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Experimental Therapeutics, Preclinical Pharmacology |
Division of Hematology/Oncology [J. W. P., C. C. B.] and Department of Anesthesia [U. B. N., J. D. M.], University of California, San Francisco, San Francisco, California 94143; California Pacific Medical Center Research Institute, San Francisco, California 94115 [K. H., D. B. K., G, C., R, S., Y. S., D. M., D. P.]; Memorial Sloan-Kettering Cancer Center, New York, New York [J. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Here we describe the pharmacokinetics and therapeutic efficacy of anti-HER2 immunoliposomes containing doxorubicin (dox) in a series of animal models.
Results: Immunoliposomes displayed long circulation that was identical to that of sterically stabilized liposomes in single- and multiple-dose studies in normal rats. Anti-HER2 immunoliposome-dox produced marked therapeutic results in four different HER2-overexpressing tumor xenograft models, including growth inhibition, regression, and cures. These results demonstrated that encapsulation of dox in anti-HER2 immunoliposomes greatly increased its therapeutic index, both by increasing antitumor efficacy and by reducing systemic toxicity. Immunoliposome-dox was significantly superior to all other treatment conditions tested, including free dox, liposomal dox, and anti-HER2 MAb (trastuzumab). When compared with liposomal dox in eight separate therapy studies in HER2-overexpressing models, immunoliposome delivery produced significantly superior antitumor efficacy in each study (P < 0.0001 to 0.04). Anti-HER2 immunoliposome-dox containing either recombinant human MAb HER2-Fab' or scFv C6.5 yielded comparable therapeutic efficacy. Cure rates for immunoliposome-dox reached 50% (11 of 21) with optimized immunoliposomes and Matrigel-free tumors and overall was 16% (18 of 115) versus no cures (0 of 124) with free dox or liposomal dox. Finally, anti-HER2 immunoliposome-dox was also superior to combinations consisting of free MAb plus free dox or free MAb plus liposomal dox.
Conclusions: Anti-HER2 immunoliposomes produced enhanced antitumor efficacy via targeted delivery.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development of stable, long-circulating liposomes has led to a new era in liposome drug delivery (2) . For example, sterically stabilized liposomes, consisting of small, neutrally charged unilamellar liposomes with a polymeric coating of PEG, display retarded clearance by the RES, resulting in substantially prolonged drug circulation (3) . In addition, the long circulation times and small sizes of these liposomes allow preferential extravasation in many solid tumors because of vascular abnormalities associated with tumor angiogenesis (4) . However, sterically stabilized liposomes do not interact directly with tumor cells in vitro or in vivo, and instead release drug for eventual diffusion into tumor cells (5) .
Similarly, progress in MAb-based therapy of cancer has finally led to clinical validation after two decades of research (reviewed in Ref. 6 ). A leading example has been the development of MAbs directed against the p185HER2 (HER2; ErbB2) receptor tyrosine kinase, the product of the HER2 (c-erbB2) proto-oncogene. HER2 plays an important role in the pathogenesis of breast and other cancers (reviewed in Ref. (7) . HER2 is highly overexpressed in a significant proportion of these cancers, and HER2 overexpression is clearly associated with poor prognosis in breast cancer (8 , 9) . Hence, HER2 is an attractive target for MAb-based therapeutic strategies. rhuMAb HER2 (trastuzumab; Herceptin), a humanized MAb directed against HER2, represents one such therapy (10) and has demonstrated clinical benefit in the treatment of advanced breast cancer. Trastuzumab can induce antitumor responses as a single agent (11 , 12) , but it is most efficacious when combined with chemotherapy (13) .
We have developed anti-HER2 immunoliposomes to combine the tumor-targeting properties of MAbs such as trastuzumab with the drug delivery properties of sterically stabilized liposomes. Anti-HER2 immunoliposomes efficiently bind to and internalize in HER2-overexpressing cells, resulting in intracellular drug delivery (14 , 15) . Here we show the PK and therapeutic properties of dox-loaded anti-HER2 immunoliposomes in a series of animal models.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of Liposomes and Immunoliposomes.
Liposomes were prepared by a lipid film hydration-extrusion method as small unilamellar vesicles (100 ± 10 nm) containing hydrogenated soy phosphatidylcholine and cholesterol as described (14
, 15)
. Liposomes of this composition have increased rigidity and decreased permeability because of the high transition temperature of hydrogenated soy phosphatidylcholine (Tm = 54°C) at 37°C. PEG (Mr 1900)-derivatized distearoyl-phosphatidylethanolamine was included at 0–7 mol % of total lipid for steric stabilization. dox was encapsulated into liposomes with high efficiency by the ammonium sulfate remote loading method (18)
. For immunoliposomes, Fab' or scFv was covalently conjugated to liposomes after drug loading. Conjugation was via a thioether linkages between the free thiol near the COOH terminus of the MAb fragment and terminal maleimide groups at the liposome surface (Ls-MAb linkage) as described (14)
. Alternatively, the MAb thiol was conjugated to maleimide groups at the termini of PEG chains (PEG-MAb linkage) as described (15)
.
Animal Studies.
PK studies were performed in healthy rats, and therapeutic efficacy studies were performed in tumor xenograft nude mouse models, as described below. Institutional approvals for animal use were obtained at each site at which animal studies were performed (Memorial Sloan-Kettering Cancer Center, University of California, San Francisco, and California Pacific Medical Center Research Institute), and the requirements were followed in each of these studies.
PK Studies.
Healthy adult Sprague Dawley rats received single or multiple i.v. injections of immunoliposomes, liposomes, or free drug via indwelling jugular venous catheters. After the injection, blood was serially sampled via catheter up to 48 h postinjection, and plasma was assayed for dox, Fab', or liposome. Dox concentration was determined by spectrofluorimetry after extraction of plasma with acidified alcohol. For two-component PK studies, plasma samples were divided and analyzed for both dox and rhuMAb HER2-Fab' concentrations. The Fab' concentration was determined by ELISA, using wells coated with recombinant HER2 extracellular domain for capture and horseradish peroxidase-linked goat antihuman IgG for detection. For multiple-dose PK studies, rats were given i.v. injections of immunoliposomes every week for 3 weeks as in the therapy studies (see below). The initial two doses consisted of "empty" (no encapsulated agent) immunoliposomes, whereas the final dose consisted of immunoliposomes stably loaded with 67Ga-DTPA chelates. Plasma levels were determined by radioactivity counting.
Tumor Cell Lines.
Cell lines BT-474, MDA-MB-453, and MCF-7 were obtained from American Type Culture Collection. These cell lines have been extensively characterized for HER2 expression by flow cytometry, ELISA, and immunohistochemistry (19)
. MCF-7/HER2 was derived by stable transfection of HER2 cDNA in MCF-7 cells as described (20)
. IHC scoring of HER2 expression on a semiquantitative scale (0–3+) was first developed for trastuzumab clinical trials (21)
and is now routinely used clinically (22)
. Recent studies have suggested that the presence of HER2 gene amplification as detected by fluorescence in situ hybridization may be more predictive of response to trastuzumab than IHC assays of HER2 (23
, 24)
.
Tumor Xenograft Models.
Highly tumorigenic clones were generated from BT-474, MDA-MB-453, MCF-7, and MCF-7/HER2 lines by in vivo selection in nude (athymic) mice. In separate studies, tumorigenic clones of BT-474 were independently derived at Memorial Sloan-Kettering Cancer Center (by J. B.) and California Pacific Medical Center (by G. C.), and the resulting xenograft models were designated BT-474/MSKCC and BT-474/SF, respectively. HER2 expression in tumor xenografts was evaluated by IHC analysis and confirmed to be still overexpressed.6
Estradiol pellets were also implanted to increase tumorigenicity as described (25)
. In initial experiments, the BT-474/SF, MDA-MB-453, and MCF-7/HER2 models involved s.c. injection of tumor cells mixed with Matrigel (Collaborative Biomedical Associates, Oxford, MA), a complex extracellular matrix substitute. Matrigel was omitted in subsequent studies (see text).
Therapy Studies.
For each model, tumor cells were implanted s.c. with or without Matrigel in the dorsum of nude mice. When tumor xenografts had become fully established and tumors were 
200 mm3, mice were randomly assigned to different treatment groups (5–15 mice/group). In the nude mice, all i.v. treatments were performed via retro-orbital injection. Anti-HER2 immunoliposomes were administered i.v. at 5.0–7.5 mg dox/kg/dose every week for 3 weeks, for a total dox dose of 15.0–22.5 mg/kg. The corresponding lipid dose was typically 0.6–1.0 µmol of phospholipid per dose and 1.8–3.0 µmol total. Additional treatment groups were included as indicated in the text. Saline (PBS) was administered i.v. at the same injection volume and schedule as immunoliposomes. Free dox (Adriamycin) was administered i.v. at its MTD of 7.5 mg/kg on the same schedule as immunoliposomes. Sterically stabilized liposomal dox was either prepared identically as immunoliposome-dox except for omission of the MAb conjugation step or consisted of commercial PEGylated liposomal dox (Doxil; Alza Pharmaceuticals). Both versions of liposomal dox were administered i.v. at the same dose and schedule as immunoliposomes. Empty immunoliposomes were administered i.v. at the same lipid dose as anti-HER2 immunoliposome-dox and by the same weekly schedule or three times per week. Free trastuzumab (Genentech, Inc.) was administered i.p. at 0.3 mg/kg twice a week for 3 weeks as described (25)
. Although all other treatments were administered i.v. (retro-orbital injection) weekly, trastuzumab was administered i.p. twice per week based on extensive preclinical optimization with this regimen (21
, 25) .
Tumors were measured twice a week by caliper, and tumor volumes were calculated using the equation: length x width x depth. Mice with complete tumor regressions by measurement were necropsied at the end of the study and classified as "cured" if no residual tumor cells were detected on histopathological examination of the tumor injection site. Although these xenograft models do not typically support metastatic tumor growth, necropsy also included gross examination of viscera to confirm absence of metastases. Studies were terminated at 
2 months after treatment initiation in these models because of their requirement for estrogen supplementation; estrogen pellets implanted at the time of tumor injection potentially lose potency over extended periods. Mice were also weighed and examined for toxicity twice a week. In most of the studies, the experimenter measuring tumor volumes was blinded to treatment condition.
For MTD studies, nude mice with established tumor xenografts were treated with various doses of free dox, liposomal dox, or anti-HER2 immunoliposome-dox (5–15 mice/group). As in the therapy studies, treatments were administered via retro-orbital i.v. injection over three weekly doses. Total dox administered ranged from 2.5 to 22.5 mg/kg. Mice were monitored for weight and gross toxicity as described over 2 months, the typical duration of the therapy studies. The MTD was defined as the highest tested dose that gave <20% weight loss and no treatment-related deaths.
Statistical Analysis.
Tumor growth delay and tumor growth ratio were determined as described in Table 2
. Confidence intervals for tumor growth ratios were based on the assumption that the log of the ratios follows a normal distribution, which was consistent with observed data. A separate analysis was performed by fitting a modified Norton-Simon model of tumor growth (26)
to the observed data using the method of maximum likelihood to estimate model parameters. In two cases, application of this model to the data did not provide an adequate fit. In these cases, data were analyzed by two-way ANOVA using two factors, treatment group and day. Scheffé post hoc comparisons were then used to compare pairs of treatments (Table 2)
. Cure rates were compared using Fisher’s exact test for comparing two proportions.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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dox-loaded anti-HER2 immunoliposomes were initially prepared as described (14) , using liposomes containing 0–7 mol % PEG and with rhuMAb HER2-Fab' conjugated to derivatized head groups of phosphatidylethanolamine on the liposome surface (Ls-MAb linkage). However, immunoliposomes combining the Ls-MAb linkage with a high-density PEG coating showed reduced binding and endocytosis because of steric inhibition (14 , 15) . This problem was circumvented by use of immunoliposomes with MAb fragments conjugated to the termini of PEG chains (PEG-MAb linkage; Ref. 15 ). Our optimized construct design therefore consisted of anti-HER2 MAb fragments conjugated via a PEG-MAb linkage to dox-loaded sterically stabilized liposomes containing 6–7 mol % PEG. MAb fragments were either rhuMAb HER2-Fab' as described (14 , 15) , or alternatively, scFv C6.5, a fully human anti-HER2 single-chain Fv cloned from a phage antibody library (17) . Conjugations were highly efficient, resulting in 30–50 MAb fragments/liposome (70–90% of added MAb fragment).
Pharmacokinetics of Anti-HER2 Immunoliposomes.
Although prolonged circulation does not ensure improved efficacy, we sought to construct immunoliposomes with long circulating properties to maximize the effects of tumor targeting. PK studies of anti-HER2 immunoliposome-dox constructs were performed in healthy adult rats. dox-loaded immunoliposomes were prepared with 0–7 mol % PEG and with MAb fragments linked either to the liposome surface (Ls-MAb linkage) or to the termini of PEG chains (PEG-MAb linkage). After a single i.v. dose, all immunoliposome-dox constructs showed a biphasic plasma PK profile for dox (Fig. 1A)
, with terminal t1/2 = 11.6–13.6 h, area under the curve of 57,539–93,133 min %, and mean residence time of 15.8–24.3 h (Table 1)
. In contrast, dox levels were undetectable beyond 5 min after administration of the equivalent dose of free dox. The long circulation times of anti-HER2 immunoliposomes are fully equivalent to previous reports of sterically stabilized liposomal dox and greatly superior to those of "conventional" liposomes (27)
.
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. The presence of MAb fragments on immunoliposomes therefore did not measurably alter clearance in normal rats.
Notably, all anti-HER2 immunoliposome-dox constructs were long circulating. Thus, immunoliposomes of this type (i.e., small unilamellar vesicles, solid bilayer, neutral charge) did not require a polymeric coating such as PEG to achieve long circulation. Nevertheless, PEGylation of immunoliposomes, although not necessary for long circulation, was associated with incremental prolongation of mean residence time (Fig. 1A
and Table 1
).
Stability of Anti-HER2 Immunoliposome-dox in Vivo.
The stability of dox-loaded anti-HER2 immunoliposomes in circulation was evaluated in two-component PK studies, in which dox and MAb levels were simultaneously assayed to evaluate possible drug leakage or dissociation of MAb fragments from liposomes. For these studies, plasma pharmacokinetics of dox (assayed by spectrofluorimetry) and of rhuMAb HER2-Fab' fragments (assayed by ELISA) were codetermined from the same plasma samples after single i.v. injection of anti-HER2 immunoliposome-dox. Both the dox and Fab' components showed concordant plasma PK values after immunoliposome administration, with terminal t1/2 = 10 h for both. In contrast, free dox was again undetectable beyond 5 min, whereas free rhuMAb HER2-Fab' has a terminal t1/2 in rats of 1–2 h.7
These results indicated that immunoliposomes greatly prolonged the circulation of both Fab' and dox components and suggested that anti-HER2 immunoliposome-dox remained intact in circulation, with negligible drug leakage or MAb dissociation.
Multiple-Dose Pharmacokinetics of Anti-HER2 Immunoliposomes.
A major limitation of many immunoconjugate strategies has been the immunogenicity of the construct because the MAb component, the effector/cytotoxic component, or the linker can elicit a host immune response that precludes repeated administrations. To circumvent this problem, anti-HER2 immunoliposomes were designed to have components of minimal immunogenicity: sterically stabilized liposomes and humanized/human MAb fragments. Trastuzumab, a humanized IgG1, induced minimal levels of circulating anti-antibodies in clinical trials (11)
. MAb fragments rather than intact IgG were used to further reduce the potential for immune recognition associated with Fc sequences. To confirm that repeated administrations of anti-HER2 immunoliposomes do not lead to accelerated clearance, multiple-dose PK studies were performed in normal rats. Radiolabeled anti-HER2 immunoliposomes were administered once a week for three doses, using the same lipid dose and schedule as in the therapy studies (see below). Anti-HER2 immunoliposomes showed equivalent plasma pharmacokinetics in rats after the third and final dose as in naive rats without prior immunoliposome treatment (Fig. 1B)
. Furthermore, anti-HER2 immunoliposomes after one or three doses demonstrated plasma pharmacokinetics that were indistinguishable from those of sterically stabilized liposomes (prepared identically except for omission of Fab' fragments). Thus, the presence of MAb fragments on immunoliposomes did not result in accelerated clearance over a multiple dose schedule, even when humanized Fab's were introduced into an immunocompetent rodent host. In humans, immunoliposomes containing humanized Fab' or fully human scFv are designed to further minimize the potential for immunogenicity.
Antitumor Efficacy of anti-HER2 Immunoliposome-dox against HER2-overexpressing Human Breast Cancer Xenografts.
The antitumor efficacy of anti-HER2 immunoliposome-dox constructs was evaluated extensively in four different HER2-overexpressing breast cancer xenograft models: BT474/MSKCC and BT474/SF, independently derived xenograft models from the BT-474 cell line (106 HER2 receptors/cell; IHC score, 3+); MCF-7/HER2, MCF-7 cells stably transfected with HER2 (106 HER2/cell; IHC score, 3+; Ref. 20
); and MDA-MB-453 (105 HER2/cell; IHC score, 2+). For each model, tumor cells were implanted in nude mice with or without Matrigel, and treatment was initiated subsequently after tumors had become fully established. Tumor volumes at the start of treatment were 200-1000 mm3 (0.2–1.0 g) in 20-g mice, thus representing a spectrum from well-established moderate tumors to very large (5% of body weight) tumors.
Anti-HER2 immunoliposomes, prepared with 0–7 mol % PEG and either the Ls-MAb or PEG-MAb linkage, were loaded with dox and administered i.v. at a total dox dose of 15.0–22.5 mg/kg, divided over three weekly doses. Immunoliposomes were directly compared with multiple other treatment conditions, including saline, free dox at its MTD of 7.5 mg/kg, and sterically stabilized liposomal dox at the same dose and schedule as immunoliposome-dox. Additional controls included empty anti-HER2 immunoliposomes (no dox), at the same lipid dose as immunoliposome-dox and free anti-HER2 MAb (trastuzumab) administered at 0.3 mg/kg i.p. twice a week as described (25)
. Other controls included dox-loaded immunoliposomes containing an irrelevant MAb fragment, rhuMAb H52-Fab' (14)
, as well as dox-loaded immunoliposomes containing inactivated anti-HER2 scFv C6.5, in which binding affinity was abrogated by reduction of internal disulfides in the antigen-binding domain before immunoliposome conjugation. Both irrelevant and inactivated immunoliposomes yielded results comparable to those of liposomal dox (data not shown). For this reason, liposomal dox was used as the nontargeted control liposomes for most studies, either by preparing sterically stabilized liposomes identical to anti-HER2 immunoliposome-dox except for omission of MAb fragments or by use of commercial PEGylated liposomal dox (Doxil). The treatment groups are summarized in Table 2
.
Each of the anti-HER2 immunoliposome-dox constructs produced marked antitumor effects, including tumor growth inhibition, tumor regression, and cures of mice. These effects were observed in all sizes of tumors. Anti-HER2 immunoliposome-dox consistently showed activity superior to that of all other treatment conditions. For example, initial studies of anti-HER2 immunoliposome-dox containing 1 mol % PEG and Ls-MAb linkage demonstrated suppression of tumor growth that persisted after treatment. Tumor inhibition was clearly superior to that associated with free dox or liposomal dox (Fig. 2
and Table 2
).
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and/or the unimpeded internalization associated with the PEG-MAb linkage (15)
. Subsequent studies therefore evaluated the optimized anti-HER2 immunoliposomes containing 6 mol % PEG and a PEG-Fab' linkage. In addition, subsequent studies used a Matrigel-free version of the BT-474/SF tumor xenograft model. Matrigel was omitted once tumor localization studies revealed that liposomes and immunoliposomes were nonspecifically trapped in Matrigel deposits.6
Establishment of tumors without Matrigel coimplantation removed this artifact and produced improved tumor penetration of liposomes and immunoliposomes.
Therapy studies using the optimized anti-HER2 immunoliposome-dox construct in the Matrigel-free BT-474/SF model further demonstrated that targeted delivery of dox was associated with enhanced efficacy (Fig. 3)
. Anti-HER2 immunoliposome-dox produced substantial tumor regressions in this model, including high rates of cured mice (complete regression of tumor confirmed histopathologically at sacrifice). Indeed, anti-HER2 immunoliposomes containing either rhuMAb HER2-Fab' or an alternative anti-HER2 antibody fragment, scFv C6.5, produced significantly superior growth inhibition than liposomal dox (P < 0.0001 for either anti-HER2 immunoliposome-dox construct versus liposomal dox). Furthermore, cure rates in this model reached 50% (5 of 10 mice) for rhuMAb HER2-Fab' immunoliposomes and 55% (6 of 11 mice) for scFv C6.5 immunoliposomes versus 0% (0 of 11 mice) for liposomal dox. These differences were also highly significant, with P < 0.0001 for either anti-HER2 immunoliposome-dox construct versus liposomal dox.
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. In fact, empty anti-HER2 immunoliposomes, administered at the same lipid dose and schedule (once a week) as anti-HER2 immunoliposome-dox, produced no effects on tumor growth (data not shown), indicating that the therapeutic activity of anti-HER2 immunoliposome-dox was attributable to targeted delivery of dox and not to the antiproliferative activity of the rhuMAb HER2-Fab' fragments on the immunoliposome itself. When given three times per week instead of once a week, empty anti-HER2 immunoliposomes containing rhuMAb HER2-Fab' showed only modest inhibition of tumor growth (Table 2)
, which again was much less than that of anti-HER2 immunoliposome-dox. These results are consistent with previous studies of trastuzumab, which requires steady-state pharmacokinetics and serum trough concentrations 10 µg/ml for tumor inhibition (11
, 28)
. The three weekly doses of anti-HER2 immunoliposome-dox did not lead to steady-state levels.
Taking all of the therapy studies together, in each of four HER2-overexpressing tumor xenograft models (BT474/MSKCC, BT474/SF, MCF-7/HER2, and MDA-MB-453), treatment with anti-HER2 immunoliposome-dox was significantly superior to all other treatment conditions (Table 2)
. In contrast to free dox, which produced modest tumor growth inhibition in these xenograft models, anti-HER2 immunoliposome-dox showed highly potent antitumor efficacy. In eight separate studies comparing anti-HER2 immunoliposome-dox versus liposomal dox, immunoliposome-mediated delivery produced significantly superior therapeutic results (P <0.0001 to 0.04). Furthermore, anti-HER2 immunoliposomes, but not free dox or liposomal dox, produced frequent cures in mice. The total cure rate with immunoliposomes in all studies was 18 of 115 mice (16%), with higher rates of 50% obtained in later studies using optimized immunoliposomes (PEG-MAb linkage) and non-Matrigel models as described. In contrast, no cures were observed in any of the mice treated with free dox (0 of 46) or liposomal dox (0 of 78). These differences were highly significant (P < 0.0001 versus free dox or liposomal dox).
Toxicity of Anti-HER2 Immunoliposome-dox.
In addition to enhanced efficacy, encapsulation of dox in either liposomes or anti-HER2 immunoliposomes was associated with markedly reduced host toxicity compared with free dox. In studies to determine the MTD in nude mice with tumor xenografts, various doses of free dox, sterically stabilized liposomal dox, or anti-HER2 immunoliposome-dox were administered i.v. once a week for three doses. The MTDs of immunoliposome-dox (22.5 mg/kg) and liposomal dox (18.8–22.5 mg/kg) were 2.5-fold greater than that of free dox (7.5 mg/kg).
Effects of Anti-HER2 Immunoliposome-dox against Non-HER2-overexpressing Tumor Xenografts.
As an additional control, anti-HER2 immunoliposome-dox was evaluated in a non-HER2-overexpressing xenograft model. MCF-7 xenografts express low or basal levels of HER2 (104 HER2/cell; IHC score, 1+). Previous studies of anti-HER2 immunoliposome binding and internalization in MCF-7 cells in vitro showed undetectable uptake by confocal microscopy and quantitative fluorimetry; furthermore, uptake of immunoliposomes was indistinguishable from that of control liposomes lacking MAb fragments (15)
. To evaluate in vivo selectivity, anti-HER2 immunoliposome-dox was administered i.v. in the MCF-7 xenograft model at the same dose and schedule as in the other therapy studies. Equivalent therapeutic effects were obtained for both immunoliposome-dox and nontargeted liposomal dox (Fig. 4)
. This result confirmed that anti-HER2 immunoliposomes require a threshold HER2 density (or functional activity) to enable appreciable drug delivery in vivo as well as in vitro.
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, anti-HER2 immunoliposome-dox showed efficacy that was significantly superior to that of the combination (P < 0.0001). Anti-HER immunoliposome-dox was also directly compared with the combination of free trastuzumab plus liposomal dox, which in this experiment consisted of commercial PEGylated liposomal doxorubicin (Doxil; Fig. 5B
). Anti-HER2 immunoliposomes again showed significantly superior efficacy than this combination (P < 0.0001).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Therapy studies in four different HER2-overexpressing human tumor xenograft models demonstrated that delivery of dox via anti-HER2 immunoliposomes greatly increased the therapeutic index of dox, both by increasing antitumor efficacy and by reducing systemic toxicity. Furthermore, anti-HER2 immunoliposome-dox was significantly superior to all other relevant treatment conditions, including liposomal dox, free trastuzumab, trastuzumab plus free dox, and trastuzumab plus liposomal dox.
These results indicated that anti-HER2 immunoliposome-dox produced enhanced antitumor efficacy via tumor-targeted drug delivery. An alternative explanation is that rhuMAb HER2-Fab' fragments provided an independent antiproliferative effect on HER2-overexpressing cancer cells, which in turn acted additively or synergistically with dox therapy. Indeed, we have previously shown that empty anti-HER2 immunoliposomes containing rhuMAb HER2-Fab', when present continuously in vitro, do possess antiproliferative activity approaching that of trastuzumab and greatly exceeding that of rhuMAb HER2-Fab' alone (14)
. However, two lines of evidence preclude the possibility that this antiproliferative mechanism contributed to the in vivo efficacy of anti-HER2 immunoliposome-dox observed in the present studies. The first consideration is that administration of empty anti-HER2 immunoliposomes at the same lipid dose and schedule as dox-loaded immunoliposomes failed to produce significant antitumor efficacy. This result is consistent with preclinical and clinical studies of trastuzumab, which required steady-state pharmacokinetics with a trough serum concentration 10 µg/ml for optimal antitumor activity (11
, 28)
. The dose and schedule for anti-HER2 immunoliposomes-dox were far below this, consisting of just three weekly doses. The second consideration is that immunoliposomes containing either rhuMAb HER2-Fab' or scFv C6.5 produced comparable therapeutic results, although scFv C6.5 does not possess antiproliferative activity as a monomer or when bivalent as a diabody (31)
.
We previously showed that anti-HER2 immunoliposomes efficiently bind and internalize in HER2-overexpressing cells in vitro, leading to intracellular drug delivery (14 , 15) . Importantly, we have recently evaluated the mechanism by which anti-HER2 immunoliposomes mediate drug delivery in vivo.8 In the same breast cancer xenograft models described here, tumor xenografts were examined histopathologically after i.v. treatment with either gold-loaded anti-HER2 immunoliposomes or gold-loaded sterically stabilized liposomes. Visualization of liposomes and immunoliposomes via a silver enhancement method revealed striking differences in intratumoral localization and mechanism of delivery. Sterically stabilized liposomes were predominantly observed in extracellular areas of tumor stroma and within tissue macrophages and were not observed within tumor cells, consistent with previous reports (32) . In contrast, anti-HER2 immunoliposomes were predominantly found internalized within tumor cells in a broad distribution throughout tumor tissue. These results confirm that anti-HER2 immunoliposomes mediate intracellular drug delivery in HER2-overexpressing tumor cells in vivo and suggest that the therapeutic advantage obtained with anti-HER2 immunoliposome-dox derives from this novel mechanism of action.
It is notable that anti-HER2 immunoliposomes constructed using an alternative methodology appear to have properties, including lack of enhanced efficacy, very different from those described here (30) . In that report, MAb N-12A5, an intact IgG1 directed against HER2, was conjugated to sterically stabilized liposomal dox. N-12A5 immunoliposomes bound to HER2-overexpressing cells in vitro, but were not internalized. Therapy studies showed that these immunoliposomes provided no additional efficacy over sterically stabilized liposomal dox. This result suggests that internalization of anti-HER2 immunoliposome-dox constructs in cancer cells was a critical factor in the enhanced efficacy that we observed.
Targeted delivery of dox by anti-HER2 immunoliposomes may represent a particularly advantageous strategy for the treatment of HER2-overexpressing cancers. Preclinical and clinical studies have raised the possibility that HER2-overexpressing breast cancers may be relatively resistant to hormone therapy and certain types of chemotherapy (reviewed in Ref. (33)
, but may be especially sensitive to anthracycline-based chemotherapy (34, 35, 36)
. In principle, anti-HER2 immunoliposome-dox can provide maximal anthracycline delivery to HER2-overexpressing cancers while reducing the toxicity of free dox to the myocardium and hematopoietic cells because HER2 expression is extremely low in these cell types (37)
and drug delivery by anti-HER2 immunoliposomes is dependent on receptor density (15)
. Another relevant issue is that trastuzumab therapy is associated with some cardiotoxicity as a single agent (12)
and prohibitive cardiotoxicity when used in combination with dox chemotherapy (13)
. Although the toxicological mechanism remains to be elucidated, it is presumed that trastuzumab-associated cardiotoxicity is related to its therapeutic antiproliferative and/or immunological effects. As discussed, these effects require high steady-state levels of trastuzumab. Hence, immunoliposomes, which can be administered intermittently, should not produce these effects. Consistent with this, empty immunoliposomes containing rhuMAb HER2-Fab' did not display antiproliferative activity in vivo with a weekly schedule. Furthermore, immunoliposomes can be constructed with scFv C6.5, a ligand devoid of antiproliferative activity, and immunoliposomes containing either Fab' or scFv lack Fc sequences to mediate immunological functions such as antibody-dependent cellular cytotoxicity. Finally, as noted, this delivery system does not accumulate in low-HER2-expressing cells such as MCF-7 (104 HER2/cell; Ref. 15
); this threshold suggests that significant delivery or accumulation is unlikely in myocardial cells containing 103 HER2/cell. Hence, we hypothesize that anti-HER2 immunoliposome-dox may represent a particularly apt strategy to deliver dox to HER2-overexpressing cancer cells, resulting in less cardiotoxicity than dox chemotherapy alone and much less than dox chemotherapy plus trastuzumab. Final determination of the safety profile of anti-HER2 immunoliposome-dox will require formal toxicological evaluation and Phase I clinical studies.
We conclude that tumor-targeted drug delivery using anti-HER2 immunoliposomes can enhance the therapeutic index of dox chemotherapy and therefore may provide a particularly potent and useful therapy for cancers that involve HER2 overexpression. In addition, the strategy of immunoliposome delivery may have broad utility for targeted delivery of other anticancer agents, such as those agents with narrow therapeutic indices, PK limitations, or a requirement for intracellular delivery.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the National Cancer Institute Specialized Programs of Research Excellence (SPORE) in Breast Cancer (Grant P50-CA 58207-01), the Department of Defense Breast Cancer Research Program (Grant DAMD17-94-J-4195), and the American Society of Clinical Oncology Young Investigator Award (to J. W. P.) sponsored by the Don Shula Foundation. 
2 To whom requests for reprints should be addressed, at UCSF Comprehensive Cancer Center, 1600 Sutter Street, 2nd Floor, San Francisco, CA 94115-1710. Phone: (415) 502-3844; Fax: (415) 353-7021; E-mail: jpark{at}cc.ucsf.edu
3 Present address: Alza Corporation, Mountain View, CA 94043.
4 Present address: Hospital General Universitari Vall D’Hebron, 08035 Barcelona, Spain.
5 The abbreviations used are: MAb, monoclonal antibody; PEG, polyethylene glycol; RES, reticuloendothelial system; PK, pharmacokinetic; rhuMAb, recombinant human MAb; dox, doxorubicin; Ls, liposome; DTPA, diethylenetriaminepentaacetic acid; IHC, immunohistochemical; MTD, maximum tolerated dose.
6 M. R. Shalaby, D. Kirpotin, K. Hong, B-M. Ljung, Z. Meng, and J. W. Park. The use of Matrigel in xenograft models: new implications, submitted for publication.
7 D. Maneval, Canji, Inc., San Diego, CA, personal communication.
8 D. B. Kirpotin, K. Hong, J. W. Park, Y. Shao, R. Shalaby, G. Colbern, C. C. Benz, and D. Papahadjopoulos. Localization of anti-HER2 immunoliposomes in HER2-overexpressing breast cancer xenografts: intracellular delivery via immunotargeting, submitted for publication.
Received 6/ 4/01; revised 1/23/02; accepted 1/30/02.
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), 7 mol % PEG, PEG-MAb linkage (
), or free dox (
) were administered i.v. at time 0. Immunoliposome dose was 5 µmol of total lipid, 0.9 mg of dox; free dox dose was 0.9 mg. Dox levels were determined at the indicated times and expressed as a percentage of the dox concentration at 5 min; dox was undetectable in plasma beyond 5 min after injection of free dox. B, Plasma pharmacokinetics of radiolabeled anti-HER2 immunoliposomes after multiple versus single i.v. injection in rats. Immunoliposomes (6 mol % PEG, PEG-MAb linkage) were administered i.v. in rats every week for 3 weeks as in the therapy studies. For the third dose, immunoliposomes were loaded with 67Ga-DTPA chelates, and immunoliposome levels were determined at the indicated times by radioactivity counting (
). For comparison, 67Ga-DTPA-loaded immunoliposomes (
) were also administered to naive rats. Plasma levels of immunoliposomes (ILs) or liposomes (Ls) are expressed as a percentage of the radioactivity present at 5 min.
), free dox (
), prepared identically to anti-HER2 immunoliposome-dox but without MAb and given at the same dose and schedule. Anti-HER2 immunoliposome-dox was significantly superior to all other treatment conditions (P < 0.0001 versus liposomal dox). Data represent mean tumor volumes; bars, SE.
) of free rhuMAb HER2 (trastuzumab; Herceptin) plus free dox. Anti-HER2 immunoliposomes were administered i.v. at a total dox dose of 15 mg/kg over three doses (arrows), free dox was administered i.v. at its MTD of 7.5 mg/kg over three doses (arrows), and trastuzumab was administered i.p. at 0.3 mg/kg twice a week over six doses (arrows and arrowheads). Anti-HER2 immunoliposome-dox was significantly superior to the combination of free MAb plus free drug (P < 0.0001). B, dox-loaded anti-HER2 immunoliposomes containing rhuMAb HER2-Fab' (