| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics |
Differentiation Therapy Unit, Laboratory of Oncology, G. Gaslini Children’s Hospital, Genoa, Italy 16148 [F. P., C. B., D. M., M. P.], and Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G2H7 [P. S., E. H. M., T. M. A.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
NB is a chemosensitive tumor and cytotoxic agents such as DXR are considered to be important and effective treatment modalities. However, the therapeutic efficacy of this anticancer drug, which is widely used in the treatment of solid tumors, is restricted by dose-limiting toxicity to bone marrow and heart tissue (4) . The selective toxicity of DXR would be greatly improved if the concentration of drug in tumors could be increased relative to that in sensitive normal tissues. NB cells express abundant amounts of the disialoganglioside GD2 at their cell surface, but the presence of this epitope on normal tissues, such as the cerebellum and peripheral nerves, is limited (5, 6, 7, 8) .
In the last few years, liposomes have been used to increase the selective toxicity of chemotherapy drugs, resulting in improved therapeutic outcomes and/or reduced toxicities (9, 10, 11) . A long circulating (sterically stabilized or Stealth) liposomal formulation of DXR (Doxil/Caelyx) has been approved for use in the treatment of both Kaposi’s sarcoma and ovarian cancer (12 , 13) . In patients receiving large cumulative doses of DXR, clinical cardiotoxicity was reduced (14) , and this liposomal formulation of DXR is now under advanced clinical trial evaluation for several human solid cancers.
We reported previously that the ability to selectively target liposomal anticancer drugs via specific ligands against antigens expressed on malignant cells could improve the antitumor activity of the liposomal formulations. Several studies have shown that the use of immunoliposomes (SIL) significantly increased target cell binding in vitro (15, 16, 17, 18, 19) and improved therapeutic efficacy in vivo (16 , 20, 21, 22) . Moreover, the coupling antibody Fab' fragments instead of whole immunoglobulin molecules to Stealth liposomes have increased their circulation times (23) and improved their in vivo efficacy (24) . Because NB tumors express abundant amounts of GD2 at the cell surface, and because this epitope is only minimally expressed by normal tissues (5 , 6) , the use of anti-GD2 as a ligand for targeting liposomal DXR to NB merits additional investigation.
In this study we examined target binding and selective cytotoxicity of aGD2-targeted formulations against GD2-positive NB cell lines in vitro. Pharmacokinetic studies were performed, and survival times were evaluated in nude mice xenografts of the human NB cell line, HTLA-230. The HTLA-230 cells, when administered i.v. in nude mice, provide an in vivo pseudometastatic model for the metastatic spread of tumor in human disease (25) . Our results clearly show that liposomal DXR targeted via Fab' fragments of aGD2 were effective in suppressing the growth of metastases in mice implanted with a human NB tumor.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
For preparation of F(ab')2 fragments, purified antibody was digested with lysyl endopeptidase (Wako Chemicals, Richmond, VA) in 50 mM Tris-HCl (pH 8.5) at 37°C for 3 h, and the reaction mix was passed through a protein-A/G column equilibrated with 50 mM Tris-HCl (pH 7.5). F(ab')2 fragments were reduced with 7.5 mM ß-mercaptoethylamine (Sigma) for 90 min at 37°C in acetate buffer (pH 6.5), after which the reducing agent was removed by passing the sample over a Sephadex G-25 column chromatography pre-equilibrated with the same buffer. The concentration of all of the mAbs was determined by spectrophotometry (
= 280 nm), and purity was assessed by SDS-PAGE. The reactivities of whole mAbs, F(ab')2, and Fab' fragments were checked by indirect immunofluorescence using FITC-labeled goat antimouse antibody and IgG-Fab'-specific (Sigma), respectively, and FACS analysis (Becton Dickinson, San Jose, CA).
Liposome Preparation and DXR Loading.
SLs and SILs were synthesized from HSPC:CHOL:DSPE-PEG2000, 2:1:0.1 molar ratio, and HSPC:CHOL:DSPE-PEG2000:DSPE-PEG2000-MAL, 2:1:0.08:0.02 molar ratio, respectively. The lipids were first dissolved in chloroform and then combined in the appropriate ratios (15
, 19 , 27)
. In some preparations, [3H]CHE was added as a nonexchangeable, nonmetabolizable lipid tracer. After evaporation under nitrogen, dried lipid films were hydrated in 25 mM HEPES and 140 mM NaCl buffer (pH 7.4). The hydrated liposomes were sequentially extruded at 65°C (Lipex Biomembranes Extruder, Vancouver, British Columbia, Canada) through a series of polycarbonate filters of pore size ranging from 0.2 µm down to 0.08 µm to produce primarily unilamellar vesicles. Liposomal size was characterized by dynamic light scattering (28)
using a Brookhaven BI90 submicron particle size analyzer (Brookhaven Instruments Corp., Holtsville, NY). The mean liposomal diameters of extruded liposomes were in the range of 98–110 nm.
DXR was loaded into liposomes via an ammonium sulfate gradient, using a method adapted from Ref. 29 and reported previously (21) . The loading efficiency of DXR was >95%, and liposomes routinely contained DXR at a concentration of 150–180 µg DXR/µmol phospholipids (PL).
For preparation of immunoliposomes, whole mAb or Fab' fragments, trace labeled with 125I (21) , were coupled to the maleimide terminus of maleimide-terminated polyethylene glycol-DSPE, as described previously (19) . After activation with Traut’s reagent (2-iminothiolane) at a molar ratio of 20:1 (Traut’s: IgG) for 1 h at room temperature in HEPES buffer (pH 8.0; 25 mM HEPES and 140 mM NaCl), unreacted Traut’s reagent was removed using a G50 column. The coupling reaction was run at molar ratio of whole mAb or Fab' fragments to phospholipid molar ratio of 1:2000 or 1:1000, respectively, under argon atmosphere for 18 h at room temperature. Uncoupled mAb or Fab' were separated from the liposomes by passing the coupling mixture through a Sepharose CL-4B column in HEPES buffer (pH 7.4). The efficiency of coupling was determined from the amount of 125I-labeled mAb or Fab' bound to the surface of liposomes and is expressed as nmol mAb/µmol PL. The coupling densities on liposomes were in the range of 0.33–0.53 nmol anti-GD2 mAb/µmol PL (0.66–1.06 nmol binding sites, because each mAb has two binding sites) and 0.54–0.91 nmol anti-GD2 Fab' fragments/µmol PL.
Cell Lines and Culture Condition.
To broadly cover the phenotypes exhibited by NB cells in vitro, we used the GD2-positive human NB cell lines ACN, GI-LI-N, HTLA-230, and IMR-32 (17
, 19)
. Four GD2-negative cell lines, the adherent cell lines HeLa (human cervical carcinoma) and A431 (human epidermoid carcinoma), and the suspension cell lines HL-60 (human myeloid leukemia) and Jurkat (human T cell) were used in some experiments as controls. All of the cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 50 IU/ml sodium penicillin G, 50 µg/ml streptomycin sulfate, and 2 mM L-glutamine, as described previously (30)
.
Cellular Association Studies.
3H[CHE]-labeled liposomes were used to measure cellular binding and uptake, as described previously (17
, 19)
. Briefly, cells (1 x 106/ml) were plated in 48-well tissue culture plates. Different formulations of [3H]CHE-labeled liposomes, with or without coupled aGD2-specific mAb or isotype-matched nonspecific mAb, anti-CD19, were added to each well (100–800 nmol PL/ml) and maintained at either 4°C or 37°C in a total volume of 200 µl. In competition experiments, a 25-fold excess of free mAb aGD2 was added 30 min before adding [3H]CHE-labeled immunoliposomes. After a 1-h incubation, the cells were extensively washed, trypsinized, and lysed with 1 N NaOH for protein evaluation and analyzed in a scintillation counter.
The cellular association with NB cells of immunoliposomes targeted via either aGD2 or Fab'-fragments was also analyzed by flow cytometry (FACS). Aliquots of cells (1 x 106/tube) were incubated for 1 h at 4°C or 37°C with different formulations of liposomes (SL, aGD2-SIL, or Fab'-SIL). The cells were washed subsequently with PBS, and then incubated with goat antimouse FITC-conjugated IgG-Fab' specific for 30 min at 4°C, and, after washing, were enumerated by flow cytometry. All of the cytometry experiments were performed using a FACScan instrument for fluorescence-activated cell sorting (Becton-Dickinson Immunocytometry Systems).
In Vitro Cytotoxicity Experiments.
The specific inhibition of the proliferation of NB cells was determined by an in vitro proliferation assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (31)
. Samples included free DXR and SL[DXR], aGD2-SIL[DXR], or Fab'-SIL[DXR]. Briefly, 3 x 104 HTLA-230 or GI-LI-N NB cells (100% and 70% GD2-positive, respectively) were plated in 96-well plates and incubated with either free DXR or liposome-encapsulated DXR as above, for 1, 24, and 48 h at 37°C. At the different time points, the cells were washed twice with PBS before replacing with fresh medium and incubating for an additional 71, 48, and 24 h, respectively, i.e., all of the plates were incubated for a total time of 72 h. At the end of the incubation, tetrazolium dye was added, plates were incubated for an additional 3–4 h, formazan crystal were dissolved in acid isopropanol, and then the plates were read on a Titertek Multiskan Plus (Flow Laboratories, Inc., Mississauga, Ontario, Canada) at the dual wavelengths of 570 and 650 nm (31)
.
Leakage of DXR from Liposomes.
Release of DXR from liposomes was measured by incubation of DXR-containing liposomes at 37°C with either PBS or human plasma in 24-well plates at a final DXR concentration of 10 µg/ml, as described previously (32)
. DXR leakage from liposomes was followed by measuring the fluorescence dequenching of DXR at selected time points (0–24 h) using fluorimetry (33)
. Complete release (100% dequenching) of DXR was obtained by treating liposomes with 5 µl of 10% Triton X-100 in distilled water/ml. The percentage of DXR released was determined from fluorescence intensity of wells relative to 100% dequenching.
Pharmacokinetic Experiments.
Six to 8-week-old outbred female BALB/c mice were purchased from the Health Sciences Laboratory Animal Services, University of Alberta. BALB/c mice in the weight range of 20–30 g were injected via the tail vein with a single dose of liposomes (0.5 µmol PL/mouse), with or without coupled aGD2 or GD2 Fab' fragments, containing approximately 2–4 x 105 cpm of the aqueous-space marker TI (34)
. At selected time points (0.5, 2, 6, 12, 24, and 48 h) postinjection, mice (3 mice/group) were anesthetized with halothane and sacrificed by cervical dislocation. A blood sample (100 µl) was collected by heart puncture and counted for the 125I label in a Beckmann 8000 gamma counter. Blood correction factors were applied to all of the samples (35)
. Pharmacokinetic parameters were calculated using the software package PKAnalyst, Version 1.1 (MicroMath Scientific Software, Salt Lake City, UT). In these experiments and the experiments, below, all of the procedures involving animals and their care were in accordance with institutional guidelines, and in compliance with national and international laws and policies.
In Vivo Therapeutic Studies.
Four to 6-week-old female athymic nude (nu/nu) mice were purchased from Harlan Laboratories (Harlan Italy-S. Pietro al Natisone, UD). Female nude mice (8 mice/group) were injected with HTLA-230 cells (3.5 x 106 cells in 200 µl of HEPES-buffered saline) in the tail vein (i.v.) on day 0, and treated on days 1, 3, 5, and 7 postinoculation of cells at a dose of 2.5 mg DXR/kg (schedule A). In an alternative dosing schedule, mice were injected with 4 x 106 HTLA-230 cells on day 0, and treated on days 1 and 3 postinoculation of cells at a dose of 5 mg DXR/kg (schedule B). For each experiments, mice received one of the following treatments: HEPES-buffered saline, free Fab' from aGD2 (amount of Fab' was equivalent to the amount coupled to the injected dose of Fab'-SIL[DXR]), Fab'-SIL, SL[DXR], or Fab'-SIL[DXR]. In another experiment mice received a single administration of 10 mg of Fab'-SIL[DXR]/kg on day 1 postinoculation of cells. Mice were monitored routinely for weight loss, and survival times were used as the main criterion for determining treatment efficacy. Histological evaluation of microscopic metastases was performed for all of the tissues. Organs were fixed in neutral-buffered 10% formalin, processed by standard methods, embedded in paraffin, sectioned at 5–10 µm, and stained with H&E.
Statistical Methods.
Results are expressed as means ±95% CIs. All of the in vitro data derive from at least three independent experiments. All of the in vivo experiments have been performed at least twice with similar results. The significance of the differences between experimental groups in the survival experiments was determined by Kaplan-Meier curves and Peto’s log-rank test by the use of StatsDirect statistical software (CamCode, Ashwell, United Kingdom).
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), A431, HL-60, and Jurkat (data not shown). The data showed that the level of cellular binding of liposomes is dependent on GD2 expression in the different cell lines. aGD2-SIL showed 5–10 times higher cellular association to all of the GD2-positive cell lines, compared with nontargeted liposomal preparations (SL). In contrast, the GD2-negative cell line, HeLa, showed very low levels of liposomes associated with the cells for both targeted or nontargeted formulations (Fig. 1A)
.
|
. aGD2-SIL increased the cell association on IMR-32 cells by a factor of 10-fold over that obtained with liposomes bearing nonspecific isotype-matched antibodies (e.g., aCD19) or with mAb-free liposomes when incubated at 37°C and by a factor of 5-fold when incubated at 4°C. These results suggest that internalization of aGD2-SIL may be occurring at 37°C. Moreover, in competition experiments we showed that the dose-dependent cellular association and uptake of the aGD2-targeted liposomes to GD2-positive NB cells could be blocked by the addition of a 25-fold excess of free mAb 30 min before adding [3H]CHE-labeled aGD2-SIL (Fig. 1B)
.
In Vitro Cytotoxicity Experiments.
The cytotoxicity of DXR, either free or encapsulated in SL[DXR] (no mAb) or aGD2-SIL[DXR], was compared as a function of time. As expected (Table 1)
, the IC50 decreased as the exposure of cells to drug increased from 1 h to 48 h. After 24 h of incubation aGD2-SIL[DXR] were 
3-fold more cytotoxic than SL[DXR] against GI-LI-N cells and 4-fold more cytotoxic against HTLA-230 cells, respectively. Similar cytotoxicities were seen against HeLa cells for SL[DXR] and aGD2-SIL[DXR] suggesting that binding and internalization of the targeted liposomes to the GD2 epitope in the other cell lines is contributing to the increased cytotoxicity. Free DXR had the highest levels of cytotoxicity against all three of the cell lines in vitro, and did not distinguish between GD2-expressing and GD2-nonexpressing cell lines (Table 1)
. With increased exposure times, the IC50 for the aGD2-SIL[DXR] became more similar to that of free DXR for the GD2-expressing cells but not for the HeLa cells. This suggests that a longer exposure of aGD2-SIL[DXR] to GD2-positive cells increases the cellular uptake of the liposomal drug package, contributing to the increased cytotoxicity. Because free DXR is rapidly and widely redistributed to tissues after in vivo administration, it is expected that the targeted formulations, with their ability to selectively bind the target cells, will have an advantage over the free drug in vivo.
|
Pharmacokinetic Profiles.
The pharmacokinetics of TI-labeled SL, aGD2-SIL, and Fab'-SIL was evaluated in 6–8-week old outbred female BALB/c mice (34)
. In Fig. 2
, the results are expressed as percentage of the administered dose remaining in blood, which corrects for leakage of the label, and represents intact liposomes at selected time points (36
, 37)
. These findings clearly indicate that liposomes coupled to Fab' fragments of aGD2 had long-circulating profiles in blood with a MRT of 26.2 h, compared with aGD2-SIL (MRT of 9.5 h) with the PK profile for Fab'-SIL being almost identical to that obtained with SL (MRT of 26.2 h).
|
, bold line) showed high levels of binding to the HTLA-230 cell line, similar to that obtained with aGD2-SIL (Fig. 3
, thin line), whereas SL did not bind to NB cells (Fig. 3)
. The GD2-negative cell line HeLa showed no differences in cell association among SL, aGD2-SIL, or Fab'-SIL, additionally confirming the selective cell association of Fab'-SIL to GD2-positive cells (Fig. 3)
. To confirm these results, [3H]CHE-labeled Fab'-SIL were also used in some cell binding experiments. Fab'-SIL showed 10-fold higher levels of both cellular binding and internalization to NB cells over that obtained with liposomes bearing nonspecific isotype-matched Fab'-fragments or with Fab'-fragment-free liposomes (data not shown).
|
). After a 1-h incubation the IC50 was similar for the Fab'-SIL to those seen for the aGD2-SIL[DXR] (Table 1)
.
To evaluate the in vivo effectiveness of Fab'-SIL[DXR], experiments were performed using two different dosing protocols, keeping in mind that the commonly used dosing of DXR in nude mice with human xenografts is 2.5–10 mg/kg mouse/week (39)
. The results are summarized in Fig. 4
. For schedule A (2.5 mg/kg DXR on days 1, 3, 5, and 7), mice receiving Fab'-SIL[DXR] showed a significant improvement (P = 0.0003) in long-term survivors compared with all of the other groups. Seven of 8 mice treated with Fab'-SIL[DXR] were still alive at 120 days after inoculation of tumor cells (Fig. 4A)
. Mice receiving schedule A also showed partial, but not statistically significant (P = 0.6), improvement in mean survival times for mice treated with SL[DXR] (Fig. 4A)
when compared with untreated animals or mice treated with free-aGD2 Fab', Fab'-SIL, and free-DXR. In addition, histological evaluation of the main organs clearly showed that Fab'-SIL[DXR] selectively inhibited NB growth in all of the examined organs in nude mice (Table 2)
. Noteworthy, mice receiving Fab'-SIL[DXR] and schedule B (5 mg/kg DXR on days 1 and 3) were still alive after 4 months and could be considered cured (P < 0.0001).
|
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The exposure of NB cells to aGD2-SIL[DXR] resulted in a significantly greater inhibition of cell proliferation, in vitro, than that obtained for nontargeted liposomes. As expected, no differences in cell association and inhibition of cell proliferation were observed for aGD2-targeted compared with nontargeted liposomal DXR on GD2-negative cell lines. These results are compatible with the interpretation that binding and internalization of aGD2-immunoliposomes contributed to the increased cytotoxicity of aGD2-SIL[DXR] against GD2-positive cells. Competition data provided an additional indication that aGD2-SIL[DXR] internalization was receptor mediated, indicating that the ability of targeted liposomes to trigger receptor-mediated endocytosis is important for the cytotoxicity of their encapsulated drugs (20 , 22 , 32) . These results are in agreement with previous reports showing that endocytosis of targeted immunoliposomes leads to release of drug from the internalized material into the cytosol and to subsequent cytotoxicity (17 , 40, 41, 42) .
Previous preclinical studies showed that lysis of NB cells by mouse and chimeric aGD2 resulted from antibody-dependent cellular cytotoxicity and complement-depended cytotoxicity (7 , 8 , 38) . Because we desired to avoid complicating the interpretation of our results by this nonspecific mechanism of cell kill, in the second part of our study we investigated a new therapeutic strategy, by using a liposomal carrier targeted by aGD2 Fab' fragments, which should allow us to obtain a therapeutic effect attributable primarily to the cytotoxic effect of entrapped DXR. The use of the Fab' fragment of aGD2 instead of whole mAb abolishes the mononuclear phagocyte system uptake of the aGD2, which takes place via an Fc receptor-mediated mechanism (43) . Moreover, it has been shown that small PEG immunoliposomes with Fab' fragments coupled at the PEG terminus have significantly longer circulation time than comparable formulations containing whole mAbs (23) . This can result in enhanced accumulation of the liposomes in solid tumor (23) and in significant suppression of tumor growth (24) . Fab'-SIL, like aGD2-SIL, showed specific cell association to GD2-positive NB cells, but unlike aGD2-SIL, were long-circulating, having MRTs almost identical to that obtained with nontargeted liposomes (SL). As shown previously (10 , 44) , long circulation times are required for liposomes to gain access to tumor sites.
The human NB cell line HTLA-230 was chosen for our investigation into the therapeutic efficacy of Fab'-SIL[DXR] because, when injected i.v. in nude mice, this xenograft model mimics the metastatic spread observed in advanced-stage NB patients (25) . A number of studies conducted on large cohorts of patients have shown that the presence of circulating NB cells in the blood and micrometastases in the bone marrow at the time of primary surgery are strong predictors of relapse (45) . Because bone marrow micrometastases are a direct measurement of the ability of tumor cells to spread systemically, the establishment of a model that closely mimics the clinical situation allows a more realistic evaluation of antitumor therapies. Thus, the experimental metastatic model used in these studies provides a consistent test for the potential use of targeted DXR in human metastatic disease. The schedule of treatment was chosen deliberately to allow for evaluation of the effects of treatments during the metastatic cascade, i.e., during the stages in which tumor cells are in intravascular circulation and endothelium-attachment take place, or when extravasation, stromal invasion, and colonization take place (46 , 47) .
The efficacy of Fab'-SIL[DXR] was evaluated in terms of metastasis growth inhibition and increasing life span, which represent the principal end points for establishing the antitumoral activity of a new drug formulation. Keeping in mind that the highest dose of DXR that can be administered to nude mice is 10 mg DXR/kg weekly (39 , 48) , together with the considerations above, we designed a schedule in which mice received 5 mg DXR/kg on days 1 and 3 after inoculation of tumor cells. Using this protocol, 100% of mice treated with Fab'-SIL[DXR] were still alive >4 months after treatment (P < 0.0001 compared with control).
In conclusion, in this work we have shown significant improvements in the therapeutic effects of the chemotherapeutic drug DXR, when encapsulated in immunoliposomes targeted with the Fab' of aGD2. Long-term survival rates approaching 100% were observed for several doses and dosing schedules of the immunoliposomes, suggesting that total inhibition of the metastatic growth of human NB in a xenograft model of nude mice was occurring. Although Ahmad et al. (49) have demonstrated previously that immunoliposomes can partially eradicate lung cancer in a metastatic murine tumor model, it is important to emphasize that this is the first work, to our knowledge, in which it has been possible to completely prevent the growth of NB cells as macro- and micrometastases with the use of immunospecific liposomes. As a note of caution, SIL appear to lose their advantage when attempts are made to treat established advanced solid tumors (20 , 35 , 49) , likely because the "binding site barrier" restricts penetration of SIL into the tumor (50 , 51) . It is our opinion that Fab'-SIL[DXR] deserve clinical evaluation as an adjuvant therapy for advanced-stage NB disease or for disease resulting from incomplete surgery or early micrometastatic lesions. Moreover, because other neuroectoderma-derived tumors, such as melanoma, express abundant amounts of the GD2 epitope (18 , 19 , 52) use of these new targeted liposomal formulations of DXR may provide an effective therapeutic strategy for additional GD2-positive human tumors.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by Fondazione Italiana per la lotta al Neuroblastoma, Costa Crociere, and Associazione Italiana Ricerca Cancro (AIRC). F. P. is a recipient of a Fondazione Italiana per la lotta al Neuroblastoma fellowship. 
2 These authors contributed equally to the work.
3 To whom requests for reprints should be addressed, at Differentiation Therapy Unit, Laboratory of Oncology, G. Gaslini Children’s Hospital, Largo G. Gaslini 5, 16148 Genoa, Italy. Phone: 39-010-5636342; Fax: 39-010-3779820; E-mail: mircoponzoni{at}ospedale-gaslini.ge.it
4 The abbreviations used are: NB, neuroblastoma; DXR, doxorubicin; SIL, sterically stabilized (Stealth) immunoliposome; SL, nontargeted Stealth liposome; CI, confidence interval; SL[DXR], DXR-loaded SL; DSPE, distearoylphosphatidylethanolamine; PEG, polyethylene glycol; mAb, monoclonal antibody; aGD2, anti-GD2 whole murine monoclonal antibody; HSPC, hydrogenated soy phosphatidylcholine; FACS, fluorescence-activated cell sorter; Fab', Fab' fragments of aGD2; aGD2-SIL[DXR], DXR-loaded aGD2-targeted sterically stabilized (Stealth) immunoliposome; Fab'-SIL[DXR], DXR-loaded Fab' fragments of aGD2-targeted sterically stabilized (Stealth) immunoliposome; MRT, mean residence time; CHOL, cholesterol; [3H]CHE, cholesteryl-[1,2-3H-(N)]-hexadecyl ether; TI, [125I]-tyraminylinulin.
5 F. Pastorino, personal observation.
Received 7/ 8/02. Accepted 10/28/02.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Hussain, A. Pluckthun, T. M. Allen, and U. Zangemeister-Wittke Antitumor activity of an epithelial cell adhesion molecule targeted nanovesicular drug delivery system Mol. Cancer Ther., November 1, 2007; 6(11): 3019 - 3027. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Pastorino, C. Brignole, D. Di Paolo, B. Nico, A. Pezzolo, D. Marimpietri, G. Pagnan, F. Piccardi, M. Cilli, R. Longhi, et al. Targeting Liposomal Chemotherapy via Both Tumor Cell-Specific and Tumor Vasculature-Specific Ligands Potentiates Therapeutic Efficacy. Cancer Res., October 15, 2006; 66(20): 10073 - 10082. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Brignole, D. Marimpietri, F. Pastorino, B. Nico, D. Di Paolo, M. Cioni, F. Piccardi, M. Cilli, A. Pezzolo, M. V. Corrias, et al. Effect of bortezomib on human neuroblastoma cell growth, apoptosis, and angiogenesis. J Natl Cancer Inst, August 16, 2006; 98(16): 1142 - 1157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Campochiaro and the First ARVO/Pfizer Institute Working Group Ocular versus Extraocular Neovascularization: Mirror Images or Vague Resemblances Invest. Ophthalmol. Vis. Sci., February 1, 2006; 47(2): 462 - 474. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Puppo, S. Pastorino, G. Melillo, A. Pezzolo, L. Varesio, and M. C. Bosco Induction of Apoptosis by Flavopiridol in Human Neuroblastoma Cells Is Enhanced under Hypoxia and Associated With N-myc Proto-oncogene Down-Regulation Clin. Cancer Res., December 15, 2004; 10(24): 8704 - 8719. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. PASTORINO, C. BRIGNOLE, D. MARIMPIETRI, D. DI PAOLO, M. ZANCOLLI, G. PAGNAN, and M. PONZONI Targeted Delivery of Oncogene-Selective Antisense Oligonucleotides in Neuroectodermal Tumors: Therapeutic Implications Ann. N.Y. Acad. Sci., December 1, 2004; 1028(1): 90 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Brignole, F. Pastorino, D. Marimpietri, G. Pagnan, A. Pistorio, T. M. Allen, V. Pistoia, and M. Ponzoni Immune Cell-Mediated Antitumor Activities of GD2-Targeted Liposomal c-myb Antisense Oligonucleotides Containing CpG Motifs J Natl Cancer Inst, August 4, 2004; 96(15): 1171 - 1180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, and L. Gianni Anthracyclines: Molecular Advances and Pharmacologic Developments in Antitumor Activity and Cardiotoxicity Pharmacol. Rev., June 1, 2004; 56(2): 185 - 229. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Sapra and T. M. Allen Improved Outcome When B-Cell Lymphoma Is Treated with Combinations of Immunoliposomal Anticancer Drugs Targeted to Both the CD19 and CD20 Epitopes Clin. Cancer Res., April 1, 2004; 10(7): 2530 - 2537. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Sapra, E. H. Moase, J. Ma, and T. M. Allen Improved Therapeutic Responses in a Xenograft Model of Human B Lymphoma (Namalwa) for Liposomal Vincristine versus Liposomal Doxorubicin Targeted via Anti-CD19 IgG2a or Fab' Fragments Clin. Cancer Res., February 1, 2004; 10(3): 1100 - 1111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Pastorino, C. Brignole, D. Marimpietri, M. Cilli, C. Gambini, D. Ribatti, R. Longhi, T. M. Allen, A. Corti, and M. Ponzoni Vascular Damage and Anti-angiogenic Effects of Tumor Vessel-Targeted Liposomal Chemotherapy Cancer Res., November 1, 2003; 63(21): 7400 - 7409. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Pastorino, C. Brignole, D. Marimpietri, G. Pagnan, A. Morando, D. Ribatti, S. C. Semple, C. Gambini, T. M. Allen, and M. Ponzoni Targeted Liposomal c-myc Antisense Oligodeoxynucleotides Induce Apoptosis and Inhibit Tumor Growth and Metastases in Human Melanoma Models Clin. Cancer Res., October 1, 2003; 9(12): 4595 - 4605. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||
), nonspecific aCD19-SIL (
), and aGD2-SIL (
). Cells were also incubated at 4°C for 1 h with aGD2-SIL (
). In competition experiments, cell association of aGD2-SIL was conducted in the presence of excess free-aGD2 (
). Data are expressed as nmol of PL/mg protein, and are the mean of four experiments, each done in triplicate ±CI.
). At different times postinjection blood was collected and counted for 125I label. Each point represents the average of 3 mice, ±CI.
), Fab'-SIL (+), and HEPES buffer (control,