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Experimental Therapeutics |
Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263 [M. S., N. K. E., S. J. Y., F-A. C., R. B. B.], and Alza Corporation, Mountain View, California 94025 [J. H., S. K. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The use of antibodies to direct the delivery of drugs carried inside of liposomes to tumors represents a potentially viable method for further increasing the specificity and enhancing the therapeutic effect of liposome drug delivery (11 , 12) . Although many laboratories have demonstrated that antibodies can be used to enhance the selective liposome drug delivery to tumors in vitro (13, 14, 15, 16, 17) , demonstrating this in vivo has proven to be a far more daunting task (18 , 19) . The problem in vivo is likely attributable to many factors, but three obvious pitfalls stand out, i.e., the inability of the targeted liposomes to gain access to the tumor, the aggregation and subsequent premature clearance of immunoliposomes by the reticuloendothelial system, and the failure of the immunoliposomes that bind to the tumor target to enter the cell and/or release their drug. The development of methods for coupling specific ligands to the PEG terminus of Stealth liposomes (20, 21, 22, 23, 24, 25) and the use of Fab' fragments of antibodies instead of complete immunoglobulin molecules to conjugate to the liposomes (25) have increased the time in circulation and improved the extravasation of the immunospecific Stealth liposomes into solid tumors in vivo. This new generation of immunospecific Stealth liposomes targeted to a cell surface integrin has been tested here and shown to be effective in suppressing the growth and metastasis of a human lung tumor in SCID mice.
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MATERIALS AND METHODS |
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Antibodies.
Monoclonal antibodies 1F11 and 2C3, both mouse IgG1, were developed in
our laboratory using technology described previously (26
, 27) . 1F11 is specific for the human integrin
ß1 subunit, and 2C3 is specific for the hapten
phthalate. For preparation of the (Fab')2
fragments, purified antibodies were dialyzed against 0.2 M
acetate buffer (pH 4.0), concentration was adjusted to 2 mg/ml, and
pepsin was added to a final concentration of 0.2 mg/ml. Digestion was
performed at 37°C for 7 h, and the reaction was terminated by
the addition of 0.1 volume 2 M Tris-base (pH 8). The
reaction mix was dialyzed against PBS (pH 8) and was passed through a
protein-G column equilibrated with the same buffer. Unbound fractions
were collected, sterilized by filtration through a 0.22 µm filter,
and the protein concentration was determined by using the standard
extinction coefficient method (A280
divided by 1.48).
Liposomes.
A long-circulating liposome formulation of doxorubicin was prepared and
characterized at ALZA Corp. (Mountain View, CA; formerly Sequus
Pharmaceuticals, Inc.) using hydrogenated soy phosphatidylcholine
(Natherman Phospholipids, Cologne, Germany), cholesterol (Croda,
Fullerton, CA), PEG-DSPE, and DL]-
-tocopherol (Hoffman
La Roche, Nutley, NJ) in a molar ratio of 56.1:38.2:5.5:0.2, as
described previously (28)
. The
(Fab')2 fragments of targeting (1F11) and
isotype-matched control (2C3) monoclonal antibodies were then
conjugated to the sterically stabilized liposomes using a recently
described postinsertion technology for transfer of ligands to preformed
liposomes (29)
. Briefly, the (Fab')2
fragments were reduced with 7.5 mM ß-mercaptoethylamine
(ß-MEA; Sigma, St. Louis, MO) for 30 min at 37°C in 25
mM HEPES/0.9% saline (pH 7.2), after which the reducing
agent was removed by passing the sample over a PD-10 desalting column
(Pharmacia, Piscataway, NJ) pre-equilibrated with the same buffer
without ß-mercaptoethanol. The freshly reduced Fab' fragment was
reacted with 5 molar excess of maleimide-PEG-DSPE (Shearwater Polymers,
Huntsville, AL) in HEPES buffer (pH 7.2) at 37°C for 5 h. The
conjugate was mixed with liposomes at a ratio that should yield 
10
Fab' molecules/liposome assuming that 25% of the Fab' conjugated to
Mal-PEG-DSPE and 100% inserted into the liposomes. The appropriate
volume of liposomes was added to the Fab'-PEG-DSPE conjugate and was
incubated overnight at 37°C. The next day, the unreacted maleimide
was reduced by incubation with 2 mM ß-mercaptoethanol for
30 min at room temperature. The Fab'-conjugated liposomes were purified
from the unconjugated Fab' by size exclusion chromatography on a
1.5 x 30-cm, A50m column (Bio-Rad, Hercules, CA)
equilibrated with 25 mM HEPES/0.9% saline (pH 7.2). The
amount of protein conjugated to the liposomes was quantitated by
SDS-PAGE on a 4–20% gradient gel (Bio-Rad Laboratory, Hercules, CA).
The protein in the gel was visualized with Sypro Orange (Bio-Rad), and
the intensities of the samples were quantitated using an AlphaImager
from Alpha Innotech Corporation (San Leandro, CA).
Tumor Cells.
The human lung squamous cell carcinoma cell line RPCI-2E9/IV4/PSA,
which is adapted for rapid orthotopic growth in the SCID mouse lung and
is genetically modified to secrete human PSA, was described by us
previously (30)
.
In Vitro Binding and Internalization Studies.
RPCI-2E9/IV4/PSA cells (1 x 106)
were mixed with the appropriate liposome formulation loaded with Texas
Red (final lipid concentration, 40 mM) in 1 ml of complete
culture medium (DMEM-F12 + 10% FCS + 1 mg/ml G418)
and were incubated on ice for 30 min. Cells were then centrifuged and
washed twice in 1 ml of culture medium and were transferred to 37°C.
After a 1-h incubation at 37°C, cells were washed with DMEM and with
PBS and were resuspended in 200 µl of PBS. Approximately 50 µl
of the cell suspension were then spotted on an Alcian blue-coated
coverslip and incubated at room temperature for 10 min in a humid
chamber. Cells were rinsed with PBS, fixed with 2% paraformaldehyde
for 7–10 min at room temperature, rinsed with water and were mounted
with Polymount. Samples for confocal microscopy were prepared either
prior to transfer to 37°C (time 0) or after incubation at 37°C
(time 60 min). Images were obtained using a Bio-Rad MRC-600 laser
scanning confocal microscope with an argon laser. The GHS filter set,
with an excitation filter at 512 nm and an emission filter at 540 nm,
was used to collect 1 µm optical sections. Projections of cells were
displayed using Bio-Rad software.
In Vitro Toxicity Studies.
RPCI-2E9/IV4/PSA cells were plated in 96-well culture plates
(1 x 104
cells in 200 µl of
culture medium/well) with the indicated quantity of liposome
formulations. Cells were incubated at 37°C, 5%
CO2 for 5 days, and inhibition of cell growth was
determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay as described (31)
.
In Vivo Therapy Studies.
All mice received i.p. injections of the monoclonal antibody TMß1
(kindly supplied by Dr. T. Tanaka of Tokyo Metropolitan Institute for
Medical Science, Tokyo, Japan) to deplete the murine natural killer
cells 1 day prior to tumor inoculations (day -1) as described
(32)
. Mice then received injections in the tail vein with
the RPCI-2E9/IV4/PSA cells (4 x 106 cells in 200 µl of PBS) on day 0.
Treatments with PBS and the indicated drug or liposome formulations
were performed on days 7, 14, 21, 28, 35, and 42 by tail vein
injections (in 200 µl of PBS). Free doxorubicin, in powder form
(Farmitalia), was solubilized in sterile saline to 2 mg/ml. The
liposome/drug formulations were diluted in sterile saline for injection
into the lateral tail veins of SCID mice at the indicated doses.
Injections were in a total volume of 200 µl/mouse.
Mice were bled through the tail vein weekly starting at week 4 and ending at week 7. With the exception of the survival experiment, all mice were sacrificed at week 8 and were analyzed for tumor burden. Lung tumor burden was determined by counting the number of visible tumor nodules under a dissecting microscope. For the determination of liver and adrenal gland tumor weights, livers and adrenal glands of mice in the experimental groups were removed, weighed, and averaged for each group. The average liver and adrenal gland weights of age and sex-matched healthy controls (five mice) were then subtracted from the experimental groups, and the difference was plotted as tumor weight.
PSA ELISA.
The ELISA for the quantification of serum PSA levels was described by
us previously (30)
. Briefly, 96-well plates were coated
with a rabbit anti-HuPSA antibody (1 µg/ml in PBS) at 4°C overnight
and washed with PBS containing 0.1% Tween 20. Plates were then blocked
with 1% BSA in borate-buffered saline at room temperature for 1 h
and then washed once with PBS containing 0.1% Tween 20. Mouse sera and
PSA standards (0–4000 pg/ml) were added to the plates, followed
immediately by the addition of 50 µl of a biotinylated mouse
anti-HuPSA (100 ng/ml), and the plates were incubated for 2 h at
room temperature. Plates were washed four times, 50 µl of
streptavidin-conjugated horseradish peroxidase (1 µg/ml Sigma A3151)
were added, and the samples were incubated at room temperature for 20
min. Plates were washed, and 3,3',5,5'-tetramethyl-benzidine substrate
(Sigma T8540) was added. Reaction was stopped by the addition of 100
µl of 0.1 N HCl, and absorbance readings (450–540 nm)
were taken using an ELISA reader.
Statistical Methods.
The significance analysis of the differences observed between different
groups regarding serum PSA levels, primary lung tumor burden, and
metastasis to liver and the adrenal gland were determined by using the
standard Student’s t test analysis. The significance of the
differences between the groups in the survival experiment was
determined using the Mantel-Cox log-rank test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, but not control
liposomes (Fig. 1, A and E
; i.e.,
liposomes without antibody or liposomes conjugated with an isotype
control antibody 2C3), bound to the cell surface of the RPCI-2E9 tumor
cells. Fluorescent immunoliposomes were diffusely distributed over the
entire surface of the tumor cells. To determine whether the bound
liposomes were internalized, the cells were incubated at 37°C for
1 h and reexamined. Confocal microscopy established that the bound
liposomes were efficiently internalized as evidenced by the large
aggregates of red fluorescence observed within the cell (Fig. 1D)
. No fluorescent aggregates were observed in the tumor
cells pulsed with control liposomes and incubated at 37°C (Fig. 1, B and F)
. These studies establish that the
ß1 integrin on human lung tumors represents
experimentally a viable target for testing the ability of
immunoliposomes to deliver cytotoxic drugs to a human solid tumor.
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demonstrate
that the anti-ß1 Fab'-doxorubicin-loaded
liposomes (1F11-s-dox) were 30-fold more effective than
no-antibody/doxorubicin-loaded liposomes (s-dox) or than control
isotype-matched Fab' doxorubicin-loaded liposomes (2C3-s-dox).
Interestingly, 1F11-s-dox liposomes were also 3-fold more effective in
inducing tumor cell death than free doxorubicin in vitro.
Anti-ß1 Fab' liposomes without doxorubicin
(1F11-s) were not cytotoxic to cells at doses where >95% of the cells
were killed by 1F11-s-dox, indicating that the observed tumor killing
was induced by intracellular delivery of doxorubicin and was not simply
attributable to the toxicity of internalized lipid (Fig. 2B)
.
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. Incubation of tumor cells with
1F11-s-dox liposomes induced complete tumor cell death, whereas the
addition of increasing amounts of free 1F11 antibody reduced
cytotoxicity of 1F11-s-dox in direct proportion to the antibody
concentration. Incubation of tumor cells with a nonspecific,
isotype-matched antibody (2C3) or with 1F11 did not affect cell
viability significantly, except that a slight (10%) but consistent
suppression of cell growth was observed with 1F11. The results
demonstrate that cytotoxicity was dependent on the specific binding of
the 1F11 Fab' to the ß1 integrin on the surface
of the cells.
Engraftment of Human Lung Tumor into Lungs of SCID Mice.
To test the ability of immunoliposomes to deliver doxorubicin to human
tumors in vivo, an orthotopic tumor xenograft model was
established in SCID mice. The RPCI-2E9 cell line was inoculated i.v.
into SCID mice, and tumor nodules forming in the lungs of SCID mice
were removed and reinoculated into another set of SCID mice. This
procedure was repeated four times, thereby establishing a tumor subline
adapted to engraft and grow well orthotopically in the lungs of SCID
mice (RPCI-2E9/IV4). Next, the tumor subline was transfected with a
mammalian expression vector containing the gene encoding human PSA. The
stably transfected tumor cell line RPCI-2E9/IV4/PSA, when injected i.v.
into SCID mice, engrafts within the lung and releases human PSA into
the serum of tumor-bearing mice. Thus, PSA in the serum serves as an
indicator of the tumor engraftment, and increasing serum PSA levels
were shown to correlate with tumor progression (30)
. This
model was used here to evaluate the ability of the
anti-ß1 immunospecific liposomes to target the
delivery of doxorubicin to the ß1-positive
human lung tumor and to compare its therapeutic efficacy to control
liposomes and to free drug.
Effect of Doxorubicin-loaded Anti-ß1 Fab'
Immunoliposomes on the Growth of Orthotopically Established Human Lung
Tumor Xenografts in SCID Mice.
SCID mice were inoculated i.v. with RPCI-2E9/IV4/PSA tumor cells. The
mice remained untreated for 1 week to allow tumors to establish within
the lung. One week after tumor inoculation, mice received weekly i.v.
injections of 1F11-Fab-dox-immunoliposomes, or control liposome
formulations for a total of six treatments. Another group of mice
received no treatment. Mice were bled weekly between weeks 4 and 7
after tumor inoculation, and tumor growth was estimated by quantifying
serum PSA levels periodically by ELISA. PSA was detectable in the sera
of untreated mice by week 4 and mice treated with control liposome
formulations by week 5. PSA was not detected in mice treated with
immunospecific liposomes until weeks 6 or 7 after tumor inoculation
(data not shown). In three independent experiments, serum PSA levels of
tumor-bearing mice treated with immunospecific liposomes were
substantially lower than in mice receiving no treatment (Fig. 3)
. The immunospecific liposomes,
i.e., 1F11-s-dox were also shown to be more effective than
free doxorubicin, i.e., f-dox (Fig. 3A)
or
doxorubicin in control liposomes (drug-loaded liposomes without
antibody), i.e., s-dox (Fig. 3, A and C)
, drug-loaded liposomes with an isotype control antibody,
i.e., 2C3-s-dox (Fig. 3)
or immunospecific liposomes without
doxorubicin, i.e., 1F11-s (Fig. 3B)
. These
results establish that in all three experiments tumor growth was
suppressed more effectively in mice treated with the 1F11-s-dox
immunoliposomes as compared with mice treated with free drug, the
control liposome treated mice or untreated mice.
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clearly establish the
superiority of immunoliposomes in suppressing the growth of established
tumor, evidence of some tumor growth (i.e., serum PSA
levels) was observed in the immunoliposome-treated groups in two of the
three experiments. Another common indicator of efficacy that is used is
enhanced survival of tumor-inoculated mice. To investigate the effect
of different treatments on survival, mice were treated 1 week after
tumor inoculation and monitored for survival for up to 19 weeks. All
treated groups received six weekly treatments. Mice experiencing one or
more of the following clinical signs were sacrificed: paralysis,
dyspnea, or cachexia. By 12 weeks, all untreated tumor-bearing mice had
died or were sacrificed. Treatment of mice with free doxorubicin
actually decreased the survival time at the dose and schedule used (all
mice were dead by week 10), possibly because of a combination of tumor
growth and toxicity of the drug (Fig. 4)
. The median survival time of the
mice that received doxorubicin-loaded control liposomes (either no
antibody s-dox or control antibody 2C3-s-dox) was similar to mice that
received no treatment (all mice were dead by week 12 or 13). The median
survival time of mice treated with the immunoliposomes (1F11-s-dox) was
increased 35%, with 80% of the mice still alive between weeks 15
and 16. We conclude that the immunoliposomes compared with either free
drug or control liposomes preparations increase survival time of
tumor-bearing mice and are therefore more efficient at delivering a
cytotoxic drug to a solid tumor in vivo.
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. In both experiments,
the number of tumor nodules in the lung was suppressed in the
1F11-s-dox-treated group compared with the no-treatment group. In one
experiment (Fig. 5A)
, there was no statistically significant
difference in the total number of lung tumor nodules in the s-dox- and
1F11-s-dox treated groups. However, the tumor nodules in the
1F11-s-dox-treated group were smaller, and 1F11-s-dox completely
blocked metastasis of the tumor to the adrenal gland and dramatically
suppressed metastasis to the liver, whereas control treatment groups
had little or no consistent effect upon metastasis to either the liver
or adrenal glands (Fig. 5, C–F)
. These results establish in
this tumor model that an additional therapeutic advantage of the
immunospecific liposomes over the nontargeted liposomes is the
suppression or prevention of the metastatic spread of the tumor.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study what is particularly interesting and potentially clinically relevant is the suppression of tumor metastases from the lung to liver and adrenal glands observed in lung tumor-bearing mice treated with the 1F11-s-dox (immunospecific) liposomes. Although there was some suppression of primary tumors observed with the nontargeted liposomes, no consistent suppression of tumor metastases was observed in tumor-bearing mice treated with these control liposomes. These results suggest that the optimal clinical use of the antibody-targeted liposomes may be in conjunction with surgery to suppress smaller tumors in the lung and especially to suppress or prevent metastatic disease.
The in vitro and in vivo efficacy of sterically stabilized immunoliposomes and their increased effectiveness when prepared using intact antibodies (but not Fab' fragments) has been reported previously using murine tumor models (33, 34, 35) . However, very little information exists on the utility of these immunospecific liposomes in the treatment of human cancer. In the one previous in vivo study of human tumors, immunoliposomes were shown to be superior to control nontargeted liposomes in suppressing the growth of a human B-cell lymphoma xenograft in SCID mice (36) . In this study, sterically stabilized liposomes were targeted to a differentiation antigen (CD19) expressed on the tumor. Mice were inoculated with the tumor either i.p. or i.v. and were treated with immunoliposomes injected i.p. or i.v. 1 or 24 h after tumor inoculation. The immunoliposomes loaded with doxorubicin were shown to significantly increase the survival time compared with tumor-inoculated mice receiving either free drug or drug-loaded, nontargeted liposomes. Although our study confirms the efficacy of immunoliposomes in vivo with another human tumor and a different tumor target, it differs in several other significant ways from the initial report with B-cell lymphomas. In the present study, treatment was not started until 7 days after tumor inoculation, and the human lung tumor xenografts were established orthotopically, i.e., in the lung of SCID mice. The design of this study also made it possible to monitor tumor growth continuously by assaying the serum of tumor-bearing mice for a tumor marker, thereby providing a quantifiable indicator of tumor progression for establishing the efficacy of the liposome treatment that is independent of survival times. In contrast to the B-cell lymphoma study, we have used Fab' fragments of the antibody in preparing the immunoliposomes. This has been shown previously to enhance the circulation time and enhance the extravasation of immunoliposomes into tumors in a mouse tumor model (37) . Another significant, and perhaps the most important, difference between the two models is that we were able to observe in the lung tumor xenograft model the metastatic spread of the lung tumors to the liver and adrenal, making it possible to establish another beneficial effect of the immunospecific liposomes, i.e., suppression of tumor metastases.
It is important to emphasize that in neither the work reported here nor in the one previous study with a human tumor (36) was it possible to completely eradicate established human tumors with the dose and schedules of immunospecific liposomes used. We have tried to eradicate tumors without success using a single high dose of or additional low doses of the drug-loaded immunoliposomes. For example, we have compared the effect of a single high dose of doxorubicin (3 mg/kg) delivered in immunospecific liposomes to six low doses of doxorubicin (0.5 mg/kg) delivered in immunospecific liposomes on the growth and metastasis of the RPC-2E9/PSA-transfected lung tumor in SCID mice. Eight weeks after tumor engraftment and 7 weeks after the initial treatment, we saw no significant difference in the serum levels of PSA (i.e., 1.9 ± 1.1 ng/ml for multiple dose and 1.4 ± 1.8 ng/ml for single dose, P = 0.548) and only a modest difference in the number of tumor nodules in the lung, i.e., 13.2 ± 4 in mice treated with multiple low doses of doxorubicin versus 18.0 ± 6.5 for the mice treated once with a high dose of the drug, P = 0.195. The mean tumor weights in the liver of mice receiving multiple doses of the drug-loaded immunoliposomes was 13 ± 21 mg and 45 ± 33 mg in mice treated with a single dose, P = 0.105. Thus, there appears to be no significant potential therapeutic benefit of giving multiple low doses to a single high dose of the drug in the immunospecific liposomes. These results are consistent with the results in previous studies in lymphoma and lung cancer (35 , 36) , which established that most of the therapeutic effect of the immunoliposomal doxorubicin is associated with the first injection, with subsequent injections having little additional effect. Thus, although immunoliposomes represent an improved drug delivery system, it is apparent that they still have limitations. The inability of immunoliposomes to completely eradicate established tumors has been recognized previously and suggested to be attributable, at least in part, to the inability of the liposomes to penetrate into the interior of existing solid tumor nodules (38 , 39) . Thus, the use of targeted liposomes alone may not be an effective treatment for cancer, but they may be useful when used in conjunction with other therapeutic modalities including surgery, radiation, or immunotherapy.
This study has also provided some important insights with respect to
the potential and the limitations of SCID mice as a model in which to
study and evaluate anticancer therapeutic modalities. Since the first
successful xenograft of a human tumor in SCID mice was reported
(40)
, the SCID mouse has become a popular host for growing
human neoplasms and has been used effectively to evaluate different
therapeutic strategies (8, 9, 10)
. However, the observation
made here that free doxorubicin treatment of tumor-bearing SCID mice
actually decreased survival compared with untreated mice (Fig. 4)
reflects one of the limitations of the SCID model. SCID mice have a
defect in repair of double-stranded breaks in DNA such as those
mediated by free oxygen radicals (41
, 42)
. Although the
exact mechanism of the antitumor activity of doxorubicin remains
unclear, some of its associated toxicities have been attributed to the
formation of free radicals (43)
, which would make this
drug even more toxic in SCID mice. We have observed previously enhanced
toxicity of doxorubicin in SCID mice (8)
and have been
able to alleviate some of the toxicity by scheduling multiple
injections at lower doses, similar to what is observed in humans with
doxorubicin (44)
. We have used this strategy in the work
presented here. One of the distinct advantages of the SCID model is the
ability of human tumors to be engrafted orthotopically and to
metastasize in SCID mice as is demonstrated here. Another advantage of
SCID mice is based upon previous studies that have established that
human immunocompetent cells can be engrafted into SCID mice
(45)
and that these cells remain functional
(46)
. Coengraftment of peripheral blood lymphocytes
(47)
or tumor-infiltrating lymphocytes (48)
into SCID mice have shown that these cells are able to generate an
antitumor response that can be augmented with cytokine immunotherapy.
It is apparent that the SCID mouse would provide a viable model in
which to evaluate a combined cancer therapeutic approach in which
tumor-bearing mice coengrafted with peripheral blood leukocytes are
treated with immunoliposomes initially followed by immunotherapy with
cytokines.
The ß1 integrin in these studies has proven to be a viable target for establishing the proof of principle for immunoliposome delivery to a human lung tumor in our experimental model. The Fab' fragments of antibodies were shown to be effective in directing the selective binding of the liposomes to a ß1 integrin expressing human lung tumor in vitro, and more importantly, the bound liposomes are shown to be subsequently internalized by the tumor. Whether this particular family of heterobifunctional adhesion molecules turns out to be a suitable target to be used in a clinical setting has not been addressed here.
In this regard, it is of interest to note that many different
tumors have been shown to up-regulate their expression of one or more
of the members of the ß1 integrin family. For
example, 1ß1 integrins
are overexpressed in some invasive bladder carcinomas but not in normal
bladder epithelium (49)
. Increased levels of the
2ß1 integrin have been
found on a number of different cancers including non-small cell lung
carcinomas (50)
, pancreatic carcinomas (51)
,
and invasive melanoma (52
, 53)
. However, these integrins
also have a relatively wide distribution pattern on many normal tissues
including epithelium and some leukocytes that may limit their
usefulness as a target for liposome delivery in a clinical setting. The
3ß1 and
4ß1 integrins have a
more restricted distribution pattern in normal cells (54
, 55)
; the 3ß1
integrin is highly expressed in melanomas with dermal invasion or
distant metastases (56)
, and both
3ß1 and
4ß1 integrins have
been found in increased levels on human lung tumors
(27)
. Preferential binding of
anti-integrin-targeted liposomes to tumors in the lung may be further
enhanced by the natural homing tendency of liposomes to the lung and
binding to integrin expressing normal tissue could possibly be avoided
further by injecting liposomes directly into the lung via the pulmonary
artery. Although we have established the feasibility and potential of
targeting liposomes to lung tumors experimentally, additional
preclinical studies are necessary to assess the real clinical utility
and safety of using one or more ß1 integrins as
targets for the delivery of drug-loaded liposomes to tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by USPHS Grants
R01 CA54491 and RO1 CA75235 and the Roswell Park Cancer Institute Core
Grant CA16056. 
2 Present address: Juntendo University School of
Medicine, First Department of Surgery, Tokyo, 113-8421 Japan.
3 Present address: Department of Otolaryngology,
Division of Head and Neck Surgery, New York University Medical Center,
New York, NY 10016.
4 To whom requests for reprints should be
addressed, at Department of Immunology, Roswell Park Cancer Institute,
Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-8668;
Fax: (716) 845-8906; E-mail: bankert{at}sc3101.med.buffalo.edu
5 The abbreviations used are: PEG-DSPE,
polyethylene glycol-derivatized distearoyl phosphatidylethanolamine;
SCID, severe combined immunodeficient; PSA, prostate-specific
antigen.
Received 3/31/00. Accepted 10/ 9/00.
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2, -3, -5, and -6 integrins receptors in pancreatic carcinoma. Int. J. Cancer, 52: 827-833, 1992.[Medline]
2ß1 (VLA-2) in the migration of human melanoma cells on laminin and type IV collagen. J. Invest. Dermatol., 100: 640-647, 1993.[Medline]
3/ßI heterodimer with tumor progression. Int. J. Cancer, 54: 68-72, 1993.[Medline]
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P. Sapra and T. M. Allen Internalizing Antibodies Are Necessary for Improved Therapeutic Efficacy of Antibody-targeted Liposomal Drugs Cancer Res., December 15, 2002; 62(24): 7190 - 7194. [Abstract] [Full Text] [PDF]
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C. M. Lee, T. Tanaka, T. Murai, M. Kondo, J. Kimura, W. Su, T. Kitagawa, T. Ito, H. Matsuda, and M. Miyasaka Novel Chondroitin Sulfate-binding Cationic Liposomes Loaded with Cisplatin Efficiently Suppress the Local Growth and Liver Metastasis of Tumor Cells in Vivo Cancer Res., August 1, 2002; 62(15): 4282 - 4288. [Abstract] [Full Text] [PDF]
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E. Waelti, N. Wegmann, R. Schwaninger, A. Wetterwald, C. Wingenfeld, B. Rothen-Rutishauser, and C. D. Gimmi Targeting HER-2/neu with Antirat Neu Virosomes for Cancer Therapy Cancer Res., January 1, 2002; 62(2): 437 - 444. [Abstract] [Full Text] [PDF]
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0.02). Bars, SD;
n = 5/group.
, no treatment; •, f-dox; 
, s-dox; 
, 2C3-s-dox;
, 1F11-s-dox.