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Research Articles: Therapeutics, Targets, and Development

Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs

Departments of ¹ Advanced Therapeutics and ² Medical Oncology, British Columbia Cancer Research Center, ³ Department of Pathology and Laboratory Medicine, Faculty of Medicine, and ⁴ Division of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada

Requests for reprints: Gigi N.C. Chiu, Department of Pharmacy, Faculty of Science, National University of Singapore, Block S4, 18 Science Drive 4, Singapore 117543, Singapore. Phone: 65-6516-5536; Fax: 65-6779-1554. E-mail: phacncg{at}nus.edu.sg

Abstract

Various methods have been explored to enhance antibody-based cancer therapy. The use of multivalent antibodies or fragments against tumor antigens has generated a great deal of interest, as various cellular signals, including induction of apoptosis, inhibition of cell growth/survival, or internalization of the surface molecules, can be triggered or enhanced on extensive cross-linking of the target/antibody complex by the multivalent form of the antibody. The goal of the studies reported here was to develop multivalent antibody constructs via grafting of antibody molecules onto liposome membranes to enhance antibody activity. Using trastuzumab and rituximab as examples, up to a 25-fold increase in the antibody potency in cell viability assay was observed when the antibodies were presented in the multivalent liposome formulation. Key cell survival signaling molecules, such as phosphorylated Akt and phosphorylated p65 nuclear factor-B, were down-regulated on treatment with multivalent liposomal trastuzumab and liposomal rituximab, respectively. Potent in vivo antitumor activity was shown for liposomal trastuzumab. The data presented here showed the potential of liposome technology to enhance the therapeutic effect of antibodies via a mechanism that modulates cell survival through clustering of the target/antibody complex. [Mol Cancer Ther 2007;6(3):844–55]

Introduction

Therapeutic antibodies have emerged as the most rapidly expanding class of pharmaceuticals for use in treating cancer as well as other diseases. The therapeutic effectiveness of antibodies can be attributed to multiple functional properties, including ligand binding competition, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), interference with receptor dimerization and signaling, and induction of apoptosis (1–3). Various strategies are being explored to increase the efficacy of antibody-based therapies, including (a) modifications to the Fc domain to enhance effector functions for improving ADCC and CDC responses, (b) modifications to the binding domain to increase affinity for target antigens, (c) attachment of toxins, drugs, radionuclides, or cytokines to the antibody, (d) pretargeting of prodrugs and radionuclides to diminish systemic toxicities of these agents, and (e) construction of multivalent antibodies or fragments via chemical conjugation or protein expression (1, 4).

Of the various strategies, multivalent antibody constructs are of particular interest. These complexes have considerable therapeutic potential and will form the basis of developing cancer-targeted nanopharmaceuticals. Many of the tumor antigens to which therapeutic antibodies have been developed function through antigen dimerization or via inducing formation of antigen clusters consisting of tens to hundreds of molecules on cell surface (5, 6). When these tumor antigens are exposed to multivalent constructs of antibodies or fragments, clustering of the target/antibody complex occurs as a result of increased valency and avidity of the construct. Multimerization of antibodies or fragments can decrease off-rate and increase the efficiency of inducing clustering of the target/antibody complex (7). Indeed, recent studies have shown that cross-linking or multimerization of several antibodies or fragments enhanced biological responses compared with the bivalent format. These responses include activation of cell death signals, inhibition of cell survival signals, and/or increased internalization of the tumor antigen (8–11). This is best exemplified by recent studies with rituximab, a therapeutic antibody targeting CD20. Under in vivo conditions, the FcR-expressing cells are thought to act as cross-linking agents that induce the clustering of CD20/rituximab and subsequent apoptotic signals, in addition to their role in mediating ADCC (2, 8, 9). Currently, multimerization of antibodies or fragments can be achieved through several techniques. For instance, the use of anti-IgG antibodies or protein A/G as cross-linking agents is commonly used in vitro because of ease and simplicity, but this approach is not well suited for in vivo applications. Alternatively, multivalent antibodies or fragments can be readily generated by protein expression technology; however, in vivo application of these constructs is challenging (7, 12). Recently, the research group led by Alan Epstein developed a dextran polymer formulation of rituximab that promoted hyper-cross-linking–induced apoptosis of CD20⁺ lymphoma cells (4).

We consider here a strategy to develop multivalent antibody constructs that involves grafting therapeutic antibodies onto liposomes. Liposomes are vesicular structures consisting of a lipid bilayer membrane enclosing an aqueous core. They have been widely applied as drug carriers for a variety of agents, including several clinically approved liposomal formulations of anticancer drugs. Improvements in therapeutic activity of the liposome-associated drug are thought to be due, in part, to liposome-mediated increases in tumor delivery (13, 14). Antibodies have been attached to liposomes for the purpose of showing target cell–specific delivery of drug-loaded carriers and the encapsulated contents (15–18). However, these studies rarely examine the potential therapeutic activity of the attached antibody or ligand. When testing was done with anti-HER2 F(ab')-conjugated liposomes, the results showed minimal or no measurable activity from these liposomes in the absence of an encapsulated drug (19). We believe there is a need to better define the activity of liposome-conjugated therapeutic antibodies partly because the methods used to generate these conjugates result in the attachment of multiple antibody copies onto each liposome and confer multivalency. We also believe that liposomes offer several additional benefits when considered for the development of multivalent therapeutic antibody constructs. Liposomes containing polyethylene glycol (PEG) are well established for their long circulation half-lives (20), and this is not compromised when using appropriately conjugated antibodies (21, 22). As the elimination kinetics of the attached antibodies is controlled by the liposome carrier, this should translate to an increase in the circulation half-life of the attached antibodies. This is a desirable attribute because infrequent antibody dosing is more convenient and acceptable for patients (23). In addition, antibodies are commonly combined with chemotherapy in treating various cancers because of beneficial clinical responses (24, 25). The use of liposomes in this case offers the potential of codelivering antibody/drug combinations (17, 18), which in turn could facilitate the close temporal association of both antibody and drug as may be required to achieve optimal therapeutic effects from the selected combinations.

In this study, multivalent liposomal antibody constructs were developed using trastuzumab and rituximab. These therapeutic antibodies were chemically conjugated to the PEG-containing liposomes with reactive maleimide groups present at the PEG₂₀₀₀ terminus (26, 27). The effects of multivalent presentation of these antibodies via liposomes on cell viability, cell cycle status, and the expression/activation of signaling molecules were analyzed. The data presented here showed that the in vitro activity of antibody was enhanced when the antibody was presented in a multivalent format, suggesting a mechanism that involves clustering of target/antibody complex.

Materials and Methods

Cell Lines
MCF-7^HER2 cells were a kind gift from Dr. Moulay Alaoui-Jamali (McGill University, Montreal, Quebec, Canada). MDA-MB-435/LCC6 cells were generously provided by Dr. Robert Clarke (Georgetown University, Washington, DC), and LCC6^HER2 cells were transfected with the human expression plasmid pREP9 containing the full-length human c-erbB-2 cDNA (provided by Dr. Ming Tan, M. D. Anderson Cancer Center, Houston, TX) as described previously (28). MCF-7^HER2 and LCC6^HER2 cells were grown in DMEM (Stemcell Technologies, Vancouver, British Columbia, Canada) supplemented with 2 mmol/L L-glutamine, 10% fetal bovine serum (Hyclone, Logan, UT), and 500 µg/mL geneticin (LCC6^HER-2) or 100 µg/mL geneticin (MCF-7^HER-2). For all experiments, MCF-7^HER2 and LCC6^HER2 cells were cultured without geneticin for 1 week before the experiment. Ramos (RA 1) cell line was purchased from the American Type Culture Collection (Manassas, VA) and maintained in culture conditions according to the American Type Culture Collection instructions. Z-138 cell line was generously provided by Dr. Zeev Estrov (University of Texas, Houston, TX) and maintained in RPMI 1640 (Stemcell Technologies) supplemented with 2 mmol/L L-glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Preparation of Liposome-Conjugated Antibodies
Liposomes, composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(PEG)₂₀₀₀]/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(PEG)₂₀₀₀]-MAL (molar ratio, 48:45:5:2), were prepared by the extrusion procedure (29). The resulting mean vesicle diameter was 100 to 120 nm as determined by quasielastic light scattering using the Nicomp submicron particle sizer model 370/270. Therapeutic antibody was conjugated to liposomes according to an established procedure using N-succinimidyl-3-(2-pyridyldithio)propionate (Pierce, Nepean, Ontario, Canada) as the bifunctional linker (27). Liposomes were labeled with [³H]CHE (0.03 µCi/µmol lipid) for the determination of liposomal lipid concentration by liquid scintillation counting (Packard scintillation counter model 1900 TR) with aliquots mixed with 5.0 mL Pico-Fluor 15 scintillation fluid (PerkinElmer BioSignal Inc., Montreal, Quebec, Canada). The Pierce Micro bicinchoninic acid protein assay kit was used to determine the concentration of free antibodies as well as the amount of antibodies conjugated to liposomes using 0.5% Triton X-100 to solubilize the liposomal lipids.

Measurement of Mitochondrial Activity
Cells (3,500 per well for LCC6^HER2 and MCF-7^HER2, 20–50,000 per well for Ramos, and 20,000 per well for Z-138) were seeded in 96-well plates. For LCC6^HER2 and MCF-7^HER2 cells, treatment was added after an overnight incubation to allow cell adherence to plates. For Ramos and Z-138 cells, treatment was added on the same day after seeding. Cell viability was estimated using measurements of mitochondrial activity, as determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as described previously (28), or Alamar Blue was added in 10 µL/well with an 18-h incubation before reading fluorescence in a Fluorstar (v 1.2) plate reader (BMG Labtechnologies, Offenburg, Germany).

Flow Cytometric Analyses and 4',6-Diamidino-2-Phenylindole Staining
Cells were harvested after treatment and fixed in cold 70% ethanol adjusted to 1 x 10⁶ cells/mL and 2 x 10⁶ cells/mL for cell cycle analyses and 4',6-diamidino-2-phenylindole (DAPI) staining, respectively. For cell cycle analyses, cells were stained in propidium iodide buffer [50 µg/mL propidium iodide, 1 mg/mL RNase A (Sigma, Oakville, Ontario, Canada), 0.1% Triton X-100 in PBS] at 37°C for 15 min as described previously (28). Cells were then chilled on ice for 1 h and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) with 20,000 events collected for each sample. For DAPI staining, cells were incubated in DAPI staining solution (0.1 µg/mL DAPI, 1 mg/mL RNase A, and 0.1% Triton X-100 in PBS) for 15 min followed by chilling on ice for 30 min. Cells were cytospun onto a glass slide and viewed with a Leica DMLB fluorescence microscope (Leica, Richmond Hill, Ontario, Canada). Images were captured with a Retiga 1300i digital camera and analyzed by the computer software OpenLab 3.5.1 (Improvision, Lexington, MA).

Western Immunoblot Analyses
The following antibodies were used in this study: anti–phosphorylated HER2-Tyr¹²⁴⁸/Tyr¹¹⁷³, anti-HER2, anti–phosphorylated Akt-Ser⁴⁷³ and anti-Akt, anti–phosphorylated p65 nuclear factor-B, p65 nuclear factor-B, Bcl-xL, Bcl-2, and Bax (all were rabbit polyclonal; New England Biolabs, Pickering, Ontario, Canada) and anti-human ß-actin (mouse monoclonal; Sigma). The secondary antibody was horseradish peroxidase–conjugated anti-mouse or anti-rabbit IgG (Promega, Madison, WI). Proteins were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) and visualized after exposure to Kodak autoradiography films (Sigma). The films were subsequently subjected to absorbance analyses for each specific protein band with background correction. Equivalent amounts of protein (30 µg/lane determined by Bradford assay) were resolved by 12% SDS-polyacrylamide premade gels (Bio-Rad, Mississauga, Ontario, Canada).

Colabeling of GM₁ and Free or Liposomal Rituximab in Z-138 Cells
Cells (4 x 10⁶/mL) were seeded in 24-well plates and incubated for 18 h with the following: free rituximab, liposome-conjugated rituximab, and liposome-conjugated irrelevant antibodies. Free rituximab was labeled with Alexa Fluor 488 according to the instructions of the manufacturer's labeling kit (Molecular Probes, Burlington, Ontario, Canada). Liposomes conjugated with rituximab or irrelevant antibodies were labeled with the fluorescent lipid tracer DiO (Molecular Probes). At the end of incubation, cells were washed in cold medium and resuspended in 2.5 µg/mL cholera toxin subunit B labeled with Alexa Fluor 647 in cold medium for 10 min on ice. Cells were washed and mounted in chilled HBSS onto a glass slide. Images were subsequently captured and analyzed with the same fluorescence microscope, digital camera, and software as mentioned above.

Animal Studies
All in vivo studies were completed using protocols approved by the Animal Care Committee of the University of British Columbia and were in accordance with the current guidelines of the Canadian Council of Animal Care. Female Rag2-M mice (20–22 g) were obtained from Taconic (Hudson, NY) and inoculated s.c. unilaterally with 5 x 10⁶ LCC6^HER2 cells. For plasma elimination studies, LCC6^HER2 tumor-bearing Rag2-M mice (tumor volume of 50 mm³) were injected i.v. with 6 mg/kg (in 200 µL) free or liposomal trastuzumab. Twelve mice were used for each study group, with 4 mice used at each time point of blood collection. At 1, 4, and 24 h after injection, blood was collected by cardiac puncture and placed into EDTA-coated microtainer tubes. Plasma was isolated from blood samples by centrifugation at 1,000 x g for 15 min. Aliquots of the plasma were then used to determine free or liposomal trastuzumab plasma levels by a colorimetric ELISA assay. Briefly, a rabbit anti-human IgG Fc fragment (MP Biomedicals, Irvine, CA) was coated onto Nunc MaxiSorp 96-well plate for capturing trastuzumab. A horseradish peroxidase–conjugated rabbit anti-human whole IgG (Sigma) was used for detection, with orthophenylenediamine (Sigma) added as the substrate. Absorbance at 405 nm was measured and compared with values from a standard curve constructed from known amounts of trastuzumab in the free or liposomal form. For tumor localization study, Rag2-M mice with a tumor volume of 50 mm³ were used. Four animals in each study group were injected i.v. with liposomes (labeled with [³H]CHE at 0.1 µCi/µmol) either with or without conjugated trastuzumab at a lipid dose of 100 mg/kg. Tumors were harvested at 24 h after liposome injection. Solvable (PerkinElmer BioSignal Inc.) was added at 0.5 mL to tumor samples, and the mixture was incubated at 50°C overnight. After cooling to room temperature, 50 µL EDTA 200 mmol/L, 200 µL hydrogen peroxide 30%, and 25 µL HCl 10 N were added, and the mixture was incubated for 1 h at room temperature. Subsequently, 5.0 mL scintillation fluid was added, and the samples were kept in the dark for 24 h before determining radioactivity of [³H]CHE by liquid scintillation counting.

For in vivo efficacy studies involving dose titration of liposomal trastuzumab, mice were inoculated s.c. unilaterally with 5 x 10⁶ LCC6^HER2 cells. On day 18 after inoculation of tumor cells, mice were divided into five study groups with six animals per group: (a) saline control, (b) 1 mg/kg liposomal irrelevant antibodies, (c) 0.25 mg/kg liposomal trastuzumab, (d) 0.5 mg/kg liposomal trastuzumab, and (e) 1 mg/kg liposomal trastuzumab. Mice were treated twice weekly for 5 weeks. Tumor measurements were made twice weekly with a caliper. Tumor volume was calculated using the following equation: V = 0.5 x (L x W²), where V, L, and W represent tumor volume (mm³), longer diameter (mm), and shorter diameter (mm) of tumor mass, respectively. The same study was repeated to include additional control arms as follows: 1 mg/kg free irrelevant antibodies, 1 mg/kg free trastuzumab, and control liposomes without conjugated trastuzumab (given at a lipid dose equivalent to that of the highest dose of liposomal trastuzumab). Six mice were used in each study group, and the animals were treated twice weekly for 5 weeks. Tumor measurements were made with a caliper at the beginning and at the end of the 5-week treatment. Fold change in tumor volume was calculated with the following equation: fold change = V_end / V_initial, where V_end is the tumor volume determined after 5 weeks of treatment and V_initial is the tumor volume determined on the day when treatment was first given. Results were presented as an inset in Fig. 5.

View larger version (17K): [in this window] [in a new window]   Figure 5. In vivo efficacy of liposomal trastuzumab in LCC6HER2 breast cancer model. Trastuzumab presented in the multivalent liposomal form was given at 0.25 mg/kg (), 0.5 mg/kg (), and 1 mg/kg () i.v. twice weekly for 5 wks starting on day 18 after inoculation of tumor cells. The liposomal irrelevant antibody construct () was given at 1 mg/kg following the same dosing schedule as liposomal trastuzumab. Saline () was used as the vehicle control. Points, representative study using six mice in each study group; bars, SE. Inset, free or liposomal trastuzumab was given i.v. twice weekly for 5 wks starting on day 18 after inoculation of tumor cells. Irrelevant antibody, as free or liposomal form, was given following the same dosing schedule. Same amount of liposomal lipid was given to the animals as in the group treated with liposomal trastuzumab. Saline was used as the vehicle control. Fold change in tumor volume was calculated as follows: fold change = Vend / Vinitial, where Vend is the tumor volume determined after 5 wks of treatment and Vinitial is the tumor volume determined on the day when treatment was first given using the equation described in Materials and Methods. Columns, representative study using six mice in each study group; bars, SE. Treatment groups: 1, saline; 2, free irrelevant antibody (1.0 mg/kg); 3, free trastuzumab (1.0 mg/kg); 4, liposomal irrelevant antibody (1.0 mg/kg); 5, liposome control; 6, liposomal trastuzumab (0.25 mg/kg); 7, liposomal trastuzumab (0.50 mg/kg); 8, liposomal trastuzumab (1.0 mg/kg). *, P < 0.05, compared with group 3 (free trastuzumab).

Results

Conjugation of Therapeutic Antibodies to 1,2-Distearoyl-sn-Glycero-3-Phosphocholine/Cholesterol/1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(PEG)₂₀₀₀] Liposomes
Trastuzumab and rituximab were conjugated to 1,2-distearoyl-sn-glycero-3-phosphocholine/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(PEG)₂₀₀₀] liposomes containing 2 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(PEG)₂₀₀₀]-MAL through a thioether linkage using N-succinimidyl-3-(2-pyridyldithio)propionate as the bifunctional linker (27, 30). The N-succinimidyl-3-(2-pyridyldithio)propionate modification did not compromise the binding of the N-succinimidyl-3-(2-pyridyldithio)propionate antibody to target cells when compared with the unmodified antibody as determined by flow cytometry (data not shown). With an initial antibody-to-lipid ratio of 75 µg/µmol, a final ratio of 68 ± 6 µg/µmol or 60 ± 2 µg/µmol was obtained for trastuzumab or rituximab, respectively. This represents a range of conjugation efficiencies of 80% to 91%. For subsequent biological evaluation, the liposomal antibody constructs had similar antibody-to-lipid ratios in the range of 60 to 70 µg/µmol, which is equivalent to approximately 38 to 45 copies of antibodies present in a single liposome, an estimate based on the molecular weight of the antibodies and the assumption that 1 µmol of liposomal lipid mixture contains 6.23 x 10¹² vesicles (31).

Reduction in Cell Viability after Exposure to Multivalent Liposomal Antibody Constructs
Standard cell viability assays, where viability was a reflection of mitochondrial activity, were used to determine the fraction of cells affected (fa) on treatment with free or liposomal antibodies. For trastuzumab, testing was conducted in LCC6^HER2 and MCF-7^HER2 breast cancer cell lines under various culture conditions (Fig. 1A and B ). The fa value for free trastuzumab tested in the presence of 10% serum following a 5-day incubation period was at most 0.12, and no improvement in the activity was observed even when the dose was increased to 1 mg/mL (data not shown). In contrast, the fa value of the multivalent liposomal trastuzumab was significantly higher in LCC6^HER2 and MCF-7^HER2 cells at dose of 10 and 100 µg/mL, respectively (P < 0.05). Consistent with previous observations (32), the activity of free trastuzumab was slightly increased when tested in the presence of 0.1% serum or heregulin (the binding ligand associated with HER2/HER3 dimerization). The fa value of free trastuzumab increased to a maximum of 0.2. The activity of multivalent liposomal trastuzumab in the presence of 0.1% serum or heregulin, however, was further enhanced relative to results obtained using serum-rich medium. The fa values approached 0.7 when tested at 200 µg/mL in both LCC6^HER2 and MCF-7^HER2 cells, and significant activity was noted at dose as low as 10 µg/mL for both cell lines. Again, these levels of activity were not observed when using free trastuzumab at doses of 1 mg/mL, thus suggesting that, under these conditions, the activity of this therapeutic antibody was increased at least 100-fold.

View larger version (17K): [in this window] [in a new window]   Figure 1. In vitro treatment of cancer cell lines with bivalent or multivalent liposomal antibody constructs. Fraction of cells affected (fa) by free or liposomal trastuzumab for LCC6HER2 (A) and MCF-7HER2 (B) breast cancer cells after exposure for 5 d under various culture conditions. , free trastuzumab; , liposomal trastuzumab. HRG, heregulin (final concentration, 5 ng/mL). Fraction of cells affected (fa) by free or liposomal rituximab for (C) Ramos (Burkitt's lymphoma) and (D) Z-138 (mantle cell lymphoma) cell lines after exposure for 3 d in 10% serum. , free rituximab; , liposomal rituximab; , free rituximab + goat F(ab')2 anti-human IgG (50 µg/mL). Fraction of cells affected (fa) was calculated as follows: fa = 1 – fu, where fu was the fraction of remaining viable cells calculated as the ratio of the absorbance readings from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for treated and untreated groups. Columns, mean of three independent experiments; bars, SE. *, P < 0.05; #, P < 0.05, compared with the free antibody treatment.

Modulation of Cell Survival Pathways by Multivalent Liposomal Antibody Constructs
To understand the enhancement in biological activity produced by multivalent liposomal antibody constructs, an assessment of antibody-mediated changes in cell survival pathways was completed. Trastuzumab activity of these pathways was conducted in LCC6^HER2 cells, and two important observations can be made based on these data, which have been summarized in Fig. 2 . First, changes in the cell cycle status and in the expression levels of HER2 and its downstream target Akt induced by both free or liposomal trastuzumab were more prominent when cells were cultured in low (0.1%) serum. This observation is consistent with a previous study showing that trastuzumab activity could be increased when cells were treated in reduced serum conditions (32). Second, although free trastuzumab was capable of inducing a small increase in the percentage of cells in G₁-G₀ and engenders down-regulation of phosphorylated HER2 and total HER2 in treated cells, it was not capable of reducing phosphorylated Akt level under the conditions used. In contrast, liposomal trastuzumab induced a reduction in phosphorylated Akt level in addition to a significant loss of phosphorylated HER2 and down-regulation of total HER2. Also of interest, cell cycle analysis suggested that the liposomal trastuzumab caused a substantial decrease in the percentage of cells in G₁-G₀ phase with a concomitant increase in the proportion of cells in S phase. These differences in the ability of free and liposomal trastuzumab to modulate Akt-mediated cell signaling pathway and to affect cell cycle status are therapeutically important (33–35).

View larger version (28K): [in this window] [in a new window]   Figure 2. Cell cycle and Western blot analyses of LCC6HER2 breast cancer cells after exposure to free or liposomal trastuzumab for 5 d in 10% or 0.1% serum. The concentration of free or liposomal trastuzumab was 200 µg/mL. Absorbance of each protein band was analyzed, and the protein-to-actin absorbance ratio was reported above each protein band. Ab, antibody.

View larger version (38K): [in this window] [in a new window]   Figure 3. Cell cycle analysis, DAPI staining, and Western blot analyses of Z-138 cells after exposure to free or liposomal rituximab (RTX) for 3 d. The concentration of free or liposomal rituximab was 15 µg/mL. For Western blot analyses: lane 1, untreated control; lane 2, free rituximab; lane 3, liposomal rituximab; lane 4, liposome control; lane 5, liposomal irrelevant antibody; lane 6, cross-linked rituximab. Absorbance of each protein band was analyzed, and the protein-to-actin absorbance ratio was reported above each protein band. For DAPI staining, yellow arrows indicate apoptotic morphology of DNA condensation or fragmentation associated with Z-138 cells exposed to various treatments. Bar, 50 µm. All images are shown at the same magnification.

View larger version (24K): [in this window] [in a new window]   Figure 4. Colabeling of GM1 glycolipid and free or liposomal form of rituximab in Z-138 mantle lymphoma cells. Free rituximab was labeled with Alexa Fluor 488, and liposomal rituximab was labeled with the fluorescent lipid DiO. Z-138 cells were incubated with fluorescently labeled free or liposomal rituximab for 18 h. After washing the cells thrice with clear HBSS, cholera toxin subunit B (CTB)-Alexa Fluor 647 (2.5 µg/mL) was added to label GM1. Cells were then washed, mounted, and imaged live. A to C, white arrows, orange regions that are indicative of colocalization. Bar, 50 µm (C). A to C, all images are shown at the same magnification. Insets 1 and 2, representative single and merged images of individual cells stained with free or liposomal rituximab (green) and cholera toxin subunit B-Alexa Fluor 647 (red).

Results from a preliminary pharmacokinetic analyses of free and liposomal trastuzumab injected i.v. into LCC6^HER2 tumor-bearing mice have been summarized in Table 1 . Importantly, liposome conjugation of trastuzumab substantially increased the mean plasma area under the curve values and decreased clearance of the liposomal form of the antibody. These data would suggest that the elimination behavior of the antibody is now dictated by the liposomes, which are likely retained in the plasma compartment for extended time frames. Consistent with liposomal formulation-mediated decreases in elimination, the results summarized in Table 1 also suggest that liposomal trastuzumab exhibited a small, yet significant (P < 0.05), increase in tumor accumulation levels when compared with liposomes without trastuzumab, a result supportive of the notion that these liposomes are targeting the HER2-positive cell population.

Discussion

Multimeric assembly or clustering of receptors on cell surface has emerged as a common theme to a wide variety of biological processes. In malignant cells, various cellular signals, including induction of apoptosis, inhibition of cell growth/survival, or internalization of surface molecules, could be triggered or enhanced on extensive cross-linking with multimeric ligand or antibody (and fragments), suggesting that receptor or silver clustering via multivalent interactions can modulate cell survival (8–11). In light of these observations, it was hypothesized that multivalent antibody constructs generated by the grafting of antibody molecules onto liposome membrane surfaces would modulate malignant cell survival signaling pathways via extensive cross-linking of target/antibody complex. Several lines of evidence from this study support this hypothesis.

First, when rituximab was presented in the multivalent liposomal form, it was capable of inducing regions of enriched GM₁ staining on the cell surface compared with the free, bivalent form (Fig. 4). Previous biophysical analyses of the interactions of biotinylated liposomes with another surface containing neutravidin or avidin showed that the liposomes could deform and spread without rupture on binding (43) and that multiple biotin-avidin-biotin cross-bridges were concentrated in the "contact zone" between the liposome surface and the interacting surface (44). The enrichment in GM₁ staining when cells were exposed to liposomal rituximab is thus in line with these previous observations and is possibly due to an increase in membrane raft size mediated through the multivalent liposomal rituximab/CD20 cross-linking.

Second, using trastuzumab and rituximab as examples, significant increases in the in vitro antibody activity were shown in the respective cancer cell lines when the antibodies were presented in the multivalent liposomal form compared with the free, bivalent form (Fig. 1). Free, bivalent antibodies are active when tested in the animal models, but it is anticipated that this activity is due, in part, to the presence of FcR-expressing effector cells, such as macrophages and natural killer cells (45). The FcR-expressing effector cells have been suggested as a mediator of the cross-linking of target/antibody complex (8, 46). Our in vivo efficacy results showed that the multivalent liposomal antibody construct was therapeutically active. It is possible that these multivalent liposomal antibody constructs could act through multiple mechanisms in vivo, including (a) apoptosis induction via hyper-cross-linking of target/antibody complex as supported by our in vitro data and (b) improved Fc effector functions, including ADCC and CDC responses. In our preliminary in vitro studies, 15-fold increase in ADCC and 3-fold increase in CDC were observed in the Z-138 cells treated with liposomal rituximab compared with those treated with the free antibody (39). Z-138 cells treated with rituximab cross-linked by a secondary antibody were similar to those treated with the free antibody alone for in vitro ADCC and CDC responses. Currently, further studies are being done to discern the in vivo mechanisms of the multivalent liposomal antibody.

Third, our results showed that key cell survival signaling molecules were down-regulated when the cancer cells were exposed to the multivalent liposomal antibody. For trastuzumab, a difference in the ability of free and liposomal form of the antibody to reduce the expression levels of the target molecule HER2 and the downstream molecule Akt was observed. The phosphatidylinositol 3-kinase-Akt signaling pathway has been shown to play a key role in cancer cell proliferation, survival, and chemoresistance in breast and other cancers and represents a pathway that can be exploited for the development of novel therapeutics (33–35). Multivalent liposomal trastuzumab was capable of down-regulating the active, phosphorylated form of Akt in addition to the target receptor HER2 (Fig. 2), showing the importance of multivalent interactions of trastuzumab with its target molecule and the therapeutic potential of liposomal antibody constructs. This is further supported by a recent study that showed that the internalization of HER2 could only be triggered on extensive cross-linking by a secondary antibody of the anti-HER2 antibody-bound receptor (10).

For rituximab, a similar trend was observed, where only on presenting the antibody in the multivalent form via either liposome conjugation or secondary F(ab')₂ cross-linking was the antibody activity significantly increased over that of the free, bivalent form. Importantly, multivalent liposomal rituximab was capable of down-regulating the phosphorylated form of nuclear factor-B, which is a transcription factor that has been suggested to play a pivotal role in the regulation of cell proliferation and survival in lymphomas, including the aggressive mantle cell lymphoma (47) as well as the downstream, antiapoptotic Bcl-xL, which has been shown to protect cells from drug cytotoxicity (48–50).

Considering that trastuzumab and rituximab are frequently combined with chemotherapy to achieve optimal therapeutic effects (51, 52), developing liposome formulations of therapeutic antibody would offer advantages in addition to those based on multivalent interactions with the target ligand. From a molecular perspective, the multivalent liposomal antibody constructs, which were able to down-regulate pivotal signaling molecules that mediate chemoresistance, could potentially sensitize cancer cells to drug-induced cytotoxicity, thus improving responses to chemotherapy. The use of the multivalent antibody constructs could also provide an effective means of screening antibody/drug combinations, as it would overcome the problem of inaccurate assessment due to the use of an inactive form of the antibody in the in vitro setting.

Although it has been suggested that the use of antibody fragments could reduce Fc-mediated rapid plasma elimination and immunogenic responses (53), we believe that fragmenting the whole antibody may not be beneficial for the preparation of antibody-liposome conjugates, especially when the antibody molecule possesses therapeutic activity, such as those used in this study. Our results showed that whole trastuzumab conjugated to liposomes exhibited potent antitumor activity compared with the trastuzumab-based F(ab')-conjugated liposomes reported previously (19). Fc-mediated plasma elimination could be reduced through conjugation of whole antibody to PEG terminus of liposomes as supported by the plasma elimination data of liposomal trastuzumab (Table 1). Immunogenicity is anticipated to be reduced through the use of humanized and fully human antibody molecules for liposome conjugation (17). Although our results indicated tumor accumulation of the liposomal trastuzumab construct, further studies are warranted to examine the penetration of these liposomal antibody constructs into solid tumors because of potential "binding site barrier" effect (17).

From a pharmaceutical and clinical perspective, liposome technology is well suited for the development of delivery system (14, 54), and we believe that this technology could be used to develop a pharmaceutical product capable of codelivering therapeutic antibodies and chemotherapeutics. Taken together, we are now pursuing the goal of screening for optimized antibody/drug combinations that exhibit synergistic interactions and have the potential to be developed as a combination product for treating cancer.

Acknowledgments

We thank Dana Masin for her excellent support for the animal studies.

Footnotes

Grant support: Canadian Institutes of Health Research (M.B. Bally, G.N.C. Chiu, and D.N. Waterhouse), Canadian Breast Cancer Research Alliance (M.B. Bally), Lymphoma Foundation of America (R. Klasa), Academy of Finland, and Cultural Foundation of Finland (A.I. Kapanen).

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.

Received 3/23/06; revised 11/14/06; accepted 1/31/07.

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