3 days in 24-well plates under standard culture conditions with calcium-supplemented medium (2 mmol/L Ca2+). On day 0, cells were incubated with 100 nmol/L fusion protein for 2 hours at 37°C. The plates were washed, and medium containing varying concentrations of selenomethionine was added. An Alamar Blue assay was performed daily with a 4-hour incubation of 10% Alamar Blue (Life Technologies) to assess viability using a fluorescence measurement at an excitation wavelength of 530 nm and emission at 590 nm. Medium containing the prodrug selenomethionine was replaced daily.

Mouse tumor models and treatments

Six-week-old female BALB/cJ and SCID mice were purchased from The Jackson Laboratory and housed in a pathogen-free facility at the University of Oklahoma Health Sciences Center (Oklahoma City, OK) in accordance with protocols approved by the Institutional Animal Care and Use Committee. Mice were injected in the mammary fat pad number 4 with 105 4T1-TdTomato or MDA-MB-231 mouse breast cancer cells suspended in 50 μL PBS and an equal volume of Matrigel. Mouse body weight and tumor volume were monitored every 3 to 4 days. Tumor volume was calculated with the modified ellipsoid formula volume = (1/2) × (length × width2) using caliper measurements of the longest dimension and perpendicular width.

Mice bearing orthotopic 4T1 or MDA-MB-231 tumors were randomized into groups (7–10/group) prior to the start of treatment on day 10 postinoculation of 4T1 cells or day 48 for MDA-MB-231 xenografts. Fusion protein was administered daily at 10 mg/kg by intraperitoneal injection. Selenomethionine (5 mg/kg i.p.), rapamycin (5 mg/kg i.p.), and cyclophosphamide (10 mg/kg i.p.) were administered daily 10 hours after fusion protein injection.

Antibody titers

Protein-specific antibody titers were determined on the basis of modified protocols (45, 46). Blood samples were collected from mice at 0, 1, 2, and 3 weeks of treatment. A sandwich ELISA assay was performed to determine protein-specific antibody titers. A 0.1 mol/L carbonate coating buffer and 20 μg/mL fusion protein were incubated overnight at 4°C on high binding capacity ELISA 96-well plates. Plates were then washed and blocked with FBS, and plasma dilutions were incubated overnight at 4°C. Following additional washes, goat anti-mouse IgG and IgM conjugated to HRP (Jackson ImmunoResearch Laboratories, Inc.) were used to develop o-phenylenediamine for quantification on a BioTek Synergy plate reader.

Tissue analysis and IHC

At the conclusion of the treatment period for mice with 4T1 tumors, 3 mice per group were sacrificed for tissue analysis. Tumors were removed and fixed in 10% neutral-buffered formalin. IHC slides were produced using 3,3′-diaminobenzidine (DAB) staining for activated caspase-3 (Abcam 2302), Ki-67 (Abcam 15580), CD-31 (Abcam 2302), and HIF1A (Abcam 82832) with hematoxylin counter staining. Heat-mediated antigen retrieval was performed at pH 6. Tumor sections for activated caspase-3 and Ki-67 staining were imaged at 20× using a Nikon Eclipse E800 microscope collecting 15 images per section. An automated ImageJ macro was developed for quantification of DAB staining and visually verified (Supplementary Figs. S2 and S3). HIF1A images were collected with a Leica stereomicroscope to capture the entire tumor section.

The lungs were removed and immediately analyzed for the presence of TdTomato-positive metastatic nodules using a Leica stereomicroscope. The size and number of metastatic nodules were quantified with ImageJ software. In addition, the spleen was removed, and the cells were mechanically dissociated from the organ in FACS buffer and passed through a 70-μm cell strainer. A Mouse Regulatory T Cell Staining Kit (eBioscience) was used to stain splenocytes for flow cytometry according to the manufacturer's instructions. A BD Biosciences Accuri C6 flow cytometer was used for data acquisition and analysis. CD4+ CD25+ Foxp3+ cells were considered to be Tregs and quantified as a percentage of total spleen lymphocytes. Gating is shown in Supplementary Fig. S4.

Statistical analysis and synergism assessment

Statistically significant differences of cell viability, tumor volume, and section staining were assessed using a one-way ANOVA and Tukey–Kramer multiple comparisons test with GraphPad Prism software. Assessment for synergism of combination therapies was performed using the Bliss independence model on primary tumor growth inhibition (47, 48). The assessment of synergism was determined by calculating a synergism assessment factor, which is defined as the percent inhibition observed for the combination therapy minus the additive probability of the % inhibitions observed for the separate therapies (see the Supplementary Data for additional information). Statistical significance for survival was assessed on the basis of Kaplan–Meier survival curves using the log-rank (Mantel–Haenszel) test with a 0.05 significance level corrected for family-wise significance based on the number of comparisons according to the Bonferroni-corrected threshold (significance level/number of comparisons).

Results

The mCTH enzyme was fused to ANXA1 and ANXA5 and confirmed to have methioninase activity that allows for the conversion of selenomethionine to methylselenol. As this enzyme prodrug therapy utilizes the conversion of l-selenomethionine to methylselenol and not l-methionine depletion alone, we evaluated the activity of the engineered mouse CTH toward l-selenomethionine. mCTH-ANXA1 and mCTH-ANXA5 exhibited an activity of 0.75 ± 0.2 U/mg and 0.95 ± 0.1 U/mg, respectively. Enzyme activities with the substrate l-methionine for mCTH-ANXA1 and mCTH-ANXA5 were 1.0 ± 0.1 and 1.3 ± 0.2 U/mg, respectively, compared with 1.0 U/mg for MGL-ANXA5 (26). Yields of purified fusion protein were achieved in excess of 120 mg/L of culture medium, with >95% purity and endotoxin levels of <10 EU/mg.

Strong in vitro binding and cytotoxic efficacy on human breast cancer cells and endothelial cells representative of the phosphatidylserine exposure of tumor vasculature were apparent and comparable among the three systems (mCTH-ANXA1, mCTH-ANXA5, and MGL-ANXA5). Figure 1A summarizes the dissociation constants, all in the nanomolar range, for the three systems, and binding to the cell membrane is shown (Fig. 1B); this binding image is consistent with the protein's being designed for Annexin V to bind to phosphatidylserine expressed on the cell membrane. A cytotoxicity analysis confirms the therapeutic potential of the enzyme prodrug system on MDA-MB-231 and 4T1 breast cancer cells (Fig. 1C and D). The Supplementary Data contain additional in vitro binding data, including the binding curves and hyperbolic fit for dissociation constant determination (Supplementary Fig. S5) and additional live cell confocal multiphoton microscopy data (Supplementary Fig. S1). Relatively minor in vitro differences between the ANXA1 and ANXA5-targeted mCTH failed to distinguish the superiority of one system over the other, warranting a comparative in vivo study.

Figure 1.
Figure 1.

In vitro analysis of fusion protein binding and enzyme prodrug system cytotoxicity. A, The dissociation constants for the fusion proteins were determined from the specific binding curves (available in Supplementary Data) on breast cancer and nonconfluent endothelial cells representative of tumor vasculature endothelium. B, Live cell imaging indicates membrane binding of Dylight 680 conjugated fusion protein the cell membrane of GFP expressing MDA-MB-231 cells. C and D, The cytotoxic effects of the mCTH-ANXA1, mCTH-ANXA5, and MGL-ANXA5 enzyme prodrug systems were compared for MDA-MB-231 (C) and 4T1 (D) breast cancer cells. Groups that received fusion protein were treated on day 0. Selenomethionine was administered daily. Viability was determined by the Alamar Blue assay on day 3, and each sample was represented as a percentage of the vehicle-treated control. Statistical analysis was performed with a one-way ANOVA test with data presented as mean ± SEM (n = 3). Statistical significance versus vehicle-treated control is denoted by * (P < 0.001).

Pharmacokinetic analysis of the mammalian fusion protein indicates rapid entrance into the bloodstream and complete clearance from circulation within 10 hours, shown in Fig. 2A. This result is consistent with similar fusion proteins and allows for daily dosing of both the fusion protein and the prodrug. IHC staining confirms accumulation of the fusion protein on the tumor vasculature and within the tumor tissue (Supplementary Fig. S6).

Figure 2.
Figure 2.

Preliminary in vivo analysis in immune competent 4T1 model. A, The pharmacokinetic profile of mCTH-ANXA5 was determined via ELISA assay of serum samples collected at the time points indicated after fusion protein administration (10 mg/kg i.p.). B, The efficacy of ANXA1 and ANXA5-targeted mCTH enzyme prodrug therapies with selenomethionine on 4T1 tumors in BALB/cJ mice were compared (arrow, treatment period). C, The mammalian (mCTH-ANXA5) and bacterial (MGL-ANXA5) enzyme prodrug systems were evaluated in BALB/cJ mice (arrow, treatment period). B and C, Fusion protein was administered daily (10 mg/kg i.p.), and selenomethionine (5 mg/kg i.p.) was administered 10 hours after fusion protein administration. Statistical significance versus vehicle is indicated by * (P < 0.001). B, No significant difference was observed between mCTH-ANXA1 and mCTH-ANXA5 groups. C, mCTH-ANXA5 and Sel versus MGL-ANXA5 and Sel is indicated by # (P < 0.001). No side effects were observed in any group. Data, mean volume ± SEM (n = 6–10). D, Blood samples were collected weekly during the treatment period and analyzed for the presence of antibodies specific to the administered protein. Antibody detection was performed via ELISA of serum samples, with a positive detection reported as a maximum serum dilution factor (antibody titer).

Evaluation of the therapeutic efficacy of the mCTH-ANXA1 and mCTH-ANXA5 enzyme prodrug systems was performed in BALB/cJ mice bearing orthotopic 4T1 tumors. Although both systems demonstrated tumor growth suppression, the effect was more pronounced with the mCTH-ANXA5 system, shown in Fig. 2B. The stronger antitumor effect of the mCTH-ANXA5 system with the 4T1 in vivo model led to the selection of ANXA5 as the primary candidate for targeting the enzymes in further in vivo studies.

Observation of tumor volumes showed 4T1 tumor progression to occur faster with the MGL-ANXA5 system compared with the mCTH-ANXA5 system (Fig. 2C). Significant tumor progression during MGL-ANXA5 and selenomethionine treatment after 1 week of treatment initiation suggests that the elicited antibody response has neutralizing capabilities, quickly rendering the bacterial enzyme prodrug system ineffective in the immunocompetent model. The mCTH-ANXA5 system does ultimately revert from tumor growth suppression to tumor progression. The cytotoxic effect generated by the targeted enzyme prodrug therapy was insufficient for sustained tumor suppression in the challenging 4T1 in vivo model, motivating the study of combination with rapamycin and cyclophosphamide.

Fusion protein–specific antibody titers were analyzed alongside antitumor efficacy for the mCTH-ANXA5 and MGL-ANXA5 enzyme prodrug systems in BALB/cJ mice, shown in Fig. 2D. The MGL-ANXA5 system was included as a positive control, expected to elicit an immune response based on the enzyme origination from Pseudomonas putida. Daily administrations of the enzyme prodrug therapy, consisting of MGL-ANXA5 or mCTH-ANXA5, elicited no anaphylactic response or observable negative reaction over a 3-week treatment period from either group. An analysis of blood samples collected weekly during the treatment revealed the presence of MGL-ANXA5–specific antibodies within 7 days of the start of the treatment (at a serum dilution of 10−4). By the third week, MGL-ANXA5–specific antibody levels were an order of magnitude higher than after 1 week of treatment. Contrary to the MGL-ANXA5 results, no mCTH-ANXA5–specific antibodies were detected through 3 weeks of treatment.

Survival and primary tumor volume were assessed using the MGL-ANXA5 system in immunodeficient SCID mice bearing orthotopic MDA-MB-231 tumors (Fig. 3). The enzyme prodrug system delayed tumor growth and increased survival compared with vehicle treatment and treatment with either the targeted enzyme or prodrug alone. Interestingly, the targeted enzyme alone does produce a mild growth delay possibly due to a local reduction in tumor methionine levels due to enzymatic activity. As observed in the immunocompetent 4T1 model, tumors ultimately progress with enzyme prodrug therapy. Upon tumor progression, the enzyme prodrug therapy was complemented with the addition of rapamycin treatment once tumor volumes reached 1,000 mm3. The MDA-MB-231 cell line is characterized as rapamycin resistant by many groups and frequently used as a control in rapamycin resistance studies (49–52). Treated mice were grouped as enzyme prodrug therapy only or enzyme prodrug with rapamycin treatment to determine whether targeting mTOR could counter the anticipated tumor progression with the enzyme prodrug therapy alone. Rapamycin addition to the enzyme prodrug therapy resulted in a dramatic reduction of MDA-MB-231 tumor volume and enhanced survival.

Figure 3.
Figure 3.

In vivo tests in the immunodeficient MDA-MB-231 model. A, The enzyme prodrug therapy in combination with rapamycin (Rap) was evaluated in SCID mice bearing orthotopic MDA-MB-231 tumors (bottom arrow, enzyme prodrug treatment period). MGL-ANXA5 was administered daily (10 mg/kg i.p.). Selenomethionine (5 mg/kg i.p.) was administered 10 hours after fusion protein administration. Rapamycin (5 mg/kg i.p.) was administered daily indicated by top arrow. Data, mean volume ± SEM (n = 5–10 at the start of the study). Statistical significance versus vehicle-treated group is indicated by * (P < 0.001). Statistical significance of enzyme prodrug treatment versus enzyme prodrug treatment with rapamycin is indicated by # (P < 0.05). B, Kaplan–Meier survival curves are shown for groups indicated from A. C, Negligible change in mouse mass observed throughout the duration of study is shown (arrow, treatment period). In addition, no side effects were observed for any treatment.

Survival of immunocompetent BALB/cJ mice and primary 4T1 tumor volume were assessed for the combination treatments with the enzyme prodrug system, rapamycin, and cyclophosphamide, with the results presented in Fig. 4A and B and Table 1, and experimental replicates are available in Supplementary Fig. S7. There were no apparent side effects or weight differences among the experimental groups (Fig. 4C), and a full pathologic analysis for treatment-related toxicities revealed no abnormalities (Department of Pathology, University of Oklahoma Health Sciences Center). The enzyme prodrug system with cyclophosphamide and rapamycin yielded the smallest tumor volumes and longest median survival. When administered alone, the individual constituents of the enzyme prodrug system have minimal effect on survival or primary tumor volume compared with the vehicle-treated mice, yet together, the enzyme prodrug system (mCTH-ANXA5 and selenomethionine) almost doubles survival. When the enzyme prodrug system is combined with rapamycin, tumor growth suppression is sustained to levels comparable with the full combination (mCTH-ANXA5 and selenomethionine, rapamycin, and cyclophosphamide); however, no further survival benefit is apparent without the inclusion of cyclophosphamide. Statistical analysis strongly supports the benefit of the full combination of the enzyme prodrug therapy, rapamycin, and cyclophosphamide, as the Kaplan–Meier survival curves are significantly improved over the vehicle-treated mice and mice receiving only the enzyme prodrug treatment (Fig. 4B; Table 1).

Figure 4.
Figure 4.

Combination therapy in the immunocompetent 4T1 model. A, The enzyme prodrug therapy in combination with rapamycin (Rap) and cyclophosphamide (Cyc) was evaluated in BALB/cJ mice bearing orthotopic 4T1 tumors (arrow, treatment period). mCTH-ANXA5 was administered daily (10 mg/kg i.p.). Selenomethionine (Sel; 5 mg/kg i.p.) was administered 10 hours after fusion protein administration. Rapamycin (5 mg/kg i.p.) and cyclophosphamide (10 mg/kg i.p.) were administered daily. Statistical significance versus vehicle treated on same day or last available point is indicated by * (P < 0.001). Data, mean volume ± SEM (n = 5–10 at the start of the study). B, Kaplan–Meier survival curves are shown for groups indicated from A. C, Negligible change in mouse mass observed throughout the duration of study is shown (arrow, treatment period). In addition, no side effects were observed for any treatment.

View this table:
Table 1.

Kaplan–Meier survival curves for all groups of mice with 4T1 tumors were analyzed for significance against vehicle-treated mice, and the combination therapies were additionally analyzed against the enzyme prodrug therapy

An analysis for synergism regarding tumor growth inhibition of 4T1 tumors (Table 1) was utilized to confirm that the antitumor effects of the enzyme prodrug system were in fact a result of methylselenol generation and not simply the additive results of the therapy constituents, selenomethionine alone or mCTH-ANXA5 alone. The highest synergism factors were found for the combination of rapamycin with the enzyme prodrug therapy (with or without cyclophosphamide).

IHC analysis of 4T1 tumor sections for apoptosis and cell proliferation, quantified in Fig. 5, confirmed the cytotoxic effect of the enzyme prodrug system. Representative images for each group are shown in Supplementary Fig. S8. Staining of activated caspase-3 for apoptosis supports the methylselenol-related activation of the caspase cascade, as all tumors receiving mCTH-ANXA5 and selenomethionine displayed a significantly increased percentage of tumor cells staining positive for activated caspase-3 (Fig. 5A). In addition, the reduction in Ki-67, a marker for proliferative activity, further corroborates the cytotoxic enzyme prodrug system activity (Fig. 5B). IHC staining of tumor sections for HIF1A expression showed increased levels of HIF1A in the enzyme prodrug therapy–treated group (Fig. 5C). As expected, all of the groups treated with rapamycin showed very low HIF1A levels.

Figure 5.
Figure 5.

IHC and cellular analyses of combination therapy in immunocompetent model. BALB/cJ mice bearing orthotopic 4T1 tumors treated for 3 weeks with the enzyme prodrug treatment result in increased staining of the apoptosis marker, activated casapse-3, and decreased staining of the proliferation marker, Ki-67. Rapamycin (Rap) reduced staining of HIF1A in the same mice. A Nikon Eclipse E800 microscope was used to capture 15 fields of view of tumor sections from 3 mice per group (necrotic tumor cores were excluded) for activated caspase-3 and Ki-67 staining. Immunostaining for activated caspase-3 (A) and Ki-67 (B) was quantified as percent of cells (hematoxylin counterstain) with DAB and is presented as mean ± SEM. C, IHC staining of HIF1A with DAB development was quantified from whole section images counterstained with hematoxylin. Data, mean ± SEM (n = 3 mice). Statistical significance between groups is indicated by * (P < 0.01). Cyclophosphamide (Cyc) reduces the number of pulmonary metastasis in the orthotopic 4T1-TdTomato mouse model despite poor correlation with Treg numbers in the spleen of BALB/cJ mice. D, A Leica stereomicroscope with an automated ImageJ macro was used to quantify fluorescent nodules in the lung. Data are shown as individual nodules from the lungs of 3 mice per group after 3 weeks of treatment on a log-normal scale, as the nodule sizes were logarithmically distributed. Median nodule size on the log scale is marked. Total nodules per group are summed and shown. Statistical significance between groups is indicated by * (P < 0.05). E, CD4+ CD25+ FoxP3+ Treg levels were quantified with flow cytometry and are presented as a percentage of spleen lymphocytes in BALB/cJ mice with 4T1 grafts after 3 weeks of treatment or healthy BALB/cJ mice with no tumor. Data, mean ± SEM (n = 3 mice). No statistical significance was found in the difference of the level of Tregs for mice with tumors in the vehicle-treated group compared with any of the treated groups.

Sections of tumors of mice in the groups indicated in Fig. 5 were analyzed by IHC for blood vessels using staining for CD31. At the conclusion of the treatment, blood vessels were found to be present in the vehicle-treated tumors at a low density but were not observed in any of the enzyme prodrug–treated tumors (Supplementary Fig. S9).

Individual metastatic nodules on the lungs of tumor-bearing mice are graphically presented as a function of nodule size with nodule quantities summarized in Fig. 5D. Both the enzyme prodrug system and cyclophosphamide independently and together provided a strong reduction in the quantity of metastatic nodules on the lung. The quantities of metastatic nodules observed for each experimental group correlate with the observed survival data for all groups, with reduced metastases and enhanced survival for cyclophosphamide-treated groups and groups treated with the enzyme prodrug therapy except the enzyme prodrug therapy combined with rapamycin. Rapamycin provided minimal benefit regarding the quantity of pulmonary metastases and in the absence of cyclophosphamide negatively impacted the benefit provided by the enzyme prodrug therapy. Cyclophosphamide proved to exhibit antimetastatic effects alone and when combined with the other therapies, including rapamycin. There was minimal correlation of Treg levels in the spleen with metastatic formation or cyclophosphamide treatment (Fig. 5E). In addition, there was no statistical significance in the difference in the level of Tregs for mice with tumors in the vehicle-treated group compared with any of the treated groups.

Discussion

The data presented provide support for the viability of targeted enzyme prodrug systems and for the design of rational therapeutic combinations that address the different hallmark characteristics of cancer. Enzyme prodrug systems, a unique therapeutic approach to minimize systemic toxicity, are traditionally limited by delivery methods and immunogenicity. The mCTH-ANXA5 system overcomes both limitations through the use of a primarily native protein and targeted delivery to phosphatidylserine that is easily accessible on the tumor vasculature. The enzymatic conversion of selenomethionine to methylselenol creates a cytotoxic effect, increases tumor hypoxia, and transiently suppresses tumor growth. Addressing the hypoxic response with rapamycin and triggering immune involvement with cyclophosphamide produces a synergistic antitumor effect and results in significant survival benefits.

The fusion of mCTH to ANXA5 effectively allows the binding to phosphatidylserine present on cancer cells and the tumor vasculature. The externalization of phosphatidylserine in most solid tumors suggests the ANXA5-targeting strategy for mCTH may be broadly applicable to a number of cancers. In addition, the utilization of a phosphatidylserine-targeted enzyme did not exhibit any perceptible off-target effects. A protein-based delivery strategy allows for a recombinant production approach and traditional administration strategies for biologics as opposed to gene delivery methods required for other enzyme prodrug systems. The reasoning behind the development of the mCTH-ANXA5 enzyme prodrug system, which consists entirely of native protein with the exception of the three amino acid substitutions, is the theorized immunogenicity in mice and anticipated immune reaction in humans of the comparable bacterial MGL-ANXA5 system. The three amino acid substitutions in the human CTH, equivalent to those performed in this study for the mouse CTH, are not anticipated to produce any immune response in humans through computational analysis (16). Although the immunogenicity analysis in BALB/cJ mice is not comprehensive, the positive results provide preliminary rationale for further evaluation. Examination of in vitro viability and in vivo confirmation with IHC analysis suggests the mCTH conversion of selenomethionine to methylselenol provides antiproliferative and proapoptotic stimuli. Achievement of cytotoxic levels of drug using targeted delivery of enzyme with ANXA5 and minimized immunogenicity with a protein-engineered approach provides a feasible and translational enzyme prodrug strategy.

HIF1A expression in the treated tumors is indicative of a hypoxic environment resultant from vessel occlusion and reduced blood flow due to the vasculature-targeted approach (27). The hypoxic response, brought about by the enzyme prodrug therapy, promotes cell survival despite restrictive conditions; therefore, its enhanced presence in the enzyme prodrug–treated group suggests that vessel occlusion does occur but is insufficient for complete tumor destruction. The hypoxic response was addressed through the incorporation of rapamycin in the treatment regimen. Rapamycin inhibits mTOR and counters the prosurvival signaling present in tumor cells following the hypoxic environment enhanced by the vasculature-directed enzyme prodrug system. The fact that the addition of rapamycin to the enzyme prodrug therapy resulted in much less tumor growth in two different mouse tumor models (Figs. 3A and 4A) is a strong indication that rapamycin countered mTOR-dependent, and likely HIF1A-related, regrowth. The reduction of HIF1A expression suggests that rapamycin could provide benefit to other therapeutic strategies that enhance the hypoxic conditions of the tumor, particularly vasculature-directed cytotoxic approaches. Reduction of HIF1A expression alone may indeed provide some therapeutic benefit, although the survival and tumor volume analysis of the rapamycin-only–treated group in the 4T1 model suggest that targeting the mTOR pathway is not sufficient for a significant survival advantage. This study focuses on the use of rapamycin as a complement to the enzyme prodrug therapy; however, it may also be of interest to examine the enzyme prodrug therapy or other vasculature-directed approaches as possible sensitizers to rapamycin treatment.

The enzyme prodrug therapy combined with rapamycin resulted in significantly reduced tumor volumes in the MDA-MB-231 and 4T1 models (Figs. 3A and 4A). However, with the 4T1 model, survival was essentially identical to when the enzyme prodrug was used by itself (Fig. 4A and B; Table 1). The triple-negative 4T1 tumor model utilized is highly aggressive and metastatic, with mortality frequently resultant from pulmonary metastasis in addition to primary tumor load. A significant extension of survival would likely require an antimetastatic approach beyond the capabilities of the enzyme prodrug therapy. The cytotoxic effect of the enzyme prodrug therapy against the tumor vasculature and the cancer cells likely enhances tumor antigen presentation systemically. As the tumor vasculature is targeted, this allows tumor antigens to be released directly into the bloodstream, resulting in improved antigen recognition and heightened host immune response. Thus, this therapy provides a unique opportunity for further stimulation of an antitumor immune response through immunomodulation and/or immunostimulation.

The combination with cyclophosphamide aims to reduce immune tolerance through preferential depletion of Tregs. Contrary to many tumor models (41), 4T1 tumor–bearing mice did not exhibit elevated levels of Treg levels, and a cyclophosphamide-related decrease was not overtly apparent. Yet, the enhanced antimetastatic benefits of cyclophosphamide are evident. It is likely that the antimetastatic effects of low-dose cyclophosphamide were due to alterations in the levels of various cytokines and/or to the emergence of tumor-infiltrating dendritic cells, which have been observed in previous studies of tumors in animal models (53). A more comprehensive immunologic evaluation would help to fully elucidate the cyclophosphamide-induced antimetastatic mechanism in the orthotopic 4T1 mammary tumor mouse model.

Together, cyclophosphamide, rapamycin, and mCTH-ANXA5/selenomethionine enzyme prodrug therapy enhance survival, reduce primary tumor volumes, reduce metastatic progression, increase apoptosis, decrease proliferation, and decrease HIF1A expression. The three-pronged approach involving a cytotoxic agent, immunomodulation, and an antihypoxic response mechanism addresses many of the challenges presented with the highly aggressive and metastatic, poorly immunogenic, treatment-resistant, triple-negative 4T1 tumor model (54). The phosphatidylserine-directed enzyme prodrug system provides a targeted cytotoxic strategy applicable to other solid tumor models that utilize the hypoxic response and immune evasion mechanisms to some extent. Rapamycin and cyclophosphamide combined with a vasculature-targeted delivery of apoptosis-inducing agents would likely produce a significant enhancement of survival and reduction in tumor progression for other models.

Disclosure of Potential Conflicts of Interest

J.J. Krais has ownership interest (including patents) in a patent application (enzyme conjugate and prodrug cancer therapy). R.G. Harrison has ownership interest (including patents) in U.S. patent number 8,986,701 (enzyme prodrug cancer therapy selectively targeted to tumor cells or tumor vasculature and methods of production thereof) and U.S. patent application number 14/865,650 (enzyme conjugate and prodrug cancer therapy). No potential conflicts of interest were disclosed by the other authors.

Authors' Contributions

Conception and design: J.J. Krais, C. Kurkjian, R.G. Harrison

Development of methodology: J.J. Krais, K.-M. Fung, V.I. Sikavitsas, R.G. Harrison

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.J. Krais, N. Virani, P.H. McKernan, Q. Nguyen, K.-M. Fung

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.J. Krais, N. Virani, P.H. McKernan, Q. Nguyen, K.-M. Fung, R.G. Harrison

Writing, review, and/or revision of the manuscript: J.J. Krais, N. Virani, P.H. McKernan, Q. Nguyen, V.I. Sikavitsas, C. Kurkjian, R.G. Harrison

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Q. Nguyen

Study supervision: R.G. Harrison

Grant Support

This work was supported by Oklahoma Center for the Advancement of Science and Technology (one grant to R. Harrison, V. Sikavitsas, and C. Kurkjian) and the University of Oklahoma Biomedical Engineering Center (one grant each to J. Krais, N. Virani, and Q. Nguyen)

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.

Acknowledgments

We thank the following for their contributions: Molecular Imaging Core at the Stephenson Cancer Center for the use of the animal imaging equipment and the laboratory of Dr. Rajagopal Ramesh for transfecting the 4T1/TdTomato/Luciferase cells; the Noble Microscopy Facility at the University of Oklahoma (Norman, OK) for use of the confocal and stereomicroscopes; and the Peggy and Charles Stephenson Cancer Center at the University of Oklahoma and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the NIH under grant number P20 GM103639 for the use of Histology and Immunohistochemistry Core, which provided histology services.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

  • Received May 2, 2016.
  • Revision received January 17, 2017.
  • Accepted May 9, 2017.

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Antitumor Synergism and Enhanced Survival with a Tumor Vasculature–Targeted Enzyme Prodrug System, Rapamycin, and Cyclophosphamide
John J. Krais, Needa Virani, Patrick H. McKernan, Quang Nguyen, Kar-Ming Fung, Vassilios I. Sikavitsas, Carla Kurkjian and Roger G. Harrison
Mol Cancer Ther September 1 2017 (16) (9) 1855-1865; DOI: 10.1158/1535-7163.MCT-16-0263
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